Book - Sex and internal secretions (1961) 5

Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.

SECTION C Physiology of the Gonads and Accessory Organs

The Mammalian Testis

A. Albert, Ph.D., M.D.

Professor 0f Physiology, Mayo Foundation, And Head Of The Endocrinology Laboratory, Mayo Clinic, Rochester, Minnesota

I. Introduction 305

II. Postnatal Development of the Testis 307

III. Descent of the Testis 309

IV. Breeding Patterns 315

V. Architecture of the Testis 317

VI. The Circulatory System of the Testis 318

VII. The Nervous System and the Testis 321

VIII. The Excretory Duct System 323

IX. The Seminiferous Epithelium.... 323

X. The Interstitial Tissue 329

XI. Hormones of the Testis 332

XII. Effects of the Pituitary on the Testis 335

XIII. Effects OF Steroids ON THE Testis. 337

A. Androgens 338

B. Estrogens 343

C. Adrenal Steriods 344

D. Miscellaneous Steroids and Mixtures of Steroids 345

XIV. Effects of Altered Endocrine States on the Testis 346

XV. Nonneoplastic Disorders of the Testis 348

XVI. Tumors of the Testis 349

XVII. Conclusion 351

XVIII. References 353

I. Introduction

The function of the testis is concerned with the preservation of the species. It accomplishes this by producing sperm and hormones. The tubular apparatus is responsible for the manufacture of sperm, and the interstitial tissue gives rise to the hormones. These two compartments are intimately associated with one another embryologically, anatomically, and functionally. Furthermore, they are controlled by separate gonadotrophic hormones of the anterior pituitary. In turn, the secretion and me 305

tabolism of the pituitary gonadotrophins are controlled by the tubules and the Leydig cells. Knowledge of this reciprocal control of pituitary-testis activity was well established by 1940; in general, this reciprocity is the basic frame of reference for the interpretation of all aspects of testicular function. JMore intimate relationships are quite complex and, as will be seen, not completely understood.

It would be gratifying to interpret all aspects of the testis within this fundamental frame of reference. This is not possible at present because the literature is too conflicting and no one has a sufficiently broad experience with testicular endocrinology to sift all of this literature competently. The extreme scatter of literature on the testis furnishes ample evidence for the discontinuity and heterogeneity of effort. Perhaps the main service of this chapter is the compilation in broad categories of the heterogeneous literature of the past 20 years, so that the student may have a handy, albeit incomplete, guide to the subject and to several of the major problems. A preview of the material to be discussed follows.

This chapter pertains to the testis in postnatal life. Acquaintance with the principal facts of the embryology of the testis and with recent developments in fetal endocrinology of the testis is presumed. Only a short description of the postnatal development of the testis is given because encyclopedic coverage is to be expected in other treatises, and because the acquisition of further details of the postnatal development of the testis in various species belongs

306

PHYSIOLOGY OF GONADS

more to the domain of comparative morphology. The basic lessons already have been learned from a few species, and only the jDrovision of an unusual specimen for study could be expected to aid the endocrinologist.

Interest in the effects of cryptorchism has shifted in the 20 years following jNIoore's (1939) summary in the second edition of this book; at that time, the main interest in the cryptorchid testis was in its capacity for hormonal production. At present, the chief concern is with its capacity for spermatogenic function. Despite some labor and much discourse, the treatment of cryptorchism in the human is not satisfactory. Controlled methods of management based on a reasonable working hypothesis have not been evolved, so that a definitive evaluation of results in terms of fertility is impossible.

The architecture of the testis has been described in terms of structural pattern and composition adapted for the formation and transport of sperm and for hormonal production. The influences of the circulatory and the nervous systems on testicular function have received uneven consideration. The former system is essential for testicular function; not only does it bring the necessary gonadotrophic hormones to the testis but, just as important, it provides foodstuffs and oxygen and carries away metabolites. The testis is extremely sensitive to derangement of its blood supply. The peripheral nervous system, however, appears to be relatively unimportant to the postnatal well-being of the testis.

The compartments of the testis are discussed in two sections of this chapter. The germinal epithelium produces sperm, and it is with regard to this compartment that major advances have been made. Quantitative cytologic studies have unraveled the spermatogenic cycle and have provided detailed information on spermiogenesis. These studies are tedious and require painstaking techniques, but there is at present no other way to obtain quantitative information.

The hormonal compartment of the testis has been further clarified by morphologic methods, but the greatest advances have been made by chemists. The biogenesis of male hormone has been worked out and is

discussed in detail in the chapter hj Villee. So far, the hormones manufactured by the testis have been shown to include only steroids. A flare of interest in a water-soluble hormone, namely inhibin, was shortlived, and this issue has been dormant in the past decade.

The next two sections of this chapter, the control of the testis by the pituitary, and the effects of male hormone and other steroids on the testis are representative of classic endocrinology. The dual concept of testicular control by means of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) for the tubular apparatus and the Leydig cells, respectively, is less secure than it was believed to be in 1939. Interest in the fractionation of pituitary gonadotrophins waned in the 1940's, and investigators were unable to obtain purified FSH and LH for experimental study. Furthermore, the discovery that testosterone and other steroids maintained spermatogenesis in the complete absence of gonadotrophins became an irritant to the dualist's composure. Intensive effort in this area has removed some difficulties, but it has not solved the problems. Recent studies have shown that male hormone is needed for spermiogenesis and gonadotrophins for copious spermatogenesis, but there the problem rests.

The effect of alterations in the endocrine system on the testis is discussed briefly. Extremely little has been done in this area except for the influence of altered thyroidal states. As will be seen, the thyroid can exert some influence on testicular function, but this depends largely on the species studied. Further understanding will evolve as more species are studied.

The last sections in the chapter deal with disorders and tumors of the testis. Disorders of the testis, chiefly hypogonadal states, are important in both veterinary and clinical medicine. Study of some of these disorders has greatly clarified normal jihysiology. A brief survey will be given of this aspect to emphasize the pituitary regulation of testicular function as shown by the effects of certain spontaneous disorders of the pituitary. Brief mention will also be made of the awareness of the increasing im])()rtanco of genie factors and of the fetal

MAMMALIAN TESTIS

30;

endocrine system in basic and clinical problems of the testis. The inherited types of infertility in males seem to be an especially rewarding field of investigation. Spontaneous tumors of the testis supply interesting and instructive material for study in both clinical and veterinary medicine. Tumors induced in the testis by experimental means have contributed nothing unique to the problem of oncogenesis. They have, however, provided material for the concept of hormonal dependence" of certain tumors; therefore, they are of importance in the field of cancer.

Not included in this review are studies on the effects of nutritional deficiency, of radiation, and toxic substances on the testis. The first is discussed in detail in the chapter by Leathern. The second has been purposely omitted because it belongs more to the sphere of interest of the radiation biologist than to that of the endocrinologist. It must not be forgotten, however, that knowledge of the relative sensitivity of the various cells of the testis to injury, the first quantitative information on the spermatogenic cycle, and the mechanism of repopulation of the germinal epithelium after severe damage were contributions of the radiation biologist. The third is a hodgepodge of material which at present defies orderly condensation. Despite this, some of the studies in this area are of potential value in providing unique experimental preparations, i.e., animals with testes containing only Leydig cells, or only Sertoli cells. Finally, a miscellany of papers dealing with the general physiology or with the general biochemistry of the testis has also been omitted.

II. Postnatal Development of the Testis

In the past 20 years, voluminous descriptive information has been compiled on the events and consequences of the postnatal development of the testis of mammals. Only a few examples will be given. The developmental anatomy and postnatal changes in the testis of the laboratory rat have been described. Various monographs on the rat (Farris and Griffith, 1949, for references) and Moore's chapter in the 1939 edition are available.

The testis of the guinea pig has a growth spurt about the 20th day of life. The accessory sex structures, such as the vas deferens, epididymis, and prostate, are stimulated somewhat later, at 30 to 40 days. The growth of these accessory organs is an indication of male hormone activity. The time at which hormonal secretion occurs varies among individual animals. Variability (48 to 70 days) occurs also in the appearance of sperm (Sayles, 1939). Under controlled conditions of breeding, Webster and Young (1951) observed that the first intromission in guinea pigs occurs at about 54 days of age. The first ejaculate occurs some 10 days later and is sterile. Fertile ejaculates begin on the average at 82 days of age. Thus, a period of adolescent sterility exists as the result of both lack of ejaculation and a period when there is an insufficiency of spermatozoa. The hamster (Bond, 1945) copulates at 30 days of age but is not fertile until 43 days of age. Adolescent sterility of the male may be a more common phenomenon than is generally apjireciated.

Tile testes of the cat are descended at bh-th. Testicular growth is slow, the combined weight of the two testes increasing from 20 mg. at birth to 100 mg. at weaning. During the 2 months following weaning, the testes attain a weight of 130 mg. A spurt in growth occurs between the third and the fifth month of life, when the testes may weigh 500 mg. This spurt is associated with the appearance of Leydig cells and an increase in the size of the epididymis. Mitotic activity of the germinal epithelium is present in testes weighing 400 to 500 mg. Spermatids appear when the testes weigh 700 mg., and sperm are found fairly uniformly when the testes are more than 1 gm. in weight. At this stage, the tubular diameter is maximal. After maturity, the weight of the testes is generally proportional to body weight. A 5-kg. cat will have testes weighing 4 gm. (Scott and Scott, 1957).

From birth to 80 days of age, the testes of goats grow at a slow but uniform rate. In the immature animal, the tubules are small, measuring 30 fjL in diameter, and are composed of a single layer of cells without any lumen. The interstitial tissue contains only mesenchymal cells. At 90 days of age,

308

PHYSIOLOGY OF GONADS

a lumen appears in the tubules and spermatogenesis begins. At 94 days, maturation of the Lej^dig cells is noted, and spermiogenesis occurs. The diameter of the tubules at maturity is about 100 jx, but the tubule continues to increase to about 160 /x when the goat is 135 days of age. Formation of sperm occurs earlier in goats than in rams, bulls, and boars (Yao and Eaton, 1954).

As in common laboratory animals, the time sequence in the testicular maturation of farm animals is determined by genie factors, which obviously is an important phenomenon economically. In different strains of the ram, for instance, there is a variation of 5 weeks in the time of appearance of primary spermatocytes, of 9 weeks in the appearance of secondary spermatocytes, and of 2 weeks for spermatids (Carmon and Green, 1952).

Among the primates, postnatal development has been studied intensely in the monkey and man. In the rhesus monkey (Fig. 5.1), the tubules attain a diameter of 70 to 80 fx during fetal life (van Wagenen and Simpson, 1954). Only spermatogonia and Stertoli cells containing basal nuclei are present. Mature Leydig cells are also identifiable. Shortly after birth, regression occurs in the tubules, which decrease to a diameter of 50 to 60 )u, and in the Leydig cells, which dedifferentiate into mesenchymal cells. The presence of mature Leydig cells and of differentiated Sertoli cells in the fetal testis

and their involution shortly after birth may be related to the secretion of a fetal morphogenic substance (c/. chapter by Burns).

During the first year after birth, the few spermatogonia increase in number and size. During the second year, they become more numerous and the tubules increase in length. However, the germinal epithelium remains quiescent until late in the third year. At this time, the Sertoli cells increase in number and differentiate. The Leydig cells mature again. The tubular diameter now is about 100 jx. A lumen appears when the tubules are 100 to 150 /x in diameter. Spermatids appear, and orderly spermatogenesis occurs. The prepubertal period in the monkey is about a fifth that of man; except for the factor of time, the sequence of development in the monkey testis resembles that in man (Figs. 5.2 and 5.3). In the pubertal monkey Leydig cells and tubules are stimulated simultaneously. Some observers have reported that maturation of Leydig cells occurs after tubular maturation in humans (Sniffen, 1952; Albert, Underdahl, Greene and Lorenz, 1953a) and in bulls (Hooker, 1948). However, this point may depend on the choice of the criteria for tubular stimulation (tubular wall versus germinal epithelium) and for function of Leydig cells (morphologic differentiation versus secretory activity) .

Some interesting details on the relative

Grams

8000 7000 6000 5000 4000 3000 2000 1000

Tubule growth slow

Centrol Sertoli nuclei ^^^ s^^^

y^ Fertile Sperm Tubule growth ropid

eo^V>^

^^/-"^^'^

Bosol Sertoli ^ — "^

^y^ Testis lenqih|n__c|]]_^^_____ — ,^

^^^^^^

-^^^^ -^

'

Cm

4 3 2

I

Age in Years

Fig. 5.1. Graphic representation of changes in testes of the rliesus monkey during development. Coordinates are body weight and age of animals, and length of testes. (From G. van Wagenen and M. E. Simpson, Anat. Rec, 118, 231, 1954.)

MAMMALIAN TESTIS

309

weight of the testis in primates have been supplied by hunting and scientific expeditions. Schultz (1938) studied 87 adult primates. The relative testicular weights (testicular weight divided by body weight X 100) varied between 0.1 and 0.4 in American monkeys. Considerably more variation was seen in Old World monkeys. The relative weight in five species of macaques varied between 0.46 and 0.92, but in langurs was only 0.06. The weight ratio in the orangutan was 0.05, in the gibbon and man 0.08, and in the chimpanzee, 0.27. Rough estimations of the ratio of the volume of interstitial tissue to tubular volume showed that the macacjue has a greater relative volume of interstitial tissue than man. Testicular weights vary according to race; Japanese men have smaller testes than American men (Schonfeld, 1943) .

The human testis at birth consists of small tubules measuring 50 to 75 yn in diameter and arranged in cords containing several rows of darkly staining nuclei. The epithelium is mostly undifferentiated, but large cells with sharp boundaries are present. These are primary germ cells. The interstitium of the testis is highly developed and contains solidly packed Leydig cells. After birth the Leydig cells disappear from the interstitium in a matter of a few weeks. From this time, the testis remains generally quiescent until puberty, when Leydig cells reappear as a result of the secretion of gonadotrophin. Only mesenchymal cells resembling fibroblasts characterize the interstitium in the period from a few weeks after birth until puberty. The germinal cords acquire a lumen at approximately 6 years of age, although this landmark shows considerable variation. The nuclei of the germinal cells at this time are arranged in two layers.

At puberty, which may occur at any age from 9 to 19 years, a great increase in size and tortuosity of the tubules occurs. The lamina propria develops, the Sertoli cells become differentiated, and the seminiferous epithelium gradually matures. The Leydig cells mature somewhat later than the changes noted in the tubules and become characteristically arranged in groups in the intertubular zones.

After maturity is attained, the adult his

tologic pattern may be maintained into old age without pronounced changes. Spermatogenic activity varies from tubule to tubule, but an over-all picture shows spermatogenesis proceeding in an orderly fashion, with sperm heads closely approximating the luminal end of the Sertoli cells. About twothirds of the tubule is occupied by the germinal epithelium. The lumen makes up about 15 per cent of the tubule. The proportionate volumes for the germinal cells are as follows: spermatogonia, 24 per cent; spermatocytes, 45 per cent; spermatids and spermatozoa, 29 per cent; various abnormal cells, 2 per cent. The Sertoli cells occupy about one third of the tubular epithelium. The Leydig cells occupy 9 per cent of the total intertubular spaces. About 66 per cent of the human adult testis is composed of tubules and about 22 per cent is made up of the intertubular spaces (Table 5.1). With age, progressive fibrosis occurs in the human testis, the width of the tubular wall increases, and thinning of the germinal epithelium occurs (SnifTen, 1952; Charny, Constin and Meranze, 1952; Albert, Underdahl, Greene and Lorenz, 1953b; de la Baize, Bur, Scarpa-Smith and Irazu, 1954; Roosen-Runge, 1956).

III. Descent of the Testis

The descent of the testis from an abdominal position in the fetus was known to the ancients (Badenoch, 1945). This change in position is a mammalian phenomenon. In the Monotremata and most of the Edentata, the testes are abdominal. Some of the Insectivora, Cetacea, and Sirenia also have abdominal testes. The testes in marsupials lie suprapubically in a pouch that has a closed vaginal process. The testes of Aplodontia rufa, the most primitive rodent extant, occupy a semiscrotal position during the breeding season; otherwise, the testes are abdominal (Pfeiffer, 1956). In some rodents, Lnsectivora and Chiroptera, the testes are intra-abdominal in the resting stage, but during the rutting season they are pulled into the scrotum by muscles. In the Ungulata. Carnivora, and Primates, the testes are extra-abdominal. Exceptions are the elephant and stag, whose testes are retracted in the nonrutting season. Thus, "cryptorchism" is normal for many mammals, and

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? To^lls of .1 96-(lav fetus Tul)ules aie shoit and stiaight. diametei is 60 to 70 /j. Oiih Sertoli cells and a few spermatogonia are present in tubules. Sertoli cells are large and fill the lumen. Intertubular spaces are wide and contain many cells, some epithelioid.

3. Biopsy of testis at birth (174-day gestation). Tubular diameter is 60 to 80 fj.. Nuclei of Sertoli cells are basal, and cytoplasmic strands fill tubular lumen. Spermatogonia are sparse. Iiitertiil)ular tissue is abundant but imdifforentiated.

MAMMALIAN TESTIS 311

the term should be restricted to designate nal cavity. Early work by Moore (reviewed an abnormal testicular position in those spe- by Moore, 1951) showed that the testes cies whose testes normally are scrotal. In of rats, rabbits, and guinea pigs become such species, cryptorchism of sufficient du- atrophic within a few weeks when placed ration results in an irreversible loss of sper- in the abdominal cavity. Moore wrapped the matogenic function and in a variable failure testes of the ram with wool batting, with of hormonal function. the result that the ram was sterilized by its In most animals the testicular tempera- own body heat. The application of water ture afforded by the scrotum is 1 to 8°C. 5°C. above body temperature to the scrotum lower than the body temperature. In man of guinea pigs causes temporary sterility, the testicular temperature is 1.5 to 2.5°C. The deleterious effects of increased temperalower than the temperature of the abdomi- ture also are observed in man. Fever, dia 4- Testis at 3 months. More coiling of tubules is present; diameter is 50 to 60 /i. Cytoplasm of Sertoli cells is developed and still fills the lumen. A few spermatogonia are present. Intertubular spaces are narrow and interstitial cells have regressed.

5. Testis at 3 months and 25 daj's. Considerable increase in length, with coiling of tubules, has occurred. Tubides are small (50 to 60 n), compact, and filled with the Sertoli nuclei. There are occasional spermatogonia. The peritubular arrangement of dark-stained nuclei of intertubular tissue is clearty seen.

6. Testis at 4 months and 24 days. Tubules are small and closely packed. The size has not changed (50 to 60 m). The Sertoli nuclei fill the lumen, only occasional spermatogonia being seen.

7. Testis at 1 year, 3 months, 24 days. Tubules are still small (40 to 50 fi). Spermatogonia are now increased in number and size. Nuclei of vmdifferentiated cells fill the lumen. Only dark-stained nuclei in rows around tubules are seen in narrow peritubular spaces.

S. Testis at 1 year, 8 months, 7 days. Tubules are still small (50 yu). The Sertoli nuclei continue to crowd the lumen. Intertubular tissue is undifferentiated.

9. Testis at 2 years, 7 months, 6 days. Note that during an entire year no appreciable advance in development has occurred except in multiplication of the Sertoli nuclei and a slight increase in tubular diameter (60 to 70 /n). Some of the interstitial cells are now lighter staining.

10. Testis at 2 years, 9 months, 21 days. Tubules are now definitely larger (90 /i). Sertoli cells have moved to the periphery of the tubule. Spermatogonia are nvmiorous and rounded. Rounded Lej'dig cells are now freciuently seen.

11. Right testicular biopsy at 2 years, 4 months, 11 days. Tubules are long, convoluted, and closely packed, measuring 50 to 70 m in diameter. Sertoli cells are poorly differentiated, but the cytoplasm is increasing and the nuclei have partially moved basally. Interstitial cells are small and sparse.

12. Right testicular biopsy at 2 years, 9 months, 16 days. The changes are slight. Tubules are somewhat larger (70 m) and the Sertoli nuclei are more basal. No differentiation of the Leydig cells is seen.

IS. Right testicular biopsy at 3 years, 1 month, 26 days. Tubules have increased slightly in diameter (80 to 90 m)- The Sertoli nuclei are now definitely basal. A few cells, recognizable as spermatocytes I by the spireme, are present. Interstitial cells are still not clearly recognizable.

14. Left testis at 3 years, 2 months, 14 days. Tubules measure 100 to 120 /a. Many spermatocytes are now present. Canalization is seen in a few tubules. A few epithelioid Leydig cells are present singly or in pairs.

15. Testicular biopsy at 2 years, 7 months, 14 days. Tubules measure 70 m. The Sertoli nuclei are basal. A few desciuamating cells are present in the meshes of the Sertoli cytoplasm. Occasional Leydig cells are recognizable.

16. Testicular biopsy at 3 years and 17 days. Tubules measure 70 to 80 m- The Sertoli nuclei are basal. Vascularity has increased, and more space is present between tubules. Epithelioid Leydig cells are present, although not of mature size. Spermatocytes are appearing.

17. Left testis at 3 years, 2 months, 14 days. Tubules measure 100 to 120 fi. Formation of spermatocytes I is abundant.

18. Testicular biopsy at 3 years, 5 months, 28 days. Tubules measure 130 to 150 ti. Many spermatids and some sperm cells are present. Some desquamation of immature cells is still present in the tubular lumen. (From G. van Wagenen and M. E. Simpson, Anat. Rec, 118, 231, 1954.)

312

PHYSIOLOGY OF GONADS

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Fig. 5.3. Developmental stages in the monkey testis {continued jrom Figure 5.2)

19. Testis of 110-day fetus. Note short, uncoiled tubules, with a diameter of 70 to 80 ti. Cytoiilasm of Sertoli cells is well developed and largely fills the lumen. The broad intertubular spaces contain abundant cells, many of which are enlarged and roun<led.

20. Testis of 110-day fetus. This shows tubules in cross section. The orientation of the interstitial tissue concentrically around the tubules and the enlargement of the cells within the concentric rings are evident.

21. Testis at birth after a 174-day gestation. Note the Sertoli nuclei remaining in a basal position and the persistent wide intertubular space; however, the size of the Leydig cells has decreased.

22. Right testis at 2 years, 10 months, 3 days. Tubular diameter is 100 to 120 fi. Spermatids have differentiated to presperm cells or almost mature sperm cells. Great amounts of cellular debris fill the lumen. Few Leydig cells are differentiated.

23. Right testis 1 month later, at 2 years, 11 months, 1 day. Tubular diameter is 100 to 140 fi. Sperm cells are present.

MAMMALIAN TESTIS

313

thcrniy, or heating of the testes by other methods produces temporary depression of the sperm count (see Hotchkiss, 1944b, for review) . These experiments support the concept of the thermoregulatory function of the scrotum. These studies also indicate that spermatogenesis in mammals can proceed only at an optimal temperature.

Cryptorchism in man has received considerable attention because it is a common clinical problem. The incidence of cryptorchism in man is given as 10 per cent at birth, 2 per cent at puberty, and 0.2 per cent at maturity (Nelson, 1953). The testis of man develops between the adrenal glands medially and the body wall laterally, ventral to the metanephros, and in the 20mm. fetus is not far from the groin. The gubernaculum develops from the plica inguinalis and attaches to a pocket of the abdominal wall which forms the vaginal process (Wyndham, 1943). At the 2nd month of fetal life, the testis is elongated, extending from the diaphragm to the site of the future abdominal inguinal ring. By the 3rd month, the cranial end of the testis undergoes involution. By the 4th to the 7th month, the testis is in the iliac fossa near the internal ring. Descent occurs in the seventh month, and the testis becomes scrotal during the 8th fetal month. The gubernaculum shortens as descent takes place and finally becomes a vestige in the adult.

Prenatal descent of the testis into the scrotum also occurs in mammals, such as the monkey, horse, bull, pig, sheep, and goat. Descent occurs postnatally in the opossum, whereas it takes place in the pubertal period in rats, mice, rabbits, and guinea pigs. The monkey occupies a somewhat intermediate position. The testes descend before birth but then migrate back into the abdominal cavity until puberty when descent occurs for the second time

TABLE 5.1

Comparison of average volume of structures in the testis of man and the rat All figures are percentages of total testicular volume. (From E. C. Roosen-Runge, Fertil. & Steril., 7, 251, 1956.)

Interstitial tissue

Leydig cells

Basement membrane

Total interstitial space

Spermatogonia

Spermatocytes

Spermatids and spermatozoa

Abnormal germ cells

Residual bodies

Total germ cells

Sertoli cells

Lumen

Space

Man

22.0 3.1 9.1

34.2

7.8 14.4 9.1 0.7

32.0 17.4 10.6

5.8

8.0 1.7 2.4

12.1 1,7

14.7

41.1 0.1 1.2

58.8 8.4

19.5 1.1

(Wells, 1944) . In seasonal breeders like the ground squirrel, the testes alternate between scrotum and abdomen with the breeding and nonbreeding seasons.

The mechanism of testicular descent is not entirely understood. The gubernaculum seems to act as a guide for the descending testis, but it is not essential for descent. Excision of the gubernaculum does not prevent descent (Wells, 1943a, b). Growth of the testis is not a determining factor in its downward migration. Martins (1943) showed that substitute testes in the form of paraffin pellets can be made to descend in castrated rats by the administration of testosterone. Therefore, it appears that descent is determined by androgens affecting the testis and accessory structures. The questions as to what androgens are responsible for testicular descent, where they are formed, and how they are controlled remain unanswered. Because Leydig cells appear in the fetal testis of man during the fourth month and because Ferner and Runge

24. Right testis after another month, at 3 years and 1 day. Tubular diameter is 120 to 150 fi. Tubules are tightly packed and it is difficult to find Leydig cells. Cellular debris has been cleared from most tubule.s, and a new generation of spermatids with orderly arrangement is present.

25. Right testis 5 months later, at 3 years, 5 months, 1 day. Tubular diameter is 150 ^l. Leydig cells are extremely rare. There are many presperm cells and a few mature sperm cells free in the lumen.

26 to 2S. Adult testis, age 11 years, 6 months, 20 days. 26. Seminiferous tubule lumen lined by j^oung spermatids. 27. Tubule lined by spermatids with spermlike heads. 2S. Tubule containing mature sperm cells about ready to be shed. (From G. van Wagenen and M. L. Simpson, Anat. Rec, 118, 231, 1954.)

314

PHYSIOLOGY OF GONADS

(1956j have .shown that these Ley dig cells give strong histochemical reactions suggestive of the presence of steroidal materials, it is presumed that the human fetal testis produces androgens responsible for its descent. Were this true, it would appear reasonable that human chorionic gonadotrophin produced throughout the pregnancy could be responsible for the formation and secretion of androgenic substances by the fetal testis. Such an action would provide a function for human chorionic gonadotrophin during the last two trimesters of pregnancy. However, Wells (1944) held androgens from adrenal sources responsible for testicular descent.

The precise control of testicular descent is not known. Gonadotrophins effective on the Leydig cells induce rapid descent. Androgens hasten descent and estrogens inhibit it (Mussio Fournier, Estefan, Grosso and Albrieux, 1947). However, Finkel (1945) concluded that descent may be a genetic phenomenon in the opossum, which seems to be different from rodents in many aspects of testicular physiology. Because hormonal secretion is not detected in the opossum until the 100th day and because descent occurs earlier, it was doubted that androgens are responsible for descent.

The effects of cryptorchism on the histologic appearance of the human testis have been the subject of many studies. However, few new observations have been made since the excellent description by Cooper (1929) of the normal and retained testis in man. Cooper studied abdominal and scrotal testes at various ages from birth to senility and concluded: (1) the farther the prepubertal testis descends, the more similar it is to its normal scrotal mate; (2) sperm cells are rare in retained testes but occasionally may be found in testes held at the external ring; (3) the younger the child, the more normal is the retained testis; (4) the Leydig cells are not affected adversely by retention, nor are they more numerous, as earlier observers had thought. As a result of these conclusions, surgical treatment of retained testes in the first two years of life was advised.

Rea (1939) concluded erroneously that the undescended testis is normal until puberty but stated correctly that it rapidly de

generates after puberty. That the retained testis may not be normal before puberty and is almost always abnormal after puberty was confirmed by Nelson (1953), and by Robinson and Engle (1954). Before the age of 5 years, the scrotal testis and the cryptorchid testis are indistinguishable histologically; after this time, the cryptorchid testis is always retarded in growth and differentiation. Puberty is accompanied by growth of both the scrotal and the retained testis, but the retained testis does not keep pace with its scrotal mate. Nelson (1951) pointed out that fewer spermatogonia are present in the retained testis than in the scrotal mate and that this difference becomes proportionally greater after puberty. The testis retained for a long time becomes fibrotic. The germinal epithelium (excepting the Sertoli cells) is destroyed but the Leydig cells are said to be normal. However, Sohval (1954) found that the number of Sertoli cells in the pubertal retained testis is less than that in the normal scrotal testis. In older men, the Sertoli cells may be obliterated, in which case the tubules eventually become hyalinized. This is not an invariable sequence in testes retained for long periods. Sohval stated that a man, aged 42, produced sperm from a retained testis.

The comments just presented are based on the supposition that failure of descent occurs in normal testes because of adhesions or abnormalities of the canal, or because of failure of the normal stimulus for descent. However, lack of descent also may result because the testis is intrinsically defective (Nelson, 1953). Three boys were described whose undescended testes did not contain germ cells; however, the descended testes also did not have germ cells, so the relationship between germinal aplasia and nondescent is not clear. Another type of abnornuility was described by Sohval ( 1954) and referred to as "tubular dysgenesis." These tubules were characterized by the absence of Sertoli cells and spermatogonia. Only undifferentiated cells were present. Furthermore, these tubules did not become fibrotic after puberty. Dysgenetic tubules were observed in about half of the cases of cryptorchism.

Little doubt exists, therefore, that the fer

MAMMALIAN TESTIS

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tility iiotential of cryptorchid human testis, regardless of the cause, is seriously damaged. Sterility is not an inevitable consequence of bilateral cryptorchism in man as indicated by the report of sperm in the ejaculate of bilaterally cryptorchid men (Sohval, 1954). The work of Gross and Jewett (1956) also indicates that cryptorchism does not always produce irreversible damage because some patients do become fertile when orchidopexy is carried out at puberty.

The studies of Engberg (1949) and Raboch and Zahof (1956) indicate that the function of the Leydig cells of cryptorchid testes also may be impaired. The former found that bilaterally cryptorchid men excreted reduced amounts of urinary androgen and estrogen and had a lessened concentration of acid phosphatase (a secondary sex characteristic) in the semen. The latter two authors, contrary to previous reports, noted a high incidence of regressive Leydig cells. Also, severe androgenic insufficiency occurs in bilateral cryptorchid men in middle and old age. The affect of cryptorchism on hormonal function is best ascertained in experimental animals and will be considered shortly.

Spontaneous cryptorchism is found in many animals. Schultz < 1938 1 found that 5 per cent of the wild gibbons shot during the Asiatic Primate Expedition of 1937 were cryptorchid. Many veterinary reports show cryptorchism in various farm animals and pets. Cryptorchism also results from a deficiency of biotin in the laboratory rat (Manning, 1950). None of these reports deals with functional aspects of retention.

Experimental cryptorchism maintained for 28 days in the rat is followed by recovery of spermatogenesis in 40 to 100 days after restoration of the testes to the scrotum. With a proportionally longer sojourn in the abdomen, fewer and fewer of the tubules recover until finally tubular damage is irreparable. Androgenic function is gradually lost. Castration cells appear in the pituitary in 75 days, the seminal vesicles are reduced in size after 240 days, and the prostate becomes atrophic in 400 days. This sequence parallels the requirement for androgen; the seminal vesicles need more androgen (2.5 times) than does the prostate for maintenance, and the prevention of cas

tration changes in the anterior pituitary requires twice as much androgen as does maintenance of the seminal vesicles (Nelson, 1937). These results were confirmed by Moore (1942), who added that the effects of cryptorchism on androgen production are dependent on the age at which it is induced. Old rats are less affected than young animals.

In the guinea pig, old experiments showed that tubular degeneration occurs rapidly but that secretion of androgen continues for more than a year. Using the condition of the accessory sex organs and sex behavior as end points for the activity of male hormone, Antliff and Young (1957) found no evidence of diminished production of androgen in animals made cryptorchid two days after birth.

In the boar, bull, and stallion secondary sex characteristics can be maintained by bilateral retained testes or by the testis made unilaterally cryptorchid after removal of the scrotal mate (Moore, 1944). Schlotthauer and Bollman (1942) removed one scrotal testis from an adult dog and placed the other in the abdomen; the prostate was maintained for two years. Hanes and Hooker (1937) found that cryptorchid testes in swine contained only half the normal amount of androgen. Kimeldorf (1948) reported that cryptorchism in rabbits results in a decrease of some 40 per cent in the total urinary 17-ketosteroids, a change similar to that after castration. He suggested that altered metabolism of testicular hormone caused by high temperatures may be responsible for this decrease. In general, then, the cryptorchid testis is capable of producing androgen but, depending on species, probably m amounts less than normal. The longer the state of cryptorchism exists, the more deficient is the capacity to secrete androgens.

IV. Breeding Patterns

The breeding pattern of adult male mammals may be divided into two major types (Moore, 1937). The first type is that of the seasonal breeder, including such animals as the ground squirrel, weasel, stoat, ferret, mole, hedgehog, and shrew (Asdell, 1946). In these animals, there is a short period of time, the duration of which varies in differ

316

PHYSIOLOGY OF GONADS

ent species, in which breeding is at its height. Sperm cells are formed only during this period. The accessory sex structures are under intense androgenic stimulation. After the breeding season, regression occurs. For the remainder of the year spermatogenesis does not take place and the state of the accessory system resembles that of a castrated animal (Fig. 5.4).

The second type is the continuous

breeder. Rat, mouse, guinea pig, rabbit (under laboratory conditions) , and man are well known examples. In this type, the testes and accessory sex structures maintain a constant state of activity, and breeding can occur at any time of the year.

The ram is an intermediate type of breeder, in which spermatogenesis is arrested in the nonbreeding season with a concomitant decrease in number of Leydig

LATERAL FAT

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Fig. 5.4. Reproductive tract of a seasonal hrrrdrr, the prairie dog, Cynumys. a. Anatomy of male reproductive tract and its abdoiinii.il i( l.ilionships during period of sexual activity. b. Diagrammatic representation of the PCNu.illy adive and the involuted reproductive tracts (dorsal view). (From A. Anthony. J. Morpliol., 93, 331, 1953.)

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cells. Abnormal forms of sperm cells appear in the semen, reflecting the degeneration of the germinal epithelium at this time. The volume of the semen decreases, and libido is depressed. Thus, the ram may be regarded as an annual breeder, but the regression in the nonbreeding season is not complete (Maqsood, 1951a). Even the laboratory rat shows some evidence of a seasonal rhythm (Gunn and Gould, 1958). The capacity of the dorsolateral prostate to concentrate injected Zn*^^ is greater in February-March and June-July than at other seasons of the year.

Various species of squirrels have been favorite subjects for the investigation of certain features of seasonal breeders (Mossman, Hoffman and Kirkpatrick, 1955; Kirkpatrick, 1955.1. The testes of the infantile fox squirrel contain small, round, lumenless cords with a single row of spermatogonia, Sertoli cells, and a few spermatocytes. The Leydig cells are undifferentiated. The prepubertal fox squirrel has larger tubules with lumina. The testes at this time have increased about five times in weight, and the tubules are twice their former diameter. Spermatids are present.

The mature fox squirrel has free sperm cells in rather wide lumina, and mature, large Leydig cells. In the nonbreeding season, the spermatids and spermatozoa degenerate, leaving only spermatogonia, Sertoli cells, and a few spermatocytes. The tubular wall becomes contracted and thickens, causing the shape of the tubule to be irregular. The Leydig cells atrophy. In December and January, when the breeding season starts, the process of sexual maturation is repeated. This process recurs annually ; however, with each year, the tubular wall becomes more irregular in shape demonstrating that periods of maturation and degeneration have taken place previously.

V. Architecture of the Testis

The testis of most mammals is divided into lobules by means of septa, and the entire testis is enveloped by the tunica albuginea, which keeps it under pressure.

The testis of the rat, however, has no septa; instead, the organ is constructed in a fan-shaped manner from a series of spiral canal arches, which arise from the rete testis (Fig. 5.5). The tubule is a Ushaped structure, open at two ends to the

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Fig. 5.5. Architecture of rat testis, showing relationship of tubules to spermatic artery. and arrangement of the Leydig cell aggregates and capillaries. (From I. MUller, Ztschr, Zellforsch. mikroscop. Anat., 45, 522, 1957.)

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PHYSIOLOGY OF GONADS

rete and mnning such a constant zigzag course within the testis that a palisade is formed. An average tubule is 30 cm. long. The tubules do not end blindly as a rule. They rarely fork or bifurcate and they never communicate with one another (Mliller, 1957; Clermont. 1958).

The suitability of architecture of the tubule for the process of spermatogenesis is obvious because it provides an epithelium with a large surface area. The arrangement of the tubules in arcs and palisades allows long tubules to be packed neatly in a small, ovoidal organ. The lumen of the tubule constitutes a pathway for the transport of sperm to the outside. Because sperm cells are transported passively, some mobile medium is needed. The obvious medium is fluid which can be transferred along the length of the tulnile. It is not known where and how such fluid enters the tubule and how it moves through the lumen as it transliorts sperm to the ductuli efferentes.

If fluid moves constantly along the length of the tubule to carry sperm, it must be reabsorbed from the excretory duct system. The ductuli efferentes, derived from the mesonephros, may play a role analogous to that of a nephron in reabsorbing large ciuantities of fluid from the seminiferous tubules (Ladman and Young, 1958). The cytologic organization of ciliated and nonciliated cells in the ductuli efferentes and rete testis of the guinea pig seems compatible with the presumption that these cells absorb fluid from the lumen and excrete it by way of the ductular system. Physiologic evidence of the transport of fluid within the excretory duct system will l)e given in Section VIII.

The architecture of the interstitium appears to be well adapted for the internal secretory function of the Leydig cells. Wedges of connective tissue are present in the interstices bounded by three tubules. The wedges contain Leydig cells, blood vessels, and connective tissue. Branches of the testicular artery feed the capillary network in the connective tissue wedges. The wedge capillaries are in close relationship to the Leydig cells. The topography of the capillary system of the rat testis is such that blood, after contact with the interstitial cells, flows by the g('nerati\'c portion

of the testis before entering the general circuation through the great veins at the hilum. This architecture apparently makes it feasible for hormones of the Leydig cells to exert local action on the tubule.

VI. The Circulatory System of the Testis

The testicular artery of mammals convolutes before reaching the testis. It is surrounded by the pampiniform plexus which is thermoregulatory, serving to preheat or to precool the blood. The convolutions of the testicular artery constitute a distinctive feature of mammals (Fig. 5.6). Lower vertebrates have segmental arteries, the testes do not descend, and the arteries do not lengthen or convolute. Marsupials differ in that the artery forms a rete marabile from which a short artery enters directly into the testis. The testicular artery in the dog forms 25 to 30 loops before entering the tunica albuginea. In the goat, the artery convolutes many times but finally branches into 3 or 4 convolutions that enter the testis from all sides. The testicular artery in the mouse has half-loops; except for man, it shows the fewest convolutions of all the mammalian testicular arteries thus far investigated by arteriography. The situation in the monkey is similar to that in the dog, whereas the artery in the cat and the guinea pig has between 5 and 10 loops.

The testicular artery of man is unique in two respects. It is the longest and thinnest artery in all the viscera, and it is also the straightest testicular artery in the 50 mammals thus far investigated. The testicular artery of man after giving off branches to the cord and epididymis generally runs on the posterior border of the testis. It bifurcates, each branch penetrating the tunica over its lateral and medial aspects (Harrison and Barclay, 1948). The testicular artery in man has a direct anastomosis with the vasal and cremasteric arteries (Harrison, 1948a, b; 1949a, b; 1952; 1953a, b).

The gradient in temperature between the i:»eritoneal and scrotal cavities varies widely among different species. A large gradient ocelli's in the goat, rabbit, rat, mouse, and ram ; a small gradient is i)resent in the monkey, (log, guinea pig. and man. The tcm

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319

.1

^frv

^^fcw^

--^-sffc:

Fig. 5.6. Vascular patterns of the testi.- ni ,i i. \\ maniinals. Roentgenograms of testicular artery injected with opaque medium from (1 ) (.log, (..^) goat, (3) ram, (4) mouse, (5) rat, (6) rabbit, (?) guinea pig, (S) cat, and (9) monkey. (From R. G. Harrison and J. S. Weiner, J. Exper. Biol., 26, 304, 1949.)

perature gradient depends on many factors, such as the convolutions of the artery, the length of the artery, the size of the testis, the relationship between veins and arteries, and the activity of the dartos muscle (Harrison and Weiner, 19491.

Inasmuch as temperature affects the testis directly, and also indirectly by way of the circulatory system, it is necessary to deal separately with the direct effects of temperature on the testis, the effects of environmental temperature, and the effects of circulatory occlusion. It is generally agreed that heat applied locally is injurious to the testis. Moore's experiments in which the testes were wrapped in insulating material

already have been mentioned. In the guinea pig, sex activity and fertility are depressed for 44 to 72 days after exposure to heat (Young, 1927). Similar effects may be obtained (Williams and Cunningham, 1940) by heating rat testes with infrared lamps or by heating dog testes with microwaves from a radar source (Williams and Carpenter, 1957). In men, a single bout of fever (MacLeod and Hotchkiss, 1941) that increased the body temperature to 40.5°C. caused a depression in the sperm count. After return of the sperm count to normal, another episode of fever induced another depression. The production of androgen is not affected by exposure to high environ

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PHYSIOLOGY OF GONADS

mental temperature (Stein, Bader, Eliot and Bass, 1949). The local application of heat does not markedly suppress the production of androgens, judging from the older work with rats and guinea pigs. To the contrary, some evidence exists that secretion of androgens may be enhanced. After scrotal insulation, bulls were more ready to serve and excreted more androgenic" steroid than normal. The amount of fructose in semen (an indicator of androgen) increased after scrotal insulation in the ram (Glover, 1956) . Increased temperature originating locally may affect spermatogenesis in man. Davidson ( 1954) studied semen in cases of oligospermia before and after removal of varicoceles. Removal of the varicocele was followed by an increased number of sperm cells and a greater incidence of fertility. Defective fertility presumably was caused by interference with normal heat transfer because of the varicocele.

Local application of cold to the testis or scrotum also results in testicular degeneration (Harris and Harrison, 1955) ; however, testicular tissue can be frozen and stored and still retain transplantability and subsequently produce hormones and sperm (Parkes, 1954; Parkes and Smith, 1954; Deanesly, 1954).

The effects of temperature when the entire animal is subjected to thermal changes depend on numerous compensatory alterations in testicular circulation. The compensatory mechanisms differ in both ciuality and degree, depending on the nature of the experimental conditions. The testicular temperature of rodents placed in a hot room does not increase to a higher level than that of the general body temperature. This is true also for the ram. Within fairly wide limits of environmental temperature (10 to 40°C.), the intratesticular temperature of the bull is constant. However, the temperature of the scrotal surface increases slightly with increases in air temperature, but remains below body temperature (Riemerschmid and Quinlan, 1941). The homeostatic mechanisms for maintaining a constant, optimal testicular temperature are several. With increasing scrotal temperature, the scrotum extends and the testes lie lower. This increases heat exchange, despite ilw absence in certain species (mouse, rat, dog.

cat, and rabbit) of scrotal sweat glands (Harrison and Harris, 1956). Optimal testicular temperature is also maintained by means of heat exchange between vessels of the pampiniform plexus. Exact data on the transfer of heat are not available, because determinations of the blood ffow have not been done.

There remains for consideration the effect on the testis of severe alterations in circulation. The histologic changes produced in the rat testis by temporary or permanent occlusion of the testicular artery were studied in great detail (Oettle and Harrison, 1952). Acute temporary ischemia (10 to 20 minutes in duration) produced only hyperchromasia of the spermatogonia. Normality was restored within 2 weeks. Ischemia of increasing duration produced correspondingly increased testicular damage. Hyperchromatic changes in the spermatogonia, loosening and exfoliation of the germinal epithelium, and desquamation of the mesothelium of the tunica occurred. The testis shrunk, the interstitium became edematous, and the Leydig cells swollen. A layer of ragged and vacuolated Sertoli cells, a few spermatogonia, and an occasional primary spermatocyte may be the only surviving elements. When the damage was extreme, the tubule became markedly atrojihic, the lumen disappeared, and the Sertoli cells became embedded in a collagenous matrix.

Permanent occlusion of the testicular artery in the rat can be accomplished by removing a segment of the artery within the abdominal cavity proximal to its anastomosis with the vasal artery (Fig. 5.7), which results in incomplete ischemia. After 1 hour of such occlusion, hyperchromasia of the spermatogonia occurs, with exfoliation of the spermatids. After 6 hours, the spermatocytes are exfoliated. One day later the testis enlarges considerably owing to edema. Multinucleated cells appear and many show pyknosis. The cytoplasm of the Sertoli cells disintegrates. After 3 days, all tubules are abnormal ; within 1 week, they are necrotic. The damage is restricted at first to the central jwrtion, but within a week practically all tnbulcs except some near the epididymal pole have been killed. Two weeks later, vacuolation occurs in the Leydig cells, with

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an accumulation of yellow pigment. By the end of a month, the interstitium becomes invaded by fibroblasts. The tubules, although not yet shrunken, show a thickened basement membrane. The necrotic contents conglomerate into a mass. After 7 months, pronounced interstitial fibrosis is present, extending from the periphery toward the center. Plasma cells are seen. Some of the Sertoli cells survive. The tubular debris is removed. Thus, it seems that the Leydig cells are most resistant to arterial occlusion. The Sertoli cells are the next most resistant, followed by the resting spermatogonia. The active differentiating cells are most susceptible to arterial occlusion.

A different type of lesion is produced by ligation of the superior epididymal artery. Focal necrosis of the initial segment of the caput occurs (Macmillan, 1956; Harrison and Macmillan, 1954). This disrupts the pathway between the vasa efferentia and the ductus epididymidis. The vasa distal to the ligature become choked with sperm within 3 days. The testes enlarge and then atrophy. In this manner, permanent atrophy of the testes occurs, with azoospermia due to obstruction.

Vn. The Nervous System and the Testis

It is difficult to see nerve endings in the parenchyma of the testis. Van Campenhout (1947, 1949a, b) described masses of paraganglionic cells in the midportion of the genital ridge of the testis during development. The fibers of these cells are intimately associated with the interstitial cells. The testes of 22-day-old pigs contain numerous neuro-interstitial connections between nerve fibers and groups of Leydig cells in the hilar zone or near the tunica.

The origin of testicular nerve fibers is not entirely clear. The general belief is that the testis receives fibers from the lumbar sympathetic chain. These nerve fibers innervate only the blood vessels in the rat and cat. Varying reports have been made of the nervous connections in man. Apart from vasomotor and sensory nerves, few fibers enter the human testis. These follow the course of the arteries to the septula and make contact with the Leydig cells. Three types of contact are made, namely (1) peri

FiG. 5.7. Diagram of arterial supply of rat testis. The testicular artery (a), as it nears the testis, becomes tortuous just after giving off a branch (c) to the head of the epididymis that also supplies the fatty body (upper right). On reaching the testis, the testicular artery goes to the deep surface of the tunica albuginea. After coursing around the inferior pole, the artery winds up the anterior border of the testis, entering the parenchyma at e to break up into its terminal branches. The vasal artery (6) passes along the vas to reach the tail of the epididymis, where it anastomoses with the descending branch of the artery (d) supplying the body and tail. In the experiments, the testicular artery was permanently interrupted at point X (in the abdomen) or temporarily occluded at point Y. In the former case, the testis still would have some blood supply via the vasal artery, the branch of the testicular artery to the tail, and the terminal part of the testicular artery. However, the testicular artery is an end-artery at point Y. (From A. G. Oettle and R. G. Harrison, J. Path. & Bact., 64, 273, 1952.)

neural, in which the Leydig cells lie alongside the nerve, (2) intraneural, in which groups of Leydig cells may be found within the perineurium, and (3) interdigitational, in which the course of the nerve breaks a cluster of Leydig cells into small groups (Okkels and Sand, 1940-1941). It is not certain that nerve fibers actually penetrate Leydig cells (Peters, 1957; Gray, 1947). Peters noted that nerve fibrils also

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PHYSIOLOGY OF GONADS

run to the walls of the tubules and enter the membrana propria to reach the Sertoli cells.

Experimental studies on the significance of the sympathetic nervous system with regard to testicular function have been only sporadically performed in lower animals. Coujard (1952,1954) found that the sympathetic ganglia along the vas deferens are most important to testicular development in the guinea pig. If these ganglia are injured, hypoplasia and aspermatogenesis of the testis follow. Unilateral removal of the prostatovesiculodeferential ganglion causes ipsilateral testicular immaturity. Defects of spermatogenesis also are noted when distant lesions in the sympathetic trunk are produced. Coujard concluded that the sympathetic system is an obligatory intermediate between gonadotrophic hormones and the testis. Somewhat similar studies have been reported on the cat (King and Langworthy, 1940) . If 7.5 cm. of the sacral and lumbar ganglionic chain are removed unilaterally, cessation of spermatogenesis occurs on the affected side within 2 or more weeks. The Leydig cells remain normal. Bilateral extirpation of strips of the ganglionic chain leads to reduction in spermatogenesis. In addition Weidenmann (1952) reported a diminution in volume of the Leydig cells after lumbar sympathectomy in cats. Destruction of the spinal cord by ultrasound in mice at levels from the eighth to the tenth thoracic segment had no effect on testicular weight, morphology, or spermatogenesis (Josimovich, 1958).

Lumbar sympathectomy in man has yielded variable results. Bandmann (1950) found atrophy of the testis and loss of potentia after unilateral lumbar sympathectomy; sperm examinations before and after operation disclosed deterioration in all of his cases. However, Alnor (1951) could not observe any effects in 14 patients after unilateral lumbar sympathectomy, and Kment (1951) found a temporary increase in potency in men after procaine block of the trunk. The effect of lumbar sympathectomy in man clearly needs more decisive study.

The poor sexual status of Iniinan paraplegics has led many authors to conclude that the nervous system controls testicular function in man. Apart from the muscular

disability of male paraplegics, such symptoms and signs as gynecomastia, loss of potency, atrophy of the testes, creatinuria, proteinuria, a decreased basal metabolic rate, loss of sex hair, and decreased excretion of 17-ketosteroids suggest testicular insufficiency (Cooper and Hoen, 1949, 1952; Cooper, Rynearson, Bailey and MacCarty, 1950; Cooi^er, Rynearson, MacCarty and Power, 1950). The extent to which these changes occur in paraplegics is debatable, and certainly not all changes are always present in any one patient. A study by Talbot (1955) of 400 paraplegic and quadriplegic patients showed that two-thirds were capable of achieving erection, and that one-third of these had successful intercourse. One-twentieth were fertile. It is obvious, then, that potency and fertility are not invariably lost. The histologic appearance of the testis in paraplegics has been determined by both biopsy and necropsy. In contrast to the variability in symptoms, the histologic appearance of the testis is more uniform. Atrophy of the tubule occurs, often with disai)pearance of all germinal epithelium except the Sertoli cells. The tubular wall is thickened. Leydig cells are present but may be found in clumps, giving the appearance of hyperplasia (Keye, 1956). Perusal of most of the illustrations showing atrophic testes in paraplegic men (Stemmermann, Weiss, Auerbach and Friedman, 1950; Klein, Fontaine, Stoll, Dany, and Frank, 1952; Bors, Engle, Rosenquist and Holliger, 1950) indicates that all stages of degeneration may be encountered. Some testes resemble those in adult seminiferous tubular failure, and others, especially those showing severe atrophy, resemble cryptorcliid testes. It is most difficult to determine from a testis containing only Sertoli cells and clumped Leydig cells what the nature of the pathologic process was, because all tyi:)es of atrophy end in the same general histologic picture regardless of cause.

jMental disease and mental stress are said to affect the testis. Jankala and Naatanen (1955) found that severely disturbed rats, presumably under "mental strain," showed marked atrophy of the testis within 6 weeks. The severity of this atrophy is evident from the finding that only Sertoli cells remained. Caged dogs, apparently under mental strain,

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have transient testicular atrophy. Hormonal secretion is not impaired (Huggins, Masina, Eichelberger and Wharton, 1939). Testicular atrophy has been noted in schizoid patients (Hemphill, 1944; Hemphill, Reiss and Taylor, 1944) and was thought to be caused by this severe mental illness. However, the histopathologic appearance of the testis in schizophrenia is not specific (Blair, Sniffen, Cranswick, Jaffe and Kline, 1952; Tourney, Nelson and Gottlieb, 1953) and it may not be stated that mental illness has any direct or specific action on the human testis.

VIII. The Excretory Duct System

The old concept that vasectomy is followed by hypersecretion of male hormone and rejuvenation has been disproved completely. Recent studies have been concerned with the effects of occlusion of the excretory ducts on the tubular apparatus. Although some reports have indicated that the testes of rats and rabbits decrease to one-half normal size after vasoligation, the majority opinion is that no change in testicular weight occurs (see Young, 1933, for review). Poynter (1939) did not observe any changes in the structure of the rat testis one year after vasoligation. Atrophy of the testes w^as obtained only when vasectomy was performed scrotally; under these circumstances, it resulted from adhesions subsequent to operation. No change was observed in the seminal vesicles, indicating no alteration in secretion of androgen. Also, no changes were evident in the Leydig cells.

Ligation of the ductuli efi'erentes, however, does produce pressure atrophy of the germinal epithelium (Young, 1933; Mason and Shaver, 1952). The testis becomes swollen and tense owing to distention of the ductuli with sperm on the testicular side of the ligature. The rete is also dilated. Peritubular fibrosis occurs, especially in tubules at the periphery of the testis. Degeneration of the germinal epithelium then ensues. Ten weeks after ligation, only Sertoli cells are left in the tubules. The Leydig cells remain unscathed (Harrison, 1953a).

The dift'erence in effect of ligation of the excretory path distal or proximal to the epididymis is attributable to the function of the excretory duct system of reabsorbing

fluid needed to carry sperm. Obstructive necrosis of the testis does not occur after ligation of the ductus deferens, because reabsorption of fluid takes place. This would also explain the absence of testicular atrophy in clinical states of inflammatory obstruction along the excretory pathway caused by gonorrhea or of obstruction caused by congenital absence of the ductus deferens.

IX. The Seminiferous Epithelium

Clarification of the spermatogenic cycle in the germinal epithelium is probably the most important development in knowledge of the testis since the second edition of this book. The difficulties in expressing spermatogenesis in quantitative terms were great. Clear identification of each type of cell was not possible. Certain basic information on the transformation of one type of cell into another, on the renewal of certain cells, and on degenerative phenomena was lacking. Despite these difficulties, the time of a complete spermatogenic cycle in the rat was estimated by several investigators using diff"erent methods. These methods were ( 1 ) time of recovery after irradiation of the testis, (2) morphologic studies of the changes in cellular population with reference to a static cell such as the Sertoli cell, and (3) turnover time of organically bound radiophosphorus in the germinal epithelium (Howard and Pelc, 1950). The introduction of the periodic acid and fuchsin sulfurous acid (PAS) stain for glycol groups such as exist in glycogen, mucoprotein, and mucopolysaccharides solved the difficulties enumerated above.

Cytologic studies have shown that the cells of the seminiferous epithelium are organized in similar associations. The development of any one generation of a certain type of cell is correlated with other generations present in the same part of the tubule. The changes in a certain zone of the germinal epithelium between two successive appearances of the same cellular association constitute a cycle. Different investigators do not use the same number of phases, the same classification of cell types, or the same points of reference. Depending somewhat on the cytologic detail and somewhat on the

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PHYSIOLOGY OF GONADS

point of reference, the cycle can be divided into 6, 8, 12, or more phases.

Roosen-Runge (1951-1955), RoosenRunge and Barlow (1953j, and RoosenRunge and Giesel (1950) used eight phases to characterize a seminiferous cycle in the rat. In phase 1, no sperm cells are present in the tubule; at the end of phase 8, the sperm cells forming over the intervening phases have disappeared from the lumen. Two types of spermatogonia are recognized; type A is a large cell with a large nucleus and little chromatin, and type B is a smaller cell with a smaller nucleus and masses of chromatin arranged peripherally. Type A spermatogonia divide simultaneously in phase 1 and again at phases 4, 6, and 7, leading to successive doublings. Type B spermatogonia form from type A in phase 6. In phase 8, a total of 98 per cent of all the spermatogonia are type B, leaving a 2 per cent quota of type A to start the cycle over again. When the spermatozoa are floated off the tubular wall, type B spermatogonia rapidly change into prespermatocytes. The prespermatocytes grow rapidly and become spermatocytes. Spermatid formation occurs in the first four phases. Spermatozoa are present from the end of phase 5 through phase 8.

Interestingly, the Sertoli cells show a cyclic variation in volume, being largest at phases 7 and 8 and smallest at phase 1. Retraction and expansion of the Sertoli cells, with cycles of spermatogenic activity, was noted by Rolshoven (1945, 1947, 1951). When the cells retract, part of the cytoplasm is lost, leaving a pars basalis. In expanding, this part of the Sertoli cell forms a fine lattice. The Sertoli cells resorb regressive spermatozoa and probably also the residual bodies during the spermatogenic cycle.

Because PAS-positive material can be traced back to the Golgi apparatus of young spermatids (Leblond, 1950), Leblond and Clermont (1952a, b) have been able to divide spermiogenesis in the rat into 19 stages. In the first 8 of these, the germinal epithelium has old spermatids, which are released when the new crop reaches stage 8. Hence, the new crop of spermatids is alone until they reach stage 15, when another generaation of spermatids appear. Therefore, stage

1 and stage 15 spermatids appear together, and the succession of cells associated with this appearance marks one cycle. These authors have divided their 19 stages of spermiogenesis into four phases (Fig. 5.8).

The first phase is the Golgi phase, which includes 3 of the stages. In stage 1, the idiosome is in the Golgi zone and two centrioles are near the chromatoid body. The fine filament from one centriole eventually becomes the tail of the sperm. In stage 2, one to four granules appear in the idiosome. In stage 3, the fusion of pro-acrosomic granules into one large one is accomplished.

The second phase is the cap phase, which consists of 4 stages. In stage 4, the acrosome granule flattens on the nucleus. In stage 5, a membrane arising from the granule spreads over the nucleus. In stage 6, a cap is formed over the nucleus. The idiosome separates from the acrosome granule, and the two centrioles move closer to the nucleus. In stage 7, the cap reaches maximal size. The proximal centriole adheres to the nucleus, and the flagellum remains attached to the distal centriole. The chromatoid body is loose in the cytoplasm.

The third phase is the acrosome phase, which includes 7 stages. The caudal tube is present, and the head caps are oriented toward the tubular wall. In stage 8, the granule and cap move toward the basement membrane, and the cytoplasm shifts to the opposite pole of the nucleus. The chromatoid body surrounds the flagellum near its insertion to the distal centriole. In stage

9, the acrosome granule elongates. In stage

10, the head cap moves toward the caudal end of the nucleus, and the apical end is pointed. In stage 11, the nucleus and head cap elongate. In stage 12, the nucleus is at its maximal size. In stage 13, the nucleus is thinner, and the distal centriole divides into a dot and ring. In stage 14, the head cap is loose over the nucleus, the cytoplasm condenses, and the sj^crmatid begins to look like a mature spermatozoon.

The fourth phase is the maturation phase, which consists of 5 stages. In stage 15, the head cap has a finlike membrane; the ring centriole separates from the centriole and forms the middle piece. In stage 16, elongation of the finlike membrane occurs. In stage 17. the acrosome and head cap move for

MAMMALIAN TESTIS

325

Fig. 5.8. Spermiogenesis in the rat. 1 to 3, Golgi phase. The idiosome produces two proacrosomic granules, which fuse into the single acrosomic granule. 4 to 7, cap phase. The acrosomic granule produces the head cap, which enlarges to cover a third of the nucleus. 8 to 14, acrosome phase. The nucleus and head cap elongate, whereas the acrosomic granule transforms into the acrosome. 15 to 19^ maturation phase. Near the end of this phase, the reactivity of the head cap and acrosome decreases considerably, and the spermatozoon is released into the lumen (19). (From C. P. Leblond and Y. Clermont, Ann. New York Acad. Sc, 55, 548, 1952.)

ward. In stage 18, the perforatorium appears. In stage 19, the staining capacity of the sperm is sharply reduced.

The behavior of the remaining cells of the germinal epithelium now can be correlated. Five peaks of mitosis occur in the spermatogonia. The first three peaks give rise to type A spermatogonia, the fourth peak to type B spermatogonia, and the fifth to spermatocytes. Spermatocytes, formed in stage 6, undergo the long meiotic division and become spermatids at stage 1 of the third cycle.

This quantitative method has been applied to three areas which are of importance

to the experimental or clinical endocrinologist: renewal of stem cells, postnatal degeneration of germ cells, and the effects of hypophysectomy on the germinal epithelium.

The renewal of spermatogonia always has been puzzling. It was postulated that they were renewed from the Sertoli cells, from unequal mitosis of a spermatogonium into a spermatocyte and another spermatogonium or from type A cells which did not difTerentiate into type B cells. Clermont and Leblond (1953) proposed a new theory for the renewal of stem cells. Three types of spermatogonia are present in the rat and

326

PHYSIOLOGY OF GONADS

SCHEMAT I C REPRESENT AT I ON

PROGENY or ONE A CELL

Of STAGE VIII

A In B R

FINAL HYPOTHESIS

|b [bIb [bIb [bIb [bIb [bIb

R Ir'rIrirIrirIriR JRiR jfilR iR|RlR!R"|RiRlRiR IrJrIr

0-12 12

12 0-24 24

Fig. 5.9. Diagrammatic representation of the most probable pattern for the development of spermatogonia (or "stem cell renewal theory"). The Roman numerals on either side of the diagram indicate the stages of the cycle. A, type A spermatogonia; Ad, dormant type A spermatogonia ; In, intermediate type of spermatogonia ; B, type B spermatogonia ; R, resting spermatocyte. In this hypothesis, the two daughter cells of the stage IX mitosis do not divide simultaneously. One of the granddaughter cells becomes a new dormant type A cell (Ad), ensuring the renewal of the spermatogonial population at the subsequent cycle, whereas the three other daughter type A cells divide again to produce intermediate tvpe cells, which in turn produce type B cells, which in turn produce spermatocytes. (From Y. Clermont and C. P. Leblond, Am. J. Anat., 93, 475. 1953.)

mouse (Fig. 5.9). Type A spermatogonia give rise to either intermediate spermatogonia or to dormant type A spermatogonia. The intermediate type of spermatogonia gives rise to the type B forms, which produce spermatocytes. The dormant type A spermatogonia are so designated because they do not divide for 8 stages. At the 9th stage, the dormant type A spermatogonium forms 4 large type A spermatogonia. In the next cycle, one of these 4 type A spermatogonia becomes another dormant type A spermatogonium; the others form 6 of the intermediate types of spermatogonia and eventually 24 spermatocytes. The cytologic details and the alterations in numbers of the three types of spermatogonia are illustrated in Figure 5.10. Full information can only be obtained by consulting the original papers.

Considerable degeneration of the primary germ cells occurs during development of the testis in the mouse and the rat (Allen and Altland, 1952). Degeneration usually ceases on the ninth day of age in the rat. Over the next 4 days, however, considerable multiplication occurs, but from day 14 to day 48 degenerating cells also may be seen in many tubules. Six different types of degeneration are evident — loss of cells in layers (exfoliation or shedding) , necrosis, loss of individ

ual cells, pyknosis, degeneration of leptotene forms, and abnormal mitosis in stem cells and spermatocytes.

The degeneration of the germ cells, or gonocytes, soon after birth had given the impression that the spermatogonia arise from the small supporting cells that also form the Sertoli cells in the adult. Gonocytes have a large, light, spherical nucleus, fine chromatin, and a sharp nuclear membrane. The supporting cells have smaller nuclei and coarse chromatin. The fourth day of life in the rat the supporting cells increase in number and form a palisaded layer along the basement membrane. The gonocytes swell and begin to degenerate; however, some of them look like type A spermatogonia. By day 6, most of the spermatogonia are tyi)e A but a few intermediate spermatogonia and type B forms appear. By days 9 to 12, gonocytes are no longer present. Primary spermatocytes appear for the first time in resting leptotene stages. By days 15 to 18, two generations of germ cells are present. By days 23 to 26, the spermatocytes are in meiotic prophase and some spermatids are being formed. By days 33 to 50, the Sertoli cells have matured. Because the supporting cells do not divide after day 15, type B spermatogonia can

MAMMALIAN TESTIS

327

-.J

8 »<

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^ tl'^^' "Xi^^'^,) ^.¥-'^^- • :^%^«^

\ > 13

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Fig. 5.10. Diagrammatic drawings of stages I to XIV of the f\cle ul ilie seminiferous epithelium. Drawings made from PAS-hematoxylin stained preparations. Numbers 1 to 19 refer to spermatids at different steps of spermiogenesis. A, type A spermatogonia; B, type B spermatogonia; Bm, mitosis of spermatogonia; R, resting spermatocytes; L, leptotene stage; Z, zygotene stage; P, pachytene, Di, diplotene and diakinesis; SI, primary spermatocytes; Sim, primary spermatocyte metaphase; SI I, secondary spermatocytes; Slim, secondary spermatocyte metaphase; S, Sertoh element; Rh, residual body. (From R. Daoust and Y. Clermont, Am. J. Anat., 96, 255. 1955.)

not arise from supporting cells (Clermont and Perey, 1957).

It was known that, after hypophysectomy, spermatids disappear but spermatogonia, Sertoli cells, and primary spermatocytes remain for long periods and spermatogonial mitosis continues. Clermont and ]\Iorgentaler (1955) noted spermatids being phagocytosed by the Sertoli cells within 3 days after hypophysectomy. Young spermatids at stages 1 through 7 are present, but at 10 days after hypophysectomy, no developing spermatid has reached stage 9. A few pachytene spermatocytes are degenerating, but primary and secondary spermatocytes are present during the first week after the operation. By the tenth day, the Sertoli cells shrink but do not disintegrate. Spermatogonial types A and B remain intact. Maximal regression after hypophysectomy is reached within 29 days.

The basement membrane is thick and there are two rows of type A, intermediate, and type B spermatogonia, a few primary and secondary spermatocytes, spermatids at stages 1 through 7, and Sertoli cells. Type B cells form spermatocytes, but the spermatocytes degenerate before and during meiosis, and only 4 per cent of them survive to produce spermatids. The spermatids develop to stage 7 and then disintegrate. Therefore, spermatogenesis up to stage 7 of spermiogenesis can occur in the absence of the pituitary gland but at a greatly reduced rate. As was surmised from early observations on the maintenance of spermatogenesis by androgen, the premeiotic phase of spermatogenesis apparently can take place without gonadotrophins ; meiosis suffers severely from gonadotrophic deprivation; and the postmeiotic phase is controlled by androgen. The observation that testos

328

PHYSIOLOGY OF GONADS

<.^

^ ; >

1^6 J^

^'^

^

r

'S

(

C V

Fig. 5.11. Spermiogenesis of the mouse as seen with PAS-hematoxyhn staining of Zenkerformol fixed testis. Drawings are arranged in a spiral to demonstrate stages which overlap in a cycle of the seminiferous epithelium. Orientation of spermatids in relationship to the basement membrane also is shown. ^ to S is the Golgi phase, 4 to 7 the cap phase, 8 to 12 the acrosome phase, and 13 to 16 the maturation phase. (From E. F. Oakberg, Am. J. Anat., 99, 391, 1956.)

terone can maintain spermatogenesis if it is administered within a month after hypophyscctomy but cannot if treatment is delayed more than a month may not be so puzzling if it is assumed that androgen protects in some way the serious depletion of spermatocytes at meiosis.

The plan of spermiogenesis in many species is essentially similar to that in the rat and mouse. Clermont (1954) found that the hamster shows the same successive stages of spermatogenesis except that five cycles may be represented simultaneously. In the mouse, Oakberg (1956a, b) described 16 stages, the first 12 of which constitute a cycle (Fig. 5.11 and Table 5.2). Four cycles constitute complete spermatogenesis and require 34.5 days. Generally, the same plan of spermiogenesis holds for the guinea pig, cat, bull, dog, ram, monkey (Fig. 5.12), and

man (Leblond and Clermont. 1952b; Clermont and Leblond, 1955) , although many differences in cytologic detail exist and have been documented (Zlotnik, 1943; Gresson, 1950; Gresson and Zlotnik, 1945, 1948; Burgos and Fawcett. 1955; Watson, 1952; Challice, 1953).

Application of quantitative studies to human spermatogenesis has, to date, been disappointing. Spermatogenesis does not proceed along a wave, nor are the various stages sharply delimited, as they are in the rat. Further, the testis of the human also differs from that of the rat in that the relative pro})ortion of differentiated germ cells to spermatogonia is less. No helpful findings in cases of human infertility have been obtained by quantitative analysis of the germinal epithelium (Roosen-Runge and

MAMMALIAN TESTIS

329

TABLE 5.2

Characteristic cell associations at each stage of the cycle of the seminiferous epithelium (From E. F. Oakberg, Am. J. Anat., 99, 391, 1956.)

Stage of Cycle

Spermatogonia

Type A

Intermediate Type B

Spermatocytes I First Layer

Second layer. . . , Spermatocytes II

Spermatids (see Fig. 1)

First layer

Second layer

15

10

Z

Dip

P Dia M-I

S Mil

12

A

In B MI

S

= Spermatogonia type A = Intermediate type sperm; = Si^erniatogonia type B = First meiotic division = Secondary spermatocyte

)goni

M-II = Second meiotic division

R = Resting L = Leptotene Z = Zygotene P = Pachytene Dip = Diplotene Dia = Diakinesis

Primary spermatocytes

Barlow, 1953; Eoosen-Runge, jNIarbergcr and Nelson, 1957).

X. The Interstitial Tissue

Although miscellaneous general information is available on the interstitial tissue of many animals, including the gorilla, the short-tailed manis, and the vampire bat (Popoff, 1947), detailed knowledge comes from common laboratory animals, such as the rat, mouse, guinea pig, rabbit, and cat. With the exception of man, however, the life history of the interstitial tissue of the testis is probably known best for the bull (Hooker, 1944, 19481.

In the 1 -month-old bull, when widely separated, lumenless tubules are present, the intertubular spaces contain only mesenchymal cells. The number of Leydig cells gradually increases up to 2 years of age; after this time, the Leydig cells become vacuolated and increase in both number and size (Fig. 5.13). From 5 to 15 years of age, loss of vacuolation and decrease in size occiu-. After 15 years of age, degeneration ensues.

^Metamorphosis of the Leydig cell begins with nuclear changes. The nucleus acquires 1 to 3 nucleoli, increases by 25 per cent in volume, and becomes spherical. Hypertrophy and hyperplasia of the cell occur. The cell still retains its stellate appearance, but becomes polygonal in shape after granules appear in the cytoplasm. After 2 years of age, vacuolation occurs and, with age, the vacuoles become larger. At 5 years of age, vacuolation is present in all Leydig cells. Regression of the Leydig cells begins at 7 years of age; it is manifested by a decrease in vacuolation and mitotic activity (Hooker, 1944, 1948), and ends in cellular disintegration (Fig. 5.14).

In addition to regression, Leydig cells may also dedifferentiate. This occurs in the rabbit. In autografts of testis to the ear, mature Leydig cells show fusion of granules, shrinkage of cytoplasm, loss of nuclear transparency, and finally cannot be distinguished as a Levdig cell (Williams, 1950).

The life history of the Leydig cell in man and monkey is in general similar to

330

PHYSIOLOGY OF GONADS

V

vv

1. 1.

Fig. 5.12. Spermiogenesis in the monkey, i to 3 is the Golgi phase. 4 to 7 the cap phase, 8 to 12 the acrosome phase, and 13 to l-i the maturation phase. (From Y. Clermont and C. P. Leblond, Am. J. Anat., 96, 229. 1955.)

that in the bull. In the human, Leydig cells are large polyhedral cells containing a large vesicular nucleus, which is not found in other cells of the interstitial tissue. The cells contain pigment, vacuoles, crystalloids, and granules. The granules vary in density, number, and arrangement within the cytoplasm. These granules contain lipides (Nelson and Heller, 1945) and, like those in common laboratory animals (Pollock, 1942), give reactions of steroids. Various types of Leydig cells can be distinguished on the basis of the size and nature of the granules and vacuoles. The medium-sized

granular cells are believed to be vigorous producers of androgen (Sniff en, 1952; Tillinger, Birke, Franksson and Plantin, 1955) . It is difficult to determine the absolute number of Leydig cells. However, rough counts made in testes of men (necropsy material) indicate that the number declines with age (Sargent and McDonald, 1948). In general, the excretion of 17-ketosteroids and the development and condition of secondary sex characteristics parallel histologic and cytologic evidence of secretory activity by the Leydig cells (Fig. 5.15).

It is generally held that the Leydig cell

MAMMALIAN TESTIS

331

■#\

2!^ 22 23 24

Undifferentioted cells

P

25

26 27

Differentioting cells

28

29

/^J)

//

ti^^

V / ^0 ^' .

Young Leydig cells

/

32

V-^

33

Mature Leydig ceils

36

(f^

Leydig cells from oid animoi

^:\

38

Fig. 5.13. Life history of Leydig cells of the bull testis. 21 to 2S, calf 1 month old. 2.'^, calf \Vz months old; note threadlike processes extending from angulation of mesenchymal cell. 25 to 21, cells of interstitium; 25 is a fibroblast, and 26 and 21 are pre-Leydig cells. 2S, bull 4 months old. ;^9 and SO, bull 2 years old, with young Leydig cells. SI and S2, bull 28 months old; note vacuoles. SS to 35, mature Leydig cells in a 5-year-old bull. SQ to S8, bull 15 years old. (From C. W. Hooker, Am. J. Anat", 74, 1, 1944.)

- ^ - ^ — ( O U #:v;(^:;-yv:;(^

Fig. 5.14. Life history of the Leydig cell of the bull. (From C. \V. Hooker, Recent Progr. Hormone Res., 3, 173, 1948.)

332

PHYSIOLOGY OF GONADS

Numbers

of

Leijdigl

cells

Fig. 5.15. Schematic summary of thv life history of the human Leydig cell. (From A. Albert, L. O. Underdahl, L. F. Greene, and N. Lorenz. Proc. Staff Meet., Mayo Clin., 29, 368, 1953; 30, 31, 1955.)

is the source of androgen. Gonadotrophin evokes secretion of androgen from the testis only if the Leydig cells are stimulated. Tumors of Leydig cells produce large amounts of androgen. Testes impaired by heat or x-rays still produce androgen even though the germinal epithelium may be destroyed. The parallelism between the number of Leydig cells, their morphology, histologic appearance, and histochemical properties (Wislocki, 1949), on the one hand, and androgenic secretion as measured chemically or as determined by the behavior of the secondary sex characteristics, on the other hand, supports the conclusion that the Leydig cell produces male hormone (Figs. 5.16 and 5.17).

XI. Hormones of the Testis

The mammalian testis produces androgens and estrogens. Because the chemistry of the hormones is discussed in Villee's chapter, only a brief account will be given here. Testosterone was first obtained from bull testes and later from horse testes (Tagmann, Prelog and Ruzieka, 1946). However, difficulties attended the isolation of testosterone from the testes of pigs. Although not obtained in crystalline form, testosterone was identified bv a characteristic in

frared absorption spectrum in extracts of hog testes (Prelog, Tagmann, Lieberman and Ruzieka, 1947). Other steroids are present in hog testes (Ruzieka and Prelog, 1943; Prelog and his associates, 1947). C21ketosteroids, such as allopregnane-3-(^)ol-20-one, allopregnane-3- (a) -ol-20-one, and 5-pregnane-3-(^)-ol-20-one, have been identified. Haines, Johnson, Goodwin and Kuizenga (1948) isolated pregneninolone from hog testes as well as several other unidentified steroids, some of which had estrogenic activity. Ketosteroids have been found in human sperm (Dirschcrl and Breuer, 1955).

The testes of deer, bulls, stallions, and humans contain estrogens. The amount present in deer testes is three times that in bulls (Cunningham, ]\Iay and Gordon, 1942). Estradiol (0.21 mg. per kg. I and estrone (0.36 mg. per kg. ) wer(> isolated from 28 kg. of hoarse testes by Beall 1 1940). Estradiol also has been isolated from hinnan testes obtained shortly after death (Goldzieher and Roberts, 1952).

Testicular tissue is able to convert acetate into cholesterol (Srere, Chaikoff, Treitman and Burstein, 1950) and also to testosterone in the hog, rat. and human (Brady, 1951). Human chorionic gonadotrophin

MAMMALIAN TESTIS

333

60- * 30

V'

Hormone assays in normal boys (Data of Greulich el al )

. Androgen

"" Gonadotropin

Estrogz

.^-^

10 is

16 20 22 24

2 4 6 8 10 12 14 16

A§e in years

Fig. 5.16. Frequencj- of puberty, measurements of testis and penis, and excretion of hormones during puberty in man. (From A. Albert, L. O. Underdahl, L. F. Greene, and N Lorenz, Proc. Staff Meet., Mavo Clin., 28, 409. 1953.)

334

PHYSIOLOGY OF GOKADS

1

2

3

4

5

6

HAIRIINE FACIAL HAIR

O

w

e

©

VOICE (Loryn,)

T

m If

■«*

BREASTS

1

^

►

« 

1 .' --.

AXILLARY HAIR BODY

f?'

\'^

III

CONFICUtATION

BODY HAIR

(i)

PUBIC HAIR

-.-,.

PENIS

Y

?

¥

t

T '. ^

LENGTH (cm.)

v^

Vl/

3.«.

45-9,

45-12

8-15

9-15

105K

CKCUMFtlENCI

(cm.)

m

•

m

• "°

• '°

^^-105

TESTES (cc)

>.•

T75^

17^B

^■■« 

6 2(^A

"•

OR

J^

-^1

PROSTATE

-"■^

-^^^

PRE.

POST

PUBESCENCE

PUBESCENCE

PUBESCENCE

Fig. 5.17. Stages of sexual development and maturation. (From W. A. Schonfeld, Ai J. Dis. Child., 65, 535, 1943.)

(HCG) increases the yield of testosterone from testicular slices incubated with acetate. Estradiol- 17-/? also has been found in the products obtained by incubating tissue slices with acetate. Human testicular tumors incubated with labeled acetate form labeled testosterone, androstenedione, progesterone, estradiol, and estrone (Wotiz, Davis and Lemon, 1955). Mevalonic acid, a precursor of cholesterol, yields estradiol when incubated with homogenates of human testis (Rabinowitz and Ragland, 1958). The biogenesis of male hormone as worked out in the stallion, rat, and human (Savard, Dorfman and Poutasse, 1952; Savard, Besch, Restivo and Goldzieher, 1958; Savard, Dorfman, Baggett and Engel, 1956) by means of radioisotopic methods shows a common pathway from 17a-hydroxyprogcsterone -^ progesterone -> 4-androstene3,17-dione —^ testosterone. Testosterone has been identified in the spermatic vein blood of dogs (West, Hollander, Kritchevsky and Dobriner, 1952). Also identified were A^androstcno(lione-3-17 and 7-keto-cholesterone.

In addition to confirming the presence of

several biologically active steroids in the testis, the studies made in the last two decades have clarified the biosynthesis of male hormone. The peripheral metabolism of testosterone and its biologic actions in the organism are described in chapters by Villee and by Price and Williams-Ashman, respectively.

In addition to these well-known steroid hormones, the presence of a water-soluble hormone in the testis has been postulated on biologic evidence. Vidgoff, Hill, Vehrs and Kubin (1939) and Vidgoff and Vehrs (1941) induced atrophy of the testis and accessory sex organs in the rat by the administration of aqueous extracts of bull testes. Because the atrophy was similar to that occurring after hypophysectomy, it was claimed that a water-soluble principle in the testis was capable of inhibiting the gonadotrophic function of the ])ituitary. This principle was called "inhibin." The theory was then constructed that the testis secretes two hormones, nnnu'ly a water-soluble hormone responsible for the integrity of the germinal epithelium by regulating the secretion of pituitary gonadotrophin, and a fat

MAMMALIAN TESTIS

335

soluble hormone (testosterone) responsible for maintaining the accessories. The observations of Vidgoff and his associates were disputed by Rubin (1941). The inhibin concept was supported by McCullagh and Hruby (1949) because testosterone did not inhibit the excretion of pituitary gonadotrophin and was not effective in correcting castration changes in the pituitary of cryptorchid rats at doses that were sufficient to stimulate the accessories. Inhibin was now identified with estrogen, and the source of estrogen was claimed to be the Sertoli cell. The new evidence for this modified concept will now be considered.

]\IcCullagh and Schaffenburg (1952) stated that estrogen is much more effective than androgen in suppressing gonadotrophin and that estrogen is present in saline extracts of bull and human sperm. Estrogen is found in the testes, but localization of its production to the Sertoli cells is uncertain (Teilum, 1956), and is doubted by Morii (1956) and Ballerio (1954). The almost complete absence of Sertoli cells in Klinefelter's syndrome, in which values for urinary gonadotrophin are high, also is considered as evidence that estrogen is manufactured by the Sertoli cells. The high excretion of gonadotrophin in Klinefelter's syndrome can be interpreted, at least in part, by the concept of Heller, Paulsen, ^lortimore, Jungck and Nelson (1952) that the amount of urinary gonadotrophin varies inversely with the state of the germinal epithelium. Utilization of gonadotrophins by the germinal epithelium could explain the levels of this hormone in various syndromes as satisfactorily as the lack of a hypothetic testicular inhibitory hormone. Furthermore, if the Sertoli cells secrete an inhibitory hormone, patients who have germinal aplasia (Sertoli cells only in the tubules) should have normal values for urinary gonadotrophin, whereas it is well known that this hormone is greatly increased in these patients. The proponents of the inhibin theory claim that aqueous extracts of testes prevent the castration changes but do not repair the accessories, whereas testosterone corrects the accessories but does not restore the normal histologic appearance of the pituitary. However, Nelson showed that cryptorchid testes produce less androgen

than normal and that the order in which the above structures are affected represents differences in the degree of their sensitivity to the amount of androgen produced. The efficacy of aqueous extracts on the cytologic appearance of the pituitary has not been confirmed. Thus, evidence deduced from cryptorchism that an inhibitory hormone is produced by the germinal epithelium is inadequate.

XII. Effects of the Pituitary on the Testis

Little information has been added in the past 20 years to the effects of acute hypophyseal deprivation on the mammalian testis. Smith (1938, 1939) had shown in the rat that spermatocytes as well as spermatogonia and Sertoli cells remain for a long time after hypophysectomy. However, in the monkey, and possibly in man, all cells of the germinal line except the spermatogonia and the Sertoli cells disappear. Even though hypophysectomy has been employed for several years as a palliative procedure in inoperable carcinoma of the prostate, no data have been obtained concerning the effects of hypophysectomy on the testis in otherwise normal man. In the dog, the testes decrease to about one-third their normal weight after surgical removal of the pituitary. Only a single row of spermatogonia remains (Fig. 5.18). The Leydig cells are reduced in size and contain abundant quantities of fat. The lack of complete involution of the Leydig cells in the dog as a result of hypophysectomy is somewhat unusual, because marked involution of these cells occurs in all other mammals thus far studied. With respect to the behavior of the germinal epithelium, the dog (Huggins and Russell, 1946) seems to be more like the monkey and man than like the rat and mouse. The total relative decrease in testicular weight of the dog is intermediate between that observed in the cat (50 per cent) and that in the rat, guinea pig, and rabbit (75 per cent). With respect to histologic features, the guinea pig and ferret are intermediate between the rat and the monkey, because occasional spermatocytes remain in addition to spermatogonia and Sertoli cells. In the mouse, the testicular weight decreases for 25 days after hypophysectomy. Mess

336

^,

PHYSIOLOGY OF GONADS

.#•

.,**^,,,i*^

• .;

«

m,gm.

^

1

Fig. 5.18. Testis of dog 60 days after hypophysectomy. (From C. Huggins and P. S. Russell, Endocrinology, 39, 1, 1946.)

(1952 ( showed that early differentiation of spermatids in the rat is affected first by hypophysectomy. Spermatids degenerate, tubuhir fluid is lost, and atrophy of the germinal epithelium finally takes place (Gothie and Moricard, 1939).

Some recent studies on comi^ensatory hyi)ertrophy of the remaining testis after unilateral orchiectomy have been made. Old investigations showed that compensatory hypertrophy occurs in boars, rabbits, and hedgehogs. Compensatory hypertrophy does not occur in mature guinea pigs or man (Calzolari, Pulito and Pasquinelli, 1950; Pasquinelli and Calzolari, 1951; Zide, 1939). In the prepubertal guinea pig and rat, however, the remaining testis shows accelerated development. The volume of the remaining testis increases in the adult rat after unilateral orchiectomy (Grant, 1956). Since the compensatory hypertrophy is suppressed by testosterone, it appears likely that the accelerated development of the remaining testis is mediated by gonadotropliins.

The effects of gonadotrophins on testicular structure and function have been studied in many species. Injection of anterior jHtuitary extract or implantation of fragments of the anterior i)ituitary into the testis of guinea pigs has residted in pronounced stimulation and hypertr()])hy of the Leydig cells

(Petrovitch, Weill and Deminatti, 1953; Petrovic, Deminatti and Weill, 1954; Petrovic, Weill and Deminatti, 1954; Marescaux and Deminatti, 1955). In hypophysectomized mice, May (1955) found that testicular grafts of anterior pituitary tissue repair the atrojihic tubules and the involuted Leydig cells.

The effects of in(li\i(lual gonadotrophins of both pituitary and placental origin have been reviewed by Greep (1937) and Fevold (1944), and in the chapter by Greep in the present volume. The established concept, as worked out in the rat, is that folliclestimulating hormone (FSH) maintains and repairs the tubular apparatus but does not affect the function or structure of the Leydig cells. Luteinizing hormone (LH) maintains the functional activity of the Leydig cells but does not dii-ectly control tubidar activity.

Urinary gonadotrophin from menopausal women stimulated the tubides (Greep, 1937) and the Leydig cells (Balm. Lorenz, Bennett and Albert, 1953a-d). Hmnan chorionic gonadotropiiin (HCG) has little oi' no effect on tiie tubules, hut it induces ])ronounced stimulation of the Leydig cells. Pr(>gnaiit marc sci'uni (PMS) stimulates spermatogenic and endocrine activities of the testis. Both LH and HCG maintain spermatogcniesis after hypophysectomy.

Ncitlier FSH nor LH hastens the appearance of sperm in the testis of immature animals. No type of gonadotrophin has induced the appearance of sperm in the rat earlier that 35 days of age.

Because interest in the chemical fractionation of animal i^ituitary tissue waned after 1945, new studies on the effects of pituitary gonadotrophins on the testis have not been performed. Instead, HCG has received attention. The well known hyperemia induced in the ovary by HCG, which is used as a pregnancy test, has been reported to occur also in the testis by Hartman, Millman and Stavorski (1950). Hinglais and Hinglais (1951 ) have not confirmed this. HCG causes increased testicular weight in young rats (Rubinstein and Abarbanel, 1939). The effect of HCG on the rat testis has been summarized by Gaarenstroom (1941), who listed the following four main actions: (1) stimulation of the Leydig cells in both normal and hypophysectomized animals to produce androgen; (2) increase in growth of the testis in the normal immature animal; (3) maintenance of testicular tubules in hypophysectomized animals; (4) potentiation of the effects of i^ituitary gonadotroi)hins in either normal or hypophysectomized animals. All effects are interpreted as being caused by the increased liberation of androgen. This explanation probably also holds for the increased fibrosis in and around the tubular wall in hypophysectomized rats after administration of HCG, for the increase in the number of primary spermatocytes (Muschke, 1953; Tonutti, 1954), and for the slight increase in testicular weight (Diczfalusy, Holmgren and Westerman, 1950).

The effects of HCG in normal men are similar to those in animals (Maddock, Epstein and Nelson, 1952; Maddock and Nelson, 1952; Weller, 1954). The Leydig cells become hyperplastic and produce more estrogen and androgen. This is reflected first by an increase in urinary estrogen of some 5- to 20- fold and later by an increase in 17-ketosteroids of about 2-fold. The increased secretion of steroids by the Leydig cells is accompanied by an increase in the frequency of erections and occasionally by gynecomastia. The increased levels of estrogen and androgen induce tubular atro

phy. The tubular diameter becomes smaller, spermatogenesis ceases, and there is an increase in necrosis and sloughing of the germinal cells. The basement membranes become hyalinized, and peritubular fibrosis develops. In certain eunuchoidal persons ( hy{)ogonadotrophic hypogonadism ) , use of HCG induces differentiation of the Leydig cells and hastens maturation of the Sertoli cells. Some spermatogenesis is obtained (Heller and Nelson, 1947, 1948; Maddock, Epstein and Nelson, 1952). If FSH also is administered to such eunuchoidal men, complete spermatogenesis occurs (Heller and Nelson, 1947).

PMS acts on the rat testis in a manner intermediate between that of HCG and FSH (Creep, 1937; Kemp, Pedersen-Bjergaard and Madsen, 1943). Tubular growth and hyperplasia of the Leydig cells result. Interstitial cell hyperi)lasia also occurs in mice (Bishop and Leathern, 1946, 1948) , although the testicular weight does not increase after the use of PMS, as it does in rats. In the opossum, PMS does not induce secretion of androgen until the the animals are 70 days of age (Moore and Morgan, 1943). PMS is able to maintain the monkey testis after hypoi)hysectomy l)ut only for 20 days, after which involution occurs. If given to a hypophysectomized monkey in which testicular atrophy already is present, PMS causes formation of spermatocytes, but it does not induce the formation of spermatids or sperm cells (Smith, 1942). In man, PMS causes an increase in testicular weight (Hemphill and Reiss, 1945).

Unfractionated extracts of pituitaries of sheep or horses induce both tubular maturation and androgenic formation (Sotiriadou, 1941 ) . Preparations of FSH in mice produce slightly heavier testes but do not cause androgenic secretion (Moon and Li, 1952). Purified preparations of LH produce atrophy of the tubules and stimulation of the Leydig cells in infantile rats, and maintenance of germinal epithelium and Leydig cells in hypophysectomized rats (Zahler, 1950).

XIII. Effects of Steroids on the Testis

Between 1930 and 1940, rapid advances were made in the understanding of pituitarv and gonadal interrelationships, and the eon

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PHYSIOLOGY OF GONADS

cept of a servomechanism controlling pituitary-testis activities was well established. According to this concept, male hormone was considered to have its major effect on the testis by inhibiting the secretion of pituitary gonadotrophins. However, it was difficult to fit into this concept the report by Walsh, Cuyler and McCullagh (1934) that testosterone was capable of maintaining spermatogenesis in the rat after hypophysectomy. If testosterone were the medium by which spermatogenesis was maintained normally, the dualistic concept of gonadotrophic control of the testis would be in jeopardy. As can be imagined, this finding stimulated much research. By 1940 the fact that spermatogenesis is maintained in hypophysectomized rats, mice, and rabbits by testosterone was amply established (Cutuly and Cutuly, 1940) .

A. ANDROGENS

The varied effects obtained by injecting male hormone into normal and hypophysectomized rats depend on the nature of the androgen, the dose, the length of the treatment period, and the age of the animals when injections are begun. Inasmuch as most of the experimental work has been done with the rat and rats of various ages and sizes were employed, it is obvious that the dose of hormone is an important factor. Doses of testosterone of 100 /xg. per day or less can be regarded as small doses, whereas doses of 1 mg. or more can be considered as large. These definitions pertain only to the doses employed in studying the action of androgen on the testis and do not necessarily have any relationship to the physiologic levels of testosterone produced by the rat testis, which is not known, or to the effects of testosterone on the accessory sex organs (Moore, 1939).

In general, testosterone has no action on the undifferentiated gonad of the mouse, rat, opossum, or guinea pig (Moore and Morgan, 1942). In the immature rat small doses of testosterone propionate depress the testicular weight (Zahler, 1947; Dischreit, 1939; Greene and Burrill, 1940). However, if small doses are continued for long periods, incomplete supi:)ression results. Because the testicular inhil^ition induced by small doses of testosterone apparently results from sup

pression of gonadotrophins, it seems that greater ciuantities of gonadotrophins are formed as rats grow; hence, escape from suppression may occur (Biddulph, 1939). The work of Rubinstein and Kurland (1941) indicates that even small doses of testosterone, as already defined, may produce dift'erent effects in the rat. These investigators compared the effects of administration of 5 and 50 fxg. testosterone propionate per day in young animals. Young rats receiving the former dose showed increased testicular weight without, however, any hastening of maturation of sperm cells. The larger dose decreased testicular weight.

The effect of androgen on mature rats is also dependent on dose. Small doses cause atrophy of the mature testis because of suppression of gonadotrophins. Large doses have the same suppressing effect, but this is overridden by a direct stimulating effect of androgen on the testis, and atrophy does not occur. In both instances, the Leydig cells are atrophic (Shay, Gershon-Cohen, Paschkis and Fels, 1941). Large doses of testosterone have a direct action on the testis as indicated by the protective effect exerted on the experimentally induced cryptorchid testis (Hamilton and Leonard, 1938) and on the transplanted testis (Klein and Mayer, 1942) .

The aftereffects of androgenic administration also depend on the age of the animal and the duration of therapy. Using fecundity, libido, potency, and the state of the reproductive tract as indices of testicular function, Wilson and Wilson (1943) examined rats 3 to 5 months after a 28-day period of injection of androgen. In rats age 1 to 28 days, androgen severely affected the reproductive system. Low libido, absence of fecundity, and atrophic accessories were noted 3 to 5 months after testosterone therapy was discontinued. However, the later this treatment was instituted in the life of the rat, the more normal was the reproductive system 3 to 5 months after administration of the hormone was stopped.

Nelson and Merckel (1937), in a series of extensive experiments, confirmed the earlier finding that various androgens maintain spermatogenesis in the rat after hypophysectomy. Furthermore, they showed that the Leydig cells are atroj^hic in the face of

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active spermatogenesis in the androgentreated, hypophysectomized rat. Comparing such steroids as testosterone, androsterone, dehydroisoandrosterone, androstenedione, and various isomers of androstenediol, they concluded that the ability of androgens to maintain spermatogenesis is not related to their androgenicity. In fact, the weaker the androgen the better is the maintenance of spermatogenesis after hypophysectomy. This observation is important for it shows that maintenance of spermatogenesis is not due to the induction by androgen of a favorable scrotal environment for the testis. In further studies, Nelson (1941) showed that spermatogenesis could be maintained for

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178 days after hypophysectomy by testosterone propionate. No difference was observed between spermatogenesis under these conditions and that which occurs normally. Motile sperm were formed, and the animals could copulate with and impregnate females. The only difference was that the testes in the hypophysectomized animals treated with testosterone were only one-sixth normal size.

As is true of other effects of androgens on the testis, the time at which rats are hypophysectomized seems to be a critical factor in the ability of testosterone to maintain spermatogenesis. Leathern (1942, 1944) showed tliat troatmcnt witli tostosterone in

Fig. 5.19A. Effect of testosterone on the testis of the rat. 4, normal rat, 30 days of age. S, normal rat, 60 days of age. 6, 30-day-old rat given 10 ^g- of testosterone propionate daily for 30 days (no inhibition of spermatogenesis). 7, 30-day-old rat given 100 ^ig. of testosterone propionate daily for 30 days (suppression of spermatogenesis).

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PHYSIOLOGY OF GONADS

rats Itypophysectomized at 27 days of age resulted in the production of spermatids, but spermatogenesis did not occur. However, if the animals were operated on at 33 days of age, testosterone induced the formation of sperm. Furthermore, if the atrophic testes of hypophysectomized rats were stimulated by a gonadotrophin (PMS), testosterone also maintained the spermatogenesis thus induced.

It is not known exactly how testosterone maintains spermatogenesis after hypophysectomy. It seems that the "maintenance type" of spermatogenesis is not the same as spermatogenesis resulting from gonadotrophin, because the seminiferous tubules of the androgenically maintained testes in hypophysectomized rats are small. The ef

fect of androgen is not produced simply by the maintenance of sperm cells already present in the testis at the time of hypophysectomy because Nelson (1941) showed that spermatogenesis can be reinstituted in the testis of a hypophysectomized rat in spite of delaying treatment with testosterone for 3 to 4 weeks after hypophysectomy. This interval of time exceeds the normal sojourn of sperm cells in the epididymis; thus the results in terms of siring young cannot be attributed to sperm cells already present in the accessory duct system at the time of hyjjophysectomy (Figs. 5.19, A and B, and 5.20).

The dose of testosterone propionate necessary for maintenance of spermatogenesis in the rat seems to be around 80 fig. per day.

9^-: v;: - n II

Fig. 5.19B. 8, 3U-day-old mt given 1000 /ug. of testosterone propionate daily for 30 days (no suppression of spermatogenesis). .9, 30-day -old rat given 8.4 mg. of estradiol daily for 30 days (])ronoun(ed inhibition of spermatogenesis). 10, 30-day-old rat given 8.4 ng. of estradiol and 1000 mS- of lestosterone i)ro])ic)nale for 30 days (no inhir)ition of spermatogenesis). (From D. J. Jjudwig, Endocrinology, 46, 453, 1950.)

MAMMALIAN TESTIS

341

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12

Fig. 5.20. Klicct oi Ti-siostcioiic on testis oi li\|M)pli\-,~(i lomi/i d lat //, testis of norm.al rat, 30 days of age. 12, testis of 60-day-old rat li^popli^x ( tomizcd at 30 days of age. 13, testis of 60-day-old rat hypophysectomized at 30 (la.\s of age and given 1000 /lig. testosterone propionate daily for 30 days. (From D. J. Liidwig, Endocrinology, 46, 453, 1950.)

However, larger doses generally have been used in experiments on the maintenance of spermatogenesis. These doses are far greater than those necessary to maintain the accessory sex organs of castrated animals. Tubules can be maintained by much smaller doses of testosterone. Dvoskin (1944) implanted pellets of testosterone intratesticularly; approximately one-tenth of the amount of testosterone needed by the parenteral route was effective by this route.

The concept that testosterone maintains spermatogenesis in hypophysectomized rats was challenged by Simpson, Li and Evans (1942, 1944) and by Simpson and Evans (1946a, b). These investigators found that gonadotrophins, including interstitial cellstimulating hormone (ICSH), maintained spermatogenesis in hypophysectomized rats at doses far lower than those needed to

maintain the Leydig cells and the accessories. The testes remained in the scrotum, and motile sperm cells were produced. Inasmuch as testosterone propionate can maintain the tubules only at doses effective in maintaining the accessories, it was doubted that maintenance of spermatogenesis occurred by way of the direct tubular action of androgen. In addition to casting some doubt on the accepted mechanism of the spermatogenic action of androgen, this work raised doubt concerning the dualistic concept of gonadotrophic control of the testis. Maintenance of the testis by ICSH after hypoj^hysectomy suggests that one gonadotrophic hormone may be sufficient to maintain testicular function in mammals. However, these findings may be interpreted conventionally; i.e., that ICSH caused the Leydig cells, even though they were not re

342

PHYSIOLOGY OF GONADS

paired morphologically, to secrete androgen which by virtue of its local action on the tubules maintained spermatogenesis (Ludwig, 1950).

Testosterone maintains spermatogenesis in other species. In hypophysectomized ground squirrels, the testes are atrophic, aspermatic, and abdominal (Wells, 1942; 1943a). Hypophysectomized animals given testosterone propionate (0.5 mg. per day for 15 to 25 days) show growth of the testes, sperm formation, and testicular descent. Leydig cells remain atrophic. Because sperm formation ceases after hypophysectomy in the ground squirrel, as it does in the monkey, rat, guinea pig, mouse, cat, and ferret,

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Fic. .').21. KIT(H't of .iihlhitiMi HI ,( liypophysoctomized inoiikoj-. 1, biop-x -iniiincn from a normal 8-kg. rhesus monkey. .'. liiii|i-\ specimen from a hypophysectomized monkey .ifici- 56 da.ys, during which 1.4 gm. of testosterone propionate was administered at a daily dose of 25 mg. 3, state of testis 20 days after use of testosterone was discontinued. Note atrophy of tubules. The Sertoli cells and spermatogonia remain. (From P. E. Smith, Yale J. Biol. & Me.l., 17, 281, 1944.)

it is obvious that androgen initiated spermatogenesis.

Testosterone propionate maintains the spermatogenic activity of the testis of the hypophysectomized monkey for 20 to 50 days (van Wagenen and Simpson, 1954). A dose of about 20 mg. per day is required. When medication is discontinued, marked involution of the testis occurs within the ensuing 3 weeks. Testosterone is effective even after a lapse of 50 days between hypophysectomy and the institution of therapy. Spermatogenesis can be restored and formation of motile sperm cells induced. As in the rat, the testes maintained by androgen are smaller than normal. Pellets of testosterone implanted locally exert a strong local action. Thus, the essential findings in the rat are duplicated in the monkey (Fig. 5.21).

In man the effects of testosterone on the testis have been studied by Hotchkiss (1944a), and by Heller, Nelson, Hill, Henderson, Maddock, Jungck, Paulsen and Mortimore (1950). The main effects were disappearance of the Leydig cells, atrophy of the tubules, arrest of spermatogenesis, and pronounced hyalinization of the basement membrane (Fig. 5.22). Complete recovery of the testis occurred 17 months after cessation of therapy. In fact, the testes were histologically more normal than before treatment. The improvement in sperm production after preliminary depression of the testis by administration of testosterone has been used widely in the treatment of male infertility. Heckel, Rosso and Kestel (1951) and Heckel and McDonald (1952a, b) obtained an increase in spermatogenic activity, as determined by sperm counts and biopsy, after cessation of treatment. This increase was termed a "rebound phenomenon"; during it, increased fertility, as determined by an increased incidence of pregnancy among infertile couples, was reported. The improved quality and quantity of sperm following therai)y with testosterone are transient. Furthermore, they occur in only a small i^-oportion of men so treated (Getzoff, 1955; Heinke and Tonutti, 1956). The suppressive effect of androgen on the human testis results from inhibition of pituitary gonadotrophin as evidenced by measurement of the amount of urinary gonadotrophin before, during, and after use of

MAMMALIAN TESTIS

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1

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Fig. 5.22. Elluci ul iC5io.stcruiie on the testis of a man with infertiUty caused by adult tubular failure. Testicular biopsies, showing the pronounced degree of sclerosis and hyalinization that occurs when an initially very poor testis is subjected to the administration of 91 consecutive injections of testosterone propionate, 25 mg. each. A, before treatment; B, at end of 91 days of treatment; C, 17 months after cessation of treatment. Note, in C, the disappearance of hyalinization, the increase in size of the seminiferous tubules, and the appearance of fairly orderly spermatogenesis. Leydig cells, not shown here, were present 17 months after treatment was stopped. (From C. G. Heller, W. O. Nelson, I. B. Hill, E. Henderson, W. O. Maddock, E. C. Jungck, C. A. Paulsen and G. E. Mortimore, Fertil & Steril., 1, 415, 1950.)

testosterone. The mechanism by which gonadotrophin is inhibited always has been assmned to be a direct effect of androgen on the pituitary. It is interesting in this regard that Paulsen (1952) showed that the use of testosterone, w^iile reducing urinary gonadotrophin, increases the amount of urinary estrogen 20-fold. Estrogen is by far the most powerful suppressant of gonadotrophin secretion known; hence, it is possible that the atrophy of the testis observed during testosterone therapy in man may be caused by estrogen. No reports of maintenance of spermatogenesis in men with pituitary insufficiency or after hypophysectomy are available.

B. ESTROGENS

Various natural and synthetic estrogens have been given to rats, guinea pigs, hamsters, cats, bulls, boars, and man. In all forms, estrogen induces atrophy of the male gonad. The histologic appearance of the atrophic rat testis after estrogen therapy has been described by Dischreit (1940). In young rats, estradiol prevents testicular descent, produces atrophy, and inhibits spermatogenesis (Pallos, 1941; Gardner, 1949). Two weeks following atrophy induced by estradiol or stilbestrol, regeneration of the testis begins (Bourg, Van Meen

sel and Compel, 1952) and is complete wuthin 6 weeks (Lynch, 1952). However, Snair, Jaffray, Grice and Pugsley (1954) noted that the accessory sex organs recover before spermatogenesis resumes. The same inhibiting effects have been obtained with methylbisdehydrodoisynolic acid (Tuchmann-Duplessis and Mercier-Parot, 1952) and hydroxypropiophenone (Lacassagne, Chamorro and Buu-Hoi", 1950). In general the effect of estrogen in the rat is to induce atrophy of the Leydig cells and germinal epithelium, so that only spermatocytes, spermatogonia, and Sertoli cells remain.

Uncertaint3^ exists concerning the general effects of estrogen in guinea pigs. Lynch (1952) noted that the Leydig cells are normal in animals treated with estrogen, but Marescaux (1950) and Chome (1956) noted that the Leydig cells are atrophic. Marescaux, in studying hypophysectomized guinea pigs, concluded that estrogen has a direct stimulating effect on the Leydig cell. Massive tubular damage occurs in the guinea pig after administration of estrogen. In the hamster. Bacon and Kirkman (1955) found that various estrogens induce testicular atrophy. In occasional animals, hyperplasia of interstitial and Sertoli cells occurs and is attributed to direct effects of estrogen. In general, atrophy of the germinal epithelium

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PHYSIOLOGY OF GONADS

is nearly complete; only a few spermatocytes remain in addition to the Sertoli cells.

The testis of the immature cat is unaffected by estrogen (Starkey and Leathem, 1939j . Severe tubular atrophy and involution of the Leydig cells are noted in bulls (Ferrara, Rosati and Consoli, 1953) and boars (Wallace, 1949j after feeding with stilbestrol.

Although Haschek and Gutter (1951) found no effect of estrogen on the testis, the consensus is that any kind of estrogen produces profound involution of the human testis. Temporary sterility is induced, of course, as well as impotence and gynecomastia (Heckel and Steinmetz, 1941). Most of the information in man has been obtained from the therapeutic administration of estrogen in cases of prostatic carcinoma (Chome, 1956; de la Baize, Mancini and Irazu, 1951 ; de la Baize, Bur, Irazu and Mancini, 1953; de la Baize, Mancini, Bur and Irazu, 1954; Schwartz, 1945; Schiiltz, 1952, to mention only a few) and from the administration of estrogen to hypersexual and homosexual men (Dunn, 1941). Estrogen induces atrophy of the tubules and the Leydig cells ; the latter revert to fibroblasts. The germinal epithelium shows an increase in lipids and a decrease in glycogen. Unless other disease is present, the atrophy proceeds so that only the Sertoli cells remain in the tubules; even these cells may disappear with the induction of peritubular hyalinization and sclerosis.

C. ADRENAL STEROIDS

Tubular diameter in the testis of the mature rat remains normal despite the presence of severe hypercortisonism resulting fi'oiii administration of 3 mg. cortisone per day for 6 weeks (Winter, Silber and Stoerk, 1950) or of 5 to 10 mg. per day (Ingle, 1950) . A few reports indicate that cortisone stimulates growth of the testes of young rats (Leroy, 1951) or causes degeneration of the germinal ei)ithelium of the rat (Leroy, 1952) and mouse (Antopol, 1950). A careful study by Hanson, Blivaiss and Rosenzweig (1957) showed that the relative growth of the testis is stimulated only slightly by cortisone.

Extremely little infoi-mation is available on the maintenance of spermatogenesis

in hypophysectomized rats by cortisone. Leroy and Domm (1952) reported maintenance at doses of 5 mg. per day. The Leydig cells involuted, and the secondary sexual apparatus was atrophic. However, these findings were not confirmed by Aterman ( 1956) , who used 5 mg. hydrocortisone per day after hypophysectomy. The scrotum became atrophic and the testes retracted. The histologic appearance of the testes of the cortisone-treated animals was indistinguishable from that of the hypophysectomized controls. In rabbits Arambarri (1956) reported only small changes in the relative weight after prolonged use of cortisone. In man, fairly large doses of cortisone given to patients with rheumatoid arthritis do not affect the histologic appearance of the testes (Maddock, Chase and Nelson, 1953). Cortisone does bring about rapid testicular maturation in boys who have congenital adrenal hyperjjlasia, but only if the bone age is near the age of puberty (Wilkins and Cara, 1954). This must not be construed as a direct effect of cortisone on testicular maturation. The action of cortisone in this instance is to inhibit the excessive release of corticotrophin (ACTH) from the pituitary, thus reducing the amount of 17-ketosteroids produced by the abnormal adrenals. Removal of the inhibiting effect of the androgenic steroids allow^s the formation of gonadotrophin, with resulting maturation of the testes.

The consensus is that cortisone does not cause any change in the histologic appearance of the testis (Cavallero, Rossi and Borasi, 1951 ; Soulairac, Soulairac and Teysseyre, 1955; Baumann, 1955). Furthermore, it causes no change in the accessory structures, or in the secretion of androgen by the testis. Cortisone has no direct effect on the prostate or seminal vesicles in castrated animals (Moore, 1953). It is doubtful whether cortisone can maintain spermatogenesis after hypophysectomy. The bearing of these studies on normal testicular physiologic function is questionable. Cortisone has been the main adrenal steroid studied in the rat but the rat adrenal secretes corticosterone, not cortisone.

Desoxycorticosterone has been administcicfl to rats in various doses. Arvy (1942) and Overzici' (1952) I'cported that the de

MAMMALIAN TESTIS

34;

velopment of the testis of the iiinnature rat was arrested by prolonged injections of this steroid. Effects from desoxycorticosterone are not evident in adrenalectomized animals (Migeon, 1952). Adult rats show atrophy of both the tubular apparatus and the Leydig cells (Naatanen, 1955; Selye and Albert, 1942a, b). Maintenance of spermatogenesis after hy])ophysectomy was described by Overzier (1952).

Because cortisone even in massive doses has little effect on the testis, it would seem unlikely that ACTH would have any dramatic effects. Li and Evans (1947) repoi'ted that ACTH depresses testicular weight and the weight of the accessories in young rats, has no effect in old rats, and does not maintain spermatogenesis or the accessories in hypophysectomized rats. Baker, Schairer, Ingle and Li (1950) reported a small reduction in testicular weight in adult rats, but spermatogenesis j^roceeded satisfactorily. Large doses of ACTH produced atrophy of the Leydig cells. Asling, Reinhardt and Li (1951) stated that large doses depress the weight of the accessory sex organs. However, Moore (1953) found that administration of 5 mg. ACTH per day for 10 days has no effect on the testis of young or old rats and has no extratesticular effect on the production of androgen.

D. MLSCELLAXEOU.S .STEROIDS AND MIXTURES OF STEROIDS

Masson (1945, 1946) studied 16 different steroids for their ability to maintain spermatogenesis. Androstenediol, methylandrostenediol, methylandrostanediol, A'^-pregneninolone, and dehydroisoandrosterone are the most active compounds in maintaining spermatogenesis after hypophysectomy. No relationship is apparent between the ability to maintain spermatogenesis and the androgenic activity of the compound as measured by stimulation of the seminal vesicles or the progestational activity (progesterone is effective in maintenance but ethinyl testosterone is not).

One compound, A^-pregneninolone, was studied in detail. It prevents testicular atrophy after hypophysectomy or following therapy with estradiol or testosterone; it does not produce atrophy of the Leydig cells. In doses of 1 to 2 mg. a day, preg

neninolone maintains spermatogenesis in young and adult hypophysectomized rats, l)ut it does not repair the tubules or Leydig cells after a 2-week delay between hypophysectomy and therapy. Pregneninolone also exerts a protective effect against the damage evoked by estradiol; however, it does not affect the regeneration that occurs after cessation of estradiol treatment. In this respect, it is different from testosterone, which hastens the recovery from the estradiol-induced damage. In fact, the acceleration of regeneration by testosterone is inhibited by pregneninolone. The chief difference between pregneninolone-progesterone and testosterone-androstenediol is that, whereas spermatogenesis is maintained by either pair after hypophysectomy, the former pair cannot restore spermatogenesis, and the latter can. ]\Iost of these effects of pregneninolone were confirmed by Dvoskin (1949). Progesterone and some new progestational compounds have been studied recently in man (Heller, Laidlaw, Harvey and Nelson, 1958). Progesterone given to normal men produces azoospermia and slight tubular atrophy, abolishes libido, and reduces potentia, but has no effect on the Leydig cells and the excretion of gonadotrophin, estrogen, and 17-ketosteroids.

Certain doses of desoxycorticosterone or estradiol have no effect on the testis singly, l)ut when mixed produce severe depression of testicular weight (.lost and Libman, 1952). The earlier work of Emmens and Parkes (1938), showing that testosterone inhibits the debilitating action of estrone, was confirmed by Joel (1942, 1945). The testes of animals treated with estradiol are one-sixth normal size; however, when testosterone propionate is added to the estrogen, the testicular weight is one-fourth normal. Furthermore, sperm cells are present in the epididymides of the group receiving testosterone. Mixtures of small amounts of androstenediol and estradiol in a constant proportion produce more profound atrophy than large doses given in the same constant proportion (Selye and Albert, 1942a, b; Selye, 1943). Furthermore, androstenediol and pregneninolone prevent the atrophy induced by small doses of testosterone. Plence, this protective action is not related to testoid activity, because the first compound is

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PHYSIOLOGY OF GONADS

a weak androgen ; the second has no androgenic action. The protective effect possibly is due to interference with the inhibiting action of testosterone on pituitary gonadotrophin.

XIV. Eflfeets of Altered Endocrine States on the Testis

Apart from the pituitary, alterations in the endocrine system do not have pronounced effects on the testis. The thyroid has been studied extensively with regard to testicular function (Maqsood, 1952). It is difficult to generalize with respect to the total impact of the thyroid on the testis except to state that there is great variability not only from species to species, but also in different individuals of any one species. Young, Rayner, Peterson and Brown (1952a) suggested that the range of thyroid activity within which normal testicular function is possible is rather wide. This may explain why many effects on the testis of altered thyroid function are marginal and why so many reports are exceedingly conflicting. Furthermore, it seems reasonable that animals having a naturally high level of thyroid activity may be impaired with respect to reproductive performance when made hypothyroid; conversely, species or individuals functioning normally at relatively low levels of thyroid activity may be adversely affected with regard to testicular activity when made hyperthyroid (Young, Rayner, Peterson and Brown, 1952b).

In laboratory animals, hypothyroidism is induced by thyroidectomy, by feeding of antithyroid substances, by administering radioiodine, or by combination of these methods. Hyperthyroidism is induced by feeding desiccated thyroid or various artificial thyroproteins, or by injecting thyroxine or triiodothyronine. Because it does not seem to matter, as far as testicular physiology is concerned, how hypothyroidism and hyperthyroidism are induced, dotails of the method of altering thyroidal status will not be given.

Hypothyroid rats show decreased spermatogenesis and have smaller accessory structures than normal rats (Smelser, 1939a). However, Jones, Delfs and Foote (1946) found that adult hypothyroid rats sire litters. Young animals, made hypothyroid at birth or shortlv thereafter, mav show

delay in sexual maturation (Scow and Marx, 1945 ; Scow and Simpson, 1945) , or may have normal reproductive tracts (Goddard, 1948). Hyperthyroid rats show testicular degeneration associated with a decrease in sperm production and androgen secretion. The deleterious effects of hyperthyroidism are attributed to an incapacity of the testis to respond to gonadotrophin. The atrophy of the accessory structures is attributed to the decrease in androgen production and to their increased requirement for androgen in states of hyperthyroidism (Smelser, 1939b). A nonendocrine explanation offered by Cunningham, King and Kessell (1941) is that testicular degeneration occurs because of the increased body heat of the animals in the hyperthyroid state. Richter and Winter (1947), however, stated that hyperthyroidism has a stimulating effect on the rat testis and accelerates the transfer of sperm through the genital ducts. Lenzi and Marino (1947) wrote that experimental hyperthyroidism causes a decrease in the number and volume of Leydig cells. Mixtures of thyroxine and testosterone in doses that have no effect on the rat testis when given singly, produce severe atrophy in normal rats (Masson and Romanchuck, 1945). Small doses of testosterone augment the debilitating effect of hyperthyroidism; large doses protect the testis (Roy, Kar and Datta, 1955). Changes in thyroidal status also appear to affect the responsiveness of the testis to gonadotrophins. Meites and Chandrashaker (1948) stated that hyperthyroidism decreases the responsiveness of the rat testis to exogenous gonadotrophin (PMS) whereas hypothyroidism increases it. The reverse holds for mice.

In growing mice, sexual development is retarded by hypothyroidism and accelerated by mild liyperthyroidism (Maqsood and R('inek(\ 1950). Moreover, the effectiveness of testosterone on the seminal vesicles of mice is increased by the concomitant administration of thyroxine (Masson, 1947). indicating an increased responsiveness of the accessory reproductive tract to male hormone in the hyperthyroid state.

Hyperthyroid guinea pigs have small testicular tubules and fewer sperm in the seminiferous tubules. As in the rat, Richter (1944) found that hyperthyroidism in the

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guinea pig was associated with a rapid discharge of sperm through the genital ducts. Hypothyroidism was found to have no effect on the structure of the testis, on the structure of the sperm cells in the ejaculate, or on fertility (Shettles and Jones, 1942). Young, Rayner, Peterson and Brown (1952a, b ) , however, observed that the degree of fertility of hypothyroid guinea pigs was slightly reduced but in general the strength of the sex drive was not altered significantly by either hypothyroidism or hyperthyroidism.

Other laboratory animals studied include the rabbit and the dog. Hypothyroidism in beagle puppies has no effect on spermatogenesis (Mayer, 1947j, whereas Maqsood (1951b) found atrophy of the seminiferous tubules and signs of decreased sexual drive in hypothyroid rabbits.

In male farm animals, alterations in thyroid function are associated with variable effects on the reproductive system. Atrophy of the tubules and Leydig cells occurs in the hypothyroid ram. Reduction of libido is noted in the hypothyroid ram, goat, and bull (Maqsood and Reineke, 1950). "Summer sterility" of sheep is explained as being due to depression of thyroid activity brought about by hot weather. Feeding thyroidal materials increases libido and spermatogenesis in bulls (Reineke, 1946; Petersen, Spielman, Pomeroy and Boyd, 1941). The reduction in testicular activity during hypothyroidism is attributed to an altered secretion of trophic hormones by the pituitary ; the excess secretion of thyrotrophin induced by thyroid deficiency in some way reduces the secretion of gonadotrophins (De Bastiani, Sperti and Zatti, 1956).

In man, Marine (1939) reported atrophy of the Leydig cells in a case of myxedema and atrophy of the tubules in a case of exophthalmic goiter; however, examination of the accompanying photomicrographs is not convincing. Many conflicting claims of the effect of thyroidal materials in infertile men have been made (c/. Dickerson, 1947) but these studies are uncontrolled and deserve no further comment. A recent study by Farris and Colton (1958), if verified, indicates that the nature of the thyroid substance used may be important after all. Thyroxine and triiodothvronine were ad

ministered to normal and subfertile men. Thyroxine depressed the number and activity of the sperm cells in the ejaculate, whereas triiodothyronine had a beneficial effect on the quality and motility of the spermatozoa.

Very little can be found on the effect of altered adrenal function on the testis. During the alarm reaction induced by the injection of formalin, no changes are evident in the testis when the adrenal cortex is undergoing its usual response (Croxatto and Chiriboga, 1951, 1952). Chronic hyperadrenalism produced by injections of epinephrine is accompanied occasionally by testicular atrophy and usually by regression of the accessories (Perry, 1941). Adrenalectomy in dogs, cats, and man is not followed by alteration in testicular structure (Morales and Hotchkiss, 1956) .

In rats rendered diabetic by removal of 95 per cent of the pancreas, a slight decrease was observed in testicular weight. In the final stages of diabetic cachexia, however, severe testicular atrophy occurs (Foglia, 1945). Horstmann (1949, 1950) concluded that the impotence of diabetic men results from the combined effects of decreased androgen production and of increased androgen destruction. This conclusion was, however, denied by Bergqvist (1954). Impotency and loss of libido are encountered frequently in association with uncontrolled diabetes; both may be corrected by adequate therapy. However, men more than 35 years of age whose diabetes is well controlled may have irreversible loss of libido and potentia. Histologic evidence of atrophy in the testes of such diabetic men can be found in the literature. The atrophy described seems no greater than that which may occur spontaneously in normal men at various ages, however.

The pineal body has long been thought to be involved in the regulation of the testis. The following conflicting statements have been made: (1) administration of pineal extracts inhibits testicular development, (2) pinealectomy causes testicular hypertrophy, (3) the concentration of cholesterol esters in the testis is lowered by administration of pineal extracts, and (4) none of the above results are obtained (Simonnet and Sternberg, 1951; Simonnet

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and Thieblot, 1951 ; Alcozer and Costa, 1954, Alcozer and Cliordano, 1954; Bailo, 1955). The reader is referred to a recent book which summarizes the literature on the pineal body (Kitay and Altschule, 1954 ) . Extensive hepatic disease is associated with testicular atrophy. Morrione (1944) induced cirrhosis in male rats by means of carbon tetrachloride. The testes of the cirrhotic rats were not affected. However, when estrogen was administered, severe testicular atrophy occurred, much greater than that induced by the same amount of estrogen in control, noncirrhotic animals. Testicular atroi^hy is said to occur in 70 per cent of men who have cirrhosis of the liver (Bennett, Baggenstoss and Butt, 1951). There is no critical information from which one could conclude that the atrophy of the testis in cirrhotic men is caused by failure of the diseased liver to inactivate estrogen.

XV. Nonneoplastic Disorders of the Testis

Study of certain hypogonadal disorders of man has provided information of general interest and bearing on the physiology of the mammalian testis. For an index to the large clinical literature on pituitary-testis relationships, the reader may consult Heller and Nelson (1948) and Albert, Underdahl, Greene and Lorenz (1953-1955). A group of spontaneously occurring disorders shows clearly the control of the testis by gonadotrophin. In pituitary dwarfism, the testis remains infantile even as late as 30 or 40 years of age, and perhaps for the entire life span of the individual so afflicted. Leydig cells are not jirescnt, and the tubules contain only undifferentiated cells and occasional spermatogonia. Pituitary dwarfism is a form of hypopituitarism in which all hormones of the anterior lobe may be absent. Anotiiei' type of hyi)ogonadism in man is restricted to the loss of only the gonadotrophic function of the pituitary. In this syndrome, the testis does not contain mature Leydig cells or mature tul)ules. This syndrome represents a condition that cannot be duplicated in lower animals. A few instances of a selective type of gonadotropliir insuflficiency have been described in which tubular maturation proceeds, with (liffei'entiation of the Sei'toli cells and the

formation of sperm. However, Leydig cells are not present. This syndrome ("fertile eunuchs"), if interpreted in terms of the dualistic concept of pituitary control of the testis, is explainable on the basis that formation and secretion of FSH have occurred but that LH is absent. If pituitary lesions occur before puberty, the testes remain immature. Pituitary lesions occurring after maturity cause atrophy of the seminiferous epithelium, not immaturity. The adult tubule of man cannot dedifferentiate as does the mature Leydig cell following hypol^hysial deprivation. The atrophy may vary in severity from hypospermatogenesis to complete sclerosis. Lack of gonadotrophin in the adult also results in thickening of the tubular wall and atroph}^ of the Leydig cells.

The most common defect in the human testis is failure of the seminiferous tubules. In contrast to the pituitary deficiencies, which generally result in both tubular and androgenic failure, disorders of sj^ermatogenesis lead only to infertility. The Leydig cells are normal, and androgenic function is unimpaired. The disordered spermatogenesis and the presence of cellular debris in the lumen are reflected by an abnormal spermogram. Depression of the sperm count to the point of azoos]M'rmia, abnormal sperm cells, and poor motility are characteristic findings. Another type of primary testicular disorder associated with azoospermia is germinal aplasia, in which the tubules contain only Sertoli cells. The Leydig cells are normal; hence, androgenic function is normal. Klinefelter's syndrome also is associated with azoospermia but the function of the Leydig cells is variable, ranging from severe insufficiency, in which the afflicted persons are eunuchoidal, to mild insufficiency, in which the liabitus is normal or almost so.

Testicular disorders are not restricted to man. They occur in common laboratory animals and in veterinary practice. Their similarity to some of the clinical entities just described will be evident.

A genito-urinary abnormality occurs in 20 per cent of males of the A x C rat (Vilar and H(n-tz, 1958). On one side, the testis is atroi)hic and the kidney, ureter, ductus deferens, epididymis, and seminal vesicle are absent; however, the coagulating gland is |)r('scnt. The testis is noi'mal ])i'epul)ertally

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u}) to 10 days of age. The lumenlcss tubules contain two types of cells; one is a small cell with one nucleolus; the other is a large round cell containing two or three nucleoli. Oval cells resembling Leydig cells are present in the interstitium. At 19 to 24 days of age, both testes are ecjual in weight. The diameter of the tubules increases, a lumen is present, and the tubular wall becomes differentiated. Sertoli cells, spermatogonia, and spermatocytes are evident, and the Leydig cells are maturing. At 30 to 38 days of age, the testis on the abnormal side is noticeably smaller. The Leydig cells remain normal, but the tubules are decreased in size. Between 45 and 47 days of age, spermatogenesis ceases and the tubules become atrophic. Thick collagenous and elastic fibers are found in the tubular wall. This disorder seems to be an inherited defect with delayed somatic manifestations. In some aspects, the pathogenesis of this testicular disorder in rats resembles that in Klinefelter's syndrome.

Congenital spermatogenic hypoplasia occurs in guinea pigs (Jakway and Young, 1958) . It ranges from germinal aplasia in most of the seminiferous tubules to a condition in which the appearance of the tubules is almost normal and the percentage of fertile matings is only slightly reduced. When sterility is present, the testes are smaller than those of normal males. The hormonal production, as reflected by the size of the penis and seminal vesicles and by sexual behavior, is normal.

The mule has a J-shaped chromosome which is contributed by the ass (Makino, 1955). Spermatogenesis in the mule does not proceed beyond meiotic prophase, degeneration occurring without formation of the metaphase of the first division. Hence, sperm cells will not form. The testes become atroi^hic, and only a few^ spermatogonia remain. The Leydig cells are normal.

Different types of hypogonadism, some of which are inherited, are encountered in bulls. Hypoplasia associated wuth urate crystals in the semen probably results from disintegration of the seminiferous epithelium (Barron and Haq, 1948) . Idiopathic necrosis of the tubule also may cause massive testicular calcification (Barker, 1956). Seven cases of hypogonadism in Belgian bulls were

reported as a form of congenital sterility (Derivaux, Bienfait and Peers, 1955) ; photomicrographs of the testes in these cases are similar to those of germinal aplasia in the human. Testicular hypoplasia occurs also in goats (Rollinson, 1950).

Captive wild animals become sterile. Bushman, the famous gorilla at the Chicago Zoo, died at the age of approximately 22 years. Necropsy revealed neuropathy, cardiopathy, hemosiderosis, and testicular sclerosis (Steiner, Rasmussen and Fisher, 1955). No cells of the germinal epithelium were present except occasional Sertoli cells. The Leydig cells were normal. The testicular atrophy of Bushman was similar to that of Bobby, at the Berlin Zoo. Whether this degenerative testicular lesion is caused by nutritional deficiency or by the "stress" of captivity is not known.

XVI. Tumors of the Testis

Testicular tumors are more common among lower animals than in man (Innes, 1942). Spontaneously occurring Sertoli-cell and Leydig-cell tumors of animals have been studied more than seminomas presumably because of the greater endocrinologic interest attached to them. Huggins and Pazos (1945) found 64 testicular tumors in 41 dogs; of these, 33 were Leydig-cell tumors, 19 were seminomas, 9 were tubular adenomas, and 3 were undifferentiated tumors. Zuckerman and McKeown (1938) found tumors in 35 of 243 dogs. A few of these were Sertoli-cell tumors which were associated with metaplasia of the prostate. The life span of dogs varies from 8 to 15 years, and testicular tumors occur most frequently at 7 years of age or older; in fact, more than half of old dogs are found to have such tumors (Scully and Coffin, 1952). The most common tumor of the dog testis is a Leydig-cell tumor. Five per cent of testicular tumors in dogs occur in undescended testes. The neoplasms in cryptorchid testes are usually Sertoli-cell tumors (Greulich and Burford, 1936; Coffin, Munson and Scully, 1952; Mulligan, 1944).

The veterinary diagnosis (Blum, 1954) of Sertoli-cell tumors is easily made, because the dogs become feminized. For this reason, the chief comjilaint of the owners is tlutt normal male dogs, after a brief olfaciMi}

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reconnaissance, attempt to mount their afflicted pets. In addition to the feminization, evidence that Sertoli-cell tumors produce estrogen comes from the finding of estrogen in the urine of tumor-bearing animals and from the extraction of estrogen from the tumor itself (Berthrong, Goodwin and Scott, 1949). In terms of estradiol, the concentration of estrogen extracted from a Sertoli-cell tumor (Huggins and Moulder, 1945) was twice that found in the ovary from an estrous bitch. Sufficient estrogen appears to be produced to cause such changes as loss of hair, depression of libido, cystic hyperplasia of the mammary glands, and atrophy of the testis.

Interstitial cell tumors in dogs are usually nonfunctional, but they may produce estrogen (Laufer and Sulman, 1956; Kahan, 1955). Leydig-cell tumors have been reported in the mule, the Brahma bull, and the saddle horse (Smith, 1954). Significantly, in the last instance, an interstitial cell tumor occurred in the undescended testis of a 7-year-old horse, the descended testis having been removed early in life.

In man the proportion of various types of testicular tumors is different from that in lower animals. Seminomas and embryonal carcinomas are the most frequent neoplasms. Interstitial cell tumors have been recorded in less than two dozen instances in the world literature. Several cases of Leydig-cell tumor have been studied by Venning (1942) , Cook, Gross, Landing and Zygmuntowicz (1952), Hertz, Cohen, Lewis and Firminger (1953), and Jungck, Thrash, Ohlmacher, Knight and Dyrenforth (1957). This tumor causes isosexual precocity in boys. Signs of androgenic activity are evident in the large penis ; scrotal maturation ; the appearance of pubic, facial, and axillary hair, and acne; increased bodily growth; maturation of the larynx; and increased excretion of 17-ketosteroids. All these findings occur when sufficient amounts of testosterone are injected into normal prepubertal boys. This tumor cannot conceivably be related to the secretion of LH (see subsequent material on experimental tumors), because the neoplasms are usually unilateral and the contralateral normal testis shows no activation of the Leydig cells.

Neoplasms classified as Sertoli-cell tu

mors are rich in lipids and are thought to secrete estrogen (Teilum, 1950). However, the histogenesis of these tumors is not clear, and there is doubt that Sertoli-cell tumors actually occur in man.

Testicular tumors have been induced in rats by transplantation of immature testes to the spleen of castrated adult animals (Biskind and Biskind, 1945) and by radiation, carcinogens, and other means (Peyron and Samsonoff', 1941). Transplantation of day-old rat testes to the spleen of castrated adult rats, normal male rats, and castrated adult female rats resulted in the formation of encapsulated and sharply circumscribed tumors. Of 29 tumors thus produced, 16 were composed entirely of interstitial cells and 13 contained other testicular elements as well. One of the tumors was transplantable into the spleen of a castrated animal. Because hyperplasia of the interstitial cells was seen in most of the transplanted testes, it was thought that the neoplasia followed the hyperplasia induced by the excess of gonadotrophin in the castrated host (Twombly, Meisel and Stout, 1949). Such Leydig-cell tumors produce estrogen (Fels and Bur, 1956).

In contrast with the rat, experimental tumors in the mouse are not induced by any of the methods already mentioned (Gardner, 1953). Spontaneous tumors of the testis in mice do occur, however. Slye, Holmes and Wells (1919) found 28 testicular tumors in some 9000 male mice. None formed metastatic lesions. Hummel (1954) reported a spontaneous tumor in an 18-month-old mouse of the BALBC strain; this neoplasm was transplantable for three generations in normal or gonadectomized adult males or females. This was a functioning tumor as evidenced by masculinization of the submaxillary glands, mucification of the vagina, hypertrophy of the clitoris, and an increase in size of the uterus of the female host and of the accessory sex organs of the male host. All these findings indicate estrogenic and androgenic secretion. In general, however, interstitial cell tumors in mice are strainlimited, occurring particularly in the AC and JK strains. Spontaneous interstitial cell tumors also occur in hybrids and are associated with mammary tumors (Gardner, Pfeiffcr, Trentin and Wolstenholme, 1953).

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This association indicates that estrogen is involved in the formation of the tumor; indeed, it is chiefly by the use of estrogen that experimental tumors in mice have been provoked.

Various natural and synthetic estrogens are effective. For example, Hooker, Gardner and Pfeiffer (1940) and Hooker and Pfeiffer (1942) using estradiol and stilbestrol have been able to produce interstitial cell tumors in the A and C strains of mice, with an incidence of 50 and 90 per cent respectively. Treatment for 8 months with 16.6 to 50 fjig. of estradiol dibenzoate or 0.25 /xg. stilbestrol weekly produces tumors, some of which metastasize to the renal, lumbar, and mediastinal lymph nodes. These tumors are transplantable if the hosts are given estrogen. They are inhibited by the simultaneous injection of testosterone. Tumors also may be induced by implantation of pellets of stilbestrol and cholesterol. The implantation of a 4- to 6-mg. pellet of 10 to 25 per cent stilbestrol in cholesterol induced tumors within 5 months (Shimkin, Grady and Andervont, 1941). Of the various natural and synthetic estrogens the triphenylethylene derivatives appear to be the most potent. Bonser (1942) and Gardner (1943) produced transplantable tumors in the JK, the A, and the C 3H strains by triphenylethylene. Tumors thus induced are generally composed of interstitial cells. They are transplantable only in the same strain of mice and only when the hosts are given estrogen. After several generations, however, the tumor may be transplanted without administration of estrogen in normal and in hypophysectomized mice (Gardner, 1945; Andervont, Shimkin and Canter, 1957).

The tumors arise from hyperplastic interstitial cells. The Leydig cells enlarge, become foamy, and degenerate. JMacrophages or, at least, cells containing a brown pigment appear and phagocytose the exhausted Leydig cells. A new crop of interstitial cells appears from the mesenchyme. These may grow faster in one zone than in another. The faster-growing Leydig cells thus constitute a nodule. The Leydig cells in the nodule also become hyperplastic and foamy. These nodules appear as white spots and cause pressure atrophy of the tubules. Leydig cells in

the tumor thus result from three generations, since the second crop of Leydig cells is followed by a third generation containing small primitive and hyperchromatic cells. These contain brown pigment and hence give the brown color to the tumor. At this stage, the tumor may become necrotic, may metastasize by way of lymph or blood, or may invade locally. Such tumors secrete both estrogen and androgen. The consensus is that estrogen induces interstitial cell tumors in mice by liberation of LH (Gardner, 1953).

The assumption that LH induces interstitial cell hyperplasia and finally a tumor has received support from studies by Simpson and van Wagenen (1954) on young monkeys. These investigators gave ICSH for 53 days. Hyperplasia of the Leydig cells took place and nodules resulted. These nodules were composed of concentric laminated peritubular cells and arose from the same type of mesenchymal cell that yields the Leydig cell under normal conditions. Under the influence of HCG, the nodules secreted androgen.

XVII. Conclusion

The postnatal development of the mammalian testis follows a fairly definite pattern. Development is slow for the variable period of prepubertal life. The testis then undergoes rapid evolution during puberty, remains fairly constant in adult life, then regresses somewhat in old age. The rapid development of the testis during puberty is brought about by the onset of gonadotrophic function of the pituitary. This developmental pattern is fixed for each species, but can be modified by genie and environmental factors. Once the adult status is attained, secretory controls of androgenic and spermatogenic functions are established. A steady state of testicular function is maintained in continuously breeding species. In those mammals which show a seasonal breeding cycle, these secretory controls, particularly those of the pituitary gland, are periodically activated and deactivated.

The testes of many eutherian mammals migrate from the abdomen during fetal life to the scrotum. This migration is regulated by hormones of the fetus, presumably arising from the fetal testis. It is not clear just

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why the testes occupy the scrotum. The explanation that scrotal residence provides "optimal testicular temperature" is not satisfying because one then wishes to know why the male gonad requires the cooler environment afforded by the scrotum. Failure of the testes to descend may occur as a consequence of defects in the testes, probably of genie origin ; or because of anatomic obstacles, representing embryologic defects, inadvertently placed along its prescribed narrow path. In either event, the testis is damaged, mildly in its endocrine function, and seriously in its spermatogenic function. Impairment of spermatogenesis of the misplaced testis is due to the relatively high temperature of the abdomen. Temperature affects the germinal epithelium directly. It also affects the testis indirectly through the circulatory system. The effect of temperature, or for that matter, of any type of injurious agent whether it be chemical or physical, is atrophy of the seminiferous epithelium. The response of the germinal tissue to deprivation of pituitary gonadotrophin likewise is atrophy. Quantitative variation among different species does of course exist, but qualitatively, atrophy is the universal response to injury. Obviously, a common denominator must exist for this fairly general reaction on the part of the germinal epithelium. If various chemical and physical stimuli act on the testis by means of suppression or interference with the action of gonadotrophins, atrophy of the Leydig cells would also result. However, many chemical and physical agents affect only the germinal epithelium, leaving the Leydig cells unscathed. Thus, the germinal epithelium can be damaged directly and the variable damage to the components of the spermatogenic epithelium must be due to different sensitivities of its cellular components. The Sertoli cell is much more resistant than the cells of the germinal line, and of the seminiferous elements, the type A spermatogonia are the most resistant. Of great importance in the interpretation of the damage induced by many substances or occurring as a result of disease is the characteristic of the germinal epithelium to reproduce in a fixed order and sequence. It follows that the extent of injury to spermatogenesis as a whole would 1)C determined

by the relative susceptibility of the various germinal cells as well as by the nature of the noxious agent. If only sperm cells are affected, spermatogenesis will proceed through the formation of spermatid. However, if spermatogonia are injured, full differentiation of the germinal epithelium will fail, and only Sertoli cells will be found in the tubule. Thus, it is possible that all sorts of injury to the testis, if sufficiently great, may result in the same end stage of testicular atrophy. In spite of this common reaction pattern to severe injury, many substances induce what seem to be specific lesions in the testis. However, these represent intermediate or partial injuries, and do not necessarily constitute exceptions to the general pattern of testicular response to injury. As more is learned about the biochemistry of the germinal epithelium, it may be possible to induce specific lesions.

Quantitative studies on spermatogenesis have greatly clarified the role played by the pituitary gland. Spermatogenesis does proceed in hypophysectomized animals but only at a low rate. Also it appears that androgen, not gonadotrophin, is responsible for the maturation of the spermatid. However, it must be remembered that the formation of androgen is dependent on pituitary gonadotrophic function. Thus spermatogenesis is regulated entirely by pituitary gonadotrophins, which exert direct supervision over the rate of the mitotic and meiotic activity of germ cells and indirect supervision by way of the Leydig cell over spermatid maturation, or spermiogenesis. The effectiveness of androgen in sperm formation is hardly equal to that of the pituitary. Addition of trophic hormones (except gonadotrophin) or of hormones of the target glands (tliyi'oid, adrenal cortical hormone, etc. I will ])robably not improve the effectiveness of androgen. The best evidence that this surmise may be correct is obtained from jnitients with hypogonadotroi)hic hyl)ogonadism. These i)atients have normal function with respect to the other trophic hormones of the pituitary and, therefore, normally functioning peripheral glands, but do not have sperm.

The quantitative studies on the spermatogenic cycle have important bearing on other |)i'o]»lciiis wliicli have been i)uzzling to endo

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crinologists. jMany unsuccessful attempts have been made to induce precocious sperm formation in the rat by chronic or massive use of various gonadotrophins. The time of a complete spermatogenic cycle is not accurately known. Estimates ranging from 20 to 40 days have been given, which reflects the difficulties and errors of present methods. If one adds to the time at which sperm formation normally occurs in common strains of the laboratory rat (around 35 days of age ) , about 10 days borrowed from fetal life, the time of a complete spermatogenic cycle is probably between 45 and 50 days. Hence, no amount of exogenous gonadotrophin could be expected to produce precocious spermatogenesis, because a certain irreducible minimum of time may be recjuired for the series of divisions which in toto constitutes a spermatogenic cycle. However, if the interval between birth and maturity is much longer than the time of a complete spermatogenic cycle, precocious spermatogenesis could be experimentally achieved, as is again indicated by an example from clinical endocrinology, i.e., the spontaneous occurrence of isosexual precocity in boys.

In another clinical area, the application of quantitative techniques to the study of testes of iKitients afflicted with infertility has so far not yielded helpful information. Restoration of fertility in men with adult seminiferous tubular failure has not been accomplished. Infertility, however, is receiving increasing attention, especially from the standpoint of genie factors. It is in this area that the only startling development of knowledge on the testis in the past 20 years has occurred, i.e., the discovery that men with Klinefelter's syndrome are "genetic females." One may, with good reason, question the suitability of the term "genetic females." It arose from the application of Barr's discovery of sex dimorphism in the heterochromatin of somatic cells (Barr, 1956; Barr and Bertram, 1949; Moore and Barr, 1955) . Normal females are "chromatin positive"; normal males are "chromatin negative." This, however, may not be absolute. Men with Klinefelter's syndrome are chromatin positive, and if chromatin positivity reflects genie constitution, it is likely that the sterility of men with this syndrome

(one of its outstanding features) represents an abnormality of chromosomal division or number during gametogenesis of one of their parents. Generally similar situations may occur in lower animals; hence, the role of genie factors in fertility can be studied experimentally.

Great advances have taken place in knowledge of the biosynthesis of male hormone by the testis. Illumination of the chemical pathway over which simple precursors (acetate) or more complex ones (cholesterol) are transformed to testosterone represents a major contribution in biochemistry. The enzymatic control of the various chemical steps will undoulitedly be disclosed before long.

XVIII. References

Albert, A., Underdahl, L. O., Greene, L. F., and LoRENz, N. 1953a. Male hypogonadism. I. The normal testis. Proc. Staff Meet., Mavo Clin., 28, 409.

Albert, A., Underdahl, L. O., Greene, L. F., and LoRENz, N. 1953b. Male hypogonadism. II. Classification. Proc. Staff Meet., Mavo Clin., 28, 557.

Albert, A., Underdahl, L. O., Greene, L. F., and LoRENZ, N. 1953c. Male hypogonadism. III. The testis in pituitary dwarfism. Proc. Staff Meet., Mayo Clin., 28, 698.

Albert, A., Underdahl, L. O., Greene, L. F., .\nd LoRENz, N. 1954a. Male hypogonadism. IV. The testis in prepubertal or pubertal gonadotrophic failure. Proc. Staff Meet., Mavo Chn., 29, 131.

Albert, A., Underdahl, L. O., Greene, L. F., .^nd LoRENZ, N. 1954b. Male hypogonadism. V. The testis in adult patients with multiple defects of pituitary function. Proc. Staff Meet., Mayo Clin., 29,317.

Albert, A., Underdahl, L. O., Greene, L. F., .od LoRENz, N. 1954c. Male hypogonadism. VI. The testis in gonadotrophic failure in adults. Proc. Staff Meet., Mayo Chn., 29, 368.

Albert, A., Underdahl, L. O., Greene, L. F., .\^d LoRENz, N. 1955. Male hypogonadism. VII. The testis in partial gonadotrophic faihne during puberty (lack of luteinizing hormone onlv). Proc. Staff Meet., Mayo Clin., 30, 31.

Alcozer, G., and Costa, U. 1954. Comportamento delle frazioni colesteroliche nel testicolo di cavia adulta trattata con estratto acquoso di pineale. Arch. "E. Maragliano" pat. e clin., 9, 355.

Alcozer, G., .^nd Giordano, G. 1954. Rapporti epifiso-testicolari ; modificazioni istologiche del testicolo di cavia adulta e di topino aduho dopo somministrazione di estratto acquoso di ghiandola pineale. Arch. "E. Maragliano" pat. e clin., 9, 433.

Allex, E., and Altlaxd, P. D. 1952. Studies of degenerating sex cells in immature mammals. II. Modes of degeneration in the normal differentiation of the definitive germ cells in the male albino rat from age twelve days to maturity. J. Morphol., 91, 515.

Alnor, p. 1951. Zur Frage der Beeinflussung der Sexualfunktion durch Resektion des lumbalen Grenzstranges. Arch. klin. Chir., 269, 506.

Andervoxt, H. B., Shimkin, M. B., axd Canter, H. Y. 1957. Effect of discontinued estrogenic stimulation upon the development and growth of testicular tumors in mice. J. Nat. Cancer Inst., 18, 1.

Anthoxy, a. 1953. Seasonal reproductive cycle in the normal and experimentally treated male prairie dog, Cynomys ludovicianus . J. Morphol., 93, 331.

Antliff, H. R., .and Young, W. C. 1957. Internal secretory capacity of the abdominal testis in the guinea pig. Endocrinology, 61, 121.

Antopol, W. 1950. Anatomic changes produced in mice treated with excessive doses of cortisone. Proc. Soc. Exper. Biol. & Med., 73, 262.

Arambarri, M. L. 1956. Die Wirkung von holen Cortisondosen auf das Hodenzwischengewebe des Kaninchens. Ztschr. ges. exper. Med., 127, 227.

Arvy, L. 1942. L'hypophyse et I'acetate de desoxvcorticosterone. Compt. rend. Soc. bioL, 136, 660.

AsDELL, S. A. 1946. Patterns of Mammalian Reproduction. Ithaca: Comstock Publishing Company, Inc.

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Smith, P. E. 1938. Comparative effects of hypophysectomy and therapy on the teste.s of monkeys and rats. In Les hormones sexuelles, Foundation Singer-Polignac Collogium, L. Bronha, Ed., vol. 3, p. 201. Paris: Herman.

Smith, P. E. 1939. The effect on the gonads of the ablation and implantation of the hypophysis and the potency of the hypophysis under various conditions. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., p. 931. Baltimore: The Williams & Wilkins Company.

Smith, P. E. 1942. Effect of equine gonadotrophin on testes of hypophysectomized monkeys. Endocrinology, 31, 1.

Smith, P. E. 1944. Maintenance and restoration of spermatogenesis in hypophysectomized rhesus monkeys by androgen administration. Yale J. Biol. & Med., 17, 281.

Snair, D. W., Jaffray, S. E., Grice, H. C, and PuGSLEY, L. I. 1954. Studies on the subacute and chronic administration of stilbestrol in the male rat. Canad. J. Biochem. & Phvsiol., 32, 41.

Sniffen, R. C. 1952. Histology of the normal and abnormal testis at pubertv. Ann. New York Acad. Sc, 55, 609.

Sohval, a. R. 1954. Histopathology of cryptorchidism; a study based upon the comparative histology of retained and scrotal testes from birth to maturity. Am. J. Med., 16, 346.

SoTiRiADou, E. 1941. Gonad stimulants in immature hypophysectomized male rats. Acta brev. neerl. Physiol., 11, 1.

S0ULAIR.\C, A., S0UL.«R.'\C, M.-L., AND TeYSSEYRE, J.

1955. Action de la corticostimuline hypophysaire, de la cortisone et de la desoxycorticosterone sur le testicule et le canal deferent du rat normal adulte. Ann. endocrinol., 16, 229.

Srere, p. a., Chaikoff, I. L., Treitm.an, S. S., and Burstein, L. S. 1950. The extrahepatic synthesis of cholesterol. J. Biol. Chem., 182, 629.

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Stein, H. J., Bader, R. A., Eliot, J. W., and Bass, D. E. 1949. Hormonal alterations in men exposed to heat and cold stress. J. Clin. Endocrinol., 9, 529.

Steiner, p. E., Rasmussen, T. B., and Fisher, L. E. 1955. Neuropathy, cardiopathy, hemosiderosis, and testicular atrophv in Gorilla gorilla. A. M. A. Arch. Path. 59, 5.

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Talbot, H. S. 1955. The sexual function in paraplegia. J. Urol., 73, 91.

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Teilum, G. 1956. The sources of testicular androgen and estrogen. Acta med. leg. et sociol., 9, 305.

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TUCHIVLANN-DUPLESSIS, H., .\ND MeRCIER-P.AROT, L.

1952. Action de I'acide 1 methyl-bis-dehydrodoisynoliqiie sur le systeme endocrine et la repousse des poils du rat. Compt. rend. Soc. biol., 146, 919.

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Zide, H. x4.. 1939. Does compensatory hypertrophy of the adult human testis occur? J. Urol., 42, 65.

Zlotnik, I. 1943. A nuclear ring in the developing male germ cells of dog and cat. Nature, London, 151, 670.

Zuckerman, S., and McKeown, T. 1938. The canine prostate in relation to normal and abnormal testicular changes. J. Path. & Bact., 46, 7.

The Accessory Reproductive Glands of Mammals

Dorothy Price, Ph.D.

Professor Of Zoology, The University Of Chicago

and

H. Guy Williams- Ashmari, Ph.D.

Associate Professor, Ben May Laboratory, The University Of Chicago

L Gross Structure, Homologies, and I. Gross Structure, Homologies, and

Occurrence in Mammalian Orders. 366 Occurrence in Mammalian

A. Introduction c566 „ ,

B. General Characteristics 367 Ureters

C. Survey of the Glands 368 j^ INTRODUCTION

1. Bulbo-urethral and bulbovestibu- ^, • , , ^ , ,

la,!- 368 The genital system of male mammals con 2. Male and female prostate glands. . 369 sists of three component parts. These are:

3. Seminal vesicles 376 ( 1) paired testes, the primary sex organs in

4. Ampullary glands ■ • ■ • ■ 376 ^^^j^j^j-, spermatozoa are formed and andro U. Evolutionarv Historv of Accessory • i j. j /ov

Reproductive Glands in Mammals 376 g^nic hormones are secreted; (2) accessory

II. Function ofMale Accessory Glands.. 377 reproductive organs, a continuous series of

A. Introduction 377 ducts in which spermatozoa are transported

B. Volumetric Studies of Secretion 378 fpo^ the testes, stored in the tail of the epi 1. Prostatic isolation operation^. 378 .^^^^ ■ ^nd finallv carried to the exterior

2. Prostatic translocation operation. 380 , - . , ,. ' ,

C. Chemical Composition of the Glandu- ^^hen ejaculation occurs, and various

lar Secretions 380 glands, the secretions of which provide the

I). Metabolism of the Prostate and Sem- carrying medium for the spermatozoa at

inal Vesicle 394 emission ; ( 3) external genitalia, the penis

K. Coagulation of Semen 396 , , , . , ,

III. Structure and Function in Relation ^r copuhitory organ and, m most mammals,

TO Hormones 398 i^ scrotum in which the testes come to lie

A. Introduction 398 more or less permanently, or only periodi B. Effects of Androgens 399 p.^Hy during the breeding season.

1. Testicular andn,gens 399 ^^ ^^jj^ -^ ^j^^ epithelial lining of the

2. Adrenal androgens 423 / . i i j r l ^ c

3. Ovarian androgens 424 ^ttcrent, epididymal, and deferent parts of

4. Progesterone 425 the duct system have secretory functions,

C. Effects of Estrogens 426 but all male mammals develop discrete and

I). Hormonal Control ()f Spontaneous ^^^ specialized glands which are associated with

1. Benign growtU.""^ '.'.'.'.'.'.'.'.'.'.'.'.'.'. 429 ^Pecific regions of the reproductive tract and

2. Prostatic cancer 430 eject their secretions into it at seminal

E. I'^ffects of Carcinogenic Aromatic Hy emission. The degree of development of

drocarbons 430 these large, conspicuous glands is a unique

F. Effects of Nonsteroid Hormones 433 .ij^r^cteristic of mammals.

1. I'rolactin (LTH) 433 _, i x- i j u

2. Growth hormone (STH) 434 The accessory reproductive glands can be

IV. References 435 grouped logically into those ^vhich arise

366

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

367

embryonically from the mesonephric or Wolffian duct (ductus deferens) i.e., the ampullary glands (glandula vasis deferentis) and seminal vesicles or vesicular glands, and those deriving from the urogenital sinus or urethra, namely the prostate and bulbo-urethral or Cowper's glands (see chapter by Burns). The anatomic relationships established in the fetus are retained to a considerable degree postnatally so that the ampullary glands and seminal vesicles are associated with the ducti deferentes. However, in some mammals the seminal vesicles empty into the pelvic urethra close to the openings of the deferent ducts but separate from them; no ejaculatory ducts are present. The prostatic and bulbourethral glands are associated with the proximal and distal urethra, respectively. The secretion of the prostate is discharged, in most cases, through multiple ducts that join the prostatic urethra at the level of the colliculus seminalis. The ducts of the bulbo-urethral glands drain into the urethra in the region of the urethral bulb.

In addition to these accessory reproductive glands, there are small mucus-secreting glands (of Littre) opening into the urethra along its length, and preputial glands (which are modified sebaceous glands) emi)tying their secretion on the prepuce.

In many female mammals, homologues of the male prostate and bulbo-urethral glands develop in the fetus. These glands may retrogress prenatally, remain vestigial, or develop postnatally and become functionally active. These homologues are the female prostate glands (para-urethral glands of Skene) and the bulbovestibular (major vestibular or Bartholin's glands). In addition, there are urethral glands (minor vestibular) which are homologous with the male urethral glands of Littre, and female preputial or clitoridal glands corresponding to the male preputials. The major vestibular, when present, and the minor vestibular and clitoridal glands are functional in many mature females. In a few cases, well developed prostate glands which are actively secretory have been found in females of four mammalian orders.

B. GENERAL CHARACTERISTICS

The male accessory reproductive glands of higher mammals have many characteristics in common. Typically, all possess (1) a secretory epithelium which is enormously increased in effective secretory area by villous infoldings, or by a compound tubuloalveolar structure, (2) an underlying layer of connective tissue (the lamina propria) and (3) smooth muscle fibers. It is now well established that the secretory activity of the epithelial cells is normally under the control of testicular hormones. The secretions pass from the cells into the lumina of the glandular alveoli where they are usually stored until ejaculation.

The sensory innervation includes various types of sensory nerve endings in the connective tissue, and free nerve endings in the epithelium. The autonomic innervation is parasympathetic (nervi erigentes) and sympathetic (hypogastric nerve) from the pelvic plexus. If the plexus is resected or the sympathetic chain above is interrupted, there is no reflex ejection of the glandular secretions. When the hypogastric nerve is stimulated, peristaltic waves of contraction occur in the ductus deferens, and there is contraction in the seminal vesicles and prostate which partially empties the stored secretion from the lumina of these glands. Stimulation of the parasympathetic system or the administration of pilocarpine results in an increased output of prostatic secretion.

There are marked dissimilarities in gross structure, character of the epithelia, and the chemical nature of the secretions in the various glands — ^prostates, seminal vesicles, bulbo-urethral, and ampullary (Mann, 19o4a). There are also differences in structure and function between homologous glands in related forms. The nomenclature that was applied to the glands in early descriptive studies was often based on anatomic relationships and gross morphologic structure in adults. This resulted in some confusion in classification, but most of the disputed points have been clarified and some of the homologies have been established by embryologic study. The extensive studies of INIann (1954a) show clearly that

368

PHYSIOLOGY OF GONADS

TABLE 6.1 Occurrence of male accessory reproductive glands and their homologues in females"

Male*

Female<^

Order

Bu

Pr

Sv

Am

Bv

Pr

Genera and species with functioning female prostates

Monotremata . . .

+

_?

_

_

+

_

Marsupiala

+

+

+

Insectivora

+

+

±

±

+

+

Erinaceoii.s europeus (Deanesly, 193-4) Hemicentetes (Lehmann, 1938) Talpa europea (Godet, 1949)

Chiropt era

+

+

±

±

+

=t

Coelura afra (Mathews, 1941) Taphozous sp. (Mathews, 1941) Nycteris luteola (Mathews, 1941) Carioderma cor (Mathews, 1941)

Primates

+

+

±

=b

+

—

Carnivora

±

+

—

zb

±

—

Perissodactyla...

+

+

+

+

+

_

Artiodactyla ....

+

+

+

±

+

—

Hyracoidea

+

+

+

—

Proboscidea

+

+

+

+

Sirenia

—

+

+

—

—

Cetacea

—

+

—

Edentata

±

+

+

—

Pholidota

—

+

+

—

Rodentia

+

+

+

=t

±

±

Arvicanthus cinereus (Rant her, 1909)

Rattus norvegicus (Marx, 1931, 1932; Korenchevsky

and Dennison, 1936; Korenchevsky, 1937; Witschi,

Mahoney and Riley, 1938; Price, 1939; Mahoney,

1940, 1942; Mahoney and Witschi, 1947)

Mastomys erythroleucus (Brambell and Davis, 1940)

Apodemus sylvaticus (Raynaud, 1942, 1945)

Microtus arvalis (Delost, i953a, 1953b)

Lagomorpha. . . .

+

+

±

+

+

±

Sylvilagus floridanus (Elschlepp, 1952)

"Compiled from Oudemans, 1892; Engle, 1926a; Retief, 1949; Eckstein and Zuckerman, 1956 and others. + indicates the presence of a well developed functioning gland. — indicates either a small vestigial gland or the absence of any rudiment.

^ Bulbo-urethral (Cowper's), prostate, seminal vesicle and ampuUary glands.

"^ Bulbovestibular (Bartholin's) and prostate glands (para-urethral glands of Skene); genera and species refer only to those in which functioning female prostates have been reported in the listed references.

homologous organs do not necessarily have the same chemical functions.

Finally, there is variability among orders of mannnals and families within orders, with respect to the accessory glands which are present (Table 6.1). The prostate is the only u;land that is found almost universally.

C. SURVEY OF THE GL.4NDS

1.

Bulbo-urethral Glands

and Bulbovestihidar

Bulbo-urethral (Cow^per'.s). The bulbourethral glands are compound tubulo-alveolar glands resembling mucous glands in some respects. Their secretion is a viscid lubricant which is em])tied into the bulbar

region of the pelvic urethra. There may be a single pair of glands as in the monotremes, primates, and rodents, or as many as three pairs (Fig. 6.1), as in some marsupials (Chase, 1939; Rubin, 1944). Their relative size, gross structure, and complexity vary widely. For example, they are small, compact, bean-shaped glands in man, relatively enormous, complicated glands in squirrels, and large, cylindrical glands in the boar.

Bulbo-urethral glands are notably lacking in Cetacea, Sirenia, and certain carnivores such as seals, walruses, sea lions, all raustelids, and the bear and dog (Oudemans, 1892; Engle, 1926a; Eckstein and Zuckerman, 1956). (Oudemans made a point of the fact that they are not present in aciuatic mam

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

369

mals, but it is rather doubtful if their absence is related to an aquatic environment. BuLBOVESTiBULAR (Bartholin's) . The bulbovestibular or major vestibular glands are also compound tubulo-alveolar glands which resemble their male homologues in structure and secrete a mucus-like substance. Their secretory function is under control of ovarian hormones and they involute when the ovaries are removed. They are widely distributed in the various orders of mammals although the information is fragmentary with respect to some groups. A single pair of glands is the general rule and they are usually much smaller than the bulbo-urethral. In the female opossum, the single pair of glands is homologous with the smallest of the three pairs of Cowper's glands. In the adult, they are well developed and filled with colloid (Rubin, 1944). In monotremes the ducts open at the base of the clitoris, in opossums into the urogenital sinus canal, and in hyenas (where they are well developed) into the urogenital canal close to the base of the clitoris (Eckstein and Zuckerman, 1956). In many other females the ducts open into the vestibule. In the adult human female, Bartholin's glands resem])le Cowper's glands closely in histologic structure.

2. Male and Female Prostate Glands

Male prostate. The prostate is a compound tubulo-alveolar gland in which the gross structure is variable and may be (1) disseminate or diffuse, in which the glandular acini remain within the lamina propria around the urethra and do not penetrate the voluntary muscle of the urethra, (2) a type in which the gland forms a '"body," sometimes lobed, outside the urethral muscle, or (3) a combination of both types. A disseminate prostate is found in some marsupials (Fig. 6.1) and edentates, and in sheep, goats, the hippopotamus, and the whale. The bull and boar prostates have a disseminate region as well as a discrete body of the gland. In mammals in which there is a glandular body, there may be a solid, compact prostate as in the dog and man, or several lobes as in rodents (Figs. 6.2 and 6.3), lagomorphs (Fig. 6.4), and insectivores.

Fig. 6.L Male opossum reproductive tract. B, bladder; C, Cowper's glands; D, ductus deferens; E, epididymis; P, penis; Pr, prostate I, II, III surrounding the urethra; T, Testis; U, ureter. (Redrawn from C. R. Moore, Phj'siol. ZooL, 14, 1-45, 194L)

A prostate gland has been found in all mammals that have been studied except monotremes, and is the only accessory gland in carnivores such as the ferret, weasel, dog, and bear, and in cetaceans — whales, dolphins, and porpoises. Oudemans (1892) considered that monotremes and marsupials lack prostate glands but possess well developed urethral glands. His classification of glands as "urethral" (glands of Littrei or "prostatic" depended on whether the glandular acini remained in the urethral stroma or penetrated the muscle to form a

370

PHYSIOLOGY OF GONADS

COAGULATING GLANDS

SEMINAL VESICLE

SEMINAL VESICLE

■1%.^

AMPULLARY GLAND DORSAL PR0STATE VENTRAL PROSTATE

Fic. 6.2. Mule hamster accessory repidiluri i aspect.

body outside. It is now recognized that marsupials such as the opossum have a disseminate prostate, and in Didelphys Virginian a there are three regions which differ

5EMINAL VESICLE .- RIGHT

DORSAL PROSTATE

^VENTRAL PROSTATE

gl.-inds. Above, ventral aspect; below, dor.sal

clearly in histologic structure (Chase, 1939). It may be considered that there are three prostatic "lobes" probably differing in function as well as in structure. Although Oude

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

371

— RIGHT SEMINAL VESICLE

COAGULATING GLAND

DORSAl PROSTATE

LATERAL

DUCTUS -DEFERENS

BLADDER

URETHRA

Fig. 6.3. Male guinea pig accessory reproductive glands. (From E. Ortiz, D. Price, H. G. Williams-Ashman and J. Banks, Endocrinology, 59, 479-492, 1956.)

mans concluded that monotremes lack prostate glands, he described a concentration of urethral glands at the neck of the bladder in the duckbill platypus. Ornithorhyncus poradoxicus. The diagrams in his monograph suggest that this concentration of complicated glands is a disseminate type of prostate.

There has been confusion in the nomenclature of the lobes of the prostate in the rat and in the descriptions of the structure of the lobes. In early studies the application of human anatomic terminology to rodents resulted in designation of the lobes as anterior (ventral), middle, and posterior (dorsal). Later terminology, more suitable for

cfuadrupedal animals, led to anterior (cranial), middle, and posterior (caudal). Unfortunately, combinations of these two systems of nomenclature still occur in the literature, and there is uncertainty as to the number of histologically distinguishable regions or lobes. In view of the current interest in the chemical composition of the glands and their secretions the subject will be reviewed.

For many years the prostate was usually described as being composed of three pairs of lobes: cranial or anterior (coagulating glands) bound to the seminal vesicles; middle or dorsolateral nearly encircling the urethra dorsolaterally, and the ^-entral or

372

PHYSIOLOGY OF GONADS

Fig. 6.4. Rabbit accessory reproductive glands; lateral aspect. Left, domestic male; center, cottontail male; right, cottontail female. B, bulbo-urethral gland; C, coagulating gland (vesicular gland); D, ductus deferens; P, paraprostate ; Pr, prostate; V , urethra; VC, urogenital canal (vestibulum) ; V, vagina. (Redrawn from J. G. Elschlepp, J. Morphol., 91, 169-198, 1952.)

lio.sterior (Moore, Price and Gallagher, 1930; Callow and Deanesly, 1935; Price, 1936). Korenchevsky and Dennison (1935) noted that the histologic structure of the dorsal lobe (or region) is quite similar to that of the coagulating glands whereas the lateral lobes more nearly resemble the ventral. This has been confirmed in histologic and functional studies (Price, Mann and Lutwak-Mann, 1955). Gunn and Gould (1957a) reported differences in histologic structure and functional activity in the two lobes.

The lateral lobes can be distinguished grossly from the dorsal by anatomic relationships and color, but the glandular lobules form a continuous mass and can be separated into distinct lateral and dorsal lobes only by dissection. This can be accomplished with considerable accuracy in immature males and young adults; in large rats it is more difficult because of distention of the alveoli and overlap of lobules in contiguous regions. The ventral tips of the lateral lobes extend down and partially underlie the ventral lobes to which they are loosely bovmd. The dorsal prostate is somewhat butterfly-shaped with a single cranial region and wings extending caudally along

the urethra much as in the hamster (Fig. 6.2). By dissection in the midline, it can be divided into right and left lobes. The dorsal and lateral prostates are drained by 50 or more ducts opening into the roof of the prostatic urethra (Witschi, Mahoney and Riley, 1938). Those from the dorsal region open more dorsally; those from the lateral lobes, laterodorsally.

Some of the confusion in prostatic terminology arises from the general application of the word "lobe" to (1) organs that are grossly anatomically distinct, (2) regions that do not form entirely discrete structures but can be distinguished histologically, (3) ])arts of the gland which contain two histologically different portions, and (4) regions that differ, not in histologic structure but in response to hormones and in the tendency to ])athologic growths (human and dog).

The lobation of the human prostate has been the subject of controversy for some time. It is of especial interest because one region, the posterior or dorsal lobe, is commonly the site for prostatic carcinoma and another, the more anterior or ventral region, for benign prostatic hypertrophy. The lobes have been described as posterior, anterior, middle, and two lateral, or as posterior and

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

373

anterior, or outer and inner (medullary). The component parts of these regions have been discussed extensively (see Moore, 1936; Huggins and Webster, 1948; Retief, 1949; Franks, 1954). Lowsley (1912) studied the embryologic development of the human prostate and concluded that the gland derives from five independent groups of tubules. A cranial posterior or dorsal group (lobe) arises from the dorsal wall of the prostatic urethra or urogenital sinus; right and left lateral lobes originate from the prostatic furrows and grow back to form the main part of the base of the gland; a middle lobe derives dorsally from the urethra between the bladder and ejaculatory ducts; a ventral or anterior lobe forms but regresses and becomes insignificant.

Although these prostatic buds or tubules form independent groups in their embryonic origin there is no clear separation into such groups in the human prostate postnatally. However, Huggins and Webster (1948) were able to distinguish clearly two different regions, a posterior and an anterior lobe, by differential response to estrogen administration. The extent of the anterior or ventral lobe, as delimited by them, apparently includes the tubules of the middle and lateral lobes as described by Lowsley.

The pioneer studies of Walker (1910a) on the coagulating function of discrete glands of the prostatic complex in rats and guinea pigs (Fig. 6.3) were followed by specific identification of coagulating glands in several rodents including mice and hamsters (Fig. 6.2) and in the rhesus monkey (van Wagenen, 1936). However, a copulation plug in the vagina of females has been reported in some marsupials, insectivores, chiropterans, the chimpanzee among the primates (Tinklepaugh, 1930), and several genera of rodents in which coagulating glands have not been identified. Eadie (1948a) found that in an insectivore, Condylura cristata, there is a peculiar prostatic secretion from paired ventral lobes. It contains an enormous number of amyloid bodies resembling the corpora amylacea present in the prostate gland of man and some other mammals. These prostatic concretions are generally considered abnormal, but Eadie suggested that this unusual se

cretion, which was found in all breeding males, might be instrumental in the formation of a unique type of copulation plug. A large urethral" gland which lies between the prostate and bulbo-urethral glands and surrounds the urethra is peculiar to certain species of bats. Mathews (1941) considered it probable that the presence of this gland is correlated with the formation of a large copulation plug, but he did not ascribe a specific coagulating function to the gland (which bears a histologic resemblance to the bulbo-urethral glands in some bats).

The difficulties of homology and classification can be illustrated by the case of the rabbit. Differences of opinion have existed concerning the nomenclature and homologies of the seminal vesicles (or prostatic utricle), vesicular glands (seminal vesicle or prostate), and paraprostate glands (or superior Cowper's glands). In studies on embryologic development and histologic structure, Bern and Krichesky (1943) clarified the problem. They established that the domestic rabbit has true seminal vesicles, vesicular glands (which are considered as probably homologous with the coagulating glands of rats), prostates, paraprostates (usually similar to the bulbo-urethrals in histologic structure but in about one-third of the cases, one or more of the paraprostates resembled the prostate histologically), bulbo-urethral glands, and glandular ampullae. Elschlepp (1952) compared the accessory glands of the cottontail, Sylvilagus floridamis, with those of the domestic rabbit, and concluded that coagulating glands (avoiding the usage of "vesicular glands" which has often been used synonymously with seminal vesicles) , dorsal prostates, and bulbo-urethral glands are homologous in the two species. The adult cottontail has neither paraprostates nor seminal vesicles (Fig. 6.4) . Classification of the glands in the hedgehog and shrew has also presented problems (see discussion in Eckstein and Zuckermann, 1956; Eadie, 1947). Among the Sciuridae, many possess a bulbar gland which differs from their true Cowper's glands (Mossman, Lawlah and Bradley, 1932). It is evident that among mammals

374

PHYSIOLOGY OF GONADS

there are many potentialities for forming accessory glands with varied anatomic structure, histologic characteristics, and functional activities.

Female prostate. In fetuses of many female mammals, small cords of cells which represent the homologues of the male prostate bud off from the epithelial lining of the urethra. These primordia normally retrogress or remain vestigial and only rarely continue to develop after birth. In the human female, these rudimentary structures are known as para-urethral glands of Skene. They have also been referred to as periurethral glands. However, it seems advisable, as Witschi, Mahoney and Riley (1938) suggested, to restrict the usage para-urethral and peri-urethral to the aggregations of mucus-secreting glands that have short ducts opening into the urethra. These clearly differ from the true female prostate glands.

In contrast to the rudimentary prostate glands which are retained postnatally by some female mammals, relatively large, well developed female prostates have been reported postnatally in some insectivores, chiropterans, rodents, and lagomorphs. The male accessory glands of many species in these orders are exceptionally well developed and the prostates are usually lobed. Female prostates are tubulo-alveolar glands, as are their male homologues, and they too form lobes, but the glands are never as large as those of the male. Their secretory activity is apparently dependent mainly on ovarian androgens, but the function, if any, of the secretion is obscure. Extensive research has shown that the administration of androgens to rodents, either to pregnant females or fetuses, to fetal lagomorphs, and to pouch-young oppossums, results in the formation and retention of prostates in females which normally do not have such glands (see chapter by Burns).

Deanesly (1934) described vaginal glands in the female hedgehog and suggested that one pair is homologous with the external prostates of the male. The female glands extend dorsolaterally on either side of the urethra and a single duct from each lobe opens into the vagina. They seem to be active during the breeding season and to retrogress in the anestrum. In another insectivore,

Hemicentetes, there is a pair of large "paravaginal glands" which are functionally active in the mature female and have large acini filled with secretion. They resemble the male prostate in histologic structure and anatomic position, but have no ducts (Lehmann, 1938). In adult European moles, most females have bilobed ventral prostate glands which undergo cyclic changes in the epithelium. The prostate of the male is also bilobed and ventral in position and the homology in the two sexes is clear (Godet, 1949).

Mathews (1941) studied the anatomy and histophysiology of the male and female genital tracts of nine species of African bats. Female prostates are well developed in four species, less conspicuous in a fifth, and absent from the remaining four. There is a marked tendency for greater development of the glands in pregnant and lactating females. In three species, the female prostates surround the urethra (as do their male homologues), but in Nycteris luteola the female prostate appears ventrally, whereas the male prostate in this species is limited to the dorsal aspect of the urethra. Mathews considered that the female prostates represent greatly enlarged female urethral glands which are homologous with the male ])yostate.

The occurrence of female prostates and their relation to hormones have been most extensively studied in rodents. The first description of a well developed female prostate gland seems to be that of Rauther (1909), who found such a gland ventral to the neck of the bladder in the African field rat, Arvicanthis cinereus. In Rattus norvegicus, Marx (1931, 1932) reported the sporadic occurrence of female prostates. Korenchevsky and Dennison (1936) and Korenchevsky (1937) found prostates in 9 of 56 females and stated that the glands wei'c atrophic, but when androgens were administered, these glands resembled the male ventral prostate. On this basis, the homology of the glands of the female with the \-entral prostate of the male was suggested. Further studies (Witschi, Mahoney and Riley, 1938; Mahoney, 1940, 1942; Malioney and Witschi, 1947) showed that the female prostate of the rat is homologous

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

375

with only the most medioventral part of the male prostate ; the lobes are bilateral or unilateral, with the right the preferred side; each lobe has a single duct which opens into the urethra. The incidence of female prostates varies markedly in different strains, and can be increased by selective inbreeding, w^iich also increases the occurrence of bilateral compared with unilateral lobes. The frequency of female prostates was increased in the Wistar stock from 28 to 99 per cent, but when selective inbreeding was stopped, the frequency declined.

In young untreated female rats, the prostate, when present, develops a histologic structure identical with that of the male homologue, but at about 6 weeks of age the epithelium undergoes regression (Price, 1939; Mahoney, 1940) and becomes histologically well developed again only during pregnancy and lactation (Burrill and Greene, 1942; Price, 1942). Thus, the female prostate of the rat is not only homologous with a part of the ventral prostate of the male on the basis of embryologic development, but during early postnatal development and in periods of pregnancy and lactation it resembles its male homologue histologically (Fig. 6.46). In addition, it is functionally equivalent (see Section II) to the male ventral prostate in the secretion of citric acid (Price, Mann and LutwakMann, 1949).

Brambell and Davis (1940) found large, well developed prostate glands in every one of 104 female African mice, Mastonujs erythroleucus Temm. These glands consist of paired lobes, each draining into the urethra by a single duct. They resemble the ventral prostate of the male in position, shape, and histologic structure. On the basis of this evidence it was concluded that the female glands are the homologues of the male ventral prostate. In some cases, the female prostates are nearly as large as their male homologues and are actively secretory. Brambell and Davis correlated hypertrophy and secretory activity with the luteal phase of the cycle and gestation.

Female prostate glands have also been described in the field mouse, Apodemns sylvaenms sylvaticus. Raynaud (1942, 1945) found bilobed prostates in 51 immature and

adult females collected in the vicinity of Vabre (Tarn) and in 3 females from three other regions of France. However, the lobes were macroscopically visible in only 10 females; in all others, the glands were identified in histologic preparations. There w^as great variability in histologic structure, but a well developed epithelium showing secretory activity was found during pregnancy and lactation. Raynaud established that the female prostate is homologous with a part of the male ventral prostate. He concluded that there is a probability that bilobed female prostates exist normally in all females of Apodemus sylvaticus.

The prostate glands in adult female field voles, Microtiis arvalis P., are considered homologous with the ventral lobes and part of the lateral lobes of the male prostate (Delost, 1953a, b). The lobes in the female are lateral in position in part of the gland, but in other regions they completely surround the urethra. The structure is identical with that of the ventral prostate of the male. The epithelium appears secretory in normal adult females, and during gestation this activity is intense.

Bilobed female prostates were found in the 37 adult cottontail rabbits examined by Elschlepp (1952). They lie on the dorsal wall of the vagina (Fig. 6.4) and are similar histologically to the prostate of the male. The glands are larger in pregnant than in nonpregnant females and contain more secretion.

In summary, well developed female prostate glands are present in immature and adult females of many species. They may occur as ventral, lateral, or dorsal lobes; the lobes may be unilateral or consistently bilateral; their occurrence may be sporadic or reach an incidence of 100 per cent; they are found both in laboratory strains and in wild populations. The genetic studies of Witschi and his collaborators show that the incidence in rodents can be increased by selective inbreeding. In certain populations of wild rodents the character has become established. A striking example of this is the presence of large prostates in all female Masto)7iys erythroleucus. The secretory activity of the glands seems to be controlled mainly by ovarian androgens (sec Section

376

PHYSIOLOGY OF GONADS

III) . No function can be ascribed to the secretion.

3. Seminal Vesicles

The seminal vesicles are paired, usually elongated glands which may appear relatively simple externally (Figs. 6.2 and 6.3) but are subdivided internally by complicated villous projections. The name refers to an old misconception that they are sperm reservoirs. The seminal vesicles are relatively enormous and distended with secretion in some mammals, for example, the rat, guinea pig, and hamster; they are large in others such as the boar, and in still others, as in man, they are small and compact.

Seminal vesicles are absent from the monotremes, marsupials, carnivores, and cetaceans that have been studied, and from some insectivores, chiropterans, primates, and lagomorphs. Variability exists among the edentates ; the sloths and the armadillo have seminal vesicles which are well developed in the two-toed sloth and armadillo, but are very small and rudimentary in the three-toed sloth. Among the lagomorphs, the seminal vesicle of the domestic male rabbit is a large unpaired gland whereas the seminal vesicles in the adult cottontail rabbit are vestigial or absent although they develop for a time in the fetus (Elschlepp, 1952).

4- Ampullary Glands

These organs are glandular enlargements arising from the ampullae of the ducti cleferentes or the posterior region of the ductus if a distinct ampullary enlargement is not present. They may be only slight glandular enlargements of the wall, or discrete glands which nearly encircle the ductus deferens as in rats, some mice, and hamsters (Fig. 6.2). They are vestigial in certain pure line strains of mice (Horning, 1947) and lacking in guinea pigs (Fig. 6.3). In some bats, they attain very large size. In general, they are absent from many mammalian orders and variable in others (Table 6.1).

D. EVOLUTIONARY HISTORY OF ACCESSORY REPRODUCTIVE GLANDS OF MAMMALS

The well developed male accessory glands which characterize the niamnialiaii class as

a whole, and form such a conspicuous part of the reproductive tract in most mammals, are not found in nonmammalian vertebrates. These glands appear as anatomically distinct organs in the primitive prototherian mammals, the monotremes, which are definitely mammalian but which also retain certain anatomic characteristics of their reptilian ancestors and still lay shelled eggs. However, it has been suggested that in the evolution of the three groups of living mammals from mammal-like reptiles, the line of descent of monotremes is entirely separate from that of marsupials and placentals. Furthermore, the last two groups are probably parallel branches of the mannnalian stock.

The accessory glands of modern mammals represent, then, the parallel evolution of discrete glands that probably began their development very early in the evolutionary history of mammals. In gross structure, size, and internal complexity they are unique accessory organs among vertebrates. Modern reptiles have no such glands; the seminal plasma is composed mainly of secretions from the epididymis and the renal tubules of the sexual segment of the long, lobulated kidney. Both these regions become highly secretory during the breeding season (see chapter by Forbes) . Parenthetically, the semen of birds (a later offshoot from the reptilian line than mammals) contains only a small amount of seminal plasma (Mann, 1954a) which is secreted in the cock almost entirely by the seminiferous tubules and vasa efferentia (Lake, 1957). In modern mammals, the epididymal epithelium is still an important accessory secretory area (see chapter by Bishop) , but the bulk of the seminal plasma comes from glandular elaborations of quite different regions, the urogenital sinus (a derivative of the primitive cloaca) and the posterior part of the Wolffian ducts, the ducti deferentes.

Modern monotremes are specialized forms but in certain characteristics they are primitive. They show almost diagramatically some of the first steps in the evolution of accessory glands. The bulbo-urethrals are already well developed but the concentration of complicated urethral glands at the neck of the bladder in the duckbill platypus almost certainlv illustrates the derivation

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

877

of a specialized gland (the prostate) from simpler glands which are nmnerous along the urethra. The probable evolution of prostates and bulbo-urethral glands from smaller, simpler urethral glands has been suggested in the past. Observations of Bruner and Witschi (1946) support this concept. In experiments on fetal hamsters, it was found that masculinized females developed prostate glands but the ducts joined the collecting ducts of the urethral glands and did not open directly into the urogenital sinus. According to these workers, this may represent an intermediate stage in the development of specialized glands.

The history of the cloaca may well be important in relation to development of accessory glands (Retief, 1949). The cloaca is retained in modern reptiles; in monotremes it is subdivided cranially into ventral urodeum or urogenital sinus and a dorsal coprodeum; it is represented by a pocket in marsupials but is lost as a discrete structure in all higher mammals. The first development of a separate urogenital duct or urethra as it occurs in monotremes may be correlated with the first appearance of discrete accessory glands from this specific region in mammals.

The marsupials illustrate a more advanced type of glandular development with three histologically distinguishable regions in the disseminate prostate and three pairs of bulbo-urethral glands. Seminal vesicles and ampullary glands are found only among higher mammals.

The size and structural complexity of these unique glands in mammals raises the question of the adaptive value of relatively large accessory glands associated with the mammalian reproductive tract. This is a matter only for speculation. The evolution of such glands with increased surface for secretion and enlarged storage space may, perhaps, have been correlated with a tendency for an increase in volume of seminal plasma in the ejaculate of mammals. Mann (1954a) pointed out the variability in the volume of ejaculated semen and in the sperm density in various species. With regard to the volume of seminal plasma, he stated, "In lower animals it may be so scarce that the emitted semen takes the

form of a very thick lump of spermatozoa, closely packed together. There is little seminal plasma in bird semen and even among some of the mammals, but on the whole, the higher mammals including man, produce a relatively dilute semen with a considerable l)roportion of seminal plasma." A second suggestion, more speculative, is that the evolution of large mammalian glands may also have compensated for loss of accessory reproductive function in the kidney. The kidney of mammal-like reptiles and ancestral mammals may have contributed to the formation of seminal plasma (as is true in modern reptiles, amphibians, and fishes), but the compact kidney of warm-blooded, metabolically active mammals may be ill adapted for such a purpose.

II. Function of the Male Accessory Glands

A. INTRODUCTION

The only known function of the male accessory glands is to secrete the seminal plasma. The proportion of this fluid which originates from the various secretory organs, or even from different lobes of the same gland, varies greatly from one species to another. There is also remarkable species variation in the volume and composition of the individual secretions. The functional activity of the accessory glands is governed primarily by hormones of testicular origin. The output of androgens is subject to the control of the anterior hypophysis, and many factors (e.g., age, light, season, temperature, and diet) affect the secretory activity of the hypophysis and testis. Thus, it is not surprising that in a given individual, there may be marked fluctuations in the cjuantity and chemistry of the secretions of the accessory glands, and hence of the seminal plasma.

The development by Charles Huggins of ingenious surgical procedures enabled the secretory activity of the canine prostate to be measured by simple volumetric methods. Such studies of prostatic secretion in the dog established the quantitative relationships between the function of prostatic epithelium and the androgenic status of the host. In other species, serial collection of the individual secretions in the same animal

378

PHYSIOLOGY OF GONADS

has not been achieved for purely technical reasons. The use of "split ejaculates" has given some insight into the glandular origin of various conii)oncnts of the seminal plasma, but this techniciue does not provide uncontaminated secretions from any one gland. However, in the last two decades extensive analyses of the chemical and enzymatic constituents of the individual secretions stored in the accessory glands, and of the whole seminal plasma, have been performed. The levels of many of these substances and enzymes are dependent on androgenic hormones. These findings have provided a basis for sensitive chemical methods for the bioassay of androgens. Moreover, knowledge of the biosynthesis of these substances by the accessory glands may point to the primary biochemical locus of action of androgenic steroids. This chemical approach to the study of the accessory glands has received great impetus from the pioneer studies of Thaddeus Mann.

The secretions of the accessory glands of many species are a repository for huge Cjuantities of substances which are present only in trace amounts in other tissues and body fluids. It is obvious that the seminal plasma must provide an ionically balanced and nutritive milieu suitable for the survival of sperm in the vagina and uterus. Certain substances secreted by one or more of the accessory glands, e.g., fructose, undoubtedly serve as a source of energy for the sperm. However, there is no evidence that any component of mammalian seminal plasma, or any one of the accessory glands, is absolutely indispensable for fertility. Artificial insemination is successful in some mammals if sperm from the epididymis are diluted in a suital)ly prepared medium, placed in a female in the correct stage of the estrous cycle, and deposited in a region of th(; female tract where there is maximal opiiortunity for their successful ascent. Removal of the coagulating glands in guinea l)igs (Engle, 1926b) , or dorsolateral prostate (Gunn and Gould, 1958) in rats does not prevent insemination and fertilization. Blandau (1945) extirpated the seminal vesicles and coagulating glands of rats and found that when these males were mated there was no copulation plug and, evidently as a result, the spermatozoa did not penetrate the

vaginal canals of the females. Thus the secretions of the coagulating gland and seminal vesicles in the rat assist the transport of sperm in the female.

The following section will consider the output and composition of the secretions, and their hormonal regulation, primarily from a chemical standpoint, rather than in relation to the anatomy and embryology of the structures from which they originate.

B. VOLUMETRIC (STUDIES OF SECRETION

1. Prostatic Isolation Operation

Volumetric studies of the secretion of canine prostatic fluid have yielded great insight into the factors which determine the functional activity of male accessory glands. The dog is devoid of both seminal vesicles and bulbo-urethral glands, and if the urine is suitably deviated, practically pure prostatic secretion can be collected from the urethra. Eckhard (1863) ligated the neck of the bladder of dogs and obtained prostatic fluid by urethral catheterization. This technique was used by a number of investigators to study the secretory activity of the i:)rostate gland (Mislawsky and Bormann, 1899; Sergijewsky and Bachromejew, 1932; Winkler, 19311. A superior modification of the operation was introduced by Farrell (1931, 1938; Farrell and Lyman, 1937). The output of prostatic fluid was increased greatly either by electrical stimulation of the nervus erigens or by the injection of cholinergic drugs such as pilocarpine. These early prostatic isolation operations suffered from the signal disadvantage that, for technical reasons, they permitted only brief experiments.

In 1939, Huggins, Masina, Eichelberger and Wharton developed a simple surgical procedure which enabled frequent collection of canine prostatic secretion over long l)eriods of time. The original technique was modified slightly by Huggins and Sommer (1953) and is depicted in Figure 6.5. The bladdei' is separated from the prostate gland, the urine voided through a supral)ubic canula, and the animals circumcised. Healing was complete within one week after surgery, and the animals were maintained in good health. Prostatic fluid could be collected at fre(iucnt intervals for as long as

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379

two years. Normal adult dogs were found to secrete 0.1 to 0.2 ml. of prostatic fluid per hour without external stimulation. Following the administration of pilocarpine, the canine prostate secreted as much as four times its weight of fluid (60 ml.) in one hour. The amount of secretion obtained in response to a standard dose of pilocarpine remained relatively constant for three months or more, and bore no direct relationship to the weight of the gland. The volume and composition of the fluid varied with the time and intensity of the cholinergic stimulus. Huggins (1947c) found that, after a single intravenous injection of pilocarpine, the volume and the content of total protein, certain enzymes (acid phosphatase, fSglucuronidase and fibrinogenase ) , and citrate were maximal in the first 15 minutes, then declined progressively in three succeeding quarter-hour periods. But the chloride content always rose initially from the low values of the resting secretion and reached maximal levels after the first 15-minute period. If the drug was administered intramuscularly, maximal values for total protein and citrate were found in the first period, whereas those for the volume and enzyme content were higher in the second and third periods. It was concluded from experiments involving the repeated intravenous injection of pilocarpine that acid phosphatase and fibrinogenase were definitely secreted and not simply washed out of the gland. However, a "washing out" process does occur after an initial stimulus with respect to total protein and citrate levels.

It was observed by Huggins, Masina, Eichelberger and Wharton (1939) that infectious diseases {e.g., pyelonephritis, distemper) often decreased the volume of stimulated fluid. This effect seemed to be due to inhibition of the hypophysis, because it could be overcome by injection of gonadotrophin. Soon after castration (7 to 23 days) the secretion ceased and was restored by the administration of testosterone propionate. Androgens also initiated secretion in immature animals. In castrate dogs maintained on testosterone, neither adrenalectomy nor removal of the thyroid and parathyroid glands affected the rate of prostatic secretion. Huggins (1947c) observed that in normal

Fig. 6.5. The canine prostatic isolation operation. The connection of the prostatic urethra with the bhulder has been severed and the prostatic secretion is collected by way of the penis. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663680, 1953.)

animals, secretion was unaffected by injection of either progesterone or desoxycorticosterone.

Cystic hyperplasia of the prostate occurs in many senile dogs. The volume of fluid secreted by such hypertrophied glands in response to pilocarpine was smaller than that obtained from young adult animals (Huggins and Clark, 1940).

Injection of diethylstilbestrol into normal adult dogs abolishes prostatic secretion. Administration of gonadotrophin restores secretion in such estrogen treated animals, which suggests that the primary effect of estrogens under these conditions is on the hypophysis (Huggins, 1947c). Estrogens also antagonize the stimulatory effects of injected androgens. In castrate dogs receiving testosterone, injection of large doses of diethylstilbestrol decreases the output of prostatic fluid to very low levels (Huggins

380

PHYSIOLOGY OF GONADS

and Clark, 1940). The neutralization of androgen action by estrogens in this situation is pronounced but not complete. Thus the acid phosphatase activity of prostatic fluid collected from animals treated with both testosterone propionate and diethylstilbestrol is of the same order of magnitude as that of normal secretion, despite the fact that the volume of the secretion is extremely low (Huggins, 1947c). The ratio of diethylstilbestrol recjuired to antagonize maximally the action of testosterone was found to be about 1 : 25. In dogs with either normal or cystic prostate glands, injection of amounts of estrogen sufficient to decrease prostatic secretion leads to shrinkage of the prostate. Large doses of estrogen cause the canine prostate gland to enlarge ; the dorsal segment undergoes squamous metaplasia and the ventral lobe becomes atrophic (Huggins and Clark, 1940; Huggins, 1947c). If both estrogen and androgen are administered simultaneously, the dorsal region becomes squamous and the ventral portion of the gland retains its columnar epithelium, although the volume of the prostatic secretion may be drastically reduced.

Fig. 6.6. The c-anine prostatic translocation operation. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663-680, 1953.)

2. Prostatic Translocation Operation

Huggins and Sommer (1953) transposed the prostate gland of the dog from its natural position to the perineum, as depicted in Figure 6.6. This procedure permitted the size of the prostate to be measured in the living animal, and provided prostatic fluid quite uncontaminated with other material. Pilocarpine was used as a secretory stimulus. Using this technique, Huggins and Sommer found that the effects of androgens and estrogens on prostatic size and secretion were similar to those obtained with dogs that had undergone the prostatic isolation operation.

C. CHEMICAL COMPOSITION OF THE GLANDULAR SECRETIONS

Electrolytes. Water is the main constituent of prostatic and seminal vesicle secretions and of seminal plasma, all of which are approximately iso-osmotic with respect to blood serum. The vesicular secretion is usually more alkaline than the prostatic secretion and has a higher dry weight, mainly because it contains more protein. The electrolyte content of the secretions varies widely between different species (Huggins, 1945; Mann, 1954a). In general, sodium is the main cation, although this is not true of boar vesicular secretion which is very rich in potassium. Chloride tends to be the main anion in those species whose accessory gland secretions do not contain large amounts of citrate. In this connection it is instructive to compare the resting prostatic fluid of man, and the pilocarpine-stimulated prostatic fluid of the dog (Huggins, 1945, 1947c). The human secretion has a much greater citrate and calcium content, and a much smaller chloride level than the corresponding canine fluid, although the total concentration of osmotically active substances is of the same order of magnitude in both secretions.

Zinc. Berti'aiid and X'hidesco (1921 » found large amounts of zinc in human semen. The highest concentration of zinc is present in the first fraction of the ejaculate, which is largely prostatic secretion (Mawson and Fischer, 1953). In the rat, the zinc content of the dorsolateral prostate is especially lii<i;li (.Mawson and Fisclicr, 1951 ). After in

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tracardiac injection, Zn*'^ is concentrated by this tissue 15 to 25 times more than any other organ, including the ventral prostate (Gunn, Gould, Ginori and Morse, 1955). The dorsolateral prostate of the rat consists of two parts which are functionally and anatomically distinct, and only the lateral portion concentrates Zn*'^ (Gunn and Gould, 1956a, 1957a). A rapid uptake of Zn^'s by slices of the rat dorsolateral prostate in vitro has been noted by Taylor (1957) .

The zinc content of the rat dorsolateral prostate, and its uptake of Zn*'^, are under hormonal control. The concentration of zinc in this gland increases 6- to 10-fold between the 35th and 100th day of life (Fischer, Tikkala and IMawson, 1955). In the adult rat, Gunn and Gould (1956b) observed a marked decrease in Zn^^ uptake after castration, which could be prevented by androgen treatment. In immature (Alihar, Elcoate and JNIawson, 1957) and hypophysectomized (Gunn and Gould, 1957b) animals, the administration of testosterone or gonadotrophin increased the zinc levels and the rate of Zn*'^ uptake, whereas estradiol was ineffective.

The physiologic function of the zinc in seminal plasma is problematical. This metal is an integral component of the enzyme carbonic anhydrase. The distribution of carbonic anhydrase among the various lobes of the prostate gland was studied by Mawson and Fischer (1952). In the rat, the posterior prostate contains about the same amount of carbonic anhydrase as the erythrocytes, whereas the ventral prostate contains very little of this enzyme. The lateral portion of the posterior prostate contains about 6 times as much zinc as the median part. But only a small portion of the zinc in the rat dorsolateral prostate, and in human semen, can be accounted for as carbonic anhydrase (Mawson and Fischer, 1953). According to Gunn and Gould (1958), dorsolateral prostatectomy, which removes most of the zinc from semen, is without influence upon either fecundity or fertility in the rat.

Fructose. The presence of a reducing and yeast-fermentable sugar in mammalian semen has been known for some time (McCarthy, Stepita, Johnston and Killian, 1928;

Huggins and Johnson, 1933). It was assumed by early workers that this sugar was glucose. Although Yamada (1933) reported that human semen contained a sugar which reacted as a ketose, it was not until 1945 that the nature of the main reducing sugar in most mammalian semens was elucidated by Thaddeus Mann. Using a highly specific enzymatic method of estimation, Mann (1946) showed that the semen of men and many other mammals contains little or no glucose. The reducing and yeast-fermentable sugar of bull seminal plasma was isolated in the pure state and identified as d( — ) -fructose on the basis of its optical rotation and formation of methylphenyl fructosazone.

Mann (1949) found fructose in the semen of the bull, ram, boar, goat, opposum, rabbit, guinea pig, mouse, hamster, and man. It is also present in the European mole iTalpa) and hedgehog {Erinaceus) (Mann, 1956). Table 6.2 shows that the fructose content of semen varies greatly from one species to another. In the five species indicated, the total fructose accounts for all the yeast-fermentable carbohydrate present, but these species also contain nonfermentable sugars. The semen of some animals contains glucose. This is true of the rabbit, in which both glucose and fructose are detectable (]\lann and Parsons, 1950), and the cock (Mann, 1954a), the semen of which is devoid of fructose. Ketoses other than fructose are found in certain semens. For example, the vesicular and ampullar secretions of the stallion possess carbohydrates which react in colorimetric tests as

TABLE 6.2 Reducing sugars of semen Values obtained from Mann (1946) and expressed in terms of milligrams of fructose per 100 ml. of semen.

Species

Total

Reducing

Sugar

Fructose

Yeast Fermentable Sugar

Nonfer mentable

Sugar

Bull

937 443 528 295 31

910 340

417

141

9

917

370

411

144

10

25

Ram

73

Rabbit"

117

149

Boar

21

" Small amounts of glucose were occasionallj' fovmd in rabbit semen by Mann and Par.-ons (1950).

ketoses, but cannot be fructose as they are not fermented by yeast (Mann, Leone and Polge, 1956) .

In most mammals, fructose is formed mainly in the seminal vesicles (Huggins and Johnson, 1933; Davies and Mann, 1947; Mann, 1949; Ortiz, Price, Williams-Ashman and Banks, 1956). But in the rat, the seminal vesicles secrete little fructose, most of which originates from the dorsal prostate and coagulating glands (Humphrey and Mann, 1949).

Metabolic pathways for the biosynthesis of seminal fructose by the accessory glands have been studied extensively. From the results of experiments on diabetic animals, Mann and Parsons (1950) concluded that blood glucose was the precursor of seminal fructose. The hyperglycemia resulting from the administration of alloxan to rabbits was accompanied by a parallel increase in the fructose content of semen. Injection of insulin into such diabetic animals led to a fall in the levels of both blood glucose and seminal fructose. Similarly, the concentration of fructose in human semen was found to be abnormally high in diabetic patients. Mann and Lutwak-Mann (1951b) incubated minced accessory gland tissue with glucose and observed the formation of small amounts of fructose. Cell-free extracts of bull seminal vesicle showed marked phosphoglucomutase and phosphohexoisomerase activity. The same preparations hydrolyzed both glucose 6-phosphate and fructose 6-phosphate to the corresponding free sugars. Slices of seminal vesicle glycolyzed glucose at much greater rates than fructose. On the basis of these facts, Mann and Lutwak-Mann (1951a, b) postulated that the conversion of glucose to fructose involved an initial phosphorylation of glucose to glucose 6-phosphate by hexokinase, with adenosine triphosphate as the ])hosphate donor. After enzymatic isomerization of glucose 6-phosphate to fructose 6-phosphate, the latter was dephosphorylated to free fructose. It was assumed that any glucose formed by the dephosphorylation of glucose 6-phosphate was rcutilized, whereas fructose was not, and hence accumulated in the secretion. This formulation is consonant with the properties of the hexokinase of seminal vesicle (Kellerman, 19551 wliicli, at low sugar concentrations, phosphorylates glucose at much faster rates than fructose.

It was suggested by Mann and LutwakMann (1951a, b) that the dephosphorylation of hexosemonophosphates by accessory glands was catalyzed by an alkaline phosphatase which attacked the 6-phosphate esters of both glucose and fructose. Kuhlman (1954) claimed, on histochemical evidence, that rat seminal vesicle contains a phosphatase specific for fructose 6-phosphate which is most active in the vicinity of pH 7. Kellerman (1955) stated that, although the microsome-bound alkaline phosphatase of guinea pig seminal vesicle hydrolyzes the 6-phosphate esters of glucose and fructose at approximately the same rate, the mitochondria of this tissue contain a phosphatase which, at pH 5.8, hydrolyzes fructose 6-phosphate ten times as rapidly as glucose 6-phosphate. However, Hers (1957a) was unable to confirm these observations.

An alternative pathway for the conversion of glucose to fructose was suggested by Williams-Ashman and Banks (1954a), who found that certain fructose-secreting accessory glands of rodents contain an enzyme, ketose reductase, which catalyzes the reversible oxidation of sorbitol to fructose. This enzyme attacks a number of higher polyols and uses diphosphopyridine nucleotide (DPN) as a specific hydrogen acceptor (Williams-Ashman, Banks and Wolf son, 1957). The presence of an active ketose reductase in male accessory sexual tissues was confirmed by Hers (1956, 1957a) who discovered another enzyme, aldose reductase, which catalyzes the reduction of glucose to sorbitol with dihydrotriphosphopyridine nucleotide (TPNH) as the hydrogen donor. Hers suggested that seminal fructose was formed from glucose by the combined action of ketose and aldose reductases, as follows:

Glucose + TPNH + H+ ;^± Sorbitol + TPN+

Sorbitol + DPN+ <=^ Fructose + DPNH + H+

Glucose + TPNH + DPN+

-^ Fructose + TPN^ + DPNH

this nu'chanism for fructose biosynthesis accounts for the following observations ( Hers. 1957a) : (1) extracts of sheep seminal vesicle convert C'^-labelcd glucose to fructose without rupture of the carbon chain; (2) TPNH and DPN are required for this transformation, during which sorbitol is produced, and becomes radioactive; (3) inhibitors of aldose reductase {e.g., glucosonej inhibit the conversion of glucose to fructose and sorbitol; and (4j sorbitol is present, alongside fructose, in the secretions of sheep seminal vesicle and of certain accessory glands of other species [vide injra) .

The relative importance of these phosphorylative and nonphosphorylative pathways for the biosynthesis of seminal fructose remains to be determined.^

The fructose content of semen and of the accessory glands is strictly controlled by testicular hormones. The experiments of Mann and Parsons (1950), depicted in Figure 6.7, show that in the sexually mature rabbit, seminal fructose levels fell dramatically soon after castration. This decrease in seminal fructose was prevented by implantation of a pellet of testosterone. Later administration of androgen to the orchidectomized animals restored seminal fructose to normal levels. Measurement of the fructose content of ejaculated semen may be a sensitive index of androgenic activity, and has the signal advantage that the timesequence of changes which result from alterations in the level of circulating androgen can be determined without sacrifice of the animal. This "fructose test" has been used to assess the production of testicular androgen, or the hormonal activity of exogenous substances, in man (Harvey, 1948; Landau and Longhead, 1951; Tyler, 1955; Nowakowski and Schirren, 1956; Nowakowski, 1957) and in other animals (Gassner, Hill and Svdzberger, 1952; Branton, D'Arensbourg and Johnston, 1952; ]\Iann and Walton, 1953; Glover, 1956; Davies, Mann and Rowson, 1957) .

The amounts of fructose in semen and in the accessory glands are not determined solely by androgenic hormones, as Mann (1954a, 1956) has emphasized. The relative size and storage capacity of the accessory

^Samuels, Harding and Mann (1960) measured the aldose and ketose reductase levels in the various accessory glands of the rat, and in the seminal vesicles of the sheep, bull, boar, and horse. They found that the level of fructose production could be correlated with the activity of the least of the two enzymes.

Fig. 6.7. Postcastrate fall and testosterone-induced rise of seminal fructose in the rabbit. The pellet contained 100 mg. of testosterone. (Redrawn from T. Mann, The Biochemistry of Semen, Methuen & Co., 1954.)

glands play an important role. Another factor which complicates the fructose test is frequency of ejaculation. In man and the stallion, a single ejaculation largely depletes the seminal vesicles (Mann, 1956). In the bull, however, the seminal vesicles have a remarkable storage capacity such that the fructose content of eight consecutive ejaculations obtained within one hour is practically the same (Mann, 1954a) . Blood sugar levels can influence seminal fructose, but there is no evidence that the abnormally large quantities of fructose in diabetic semen (Alann and Parsons, 1950) result from increased output of androgenic hormones. Interruption of the blood supply to an accessory gland is another factor which affects the amount of fructose it secretes (Clegg, 1953). In immature animals, there seems to be a short time after birth when the accessory glands will not produce fructose in response to testosterone (Ortiz, Price, Williams-Ashman and Banks. 1956). Obstruction of the ejaculatory ducts will, of course, prevent the appearance of fructose in semen, as Young (1949) found in a patient with congenital bilateral aplasia of the vas deferens.

The anterior hypophysis may determine indirectly the secretory activity of accessory glands, and the amounts of fructose and other chemical substances which accumlate therein. Mann and Parsons (1950) showed that hypophysectomy in the rabbit results in a decline in the fructose content of semen, and of the prostate gland and glandula vesicularis. These changes were similar to those induced by castration, and could be reversed by treatment with androgens or with gonadotrophin. The deleterious effect of inanition, or a deficiency of certain B vitamins, on fructose formation in the accessory glands is almost certainly related to a concomitant depression of gonadotrophin secretion by the anterior pituitary gland (Lutwak-Mann and Mann, 1950 ) . In the bull (Davies, Mann and Rowson, 1957j, underfeeding leads to a greater depression of fructose levels in semen than of sperm formation.

Analysis of fructose in excised ]irostate gland or seminal vesicle has been used widely as an indicator of androgenic activity. This procedure has yielded much information concerning the relationship between the time of onset of androgen secretion by the testes and the initiation of spermatogenesis. In the rabbit (Davies and Mann, 1949), rat (Mann, Lutwak-Mann and Price, 1948), bull (Mann, Davies and Humphrey, 1949), boar (Mann, 1954b), and guinea pig (Ortiz, Price, Williams-Ashman and Banks, 1956) , fructose can be detected in the accessory glands before spermatozoa are produced. The androgenic potency of exogenous substances can be determined by application of the fructose test to the accessory glands of animals castrated before or after puberty. The increase in fructose content of the coagulating gland of castrated rats in response to testosterone is greater than the corresponding change in organ weight (Mann and Parsons, 1950) . The prostate gland and seminal vesicle of the rat (Rudolph and Samuels, 1949; Rudolph and Starnes, 1955; Rauscher and Schneider, 1954) and the seminal vesicle of the guinea pig (Levey and Szego, 1955b; Ortiz, Price, Williams-Ashman and Banks, 1956) behave in a similar fashion. This technique has provided evidence for the slight androgenic activity of progesterone (Price, Mann and Lutwak-Mann, 1955), and for the antagonistic (Parsons, 1950) or synergistic (Gassner, Hill and Sulzberger, 1952) influence of estrogens on the action of androgens.

Intact vascular and neural links are not necessary for the male accessory glands to accumulate fructose after androgenic stimulation. Subcutaneous transplants of these tissues into male rats will grow and produce fructose. After castration, the fructose content falls and can be restored by testosterone therapy (Mann, Lutwak-Mann and Price, 1948). Fructose secretion was also observed in accessory tissues transplanted into female hosts which had received androgens (Lutwak-Mann, Mann and Price, 1949), or were injected with gonadotrophins. which probably stimulate the secretion of ovarian androgens (Price, Mann and Lutwak-]Mann, 1955).

The fructose in seminal i^lasma serves as a source of energy for spermatozoa under both anaerobic and aerobic conditions (Mann, 1954a).

Sorbitol. Sorbitol has been detected in the seminal vesicles of the sheep ( Hers, 1957a, b) and the coagulating gland of the rat (Wolfson and Williams-Ashman, 1958), as well as in the semen of many species ( King, Isherwood and Mann, 1958; King and Alann, 1958, 1959). The sorbitol content of semen tends to be high in those animals which exhibit high levels of seminal fructose, although it is also present in the semens of the stallion and cock, which are virtually devoid of fructose (Table 6.3). Sorbitol can be synthesized in the accessory glands by the action of either ketose reductase (WilliamsAshman and Banks, 1954a; Williams-Ashman, Banks and Wolfson, 1957; Hers, 1956, 1957a) or aldose reductase (Hers, 1956, 1957a). Under anaerobic conditions, spermatozoa will not glycolyze sorbitol (unlike fructose) to lactic acid, but will reduce both fructose and glucose to sorbitol. In oxygen, spermatozoa readily oxidize sorbitol (Mann and White, 1956), and also form sorbitol from glucose and fructose. The interconversion of fructose and sorbitol by spermatozoa is catalyzed by a DPN-specific ketose re(Uictas(> (sorbitol dehydrogenase) which is similar to that of the accessory glands (King and ^Nlann, 1959). Although the spermatozoa can affect the ratio of the levels of soi'bitol and fi-uctose in seminal plasma, most of the seminal sorbitol is probably derived from the accessory glands.

Inositol. During his studies on the vesicular secretion of the boar, Mann (1954b) isolated large amounts of a crystalline, nonreducing carbohydrate which he identified rigorously as ?weso-inositol. This cyclic polyol was found only in the seminal vesicle, being absent from the epididymis and Cowper's gland. The concentration of inositol in boar vesicular secretion was as high as 2.6 gm. per 100 ml., and constituted as much as 70 per cent of the total dialyzable material therein. Using a specific microbiologic method of estimation, Hartree (1957) found that in the boar, the inositol content of seminal plasma was usually greater than 600 mg. per 100 ml., although much smaller quantities (less than 60 mg. per 100 ml.) were present in the bull, ram, stallion, and man. In all of the species examined, the bulk of the inositol in seminal plasma was in the free state, and in amounts much greater than those in blood or cerebrospinal fluid. In most animals, seminal inositol originates from the seminal vesicles, but it has been detected in the prostate gland of the hedgehog, and in the ampullar secretion of the stallion.

The levels of inositol in human semen, together with those of fructose, are increased after the administration of testosterone according to Kimmig and Schirren (1956).

The physiologic function, if any, of the inositol in seminal plasma is unknown. Since boar vesicular secretion, unlike other body fluids of the pig, contains immense amounts of inositol and very little sodium chloride, j\Iann (1954b) suggested that inositol is concerned with the maintenance of the osmotic ecjuilibrium of boar seminal plasma.

Ascorbic acid. Deproteinized extracts of the seminal plasma of many species reduce 2,6-dichlorophenol indophenol in the cold. This property has been attributed to the presence of ascorbic acid in the semen of the bull (Phillips, Lardy, Heiser and Ruppel, 1940), guinea pig (Zimmet, 1939), and man (Nespor, 1939; Berg, Huggins and Hodges, 1941; Huggins, Scott and Heinen, 1942). However, it is now established that ascorbic acid does not always account for the total reducing power of semen. In some animals, e.g., the boar, ergothioneine is responsible in

TABLE 6.3 Sorbitol and fructose content of fresh seminal

plasma In some oases the .samples represented semen which had lieen pooled; the number of individuals is given in brackets. (From T. E. King and T. Mann, Proc. Roy. Soc. London, ser B, 151, 2262-13, 1959.)

Number of Species \ Samples Sorbitol ; Fructose

Analyzed

Ram. . . Rabbit. Bull . . . Boar. . . Stallion Dog .. . Cock... Man . . .

mg./lOO ml.

150-600 (12)

40-150 (4)

120-540 (14)

20-40 (4)

<1 (4)

<1 (5)

<1 (14)

154 (3)

large i^art for the reduction of indophenol [vide infra), and bull semen contains sulfite and another, unidentified, reducing substance (Larson and Salisbury, 1953) . Nevertheless, ascorbic acid is undoubtedly present in seminal plasma. Employing a specific analytical method based on the formation of its dinitrophenylhydrazone, ]\Iann (1954a) found that the seminal vesicle secretion of the rat, bull, guinea pig, and man contains ascorbic acid in amounts varying from 5 to 12 mg. per 100 ml. Mann's values for the ascorbic acid content of human semen (10 to 12 mg. per 100 ml.) agree well with those reported by Berg, Huggins and Hodges (1941), which were based on indophenol reduction.

Amino sugars. After hydrolysis with acid, boar semen contains considerable amounts of amino sugars (Mann, 1954a). The epididymal "semen" contains more amino sugar than the vesicular secretion.

Ergothioneine. The vesicular secretion of the boar (Leone and Mann, 1951 ; Mann and Leone, 1953) is a rich source of ergothioneine. This sulfur-containing base is also found in the accessory glands of the European hedgehog and mole (Mann, 1956) , and in the ampullar secretion of the stallion (Mann, Leone and Polge, 1956). Little or no ergothioneine is present in the semen of the bull, ram, and man.

Experiments with S^^-labeled precursors suggest strongly that seminal ergothioneine is not synthesized in the animal V)ody (Alelville, dtken and Kovalenko, 1955; Heath, Rimington and Mann, 1957). Because orally ingested S'^'"'-labeled ergothioneine l)asses into the seminal plasma of the boar (Heath, Rimington, Glover, ]Mann and Leone, 1953) , it is possible that those accessory glands which secrete ergothioneine concentrate this substance from the blood.

Mann and Leone (1953) are of the opinion that the function of ergothioneine in seminal plasma is to protect the spermatozoa from the poisonous action of oxidizing agents. It is remarkable that the seminal fluids of the boar and the stallion, both of which contain ergothioneine, have common characteristics which would render their spermatozoa especially sensitive to oxidizing agents, viz., large volume, low sperm density, and small content of glycolyzable sugars.

PoLYAMiNES. Large amounts of spermine and spermidine are present in the prostate gland of many species (Harrison, 1931 ; Rosenthal and Tabor, 1956). The chemical structure of these polyamines was elucidated by Dudley, Rosenheim and Starling (1926, 1927). Human seminal plasma contains as much as 300 mg. spermine per 100 ml., most of which is derived from the prostate gland. If human semen is allowed to stand for a few hours at room temperature, the spermine present crystallizes in the form of spermine phosphate ("Boettcher's crystals").- Both spermine and spermidine are oxidized by the diamine oxidase of human seminal plasma (Zellcr, 1941; Zeller and Joel, 1941). These polyamines via their degradation products are highly toxic to spermatozoa (Tabor and Rosenthal, 1956) , and it seems unlikely that

"In a letter written to llie Royal Society of London in November 1677, Antoni van Leeuwenhoek described for the fir.'^t time the presence and movement of spermatozoa in human semen. In the same letter, he also mentioned that "threesided bodies," which were "as bright and clear as if they had been crystals," were deposited in the aged semen of man. These crystals were undoubtedly composed of spermine phosphate. The liistory of the discovery of spermine in semen is admirably summarized by Mann (1954a), with special reference to the contributions of Louis Vauquelin (see footnote 3), and also of Alexander von Pcihl, whose claims for the therapeutic proiinities' of spermine aroused much interest aiul controversy at the end of the 19th centurv.

their presence in seminal plasma is of functional value.

Choline DERIVATIVES. Florence (1895) described the formation of brown crystals upon the addition of a solution of iodine in potassium iodide to semen. This reaction was used as the basis of a medico-legal test for semen stains. Bocarius (1902) showed that choline was responsible for the formation of this material. In the rat, the seminal fluid is by far the richest source of choline of any tissue or body fluid (Fletscher, Best and Solandt, 1935). A series of careful studies by Kahane (Kahane and Levy, 1936; Kahane, 1937) revealed that human semen contains very little free choline immediately after ejaculation, but that large amounts of the free base are formed if the semen is allowed to stand at room temperature. Lundquist (1946, 1947a, b, 1949) isolated phosphorylcholine from human seminal plasma and showed that it was converted to choline and inorganic phosphate by seminal acid phosphatase. However, the French investigators (Diament, Kahane and Levy, 1952. 1953; Diament, 1954) isolated a-glycerophosphorylcholine from the vesicular secretion of rats, and suggested that this substance, rather than phosphorylcholine, was the precursor of free choline in aged semen. Lundquist (1953) also found glycerophosl^horylcholine in the vc'sicular secretion of the rabbit, rat, and guinea pig. WilliamsAshman and Banks (1956) showed that the amount of glycerophosphorylcholine in rat vesicular secretion falls rapidly after castration, and can be restored to normal levels by administration of testosterone. Rezek and Sir (1956) found both ]')hosi)liorylcholinf and glyceroi)hospliofylclu)line in lunnan ejaculates.

A thorough study of the wat('i'-s()lul)lf choline (lerivati\-cs in seminal plasma was made by Dawson, Maiui and White (1957). They foimd (Tabh'().4i that in most species, gh^cerophosphoryh-holine is the only derivati\'e preseiu, but ill man there are consider■a\)\v (|uantities of phosphoiyh'lioline as well. The lattei- substance is rai)idly dephospholylated after ejaculation, but glycerophosphoryh'holine is not degradeil by enzymes in seminal phisnia or \'esiculai' secretion. In

TABLE 6.4

Phosphor ylchoUne and a-glycerophosphorylcholine in semen and in secretions of accessory reproductive glands

Species

Concentration (mg. per 100 gm.) of:

Phosphorylcholine a-Glycerophosphorylcholine

Ram

Ram

Bull

Bull

Bull

Bull

Bull

Cioat

Boar

Boar

Boar

Stallion. . . Stallion. . .

Man

Rat

Rat

Rabbit... Hedgehog. Hedgehog. Monkey. . . Cock..'....

Semen

Seminal pla.sma

Semen

Seminal plasma

Vesicular secretion

Epididymal secretion

Ampullar secretion

Semen

Seminal plasma

Vesicular secretion

Epididymal secretion

Semen

Ampidlar secretion

Semen

Vesicular secretion

Seminal vesicle

Semen

Secretion of "Prostate I and 11"

Secretion of "Prostate III"

Vesicular secretion

Semen

Present

256-380

Present Present Present Present

1185-1942

1601-2040

237-460

110-496

1490

94

1382-1550

108-235

190

3060

38-113

120

59-90

654; 530-765"

190-515"

215-370

Present

° Results from Williams-Ashman and Banks (1956); all other values from Dawson, Mann and White

lf57).

the bull and boar, the epidiclymi.^ is the ])rincii)al source of the glyceroiihosi)horylcholine of the seminal plasma.

Williams-Ashman and Banks (1956) in-ovided evidence that the choline moiety of the glycerophosphorylcholine in vesicular secretion is not derived from a direct reaction between glycerol and cytidine diphosphate choline. The latter nucleotide was shown to be a precursor of lecithin in rat seminal vesicle tissue. The glycerophosphorylcholine of seminal plasma may originate from the enzymatic degradation of the choline-containing lipids of the seminal vesicle epithelium.

Choline and glycerophosjihorylcholine are not metabolized by spermatozoa, and do not affect their respiration (Dawson, Mann and White, 1957). There is no evidence that the water-soluble choline derivatives of seminal plasma serve any useful function.

Lipids. That lipid-containing granules are l^resent in human seminal plasma has been known for more than a century. They are found in prostatic secretion (Thompson, 1861 1, and were termed "lecithin-kornchen" by Fuerbringer (1881). However, Scott ( 1945 ) showed that lecithin is absent from both of these fluids, and that the majority of the phospholipid therein is phosphatidyl ethanolamine. Neutral fat is virtually absent from human seminal plasma and prostatic secretion, one-third of the total lipid of which can be accounted for as cholesterol.

According to Boguth (1952), about onethird of the total plasmalogen in bull semen (30 to 90 mg. per 100 ml.) is in the seminal plasma. In the ram, only 10 per cent of the seminal plasmalogen is found outside the spermatozoa (Hartree and ]Mann. 1959).

Large amounts of 7-dehydrocholesterol were found in the preputial gland and epididymis of the rat (Ward and ]\Ioore, 1953) . One gram of the hydrocarbon heptacosane (CH3(CHo)o5CH3) was isolated from an alcoholic extract of 18 liters of human semen by Wagner-Jauregg (1941). The partition of heptacosane, and of steroidal estrogens (Diczfalusy, 1954) and androgens (Dirscherl and Kniicliel, 1950) between the sperm and plasma of human semen remains to be determined.

Citric acid. Citric acid was first detected in human semen by Schersten (1929). The distribution of citric acid in the semen and in the secretions of the accessory glands of various species is summarized in Table 6.5. In some animals, {e.g., the rat and man), citric acid is produced mainly by the prostate gland, and in others {e.g., the bull, boar, and guinea pig), most of it originates from the seminal vesicles.

The citric acid content of the seminal plasma and of the secretions of accessory glands depends on androgenic hormones. Citric acid disappears from these fluids after castration, and is formed again after treatment with testosterone. This citric acid test" has been used to determine the time of onset of secretory function in accessory glands (Mann, Davies and Humphrey, 1949; Ortiz, Price, Williams- Ashman and Banks, 1956), hormonal influences on secretion in subcutaneous transplants (Mann, LutwakMann and Price, 1948; Lutwak-jNIann. Mann and Price, 1949 L the androgenic ac

TABLE 6.5

Citric acid in semen and in the secretions of accessory reproductive glands

Species

Material

Citric Acid

Reference

(tng./lOO gm.)

Man

Semen

140-637

Huggins and Xeal (1942)

Man

Prostatic secretion

480-2()88

Huggins and Neal (1942)

Man ....

Seminal vesicle secretion Hypertrophic adenoma of the

15-22 201-1533

Huggins and Neal (1942) Barron and Huggins (1946a)

Man

prostate gland

Man

Carcinoma of the prostate gland

12-137

Barron and Huggins (194()a)

Bull

Semen

510-1100

Humphrey and Mann (1949)

Bull

Seminal gland secretion

670

Humphrey and Mann (1949)

Bull

Ampullar semen

550

Humphrey and Mann (1949)

Bull

Epididvmal semen

Humphrey and IVIann (1949)

Bull

Epididymis

18

Humphrey and Mann (1949)

Boar

Semen

130

Humphrey and Mann (1949)

Boar

Cowper's gland secretion Epididymal semen Seminal yesicle secretion

Humphrey and Mann (1949)

Boar

Humphrey and Mann (1949)

Boar

580

Humphrey and Mann (1949)

Ram

Semen Semen

110-260 110-550

Humphrey and Mann (1949) Himiphrey and Mann (1949)

Rabbit

Rabbit

Epididymis

54

Humphrey and Mann (1949)

Rabbit

Prostate (I, II and III)

62

Himiphrey and Mann (1949)

Rabbit

Cowper's Gland

42

Humphrey and Mann (1949)

Rabbit

Ampulla

273

Himiphrey and Mann (1949)

Rat

Seminal vesicle Coagulating gland Ampulla Dorsolateral prostate

39

Humi)hrey and :\Iann (1949) Hunii)hrc\ and Mann (1949)

Rat

Rat

Humplucv and Mann (1949)

Rat

20

Humphrey and Mann (1949)

Rat

Ventral prostate Semen

122

Humphrey and Mann (1949) Mann, Leone and Polge (1956)

Stallion

8-53

Stallion

Seminal vesicle

77

Mann, Leone and Polge (1956)

Guinea pig. . . .

Seminal vesicle

153-216

Ortiz, Price, Williams-Ashman Banks (195())

Guinea pig. . . .

Semiiuil vesicle sec'retion

320-357

Ortiz, Price, Williams-Ashman Baidvs (1956)

Guinea pig. . . .

Coagulating gland

26-40

Ortiz, Price, Williams- Ashman Banks (1956)

Guinea pig. . . .

Lateral i)rostate

16-20

Ortiz, Price, Williams- Ashman Banks (1956)

Guinea i)ig. . . .

Doi'sal ])i(is1at(>

47-75

Ortiz, Price, Williams-Ashman Banks (195(i)

Dog

Prostatic secretion

0-30

Barron and Huggins (1946a) Barron and Huggins (1946a) Mann, Leone and Polge (1956)

Dog

Prostate gland Vesicular secretion

8

Jackass

22-82

tivity of various hormones (Mann and Parsons, 1950; Price, Mann and Lutwak-JMann, 1955; Ortiz, Price, Williams-Ashman and Banks, 1956), and the effect of nutrition on the onset of androgen secretion and sperm formation in bull calves (Davies, Mann and Rowson, 1957).

The androgen-induced changes in the citrate levels in semen and various accessory glands are reminiscent of similar alterations in the fructose content of these tissues. However, the concentrations of these substances do not necessarily parallel one another in response to hormonal stimulation. In the postcastrate animal, the fall in citric acid and its reappearance after androgen treatment is usually more sluggish than that of fructose. Also, the seminal fructose of some species may not be secreted by the same accessory organ (or lobes of the gland ) which produces citric acid. Thus in the rat, fructose is secreted by the anterior and dorsolateral prostate, whereas citric acid is derived from the seminal vesicles and dorsolateral and ventral prostates, but is totally absent from the anterior prostate (Humphrey and Mann, 1949). In the guinea pig, however, the seminal vesicles are the principal source of both fructose and citric acid (Ortiz, Price, Williams-Ashman and Banks, 1956).

Using a strain of rats in which the incidence of the female prostate is very high, Price, ]Mann and Lutwak-JVIann (1949) showed that the growth of this gland which follows the injection of testosterone is accompanied by a tremendous increase in its content of citric acid. In this way the female prostate resembles the ventral prostate gland of the male rat.

Citric acid is synthesized in the i)rostate gland by the usual reactions of the tricarboxylic acid cycle (Williams-Ashman, 1954; Williams-Ashman and Banks, 1954b). No other organic acids are present in more than trace amounts in the secretions of those accessory glands that accumulate citrate. The enzymatic machinery for the degradation of citric acid via the tricarboxylic acid cycle is jH-esent in the rat ventral prostate gland OVilliams-Ashman, 1954; Williams-Ashman and Banks, 1954b; Williams-Ashman, 1955) and there is no evidence, despite suggestions to the contrary (Awapara, 1952a), that citric acid accumulates because it cannot be oxidized. It has been suggested that a common denominator affecting the androgendependent accumulation of citric acid and fructose in the accessory glands is the intracellular balance between the oxidized and reduced forms of DPN and TPN (Talalay and Williams-Ashman, 1958).

^lann (1954a) has summarized the ideas of various authors concerning the possible functional role of citric acid in seminal plasma. All of these suggestions are based more upon conjecture than experimental fact.

Catecholamine^. There is evidence for the presence of both epinephrine and norepinephrine in seminal pla.sraa (Brochart, 1948; Beauvallet and Brochart, 1949). Extracts of human prostate and seminal vesicle contain a monoamine oxidase which oxidizes catecholamines (Zcller and Joel, 1941). Katsh (1959) detected serotonin and histamine in human ejaculates.

Amino acids. Chromatographic studies have revealed the presence of many free amino acids in human semen (Jacobbson, 1950; Lundquist, 1952), from which crystalline tyrosine was isolated by WagnerJauregg (1941). According to Barron and Huggins (1946b), human prostatic adenoma is very rich in free glutamic acid, and the nonprotein amino-nitrogen of this and dog prostatic tissue is high. Bovine seminal plasma contains free serine, alanine, glycine, and aspartic and glutamic acids (Gassner and Hopwood, 1952). A similar distribution of amino acids is found in the vesicular and ampullary secretions of the bull. The free amino acid levels of bull seminal plasma fall greatly after castration. In the rat, Marvin and Awapara (1949) found that the concentration of free amino acids in the whole prostate decreased markedly following orchidectomy, and could be restored to normal levels in the castrate animal by treatment with androgen. In this species, Awapara (1952a) observed that the content of free amino acids in the ventral lobe of the prostate was much higher than in the dorsal lobe. After castration, there was a marked drop in the content of most amino acids with the exception of aspartic and glutamic acids, which seemed to remain at almost normal levels (Awapara, 1952b j.

Seminal plasma and the secretions of the male accessory glands contain a battery of proteolytic enzymes [vide infra). For this reason, changes in the levels of free amino acids in these fluids resulting from hormonal treatments should be interpreted with caution. Jacobbson (1950), for example, has shown that in human semen, the nonprotein nitrogen and amino-nitrogen content increases many fold within 60 minutes after ejaculation.

Prostaglandin. A vasodepressor substance, designated j^rostaglandin, was found by von Euler (1934, 1936) in the prostatic and vesicular secretions of man, and also in the accessory glands of sheep (von Euler, 1939). The prostaglandin of ram prostate was i^urified by Bergstrom ( 1949), who suggested that it was an unsaturated fatty acid devoid of nitrogen. According to Eliasson (1957), the prostaglandin of human semen and of the prostate gland of sheep are identical.

The pharmacologic effects which result from the injection of seminal plasma icf. Kurzrok and Lieb, 1931; von Euler, 1934, 1936, 1939; Goldblatt, 1935; Cockrill, Miller and Kurzrok, 1935; Asplund, 1947) are complex, and are probably due to the combined action of many constituents of this fluid. The hypotensive action of protein fractions of the secretions of some accessory organs (Freund, Miles, Mill and Wilhelm, 19581 is discussed below.

Uric acid. Bull seminal vesicles may contain as much as 70 mg. per cent of uric acid (Leone, 1953). The uric acid content of the semen of other animals is much lowci' (Mann, 1954a).

Urea. The urea content of human and ram semen is much higlici' than that found in the bull, boar, and stallion (Mann. 1954a).

Major protein constitiiknts. Human seminal plasma contains from 3.5 to 5.5 gm. of protein-like material per 100 niL (Huggins, Scott and Heinen, 1942). Less than 18 per cent of this material is coagulable by heat, and as much as 68 per cent of it is dialyzable. Thus the majority of the seminal proteins of man can be classified as proteoses. Electrophoretic analyses of the nondialyzable proteins of human seminal plasma have been performed by Gray and Huggins ( 1942 1 and by Ross, Moore and Aliller (1942). The major components bore some correspondence to those of blood serum, although the amount of albumin was small. The proteins of bovine seminal plasma are less dialyzable, and more coagulable by heat, than those of man (Larson and Salisbury, 1954). Electrophoretic studies showed the presence of three major and eight minor constituents, which seemed to be distinct from the proteins of bovine blood serum. In this species giycoor lipoproteins were present only in very low concentrations. Larson, Gray and Salisbury (1954) found that the bovine seminal plasma proteins are highly antigenic. They obtained immunologic evidence that the major protein constituents of this fluid are distinct from any of the main ])rot(>ins of either blood or milk.

Enzymes, (i) Acid phosphatase. Kutscher and Wolbergs (1935) discovered that human semen and prostate contain a very active phosphatase which is optimally active at pH 5 to 6. This enzyme is resjjonsible for the greater phosphatase activity of male as compared with female urine. Its secretion by the prostate accounts for the fact that male urine collected from the renal pelvis exhibits very little enzyme activity (Scott and Huggins, 1942). Human prostatic acid phosjihatase hydrolyzes a number of phosphate monoesters (Kutscher and Worner, 1936; Kutscher and Pany, 1938). The enzyme has been purified extensively (London and Hudson, 1953; Boman, 1954; London, Sommer and Hudson, 1955). In addition to hydrolyzing phosphate esters, human prostatic acid phosphatase catalyzes the transfer of phosphate from various donors to alcohols such as glucose, fructose, and methanol (London and Hudson, 1955; Jeffree, 1957). L-Tartrate inliibits the enzyme competitively (AbulKadl and King, ^1948).

'I'hc activity of acid phosphatase in the human jjrostate is low in childhood and increases about 20 times at i)uberty (Gutman and Gutman, 1938a). In adult men, the acid phosphatase content of semen seems to reflect the circulating levels of androgenic hoi-niones (Gutman and Gutman, 1940). High levels of acid })hosphatase are also present in osteoplastic metastases of prostatic carcinoma (Gutman, Sproul and Gutman, 1936). Acid phosphatase does not seem to enter the circulation from the prostate gland in healthy individuals unless they are subject to prostatic massage. But in about 65 per cent of men with metastatic carcinoma of the prostate, the serum levels of this enzyme are abnormally high (Gutman and Gutman, 19381); Robinson, Gutman and Gutman, 1939; Huggins and Hodges, 1941). The diagnosis and prognostic evaluation of carcinoma of the prostate in men has been aided greatly by measurements of the acid phosphatase levels of serum. Inhibition of the acid phosphatase activity of blood serum by L-tartrate has been used as an index for the outflow of prostatic acid phosphatase into the serum in neoplastic diseases of the prostate gland (Abul-Fadl and King, 1948; Fishman and Lerner, 1953).

The prostate gland of the monkey (Gutman and Gutman, 1938a) and dog (Huggins and Russell, 1946), and the seminal vesicle of the guinea i)ig (Bern and Le\y, 1952) exhibit powerful acid phosphatase activity, whereas the levels of this enzyme in the prostate of the rabbit (Bern and Levy, 1952) and rat (Huggins and Webster, 1948) are relatively low. The properties of these enzymes from different species are strikingly similar (Novales and Bern, 1953) . In the monkey and dog, the prostatic acid jihosphatase activities are controlled by androgenic hormones. This is also true in the rat (Stafford, Rubinstein and Meyer, 1949), and guinea pig (Ortiz, Brown and Wiley, 1957).

{2} Alkaline phosphatase. An enzyme, activated by magnesium ions, which hydrolyzes a variety of phosphate monoesters at pH 9, is present in the seminal fluid and accessory glands. In some species, e.g., the l)ull (Reid, Ward and Salisbury, 1948 ) , the levels of seminal alkaline phosphatase are much greater than those of the acid phosphatase. In the rat, alkaline phosphatase activity in the prostate and seminal vesicles decreases markedly after castration (Stafford, Rubinstein and Meyer, 1949).

(3) 5'-Nucleotidase. Reis (1937, 1938) noticed that human seminal plasma dcphosphorylated adenosine 5'-phosphate and inosine 5'-phosphate very rapidly. He proposed the term 5'-nucleotidase" for enzymes which specifically hydrolyze the 5'-monophosphates of ribose and its nucleosides. Mann (1945) reported that bull seminal plasma is exceedingly rich in 5'-nucleotidase. The enzyme was purified from this source by Heppel and Hilmoe (1951a). It was inactive towards adenosine 2'- and 3'phosphates, but catalyzed the hydrolysis of the 5'-monophosphate esters of adenosine, inosine, cytidine, uridine, and ribosyl nicotinamide. The 5'-nucleotidase of bull semen is optimally active at pH 8.5, and requires magnesium ions for maximal activity.

(4) Inorganic pyrophosphatase. Heppel and Hilmoe (1951b) reported the presence of an inorganic pyrophosphatase in bull seminal plasma. The enzyme was not purified extensively, and it is not clear whether it is different from other in'rophosphatases in semen.

(5) Nucleotide pyrophosphatases. The enzymatic hydrolysis of adenosine triphosphate (ATP) by seminal plasma was observed by Mann (1945) and by MacLeod and Summerson (1946). Three distinct ATPases were isolated from bull seminal plasma by Heppel and Hilmoe (1953). The first of these enzymes catalyzed the hydrolysis of ATP to inorganic pyrophosphate and adenosine 5'-phosphate. The other two catalyzed the liberation of inorganic orthophosphate from ATP, and were active at pH 5 and pH 8.5 respectively. The possible identity of any of these proteins with other enzymes which hydrolyze the pyrophosphate linkage of ]iyridine nucleotides (Williams-Ashman, Liao and Gotterer, 1958) and cytidine diphosphate choline (Williams-Ashman and Banks, 1956) remains to be established.

The physiologic function of any of the phosphatases in seminal plasma is unknown.

{6) Proteolytic enzymes. The proteolytic activity of human semen was first noted by Huggins and Neal (1942), and has been studied extensively by Lundquist and his collaborators. An enzyme similar to pepsinogen, and probably secreted by the seminal vesicles, was discovered in human seminal plasma by Lundquist and Seedorf (1952). Three other proteolytic enzymes were partially purified from human semen by Lundquist, Thorsteinsson and Buus (1955). The first enzyme resembled chymotrypsin, and the second was an aminopeptidase. The third enzyme hydrolyzed benzoylarginine ethyl ester, and seems to be identical with the arginine ester hydrolyzing enzyme described in male accessory reproductive glands by Gotterer, Banks and Williams-Ashman (1956). The relationship of these enzymes to the hydrolysis of fibrin or fibrinogen by prostatic secretion is discussed below with reference to the coagulation and liquefaction of semen.

(7) Glycosidases. Using phenolphthalein glucuronide as a substrate, Talalay, Fishman and Huggins (1946) determined the /?-glucuronidase activity of the male accessory glands of the rat. The levels of this enzyme in the epididymis fall about 50 per cent after castration, and can be restored to normal levels by the administration of testosterone (Conchie and Findlay, 1959). When the corresponding phenol- or p-nitrophenol-glycosides were employed as substrates, Conchie, Findlay and Levvy (1956) showed that the epididymis of the rat is particularly rich in y3-iV-acetylglucosaminidase. The levels of this enzyme were found by Conchie and Mann (1957) to be very much greater than those of seven other glycosidases in male accessory secretions. The levels of various glycosidases in the epididymis of rodents increases enormously at puberty. In adult animals the activity of some of these enzymes {e.g., a-mannosidase and /?-iV-acetylglucosaminidase) fell to negligible values after castration, and were restored only partially by treatment with testosterone.

(8) Miscellaneous enzipnes. The kneels of a number of oxidizing enzymes in human seminal plasma were studied by Rhodes and Williams-Asluiuiii (1960», who noted the presence of a x'cry active TPN-linked isocitric dehydrogenase. The ability of luiinan semen to hydrolyze acetylclioline is rather fe(>!)le, and the bulk of the activity resides in the seminal plasma (Zeller and Joel, 1941). According to Sekine (1951), boar semen exhibits powerful choline esterase activity, wliicli is confined mainly to the s])ermatozoa. The activity of phosphohexoisomerase (Wiist, 1957) and lactic dehydrogenase (MacLeod and Wroblewski, 1958) in human seminal plasma has been documented.

The levels of the following soluble enzymes have been determined in the accessory glands of male rodents: phenol sulfatase (Huggins and Smith, 1947). nonsi)ecific esterase (Huggins and ]\Ioulton, 1948), enolase, and dehydrogenases for lactate, malate, glucose 6-phosphate, 6-phosphogluconate and isocitrate (Williams- Ashman, 1954; Rudolph, 1956), aldolase and a-glycero])hosphate dehydrogenase (Butler and Schade, 1958). The nucleoside phosphorylase and adenosine deaminase activities of bull seminal vesicle were measured by Leone and Santoianni (1957). The vesicular secretion of the bull is rich in flavins, and exhibits strong xanthine oxidase activity (Leone, 1953). Leone and Bonaduce (1959) described a very active diphospho]5yridine nucleotidase in the vesicular secretion of the bull.

Conclusions. The foregoing survey indicates that, just as the size and morphology of the accessory glands differ profoundly, so there are wide species variations in the chemistry of their secretions, which comprise the seminal plasma. Some seminal constituents {e.g., fructose) are found in many mammals. Other substances, such as ergothioneine, are present in appreciable amounts in the seminal plasma of only a few species. The biochemistry of the accessory glands is still in its infancy, and it may be expected that future research will disclose other species-restricted comjionents of seminal plasma. Mann (1954a, 1956) I'ightly emphasizes that the finding of substantial concentrations of certain substances in the semen of only relatively few species does not necessarily detract from their physiologic value. The high levels of ci-gotliioneine in the seminal plasma of the boar and stallion is a case in point. The cjacuhitcs of these species have peculiarities which may render their spermatozoa particularly susceptible to the immobilizing action of oxidizing agents, and the suggestion (Mann and Leone, 1953; IMann, Leone and Polge, 1956) that ergothioneine, in virtue of its reducing properties, serves a protective function in boar and stallion semen seems an eminently reasonable one. However, the accessory glands of many animals secrete certain substances {e.g., glycerophosphorylcholine, spermine, citric acid) that do not appear to be of any particular value for the survival of spermatozoa in the male or female genital tracts. Perhaps these substances are simply by-products of the secretory mechanisms of the glands from which they originate, or represent biochemical vestiges.

The widespread occurrence of fructose in accessory gland secretions deserves further comment. The only other situation where large amounts of fructose are present in mammalian extracellular fluids under normal physiologic conditions is in the fetal blood of ungulates (Bernard, 1855; Bacon and Bell, 1948; Alexander, Huggett, Nixon and Widdas, 1955). Mammalian spermatozoa metabolize glucose just as well as fructose as a source of energy under anaerobic and aerobic conditions. Indeed, glucose has been used widely as the sole glycolyzable sugar in artifi^l diluents employed in the storage of semen for artificial insemination (Mann, 1954a). Thus fructose does not seem to be more beneficial than glucose to the well being of spermatozoa. There is evidence that the utilization of fructose, in contrast to glucose, is not impaired in the diabetic state (Chernick, Chaikoff and Abraham, 1951 ; Renold, Hastings and Nesbett, 1954) . It is conceivable that the presence of fructose in semen w^ould render the spermatozoa relatively insensitive to insulin. But it would seem more probable that the physiologic value of seminal fructose is related to factors other than the maturation or survival of spermatozoa. Mann (1954a) has pointed out that if glucose were the only glycolyzable sugar in semen, its concentration would not be expected to exceed that of blood. The transformation of blood glucose into seminal fructose by the accessory glands permits the establishment of very high levels of fructose in semen. Furthermore, the formation of seminal fructose is strictly controlled by androgenic hormones, and it would be hard to conceive of a similar hormonal dependence of glucose levels in semen.

Although the volume and chemical composition of seminal plasma are influenced by many factors, androgenic hormones are undoubtedly the principal determinants of the secretory activity of the accessory glands. Chemical and enzymatic constituents of accessory gland secretions such as fructose, citric acid, and acid phosphatase have proved to be exquisitely sensitive indicators of androgenic activity. The application of such "chemical tests" for androgen action has provided important corroborative evidence for previous conclusions, based on purely morphologic studies, that the initiation of mature secretory function of the accessory glands precedes the appearance of sperm in the seminiferous tubules, and also that the adverse effects of malnutrition on the functional activity of the prostate gland and seminal vesicle are mediated via the hypophysis. Chemical investigations have established that the major portion of certain components (glycerojihosphorylcholine, glycosidases ) of the seminal plasma of some species originates from the epididymis. The way to the successful treatment of metastatic carcinoma of the prostate in man by antiandrogenic measures was paved by the availability of a chemical systemic index of the hormonal dependence of many of these neoplasias, viz., the acid phosphatase of blood serum. Changes in the chemistry of some accessory organs (e.g., the fructose content of the rat coagulating glandj seem to l)e more sensitive indicators of the action of exogenous androgens in castrated animals than the weights or histologic structure of these organs. The application of such chemical methods to the bioassay of androgens holds much promise for the future. Finally, it may be mentioned that chemical studies of the secretions of the accessory glands have given insight into the homology of these organs. The finding of high concentrations of citric acid, but not of fructose, in the rat female prostate after stimulation with androgens shows that the secretion of this tissue resembles that of the ventral prostate gland of the male rat. On the other hand, structures which are usually considered to be anatomically and functionally homologous may secrete quite different substances. Thus in the guinea pig and bull, both citric acid and fructose are secreted by the seminal vesicles, whereas in the rat, citric acid is produced by the seminal vesicles and fructose is formed only in the dorsolateral prostate and coagulating glands.

D. Metabolism of the Prostate and Seminal Vesicle

The metabolism of the male accessory reproductive glands, and the activity of many enzymes therein, are influenced profoundly by steroid hormones. In adult animals, excision of the testes results in a rapid decline in the respiration, but not of the anaerobic glycolysis, of slices of the prostate gland of the dog (Barron and Huggins, 1944), and of the rat prostate (Homma, 1952; Nyden and Williams-Ashman, 1953; Bern, 1953; Rudolph and Starnes, 1954; Butler and Schade, 1958) and seminal vesicle (Rudolph and Samuels, 1949; Porter and Melampy, 1952; Rudolph and Starnes, 1954j. The post-castrate fall in oxygen consumption by these tissues can be reversed by the administration of testosterone. The respiration of the epithelium (but not of the muscle) of the guinea pig seminal vesicle responds in a similar way to androgen deprivation (Levey and Szego, 1955b). The stimulatory effect of testosterone on the respiration of the prostate gland and seminal vesicle of castrated rats is not prevented by the simultaneous administration of hydrocortisone (Rudolph and Starnes, 1954).

The activity of a number of respiratory enzymes in the rat prostate gland is decreased by castration to about the same extent as the respiration of slices of this tissue. This is true for the succinic and cytochrome oxidase systems (Davis, Meyer and McShan, 1949), and for fumarase, aconitase, and malic dehydrogenase (Williams-Ashman, 1954). But the succinic oxidase levels in two other androgen-sensitive tissues are uninfluenced by castration, viz., the epithelium of the guinea pig seminal vesicle (Levey and Szego, 1955b), and the levator ani muscle of the rat (Leonard, 1950). In the rat prostate, androgens have little influence on the activity of the glycolytic enzymes enolase and lactic (l(>hydrogenase, and of the TPN-specific enzymes which oxidize isocitrate, glucose 6-phosphate and 6-phosphogluconate (WilliamsAshman, 1954; Rudolph, 1956). The enzymatic machinery responsible for the respiration of the male accessory glands seems to be similar to that of other mammalian tissues (Barron and Huggins, 1946a, b; Nyden and Williams-Ashman, 1953; WilliamsAshman, and Banks, 1954b; Williams-Ashman, 1954, 1955; Levey and Szego, 1955a). Glock and McLean (1955) have shown that, as in most other mammalian tissues, the levels of DPN in rodent prostate and seminal vesicle are higher than those of DPNH, whereas the content of TPNH is much greater than that of TPN.

Nyden and Williams-Ashman (1953) found that the respiration-coupled synthesis of long-chain fatty acids from acetate by ventral prostate slices m viti'o was depressed by castration to a greater extent than the respiration, and could be restored to normal levels by testosterone therapy. Certain other synthetic reactions (the incorporation of P^--labeled inorganic phosphate into phospholipids, total nucleic acids, and phosphoproteins) were less sensitive to androgens under these conditions. However, in experiments involving the injection of P-^--labeled inorganic phosphate into animals, the administration of androgen increased the turnover of various acidinsoluble phosphorus containing fractions. Thus Levin, Albert and Johnson (1955) observed that testosterone increases the turnover of various phospholipids in the lirostate gland and seminal vesicle. In the seminal vesicle, Fleischmann and Fleischmann (1952) found that the entry of P-'into the desoxyribonucleic acid fraction was increased 100-fold by androgen administered to castrate rats, whereas the sjiecific radioactivity of the ribonucleic acid was increased only 2-fold. Cytoplasmic basophilia in the rat seminal vesicle (Melampy and Cavazos, 1953), and the endoplasmic reticulum of the ventral prostate gland (Harkin, 1957a), which are intimately associated with cytoi)lasmic ribonucleic acid, are influenced profoundly by androgenic hormones.

Transamination bet^^■een glutamate and cither pyruvate or a-ketoglutarate was shown by Barron and Huggins (1946b) to proceed rapidly in canine and human prostate tissues. Awapara (1952a. bl reported that the ahmine (but not aspartic) transaminase activities of the ventral prostate gland of the rat were decreased by castration, and increased by testosterone therapy.

Rudolph and Starnes (1954) studied the water distribution in the rat accessory glands. The extracellular water in normal seminal vesicles and prostates was 13.8 per cent and 8.5 per cent, respectively. The corresj^onding values in castrate animals were 37.0 per cent and 31.8 per cent. The growth of the glands which resulted from treatment with testosterone was accompanied by a greater increase in the intracellular water than in extracellular water. Rudolph and Samuels (1949) provided evidence that changes in the water content of seminal vesicles induced by treatment of castrate rats with testosterone did not \)recede metabolic changes (e.g., fructose synthesis) in this tissue.

The pronounced effects of androgen administration in vivo on the metabolism and enzymatic activity of the accessary glands cannot be mimicked by the addition of androgens in vitro. Dirscherl, Breuer and Scheller (1955) reported that low levels of testosterone stimulated the respiration of mouse seminal vesicles if the control respiration was low. But others have found that the respiration and glycolysis of male accessory glands are uninfluenced by the direct addition of androgens /// I'itro except at high concentrations (>5 X 10~^ m), at which testosterone is inhibitory (Bern, 1953; McDonald and Latta, 1954, 1956; Andrewes and Taylor, 19551. According to Farnsworth (1958), the direct addition of testosterone to prostate tissue impedes citrate synthesis to a greater extent than oxygen consumption. Williams-Ashman (1954) found that the in vitro addition of testosterone did not affect the activity of a number of respiratory and glycolytic enzymes in the rat A-entral prostate gland.

The mechanism of action of androgenic hormones at a molecular level is not known. There is no evidence that androgens are directly involved in the large changes in the activity of some enzyme systems in accessory glands which follow the administration or deprivation of these hormones. Recent studies wliich indicate that minute concentrations of certain steroid hormones can stimulate the transfer of hydrogen between pyridine nucleotides by isolated enzyme systems deserve further comment. A soluble enzyme in human placenta catalyzes an estradiol- 17y8-dependent exchange of hydrogen between TPNH and DPN (Talalay and Williams-Ashman, 1958). There is evidence in favor of the hypothesis (Talalay, Hurlock and Williams-Ashman, 1958; Talalay and Williams-Ashman, 1960) that estradiol- 17^ transports hydrogen in this reaction by undergoing reversible oxidation to estrone:

Estrone + TPNH + H+ ^ Estradiol-17/3 + TPN EstradioI-17/3 + DPN ^

Estrone + DPNH + H+ TPNH + DPN ^ TPN + DPNH

Hagerman and Villee (1959), however, believe that estradiol- 17/;^ and estrone mediate transhydrogenation between TPXH and DPN by a mechanism which does not involve oxido-reduction of the steroids. Hurlock and Talalay (1958) showed that a soluble 3a-hydroxysteroid dehydrogenase isolated from rat liver catalyzes hydrogen transfer between pyridine nucleotides in the presence of catalytic levels of androsterone and some other 3a-hydroxysteroids. In this instance also, it seems that the steroids act in a coenzyme-like manner by undergoing alternate oxidation and reduction. However, biologically inactive steroids such as etiocholan-3a-ol-17-one are even more active than androgenic substances such as anch'osterone in this isolated enzyme system. Hurlock and Talalay (1959) reported that the particle-bound 3a- and 11^-hydroxysteroid dehydrogenases of rat liver react at comparable rates with both TPX and DPN, and they suggest that these dehydrogenases might function as transhydrogenases in the presence of their appropriate steroid substrates. The hydroxy steroid dehydrogenases for which there is direct or circumstantial evidence for their ability to function as transhydrogenases are localized either in the microsomes (endoplasmic reticulum) or in the soluble cell sap. Other enzymes that catalyze the transfer of hydrogen between pyridine nucleotides are bound to the mitochondria of many animal tissues (Stein,

Kaplan and Ciotti, 1959; c/. Talalay, Hurlock and Williams-Ashman, 1958). These mitochondrial transhydrogenases do not require steroid hormones as cof actors. According to Hmiiphrey (1957), the large cytoplasmic particles of rat prostate gland and seminal vesicle are devoid of transhydrogenase activity. Slices of human prostate gland convert testosterone to androst-4ene-3,17-dione (and other metabolites) (Wotiz and Lemon, 1954; Wotiz, Lemon and Voulgaropoulos, 1954). This suggests that the human prostate contains a 17/3hydroxysteroid dehydrogenase which could conceivably function as a transhydrogenase in the presence of low levels of testosterone. Baron, Gore and Williams (1960) reported the presence of androsterone-stimulated transhydrogenase reactions in the prostate gland of rodents and man. On the contrary, Williams-Ashman, Liao and Gotterer (1958), and Samuels, Harding and Mann (1960) were unable to demonstrate any activation by testosterone of hydrogen transfer between TPNH and DPN in rat prostatic tissue. DPNH and TPNH serve rather different metabolic functions (c/. Talalay and Williams-Ashman, 1958), and it is possible that steroid-mediated transhydrogenations might exert a controlling influence over the balance between the oxidized and reduced forms of pyridine nucleotides in the extramitochondrial regions of certain cells. However, at present there is no direct evidence in support of this hypothesis (cf. Talalay and Williams-Ashman, 1960).

E. Coagulation of Semen

Mammalian semen is emitted from the urethra as a liquid. In some species, e.g., the bull and the dog, the semen remains permanently in the liquid state. But the seminal fluid of many other mammals may undergo remarkable changes in its physical IM^operties on standing. Rodent semen clots rapidly and, if ejaculated into the vagina, forms a solid vaginal plug. This structure assists fertilization by preventing an outflow of semen from the vagina after copulation (Blandau, 1945). The subsequent dissolution of the vaginal plug, probably as the result of the action of leukocytic enzymes, was studied by Stockard and Pajianicolaou (1919). A copulatory plug lias also been described in certain Insectivora, Chiroptera, and Marsupiala (Camus and Gley, 1899; Engle, 1926a; Courrier, 1925; Eaclie, 1948a, bj.

It has been stated that in the opposum (Hartman, 1924) and in the bat (Courrier, 1925), the vaginal plug results from the coagulation of the female secretions by seminal plasma. However, the semen of many other species clots on its own accord. Camus and Gley (1896, 1899) were the first to recognize that in the rat and guinea pig, the clotting process involves the solidification of the vesicular secretion by an enzyme of prostatic origin, which they termed vesiculase. The classical experiments of Walker (1910a, b) showed that this enzyme is secreted solely by the anterior prostate or "coagulating" gland. In the rhesus monkey, the secretion of the cranial lobe (but not of the caudal lobe) of the prostate gland coagulates the vesicular secretion (van Wagenen, 1936) . The "soft calculus" frequently present in the urinary bladder of male but not female rats is l^robably formed by clotting of the seminal vesicle secretion by the action of enzymes from the coagulating gland (Vulpe, Usher and Leblond, 1956) .

More recently, the mechanism of action of vesiculase has been studied in considerable detail. A crude preparation of the proteins of the vesicular secretion that are clotted by this enzyme can be obtained in a stable form, and the clotting process may l)e measured quantitatively by simple spectrophotometric procedures (Gotterer, Ginsburg, Schulman, Banks and Williams-Ashman, 1955; Gotterer and Williams-Ashman, 1957; Zorgniotti and Brendler, 1958). The over-all coagulation process is extremely sensitive to the ionic strength of the solution in which it takes place, and is abolished by the addition of metal chelating agents such as Versene (ethylcnediaminetetraacetic acid), o-i)henanthroline, and a,adipyridyl, and also by heavy metals such as mercuric ions. The inhibitory action of Versene can be overcome by manganous ions, or by somewhat higher concentrations of calcium ions. Experiments involving the delayed addition of either heavy metal ions or of metal chelating agents established that till' coagulation process can be separated into two distinct phases (Gotterer and AVilliams-Ashman, 1957). The first of these requires a metal ion such as Mn++, is inhibited by Versene, and does not necessarily involve the precipitation of insoluble material. The second phase, which is insensitive to the action of metal chelating agents, is inhibited by mercuric ions and leads to the formation of a coagulum. The coagulated material is protein in nature.

Further fractionation of the vesicular secretion by Speyer (1959) led to the isolation of a heat-stable protein, coagulinogen, which is the precursor of the insoluble material of the vaginal plug, but is not clotted by vesiculase. Speyer (1959) isolated another, heat-labile protein from vesicular secretion which he designated procoagulase, and which is converted into a clotting enzyme coagulase by the action of vesiculase. The coagulation of the seminal vesicle secretion by the prostatic enzyme vesiculase thus seems to take place by the following reactions:

„ , Vesiculase „ ,

rrocoagulase > Coagulase

Coagulinogen °^^" '^^^ — ^ Coagulated protein

Only the first reaction is inhibited by Versene.

Partial purification of vesiculase has l>een achieved (Gotterer, Ginsburg, Schulman. Banks and Williams-Ashman, 1955). Vesiculase is quite distinct from another enzyme in the secretion of the coagulating gland of guinea pigs which hydrolyzes, inter alia, tosyl-L-arginine methyl ester (TAMe) (Gotterer, Banks and WilliamsAshman, 1956). Unlike thrombin, vesiculase does not hydrolyze TAMe and does not clot fibrinogen. The dissimilarity between the coagulation of blood and of semen is further borne out by the failure of thrombin to clot the proteins of the vesicular secretion, and by the inability of TA]\Ie (which depresses the action of thrombin) to inhibit vesiculase action.

Electrical stimulation of the head of the guinea pig induces ejaculation without voiding of either urine or feces (Batelli, 1922). Ejaculates obtained in this manner from normal, sexually mature guinea pigs coagulate rapidly. After castration, the semen is no longer coagulable, but becomes so a few days after treatment with androgens (Moore and Gallagher, 1930). This "electric ejaculation test" can be used as an indicator for androgenic activity (c/. Sayles, 1939, 1942).

It is generally believed that human semen is ejaculated as a fluid, and then coagulates (Lane-Roberts, Sharman, Walker and Wiesner, 1939; Joel, 1942; Huggins and Neal, 1942; Lundquist, 1949), although some authors state that it is emitted in a gelatinous form (Pollak, 1943; Hammen, 1944; Oettle, 1954). But there is no doubt that the semen from normal men subsequently liquifies if kept at room temperature.^ Human semen possesses strong fibrinolytic activity (Huggins and Neal, 1942; Harvey, 1949; Ying, Day, Whitmore and Tagnon, 1956). The prostate gland of men secretes a proteolytic enzyme, fibrinolysin, which is probably responsible for the phenomenon of liquefaction. Prostatic fibrinolysin is produced in large amounts by certain cancers of the prostate in man, and seems to enter the circulation since there is a pronounced bleeding tendency in such patients (Tagnon, Schulman, Whitmore and Leone, 1953; Scott, Matthews, Butterworth and Frommeyer, 1954; Swan, Wood and Owen, 1957). Canine semen, which does not clot, contains little fibrinolysin, but is rich in another proteolytic enzyme, fibrinogenase, which hydrolyzes fibrinogen. Little fibrinogenase is present in human semen (Huggins and Neal, 1942). The presence of related proteolytic enzymes in the secretions of the male accessory glands is described above.

^ Louis Nicolas Vauquelin published the first paper on the chemistry of seminal fluid (Vauquelin, 1791). This remarkable study includes a detailed and accurate account of the liquefaction of human semen which is quite unexcelled by later writings. It also describes the formation, in ejaculates which had stood for three or four days, of "cristaux transparens, d'environ une hgne de long, tres-minces, et qui se croisent souvent de maniere a representer les rayons d'une roue. Ces cristaux isoles nous ont offer, a I'aide d'un verre grossissant, la forme d'un solide a quatre pans, termines par des pyramides tres-allongees, a quatre faces." Although Vauquelin believed that these crystals were composed of calcium phosphate, Mann (1954a) has pointed out that he had, in reality, obser\ed the deposition of spermine phosphate in aged semen.

Freund and Thompson (1957) reported that intravenous injection of crude guinea pig coagulating gland secretion into rabbits or guinea pigs induces hypotensive shock. Edema results if the secretion is injected locally. The secretion of the coagulating gland of the rat does not possess these properties. Further studies by Freund, Miles, Mill and Wilhelm (1958) showed that two main protein fractions can be separated from the secretion of the guinea pig coagulating gland by preparative starch electrophoresis. Fraction I was hypotensive and a potent permeability factor in rabbits and guinea pigs. It hydrolyzed TAMe rapidly and may be identical with the TAMehydrolyzing enzyme described in guinea pig coagulating gland l)y Gotterer, Banks and Williams-Ashman (1956). The latter enzyme is not present in the coagulating gland of the rat. Fraction II isolated by Freund and his associates is ]n"obably vesiculase.

III. Structure and Function in Relation to Hormones

A. INTRODUCTION

Some of the effects of removal of the testes in males have been recognized ever since castration was first practiced on man and domestic animals. Aristotle's writings include accurate descriptions of the effects of castration on secondary sex characters in birds and in man. The classical studies of John Hunter (1792) laid the basis for an understanding of the relation between the presence of the testes and the size and functional state of the accessory reproductive glands of mammals, although he did not postulate the existence of testicular hormones.

Hunter demonstrated experimentally that the seminal vesicles of guinea pigs are not reservoirs for semen and concluded that this a])plies to the seminal vesicles in man and in other mammals. He not only described the gross anatomy of the seminal vesicles in many species (hedgehog, iiiole. man, boar, bull, horse, buck, mouse, rat, beaver, guinea pig) and their absence fi'om others, but he observed tliat they arc smaller in the gelding than in the stallion. In refei'ence to other glands, he generalized

that "the prostate gland, Cowper's glands and the glands along the urethra . . . are in the perfect male large and pulpy, secreting a considerable quantity of slimy mucus which is salt to the taste . . . while in the castrated animal these are small, flabby, tough and ligamentous, and have little secretion." In addition, he made the equally important discovery that the testes of mammals (and birds as well) are very small in winter in animals which have their seasons of copulation" and the seminal vesicles and prostates are "hardly discernable." He concluded that "from these observations it is reasonable to infer that the use of the vesiculae in the animal oeconomy must, in common with many other parts, be dependent upon the testicles."

Over 100 years later, many of his observations were rediscovered, extended, and interpreted in the light of the first demonstration that the testis is an endocrine organ (Berthold, 1849). In the early part of the 20th century the interest in attempting to isolate and characterize androgens from testis tissue and urine led to a search for rapid and dependable bioassay methods. The cock's comb provided a sensitive and convenient test object (Pezard, 1911). In addition, some of the accessory reproductive glands of mammals were found to atrophy rapidly after castration and proved also to be sensitive indicators for the presence of androgenic hormones. Cytologic tests using the rat prostate, seminal vesicles, and Cowper's glands were developed and an electric ejaculation test in the guinea l)ig was devised (Moore, 1932, 1939). Weights, sizes, cytologic structure, and mitotic activity in mouse seminal vesicles were suggested as bioassay methods for androgenic hormones (Deaneslv and Parkes. 1933).

After th(> successful isolation and chemical characterization of androgens and estrogens from various sources, interest centered on the fundamental relationships of androgens to normal develojjment, histologic structure, and secretory activity of the accessory glands in many species of inainmals. The effects of estrogens and gestagens and the competitive and synergistic i'elationshii)s of steroid hormones were examined. The results of this early work contributed extensively to the fields of biochemistry, biology, and medicine. More recently there have been studies on the relation of hormones to the ultrastructure, histochemistry, and metabolism of the glands, and to the chemical composition of their secretions ( Section II I .

In the following section, the hormonal control of structure and function will be discussed with particular reference to the luniierous studies on the prostate glands and seminal vesicles of rats and mice.

B. EFFECTS OF ANDROGENS

The term androgen will be used in the collective sense for substances that are capable of stimulating accessory reproductive glands in castrated animals and maintaining normal histologic structure and secretory activity in the epithelium. Androgenic substances are formed by the testes, ovaries, and adrenal cortex. All androgens which have been characterized are steroids. The urine contains many androgen metabolites, mainly in the form of their conjugates with either glucuronic or sulfuric acids. Testosterone is the principal androgen secreted by the testis and this substance, or tiie longer acting testosterone propionate, is most commonly used as a replacement for testicular androgen. In the last two decades a number of unnatural androgens [e.g., 17a-methyl testosterone) have been synthesized and found to possess strong biologic activity. The relationship between chemical structure of steroids and andro

genic activity in a variety of bioassay procedures is discussed by Dorfman and Shipley (1956).

1. Testicular Androgens

The effects of endogenous and exogenous androgen on weight, histologic structure, and secretory activity of the accessory glands have been reviewed by Moore (1939), Price (1947), Burrows (1949), Dorfman (1950), Dorfman and Shipley (1956) and many others. Aspects of metabolic activity have been treated by Roberts and Szego (1953) and Mann (1954a).

The first detailed cytologic studies of male accessory glands and the changes following castration and hormone administration were made on the prostates, coagulating glands, and seminal vesicles of adult rats (Moore, Price and Gallagher, 1930; Moore, Hughes and Gallagher, 1930). Extensive research on structure and function of these and other accessory glands in many species followed this early work, but the cytologic structure of prostates and seminal vesicles of rats and mice remains one of the most sensitive indicators for androgenic hormones.

Rat PROSTATE AND SEMINAL VESICLES. Ventral prostate. In the normal adult gland, the columnar secretory epithelium has basal nuclei with conspicuous nucleoli and chromatin particles, and a supranuclear clear zone or light area in the cytoplasm corresponding to the position of the Golgi zone (Figs. 6.8, 6.9, and 6.14). In osmium preparations, the Golgi apparatus appears as

Fics. (3.8 Axn 6.9. Rat ventral prostate from a normal adult male. X 5UU and lOUU. Boinnhematoxylin preparations. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)

400

PHYSIOLOGY OF GONADS

heavy strands or networks (Figs. 6.19 and 6.22) which do not conform precisely to the shape or area of the cytoplasmic clear zone. Mitochondria are distributed as rods or granules in all parts of the cell. The secretion in the lumina of the alveoli is eosinophilic and mainly granular. A basement membrane rests on a stroma of connective tissue containing smooth muscle strands and blood vessels. Occasional small basal cells are wedged between the tall secretory cells. These observations were made by light microscopy of tissues fixed and stained by routine methods (Moore, Price and Gallagher, 1930).

Electron microscopy (Harkin, 1957a) shows that the epithelial cells have an endoplasmic reticulum or ergastoplasm composed of membrane-lined sacs with a finely granular component in the spaces between them (Figs. 6.27 to 6.29) ; the outside of the thin membrane is studded with Palade's granules (Palade, 1955). The arrangement of the sacs tends to parallel the long axis of the cells, but in cross section the pattern ai)pears concentric or lamellar, particularly in the supranuclear region (Fig. 6.29). The ergastoplasmic sacs occupy more space than the matrix apically, but basally the two are equally prominent (Fig. 6.28). The mem

TABLE 6.6

Summary of the effects of testicular androgen, on the rat prostate and coagulating glands

Normal Males

Castrated Males

General Characteristics

All lobes alveoli witli folded inueosa; secretion in the lumina.. columnar epithelial cells: cytoplasm granular or foamy. supranuclear clear zone in cytoplasm (ventral lobe)

Golgi supranuclear networks

mitochondria as rods or granules

nuclei basal or central

stroma of connective tissue and smooth muscle

Size reduced; villi lost; secretion reduced

Size reduced; pseudostratified; cytoplasm less dense

Clear zone lost

Reduced in amount; fragmented

Still numerous but reduced in relative numbers

Shrunken and pyknotic

Increased fibromuscular tissue

Specific Characteristics

Ventral lobes

Histochemical observations:

secretion in lumina strongly PAS- and alkaline phosphatase-positive . . .

cytoplasm weak PAS, strong alkaline phosphatase activity;

basophilic reaction except in clear zone

Golyi accumulations of PAS-positive granules

stroma some alkaline phosphatase activity

Electron microscopic observations:

cytoplasm moderately distended ergastoplasmic sacs

Golgi supranuclear microvesicular complex

mitochondria numerous, prominent apically

Lateral lobes

Histochemical observations:

cyioplasm luminal border organelle with high concentrations of zinc and basophilic material; osmiophilic, argentophilic

nucleoli high concentrations of zinc and marked basophilia

stroma high concentrations of zinc; basojihilic material (jresent Dorsal lobes Histochemical observations:

cytoplasm in apical region strongly basophilic; basally, some zinc

nucleoli high concentrations of zinc and marked basophilia

stroma basophilic material; strong alkaline i)hosphatase reaction

Electron microscopic observations:

cytoplasm distended ergastoplasmic cisternae;

Coagulating glands (anterior prostate) Histocheinical observations:

secretion strongly PAS-positive

cytoplasm weak P.\S reaction

stroma some alkaline phosphatase activity

Electron microscopic observations:

cyioplasm extremely dilated ergastoplasmic cisternae

Some phosphatase activity retained Phosphatase activity low

Sacs collapsed; granvilar component reduced

Reduced in size

Reduced in relative numbers

Cisternae collapse

granules reduced

pears unalterc

Cisternae collapsed; granules reduced

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

401

hrane is continuous with the outer nuclear membrane. The Golgi complex is conspicuous as microvesicles midway between nucleus and lumen. Mitochondria lie in the matrix between the sacs and are very prominent in the most apical region. Microvilli project into the lumina of the alveoli with no interruption of the cytoplasmic, or plasma, membrane. The membrane at the base of the cell is double (see Fig. 6.28) ; one component, the basement membrane, continues unbroken under adjacent cells; the second forms a part of the double plasma membrane between cells. Brandes and Groth (1961) have confirmed Harkin's findings and added further observations. Nuclei contain patches of granules which are frequently along the inner nuclear membrane; the Golgi complex consists of vesicles, vacuoles, and parallel meml)ranes; vesicles and granules surrounded by smooth-surfaced membranes are disposed in the cytoplasmic matrix and are more numerous apically; the dilated sacs or cisternae of the supranuclear region seem to intercommunicate.

Histochemical studies of basophilia, alkaline phosphatase activity, and the localization of i^eriodic acid-reactive carbohydrates (Periodic acid-Schiff or PAS reaction) add further information (Table 6.6). Davey and Foster (1950) found basophilia (which was abolished by ribonuclease) distributed through the cytoplasm except in the clear area described by Moore, Price and Gallagher (1930) as corresponding to the position of the Golgi zone. Stroma of the ventral prostate shows some degree of alkaline phosphatase activity but luminal secretion and epithelial cells are strongly positive (Bern, 1949a), especially at the luminal and basal borders (Stafford, Rubenstein and Meyer, 1949). The secretion also gives a fairly intense PAS reaction whereas the epithelial cells are only slightly reactive; occasionally the Golgi apparatus is visible as PAS-positive granules (Leblond, 1950).

After castration, there is reduction in cell height and loss of the cytoplasmic clear zone (Fig. 6.15) within 4 days. On subsecjuent days, cell size continues to decrease and nuclei become small and pyknotic (Figs. 6.10. 6.16 to 6.18). The Golgi ap

paratus begins to fragment by 10 days; by 20 days it consists of granules much reduced in amount (Fig. 6.20) and the basement membrane of the cells disappears (Moore, Price and Gallagher, 1930).

Harkin (1957a) reported changes observable by electron microscopy within 24 hours after castration; distention of apical ergastoplasmic sacs and reduction in size and number of microvilli. By 2 days, there is dilation of Golgi microvesicles, collapse of the apical ergastoplasmic sacs, and reduction in mass of apical cytoplasm; at 4 days, massive collapse of sacs, reduction in mitochondrial number, and increase in electron-dense bodies (Fig. 6.30). The granular component is not reduced until 8 days after castration or longer. Brandes and Portela (1960a) noted, briefly, collapse in the cisternae of the ergastoplasm, loss of the ribonucleic acid- (RNA) rich granules from the membranes of the endoplasmic reticulum, and apparent increase in mitochondria but with a reduction in their size (Table 6.6).

The distribution of alkaline phosphatase in the stroma, epithelium, and secretion is unchanged 32 days after castration; the stroma is still reactive at 120 days but the epithelium is completely atrophic (Bern and Levy, 1952). (Quantitative determinations of alkaline and acid phosphatases showed, however, that activities of both enzymes are reduced markedly by 8 days (Stafford, Rubenstein and Meyer, 1949). The epithelium loses the ability to secrete citric acid (see Section IT).

Changes after gonadectomy are prevented or reversed by administration of androgenic substances. Extracts of bull testes (:\Ioore, Price and Gallagher, 1930) prevented involution of the epithelium in castrates (Fig. 6.11 and 6.21) and androsterone, testosterone, and testosterone propionate prevented or repaired castration changes CMoore and Price, 1937, 1938). The response of the castrate to androgen is rapid; cell hypertrophy begins within 23 hours after a single injection of testosterone propionate into males castrated for 40 days ; at 35 hours mitotic activity begins and reaches a maximum at 43 hours (Burkhart, 1942).

Ergastoplasmic sacs in the epithelial cells

402

PHYSIOLOGY OF GONADS

^.. Da .^.^^

I I'- <; II) 0.13. Rat ventr;il lu-o-tatc (Figs. 6 10. H.U) and coagulal iim aland ( Fm- C) 12, (i.lo). All pliotomicrographs , lOUU. Fig. 6.10. 20-day cabtiatc. Fig. 6.11. 20-day ca.^tratf injected with testis extract. Fig. 6.12. 20-day castrate. Fig. 6.13. 20-day castrate injected with testis extract. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)

are prevented from collapsing by treatment of castrates with testosterone and the process is reversed if the androgen is given after castration changes have developed (J. C. Harkin, personal communication). Alkaline and acid phosphatase levels are essentially normal in castrates injected with testost(M'one propionate (Stafford, Rubinstein and Meyer, 1949).

Lateral prostate. The epithelial cells in normal adult glands are columnar and the nuclei are basal, but cell size and nuclear position arc more variable than in the ventral prostate ( Korenchevsky and Dcnnison. 193.51. The Golgi apparatus appears as

l^rominent supranuclear networks in osmium stainecl preparations (Rixon and Whitfield, 1959).

Histochemical studies employing a dithizone zinc stain demonstrated high concentrations of zinc in the apical jiart of the cells (Gunn and Gould, 19o6a). Fleischhauer (1957) observed (macroscopically) heavy staining that was visible in this lobe after intravenous or subcutaneous injections of dithizone. In mifix(Hl frozen sections, he found in tlic basal regions of all epithelial cells numerous stained granules which lie interpreted as zinc-positive material. 'Hie nature of a rather wide diffusely

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

403

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m

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IB

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Figs. 6.14-6.26

Figs. 6.14-6.22. Rat ventral prostate. Figs. 6.14-6.21. Camera lucida drawings X 3000. Figs. 6.19-6.22. Mann-Kopsch preparations for Golgi apparatus. Fig. 6.14. Normal male. Figs. 6.156.18. From males castrated for 4, 10, 20 and 90 days. Fig. 6.19. Normal male. Fig. 6.20. 20-day castrate. Fig. 6.21. 20-day castrate injected with testis extract. Fig. 6.22. Normal male ; photomicrograph X 1000. (From C. R. Moore, D. Price and T. F. Gallaglier, Am. J. Anat., 45, 71107, 1930.)

Figs. 6.23-6.26. Rat coagulating gland. Figs. 6.23-6.25. Camera lucida drawings X 3000. Fig. 6.23. Normal male. Fig. 6.24. 20-day castrate. Fig. 6.25. 20-day castrate injected with testis extract. Fig. 6.26. Normal male; photomicrogaph X 1000; Mann-Kopsch preparation for Golgi apparatus. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat.. 45, 71-107, 1930.)

404

PHYSIOLOGY OF GONADS

■^f

«#

Fig. 6.27. R:it xcniinl |.in~i,ii( ikhihiI mil. I ,li . 1 1 uiiiuicrograpli X 8500; LuftV ])prmanganate fixative. Aliciuvilli lxIlikI a^ i)iul()n^atioii.-. ol the cytoplasm into the lumen; a major part of the cytoplasm is a labyrinth of ergastoplasmic sacs with scattered mitochondria ; nuclei are basal ; half-way between nucleus and cell apex is a zone of small vesicles and canals, the Golgi complex (From J. C. Harkin, un])u})lishe(l.)

stained area in the ai)ical cytoi)lasin was not clear. Ki.xoii and Whitfield 11959) reported high concentrations of zinc in the apical cytoplasm, nucleoli, and stroma in fixed tissues stained with dithizone. Tn the apical cytoplasm, the zinc is conccnti atcd at the tip of the cells in a "luminal l)order organelle" which is osmiophilic (distinct from the (Jo].o;i apparatus), argent()i)hilic,

and basophilic. Nucleoli and subepithelial sti'oma are basophilic.

Castration results in a typical pattern of involution in the epithelial cells: size is !•(■( bleed, nuclei become small and jwknotic, and changes occur in the density of the cytoplasm (Korenchevsky and Dennison, 1935; Price, Mann and Lutwak-Mann, 1955). The zinc content of the gland (dor

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

405

solatcral or lateral prostate) and the rate of Zn*^^ uptake decrease after gonadectomy as does the secretion of citric acid and fructose (see Section II).

Dorsal prostate. The epithelium in the dorsal prostate of normal rats is columnar or cuboidal depending on distention of the alveoli; nuclei are basal and stain heavily; there is cytoplasmic vacuolization which is usually limited to the basal region (Korenchevsky and Dennison, 1935) .

Brandes and Groth (1961) described the ultrastructure of two different cell types in the dorsolateral (or dorsal) lobe. These types differ in the relation of cytoplasmic matrix to endoplasmic reticulum. In both, the matrix is moderatelv homogeneous and

contains small particles, but in cell type 1, the matrix appears as separate profiles and the reticulum as membrane-bounded individual cavities. In cell type 2, the reticulum forms dilated membrane-bounded cisternae which are intercommunicating and the cytoplasmic matrix is reduced mainly to thin strands appearing isolated within the cisternae.

Gunn and Gould (1956a) reported a zinc-negative histochemical reaction in the epithelium of the dorsal prostate. Fleischhauer (1957) observed a slight dithizonestain macroscopically, and in unfixed frozen sections, individual groups of cells contain the distinctive basal zinc-positive granules that are characteristic of all epithelial cells

...\

Fl(.i. li..'N. l;;il v,l,ii;,l |Mm-;;:h, n,uM,:,l in.ih. 1 ,1. r i ,, ,i i li i in . ,-i:i [ .1, . 2(;,()()(); 1 ),-| ll ( )ll's chrome o^niic and hxatixi . Il;i>:il pari ot epithelial cell to show the character of the granular component and ergastopla>iiiic -acs which are essentially equal in amount in this region. Double basement membrane indicated by arrow. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.)

406

PHYSIOLOGY OF GONADS

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l'"i(..G.2U. Rat \(;iilial lUo.-Uilu, iiuinial iiiak;. I'^lcctionnu' acid fixative with sucrose. Supranuclear region of epithelia plasmic sacs. (From J. C. Harkin, unpublished.)

.-laph Is.iiOO; Paladps osmic •ell sliowing laniellatetl ergasto

in the lateral lolje. Tiiere is no diffuse staining of the apical cytoplasm. Nucleoli arc intensely zinc-positive after fixation and staining with dithizone; nucleoli, apical cytoplasm and stroma are basophilic fRixon and Whitfield, 1959). The stroma is also strongly alkaline phosphatase - positive (Bern, 1949a).

Epithelial cells respond to castration l»y reduction in cell and nuclear size, and loss of granulation in the cytoplasm (Korenchevsky and Dennison, 1935). Brandes and Portela (1960a) observed in electron micrographs the V)eginning of collapse of the

cisternae of the endoi)lasmic reticulum, reduction in RNA-rich particles, and changes in mitochondria. Histochemical studies ( Iicni and Levy, 1952) indicate that distribution of alkaline phosphatase activity remains unchanged. Fructose content is reduced in the gland after castration (Price, Mann and Lut\vak-]\Iann, 1955).

CocKjulating gland {anterior prostdte) . In normal males, the ei)ithelium is columnar and rests on a well marked basement membrane; nuclei stain heavily and homogeneously and ai'e situated midway between the basement meml)i';iiie and lumen. The cvto

ACCESSORY MAMMALIAN REPRODUCriVE GLANDS

407

plasm is not as granular as in the ventral prostate and appears vacuolated, particularly in the basal region and around the nuclei; the apical cytoplasm is condensed and granular (Fig. 6.13, a gland from a castrated male injected with testicular extract, illustrates essentially the characteristics of the normal epithelium). Golgi bodies ( Fig. 6.26) form large networks close to the

luminal end of the cells (Moore, Price and Gallagher, 1930).

The striking characteristic of these cells in electron microscopy (Brandes, Belt and Bourne, 1959; Brandes and Groth, 1961) is the great dilation of the cisternae of the endoplasmic reticulum (Fig. 6.31) which fill the greatest part of the cell and are particularly distended in the basal region. The

Fig. 6.30. Rut \entral prostate, 4-day castrate. Electronmicrograph X 26,000; Dalton's chrome osmic acid fi.xative. Portion of nucleus and di.stal region of epithelial cell. An electron dense body lies above the nucleus and below dilatated Golgi microvesicles. Arrow points to collapsed ergastoplasmic sacs. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.)

408

PHYSIOLOGY OF GONADS

Fig. 6.31. Rat coagulating gland, normal male. Electronmiciogiaplis, lefl X 7200; upper and lowei- right X 39,000. Caulfield's modification of Palade's osmic acid fixative. Left, ba.sal portions of two epithelial cells; right, details of basal region: bni, basement membrane; ci, dilated cisternae; cm, plasma membrane; cy, cytoplasmic matrix; G, Golgi complex; bn, limiting membrane of endoplasmic reticulum; w, mitochondria; n, nucleus. (From D. Brandes, unpublished.)

cytoplasmic matrix appears as strands within the cisternae. The Golgi complex is represented by parallel rows of membranes, vacuoles, and smaller vesicles.

Histochemically (Table 6.6), the secretion is intensely PAS-positive and the cytoplasm is slightly reactive (Leblond, 19501. The stroma is strongly alkaline phosphatase-positive (Bern, 1949a).

The effects of castration arc not ai)i)arent by light microscopy as early as in the ventral prostate and seminal vesicles. At 10 days after castration the cells are slightly smaller and the cytoplasm less dense; by 20 days, the cells are markedly reduced in size, nuclei smaller, cytoplasm clear, basement membrane absent or less well defined (Figs. 6.12 and 6.24). The Golgi apparatus is reduced in amount but not fragmontcnl. It still retains the shape of strands or threads which cap around tlic nucleus at

90 days of castration but the mass is reduced (Moore, Price and Gallagher, 1930).

Brandes and Portela (1960a) state that castration produces gradual and slow collapse of cisternae in the endoplasmic reticulum, changes in mitochondria, and reduction and loss of RNA-rich particles from the membranes. Studies of functional activity show that the ability to secrete fructose and vesiculase is lost (see Section II).

Depending on the length of the interval between the operation and administration of the hormone, treatment of castrates with testis extracts (Figs. 6.13 and 6.25) or testosterone prevents or repairs histologic and functional changes.

Seminal vc.'iicles. The secretory epithelium is colunuiar in normal males; nuclei are basal and contain one or two conspicuous nucleoli and smaller chromatin masses (Table 6.7). Seci-etion granules, surrounded

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

409

TABLE 6.7 Summary of the effects of testicular androgen on rat and mouse seminal vesicles

Normal Males

Castrated Males

General Characteristics

Rat and mouse

mucosa folded; acidophilic secretion in lumen Villous folding reduced; secretion greatly reduced

columnar epithelial cells: secretion granules supranuclear Cell size reduced; granules lost

Golgi supranuclear networks \ Reduced in volume; fragmented

mitochondria as rods or granules ' Apparently reduced in relative numbers (rat)

nuclei basal; nucleoli prominent I Nuclei shrunken and pyknotic; nucleoli disappear

stroma of connective tissue and smooth muscle Amount appears increased

Specific Characteristics

Rat Histochemical observations : secretion in lumen PAS-positive, intensity variable; acidophilic

ci//opZasm slight PAS reaction; strongly acid phosphatase-positive;

strongly basophilic

secretion granules in epithelium strongly acid phosphatase-positive

nuclei strong acid phosphatase reaction

stroma slight PAS reaction; acid phosphatase-positive; strong alkaline

phosphatase reaction

Mouse Histochemical observations :

secretion in lumen moderately PAS-positive and acidophilic

cytoplasm moderately basophilic at base and lateral margins of cells;. . .

apical granules acid phosphatase-positive secretion granules in epithelium weakly PAS-positive and acidophilic... Golgi region; granules PAS-positive and acidophilic stroma intensely PAS- and alkaline phosphatase-positive Electron microscopic observations: cytoplasm complex pattern of basal and lateral ergastoplasmic membranes;

Phosphatase activity reduced

Slight basophilia

Activity lost

Remained weakly acid phosphatase-positive

Acid and alkaline phosphatase activity reduced

Secretory granules less acidopliiliWeakly basophilic

abundant RNA-rich granules

Golgi region; parallel arrays of smooth-surfaced membranes and vesicles

Reduced in number; less acidophilii Phosphatase activity reduced

Ergastoplasmic channels less distended and contorted Relative number reduced

by vesicular zones are present in the supranuclear region (Fig. 6.36) and resemble the secretion in the lumen in staining reactions. The Golgi complex appears in osmium preparations as irregular networks or a vesicular structure (Fig. 6.32); the basement membrane is poorly defined or absent (Moore, Hughes and Gallagher, 1930).

The extracellular secretion is only slightly PAS-positive but varies in the intensity of reaction; there is little reaction in the epithelial cells except in some cells with stained granules; fibers of the lamina propria, smooth muscles, and walls of arterioles are weakly reactive (Leblond, 1950; ]\Ielampy and Cavazos, 1953). Stroma and capillaries are strongly alkaline phosphatase-positive (Bern, 1949a; Dempsey, Greep and Deane, 1949; :\lelarapy and Cavazos, 1953). The cytoplasm, secretion granules, nuclei, and stroma give an intense acid phosphatase reaction; the cytojilasm is strongly baso

philic and the reaction is abolished by ribonuclease (]\lelampy and Cavazos, 1953).

The response to castration is rapid. In 2 days the cells are reduced in height mainly by reduction in apical mass ; secretion granules are few, small, and indistinct. By 10 days, cells are small, secretion granules are gone, nuclei are small with heavily staining chromatin (Fig. 6.35), and Golgi bodies have begun to fragment; at 20 days, these changes are more advanced and the remnant of the Golgi bodies (Fig. 6.33) occupies almost the entire supranuclear region (Moore, Hughes and Gallagher, 1930). In a cytometric study, Cavazos and ]\Ielampy (1954) found a statistically significant reduction in cell height by 6 hours after castration; by 48 hours many nucleoli are smaller than normal and by 60 hours most nucleoli are small; nuclear diameters are reduced but change more slowly.

410

PHYSIOLOGY OF GONADS

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Figs. 6.32-6.36. Rat seminal vesicle; camera lucida drawing.s ,: 3000. Figs. 6.32-6.34. ManuKopsch preparations for Golgi apparatus. Fig. 6.32. Normal male. Fig. 6.33. 20-day castrate. Fig. 6.34. 20-day castrate injected with testis extract. Fig. 6.35. 10-day castrate. Fig. 6.36. 20day castrate injected with testis extract. (From C. R. Moore, W. Hughes and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)

Gonadectomy causes gradual reduction and disappearance of alkaline phosphatase activity (Dempsey, Greep and Deane, 1949; Melampy and Cavazos, 1953) and hypophysectomy, with consequent diminution of testicular hormones, gives similar results (Dempsey, Greep and Deane, 1949). Bern and Levy (1952) reported some retention of alkaline phosphatase activity in the fibromuscular tissue of castrates. Acid phosphatase activity decreased within 10 days following castration (Melampy and Cavazos, 1953).

In early experiments (Moore, Hughes and Gallagher, 1930) , administration of bull testis extracts to castrated rats maintained normal histologic structure or repaired involutional changes (Figs. 6.34 and 6.36),

and androsterone, testosterone, and testosterone propionate gave similar resvdts (Moore and Price, 1937, 1938). Androgen treatment in castrates produced detectable changes within 2 days. Burkhart (1942 1 observed cell hypertrophy ahd enlargement of nuclei 23 hours after a single injection of testosterone propionate into 40-day castrates; mitotic activity began at 35 hours and reached a maximum at 43 hours. Cavazos and Melampy (1954) treated castrates with testosterone propionate and found increases in nuclear diameter within 12 hours, cell height within 24 hours, and nucleolar size l)y 36 hours; mitotic activity was evident at 48 hours. The same hormone restoi'cd normal alkaline and acid phosphatase activity in castrates within 10 days

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

411

(Dempsey, Grecp and Deane, 1949; Melampy and Cavazos, 1953).

Mouse prostate and seminal vesicles. Ventral prostate. The epithelial cells in the adult gland are low to moderately tall columnar; acini are surrounded by a thin fibromuscular layer; lumina contain finely granular, acidophilic secretion. The cytoplasm appears somewhat foamy with a clear zone in the supranuclear Golgi region and rather dense basophilia near the lumen (Franks, 1959). In approj)riate histologic preparations, the Golgi apparatus is visible as a network in the apical cytoplasm close to the nucleus and the twisted strands are oriented parallel to the long axis of the cell (Horning, 1947).

Brandes and Portela (1960c) observed by electron microscopy an endoplasmic reticulum of cisternae or vesicles that are usually flattened but have dilations. RNA-rich granules are attached to the outer surface of the thin membranes bounding the vesicles and occur also in the cytoplasmic matrix; the arrangement of cisternae may be parallel or in a random pattern. The luminal margin of cells exhibits small cytoplasmic projections covered by the cell membrane. There are also extensions of the margin which ai)pear similar to fragments of cytoplasm that seem to lie free in the lumen, and are presumably detached from the apical tips of cells. The lumina also contain structures that resemble profiles of the endoplasmic reticulum and mitochondria. The supranuclear Golgi complex consists of vacuoles of various sizes and flattened vesicles; endoplasmic reticulum and mitochondria are present in the Golgi zone.

Histochemical findings (Table 6.8) indicate alkaline phosphatase activity in the stroma with a positive reaction in the basement membrane, endothelium of blood vessels, and sheaths of smooth muscle fibers (Brandes and Bourne, 1954). With longer incubation periods of the tissue (Bern, 1949a), the epithelium and secretion in the lumina are strongly reactive and the stroma shows some activity. Brandes and Bourne (1954) reported acid phosphatase activity in the epithelium; the Golgi region gave the strongest reaction and the nuclei were moderately positive. Luminal secretion, ag

gregations of granules in the Golgi region and apical cytoplasm, basement membranes, and capillary endothelium were PA8-positive. Sulfhydryl and disulfide reactions were moderately strong in epithelial cells and basement membranes.

Gonadectomy results in typical cell retrogression with reduction in cell height and nuclear size. Brandes and Bourne (1954) summarized their results as follows: after gonad removal, the Golgi apparatus showed some fragmentation and was not so dense by 12 to 14 days; alkaline phosphatase activity was slightly less intense by 4 days; changes in acid pliosphatase activity in the Golgi region were evident by 4 days and marked by 21 to 22 days; the PAS reaction was reduced by 8 days and almost lost in the epithelium by 21 to 22 days. Subcutaneous implantation of pellets of testosterone propionate 13 to 32 days after castration produced a rapid return to normal of Golgi apparatus and phosphatase activity and a gradual recovery of normal PAS reactions. Allen (1958) reported a significant increase in mitotic activity in the epithelium of 30-day castrates within 30 to 36 hours following a single injection of 16 /xg. of testosterone propionate; peak activity was reached in 42 to 48 hours.

Dorsal prostate. The epithelial cells resemble rather closely those of the coagulating gland but the cytoplasm is more granular, the centrally placed nuclei darker, and the Golgi apparatus in the apical cytoplasm (in close contact with the nucleus) is less dense than the Golgi networks in the coagulating gland (Horning, 1947). Histochemically, the distribution of phosphatase activities and PAS reaction in normal males, castrates, and castrates treated with testosterone propionate are similar to the findings in the coagulating gland (Bern, 1949a, 1951; Brandes and Bourne, 1954).

Coagulating gland {anterior prostate). The secretory cells are columnar and the nuclei are approximately midway between basement membrane and lumen. The cytoplasm is granular and the condensed Golgi apparatus is a flattened network oriented transversely in the most apical region of the cytoplasm (Horning, 1947).

Electron microscopic studies by Brandes

412

PHYSIOLOGY OF GONADS

TABLE 6.8

Summary of the effects of testicular androgen on the mouse prostate and coagulating glands

Normal Males

Castrated Males

General Characteristics

All lobes alveoli with folded

lucosa; secretion in luniina.

columnar epithelial cells

cytoplasm of epithelial cells granular or foamy

nuclei basal .

stroma of connective tissue and smooth muscle

Histochemical observations:

secretion in lumina PAS-positive

cytoplasm acid phosphatase-positive; sulfhydryl reaction

intracytoplasmic granules PAS-positive; near luminal border

Golgi region PAS-positive granules; strong acid phosphatase activity.

basement membrane PAS- and alkaline phosphatase-positive

Stroma PAS- and alkaline phosphatase-positive

Electron microscopic observations:

epithelial cells with microvilli

Golgi complex smooth surfaced membranes and vesicles

Iveolar size; loss of villi and bulk of

Reduction i

secretion

Reduction in cell size; pseudostratification .\ppears less dense Shrunken and pyknotic Fibromuscular increase

Almost completely negative

Phosphatase activity reduced

Almost completely negative

Almost completely negative

Activity retained; less intense

Activity retained in sheaths of smooth muscles

Cell size reduced

Specific Characteristics

Ventral lobes

Histochemical observations : secretion in lumina strong alkaline phosphatase activity cytoplasm alkaline pliosphatase-positive

Golgi loose networks in apical cytoplasm

Electron microscopic observations: cytoplasm; ergastoplasm with generally flattened cisternae.. Dorsal lobes Histochemical observations :

Golgi compact networks in apical cytoplasm

Coagulating glands (anterior prostate) Histochemical observations : Secretion in lumina intense protein reaction; PAS-positive;.

sulfhydryl reaction

cytoplasm high concentrations of RNA basally and apically

protein reactions, intense apically

sulfhydryl reaction, especially strong apically

Golgi condensed apical networks

Electron microscopic observations: cytoplasm extremely dilated ergastoplasmic cisternae

Reduced in amount; fragmented Cisternae collapsed; reduced granules

Retluced in amount; fragmented

Some PAS reaction retained

Sulfhydryl reaction lost

Markedly decreased

Greatly reduced

Reaction lost

Reduced in amount; fragmented

Cisternae collapsed; granules reduced

and Portela (1960b) show that these epithelial cells are characterized by an endoplasmic reticulum with greatly dilated cisternae. This dilation is more marked in the middle of the cell and in the basal region (Fig. 6.37) where the dilated cisternae appear as intercommunicating channels in which the cytoplasmic matrix forms isolated profiles or strands containing mitochondria and other organelles. The matrix is more abundant in the Golgi region and protrudes from the luminal margin of the cells as microprojections covered by tlu; cell membrane.

Alkaline phosphatase activity is localized in the stroma (Bern, 1949a, 1951 ; Brandes

and Bourne, 1954; Bern, Alfert and Blair, 1957) ; acid phosphatase activity (Brandes and Bourne, 1954) is found in the epithelium and is particularly strong in the Golgi zone. Brandes and Bourne (1954) and Bern, Alfert and Blair (1957) reported PAS-positive reactions in the epithelial cells in the Golgi region and apical cytoplasm, and intense reactions in luminal secretion, basement membrane, and stroma. Sulfhydryl and disulfide reactions are evident in luminal secretion, epithelium (especially in the a]ucal region), basement membrane, and fibromuscular tissue. The reactions are stronger than in the ventral prostate. Bern, Alfert and Blair (1957) found high con

ACCESSORY MAMMALIAN REPRODTTCTIVE GLANDS

4i;

cy

/Jf-!^

pc

N.pc

y

. .... ^

cy

\

m

..^cy

^"^^

^m

V*'

^

cy

IS

Fig. 6.37. Mouse coagulating gland, normal male. Electronmicrograph X 39,000. Caulfield's modification of Palade's osmic acid fixative. Basal portion of an epithelial cell; insert, details of basement membrane region: bm, basement membrane: ci, dilated cisternae: cm, plasma or cell membrane; cy, cytoplasmic matrix; ?n, mitochondrion: /;, nucleus. (From D. Brandes, unpublished.)

centrations of RNA basally and apically in the epithelial cells. Strong protein reactions are present in luminal secretion and apical regions of the cells.

The response to gonadectomy (Brandes and Bourne, 1954) includes reduction of alkaline and acid phosphatase activity within 4 days, PAS reactions by 8 days, and slight

fragmentation and loss of density of the Golgi apparatus by 12 to 14 days. Changes are more marked after longer periods of castration (Table 6.8), although Bern (1951) observed retention of stromal alkaline phosphatase for long periods. RNA concentrations in the cell (Bern, Alfert and Blair, 1957) are greatly decreased but some

414

PHYSIOLOGY OF GONADS

Fig. 6.38. Mouse seininul \esick\ norniMl male. Pliotomicrograpli preparation. (From E. Howard, Am. J. Anat., 65, 105-149, 1939.)

50. Houm-liematoxvlin

accunuilation remains in the apical region. The sulfhydryl reaction is partially lost luit the cells retain apical reactivity.

Testosterone propionate implanted subcutaneously 13 to 32 days after testis removal (Brandes and Bourne, 1954) rapidly restored the Golgi apparatus and enzyme activity to normal, and the PAS reaction returned gradually. Allen (1958) showed that the epithelium of 30-day castrates responds to a single injection of 16 fig. of testosterone propionate by an increase in mitotic activity within 30 to 36 hours.

Se7ninal vesicles. The epithelial cells in adult glands are colunniai- with basal nuclei and secretory granules surrounded by halos in the supranuclear cytoplasm (Fig. 6.35) ; the epithelium rests on a layer of smooth muscle and connective tissue stroma (Howard, 1939).

In electron micrographs (Deane and Porter, 1959), it can be seen that the surface membranes of secretory cells Ivdvv microvilli which extend into the lumen. The supranuclear region contains secretory granules enclosed in vesicles or cisternae, smaller membrane-bound vesicles, and parallel arrays of smooth Golgi membranes. Moderately distended ergastoplasmic cliannels with membranes studded with pi'csumed libonucleoprotein ])articles lorm comi)lex convolutions along the latci'al luai'gins and at the base of cells. The nucleus possesses clumped chromatin along the meml)rane (Figs. 6.39, 6.42, and 6.43). Fu

jita (1959) described essentially the same type of endoplasmic reticulum and the presence of microvilli and secretion granules. In addition, small granules in the Golgi region were interpreted as precursors of secretory granules.

Histochemical preparations (Table 6.7) show strong alkaline phosphatase activity in stromal elements (Atkinson, 1948; Bern. 1951 ) ; acid phosphatase activity is present in the apical or Golgi region of the epithelial cells (Deane and Dempsey, 1945). Secretory material and secretory granules in the lumen and cytoplasm are acidophilic and weakly PAS-positive, whereas the reticulum in the lamina propria is intensely PAS reactive (Fig. 6.44). Lateral margins and basal regions of the cells (Fig. 6.45) are moderately basophilic (Deane and Porter, 1959).

Following castration of adult mice, the secretory epithelium retrogresses with loss of secretion granules and reduction in cell heiglit and nuclear size. Effects of gonadectomy may be retarded and not uniform in all cells (Howard, 1939), but changes within 5 days have been rei:)orted for cell and nucleai- size (Martins and Rocha, 1 929 ) .

{electron mici'osco])ic and histochemical studies (Table ().7l reveal marked changes within a week aftei' gonad removal (Deane and Porter, 1959). Cell size is reduced, there are fewer secretory granules, ergastoplasmic channels are less distended and

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

415

Fig. 6.39. Mouse seminal vesicle, normal male. Electronmierograph X 4200; osmic acid fixation with sucrose. Epithelial cells showing basal and lateral ergastoplasmic channels and membranes, and supranuclear \-psicles containing .secretory granules. (From H. W. Deane and K. R. Porter, unpubli.shed.)

coin-oliitcd (Fig. 6.40). The relative number of riboniicleoprotein particles is somewhat leduced, secretory granules are less acidophilic, and the cytoplasm is only weakly basophilic (Fig. 6.45). Secretion granules were still visible by electron microscopy 10 days after castration but they were not visible at 25 days (Fujita, 1959) .

Atkinson (1948) found that alkaline phosphatase activity disappears almost completely from the stroma within 10 days, but Bern, Alfert and Blair (1957) observed retention in the fibromuscular tissue.

Martins and Rocha (1929) reported complete prevention of castration effects by injection of extracts of bull or goat testes. The epithelium of castrates responds readily to androgens. A single dose of 16 /x,g. of testosterone propionate in 30-day castrates resulted in increased mitotic activity beginning 30 to 36 hours after treatment and

reached a peak at 42 to 48 hours (Allen, 1958). Administration of testosterone to castrates completely restored the fine structure to normal (Fujita, 1959). Alkaline phosphatase activity in the stroma returned to normal within 10 days with testosterone propionate administration (Atkinson, 1948). The same hormone given to normal males for one week resulted in increased cell height, more abundant and acidophilic secretion and secretory granules, increased basophilia (Fig. 6.45), more distended and convoluted ergastoplasmic channels (Fig. 6.41), and a relative increase in ribonucleoprotein particles (Deane and Porter, 1959).

Discussion. The secretory cells in the epithelia of rat and mouse prostatic lobes and seminal vesicles have many histologic characteristics in common and some marked dissimilarities. In light microscopy with

416

PHYSIOLOGY OF GONADS

V\v, 6 40 Mouse seminal vesicle, 7-(lay castrate. Electronmicrograph X 4200; osmic acid fixation wjtli sucrose. Note the reduction in cell height, number of secretory granules, and contortion of the ergastoplasmic membranes. Arrow indicates microvilli. (From H. W. Deane and K. R. Porter, unpublished.)

routine fixation and stains, the most obvious differences are in cell height, position and staining intensity of the nuclei, presence or absence of secretory granules, and in such cytoplasmic characteristics as the supranuclear clear zone in the rat ventral prostate and the basal vesicular region in the coagulating gland. The Golgi apparatus varies in density, structure, and position in the apical cytoplasm. Studies on ultrastructure reveal striking differences in the degree of dilation and the disposition of the endoplasmic reticulum. In the rat ventral prostate the most dilated cisternae are in the supranuclear region; in the dorsal lobe, generally distended vesicles are disposed throughout the cytoplasm in both cell types; in the coagulating gland, there is extreme dilation of the sacs, particularly in the basal region. The flattened vesicles of the mouse ventral prostate are disposed at random; the coagulating gland, like that of the rat, shows greatly dilated cisternae, especially basally. Moderately distended ergastoplasmic channels in the basal and lateral regions of cells are characteristic of the mouse seminal vesicles.

Changes following castration are detect

able by light microscopy within 2 days in the rat seminal vesicle; 4 days in the ventral prostate; 10 days in the coagulating gland.

Harkin (1957a) suggested a correlation between distention of the sacs and secretory activity of the cells in the rat ventral prostate. Within 24 hours after gonadectomy, he observed dilation of the sacs, but within 2 days, collapse of the apical sacs was marked and by 4 days, there was general collapse of the vesicles in other regions of the endoplasmic reticulum. At this stage secretory activity of the cells was apparently reduced.

Brandes and Portela (1960a, b, c) discussed the relation of the cisternae to secretion in the mouse glands. They proposed that the extremely dilated cisternae of the coagulating glands contain secretory products which are released into the lumina of acini by some undetermined mechanism. They found no evidence that the Golgi complex is involved in the elaboration of secretory material in the cisternae, but histochemical findings suggest that it might take part in formation of secretory products that are not intracisternal. The ventral prostate

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

417

Fig. 6.41. Mouse seminal vesicle, intact male treated with testosterone propionate for 7 days. Electron micrograph X 4200; osmic acid fixation with sucrose. Note increase in cell height, abundance of secretory granules and contortion of the ergastoplasmic membranes. (From H. W. Deane and K. R. Porter, unpublished.)

is characterized by flattened vesicles. Brandes and Portela doubted that there is transport of secretory material to these cisternae and release from the intracisternal spaces into the acinar Imnen. They suggested an apocrine type of release involving extrusion of portions of the apical cytoplasm from the

free margin of cells. The possibility of implication of the Golgi apparatus in the production of secretory material was considered.

From a study of the rat, Brandes and Groth (1961) concluded that the dilated cisternae of coagulating glands, dorsolat

418

PHYSIOLOGY OF GONADS

Fig. 6.42. Moil. M - :,,:ii il , i -id, , nui n, il n, ,i , I i_ (,;(,) l-.l, ,ii,m micrograpli X 36,000: omiik- ;ici(l tixation; section liiat((l with iii.myl cicclatc to enhance the density of nucleoi)ro1(Mn.-^. Infianuclear region of an epithehal cell; nucleus at the top ; ergastopla.sniic channel.s with inomhianes studded with particles; arrow indicates a mitochondrion. (From H. W. Deane and K. R. Porter, unpublished.)

eral and ventral prostates contain secretory products, and that it is probable that the membranes of the endoplasmic reticulum (or the granules associated with them) play an active role in the syntheses of the proteins present in the glandular secretions. Attemi)ts have been made to correlate

structure of cells as observed by light and electron microscopy with histochemical localizations of mucoproteins (PAS reaction), alkaline and acid phosphatase activity, and basophilic material. Various interpretations have been offered for the functional significance of these substances, all of which

ACCESSORY MAMMALIAN REPRODUCTIVK GLANDS

419

are under control of andi'ogcnic hormones of testicular origin. Leblond (1950) stated that the presence of PAS-positive granules in the Golgi region supjiorts the concept of participation of the Golgi apparatus in the secretory process. Brandes and Portela (1960b) suggested that the vesicles and vacuoles in the Golgi zone might represent presecretory or secretory material. The possibility that collapse of ergastoplasmic sacs after gonadectomy might be correlated with reduction in PAS-positive secretory material was proposed by Harkin (1957a).

Alkaline and acid phosphatase activity is also found in the Golgi region, but the significance of this localization is not clear. Harkin (1957a) suggested that reduction in acid phosphatase activity following castration might be correlated with the decrease in numbers of mitochondria.

The histochemical pattern of enzyme activity has been discussed by Brandes and Bourne (1954), and the functional significance of distribution of epithelial and stromal alkaline phosphatase activity has been treated by Bern (1949a).

Cytoplasmic basophilia that was abolished by ribonuclease was demonstrated in the epithelial cells of the rat seminal vesicle (Melampy and Cavazos, 1953). In the mouse seminal vesicle, Deane and Porter (1959) found cytoplasmic basophilia (all of which was attributable to ribonucleic acid) localized in regions which corresponded to the distribution of ergastoplasmic membranes with their associated particles of presumed ribonucleoprotein. The relative number of particles was apparentlj^ reduced after one week of castration, and increased with testosterone propionate administration to normal males. These changes were not considered marked enough to account for the pronounced reduction in basophilia following gonadectomy, and the increase with androgenic hormone treatment of normal males.

Rixon and Whitfield (1959) found high concentrations of zinc, lipid, and basophilic material in a luminal border organelle in the lateral prostate of rats. Silver staining demonstrated fibrils, and it was suggested that zinc may be involved in the ergastoplasmic reticulum, possibly with lipopro

tein, and would be associated with the microsome fraction in homogenatcs.

In the discussion of changes in structure and histochemical localizations of substances after gonadectomy and with hormone administration, no specific mention was made of differences in response among the glands. There arc, however, pronounced differences in rate of regression following withdrawal of testicular hormone, and in rate and degree of response to administered androgen. These differences in hormone sensitivity or threshold have been established by such end points as changes in histologic structure, weight (which includes increase in mass of cells and accumulation and storage of secretion), and secretion of specific substances such as fructose and citric acid (Mann, 1954a). In order of sensitivity they are first, secretory function, second, histologic structure, and finally, weight, whichis frecjuently used as an end point (Dorfman and Shipley, 1956).

Responsiveness of the epithelial cells depends on many factors and varies with specific glands, age of the animal, genetic strain, and species. A few examples will illustrate these points. Following castration of adult rats the seminal vesicles retrogress more rapidly than the ventral prostate and recjuire higher doses of testosterone propionate to restore normal histological structure (Price, 1944a). The ability of the seminal vesicles and the ventral ])rostate in young rats to respond to testosterone propionate increases with age to a peak which is specific for the organ (Price and Ortiz, 1944; Price, 1947). This is true also for the female prostate (Price, 1944b) and the accessory glands in young male hamsters (Ortiz,' 1947). The effect of age on responsiveness in the mouse ventral prostate was studied by Lasnitzki ( 1955a ) who cultured glands from mice 4 to 6 weeks of age and 6 months old in normal control medium and in the presence of testosterone propionate. Young prostates regressed on the control medium but retained normal histologic structure when the hormone was added. In the control medium, older glands maintained normal structure and became hyperplastic with addition of the androgen. Franks (1959) also found differences re

420

PHYSIOLOGY OF GONADS

. (i 1.; .Mnii-i -(iiiiiiit Ill III ill - iiiir ~|icciiiii'ii. Ill, muilii Ml Kill, and preparation as Fig. 6.39). Suprunuclcur icgiuu of au epilht'lial c-ell ; nucleus at the hottoui; numerous membrane-bound vesicles with secretory granules in the larger vesicles; arrows indicate some of the parallel arrays of smooth-surfaced Golgi membranes. (From H. W. Deane and K. R. Porter, unjiuhlished.)

lated to age in the response of cultured mouse ventral prostate to testosterone propionate. The Long-Evans and SpragueDawlcy strains of rats differ in responsiveness of the ventral prostate of adult hypojihysectomized castrates to testosterone

propionate (Lostroh and Li, 1956). Species differences in rate of retrogression of accessory glands in adult castrates are marked. As reported above, changes in histologic structure occur rapidly in rats, more slowly and less uniformly in mice (Howard, 1939),

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

421

Fig. 6.44. Mouse seminal vesicle, normal male. Photomicrofirapli - 250. Carnoy's fixative, stained by the periodic acid-Schiff method, counterstained with hematoxylin. Note the intense PAS-reaction in the reticulum in the lamina propria and surrounding the smooth muscle fibers; secretory material in the lumen is moderately reactive. (From H. W. Deane and K. R. Porter, unpublished.)

ctncl slowly and somewhat incompletely in guinea pigs (Sayles, 1939, 1942) and hamsters (Ortiz, 1953). It should be noted that in adult castrated guinea pigs the ability to secrete fructose and citric acid is apparently lost in cells which show only partial retrogression histologically (Ortiz, Price, Williams-Ashman and Banks, 1956).

Another type of variation in responsiveness is demonstrated when weights of seminal vesicles and ventral prostrates in immature castrated rats are used as end points for the potency of various C19 steroids (Dorfman and Shipley, 1956 L When lower dosages of testosterone and 17aniethyl-A^-androstene-3^ , 17/?-diol are given the ventral prostate is more responsive than the seminal vesicles, but at high dosage levels the percentage increase in seminal vesi

cle weight equals or exceeds that of the prostate. Three other C19 steroids are far more effective on the ventral prostate than on seminal vesicles.

The female prostate in adult rats responds histologically and gravimetrically to a number of C19 steroids (Korenchevsky, 1937). These findings have been confirmed and extended by Huggins and Jensen

(1954) and Huggins, Parsons and Jensen

(1955) in hypophysectomized female rats. These workers examined the relation of molecular structure to the growth-promoting ability of the steroids.

Atrophy in male accessory glands of rats and mice has been reported under conditions of inanition and vitamin deficiency. These results are not usually attributable to reduction in responsiveness of the glands

422

PHYSIOLOGY OF GONADS

Fig. 6.45. Moil-^c .^einiiial mmcIi- l'li(iiniiiicinmn|ilis x 7UU. C'ain(>\-'- ti\ati\ c-nictliylcnie blue. Top, normal mali' ; in k Idle 7-ila\ ca-tiaN , iMJitoin, intact male ticatcil wil li ic.-tostcionp proprionate for 7 days. Hasoi)lidic inatciial ( ciga-tDplasm) occurs at the hasc and along lateral margins of cells; the Golgi zone appears clear; secretory granules are unstained. Basophilic material and Golgi zone are less evident after castration and more highly developed after testosterone treatment than normal. (From H. W. Deane and K. R. Porter, unjniblished.)

themselves Init to (liiuiniition in <2;onadotrophin titer by way of pituitary inhibition. Moore and Samuels (1931) showed that gonadotrophin or androgen treatment repaired the atrophied accessory glands in vitamin B-deficient rats and in those on limited food intake. Further, Lutwak-Mann and Mann (1950) demonstrated reduction of fructose and citric acid in accessory glands of rats on a vitamin B-deficient diet, but treatment with chorionic gonadotrophin not only restored the levels of these substances to normal but produced hypersecretion. Grayhack and Scott ( 1952 ) reported that the growth response to testosterone propionate of the ventral and posterior prostate in castrated rats on reduced food intake, vitaminfree casein, or glucose was little different from normally fed rats at lower dosages, but at higher levels there was less resj^onse in rats on limited dietarv intake. Testosterone

propionate did not produce normal stimulation of the accesisory glands in castrated mice on limited food intake (Goldsmith and Nigrelli, 1950) . In adult rats, a folic acid antagonist (Aminopterin) partially prevented the reduction in jirostatic weight produced by estradiol but did not interfere with testosterone stimulation of the prostate in castrated adults or intact immature animals (Brendler, 1949).

The senile changes which occur in adx'anced age in the prostate glands of the rat, mouse and of man have been describetl by Moore (1936) and interjireted on tht. basis of decrease in testicular androgen. Presenile variations in histologic structui'e are pr()l)al)ly ivhited to changes in responsiveness to andi'ogens. In a brief report on electron microscopy (Harkin, 1957b), involutional changes in the rat ventral prostate were described. With increasing age.

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

423

eloctron-denye material is deposited in the Golgi region of epithelial cells and these bodies are said to resemble structures found in hyperplastic prostates in man.

2. Adrenal Androgens

A large Ixxly of evidence jioints to effects of hormones from the adrenal cortex on the accessory reproductive glands of male rats and mice and on prostate glands of female rats. The significance of this relationshi}) is unknown and the effects are slight in many cases. Reviews by Parkes (1945), Ponse (1950), Courrier, Baclesse and Marois (1953), Moore (1953) and Delost (1956) deal extensively with the subject. In man, a relationship between pathologies of the adrenal cortex and virilism is well recognized (Dorfman and Shipley, 1956).

The marked development of the ventral prostate in young castrated rats (Price, 1936) was attributed by Howard (1938) to the action of androgen from the adrenal cortex. The same explanation was suggested for the extensive development of the seminal vesicles and prostate in young castrated mice (Howard, 1939). The ventral prostate does not develop in immature castrated-adrenalectoraized rats according to Burrill and Greene (1939a) and Howard (1941), but Gersh and Grollman (1939) did not confirm these findings. The impairment of prostate and seminal vesicle development in young castrated-adrenalectomized mice (Howard, 1946) was considered to be the result of poor physical condition rather than loss of adrenal androgen. Gonadectomy in young male mice of an inbred strain (Woolley and Little, 1945a. b) produced adrenal cortical carcinoma correlated w^ith strong stimulation of the prostate and seminal vesicles. Spiegel (1939) castrated young guinea pigs and found the development of adrenal-cortical tumors and evidence of stimulation of prostates and seminal vesicles.

In the field vole {Microtus arvalis P.), Delost (1956) observed extensive development of the ventral prostate in young castrated males. Gonadectomy of adult males during the breeding season results in atrophy of seminal vesicles, and dorsal and lateral prostate, whereas the ventral prostate

shows an intense secretory activity by one month after testis removal. Adrenalectomy of castrates produces complete involution of the ventral prostate. Outside the breeding period, there is atrophy of all accessory glands except the ventral prostate which exhibits strong activation that can be prevented by adrenalectomy.

The prostate gland of young female rats undergoes development and differentiation, and resembles the male ventral prostate with which it is homologous (Price, 1939; Mahoney, 1940). Development still occurs following ovariectomy (Burrill and Greene, 1939b; Price, 1942) or adrenalectomy (Burrill and Greene, 1941), but not in ovariectomized-adrenalectomized females. A comparison of the responsiveness of female and male prostates indicated that the male gland is more sensiti\'e to adrenal androgens (Price, 1942).

Autotransplants of adrenals into one seminal vesicle of adult castrated rats produced slight local stimulation of the gland and also androgenic effects on the other seminal vesicle and on the ventral prostate (Katsh, Gordon and Charipper, 1948). But androgenic action was local and barely discernible in somewhat similar experiments (.lost and Geloso, 1954). Price and Ingle (1957) autotransplanted adrenals into seminal vesicles and ventral prostates of adult castrates and observed definite but local stimulation of seminal vesicles, coagulating glands, and ventral prostates. Negative results of adrenal transplants in seminal vesicles of nonadrenalectomized rats were reported by Moore (1953). Takewaki (1954) failed to detect any androgenic effect of autotransplants of adrenals placed subcutaneously in contact with seminal vesicle grafts in castrated males.

The finding that treatment of young castrated male rats with adrenocorticotrophin caused stimulation of the ventral prostate (Davidson and Moon, 1936) has been confirmed by Deanesly (1960) who observed, in addition, a slight stimulation of the seminal vesicles. Nelson (1941) also found androgenic effects on accessory glands following ACTH treatment but Moore (1953), van der Laan (1953), and Takewaki (1954) obtained negative results. In hypophysec

424

PHYSIOLOGY OF GONADS

tomized-castrated rats, ACTH was reported ineffective in increasing ventral prostate weight (van der Laan, 1953; Grayhack, Bunce, Kearns and Scott, 1955) , but Lostroh and Li (1957) obtained some growth of ventral pi'ostates and seminal vesicles at certain dosage levels. They emphasized that dosage is a critical factor in demonstrating the androgen-secreting ability of the adrenal cortex under ACTH stimulation. Administration of ACTH to hypophysectomized-castrated-adrenalectomized rats does not affect the accessory glands.

There has been no general agreement on the androgenicity of desoxycorticosterone on the accessory glands (see reviews by Parkes, 1945 and by Courrier, Baclesse and Marois, 1953). Lostroh and Li (1957) reported that ll-desoxy-17-hydroxy-corticosterone and 11-dehydro-corticosterone displayed an androgenic activity equivalent to 4 fxg. of testosterone propionate on the ventral prostates and seminal vesicles of hypophysectomized-castrated adult rats. Corticosterone, cortisone, and hydrocortisone were ineffective. Grayhack, Bunce, Kearns and Scott (1955) found cortisone ineffective on the weight of ventral prostates in hypophysectomized castrates. In the field vole (Microtus arvalis P.), Delost (1956) produced effects on the ventral prostate by cortisone administration.

3. Ovarian Androgens

It has long been known that mammalian ovaries can secrete androgenic hormones which have virilizing effects in females. Discussion of the evidence for androgenic activity of the ovary has been presented by Ponse (1948) and Parkes (1950). More recently the subject has been extensively reviewed (Ponse, 19541), 1955). Much of the interest in ovarian androgens in the rodent has centered on the question of their site of origin in the ovary and the effects of temperature and gonadotrophin administration on androgen production (see reviews, and Chapter 7 by Young) . However, the use of male accessory glands as bio-indicators for ovarian androgen has contributed to our knowledge of the responsiveness of the glands.

Methods for approaching this problem include transplantation of ovaries into vai'i

ous sites in castrated male rats and mice; ])lacing ovarian autotransplants into the ears of females ; transplantation of male accessory glands into females; and observations of prostate glands in females of the so-called female prostate strains of rats. The use of such females as hosts for grafts of male prostatic tissue has permitted a direct comparison of responsiveness in these homologous glands.

The first observations that ovarian grafts maintain normal prostates and seminal vesicles in castrated males w^ere made in guinea pigs (Lipschiitz, 1932) and mice (de Jongh and Korteweg, 1935). Hill (1937) transplanted ovaries into the ears of castrated male mice and obtained stimulation of the prostate and seminal vesicles. Deanesly ( 1938) reported similar findings in rats. Local effects from ovaries grafted into seminal vesicles of castrated rats w^ere shown by Katsh (1950). Takewaki (1953) also found local stimulating effects on seminal vesicles when rat ovaries and seminal vesicles were transplanted close together into the spleens of gonadectomized males and females.

In experiments in which ventral prostates and seminal vesicles were transplanted subcutaneously into adult female rats (Price, 1941, 1942) it was sliown that the ventral prostate is well maintained in virgin females (Fig. 6.46) and highly stimulated during jiregnancy and lactation in the host. It is comi)letely retrogressed in spayed females. Seminal vesicle grafts, however, are stimulated only rarely. This occurs only in females that have littered repeatedly and have been lactating for long periods. This indicates that the threshold of response of the ventral prostate to ovarian androgens is lower than that of the seminal vesicles. Evidence of functional stimulation with production of fructose and/or citric acid was obtained in coagulating glands, and ventral, lateral, and dorsal prostates trans])lanted into female rats in which the ovaries were stimulated by gonadotrophin treatment (Price, Mann and LutwakMann, 1955). It may be assumed that the effects were attributable to ovarian androgens since ventral prostate grafts in spayed females (Greene and Burrill, 1939) are not stimulated histologicallv when gonado

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Fig. 6.46. Rat male and female prostates. Photomicrographs X 650. Bouin-hematoxylin preparation. Top, host prostate from an adult virgin female; middle, male prostate graft from the above female host; bottom, prostate from a pregnant female. Note the semi-regressed epithelium in the female prostate of the virgin female host compared with the columnar epithelium and light areas in the male prostate graft and the prostate from a pregnant female. (From D. Price, Anat. Rec, 82, 93-113, 1942.)

trophin is administered. Great stimulation of ventral prostate grafts in the ovarian bursa of females was obtained by Ponse (1954a).

The female prostate gland is normally jiartially retrogressed in adult females except during pregnancy and lactation (Fig. 6.46) when it appears stimulated (Burrill and Greene, 1942; Price, 1942); in spayed females it is atrophic (Price, 1942). The striking development of the female prostate in pregnancy and lactation in a number of species of mammals is discussed in Section I. Hernandez (1942) obtained stimulation of female prostates by autotransplants of oA-aries into ears, hind legs, or tails of rats.

Transplantation of rat ventral prostates into virgin females shows that the male

prostate has a lower threshold to ovarian androgens than the female gland and maintains high epithelium and cellular light areas whereas the epithelium of the host prostate (Fig. 6.46) is low and retrogressed (Price, 1942).

4- Progesterone

The administration of progesterone in relatively enormous doses has stimulating effects as determined by weight, histologic structure, and function of some of the accessory glands in castrated male rats, mice, and guinea pigs. The literature has been reviewed by Greene, Burrill and Thomson (1940), Parkes (1950), and Price, Mann and Lutwak-Alann (1955).

Burkhart (1942) treated adult 40-day

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PHYSIOLOGY OF GONADS

castrated rats with one or two 20-mg. doses of progesterone and observed a slight stimulation of mitotic activity in the ventral prostate and seminal vesicles after 55 hours but a pronounced hypertrophy of epithelium and connective tissue in both glands. The ventral prostate is more sensitive to progesterone than the seminal vesicles.

In castrated rats (Price, IMann and Lutwak-Mann, 1955), treatment with 25 mg. of progesterone daily stimulated the secretion of fructose or citric acid in seminal vesicles, coagulating glands, and ventral, lateral and dorsal prostates, and produced histologic changes in the last three glands. The effect, however, was only equivalent to that of about 5 /xg. of testosterone propionate. The lowest threshold to the hormone is in the ventral prostate and the highest in the seminal vesicles. Lostroh and Li (1957) found no effects of 0.5 mg. of progesterone daily on the ventral prostate and seminal vesicles of hypophysectomizedcastrated adult rats, but the same dose of 17a-hydroxy progesterone was the androgenic equivalent of 4 /xg. of testosterone propionate. It may be noted that the following transformations are involved in the biosynthesis of testicular androgens: cholesterol —> pregnenolone — > progesterone — > 17a-hyclroxy progesterone — > androst-4-ene3,17-dione -^ testosterone (Dorfman, 1957). The slight androgenic actions of progesterone and 17a-hydroxy progesterone may result from their conversion to androstane derivatives by extragonadal tissues. The findings of Katsh (1950) that progesterone crystals implanted directly into the seminal vesicles of castrated rats have no stimulating influence may be significant in this regard.

C. EFFECTS OF ESTROGENS

Administration of estrogenic hormones to normal males affects the accessory glands both indirectly and directly. The effects fall into three categories: inhibition as evidenced by weight changes, involution of the epithelium, and loss of secretory activity (attributable to inhibition of pituitary gonadotro])hin and reduction in endogenous androgen) ; direct stimulation of fibromuscular tissue; and stimulation of hyi)erplasia and stratified squamous metaplasia of the

ei)ithelium with possible keratinization. In no case does estrogen induce secretory activity of epithelial cells. The reduction in seci'etion as determined cjuantitatively (see Section II) may result from castration atrophy of secretory cells, or from hyperplastic and metaplastic transformations of the e])ithelium witli resultant loss of normal secretory function.

The observed responses to estrogen treatment in glands of intact and castrated males, and in organ cultures of prostatic tissue, represent the dual effects of androgen withdrawal and estrogen addition. The extensive literature on the effects of estrogen and the evidence for so-called antagonistic, cooperative and synergistic effects of simultaneous administration of androgen and estrogen have been discussed extensivelv (Zuckerman, 1940; Emmens and Parkes, 1947; Ponse, 1948; Bern, 1949b; Burrows, 1949).

The observation that administration of estrogen to intact male rats causes atrophjof the accessory glands which is mediated by way of reduction of pituitary gonadotrophin and failure of secretion of testicular hormones was made by Moore and Price (1932). Estrogen-induced atrophy was prevented by simultaneous treatment with gonadotrophin or androgen. Direct stimulating effects were reported by Freud

(1933) and David, Freud and de Jongh

(1934) who observed fibromuscular growth in seminal vesicles of estrogen-treated castrated rats and stratification in the duct epithelium of the lateral prostate. Simultaneous treatment with androgen enhanced the hypertrophic effect of estrogen on the fibromuscular wall of the seminal vesicle but prevented epithelial change in the lateral prostate ducts. Korenchevsky and Dennison (1935) found estrogen stimulation of the muscular layer of the rat seminal vesicle with no effect on the epithelium, but in coagulating glands (and to a lesser degree in the dorsal prostate ) there was not only fibromuscular hypertrophy but also metaplastic transformation of the epithelium with stratification; changes in the ventral and lateral lobes were slight and the epitiieHum was unaffected. Androgen treatment prevented the jxithologic changes induced by estrogen. Harsh, Overholser and Wells

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(1939) noted stratified, sqiuinious epithelium in the ducts of the seminal vesicles, and ducts and acini of coagulating glands following estrogen administration. But a slight delay in castration atrophy and a weak stimulating effect of estrogen on seminal vesicle epithelium were observed by Overholser and Nelson ( 1935 ) and Lacassagne and Raynaud (1937).

Ovaries transplanted into castrated male rats (Pfeiffer, 1936) induce fibromuscular hyi^ertrophy in host seminal vesicles and coagulating glands; stratified sciuamous cornified epithelium appears also in coagulating glands, and hyperplasia and metal^lasia are present in lateral prostates. Estrogenic stimulation of fibromuscular tissue occurs (Price, 1941) in seminal vesicle grafts in normal female hosts but no such effects are evident in ventral prostate grafts.

Burkhart (1942) injected a single dose of estradiol benzoate into 40-day-castrated rats and observed no effect on the ventral prostate. But in the seminal vesicles, hypertrophy of epithelial cells occurred by 27 liours after treatment and by 55 hours, mitotic activity was evident in the epithelium and to some extent in the connective tissue.

In histochemical studies (Bern and Levy, 1952) , metaplastic changes were observed in the seminal vesicle epithelium after estrogen treatment but no cornification occurred; the replacing epithelium was alkaline phosphatase-positive in contrast to the negative reaction in the original epithelium (Table 6.7). Fibromuscular hypertrophy was found but no definite alteration in enzyme concentrations except an absence of activity in edema of the subepithelial stroma. No metaplastic changes appeared in the coagulating gland epithelium, but the ducts of the dorsal prostate underwent metaplasia; alkaline phosphatase activity of the stroma in both glands was retained as in the castrate. The ventral prostate ejnthelium was atrophic but still enzyme-active after 120 days of treatment and the stroma reacted positively.

The effects of estrogen on the accessory glands of mice are far more marked than in rats. Long continued and strong doses of ■estrogen cause hyperplasia, metaplasia and

keratinization in the epithelium of mouse coagulating glands (Lacassagne, 1933 ) . The same effects, with fibromuscular hypertrophy, were described in coagulating glands and prostates by Burrows and Kennaway (1934), Burrows (1935a), and de Jongh (1935) who prevented epithelial metaplasia in prostates by simultaneous treatment with androgen. Burrows (1935b) studied the localization of responses to estrogenic compounds and found that in order of time of response, the coagulating gland is first, seminal vesicles next, and finally the prostatic lobes. Changes begin in the urethral ends of ducts and jirogress peripherally into the acini. Li the degree of response, the coagulating glands and seminal vesicles show the most drastic changes with the appearance of stratified, squamous, keratinizing epithelium and ultimate loss of acini. The effects on the lobes of the prostate include stratified, cornifying epithelium but the changes are not so i)ronounced. Some hypertroj^hy of fibromuscular stroma occurs in all the glands and hyperplasia is marked in the fibromuscular wall of the seminal vesicles.

Tislowitz (1939) found stimulation of mitotic activity in muscle and connective tissue of seminal vesicles and ventral prostate glands of immature castrated mice treated with estrogen. Stratification and cornification appear in the ventral prostate epithelium, with mitoses in the basal cell layers and also in seminal vesicle epithelium. Allen (1956) compared the mitogenic activity of a single dose of 16 /xg. of estradiol benzoate on seminal vesicles, coagulating glands, and ventral prostates of 30-daycastrated mice. Significant increases in mitotic activity occur in seminal vesicles and coagulating glands about 24 hours after treatment; the ventral prostate does not respond significantly until 72 hours and gives a low absolute value of mitoses.

Horning (1947) studied some of the initial changes in prostatic epithelium of intact mice receiving estrogen. Slight hypertrophy of epithelial cells and extensive fragmentation and dispersal of hypertrophied portions of the Golgi network occur by 8 days in the coagulating gland. At the same period, hypertrophic changes are less pronounced in the ejuthelium of the dorsal

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PHYSIOLOGY OF GONADS

prostate and there is only slight fragmentation of the Golgi apparatus. In the ventral prostate no epithelial hypertrophy is found but the Golgi network hypertrophies without fragmentation or dispersal. The ventral prostate is definitely less sensitive to estrogen than the other two glands.

After longer periods of estrogen administration, Bern (1951) observed fibromuscular hypertrophy of the seminal vesicle and intense alkaline phosphatase activity as in untreated intact males (Table 6.7) ; the epithelium, which is normally negative in enzyme activity, becomes positive and the beginning of metaplastic changes is occasionally visible. In the coagulating gland, stratified scjuamous metaplasia with masses of keratin is found and the metaplastic epithelium is strongly alkaline phosphatasepositive; enzyme activity is retained in the stroma but is variable. Bern, Alfert and Blair (1957) reported that the metaplastic coagulating gland epithelium is strongly alkaline phosphatase reactive, virtually PAS-negative, has dense homogeneous RNA concentrations decreasing in amount from base to lumen, and a dense homogeneous cytoplasmic protein reaction with a gradient of increasing intensity from base to lumen. The enlarged vesicular nuclei of the metaplastic epithelium have lower concentrations of deoxyribonucleic acid (DNA) than the nuclei of normal epithelial cells. The cytoplasm of these cells is as reactive as normal cells for sulfhydryl groups, and the newly formed keratin is intensely reactive; the greatest concentrations of disulfide groups are in superficial keratin.

In the dorsal prostate (Bern, 1951), estrogen causes fibromuscular hypertrophy with variable retention of alkaline phosi)hatase activity. Metaplastic changes involving basal cell proliferation and stratification begin and the metaplastic epithelium is intensely alkaline phosphatase active, a rvversal of the normal reaction.

Brandes and Bourne (1954), using diethylstilbestrol, observed an increase in fibromuscular stroma in coagulating glands. dorsal and ventral prostates, and epithelial hyperplasia and stratification in varying degrees. The most pronounced changes occurred in the coagulating gland. The effects of estrogen on Golgi networks, and on PAS

and acid and alkaline phosphatase reactions are in general similar to the results of castration (Table 6.8).

Ventral prostate glands have been grown in culture by the watch glass method, with estrogens added to the medium. Lasnitzki (1954, 1958) reported hyperplasia and squamous metaplasia of the epithelium in young i^rostate tissue from C3H mice. In older glands, stimulation of fibromuscular tissue occurred. Franks (1959) using the C57 strain and a different culture medium observed no epithelial hyperplasia and metaplasia, but obtained increases in stroma and muscle. He attributed atrophic changes in the epithelium, which appear more marked in estrogen-treated than in control cultures, to direct inhibition by the hormone. Ventral prostate tissue from young mice is more sensitive to estrogen than tissue from adult or old males.

In the dog, Huggins and his collaborators demonstrated the effects of estrogen and combinations of estrogen and androgen on histologic structure and secretion in the prostate (see Section II). Estrogen causes decrease or increase in prostatic size depending on the dosage and on the levels of endogenous or exogenous androgen (Huggins and Clark, 1940) . Sciuamous metaplasia of the epithelium of ducts and acini occurs with estrogen treatment, but only in the posterior lobe.

Discussion. The results of estrogen administration to rats and mice vary with species, age of animal, specific gland under consideration, dosage, duration of treatment, and presence or absence of endogenous or exogenous androgen. Interpretation of the findings rests on the understanding that androgen directly stimulates mitotic and secretory activity in the epithelial cells. Estrogen inhibits i)ituitary function and thus i-eduees testicular androgen in intact males. It directly increases mitotic activity in th(> epithelium of the accessory glands, and inckices (>pithelial liy|)erplasia and iiietaphisia, and fibromuscular hyperplasia. Whether the effects of simultaneous presence of androgen with exogenous estrogen are classified as protective, competitive (antagonistic), or cooperative (synergistic) on the acce.ssoiy glands depends on the

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relative levels of the two hormones. Both affect mitotic activity directly.

In a comparison of the effectiveness of androgen and estrogen on mitotic activity, Allen (1956, 1958) showed that a dose of 16 fjig. of testosterone propionate induces statistically significant increases in mitotic activity of the epithelium of seminal vesicles, coagulating glands and ventral prostates of castrated mice in 30 to 36 hours. The same dose of estradiol benzoate increases mitotic activity in 24 hours in seminal vesicles and coagulating glands, but not until 72 hours in ventral prostates.

Differences in responsiveness to estrogen are evident between rats and mice but in both species there is a gradient of reactivity with coagulating glands showing the most marked changes, seminal vesicles next, and prostatic lobes least. In glands of both species, the duct epithelium is more sensitive than acinar epithelium and the first observable effects are on urethral ends of ducts. Hyperplastic and metaplastic responses to estrogen occur also to varying degrees in accessory glands of other mammals — man, monkey, dog, cat, ground squirrel, and guinea pig. Zuckerman (1940) reviewed the effects of estrogen in male and female rodents and other mammals and suggested from the evidence that "stratified squamous proliferation or metaplasia is usually a primary response of tissue in whose development oestrogen-sensitive entodermal sinus epithelium has played a part."

On the basis of the pathologic effects of estrogen on the mouse coagulating glands and the protective action of androgen, several workers originally suggested that benign prostatic hypertrophy in man might result from a primary imbalance in the normal ratio of estrogenic to androgenic hormones in the male organism (Zuckerman, 1936). Further study has not supported this concept.

D. HORMONAL CONTROL OF SPONTANEOUS PROSTATIC NEOPLASMS

Spontaneous tumors of the prostate occur in rodents rarely if at all, but benign growths are extremely common in aging dogs and men, and prostatic cancer is a major prol^lem in man. It is noteworthy that

neoplasms of the seminal vesicles in man are rare (Dixon and Moore, 1952).

1. Benign Growths

In the dog, prostatic enlargement which is essentially due to cystic hyperplasia of the epithelium occurs in almost all senile males with functioning testes, but is not found in castrates (Huggins, 1947b). In these prostatic growths, which characteristically involve the entire gland, tall columnar secretory epithelium is always present in some acini. Canine prostatic hyperplasia is under control of testicular androgens (Huggins and Clark, 1940) and marked involution of these tumors as evidenced by their size and secretory activity (see Section II) can be induced by gonadectomy or treatment with suitable dosages of estrogen. Estrogen overdosage, however, causes prostatic enlargement and a metaplasia of the posterior lobe which does not resemble cystic hyperplasia. Huggins and Moulder (1945) reported that dogs feminized by estrogen-secreting Sertoli cell tumors of the testis do not have cystic hyperplasia. The important factors in this pathologic growth seem to be age and testicular androgens (Huggins, 1947b), but prolonged administration of testosterone propionate to aged castrate dogs results in normal-appearing prostates and not cystic hyperplasia.

Benign prostatic hypertrophy in man is rarely encountered before the age of 40 (Moore, 1943; Huggins, 1947b) but it is extremely common in old men. It differs markedly from prostatic hyperplasia in dogs; the lesions are limited to the medullary region of the prostate and are spheroidal neoplastic nodules involving, usually, both epithelium and fibromuscular tissue; other nodular types occur but are less frequent (Huggins, 1947b; Franks, 1954). The prostatic epithelium is composed of tall secretory cells (Huggins and Stevens, 1940). Despite the fact that castration may be followed by some shrinkage of hypertrophied human prostate tissue (White, 1893; Cabot, 1896; Huggins and Stevens, 1940), it is generally admitted that this treatment is of little value. Estrogen treatment results in changes in the acini of the inner or medullary (periurethral) part of the prostate, and stratification with squamous metaplasia

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PHYSIOLOGY OF GONADS

of the duct epithelium, but there is little effect on nodular stroma and acini (Huggins, 1947bj. Since benign prostatic hypertrophy has not been observed in men castrated early in life, testicular androgen is presumably involved in its etiology (Huggins, 1947b). However, it is doubtful whether androgens are causative agents for this disease. Lesser, Vose and Dixey (1955) found that in men over the age of 45 who had received androgen treatment for noncancerous conditions, the incidence of benign enlargement of the prostate was no greater than in untreated controls.

2. Prostatic Cancer

Prostatic cancer is a common disease in elderly men. This carcinoma, which characteristically arises in the posterior (outer) region of the prostate, consists of an abnormal growth of cells resembling adult prostatic epithelium rather than undifferentiated tissue (Huggins, Stevens and Hodges, 1941 ). It was found that these neoplasms are hormone-dependent and usually are influenced by anti-androgenic therapy; those which fail to respond are not adenocarcinomas with acini present in the tumor, but are undifferentiated carcinomas with solid masses of malignant cells (Huggins, 1942). However, the two types intergrade and both contain large amounts of acid phosphatase and are considered cancers of adult prostatic epithelium. The beneficial effects of castration or estrogenic treatment or both simultaneously, on metastatic carcinoma of the prostate in man were first demonstrated by Huggins and his collaborators (Huggins and Hodges, 1941 ; Huggins, Stevens and Hodges, 1941 ; Huggins, Scott and Hodges, 1941 ; Huggins, 1943, 1947a). This discovery was facilitated by the availability of a chemical index of the activity of the neoplasm, namely, the acid phosphatase activity of blood serum.

Although testosterone increases the level of serum acid phosphatase in patients with prostatic cancer (Huggins and Hodges, 1941 ; Sullivan, Gutman and Gutman, 1942 1 , androgen treatment does not always exacerbate the growth of the tumor (Trunnel and Duffy, 1950; Brendler, Chase and Scott, 1950; Brendler, 1956; Franks, 1958) or may even decrease it (Pearson, 1957). It is ciues

tionable if androgens induce prostatic cancer, inasmuch as their prolonged administration neither increases the incidence of this disease in man (Lesser, Vose and Dixey, 1955) nor induces it in dogs (Hertz, 1951).

The response of human metastasizing prostate cancer to anti-androgenic therapy is often very dramatic, but neither castration nor treatment with estrogens cures this disease. The tumor may regress for considerable periods of time, but eventually it recurs and begins to grow again. A small proportion of cases do not benefit at all (Huggins, 1957; Franks, 1958). Nevertheless, castration and/or estrogen therapy remain the best treatment for prostatic carcinoma in man (Nesbit and Baum, 1950; Huggins, 1956; O'Conor, Desautels, Pryor, Munson and Harrison, 1959).

Huggins and Scott (1945) suggested that the failure of some patients with prostatic cancer to obtain long lasting improvement from castration or estrogen treatment, or the two combined, lay in the secretion of androgenic substances by the adrenal glands. Early attempts to study the effect of bilateral adrenalectomy on human prostatic cancer were thwarted by the lack of suitable adrenal cortical steroids for adequate substitution therapy. But with the advent of cortisone, bilateral adrenalectomy could be accomplished with ease (Huggins and Bergenstal, 1951, 1952). It seems, however, that adrenalectomy is of limited value to patients with prostatic cancer in relapse after orchiectomy and/or treatment with estrogens (Whitmore, Randall, Pearson and West, 1954; Huggins, 1956; Fergusson, 1958).

E. EFFECTS OF CERTAIN AROMATIC HYDROCARBONS (CARCINOGENS)

Spontaneous tumors have not been found in the prostate glands of rodents, but tumors can be induced in rats and mice by treatment with carcinogenic chemicals such as benzpyrene and metiiylcholanthrene. There lias been considerable interest in inducing such tvnnois and studying their inception and growth, and the iH'lation of steroid hormones to their de^•elopment. Such investigations have contributed to an understanding of early neoi)lastic changes in the rodent prostate, but have had limited applicability

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to the })rol)lcm of hormonal control of prostatic cancer in man.

The first induction of prostatic cancer in rodents was accomplished by Moore and Melchionna (1937) who injected benzpyrene in lard directly into the rat anterior prostate (it should be noted that Moore and ]\Ielchionna used "anterior" in the sense of ventral as indicated by their histologic descriptions of a characteristic clear zone in the peripheral cytoplasm of the epithelial cells; this is typical only of the ventral lobe). The treatment was followed within 210 days by the development of squamous cell carcinomas in 72 per cent, and sarcomas in 5 per cent, of intact rats. Essentially similar results were obtained in an equivalent number of rats castrated at the time of carcinogen injection. Castration after tumors had developed did not cause atrophy of tumor cells. No metastases were found but there was anaplasia of cells and the tumors were invasive. The squamous metaplasia occurred in columnar secretory epithelium which was close to, or in contact with, benzpyrene cysts. The sequence of changes was reduction in cell height, loss of the clear area in the peripheral cytoplasm, pseudostratification, true stratification, development of intercellular bridges, and formation of keratohyaline. It was concluded that testicular androgen is not an important factor in the development of these squamous cell carcinomas, but on the basis of a small series of experimental animals it was suggested that exogenous androgen treatment in castrates may increase the incidence of sarcomas.

In 1946, Dunning, Curtis and Segaloff implanted compressed methylcholanthrene l)ellets into rat prostates (lobe not specified) and induced metastasizing squamous cell carcinomas. The tumors were transplantable and metastasized equally well in male and female hosts. Bern and Levy (1952) injected methylcholanthrene in lard into ventral prostates of intact Long-Evans rats and induced extensive neoplasms within 7 to 9 months. All but one were squamous cell carcinomas; the exception was a sarcoma. Quantitative determinations of enzyme activity showed a loss of alkaline phosphatase in cancerous prostates but no significant changes in acid phosphatase activity. Histo

chemically, the stroma and capillaries were alkaline phosphatase reactive, but the carcinomas had virtually lost the strong alkaline phosphatase activity of the epithelium of origin (Table 6.2). There was some pseudoreaction or reaction in sloughed keratin and necrotic areas.

Allen (1953) injected a suspension of methylcholanthrene in distilled water into ventral prostates or coagulating glands of intact and castrated rats. All were autopsied 180 days later. A high percentage of squamous cell carcinomas and a few sarcomas developed; metastases occurred in a few cases. There was no statistically significant difference between tumor incidence in the ventral prostate and coagulating gland. Tumors of the ventral prostate were found in 70.6 per cent of the intact rats and in 100 per cent of the castrates; in castrates injected with testosterone propionate there were tumors in 57.7 per cent of the animals, and in castrates treated with estradiol benzoate, 77.8 per cent. It was concluded that tumor incidence was highest in castrates and lowest in intact males or castrates treated with testosterone propionate, and that estrogen did not affect tumor incidence. ]\Iirand and Staubitz (1956) placed methylcholanthrene crystals in ventral prostates of 99 intact Wistar rats and observed the effects for over 300 days. The resulting tumors were classified as 30 squamous cell carcinomas, 3 leiomyosarcomas, and 2 adenocarcinomas; squamous cell carcinomas and adenocarcinomas metastasized. Fragments of squamous cell carcinomas were transplanted and survived and metastasized more successfully in males than in females.

Horning (1946) imjiregnated strips of tissue from mouse dorsal prostates and anterior prostates (coagulating glands) with crystals of methylcholanthrene and inserted them as subcutaneous homografts into intact males. By this method adenocarcinomas were induced in grafts of both dorsal prostate and coagulating gland.

In mice of the RIII and Strong A strains (Horning and Dmochowski, 1947) methylcholanthrene in lard was injected into dorsal and anterior prostates (coagulating glands). Squamous cell carcinomas and sarcomas developed in Strong A mice, but only sarcomas in RIII. Squamous metaplasia of

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PHYSIOLOGY OF GONADS

the epithelium occurred in the RIII strain but no malignant proliferation of metaplastic cells followed. It was noted that the epithelial changes which occurred with raethylcholanthrene treatment were "almost identical" with the secjuence of changes following prolonged estrogen administration in Strong A mice (Horning, 1947).

Horning (1949, 1952) studied the effects of castration, diethylstilbestrol, and testosterone propionate on growth rates of prostatic tumors transplanted as grafts. Tumors were induced in ventral prostate, dorsal prostate, and coagulating gland tissue of Strong A mice by wrapping pieces of epithelium around crystals of methylcholanthrene and transplanting the grafts su!)cutaneously into 75 intact males. Of the 54 tumors which developed, 42 were adenocarcinomas or secreting glandular carcinomas, 10 were squamous cell carcinomas, and 2, spindle cell sarcomas. Neoplastic development began, apparently, in epithelium in a nonsecretory phase, and hyperplastic changes followed the sequence of mitosis, abnormal cell division, and pyknosis accompanied by an increase in fibromuscular tissue. Three distinct types of epithelial proliferation then occurred; one, with tonguelike groups of early malignant cells, gave rise to secretory glandular carcinomas; the second, from acinar ejMthelium, and the third, from duct epithelium, developed into squamous cell carcinomas with keratinization and formation of keratin pearls. Some grafts had foci of the first and third type and the evidence suggested that the tumors subsequently became squamous cell carcinomas. Both tumor types were transplantable and were cari'icd through many serial transi)lantations without losing their histologic characteristics.

In an effort to study effects of testicular androgen on growth, transplants of an adenocarcinoma were made into intact and castrated males. The tumors grew rapidly and progressively in intact mice but regressed in castrates. Testosterone propionate administration to castrated males bearing regressed tumors resulted in a resumption of tumor growth in some cases. Gonadectomy of the host had little effect on the growth of transplanted s(|uamous cell carcinomas. An

drogen-dependence of secreting glandular carcinomas was suggested.

When stilbestrol pellets were implanted into one flank of intact males and a glandular carcinoma into the opposite flank, the effects varied from slight to pronounced retardation of the tumor but complete regression did not occur. The squamous cell carcinomas were insensitive to stilbestrol.

Additional experiments (Horning, 1952) involved transplanting pieces of prostatic epithelium impregnated with methylcholanthrene alone, or with the carcinogen combined with stilbestrol or testosterone propionate into intact males (groups of 35 for each treatment). The carcinogen alone induced 8 adenocarcinomas and 5 squamous cell carcinomas; carcinogen and stilbestrol, 23 squamous cell carcinomas and 3 sarcomas; carcinogen and testosterone propionate, 2 squamous cell carcinomas and 1 sarcoma. The increased tumor incidence with estradiol was interpreted as an inhibitory action of the estrogen on secretory epithelial cells, making them more susceptible to methylcholanthrenc.

Brandes and Bourne (1954) made homografts of pieces of ventral and anterior prostate (coagulating gland) impregnated with methylcholanthrenc into intact males of the Strong A strain, and studied histochemical changes. The grafts underwent squamous metaplasia and the processes of epithelial proliferation, stratification, and keratinization were completed within 10 days in some cases. Histochemical changes from the normal i)attern (Table 6.8) occurred concurrently. Alkaline phosphatase activity disappeared early; acid phosphatase activity became weak in nuclei and cytoplasm but keratohyalin granules were strongly reactive; PAS-positive reactions were gradually lost in luminal secretion and intracytoplasmic granules but retained in the basement membrane. In some grafts there was transformation into squamous cell carcinomas and when this happened phosjihatase activity was lost but the basement membranes were still PAS-positive.

Lasnitzki (1951, 1954, 1955a. b, 1958) grew ventral jirostate glands from C3H and Strong A mice in culture by the watch-glass t('chnif|ue and added methylcholanthrenc to the medium. Hyperplasia and squamous metaplasia resulted in glands ex]ilanted

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from both young and older mice. When estrone and carcinogen were added simultaneously to cultures of young glands, squamous metaplasia was increased; with older glands, hyperplasia was inhibited and stromal increase occurred. The relation of vitamin A to the response of prostates to methylcholanthrene was studied (Lasnitzki. 1955b). Vitamin A added to the medium caused an increase in secretion and deposition of PAS-positive material in the secretory cells but did not influence growth or development; the vitamin added simultaneously with methylcholanthrene did not influence hyperplasia, but did prevent keratin formation and degenerative changes in the secretory epithelium; excess vitamin A following the carcinogen prevented formation of keratin and decreased hyperplasia.

Summary. Treatment of rat and mouse accessory glands with benzpyrene or methylcholanthrene has induced precancerous and cancerous changes which led to the development of adenocarcinomas, squamous cell carcinomas, and sarcomas. The first type of tumor has been induced in large numbers only in mice and by the homograft method.

The evidence suggests that, in mice, growth of adenocarcinomas is androgendependent, but squamous cell carcinomas are little affected by androgen loss or estrogen treatment. The incidence of tumors has been increased by simultaneous administration of estrogen and carcinogen but reduced by the administration of androgen with carcinogen. In rats, it has been affirmed and denied that incidence and growth of squamous cell carcinomas are reduced in intact males ; estrogen has not affected tumor incidence. Species and strain differences in response are marked.

F. EFFECTS OF NONSTEROID HORMONES

There is e-\'idence that hormones from the anterior pituitary may directly affect the weight and histologic structure of accessory glands, or act synergistically with androgen. However, the findings have been somewhat conflicting. Dosage level, age, and strain of rats have varied, and questions have been raised with respect to the purity of the hormone preparations.

Attention was focused on the pituitary in relation to accessory glands when Huggins and Russell (1946) observed that prostatic

atrophy is more marked in the hypophysectomized than in the castrated dog. Van der Laan (1953) found the ventral prostates of hypophysectomized-castrated immature rats less responsive to testosterone propionate than the glands of castrates; a crude extract of beef pituitaries restored responsiveness in hypophysectomized-castrates. Prostates of young adult hypophysectomized-castrated Sprague-Dawley rats were also less responsive (total weight of dorsal and ventral prostates) to testosterone propionate than those of castrates (Grayhack, Bunce, Kearns and Scott, 1955). Paesi, de Jongh and Hoogstra (1956) administered pituitary extracts simultaneously with a low dose of testosterone propionate to hypophysectomized-castrated rats and reported a slightly greater ventral prostate weight than with the androgen alone.

To identify the hormones of the anterior pituitary that are capable of affecting the accessory glands or influencing their responsiveness to androgen, the following hormone preparations have been injected alone and in various combinations into hypophysectomized-castrated rats: prolactin (luteotrophin; LTH), growth hormone (somatotrophin; STH), adrenocorticotrophin ( ACTH ) , interstitial cell-stimulating hormone (luteinizing hormone; ICSH; LH), follicle stimulating hormone (FSHl. In addition, chorionic gonadotrophin and thyroxine have been administered. Of these hormones, only prolactin and growth hormone have been shown to act directly on accessory glands (for comprehensive data on negative and positive results of these hormones see Grayhack, Bunce, Kearns and Scott, 1955; Lostroh and Li, 1956, 1957 ». The degree to which contamination with prolactin or growth hormone might influence the assay of ICSH preparations by the ventral prostate test has been examined by Lostroh, Squire and Li (1958).

1. Prolactin {LTH)

When Pasqualini (1953) treated castrated adult rats with testosterone propionate followed by administration of a lower dose of androgen plus LTH, the amount of secretion in the seminal vesicles was greater than with androgen alone. Prostate weights were increased slightly by LTH with androgen. Van der Laan (1953) reported that in adult

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hypophysectomized-castrated rats LTH had no effect on ventral prostate weiglit. Grayhack, Bunce, Kearns and Scott (1955) made the same observation for prostate weights in young adult Sprague-Dawley rats, but found that LTH augmented the effect of testosterone propionate on prostate weight.

A difference in response between LongEvans and Sprague-Dawley strains of rats was observed by Lostroh and Li (1956). In immature hypophysectomized-castrated Long-Evans rats, LTH alone had no effect on ventral prostate or seminal vesicle weights, and no synergistic effect when administered with a low dose of testosterone propionate; in Sprague-Dawleys, however, the weights of ventral prostates and coagulating glands were increased by LTH but, again, no synergism occurred with exogenous androgen. Chase, Geschwind and Bern (1957) reported that in immature hypophysectomized-castrated Sprague-Dawleys, LTH did not affect weights of ventral prostates or coagulating glands but it did increase seminal vesicle weight. When LTH was administered with testosterone propionate, glandular tissue in the ventral prostate was increased and weights of coagulating glands (in some cases) and seminal vesicles were significantly higher than with androgen alone.

In the immature hypophysectomized Sprague-Dawley rats that were not castrated, LTH alone did not affect ventral prostate weight but when given simultaneously with ICSH it acted synergistically (Segaloff, Steelman and Flores, 1956). These results were confirmed by Lostroh, Squire and Li (1958) for the Sprague-Dawley strain, but in Long-Evans rats, LTH neither increased prostatic weight, nor augmented prostatic response to ICSH.

Antliff, Prasad and Meyer (1960) have shown that in the guinea pig, LTH had no effect on seminal vesicles of castrated or hypophysectomized males, but when it was administered with subminimal doses of testosterone propionate, seminal vesicle weight and epithelial height were increased.

2. Growth Hormone {STH)

Van der Laan (1953) found no effects of STH on ventral prostate weights in young hypophysectomized-castrated rats. Huggins,

Parsons and Jensen ( 1955) observed only slight effects on weights of ventral prostates and seminal vesicles with administration of STH to young hypophysectomized-castrated Sprague-Dawley rats, but a synergistic effect on weight was evident with simultaneous treatment with STH and testosterone propionate.

In hypophysectomized-castrated LongEvans rats (Lostroh and Li, 1956, 1957), STH produced slight histologic changes and significant weight increases in the ventral jirostate; when administered with testosterone propionate, an additive effect on weight was obtained. The changes in the seminal vesicles were less evident. The effects on Sprague-Dawley rats included weight increases in ventral prostates and seminal vesicles and a greatly enhanced weight response when STH and testosterone propionate were administered simultaneously (hypophysectomized-castrates in this strain gave a limited response to the androgen I . Chase, Geschwind and Bern (19571 found no consistent weight increases of ventral prostates, coagulating glands or seminal vesicles in young hypophysectomized-castrated Sprague-Dawleys treated with STH or STH and testosterone propionate. Simultaneous administration of STH, LTH and testosterone, however, induced significant increases in all accessories above the weights produced by the androgen alone.

Lostroh, Squire and Li (19581 determined that STH had no effect on the ventral prostate response to ICSH in hypophysectomized Long-Evans rats, but })roduced an enhanced response in Sprague-Dawleys. It was concluded that the Long-Evans strain is jjreferable for the testing of crude ICSH extracts, inasmuch as neither STH, LTH, nor both simultaneously, affect the response of the ventral prostate to ICSH.

With regard to the action of STH on histologic structure of the prostate in hypopiiysectomized-castrated rats, it should be noted that the effects are slight; nuclei api)ear vesicular, and the connective tissue stroma is increased (Lostroh and Li, 1957). The synergistic action of STH on prostate growth in hypoi)hysectomized-castrated rats when administered simultaneously with testosterone is more striking. In a general discussion of the many biologic effects of

ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS

435

growtli hormone, Li (1956) wrote, Does this ability to act as a synergist mean that growth hormone plays a permissive or supporting role in the biological action of a hormone or of a biological agent? It is not unreasonable to assume that growth hormone creates the necessary and sufficient environment for other biological agents to exercise the full scope of their functions."

Acknowledgments. We are greatly indebted to Drs. Thaddeiis Mann, James Harkin, Helen Deane, Keith Porter, and David Brandes for generously supplying luipublished data and electronmicrographs. Mrs. Eva Brown provided invaluable assistance in the preparation of the manuscript. We wish to thank our artist. Mr. Kenji Toda, for many of the original figures. The researches of one of us (D. P.) cited in the chapter were supported in part by grants from the Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago and by Research Grants 2912 and 5335 from the National Institutes of Health, Public Health Service.

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1938. Occurrence of prostatic lobes in the female rat. Biol. Zentralbl., 58, 455-464.

Wolfson, S. K., Jr., and Williams-Ashman, H. G. 1958. Enzymatic determination of sorbitol in animal tissues. Proc. Soc. Exper. Biol. & Med., 99, 761-765.

Woolley, G. W., AND Little, C. C. 1945a. The incidence of adrenal cortical carcinoma in gonadectomized male mice of the extreme dilution strain. Cancer Res., 5, 211-219.

Woolley, G. W., and Little, C. C. 1945b. The incidence of adrenal cortical carcinoma in male

mice of the extreme dilution strain over one year of age. Cancer Res., 5, 506-509.

WoTiz, H. H., AND Lemon, H. M. 1954. Studies in steroid metabolism. I. Metabolism of testosterone bv human prostatic tissue slices. J. Biol. Chem., 206, 525-532.

WoTiz, H. H., Lemon, H. M., and Voulg.\ropoulos, A. 1954. Studies in steroid metabolism. II. Metabolism of testosterone by human tissue slices. J. Biol. Chem., 209, 437^45.

WusT, H. 1957. Glykolytische Enzyme im menschlichen Seminalplasma bei Xormo-, Oligound Azoospermie. Arch. Klin. Exper. Dermat., 205, 351-356.

Yamada, K. 1933. fber den Zuckergehalt des Samens. Japan. J. Med. Sc. (II. Biochem.), 2, 245.

YiNG, S. H., Day, E., Whitmore, W. F., Jr., and Tagnon, H. J. 1956. Fibrinolytic activity m human prostatic fluid and semen. Fertil. & Steril., 7, 80-87.

Young, D. 1949. Bilateral aplasia of the vas deferens. Brit. J. Surg., 36, 417-418.

Zeller, E. a. 1941. Uber das Vorkommen der Diamin-oxydase in menschlichen Sperma. Helvet. Chim." Acta, 24, 117-120.

Zeller, E. A., and Joel, C. A. 1941. Beitriige zur Fermentchemie des miinnlichen Gesehlechtsapparats. 2. Mitteilung. Uber das Vorkommen der Cholinesterase, der Mono- und Diamin-oxydase in Sperma und Prostata, und iiber die Beeinflussung der Spermien-Beweglichkeit durcli Fermentinhibitoren. Helvet. Chim. Acta, 24, 968-976.

ZiMMET, D. 1939. Vitamine C et liquide d'ejaculation du cobaye. Effets sur les caracteres generaux de lejaculat, les spermatozoides et la reproduction. Compt. rend. Soc. l)iol., 130, 1476-1479.

ZoRGNioTTi, A. W., AND Brendler, H. 1958. Studies in semen coagulation. Proc. Soc. Exper. Biol! & Med., 96, 195-197.

ZucKERMAN, S. 1936. The endocrine control of the prostate. Proc. Roy. Soc. Med., 29, 81-92.

ZucKERMAN, S. 1940. The histogenesis of tissues sensitive to oestrogens. Biol. Rev., 15, 231-271.

THE MAMMALIAN OVARY

William C. Young, Ph.D.

PROFESSOR OF ANATOMY, UNIVERSITY OF KANSAS, LAWRENCE, KANSAS

I. Introduction 449

II. FOLLICULOGENESIS 451

A. Growth of Primary and Small Ve sicular Follicles 451

B. Growth of Vesicular Follicles 455

C. Preovulatory Swelling 455

1). Ovulation . ." 456

E. Folliculogenesis in Pregnancy and

Lactation 457

III. Corpus Luteum 459

IV. Follicular Atresia 4(11

V. Hormones of the Ovary 4(i4

A. Sites of Origin 466

B. Amounts of Hormone Produced. . . . 471 VI. Age of the Animal and Ovarian

Functioning 476

VII. Other Endocrine Glands and the

Ovaries 478

A. Thyroid 478

B. Adrenal Cortex 480

VIII. Concluding Remarks 483

IX. References 484

I. Introduction

Despite the impetus given to the study of ovarian structure and physiology by the work of Edgar Allen and Edward A. Doisy in the 1920's, knowledge of the mammalian ovary has hardly progressed beyond a descriptive phase. This cannot be attributed to lack of effort, although it must be realized that the ovary as an object of investigation has not held its own with the hypophysis, the thyroid, the adrenal, and the testis. Nor lias there been any failure to apply new techniques to the many problems of ovarian structure and physiology. Histochemical and cytochemical techniques were seized ujion for what they might contribute to the prol)lem of the site of hormone production,^ and,

' Demp.sey and Bassett, 1943; Dempsey. 1948; Claesson, 1954; Claesson and Hillarp, 1947a-c ; Claesson, Diczfalusy. Hillarp and Hogberg, 1948; Claesson, Hillarp, Hogberg, and Hokfelt. 1949:

in at least one series of studies (Zachariae, 1957, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958), to the mechanism of ovulation. Methods for obtaining blood from the ovarian vein have been devised (Paschkis and Rakoff, 1950; Rakoff and Cantarow, 1950; Xeher and Zarrow, 1954; Edgar and Ronaldson, 1958) and refined techniques for the assay of secreted estrogens and progesterone have been developed (Reynolds and Ginsburg, 1942; Hooker and Forbes, 1947; Emmens, 1950a, b; Haslewood, 1950; Wolstenholme, 1952; Zander and Simmer, 1954; Brown, 1955; Loraine, 1958; Sommerville and Deshpande, 1958j . The collection of follicles and corpora lutea timed more accurately with respect to the moment of ovulation has become possible, and distinction between the normal and the pathologic has become clearer (Deane, 1952). Recently, the electron microscope has been found to have a place, in an investigation of the finer structure of the cells of the corpus luteum (Lever, 1956), in the unraveling of the jirocesses whereby the zona pellucida is formed around the developing oocyte (Chiquoine, 1959; Odor, 1959), and in studies of ovarian oocytes and unfertilized tubal ova (Odor, 1960; Odor and Renninger, 1960) (see Figs. 14.6 to 14.8). As Villee has indicated in his chapter, great strides have been taken toward an understanding of the metabolic pathways in estrogen and progesterone synthesis and degradation.

Two factors may have contributed to the

McKay and Robinson, 1947; Meyer and McShan, 1950; Barker, 1951; Rockenschaub, 1951; White, Hertig, Rock and Adams, 1951 ; Deane, 1952; Nishizuka, 1954; Ford and Hirschman, 1955; Noach and \an Rees, 1958.

449

450

PHYSIOLOGY OF GONADS

disappointment that has been expressed. First, the purification and synthesis of the hormones in the 1930's (Allen, 1939; Doisy, 1939) and the later successful development of synthetic estrogens and gestagens ( Solmssen, 1945; Dodds, 1955; Rock, Garcia and Pincus, 1956; St. Whitelock, 1958) provided a means whereby much of ovarian physiology could be studied out of context with the processes by which this organ functions. Specifically, there are many effector actions of ovarian hormones, many interrelationships with other hormones and with each other, many problems of tissue responsiveness, and many questions bearing on processes of ovarian hormone metabolism, all of which can be studied in ovariectomized animals.

Secondly, there were many practical reasons why chemists should have striven to synthesize estrogenic substances and gestagens which are suitable for replacement therapy. Once prepared, these synthetic substitutes are of interest, but their development and therapeutic application may well have diverted attention from studies of the ovary.

If there is disappointment with the progress that has been recorded, we would direct attention to substantial accomplishments which should stand us in good stead in the future. Among these are the numerous careful descriptions of the growth and maturation of ovarian follicles and the meticulous accounts of corpus luteum formation, structure, and involution. In a general way it has become clear that in many species estrogen and progesterone are produced while the follicles are maturing, and that, during the functional life of the corpus luteum, progesterone and estrogen are secreted. Estimates of the amounts produced have been numerous and of more than ordinary interest. In addition, they probably represent steps toward the determination of additional important information : the day-to-day rate of production correlated with the growth of the follicles and the development of the corpora lutea, and, in species in which variable numbers of follicles and corpora lutea develop, steps in an effort to ascertain whether, for example, 10 follicles in an individual produce more hormone than 5. This knowledge, if we possessed it, might

contribute significantly to current theories of gonadal-pituitary relationships because thresholds are involved in the regulatory processes <see chapters by Everett and Greep ) .

It could be disappointing that, on the basis of evidence which is largely circumstantial and inferential, almost every tissue component of the ovary, membrana granulosa, theca interna, and interstitial cells, has been claimed to be the source of estrogen and progesterone. But it is encouraging that information has been obtained which prompts us to recognize that it may be futile and unrealistic to attempt to identify specific cell types as the sources of hormones in the ovary. Current thought, stimulated by the discovery that testicular cells, placental tissue, and occasionally the adrenal cortex are sources of estrogen and progesterone, and that ovarian tissue produces androgens, is leaning toward the view that the several tissues involved in steroid hormone biosynthesis may be subject to metabolic aberrations which change their hormone production either in rate or in kind. The a])i)roach to the problem now seems to be through enzymatic biochemistry rather than through gross or finer morphology. Examples of this approach which are suggestive for further work on the ovaries are jirovided by the numerous studies by Samuels and his associates (Samuels, Helmreich, Lasater and Reich, 1951 ; Huseby, Samuels and Helmreich, 1954; Beyer and Samuels, 1956; Samuels and Helmreich, 1956; Slaunwhite and Samuels, 1956).

It is known in a general way that follicular maturation, ovulation, and corpus luteum formation are controlled by gonadotroi)hic hormones from the pituitary. However, as Greep emphasizes in his chapter, the specific gonadotrophic hormones have not yet been isolated and identified, nor have their specific roles in ovarian physiology b(»en demonstrated. To be sure, ovulation has been repoited following the injection of allegedly purified pituitary gonadotrophins into hypojihysectomized rats (Velardo, 1900), but until stages normally seen ill the jn'ocess of folliculogenesis and ()\-ulati()ii can he rejn'oduced consistently by the use of pituitary gonadotroI)hins, and until target organ responses simi

MAMMALIAN OVARY

451

lar to those in intact cycling animals are being evoked, no satisfactory conceptualization of ovarian functioning will be possible.

Chorionic gonadotrophins are not without practical value in the stimulation of ovulation (Cole and Miller, 1933; Folley and Malpress, 1944; Folley, Greenbaum and Roy, 1949; Marden, 1951; Umbaugh, 1951; Robinson, 1954; and others); nevertheless, their contribution to ovarian physiology may be limited. Bradbury has pointed out in a personal communication that the human placental hormone (HCG) is exotic for laboratory animals. It has practically no effect on the ovaries of guinea pigs or field mice. In rats its biologic effects arc finite different from those of pituitary luteinizing hormone (LH) or interstitial cellstimulating hormone (ICSH) (Selye, Collip and Thomson, 1935; Evans, Simpson, Tolksdorf and Jensen, 19391. HCG is so ineffective in the hypophysectomized rat that it has been assumed, in the case of the intact animal, either that it acts through the pituitary or that it requires the presence of the pituitary to be effective (Aschheim, Fortes and Mayer, 1939; Noble, Rowlands, Warwick and Williams, 1939) . If HCG and other chorionic gonadotrojihins are exotic, as such results would indicate, the extrapolation of effects which have followed their use to the normal functioning of the ovary could be seriously misleading.

What we have written is intended to set the tone for what follows. Many of the solid accomplishments of the past two decades will be recounted, but the areas of uncertainty and the difficulties which slowed down the progress of the twenties and thirties will be enumerated in the hope that the curiosity of a new generation will be aroused and guide us into a period of even more productive eft'ort.

II. Folliculogenesis

A. GROWTH OF PRIMARY AND SM.\LL VESICULAR FOLLICLES

In mammals, by the time of birth, oogonia have completed their proliferative activity and become primary oocytes. The serosal surface of the ovary is covered by a layer of cells known as the germinal epithelium. There has been and still is considerable

speculation whether adult germinal epithelium contains, or gives rise to, any germ cells (Sneider, 1940; Mandl and Zuckerman, 1950, 1951a-d, 1952b; Zuckerman, 1951; Green and Zuckerman, 1951). The subject is reviewed extensively by Brambell ( 1956 ) and in the chapter by Blandau. Careful studies of human ovaries have been made by Block (1951a, b, 1952, 1953). From all this material, it is clear that a satisfactory answer has not been given. The latter may be awaiting the development of a fresh approach and until then another review of the many conflicting reports and opinions would be repetitious.

]\Iore relevant to the present review is the relationship between ovarian estrogens, on the one hand, and germ cell proliferation and follicle development, on the other. It has been claimed that exogenous estrogen stimulates mitotic activity in the germinal epithelium in mice, rats, and the minnow, Phoximis laevis L. (Bullough, 1942a, 1943; Stein and Allen, 1942; de Wit, 1953; von Burkl, Kellner, Lindner and Springer, 1954) . Bullough (1943) suggested that new cycles of oogenesis are intiated by estrogen in the follicular fluid of large and rupturing follicles. Mandl and Zuckerman (1950) and Dornfeld and Berrian (1951) were less certain. The former expressed the belief that the direct effect of estrogenic stimulation cannot be measured by comparing the total number of oocytes in the ovary. The latter, after finding that isotonic saline, gelatine, or agar injected into the perovarian capsules of immature rats elicited mitoses in the germinal epithelium, concluded that the reaction was in response to injury rather than to the substance injected. Notwithstanding the precautionary notes sounded by ]\Iandl and Zuckerman and by Dornfeld and Berrian, the number of reports of increased mitotic activity near the site of ovulation or when estrogen is placed in contact with the germinal epithelium remains impressive. Particularly because of the analogy with the androgenic control of spermatogenesis pointed out by Bullough ( 1942b) and others, the possibility should be tested further.

As the oocyte starts to grow, the flat investing cells proliferate and form the membrana granulosa. By the time the rat oocyte has completed its growth it has acciuired

452

PHYSIOLOGY OF GONADS

^l!:

I--|(..7.1. Munlry l,yi„,pliy>r,-ini,iiz<Ml 1 y,-uv | hvm, ,u^ly . I'., Hide- :nv ,n ) ,i n^i .—in . ' ~::,-i>s (jl (le\f'lu]iiiieiil. C<nic('iitr;itiun ul' uorylcs m ihc corlcx n'S('inl)lfs llial in iVlal or jusciiilc o\aiie8. No evidence of estrogen production in this animal. (Courtesy of Dr. Ernest Knobil.)

foiii' layers of granulosa cells (Mandl and Zuokerman, 1952a). The increase in size of the growing follicle is relatively constant until the stage of antrum formation (Paesi, 1949a ». Follicles grow and develop to this stage in rats and guinea pigs even after hypophysectomy (Dempsey, 1937; Paesi, 1949b). It is thus apparent that the gonadotrophic hormones of the pituitary are not essential for the early growth of ovarian follicles (Fig. 7.1). The rate of this early follicular growth may, however, be accelerated in the presence of certain, but perhaps not all, gonadotrophic hormones (Pencharz, 1940; Sim])son, Evans, Fraenkel-Conrat and Li, 1941 ; Gaarenstroom and de Jongh, 1946; Payne and Hellbaum, 1955).

As the granulosa of the growing follicle ])roliferates, the surrounding tissue differentiates into theca interna. Dubreuil (1942, 1948, 1950) postulated that the granulosa j)roduces an inductor substance which causes the differentiation of the theca interna. Hisaw (1947), in a review of the literature bearing on this point, also suggested that there must be organizers within

the developing gramdosa cells which stimulate tlifferentiation of the theca interna. Furthermore, this autonomous process continues until the follicle reaches a stage of development at which it becomes responsive to gonadotrophic hormones. Hisaw called this the stage of "comjietency."

Somewhere in this process estrogens seem to have a role, or, if the ojjinions expressed by Bullough (1943) and the other investigators whose work has just been cited can be confirmed, perhaps there is a continued stimulation by these substances. If large doses of estrogen are administered to imnuitui'c or to liypopliyscctomi/ed immature rats, many follicles (k'\x'lop to the early antrum stage within 72 hours. The theca interna differentiates ai'ound these estrogenstimulated follicles. When immature rats ai'e giv(m large doses of estrogen there is a <le(iiiil(' incicasc in ox'arian weight and in the iiuiiihei' of medium-sized follicles ( ]ig. 7.2 1 ; small amounts, on the othei' hand, ai'e iiihihitoiy (Paesi, 1952). Increased giowth of large follicles, or at least a retai'dation of their degeneration, has

MAMMALIAN OVARY

453

y^

Fig 7.2. Intact immature lat given estrogen for 3 days. Active proliferation of granulosa in many follicles. Decreased incidence of atresia and hypertrophy of theca. (Courtesy of Dr. J. T. Bradburv.)

Ijeen reported in hypuphyscctoiuizcd I'uts given stilbc-^trol (Pencharz, 1940; Williams, 1944; Desclin, 1949; Payne and Hellbaum, 1955; Ingram, 1959 1. Histologically, when estrogen is given, there is a marked increase in mitotic activity of the granulosa cells and a decrease in atresia (Williams, 1945a; de Wit, 1953; Payne and Hellbaum, 1955; Payne, Hellbaum and Owens, 1956; Williams, 1956). The fact that estrogen stimulates the follicle and protects it against atresia suggests that the granulosa is not a significant source of estrogen. On the other hand, if estrogen is jiroduced by the theca interna, it could exert a localized stimulatory action on the membrana granulosa (Corner, 1938; Bullough, 1942b, c, 1943). The differentiation and development of the theca interna is nicely timed for a localized jiroduction of estrogen around each Graafian follicle.

Estrogen not only stimulates the granulosa to proliferate, but also renders the follicles more responsive to exogenous gonadotrophins. Williams (1945b) found that the ovary in the stilbestrol-treated hypol^hysectomized rat was more responsive to small doses of pregnant mare serum (PMS) than was the ovarv in the intact

immatui'e rat. Payne and Runser (1958) found that stilbestrol augmented the response of hypophysectomized immature rat ovaries to exogenous pituitary extracts. In Bradbury's experience at Iowa 48 hours of stilbestrol pretreatment rendered the ovaries of both intact immature and hypophysectomized rats more responsive to Armour's LH (Lot No. R377242H), but not to Armour's follicle-stimulating hormone (FSH) {Ah 1027) . Furthermore, increasing the dosage of stilbestrol from 0.02 mg. to 0.2 mg. markedly increased the response of the ovaries to a given dose of LH. He suggested that the possibility should be exi)lored that the local (})erifollicular) concentration of estrogen determines the resjionsiveness of the maturing follicles. Thoughtful discussions of the subject are given by Paesi (1952) and by Bradbury (1961). The former suggested that 2 or 3 types of estrogen action may be involved in the stimulation of the ovary which is seen wdien estrogen is administered. Bradbury applied estradiol or stilbestrol to one ovary of the immature rat, leaving the other ovary untreated. The various unilateral responses — increase in weight, formation of corpora lutea, greater ■ reactivity to gonadotrophins — demonstrated

454

PHYSIOLOGY OF GONADS

.Sj^cr^r

•■/>:

ikm

Fig. 7.O. Imuiuturu li.ypophy.sectomized rat treated with Arniuui - i>il. (iiauulo.sa ha^ proliferated and follicles have developed antra. The theca is diffeientiated but the interstitium is deficient. (Couitesy of Dr. R. M. Melampy.)

clearly the local stimulating effect of estrogens with the ovary, as well as the systemic effect by way of the pituitary.

If estrogen administration to immature or to hypophysectomized immature rats is continued 7 to 10 days, the granulosa of the stimulated follicles degenerates. This atretic process differs from natural atresia in that it seems to start peripherally rather than centrally. The oocytes do not fragment or give off polar bodies as frecjuently as do oocytes in normally atretic follicles. It seems that the stimulatory effect of estrogen on the granulosa is very temporary. Its din-ation, however, is long enough to be coni])atible with the noi'inal pi'ocess of maturation and ovuhitiou.

Before concluding the subject, a certain amount of back-tracking may be dcsii'able. One of the first suggestions to be made by Edgar Allen (1922; see also the biogi'aphic;il sketch in this book) was tliat the ovum is

the dynamic center of the follicle. If the suggestion is placed in the context that has since been developed, the sequence of events would l)e an inductive influence of the oocyte on the membrana granulosa, a continued inductive influence (oocyte or membrana granulosa?) on the surrounding connective tissue cells until the theca interna is formed,- and then the secretion of estro 'Resuhs obtained by Genther (1931), Schmidt (1936), Humphreys and Zuckerman (1954). and Wcstman (1958) suggest that a similar functional iclationship exists between the granulosa and interstitial cells. According to Genther, x-ray-iujured o\aries composed of interstitial cells produced estrogen only if a growing follicle was present. The in\oluted condition of the uteri in rabbits in wliich all oocytes and f()llicl(>s iiad been destroyed by x-rays led Humphreys and Zuckerman to conclude that the ovaries of these animals were not producing estrogen. The results reported by Westman suggested tliat interstitial cell function continues only lor .1 limited jieriod after x-ray-induced degeneration of the granulosa cells. The results from an ingenious investigation by Ingram (1957) ar(

MAMMALIAN OVARY

455

gen wliich feeds back to stimulate further growth of the meinbrana granulosa and follicle.

Attractive as such a hypothesis is, it has at least one weakness. If gonadotrophic extract rich in FSH is administered to immature rats for three days, there is a generalized stimulation of granulosa tissue (Fig. 7.3). Small follicles increase in size, medium sized follicles develop an antrum, and Graafian follicles become large and vesicular (Parkes, 1943), but ovulation is uncommon. At autopsy the ovaries are pale and edematous. The ovaries are markedly increased in size from numerous follicles becoming vesicular. Histologically there is little stimulation of the theca interna. Gaarenstroom and de Jongh (1946) recognized this ovarian response when they suggested that FSH be designated as Ge (gonadotrophin e])ithelial). This tissue response offers evidence that FSH is primarily f'oncerned with growth and proliferation of the granulosa cells, but there is no explanation to account for the failure of these granulosa cells to stimulate the differentiation of the theca interna and the eventual secretion of estrogens.

During all of follicular growth, the presence of estrogen has come to l^e assumed and its production by the growing follicle is thought to begin with the appearance of the theca interna (see below). On the other hand, the amount produced and the rate of production are unknown. The amount must increase with the growth of the follicles. Gillman and Gilbert (1946) found during their investigation of perineal turgescence in the baboon that, once the perineum reaches maximal turgescence, additional estrogen is required to maintain it. They concluded that in normal animals, during the second part of the phase of turgescence, there must be an increased output of ovarian estrogen. Direct studies have yielded little

similarly suggestive. Autografts of ovarian medulla without cortical tissue or oocytes, and autografts of cortical tissue were transplanted to various sites in .sexually mature rabbits. The grafts of cortical tissu(> p(>rsisti'(l after the medullary grafts had disappeared. Ingram concluded that medullary tissue containing interstitial tissue but no follicles cannot survive.

information. Foi'd and Hirschman (1955) estimated alkaline phosphatase activity in the ovary of the rat, but the concentrations in the theca interna and ovarian tissue as a whole were relatively constant during the phases of the cycle.

B. GROWTH OF VESICULAR FOLLICLES

The growth of the follicle which is dependent on stimulation by hypophyseal gonadotrophins has been described for a number of animals and, for a few (cow, sow, ewe, guinea pig, rat) plotted with respect to the time of the preceding ovulation (McKenzie, 1926; Hammond, 1927; Grant, 1934; Myers, Young and Dempsey, 1936; Boling, Blandau, Soderwall and Young, 1941 ; von Burkl and Kellner, 1956). Data of the latter sort are especially valuable for the baselines they provide for experimental studies of the factors affecting the pituitary-gonadal relationships. Deviations in the shape of the curve of follicular growth, and disparities in the size of the growing follicles and in the size and structure of the corpora lutea, are clear indicators of abnormalities in function which have been too little used.

C. PREOVULATORY SWELLING

Without exception in the animals listed above, and probably in the horse, goat, and bat, if we may judge from the data presented by Hammond and Wodzicki (1941), Wimsatt (1944), and Harrison (1946, 1948b), a linear period of growth during most of the diestrum is followed by a positive acceleration (preovulatory swelling) ; shortly before estrus and ovulation. The point at which this acceleration occurs is the point in the development of the follicle where physiologic evidence for the production of progesterone by the unruptured follicle was first found (Dempsey, Hertz and Young, 1936; Astwood, 1939). As we will see later, however, the ^'moment" the preovulatory swelling begins is not necessarily the point in time when the first progesterone is produced.

In most sjiecies in which the course of the preovulatory swelling has been followed, it is a 10- to 12-hour process (Hammond, 1927; Grant, 1934; Myers, Young and

456

PHYSIOLOGY OF GONADS

Dempsey, 1936; Doling, Blandau, Soderwall and Young, 1941 ; Rowlands and Williams, 1943; Rowlands, 1944), although in the cat and ferret the process is triggered by mating and extends over 25 to 30 hours. The preovulatory swelling can be initiated by injecting gonadotrophins of the LH or ICSH type, but they are effective only on well matured follicles (Hisaw, 1947; Talbert, Meyer and McShan, 1951). The younger follicles are not stimulated and, on the contrary, they may show an accelerated atresia. It could be postulated that the follicules with well developed theca interna were "competent" and that stimulated theca interna produced estrogen which favored the development of these follicles. The smaller follicles were "incompetent" in the absence of a thecal investment and became atretic. If HCG is injected into immature rats, the theca interna around the vesicular follicles hypertro])hies within 24 hours and these follicles enlarge rapidly. The vasodilation of the theca blood vessels is grossly evident within a few hours (Kupperman, McShan and Meyer, 1948; Sturgis and Politou, 1951; Odcblad, Nati, Selin and Westin, 1956).

Explanation has been sought for the nature of the changes within the follicle which lead to the accelerated enlargement culminating in ovulation. Studies of the staining qualities of such follicles reveal that the metachromatic polysaccharides of the granulosa (hyaluronic acid and chondroitin sulfuric acid) become progressively depolymerized and orthochromatic. This hydrolysis of the mucopolysaccharides gives rise to an increased osmolarity which may be the major factor in the preovulatory swelling of the follicle (Harter, 1948; Catchpole, Gersh and Pan, 1950; Odeblad, 1954; Zachariae, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958). Accompanying the swelling is a dispersal of the cells of the cumulus oophorus. This may be a cons(^quence of the breakdown of the intcrcclhihii' substance in the stinudated niciubraiia gi'aiiulosa.

The time r('(|uirc(l for follicular growth and maturation fi'oin the stage when its further development is dependent on pituitary gonadotrophin stimulation to ovulation is related to the length of the cycle and

therefore varies greatly from species to species. Somewhat less than 4 to 5 days are required in the rat (Boling, Blandau, Soderwall and Young, 1941 ) , somewhat less than 16 days in the guinea pig (Myers, Young and Dempsey, 1936), somewhat less than 21 days in the cow (Hammond, 1927), and ])resumably comparable intervals in other species. Vermande-Van Eck (1956) estimated that in the rhesus monkey the average time required for the growth of a mature follicle from the large follicle without an antrum is 4 to 6 weeks; 11 days are estimated to lie necessary for the complete development of a follicle in the rabbit (Desaive, 1948) . Ovulation occurred earlier than normal when the corpora lutea from the ])receding cycle were removed, but the rate of follicular development was not altered (Dem])sey, 1937). Presumptive evidence exists, however, that the rate of growth may be slower in pubescent chimpanzees (Young and Yerkes, 1943), baboons (Gillman and Gilbert, 1946), and guinea jngs (Ford and Young, 1953).

D. OVULATION

Ovulation, under normal circumstances, ))robably is explosive (Hill, Allen and Kramer, 1935, in the rabbit; Blandau, 1955, in the rat). In 1 of 2 human i)atients Doyle (1951) saw a gush of follicular fluid at the time of ovulation. In 163 ovulations timed l)y Blandau the interval between the rupture of the stigma and the escape of the ovum was 72 seconds when most of the folliculai' fluid escai)ed in advance of the ovum, and 216 seconds when the cumulus oophorus preceded the follicular fluid. The slower, steady, continuous flow of the liquor folliculi which has been described by Walton and Hammond (1928) in the cow, Markee and Hinsey (1936) in the rabbit, and by Doyle (1951) in one human sul)ject could be an artifact of the procedures used in watching the jM'Ocess.

The mechanisiu heading up to formation of the stigma and rupture of the follicle is unknown. Claesson (1947), using the submicroscoi)ical differences which can be ol)served in ])olarized light, distinguished smooth muscle from connective tissue cells and rep()rte(l that no bundles of smooth muscle or isolated cells were found in the

MAMMALIAN OVARY

457

theca externa in ovaries from the cow, pig, rabbit, and guinea pig. The earlier contradictory results he reviewed were attributed to the nonspecificity of the older staining methods. A possible clue to the mechanism of ovulation wliich does not seem to have been explored was given by the observations of Boling, Blandau, Soderwall and Young ( 1941 ) when they were studying follicular growth in the rat. Immediately before ovulation, but at no other time, a large pocket at the base of the cumulus, and described as an invagination of the granulosa, is a constant feature of follicular structure (Fig. 9 in their article). No guess w^as made as to its significance.

In all the spontaneously ovulating infrahuman mammals that have been studied, except the dog (Evans and Cole, 1931 ) , possibly other Canidae, and the mouse (Snell, Fekete, Hummel and Law, 1940) in which it takes place early in estrus, ovulation occurs toward the end of heat (see reviews in Young, 1941; Dukes, 1943; and more recent articles on the chimpanzee, rhesus monkey, baboon, cow, and mare by Young and Ycrkes, 1943; van Wagenen, 1945, 1947; Gillman and Gilbert, 1946; Cordiez, 1949; and Trum, 1950; respectively ) . Only in the human female in which cyclic waxing ^nd waning of sexual desire is not easily detected does uncertainty exist.

Since an early period, when emphasis was given to the opinion that ovulation occurs about midway in the intermenstrual interval (Knaus, 1935; Hartman, 1936; Farris, 1948), much evidence has been produced indicating that it may occur at other times as well, even during menstruation (Teacher, 1935; Rubenstein, 1939; Sevitt, 1946; Bergman, 1949; Stieve, 1952; and many others). If we may judge from what has been found in the chimpanzee (Young and Yerkes, 1943), baboon (Gillman and Gilbert, 1946), ihcsus monkey (Rossman and Bartclmez, 1946), and man (Bergman, 1949; Buxton, 1950), irregularities in the length of the preovulatory and postovulatory phases of the cycle complicate the problem and could account for some of the confusion. In the chimjianzee, baboon, and human female, in which the irregularities can be located wdth respect to the time of ovulation, age influences the length of both phases, and fol

lowing pregnancies there are similar irregularities. In the baboon, environmental stresses result in temporary or even prolonged inhibition of ovarian activity. There is no reason for believing that the same factors have less effect on folliculogenesis in the human female; irregularities in adolescence (Engle and Shelesnyak, 1934) and following pregnancy (Sharman, 1950, 1951) are common and there are many reports of psychic effects (see reviews by Kelley, 1942; Kelley, Daniels, Poe, Easser and Monroe, 1954; Kroger and Freed, 1950; Randall and McElin, 1951; Bos and Cleghorn, 1958). In all cases follicular growth is interrupted and amenorrhea follows. But if the estimates are correct that the average fertile woman ovulates normally about 85 per cent of the time (Farris, 1952), or that perfectly healthy women may have 3 or 4 anovulatory cycles a year (de Allende, 1956; also see table in Bergman, 1949), there must also be cases in which much of follicular growth is normal, or at least adequate to stimulate growth changes in the uterus, but ovulation does not occur. As if the complications noted above are not enough, the reviews of the methods used in determining the time of ovulation (D'Amour, 1934; Cohen and Hankin, 1960) and the critical study of Buxton and Engle (1950) in which an attempt was made to correlate basal body temperature, the condition of the endometrium, and the stage of folliculogenesis in the ovary, suggest either that a really sensitive indicator of the time of ovulation has not been found, or if one exists, that it has not been used in a study sufficiently systematic to reveal the true situation in the human female. The problem is one of the many that is with us very much as it was 20 years ago.

E. FOLLICULOCJENESIS IN PREGNANCY AND LACTATION

Before leaving the subject of follicular growth, its course in pregnancy and lactation should be reviewed. Information has been obtained from many species, but in most cases it is not complete and a considerable amount of conjecture is necessary. What is certain is that pregnancy affects the process of folliculogensis in many ways ; each must be the reflection of a different in

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terrelationsliip betwen pituitary, gonads, and placenta. In the mare, and presumably other species in which multiple ovulations occur early in pregnancy, the involvement of chorionic gonadotrophins, pituitary gonadotrophins, and estrogen of placental origin has been suggested (Rowlands, 1949). When folliculogenesis is inhibited just before the stage of the preovulatory swelling, as it is in many pregnant animals (see below), the nervous system may be involved. An unusually significant investigation in which the threshold of stimulation to ovulation in the rabbit was correlated with threshold changes in cerebral activity has recently been completed (Kawakami and Sawyer, 1959). It was demonstrated that pregnancy or prolonged treatment with progesterone maintains the electroencephalogram (EEGj after-reaction threshold to low frequency stimulation of hypothalamic or rhinencephalic nuclei at an elevated level. At this level, gonadotrophin release does not occur in response to coitus or other ovulatory stimuli. The discovery of this fact has provided a basis for understanding the various ovarian conditions associated with pregnancy and lactation, or at least those in which follicular development proceeds to the point of preovulatory swelling and then stops. It may be that some other mechanism of inhibition accounts for the more severe retardation of folliculogenesis in si)ecies in which this occurs.

The European hares, Lepus tiniidus L., and L. ciiniculus L., are reported as mating during pregnancy with the occurrence of superfetation (Lienhart, 1940). Pregnancy, therefore, has little or no effect on any stage of folliculogenesis in these species. The domestic rabbit appears to he somewhat more affected and perhaps more variable. Claesson, Hillarj), Hogberg and ll()kfeh (1949) state that the ovaiics of pii'gnant rab})its are composed almost entirely of interstitial gland, except for the corpoi'a lutea, but, according to Hannnond and Marshall (1925) and Dawson (1946), mature follicles ai'e present and pregnant animals will occasionally mate. However, if we may assume that the reaction of i)regnant animals is similar to that of ])seudopregnant animals (Makepeace, Weinstein and Friedman, 1938), pituitary gonadoti'opliin is not

released and ovulation does not occur. From examination of the ovaries and from the fact that fertile matings can occur within a very few hours after parturition (Dempsey, 1937; Boling, Blandau, Wilson and Young, 1939; Blandau and Soderwall, 1941), it is clear that follicular development in the pregnant guinea pig and rat proceeds to a point just short of the preovulatory swelling. According to Nelson (1929) and Swezy and Evans (1930), cycles of oogenesis occur in laboratory rats, and, although the follicles may form small corpora lutea (Swezy and Evans), ordinarily they do not rupture. The musk-rat. Ondatra zibethica, and the African bat, Xycteris luteola, must display an advanced follicular development during pregnancy because there is evidence of postl)artum estrus (Warwick, 1940; Matthews, 1941, respectively). Brown and Luther (1951) state that postpartum estrus occurs within 3 days after farrowing in the sow, if the young pigs are removed. We assume, from this latter statement and from the rel^ort that estrus and service may occur during pregnancy in this species (Perry and Pomeroy, 1956), that large follicles are present in the ovaries of the pregnant sow.

Heat ])eriods in the jjregnant ewe are associated with follicular growth, but ovulation does not occur, and late in pregnancy follicle size decreases significantly (Williams, Garrigus, Norton and Nalbandov, 1956 ) . The first heat after {parturition was an average of 23.9 days later, range 1 to 61 days. According to Harrison (1948b), widespread atretic changes can be seen in all the follicles in the goat, beginning the 40th day of |)regnancy. By the (iOth day. no healthy follicles can be found.

Hammond (1927) was of the opinion that during jircgnancy in the cow, follicles develop to the size at which the jireovulatory swelling begins, but Dukes (1943), citing a study by Weber, wrote that cows come into heat 3 to 7 weeks after parturition. Support for this \-iew comes from the report l)y Hafez ( 1954) that the average interval to the postpartum esti'us in another bovine, the Egyptian buffalo, is 43.8 days, range 16 to 76 days. The iclatixcly long postpartum inter\al in these two species is presumptive evidence that follicles are relatively small at the end of pregnancy in bovines.

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Between the 40th and 150th day of pregnancy in the mare the ovaries contain numerous actively growing follicles and several functional corpora lutea (Cole, Howell and Hart, 1931; Rowlands, 1949). However, from the 150th day until the late stages, there is a regression of all the corpora lutea and an absence of large follicles. In the late stages only minute vestiges of corpora lutea and small follicles remain. If the latter is true, follicular growth must be rapid after parturition, because the first heat following foaling was between the 7th and 10th days in 77 per cent of the many mares Trum (1950) studied. In the African elephant, Loxodonta africana, there is also a replacement of the corpora lutea (one plus several accessory corpora lutea j about midway through pregnancy (Perry, 1953). Some are formed following ovulation and some not. They persist until term when they involute rapidly. During the late stages of pregnancy no follicles with antra are founcl. Dawson ( 1946) wrote that the domestic cat does not possess mature follicles at the time of parturition. In nonlactating animals the proestrous level is reached the 4th week after parturition.

Presumptive evidence exists that the follicles in the parturitive chimpanzee are small (Young and Yerkes, 1943 ) . In the human female the appearance of the first ovulatory cycle after pregnancy is irregular (Sharman, 1950, 1951 ; AlcKeown, Gibson and Dougray, 1954). According to Sharman, it may occur about 6 weeks after delivery in nonlactating women. This suggests that follicles are small at the end of ju-egnancy in the human female.

Inhibitory effects of lactation on follicular development are indicated by the substance of many of the reports cited above (Dawson; Dukes; Perry; Schwartz; Sharman; Williams, Garrigus, Norton and Nalbandov) and by much other information. As would be expected, the intra- and interspecies variations are great. Studies in progress at Iowa (Bradbury, personal communication) are revealing that some women experience an atrophy of the vaginal epithelium during the second and third month of lactation. The atrophy is indicative of a lack of ovarian estrogen and suggests that follicular development is not normal. Observations that are

similarly suggestive have been made in other species. The absence of estrogen in significant ciuantities during lactation in the mouse (Atkinson and Leathem, 1946) and guinea pig (Rowlands, 1956) is believed to be the reflection of a delay in the resumption of follicular growth and ovulation. Mother rats and mice may copulate and conceive within 24 hours after delivering a litter of young. AVhile the mother is nursing the newborn litter, the fertilized eggs of the new pregnancy develop into blastocysts, but these blastocysts fail to implant in the uterus at the usual time (Talmadge, Buchanan, Kraintz, Lazo-Wasem and Zarrow, 1954; Whitten, 1955; Cochrane and Meyer, 1957). This delay in implantation is apparently due to a lack of estrogen, because an injection of estrogen will result in implantation of the blastocysts. The suppression of estrous cycles during lactation in the mouse and rat is influenced in part by the size of the litter. A litter of 8 to 10 young will inhibit cycles, whereas cycles are displayed if the litter is reduced to 2 or 3 young (Parkes, 1926a; Hain, 1935). The cottontail rabbit [Sijlvilagus floridamis) seems to be a species in which ovarian follicular development is little if any affected by lactation, for Schwartz ( 1942 ) stated that suckling does not prevent ovulation after coitus, at least in the early stages of lactation.

III. Corpus Liiteuni

The formation of the corpus luteum has been described for many species ( see reviews in Corner, 1945; Harrison, 1948a; Brambell, 1956). In general, after rupture of the follicle and discharge of the ovum, the granulosa is invaded by blood vessels from the theca interna (Bassett, 1943). They form a rich network among the enlarging granulosa lutein cells. The extent and nature of the contribution from the theca interna varies from species to species, but, as Corner states, the origin of the major part of the epithelioid cells of the corpus luteum from the granulosa may now be considered a fact.

Whereas there may be a fairly uniform pattern of development and control of ovarian follicles in mammalian forms, there are diverse mechanisms for the formation and maintenance of corpoi'a lutea; consequently

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specific examples must be presented in order to avoid the dangers of generalization.

In the rabbit copulation triggers a neurohumoral mechanism which releases gonadotrophin from the pituitary which subsequently induces ovulation in 10 to 12 hours. The ruptured follicles form corpora lutea which have a functional span of about 28 days if pregnancy ensues but only 14 days if the mating is infertile. Crystals of estrogen implanted into a corpus luteum of a rabbit will cause its persistence while other corj^ora lutea regress (Hammond and Robson, 1951 ). This suggests that either estrogen makes the corpus luteum more sensitive to pituitary maintenance (Hammond, 1956) or estrogen protects the corpus luteum from luteolytic action. In the cat, copulation induces ovulation about 25 hours after mating; the corpora lutea function for 36 days after an infertile mating, but gestation lasts 62 to 64 days. The ferret ovulates about 30 hours after copulation and the corpora lutea are functional for 42 days, w^hether the mating is fertile or infertile (Brambell, 1956 1.

In the unmated rat and mouse ovulation is spontaneous, but the resulting corpora lutea are nonfunctional and begin to regress within 2 days. After copulation the corpora lutea persist for 18 days if impregnation has occurred, but for only 12 days after an infertile mating. Copulation probably results in the release of enough additional gonadotrophin (LH or LTH) to activate the corpora lutea. In rats and mice the pituitary hormone, prolactin, is luteotrophic (LTH) (Desclin, 1949; Everett, 1956). These species have functional corpora lutea throughout lactation— actually two sets, that of pregnancy and that of the postpartum ovulation. Using the rat and taking weight and levels of ovarian enzymes as measures of activity, these corpora lutea were studied by Meyer and McShan and their associates and the results summarized in a ic\'i('\v (]\Ieyer and McShan, 1950). They found that "the weight of the corpora lutea of pregnancy increased greatly during the latt(>r half and that the amount of enzymes per corjius luteum was also greater. With some caution, they concluded that these corpora lutea are more highly functional during this phase of pregnancy than during the first half.

Not only copulation, but also injection of

estrogen at estrus is followed by the formation of functional corpora lutea. The estrogen maintenance of corpora lutea in rabbits and mice is offset by hypophysectomy (Hohn and Robson, 1949); presumably, therefore, maintenance is mediated through the anterior })ituitary. Reece and Turner (1937) showed that estrogen stimulates the rat i^ituitary to produce prolactin so the latter may be the luteotrophic agent in this s]:)ecies. Moore and Nalbandov (1955) found that prolactin is luteotrophic in sheep. To date this is the only species other than the rat and mouse in which prolactin has been shown to have luteotrophic activity.

In the guinea pig, monkey, man, and many other species, ovulation and the formation of functional corpora lutea are spontaneous. Copulation is not known to have any neurohumoral influence in these species. The corjiora lutea of the human female function for 2 to 3 months in pregnancy and for only 12 to 14 days in. an infertile cycle. Bergman ( 1949 ) states that the duration of the luteal i^hase is limited to a maximum of 16 days. In the rhesus monkey the functional life has been estimated to be about 13.5 days in the normal cycle, and approximately 30 days when pregnancy intervenes (Hisaw, 1944) . In the bitch, ovulation is spontaneous and the corpora lutea remain functional for 6 weeks irrespective of mating or pregnancy. In the lactating African elephant the corpora lutea degenerate soon after parturition (Perry, 1953) ; in the lactating domestic cat they not only persist, l)ut they become rejuvenated" (Dawson, 1946).

As a general statement, it can be said that the functional span of the corpora lutea is cithei' adequate to permit implantation or it is prolonged by copulation (as in rats and mice) so that imjilantation can occur. But inasmuch as imj:)lantation occurs in many species, including man, about the sixth day after ovulation and fertilization, the margin of safety is not great and a delay in the secretion of chorionic gonadotrophin by the tro])hoblast must reduce the chances of a successful pregnancy.

Ill some species, e.f/., rats, mice, rabbits, an ill felt ih' mating prolongs the life of the corpora hitea. This prolonged interval of functional luteal activity is known as pseudoj)regnancy. As Everett has noted in his

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chapter, in pseudopregnancy the hormonal aspects of pregnancy are duplicated, but no fetal tissues are present. In the pseudopregnant bitch, for example, the hormonal aspects of pregnancy are so nearly duplicated that lactation begins at the time a normal gestation would have terminated.

The duration of pseudopregnancy in different species offers evidence of adaptive or evolutionary mechanisms to control the duration of corpus luteum function, mechanisms that must be endogenous to the uterus. Rats and mice have a pseudopregnancy of 12 days duration after a sterile mating, cervical stimulation, or injection of estrogen at estrus. There is no comparable condition in guinea pigs, monkeys, or man. However, if rabbits, rats, or guinea pigs are hysterectomized, any subsequent corpora lutea will function for a time equivalent to the duration of gestation in each species (Chu, Lee and You, 1946; Bradbury, Brown and Gray, 1950), although Velardo, Olsen, Hisaw and Dawson (1953) stated that, in the rat, hysterectomy has no effect on the length of pseudopregnancy. Hysterectomy in the cow and sow will prolong the life of the corpus luteum (Melampy, personal communication). Experimental distention of the uterus l)y beads has resulted in alteration of the length of the estrous cycle in ewes (Nall)andov, Moore and Norton, 1955). The only explanation which seems to account for these results is that there is a luteolytic agent in the uterus (probably in the endometrium) of some polyestrous species which shortens the life of the corpora lutea in nonpregnant animals. In pregnancy, or when massive deciduomas are present, if Velardo, Olsen, Hisaw and Dawson are correct, the conversion of endometrium to decidual tissue may cause it to lose its luteolytic ability. In future studies on the duration of the functional span of cor]5ora lutea, the possibility of luteotrophic and luteolytic mechanisms should be considered. On the other hand, a fresh start may be advisable. Fewproblems in reproductive and clinical endocrinology (Marx, 1935) seem to have been as resistant to clarification.

In unmated females of species not having a spontaneous "pseudopregnancy," the corlius luteum involutes shortly after its formation. The rat, in which 4 to 8 corpora lutea

are formed in each ovary at intervals of 4 to 5 days, has recognizable involuting corpora lutea from the two preceding cycles, but no remnants of older ones. The early stages of involution of the corpus luteum have been described (Brewer, 1942; Boling, 1942; Dawson, 1946; Duke, 1949; Moss, Wrenn and Sykes, 1954; Corner, Jr., 1956; Rowlands, 1956; Dickie, Atkinson and Fekete, 1957). The timetables of cellular changes given by Brewer and by Corner, Jr. are of interest for the comparison they permit with physiologic estimates of the duration of secretory activity by the human corpus luteum. On day 7 the corpus luteum seems to have reached its peak of activity, as judged by the vacuolation of its cells in Bouin's or Zenker's fluid-fixed and hematoxylin and eosin-stained preparations. Corpora lutea of days 9 to 12 show evidence of i)rogressive secretory exhaustion.

The later stages of cori:)ora lutea degeneration have not received the same careful attention. In women the corpus luteum undergoes a slow hyaline degeneration and the corpora albicantia persist as old scars for months or years. They may be present in ovaries 15 to 20 years after the menopause. Whether the final stage of degeneration is a process of lysis, phagocytosis, or transformation into connective tissue has not been studied.

IV. Follicular Atresia

It was long ago estimated that the infantile liuman ovary contains about 400,000 oocytes (Fig. 7.4). In the 30 years of reproductive life about 400 ova may mature and ovulate. On this basis about 1 oocyte in 1000 achieves ovulation; the other 999 are lost through a degenerative process known as atresia. The problem is not different in any other species. Whether it is monotocous or polytocous, there is always an enormous wastage of oocytes in each cycle of folliculogenesis. Atresia may have its onset at any stage of follicular growth or maturation and oocytes may degenerate before they have acquired a distinct membrana granulosa (Mandl and Zuckerman. 1950; de Wit, 1953; Payne, Hellbaum and Owens, 1956; Williams, 1956). In advanced stages of follicular development the granulosa cells may show pycnotic changes before any degenera

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PHYSIOLOGY OF GONADS

Fig. 7.4. Ovary from 28-moiith-old child. Many primordial follicles just beneath the tunica albuginea. (Courtesy of Dr. J. T. Bradbury.)

Fill. 7.5. Iimuaturc rat h grow to medium size. Many become atretic and leave stitial tissue. (Courtesy of Dr. R. M. Melampy.)

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Fig. 7.6. Intact immature rat given progesterone for 3 days. The various-sized follicles in this area are in early stages of atresia. The pycnotic granulosa cells are dispersing into the follicular fluid and the oocyte in the small follicle at the upper left is denuded. (Courtesv of Dr. J. T.Bradburv.)

tive clianges are evident in the oocyte. When vesicular follicles become atretic the granulosa disintegrates and the cells disperse into the liquor folliculi (Knigge and Leathern, 19.56 ( . If the follicle has developed a distinct theca interna, the theca regresses after the granulosa has disintegrated. In rats the atretic follicles leave no recognizable histologic remnants. In some species the theca regresses back to ovarian interstitial tissue (Dawson and AlcCabe, 1951; Williams, 1956). In the ovary of the human female and rhesus monkey the atretic follicle leaves a scar (corpus atreticum) in which the meml)rana propria persists for months as a folded hyaline membrane within the loose fibrous remnant of the theca.

The cause of follicular atresia is not known. Immediately after hypophysectomy there is a wave of atresia in the ovary of the immature rat (Fig. 7.5) and rabbit (Foster, Foster and Hisaw, 1937). In adult rats the postovulatory wave of follicular atresia has

l)een attrilnited to an action of the corjiora lutea (Atkinson and Leathem, 1946). Injections of androgen or of progesterone increase the incidence of atr(>sia in rat ovaries (Fig. 7.6) (Paesi, 1949b; Barraclough, 1955; Payne, Hellbaum and Owens, 1956). Further study is necessary to determine whether the atresia is due to a direct effect of androgen or progesterone on the follicles, or whether the postovulatory decline in gonadotrophins, like hypophysectomy, withdraws a supi:)orting influence and permits the follicles to degenerate. The supporting influence may be estrogenic because injections of estrogen at the time of hypophysectomy will prevent, or at least delay, the expected follicular atresia (Fig. 7.7). Some months after hypophysectomy the number of remaining oocytes is greater than in the ovaries of normal litter-mate sisters (Ingram, 1953). This suggests that vegetating oocytes are less liable to undergo

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PHYSIOLOGY OF GONADS

It:

'^l&^h

■ ■;%.%,

Fig. 7.7. iiniuaiKi. li.\ pupliy.sectomized rat treated with estrogen (dit'thylstilbe.Ntrol). Many follicles have developed to a size appropriate for antrum formation. The interstitium i.s atrophic but the theca is differentiated. One follicle is obviously atretic. (Courtesy of Dr. J. T. Bnidburv.)

atresia than those which have entered the growth phase.

Hisaw (1947) discussed the problem and marshaled a number of facts in support of the idea that atresia is due to a defective differentiation of the theca interna, with a ^resulting deficiency of estrogen which is considered necessary for growth and differentiation of the granulosa. Ultimately the ])ituitary is involved because the success which has been achieved in the j^roduction of sui)erovulation reveals that the number of follicles maturing and ovulating is a measure of the amount of gonadotrophic hormone. But it is eciually true that there is an optimal dosage and time beyond which defects appear in the form of cystic follicles and premature luteinization (see review by Hisaw and more recently Zarrow, Caldw(>ll, Hafez and Piiicus, 19581.

The disintegration of the discus proligerus of the nuiture follicle and the dissolution of the granulosa in an atretic follicle have led several investigators to suggest that the

stimulus to ovulation and/or atresia is identical (Harman and Kirgis, 1938; Dawson and McCabe, 1951 ; Moricard and Gothie, 1953; Williams, 1956). Moricard and Gothic found that intrafollicular injection of HCG or P]\1S caused first polar body formation within 4 liours. Control injections of estrogen or scrum were ineffective. Dempsey (1939) noted that maturation spindles were present in ne:iily all the oocytes in medium and large follicles which had undergone atresia shortly after ovulation had been induced by luteinizing hormone. Inasmuch as the tubal egg ntay give off the second polar body about the time the corona radiata is lost, it is interesting to speculate on the significance of the fact that the eggs in atretic follicles may also give off polar bodies just as they are denuded of granulosa (Fig. 7.8).

V. Hormones of the Ovary

The hoi'mones of the ovary are the estrogens, pi-ogesterone. androgen, and relaxin. The first three are the o\'ai"ian steroid hor

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Vu,. 7.S. Aticiic lolliric in immature rat gi\en progesterone for 3 day: and second metaphase si>indle. (Courtesy of Dr. J. T. Biadbury.)

mones and are among the most important hormones participating in the regulation of reproductive physiology. In the present hook, as in editions 1 and 2, the discussions of their many actions consititute one of the central themes.

Relaxin is a protein rather than a steroid hormone and has always been considered more or less apart from the latter. The steps in proving its existence were reviewed by Hisaw and Zarrow in 1950, and the present status of the subject is discussed in the chapter by Zarrow. Unlike the other ovarian hormones, its clinical value is still uncertain (Swann and Schumacher, 1958; Stone, 1959).

Ovarian androgens present a perplexing problem. Evidence for their production is abundant (Guyenot and Naville-Trolliet. 1936; Hill, 1937a, b; Deanesly, 1938a; Bradbury and Gaensbauer, 1939; Greene and Burrill, 1939; Chamorro, 1943; Price, 1944; Katsh, 1950; Desclin, 1955; Johnson, 1958). l)ut the extent to which they are produced l)y the ovaries of normal females, and the nature of their action in normal females are

uncertain (Parkes, 1950). It has been postulated that they are produced during the normal cycle in the rat. Payne, Hellbaum and Owens (1956) suggested that the interstitial androgens produced at estrus are responsible for the postovulatory atresia of the partially developed follicles. The production of ovarian androgens has been demonstrated most effectively under abnormal conditions of stimulation. The newborn rat ovary will produce androgens when stimulated by human chorionic gonadotrophin (Bradbury and Gaensbauer, 1939). The treated infant rat shows a marked enlargement of the clitoris and may even develop the cartilage anlage of the os penis. Female guinea pigs are masculinized by injections of HOG (Guyenot and Naville-Trolliet, 1936). Ovaries transplanted to the ear of the castrate male mouse will produce enough androgen to maintain the prostate and seminal vesicle (Hill, 1937a, b). Ovarian transl^lants into the seminal vesicle may exhibit a localized androgenic stimulation of that tissue (Katsh, 1950). Ovaries of a rat in

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PHYSIOLOGY OF GONADS

parabiosi;? with a castrate partner become hypertrophied and produce enougli androgen to stimulate prostatic tissue (Jolmson, 1958) ; associated with the condition is an unusual thecal and interstitial tissue hypertrophy. The masculinizing features of the Stein-Leventhal syndrome ( polycystic ovary) have been attributed to the presence of androgens, having their source perhaps in the hilar cells of the ovaries (Lisser and Traut, 1954) , but the action of other steroids with masculinizing properties also has been suggested (Fischer and Riley, 1952). The latter possibility might well be checked in any case when the production of androgens by the ovary is suspected.

Estrogen and progesterone are the ovarian hormones whose action in the female has been studied the most extensively. The steps which led to their extraction and chemical identification were described by Doisy and by Willard Allen in the 1932 and 1939 editions and will not be repeated here. Attention, however, will be directed to the isolation and identification of several naturally occurring gestagens in addition to progesterone (Davis and Plotz, 1957; Zander, Forbes, von Miinstermann and Neher, 1958). These are metabolic degradation products which still retain progestational activity.

Now as in the period 1932 to 1939, there are many unsolved problems, but it is equally true that much of interest and value has been learned. Not the least of these contributions has been the clarification of the l)athways of their biosynthesis (see chapter by Villeej . In the sections which follow particular attention will be given to the problem of their origin, to the rate of production, the manner of transport and storage, and to their "half-life."

A. CELLULAR ORTOIX

As Villee points out in his chajiter, estrogen and progesterone are apparently derived from cholesterol by a series of chemical changes. Within the ovary and in the corpora lutea of rats, there is a definite reduction in the concentration of ascorbic acid and cholesterol after gonadotrophin administration. These changes have been considered (ividence for tiie activation of hormone synthesis (Everett, 1947; Miller and Everett, 1948; Levin and Jailer, 1948; Aldman,

Claesson, Hillarp and Odeblad, 1949; Claesson, Hillarp, Hogberg and Hokfelt, 1949; Noach and van Rees, 1958).

Efforts to identify the cell types in which these processes take place have not been altogether successful. We have noted, for example, that several tissues such as testis,, placenta, and occasionally the adrenal cortex can produce estrogen. These are tissues,, then, which produce more than one hormone. Gardner emphasized this during a discussion of Parkes' (1950) review of androgenic activity of the ovary, when he called attention to Dr. Furth's observations that some ovarian tumors possess potentialities for bisexual hormone production. About the same time, Shippel (1950) postulated that thecal cells may be a source of estrogen and androgen and that the type of hormone produced may depend on particular stresses or stimuli. On the other hand, many investigators,, and particularly those interested in the apjilication of histocheraical procedures,^ have l^roceeded under the assumption that there are tissues of the ovary in which one hormone is iH'oduced predominantly. They found, for example, that the reactions of follicular granulosa and theca cells are strikingly different (Demi)sey and Bassett, 1943; Dempsey, 1948; Shij^pel, 1950). To be sure, the results w^iich have been obtained have not led to agreement with respect to details, but there is much evidence from the reactions which have been described, as well as from the older morphologic studies, that theca interna, interstitial, and luteal cells and, perhaps to a lesser extent, granulosa cells, are active in steroid hormone synthesis. What is less certain than the fact that these cells, and consequently the ovaries, produce estrogen, progesterone, and androgen is their relative role compared with that of other tissues. Presumably it is major; nevertheless, evidence for the extra-ovarian origin of estrogenic substances is provided by the occurrence of cyclic vaginal activity in ovariectomized animals (Kostitch and Telebakovitch, 1929; Mandl, 1951; Veziris,

"Dempsey and Bassett, 1943; Dempsey, 1948; Claesson and Hillarp, 1947a-c ; Claesson, Diczfahisy, Hillarp and Hogl)erg, 1948; McKay and Robinson. 1947: Sliii)iH>l. 1950: Barker. 1951: Rockenscliauh. 1951: Wliite, Heitis, Rock and Adams. 1951: Deane. 1«)52: Fuiulue-lm, 1954: Xisliizuka. 1954.

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1951) and by the high titer of estrogens in the urine from ovariectomized rats on a high fat diet (Ferret, 1950).

Corner, as long ago as 1938, in a consideration of the subject, emphasized that there is only circumstantial evidence that the ovary is the major site of estrogen production. Attempts to extract estradiol or any other estrogen from ovarian tissue had yielded very small amounts. MacCorquodale, Thayer and Doisy (1936) processed 4 tons of hog ovaries and recovered about 6 mg. estradiol from each ton. They estimated that the concentration in liquor folliculi was of the order of 1 part in 15,000,000 and that about 0.1 of this concentration is in the rest of the ovarian tissue. There are much better sources from which "ovarian hormone" can be extracted than from the ovary, i.e., placentas, pregnancy urine, the urine of the stallion or boar. The adrenal is also a source and there may be other tissues as well, for Bulbrook and Greenwood (1957) reported that urinary estrogen continued to be excreted after oophorectomy and adrenalectomy of a breast-cancer patient.

Whatever the relationships are quantitatively between the follicles and the other estrogen-secreting tissues, the evidence that the follicles are a major source of estrogenic substances remains impressive. This is also true of the corpus luteum. The human corpus luteum produces as much, or more, estrogen than was produced during the follicular phase. In the rat and mouse, estrogen production during the luteal phase must be low because more than 1 part of estrogen nullifies the action of 1000 parts of progesterone in these species ( Velardo and Hisaw, 1951). By contrast, ratios as high as 100 parts of estrogen to 1000 parts of progesterone enhance the progestational reactions in women (Long and Bradbury, 1951).

The different tissues of the ovary — membrana granulosa, theca interna, and interstitial cells — have been studied in efforts to ascertain whether they are sites of the production of specific hormones. Inasmuch as unruptured follicles and corpora lutea secrete both hormones, the two structures must be considered. It has long been known that corpora lutea secrete estrogen as well as progesterone. Evidence supporting the conclusion that jirogesterone is secreted by

the preovulatory follicle is more recent, but it comes from many sources. The possibility was first suggested following the discoveries that the beginning of mating behavior (Dempsey, Hertz and Young, 1936) and the decrease in tissue uterine fluid (Astwood, 1939) , which depend on the presence of small amounts of progesterone, coincide with the beginning of the preovulatory swelling. More recently, progesterone has been found in the follicular fluid from sows, cows, and the human female, and, in small quantities in blood plasma of the rabbit, human female, and rhesus monkey during the follicular phase of the cycle (Duy vene de Wit, 1942 ; Forbes, 1950, 1953; Bryans, 1951; Kaufmann, 1952; Edgar, 1953b; Buchholz, Dibbelt and Schild, 1954; Zander, 1954).

Earlier in this section it was noted that there is much evidence from both histochemical and the older morphologic studies that theca interna, interstitial tissue, and luteal cells, and to a lesser extent, granulosa cells secrete the ovarian steroid hormones. Many who have used histochemical methods still feel that, even though these methods demonstrate steroids and their precursors, the reactions are not sufficiently specific for identification of the individual hormones. Others, however, have been more confident, and when the evidence they have presented is combined with that reported in some of the more conventional morphologic studies the following summarization of opinion seems justified. Granulosa cells of the follicles and granulosa lutein cells of the corpora lutea contain progesterone or a precursor and secrete this hormone (Nishizuka, 1954; Green, 1955). Cells of the theca interna, theca lutein cells, and interstitial cells are believed to secrete estrogen and possibly androgen (Corner, 1938; Deanesly, 1938a; Pfeifter and Hooker, 1942; Hernandez, 1943; Claesson and Hillarp, 1947a, b; Rockenschaub, 1951 ; Aron and Aron, 1952; Furuhjelm, 1954; Nishizuka, 1954; Fetzer, Hillebrecht, Muschke and Tonutti, 1955; Johnson, 1958). The interstitial cells have been the object of much study and will be given especial attention.

Interstitial tissue or cells in the ovary is not as clear a concept as it is in the teste.-. In the latter interstitial or Leydig cells are derivatives of connective tissue elements

468

PHYSIOLOGY OF GONADS

irf^'t'H-'^

. 7.'.). Aii< ^lioii.-. ral)l)U. J^aigc lulliclf^ lUulcifioinK ati< Ma Iiilcr^l il luui i,> \i>ilil<- only as the narrow wedge of granular tissue extending from the cortex into the intrafollit-ular septum. (Courtesy of Dr. J. T. Bradbiuy.)

and can (ledift'erentiate to form connective tissue cells (Esaki, 1928; Williams, 1950). The role of Leydig cells as secretors of testicular androgen or a precursor is not (juestioned. In the ovary it is also presumed that undifferentiated connective tissue elements exist, indeed much of the stroma must be composed of such cells. It is believed rather generally, although unequivocal proof has not been given, that the theca interna is derived from connective tissue elements and that, as a component of the Graafian follicle, it secretes estrogen and possibly androgen Hoc. cit.). After ovulation and corpus luteura formation, and after atresia in the case of follicles not rui)turing, the cells of the theca interna may pcrhajjs I'csuinc their place as connective tissue cells or they may become interstitial cells (Mossman, 1937; Dawson and McCabe, 1955; Rennels, 1951 ; Nishizuka, 1954; Williams, 1956). The |)rominence of interstitial tissue varies from species to species and also with stages of the

rei)roductive cycle. Whether it is functional in i^roducing hormones has been controversial, but most contemporary investigators seem to feel that internal secretory capacity has been demonstrated. In all the work that has been done, supporting evidence is varied; in some cases it is circumstantial, but in others it is quite substantial.

Interstitial tissue is deficient in the anestrous rabbit, and even though there may be considerable follicular development, there seems to be little or no estrogen production (Claesson and Hillarp, 1947a) (Fig. 7.9).^ The liypei'ti'opliied iiitei'stitium of the estroiis i;il)l)it (Fig. 7.10) undergoes further de\-el()pnicnt (hii'ing prc'gnancy and seems almost as luteinized as th(^ corpora lutea

' Rogr('ssi\-e changes in tlic rciirodiictixe tract and accessory structures following ovariectomy of the anestrous opossimi were taken to indicate that these parts receive estrogenic stimidation of ovarian origin during th(^ anestnun (Morgan, 1946; Risman. 1946).

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469

Kid, 7,10. l'(isto\nl.-,l,,iy stigma is evident. Note tli of Dr. J. T. Bradburv.)

epithelioid natiii

of the hypertiopliied mterstitiuin. (Courtesy

(Fig. 7.11). Grossly the ovary in the anestrous rabbit is translucent whereas the estrous ovaries and the ovaries during pregnancy have a chalky white opacity due to the development of the interestitium. After hypophysectomy the interstitial cells of the rat ovary exhibit a deficiency condition and the nuclear appearance has suggested the name "wheel cells." If pituitary ICSH is administered, the deficiency cells are restored to normal (Fig. 7.12). Hyperplastic ovarian interstitium in older women has been considered a probable source of estrogen in some cases and of androgen in others. The stimulation of interstitium by injected gonadotrophins may be associated with the formation of estrogens and/or androgens (Bradbury and Gaensbauer, 1939; Marx and Bradbury, 1940). Some rats displayed a permanent estrus; others, during a period of androgenic function, were masculinized. During this period, the theca and interstitium were not luteinized in many cases and it was concluded from the responses of accessory organs that these small

immature cells had secreted male hormone and perhaps female hormone, too. In rats with fully luteinized theca and interstitium and the pronounced estrous symptoms, it was considered that the androgenic effect was no longer apparent. Information obtained recently, however, suggests that the permanent estrus, when it was shown, may have been a consequence of an androgenic effect. Cystic follicles which might have stimulated a permanent estrus had a vagina been present, were found in many adult guinea pigs which had received androgen prenatally (Tedford and Young, 1960).

Without necessarily excluding the possibility that the heterotypical hormone is also jiroduced, many articles contain suggestions that interstitial tissue has specific estrogenic or androgenic activity. There is the report that an ovarian interstitial-cell tumor was producing estrogens (Plate, 1957). The observation that estrogen continues to be secreted by ovaries in which the follicles have been destroyed by x-rays was reported by Parkes (1926b, i927a,^b), Brambell and

470

PHYSIOLOGY OF GONADS

V J ' *

k •'

6^i;>ir/

^ V

^,

Fi(. 7 11 ()\,ii\ liMiii jir. ^uaiii i.ililni l.amc luu uiiz. d < . IK m liiu. ih.niiiii ( uipu- liiii iim. Hvi)f'itioi)lue(l intPi-titium. Pninoidial t'olhcles in cortex. (Couitc^y of Di. J. T. Bra(U)iuy.)

Parkes (1927), Genther (1931), Schmidt (1936), Mandl and Zuckerman (1956a, b), and others. This conclusion w^ould seem to be .strengthened by the recent report that there is no intensification of the secretion of gonadotrophins by x-rayed rats in which there was an apparent destruction of the ova and folhcles (AVestman, 1958). Evidence of an entirely different sort for the secretion of estrogen by interstitial tissue has been i)resented by Ingram (1957). Autografts of medullary tissue containing interstitial tissue but no follicles were made in rabbits. Five animals from which this tissue was recovered had uteri which were not as atroi)hic as the uteri of spayed animals. He noted, however, that in the absence of the follicular apparatus the capacity to secrete estrogen is soon lost. As we have seen,- Ingram is one of several investigators who have related the functioning of interstitial tissue to granulosa elements.

Histochemical staining procedures for cholesterol indicated to Dempsey (1948) that the theca interna is a possible source of estrogen. The results obtained during a more extensive utilization of histochemical reactions in studies of the ovaries of nonpregnant, psoudopregnant, and pregnant rabbits, and in the ovaries of rats and guinea pigs were consistent with the conclusion that a li])i(l i)recursor of estrogenic substances is ]:)resent in interstitial tissue (Claesson, 1954; Claesson and Hillarji, 1947a, b; Claesson, Diczfalusy, Hillarp and Hclgberg, 1948).

Rennels (19511, on the basis of histochemical reactions in the oxaries of innnature rats, advanced the liypothesis that interstitial tissue has a dual origin. There is a primary type present between 10 and 18 (lays aftei- bii'th wliicli is dosc^ly associated with gi'anulosa ()Ut<j;i()wtlis and ingrowing cords of cells from the germinal epithelium. A secondary type is formed later from the

MAMMALIAN OVARY

471

-i:-* :^-"!r

t:^-^-!..

4"

jP^-'-^i'

Fig. 7.12. Immature hypoi)liyspftomized rat alter 3 days treatment with Armour's IC8H. The interstitium is restored and is mildly hyperplastic. Nearly all of the vesicular follicles are atretic. The theca blends into the interstitimn. Two of the oocytes contain maturation spindles. (Courtesy of Dr. R. M. Melampy.)

theca interna of atretic follicles. He presented no evidence for the production of estrogen by the latter tissue, but expressed the opinion that Claesson and Hillarp (1947b) had done so. Under the assumption that estrogen rather than androgen is produced by o^'aries of untreated rats during the early juvenile period (10th to 18th day), he interpreted the presence of histochemically reactive materials in the iirimary interstitial tissue as an indicator of estrogenic activity. To Huseby, Samuels and Helmreich (1954), the steroid-3/?-ol dehydrogenase activity in interstitial cell tumors having androgenic activity, suggested a relationship between the presence of this enzyme and the production of the androgen.

B. AMOUNTS OF HORMONE PRODUCED

Dependable estimates of the rate of estrogen and progesterone production would provide investigators of reproductive physiology with interesting and valuable information. It must be recognized, however, that there are many pitfalls and outright difficid

ties; consetiuently the estimates which have been made must be regarded as tentative. Furthermore, they are of limited value. The amounts produced probably deviate greatly from the quantities which are effective in meeting the threshold requirements of the tissues the ovarian hormones stimulate. This latter information would make the greater contribution to an understanding of the functioning of these substances in the regulation of rei^roductive processes. Most efforts to estimate the rate of secretion of estrogen have involved measurements of the amount of hormone given subcutaneously that will restore normal structure or function in ovariectomized animals. As Corner (1940) emphasized, estimates obtained in this manner are based on the assumption that a hormone injected once daily in an oil solution is utilized by the body as efficiently as a hormone produced by an animal's own ovaries. Gillman (1942), Barahona, Bruzzone and Lip.schutz (1950), and Zondek (1954) are among the many who have directed attention to the fact that the amount of an esiro

472

PHYSIOLOGY OF GONADS

gen required to e^'okc one response often is different from that required for a second response. For example, the amount of estradiol necessary to produce perineal enlargement in the baboon is less than that required to produce withdrawal bleeding. In the human female, the order of sensitivity of three tissues to estrogen is uterine cervix, vaginal mucosa, endometrium (Zondek, 1954). Theoretically, therefore, if the response of tissues is to be used in estimating the amount of hormone produced, the investigator should follow the response having the highest threshold value. Gillman also called attention to another complication. The minimal amount of estrogen necessary to produce a response does not necessarily approximate the amount being produced, because, in the instance he cites, larger amounts do not produce a larger perineum.

Another comi^licating circumstance is the occurrence of inherent" cycles (in some cases the length of a reproductive cycle) of responsiveness which have been demonstrated in ovariectomized females receiving constant amounts of estrogen from exogenous sources. Many such animals have displayed cyclic vaginal changes (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941a), uterine l)leeding (Zuckerman, 1940-41 ), and running activity (Young and Fish, 1945). This unknown factor must be taken into consideration in any attempt to estimate the rate of estrogen production. Existence of this factor gives emphasis to the importance of direct determinations, wiien they can be made, either from follicular fluid or from freshly drawn blood. Corresponding considerations would hardly be tliough of as applying to progesterone. Most, if not all, of its actions are synergistic or potentiating. Presumably, therefore, they are directly dependent, not so much on any changes in the inherent responsiveness of the tissues as on the extent to which the tissues have been conditioned or primed by the estrogen.

A part of the picture which must be brought into context with the problem of estrogen secretion comes from a review of the temporal factors in a normal cyclic animal. In animals with short estrous cycles (4 or 5 days in the rat, mouse, and hamster) . and in

species in which the cycles are longer as in the guinea pig it has generally been assumed that estrogen is produced maximally at the time of estrus. This is an assumption based on the simultaneous occurrence of the cornified vaginal smear and the display of estrous behavior. When one considers that 48 to 72 hours are necessary for vaginal epithelium to proliferate and then degenerate into cornified cells, it is obvious that the estrogen which starts these changes must be elaborated 2 or 3 days before estrus and therefore before the size of the follicles is maximal. Zondek (1940) demonstrated that if an immature rat was injected with HCG the ovaries could be removed 27 hours later and the rat would exhibit vaginal cornification in 84 to 96 hours. This emphasizes that the estrogen which caused the cornified smear had been elaborated 2 or 3 days before vaginal estrus (Zondek and Sklow, 1942; Green, 1956). The dilation of the uterus with fluid late in the proestrum (Astwood, 1939) is also evidence that the efi^ective estrogen had been elaborated 24 to 30 hours earlier.

Problems of assay are involved in any attempt to estimate secreted estrogen and are discussed briefly by Emmens (1950a, b). They are more serious in the case of the estrogens than in the case of progesterone. The bioassay of estrogens is usually based on vaginal cytology or change in uterine weight. The vaginal cytology or smear method is essentially the original method of Allen and Doisy(kahnt and Doisy, 1928; Allen, 1932). Ovariectomized rats or mice are given subcutaneous injections of the substance being tested for estrogenic i)otency. Smears are made of the vaginal contents 24, 48, 60, and 72 hours later. If the smear reveals the presence of cornified cells 60 to 72 hours after the first injection, the tested substance is judged to be estrogenic. By using groups of animals at each of several dose levels, the minimal eft'ectiA-e dose can be judged. The smallest amount of substance which will jiroduce cornified smears in 50 to 70 ]ier cent of the test grouj) is usually designated as the rat or mouse unit. Intravaginal tests, introduced by Berger (1935) and by Lyons and Templeton (1936), and refined, especially by Biggei's ( 1953) and by Biggers and Claringbold (1954, 1955), are 200 times more

MAMMALIAN OVARY

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sensitive than tliose in which the estrogen is administered subcutaneously. When the mean number of arrested mitoses was used as the measure of estrogenic activity, the sensitivity was increased another ten times ( Martin and Claringbold, 1958).

Immature rats or mice can also be used foi' the assay of estrogens. The establishment of vaginal patency and mucified or cornificd vaginal smear denote estrogen effects. Vaginal patency per se, however, is not specific for estrogen because androgen will also induce precocious vaginal patency (Rubinstein, Abarbanel and Nader, 1938; Marx and Bradbury, 1940). Injection of estrogen into immature animals causes a rapid increase in the weight of the uterus, due to water imbibition. Astwood (1938) found that the immature rat uterus increases in weight as early as 6 hours after an injection of estrogen; however, the optimal response was at 30 hours. Just above threshold levels graded doses produce graded increases in uterine weight. This makes it possible to plot a dose-response curve so that closer approximations of potency can be achieved. Some authors have used ovariectoraized animals for the uterine response method. This requires a prior operation and subsequent adhesions may make it difficult to strip out the uterus cleanly at the end of the test.

Whatever the test, each estrogen derivative or synthetic estrogen has an optimal assay interval for maximal effect depending in part on its solubility, rate of absorption, and utilization (Hisaw, 1959). For this reason a standard assay may not be an accurate indicator of the estrogenic potency of several comijounds. This factor plus some competitive antagonism make this method impractical for the assay of mixtures of estrogens (Merrill, 1958). Whatever their faults, the bioassay methods in general are very sensitive and will detect estrogens in 0.1 to 1.0 ixg. quantities.

The chemical assay methods for estrogen are rather involved and have usually been relial)le only in milligram quantities. Fluorometric methods were tried and generally discarded because frequently small amounts of contaminants were strongly fluorescent; consequently fluorescence was being obtained in the absence of biologic activity

(Bitman, Wrenn and Sykes, 1958) . Recently paper chromatographic methods have permitted sufficient purification and isolation to make identification and quantification of estrogens in microgram quantities (Brown, 1955; Smith, 1960; Svendsen, 1960).

The most common bioassay methods for progesterone are still the Corner-Allen and the Clauberg tests which utilize the rabbit. The animal is primed with estrogen and then given progesterone after an appropriate interval. A portion of the uterus is removed and examined histologically for the degree of glandular develojiment in the endometrium. The test is relatively insensitive since it requires about 1 mg. progesterone per rabbit. McGinty, Anderson and McCullough (1939) increased the sensitivity of the test to 0.5 to 5.0 /xg. by injecting the progesterone into the lumen of an isolated segment of the rabbit uterus. The histologic response of the endometrium in this isolated segment was then judged.

Hooker and Forbes (1947) adapted the McGinty intra-uterine technique to the uterus of the ovariectomized mouse. The end result is judged histologically by the characteristics of the endometrial stromal nuclei of the isolated uterine segment. The sensitivity is of the order of 0.3 fxg. per ml. and the method has been used widely. This advantage of the Hooker-Forbes technique is that it is sensitive enough to detect gestagens in l)lood plasma and liquor folliculi. Disadvantages are that the test is not specific for progesterone, and that certain gestagens such as 17-a-hydroxyprogesterone which is devoid of progestational activity in some species (rabbit, guinea pig, man) are very active in the mouse test (Zarrow, Neher, Lazo-Wasem and Salhanick, 1957; Short, 1960).

There are spectrophotometric techniques for jn'ogesterone assay (Reynolds and Ginsburg, 1942; Zander and Simmer, 1954; Short, 1958; Sommerville and Deshpande, 1958 ) . These methods have the advantage of instrumental precision, but require rather tedious initial chemical purification. However, they have proved of especial value in comparative studies of the blood levels of progesterone in sheep with active and inactive ovaries, in studies of the progester

474

PHYSIOLOGY OF GONADS

one content of unruptured follicles in the ovaries of cows and sows, and in determinations of the cjuantities of progesterone secreted by 1 or 2 corpora lutea in sheep ( Edgar, 1953a, b; Edgar and Ronaldson, 19581.

After allowance was made for these considerations tentative estimates were given: rhesus monkey, 200 I.U. or 20 y estrone daily; human' female, 3000 I.U. or 300 y estrone daily (Corner, 1940); baboon {Papio porcarius) , somewhat more than 0.04 mg. estradiol benzoate daily (Gillman, 1942) ; guinea pig, less than the equivalent of 1.8 fjig. estradiol daily (Barahona, Bruzzone and Lipschutz, 1950). The variation in the responsiveness of individual animals was recognized by all the investigators. Two important variables were not considered, the cyclic growth of follicles, and the number of developing follicles. Presumptive evidence that the amount of secreted estrogen increases as the follicle enlarges was provided by the demonstration that more and more estrogen is required to maintain perineal turgescence (Gillman and Gilbert, 1946). The number of developing follicles may vary greatly within a species, in the guinea pig, for example, from 1 to at least 6. The fact that two corpora lutea in the ewe do not produce more progesterone than one (Edgar and Ronaldson, 1958) could prepare us for a corresponding finding with respect to estrogen.

The problem of estimating the rate of progesterone secretion is l)eset by many of the difficulties that confront an investigator attempting to estimate the rate of estrogen production, but one circumstance especially has facilitated progress by those especially interested in progesterone. It is that the amount of excreted free prcgnanediol or excreted sodium pregnanediol glucuronidate is about 1/7 the amount of injected progesterone (Trolle, 1955a, b) ; from determinations of cither of tlic former, therefore, the amount of the latter can be estimated with what is believed to be a reasonable degree of accuracy. The pioneer attempt of Corner (1937) to calculate the amount of progesterone secreted by the rabbit, sow, and human female resulted in estimates (60 mg. during the luteal phase of the cycle in the latter) wliicli are much lower than those made more recentlv lObei

and Weber, 1951, 200 mg.; Kaufmann, 1952, 200 mg.; Trolle, 1955a, b, 260 to 440 mg.). It now seems that the lower estimate made by Corner can be attributed to the uncontrolled loss of sodium pregnanediol glucuronidate during storage, owing to bacterial hydrolysis (Trolle, 1955a). The rise in the 24-hour values after ovulation, the attainment of a peak the 7th and 8th days, and the decline between then and menstruation, are shown nicely in Trolle's (1955a) study. His data provide an excellent confirmation of the estimates based on structural changes within the cell (Brewer, 1942; Corner, .Jr., 1956). The amount of free pregnanediol excreted during the cycle and therefore the amount of secreted progesterone varied from woman to woman, and in the same woman there was variation from one cycle to another.

Data obtained by Duncan, Bowerman, Hearn and Alelampy (1960) from their chromatographic study have provided the basis of an estimate that the following average amounts of jn-ogesterone are present in the luteal tissue from swine: 23 /Ag. on day 4 of the cycle; 213 /^g. on day 8; 335 yug. on day 12; 311 [xg. on day 16; /xg. on day 18. From day 16 to day 102 of pregnancy, the amount rose from 477 [xg. on day 16 to 578 fxg. on day 48 and then decreased to a low of 120 ixg. on day 102.

Edgar and Ronaldson (1958) made direct measurements of the progesterone in lilood collected from the ovarian vein in ewes. The assay method consisted of extraction of 20 ml. of blood by, and partition between, organic solvents, final separation by chromotographic i)artition on filter paper, and subsequent estimation of the hormone by ultraviolet absorjjtion spectroscopy. They reported that there is great variability from animal to animal. The concentration in yearling sheep was not lower than that in older animals. Because of the liypothesis advanced by Young and Yerkes ( 1943) that the amount of secreted progesterone is low in adolescent chimpanzees, an extension of the Edgar and Ronaldson jirocedures to |)iimates would be of interest. In this connection, Edgar and Ronaldson postulated what has been brought out as a generalization in so many studies of the steroid hormones. The absolute amount of progesterone

MAMMALIAN OVARY

475

circulating in the fluids of the body may be less important than the minimal amount. The ewes secreting less than the minimum may be unable to maintain pregnancy, whereas those secreting more may simply have surpluses which are of little significance. The same principle may be extended to the human female (Davis, Plotz, Lupu and Ejarque, 1960 ».

An observation new to the reviewer could l)e important. When two corpora lutea were present in one ovary the concentration of I)rogesterone was in the same range as that for the ewes with one corpus luteum (Edgar and Ronaldson, 1958). In another ewe there were 2 corpora lutea in one ovary and 1 in the other, but the concentrations in the blood from the 2 ovarian veins were almost the same.

A facet of the problem of the rate of production of ovarian estrogen and progesterone which has become apparent is that endogenously and exogenously administered estrogen (Rakoff, Cantarow, Paschkis, Hansen and Walkling, 1944; Pearlman, 1957) and progesterone (Haskins, 1950; Zander, 1954; Rappaport, Goldstein and Haskins, 1957; Davis and Plotz, 1957; Plotz and Davis, 1957; Pearlman, 1957; Cohen, 1959) disai)i:)ear from the blood very cjuickly, in the human female and in such laboratory mammals as the dog, rabbit, and mouse. Zander, for example, injected 200 mg. of progesterone intravenously into menopausal and ovariectomized women. The concentration of this hormone in the blood was 1.44 /xg. per ml. after 3.5 minutes and 0.116 yu,g. per ml. after 2 hours; 24 hours after the injection progesterone could not be found by the method he employed. The data obtained by the other investigators were similar.^ Using some of these data obtained from reports in the literature and from his own studies, Pearlman (1957) divided the total amount of circulating hormone (M) by the

■'To a cpitain extent, and possibly to a considerable extent, the rapid disappearance of progesterone from the blood is explained by its storage in the fat tissue of the body (Davis and Plotz, 1957; Davis, Plotz, Lupu and Ejarque, 1960). Following intramuscular injection of C"-4-progesterone, and assuming an even distribution of radioactivity in the fat of the body, about 17.7 per cent, 33.7 per cent, and 19.6 per cent of the administered dose was present 12, 24, and 48 hours, respectively, after the administration of the labeled hormone.

endogenous production rate (r) as a means of obtaining the turnover time iT), i.e., the time refjuired for a complete replacement of the circulating hormone by a fresh supply from the endocrine gland. His method was not free from criticism by discussants; nevertheless, informative estimates were made. The turnover time of the various estrogens was calculated to be about 6 minutes or less, that of progesterone, about 3.3 minutes.

Not unrelated to the i)roblem of the amounts of hormone produced is the sul)ject of plasma (and erythrocyte) binding of the ovarian hormones. Especial attention was given the subject by Rakoff, Paschkis and Cantarow ( 1943 ) who reported that as much as 50 per cent of the total estrogen content of the serum of women is present in a combined or conjugated (bound) form, and that almost all of the estrogens of pregnancy are bound to the protein fractions of the serum. Shortly thereafter, Szego and Roberts (1946) reported that two-thirds of the total estrogen in the blood in human l)lasma is normally associated with jn-otein constituents, and in a subsequent series of publications (Roberts and Szego, 1946, 1947; Szego, 1953, 1957; and others) that the liver is the site of the formation of the protein-estrogen complex or estroprotein. The nature of the complex soon become controversial and has not yet been resolved (Eik-Nes, Schellman, Lumry and Samuels, 1954; Antoniades, McArthur, Pennell, Ingersoll, Ulfelder and Oncley, 1957; Sandberg, Slaunwhit(> and Antoniades, 1957; Daughaday, 1959). In the jiresent context, however, othei- considei'ations are more important.

Protein-liinding is not confined to the estrogens and their metabolites, but other steroidal hormones, progesterone, testosterone, and corticosteroids, are also present in the blood in a bound-state. In studies of the binding relationships of serum albumin, the link to the esti'ogens was found to be strongest, that to the corticosteroids relatively weak, and that to ])rogesterone and testosterone intermediate (Sandberg, Slaunwhite and Antoniades, 1957; Slaunwhite and Sandberg, 1958; Daughaday, 1959). The i;lationships in the case of other components of i^rotein mixtures have been sliown to be

47()

PHYSIOLOGY OF GONADS

different, but they are ai)parently eriually specific (Daughaclay, 1959; Slaunwhite and Sandberg, 1959). A considerable specificity of the binding sites may be involved ( Sandberg, Slaunwhite and Antoniades, 19571. Daughaday (1959) states that separate binding sites may exist for each of the steroid hormones studied, and Szego (1957) suggested that a competition for these sites may be the basis for antagonisms which are known to exist in many steroid interactions (Courrier, 1950; Hisaw and Velardo, 1951; Roberts and Szego, 1953; Velardo, 1959; Velardo and Hisaw, 1951 ; Zarrow and Neher, 1953).

The most important consideration has to do with the significance of protein binding for the steroid hormones, and, in the present chapter, the significance for estrogen and progesterone. Roberts and Szego (1946, 1947) and Szego (1957) proposed that formation of the estrogen-protein complex is necessary for the transport and activity of endogenous and exogenous estrogens. Riegel and Mueller (1954), on the other hand, found that the protein-estrogen complex they used had only a slight, if any, estrogenic activity, and Daughaday (1958, 1959) expressed the opinion that the unbound steroid hormones of the plasma are probably the biologically significant moieties. He suggested that the degree of protein binding imposes a major restraint on the passage of hydrocortisone (and presumably other steroids) through the cajiillary membranes, but pointed out that this view has not yet been established. He then asked, in the event that the steroid-protein complex does not function in the transi)ort of hormones from the vascular component to the cell, is it likely that the presence of a steroid-protein complex stabilizes the pliysiologically significant concentration of unbound steroid very much as buffer salts stabilize the small concentration of hydrogen ion? In this way, he continued, the organism would l)c protected against the rapid changes in concentration which characterize an unbuffered system.

At tiic ]ir(>sent stage in this controversial sul).iect, any hypothesis with res])ect to the significance of the protein binding of steroid hormones must be tentative. It would seem, however, that whatever emerges will have

validity only if it is compatible with the cyclic waxing and waning of reproductive phenomena. If the unbound, rather than the bound fractions, are the active fractions, the functioning of the ovarian steroid hormones must dei)end on the presence of unbound fractions, in some way made available at cyclic intervals to the tissues on which these hormones act. It would seem, too, that the significance of the increased capacity for l)inding in i)rcgnancy (Rakoff, Paschkis and Cantarow, 1943; Baylis, Browne, Round and Steinbeck, 1955; Daughaday, 1959; Slaunwhite and Sandberg, 1959) should be a ])art of the picture. Tentatively, this greater binding capacity on the part of the ])regnant adult, coupled with an inability of the developing fetuses to bind androgens, might account for the failure of the adult to be affected by the presence of androgen at a time when the genital tracts and neural tissues of the female fetuses she is carrying are undergoing profound modifications (Phoenix, Goy, Gerall and Young, 1959; Diamond, 1960).

VI. Age of the Animal and Ovarian Functioninij

The position of the ovary is such — at one and the same time being dependent on the pituitary, possessing its own varying capacity to function, and having an effectiveness which is limited by the responsiveness of the tissues on which its hormones act — that no simple consideration of the relationshii) between the age of the animal and ovarian functioning can be given. An investigation, therefore, should be planned accordingly and we find experiments in which the amount of gonadotrophic stimulation was varied when age was constant, and experiments in which age was the variable and the amount of gonadotrophin the constant. If hypo- or hyper-responsiveness of the tissues is suspected, the point can be checked by the use of spayed animals given variable amounts of ()\-arian hormones. When information of these sorts is brought together, a fairly accurate account of the relationship between age of the animal and ovarian activity can l)e ])repared.

The results fi'oni many studies have revealed that the ovaries in both inunature and senescent females are potentially able

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to secrete hormones, both estrogen and progesterone, in amounts which are in excess of those secreted by untreated animals. The secretion of these hormones was elevated in rats, mice, and hamsters by the implantation of whole pituitaries (Smith and Engle, 1927 » or by the administration of chorionic gonadotrophin (Price and Ortiz, 1944; Ortiz, 1947; Green, 1955). The reactivation of senile ovaries was first demonstrated by Zondek and Aschheim (1927) following the insertion of hypophyseal imjilants, and later by numerous other investigators listed in the review of the subject l)y Tlmng, Boot and Miihlbock (1956). In more recent experiments an enhanced secretion of estrogen and progesterone followed the injection of old hamsters with chorionic gonadotrojihin (Peczenik, 1942; Ortiz, 1955).

In women fertility may be lost before the menopause (Engle, 1955). Studies in progress at Iowa (Bradbury, personal communication) show that urinary gonadotroi')hins may be elevated before the menopause and the last ovarian cycles are achieved in the presence of excessive amounts of pituitary gonadotrophin. As a rule the human ovary is devoid of oocytes and produces relatively little estrogen at the time of the menopause. Frequently, however, there is enough residual ovarian activity (estrogen production) to maintain the vaginal epithelium for 10 to 15 years after the menopause. These observations on women suggest that as the supply of oocytes l)ecomes depleted, less estrogen is produced and more gonadotrophin is released to stimulate the aging ovary. This secjuence is in harmony with the concepts of Dubreuil (1942) and Hisaw (1947), because with fewer areas of granulosa there would be fewer centers of organizer to bring al)out the differentiation of thecal tissue competent to produce estrogen.

Ovarian stromal hyperplasia has been found in association with endometrial hyperplasia after the menopause (Morris and Scully, 1958). Sherman and Woolf (1959) suggested that the postmenopausal ovary may produce abnormal sexogens which bring about an endometrial proliferation and ultimately adenocarcinoma of the endomi'trium. Their urinarv l)ioassay studies

indicate that the patients were excreting ICSH-type gonadotrophin. The observation has been made at Iowa that a few postmenopausal women with endometrial carcinoma were maintaining an estrogenic vaginal epithelium when they were ovariectomized at ages varying from 65 to 70 years. Subsequently the gonadotrophin excretion increased to the ciuantities usually seen after the menopause. In these unusual cases the aging ovaries produce estrogen, or possibly estrogen and androgen, in quantities sufficient to suppress the usual excess production of gonadotrophins.

The responsiveness or sensitivity of the ovary to gonadotrophic stimulation is not constant throughout the life of an individual. If we may judge from the studies of Corey (1928) and Selye, Collip and Thomson (1935) on newborn and 10- to 15-day-old rats, Moore and Morgan (19431 on young opossums, and Price and Ortiz (1944) and Ortiz (1947) on rats and hamsters, the prepubertal period is characterized by very rapid and great increases in responsiveness to gonadotrophic stimulation. Species differences are great. The opossum ovaries do not respond to gonadotrophic stimulation until about 100 days of age (Moore and Morgan), whereas responsiveness was first detected in the rat ovary at 4 to 10 days (Price and Ortiz) and in the hamster ovary by the 10th day (Ortiz). Such data, coupled with the appearance of the ovaries at birth, would seem to exclude the possibility of gonadotrophic stimulation during the prenatal period, and perhaps the capacity for being stimulated as well.

Certain other species are different and present problems. There is an extensive follicular development and luteinization in the fetal ovaries of the giraffe which is the basis for the suggestion that the ovaries of this species are responsive to gonadotrophin before birth (Amoroso, 1955). Such a conclusion is predicated on the assumption either that serum gonadotrophin crosses the placental membrane or that the fetal pituitary secretes gonadotrophin. Neither hypothesis has been proved. Evidence exists that the ovaries of the horse and seal are strongly stimulated before birth (Cole, Hart, Lyons and Catchpole, 1933; Amoroso,

47!

PHYSIOLOGY OF GONADS

Harrison, Harrison-Matthews and Rowlands, 1951; Amoroso and Rowlands, 1951), but an unusual structural condition is found in these ovaries. No vesicular follicles are present and the ovaries, which are larger than those of the adult, are composed mostly of interstitial tissue which is enclosed by a thin cortex containing short chains of germ cells and a few oocytes surrounded by a single layer of epithelial cells. Comparable information does not exist for the seal, but in the horse the development of this condition is reached during the estrogenic phase and after the gonad-stimulating hormone is no longer detectable in the blood of the pregnant adult. As a result, and cjuite apart from the belief that serum gonadotrophin does not cross the placenta (Amoroso and Rowlands, 1955 j, the massive interstitial tissue liyperplasia is thought to have been stimukited by estrogenic rather than by gonadotrophic action.

The immediate jiostpubertal jieriod and middle age are periods of relative stability. The period of old age has been too little studied and is in need of attention. In old mice the ovaries are reported to become unresponsive to exogenous gonadotrophin (Green, 1957). Ortiz (1955), on the other hand, stated that although a certain degree of ovarian sensitivity is lost in old hamsters, there is a surprising degree of responsiveness present until death, not only after the animal is no longer fertile, but even in animals with ovaries almost completely atrophic.

In young animals and in old animals there are irregularities of ovarian function and irregularities in the character of the cycles which probably can be related to imbalances in the pituitary-ovarian relationship. In polytocous sjK'cies, fewer follicles ovulate in young animals (Young, Dempsey, JMyei-s and Hagquist, 1938; Ford and Young, 1953, the guinea pig; Perry, 1954, the domestic pig; Ingram, Mandl and Zuckerman, 1958. the mouse and rat), and in old animals (Perry; Ingram, Mandl and Zuckerman). These statements of fact, iiowcx-cr. do not reveal what is presumed to be more important. In young and old animals the nature of the irregularities, particularly those of ovarian function, seem to differ. Evidence collecte(l fi'om rhesus monkeys (Hartman,

1932) and chimpanzees (Young and Yerkes, 1943) suggests follicular growth without ovulation and luteinization, or in the guinea pig a sluggishness of follicular growth which is followed by ovulation and the formation of functional corpora lutea (Ford and Young, 1953) . In old animals there may also be abnormalities of follicular growth, but as the numerous reports are read, the impression is given that abnormalities of luteinization are more prominent (Deanesly, 1938b; Wolfe, 1943; Wolfe and Wright, 1943; Loci), 1948; Thung, Boot and Miihlbock, 1956; Dickie, Atkinson and Fekete, 1957; Green, 1957). Additional investigation will be necessary before we can be sure that the pituitary-gonadal imbalance in young animals differs from that in old animals. On the whole, the possibility seems to have received little attention, but its importance justifies more careful study.

VII. Other Endocrine Glands and the Ovaries

A. THYROID

The relationship of the thyroid to the functioning of the ovaries was one of the first subjects of modern endocrinologic investigation. Notwithstanding, disappointment must be expressed that after more than 50 years of effort, little more than cautious generalization is possible. This admission is not a confession of defeat; to be sure, there is an unfortunate number of uncertainties, but we have come to know what is necessary in the way of experimental design and techniques to enable us to proceed with the confidence that a gratifying clarification can be achieved. The greatest obstacle could be, not the lack of means, but rather the failure to use the means which are alnmdantly at liand for more coordinated eft'orts than many which lia\e cliaracterized this field in the past.

The general l)elief that the thyroid is invoh-ed in reproductive function is grounded in two categories of observations. The first includes those demonstrating that ovarian lioi'inones exert an action on the thyroid. 'I'lieic lia\-e been many I't'ports that in the human female the thyi'oid enlarges at puberty, at menstruation, and during pregnancy (Gamier, 1921; Marine, 1935; Neu

MAMMALIAN OVARY

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mann, 1937; and others). Modern counterparts are the reports of the increase during pregnancy in the concentration of serum precipitable iodine (Heinemann, Johnson and Man, 1948; Dowling, Freinkel and Ingbar, 1956a; Tanaka and Starr, 1959), in serum thyroxine (Danowski, Gow, Mateer, Everhart, Johnson and Greenman, 1950), and in the accumulation of radioiodine (Pochin, 1952). Some conflicting reports should be noted. There was said to be no consistent alteration in the concentration of serum precipitable iodine in oophorectomized women (Stoddard, Engstrom, Hovis, Servis and Watts, 1957), and Pochin (1952) found no detectable variation in P^^ uptake during the menstrual cycle in 5 women he studied. Comparable observations have been made on laboratory mammals (Greer, 1952; Soliman and Reineke, 1954; Soliman and Badawi, 1956; Feldman, 1956a) and the baboon, Papio ursinus (Van Zyl, 1957), except that Brown-Grant (1956) could not agree from his findings in the rat and rabbit that the level of gonadal function exerts any striking influence on thyroid activity in the normal experimental animal.

In man (Engstrom, Markardt and Liebman, 1952; Engstrom and Alarkardt, 1954; Bowling, Freinkel and Ingbar, 1956b) and in laboratory mammals (chiefly the rat) (Money, Kraintz, Eager, Kirschner and Rawson, 1951; Feldman, 1956a; Feldman and Danowski, 1956) the enhancement of thyroid activity is attributed to the level of circulating estrogen, whether it be endogenous or exogenous in origin. On the other hand, many who have worked with laboratory mammals have not found evidence of augmented thyroid activity, and not infreciuently decreases were reported (see Paschkis, Cantarow and Peacock, 1948; and the numerous articles cited by Farbman, 1944; and Feldman, 1956a). The conflicting results may perhaps be accounted for by the circumstance that the response of the thyroid seems to be related to the duration of the estrogen treatment and to the estrogen that was used. Decreases in thyroid activity have been reported when the estrogen treatment was prolonged (Feldman, 1956a), and Money and his associates showed clearly that estrone and some other

components increased the collection of P-^^ by the thyroid of rats whereas estradiol, estriol, and diethylstilbestrol decreased the collection. ]\Iany attempts have been made to ascertain the nature of the mechanism whereby the effective estrogenic substances exert their action on the thyroid (Noach, 1955a, b; Feldman, 1956b; Dowling, Freinkel and Ingbar, 1956a, b; Bogdanove and Horn, 1958). but they are so varied and speculative that they will not be reviewed here.

The second category of observations related to the thyroid and ovarian functioning includes those in which there is evidence of action of thyroid hormone on the ovary. Reviews of this work are contained in the articles by Peterson, Webster, Rayner and Young (1952), Hoar, Goy and Young (1957), and Parrott, Johnston and Durbin (1960) and most of their citations of work done on the relationship of the thyroid to the ovary will not be repeated here. As they point out, many investigators have reported that thyroidectomy is followed by ovarian degeneration, arrested folliculogenesis, and failure of ovulation. Irregularity of the reproductive cycles was common and much of this in the guinea pig could be attributed to retarded and sporadic follicular development (Hoar, Goy and Young, loc cit.) . The latter investigators gave especial attention to the condition of the ovaries in their hypothyroid guinea pigs. In 10 pairs from thyroidectomized animals (oxygen consumption and heart rate were depressed) follicular development was good in the sense that the follicles appeared healthy, but a generation of corpora lutea was missing in four. This absence of corpora lutea, which is not seen in normal adult guinea pigs, was believed to be a consequence of the involution of the older generation during the longer than normal interval between ovulations. It is considered significant in terms of the functional capacity of such ovaries, that although the percentage of sterile matings was higher than in the controls, that, in the course of the two studies at Kansas, 29 of 38 matings were fertile. This experience may perhaps account for the many reports (cited in tlu papers from the Kansas laboratory) that thvroidectomv or treatment with antithv

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PHYSIOLOGY OF GONADS

roid drugs have no, or at the most rehitively little, effect on the ovary. To these, several additional reports should be mentioned. In thyroid-deficient female mice, fertility and litter frequency were affected only to the extent that the estrous cycles were prolonged (Bruce and Sloviter, 1957). In the rabbit, thyroidectomy did not interI'upt or alter the periodicity of follicular development, but it did eliminate the final stages (Desaive, 1948). Parrott, Johnston and Durl)in (1960) express the opinion that the long i)hysiologic life of thyroid hormone may account for many of the contradictions in the reports of the relationship between thyroid deficiency and reproduction.

Except as it is speculative, an unexplained action of thyroidectomy or the administration of goitrogenic drugs is the augmentation of the ovarian response to gonadotrophins and to anterior pituitary im])lants (citations in Peterson, Webster, Rayner and Young, 1952; and see in addition Janes, 1954; Janes and Bradbury, 1952; Kar and Sur, 1953). Thyroid substances, on the other hand, were inhibitory. Of alternative hyjiotheses, Janes favored the suggestion that during the period of propylthiouracil treatment, provided it was short rather than long, there was an accumulation of gonadotrophin in the blood and the ovarian response varied for some unknown reason according to the concentration of this latter substance in the body fluids. To Kar and Sur (1953) direct involvement of the hypophysis could be eliminated; instead a direct role of the thyroid seemed more plausible. They postulated that the absence of thyroid hormone reduced the utilization of gonadotrophic hormones by the ovary.

The reported effects of the hyi)erthyroid state or of administered thyroid hormone on the ovary are equally conflicting. The ovaries are described as being atrophic or exhibiting incomplete folliculogenesis, or as being essentially normal or even hypertrophied (citations in Peterson, Webster, Rayner and Young, loc. cit., Hoar, Gov and Young, loc. n't.). Irregular cycles are said to have occuitcmI in the rat and mouse. but no irregularity was detected in guinea pigs given thyroxine.

A tentative explanation can be given for

the many divergent reports of the relationship between the thyroid and the ovary, divergencies which are found in the clinical literature as well as in laboratory studies. In doing so, we will recall that there is abundant evidence that the ovary is a locus of action of thyroid hormone. The action may not be directly trophic, as is that of the pituitary, but it is assumed to be su])l)ortive, jiossil)ly directly so. or jiossiljly indirectly through regulation of the general metabolic level. Whatever its nature, there must be great interspecies and even intraspecies variation in the need of the ovary for such action. In addition, within a species there appears to be a wide range of tolerance, for Peterson, Webster, Rayner and Young (1952) found in their study, in which the thyroid state was estimated from measurements of oxygen consumption and heart rate, that reproduction occurred in females in which oxygen consumption ranged from an average of 50.0 to 93.5 cc. per 100 gr. i)er hr. (52.9 in the controls), and heait rate from 238 to 316 beats per minute (272 in the controls). In females that failed to rejiroduce, the lowest values were lower than in the animals which did reproduce; nevertheless, there was much overlai)i)ing, for in this group oxygen consumption ranged from an average of 46.7 to 94.1 cc. per 100 gr. l)er hr., and heart rate from 202 to 330 beats j)er minute. Within sucli a framework, there are bound to be more divergent results than when normal functioning depends on nioi'e narrowly circumscribed conditions, and the failure to replicate a result does not have the same significance. As a part of the investigation of sucli a problem, more and better correlated infoi'niation is re(|uiicd, and this could be the most pressing ne('(l in the field of oxarian (and icproducti\-cl functioning and the thAM'oid.

B. ADRENAL CORTEX

Tlu^ adrenal cortex elaborates its steroid )nn()iu's in a biosyiU lictic scciuencc \H'ry

siiiiilai' to that in the o\aiy. In fact, i)rogcstci'onc is an intermediate substance in the synthesis of glucocorticoids. Estrogen has been found in extracts of adrenal cortical tissue, but whether it represents a degradation pi'oduct within the adrenal or an artifact resulting from the chemical i)i'o

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cedurcs is not clear. Occasionally adrenal tumors produce physiologically significant amounts of estrogen, but normally the adrenal production of estrogen, if any, is not of physiologic significance. The atrophy of the female genital tract after bilateral ovariectomy suggests strongly that this is so.

The major hormone of the adrenal cortex, hydrocortisone or corticosterone, depending on the species, has a profound effect on protein and carbohydrate metabolism. An overproduction, as manifested by Cushing's disease, results in a wasting of body protein and other metabolic disturbances which by nonspecific influences tend to reduce gonadal function. Similarly a loss of adrenal function (Addison's disease) leads to anemia, electrolyte imbalance, and hypoglycemia. There is usually a decrease in ovarian function but some Addisonian patients have conceived and carried their pregnancies with only sodium and fluid supplements.

There is a hereditarv metabolic defect

of the human adrenal which renders it defective in i)roducing hydrocortisone. This is the adrenogenital syndrome. In these jiatients the adrenals produce excessive amounts of intermediate products which are excreted in the urine. Some of these compounds are androgenic 17-ketosteroids and may cause virilization (Bradbury, 1958). These androgens tend to inhibit the gonadotrophic activity of the pituitary and leave the ovaries unstimulated and infantile (Fig. 7.13). Replacement therapy with corticoids reduces the adrenocorticotrophic hormone ( ACTH) activity of the pituitary and then the adrenal production of androgen ceases. This then permits the pituitary to stimulate normal cyclic activity in the ovaries (Fig. 7.14). The adrenogenital syndrome thus has a profound effect on ovarian function which is specific through its production of androgen. More complete descriptions of the condition and of the rationale of treatment have been prepared by Wilkins ( 1949) ,

CHOLESTEROL

PREGNENOLONE

PROGESTERONE

HYDROXYPROGESTERONE

Fig. 7.13. Scliematic representation of the interaction of the adrenal and the ovary in the adrenogenital syndrome. The process of hormone biosynthesis is defective in the adrenal (BLOCK) and the degraded by-products (17-ketosteroids) being androgenic suppress the formation of gonadotrophins (GTH). (Courtesy of Dr. J. T. Bradbury.)

482

PHYSIOLOGY OF GONADS

CHOLESTEROL

CORTISOL

CHOLESTEROL

GONAD

\

ANDROGEN ESTROGEN

PREGNENOLONE

PREGNENOLONE

ADHeNAL

^

\

17-HYDROXYPROGESTERONE

PROGESTERONE

PROGESTERONE

17-HYDROXYPR06ESTER0NE

Fit. 7 14. Tieatnieiit witli (oiti>one (or other glucocorticoid) reduces ACTH production and adrenal hormone .synthesis subside-^. This permits the normal pituitaiy-gonadal interactions to be established. (Courtesy of Dr. J. T. Bradbury.)

A\'ilkins, Crigler, Silverman, Gardner and Migeon (1952), and Bradbury (1958). Milder categories of what is believed to be adrenal cortical hyperplasia have also been described and are responsive to treatment with cortisone. They are characterized by amenorrhea or oligomenorrhea, hirsutism, slightly elevated 17-ketosteroid excretion values, and difficulty in becoming pregnant (Jones, Howard and Langford, 1953; Jefferies, AYeir, Weir and Prouty, 1958; Jefferies, I960). Indications arc that these abnormalities, like those typical of the adrenogenital syndrome, affect the ovary, not directly, but rather by the creation of a pituitary gonadotrophic-ovarian imbalance.

Some evidence exists for more direct relationships between the adrenal cortex and the ovary. These may involve actions of ovai-iaii liormones on the adrenal, and ac

tions of adrenal cortical hormones on the ovary. In general, however, the relationships are tenuous or at least not sharply defined. It is evident from the review by Parkes (1945) that sexual dimorphism in adrenal cortical structure has been demonstrated in a number of species, notably the mouse and rat and possibly the guinea pig, but it has not been detected in a number of other species. The effects of gonadcctomy and the injection of hormones, particularly estrogens, into gonadectomized animals are less clear, but they are suggestive of an action on the adrenal, however ill defined and variable it seems to be. A seasonal hypertrophy of the adrenal has been reported as occurring in the mole, Talpa europaea, (Kolmer, 1918) and the ground squirrel, Citellus tridecemlineatus (Mitchill), (Foster, 1934), as has enlargement at the time of estrus in the rat (Andersen and Kennedy,

MAMMALIAN OVARY

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1932; Bourne and Ziickennan, 1941bj. More recently, a significantly higher excretion of 17-hydroxy corticosteroids has been found during the second and third weeks, and therefore during the luteal phase, of the menstrual cycle (Maengwyn-Davies and Weiner, 1955).

Whether a causal relationship exists between these indications of a fluctuating activity within the adrenal cortex, and seasonal and cyclic changes within the ovaries remains to be determined. Little information exists. Bourne and Zuckerman {loc. cit.) described the changes in the adrenals of ovariectomized rats injected with estrone and concluded that the changes are inde|)endent of the gonads. Foster's observation that the active appearance of the adrenal can be seen during pregnancy as well as during estrus suggests, but does not prove, that there is a hormonal regulation in the ground squirrel which is dependent on reproductive processes.

Data with respect to possible direct effects of adrenal cortical secretions on the ovary ai'e ambiguous. Cortisone acetate administered to rabbits 5 to 33 days in daily doses of 5 to 20 mg. did not inhibit the ovulation which occurs after mating or after the injection of copper acetate (De Costa and Abelman, 1953). The ability of the ovary of the rat to respond after adrenalectomy was tested by the administration of gonadotrophic extracts (Brolin and Lindl)ack, 1951). They found that the ovaries could be stimulated to increase the weight of the uterus without the cooperation of the adrenals and considered that this result does not support the view that there is a direct relationship between adrenal corticoids and the biosynthesis of ovarian (also testicular) hormones. In other experiments (Payne, 1951; Smith. 1955), adrenalectomy abolished (Payne) or interfered significantly (Smith) with the ovarian hyperemia response to injections of HCG and pituitary extract (Antuitrin T) , the response utilized by Farris (1946) as a test for early pregnancy. Cortisone and hydrocortisone were partially effective in restoring the response in adrenalectomized animals. According to Payne, isocortisone acetate and compound A acetate were also effective, but in larger doses. No report of the use of corticosterone

(comi)ound B) is gi\'en; replacement therapy with this hormone would have been more physiologic because it is the natural corticoid of rats. It was concluded that the hyperemia response is more nearly normal in animals with normal adrenal function; Payne believes that the response is mediated through this gland. Despite what seem to be clear-cut results which have been confirmed, it is felt that additional closely controlled exi^eriments must be done in order to show whether these adrenal hormones affect the ovary directly or whether most of the effects are nonspecific metabolic alterations.

VIII. Concluding Remarks

The avenue followed by investigators interested in the functioning of the mammalian ovary has long carried a two-way traffic. In addition, there has been movement into the out of many side streets. No understanding of the pattern of the traffic in such a situation is possible and no satisfactory regulation can be achieved unless something is known about the nature, origin, and destination of the vehicles composing the traffic. Equally important, this information cannot l)e obtained by standing on one spot. This analogy contains much that is relevant for what has been attempted in this book. The problem of the ovary has been approached from the vantage point of forces and substances originating in the pituitary and the environment which act centripetally on it (Creep, Everett), and from the vantage j^oint of many of the tissues and organs on which the hormones we associate with it exert their action (the Hisaws, Cowie and Folley, Zarrow, Young in his chapter on mating behavior). In this chapter and that prepared l)y Dr. Villce positions have been taken near the ovary and attemj^ts made to bring together much of the information gathered by investigators who were in a sense looking right at it.

Whether our perspective is developed from a familiarity with all the material which has been brought together or whether it is restricted by the narrower treatment given here, it is obvious that the unsolved problems outnumber by far any that have been solved, if indeed there are such. Wc have learned much about the functioning

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PHYSIOLOGY OF GONADS

of the o\'ary, but there is little we can explain. As we indicated earlier, a part of this failure can be ascribed to the lack of gonadotrophic preparations which either singly or in combination will evoke changes identical with those in untreated normal animals, but this chapter alone contains an enumeration of many other problems solution of which does not depend on this particular advance. The disappointment we express may be a reflection of what seems to be the modus operandi in science. The extent of our application to unsolved problems is very unequal, but more often than not it can be traced to an investigator's success in achieving a "breakthrough"*' as Edgar Allen, Doisy, Smith and Englc, Willard Allen and Corner, Hisaw and other colleagues did in the twenties. At such a time, enthusiasm is intense and there follows a period of gratifying accomplishment, but obstacles are encountered and often interest lags, until another breakthrougli occurs. In the meantime, effort may have been diverted by discoveries elsewhere and the area of investigation which attracted so many is neglected and suffers. Ovarian physiology should not remain in this state for long. There is no tissue of the body in which the changes are as conspicuous and as dramatic as those in the ovary and there is no tissue which presents more variable aspects. Many of the stages in the cycle of ovarian structure and functioning are related to changes elsewhere in the body — changes in growth, in motility, in secretion, and in beliavior. All these changes, including those within the ovaries, offer excellent end points for continued quantitative and qualitative studies.

IX. References

Aldman, B., Claesson, L., Hillarp, N.-A., and OdeBLAD, E. 1949. Studies on the storage mechanism of oestrogen-precursors. Acta enclocrinol., 2, 24-32.

Allen, E. 1922. The oestrous cycle in the mouse. Am. J. Anal., 30, 297-371.

Allen, E. 1932. The ovarian foUirulai- lioriiioiic. theehn; animal reactions. In Sex and hih iinil Secretions, 1st ed., E. Allen, Ed., pp. 392-t,S0. Baltimore: The Williams & Wilkins Company.

Allen, W. M. 1939. Biochemistry of the corpus

" The word was not a part of the language of science at that time and prolial)ly was never used l)y them

luteum hormone, progesterone. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 901-928. Baltimore: The Williams & Wilkins Company.

Amoroso. E. C. 1955. Endocrinologv of pregnancy. Brit. Med. Bull., 11, 117-125.

Amoroso, E. C, Harrlsox, R. J., Harrlsox-M.\tthews, L., and Rowlands, I. W. 1951. ReIjroductive organs of near-term and new-born seals. Nature, London, 168, 771-772.

Amoro.so, E. C, and Rowlands, I. W. 1951. Hoimonal effects in the pregnant mare and foetal foal. J. Endocrinol., 7, 1-liii.

Andersen, D. H., and Kennedy. H. S. 1932. Studies on the physiology of reproduction. IV. Changes in the adrenal gland of the female rat associated with the oestrous cycle. J. Physiol., 76, 247-260.

Antonl\des, H. N., McArthur, J. W., Pennell, R. B., Ingersoll, F. M., Ulfelder, H., and Oncley, J. L. 1957. Distribution of infused estrone in human plasma. Am. J. Physiol., 189, 455-459.

Aron, M., and Aron, C. 1952. La glande thecale de lovaire de cobaye. Arch. Anat. Histol. et Embryol., 34, 27-41.

ASCHHEIM,, S., PORTES, L., AND M.\YER, M. 1939.

Les hormones gonadotropes. Etude critique de quelques points relatifs a leur role dans la physiologie et la pathologie des fonctions de I'ovaire. Ann. endocrinoL, 1, 42-54.

AsTwooD, E. B. 1938. A six-hour assay for the quantitative determination of estrogen. Endocrinology, 23, 25-31.

AsTW^ooD, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Am. J. Physiol., 126, 162-170.

Atkinson, W. B., and Leathem, J. H. 1946. The day to day level of estrogen and progestin during lactation in the mouse. Anat. Rec, 95, 147-155.

Barahona, M., Bruzzone, S., and Lipschutz, a. 1950. On the control of follicular development in intrasplenic ovarian grafts by minute (|uantities of oestrogen. Endocrinologv, 46, 407-413.

Barker, W. L. 1951. A cytochemical study of lipids in sows' ovaries during the estrous cycle. Endocrinology, 48, 772-785.

Barr.aclough, C. a. 1955. Influence of age on the response of prcweaning female mice to testosterone i)ro])i()nate. Am. J. Anat., 95, 493521.

Bassett, D. L. 1943. The changes in the va.scular pattern of the ovary of the albino lat during the estrous cycle. Am. J. Anat., 73, 251291.

Bavlis, H. I., Browne, J. C, Round, B. P., .and Steinbeck, A. W. 1955. Plasma 17-hydroxycorticosteroids in pregnancy. Lancet, 1, 62-67.

Beroer, M. 1935. Besonders hohe Wirk.'^amkeit des Follikelhormons bei vaginaler Instillation. Klin. Wchnschr., 14, 1601-1602.

Behcman, p. 1949. Sexual cycle, time of ovulation, and time of optimal fertility in women.

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485

Acta obst. et gvnec. scandinav., Supi)l. 29, 4, 1-139.

Beyer, K. F., AND Samuels, L. T. 1956. Distrilmtion of steroid-3^-ol-dehydrogenase in cellular structures of the adrenal gland. J. Biol. Chem., 219, 69-76.

BiGGERS, J. D. 1953. The characteristics of the dose-response line in Allen-Doisy tests obtained by the intravaginal administration of oestrone in distilled water and in 50 i)cr cent aciueous glycerol. J. Endocrinol., 9, 145-154.

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Shippel, S. 1950. The ovarian theca cell. J. Obst. <k Gynaec. Brit. Emp., 57, 362-387.

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Slaunwhite, W. R., Jr., and Samuels, L. T. 1956. Progesterone as a precursor of testicular androgens. J. Biol. Chcm., 220, 341-352.

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Smith, O. W. 1960. Estrogens in the ovarian fluids of normally menstruating women. Endocrinology, 67, 698-707.

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Sneider, M. D. 1940. Rhythms of ovogenesis before sexual maturity in the rat and cat. Am. J. Anat., 67, 471-499. "

Snell, G. I).. Fekete, E., Hummel, K. P., and L.\w, L. W. 1940. The relation of mating, ovulation and the estrous smear in the house mouse to time of day. Anat. Rec, 76, 39-54.

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MAMMALIAN OVARY

495

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Tanaka, S., and Starr, P. 1959. Clinical observations on serum globulin thyroxine-binding capacity using a simplified technique. J. Clin. Endocrinol., 19, 84-91.

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Thung, p. J., Boot, L. M., and Muhlbock, O. 1956. Senile changes in the oestrous cycle and in ovarian structure in some inbred strains of mice. Acta endocrinol., 23, 8-32.

Trolle, D. 1955a. Experimental and clinical investigations of the pregnanediol excretion in the human urine. Acta endocrinol., 19, 217-232.

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Trum, B. F. 1950. The estrous cycle of the marc. Cornell Vet., 40, 17-23.

Umbaugh, R. E. 1951. Superovulation and ovum transfer in cattle. Fertil. & Steril., 2, 243-252.

VAN Wagenen, G. 1945. Optimal mating time for pregnancv in the monkey. Endocrinology, 37, 307-312.

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Van Zyl, A. 1957. Serum protein-bound iodine and serimi lipid changes in the baboon (Papio ursinus). I. During the menstrual cycle. J. Endocrinol., 14, 309-316.

Velardo, J. T. 1959. Steroid hormones and uterine growth. Ann. New York Acad. Sc, 75, 441-462.

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Velardo, J. T., and Hisaw, F. L. 1951. Quantitative inhibition of progesterone by estrogens in de\-Glopment of deciduomata. Endocrinology, 49, 530-537.

Vel.ardo, J. T., Olsen, a. G., Hisaw, F. L., and Dawson, A. B. 1953. The influence of decidual tissue upon pseudopregnancy. Endocrinology, 53, 216-220.

Vermande-Van Eck, G. J. 1956. Neo-ovogenesis in the adult monkey. Consequences of atresia of ovocytes. Anat. Rec, 125, 207-224.

Veziris, C. D. 1951. Cycle vaginal chez les femmes castrees ou menopausees. Ann. endocrinol., 12,915-921.

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496

PHYSIOLOGY OF GONADS

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Zacharl\e, F. 1958. Studies on the mechanism of ovulation. Permeability of the blood-li(|uor barrier. Acta endocrinol., 27, 339-342.

Zachariae, F.. and Jensen, C. E. 1958. Studies on the mechanism of ovulation. Histochemical and physicochemical investigations on genuine follicular fluids. Acta endocrinol., 27, 343-355.

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Zander, J., Forbes, T. R., vox Munstermann, A. M., AND Neher, R. 1958. Two naturally occurring metabolites of progesterone — isolation, identification, biologic activity, and concentration in human tissues. J. Clin. Endocrinol., 18, 337-353.

Zander, J., and Simmer, H. 1954. Die chemische Bestimmimg von Progesteron in organislien Substraten. Klin. Wchnschr., 32, 529-540.

Zarrow, M. X., AND Neher, G. M. 1953. Studies on the validity of the Hooker-Forbes test for the determination of progesterone in untreated blood. J. Clin. Endocrinol., 8, 203-209.

Zarrow, M. X., Neher, G. M.. Lazo-Wasem, E. A., AND Salhanick, H. A. 1957. Biological activity of certain progesterone-like compounds as determined by the Hooker-Forbes bioassav. J. Chn. Endocrinol., 17, 658-666.

Zarrow, M. X., Caldwell, A. L., H.\fez, E. S. E., AND PiNCus, G. 1958. Superovulation in the immature rat as a possible assav for LH and HCG. Endocrinology, 63, 748-758.

Zondek, B. 1940. On the mechanism of action of gonadotrophin from pregnancy urine. J. Endocrinol., 2, 12-20.

Zondek, B. 1954. Some problems related to ovarian function and to pregnancy. Recent Progr. Hormone Res., 10, 395-423.

Zondek, B., and Ascheim, S. 1927. Hypophysen^•OI•del•lappen und Ovarium. Beziehungen der cndokrinen Driisen zur Ovarialfunktion. Arch. Gynak., 130, 1-45.

Zondek, B., and Sklow, J. 1942. Further investigations on the mechanisms of oestrone production in the ovary. J. Endocrinol., 3, 1-4.

Zuckerman, S. 1940-41. Periodic uterine bleeding in spayed rhesus monkeys injected thiily with a constant threshold dose of oestron(>. J. Endocrinol., 2, 263-267.

Zuckerman, S. 1951. The number of oocytes in the mature ovary. Recent Progr. Hormone Res.. 6, 63-109.

8

THE MAMMALIAN FEMALE REPRODUCTIVE

CYCLE AND ITS CONTROLLING

MECHANISMS

John W. Everett, Ph.D.

PROFESSOR OF ANATOMY, DUKE UNIVERSITY DURHAM, NORTH CAROLINA

I. Introduction 497

II. Cycles Spontaneously Interrcpted. 498

III. PiTUITARY-OVARIAN DORMANCY 499

A. The Ovary in Anestrum 500

B. The Hypophysis 500

C. Relationship of the Anestrum to the

Seasons 501

IV. Attainment of Maturity. Emergence OF Full Ovarian Function. . 502 V. Follicular Cycles. Growth and

Atresia 504

A. Correlation of Ovarian Secretion

with the Follicular Cycle 507

B. Cyclic Manifestations after Ovari ectomy or Hypophysectomy 509

C. Cyclic Manifestations in the Ab sence of Ovarian Follicles 509

D. Hypothalamus and Gonadotrophin

Secretion. General Considerations 510

VI. Follicle Maturation and Ovulation 513

A. Time of Ovulation 513

B. Ovarian Steroids and Ovulation. . . 514

1. Estrogens 514

2. Gestagens 517

C. Role of the Nervous System in Ovu lation " 520

1. The hypophyseal portal veins and

the chemotransmitter hypothesis ' 523

2. Central depressants and ovula tion 526

3. The central nervous system as a

timing mechanism for ovulation 520

D. Persistent Follicle 529

VII. The Luteal Phase 530

1. Luteotrophic substances 530

2. "Nonfunctional" corpora lutea. . 531 A. Pseudopregnancy 532

1. Duration of pseudopregnancy. .. . 533

2. Neural factors in pseudopreg nancy 534

B. Luteolytic Mechanisms 537

C. Effect of the Uterus on Luteal Func tion 538

VIII. Concluding Comments 540

IX. References 541

I. Introduction

The chain of events that constitutes the female reproductive process is characteristically repeated from time to time with considerable regularity during the adult life of an individual, and is therefore a cycle. In the broad sense, this sequence begins with ovogenesis and terminates when the progeny require no further shelter and nurture. In mammals this has become a highly complex process, involving profound maternal adjustments synchronized with successive stages in development of the ovum, fetus, and offspring. The complete mammalian cycle comprises a sequence of stages which may be identified as follows: (II follicle growth, including growth of the ovocyte; (2) ovulation, a progressive process including preovulatory maturation of follicles and ova, and the structural change of ruptured follicles to corpora lutea; (3) progravidity; (4) gravidity; (5) parturition; and (6) postpartum nurture, including lactation, protection, and training. Although it is obvious that this full sequence is often realized, it may nevertheless be retarded or frankly interrupted at almost any point.

In advanced human societies economic and social factors have diminished the number of complete cycles to such degree that they are rarities in the lifetime of an in

497

498

PHYSIOLOGY OF GONADS

dividual and infertile ("menstmal") cycles are the rule. Inasmuch as corresponding factors operate among domesticated animals, the expression "female reproductive cycle" commonly refers to those truly abortive cycles that succeed one another in the absence of insemination. The term is used in that restricted sense in this chapter.

With even that restriction, the female cycle is actually a multiplicity of interlocking cycles, in which the rhythmic interplay between hypophysis and ovary is fundamental. Attention must therefore be focused on the physiology of the ovary and on the hormonal and neural mechanisms that integrate hypophysis and ovary as a functional system. Cyclic alterations in sex accessories and other nongonadal tissues are considered mainly as indicators. The "menstrual cycle," being strictly a uterine cycle, comes in this category, together with changes in behavior.

No attempt is made to present an exhaustive description of the varied adaptive modifications of the ovarian cycle among the several mammalian orders. The reader may consult works of the late F. H. A. Marshall whose full bibliography is given by Parkes (1949 1. Asdell's Patterns of Mammalian Reproduction (1946» is anotlu'i' A-alual)lo source.

II. Cycles Spontaneously Interrupted

Cycles in the natural state are only imperfectly known, from random and often erratic sampling. One may safely assume that, as a rule, under optimal conditions they are complete, fertile cycles. There are, then, relatively few subhuman species in wdiich the characteristics of incomplete cycles have been studied. These species are necessarily the very ones that have been amenable to some form of human restraint.

Segregation of the sexes or any other interference with insemination should be regarded as a first experimental approach to understanding the complete cycle. Such factors unriuestionably operate in nature on occasion. Controlled changes of environmental conditions afford another approach in which natural factors are simulated.

The statement was made earlier that the complete cycle may conceivably be interrupted at almost any point. It has been

learned that in different species segregated females interrupt their cycles at different stages and that usually the point of interruption is species-characteristic. These facts have been of great service to the study of reproduction, first, by arousing the curiosity of the investigator and, second, by supplying a variety of ready made conditions individually appropriate for particular experimental studies.

Examples of mammalian cycles are schematically diagrammed in Figure 8.1. It is customary to state that the usual, or standard, infertile cycle is like that in primates or the guinea pig. The follicular phase culminates in spontaneous ovulation, after which corpora lutea are organized and become spontaneously functional for a period of time that is usually considerably shorter than in pregnancy.

In a few animals (rat, mouse, hamster) the cycle terminates shortly after ovulation before the corpora lutea become fully functional. Such corpora lutea are said to be inactive, in the sense that they cannot produce a decidual response to uterine trauma (Long and Evans, 1922). Sterile mating or analogous stimulation induces a luteal phase which corresponds to that of the "standard" mammal. This phenomenon is not entirely limited to the small rodents, having been described in the European hedgehog (Deanesly, 1934).

To this writer's knowledge there have not been described any mammalian species in which it is the rule that in isolated females the process of ovulation begins (follicle maturation, prelutein changes in granulosa, secretion of secondary liquor folliculi, and so on) without proceeding to eventual rupture of the follicles. Many cases could l3e cited, however, in which this has occurred "abnormally." Characteristically, some degree of luteinization occurs in the wall of such a follicle and a lutein cyst is formed.

On the other hand, there are numerous species (reflex ovulators) in which the preovulatory maturation of follicles and ovulation nearly always fail in the absence of the male. The known species in which this is true are widely distributed among the mammalian orders and are often closely iclated to other species in which spontaneous ovulation is usual. The domestic rabbit

MAMIMALIAN REPRODUCTIVE CYCLE

499

GUINEA PIG

PRIMATE

Fig. 8.1. Diagrams of cycles of representative, familiar mammals. , the follicular phase,

highly schematized and inaccurate in detail ; , atresia ; i , ovulation ; • , fully active

corpora lutea; O, corpora lutea regressing or otherwise not fully active. When sterile mating or equivalent stimulation (SM) is introduced, the cycles of the rat, rabbit and cat become directly comparable with those of the other species.

furnishes the classic exaini)le of reflex ovulation. Other reflex ovulators are the domestic cat (Greulich, 1934), the ferret (Hammond and Walton, 1934), mink (Hansson, 1947), marten (Pearson and Enders, 1944), the 13-lined ground squirrel (Foster, 1934), and the mole shrew (Pearson, 1944). To this list have been added the muskrat (Miegel, 1952) and a field mouse, Microtus californicus (Greenwald, 1956). Even among the marsupials, the female Didelphijs azarae is said not to form corpora lutea in the absence of the male (Martinez-Esteve, 1937). A few of these species display nearly constant estrus (rabbit, ferret), competent follicles being present most of the time in the isolated female during the breeding season.

Among even the spontaneous ovulators the cycle may sometimes not progress beyond the follicular phase. Thus, at the approach of puberty, waves of advanced follicle development and secretion of estrogen may take place without, however, leading to ovulation or corpus luteum forma

tion. The first cycles of primates are often anovulatory ones. In the adult macacjue, at least in some colonies, such cycles are characteristic during the summer months (Hartman, 1932) . A somewhat comparable seasonal effect has been reported in girls soon after the menarche (Engle and Shelesnyak, 1934). Menstrual cycles without ovulation have frecjuently been recognized in adult women in recent years, bearing no evident relationship to seasonal factors (Lopez Colombo de Allende, 1956). Anovulatory cycles were described in the mouse by Allen (1923) and have been noted occasionally in other species, but without clear measure of their incidence.

III. Pituitary-Ovarian Doriiianey

Varying levels of pituitary-ovarian dormancy are expressed in different ways from species to species or even from habitat to habitat within a given species. A general similarity exists between the anestrum of seasonal breeders and the prepubertal state. In fact, in animals that have a distinct sea

500

PHYSIOLOGY OF GONADS

son, puberty occurs at the very time when older females are emerging from anestrum. Whereas anestrum is often correlated with season of the year, there are exceptions, notably among dogs, in which the correlation is ill defined (Engle, 1946).

In its shortest form ovarian quiescence lasts for only a few days, probably often without being recognized, between the end of one cycle and the active follicular phase of the next. In the chimpanzee it is thought to be the chief factor in the irregularity of length of the cycle (Young and Yerkes, 1943). Rossman and Bartelmez (1946) described a comparable occurrence in monkeys. At the other extreme, anestrum may occupy the major part of the year in monestrous animals that have a very limited breeding season.

A. THE OVARY IN ANESTRUM

Generally speaking, depression of ovarian function is most extreme in greatly prolonged periods of quiescence. In the ferret, Hammond and Marshall (1930) reported that in the anestrous ovary follicles can hardly be recognized with the naked eye, because they remain small and deeply placed. The largest follicles at the "end of the season" averaged 460 /x in diameter whereas a "long time after" the average was only 240 jx, increasing again to 720 yu, at the api^roach of a new season. By contrast, the largest follicles of animals in full heat ranged between 1220 and 1440 /x. Follicle atresia abounds in the anestrous ovary of the 13-lined ground squirrel (Johnson, Foster and Coco, 1933). In sheep, however, follicles of large size may be present at any time during anestrum (Kammlade, Welch, Nalbandov and Norton, 1952).

Some moderate degree of secretory activity of the ovary is indicated even at the depth of i^rolonged seasonal anestrum (13lincd ground squirrel, Moore, Simmons, Wells, Zalesky and Nelson, 1934; ferret, Hill and Parkes, 1933; opossum, Risman, 1946). Although at this time uterus, vagina, and vulva are small, ovariectomy or hypol^hysectomy causes a further reduction. On the other hand, these structures are readily stimulated by injection of estrogens.

It may be said that low-grade follicular cycles proceed throughout the anestrous

interval, but whether there is any synchronization of one follicle with another is unknown. Some insight into this problem is furnished by study of (1) the transition from anestrum to the breeding season, and (2) the closely analogous phenomena of adolescence. In the report by Hammond and Marshall, it was shown that in ferrets during anestrum and proestrum there is a progressive increase in size of the vulva which directly parallels the diameter of the largest follicles. The absence of overt cyclic change is not surprising in view of the fact that estrus is continuous in this species. In polyestrous animals, on the other hand, it might be expected that during anestrum follicle growth and accompanying estrogen secretion are cyclic, at least at the approach of puberty or of "the season." Important information on this question has been obtained from some of the primates, notably the macaque (Allen, 1927; Hartman, 1932) and the chimpanzee (Zuckerman and Fulton, 1934; Schultz and Snyder, 1935).

Slight transitory reddening of the skin of the perineum ("sex skin") of the monkey may occur at intervals for several months preceding the onset of menses, accompanied by moderate desquamation of vaginal epithelium. During the long intervals of amenorrhea that some individuals exhibit during the summer, there is a tendency toward cyclic vaginal desciuamation (Fig. 8.2). The sex skin of the chimpanzee may begin to swell more than a year before the first menstruation. During the ensuing months the swelling may be irregularly cyclic or continuous. Thus, one may judge that lowgrade follicular cycles, accompanied by periodic increases in estrogen secretion, may succeed one another during seasonal or ])repubertal anestrum, but that in certain cases these cycles may overlap to such degree that rather continuous estrogen secretion takes place.

B. THE HYPOPHYSIS

The secretory activity of the anestrous ovary is apparently adequate to prevent "castration" changes in the adenohypophysis, for as shown by Moore, Simmons, \^'ells, Zalesky and Nelson (1934) removal of the ovary of anestrous ground squirrels I'esults in hypertrophy of the hypophysis,

MAMMALIAN REPRODUCTIVE CYCLE

501

z

28 days

1

28 days A

28 days

< "

3 s UJ . Q

III Menses

"^ v-^^/^

A>

^ Mensesllll

21 ' 15 JUNE JULY

' 15 AUGUST

15 SEPTEMBER

15 OCTOBER

Fig. 8.2. Vaginal cycles during seasonal amenorrhea in a monkey. (A portion of the record of monkey ^38 from C. G. Hartman, Contr. EmbryoL, Carnegie Inst. Washington, 13, Fig. 26, p. 121, 1932.)

increased gonadotrophin content thereof, and increased numbers of basophile cells. Warwick (1946) reported a highly significant increase of pituitary potency in spayed anestrous ewes. This is closely analogous to the results of ovariectomy in immature animals (Hohlweg, 1934). As measured by ovarian activity, gonadotrophin secretion (release) may be greatly diminished during profound anestrum. The actual hypophyseal content of gonadotrophin seems to be markedly reduced during anestrum in some species (Moore, Simmons, Wells, Zalesky and Nelson, 1934), but possibly not in others. Cole and Miller (1935) and Warwick (1946) reported that there is no seasonal variation in sheep. A study by Kammlade, Welch, Nalbandov and Norton (1952) indicates that the average content is somewhat higher during anestrum than it is in cycling ewes. The major factor in this difference, however, seems to be that during the cycle the potency of the pituitary drops during estrus and the early luteal phase.

Somewhat similarly the potency of the immature rat hypophysis has been stated to be as high as that of the sexually active adult (Clark, 1935). The fact that the ovaries of the immature female or of the anestrous adult can be stimulated by injection of gonadotrophin indicates that gonadotrophin content of, the hypophysis in these cases is not a fair measure of liberation of the hormone into the blood stream. Therefore, it seems justifiable to assume, as Robinson (1951) did in the interpretation of anestrum in the ewe, that, in spite of the possible absence of seasonal assay variation.

there is, nevertheless, a depression of hypophyseal gonadotrophin release during anestrum. We may further assume that it is not completely depressed, for the ovary remains slightly active. Ovary and hypophysis are evidently in a state of equilibrium at a relatively low level of function. It seems likely that this state of affairs is brought about by the central nervous system, inasmuch as the seasonal depression in some species is closely dependent on the daily ratio of light to darkness.

C. RELATIONSHIP OF THE ANESTRUM TO THE SEASONS

This relationship is so varied among different species that many interesting questions are raised. In many cases the midpoint of anestrum coincides approximately with the shortest days of the year (Fig. 8.3). There are other examples, however, largely among the Artiodactyla, in which it coincides with the longest days. Sheep are notable examples (Robinson, 1951). Others, like the European common hare, experience a short anestrum during the time of rapidly decreasing daylight (Asdell, 1946). The Russian yak, on the other hand, is said to experience anestrum from December to May (i.e., while day length is increasing). A general explanation of these varied adaptive manifestations is elusive. There is reason to believe that although illumination, or the light/darkness ratio, (Kirkpatrick and Leopold, 1952; Hammond, Jr., 1953) has a rather direct and primary effect in some cases, its role is more or less indirect in others where such things as temperature, humidity, availability of food and water assume major importance (Marshall, 1942).

502

PHYSIOLOriY OF GONADS

NDJFMAMJJASON

INSECTIVORA

MOLE SHREW

COMMON SHREW CARNIVORA

BROWN BEAR (EUR.)

FERRET

COYOTE

WILD CAT (EUR.)

BAD3ER (AMER.) LAGOMORPHA

HARE (ENG.)

COTTONTAIL (N . ENG.) RODENTIA

13-L. GROUND SQUIRREL

WOODCHUCK

GRAY SQUIRREL

DORMOUSE

FIELD MOUSE (EUR. )

MUSKRAT (MARYLAND) (IOWA)

PORCUPINE ARTIODACTYLA

LLAMA

ROE DEER

MULE DEER

GIRAFFE

SHEEP (HAMP )

BIGHORN

GOAT

YAK (RUSS. )

INDIAN ANTELOPE PERISSODACT YLA

HORSE

Fig. 8.3. Some representative seasonal breeders. Solid bars indicate breeding seasons (according to Asdell, 1946); blank intervals, periods of anestrum. Months of the year represented by letters at top of chart ; winter and summer solstaces marked by wavy lines. Southern hemisphere seasons converted to corresponding ones of the northern hemisphere. End of season for the Bighorn is vmcertain.

The complexity of the i)rol)lem is well illustrated by the 13-lined ground squirrel whose breeding season, like that of a multitude of small rodents, comes in the spring. Moore, Simmons, Wells, Zalesky and Nelson (1934) reported that increasing illumination, elevated temperature, and feeding all failed to bring the females into estrus out of season. If, however, hibernation was first induced by low temperature and darkness, premature estrus would follow. The conclusion was reached that hibernation itself is a necessary prerequisite. Ovarian development actually begins, under natural conditions, in early January in the midst of hibernation. Females exix'i-imentally maintained "continually for several months in cold and darkness, with more or less normal hibernation, [exhibit] sexual development at any time of the year, and periods of estrum have thus been . . . maintained for many months. ..." The impres

sion is given tliat the conditions favoring hibernation also favor sexual development to such extent that breeding potentiality continues for a few months after emergence, in spite of elevated temperatures and long periods of illumination. In another rodent, Peromyscus leucopus, however, the length of daily illumination is of paramount importance. Temperature changes (4 to 25°C. ) have no effect on rejiroduction when lighting is adequate (Whitaker, 1940). Whereas a similar primaiy dependence on lighting can be shown in a number of other species from several orders, it is unwise to generalize that this is usually true.

IV. Attainment of Maturity. Emergence of Full Ovarian Function

Ahhough ('merg(>nce of the ovary from the state of quiescence is gradual, there is usually some outward sign that allows the observer to say that puberty has ari'ived or the breeding season has begun. In ])ri

MAMMALIAN REPRODUCTIVE CYCLE

503

mates the accepted sign is the first menstruation ; in rats it is the opening of the vagina ; in many animals it is the swelling and reddening of the genitalia heralding the initial l^roestrum. In other eases, e.g., sheep, the only clear indication may be the behavior of the female toward the male. From these facts it is readily apparent that any one sign is employed simply because it happens to be accessible to easy observation. Yet the increasing output of estrogen, whether steady or cyclic, affects many parts of the organism at the same time. Furthermore, in any one individual the threshold for exl)ression of a given sign may be relatively liigh with respect to that of some other manifestation. Thus, in Hartman's monkeys (1932), some were noted in which desquamation of vaginal epithelium occurred in wave-like manner for a long time before menstruation. In others "menstrual" bleeding occurred with regularity while the uterus remained very small and A'aginal desquamation was negligible.

Hartman summarized the step-wise manner of maturation of ovary and accessory organs of the monkey during adolescence or following amenorrheic episodes somewhat as follows. The color of the sex skin may be the first to appear. A slight menstrual flow usually takes place before desquamation of vaginal epithelium becomes measurable. "More rarely there may be one or more low desquamation cycles before a bleeding is recorded. Whole cycles marked liy jieriodic bleeding and some vaginal desquamation may occur before there is any noticeable increase in size of the ovaries and uterus. These organs increase also in a saltatory manner, hence the term 'staircase' phenomenon for the process. Finally, the endocrines effect the acme of the reproductive process — ovulation."

Individual variation in the degree of abruptness with which the first ovulation is achieved is well illustrated in a study of puljertal guinea pigs by Ford and Young (1953). In most cases the first period of vaginal opening was much longer than in subsequent cycles. Whatever the duration, ovulation was more closely related to the end than to the beginning of the period, as indicated by histologic study of ovaries.

Even ovulation and corpus luteum for

mation do not signify that full power of reproduction has arrived. For example, the first cycle of the adolescent rat may culminate in ovulation without sexual receptivity (Blandau and Money, 1943). In the ewe, an ovulation without overt signs of heat may at times take place during the anestrum, especially just before and just after the breeding season (McKenzie and Terrill, 1936). The phenomenon is occasional in ewes during the season and has also been described in cattle (Hammond, 1946). In fact, the full manifestation of estrus in sheep seems to require the presence of a "waning" corpus luteum (Robinson, 1951). In sheep the transition from seasonal or prepubertal anestrum to the breeding season may involve relatively minor changes in hypophyseal activity. Even in the immature rat both the hypophysis and the ovary are capable of far greater secretory function than they normally display. In the equilibrium that prevails, the ovary appears to hold the upper hand by reason of a low hypophyseal threshold at which estrogen suppresses gonadotrophin secretion in the immature individual (Hohlweg and Dohrn, 1932; Byrnes and Meyer, 1951b) and a low ovarian threshold at which gonadotrophin stimulates estrogen secretion. Byrnes and Meyer (1951a) reported that suppression of hypophyseal gonadotrophin content in immature rats can be accomplished with doses of estrogen much smaller than those that affect uterine growth. It is also known that the immature ovary can be induced experimentally to secrete estrogen by injection of amounts of gonadotrophin that are too small to produce significant increase of ovarian weight or follicle development (Levin and Tyndale, 1937; Moon and Li, 1952). When a gonadectomized immature rat is united in parabiosis (Kallas, 1929, 1930 » with a normal or hypophysectomized female littermate, precocious puberty is induced in the latter animal because insufficient estrogen passes to the first partner to inhibit gonadotrophin secretion (see Finerty, 1952). The somewhat analogous experiment of transplanting ovaries to the spleen produces ovarian hypertrophy in much the same way. Here again, it is thought that the hypophysis becomes hyperactive because the amount of estrogen

504

PHYSIOLOGY OF GONADS

reaching the ghmd is greatly diminished, through inactivation bj^ the liver (Biskind, 1941).

Although it is true that estrogens have a suppressing action on gonadotrophin secretion, it has become increasingly evident that they can also stimulate hypophyseal function in certain ways, as Engle pro{)osed in 1931. Thus short-term injection of estrogen into intact immature rats and mice will invoke precocious puberty not only by stimulating the sex accessories, but also by increasing gonadotrophin secretion and thus causing ovarian growth and even ovulation. Frank, Kingery and Gustavson (1925j reported that after such treatment regular cycles continued after treatment was withdrawn. Lane (1935) found that when 22day-old female rats were injected daily with estrogen there was an early increase in number of ovarian follicles, including vesicular stages. After the first 10 days the nonvesicular follicles became depressed although vesicular follicles were retained. This was interpreted to mean that for a short time estrogen actually stimulates the follicle-stimulating hormone (FSH) l)ut eventually suppresses it, although luteinizing hormone (LH) secretion remains elevated. Hohlweg (1934) had already demonstrated that when somewhat older prepubertal rats are given single, rather large injections of estrogen, ovulation and corpus luteum formation are induced within a few days (p. 514). Obviously LH secretion is greatly increased.

Various bits of evidence implicate the nervous system in the processes leading to puberty and to the onset of estrus in seasonal breeders. This will be discussed in the following section with respect to the general (juestion of the relationship of the hypothalamus to gonadotrophin secretion.

V. Follicular Cycles. Growth and Atresia

Attention will be focused here on the dynamic pattern of follicle development throughout the cycle, the extent to which this i)attern depends on hyi)o[)hyseal conti'ol, and the functional changes in the o\aiy associated with estrus in preparation foi' the more specialized events that lead to ovuhition and corpus luteum formation.

Production of primordial follicles and the early growth stages have been said to be independent of the hypophysis (Smith, 1939; Hisaw, 1947). This view derives from the fact that following hypophysectomy the ovaries retain large numbers of healthy proliferating follicles below the stage of antrum formation. There are, however, several indications that these developmental stages may be accelerated by gonadotrophic stimulation. It was briefly reported by Simpson and van Wagenen (1953) that administration of purified FSH to immature monkeys caused not only a 10- to 20-fold 'increase of ovarian weight, but also stimulation of granulosa in follicles of all sizes. Indirect evidence comes from the fact that follicle atresia generally becomes maximal late in estrus or metestrum, when depressed FSH might be expected on theoretical grounds. Harrison (1948) reported tliat in ovaries of goats killed on the third or fourth days of estrus healthy primary ovocytes are rare. Some few, however, presumably remain. Myers, Young and Dempsey (1936) stated that in the estrous guinea pig there are few nonatretic follicles aside from those destined for ovulation. However, small numbers of normal ai)pearing nonvesicular follicles were found.

There seems to be general agreement that, very quickly after this catastrophic elimination of follicles, renewed growth promptly ensues. Whether or not the wave of atresia represents a depression of FSH secretion, no one would deny that the new growth reflects this type of gonadotrophic stimulation. Characteristically the population of small and medium follicles is restored early in the luteal phase of the polyestrous cycle. This is clearly indicated for the guinea pig ovary (Myers, Young and Dempsey, 1936) when the data are converted from average volumes to average diameters (Fig. 8.4). Beginning on the fourth day after estrus, when the largest follicles are approximately 300 fx in diameter and when theca interna and antra have formed, rapid growth of granulosa, theca, and antra continues for several days. This is confirmed by counts of mitotic figures obtained by the colchicine technique (Schmidt, 1942), indicating greatest mitotic activity in theca and granulosa of follicles between 300 fi and 600 fx in diameter. By the

MAMMALIAN REPRODUCTIVE CYCLE

505

4 8 12

DAYS AFTER BEGINNING OF ESTRUS

Fig. 8.4. A schematic repiesentation of the folhcuhir cycle in the guinea pig. The heavj^ sohd curve represents the diameters of the largest follicles, recalculated from the data of Myers, Young and Dempsey (1936). The arrow point indicates ovulation. The other solid curves and broken lines represent impressionistically the growth and atresia, respectivelj^ of other groups of follicles that are not ordinarily destined for ovulation.

11th or 12th day the largest follicles (ca. 800 /x) are "competent," i.e., capable of being ovulated (Dempsey, Hertz and Young, 1936; Dempsey, 1937). While the largest follicles are developing to this stage, multitudes of others begin to grow, being carried on to various stages of development before regression sets in.

This pattern of the follicular cycle seems to be generally true among mammals that have been carefully studied, when allowance is made for the fact that from one species to another the characteristic maxima of follicle diameter are extremely variable (shrew, 350 fjL-, rat, 900 /*; cow, 19,000 /x; mare 70,000 /x; Asdell, 1946). In ovulatory cycles of polyestrous animals the greater part of follicle growth is accomplished while the luteal phase of the preceding cycle is in progress. In successive anovulatory cycles like those of the cat the patterns of the follicular cycles are probably much the same (Evans and Swezy, 1931 » . In the rabbit and ferret, where more or less constant estrus char

acterizes the isolated females in season, there is probably considerable telescoping of successive waves of follicle growth such that as one set of follicles begins to undergo atresia another set is ready to take its place (Hill and White, 1933). The difference between cat cycles and rabbit cycles seems to be chiefly one of degree. The writer has seen both types represented in persistent-estrous rats, among litter mates of inbred strains (Everett, 1939, and unpublished).

At the end of the luteal phase of the cycle in polyestrous animals there are already present several competent follicles among an extensive population of smaller ones. For example, the guinea pig corpus luteum usually shows signs of regression on day 13 of the cycle. It has been proved that ovulation can be induced as early as day 12 by injection of LH (Dempsey, 1937), several days earlier than it would normalh^ occur (Fig. 8.5). In the human and monkey it is possible that the "preferred" follicles are recognizable by their larger size during

506

PHYSIOLOGY OF GONADS

1000

750

500

Volume (lO^cu.//)

250

Normal Cycle

Cycle After Removal of Corpora Luteo

Progesterone Treated or Pregnant

'Corpora Luteo Removed and Oestrin Injected

Fig. 8.5. The guinea pig follieular cycle and some of its experimental modifications. (After E. W. Dempsey, Am. J. Physiol., 120, 126-132, 1937.)

or soon after menstruation (Allen, Pratt, Newell and Bland, 1930; Hartman, 1932). In many mammals competent follicles may be present much earlier. Ablation of corpora lutea soon after ovulation in sheep (McKenzie and Terrill, 1936) and cattle (Hammond, Jr., and Bhattacharya, 1944) is followed in 2 to 4 days by another ovulation, much sooner than in the guinea pig (Fig. 8.5). Removal of the primate corpus luteum, at the other extreme, produces no such immediate response, judging from the details of three cases among Hartman's (1932) protocols (#40, #41, and #99). Whereas the next ovulations took place earlier than expectation, the intervals between unilateral ovariectomy and ovulation were 16, 14, and 22 days, respectively.

From detailed investigations in the rat, only the earlier stages of follicle growth may properly be regarded as pure FSH effects (Lane, 1935). Lane and Greep (1935) found that addition of Lli to FSH causes a marked increase in the proportion of vesicular follicles to follicles without antra. The use of more highly purified materials ((irecp, van Dyke and Chow, 1942; Fraenkel-Conrat, Li and Simpson, 1943) has amply confirmed the necessity for combination of the two gonadotrophins to yield maximal follicle growth and estrogen secretion ill rats. Morphologic evidence indicates that

LH acts selectively on thecal tissue and, therefore, on the interstitial tissue derived therefrom. Inasmuch as thecal tissue is the presumptive major source of ovarian estrogen (see below), it follows, perhaps, as Hisaw (1947) suggested that "the theca interna through the action of LH acquires competence to respond to FSH" (by secreting estrogen) .

Convincing evidence that thecal tissue and its derivatives are the principal sources of ovarian estrogen was assembled by Corner (1938). The status of this question remains essentially the same today. Few endocrinologists, however, would assume that no other ovarian cells have this capacity (see discussion in the chapter on the ovary). Nevertheless, there is a direct correlation in time between the marked rise in estrogen secretion as the follicular jihase of the cycle advances, on the one hand, and the organization of tlicca interna of the largest follicles into organs of obvious endocrine character, on the other. "When especially ])rominent the theca interna is referred to as the "thecal gland" (Mossman, 1937; Stafford, Collins and ]\Iossman (1942).

Thecal tissue from the multitudes of atretic follicles should not be neglected as a possible additional source of estrogen. From the standpoint of chronologic relations to the cycle tiiis (iiiestioii has hardly

MAMMALIAN REPRODUCTIVE CYCLE

507

been touched. Pointing up our ignorance, Sturgis (1949) in a careful study of atresia of large follicles in the monkey ovary, speculated that their hypertrophied thecal tissue may serve the useful purpose of estrogen secretion during the interim between follicle rupture and organization of the corpus luteum.

We are in need of ciuantitative appraisals not only of the total numbers of healthy and atretic follicles of all categories present in representative species at progressive stages of the cycle, as in the work on the rat by Mandl and Zuckerman (1952), but also of the respective volumes of theca, granulosa, interstitial tissue, and corpora lutea. Lane and Davis (1939) determined in rat ovaries at four stages of the cycle the respective total volumes of theca, granulosa, and antra in all healthy follicles, as well as the separate mitotic indices of theca and granulosa. Such differential information on multiplication of cells and increase of antral volume is important. Although the latter accounts for a major part of the increase in volume of the larger follicles, it represents a function quite apart from protoplasmic growth per se.

There is now considerable evidence that estrogen itself exerts a growth -promoting influence on the follicle and, furthermore, sensitizes it to gonadotrophic stimulation. Details may be found in papers by Pencharz (1940), Williams (1940, 1944, 1945a, b), Simpson, Evans, Fraenkel-Conrat and Li (1941) , Gaarenstroom and de Jongh (1946) , and Desclin (1949a,) . Although it seems that these effects have not been elicited by physiologic doses, the possibility remains that estrogen operates within the confines of the ovary as a mediator of some of the effects of the gonadotrophins. In the neighborhood of cells that produce it the estrogen concentration is probably far above that which would be considered physiologic for the remainder of the body.

A. CORRELATION OF OVARIAN SECRETION W^ITH THE FOLLICULAR CYCLE

Knowledge of the secretory output of the ovary during the cycle is almost entirely indirect and derives chiefly from (1) substitution experiments carried out in a vari

ety of si)ecies, and (2) assays of urine, mainly human but occasionally from other forms. Satisfactory assays of blood estrogen have been very limited and chemical analysis of the steroid content of ovarian venous blood is in only its preliminary stages.

The early substitution experiments are chiefly of historic interest (Allen, Danforth and Doisy, 1939). In great measure these investigations constitute crucial steps in proof that the ovary secretes steroid hormones which are fundamentally responsible for the manifestations of estrus. Conversely, then, these manifestations might be considered to reflect an increase of estrogen secretion and their absence a relative decrease. It has been learned, however, that the action of estrogen in certain instances may be greatly modified by progesterone, androgens, and certain adrenocortical steroids (notably desoxycorticosterone). Androgens are known to be secreted in the female by the adrenal cortex (Dorfman and van Wagenen, 1941 ; Gassner, 1952) and by the ovaries (Hill, 1937a, b; Parkes, 1950; Deanesly, 1938; Burrill and Greene, 1941; Pfeiffer and Hooker, 1942; Alloiteau, 1952). Progesterone secretion is probably not confined to the luteal phase of the cycle (see p. 519j. Evidence for its secretion during follicle maturation is considerable and its possible production even earlier cannot be excluded. These considerations make it unwise, therefore, to regard phenomena such as vaginal cornification, turgescence of vulva and sex skin, uterine growth, as direct ciuantitative measures of estrogen output. This point may be illustrated by certain observations made in chim])anzees by Fish, Young and Dorfman (1941) and illustrated in Figure 8.6. Assays of urinary estrogens during the cycle exhibited two peaks, only the first of which coincided with the swelling of sex skin. The second peak of estrogen excretion was unaccompanied by swelling, presumably because of the coordinate increase of progesterone secretion. Had swelling been the only guide only the first peak would have been apparent.

Assays of urinary estrogen in primates have often shown double peaks such as illustrated for the chimpanzee. PedersenBjergaard and Pederson-Bjergaard (1948i.

508

PHYSIOLOGY OF GONADS

I.U. '00

I.U.

( »ott

Androgens Curve of genital swelling

25 27 29

Day of cycle

Fig. 8.6. E.strogen and androgen excretion by a female chimpanzee, Mamo. , total

estrogens; , estradiol; -•-•, estrone; , estriol. Menstruation indicated by solid

areas on base line. (From W. R. Fish, W. C. Young and R. I. Dorfman, Endocrinology, 28, 588,1941.)

studying one woman for 2 years, found single peaks at midinterval in 8 cycles and double peaks in 12 cycles. On the average the first peak was reached on day 12 and the second on day 21. Similar double peaks were noted in blood estrogen assays in a large group of normal young women (Markee and Berg, 1944). An additional lesser rise was observed during menstruation.

None of the available assays of urinary or blood estrogen can be accepted as an absolute measure of the rate of hormone production. Urinary assays have certain advantages, in spite of the fact that probably only a variable fraction of the ovarian product is measured. Intrinsically they are measures of rate, whereas assays of blood estrogen measure concentration alone at the moment of bleeding. Attempts have been made to measure estrogens in ovarian venous blood, but with little success because of the extreme dilution (Rakoff and Cantarow, 1950). We may hope that development of sufficiently sensitive methods of detection will soon allow systematic evaluation of ovarian output by such direct means. Tracer techniciucs have shown (Werthessen, Schwenk and Baker, 1953) in perfused ovaries of the sow that C^^-acetate enters into the synthesis of estrone and /^-estradiol.

Several years ago Corner (1940) esti

mated, from the known amounts of injected estrone required to maintain the normal status of sex skin and endometrium in castrates that the ovaries of an adult rhesus monkey secrete the equivalent of about 20 fig. estrone daily. On a weight basis the estrone equivalent secreted by the ovaries of a woman would then be on the order of 300 /Ag. per day. Actual substitution data from castrated women gave an estimate of the same order of magnitude (420 ;u,g. per day). Whatever the rate of secretion may be at different times, it would seem a 'priori that effects on extra-ovarian tissues should be more directly related to amount of estrogen in circulation. The assays of human bloodestrogen in normal women by Markee and Berg (1944) and in gynecologic patients by Fluhmann (1934), although differing in absolute values, agree in indicating that the variation of blood estrogen concentration from one stage of the cycle to another may be relatively small. If this is true, then it nuist be supposed that cyclic changes in the accessory organs are brought about by relatively moderate changes in circulating estrogen. In support of this view Markee (1948) demonstrated in the macaque that a mere 50 per cent reduction in the daily dose of estrogen can invoke menstruation if the change is abrupt.

MAMMALIAN REPRODUCTIVE CYCLE

509

B. CYCLIC MANIFESTATIONS AFTER OVARIECTOMY OR HYPOPHYSECTOMY

Residual cyclic changes in the vagina liave been reported in ovariectomized mice (Kostitch and Telebakovitch, 1929) and rats (Mandl, 1951). The periodicity is very nearly that of the normal cycles, at least in the latter species. Vaginal cycles of similar duration with more extreme estrous changes are found in ovariectomized rats receiving daily injection of threshold doses of estrogens (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941). The same was remarked in mice by Emmens (1939) and a report by Veziris (1951) indicates that vaginal periodicity may obtain in castrated or menopausal women receiving estrogen. Although sucli events have been called threshold cycles," the term may simply express the fact that they are most easily recognized when estrogen is given at threshold level. Hartman (1944), employing a modified Shorr stain for vaginal smears, found that castrated rats given large amounts of estrogen daily (5 to 100 fig. estradiol dipropionate) displayed complete cornification at 4- to 5-day intervals. During the time intervening there was admixture of Shorr cells, smaller epithelial cells, and leukocytes.

Analogous phenomena have been recognized in the endometrium of castrated monkeys (Zuckerman, 1937, 1941) injected daily for as long as 1 year with threshold doses of estrone (10 fig.). Larger doses prevent cyclic bleeding (see Hisaw, 1942). From the report of Veziris (1951) it may be judged that threshold endometrial cycles also occur in women and that the vaginal and endometrial cycles are synchronized in considerable extent.

Full explanation of these phenomena is not at hand. From the standpoint of the present discussion certain considerations are especially noteworthy. (1) Vaginal "threshold cycles" have been obtained in castrated rats in the absence of either hypophysis or adrenals (Bourne and Zuckerman, 1941 ; del Castillo and di Paola, 1942) . The former authors encountered the phenomenon in two rats from which both the hypophysis and adrenals had been removed. It is important

to remember, however, that the pars tuberalis remains in situ after the usual hypophysectomy procedure, that accessory adrenocortical tissue is frequent in rats, and that gonadal rests might remain unrecognized. (2) The reported lengths of vaginal and endometrial cycles agree favorably with the cycle lengths in intact individuals of the respective species. The degree of conformity between vaginal and uterine cycles indicated by Veziris {loc. cit.) suggests some sort of integrating mechanism. Much more information is required, however, before one may reject the alternative view that rhythmic activity is an innate characteristic of these organs.

C. CYCLIC MANIFESTATIONS IN THE ABSENCE OF OVARIAN FOLLICLES

Many years ago Parkes (1926a, b) and Brambell, Parkes and Fielding (1927a, b) reported vaginal and uterine cycles in mice in which the entire follicular apparatus had been destroyed by x-radiation. Schmidt (1936) described the phenomenon in the guinea pig, noting that, although most of her estrous animals had one or more large atretic or cystic follicles, as she had earlier reported (Genther, 1931), a few animals exiiil)ited periodic vaginal opening of short duration and correlated proestrous vaginal smears, in the absence of follicles. Her assays of urinary estrogen were negative in these animals, unlike the positive assays in those in which one or more follicles were demonstrable. Attempts by several workers (Drips and Ford, 1932; Levine and Witschi, 1933; Mandel, 1935) to reproduce in rats the results that Parkes and Brambell had found in mice, were unsuccessful, a fact indicating no estrogenic activity in ovaries completely lacking follicles and ova. Parkes (1952) more recently returned to this problem, reporting vaginal cycles and "fully functional" uteri in castrated rats bearing grafts of ovaries in which all organized follicles and ovocytes had been destroyed by deep freezing. These were true estrous cycles, in the sense that the animals would mate.

Many questions are posed by these observations. The fundamental one seems to be whether these cycles express periodicity of hypophyseal gonadotrophin secretion.

510

PHYSIOLOGY OF GONADS

The answer may be long in coming. Meanwhile, one would like to know whether castration changes are visible in the hypophysis and whether constant estrus may be invoked by exposure to continuous light or by postnatal treatment of the host with androgen or other steroids (see p. 529 1 .

D. HYPOTHALAMUS AND GONADOTROPHIN SECRETION. GENERAL CONSIDERATIONS

Experimental studies, ostensibly addressed to the general problem of neural control of gonadotropin secretion, have in fact often been concerned with the special problems of reflex ovulation <p. 520) or of provoked pseudopregnancy (p. 532). Whereas substantial information is now available w^ith respect to these special phenomena, particularly ovulation, information is limited about control of the day-to-day secretion of gonadotrophin that in the female is responsible for follicle stimulation and estrogen secretion (Benoit and Assenmacher, 1955; Harris, 1955). However, evidence in regard to induction of precocious puberty and early onset of estrus in seasonal breeders leaves no doubt that the nervous system is in some manner a regulator of follicle-stimulating activitv of the i)ars distalis.

Numerous reports associate precocious l)uberty with lesions in the hypothalamus (Weinberger and Grant, 1941; Bauer, 1954; Harris, 1955). Donovan and van der Werff ten Bosch (1956) reported off-season estrus in ferrets and precocious puberty in rats following retrochiasmatic lesions in the hyjiotlialamus. Exposure of immature rats to continuous light causes the vagina to open prematurely (Fiske, 1941). When 22-dayold female rats were given electrical stimulation of the cervix uteri daily for 10 days (Swingle, Seay, Perlmutt, Collins, Fedor and Barlow, 1951), a large proportion exhibited significant increase in uterine weight beyond that found in control animals, without change in ovarian weight. In fact, 7 of 50 rats ovulated or at least formed "several well-developed corpora lutca." Somewhat similarly, according to Aron and AronBrunetierc (1953), mechanical stimulation of the vagina or the adjacent segment of the uterus in innnatur(> guinea pigs regularly

provoked follicle growth and estrogen secretion. In gregarious birds the development of ovulable follicles requires that other individuals of the species be present. In the pigeon, even the mirror image of the female constitutes a sufficient stimulus (Matthews, 1939).

Studies by Flerko and his associates (1954-1957) present consistent evidence that restricted bilateral lesions in the region of the paraventricular nuclei serve to liberate the hypophysis from inhibitory effects of estrogen and androgen. This work is in agreement with that of Donovan and van der Werff ten Bosch in that somewhat similarly located lesions brought on precocious puberty. As noted elsewhere, gonadectomy in immature rats quickly results in hypersecretion of gonadotrophin.

Transplantation of the hypophysis to sites remote from the hypothalamus has produced divergent results. At the present writing, the chief divergence seems to rest between the sexes. In male guinea pigs and rats several workers have reported maintenance of male reproductive tracts by intra-ocular transplants of hypophyses (May, 1937; Schweizer, Charipper and Kleinberg, 1940; Cutuly, 1941a; Courrier, 1956; Goldberg and Knobil, 1957). Quite to the contrary, however, there has at best been only equivocal evidence of maintenance of female tracts, a matter of sex difference which needs full investigation. JNIay's (1937) report of 2 fertile female rats is unacceptable because of inadequate controls. Schweizer, Charipper and Haterius (1937) found in several hypophysectomized guinea pigs that intra-ocular pituitary grafts produced constant estrus and significant follicle stimulation, accompanied by uterine and mammary gland develoi)inent. Although the search for pituitary remnants in the sella turcica was reported negative, the histologic check was limited to scrapings from the sella floor. Other authors, notably Phelps, Ellison and Burch (1939), Westman and Jacobsohn (1940), Harris and Jacobsohn (1952), and Elverett ( 1956a) obtained in female rats little or no evidence of gonadotrophin secretion from apparently healthy, well vascularized grafts. The respective sites were intraniusculai', intra-ocular, in the sub

MAMMALIAN REPRODUCTIVE CYCLE

511

arachnoid space under the temporal lobe of the brain, and beneath the renal capsule — all distant from the hypothalamus.

Transplantation of the pars distalis into sites close to the hypothalamus, on the other hand, is characteristically followed by maintenance of the female reproductive tract and essentially normal sex functions. Greep (1936) found that re-implantation of hypophyses into the (presumably) emptied capsule was frequently followed in both male and female rats by return of virtually normal reproductive powers. Females exhibited cycles and even went through successful pregnancy and lactation. The result observed in male rats was confirmed by Cutuly (1941a). The obvious difficulty of establishing completeness of hypophysectomy has been the only criticism of these instructive experiments. This fault has been eliminated by an improved procedure devised by Harris and Jacobsohn (1952). Hypophysectomy was performed by the parapharyngeal route, after which the tissue to l)e grafted was introduced by a transtemporal approach to a site immediately beneath the median eminence. This permitted later histologic search for remnants of the original gland in its capsule. In many cases, including all in which the graft comprised several hypophyses from the animal's own newborn young, entirely normal gonadotrophic function was recorded. This included resumption of regular estrous cycles, typically during the 2nd or 3rd postoperative week. Several of the rats became pregnant and delivered viable litters. In marked contrast, none of the grafts that were placed under the temporal lobe gave any indication of gonadotrophin secretion, although they were as well preserved and richly vascularized as the others. Explanation of the difference seems to be that grafts under the median eminence acquire blood supply from regenerated hypophyseal portal veins and iience a neurovascular linkage with the hypothalamus. The importance of this relationship has been amply confirmed by Nikitovitch-Winer and Everett (1957, 1958d) in studies described below.

In lieu of significant numbers of nerve fibers entering the pars distalis (see Rasmussen, 1938; Harris. 1948a I, the hypophyseal

portal veins afford the most likely means by which the gland is brought under hypothalamic control. Recently it was demonstrated in rats and monkeys that these vessels have the power of rapid regeneration after simple stalk-section (Harris, 1949, 1950a, b). This fact at once gives a ready explanation of many of the discordant results of stalk-section experiments reported in the past. Harris (1950b) explored in rats the efficacy of various materials as barriers to regeneration, with the result that numerous examples of partial regeneration were produced. Degree of recovery of gonadotropliic activity by the hypophysis was strikingly correlated with degree of anatomic vascular recovery. Restoration of normal ovarian function after simple interruption of the stalk, as reported in the guinea pig by Dempsey (1939), in rats by Dempsey and Uotila (1940) , and in the human by Dandy (1940), is thus explained by the assumption that portal vein regeneration had taken place. On the other hand, Westman and Jacobsohn (1937-1938), who always inserted a barrier of metal foil between the median eminence and hypophyseal capsule, consistently found ovarian atrophy, as did Harris when portal vein regeneration was completely obstructed. Attempting to prove that the portal vessels are not essential in regulating the hypophysis, Thompson and Zuckerman (1954) stated that increased illumination induced estrus in two ferrets after stalk-section and in the absence of demonstrable regeneration of portal vessels. Donovan and Harris (1954), however, examining the histologic sections prepared from 1 of the 2 animals, found many such vessels that were uninfected. In their own experimental series, an estrous response to light was always associated with regeneration of the portal veins.

Greep and Barrnett (1951) rightly emphasized the prime importance of a good vascular supply for recovery of function by the pars distalis after either transplantation or stalk-section. They pointed to the extensive central infarction and scarring that characteristically followed stalk-section by their technique, an obvious factor contributing to hypopituitarism. Harris (1950a I, however, reported good function from sev

512

PHYSIOLOGY OF GONADS

eral hypophyses in which there was pronounced central necrosis in company with well regenerated portal vessels. A study by Nikitovitch-Winer and Everett (1957, 1958b) established beyond doubt that qualitative functional losses after stalk-sectron or transplantation of the pars distalis result, not from impaired blood supply per se, but from the loss of the intimate neurovascular relationship with the hypothalamus. Hypo])hyseal autografts, after first being placed under the kidney capsule for several weeks with the usual atrophy of the ovarian follicular apparatus and interstitial tissue, were later retransplanted to a site immediately under the median eminence. In the definitive series of 14 such experiments, 13 rats resumed estrous cycles spontaneously 8 to 68 days after retransplantation ; 7 were fertile and carried litters to term. A correlated study (Nikitovitch-Winer and Everett, 1959) demonstrated clearly that on the occasion of each of these successive transplantations there was massive necrosis of the interior of the glandular mass, leaving but a thin shell from which the functional tissue of the graft was reconstituted. In spite of this double insult some special influence of the hypothalamus brought about renewed function in a surprising number of cases. Together with the restoration of gonadotrophic activity there was significant improvement in thyroid-stimulating hormone (TSH) and adrenocorticotrophic hormone (ACTH) secretion. The considerable net loss of hypophyseal parenchyma resulting from the two operations was reflected only quantitatively in the effects on the various target organs. Ovarian weights, numbers of follicles and corpora lutea, adrenal weights and extent of adrenal hypertroi)liy after unilateral adrenalectomy, and thyroid uptake of P^^ were all intermediate between those of the normal female rat and control animals in which the graft remained on the kidney or was retransplanted under the temporal lobe of the brain.

Regulation of pars distalis secretion by means of the stalk vessels may conceivably be carried out either by regulation of blood How or by transmission of chemical mediatoi-s from the proximal capillary plexus in the median eminence to the pars distalis. An

experiment describetl by Swingle, Seay, Perlmutt, Collins, Fedor and Barlow (1951) suggested that a mediator subject to Dibenamine blockade might be involved in precocious puberty. Although significant uterine enlargement was produced in immature rats by daily stimulation of the cervix uteri for 10 days, no such effects were observed in similar rats given Dibenamine daily by stomach tube. Unfortunately, there were no controls for the possible effect of Dibenamine in nonstimulated or gonadotrophininjected animals.

Fluhmann (1952) invoked precocious vaginal opening and ovarian stimulation in immature rats by injection of neostigmine. The locus of such cholinergic action is unknown. Parenthetically, Barbarossa and di Ferrante (1950) reported follicle stimulation in immature rats after injection of intermedin, an effect not found in hypophysectomized subjects. Benoit and Assenmacher (1955) proposed that, in the drake, gonad-stimulating activity is governed by an agent contained in neurosecretory substance, which is demonstrable in abundance in the retrochiasmatic region of the median eminence. Capillaries there drain selectively into an anterior set of portal venules. Oxytocin has been suggested as a possible mediator for gonadotrophin secretion (Shibusawa, Saito, Fukuda, Kawai, Yamada and Tomizawa, 1955; Armstrong and Hansel, 1958). There is much interest as this is being written (1958) in the jiossibility that vasopressin, oxytocin, or other agents associated with neurosecretory substances of the neui-ohyjioiihysis are responsible for control of production and release of the various trophic hormones of the pars distalis. As an alternative or even a supplement to neurochemical regulation, a vasomotor mechanism cannot be denied (Green, 1951), for conceivably only a slight shift in blood flow through the jiars distalis might tip the balance of hormone production one way oi- anothei-. Thus the matter stands: whereas it is apjiarent that the hypothalamus intervenes in follicle growth and estrogen secretion, how it does so is little more than speculati\'e.

MAMMALIAN REPRODUCTIVE CYCLE

51)5

VI. Follicle Maturation and Ovulation

A variety of evidence indicates discontinuity between growth of large follicles, on the one hand, and their preovulatory maturation, on the other. Such is clearly the case among "reflex ovulators." Evidence that the same is true for spontaneous ovulators will be outlined below. Follicle maturation, ovulation, and structural transformation of the follicles to corpora lutea seem to represent successive stages in a distinct physiologic process, superimposed on the follicle growth cycle and brought about by a relatively abrupt increase in circulating gonadotrophin (theoretically LH). Since there is evidence (p. 519) that progesterone secretion may become detectable as this process begins, there might be justification for regarding it as merely the first portion of the luteal phase. However, the fact that luteinization ( i.e., the organization per se of luteal tissue) does not necessarily lead to functional cor|)ora lutea warrants treatment of the ovulation-luteinization phase as a distinct phenomenon.

Although it is customary to state that the hypopliysis invokes ovulation by release of LH, there is considerable question about the auxiliary roles played by other gonadotrophic hormones (Hisaw, 1947). Inasmuch as the time of release has been known in only the reflex ovulators, one might look to them for information. However, the available data (Hill, 1934) pertain only to the ovulating i)otency of the total gonadotrophin content of the hypophysis at various times after coitus. Substitution experiments are unsatisfactory because the presence of competent follicles implies the presence of l)oth FSH and a small amount of LH. The substitution of even the purest hormone preparations immediately after hypophysectomy leads to equivocal results inasmuch as it must be assumed that some FSH and LH of intrinsic origin remain in circulation. Talbert, Meyer and McShan (1951) determined that in rats, when hypophysectomy is performed at the onset of proestrum, the follicles remain capable of responding to injected LH for about 6 hours. Morphologic signs of follicle deterioration do not appear until nuich later. Adding to the uncertainty

is the fact that relatively pure preparations of either FSH or LH will ovulate an estrous rabbit (Greep, van Dyke and Chow, 1942). On the other hand, until the recent use of species-specific gonadotrophins (van Wagenen and Simpson, 1957), the primate ovary was notoriously difficult to ovulate therapeutically. Until effluent blood from the hypophysis can be assayed, there is little likelihood that the gonadotrophin complex that is normally responsible for ovulation can be known. Thus, whereas the expression, LH-release, will be employed occasionally to refer to the release of gonadotrophin that in^•okes ovulation, the term is used purely for convenience and brevity, and should be ai^propriately qualified by the reader.

A. TIME OF OVUL.\TION

The time of ovulation with respect to other events of the cycle is relatively easy to determine in reflex ovulators, but in spontaneous ovulators has proven to be more elusive. In the former, laparotomy at various intervals after the stimulus enables exact measure to be made of the time required to accomplish ovulation. For most of the spontaneous ovulators, save the few in which the ripening follicles can be palpated as in monkeys and cattle, it has been necessary to attempt to correlate ovulation with some easily detectable external sign. Inasmuch as the ovulation stimulus to the hyj)oiihysis in these animals is probably invoked by ovarian hormones and these are equally responsible for phenomena such as vaginal cornification and behavioral estrus, a considerable degree of correlation might be expected between ovulation and a given change in the vaginal smear or onset of estrous behavior. The predictability of the relationship, however, must depend in great measure on the degree of correlation among thresholds of response in the various tissues concerned. In the primates that have no sharply limited period of sex desire the i:)roblem is even more troublesome. When reference is made to the date of the last menstruation, prediction is erratic because of the variable occurrence of postmenstrual quiescence (Rossman and Bartelmez, 1943; Young and Yerkes, 1943). Consequently, attempts must be made to find indicato:

514

PHYSIOLOGY OF GONADS

such as basal body temperature fluctuations which may bear some intrinsically closer relationship to the event in question. (See Hartman, 1936, and Buxton and Engle, 1950, for discussion of this very practical ]iroblera. )

Among mammals generally, si)ontaneous ovulation takes place sometime during estrus (Asdell, 1946) . It is found during early estrus in the opossum, red fox, dog, mouse and hamster. In the rat some authors have placed it early (Young, Boling and Blandau, 1941) and others late (Long and Evans, 19221 with respect to vaginal estrus. In the writer's colony both relations hold, in 4-day and 5-day cycles, respectively. Ovulation in late estrus is reported for the cotton rat, bank vole, guinea pig, pig, horse, and ass. Sheep usually ovulate shortly before the end of heat, sometimes a few hours afterward. As stated earlier, ovulation may even occur in guinea pigs, rats, sheep, and cattle without overt estrus. The cow usually ovulates several hours after the end of heat. The marsupial cat is said to ovulate 5 days afterward (Hill and O'Donoghue, 1913). The extreme is represented by certain bats (Asdell, 1946) which copulate in autumn and ovulate in the spring after a prolonged state of subestrus. These variations probably express several factors.

Among reflex ovulators there is considerable interspecies variation in the interval between the stimulus that invokes release of gonadotroj^hin from the hypophysis and the eventual rupture of the Graafian follicles (rabbit, ca. 10 hours; ferret, ca. 30 hours; cat, 24 to 54 hours; 13-lined ground sciuirrcl, 8 to 12 hours; mink, 36 to 50 hours) . Among spontaneous ovulators the comparable interval is clearly defined for only the rat, 10 to 12 hours (Everett, Sawyer and Markce, 1949) . In the cow the data obtained by Hansel and Trimberger (1951) and Hough, Bearden and Hansel (1955) i)lace the outside limit at about 30 hours. Here again, threshold differentials among tlic various tissues of the individual are piobably of gicat importance. 'I'hus in one species the threshold foi' gonadoti'ophin release may be lower than that foi' estrous behavior with the result tliat by the time the latter makes its ai)pearance the former has already transpired and

ovulation will shortly take place. The rat, for example, releases LH during the afternoon, begins to show estrous behavior around 8 p.m., and ovulates around 2 a.m. (Everett, 1948, 1956b). In other species these time relationships may be reversed. In the cow, activation of the hypophysis apparently occurs several hours after the onset of estrus (Hansel and Trimberger, 1951). The cow remains in heat 10 to 18 hours and ovulates 13!/2 to 151/2 hours after going out of heat (Asdell, 1946). The early termination of estrus apparently reflects a refractory state which sets in after estrogen activity has continued for a time, for castrates receiving continued estrogen therapy remain in estrus for similarly brief periods. In the mare, ovulation is delayed until a few hours before the end of estrous periods that may extend for 5 to 10 days or longer. This suggests a relative refractoriness of the LHrelease mechanism in this animal. Such a state of affairs approaches that in persistent estrus or in the anovulatory cycle.

B. OVARIAN STEROmS AND OVULATION

1. Estrogens

Chronic administration of estrogen to the intact animal eventually produces ovarian atrophy by suppression of gonadotrophin secretion. However, some moderate basic level of continuous estrogen secretion must be compatible with normal function of the hypophyseal-ovarian system; witness the fact that blood estrogen assays in normal women (]Markee and Berg, 1944) indicate only a 2-fold increase at midinterval above a base value of considerable magnitude.

Induction of corpus luteum formation by injected estrogen was first demonstrated by Hohlweg (1934) in prepubertal rats^ and the phenomenon has been repeatedly observed by other woi'kers (Desclin, 1935; Mazer, Israel and Aljjcrs, 1936; Westman and Jacobsolm, 1938b; Herold and Effkemann, 1938; Price and Ortiz, 1944; Cole, 1946). The fact that the effect was not obtained in rats younger than 30 to 36 days l>y Piice and Ortiz, whereas Cole observed it in the age-range of 21 to 28 days, demon ' Tlic eH'cct was later ol)1aiii(^(l witli androgens (Holilwpg, 1937; Salmon, 1938; Xathanson, Fianspen and Sweenev, 1938).

MAMMALIAN REPRODUCTIVE CYCl.E

515

B

^,

^ >

1 2

3 4 5

e

A

12 3 4 5

12 3 4 5

Fig. 8.7. Experimental modifications of the 5-day cycle in rats. Two units of the ordinate represent full vaginal estrus. Time in days on abscissa, each unit 24 hours (midnight to midnight). X, ovulation time; -p, progesterone, usually 1 to 2 mg.; e, estradiol benzoate, standard do.se 50 /xg. (From J. W. Everett, Endocrinology, 43, 393, 1948.)

strates the existence of strain differences in the age factor. This probably explains the absence of luteinization in the experience of Lane (1935) and Merckel and Nelson (1940). Hohlweg and Chamorro (1937) demonstrated the importance of the hypophysis in the response. When hypophysectomy was performed 2 days after injection of estrogen no corpora liitea developed, but hypophysectomy on the 4th day did not interfere with corpus luteum formation. The effect could be produced in 50-gm. rats with as little as 4 |U,g. estradiol benzoate. Westman and Jacobsohn (1938b) reported that transsection of the hypophyseal stalk less than 2Vt days after injection prevented the reaction, but after that time the operation did not interfere. Bradbury (1947) assayed the gonadotrophin content of hypophyses of normal and castrated immature rats (30 to 32 days old at autopsy) 2 to 5 days after injection of estrogen or other steroids. These rats were apparently too young to form corpora lutea in response to the treatment, l)ut marked interstitial-cell stimulation, indicative of LH (ICSH) activity, was observed as early as 96 hours. In the intact animals significant loss of potency occurred 72 to 96 hours after injection, in agreement with the hypophysectomy data of Hohlweg and Chamorro (1937). In castrated rats, however, there was no loss of potency, thus suggesting that some ovarian factor in addition to estrogen is essential for stimulation of the hypophysis. It is unfortunate that the study was confined to animals too young to give the full response of luteinization.

Induction of ovulation in adult animals by estrogen was first reported by Hammond, Jr., Hammond and Parkes (1942) and by Hammond, Jr. (1945) in the anestrous ewe. Whereas the s])ontaneous occurrence of occult ovulation was approximately 5 per cent, injection of stilbestrol was followed by corpus luteum formation in 4 of 11 ewes, with recovery of ova in 3. Injection of stilbestrol di-n-butyrate was followed by corpus luteum formation in 5 of 6 ewes and ova were recovered in 3. The finding was confirmed by Casida (1946) who stated that in cycling ewes ovulation can be invoked by injection of diethylstilbestrol on the 4th day of the cycle, but not at other times. In 1947 Everett reported the induction of ovulation in pregnant rats within 40 hours after injection of estradiol benzoate (as little as 2 or 3 /xg.) or implantation of estradiol crystals or pellets. The response was not obtained in animals autopsied 24 hours after treatment nor in other animals hypophysectomized at 24 hours and autopsied the following day. In other studies with adult rats it was demonstrated (Everett, 1948) that in 5-day cyclic rats the injection of estrogen at mid-diestrum will regularly induce ovulation 24 hours earlier than exi:)ected (Figs. 8.7D, 8.8F|. Persistent-estrous rats were refractory to estrogen in this respect.- Nevertheless, when such animals were made pseudopregnant by daily injection of progesterone,

"The tendency toward refractoriness of similar animals with respect to induction of estrous 1) ha\'ior had earlier been reported by Boling, Blandau, Rundlett and Young (1941).

516

PHYSIOLOGY OF GONADS

B

12 3 4

12 3 4

T%

^'

2 3 4 5

1 P

2 3 4 5 e v^^ / y

r-U^'f*^ 1 tA 1

12 3 4 5

Fig. 8.8. Experimental modification of the 4-day cycle in rats. Same key as in Figure 8.7. Progesterone dosage 1.5 mg. per day. Artificial 5-day cycles in D, E, and F indicated by dotted lines and numbering. (From J. W. Everett, Endocrinology, 43, 395, 1948.)

B

e NO OVULATION

j~~r~r~r

Fig. 8.9. Experiment with persistent-estrous rats. Units of ordinate and abscissa have same meaning as in Figure 8.7. A. Secjuence of "progesterone cycles." Each dose of progesterone (p) is 1.0 mg. Ovulation (x) in about 70 per cent of the cycles. B. Progesterone cycle followed by unsuccessful attempt to induce ovulation by estrogen during the second c-ycle. C . Pseudopregnancy maintained by daily iiijoctinn of 1.5 mg. ]irogosteronr. Ovulation induced by estrogen in several such cases. (From .1. \V. Everett, EiKhxTinolojiy. 43, ;5i»9, 194S.)

ovulation and corpus luteuni loiniatioii were induced by estrogen (Fig. 8.9 1.

Early attempts to induce luteinization in the guinea pig with estrogen were unsuccessful ( Dempsey, 1937; see Fig. 8.5), but iiioiv recently Lipschutz, Iglesias, Bruzzone, 11 uniercz and Penaranda (1948) have shown by the use of intrasplenic ovarian autografts

that luteinization is a reguhir feature in experiments in which estrogen is administered systcniically. Interestingly enough the implantation of estrogen jiellets in or near the ox'ariaii grafts had tlic coiitrai'y effect of pi'cvcnliiig luteinization.

it was early I'cportcd that rabbits fail to ovulate in response to estrogen injection

MAMMALIAN REPRODUCTIVE CYCLE

517

(Bachman, 1936; Mazer, Israel and Alpers, 1936 ». Hisaw (1947j inferred that this is generally true for reflex ovulators. Nevertheless, it was found by Klein and Mayer ( 1946) and Klein (1947) that when pseudol^regnant or pregnant rabbits were treated with estrogen and then mated, new ovulation resulted and new corpora lutea were formed, events that do not otherwise occur. The phenomenon was further explored by Sawyer (1949). Whereas untreated rabbits, unlike cats, do not ovulate in response to mechanical stimulation of the vagina, treatment with estrogen on the preceding 2 days results in a positive response to this stimulus. In fact, his later observations (1959) indicate that estrogen priming for a longer period (4 days) occasionally results in "spontaneous" ovulation, especially during the winter and spring.

In the anestrous cat, in the response to mechanical stimulation of the vagina, estrogen facilitates the ovulation of follicles primed with equine gonadotrophin (Sawyer and Everett, 1953).

Induction of ovulation by estrogen in primates remains to be demonstrated. It is of interest in this connection that Funnell, Keaty and Hellbaum (1951) observed in menopausal women an increased excretion of LH during estrogen therapy, in contrast to FSH excretion at other times. The general experience has been that injection of estrogen during the early part of the cycle significantly postpones the next expected ovulation and menstruation (monkey, Ball and Hartman, 1939; baboon, Gillman, 1942; human, Sturgis and ^leigs, 1942; Brown, Bradbury and Jennings, 1948). Gillman reported that a single injection of estrogen precipitates widespread atresia of vesicular follicles. Brown and Bradbury (1947) reported IH-eliminary data that in 4 of 6 women there was increased gonadotrophin excretion during the 24 hours following estrogen administration. They proposed that delay of ovulation by estrogen given early in the primate cycle may be the result of premature discharge of gonadotrophin before the Graafian follicle is competent. Sturgis and Meigs had suggested, on the contrary, that the estrogen suppresses hypophyseal function. D'Amour (1940), finding in urinary assays that the initial peak of estrogen ex

cretion preceded the peak excretion of urinary gonadotrophin, postulated that the increase of estrogen stimulates the gonadotrophin release that is responsible for ovulation. O. W. Smith (1944) proposed that not estrogen itself, but some metabolite resulting from inactivation by the liver, is responsible for LH release. This interesting hypothesis has not been substantiated.

3. Gestagens

Suppression of estrus and ovulation by functional corpora lutea, suggested by Beard (1898), was experimentally demonstrated in the guinea pig by Loeb (1911). It is now well established in several species that removal of the corpora lutea results in early resumption of estrus and ovulation (see p. 506), and that administration of progesterone suppresses these events. There is considerable evidence favoring the view that the primary effect is to selectively suppress the secretion of LH. Dempsey (1937) noted that in guinea pigs receiving daily injection of progesterone (50 /^g.) all stages of follicle development proceeded except the maturation enlargement that heralds LH release (Fig. 8.5). Astwood and Fevold (1939) and Cutuly (1941b) found similar results in rats. Essentially the same phenomenon has been noted in sheep by Dutt and Casida (1948). Bradbury (1947) reported that in immature rats the injection of progesterone at the time of estrogen injection prevented the release of gonadotrophin (LH?) which otherwise followed estrogen injection by 72 to 96 hours. In ovariectomized guinea pigs containing intrasplenic autografts, preparations in which luteinization can be induced by estrogen (see above) , the simultaneous administration of gestagens prevented this action (Lipschutz, Iglesias, Bruzzone, Humerez and Pefiaranda, 1948; Iglesias, Lipschutz and Guillermo, 1950; Mardones, Bruzzone, Iglesias and Lipschutz, 1951). Mardones and co-w^orkers also made the interesting observation that among several steroids having progestational activity, "antiluteinizing activity is not concomitant with, or subordinated to" the former function. Proportionately very large amounts of ethinyl testosterone and ethinyl-A-'^-androstenediol exhibited very little antiluteinizing activity. There is evi

518

PHYSIOLOGY OF GONADS

denceinmice (Solve,, 1939) that siippreijsion of FSH secretion may occur when as much as 1 mg. of progesterone is injected daily. Alloiteau ( 1954 ) believes that this also occurs in the rat, although Cutuly (1941b) found only slight evidence of FSH suppression when as much as 6 mg. progesterone were given daily for several weeks.

So much emphasis has been placed on the suppressing effect of progesterone that its facilitating actions were recognized only in recent years. The first indication that progesterone can promote ovulation and corpus luteum formation in mammals was encountered in a study of persistent-estrous rats (Everett, 1940a, b). Daily injection of 0.25 to 1.0 mg. caused the prompt interrujition of the state of persistent follicle and the resumption of outwardly normal cycles. Corjiora lutea were formed in approximately 70 per cent of these cycles.^ The effect was obtained not only in older rats in which persistent estrus had developed spontaneously, but also in young rats in which the condition had been induced by continuous illumination. The dose level employed is below that required to suppress cycles in normal rats (1.5 mg. daily; Phillips, 1937). Subsequently, it was found (Everett, 1943) that the daily injection could be avoided if a single "interrupting" dose was given, followed by a single injection during proestrum or early estrus of each recurrent cycle (Fig. 8.9.4). The histologic appearance of the ovaries reverted toward the normal after a succession of "i)rogesterone cycles" and, significantly, the interstitial-cell nuclei were "repaired." Attempts in normal rats to invoke o\'u]ati()n earliei' than the expected time were sticcessful in the 5-day cycle (Figs. S.7B, 8.8^). Injection of from 0.5 mg. to 2 mg. on the "third day of diestrum" regularly (4 mg. occasionally) invoked ovulation (luring the coming night (Everett, 1944a, 1948) unless the treatment was given too late in the dai/ (EA'erett and Sawyer, 1949; see discussion on ]). 526 I'egarding the diurnal rhythm and ovulation). Attempts to advance ovulation in the 4-day cycle were unsuccessful, possibly because the follicles were not competent or the animals'

Marvin (1947) described a similar rosull willi desoxycorticosterone acetate.

intrinsic estrogen levels were not elevated sufficiently early.

Ovulation induced by progesterone has been reported in several species. A direct action on the excised ovary of the toad Xeno-pns was early demonstrated by Zwarenstein (1937) but such action is apparently not found in higher vertebrates. In the domestic hen injection of progesterone can invoke ovulation several hours ahead of schedule (Fraps and Dury, 1943; Rothchild and Fraps, 1949). Pfeiffer (1950) observed new corpora lutea in 10 rhesus monkeys treated with progesterone during presumptive anovulatory cycles of the summer months. Similar attempts have been made in women (Rothchild and Koh, 1951); although there were said to be definite indications of induced ovulation, the evidence is equivocal. On the other hand, a rei~)ort (Hansel and Trimberger, 1952) states that in heifers the injection of small doses of progesterone (5 to 10 mg. ) at the beginning of estrus significantly advances ovulation time. This is in contrast with the inhibitory effect of larger doses (50 mg.) beginning before the onset of estrus (Ulberg, Christian and Casida, 1951). Even in the rabbit (Sawyer, Everett and Markee, 1950), spontaneous ovulation was noted in 4 of 10 animals after combined estrogen and progesterone injection.

From certain of the foregoing statements it may be inferred that whether suppression or stimulation follows administration of ju-ogestcrone depends critically on the time of injection, on the amount given, on the status of the ovary, and probal)ly on a l)riming action of estrogen. A significant illustration of the critical nature of the time factor in rats is given by the experiments represented in Figure 8.8r and E. If after the first injection of 1.5 mg. progesterone on the first day of diesti'uni, a second injection follows in. about 24 lioui's. the imjiending estrus and o\-ulation are retarded an additional 24 hours. However, if the second injection is given 48 hours after the first, the impending estrus and ovulation are advanced. Evidence^ of the synergistic action of estrogen and progesterone in evoking oA'ulation is given by the ex]ieriments represented in Figuiv 8.9/> and (\ Sawver (1952)

MAMMALIAN REPRODUCTIVE CYCLE

519

investigated the synergism in rabl^ts. Employing estrogen-primed animals, he found that ovulation was facilitated when progesterone was injected less than 4 hours before either mating, mechanical stimulation of the vagina, or intravenous injection of copper acetate. Inhibition was obtained when progesterone was injected 24 hours before such stimulation, thus confirming the often-cited observations of ]Makepeace, Weinstein and Friedman (1937 » and Friedman (1941) that progesterone inhiijits ovulation in rabbits.

Preovulatory secretion of gestagens now seems likely. Morphologic luteal changes in preovulatory follicles are considered in the chapter on the ovary. A variety of evidence in primates indicates that progestational clianges in the endometrium begin before ovulation (Bartelmez, Corner and Hartman, 1951). Several workers have reported tiie excretion of small amounts of pregnanediol during the follicular phase of the human cycle (Wilson, Randall and Osterberg, 1939; Smith, Smith and Schiller, 1943; Davis and Fugo, 1948). Determination of plasma progesterone in women by the Hooker-Forbes test indicates the presence of significant amounts a day or two before a major rise in basal body temperature (Forbes, 1950). In monkeys a small quantity (ca. 0.5 to 1.0 ixg. per ml.) was detected l)etween the 4th and 9th days, rising in the 10- to 15-day period to concentrations of 2 to 6 jxg. per ml. (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). In both species a transient decline seems to intervene before the marked rise to still higher concentrations during the luteal phase. In the rat, Constantinides (1947) studied the stromal nuclei of the endometrium at different stages of the cycle and found that by the Hooker-Forbes criteria there is evidence of progesterone secretion during proestrum. Astwood (1939) on the basis of water content of rat uteri concluded that progesterone secretion begins with proestrum. In the rabl)it, Forbes (1953) assayed peripheral blood at various intervals after mating or gonadotrophin injection. Although no progesterone was detectable in controls, significant amounts appeared an average of 97 minutes after mating and 66 minutes after gonado

trophin injection. As much as 2.5 /xg. ])er ml. was found during the first 8 to 10 hours, although marked fluctuations were noted from time to time in the blood of individual animals. Verly (1951) reported that soon after mating the urine of rabbits contains significant amounts of pregnanediol.

It has become customary to state that the gestagen that appears during the follicular phase of the cycle is probably formed by the maturing follicle itself. Indeed, assays of fluid from Graafian follicles and cysts have indicated the presence of the hormone (Duyvene de Wit, 1942; Hooker and Forbes, 1947; Edgar, 1952, 1953). However, if it is to take part in the release of ovulating hormone gestagen must be secreted earlier than preovulatory maturation. For this also there is some evidence. Two reports cited above indicate that in monkeys, at least, there is a detectable amount present in the blood during the early follicular phase. The known fact that a waning corpus luteum favors the experimental induction of estrus and/or ovulation in sheep and cattle (Hammond, Jr., 1945; Robinson, 1951 ; Alarden, 1952) is suggestive. Although Hammond, Jr., Hammond and Parkes (1942) tested this possibility by progesterone sul)stitution with negative results, the amount given may have been too small, as Robinson suggested. A waning corpus luteum in the rat favors ovulation, as disclosed in persistent-estrous animals in which pseudopregnancy had been induced (Everett, 1939). Each of three pseudopregnancies was followed b}' a short cycle before the animals returned to persistent estrus.

In the course of studies growing out of this observation evidence was advanced (Everett, 1945) which indicated that corpora lutea of the normal rat are not wholly inactive during the short cycle. Transient depletion of cholesterol was observed in such corpora lutea during the proestrum that followed their formation. This implies a transient increase of luteotrophin secretion. Significantly this occurs before the release of LH. It is this writer's opinion that gestagen from such sources is not essential for the induction of ovulation but that it does facilitate the action of estrogen.

520

PHYSIOLOGY OF GONADS

C. ROLE OF THE NERVOUS SYSTEM IN OVULATION

Historically, the fact of neural control of reflex ovulation has been recognized in the ral)bit for many years. The comparable role of the nervous system in spontaneous ovulation, on the other hand, has more recently become apparent. It now seems justifiable and useful to postulate in the hypothalamus of reflex ovulators and spontaneous ovulators alike the existence of a mechanism that is peculiarly concerned with release of ovulating hormone. Whether it consists in a discrete anatomic entity is immaterial for the present.

The suggestion has been made that the outstanding difference between reflex and sjiontaneous ovulation may be in the kinds of afferent impulses that most readily excite the hypothalamus (Sawyer, Everett and Markee, 1949). The difference is not absolute, for spontaneous ovulation has been induced in rabbits by estrogen-progesterone injection (see p. 519) and reflex ovulation has been demonstrated under special circumstances in rats (Dempsey and Searles, 1943; Everett, 1952a) and cattle (Marion, Smith, Wiley and Barrett, 1950). The random distribution of reflex ovulators and spontaneous ovulators among mammalian orders becomes more understandable if one assumes that dual controls are widely represented and that special adaptations favor one or the other in given species.

The ovulation reflex in rabbits is apparently initiated by afferent impulses of multiple origin, among them not only impulses from the genitalia, but also propriocejUive impulses from muscles that are utilized in coitus. Brooks (1937, 1938) found that, although the sacral segments of the spinal cord and the abdominal sympathetic chains could be eliminated without jM'eventing ovulation, the luml)ai' cord must remain. Only by paralysis of the lower half of the body so that the female could not take pai't in coitus was ovulation pi'cvcntfMl. The neocortex could be removed, together with the olfactory bulbs, labyrinths, auditory apparatus and eyes without impairing the ovulation response. Even after complete decortication, ovulation followecl coitus in 1 out of 3 j'abbits. It must l)e admitted, how

ever, that, although various parts of the nervous system may thus be eliminated without changing the end result, some of them may normally play a considerable role. With little cjuestion, direct stimuli from the genitalia play a part in the natural reflex. Under certain experimental conditions detectable electrical activity in the rabbit rhinencephalon is associated with the induction of ovulation (Sawyer, 1955). Electrical stimulation of the amygdala will induce ovulation in rabbits and cats (Koikegami, Yamada and Usei, 1954; Shealy and Peele, 1957 ) .

In rats, Davis (1939) found that removal of the neocortex had no effect on the estrous cycle and ovulation. Removal of portions or of the entire sympathetic chains of rats likewise did not interfere with ovulation (Bacq, 1932). Bunn and Everett (1957) reported ovulation in constantestrous rats after electrical stimulation of the amygdala.

The importance of the dorsal thalamus is unknown. The reticular activating system has been implicated as a component of the ovulation mechanism (Sawyer, 1958), but the manner of its contribution is not clear. There is little cjuestion, on the other hand, of the indispensability of the hypothalamus and its neurovascular connection to the adenoliyi:)ophysis through the median eminence and the hypophyseal portal veins.

Although the observation by ]\Iarshall and Verney (1936) that ovulation can be induced by passing an electric current through the heads of estrous rabbits hardl}^ limited the effect to the hypothalamus itself, it was later shown that more localized electrical stimuli api)lied to certain hypothalamic regions are consistently effective (preoptic area, Haterius, 1937; Christian. 1956; posterior hypothalamus or tuber cinereum, Harris, 1937, 19481); tuber cinereum or adjacent hypothalamic areas, Markee, Sawyer anirHollinshead. 1946; medial hypothalamus fi'om ])i-eo])tic area to mammillai'V bodies. Kui'otsiu Kurachi and Ban, 195o"; Kuiotsu, Kurachi, Tabaya>hi and Ban, 1952).

Ahliougli liypotliahimic lesions. l)oth natural and expeiimental. hax'c frecjuently been reported to interfere with normal

MAMMALIAN REPRODUCTIVE CYCLE

sex function (.see Harris. 1948a, 1955, for references), the majority of these reports do not api^ly to the question at issue — control of ovulation. When ovarian atrophy occurs, as it frequently did in these cases, it reflects a profound depression of gonadotroi)hin secretion and absence of competent follicles. However, Dey, Fisher, Berry and Ranson (1940) and Dey (1941, 1943) 'found in guinea pigs that gross bilateral electrolytic lesions placed in the rostral hypothalamus resulted in persistent follicles with continuous estrogen secretion. Similar results were obtained in rats by Hillarp (1949) when small bilateral electrolytic lesions were placed in the anterior hypothalamic area near the paraventricular nuclei or between this region and the median eminence. Greer (1953) reported continuous estrus in rats after placing certain small lesions in the ventromedial nucleus, provided they were bilateral. There was no correlation with obesity. There are at least four significant points in common among these several ablation experiments. ( 1 ) The effective lesions were rost rally placed and either were limited to or included the medial group of nuclei. (2) The tuber cinereum, median eminence, and stalk connection to the hypophysis were intact. (3) Although development of competent follicles was not evidently impaired, estrogen secretion became continuous instead of cyclic. (4) The proper impetus for release of ovulating hormone from the hypophysis was absent. It would be most instructive to learn whether ovulation can be invoked in such animals by reflex stimulation or by direct electrical stimulation of the tuber. AUoiteau and Courvoisier (1953) reported that rats in constant estrus as a result of hypothalamic lesions did not undergo pseudopregnancy after stimulation of the cervix uteri. This observation, confirmed by Greer (1953), could be construed as indirect evidence of failure of reflex ovulation, for Greer regularly obtained pseudopregnancy by cervical stimulation, once corpora lutea had been formed by other means.

Other findings by Greer are important because of their bearing on the location and character of a presumptive ovulation center. Althougli the onset of persistent estrus after

making the lesions was sometimes almost immediate (following a brief anestrous interval), in other cases it was preceded by several apparently normal cycles. In any event, once the condition had become established, the daily injection of small amounts of progesterone brought about the recurrence of cycles and corpus luteum formation. In about half of the cases these cycles continued for awhile after withdrawal of treatment, whereas in the remainder there was a prompt return to persistent estrus. Essentially the same results were reported by AUoiteau (1954), and the observations suggest that the areas involved in such lesions may be of only secondary importance.

The use of radioactive phosphorus for estimating energy exchange in tissues affords a different approach to the problem of neural control of ovulation (Borell, Westman and Orstrom, 1947, 1948). This method has the virtue that the experimental subject remains undamaged until injection of P'*compounds 30 minutes before the end of the experiment. In rabbits there is a marked increase in phosphorus turn-over in the tuber cinereum within 2 minutes post coitum, and continuing for about an hour thereafter (Table 8.1). The adenohypoi)hysis shows increasing activity during the first 10 minutes which reaches a peak at about 1 hour and then regresses somewhat, although it remains relatively high for 24 hours. Response of the ovary to gonadotroi:)hin release is marked by a rapid rise during the second half-hour and another pronounced increase near the time of ovulation. In rats, at various stages of the estrous cycle, phosphorus exchange in both tuber cinereum and adenohypophysis is maximal during proestrum. In the ovary high values were reported during diestrum and proestrum, somewhat lower values during estrus and metestrum.

Possibly correlated with the above information is the observation (Gitsch, 1952b) that in rats the acetylcholine (ACh) content of the tuberal region becomes elevated during proestrum and estrus. It is said that ACh synthesis requires high energy phosphate (see Bain, 1952). Further investigation by Gitsch (1952a) and Gitscl: and Reitinger (1953) revealed that ACh

TABLE 8.1 Sequence of events in rabbit ovulation

Time Post Coitum Central Nervous System

Hypophysis

Ovary

Circulating Blood

<30 sec.

<2 mill.

10 mill. 30 mill.

60 mill.

75-90 mill.

13^-2 hrs. 3-5 hrs.

6-7 hrs.

7-8 hrs. 9-11 hrs.

Barbiturate-sensitive and atropinesensitive mechanisms^

t Phosphorylation in tuber cinereum^

t Phosphorylation

in tuber ciner reumt Phosphoryhition

in tuber cine reum^

t Phosphorylation in tuber cinereum

t Phosphoryhition^

Release of LH ca. 20 per cent. 6 Hypophysectomy prevents ovulation^' 12

Release of LH nowsufficient for ovulation."' 12 Phosphorylation at peak^

I Phosphor\iatioii

i Animal may be bled and transfused without prevent ! ing ovulationi2

folliculi. in egg

f Liquor Tetrad.' nuclei*

Cholesterol depletion in interstitial gland. ^ Egg nucleus migrates, membrane dissolves.** • " Prominent corona*

Liquor folliculi increasingly viscous*

First polar hotly'*

Marked .swelling of follicles. Thecal hypertrophy,! ■ i° hyperemia

OvuL.^Tion." t Phosphorylation2

Bleeding and transfusion now prevent ovulationi2

Progesterone detectable ^

Increased estrogen (endometrial hj-peremia)'

' Asdell, 1946.

2 Borell, Westman and Orstrom, 1947.

3 Claesson and Hillarp, 1947a. " Fee and Parkes, 1929.

'^ Forbes, 1953.

« Hill, 1934.

^ Sawyer, Markee and Hollinshcad, 1947.

• Pincus and Enzmann, 1935.

^ Sawyer and associates, 1947, 1949, 1950.

1" Walton and Hammond, 192S.

" Waterman, 1943.

'2 Weslmaii and .lacohsohn, 1936.

522

MAMMALIAN REPRODUCTIVE CYCLE

523

in the rat hypothalamus is increased also by administration of estrogen or by castration, conditions that similarly increase lihosphorus exchange (Borell and Westman, 1949). The ACh content is depressed during pregnancy or when the rat has been injected with progesterone. It is also lowered by Pentothal anesthesia, a matter of interest in relation to the fact that the barbiturates suppress ovulation (see p. 526).

The location and measurement of activity in discrete nuclei and pathways are largely in the future, although a beginning has been made in the rabbit, cat, rat, and mouse. Sawyer (1955) found in rabbits, after the combined administration of pentobarbital intravenously and histamine by way of the 3rd ventricle, that there was associated with induction of ovulation a characteristic change in intrinsic electrical activity of the rhinencephalon, extending into the preoptic area. If the olfactory tracts were cut, however, this activity could not be elicited and ovulation failed. According to Porter, Cavanaugh and Sawyer (1954), vaginal stimulation of estrous cats caused altered electrical activity in two hypothalamic regions:

(1 ) in the lateral hypothalamic area at the anterior tuberal level during stimulation and for 15 to 45 seconds afterward; and

(2) in the anterior hypothalamic area near the medial forebrain bundle, where response was delayed as much as 5 n:iinutes after stimulation. According to a i)reliminary account (Critchlow and Sawyer, 1955) in curarized, proestrous rats, there were i)eriods lasting approximately 20 minutes in the midafternoon, during which altered electrical activity appeared differentially in the preoptic area or anterior hypothalamus.

Another approach to localization has been described by Hertl (1952, 1955). On the pro])Osition that increased function of particular cells is reflected by increased volume of their nuclei, cell nuclear volumes were measured in hypothalamic nuclei of female mice at different stages of the estrous cycle. During proestrum and estrus there was said to be a functional edema in hypothalamic nucleus 20 of Griinthal (possibly the pars posterior of the ventromedial nucleus of Krieg) and to lesser extent in nucleus 16 (Nucl. arcuatus).

1. The Ilypophi/seal Portal Veins and the Chemotransmitter Hypothesis

As noted elsewhere, hypothalamic control of the jiars distalis is probably mediated by the hypophyseal portal circulation. Evidence for this has been especially convincing with respect to control of ovulation, although indications are that other phases of the cycle are also regulated by this means. Pertinent data from numerous transplantation and stalk-section experiments may be summarized by the following statement. Aside from a questionable grafting experiment (2 rats) reported by May (1937), in no case has ovulation or luteinization been reported in the absence of vascular linkage of the pars distalis with the median eminence; on the other hand, ovulatory cycles have often been cjuickly restored when the gland has been revascularized by the portal vessels (see especially, Harris, 1950a; Harris and Jacobsohn, 1952; Nikitovitch-Winer and Everett, 1957, 1958b).

Although the importance of local vasomotor regulation in the stalk vessels remains to be evaluated (Green, 1951), there is extensive support for the hypothesis that ovulatory release of gonadotrophin is invoked by a chemotransmitter (Harris, 1948a, 1955). If one accei)ts the prevailing opinion that nerve fibers entering the pars distalis are too few to account for its secretomotor control and that the flow of blood in the hyi^ophyseal portal vessels is toward the gland (Wislocki and King, 1936; Green, 1947; Green and Harris, 1947, 1949; Barrnett and Greep, 1951 ; Landsmeer, 1951 ; ]McConnell, 1953; Xuereb, Prichard and Daniel, 1954; Worthington, 1955), the plausibility of the chemotransmitter hypothesis becomes inescapable.^

Evidence that the transmitter may l)e

^ For a dissenting view, see Zuckerman (1952). Reference should also be made to the hypothesis formulated by Spatz (1951) and associates (see Nowakowski, 1950, 1952). They postulated that a descending pathway in the spinal cord is the connecting link between hypothalamus and ovaries. With respect to ovulation, this is clearly denied by the fact that local stimulation of the hypotlialamus provokes ovulation in rabbits in which the thoracic spinal cord has been transsected (Christian, Markee and Markee, 1955).

524

PHYSIOLOGY OF GONADS

adrenergic was presented by Markee, Sawyer and Hollinshead (1948), who provoked ovulation in rabbits by instilling epinephrine directly into the pars distalis. Detailed experiments supporting this w^ere fully reviewed by Markee, Everett and Sawyer (1952). In discussion following that paper, Sawyer reported the induction of ovulation in rabbits by the injection into the third ventricle of either epinephrine or norepinephrine, and suggested that the latter is "more closely related to the natural mediator than is epinephrine." Donovan and Harris (1956), from studies in which the rabbit hypophysis was slowdy infused in situ with solutions of epinephrine or norepinephrine, concluded that neither substance is the agent in question, and that the positive results of Markee, Sawyer and Hollinshead (1948) were the effects of low pH and not of the drugs per se. Proof of the negative is elusive, however, and one must note that Donovan and Harris did not meet the conditions of timing and drug concentration that obtained in the earlier work.

Intravenous injection of Dibenamine or its congener, SKF-501,-^ will usually prevent ovulation in rabl)its when injection is completed within 1 minute after coitus (Sawyer and associates, 1947-50). On the other hand, when injection is delayed until 3 minutes or later, ovulation is unaffected. The nonadrenergic hydrolysis product of Dibenamine, 2-dibenzylaminoethanol, does not have the blocking action, although its central excitatory powers are much like those of the parent substance. The failure of blockade by Dibenamine, if injection is withheld for 3 minutes, demonstrates that the drug does not interfere with the actual discharge of ovulating hormone into the l)lood stream, for that process recjuires about an hour (Fee and Parkes, 1929; Westman and Jacobsohn, 1936). The Dibenamine-sensitive mechanism thus serves as a trigger, the gland being adequately stimulated within 1 01' 2 minutes post coitum. This estin^ate is in remarkal)le agreement with the earlier mentioned obscMA'ations on

•'■' Dibenamine is iV,iV-dibenzyl-/:i-cliloioetliy lamine. SKF-501 is A'-(9-fluorenyl)-.V-ethyl-/i-chlor()ethylamine hydrochlorifle. Banthine is /i-dictliylaminuc'tliyl-.\anthene-9-cai'l)Oxvlak' niclliohroiniilc

l)hosphorus exchange in the tuber cinereum and hypoi)hysis (p. 521, and Table 8.1 1.

A mechanism that is subject to blockade by atropine or Banthine^ evidently precedes the Dibenamine-sensitive process — temjDorally if not anatomically. To accomplish blockade in rabbits, these anticholinergic drugs must be injected intravenously within about 30 seconds after coitus (Sawyer and associates, 1949-1951). It should be recalled that Foster, Haney and Hisaw (1934) reported failure of ovulation in several rabbits treated with small amounts of atropine before mating. ]\Iakepeace (1938), however, was unable to confirm the effect with somewhat larger doses and the former observation was forgotten.

A seemingly crucial experiment devised by Sawyer gives conclusive evidence that the atropine-sensitive process is antecedent to the Dibenamine-sensitive one. It was based on two facts: (1) intravenous injection of nearly lethal doses of epinephrine does not induce ovulation in estrous rabbits, and (2) atropine protects rabbits against fatal pulmonary edema after injection of large amounts of epinephrine. In rabbits protected by atropine in dosage that was also sufficient to block the ovulation reflex, the injection of twice-lethal doses of epinephrine caused ovulation or significant degrees of follicle maturation in 5 of 7 cases. These effects were not found in rabbits protected by Dibenamine. Supporting evidence was adduced by Christian (1956i who found that atropine would not prevent ovulation in response to electi'ical stimulation of the medial preoptic area or adjacent parts of the hypothalanuis, whereas in a significant number of such rabliits ovulation was blocked by SKF-501.

Extension of the blocking experiments to the rat, as an example of a spontaneous ovulator, disclosed that in this species also ovulation can be blocked by Dibenamine, SKF501, atropine, and Banthine, when the injections ai'c appropriately timed with respect to the stage- of the cycle and time of day (Sawyer, Everett and Markee, 1949; Everett, Sawyer and Markee, 1949; Everett and Sawyer, 1949, 1950, 1953: see \). 526). Furthermore, blockade of ])oth estrogenimhiccd and pi'ogcstci-oiic-induced ovuhition

MAMMALIAN REPRODUCTIVE CYCLE

525

was aceomi)lished with cither Dibonamine or atropine. Neither agent, however, prevented ovulation after injection of sheep liypophyseal LH. A report by Hansel and Trimberger (1951) stated that in cattle a significant delay of ovulation (as great as 72 hours) followed atropine administration. In control experiments the simultaneous injection of atropine and human chorionic gonadotrophin was followed by ovulation slightly earlier than the normally expected time. Treatments were begun 1 to 5 hours after the onset of estrus. This work was confirmed and extended by Hough, Beardon and Hansel (1955). In the hen, blockade of ovulation, normal or induced by progesterone, has been reported after administration of Dibenamine, Dibenzyline, SKF-501 or atropine (Zarrow and Bastian, 1953; van Tienhoven, 1955). According to van Tienhoven, the drugs did not interfere with the ovulating action of extrinsic gonadotrophin.

It is important that the same drugs will block ovulation in lioth rabbits and rats (Table 8.2) . Of ec^ual significance is the fact that several agents that are ineffective in rabbits are also ineffective in rats (notably 2-dibenzylaminoethanol, the imidazoline adrenolytic drugs, and the ganglion blocking agents). These considerations are interjireted to mean that spontaneous ovulation is invoked by neurohumoral mechanisms that are very like those in the reflex ovulation of rabbits.

The suggestion that the l)locking effects might result from nonspecific stress, causing the hypophysis to be so actively secreting ACTH that gonadotroiihin secretion is interfered with (Dordoni and Timiras, 1952), is clearly denied by several facts. ( 1 ) In the rabbit studies, none of the various agents prevented ovulation when injected more than a minute post coitum. (2) In one study (Sawyer, Markee and Everett, 1950b) ovulation was actually induced by the intravenous injection of "lethal" doses of epinephrine when the animals WTre protected by atropine. (3) In rats ovulation is unaffected by massive intravenous doses of either the imidazoline drugs or 2-dibenzylamionethanol in amounts known to be stressing (Sawyer and Parkerson, 1953).

TABLE 8.2 Pharmacologic Agents and Blockade of Ovulation

Antiadrenergics

/3-Haloalkylamines

Dibenamine

SKF-501

Dibenzyline

ImidazoHnes

Priscoline

Regitine

Yohimbine

Anticholinergics

Atropine

Banthine

Antihistaminics

Neo-antergan

Ganglion blockers

Tetraethvlammonium. .

SC-1950/

Barbiturates

Nembutal

Dial

Ipral

Amvtal

Barbital

Phenobarl)ital

Prominal

Others

Morphine

Procaine, locally near tuber

Procaine, systemically .

Chlorpromazine

Reserpine

Ether

2,4-Dimtrophenol

Rabbit

Rat

Cow

Bi

Bi

Bi

Bi

01

01

0^

01

■?4. 5

Bi

BI

B6

Bi

Bi

01

01

01

01

Bi

Bi Bi Bi Bi B' Bi 8

B9

BIO

Bii

01

B12 B13 B14 B16

B3

I Sawyer and associates, 1947-1951; Everett and associates, 1949-1950; Christian, 1956; and see present text.

^ van Tienhoven, 1955; van Tienhoven, Nalbandov and Norton, 1954.

3 Zarrow and Bastian, 1953.

•• Fugo and Gross, 1942.

5 Sulman and Black, 1945.

^Hansel and Triml)erger, 1951; Hough, Beardon and Hansel, 1955.

' Fraps and Case, 1953.

• Doring and Goz, 1952.

» Westman, 1947.

1" Barraclough and Sawyer, 1955.

II Westman and Jacol)8ohn, 1942. 1- Barraclough, 1956.

13 Barraclough, 1955.

" Unpublished. Temporary, during deep anesthesia.

1^ Unpublished. EDso : 25 mg. per kg. subcutaneously.

Key: B = Blockade.

= No blockade.

1 = Ovulation induced by the drug.

526

PHYSIOLOGY OF GONADS

Nor is it influenced by severe trauma, heat, cold, or formalin injection coincident with the known "critical period" (Everett, unpublished).

2. Central Depressants and Ovulation

Reported evidence of a blocking action of barbiturates on ovulation traces back to experiments by Westman (1947) who injected female rats with Prominal twice daily for 3 weeks. Approximately 30 per cent of these rats experienced prolonged vaginal estrus and had ovaries containing only follicles at the end of the experiment. Almost identical results were reported by Doring and Goz (1952) when rats were treated daily with phenobarbital. The agreement is not unexpected in view of the fact that in the body Prominal is quickly demethylated to phenobarbital. It was shown by Everett and Sawyer (1950) that when administration of barbiturates to rats is critically timed with respect to stage of the cycle and the time of day, blockade of ovulation can be accomplished at will in shortterm experiments (Fig. 8.10). Chronic administration introduces considerable uncertainty for reasons that are not yet clear (Everett, 1952b). In the rabbit, the rapid intravenous injection of pentobarbital or Pentothal, within as short a time as 12 seconds after coitus, generally failed to block ovulation (Sawyer, Everett and Markee, 1950) . However, it was later shown that barbiturate anesthesia will prevent the ovulation that is otherwise caused in the estrogen-primed rabbit by mechanical stimulation of the vagina, the anesthesia t)eing induced in advance of the stimulation (unjuiblished i.

Other central dcpi-es^ants reported to block ovulation in the rat are morj^hine, reserj^ine, chlorpromazine (Barraclough and Sawyer, 1955; Barraclough, 1955, 1956), and even meprobamate acting synergistically with an anticholinergic drug (Gitscli, 1958). Special interest attaches to the mori)hine work, in that anicnoi'i lica and sterility often a('C()in|)any moiphinc addiction in the human female. Related studies (Sawyer, Critchlow and Barraclough, 1955), in which recordings were made of electi'ical acti\ity in various regions in the bi-ain.

demonstrated in rats that morphine acts nmch like the barbiturates in depressing activity in the reticular activating system. The effect was also shown by atropine, in doses that would block ovulation. The inference is that all three agents block by striking at the same central elements of the LH-release apparatus.

An interesting peculiarity of domestic hens with respect to barbiturates was encountered by Fraps and Case (1953), who noted that pentobarbital induces ovulation jirematurely, and that pentobarbital and progesterone supplement each other in this capacity. Although these developments may represent pharmacologic curiosities limited to the bird, the possibility should be seriously considered that similar effects may occur in other animals. In fact, pentobarbital in rabbits facilitates the release of hypophyseal gonadotrophin in response to intraventricular injection of histamine, seemingly by an effect in the rhinencephalon (Sawyer^ 1955).

3. The Central Xervous System as a Timing Mechanism for Oi'ulation

In the rat and the hen and probably many other species the pro-ovulatory excitation of the hypophysis is dejiendent in large measure on time of day.

In the rat, the blocking agents have served to delimit a critical period on the day of proestrum, before which ovulation can be blocked and after which it will occur in sjiite of injection of the blocking agent. Under controlled illumination for 14 hours daily, this critical period extends from about 2 P.M. to 4 P.M. Administration of either atroi^ine or i^entobarbital at 2 p.m. consistently blocks ovulation (Fig. 8.10), whereas injections later in the period are progressively less eff"ecti\-e ( l']\-erett and Sawyer, 1950, 1953; Everett, 1956b). Such l)redictability of the hour of pituitary activation is, in itself, evidence of a relationship between this event and diunial physiologic I'liythnis.

Furthei' e\-i(lence is seen in the seciuelae of pentobarbital injection (Fig. 8.11). Repetition at 2 P.M. on successive days results in a follicular cycle and prolonged vaginal estrus with eventual atresia of all

MAMMALIAN REPRODUCTIVE CYCLE <- — 24hr.— >

527

DAY NIGHT

Fig. 8.10. Deinoni^lration of 24-lioiir penodu-ity in the luteinizing hormone-release apparatus of female rats (Vanderbilt strain, 4-day cycle, controlled lighting: 14 hours per day). Schematic representations of the normal cycle (A) and of characteristic results of different regimes of Nembutal treatment (B to F). Vaginal stages indicated by Roman numerals over each time scale; symbols above these show the corresponding follicle and corpus luteum stages. The device marked l prolonged the functional life of the coi'pus luteuni in nonpregnant women. Lyon rejjorted such prolongation using lactogen alone. The Squibl) lactogen was also used by Moore and Nalbandov (1955) in prolonging the luteal phase of the cycle in the ewe. As in the human experiments, howe\-er. one would like to know whether lactogen is capable of initial stimul.'iiioii of secretory activity of corpora lutea and of maintaining their function in the ab

MAMMALIAN REPRODUCTIVE CYCLE

531

sence of the hypophysis. There is no evidence for or against the lactogenic liormone in this capacity, except in rats.

Estrogens have direct hiteotrophic action in the rabbit (Robson, 1937, 1938, 1947). The effect does not depend on the hypophysis and has been produced by impLantation of estrogen crystals within corpora liitea (Hammond, Jr., and Robson, 1951; Hammond, Jr., 1952). Westman (1934) had earlier shown that operative reduction of ovarian stroma in pseudopregnant rabbits results in corpus luteum regression and that this can be prevented by administration of estrogen. Corpora lutea induced by gonadotrophin injection or by mating, as the case may be, require the presence of the hypophysis for their continued function ( Smith and White, 1931; Westman and Jacobsohn, 1936). Theoretically, then, in rabbits the hypophysis liberates FSH and LH which act on the interstitial tissue to cause estrogen secretion. This in turn stimulates the corpora lutea to secrete progesterone.

The effect of estrogen on the corpora lutea of rats is largely indirect and requires the presence of the hypophysis. Massive dosage with estrogen beginning soon after ovulation results in the enlargement of the corpora lutea and the production of sufficient amounts of progesterone to mucify the vaginal mucosa (Selye, Collip and Thomson, 1935; Wolfe, 1935; Desclin, 1935; Merckel and Nelson, 1940). In fact, a single injection of 50 /Ag. estradiol bcnzoate on the day after ovulation is sufficient to cause pseudopregnancy. These effects are now judged to be the result of induced liberation of hypophyseal luteotrophin. vSimilar effects have been reported after administration of androgens (McKeown and Zuckerman, 1937; Wolfe and Hamilton, 1937; Freed, Greenhill and Soskin, 1938; Laqueur and Fluhmann, 1942).

Desclin (1949b) stated that in hypophysectomized rats the administration of estrogen augments the hiteotrophic action of lactogen, producing functional corpora lutea in the presence of subthreshold doses of the latter hormone. A physiologic synergism of the two substances has thus been indicated. Mayer (1951) suggested that this may explain the stimulation of corpora lutea of lactation which follows estrogen treat

ment in this species. Greep and Chester Jones (1950) postulated that estrogen favors corpus luteum function in the rat by causing the luteal cells to produce cholesterol as a precursor of progesterone. Their actual data, however, indicate that the increase of visible cholesterol after estrogen treatment was confined to the interstitial tissue.

Factors responsible for cholesterol storage and mobilization in corpora lutea of the rat were analyzed by Everett (1947). In hypophysectomized rats in which corpora lutea were maintained by lactogen the injection of pituitary LH induced the storage of cholesterol, but this effect did not occur in hypophysectomized rats in the absence of lactogen. It could be induced during pregnancy or pseudopregnancy by estrogen if the hypophysis remained in place. Addition of an excess of lactogen prevented cholesterol storage. Lactogen thus tends to deplete cholesterol content of rat luteal tissue as ACTH tends to deplete adrenocortical cholesterol.

2. XonfunctionaV' Corpora Lutea

In the short cycles of the rat, mouse, hamster, and so on, the corpora lutea are commonly said to be nonfunctional. The meaning of this statement, of course, is that they are incapable of supporting a decidual reaction (Long and Evans, 1922), or of lireventing ovulation. They need not be totally inactive, however, to fail to cause these manifestations. Whereas daily injection of 1.5 mg. or more of progesterone into intact female rats will simulate pseudopregnancy and indefinitely delay ovulation (Selye, Browne and Collii^, 1936; Phillips, 1937), smaller amounts of 1.0 rag. or less are compatible with the short cycle (Lahr and Riddle, 1936; Phillips, 1937; Everett, 1940a, b; and unpublished). In the absence of estrogen in castrated females, daily injection of as little as 0.25 rag. progesterone will support deciduomata (Velardo and Hisaw, 1951). Very small amounts of estrogen augment this action of progesterone (Rothchild, Meyer and Spielman, 1940) but somewhat larger amounts are inhibitory unless the progesterone dose is proportionately increased (Velardo and Hisaw, 1951). In the intact animal the progestational effects of

53^

PHYSIOLOGY OF GONADS

less than 1.0 mg. progesterone would be inhibited by the periodic rise in estrogen secretion.

Evidence that some rats, but not all, actually experience low-grade corpus luteum activity during the short cycle was furnished by Everett (1945). In the comparison of ovaries from females of two strains of rats, it was noted in the supposedly normal Vanderbilt strain that on the two days immediately following ovulation the corpora lutea of the next youngest generation contained a great quantity of cholesterol, giving a strong Schultz reaction. By contrast, comparable corpora lutea of the DA strain were usually free of visible lipid in Sudan preparations or the Schultz test. Administration of small amounts of lactogen (luteotrophin I during the cycle preceding the current one, amounts inadequate to cause l)seudopregnancy, resulted in the rich deposition of cholesterol in these otherwise lipidfree corpora lutea. The conclusion was reached that the corpora lutea of the Vanderbilt rat must be slightly active during the short cycle and those of the DA rat less so, if at all. This would easily explain the relative indifference of the Vanderbilt rat to continuous light and the ease with which persistent cstrus could be induced in the DA rat by such treatment (Everett, 1942a, b). In fact, the low dosages of lactogen mentioned substituted for progesterone treatment in maintaining regular cycles in persistent-estrous rats of the DA strain (Everett, 1944b). Significantly, the treatment was effective in only those animals in which a set of corpora lutea had been induced by other means at the beginning of the experiment.

To be correlated with the above indications of low-grade function during the short cycle, is the finding that corpora lutea of the Vanderl)ilt rat retain full responsiveness to luteotrophin throughout most of the diestrous interval (Nikitovitch-Winer and Everett, 1958a). Responsiveness diminishes near the onset of i:)roestrum. Once the rat has entered proestrum these older corpora lutea are not capable of sustained function. The loss is not a function of time per se, but of stage of the cycle.

.\. I'.^KTI)OPRE(;\.\XrY

The terms coi-pu,^ hitcuni of ()^•ulation and corpus luteum of pscudojji'cgnancy are com

monly u.sed to differentiate the luteal bodies occurring during the normal cycles from those found during some unusually long period of luteal activity. However, the terms deny the fundamental similarity of the luteal phase in the cycles of such animals as the guinea pig and the luteal phase induced by sterile mating or its equivalent in animals like the rat. In the unmated bitch the spontaneous luteal phase of the cycle is commonly called pseudopregnancy, yet it is equally common to say that the guinea pig does not experience pseudopregnancy. The truth is that the luteal phase of the canine cycle is simply longer than the luteal phase in the guinea pig and may be marked by a period of lactation near its close. In the present discussion, the expression pseudopregnancy will be equivalent to saying the luteal phase of the infertile cycle. Under experimental conditions it will refer to any period of sustained luteal function similar to that of the normal progestational state. Wherever appropriate, the distinction will be made between a pseudopregnancy that is spontaneous and one that is induced.

In most of the familiar animals that ovulate spontanously corpus luteum function also begins spontaneously and continues for at least several days after ovulation. With respect to the rabbit, cat, and ferret, it is often said that pseudopregnancy is invoked by sterile copulation, whereas strictly speaking it is only ovulation and corpus luteum formation which are invoked. The pseudopregnancy then follows automatically. This interpretation seems appropriate, inasmuch as in all three species the formation of corpora lutea by l)rief treatment with hypophyseal or chorionic gonadotrophin is followed by long periods of progesterone secretion which can hardly be the direct effect of the injected substances (Hill and Parkes, 1930a, b; Foster and Hisaw, 1935; van Dyke and Li, 1938). Quite different is the pseudopregnancy of the rat, mouse, and hauistei', in \\liich progestational activity is invoked by stinudation of the cervix uteri, l^verett (1952a) described the ex])eriiuental dissociation in rats of the o^•^llati()n and luteotr()])hic mechanisms. respectively. When o\-uhition is blocked (by pentobarbital) in the cycle dui'ing which controlled uiatinu occurs, pseudopi-egnancy begins

MAMMALIAN REPRODUCTIVE CYCLE

533

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Fig. 8.12. Experimental dissociation in rats of the ovulation mechanism and that causing pseudopregnancy. A. Control cycles for comparison with B and C . Points on base line represent diestrum, on ascending lines proestrum, on highest level full vaginal estrum. X, ovulation. B. Blockade with Nembutal {'NB) on day of proestrum and following day (see Fig. 8.11E'). Ovulation during third night. C. Same basic procedure as B, but with copulation during first night (M). Ovulation usually failing in this cycle (contrast with B) . Corpora lutea formed after spontaneous ovulation in second cycle regularly become functional without further stimulation : the wavy line represents pseudopregnancy. The early copulation has introduced some change in the animal such that this pseudopregnancy "spontaneously" follows ovulation as in the standard mammalian cycle. (From J. W. Everett, Ciba Foundation Collofiuia Endocrinol.. 4, 172. 1952.)

"spontaneously" a]ter the next cyclic estrus (Fig. 8.12). Dissociation of the two mechanisms is expressed in another way by certain Mustelidae, e.g., the mink and marten. Ovulation in these forms is invoked by mating, whereas corpus luteum activation awaits appropriate environmental conditions, i.e., temperature and length of daily illumination (Pearson and Enders, 1944; Hansson, 1947). In the mink, during the period of relative luteal inactivity that follows mating early in the season, recurrent estrus continues. If reraating takes place at an interval of 6 days or more, new ovulations are induced (Hansson, 1947). Matings late in the season are immediately followed by luteal activity. The pseudopregnant cycles of a representative series of mammals are much alike when conditions appropriate to the respective species are applied (Fig. 8.1).

1. Duration of Psciidopregndncn

The length of time that corpora lutea remain functional in the pseudopregnant cycle is thought to be relatively uniform in the great majority of mammals, usually about 10 to 15 days. Rarely it is shorter,

e.g., the hamster, 7 days, although usually 9 to 10 days (Asdell, 1946). At the other extreme, the corpora lutea remain functional for periods corresponding to the duration of pregnancy, as in the ferret, 5 to 6 weeks. In fact, corpus luteum function lasting over a month is usual in the other two carnivores for which information is at hand: cat, 30 to 44 days (Foster and Hisaw, 1935) ; and dog, 30 days or more (Evans and Cole, 1931).

These figures are only approximations, however, as the criteria on which they are based differ. In the rat, in which pseudopregnancy is said to last 12 to 14 days, its termination is taken to be the onset of the next estrus, whereas the corpora lutea must have undergone a decline of activity 2 or 3 days earlier (Everett, 1948). The decline is probably not abrupt, inasmuch as the vaginal smear during the next estrus is very strongly mucified and, as mentioned earlier (p. 519), enough progesterone seems to be secreted by the waning corpora lutea to facilitate ovulation. Morphologic criteria are often employed as indicators of corpus luteum regression: characteristically, fatty vacuolation of luteal cells, decrease in size of the individual cells or of the entire corpus

534

PHYSIOLOGY OF GONADS

luteuni. and changes in the smusoidal pattern suggesting reduced circulation. Such changes are first observed in the guinea pig corpora lutea on about the 13th day of the cycle. One has the choice of taking this date as the end of the pseudopregnant phase or, alternatively, the date on which the first indications of estrus are noted. Either choice is arbitrary, but the former seems preferable as it suggests that progesterone secretion is diminishing and probably is no longer sufficient to maintain progestational changes in the uterus. In fact, regression of the endometrium sets in about a day earlier than frank degenerative changes in the corpora lutea. In the cat, according to van Dyke and l.i (1938) the corpora lutea 20 days after ovulation no longer secrete enough progesterone to cause motor effects of epinephrine in the myometrium, the so-called "epinephrine-reversal" effect, yet by histologic criteria corpus luteum regression is not apparent until 28 days or later (Liche, 1939; Foster and Hisaw, 1935). In the bitch the uterus begins regression 20 to 30 days after heat, l)ut the corpora lutea are said to remain in good condition for a longer time (see Asdell, 1946, for references). Regression is so gradual that anestrum is not reached until about 85 days. In the primates the beginning of menstruation offers a means of delimiting the luteal phase, inasmuch as menstruation in the ovulatory cycle reflects a marked reduction in corpus luteum function. Nevertheless, this reduction probably occurs a few days before bleeding begins. The i^eak of pregnanediol excretion in women (Venning and Browne, 1937) and of plasma progesterone concentration in women and monkeys (Forbes, 1950; Bryans. 1951 ) is passed about midway between ovuhition and menstruation.

2. Xciivdl Factors in Pseudoprciindiicji

The importance of the nervous system in control of pseudopregnancy is well recognized in only the few species re])resented by the rat. .\ neural effect in the mink and similar Mustelidae is implied by the relation of hiteal function to daily illumination, as mentioned earlier. Beyond that fact, however, no information is available. Att(>ntion will therefore be directed largely to the rat.

Not only sterile mating, but se\'eral other

procedures involving neural stimulation will cause rats to become pseudopregnant. Stimulation of the cervix by mechanical means (Long and Evans, 1922) or electric shock (Shelesnyak, 1931) have become standard methods. In fact, Greep and Hisaw (1938) obtained pseudopregnancies after electrical stimulation during early diestrum, several days before ovulation. Pseudopregnancy is also invoked by continuous stimulation of the nipples for several days (Selye and McKeown, 1934). According to Harris (1936) electric shock through the head is effective. His negative results with "spinal shock" are difficult to explain. From the description of position of the electrode it seems doubtful that the current passed through the cord itself, yet the sacral plexus must have been stimulated.

Al)dominal sympathectomy or superior cervical ganglionectomy are said to diminish the numbers of animals responding to electrical or mechanical stimulation of the cervix (Vogt, 1931; Haterius, 1933; Friedgood and Bevin, 1941). On the other hand, there is no diminution of response to sterile copulation, which shows that the sympathetic chains are not essential. Ball (1934) emphasized the quantitative aspects of the problem, noting that partial resection of the uterus or excision of the cervix diminished the response to sterile copulation, but only when "single-plug" matings were allowed. Multiple plugs gave pseudopregnancy in 100 per cent of the animals. It may be assumed that Vogt's (1933) negative results after hysterectomy resulted from single-jilug copulations. Kollar (1953) re-opened the cjuestion and found that pelvic nerve resection usually pi-evented the response to mating. It is not clear, howevei', whether multiple copulations wnv the rule, although it seems that the I'outiiie procedure was to leave the male with the female overnight. His contention was that cervicectomy fails to abolish the resjionse completely because the vagina remains sensitive.

Anesthesia with ether, nitrous oxide, or ethylene ( Mcyei', Leonard and Hisaw, 1929) diniiiiished the ficciuency of response to inecliaiiical stimulation of tlu> (•er\-ix. The statement was made, although without v\\(len(c, that spinal anesthesia |)re\-ents pseudopreiinanev. Aeeoi'ding to A'ogt (1933),

MAMMALIAN REPRODUCTIVP] CYCLE

535

local anesthesia of the vagina and cervix by cocaine or procaine prevented the response to sterile copulation in 23 of 35 rats.

Removal of neocortex (Davis, 1939) did not interfere with the pseudopregnancy response to electrical stimulation of the cervix, although there was slight impairment of the response to mechanical stimulation or sterile mating (single-plug?).

These results taken together have been construed to mean that induction of pseudopregnancy in rats involves a reflex similar to the ovulation reflex in rabbits. Certain considerations, however, raise the possibility that it may not be a "trigger" stimulus to the hypophysis as long believed (Everett, 1952a). In the first place, it seems doubtful that a trigger stimulus would result in continuation of a new pattern of secretion (luteotrophin) for as long as 10 to 12 days. Furthermore, as noted above, cervical stimulation during the diestrum preceding ovulation may induce pseudopregnancy (Greep and Hisaw, 1938). Similarly, copulation during a cycle in which ovulation is blocked by pentobarbital results in a pseudoi)regnancv that begins after the next estrus (Fig. 8.12)^.

We turn now to experiments concerned directly with the hypothalamo-pituitary system and pseudopregnancy. Westman and Jacobsohn (1938c) cut the pituitary stalks of estrous female rats. Barriers of metal foil were inserted to prevent regeneration of nerve fibers assumed to innervate the adenohypophysis. Regeneration of blood vessels must have been equally impossible. Controls were simply hypophysectomized. Two to 5 hours after the operations electrical stimulation of the cervix was administered to all animals. Pseudopregnancies were demonstrated by deciduomas in traumatized uteri of all the stalk-sectioned animals but not in the completely hypophysectomized rats. Desclin (1950) reported the maintenance of pseudopregnancy in estrogen-treated rats in which the only remaining hypophyseal tissue was in the form of grafts in the kidney. Whereas in hypophysectomized controls the estrogen treatment (stilbestrol pellets) produced cornification of the vagina and no enlargement of corpora lutea, the engrafted-estrogenized rats developed mucified vaginas and enlarged corpora lutea as

in intact rats similarly treated with estrogen. Desclin concluded that the grafted hypophysis is able to respond to estrogen by liberating luteotrophin.

It is now apparent, however, that neither cervical stimulation nor estrogen treatment is needed to invoke pseudopregnancy when the gland is isolated from the hypothalamus (Everett, 1954, 1956a; Nikitovitch-Winer and Everett, 1958a; Sanders and Rennels, 1957; Desclin, 1956a, b). When autografts of anterior hypophysis were made to the renal capsule or near the common carotid artery on the day after ovulation in adult cyclic rats, corpus luteum function was invoked and maintained without any stimulus other than the operative procedures themselves. In short-term experiments in which the uteri were traumatized 4 days after the transplantation large deciduomas were regularly found at 8 days in the proven absence of residual hypophyseal tissue at the original site (Everett, 1954). Hypophysectomized controls were negative. In longterm experiments, continuing luteal function was demonstrated for as long as 3 months. Here the test for luteal function was vaginal mucification in the presence of massive amounts of estrogen administered during the final week of the experiment (Everett, 1956a). Controls in which the grafts or the ovaries were removed at the beginning of such estrogen treatment responded with full vaginal cornification. Follicular apparatus and interstitial tissue of the ovaries atrophied promptly after the grafting operations, whereas corpora lutea forming at that time were maintained for the long periods without histologic sign of deterioration. In later work, the decidual reaction was used as the test for luteal function, positive reactions being elicited as late as 2 months after the transplantation. It was discovered that function of the graft is not influenced by stage of the cycle at which transplantation is carried out and that grafts in the anterior chamber of the eye secrete luteotrophin like those on the kidney (Nikitovitch-Winer and Everett, 1958a). Transsection of the pituitary stalk is sufficient in itself to provoke pseudopregnancy. If an effective barrier to vascular regeneration is inserted, the pseudopregnancy will heorve permanent, but otherwise it will l:i •■ i;!*'

536

PHYSIOLOGY OF GONADS

usual length of time (Xikitovitch-Winer, 1957 j. In fact, there is reason to suspect that even a transient impairment of circulation in the median eminence-hypophj'seal linkage can be a sufficient impetus to pseudopregnancy. The experiments of Taubenhaus and Soskin ( 1941 ) in which application of an acetylcholine-prostigmine mixture to the exposed hypophysis was followed by pseudopregnancy, may well be explained in some such way.

It thus seems that in the rat the deprivation of, or interference with, the normal connection of the pars distalis with the median eminence facilitates secretion of luteotrophin and at the same time eliminates luteolytic mechanisms. It is significant that transplants of pars distalis into the pituitary capsule or to the immediate vicinity of the median eminence resume cyclic function (see page 512). The hypothalamus may have an inhibitory effect on luteotrophic activity of the pars distalis during the short cycle in this species. Greep and Chester Jones (1950) made the pertinent suggestion in attempting to explain the induction of pseudopregnancy by estrogen treatment, that the fundamental action of estrogen here is the suppression of FSH and LH, after which luteotrophic secretion may "proceed apace."

There is necessarily some uncertainty concerning the amount of luteotrophin secreted by the pars distalis when dissociated from the brain. Three sets of facts indicate that the output is larger than that in the cycling animal. (1) Sufficient gestagen is secreted by the engrafted animal to maintain a ])regnancy (Everett, 1956c; Meyer, I'rasad and Cochrane, 1958) when the pituitary is trans])lanted on the day after mating. (2) After stalk-transsection, in which there is less initial destruction of glandular parenchyma than in transplantation experiments, the corpora lutea enlarge to a diameter like that usually found in late pregnancy rather than remaining like those of pseudopregnancy (Nikitovitch-Winer, 1957). (3) A single homotransplant of pars distalis placed subcutaneously in an otherwise normal female mouse will maintain a se(iueiicc of pseudopregnancies that override the short cycles expected of the animal's own hypophysis (Miihlbock and Boot, 19591. This is also true of rats (Nikitovitch

Winer, uni)ublislie(l). To avoid the conclusion that in such preparations the grafted gland is secreting luteotrophin at an increased rate, one must assume that the outl)ut of this hormone from the intact gland is only slightly below threshold and that the graft adds just enough to make the total output effective. To explain the maintenance of pregnancy one might assume that the luteotrophin output and the resulting gestagen secretion are no greater than during the normal short cycle and that the formation of deciduomas takes place because of the deficiency of estrogen. The results of stalksection, however, cannot easily be explained away. The weight of evidence, then, is in favor of increased luteotrophin secretion by hypophyses isolated from the brain by severance of the stalk or transplantation.

Under certain experimental conditions it has seemed that to establish pseudopregnancy all that is necessary is to block out the forthcoming estrus and ovulation. Thus, in cycling rats, when the hypophysis is transplanted to the kidney as late as 60 hours after ovulation, the current diestrum transforms into a permanent pseudopregnancy supported by the existing set of corpora lutea (Nikitovitch-Winer and Everett, 1958a ». Similarly, injections of chlorpromazine (Barraclough, 1957) or Pathilon (ditsch and Everett, 1958) begun during diestrum, may transform it into a pseudopregnancy by blocking out the expected estrus.

The Miihlhock-Boot experiment mentioned above furnishes an instructive model of the standard mammalian cycle, in which both ovulation and pseudopregnancy are spontaneous events. Given the extra pituitary tissue producing luteotrophin at a presumably constant rate, with the output of the normal ghmd fluctuating (juantitatively and (jualitatively, the mouse or rat undergoes one ])seudopregnancy after another. Possibly, in animals that normally liave a spontaneou.- hiteal phase, thei'c is a considei'ahle poUion ot' the pars distalis which I'unctions somewhat indepeiidentl}- of the hyputhalaiiius. with a continuous output of luteotrophin as from the grafted gland in the Mvihlb()ck-P)Oot pi-epaiation. The portion nioic (Hrectly under control of the me(lian eminence I zona tuberaHs'.'l would

MAMMALIAN REPRODUCTIVE CYCLE

537

then act like the intact hypoiihysis of the Miihlbock-Boot mouse. Such a view, unfortunately, continues to set apart species in which the luteal phase is not spontaneous, by suggesting that only in them are special neui'al nieclianisnis involved.

B. LUTEOLYTIC MECHANISMS

By luteolysis we shall refer to corpus luteum regression in any of its manifestations. Supposedly the initial change is functional, after which overt cytologic and histologic changes appear, leading eventually to the total loss of glandular tissue. Very likely the initial stages are occult and only gradually reach recognizable proportions. Mention was made earlier of the fact that in women and monkeys the peak of gestagen secretion is about midway between ovulation and menstruation. In rats indirect evidence from progesterone injection experiments leads to the deduction that toward the close of a pseudoi)regnancy gestagen secretion must drop below the estrus-suppressing level several days before estrous changes appear in the vaginal smear (see Fig. 8.8). A more al)rupt drop is reported for the ewe l)etween the 16th and 17th (last) days of the cycle (Edgar and Ronaldson, 1958).

Long-term experiments with pituitary autotransplants indicate that at least in the rat the life span of the corpus luteum is not limited by intrinsic factors. Some agent (s) of extra-ovarian origin must, therefore, be res]5onsible for at least the initial luteolytic changes. Various bits of information suggest that the agent is associated with, if not identical with, FSH and/or LH. Greep (1938) noted that after hypophysectomy in rats the daily injection of LH over a period of 10 days caused the corpora lutea to regress more rapidly than otherwise. Greep, van Dyke and Chow (1942) later were unable to repeat this with a more highly purified LH C'metakentrin") , a fact suggesting that the earlier material was effective because of impurity. During the short cycle of the rat, luteolysis is interrupted by translilantation of the pars distalis (Everett, 1954; Nikitovitch-Winer and Everett, 1958a). Whatever regressive changes are in progress at that moment are evidently suspended forthwith. They are first appar

ent during the third day of diestrum and become increasingly pronounced during proestrum and estrus. In this connection, it should be recalled that during late diestrum and proestrum patches of cells undergoing fatty necrosis are first recognizable histologically (Boling, 1942; Everett, 1945).

Why is it that, in the face of a continuing supply of luteotrophin in the MiihlbockBoot preparation, or in intact animals injected daily with lactogen (Lahr and Riddle, 1936; Aschheim, 1954), luteolysis sets in during the 2nd week? The question obviously cannot be answered from present knowledge. Nevertheless, it is clear that the pseudopregnancy that transpires when a significant i)ortion of hypophyseal tissue remains in normal relation to the hypothalamus is far from the steady state of that which becomes established by total removal of the pars distalis to an extracranial site. It is also apparent that the onset of luteolysis may be postponed by such means as hysterectomy or production of artificial deciduomas (see p. 538). Furthermore, during lactation-pseudoi)regnancies in rats, Canivenc and Mayer (1953) prolonged luteal function to 34 days by substituting successive new litters of suckling young. This technique should prove especially valuable in experimental analysis of both luteotrophic and luteolytic mechanisms.

Benson and Folley (1956) suggested that lactogen secretion is activated by oxytocin, inasmuch as its injection prevented the normal inv'olution of the mammary glands after withdrawal of the litters from lactating rats. This observation has been confirmed by McCann, Mack and Gale (1958), who also noted the interruption of lactation by lesions of the sui)raoi)tico-hyiiophyseal tract. Selye and ]\lcKeown (1934) long ago found that pseudopregnancy could be induced in cycling rats by the introduction of a suckling litter. Although all this is consistent with the above-mentioned observation by Canivenc and Alayer, other workers have observed luteolytic effects of gonadotrophinfree oxytocin administered to cycling dairy heifers (Armstrong and Hansel, 1958). Furthermore, Grosvenor and Turner (1958), after first noting that the administration of Dibenamine, atropine, or pentobarbital to rats prevented the expected drop in assay

588

PHYSIOLOGY OF GONADS

able pituitary lactogen at nursing, found no decrease when pentobarbitalized mothers were injected with physiologic doses of oxytocin intravenously. When the dosage was increased 30 to 60 times there was apparently some moderate discharge, but the authors regard it as insignificant.

C. EFFECT OF THE UTERUS ON LUTEAL FUNCTION

This subject has been reviewed by Bradbury, Brown and Gray (1950). In three species (Fig. 8.13) hysterectomy results in significant prolongation of the functional life-span of corpora lutea (guinea pig, Loeb, 1927; rabbit, Asdell and Hammond, 1933; rat, Bradbury, 1937). In each case the period of luteal function approximates that of normal pregnancy. The fact that corpora lutea in the pseudopregnant ferret normally function as long as in the pregnant animal may be a clue to the noncffect of hysterectomy in that species (Deanesly and Parkes,

19331. Ahhough Burford and Diddle (1936) rej^orted that in monkeys total hysterectomy was followed by vaginal cycles of normal length, examination of their protocols shows that during the several postoperative months just 1 corpus luteum was produced among all 5 animals. The experiment thus seems inconclusive. Impairment of pelvic circulation seems to be a common factor complicating the results of hysterectomy in women and may have been one cause of the failure of luteinization in these monkeys.

An interpretation given by Loeb (1927) and Bradbury, Brown and Gray (1950) for the prolongation of luteal function by hysterectomy is that in species in which the effect is demonstrable the uterus secretes a specific substance which abbreviates the life of the corpus luteum. Hechter, Fraenkel, Lev and Soskin (1940) found in rats that grafts of estrous uteri shortened the pseudopregnancies of hysterectomized animals to normal length. Implantation of similar tis

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MAMMALIAN REPRODUCTn'E CYCLE

539

SIR' which had been killed by freezing had no such effect, nor did successful grafts of uteri from diestrous donors. Bradbury, Brown and Gray (1950) found that partially hysterectomized rats in which the remaining uterine tissue was continuous with the cervix, hence properly drained, experienced pseudopregnancies of normal length. However, when the continuity was interrupted the uterine remnants became greatly distended, the endometrium was destroyed, and the animals had prolonged pseudopregnancies. Possibly the endometrium is the source of the hypothetical "luteolytic" substance.*'

Under other circumstances the endometrium of rats has a luteotrophic rather than luteolytic effect, for when deciduomas are induced by trauma the pseudopregnancies of otherwise normal animals are lengthened to 22 days or more (Ershoff and Duell, 1943; Velardo, Dawson, Olsen and Hisaw, 1953). This is not true in mice, however, and Kamell and Atkinson (1948) suggest that the I'eason may lie in the shorter life-span of the decidual tissue in that species. Loeb (1927) reported that deciduomas in cyclic guinea pigs ])rolonged the luteal phase, i.e., delayed the next ovulation from 3 to 7 days, which is far less than the prolongation after hysterectomy.

As an alternative to the concept of conI rol of the corpus luteum by humoral agents

" The denial by Velardo, Olsen, Hisaw and Dawson (1953) that hysterectomy in rats prolongs l)seiidopregnancy is based on operations performed later in the luteal phase than those of Bradbury, Brown and Gray and of Hechter, Fraenkel, Lev and Soskin. Whereas the latter workers had operated in the range from the 4th to the 7th day.^^ of p.seudopregnancy and many of Bradbury's cases lacked uteri when they entered pseudopregnancy, Velardo and associates excised the uteri on the 9th day. It seems possible that this difference in time may be crucial, for by the 9th day of a 12-day pseudopregnancy the corpora lutea must be on the verge of regression, if that in'ocess has not already been initiated. After maintaining pseudopregnancy in estrogenized, liypophysectomized rats b}^ means of lactogen, its withdrawal is followed about 3 days later by estrous smears (Nelson and Bichette, 1943; Nelson, 1946). A slightly longer delay occurs in nonestrogenized, intact rats at the end of a lactogeninduced pseudopregnancy (Everett, unpublished) or after withdrawal of progesterone treatment (Fig. 8C).

formed in the uterus, Loeb considered the jjossibility that neural mechanisms are involved. The idea was not acceptable, he felt, because in partial hysterectomies the result was not detennined by the locus of the part removed. The finding by Hechter, Fraenkel, Lev and Soskin (1940) that grafts of uterine tissue in hysterectomized rats return the duration of pseudopregnancy to normal, is significant evidence pointing in the same direction.

A third hypothesis was advanced by Heckel (1942) who found in rabbits that the extent of prolongation of luteal function by subtotal hysterectomy is roughly proportional to the amount of uterine tissue removed. The suggestion was offered that removal of the uterus has an estrogensparing effect. The greater amount of estrogen thus available to the corpora lutea prolongs their life, according to this view.

Later investigations by Moore and Nalbandov (1953) revive the possibility that the uterus influences the ovary by way of the nervous system. In sheep the implantation of a plastic bead in utero during the early luteal phase shortened the cycle by several days. Successive cycles tended to be unusually short. When the uterine segments containing the beads were denervated, however, the cycles were essentially normal. Other work from the same laboratory (Huston and Nalbandov, 1953) which indirectly may bear on this problem, indicates that the presence of a mechanical irritant (a thread) in the oviduct of the domestic fowl tends to block ovulation. The blockade may extend for as long as 20 days if the thread is placed in the isthmus (van Tienhoven, 1953). The ovaries remain functional, producing large follicles which may be ovulated at will by injection of LH. The authors feel that this phenomenon, like the effect of the bead in the sheep uterus, involves a neural mechanism, Init crucial information is lacking. It may be significant that stimulation of the ovaries was found in some hens, in place of blockade.

We may hope that as more information becomes available the assortment of facts given in these paragraphs will fit into a rational system. Not until this is realized can we hope to understand the regulation of the luteal phase.

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PHYSIOLOGY OF COXADS

VIII. Concluding Comments

From what has been written here it is readily apparent that present knowledge of the mechanisms controlling the reproductive cycle is extremely spotty. The number of assumptions necessary to knit the various items of factual information into an orderly pattern is disturbing. In spite of a voluminous literature which has grown during the last 60 years, we are really only a few steps ahead of our predecessors at the turn of the century in terms of fundamental understanding. A brief recounting of some of these steps may be desirable.

The first three decades saw the gradual development of proof that the ovary is a gland of internal secretion as well as the producer of eggs, governing the uterus and other accessory organs by secretion of hormones into the blood stream. For a while it seemed that the events of the reproductive cycle could be neatly explained, with the ovary in the capacity of controlling agent. Yet there were indications that the ovary itself is not independent. As early as 19091910 (Aschner; Crowe, Gushing and Honuins) it was noted that destruction of the hypophysis is accompanied by atrophy of the gonads and reproductive tract. In 1927 the separate investigations of Smith and Engle and of Zondek and Aschheim demonstrated conclusively that function of the ovary depends vitally on the anterior hyjiophysis. Promptly it was learned that the hypophyseal secretion of gonadotrophic hormone is in turn modified by estrogens. In the early 1930's the "push-pull" hypothesis of pituitary-ovarian interaction was separately stated by Brouha and Simonnet and by Moore (see Moore and Price, 1932). Modified in detail as new facts appeared, this hypothesis is held to the present day in some quarters as a simple exi)lanation of how the cycle comes about in polyestrous, continuous breeders in which ovulation takes l)lacc spontaneously. Much of the investigation of pituitary-ovarian i)hysiology during the 1930's was performed within the framework of this hypothesis.

For seasonal breeders and reflex ovulators, however, the assumption was necessary that special controlling mechanisms are superimjiosed. It bccaiiie iccognizcd also that in

some vaguely defined manner even the human cycle is subject to intervention of the nervous system. The possible importance of the hypothalamus was debated at some length in the twenties. In 1932 the existence of a sex center there was proposed by Hohlweg and Junkmann. In an attempt to explain the coital excitation of the rabbit hypophysis which causes the liberation of gonadotrophin, Hinsey and Markee (1933) suggested diffusion of a chemical substance from the posterior lobe to the anterior lobe. Hinsey (1937) later elaborated on this possibility and mentioned the hypophyseal portal vessels as a plausible route by which the substance might travel. We have seen the later history of these ideas.

The "Sexualcentrum" of Hohlweg and Junkmann was proposed as a mediator of the effects of estrogen on the anterior hypophysis of rats. Westman and Jacobsohn (1936-1940), on the other hand, believed that through its stalk connection with the hypophysis the rat hypothalamus governs gonadotrophin synthesis, not release. The latter they regarded as a direct effect of estrogen on the gland. These views did not afford a common basis for spontaneous and refiex ovulation.

Schweizer, Chari])i)er and Haterius ( 1937) offered the first surmise of similarity, after finding that guinea pigs bearing intraocular pituitary grafts developed persistent estrus and large follicles that failed to go through maturation changes. Their feeling was that the normal connection of hypophysis with hypothalamus may be necessary for cyclic liberation of LH. Almost concurrently, Dempsey (1937) expressed a similar view as one alternative explanation of his experimental results with the guinea i)ig cycle. Suggesting cautiously that release of luteinizer may be brought about l)y a "rhythmic discharge" from the central nervous system, he went on to mention the "possibility that a high level of oestrin is necessary but not directly responsible for the release of luteinizer" (italics added). From this it is only a short transition to certain concepts set forth in the present exposition.

Accoi'ding to current views: (1) Reflex ovulation and spontaneous ovulation alike are go\-enicd by a liypothalamo-pituitary

MAMMALIAN REPRODUCTIVE CYCLE

541

apparatus whose final link to the pituitaiy is neurohumoral by way of the hypophyseal portal veins and whose activity precipitates release of LH. (2) The apparatus includes a hypothalamic center or centers whose excitation depends on estrogen-progesterone levels and afferent impulses of various kinds. (3) The sensitivity of the center (s) is influenced not only by the sex steroids, but by other poorly understood factors that vary from species to species and from time to time in individuals, e.g., the diurnal rhythm in rats. Here in bare outline is a plausible hypothesis that may be generally applied to the events immediately relating to ovulation.

Satisfactory hypotheses respecting other phases of the cycle must await future developments. The extent and manner of intervention of the nervous system in the follicular and luteal phases remain unsettled. Although the hypophyseal hormones concerned in ovarian follicle development have been characterized, their exact chemical descrijition has not been accomplished. The rate of their output at different stages of the cycle is largely a matter of conjecture. Structural changes that they jiroduce in the ovary are well known, but in chemical terms only the end products of ovarian activity are well recognized, and these probably incompletely. The fact that the estrogens, in turn, have a regulating effect on follicle-stimulating activity of the hypophysis is known, but the mechanisms by which this effect is accomplished are uncertain. The hypophyseal hormones that maintain the luteal phase are recognized with any certainty in only three species and there is a wide difference between rabbits, on the one hand, and rats and mice, on the other. For mammals generally, the luteotrophic factors have not been identified. Whether the hypothalamus is actively concerned in maintenance of the luteal i)hase in the majority of mammals is unknown. The morphologic effects of luteotrojihic stimulation on corpora lutea are well recognized, but here again the chemical mechanisms leading to the end products are obscure. The action of the corpus luteum hormone in regulation of the cycle is partially known, including the well established fact that its continual jiresence in large amount will suiijiress the estrogenic and

ovulatory phases. Yet, one cannot say whether this effect is accomplished by direct action on the hypophysis or by indirect action through the central nervous system. Nor can one state how the hypophysisgonad equilibrium of the luteal phase is interrupted in the absence of a conceptus. With respect to the ovulation mechanism itself, the hypothesis outlined above requires verification in additional species. Assuming its validity, many details remain to be studied, e.g., the neural pathways and nuclei involved, identification of neurochemical activators of the pars distalis and their sources and loci of action, the precise nature of mechanisms whereby the gonadal steroids excite or suppress, the cellular mechanisms by which ovulating hormone is released into circulation by the hypoi^hysis, and the cytochemical effects within the ovary. All too evidently an encompassing theory of the female reproductive cycle is far from realization.

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VoGT, M. 1933. Uber den Mecli.iiiismus der Ausl().sung der Graviditiit. II. Mitlcilunji- Arcli. exper. Path. Pharmakol., 170, 72-83.

Walton, A., and Hammond, J. 1928. Observations on ovulation in the rabbit. Brit. J. Exper. Biol., 6, 190-204.

W.\RWiCK, ]•]. J. 1946. Gonadolrophic potc^ncy of ewe pituitary glands as affected by spaying, .season, and breed. Proc. Soc. Exper. Biol. <fe Med., 63, 530-533.

W.\ti;r.\ia\, A. J. 1943. Studies of normal de

velopment of the New Zealand White .strain of rabbit. Am. J. Anat., 73, 473-515.

Weinberger, L. M., .\nd Grant, F. C. 1941. Precocious pubert}^ and tumors of the hypothalamus. Arch. Int. Med., 67, 762-792.

Werthessen, N. T., Schwenk, E., and Baker, C. 1953. Biosynthesis of estrone and /3-estradiol in the perfused ovary. Science, 117, 380-381.

Westman, A. 1934. Untersuchung iiber die Abhangigkeit der P^unktion des Corpus luteum von den 0\arialfollikeln und iiber die Bildungsstiitte der Hormone im Ovarium. Arch. Gynak., 158, 476-504.

Westman, A. 1947. The effect of Prominal on the sexual cvcle. Acta med. scandinav., 128, Suppl. 196, 111-115.

Westman, A., and J.\cobsohn, D. 1936. tjber Ovarialveranderungen beim Kaninchen nach Hj'poph3^sektomie. Acta obst. gynec. scandinav., 16, 483-508.

Westman, A., and Jacobsohn, D. 1937a. Uber Oestrinwirkung auf die Corpus luteum-Funktion. I. Acta obst. gynec. scandinav., 17, 1-12.

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W^estman, a., and Jacobsohn, D. 1937c. Experimentelle Untersuchungen iiber die Bedeutung des Hypophysen-Zwischenhirnsystems fiir die Produktion gonadotroper Hormone des Hypophysenvorderlappens. Acta obst. gynec. scandinav., 17, 235-265.

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MAMMALIAN REPRODUCTIVE CYCLE

555

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ZucKERMAN, S. 1937. The menstrual cycle of primates. X. The oestrone threshold of the uterus of the rhesus monkey. Proc. Rov. Soc. London, ser. B, 123, 441-471.

ZucKERMAN, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with a constant threshold dose of oestrone. J. Endocrinol., 2, 263-267.

ZucKERMAN, S. 1952. The influence of environmental changes on the anterior pituitary. Ciba Foundation CoUociuia Endocrinol., 4, 213-228.

ZucKERMAN, S., AND FuLTON, J. F. 1934. The

menstrual cycle of the primates. VII. The sexual skin of the chimpanzee. J. Anat., 69, 38-46. ZwARENSTEiN, H. 1937. Experimental induction of ovulation with progesterone. Nature, London, 139, 112-113.

9

ACTION OF ESTROGEN AND PROGESTERONE

ON THE REPRODUCTIVE TRACT OF

LOWER PRIMATES

Frederick L. Hisaw, Ph.D.

THE BIOLOGICAL LAB0RAT0RIF:S, HARVARD TNIVERSITY, CAMBRIDGE, MASSACHUSETTS

and ^

Frederick L. Hisaw, Jr., Ph.D.

DEPARTMENT OF ZOOLOGY, OREGON STATE COLLEGE, CORVALLIS, OREGON

I. Introduction 556

II. Ovarian Hormones and Growth of

THE Genital Tract 558

III. Effects of Progesterone on the

Uterus 565

IV. Synergism between Estrogen and

Progesterone 567

V. Experimentally Produced Implantation Reactions 571

VI. The Cervix Uteri 572

VII. The Vagina 575

VIII. Sexual Skin 576

IX. Menstruation 578

X. The Mechanism of Menstru.^tion. . 583 XI. References 586

I. Introduction

Cyclic menstruation is the most characteristic feature of primate reproduction, and distinguishes it from the estrous cycle of lower mammals. This cardinal primate event is heralded by the bloody uterine effluent emanating from the vagina, whereas in estrus the dominant characteristic is a sudden modification in behavior featuring an intense mating drive. However, the internal secretions that regulate the various events in the menstrual and estrous cycles are the same, and this similarity is fundamentally more significant than the key descriptive differences just mentioned. Estrus comes at the peak of the growth phase of the cycle and is associated with ovulation. In

contrast, menstruation occurs in the cycle midway between times of ovulation and is not accompanied by an increase in sexual activity. From earliest times menstruation has been recognized as degenerative: the characteristic odor, and the necrotic changes in the lining of the uterus, part of which is cast off at this time, sustain this interpretation. Therefore, menstruation is at the opposite phase of the cycle from estrus. It is such an obvious event that menstrual cycles are dated from the onset of bleeding. Menstruation is not analogous to the proestrous bleeding in the dog or cow nor to the slight bleeding of primates at midpoint between menstrual periods (Hartman, 1929). The study of menstruation was at first almost entirely the province of the clinician and the material for investigation limited to w^omen. Hitschmann and Adler (1907), Meyer (1911), Schroder (1914). Novak and Te Linde (1924), and Bartelmez (1933) are among many of the earlier investigators who contributed descriptions of the cyclic changes in the human endometrium. The physiology of the menstrual cycle and attendant morphologic changes have continued to be an area of active research interest in science and medicine. Among the many more recent contributors are Bartelmez

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(1937), Latz and Reiner (1942), Haman (1942), Knaus (1950), Mazer and Israel (1951), and Crossen (1953).

The earlier concepts regarding the menstrual cycle were based primarily on the changes occurring in the human endometrium and for convenience of description the cycle was divided into four stages or periods. The first of these was the period of active menstruation, and the length of the cycle was dated from its onset. Most authors agreed that menses began by leaking of blood from superficial vessels to form lakes under the surface epithelium and that there was some sloughing of tissue after the beginning of bleeding. There was considerable disagreement as to the amount of destruction and loss of tissue; estimates of various authors ranged from very little to almost complete denudation of the surface. Bartelmez (1933) emphasized both the wide individual variability of the amount of tissue lost and differences in the stage of development of the endometria at the time of menstruation.

The second period immediately following menstruation began with regeneration of the surface epithelium, which started sometimes before menstrual bleeding had ceased and was completed in a very short time. This lieriod included the 5 to 7 days after cessation of menses, during which the endometrium grew in thickness. Frequent mitoses were recognized, especially in the glands which lengthened but remained straight and tubular.

The third ("interval") period, lasting 6 to 10 days, was characterized by a somewhat thickened endometrium, still with straight glands and showing little evidence of secretory activity. At first this was considered a quiescent period as indicated by the term "interval." However, as will be shown later, such a characterization was not justified from the physiologic viewpoint.

The fourth period, called the premenstrual period, included the 10 days or 2 weeks before menstruation. During this phase the glands continued to increase in size and became distended, coiled, or even sacculated. The glandular cells increased in height, and there was evidence of glycogen mobilization and secretion. Next, the epithelium became "frayed out" along the

outer borders, then decreased in height, indicating secretory depletion. Decidual cells appeared in the stroma at this time. The endometrium was much thickened and extremely hyperemic. At the height of this period the endometrium was approximately 5 mm. in thickness, as compared with V2 mm. toward the end of menses. The term premenstrual was usually applied to this phase but today the term progestational would seem preferable.

During and after these descriptions of the changes in the human endometrium, many attempts were made to locate the time of ovulation in the menstrual cycle. When it was found, as will be discussed later, that ovulation occurred approximately midway between two menses, and was preceded by follicular growth and followed by development of a corpus luteum, it became customary to refer to the two halves of the menstrual cycle as the follicular phase and the luteal phase. One advantage of this descriptive terminology was the emphasis it placed on the homology of the two phases of the menstrual cycle in primates with the follicular and luteal phases of the estrous cycles of lower mammals.

A theory to explain menstruation, widely adopted in 1920, was formulated from this morphologic evidence. The essentials were that menstruation occurs because the lining of the uterus, prepared for implantation of the ovum, degenerates if fertilization of the egg does not occur. This required that ovulation and corpus luteum formation precede the i^remenstrual changes in the endometrium. Subsequent research disclosed that menstrual cycles frequently occur in which ovulation does not take place and bleeding results from the breakdown of an "interval" rather than a progestational endometrium.

The discovery of anovulatory cycles not only brought about a revision of ideas regarding an explanation for menstruation but also raised questions as to what constituted a normal menstrual cycle. The length of the cycle and the amount and duration of bleeding are approximately the same regardless of whether or not ovulation has taken place. The gross features of menstruation under these two conditions are indistinguishable one from the other. However, the biologic purpose of the menstrual cycle

558

PHYSIOLOGY OF GONADS

is reproduction wliicli obviously cannot l)0 fulfilled unless an ovum is made available for fertilization. Therefore, in this sense it seems quite clear that anovulatory cycles should be considered incomplete and abnormal.

The investigation of changes taking place in the uterine endometrium at various periods of the menstrual cycle in women was confronted with many difficulties, the chief one being that of obtaining normal tissue representative of specific times of the cycle. The entire uterus and both ovaries are essential for proper evaluations and it was rarely possible to meet these requirements. The material for such studies came from autopsies and surgery and tissues usually had suffered postmortem changes or the surgical condition was one involving serious pelvic disease. There have been, however, a goodly number of instances in which these difficulties were adequately overcome (Stieve, 1926, 1942, 1943, 1944; Allen, Pratt, Newell and Bland, 1930) and the clinic will continue to make important contributions (Rock and Hertig, 1942; Hertig and Rock, 1944), but quite early the need became obvious for a suitable primate that could be used as an experimental animal for research on the different aspects of the physiology of reproduction.

Since the initial observations by Corner (1923) on the menstrual cycles of captive rhesus monkeys iMacaca mulatta) , more has been learned about the physiology of reproduction of this animal than any other primate. Monkeys of this species thrive under laboratory conditions, which has made it possible to devise accurately controlled experiments on normal healthy animals and obtain reliable information on the menstrual cycle, gestation, fetal development, and the interaction of hormones concerned with regulating reproductive processes.

Other features that make the rhesus monkey such an attractive animal for these purposes are the many morphologic and physiologic attributes that are strikingly like those of the human being. Tiic modal length of their menstrual cycles is 28 days but there is wide variation (Corner, 1923; Hartman, 1932; Zuckcrman, 1937a). From an analysis of 1000 cycles recorded for some 80 females of different ages, Zuckerman

(1937a) found an average cycle length of 33.5 ± 0.6 days, and the mode 28 days with an over-all range of 9 to 200 days. Ovulation occtu's api:)roximately midway between two menstrual periods, most between the 11th and 14th days (Hartman, 1932, 1944; van Wagenen, 1945, 1947), and although these animals breed at all seasons of the year many cycles are anovulatory, especially during the hot summer months (Eckstein and Zuckerman, 1956). A method developed by Hartman for detecting the exact time of ovulation by palpation of the ovaries in the unanesthetized animal greatly facilitated the timing of events of the menstrual cycle. This procedure also made it possible to determine the age of corpora lutea with great accuracy (Corner, 1942, 1945) and correlate their develojiment and involution with corresponding changes in the endometrium (Bartelmez, 1951 ) and, in ju-egnancy, with the exact age of developing embryos ( Wislocki and Streeter, 1938; Heuser and Streeter, 1941).

The primary purpose of the present discussion is to review the results of experimental investigations of physiologic processes occurring in the female reproductive tract of lower primates during the menstrual cycle, and particularly those processes that are under hormonal control. The brief introductory presentation of basic observations could be greatly extended and we take up the discussion of endocrine problems knowing that we must return often to the work of these authors and that of others to be cited, as conclusions based on experimental data take on meaning only in terms of normal function.

II. Ovarian Hormones and Growth of the Genital Tract

The changes that are repeated in different parts of the reproductive tract with each menstrual cycle are produced by ovarian hormones, estrogens, and progesterone. The dominant hormone of the follicular phase is estradiol-17/y, which is secreted by the Graafian follicle, and in the tissues is readily transformed in i)art to estrone, an estrogenic metabolite. Progesterone, secreted by the corinis luteum, is jirimarily a hormone of the luteal phase of the cycle. However, small amounts of progesterone may appear

ESTROGEX AND PROGESTERONE

559

ill the blood of monkeys as early as the 7th (lay and attain a concentration of 1 fxg. per ml. of serum at ovulation, whereas a maximal concentration of 10 /xg. per ml. is reached at approximately the middle of the luteal phase (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). Also, some estrogen is present during the luteal phase, probably secreted by the corpus luteum (estrogens can be obtained from luteal tissue) or it may be partially derived from developing follicles. It is unlikely that estrogen is ever entirely absent during a normal menstrual cycle or that the presence of progesterone is completely restricted to the luteal phase.

The dependence of the reproductive tract on ovarian hormones is strikingly demonstrated by the profound atrophy that follows surgical removal of the ovaries. A progressive decrease in size of the Fallopian tubes, uterus, cervix, and vagina takes place, and usually, the involution of the uterine endometrium involves tissue loss and bleeding, commonly referred to as post castrational bleeding. Dramatic as these effects may seem, it is equally dramatic to find that these atrophic structures can be restored entirely to their original condition by the administration of ovarian hormones. Therefore, it is seen at the beginning that investigations dealing with the physiology of the female reproductive tract of primates in large measure involve a study of the independent and combined actions of estrogens and progesterone on the activities of the various structures concerned.

Much can be learned about the action of ovarian hormones by observing the changes they produce in the gross appearance of the atrophied reproductive tract of castrated animals. Daily injections of an estrogen in doses equivalent to 1000 I.U. or more for 10 days or a fortnight will restore the uterus, cervix, and vagina to a condition comparal)le with that found in a normal monkey at the close of the follicular phase of a menstrual cycle (Fig. 9.1A). If a similar castrated monkey is given 1 or 2 mg. of progesterone daily for the same length of time, very little if any change in size of the reproductive organs results. However, if first the normal condition is restored by giving estrogen and then is followed by the progesterone treatment, the size of the uterus

is maintained but that of the cervix and vagina decreases to an extent approaching that in a castrated animal (Fig. 9.1B). Such experiments show that an estrogen promotes growth of the reproductive tract whereas progesterone is comparatively ineffective when given alone. Yet progesterone can maintain the size of the uterus when administered following an estrogen treatment but it does not prevent involution of the cervix and vagina.

An additional feature of the growth-stimulating action of the ovarian hormones is brought out when estrogen and progesterone are administered concurrently. If, after repair of the reproductive tract of a castrated animal has been accomplished by a series of injections of estrogen, both estrogen (1000 I.U.) and progesterone (1 or 2 mg.) are given daily for 20 days, it will be found that the uterus is larger than when either hormone is given alone for a similar length of time, whereas the cervix and vagina have involuted and are approximately the size found in animals given only progesterone. Thus it can be demonstrated that a synergistic effect on growth of the uterus occurs when the two hormones are given simultane

FiG. 9.1. Reproductive tracts of three adolescent monkeys which were castrated and given estrogen daily for approximately 3 weeks. A shows the condition at the conclusion of the estrogen treatment, B the condition following the injection of progesterone for an additional three weeks, and C the effects of continuing the treatment with both estrogen and progesterone for a like period.

560

PHYSIOLOGY OF GONADS

ously whereas in the cervix and vagina the growth -stimulating action of estrogen is inhibited by progesterone (Fig. 9.1C). This presents a most interesting situation in which the combined actions of two hormones on three closely associated structures of the reproductive tract join in a synergistic effort in promoting the growth of the uterus although progesterone prevents estrogen from affecting growth of the cervix and vagina.

These changes in gross morphology in response to the ovarian hormones are reflected in the histology of the responding tissues. Also, the character of the response differs depending upon the physiologic nature of the tissue concerned; therefore, for the sake of clarity each will be discussed separately. The first of these to be considered is the uterus and particularly growth of the endometrium.

It has been reported (Hisaw, 1935, 1950) that growth of the endometrium, as induced by estrogen, is limited. That is, a dosage of estrogen capable of maintaining the endometrium of a castrated animal for an indefinite period without the occurrence of bleeding, stimulates rapid growth for approximately the first 2 weeks. Within this time a maximal thickness of the endometrium is attained which remains constant or may become less during the course of treatment (Fig. 9.2). Engle and Smith (1935) made similar observations. They found that the endometria of castrated monkeys receiving estrogen for 100 days or longer were thinner than endometria of animals on estrogen for a much shorter time. Also, on prolonged treatment, the stroma of the endometrium becomes dense and the lumen small whereas the size of the uterus remains about the same. In fact, they state that in

I'll,. (1.2. I'll n Ml lour i-a.-^tra(c>d monkeys wliicli were givi^i 10 /ug. estradiol daily for 10 to 78 days. A was given estrogen for 10 days, B for 30 days, C for 60 days, and D for 78 days. Depression in the endometrium of anterior wall of D is the result of a biopsy taken a year previously. (From F. L. Hisaw, in A Si/niposium on Steroid Hormones, University of Wisconsin Press, 1950.)

ESTROGEN AND PROGESTERONE

561

four experimental animals the only well developed fundus was found in the animal on the shortest treatment, i.e., 60 days.

The mitotic activity in the epithelium of the glands and surface mucosa also indicates a limited effect of estrogen. This can be demonstrated to best advantage in the endometria of castrated monkeys that have been on estrogen for different lengths of time and have received an injection of colchicine 8 hours before their uteri were removed. A comparison of the number of cells in mitosis per square centimeter of surface mucosa at 10, 30, 45, and 60 days is shown in Figure 9.3. From this it can be seen that mitotic activity approaches that in a castrated animal. Although the five points used in drawing the curve are quite inadequate for an accurate analysis of the mitotic response in the epithelial components of the endometrium, they do show that cell division is most rapid soon after the beginning of an estrogen treatment and subseciuently declines.

The loss of responsiveness of the endometrium to estrogen seems related more to the length of treatment than to dosage of hormone. An endometrium of normal thickness can be produced in 2 or 3 weeks at a dosage level of estrogen that will not maintain the growth induced for longer than about 40 days without bleeding (Hisaw, 1935; Engle and Smith, 1935; Zuckerman, 1937b). The response to a low dosage of estrogen that will prevent bleeding during the course of treatment (about 10 /xg. estradiol-17/8 daily) is one of rapid endometrial growth at first, as has been described, followed by a thinning of the endometrium. The refractoriness of the endometrium to estrogen becomes so pronounced after about 100 days of treatment that very few cell divisions are seen in the epithelium of the glands and surface mucosa. The general morphology of the endometrium retains the characteristic appearance of the follicular phase of the menstrual cycle except that the stroma is usually more dense and the cells of the glandular epithelium have large deposits of glycogen between the nucleus and the basement membrane. However, metabolically such endometria are surprisingly inactive. Although they are dependent on the presence of estrogen and may bleed within about

Mitotic Response of Uterine Lpithelium TO looo I. u. Estrogen PER D«y.

Fig. 9.3. The number of mitoses per square centimeter of surface epithelium of the endometrium in a castrated monkey and in four castrated animals given 10 /xg. estradiol daily for 10, 30, 45, and 60 days, respectively. One-tenth of actual number of mitoses is shown on the ordinate. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

48 hours if the treatment is stopped, the activity of their oxidative enzymes and the ratio of nucleoproteins (RNA:DNA) are about the same as in the involuted endometria of castrated animals.

The effects that accompany moderate estrogenic stimulation become exaggerated in several respects when large doses of estrogen are given for an extended period. The disparity between the area of myometrium and endometrium becomes greater as the treatment progresses (Fig. 9.4). Kaiser (1947) described the destruction of the spiraled arterioles of the endometrium in monkeys given large doses of estrogen and Hartman, Geschickter and Speert (1941) reported the reduction of the reproductive tract to the size of that of a juvenile animal by the end of 18 months during which injections of large doses of estrogen were supplemented by subcutaneous implantation of estrogen pellets. These observations not only show that the endometrium becomes unresponsive to estrogen when the treatment is prolonged but that large doses produce injurious effects.

562

PHYSIOLOGY OF GONADS

Fig. 9.4. .4. i'v'< — ' ri:,,ii of the uterus of a castrated monkey which had received 1.0 mg. estradiol daily for 35 days. Compare with B which shows the effects of 1/10 this dosage (100 fig. daily) when gi\en for 185 days.

The limited response of the endometrium to estrogen is in some respects surprising in view of its remarkable growth potentialities and regenerative capacity. These qualities were dramatically demonstrated by Hartman (1944) who dissected out as carefully as possible all of the endometrium from the uterus of a monkey and wiped the uterine cavity with a rough swab and yet the undetected endometrial fragments that remained were capable of restoring the entire structure. Also, considering the enormous increase in size of the uterus during gestation, it is even more difficult to account for the rather sharp limitation of growtli under the influence of estrogen.

The increase in tonus of the uterine musculature, a known effect of estrogen, has been considered as possibly exercising a restrictive influence on growth of the endo

metrium. An attempt has been made to remove this containing influence the muscle may have by making an incision through the anterior wall of the uterus (Hisaw, 1950) . A castrated monkey was given 10 ixg. estradiol daily for 21 days at which time the operation was performed and the treatment continued with 30 ^g. estradiol daily for 40 days. The uterus was laid open by a sagittal incision from fundus to cervix and most of the endometrium was removed from the anterior wall. This caused gaping of the incised uterus and exposure of the endometrium on the posterior wall. The incision was not closed and after hemorrhage was completely controlled the uterus was returned to the abdomen.

Examination of the uterus at the conclusion of the experiment showed no indications that endometrial growth had been enhanced. The muscularis had reunited and only a few small bits of endometrium were found in the incision (Fig. 9.5). It seemed probable that the purpose of the experiment had been defeated by rapid repair of the uterus. Therefore, a similar experiment was done in which the musculature of the incised uterus was held open by suturing a wire loop into the incision. Yet the incision closed and no unusual growth of the endometrium was detected (Fig. 9.6).

Observations under these conditions are necessarily limited to those made on the uterus when it is removed at the conclusion of an experiment and comparisons must be made between uteri of different animals. Obviously, it would be more desirable if the response of an individual endometrium could be followed during the course of treatment. It is possible to meet most of these requirements under conditions afforded by utero-abdominal fistulae, exteriorized uteri, and endometrial implants in the anterior chamber of the eye. In continuing our discussion we first shall present information obtained by such techni(iues that have a bearing on the response of the endometrium to estrogen.

The surgical procedure used by Hisaw ( 19501 for preparing utero-abdominal fistulae foi' studies of the exj^erimental induction of endometrial growth by estrogen and progesterone was a modification of that used by van Wagenen and Morse (1940) for ob

ESTROGEN AND PROGESTERONE

563

Figs. 9.5 and 9.6

The uteri of these two castrated monkeys were opened from fundus to cer\'ix by an incision through the anterior wall while the animals were receiving estrogen. Part of the endometrium of the anterior wall was removed and the incision in the myometrium was not closed.

Fig. 9.5. Estradiol, 10 fig., was given daily for 7 days; the uterus was opened and the animal continued on 30 /xg. estradiol daily for 40 days. (From F. L. Hisaw, in A Sy7nposiii7n on Steroid Hormones, University of Wisconsin Press, 1950.)

Fig. 9.6. Estradiol, 10 ^g., was given daily for 7 days; the uterus was opened and the treatment continued at a dosage of 30 /ig. estradiol daily for 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

serving changes in the endometrium (luring the normal menstrual cycle. This procedure makes frequent inspections possible either by hand lens or dissecting microscope, of most of the upper part of the endometrium on the anterior and posterior walls of the uterus. The elliptic slit formed by the endometrium of the two opposing walls can be located easily, the two sides pressed apart by any small smooth instrument, and the surface of the endometrium examined. Changes in thickness of the endometrium cannot be ascertained without resorting to biopsies but it is free to grow out of the opened uterus if it is so inclined. However, in such preparations the growth produced in the endometrium by daily injections of 10 fjig. estradiol for periods of 2 or 3 weeks is not sufficient to show any tendency whatever to grow out through the fistular opening or obstruct examination of the walls of the uterus. The limited growth observed in these experiments is in agreement with that obtained with estrogen on intact and incised uteri.

The cervix uteri of the rhesus monkey is sufficiently long to make it possible to bring the entire fundus to the exterior through a midal)dominal incision. Advantage of this

was taken in an attempt to exteriorize the uterus and maintain it outside the body for long enough periods to make it possible to study the growth responses of the endometrium (Hisaw, 1950). These preparations did not i)rove satisfactory in all respects but they did contribute a number of interesting observations.

The operational i^rocedure used in these experiments involved dividing the uterus transversely from fundus to cervix so that the anterior wall was deflected downward and the posterior wall upward (Fig. 9.7). The endometrium of the exteriorized uterus is difficult to maintain but with proper care it seems to retain its normal condition for at least the first few days after the uterus is opened. Small localized areas of ischemia can be seen to come and go, probably action of the coiled arteries, and there is a periodic general blanching of the endometrium associated with rhythmic contractions of the muscularis. This, however, does not seem true of the whole endometrium. A zone surrounding the internal os of the cervix tends to retain its blood-red color even during strong contractions of the uterus and the growth reactions of the endometrium in this area are of particular interest.

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Fig. 9.7. Exteriorized uteii. The uteri were divided transversely from fundus to cervix. The anterior half is seen deflected to the right and the posterior half to the left. A and B are of the same uterus taken 13 days after exteriorization showing the "blush" and "blanch" reaction of uterine contractions. It can be seen that during blanching the endometrium of the cervix does not become ischemic. C. Uterus 18 days after exteriorization, showing response to estrogen. Ridges formed by growth of the endometrium surrounding the internal OS of the cervix can be seen at the upper edge of the photograph. D, taken 83 days after exteriorization, shows response produced by a series of injections of 10 ^ig. estradiol and 1 mg. progesterone daily. The transverse ridge is formed by the two opposed lips of endometrium derived from the area surrounding the internal os of the cervix. Growth when the two hormones are given is greater than when only estrogen is injected. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

The endometrium on the exposed anterior and posterior halves of the uterus underwent deterioration despite the best of care that could be given, but that surrounding the internal OS of the cervix survived and retained its capacity to grow, in one animal, for as long as 9 months. When estrogen was given this endometrium grew rapidly and within a few days stood out as large elliptic lips surrounding the internal os (Fig. 9.7). Within 2 to 3 weeks the lips appeared to reach their full size and further growth was slow or absent. When estrogen treatments were discontinued the endometrial lips underwent bleeding within a few days and were entirely lost. At no time were activities observed that could be ascribed to coiled arterioles, nor did ischemia occur during involution previous to bleeding. It seems that the response of this tissue to estrogen is like that found in other experiments but the absence of ischemia preceding bleeding is ex

ceptional. The endometrium on the anterior and posterior walls of uterine fistulae invariably showed ischemia for several hours before active bleeding following the withdrawal of estrogen.

Markee (1940) approached the problem of endometrial growth in monkeys by studying the changes that occur in bits of endometrial tissue transplanted to the anterior chamber of the eye. Such transplants retain in large measure the normal morphology of endometrial tissue and changes in their cyclic growth parallel those going on simultaneously in the uterus. So much so that if the animal has an ovulatory cycle, the ocular implants show conditions characteristic of both the follicular and luteal phases, but if ovulation fails to occur then the luteal phase is omitted. Also, the morphologic events taking place at menstruation can be seen and recorded, since the transplants regress and bleed at each menstrual period.

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These ingenious experiments will be referred to often in the course of our discussion but at present the response of endometrial transplants in the eye to estrogen is of primary interest.

Monkeys having ocular transplants were given 200 to 300 R.U. of estrone daily for about 1 to 3 months. The transplants did not grow to a certain size and then remain stationary, but instead periods of rapid growth were interrupted by periods of regression which usually involved a marked decrease in size, and if regression was extensive and rapid, bleeding ensued. It also was found that these episodes of regression in the transplants were usually accompanied by a decrease in the size of the uterus.

Comparisons between the results of these experiments and those we have discussed previously may be misleading since it seems that only 1 of the 5 animals (no. 295) used was castrated. Also, the dosage of estrogen was not sufficient to maintain the endometrium of the uterus for an indefinite period without bleeding and this also was reflected in the transplants. It seems questionable that the growth capacity of endometrial transplants in the eye can be determined unless sufficient estrogen is given to prevent bleeding in the uterus. Therefore, it would seem that these experiments contribute less to an analysis of the effects of estrogen on endometrial growth than they do to an understanding of the events that precede and accompany menstruation.

In summary, it seems clear that the outstanding effect of estrogen on the uterus of the monkey is one of growth (Allen, 1927, 1928). The involuted uterus of a castrated animal can be restored to its normal size in 2 or 3 weeks by daily injections of adequate amounts of estrogen. At this time there is an increase in vascularity, a clear-cut hyperemia as seen in rodents. There also is secretion of luminal fluid (Sturgis, 1942) but this does not distend the uterus as in the mouse and rat. This is accompanied by an increase in tissue fluid, especially in epithelial tissues (surface epithelium and glands) , and in the connective tissue of the stroma. Glycogen may be present at the basal ends of epithelial cells beneath the nuclei (Overholser and Nelson, 1936) but it apparently is not readily released under the action of estrogen

alone (Lendrum and Hisaw, 1936; Engle and Smith, 1938) . The glands of the endometrium maintain a straight tubular structure with some branching near the muscle layers. The condition produced experimentally in the monkey's uterus by short term treatments with estrogen is equivalent to that present in the normal animal at midcycle, or even a few days later if ovulation does not occur.

If, however, an estrogen treatment is continued for several months conditions develop in the uterus that are not found during the follicular phase of a normal menstrual cycle. When the daily dose of estrogen is small menstruation occurs at intervals during the treatment (Zuckerman, 1937b) and probably marks periods of endometrial regression as observed by Markee (1940) in eye transplants, but if the dosage is increased by a sufficient amount (about 10 /xg. estradiol- 17/3 daily) injections may be continued for a year or longer without bleeding. Although the size of the uterus remains within the range of normal variation as the injections are continued, the myometrium tends to increase in thickness and the endometrium becomes thinner, a condition not corrected by further increases in dosage or by prolonging the treatment. The cause responsible for the limited response of the endometrium under these conditions is not known but apparently is not a restrictive influence of the myometrium as similar responses are given when the endometrium is exposed by incising the uterus, in abdominal fistulae, and in exteriorized uteri.

III. Effects of Progesterone on the Uterus

It has been mentioned that a menstrual cycle, in which ovulation occurs, can be conveniently divided into a follicular and a luteal phase. The follicular phase extends from menstruation to ovulation and the luteal phase from ovulation to the following menstruation. It has been shown in the previous discussion that the endometrial modifications characteristic of the follicular phase of the cycle can be duplicated in a castrated monkey by the injection of estrogen. Likewise, the progestational condition characteristic of the luteal phase can be developed by giving progesterone. In fact, all

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PHYSIOLOGY OF GONADS

the morphologic and physiologic features that are known for anovulatory and ovulatory cycles can be reproduced in castrated monkeys by estrogen and progesterone.

If one designs an experiment to simulate the normal cycle in a castrated monkey then estrogen should be given first to develop the conditions of the follicular phase followed by progesterone for the progestational development of the luteal phase. Experience has shown that this is the most effective procedure for the production of a progestational endometrium. Progesterone, as compared with estrogen, is a weak growth jH'omoter and although it can produce progestational changes in the atrophic endometrium of a castrated monkey when given in large doses, its action is greatly facilitated when preceded by estrogen. The first experiments in which progesterone was used for this purpose were planned on this principle (Hisaw, Meyer and Fevold, 1930; Hisaw, 1935; Engle, Smith and Shelesnyak, 1935).

The first noticeable effect of progesterone is an elongation of the epithelial cells of the surface membrane and necks of the glands. When the treatment is continued, this effect progresses down the gland towards the base. This change is followed closely by a rearrangement of the nuclei which is more pronounced in the glands than in the surface epithelium. The nuclei under the influence of estrogen in doses which reproduce the conditions of the follicular phase of a normal cvcle, are situated niostlv in the basal

Fig. 9.8. Uterus of a castratefl monkey which was given 2 mg. progesterone daily tor 113 days. The endometrium is thin but bleeding occiu\s when such treatment is stopped. The myometrimn is soft and pliable and ilir l)lood vessels are cnlarsed and have thick wails.

half of the cells, some of them touching the basement membrane. The nuclei retreat from the basement membrane when progesterone is given leaving a conspicuous clear zone. This zone is produced by intracellular deposits of glycogen. These early changes usually appear before pronounced spiraling and dilation of the glands.

Secretion begins in response to estrogenic stimulation and increases greatly as progestational changes are established. It appears first in the necks of the glands and progresses basalward. The surface epithelium takes a less conspicuous part in secretion and is usually reduced to a thin membrane when injections of progesterone are continued until a fully developed progestational endometrium is established. This progressive action of progesterone is such that it is possible to find all conditions in a single gland from active secretion and fraying in the neck region through primary swelling to an unmodified condition at the base.

When treatment is continued for 25 to 30 days at doses of about 2.0 mg. daily, the glands enter a state that has been called "secretory exhaustion" (Hisaw, 1935). This condition also is seen first in the necks of the glands and progresses toward the base. The glandular epithelium decreases in thickness, and active secretion, as judged by fraying of the cells, is absent. The glands may become narrow and straight and the endometrium may resemble that in castration atrophy. These involutionary changes become even more pronounced if the treatment is continued for several months or a year (Fig. 9.8). The endometrium by this time is extremely thin. The glands are straight, short, and narrow, and the stroma very dense. The myometrium is thick in proportion to the endometrium and the uterine blood vessels are large and have greatly thickened walls. Such uteri tend to be somewhat smaller than normal and are soft and pHable.

Thus, it is seen that when growth is produced in the endonietiiuni of a castrated monkey by giving estrogen and then continued on injections of progesterone, there follows a sequential development of all stages of the luteal phase of a normal menstiual cycle terminating in secretory exhaustion. However, this condition cannot

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567

be maintained by continuing the progesterone treatment, and involutionary processes set in and the endometrium is reduced to a thin structure. Yet, such degenerate endometria are dependent upon progesterone and will bleed within about 48 hours if the injections are stopped. It also was found that after discontinuence of progesterone daily injections of 10 /i.g. estradiol may not prevent bleeding.

IV. Synergism between Estrogen and Progesterone

There is considerable evidence that in primates progesterone under normal conditions rarely if ever produces its effects in the absence of estrogen. Large quantities of estrogen are present in human corpora lutea (Allen, Pratt, Newell and Bland, 1930) and during pregnancy the placenta secretes estrogens as well as progesterone (Diczfalusy, 1953) . This apparently is a common feature of primates, as indicated by the excretion of estrogens in the urine of pregnant chimpanzees and rhesus monkeys (Allen, Diddle, Burford and Elder, 1936; Fish, Young and Dorfman, 1941 ; Dorfman and van Wagenen, 1941). Also, correlated with this is the observation that estrogen and progesterone when given concurrently produce a greater effect on the uterus of castrated monkeys than either alone (Hisaw, Greep and Fevold, 1937; Engle, 1937; Hisaw and Greep, 1938; Engle and Smith, 1938) and that an ineffective dose of progesterone is greatly potentiated by estrogen. This synergistic effect of the two hormones on the uterus of monkeys is quite different from their action on the uteri of laboratory rodents and rabbits. In these animals the effects of progesterone can be inhibited quite easily by a surprisingly small dose of estrogen (see chapter 7).

The synergism between estrogen and progesterone in the promotion of endometrial growth can be demonstrated to best advantage under the conditions of some of the physiologic preparations that have been discussed. For instance, it was shown (Fig. 9.5) that growth of the endometrium under the influence of estrogen was not enhanced by relieving muscle tension by a midline incision through the anterior wall of the uterus. Now, if a similar operation is per

FiG. 9.9. Uterus of a castrated monkey that received 10 fig. estradiol and 1 mg. progesterone daily for 18 days, at which time the uterus was opened from fundus to cervix and most of the endometrium of the anterior wall removed. The incision was not closed and the treatment was continued for an additional 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

formed on the uterus of a monkey that is receiving 10 /tg. estradiol daily and the treatment continued with the addition of a daily dose of 1 mg. progesterone, there usually follows a rapid growth of endometrial tissue out through the incision until by about 3 weeks a mass is formed which approximates the size of the entire uterus (Fig. 9.9). If this experiment is repeated and the same dosage of progesterone is given without estrogen, there is no outgrowth of the endometrium (Fig. 9.10).

A similar synergistic action can be seen in utero-abdominal fistulae. We have mentioned that estrogen does not cause excessive growth of the endometrium under these conditions. However, endometria that have reached their maximal response to estrogen will show a resumption of growth if 1 or 2 mg. progesterone are added daily to the treatment. By the 4th or 5th day lobes of blood-red endometrium begin to protrude

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PHYSIOLOGY OF GONADS

Fig. 9.10. Uteru.s of a castrated monkey which was given 1 mg. progesteione (lail>' for 18 days following an estrogen treatment. The uterus was opened as described for Figure 9.9, and the injections of progesterone continued for 20 days. (From F. L. Hisaw, in A Syiyiposium on Steroid Hormones, University of Wisconsin Press, 1950.)

through the opening of the fistula. Within a few days tongue-like processes of endometrial tissue are thrust out of the opening with each uterine contraction and are entirely or i^artially withdrawn at each relaxation.

Such outgrowths are difficult to protect from mechanical injury and consequent tissue loss so it is not possible to determine accurately how much endometrium is produced in a given time. In one experiment an animal was kept on 10 fxg. estradiol and 2 mg. progesterone daily for 98 days and it was found that the endometrium continued to grow, but the rate seemed considerably slow^er toward the conclusion of the treatment than at the beginning. How long an endometrium would continue to grow under these conditions was not determined, but it is obvious that much more endometrial tissue was produced by the treatment than is ever found at one time in the uterus of a monkey during a normal menstrual cycle. This takes on added significance when it is compared with the endometrial response in the intact uterus of an animal given the same dosage of estrogen and progesterone for a similar length of time.

The progestational development of the endometrium, when both hormones are given, passes through the same stages as those following the injection of only progesterone; i.e., presecretory swelling of the glandular epithelium, active secretion, and

secretory exhaustion. The endometrium, however, is considerably thicker than when a comparable dose of progesterone is given alone, and secretory exhaustion may not be so pronounced by the 30th day (Fig. 9.11). The glandular epithelium in the necks of the glands may be reduced to a thin membrane scarcely thicker than the nuclei whereas some secretion is usually present in the dilated basal parts of the glands. Also dilation of the glands in the basalis is more pronounced following a 30-day estrogen-])rogesterone treatment than when the same amount of progesterone is given separately.

Secretory exhaustion appears to be the initial indication of an involutionary process that ensues when an estrogen-progesterone treatment is continued for a long time (Hisaw, 1950). When a combination of the two hormones, known to be capable of producing a large uterus with a thick, fully develojied, progestational endometrium within al)out 20 days, is given for 100 days, an astonishingly different endometrium results (Fig. 9.12). It is thin, the stroma is dense and the narrow straight glands are reduced to cords of cells in the basal area. The condition is one suggesting inactivity and atrophy.

When such dosages of estrogen and i)rogesterone are given to castrated monkeys for 200 days or a year further changes in the endometrium occur. By 200 days the epithelium of the surface mucosa and glands

Fk;. 9.11, .\ late i)r()ges1ati()iial condition produced in the endometrium of a castrated monkey by giving 10 (ig. estradiol daily for 18 days followed by 10 /xg. estradiol and 2 mg. progesterone daily for 31 davs.

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569

Fig. 9.12. The endometnuin of a castrated monkey that had received 10 /xg. estradiol and 1 mg. progesterone daily for 99 days.

is lost except for small glandular vestiges along the musciilaris at the base of the endometrium. There are no glands, coiled arteries, or large blood vessels in what one might yet call the functionalis. All that remains is a modified stroma that resembles decidual tissue (Fig. 9.13.4 and B). It is also of interest that these endometria will menstruate if the treatment is discontinued and in most if the injections of progesterone are stopped and estrogen continued, but not if estrogen is stopped and progesterone continued.

Even though in such experiments the endometrium has been under the influence of both estrogen and progesterone for a year and has undergone extremely abnormal modification, it yet is capable of responding to estrogen in a more or less characteristic way when progesterone is stopped and in

jections of estrogen continued. Apparently within about three weeks the modified endometrium is replaced, under the influence of estrogen, by one that has few glands which tend to be cystic, a mesenchymatous stroma, and no coiled arteries (Fig. 9.14).

Under similar circumstances, if estrogen is stopped and jjrogesterone is continued, the modified endometrium is lost without bleeding and there is almost no repair of the endometrium even after a period of 3 weeks. There seems to be an incompatability between the epithelial outgrowths from the mouths of the glands and the underlying stroma of the denuded surface. Consequently the epithelium crumbles away and epithelization of the raw surface is not accomplished (Fig. 9.15j. How long this condition could continue has not been determined.

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PHYSIOLOGY OF GONADS

Fk;. 9.13. The endometrium shown m .4 is (h;i( from a castraled mdiik.N wlml, l,a,l received 10 /xg- estradiol and 2 mg. progesterone daily for 200 days. In B, jiart of ilie endometrium of a snndai animal given the same treatment for 312 days is shown at a higher magnification. The endometrium is almost entirely a modified stroma in which glandular epithelium and coiled arteries are absent. Only vestiges of glands are present in the basal area next to the myometrium.

One of the most interesting aspects of these observations is that these effects were jiroduced by dosages of estrogen and progesterone that are very probably within the range of normal physiology. From this it appears that although growth of the endometrium is greater when the two hormones are given together, due to their synergistic interaction, this does not prevent involutionary changes from setting in when the treatment is continued for a period of weeks or months. In fact, greater damage to the endometrium occurs under the simultaneous action of the two hormones than when either is given alone. Also, increasing the dose intensifies the damaging action of both estrogen and progesterone, so much so that very large doses will almost completely destroy the endometrium.

The myometrium, however, shows a different response to these treatments. Estrogen stimulates myometrial growth, which is

Fir;. 9.14. Uterus of a castrated monkey which was given 10 ixg. of estradiol and 2 mg. progesterone daily for 307 days at which time the injections of progesterone were stopped and estrogen continued for 20 days. Bleeding occurred the second day following discontinuance of progesterone. The absence of coiled arteries and the presence of cystic glands and a mesenchymetous stroma characterize the endometrium.

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Fig. 9.15. Uterus of a castrated monkey which was given 10 yug. estradiol and 2 mg. progesterone daily for 275 days at which time estrogen was stopped and progesterone was continued for 21 days. A shows the thin endometrium and dense stroma whereas B shows failure of formation of a surface epithelium following the loss of the modified functionalis presumably present at the conclusion of treatment with both hormono.^^ (see Fig. 9.13).

intensified both by cln-onic treatment and high dosage, and seems to be equally effective when it is given alone or in combination with progesterone. Progesterone also promotes growth of the muscularis but seems less effective than estrogen and differs from it by causing pronounced thickening of the walls of the arcuate blood vessels. These vascular changes extend to the coiled arteries of the endometrium, which are also affected by high dosages of estrogen. It seems remarkable that estrogen is capable of preventing the action of progesterone on the myometrial blood vessels and correcting such effects after they are produced and yet at the same time it assists in the destruction of the coiled arteries in the endometrium.

V. Experimentally Produced Implantation Reactions

Progestational endometria of the normal menstrual cycle or those produced in castrated monkeys by progesterone, if mechanically traumatized, will develop endometrial proliferations which seem identical with those found at normal implantation sites of fertilized ova (Figs. 9.16 and 9.171 (Hisaw,

1935; Hisaw, Creep and Fevold, 1937; Wislocki and Streeter, 1938; Rossman, 1940). The proliferated cells originate from the surface and glandular epithelium and grow into the surrounding stroma. The reaction spreads from the point of injury and within a few days may involve the entire inner l)ortion of the endometrium bordering the lumen. The implantation plaques on the 3rd or 4th day present a fairly homogeneous appearance but soon thereafter certain cells attain the proportions of giant cells and many are multinucleated.

The development of the plaques is most rapid during the first week, by the end of which cell division is found only in the basal half of the proliferation and evidence of regression is seen in the superficial portion adjoining the uterine lumen. After 10 days degenerative and phagocytic processes are the dominant features and by 24 days the ut'prus contains few or no ]iroliferation cells. Wislocki and Streeter ( 1938,1 found that implantation plaques during pregnancy and those experimentally induced underwent ajjl^roximately the same development arid subsequent degeneration except for modifications produced by the invading troplio

PHYSIOLOGY OF GONADS

^a.

^^--.^

tx: ^ .

Fig. 9.16. An area of the normal implantation site of a developing ovum. (From Carnegie Institution, No. C467.)

Fig. 9.17. An experimentally induced implantation reaction in a castrated monkey showing condition 6 days after mechanical traumatization of the endometrium.

blast. Rossman (1940j made an extensive morphologic study of these epithelial proliferations and concluded that they should be regarded as typical metaplasias \vith an embryotrophic function.

VI. The Cervix Uteri

The cervix uteri of the rhesus monkey is remarkable for its size and complexity. It forms a large segment that is set off from the fundus by a conspicuous constriction at the level of the internal os (Fig. 9.1). A sagittal section (Fig. 9.18) shows the cervical canal not straight but thrown into several sharp turns by colliculi that extend from its walls into the lumen. The largest of these projects from the midventral wall. The functional advantage of such tortuosity of the cervical canal is not obvious but since the cervix probably serves as a barrier between the bacterial flora of the vagina and the corpus uteri, this may be a useful adaptation.

The physiology of the cervix has received much less attention than has been given the uterus. This is regrettable in view of the consideration it must receive in practical obstetrics and gynecology, as well as the possibility that physiologically the monkey cervix may be homologous with that of the human regardless of morphologic difTerences. Recent observations indicate that this is indeed quite probable.

Fig. 9.18. Sagittal section of the cervix from a normal monkey. The vagina and the external os of the cervix are shown at the left and the entrance to the fundus is at the right.

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Fig. 9.19. Sagittal section of the cervix of a pregnant monkey showing conditions present just previous to parturition on the 154th day of gestation. The dominant features are dilation of the cervical canal and reduction of the cervical lips (shown at the left) and the coUiculi. (From Carnegie Institution, No. C713.)

Hamilton (1949) made a detailed study of the changes in the cervix of rhesus monkeys during the menstrual cycle, paying particular attention to alterations that took place in the cells of the surface epithelium of the endocervical canal and the cervical glands. It was found that heights of the cells showed consistent increases and decreases during the cycle. The peaks came on the 3rd, 13th to 15th, and 22nd days, the greatest of these being the 14th day which is approximately the time of ovulation. It also was observed that, following a peak, secretion was associated with the decline.

Attention was called b3^ Hamilton to the rather close correlation between the fluctuations in height of the cervical epithelium in monkeys and the fluctuations observed by Markee and Berg (1944) in the blood estrogens of the human menstrual cycle. It was concluded that, if similar changes in estrogen levels also occur in monkeys, one would be justified in concluding that the increase in cell height in the cervical mucosa was due to the action of estrogen and the sudden periodic drops in blood estrogen caused secretion and consequent regression. However, it is not clear how this could account for the abundant secretion of the cervical glands in

the presence of high levels of estrogen during late pregnancy (Fig. 9.19).

Much has been learned regarding the physiology of the primate cervix from experiments on castrated monkeys. The cervical mucosa is very responsive to estrogen and castration atrophy can be repaired and a normal condition maintained by daily injections of small doses. Cervical secretion may become abundant when an estrogen treatment is prolonged and especially if large doses are injected. However, the amount of secretion induced by estrogen never equals that of the last half of pregnancy, and it usually subsides if the injections are continued for several months.

Under conditions of chronic treatments with estrogen metaplastic aberrations invariably appear in the epithelium of the endocervix. This reaction was first reported in monkeys by Overholser and Allen ( 1933, 1935) and has been confirmed by many investigators (Engle and Smith, 1935; Hisaw and Lendrum, 1936; Zuckerman, 1937c (. Similar lesions may be found in the cervix uteri of women (Fluhmann, 1954). They seem especially prone to occur under conditions characterized by excessive production of estrogen, such as hyperplasia of the

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PHYSIOLOGY OF GONADS

endometrium (Hellman, Rosenthal, Kistner and Gordon, 1954) and granulosa-cell tumors of the ovary. Various degrees of metaplasia may occur in the cervix during pregnancy both in the mother and newborn but Fluhmann (1954) did not find it as frequently as in nonpregnant women.

This reaction to estrogen as seen in the cervix of castrated monkeys is initiated by growth of small undifferentiated cells below the columnar mucous cells of the secretory epithelium. Fluhmann (1954) suggests that these cells are really undifferentiated cells of the cervical mucosa which have the potentiality of becoming columnar or squamous or simply undergoing multiplication and remaining as indifferent or reserve cells. These cells accumulate, in response to estrogen, to form aggregates of several cells in thickness and, although this may occur in any area of the endocervix, it is generally more pronounced below the base of the glands. As this process proceeds the columnar mucous cells are pushed outward and are finally desquamated thus exposing the underlying metaplastic cells to the lumen of the gland (Fig. 9.20).

Fig. 9.20. Al.iaph

in a castrated monkey that li.-id rci-civcd 1 nig. estriol daily for 48 days.

The cells of these lesions undergo a characteristic differentiation. When first formed they are small, cuboidal, and have spherical nuclei with dense chromatin. As they increase in number those in the center of the cellular mass become larger and acquire an eosinophilic cytoplasm. Such collections, as seen at the base of the cervical glands, may grow in height and form cone-shaped masses with the apexes protruding through the mucous epithelium into the lumen or they may remain as more or less compact structures. This difference in growth seems to have a general relation to the dosage of estrogen. Large doses cause more rapid growth and cone formation with the loss of cells from the apex either singly or in groups, whereas small doses produce slower growth and desquamated cells are seldom seen in the lumen. However, regardless of the rate of growth, the cells at the base of the lesion remain undifferentiated and continue as the principal area of cell proliferation.

Pearl formation is occasionally seen and may be quite common in animals on low dosages of estrogen. Under strong estrogenic stimulation and consequently rapid growth, these structures apparently are desquamated before they are completely formed. However, very early stages are frequently seen and may even be present in small clumps of metaplastic cells, but they are more commonly found in the larger collections at the base of the glands. Their appearance is initiated by swelling and disintegration of one or more adjacent cells that form a center around which epidermidization takes place. Further development does not proceed under the influence of esti'ogen, beyond the formation of a small central cavity.

The most conspicuous difference between the metaplastic growths produced by estrogen and true cancer of the cervix in the monkey (Hisaw and Hisaw, Jr., 1958) is that the former remain noninvasive even when the treatment is continued well over a year. They also involute when the treatiiiciit is discontinued and they do not appeal' when progesterone is given simultaneously with estrogen. When the injections of progesterone are started after metaplastic growths have been formed in response to estrogen, further growth is inhibited and

ESTROGEN AND PROGESTERONE

the keratinized cells of the lesion become vacuolated and are lost.

In contrast with the effects of estrogen on the cervix, the modifications that occur as pregnancy advances are remarkable. The cervix becomes a soft thin-walled structure, the glands increase in number, and their lumina become greatly enlarged, pressing the stroma into thin partitions between them, and the amount of mucus secreted is enormous (Fig. 9.19). Attempts at duplicating these changes in castrated animals by hormone therapy have been only partially successful. Estrogen produces a solid thickwalled cervix that tends to be larger than normal, an effect that is especially noticeable in young animals. Progesterone does not promote cervical growth and repair of the glands unless large doses are given and even then there is little if any secretion. The best results were obtained when both estrogen and progesterone were given and especially so when relaxin was added to the treatment (see chapter by Zarrow).

VII. The Vagina

The general features of the vaginal smear of rhesus monkeys have been described by several investigators (Allen, 1927; Hartman, 1932; Westman, 1932) and a detailed study of the cellular components at different times of the menstrual cycle has been made by Lopez Columbo de Allende, Shorr and Hartman (1945). The changes in the vagina of a monkey are in most respects like those found for the human being (Papanicolaou, Traut and Marchetti, 1948; Lopez Columbo de Allende and Orias, 1950). Epithelial growth and desquamation of cornified cells continue at all stages of the cycle but at various rates. The epithelium is thinnest at menstruation and gradually increases in thickness during the follicular phase, reaching a maximum at ovulation. At this time there is a well developed basal area in which numerous mitoses can be seen and from which many papillae or bulbs" extend into the underlying stroma. Above this is an intermediate zone, an interepithelial zone of cornification (so called Dierk's layer), and a heavily cornified outer zone (Fig. 9.21).

Cellular proliferation is less rapid during the luteal phase and apparently cells are

desquamated more rapidly than they are replaced. Consequently there is a decrease in the thickness of the epithelium in the luteal phase which may include an almost complete loss of the cornified zone (Davis and Hartman, 1935). The effects are probably due to progesterone because similar changes are seen following the introduction of progesterone into a treatment in which estrogen is being given.

The vaginal epithelium of a castrated monkey is remarkably sensitive to estrogen. A small daily dose of 5 to 10 /xg. estradiol will stimulate growth of an atrophic epithelium of 4 to 8 cells in thickness to one of 60 or even 80 layers thick within 3 weeks. One of the first things that is noticed as the vaginal epithelium thickens is the numerous mitotic figures in the stratum germinativum followed by a marked increase in the number of epithelial papillae along the basement membrane. This condition of rapid growth, cornification, and loss of cells into the vaginal lumen is typical of the follicular phase of the menstrual cycle and can be maintained indefinitely.

li(.. U.21. the vaginal epithelium of a castuUMJ monkej' showing growth antl cornification induced by estrogen.

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PHYSIOLOGY OF GONADS

Progesterone, in contrast with estrogen, does not produce rapid growth of the vaginal epithelium but at the same time it is not without an effect. The vaginal epithelium, weeks or months after castration, has relatively few papillae projecting from its basal border into the underlying stroma. When progesterone is given, this condition is changed but not in a spectacular way. There is very slow growth without cornification. The epithelium remains thin but the papillae become more numerous. These are mostly small epithelial buds which tend to remain solid but may show enlargement of the cells in their centers.

When estrogen and progesterone are given concurrently, the effects of estrogen on the vaginal mucosa are modified. If an estrogen treatment has continued for a sufficient time to produce full cornification and then progesterone is added, the first indication of an inhibition of estrogen is a decrease in mitotic activity. This is followed by a continuation of cornification and loss of cells faster than they are replaced ; consequently, most of the functionalis is lost and the epithelium becomes thinner. There is also a noticeable decrease in the intensity of cornification, which in the monkey is never as pronounced as in rodents, and under these conditions is quite incomplete, each cell retaining a conspicuous nucleus. Partly cornified cells may be present for several weeks

.-•^ss;^

4* ' V ^ §

Fig. 9.22. ^^•tgi^al epithelium of a pregnant monkey showing condition on the 154th day of gestation. (From Carnegie Institution, No. C713.)

when both estrogen and progesterone are given, but eventually they almost entirely disappear and the epithelium attains a condition resembling that of late pregnancy.

The inhibitory effect of progesterone on the action of estrogen is shown perhaps even better when a castrated monkey having a fully involuted reproductive tract is first given progesterone for a few days and then (>strogen is added to the treatment, or when injections of the two hormones are started at the same time. In such experiments estrogen has little effect on the vaginal mucosa even in doses that would produce marked cornification if given alone. These observations show that a fully cornified vaginal epithelium cannot be produced or maintained by estrogen when an effective dosage of progesterone is included in the treatment (Hisaw, Greep and Fevold, 1937).

Estrogens and progesterone are the dominant hormones of gestation and their simultaneous action is reflected by the changes in the vaginal epithelium. The fully cornified vagina, present at the time of ovulation, is gradually modified as pregnancy progresses into a condition strikingly like that seen in experiments when estrogen and progesterone are given concurrently. In late pregnancy the most striking feature of the thin, uncornified epithelium is the presence of numerous epithelial buds extending deeply into the underlying stroma. They may branch and rebranch and along their course there is conspicuous enlargement of the more centrally situated cells among which cavities ai^pear, enlarge, and join each other (Fig. 9.22). It seems quite probable that this process may be of considerable importance in increasing the diameter of the vagina.

VIII. Sexual Skin

A so-called sexual skin is jiresent in most catarrhine monkeys, is not found in platyriliine monkeys, and among the anthropoids occurs regularly only in the chimpanzee I l^]ckstein and Zuckcrman, 1956). Changes in the sexual skin during the menstrual cycle have been observed most extensively in the monkey (Macaca), the baboon (Papio), and the chimpanzee (Pan). The sexual skin of t!ie baboon and chimpanzee undergo jiro

ESTROGEN AND PROGESTERONE

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nounced swelling during the follicular phase of the cycle. A maximal size is attained by the middle of the cycle followed by a rapid regression and loss of edema which at least in the baboon is associated with a marked increase in the output of urine (Gillman, 1937a; Krohn and Zuckerman, 1937). The subsidence of the sexual skin begins approximately at the time of ovulation and remains in the reduced condition throughout the luteal phase, followed by a subsequent initiation of swelling during or soon after menstruation (Zuckerman, 1930, 1937e; Zuckerman and Parkes, 1932; Gillman and Gilbert, 1946; Young and Yerkes, 1943; Nissen and Yerkes, 1943).

A w^ell developed sexual skin is present in the monkey {Macaca mulatta) only during adolescence. With the appearance of the menstrual cycles the sexual skin undergoes a process of maturation into the adult condition in which cyclic changes in edema are absent and the most noticeable feature is a vivid red color. Such coloration is due to vascular engorgement rather than pigment (Collings, 1926) and involves the perineum, the buttocks, and may extend for various distances down the legs and over the symphysis pubis. The development and maturation of the sexual skin have been described in considerable detail by several investigators (Hartman, 1932; Zuckerman, van Wagenen and Gardiner, 1938) .

The sexual skin has been of considerable interest both as to the nature of its responsiveness to ovarian hormones and the manner in which its grossly visible changes during the menstrual cycle parallel events occurring in the reproductive tract. The sudden loss of edema at the conclusion of the follicular phase not only signals ovulation but also raises the question as to whether the loss of tissue fluid is due to a decrease in estrogen or is the direct effect of progesterone. The importance of this becomes obvious when it is considered that a similar process also goes on simultaneously in the endometrium and raises the question again as to the respective roles played by estrogen and progesterone in endometrial growth and menstruation.

That the development and edema of the sexual skin of adolescent rhesus monkeys depend on the ovaries was first demon

strated by Allen ( 1927 ) . Involution and loss of color follow castration, and the normal condition can be restored by the injection of estrogen. Also, when estrogen treatment is continued for several weeks maturation of the sexual skin occurs and a condition characteristic of that in the adult is established (Zuckerman, van Wagenen and Gardiner, 1938). The genital area loses its edema and develops a brilliant red color which is retained as long as estrogen is administered. Once this mature condition is established the response of the sexual skin to subsequent estrogen treatments is limited to a change in color.

Similar experiments have been performed on the chacma baboon, Papio porcarius (Parkes and Zuckerman, 1931; Gillman, 1937b, 1938, 1940a). The large sexual skin of these animals is very responsive to estrogen and development equal to that of the follicular phase of the menstrual cycle can be readily induced by daily injections for about 2 weeks. However, the perineal swelling of the baboon differs from the sexual skin of the genital area of the rhesus monkey in that it does not "mature" under the influence of estrogen.

When large doses of estrogen are given to a rhesus monkey a generalized edema of the skin occurs beyond the genital area. This first appears as deeply indented swellings along the sartorii from groin to knee, and next appears at the base of the tail and spreads gradually upward until it involves the entire dorsal portion of the trunk. At the same time, the skin of the face, scalp, and supraorbital ridges becomes swollen and finally the edema may extend out on the arms and down the legs to the ankles (Bachman, Collip and Selye, 1935; Hartman, Geschickter and Speert, 1941). A daily dose of 500 /xg. or more of estriol or estradiol w^ll produce this condition within 2 to 3 weeks and, when the treatment is continued for an extended period the effect tends to subside.

Progesterone has a strong inhibitory action on the effects produced by estrogen on both the genital and extragenital sexual skin of the monkey. If daily injections of progesterone are added to the treatmeiu after full development of the sexual skin has been induced by estrogen, there is a

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PHYSIOLOGY OF GONADS

noticeable loss of edema by the 4th or 5th day followed by rapid involution and reduction of the turgid folds of skin to loose, flabby wrinkles within about 10 days. When estrogen and progesterone are given concurrently to a castrated monkey from the beginning of treatment edema does not appear but the sexual skin regains its normal color. In fact, progesterone alone, like estrogen, can restore the color to the sexual skin of castrated adult monkeys (Hisaw, Greep and Fevold, 1937; Hisaw, 1942).

The interaction of estrogen and i)rogesterone on the sexual skin of rhesus monkeys can best be demonstrated by the reaction of the skin of the sexual area in adolescent animals. The most striking effect and probably the most important is the sequence of events initiated by a single dose of progesterone when given to an animal on continuous estrogen treatment. Under such treatment a full response of the sexual skin is obtained by the end of 20 days. If at this time 1 mg. progesterone is given in a single dose and the estrogen treatment continued uninterruptedly, the first indication of an effect of the luteal hormone is a slight loss of edema and color of the sexual skin on the 4th or 5th day thereafter. The sexual skin is markedly reduced by the 8th day, almost gone by the 9th, and at the end of about a fortnight regains its ability to respond to estrogen as shown by a return of color and swelling. However, the most remarkable eventuation of such treatment is menstruation which usually begins on about the 10th day (Hisaw, 1942).

Involution of the sexual skin and menstruation following a single injection of progesterone also have been produced in the baboon by Gillman (1940a). He found that 5 mg. progesterone, when given on the 8th day of a normal menstrual cycle, would cause an appreciable loss of edema of the swollen perineal sexual skin by the day after injection. This was followed by a progressive involution of the perineum until the 13th day and swelling was re-initiated by the end of the 15th day. Reduction of the sexual skin at this dosage of progesterone was not associated with menstruation. However, when the dose was increased to 20 mg. both deturgescence of the sexual skin and menstruation occurred. These effects pro

duced by progesterone in the presence of endogenous estrogen have much in common with those described above as occurring in castrated monkeys on continuous estrogen treatments.

IX. Menstruation

An experimental ai^proach to the physiology of menstruation dates from the observations of Allen (1927) that uterine bleeding would occur in castrated monkeys following the discontinuance of an estrogen treatment. He suggested that normal menstruation is due to a fluctuation in estrogen secretion and proposed the "estrogen-withdrawal" theory to account for the observed facts. This concept led to an extensive investigation of the effects of estrogens on the endometrium and of conditions that modify their action. It was soon found that in both castrated monkeys and human beings there was a quantitative relationship between the dosage of estrogen given and the maintenance of the endometrium. Bleeding occurred during treatment when the daily dose of estrogen was small, but with larger doses a point was reached at which the injections could be continued for months or even years without bleeding (Werner and Collier, 1933; Zuckerman, 1937b, d).

Estrogen also will inliihit i)ostop('rative bleeding which usually follows total castration, provided the ovaries are removed before or soon after ovulation (Hartman. 1934). With the advent of a corpus luteum and development of a progestational endometrium it becomes progressively more difficult, following castration, to prevent menstruation by injecting estrogen. Similar results are obtained when estrogen is given during a normal menstrual cycle. Small doses may not prevent the onset of menstruation, but if continued, subsequent menstrual periods are delayed (Corner, 1935). Large doses when given during the luteal phase of the cycle do not disturb the normal menstrual rhythm, but may do so if the treatment is started during the follicular phase (Zuckerman, 1935. 1936a).

Progesterone, in contrast with estrogen, will prevent menstruation from an endometrium representative of any stage of the normal cycle. It will delay onset of the next menses even when the treatment is started only

ESTROGEN AND PROGESTERONE

579

a few days before the expected menstruation (Corner, 1935; Corner and Allen, 1936) . Also, the bleeding that invariably follows the discontinuance of a long treatment with estrogen can be inhibited indefinitely by giving progesterone (Hisaw, 1935; Engle, Smith and Shelesnvak, 1935; Zuckerman, 1936b).

An impression held by many of the earlier investigators was that progesterone could not produce its effects on the primate endometrium unless it w^as preceded by the action of estrogen. It is true, of course, that progesterone is a comparatively weak growth promoter and its effects can be demonstrated to best advantage on an endometrium that has been developed by estrogen. However, Hisaw, Greep and Fevold (1937) produced a progestational endometrium in a monkey that had been castrated 242 days previously by giving synthetic progesterone. Also, the endometrium of this animal was found capable of forming a decidual plaque upon traumatization. Soon afterwards Hartman and Speert (1941) observed menstruation following the withdrawal of progesterone in castrated monkeys that had not been given estrogen and more recently similar results have been reported by Eckstein ( 1950) . At the same time it has l)een found that progesterone will induce menstruation in women suffering from amenorrhea and also that uterine bleeding can l)e jirecipitated l)y similar treatment (hiring the follicular j^hase of the cycle (Zondek and Rozin, 1938; Rakoff, 1946).

These observations have been confirmed and extended by Krohn (1951; 1955) who finds that menstrual bleeding can be induced in monkeys wdth secondary amenorrhea by the injection of 5 daily doses of progesterone. Progesterone (5 mg. daily for 5 days) also precipitates uterine bleeding in castrated monkeys at intervals of about 8 days provided the treatment is started innnediately a menstrual bleeding has been induced either by removel of the ovaries or withdrawal of estrogen. The most interesting aspect of these observations is that the number of short 8-day cycles that can be obtained in this way in a castrated animal seems to be related to the size of the initial dose of estrogen used to induce withdrawal bleeding. This also applies to pro

gesterone-withdrawal bleeding, so the effect does not depend upon the particular hormone used to obtain the bleeding. It also is of interest that such conditioning of the endometrium to subsequent responses to the 5-day treatments with progesterone may last for several months on a continuous regime. It is surprising that such a series of responses cannot be initiated unless the first injection of progesterone is given within 6 days following the initial withdrawal bleeding. These observations have much in common with those of Phelps (1947) who also studied the influence of previous treatment on experimental menstruation in monkeys.

There seems to be a quantitative relationship between the dosage of progesterone given in combination with estrogen and the ability of estrogen to prevent bleeding after the injections of progesterone are stopped. It has been mentioned that once a fully developed i^rogestational reaction has been produc(Hl l)y progesterone, it is extremely difficult, if not impossible, to inhibit menstruation by giving estrogen following the withdrawal of progesterone. However, Hisaw and Greep (1938) found that progestational endometria produced ijy small doses of estrogen plus api^roximately 0.5 mg. progesterone daily for 18 to 21 days did not bleed following progesterone withdrawal when continued on 10 to 20 times the original dosage of estrogen. In fact, such endometria were brought back to a condition typical for the action of estrogen and again transformed into a presecretory progestational state without the intervention of bleeding. Similar observations were made previously by Zuckerman (1936a, 1937d).

These experimental results give grounds for some doubt as to the adequacy of the estrogen-withdrawal theory to account fully for menstruation. Not only can progesterone bring about menstruation without the intervention of estrogen but other steroid hormones are capable of pi'oducing similar effects. Desoxycorticosterone in large doses can inhibit estrogen-withdrawal bleeding in castrated monkeys (Zuckerman, 1939, 1951 ) and induce phases of uterine bleeding in rapid succession in normal monkeys (Krohn, 1951). So too can testosterone prevent estrogen-withdrawal bleeding (Hart

580

PHYSIOLOGY OF GONADS

man, 1937; Engle and Smith, 1939; Duncan, Allen and Hamilton, 1941) and inhibit progesterone-withdrawal bleeding as well (Engle and Smith, 1939). Testosterone also will precipitate bleeding during an estrogen treatment (Hisaw, 1943) and in normal monkeys if given early in the cycle (Krohn, 1951). Just what specific action these compounds have in common that enables them to produce these effects or whether there are different modes of action that lead to the same results is not known, but, before mentioning certain possibilities, it may be helpful to consider information regarding the influence of estrogen-progesterone interactions on menstruation.

Among the most significant observations regarding primary causes of menstruation are a few indications that there may be an intrinsic difference in the ways in which estrogen and progesterone produce their effects on the endometrium. One of the first indications of this was the discovery that a short series of injections of progesterone during treatment with estrogen will precipitate menstruation (Corner, 1937; Zuckerman, 1937d; Hisaw and Greep, 1938). This can be demonstrated by giving a castrated monkey a maintenance dose of estrogen daily for 2 or 3 weeks, then adding a daily injection of progesterone for 5 to 10 days and continuing the estrogen treatment. As a rule bleeding appears within 2 or 3 days after stopping progesterone. The most interesting point brought out by such experiments is that bleeding can occur under these conditions in the presence of an otherwise maintenance dosage of estrogen.

Perhaps the most surprising as well as most important fact brought out by subsequent experiments was the small amount of progesterone required to bring about bleeding under these conditions. It was found that only a single injection of 1 mg. was required for animals on chronic treatment with a maintenance dose of estradiol (1000 LIT.) and some bled when 0.5 mg. progesterone was given (Hisaw, 1942). The sequence of events following the injection of progesterone can be seen to best advantage in an adolescent monkey whose sexual skin also respond" to the estrogen treatment. When the 1 mg. ]irogesterone is given on the 20th day of estrogen treatment the edema of th(^

sexual skin will have attained its maximal development. The first indication of an effect of progesterone is a slight loss of edema and color of the sexual skin which appears on the 4th or 5th day and by the 9th or 10th day the edema is almost gone and the sexual skin is pale. Blood apjiears in the vaginal lavage between the 7th and 10th days, of about 70 per cent of the animals on this dosage. The sexual skin may remain markedly reduced and pale until about the 15th day after which both color and edema rapidly return. These effects can also be seen when 1 mg. progesterone is given for a series of days. However, neither the time of appearance nor loss of edema of the sexual skin is significantly hastened, and if the injections extend over no more than 5 days the time between the first injection and bleeding remains approximately the same.

Similar observations have been made by Gillman and Smyth (1939) on the South African baboon iPapio porcarius). They found that 3 mg. or more of progesterone when given in a single injection during the follicular phase of the cycle would cause the relatively enormous perineal swellings to pass rapidly through deturgescence and reach a flabby resting condition within 5 to 7 days, and after a delay of about 24 hours once again begin to swell. As much as 10 or 15 mg. in a single dose caused perineal deturgescence without bleeding, whereas 20 mg. in a single dose or a total of 15 mg. if divided into 2 or 3 injections and given at 3 or 4 day intervals, produced both deturgescence and bleeding (Gillman, 1940b). The l)aboon diff"ers from the monkey in that larger doses of progesterone are required to produce the effects and the sexual skin does not mature" on repeated treatments and lose its responsiveness; otherwise the basic physiology of the reaction in both animals seems to be the same.

The most important fact l)rought out by these experiments is that the effects of a single injection of progesterone can continue in the presence of estrogen for as long as 10 to 15 days. It is highly imi^robable that progesterone lingers in the body for so long a time (Zarrow, Shoger and Lazo-Wasem, 1954). In general it is considered the most ephemeral of the sex steroids and is probablv inactivated within at least a few hours

ESTROGEN AND PROGESTERONE

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after it is administered. It seems more lilvely that progesterone modifies the sexual skin in a way that renders it unresponsive to estrogen and that about a fortnight is required to recover the original condition.

This takes on added significance when the possibility is considered that effects similar to those seen in the sexual skin might also be going on simultaneously in the uterine endometrium. An appreciable dehydration of the endometrium occurs just previous to menstruation (van Dyke and Ch'en, 1936) and a loss of interstitial fluid before bleeding has been observed in endometrial implants in the eyes of monkeys and described in detail by Markee (1940) . This was shown by periodic regression in size and compactness of the grafts which resulted in a decrease in area of 25 to more than 75 per cent. Because cyclic changes in endometrial grafts in the eye are correlated with events of the menstrual cycle there is reason to believe that similar reactions were going on in the endometrium of the uterus.

Endometrial regression, as described by Markee, did not always lead to menstruation although it invariably preceded, accompanied, and followed menstrual bleeding. Menstruation occurred only when regression was rapid and extensive. This was seen in the endometrial grafts in the eye during a normal menstrual cycle at the time of involution of a corpus luteum and during an anovulatory cycle soon after the involution of a large follicle. It also begins soon after the last of a series of injections of estrogen or i^'ogesterone. A slow decrease in size of the ocular grafts, without concomitant bleeding, can be induced in castrated monkeys by gradual withdrawal of estrogen, and when estrogen is given in amounts that are inadequate for maintaining the endometrium for an extended period the "break through" bleeding that eventually ensues is preceded by a rapid and extensive endometrial regression. Because this reaction also occurs w^hen menstruation is induced by such an unusual procedure as spinal transection (Markee, Davis and Hinsey, 1936), it probably is a phenomenon that always precedes menstruation.

It seems from these observations that the changes in the endometrium preceding menstruation are initiated by a sudden with

drawal of a stimulus on which the endometrium at the time relies for the maintenance of a particular physiologic condition, and bleeding and tissue loss are incidents that occur during the readjustment necessary for the return to an inactive state. What this involves is only partly known, but an understanding of the initial changes in the endometrium that usher in menstruation most certainly holds the explanation of the real cause. This has been a perennial subject for discussion and many suggestions and theories have been set forth in an extensive literature to account for various aspects of menstruation. Among the more recent general discussions are those by Zuckerman (1949, 1951), Corner (19511, and Zondek ( 1954 ) .

The estrogen- withdrawal or estrogen-deprivation theory proposed by Edgar Allen has received more attention than any other. From what has been mentioned earlier it is clear that this theory can account for uterine bleeding subsequent to the discontinuance of a series of estrogen injections and also perhaps menstruation at the conclusion of an anovulatory cycle. However, it is not so obvious as to how this theory can explain the occurrence of menstruation at the close of the luteal phase of a normal cycle. Estrogen in large doses will not inhibit such bleeding, but it is postponed if progesterone is given. It is equally difficult to see how this theory is helpful in accounting for the fact that a small dose of progesterone will precipitate bleeding in the presence of a maintenance dosage of estrogen. As little as 2 /xg. progesterone will induce bleeding when applied topically to the endometrial lips of an exteriorized uterus (Fig. 9.7) in a monkey that is receiving 10 fig. estradiol daily (Hisaw, 1950).

Uterine bleeding precipitated by administering progesterone during an estrogen treatment has been explained on the grounds that progesterone in some way interferes with the action of estrogen on the endometrium. Therefore, it is assumed that an animal receiving both estrogen and progesterone is in a sense "deprived" of estrogen. That is, when the two hormones are given simultaneously, progesterone itself is capable of maintaining the endometrium without bleeding; but when it is stopped, the

582

PHYSIOLOGY OF GONADS

suggestion is that the animal is physiologically deprived of estrogen and literally deprived of progesterone (Corner, 1951). Although this view is in descriptive agreement with the observed facts the idea of the inhibitory effect of progesterone does not take into consideration the synergistic interaction of the two hormones on the endometrium.

The physiologic function of progesterone is the conversion of an estrogen-endometrium into a progestational endometrium suitable for receiving and nourishing a developing blastocyst. Such an endometrium is adapted for this specific reproductive function and accordingly its physiologic nature must be quite different from that of the follicular phase of the cycle. Indeed, it is known that these two structures (follicular and luteal phase endometrial are morphologically and biochemically unlike in a number of respects. This gradual transformation, following ovulation, occurs as progesterone becomes the dominant hormone, and consequently, as this proceeds, the endometrium progressively loses competence to respond to estrogen. However, this does not imply that estrogen is without effect in the general economy of the progestational endometrium. It has been shown in a number of ways that the action of progesterone on the primate endometrium is greatly facilitated by the presence of estrogen. In fact, it seems probable that rarely if ever does progesterone perform its function in the absence of estrogen (Hisaw, 1959; chapter by Zarrow).

After consideration of the endometrial specializations brought about by ]irogesterone, it seems rather jwintless to hark back to the follicular phase and inject the past recoi'd of accomjilishments and prerogatives that estrogen had at that time into the explanation of an entirely different hormonal situation. It seems more in keeping with the facts to state outright that menstruation following the involution of a corpus luteum or the discontinuance of progesterone, even though estrogen is present, is due to a decrease or absence of progesterone.

It also has become less certain that menstruation at the conclusion of an anovulatory cycle is really an estrogen-withdrawal bleeding. This is possible, of couisc, but at

the same time the exceedingly small amount of progesterone required to induce bleeding in the presence of estrogen makes it difficult to be sure what the situation might be. Even a negative test for progesterone in the blood, by our present methods, does not necessarily indicate the absence of a physiologically effective amount of progesterone. Zarrow, Shoger and Lazo-Wasem (1954) found that in rabbits an intramuscular injection of 40 mg. progesterone was required to produce an appreciable concentration of the hormone in the blood as determined by the Hooker-Forbes method. Yet, 0.2 mg. progesterone daily for 5 days will produce a progestational reaction in the uterus equivalent to that of the 5th day of normal pseudopregnancy. In monkeys 0.5 mg. daily when given with 10 fxg. estradiol is an adequate dosage of progesterone to induce unquestionable progestational changes in the endometrium and much less will cause bleeding. These observations indicate that the minimal effective concentration of progesterone in the blood may be less than is possible to detect by our present methods.

This also seems to hold for the human being. Estimates of secretion and metabolism of progesterone in the human being have been based primarily on the recovery of its excretory product sodium pregnanediol glucuronidate in the urine. It seems obvious that such determinations must be only general approximations because only about 20 per cent of the progesterone secreted or injected can be accounted for by the pregnanediol in the urine. Also, it is generally known that a physiologically effective dosage of progesterone does not necessarily lead to the excretion of pregnanediol (Hamblen, Cuylcr, Powell, Ashley and Baptist, 1939; Seegar, 1940). In other words, the threshold dose of progesterone for endometrial stimulation is l)elow that at which the hormone is excreted as pregnanediol. In fact, it has been suggested by some investigators that there is no quantitative relationship between the l)rogesterone present in the blood and the pregnanediol excreted in the urine (Buxton, 1940; Sommerville and Marrian, 1950; Kaufmann, Westphal and Zander, 1951).

These findings and the wide variation in the amount of prc'gnancdiol excreted during

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58.3

a menstrual cycle (Venning and Browne, 1937) suggest that, even in the absence of ovulation, sufficient progesterone may be present to influence menstruation. There also is the possibility of progestational hormone from some extra-ovarian source, such as the suprarenal cortex. This was suggested by Zuckerman (1937b, 1941 j as a possible explanation for periodic bleeding in monkeys on a constant submaintenance dose of estrogen. This thought becomes more plausible in view of the fact that progesterone is one of the precursors in the metabolic synthesis of androgens, estrogens, and adrenal cortical steroids (Dorfman, 1956). Also, it has been shown that desoxycorticosterone acetate is converted to progesterone in vivo (Zarrow, Hisaw and Bryans, 1950). Therefore, progesterone is not restricted to ovarian luteal function but instead is of rather general occurrence in the body and the likelihood is that small amounts are a constant constituent of the blood.

Also, the amount of progesterone from extra-ovarian sources may fluctuate, as suggested by Zuckerman (1949), and consequently disturb the normal menstrual rhythm and probably cause bleeding in monkeys on a continuous submaintenance dose of estrogen. However, as to the latter, there is an alternative explanation. Castrated monkeys on a continuous treatment of 10 fxg. of estradiol daily do not show "break-through" bleeding, and a synergistic effect on growth of the uterus is seen when 0.5 mg. or more of progesterone daily is introduced into the treatment. However, the simultaneous administration of 0.25 mg. or even 0.125 mg. progesterone daily in similar exj^eriments results in bleeding between a!)out the 10th to 16th day of the combination treatment. Thus, a dosage of progesterone less than that required for synergism or prevention of bleeding when given alone, modifies the endometrium so that it can no longer be maintained by 10 fxg. estradiol daily (Hisaw, Jr., unpublished). When it is considered that the endometrium becomes increasingly dependent on estrogen during a chronic treatment, even after maximal growth is attained (Hisaw, 1942), it seems plausible that the effectiveness of a dosage of estrogen only slightly alcove the thresh

old for bleeding may be decreased sufficiently by the endogenous progesterone from extra-ovarian sources to precipitate bleeding.

Although it is obvious that the normal menstrual cycle is primarily under the control of the ovarian estrogens and progesterone, it is also equally clear that menstruation is not due to a specific hormonal action. Experimental evidence indicates that any natural or synthetic compound having the capacity for promoting growth or sustaining an existing metabolic state in the endometrium is also capable of inducing withdrawal bleeding. However, this does not imply that all compounds capable of inducing menstruation do so by the same biochemical action; in fact, there is considerable evidence that this is not so (see chapter by Villee). Yet in each instance a series of events is set in motion that leads up to active bleeding.

X. The Mechanism of Menstruation

The immediate cause and mechanism of menstruation has continued to be a topic of special interest for many years and the subject of frequent general discussions. A generalization in keeping with our present knowledge is that no gross morphologic feature of the endometrium is distinctive of menstruation. A menstruating endometrium may be representative of any stage of the follicular or luteal phase of the cycle. The most frequently discussed hypothesis regarding the mechanism of menstruation is that proposed by Markee (1940, 1946) which is based on direct observations of vascular changes in endometrial grafts in the anterior chamber of the eye of monkeys (see p. 564). The changes observed in the endometrium shortly before bleeding are, briefly, as follows. (1) There is extensive and rapid regression of the endometrium due to loss of ground substance from the stroma (Fig. 9.23). (2) The rapid regression brings about a disproportion between the length of the coiled arteries and thickness of the endometrium with the formation of additional coils. (3) The increased coiling of the arteries retards the circulation of blood through them and their branches. This stasis begins 1 to 3 davs before the onset of the

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PHYSIOLOGY OF GONADS

Repair

Fig. 9.23. A diagram indicating correlated changes in ovary and endometrium during an ovulatory cycle of rhesus monkey. Thickness of endometrium, density of stroma, gland form, and three types of arteries are indicated. There is a gradual rise in thickness up to the time of ovulation, and a brief decline followed by development of the luteal or progestational phase with accumulation of secretion in the glands due to relaxation of the myometrium. This is followed by loss of ground substance from the stroma, which is the primary factor in the premenstrual regression of the ischemic phase. This is a prelude to extravasation and shedding of tissue. Incidentally, secretion is extruded and glands collapse. There is further regression throughout the phase of menstruation. More than the basal zone (coarse stipple) survives menstruation. During repair, thickening of the endometrium is associated with increase in ground substance in the stroma and growth in the glands. (From G. W. Bartelmez, 1957, Am. J. Obst. & Gynec, 74, 931-955, 1957, with some modification of description.)

flow, and is associated with leukocytosis in the endometrium. (4) The portion of the coiled arteries located adjacent to the muscularis constricts 4 to 24 hours before the onset of the flow. This vasoconstriction persists throughout the menstrual period except when individual coiled arteries relax and blood circulates through them for a few minutes. Markee postulated that the immediate cause of menstruation under these conditions was the injurious effect of anoxemia upon the tissues of the endometrium l)rought about by mechanical compression and constriction of the coiled arteries. Therefore, the coiled arteries and their modifications become the central feature upon which the theory is based.

Although this offers an explanation for many of the facts, it falls short in that now it is known that menstruation can occur in the absence of coiled arteries. Kaiser (1947) showed that no spiral arteries are present in

the endometrium of three species of South American monkeys known to menstruate. He also found that the coiled vessels of the endometrium could be destroyed almost completely by giving large doses of estrogen and yet bleeding followed estrogen withdrawal.

Several experimental conditions under which the coiled vessels of the endometrium are destroyed have been mentioned in the present discussion and in each instance bleeding invariably followed withdrawal of the supporting stimulus. The extremely atrophic endometrium present at the conclusion of a prolonged treatment with progesterone (Fig. 9.8) will bleed when the injections are stopped, and if estrogen injections are started immediately thereafter the endometrium that develops is normal with the exception of the absence of coiled arteries; even so, it also will bleed when the treatment is stopped. Even a more

ESTROGEN AND PROGESTERONE

58c

drastic destruction of endometrial structures occurs when both estrogen and progesterone are given for several months. Not only are the coiled arteries destroyed but also the glands and the luminal epithelium. All that remains is a modified stroma penetrated by a few small blood and lymph vessels and scattered glandular rudiments along the myometrium (Fig. 9.13). Yet, in spite of this, bleeding follows discontinuance of the treatment.

These observations prove conclusively that the spiral arteries of the endometrium do not hold the solution to the menstrual process. However, the descriptive account by Markee of the events that take place in the endometrium during the cycle remains one of the major contributions to our knowledge of the primate endometrium. Phelps (1946) also made a very careful study of the vascular changes in intraocular endometrial transplants in ovariectomized monkeys receiving estrogen and progesterone, and concluded that the primary function of the coiled arteries is concerned with vascularization of the implantation site of a developing embryo.

There also is reason for doul^ting that ischemia is a determining factor in the menstrual process. That constriction of the endometrial vessels does occur is well established, but that tissue destruction and bleeding are consequences of prolonged anoxemia may be questioned. The endometrium around the internal cervical os as seen in incised exteriorized uteri (Fig. 9.7) contains very few coiled arteries and does not take part in the periodic blushing and blanching of the fundus, but instead remains blood-red even during menstruation. Also, certain tongues of endometrium in a uterine fistula may become crowded by their neighbors to an extent of being partly or completely deprived of blood, yet they do not bleed even though their unfavorable situation leads to deterioration within a few days.

Emmel, Worthington and Allen (1941) attempted to induce menstruation in monkeys by operative ischemia. Circulation to the fundus of the uterus was interrupted by means of a tourniquet for periods of 1 to 8V4 hours, and in two instances for 19 hours. This procedure did not precijiitate uterine

bleeding nor did it hasten the onset of an expected bleeding following estrogen withdrawal. In fact, when the uterus was deprived of blood for periods longer than 3 hours impairment of the bleeding response to estrogen withdrawal was observed, and 19 hours of ischemia caused atrophy of the uterus without bleeding.

It also has been reported that a toxic substance formed in the endometrium is responsible for menstruation. This menstrual toxin is supposed to be present in the endometrium just previous to and during menstruation, and to be a substance resembling or identical with necrosin, a material found in pleural exudate following an inflammatory reaction (Smith and Smith, 1951). Zondek (1953) reports that menstrual blood, when obtained under relatively sterile conditions, is no more toxic to experimental animals than sterile tissue extracts. He also found that death of animals given injections of menstrual blood was due to bacteremia, an effect that could be prevented by giving antibiotics. Nor was he able to demonstrate a toxic substance in the premenstrual or menstrual endometrium. It might be mentioned in this connection that endometrial tissue destroyed by experimental ischemia in the experiments by Emmel, Worthington and Allen (1941), obviously did not influence menstruation nor did involuting endometrial tissue in uterine fistulae (p. 564). Therefore, the presence of a specific toxin that may induce menstruation has not been conclusively demonstrated.

Regardless of the specific cause of menstruation, the evidence shows that it can occur in the absence of coiled arteries, endometrial glands, or surface mucosa, and is unrelated to the thickness of the endometrium. This statement is based on conditions that have been experimentally induced in the monkey and they strongly indicate that menstruation, whatever the cause, is a stromal phenomenon. This view seems to be in agreement with the observations reported by Bartelmez in his elegant studies of the morphology of the endometrium of both monkeys and the human being. He emphasizes changes taking place in the connective tissue elements of the stroma and points out that much less tissue is lost at menstruation

586

PHYSIOLOGY OF GONADS

than i.< commonly thought (Bartehnez, 1957). The reduction in thickness is clue primariiy to loss of ground substance from the stroma, and conversely, the outstanding feature of repair is the increase in stromal ground substance (Fig. 9.23). ^Mitoses are rarely seen in the stroma during repair and arc not abundant enough in any phase according to Bartelmez to account for the observed increase in thickness of the endometrium. Our present knowledge indicates that an explanation of menstruation may be found in the metabolic effects induced in the stromal connective tissue of the endometrium by a sudden withdrawal of a supporting hormonal stimulus.

XI. References

Allen, E. 1927. The menstrual CA'cle in the monkey, Macacus rhesus: observations on normal animals, the effects of removal of the ovaries and the effects of injections of ovarian and placental extracts into the .spayed animals. Contr. Embrvol., Carnegie Inst. Washington, 19, 1-44.

Allen. E. 1928. Further experiments with an ovarian hormone in the ovariectomized adult monkey, Macacus rhesus, especially the degenerative phase of the experimental menstrual cycle. Am. J. Anat., 42, 467^87.

Allen, E., Diddle, A. W., Burford, T. H., .and Elder, J. H. 1936. Analyses of urine of the chimpanzee for estrogenic content during various stages of the menstrual cycle. Endocrinology, 20, 546-549.

Allen, E., Pr.\tt, J. P., Xewell, Q. U., .and Bl.and, L. J. 1930. Human tubal ova ; related early corpora lutea and uterine tubes. Contr. Embryol., Carnegie Inst. Washington, 22, 45-76.

B.ACHMAN, C, CoLLip, J. B., .\ND Selye, H. 1935. The effects of prolonged estriol administration upon the sex skin of Macaca mulatta. Proc. Roy. Soc, London, .■^er. B., 117, 16-21.

B.artel.mez, G. W. 1933. Histologic studies on the menstruating mucous membrane of the human uterus. Contr. Embryol.. Carnegie Inst. Washington, 24, 141-186.

B.artel.mez, G. W. 1937. ^Menstruation. Plnsiol. Rev.. 17, 28-72.

B.ARTELMEZ, G. W. 1951. Cyclic changes in the endometrium of the rhesus monkey {Macacus mulaltn). Contr. Embryol., Carnegie Inst. Washington. 34, 101-144.

B.ARTELMEz, G. W. 1957. The pha.scs of the menstrual cycle and their interpretation in terms of the pregnancv cycle. Am. J. Obst. & Gvnec, 74, 931-955.

BRY.AN.S, F. E. 1951. Progesterone of the lilood in the menstrual cycle of the monkey. Endocrinology, 48, 733-740.

BrxTo.N, C. L. 1940. Pregnanediol determina

tions as an aid in clinical diagnosis. Am. J. Obst. & Gynec, 40, 202-211.

CoLLiNGS, M. R. 1926. A study of the cutaneous reddening and swelling about the genitalia of the monkev, Macacus rhesus. Anat. Rec, 33, 271-287.

Corner, G. W. 1923. Ovulation and menstruation in Macacus rhesus. Contr. Embrj^oL, Carnegie Inst. Washington, 15, 73-101.

Corner, G. W. 1935. Influence of the ovarian hormones, estrin and progestin, upon the menstrual cvcle of the monkev. Am. J. Phj'siol., 113,238-250.

Corner, G. W. 1937. Experimental menstruation. Science, 85, 437-438.

Corner, G. W. 1942. The fate of the corpora lutea and the nature of the corpora aberrantia in the rhesus monkey. Contr. Embryol., Carnegie Inst. Washington, 30, 87-96.

Corner, G. W. 1945. Development, organization and breakdown of the corpus luteum in the rhesus monkey. Contr. Embrj'ol., Carnegie Inst. Washington, 31, 119-146.

Corner, G. W. 1951. Our knowledge of the menstrual cycle, 1910-1950. Lancet, 1, 919-923.

Corner, G. W., and Allen, W. M. 1936. Inhibition of menstruation by crj-stalline progesterone. Proc. Soc. Exper. Biol. & Med.. 34, 723724.

Crossex, R. J. 1953. Diseases of Women. St. Louis: C. V. Mosby Company.

Davls, M. E., and H.art^lan, C. G" 1935. Changes in vaginal epithelium during pregnancy in relation to the vaginal cvcle. J. A. M. A., 104, 279-285.

Diczfall'.sy, E. 1953. Chorionic gonadotrophin and estrogens in the human placenta. Acta endocrinol., Suppl. 12, 87-167.

DoRFMAN, R. I. 1956. Metabolism of androgens, estrogens and corticoids. Am. J. Med., 21, 679687.

DoRFMAN, R. I., AND VAN W.AGENEN, G. 1941. The

sex hormone excretion of adult female and pregnant monkevs. Surg. Gvnec. & Obst., 73, 545-548.

Duncan, P. A., Allen. E., and Ha.milton. J. B. 1941. The action of testosterone proprionate on experimental men.struation in the monkev. Endocrinology. 28, 107-111.

Eckstein, P. 1950. The induction of progesterone withdrawal bleeding in spayed monkeys. J. Endocrinol., 6, 405-411.

EcK.STEiN, P., AND ZucKERMAN, S. 1956. In Marshall's Physiology of Reproducliori. A. S. Parkes, Ed. Vol. 1, ]\ 334. London: Longmans Green & Company.

EmMEL, V. M., WORTHINGTON, R. V., AND AlLEN. E.

1941. Attempts to induce menstruation by operative ischemia in monkey's. Endocrinologv, 29, 330-335.

Engle. E. T. 1937. Problems of experimental menstruation. Cold Spring Harbor Svmposia Quant. Biol., 5, 111-114.

Engle, E. T., .and S.mith, P. E. 1935. Some uterine effects obtained in female monkeys during continued estrin administration, with especial

ESTROGEN AND PROGESTERONE

587

reference to tlio r('i\ ix uteri. Auat. Rec, 6, 471-483.

Encle, E. T., Smith, P. E., and Shelesnyak, M. C. 1935. The role of estrin and progestin in experimental menstruation. Am. J. Obst. & Gynec. 29, 787-797.

Engle, E. T., and Smith, P. E. 1938. The endometrium of the monkey and est rone-progesterone balance. Am. J. Anat., 63, 349-365.

Engle, E. T., and Smith, P. E. 1939. Certain actions of testosterone on the endometrium of the monkey and on uterine bleeding. Endocrinology, 25^ 1-6.

Fish, W. R., Young, W. C, and Dorfman, R. I. 1941. Excretion of estrogenic and androgenic substances by female and male chimpanzees with known mating behavior records. Endocrinology, 28, 585-592.

Fluhmann, C. F. 1954. Comparative studies of squamous metaplasia of the cer\ix uteri and endometrium. Am. J. Obst. ct Gynec, 68, 1447-1462.

Forbes, T. R., Hooker, C. W., and Pfeiffer, C. A. 1950. Plasma progesterone levels and the menstrual cycle of the monkey. Proc. Soc. Exper. Biol. & Med., 73, 177-179.

GiLLMAN, J. 1937a. The cyclical changes in the vaginal smear in the baboon and its relationship to the perineal swelling. South African J. M. Sc, 2, 44-56.

GiLLMAN, J. 1937b. Experimental studies on the menstrual cycle of the baboon (Papio porcarhis). South African J. M. Sc, 2, 156-166.

GiLLMAN, J. 1938. Experimental studies on the menstrual cycle of the baboon (Papio porcarius). South African J. M. Sc, 3, 6&-71.

GiLLMAN, J. 1940a. Experimental studies on the menstrual cycle of the baboon (Papio porcarius). VI. The effect of progesterone upon the first part of the cycle in normal female baboons. Endocrinology, 26, 80-87.

GiLLMAN, J. 1940b. The effect of multiple injections of progesterone on the turgescent perineum of the baboon (Papio porcarius). Endocrinology, 26, 1072-1077.

GiLLMAN, J., AND GILBERT, C. 1946. The reproductive cycle of the chacma baboon (Papio itrsiDiis) with special reference to the problems of menstrual iriegularities as assessed by the behaviour of the sex skin. South African J. M Sc, Biol. Suppl., 11, 1-54.

GiLLMAN, J., .AND Smyth, G. S. 1939. The hormonal content of the human luteal follicle of pregnancy as determined by its effect on the perineum of the baboon. South African J. M. Sc, 4, 3&-45.

Haman, J. O. 1942. The length of the menstrual cvcle. A study of 150 normal women. Am. J. Obst. & Gynec, 43, 870-873.

Hamblen, E. C., Cuyler, W. K., Powell, N. B., Ashley, C, and B.aptist, M. 1939. Some clinical observations upon the metabolism and utilization of crystalline progesterone. Endocrinology, 25, 13-16.

Hamilton, C. E. 1949. Observations on the cervi

cal mucosa of the Rhesus monkey. Contr. EnibryoL, Carnegie Inst. Washington, 33, 81-101.

H.artman, C. G. 1929. Three types of uterine bleeding in the monkey and the homology of menstruation (Abstr.). Anat. Rec, 42, 19.

Hartman, C. G. 1932. Studies in the reproduction of the monkey, Macacus (Pithecus) rhe.S-//.S, with special reference to menstruation and piegnancy. Contr. Embryol., Carnegie Inst. Washington, 23, 1-16.

H.ARTMAN, C. G. 1934. Some attempts to influence the menstrual cvcle in the monkev. Am. J. Obst. & Gynec, 27, 564-570.

Hartman, C. G. 1937. Menstruation inhibiting action of testosterone. Proc Soc. Exper. Biol. & Med., 37, 87-89.

H.ARTMAN, C. G. 1944. Regeneration of the monkey uterus after surgical removal of the endometrium and accidental endometriosis. Western J. Surg. Obst. & Gynec, 52, 87-102.

Hartman, C. G., .and Speert, H. 1941. Action of progesterone on the genital organs of the unjirimed Rhesus monkev. Endocrinology, 29, 639-648.

H.ARTMAN, C. G., GeSCHICKTER, G. F., AND SpEERT, H.

1941. Effects of continuous estrogen administration in verv large doses. Anat. Rec, Suppl. 2, 79, 31.

Hellman, L. M., Rosenthal, A. H., Kistner, R. W., AND Gordon, R. 1954. Some factors influencing the proliferation of the leserve cells in the human cervix. Am. J. Obst. & Gvnec. 67, 899-915.

Hertig, a. T., and Rock, J. 1944. On the development of the early human ovum, with special reference to the trophoblast of the previllous stage ; a description of 7 normal and 5 pathologic human ova. Am. J. Obst. & Gvnec, 47, 149-184.

Heuser, C. H., and Streeter, G. L. 1941. Development of the macaque embryo. Contr. Embryol., Carnegie Inst. Washington, 29, 17-55.

Hisaw, F. L. 1935. The physiology of menstruation in Macacus rhesus monkevs. Am. J. Obst. & Gynec, 29, 638-659.

HiSAW% F. L. 1942. The interaction of the ovarian hormones in experimental menstruation. Endocrinology, 30, 301-308.

HiSAW, F. L. 1943. Androgens and experimental menstruation in the monkey (Macaca viulatta). Endocrinology, 33, 39-47.

HisAW, F. L. 1950. Factors influencing endometrial growth in monkeys (Macaca mulatta). In A Symposium on Steroid Hormones, E. S. Gordon, Ed., pp. 259-276. Madison: University of Wisconsin Press.

Hisaw, F. L. 1959. Endocrine adaptations of the mammalian estrous cycle and gestation. In Columbia University Symposium on Comparative Endocrinology, pp. 533-552.

Hisaw, F. L., Creep, R. O., and Fevold, H. L. 1937. Effects of progesterone on the female genital tract after castration atrophy. Proc Soc. Exper. Biol. & Med., 36, 840-842."

His.\w, F. L.. AND Creep, R. O. 1938. The inhibition of uterine bleeding with estradiol and

588

PHYSIOLOGY OF GONADS

progesterone and associated endometrial modifications. Endocrinology, 23, 1-14. His.\w, F. L., .^ND His.wv, F. L., Jr. 1958. Spontaneous carcinoma of the cervix uteii in a monkey (Macaca mulatto). Cancer, 11, 810816. HiSAW, F. L., .4ND Lendrum, F. C. 1936. Squamous metaplasia in the cervical glands of the monkey following oestrin administration. Endocrinology, 20, 228-229. HiSAW, F. L., Meyer, R. K., axd Fevold, H. L. 1930. Production of a premenstrual endometrium in castrated monkeys by ovarian hormones. Proc. Soc. Exper. Biol. & Med., 27, 400-403. HiTSCHMANN, F., AND Adler, L. 1907. Die Lehre von der Endometritus. Ztschr. Geburtsh. u. GynJik., 60, 63-86. Kaiser, I. H. 1947. Absence of coiled arterioles in the endometrium of menstruating New World monkeys. Anat. Rec, 99, 353-363. Kaufmann, C, Westphal, U., and Zander, J. 1951. Untersuchungen liber die biologische Bedeutang der Ausscheidungsprodukte des Gelbkcirperhormons. Arch. Gynak., 179, 247-299. Knaus, H. 1950. Die Physiologie der Zeugung

des Menschen. Wien: Wilhelm Maudrich. Krohn, p. L. 1951. The induction of menstrual bleeding in amenorrhoeic and normal monkeys by progesterone. J. Endocrinol., 7, 310-317. Krohn, P. L. 1955. The induction of cyclic uterine bleeding in normal and spayed rhesus monkeys by progesterone. J. Endocrinol., 12, 6985. Krohn, P. L., and Zuckerman, S. 1937. Water metabolism in relation to the menstrual cycle. J. Physiol., 88, 369-387. Latz, L. J., and Reiner, E. 1942. Further studies on the sterile and fertile periods in women. Am. J. Obst. & Gynec, 43, 74-79. Lendrum, F. C., .and Hisavv^ F. L. 1936. Cytology of the monkey endometrium under influence of follicidar and corpus luteum hormones. Proc. Soc. Exper. Biol. & Med., 34, 394-396. Lopez Columbo de Allende, I., and Orias, O. 1950. Cytology of the Human Vagina. New York: Paul B. Hoeber, Inc. Lopez Colu.mbo de Allende, I., Shorr, E., and Hartman, C. G. 1945. A comparative study of the vaginal smear cycle of the rhesus monkey and the human. Contr. Embryol., Carnegie Inst. Washington, 31, 1-26. M.arkee, J. E. 1940. Menstruation in intraocular endometi'ial tr;msplants in the I'hesus monkey. Contr. Embrvol., Carnegie Inst. Washington, 28, 219-308. Markee, J. E. 1946. Morphologic and endocrine basis foi' menstrual bleeding. In Progress in Gynecology, Meigs and Sturgis, Eds. Vol. II, pp. 37-47. New York: (hune and Stiattdii. Markee, J. E., and Berg, B. 1944. Cyclic fluctuations in blood estrogen as a possible cause of menstruation. Stanford Med. Bull., 2, 55-60. Markee, J. E., D.wis, J. H., and Hinsf.y, J. C. 1936. Uterine bleeding in spinal iii()nk(>vs. Anat. Rec, 64, 231-245.

Mazer, C, and Israel, S. L. 1951. Diagnosis and Treatment of Menstrual Disorders and Sterility. New York: Paul B. Hoeber, Inc.

Meyer, R. 1911. Uber Corpus luteum-Bildung beim Menschen. Arch. Gynak., 93, 354-404.

Nissen, H. W., and Yerkes, R. M. 1943. Reproduction in the chimpanzee: report on 49 births. Anat. Rec, 86, 567-578.

Novak, E., and Te Linde, R. W. 1924. Endometrium of menstruating uterus. J. A. M. A., 83, 900-906.

OvERHOLSER, M. D., AND Allen, E. 1933. Ovaiiau hormone and traumatic stimulation of monkey's cervix to a condition resembling early cancer. Proc Soc. Exper. Biol. & Med., 30, 1322-1326.

OvERHOLSER, M. D., AND Allen, E. 1935. Atypical growth induced in cervical epithelium of monkey by prolonged injections of ovarian hormone combined with chronic trauma. Surg. Gynec. & Obst., 60, 129-136.

OvERHOLSER, M.D.,. AND Nelson, W. 0. 1936. Migration of nuclei in uterine epithelium. A monkey following prolonged estrin injections. Proc Soc Exper. Biol. & Med., 34, 839-841.

P.APANicoL.Aou, G. N., Traut, H. F., and M.archetti, A. A. 1948. The Epithelia of Woman/s Reproductive Organs. New York: Commonwealth Fund.

Parkes, a. S., and Zuckerm.an, S. 1931. The menstrual cycle of the Primates. II. Some effects of oestrin on baboons and macaques. J. Anat., 65, 272-276.

Phelps, D. 1946. Endometrial vascular reactions and the mechanism of nidation. Am. J. Anat., 79, 167-197.

Phelps, D. H. 1947. The factor of previous treatment in experimental menstruation. J. Clin. Endocrinol., 7, 611-623.

Rakoff, a. E. 1946. Studies on high dosage progesterone therapy of amenorrhea. Am. J. Obst. & Gynec, 51, 480-491.

Rock, J., and Hertig, A. 1942. Some aspects of early human development. Am. J. Obst. & Gynec, 44, 973-983. RossM.AN, I. 1940. The decidual reaction in the rhesus monkey {Macaca mulatta). I. The epithelial proliferation. Am. J. Anat., 66, 277365. Schroder, R. 1914. Uber das Verhalten der Uterusschleimhaut um die Zeit der Menstruation. Monatsschr. Geburtsh. u. Gynak., 39, 3-21. Seeg.ar, E. G. 1940. The histologic effect of progesterone on hyperplastic endometria. Am. J. Obst. & Gynec, 39, 469-476. S.mith, O. W., and S.mith, G. V. 1951. Endocrinology and related phenomena of the human menstrual cvcle. Recent Progr. Hormone Res., 7, 209-253.

So.MMKKVILLK, I. V ., AND MaRRIAN, G. F. 1950.

I'rinary excretion of prcgnanediol in human subjects following the administration of progesterone and of pregnane-3a:20a-diol. I3iochem. J., 46, 285-289. Stieve, H. 1926. Di(> regelmassigen Verliinderungen der Muskulatur und des Bindegewebs in

ESTROGEN AND PROGESTERONE

589

der meuschlichen Gebarmutter in ihier Abhangigkeit von der Follikelreife und der Aiisbildung eines gelben Korpers, nebst Beschreibung eines menschlichen Eies im Zustand der ersten Reifteilung. Ztschr. mikroskop.-anat. Forsch, 6, 351-397.

Stieve, H. 1942. Der Einfluss von Angst und psychischer Erregung auf Bau und Funktion der weiblit'hen Geschlechtsorgane. Zeutralbl. Gynak., 66, 1698-1708.

Stieve, H. 1943. Weitere Tatsachen zur Kliirung der Frage: Wann wird das Ei aus dem Eierstock ausgestossen? Zentralbl. Gvnak., 67, 5877.

Stieve, H. 1944. Paracyclische Ovulationen. Zentralbl. Gynak., 68, 257-272.

Sturgis, S. H. 1942. Method for obtaining uterine fluid from the monkey : effect of pilocarpine, atropine, physiologic salt solution and adrenalin. Endocrinology, 31, 664-672.

VAN Dyke, H. B., and Ch'en, G. 1936. Observations on biochemistry of genital tract of female macacjue particularly during menstrual cycle. Am. J. Anat., 58, 473-499.

VAN Wagenen, G. 1945. Mating in relation to pregnancy in the monkey. Yale J. Biol. & Med., 17, 745-760.

VAN Wagenen, G. 1947. Early mating and pregnancy in the monkev. Endocrinologv, 40, 3743.

VAN W.^genen, G., and Morse, A. H. 1940. Cyclic changes in the exteriorized uterus. Endocrinology, 27, 268-273.

Venning, E. H., and Browne, J. S. L. 1937. Studies on corpus luteum function. I. The vn-inary excretion of sodium pregnandiol glucuronidate in the human men.^trual cvcle. Endocrinologv, 21,711-721.

Werner, A. A., and Collier, W. D. 1933. The effect of theelin injections on the castrated woman. J. A. M. A., 100, 633-640.

Westman, a. 1932. Studien iiber den Sexualzyklus bei Makakus-Rhesus-Affen, nebst einigen Bemerkungen liber den menstruellen Blutungs-mechanismus. Acta obst. et gynec. scandinav., 12, 282-328.

WiSLOCKi, G. B., and Streeter. G. L. 1938. On the placentation of the macaque {Macaca mulatta), from the time of implantation until the formation of the definitive placenta. Contr. Embryol., Carnegie Inst. Washington, 27, 166.

Young, W. C, and Yerkes, R. M. 1943. Factors influencing the reproductive cycle in the chimpanzee; the period of adolescent sterility and related problems. Endocrinology, 33, 121-154.

Z.ARRow, M. X., Hisaw, F. L., and Bryans, F. 1950. Conversion of desoxycosterone acetate to progesterone in vivo. Endocrinology, 46, 403-404.

Zarrow, M. X., Shoger, R. L., and Lazo-Wasem, E. A. 1954. The rate of disappearance of exogenous progesterone from the blood. J. Clin. Endocrinol., 14, 645-652.

Zondek, B. 1953. Does menstrual blood contain a specific toxin? Am. J. Obst. & Gvnec, 65, 1065-1068.

-Zondek, B. 1954. On the mechanism of uterine bleeding. Am. J. Obst. & Gynec, 68, 310-314.

Zondek. B., and Rozin, S. 1938. Production of uterine haemorrhage in the normal cycle and hi amenorrhoea through progesterone. J. Obst. & Gynaec. Brit. Emp., 45, 918-931.

Zuckerman, S. 1930. The menstrual cycle of the Primates. I. General nature and homology. Proc. Zool. Soc, London, 1930, 691-754.

Zuckerman, S. 1935. The menstrual cycles in the Primates. VIII. The estrin-vvithdrawal theory of menstruation. IX. The effect of estrin on the denervated sexual skin. Proc. Rov. Soc, London, ser. B., 118, 13-33.

Zuckerman, S. 1936a. Inhibition and induction of uterine bleeding bv means of estrone. Lancet, 2, 9-13.

Zuckerman, S. 1936b. The interrelation of estrone and progestin in the menstrual cvcle. J. PhvsioL, 86, 31-33.

Zuckerman, S. 1937a. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc, London, ser. A., 1937, 315-329.

Zuckerman, S. 1937b. The menstrual cycle of the Primates. X. The oestrone threshold of the uterus of the rhesus monkey. XL The part played by oestrogenic hormone in the menstrual cycle. Proc. Roy. Soc, London, ser. B., 123,441-471.

ZucKER.MAN, S. 1937c Effects of prolonged oestrin-stimulation on the cervix uteri. Lancet, 1, 435-437.

Zuckerman, S. 1937d. Further observations on endocrine interaction in the menstrual cvcle. J. Physiol., 89, 49-51.

Zuckerman, S. 1937e. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc London, ser. A., 1937, 315-329.

Zuckerman, S. 1939. The effect of sex hormones, cortin, and vasopressin on water-retention in the reproductive organs of monkeys. J. Endocrinol., 1, 147-155.

Zuckerman, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with constant threshold dose of oestrone. J. Endocrinol., 2, 263-267.

Zuckerman, S. 1949. The menstrual cvcle. Lancet, 2, 176.

Zuckerman, S. 1951. The hormonal basis of uterine bleeding. Acta endocrinol., 7, 378-388.

Zuckerman, S., and P.arkes, A. S. 1932. The menstrual cycle of the primates. V. The cycle of the baboon. Proc Zool. Soc. London, 1932, 139-191.

Zuckerman, S., van W.agenen, G., and Gardiner, R. H. 1938. The sexual skin of the rhesus monkey. Proc Zool. Soc, London, ser. A., 108, 385-401.

10

THE MAMMARY GLAND AND LACTATION

A. T. Cowie and S. J. FoUeij

NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING, SHINFIELD, READING, ENGLAND

I. Introduction

I. Introduction 590

II. Development of the Mammary

Gland 591

A. Histogenesis 591

B. Normal Postnatal Development . 593

1. Methods of assessing mammary

development 593

2. Mammary development in the

nonpregnant female 594

3. Mammary growth in the male . . 595

4. Mammary development during

pregnancy 596

5. Mammary involution 598

C. Experimental Analysis of Hormonal

Influences 598

1. Ovarian hormones in the animal

with intact pituitary 598

2. Anterior pituitary hormones. . . 601

3. Metabolic hormones (corticoids,

insulin, and thyroid hormones) 604 III. Endocrine Influences in Milk Secretion 606

A. Anterior Pituitary Hormones 606

1. Initiation of secretion (laeto genesis) 606

2. Maintenance of milk secretion —

galactopoiesis 609

3. Suckling stimulus and the main tenance of lactation 611

B. Hormones of the Adrenal Corte.x . . 612

C. Ovarian Hormones 613

D. Thyroid Hormones 617

E. Parathyroid Hormone 618

F. Insulin 619

IV. Removal of Milk from the Mammary

Glands: Physiology of Suckling AND Milking 619

A. Milk-Ejection Reflex 619

B. Role of the Neurohypophysis 621

C. Milk-Ejection Hormone 622

D. Effector Contractile Mechanism of

the Mammary Gland 623

E. Inhibition of Milk Ejection 624

F. Neural Pathways of the Milk-Ejec tion Reflex 625

G. Mechanism of Suckling 626

V. Relation between the Reflexes

Concerned in the Maintenance of Milk Secretion and Milk Ejection 627 VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance OF Milk Secretion and

Milk E.tection 630

VII. Conclusion 632

VIII. References 632

This account of the hormonal control of the mammary gland is in no way intended as an exhaustive treatment of mammary gland physiology, but rather an attempted synthesis of current knowledge which it is hoped will be of interest as an exposition of the authors' conception of the present status of the subject. Since the publication of the second edition of this book, the emphasis in the field under review has tended to shift towards the development of quantitative techniques for assessing the degree of mammary development, towards attempts at a ])enetration into the interactions of hormones with the biochemical mechanisms of the mammary epithelial cells, and towards an increasing preoccupation with the interplay of nervous and endocrine influences in certain phases of lactation. The reader's acquaintance with the classical foundations of the subject as described in the second edition of this book (Turner, 1939) and in other subsequent reviews (Follcy, 1940; Petersen, 1944, 1948; Folley and Malpress, 1948a, b; Mayer and Klein. 1948, 1949; Follev, 1952a, ]9r)6; Dabelow. 1957) will

590

MAMMARY GLAXD AND LACTATION

591

therefore be assumed and used as a point of departure for the present account which can most profitably be concerned mainly with developments which have occurred since the last edition was published. Reference will freciuently be made to these reviews in which authority will be found for the many ex cathedra statements that will be made, but original sources will be cited wherever appropriate.^

As an aid to logical treatment of the subject the scheme of classification proposed by Cowie, Folley, Cross, Harris, Jacobsohn and Richardson (1951) will be followed in this chapter. Besides introducing a system of terminology in respect of the physiology of suckling or milking, these writers have put forward a classification scheme which is an extension of one previously proposed by one of the present authors (Folley, 1947). This scheme considers the phenomenon of lactation as divisible into a number of phases as follows:

[ [Milk synthesis

I Milk secretion ■! Passage of milk from I I the alveolar cells

Lactation<J [Passive withdrawal of

ij milk

JThe milk-ejection re[ Hex

Milk removal

I

As is logical and customary, discussion of lactation itself will be preceded by consideration of mammary development.

II. Development of the Mammary Gland

A. HISTOGENESIS

References to the earlier work on the histogenesis of the mammary gland in various species will be found in Turner ( 1939,

^ Within the last 10 years there have been several symposia devoted to the problems of the physiology of lactation. The proceedings of these symposia have been published: Mecanisme physiologie de la secretion lactee. Strasbourg, 1950, Colloqvies Internationaux du Centre National de la Recherche Scientificiue. XXXII, 1951, Paris; Svmposium sur la physiologie de la lactation, Montreal, 1953, Rev. Canad. Biol., 13, No. 4. 1954; .Symposium sur la physiologie de la lactation, Brussels, 1956, Ann. endocrinol. 17, 519; A Discussion on the Physiology and Biochemistry of Lactation. London. 1958, Proc. Roy. Soc, .ser. B, 149, 301.

1952,) and Folley (1952a). There have also been studies on the opossum (Plagge, 1942) , the mouse and certain wild rodents (Raynaud, 1949b), the rhesus monkey (Speert, 1948), and man (Williams and Stewart, 1945; Tholen, 1949; Hughes, 1950).

A question which in the last decade has been receiving attention is whether the prenatal differentiation and development of the mammary primordium is hormonally controlled. According to Balinsky (1950a, b), the mitotic index of the mammary bud in the embryo of the mouse and rabbit is lower than that of the surrounding epidermis and he concludes that differentiation of the bud is due not to cellular proliferation (growth) but to a process of aggregation ("morphogenetic movement") of epidermal cells. This author also reports that for some time after its formation, the mammary bud is cjuiescent as regards growth, thus exhibiting negative allometry compared with the whole embryo, until the sprouting of the primary duct initiates a phase of positive allometry. The cjuestion is, what is the stimulus responsible for the onset of this allometric phase? Is the growth and ramification of the duct primordium, like that of the adult duct system, due to the action of estrogen emanating from the fetal gonad or from the mother?

Hardy (1950) has shown that dift'erentiation and growth of the mammary bud of the mouse could proceed in explants from the ventral body wall of the embryo, cultured in vitro, even when no primordia were present at the time of explantation (10-day embryo). Primary and then secondary mammary ducts and a streak canal differentiated and a developmental stage similar to that in the 7-day-old mouse could be reached. Balinsky (1950b) was also able to observe the formation and growth of mammary buds in approximately their normal locations in a minority of cases in which body-wall explants of 10-day mouse embryos were cultivated in vitro. Discounting the rather remote possibility that the effects were due to minute amounts of sex hormones present in the culture media, these observations indicate that hormonal influences are not necessary for the prenatal stages of mammary develo]iment, and in accord with

592

PHYSIOLOGY OF GONADS

this Balinsky ( 1950b j found that addition of estrogens or mouse pituitary extract to the culture medium had no effect on the growth of the mammary rudiment in vitro.

On the other hand, extensive studies by Raynaud (1947c, 1949b) of the sex difference in the histogenesis of the mammary gland in the mouse, first described by Turner and Gomez (1933), indicate that the mammary rudiment is sensitive to the influence of exogenous gonadal steroids during the prenatal stages. The mammary bud in the strain of mouse studied by Raynaud shows no sex differences in development until the 15th to 16th day at which time the genital tract, hitherto indifferent, begins to differentiate. Coincident with this the mammary bud in the male becomes surrounded by a condensation of special mesenchymal cells the action of which constricts the bud at its junction with the epidermis from which it ultimately becomes completely detached (Fig. 10.1). The inguinal glands seem particularly susceptible to this influence because they exhibit this effect earlier than the thoracic glands and in some strains the second inguinal bud in the male tends to disappear completely. Sex differences in the prenatal development of the mammary rudiment in certain species of wild mouse were also described by Raynaud (1949b).

The fact that, after x-ray desti'uction of the gonad in the 13-day male mouse embryo, the mammary bud remains attached to the epidermis and the duct primordia ramify in a manner similar to the primordia in the female shows that this phenomenon of detachment of the mammary bud is due to the action of the fetal testis (Raynaud and Frilley, 1947, 1949). That the masculinizing action of the fetal testis seems to be due to the hormonal secretion of a substance having the same effect as testosterone is suggested by the fact that injection of testosterone into the pregnant mother causes the mammary buds in the female embryo to undergo the male type of development (Fig. 10.1). Here again the inguinal glands seem most sensitive because sufficiently high doses in many cases cause complete disappearance of the primordia of the second inguinal glands (Raynaud, 1947a. 1949a).

On the other hand, destruction of the fetal gonad in the female has no effect on the development of the mammary bud (Raynaud and Frilley, 1947, 1949), yet the lattW is not completely indifferent to the action of estrogen because high doses of estrogen administered to the mother, or lower doses injected early into the embryo itself inhibit the growth of the mammary bud (Raynaud. 1947b, 1952; Raynaud and Raynaud, 1956, 1957), an effect reminiscent of the well known action of excessive doses of estrogen on the adult mammary duct system (for reference see Folley, 1952a) . In pouch young of the opossum, on the other hand, Plagge (1942) found that estrogen treatment stimulated growth of the mammary duct primordia. Similarly in the fetal male mouse low doses of estrogen stimulate growth of the mammary bud (Raynaud, 1947d), but this may be an indirect effect ascribable to estrogen's antagonizing the inhibitory action of the fetal testis.

The problem of the histogenesis of the teat has also come under experimental attack. Raynaud and Frilley (1949) showed that the formation of the epithelial hood," the circular invagination of the epidermis surrounding the mammary bud which constitutes the teat anlage in the mouse, is not hormonally determined since its appearance was not prevented by the irradiation of the fetal ovary at the 13th day of life. In the male mouse the epithelial hood does not normally appear and the male is born without teats. This is undoubtedly due to the action of the fetal testis inasmuch as the teat anlagen develop in the male embryos whose testes are irradiated at 13 days (Raynaud and Frilley, 1949).

The foregoing observations jioint to an ahormonal type of development for the teat and mammary bud in the female fetus, at least in the mouse, although the mammary bud is specifically susceptible to the action of excess exogenous estrogen which can inliibit its development without affecting that of other skin gland ])rimordia. The mammary hud is a'so sus('ei)tible to the action of anch'ogen which in the normal male fetus not only dii-ects its development along charact(M-istic lines, but also suppresses the formation of the teat.

MAMMARY GLAND AND LACTATION

593

PwokcTiL del

Fig. 101. Sex difference in the development of the mammaiy bud of the fetal mouse and effect of androgen on the histogenesis of the female mammary bud. A. First inguinal gland of female fetus (15 days, 17 hours). B. First inguinal gland of male fetus (15 days, 17 hours). C. Second inguinal gland of female fetus (15 days, 16 hours) from a mother receiving testosterone propionate. D. First inguinal gland of female fetus from the same litter as that in C. (From A. Ravnaud, Ann. endocrinol., 8, 248-253, 1947.)

For further information on the morphogenesis of the mammary ghmd, the reader is referred to the recent detailed accounts by Dabelow (1957) and Raynaud (1960).

B. NORM.\L POSTNATAL DEVELOPMENT

1. Methods of Assessing Mammary Development

In the last two decades the increasing availability of the ovarian hormones in pure form and the prospect of the large scale practical application of fundamental knowledge of the hormonal control of the mammary gland to the artificial stimulation of udder growth and lactation in the cow, have together effected a demand for greater accuracy in studying and assessing the degree of mammary development. Various quantitative and objective procedures have now been evolved which allow results of developmental studies to be subjected to statistical investigation. These methods have been re

viewed recently (Folley, 1956) and we need but mention them briefly.

In those species in which, save in late pregnancy, the mammae are more or less flat sheets of tissue, the classical wholemount preparations have been the basis for several quantitative studies. From such preparations the area covered by the duct systems can be measured by suitable means (e.g., as in our studies on the rat mammary gland; Cowie and Folley, 1947d), thus providing an accurate measure of duct extension. Such measurements, however, give no information on the morphologic changes within this area and so a semiquantitative scoring system to assess the degree of duct complexity has been used in conjunction with the measurements of area (see Cowie and Folley, 1947d) . More reliable and objective techniciues for measuring duct complexity were later developed in our laboratory by Silver (1953a) and Flux (1954a). Species such as the guinea pig in which the gland,

594

PHYSIOLOGY OF GONADS

even when immature, is three-dimensional demand other methods. For such cases a precise but rather tedious method has been described by Benson, Cowie, Cox and Goldzveig (1957) which involves the determination of the volume of glandular tissue from area measurements of serial sections of the gland in conjunction with semiquantitative scoring procedures for assessing the morphologic characteristics of the tissue.

Particularly applicable to the lactating gland is the procedure developed by Richardson (see Cowie, Folley, Malpress and Richardson, 1952; Richardson, 1953) for assessing the total internal surface area of the mammary alveoli. It is of interest to note in passing that this technique is based on that developed by Short (1950) for measuring the surface area of the alveoli in the lung, the similarity in the geometry of the two organs allowing ready transference of the method from one to the other.

At present these quantitative procedures have the disadvantage of being slow and time consuming, and it seems likely that their further development will involve the use of electronic scanning methods to speed up the examination of the tissues. Of recent introduction are some biochemical procedures for assessing changes in mammary development. The desoxyribonucleic acid (DNA) content of any particular type of cell is said to be remarkably constant (see Vendrely, 1955, for review) and the amount of DNA in a tissue has been used as a reference standard directly related to the number of cells present in a tissue and to provide an estimate of the number of cells formed during the developmental phases of a gland or tissue (see Leslie, 1955, for review). Studies on DNA changes which occur in the mammary gland during pregnancy and lactation have been made in the rat by Kirkham and Turner (1953), Grecnbaum and Slater (1957a), Griffith and Turner (1957), and Shimizu (1957). It should be noted, however, that some authorities have doubts as to the constancy under all conditions of the DNA content of a cell (see Brachet, 1957) and results obtained by this technique should be interpreted with some caution (see also Griffith and Turner, 1957). Other chemical methods for assessing mammary development include (a) the determination

of the iron content of the gland, based on the observation that iron retention occurs in the epithelium of the mammary glands of mice (Rawlinson and Pierce, 1950) ; (b) whole-mount autoradiographs using P^(Lundahl, Meites and Wolterink, 1950) ; and (c) determination of the total content of alkaline phosphatase in the mammary gland (Huggins and Mainzer, 1957, 1958).

In view of the relative rapidity of the biochemical methods it seems likely that they will be used increasingly in the future.

A technique of clinical interest allowing the qualitative assessment of changes in mammary structure in the breast of pregnant and lactating women is the radiographic method described by Ingleby, Moore and Gershon-Cohen (1957).

To those seeking information of the microscopic anatomy of the human mammary gland we would recommend the excellent and beautifully illustrated review by Dabelow (1957), and new facts on the cytologic changes occurring during milk secretion will be found in the electron microscopic study of the rat mammary gland by Bargmann and Knoop (1959), and of the mouse mammary gland by Hollmann (1959).

Having briefly outlined the various quantitative methods of assessing mammary development we will now consider recent studies on normal mammary growth.

2. Mammary Development in the X on pregnant Female

It has been the general belief that until puberty the mammary ducts show little growth, but more precise studies in which the rate of increase in mammary gland area has been related to the increase in body size have now shown that in the monkey, rat, and mouse a phase of ra])id duct growth is initiated before puberty.

The first use of this procrdure, relative gi'owth analysis (for terminology see Huxley and Teissier, 1936), for the quantitative investigation of mammary duct growth was made by Folley, Guthkelch and Zuckerman (1939), who showed that over a wide range of body weights, the breast in the nonpregnant female rhesus monkey grows faster than the body as a whole. Subsequently, more detailed studies of the dynamics of mammary growth using relative growth

MAMMARY GLAXD AND LACTATION

595

olO.

rCMALC RATS

ACt$ : i - lOO DAYS

C 22 NO DAY

LOC„ CBOOY WtlCHT C>

Fig. 10.2. Relative mammary gland growth in the female hooded Norway Cowie. J. Endocrinol.. 6, 145-157, 1949.)

(From A.T.

analysis were made in the rat by Cowie (1949) and Silver (1953a, b) and in the mouse by Flux (1954a, b), and their results will now be summarized. In the rat the total mammary area increased isometrically with the body surface (a = 1.1 as compared with the theoretic value of 1.0) until the 21st to 23rd day when a phase of allometry (a = 3.0) set in. The onset of the allometric phase could be prevented by ovariectomy on the 22nd day (see Fig. 10.2). Since estrous cycles do not begin until the 35th to 42nd day in this strain of rat, it is clear that the rapid extension of the mammary ducts began well before puberty. In the immature male rat the increase of mammary area on body surface was slightly but significantly allometric; this was not altered by castration at the 22nd day. Earlier ovariectomy, i.e., when the pups were 10 days old, was followed by a phase of slightly allometric growth of the mammary glands in the fe

males (a = 1.5). With regard to the female mouse (CHI strain) a i)hase of marked allometry in mammary duct growth set in about the 24th day (a = 5.2) which could also be prevented by prior ovariectomy.

It is clear that the presence of the ovary is essential for the change from isometry to allometry, but the nature of the mechanisms governing the change is still uncertain (for further discussion, see Folley, 1956).

3. Mammary Growth in the Male

The testes have apparently little effect on mammary duct extension in the rat inasmuch as the gland in the male grows isometrically or nearly so and its specific growth rate is unaffected by castration. Castration at 21 days, however, does prevent for a time development of the lobules of alveoli, first described by Turner and Schultze (1931 ) , which are characteristic of the mammary gland in the male rat. Eventually.

596

PHY,SI(3L0GY OF GONADS

however, some alveoli do develop in the mammae of immaturely castrated male rats (Cowie and Folley, 1947d; Cowie, 1949; Ahren and Etienne, 1957) and it has been ])Ostulated that these arise from the enhanced production by the adrenal cortex of mammogenic steroids (androgens or progesterone) due to the hormone imbalance brought about by gonadectomy (see Folley, 1956 L

In a recent study, Ahren and Etienne (1957) have shown that the ducts and alveoli in the mammary gland of the male rat are remarkable in that their epithelial lining is unusually thick, being composed of several layers of cells. It had been previously noted by van Wagenen and Folley (1939) and Folley, Guthkelch and Zuckerman (1939) that testosterone caused a thickening of the mammary duct epithelium in the monkey and sometimes papillomatous outgrowths of epithelium into the lumen of the duct. It would thus seem that, although the hormone of the testis is capable of eliciting alveolar development, these alveoli and ducts differ from those occurring in the female in the nature of their epithelium. It w^as further observed by Ahren and Etienne (1957) that in the castrated male rat the alveoli, which eventually developed, had a simple epithelial lining somewhat similar to that seen in the normal female rat, suggesting that, if the adrenals are responsible, the mammogenic steroid is more likely to be progesterone than an androgen.

A study of considerable clinical interest is that of Pfaltz (1949) on the developmental changes in the mammary gland in the human male. The greatest development reached was at the 20th year; by the 40th year there occurred an atrophy first of the l)arenchyma and later of the connective tissue. In the second half of the fifth decade there was renewed growth of the parenchyma and connective tissues. The hormonal background of these changes and the possible relationship with prostatic hyjiertrophy are discussed by Pfaltz. (Further details of the microscopic anatomy of the mammary gland of the human male may be found in the studies by Graumann, 1952, 1953, and Dabclow, 1957.)

4- Mammary Development during Pregnancy

It has been customary to divide mammary changes during pregnancy into two phases, a phase of growth and a secretory phase. In the former there occurs hyperplasia of the mammary parenchyma whereas, in the latter, the continued increase in gland size is due to cell hypertrophy and the distension of the alveoli with secretion (see Folley, 1952a j . Although it was realized that these two phases merged gradually, recent studies have confirmed earh^ reports {e.g., those of Cole, 1933; Jeffers, 1935) that a wave of cell division occurs in the mammary gland towards the end of parturition or at the beginning of lactation. Al'tman (1945) described a doubling in number of cells per alveolus, in the mammary gland of the cow at parturition, but the statistical significance of his findings is difficult to assess. More recently, how^ever, Greenbaum and Slater (1957a) found that the DNA content of the rat mammary gland doubled between the end of pregnancy and the 3rd day of lactation, a finding which they interpret as resulting in the main from hyperplasia of the gland cells. Likewise in the mouse mammary gland, Lewin (1957) observed between parturition and the 4th day of lactation a great increase both in the DNA content of the mammary gland and in the total cell count. Studies on the factors controlling this wave of cell division are awaited with interest. Also associated with the onset of copious milk secretion is a considerable increase in cell volume and coincident ally the mitochondria elongate and may increase in diameter (Howe, Richardson and Birbeck, 1956). Cross, Goodwin and Silver (1958) have followed the histologic changes in the mammary glands of the sow, by means of a biopsy technique, at the end of pregnancy, during parturition, and at weaning. At the end of pregnancy there was a ])i'()gr('ssi\-c' distension of the alveoli, the existing hyaline eosinoi)hilic secretion within the alveoli was gradually replaced by a basophilic material, and fat globules appeared. At i)arturition the alveoli were contracted and their walls appeared folded (Fig. 10.3).

MAMMARY GLAND AND LACTATION

597

Fig. 10.3. Sections of biopsy specimens from the mammary gland of a sow before and din-ing parturition. A. Six days before parturition: the mammary alveoh are small and contain a nongranular eosinophilic secretion. B. Two days before parturition: alveoli have increased in size and fat globules are conspicuous. C. Fifteen hours before parturition: alveoli are now distended with secretion which consists of an outer zone of eosinophilic material and fat globules, and a central zone of basophilic granular secretion. D. During parturition: alveoli contracted with folded epithelium and sparse secretion. (From B. A. Cross, R. F. W. Goodwm and L A. Silver, J. Endocrinol., 17, 63-74, 1958.)

598

PHYSIOLOGY OF GONADS

5. Mam /nary Involution

The involutionary changes which occur in the mammary gland after weaning in various species were described in the previous edition of this book (Turner, 1939) and in a later review by Folley (1952a). Since that time, a few further studies have appeared.

There is evidence that the course of the histologic changes in the regressing mammary gland may differ according to whether the young are weaned after lactation has reached its peak and is declining, or whether they are removed soon after parturition, when the effects of engorgement with milk seem to be more marked (see, for example, Williams, 1942, for the mouse). In rats whose young were weaned soon after parturition Silver (1956) was able to re-establish lactation provided suckling was resumed within 4 or 5 days; after that time irreversible changes in the capillary blood supply to the alveoli had set in. A further point arises from a study on the cow by Mosimann (1949) which indicates that the course of the regressive changes in a gland which has undergone one lactation only may differ from those seen in glands from muciparous animals. Oshima and Goto (1955) have used quantitative histometric methods in a study of the involuting rat mammary gland ; the values which they obtained for the percentage parenchyma 7 to 10 days after removal of the young agree quite well with tiiose reported by Benson and Folley ( 1957b) for rats weaned at the 4th day and killed 9 days later.

The biochemical changes occurring in mammary tissue during involution arc of some interest and have been studied in our laboratory by McNaught (1956, 1957). She studied mammary slices taken from rats whose young were removed at the 10th day and also slices from suckled glands, the escajie of milk from which was prevented by ligation of the galactophores, the other glands in the same animals remaining intact and serving as controls. Her results, some of whichare summarized in Figure 10.4, suggest that functional changes which may be taken as indicative of involution (decrease in oxygen up-take, respiratory quotient (R.Q.), and glucose up-take; increase in lactic acid prcxUiction ) are seen as early as

8 to 12 hours after weaning. Continued suckling without removal of milk retards the onset of these changes, but only for some hours. Injections of oxytocin into the rats after weaning (see page 607) did not retard these biochemical changes. Essentially simihii' results were independently reported by Ota and Yokoyama (1958) and Mizuno and Chikamune (i958).

C. EXPERIMENTAL ANALYSIS OF HORMONAL INFLUENCES

1. Ovarian Hortnones in the Animal with Intact Pituitary

We shall see later (page 602) that the mammogenic effects of the ovarian hormones are largely dependent on the integrity of the a'nterior pituitary and thus to analyze accurately the role of hormones in mammary development it is necessary to use hypophysectomized animals. Information of considerable academic and practical importance has been obtained, however, from studies in the animal with intact pituitary and these we shall now consider.

Early studies involving hormone administration pointed to the conclusion that estrogens were in general resi)onsible for the growth of the mammary (hicts, whereas progesterone was necessary for complete lobulealveolar growth (see reviews, l)y Turner, 1939; Folley and Malpress, 1948a; Folley, 1952a). The foundation for i^liis general statement is now more sure, for as a result of experimental studies over the last 10 years, what seemed to be exceptions to this generalization have been shown to be otherwise. In some species (mouse, rat, guinea \)ig, and monkey) it is true that progesterone alone, if given in sufficiently large doses, will evoke duct and alveolar development in the ovariectomized animal, but this is probably a pharmacologic rather than a physiologic effect. There are great differences in the response of the mammary ducts to estrogen and on this basis it has become usual to divide species into three broad categories (see FoUey, 1956). It is, however, necessary to add the warning that in the estrogentre.'ited spayed animal progesterone from the a(h'eiial eoiiex may synergize with the exogenous estrogen (see Folley, 1940; Trentin and 1'ui'iier, 1947; Hohn, 1957) and it mav

MAMMARY GLAND AND LACTATION

599

O2 Uptake

G\

ucose

uptake

Lactic acid production.

s 12 ■Hours

Fig. 10.4. Oxygen uptake, respiratory quotient, glucose uptake, and lactic acid production of mammary gland slices from lactating rats killed at various times after weaning (A — A) and from rats in which svickling was maintained, but in which the galactophores of certain

glands were ligatured (• •) to prevent the escape of milk, the nonligatured glands

(O O) acting as controls. (Courtesy of Dr. M. L. McNaught.)

be that the I'eal basis for the categories is to be found largely in differences in endogenous progesterone production by the adrenal cortex.

The first category comprises those in which estrogens, in what are believed to be physiologic doses, evoke primarily and mainly duct growth; alveoli may appear, but only if high doses are given and the administration is prolonged. Examples of this class are the mouse, rat, rabbit, and cat. Silver (1953a), using the relative-growth technique, has obtained information on the

levels of estrogen necessary for normal mammary duct growth in the nonpregnant rat. In the young ovariectomized rat, the normal mammary growth rate was best imitated by injecting 0.1 ;u,g. estradiol dipropionate every second day (from 21 days of age) and increasing the dose step- wise with body weight. In the ovariectomized mouse, Flux (1954a) found it necessary to give 0.055 /jLg. estrone daily to attain mammarv duct growth comparable with that obser\-( . i in intact mice.

In the second category are those s]:»ecies

(JOO

PHYSIOLOGY OI-' GONADS

in which estrogen in physiologic doses causes growth of the ducts and the lobule-alveoL^r system, the classical example being the guinea pig in which functional mammae can be developed after gonadectomy in either sex by estrogen alone. A recent study by Hohn (1957), however, strongly suggests that progesterone from the adrenal cortex participates in the effect. The earlier view, moreover, that complete mammary growth can be evoked in the gonadectomized guinea l)ig by estrogen alone (Turner and Gomez. 1934; Nelson, 1937.) does not find support in the recent study of Benson, Cowie, Cox and Goldzveig (1957), who, using both subjective and objective methods of assessing the degree of mammary development, found that over a wide dose range of estrone, further development of the mammary gland was obtained when jirogesterone was also administered; essentially similar conclusions have been reached by Smith and Richterich (1958).

Also in this second category are cattle and goats in which, however, the male mammary gland is not equipotential with that of the female. The early studies on these species have been reviewed at length by FoUey and Malpress (1948a) and Folley (1952a, 1956). Briefly it may be said that these studies clearly showed that estrogen alone induced extensive growth of lobule-alveolar tissue of which the functional capacity was considerable although the milk yields in general were less than those expected from similar animals after parturition. The response to estrogen treatment was, moreover, very erratic. It was generally believed that the deficiencies of this treatment could be made good if progesterone were also administered, a view supported by the observations of Mixner and Turner (1943) that the mammary gland of goats treated with estrogens, when examined histologically, showed the i)resence of cystic alv(>oli, an abnormality which tended to disappear when jirogestcrone was also administered.

When progesterone became more readily available, an extensive study of the role of estrogen and progesterone in mammary development in the goat was carried out (Cowie, Folley, ^lalpress and Richai'dson.

1952; Benson, Cowie, Cox, Flux and Folley, 1955). The mammary tissue was examined histologically and the procedure devised by Richardson (see page 594) used to estimate the area and "porosity" of the alveolar epithelium. The udders grown in immaturely ovariectomized virgin goats by combined treatment with estrogens and progesterone in various proportions and at different absolute dose levels were compared with udders resulting from treatment with estrogen alone. As in the earlier observations of Mixner and Turner (1943) , histologic abnormalities were noted, the more widespread being a marked deficiency of total epithelial surface, associated with the presence of cystic alveoli, in the udders of the estrogen-treated animals. The addition of progesterone prevented the appearance of many of these abnormalities and increased the surface area of the secretory epithelium. JMoreover, when estrogen and progesterone were given in a suitable ratio and absolute level the milk yields obtained were remarkably uniform as between different animals and the glandular tissue was virtually free from abnormalities.

Studies in the cow have been less extensive, but there is evidence that both estrogen and progesterone are necessary for complete normal mammary development (Sykes and Wrenn, 1950, 1951; Reineke, INIeites, Cairy and Huffman, 1952; Flux and Folley, cited by Folley, 1956; Meites, 1960).

The case for the inclusion of the monkey in the present category has been strengthened by the excellent monograph of Speert ( 1948) who has had access to more extensive material than many of the earlier workers whose results are reviewed by him (see also Folley, 1952a). The sum total of available evidence now justifies the conclusion that estrogen alone will cause virtually complete growth of the duct and lobule-alveolar systems of the monkey breast. Extensive lobulealveolar development in the monkey breast in response to estrogen is shown in Figure 10.5. The synergistic effect of estrogen and jirogesterone on the monkey breast has not yet been adequately studied, but from available evidence it does not seem to be very dramatic. If it is permissible to argue from pi'iinates to man. it seems jiossible that coidd

MAMMARY GLAND AND LACTATION

601

Fig. 10.5. Wliole mounts of breast of an ovariectomized immature female rhesus monkey before (left) and after (right) e.strogen treatment. (From H. Speert, Contr. Embrvol., Carnegie Inst. Washington, 32, 9-65, 1948.)

the necessary experiments be done the human breast would show a considerable growth response to estrogen alone.

Finally, in the third category are those species in which estrogen in physiologic doses causes little or no mammary growth. The bitch and probably the ferret seem to belong to this class (see Folley, 1956).

There has been considerable discussion in the past regarding the ratio of progesterone to estrogen optimal for mammary growth. Only recently, however, has this question been fully investigated in any species. Benson, Cowie, Cox and Goldzveig (1957) have shown that in the guinea pig the absolute quantities of progesterone and estrogen are the crucial factors in controlling mammary growth; altering the dose levels but maintaining the ratio gave entirely different growth responses. In view of the varying ability of the different estrogens to stimulate mammary duct growth (Reece, 1950) it is essential in discussing ratios to take into consideration the nature of the estrogen used, a fact not always recognized in the past.

2. Anterior Pituitary Hormones

Soon after the discovery by Strieker and Grueter (1928, 1929) of the lactogenic effects of anterior iiituitarv extracts, it was

shown that anterior i)ituitary extracts had a mammogenic effect in the ovariectomized animal and that the ovarian steroids had little or no mammogenic effect in hypophysectomized animals. C. W. Turner and his colleagues postulated that mammogenic activity of the anterior pituitary was due to specific factors which they termed "mammogens"; other workers, in particular W. R. Lyons, believed the mammogenic effect was due to prolactin. The theory of specific mammogens has been fully reviewed in the past (Trentin and Turner, 1948; Folley and Malpress, 1948a) and we do not propose to discuss it further for there is now little evidence to support it. Damm and Turner ( 1958) , while recently seeking new evidence for the existence of a specific pituitary mammogen, concur in the view expressed by Folley and Malpress (1948a) that final proof of the existence of a specific mammogen will depend on the development of l)etter assay techniques and the characterization or isolation of the active principle.

The mammogenic effects of prolactin were observed in the rabbit by Lyons (1942) who injected small quantities of prolactin directly into the galactophores of the suitably prepared mammary gland. IV'Iilk secretion occurred but Lyons also noted that the l)rolactin caused active growth of the alveo

602

PHYSIOLOGY OF CIOXADS

lar epithelium. Recently, Mizuno, lida and Naito (1955) and Mizuno and Naito (19561 have confirmed Lyons' observations on the mammogenic effect of intracluct injections of prolactin in the rabbit both by histologic and biochemical means (DNA estimations) and there seems little doubt that the prolactin is capable of exerting a direct effect on the growth of the mammary parenchyma, at least in the rabbit whose pituitary is intact.

In the last 18 years much information on the role of the anterior pituitary in mammary growth has been obtained by Lyons and his colleagues in studies on hypophysectomized, hypophysectomized-ovariectomized, and hypophysectomized-ovariectomized-adrenalectomized (triply operated) rats of the Long-Evans strain. In 1943 Lyons showed that in the hypophysectomized-ovariectomized rat, estrogen + progesterone + prolactin induced lobulealveolar development, but the degree of development was less than that obtained in the ovariectomized rat with intact pituitary receiving estrogen and progesterone. When supplies of purified anterior-pituitary hormones became available the experiments were extended (Lyons, Li and Johnson, 1952) and it was shown that if somatotrophin (STH) was added to the hormone combination of estrogen -f progesterone + prolactin, the degree of lobulealveolar development obtained in the hypophysectomized-ovariectomized rat was much enhanced. The omission of prolactin from the hormonal tetrad prevented lobulealveolar development from occurring. In the hypophysectomized-ovariectomized-adrenalectomized rat the above hormonal tetrad could also evoke lobule-alveolar development, provided the animals were given saline to drink (Lyons, Li, Cole and Johnson, 1953). In yet more recent experiments Lyons, Li and Johnson (1958) observed that somatotrophin has a direct stimulatory effect on duct growth, but in the hypophysectomized-ovariectomized rat, the presence of estrogen is also necessary to evoke normal duct development (Fig. 10.6a, b, c) ; Likewise, in the triply operated rat, STH plus estrogen is mammogenic, but the presence of a corticoid is r('([ui]'ed to o])tain full duct de

velopment (Fig. 10.6r/). Lyons and his colleagues were able to build up the mammary glands of triply operated rats from the state of bare regressed ducts to full prolactational lobule-alveolar development by giving estrogen + STH + corticoids for a period of 10 days to obtain duct proliferation followed by a further treatment (for 10 to 20 days) with estrone + progesterone -I- STH -I- prolactin + corticoid to induce lobulealveolar development. Alilk secretion could then be induced by a third course of treatment lasting about 6 days in which only prolactin and corticoids were given (Fig. 10. 6e, /). Essentially similar results have been obtained in studies with the hooded Norway rat (Cowie and Lyons, 1959).

Studies on mammogenesis in the hypophysectomized mouse have revealed some differences in the response of the mammary gland of this species in comparison with that of the rat and indications of strain differences within the species. The mammary gland of the hypophysectomized male weanling mouse of the Strong A2G strain shows no response to the ovarian steroids alone, to prolactin, or to STH alone, but it responds with vigorous duct proliferation to combinations of estrogen + progesterone + prolactin, or of estrogen 4- progesterone + STH (Hadfield, 1957; Hadfield and Young, 1958). In the hypophysectomized male mouse of the CHI strain slight duct growth occurs in response to estrogen + jirogesterone and this is much enhanced when STH is also given; the further addition of prolactin then results in alveolar development (Flux, 1958). Extensive studies in triply operated mice of the C3H 'HeCrgl strain have been reported by Nandi (1958a, b). In this strain some duct growth was observed in triply operated animals in response to steroids alone (estrogen -I- progesterone + corticoids), but normal duct develojmient was believed to be due to the action of estrogen + STH + corticoids, a conclusion in agreement with Lyons' observations in the rat. Extensive lobuleahcohii' development could be induced by a number of hormone coml)inations, one of the most effective being estrogen + progesterone + corticoids + prolactin + STH, milk secretion occurring when the ovarian

MAMMARY C5LAND AND LACTATION

603

Fig. 10.6. Typical areas of whole mounts of the abdominal mammary gland of rat.s after the following treatments: A. Untreated rat on day 31, 14 days after hypophysectomy. The gland has regressed to a bare duct system. B. Rat hypophysectomized and ovariectomized on day 30 and injected daily with 2 mg. somatotrophin (STH) for 7 days. Note the presence of end clubs, r. Rat treated as in B but which received, in addition to the STH, 1 ^g. estrone. Note profuse eiid-rhil' ] iroliferatiou. D. Rat li.\|M)]ili\s(>ctomized on day 30. ovariectomized and adri'nali^ctoinized on day 60, and injected daily from days 60 to 69 with 1 mg. STH + 0.1 mg. DCA + 1 fig. estrone. Note again the profuse number of end buds indicative of duct proliferation. E. Same treatment as in D followed by 10 days treatment with 5 mg. prolactin + 2 mg. STH + 1 /xg. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate. Note excellent lobule-alveolar growth. F. Same treatment as in D followed by 20 days treatment with 5 mg. prolactin + 2 mg. STH + 1 fig. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate; thereafter given 0.1 mg. prolactin locally over this gland and 0.1 mg. DCA + 0.1 mg. prednisolone acetate systemically for 6 days. Note fully developed lobules with ah'eoli filled with milk. (All glands at the same magnification.) (From W. R. Lyons. C. H. Li and R. E. Johnson, Recent Progr. Hormone Res., 14, 219-254, 1958.)

604

PHYSIOLOGY OF GONADS

steroids were withdrawn, while the })rohictin, STH, and Cortisol were continued. A further interesting observation made by Nandi is that in the C3H/HeCrgl mouse STH can replace prolactin in the stimulation of all phases of mammary development and in the induction of milk secretion; enhanced effects were obtained, however, when prolactin and STH were given together. Nandi also considers that progesterone plays a greater role in duct development in the mouse than in the rat.

The above experiments clearly indicate that both in the triply operated rat and mouse, it is possible to build up the mammary gland to the full prolactational state by injecting the known ovarian, adrenal cortical, and anterior pituitary hormones. There would thus seem to be no necessity to postulate the existence of other unidentified pituitary mammogens. It must be recognized, however, that in normal pregnancy the placenta may be an important source of mammogenic hormones. The placenta of the rat contains a substance or substances possessing luteotrophic, mammogenic, lactogenic, and crop-sac stimulating properties, but it is uncertain whether this material is identical with pituitary prolactin (Averill, Ray and Lyons, 1950; Canivenc, 1952; Canivenc and Mayer, 1953; Ray, Averill, Lyons and Johnson, 1955). There is also some evidence of the presence of a somatotrophin-like principle in rat placenta (Ray, Averill, Lyons and Johnson, 19551.

3. Metabolic Hormones {Corticoids, Insulin, and Thyroid Hormones)

We have already noted that Lyons and his colleagues were able to obtain full duct development in the triply operated rat only when corticoids were given. Early studies of the role of the adrenals in mammary development have given conflicting and uncertain results (see review by Folley, 1952a). Recent studies have not entirely clarified the position. Flux (1954b) tested a number of 11 -oxygenated corticoids, and found that not only were they devoid of mammogenic activity in the ovariectomized virgin mouse, but that they inhibited the gi'owth-promoting effects of estrogen on the mammary ducts, whereas 11-desoxycorticosterone acted synergistically with estro

gen in promoting duct growth. In subsequent studies it was shown that injections of adrenocorticotrophin (ACTH) into intact female mice did not influence mammary growth (Flux and ]\lunford, 1957), but that Cortisol acetate in low doses (12.5 /^g. l)er day) stimulated mammary development in ovariectomized and in ovariectomized estrone-treated mice, whereas at higher levels (25 and 50 ftg. per day) it was without effect (Munford, 1957). In the virgin rat, on the other hand, glucocorticoids are said to stimulate mammary growth and to induce milk secretion (Selye, 1954; Johnson and Meites, 1955). Some light on these conflicting results has been shed by the studies of Ahren and Jacobsohn (1957) who investigated the effects of cortisone on the mammary glands of ovariectomized and of ovariectomized-hypophysectomized rats, both in the presence and absence of exogenous ovarian hormones. In the hypophysectomized animals, cortisone promoted enlargement and proliferation of the epithelial cells lining the duct walls, but normal growth and differentiation did not occur, nor did the addition of estrogen and progesterone appreciably alter these effects ; in rats with intact pituitaries, however, cortisone stimulated secretion but not mammary growth, whereas the addition of estrogen and progesterone promoted both growth and al)undant secretion. Ahren and Jacobsohn concluded that "the effect elicited by cortisone in the mammary gland should be analysed with due regard to the endocrine state of the animal both as to its effects on the structures of the mammary gland and to the consequences resulting from an eventual upset of the general metabolic equilibrium." They consider that in circumstances optimal for mammary gland growth and maintenance of homeostasis the predominant actions of cortisone are enhancement of alveolar growth and stimulation of secretion, whereas under conditions ill which the metabolic actions of cortisone are not efficiently counteracted, gland growth is either inhibited or an abnormal development of certain iiianimaiy cells may be e^■()ked.

That the general metabolic milieu may indeed profoundly influence the response of the iiuuiimarv gland to hormones has

MAMMARY GLAND AND LACTATION

605

been emiiha.-^ized by the recent experiments of Jacobsohn and her colleagues. Following on the work of Salter and Best (1953) who showed that hypophysectomized rats could be made to resume body growth by the injections of long-acting insulin, Jacobsohn and her colleagues (Ahren and Jacobsohn, 1956; Ahren and Etienne, 1958; Ahren, 1959) found that treatment with estrogen and progesterone would stimulate considerable mammary duct growth in hypophysectomized-gonadectomized rats when given with suitable doses of long-acting insulin (Fig. 10.7). This growth-supporting effect of insulin could be nullified if cortisone was also administered (Ahren and Jacobsohn, 1957) but could be enhanced by giving thyroxine (Jacobsohn, 1959).

The thyroid would thus appear to be another endocrine gland whose hormones affect

mammary growth intlirectly by altering the metabolic environment. Studies in this field, reviewed by Folley (1952a, 1956), indicate that in the rat some degree of hypothyroidism enhances alveolar development wdiereas in the mouse, hypothyroidism seems to inhibit mammary development. Chen, Johnson, Lyons, Li and Cole (1955) have shown that mammary growth can be induced in hypophysectomized - adrenalectomized-thyroidectomized rats by giving estrone, progesterone, prolactin, STH, and Cortisol, no replacement of the thyroid hormones being necessary.

These investigations on the effect of the metabolic environment on mammary development seem to ])e opening up new avenues of approach to the advancement of our understanding of the mechanisms of mammary growth and we would recommend.

0-5 cm.

Fig. 10.7. Whole mount preparation of .second thoracic mammary gland of : ^. Ovariectomized rats injected with estrone and progesterone. B. Hypophysectomized-ovariectomized rat injected with estrone and progesterone. C. Hypophysectomizcd-o\ariectomized rat. D. Hypophysectomized-ovariectomized rat injected with estrone, progesterone, and insulin. (From K. Ahren and D. Jacobsohn, Acta physiol. scandinav., 37, 190-203, 1956.)

GOG

PHYSIOLOGY OF GONADS

to those seeking further information about this important new fiekl, the recent review by Jacobsohn (19581.

III. Endocrine Influences in Milk Secretion

A. ANTERIOR PITUITARY HORMONES

1. Initiation of Secretion iLactogenesis)

The early experiments leading to the view that the anterior pituitary was not only necessary for the initiation of milk secretion, but in fact i)rovided a positive lactogenic stimulus, are now well known and the reader is referred to the reviews by Folley (1952a, 1956) and Lyons (1958) for further particulars. That pituitary prolactin can evoke milk secretion in the suitably de\-eloped mammary gland of the rabbit with intact pituitary has been amply confirmed, and the original experiments of Lyons (1942) involving the intraduct injection of prolactin have been successfully repeated by Meites and Turner (1947) and

Fk;. 10.8. Liictation.'il lespon.scs in pseudoincgnant rabbit to different doses of prolactin injeclcd intraductallv. (Fiom T. R. Bradley and P. M. Clarke, J. Endo.ninol., 14, 28-36, 1956.)

Bradley and Clarke (1956) (Fig. 10.8). However, endogenous pituitary hormones may have participated in the response in such experiments and in the last 20 years there has been considerable discussion as to whether prolactin should be regarded as the lactogenic hormone or as a component of a lactogenic complex. This whole question has been fully discussed in recent years (see Folley, 1952a, 1956) and it now seems reasonably certain that lactogenesis is a response to the co-operative action of more than one anterior pituitary hormone, that is, to a lactogenic hormone complex of which prolactin is an important component, as first suggested by Folley and Young (1941 ) . The recent reports by Nandi (1958a, b) that STH -I- Cortisol can induce milk secretion in triply operated mice with suitably developed glands is further strong evidence against regarding prolactin as the lactogenic hormone.

Secretory activity is evident in the mammary gland during the second half of pregnancy, but abundant milk secretion does not set in until parturition or shortly thereafter. The nature of the mechanism controlling the initiation of abundant secretion has been the subject of speculation for many years. The earlier theories w^ere discussed l)y Turner ( 1939 ) in the second edition of this book, and included the theory put forward by Nelson with reference to the guinea pig, that the high levels of blood estrogen in late pregnancy suppressed the secretion or release of prolactin from the pituitary and had also a direct inhibitory cttcct on the mammary parenchyma, the fall in the levels of estrogen occurring at parturition then allowing the anterior pituitary to exert its full lactogenic effect. This concept proved inadequate to exjilain observations in other species and it was later extended by Folley and Malpress (1948b) to embrace the concept of two thresholds for oi:)posing influences of estrogen upon jiituitary lactogenic function, a lower threshold for stimulation and a higher one for inhibition. Subsequent observations on the inhibitory role of progesterone, in the pix'sence of estrogen, on milk secretion, however, necessitated further modification of the theorv. Before discussing these modifica

MAMMARY GLAND AND LACTATION

607

tions it is convenient to refer to the ingenious theory put forward by Meites and Turner (1942a, b; 1948) which was based on their extensive investigation of the prolactin content of the pituitary in various physiologic and experimental states. According to Meites and Turner, estrogen elicits the secretion of prolactin from the anterior pituitary thereby causing lactogenesis, whereas progesterone is an inhibitory agent, operative in pregnancy, inhibiting or over-riding the lactogenic action of estrogen. The induction of lactation was thus ascribed to a fall in the body level of progesterone relative to that of estrogen heheved to occur at the time of parturition. Subsequent studies in the rabbit by jVIeites and Sgouris (1953, 1954) revealed that combinations of estrogen and progesterone could inhibit, at the mammary gland level, the lactogenic effects of exogenous prolactin. This effect was, however, relative and by increasing the prolactin or decreasing the steroids, lactogenesis ensued. Inasmuch as the theory of Meites and Turner did not take into account the eventuality that estrogen and progesterone act at the level of the mammary gland, Meites ( 1954) modified the con('ei)t, postulating that milk secretion was held in check during pregnancy first by the combined effect of estrogen and progesterone which make the mammary gland refractory to prolactin and, secondly, by a low rate of prolactin secretion. The role of progesterone in over-riding the stimulatory effect of estrogen on the pituitary he now considered to be of only minor importance. Meites also explained the continuance of lactation in pregnant animals by postulating that the initial level of prolactin was sufficiently high as a result of the suckling stimulus to overcome the inhibitory action of the ovarian hormones on the mammary gland. One of us (Folley, 1954, 1956) put forward a tentative theory, combining various features of previous hypotheses, which seemed capable of harmonizing most of the known facts regarding the initiation of milk secretion. In this it was emphasized that measurements of the prolactin content of the pituitary were not necessarily indicative of the rate of prolactin release (a recent study bv Grosvenor and Turner (1958c)

lends further support to this contention) and were best considered as largely irrelevant; low circulating levels of estrogen activate the lactogenic function of the anterior pituitary whereas higher levels tend to inhibit lactation even in the absence of the ovary; lactogenic doses of estrogen may be deprived of their lactogenic action by suitable doses of progesterone, the combination then acting as a potent inhibitor of lactation, this being the influence operating in pregnancy; at parturition the relative fall in the progesterone to estrogen ratio removes the inhibition which is replaced by the positive lactogenic effect of estrogen acting unopposed.

It was observed by Gaines in 1915 that although a colostral secretion accumulated in the mammary gland during pregnancy, the initiation of copious secretion was associated with functioning of the contractile mechanisms in the udder responsible for milk ejection; later Petersen (1944) also suggested that the suckling or milking stimulus might be partly responsible for the onset of lactation. Recent studies have provided evidence that this may well be so, and these will be considered later when discussing the role of the suckling and milking stimulus in the maintenance of milk secretion (see page 611).

During the past decade a fair amount of information has been obtained about the biochemical changes which occur in mammary tissue near the time of parturition, and which are almost certainly related to lactogenesis. The earlier work has been reviewed in some detail by one of us (Folley, 1956) and need only be referred to briefly here.

Folley and French (1949), studying rat mammary gland slices incubated in media containing glucose, showed that — QOo increased from a value of about 1.3 in late pregnancy to a value of about 4.4 at day 1 of lactation, and thereafter increased still further. At the same time the R.Q. which was below unity (approximately 0.83) at the end of pregnancy, increased to unity soon after parturition, and by day 8 had reached a value of 1.62 at approximately which level it remained for the rest of the lactation period. In accord with the

G08

PHYSIOLOGY OF GONADS

increased respiratory activity of the tissue about the time of parturition in the rat mammary gland, Moore and Nelson (1952) reported increases in the content of certain respiratory enzymes, succinic oxidase and cytochrome oxidase, in the guinea pig mammary gland at about this time. Greenbaum and Slater (1957b) made similar observations about mammary gland succinic oxidase in the rat. Recent work is beginning to throw light on the metabolic pathways involved in this increase in respiratory activity. Thus McLean (1958a) has adduced evidence indicating an increase in the activity of the pentose phosphate pathway in the rat mammary gland at about the time of parturition. Mammary gland slices taken from rats at various stages of the lactation cycle were incubated in media containing either glucose 1-C^^ or glucose 6-C^-^, and the amount of radioactivity appearing in the respiratory CO2 was determined. The results given in Figure 10.9 show that although the recovery of C^^'Oo from C-6 was relatively unaffected by the initiation of lactation, the C^^Oo originating from C-1 began a striking increase at the time of parturition (see also Glock, McLean and Whitehead, 1956, and Glock and McLean, 1958, from which Figure 10.9 was taken).

pregnancy

in\'oliition

Imc;. 1().<», The relative amounts of C'Oi; formed fioin iiiilucosc 1-C'^ and glucose 6-C" by rat niani maiy gland slices. O O, C'^Oi formed from

glucose 1-C^'. • • . C^'Oi! formed from glucose

6-C". (From G. E. Glock and P. McLean, Proc. Roy. Soc, London, ser. B, 149, 354-362, 1958.)

Despite the well known pitfalls which surround the interpretation of C-1: C-6 quotients in experiments such as these, it seems clear that lactation is associated with an increase in the metabolism of glucose by the pentose phosphate cycle, whereas the proportion going by the Embden-Meyerhof jmthway would appear to be relatively unaffected. These conclusions are supported by the fact that the levels in rat mammary tissue of two enzymes concerned in this pathway of glucose breakdown, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, show very striking increases at the time of parturition (Glock and McLean, 1954; McLean, 1958a). Other enzymes concerned in glucose breakdown whose activities in mammary tissue begin to increase at parturition are hexokinase and phosphoglucose isomerase (]\IcLean, 1958a). In connection with the glucose metabolism of rat mammary tissue it may be noted that addition of insulin to the incubation medium markedly increases the — QOo and R.Q. of rat mammary slices metabolizing glucose or glucose plus acetate (see page 619), and that this tissue only becomes sensitive to insulin just after parturition (Balmain and Folley, 1951). It is interesting to speculate which of the two above-mentioned pathways of glucose breakdown in mammary tissue resjjonds to the action of insulin. According to Abraham, Cady and Chaikoff (1957) addition of insulin in vitro increased the production by lactating rat mammary slices of C^'^Oo from glucose l-C^'*, but not from glucose 6-C^'*, which might indicate that insulin stimulates preferentially the pentose phosphate pathway. Against this, insulin increased the incorporation of both these carbon atoms (and also the 3:4 carbon atoms of glucose) into fatty acids of the slices to about the same extent. McLean (1959) believes that the stimulatory effect of insulin on the pentose jihosphate pathway in the lactating rat mammary gland is secondary to its stimulating effect on lipogenesis. The latter l)rocess generates the oxidized form of tril)hosphopyridine nucleotide (TPN) which is needed for the first two steps of the pentose phosphate cycle.

The inci-casc in the R.Q. of mammary

MAMMARY GLAXD AND LACTATION

009

tissue beginning at parturition observed by Folley and French (1949) was interi:)reted as indicating that this tissue assumes the power of effecting net fatty acid synthesis from ghicose at this time. Much subsequent evidence confirming this idea has been reviewed by Folley (1956). It only rt'mains to add that Ringler, Becker and Nelson (1954), Lauryssens, Peelers and Donck (1956), and Read and Moore (1958) ha^-e shown that the amount of coenzyme A in mammary tissue undergoes an increase at parturition. Moreover, the recent findings of McLean (1958b), who showed that the levels of pyridine nucleotides in the mammary gland of the rat begin to increase at parturition, reaching a high level by the end of lactation, may be significant in this connection. McLean found that although the increase in the tissue levels of diphosl^hopyridine nucleotide was almost entirely due to an increase in the oxidized form (DPN), in the case of TPN it was the reduced form (TPNH) which increased. The latter might well be used for reductive syntheses such as lipogenesis.

The rate of synthesis of milk constituents other than fat must also begin to increase at parturition, and Greenbaum and Greenwood (1954) showed that an increase in the levels of glutamic aspartic transaminase and of glutamic dehydrogenase in rat mammary tissue occurs at this time. The authors believe these enzymes are concerned in the provision of substrates for the synthesis of milk protein. It is significant in connection with milk protein synthesis that the mammary gland ribonucleic acid (RNA) in the rat undergoes a marked rise at parturition (Greenbaum and Slater, 1957a).

The above - mentioned biochemical changes in mammary tissue which occur at al)out the time of parturition are almost certainly closely related to the effect on this tissue of members of the anterior pituitary lactogenic complex, and particularly prolactin. Attempts have been made to elicit the characteristic respiratory changes, described above, in mammary slices in vitro by addition of prolactin and adrenal glucocorticoids to the incubation medium (see Folley, 1956). So far, however, definitive results luive not been obtained and it is doubt

ful whether any biochemical changes in lactating mammary gland slices in vitro have been demonstrated which could with certainty be ascribed to the action of prolactin (in this connection see also Bradley and Mitchell. 1957).

2. Maintenance of Milk Secretion — Galactopoiesis

It is well known that the removal of the pituitary of a lactating animal will end milk secretion (for references see Folley, 1952a). The cessation of milk secretion has been generally ascribed to the loss of the anterior lobe, but when the importance of the neurohypophysis in milk ejection became established (see page 621), it was clear that in the hypophysectomized animal it was necessary to distinguish between a failure in milk secretion and a failure in milk ejection, since either would lead to failure of lactation. It has now been shown in the rat that adequate oxytocin therapy ensuring the occurrence of milk ejection after hypophysectomy will not restore lactation (Cowie, 1957) and it may thus be concluded that the integrity of the anterior lobe is essential for the maintenance of milk secretion. The effect of hypophysectomy on milk secretion is dramatic, because in the rat, milk secretion virtually ceases within a day of the operation and biochemical changes in the metabolic activity of the mammary tissue can be detected within 4 to 8 hours (Bradley and Cowie, 1956). It is of interest to note that these metabolic changes are similar to those observed during mammary involution (see page 598).

Since the second edition of this book, there have been surprisingly few studies on replacement therapy in hypophysectomized lactating animals. In such studies we would stress the need for rigorous methods of assessing the efficacy of treatment. In the past the presence of milk in the gland as revealed by macroscopic or microscopic examination has been regarded as an indication of successful replacement. This, however, gives no measure of the degree of maintenance of lactation and some measure of the daily milk yield of such animals should be obtained (see also Cowie, 1957).

GIO

PHYSIOLOGY OF GONADS

It is abo now obvious that oxytocin may have to be injected to ensure milk ejection; under certain circumstances, however, the neurohypophyseal tissue remaining after the removal of the posterior lol^e may be capable of releasing oxytocin and permitting milk ejection (see Benson and Cowie, 1956; Bintarningsih, Lyons, Johnson and Li. 1957, 1958).

The earliest report on the maintenance of lactation after hypophysectomy is that of Gomez (1939, 1940), who found that hypophysectomized lactating rats could rear their litters if given anterior-pituitary extract, adrenal cortical extracts, glucose, and posterior pituitary extract. These experiments are difficult to assess because they are reported only in abstract, but the use of posterior pituitary extract at a time when the role of oxytocin in milk ejection was not generally recognized is worthy of note. Recently, slight maintenance of milk secretion in hypophysectomized rats has been obtained with prolactin alone, and greater maintenance when adrenocorticotrophic hormone ( ACTH I or STH was administered with prolactin (Cowie, 1957). Similar studies were reported by Bintarningsih, Lyons, Johnson and Li (1957, 1958) (see also Lvons, Li and Johnson, 1958) in which

I « 

c

-^ 4

-0

I 1

£

Z -6

z

J

E

^ 2 ^^,

TV

2 ^

Fig. 10.10. Effect on the luilk yield of the cow of injected hormones of the anterior pituitary. (From the results of P. M. Cotes, J. A. Crichton, S. J. Folley and F. G. Young, Nature, London. 164, 992-993, 1919.)

considerable maintenance of milk secretion was obtained in hypophysectomized rats with prolactin and certain corticoids. Of related interest is the observation by Elias (1957) that Cortisol and prolactin can induce secretory activity in explants of mouse mammary gland growing on a synthetic medium. (Tissue culture techniques have been little exploited in mammary studies and further developments in this field may be expected.)

The evidence to date suggests that, in the rat, prolactin is an essential component of the hormone complex involved in the maintenance of lactation with ACTH and STH also participating, but further studies are recjuired to determine the most favorable balance of these factors.

Preliminary studies on the maintenance of lactation in the goat after hypophysectomy suggest that both prolactin and STH are important in the initiation and maintenance of milk secretioii (Cowie and Tindal, 1960). Our knowledge of the process in other species is derived from studies on the effect of exogenous anterior pituitary hormones on established lactation in intact animals— galactopoietic effects (for reference see Folley, 1952a, 1956). In the cow, considerable increase in milk yield can be obtained by injecting STH (Cotes, Crichton, Folley and Young, 1949), whereas prolactin has a negligible galactopoietic effect (Fig. 10.10; for discussion see also Folley, 1955). Recently the precise relationship between the dose of STH (ox) and the lactational response in the cow was established in our laboratory by Hutton (1957) who observed a highly significant linear relationship between log doses of STH (single injection) and the increase in milk yield obtained (Fig. 10.11 ) ; increases in fat yield relative to the yield of nonfatty solids also occurred. In the lactating rat, on the other hand, STH has no galactopoietic effect (Meites, 1957b; Cowie, Cox and Naito, 1957), whereas prolactin has (Johnson and Meites, 1958). Such studies must be interpreted with caution as endogenous pituitary hormones were present ; nevertheless, it seems reasonable to conclude that STH is likely to be an impoi'tant factor in the maintenance of lactation in the row.

MAMMARY GLAND AND LACTATION

611

mq qro\Om hormone (onthmeTTc scale)

fa-25 12-5 25-0 50-0

100-0

200-0

S-«^0

'Zoo-o

Fig. 10. IL Effect of graded doses of growth hormone on milk yield of row. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 16, 115-125, 1957.)

C)ther hormones of the anterior pituitary in all probability influence milk secretion through their target glands and these will be dealt with later.

3. Suckling Stimulus and the Maintenance of Lactation

It has been long believed that regular milking is an important factor in maintaining lactation and that if milk is allowed to accumulate in the gland, as occurs at weaning, atrophy of the alveolar epithelium and glandular involution occur. Evidence in support of this concept was obtained in studies showing that ligature or occlusion of

the main ducts of some of the mammae of a lactating animal resulted in atrophy of the glands concerned although the other glands were suckled normally (Kuramitsu and Loeb, 1921; Hammond and Marshall, 1925; Fauvet, 1941a). Studies by Selye and his colleagues, however, revealed that such occluded glands did not atrophy as quickly as did glands of animals in which the suckling stimulus was no longer maintained (Selye, 1934; Selye, Collip and Thomson, 1934) and it was postulated that the suckling stimulus evoked from the anterior pituitary the secretion of prolactin which maintained the secretory activity of the gland. This theory has been widely accepted

012

PHYSIOLOGY OF GONADS

although it has been suggested that a complex of hormones rather than prolactin alone is released (Folley, 1947). Williams (1945) showed that prolactin could in fact maintain the integrity of the mammary gland in the unsuckled mouse thus mimicking the effects of the suckhng stimulus; other supporting evidence has been reviewed by Folley (1952a). Recent studies in goats, however, have shown that milk secretion may continue more or less at the normal level after complete denervation of the udder (Tverskoi, 1958; Denamur and Martinet, 1959a, b, 1960) and it may be that in some species the suckling or milking stimulus is loss important in the maintenance of milk secretion.

Milk secretion is essentially a continuous process whereas the suckling or milking stimulus is intermittent ; indeed the milking stimulus may be of remarkably brief duration (in the cow about 10 minutes in all per 24 hours) and it is therefore likely that the stimulus triggers off the release of sufficient galactopoietic complex to maintain mammary function for some hours. Grosvenor and Turner (1957b) reported that suckling causes a rapid drop in the prolactin content of the pituitary in the rat, and that the prenursing level of prolactin in the pituitary is not fully regained some 9 hours later. It is difficult, however, to relate pituitary levels of prolactin to the rate of its secretion into the circulation and, although these observations are interesting, further advances are unlikely until a method of assay for blood prolactin becomes available and the "half-life" of prolactin in circulation is known.

The experiments of Gregoire (1947) on the maintenance of involution of the thymus during nursing suggests that the suckling stimulus releases ACTH which, as we have seen, is galactopoietic in the rat; thus, so far as the rat is concerned, there would appear to be good evidence that the suckling stimulus releases at least two known important components of the galactopoietic complex.

The milking and suckling stimulus is also responsible for eliciting the milk-ejection reflex and the relation between the two reflexes will be discussed later in this chapter (sec ])age 619 1.

B. HORMONES OF THE ADRENAL CORTEX

Adrenalectomy results in a marked inhibition of milk secretion and the early experiments in this field were reviewed by Turner in 1939. Since then, however, purified adrenal steroids have become available enabling further analysis to be made of the role of the adrenal cortex in lactation.

Gaunt, Eversole and Kendall (1942) considered that in the rat the defect in milk secretion after adrenalectomy could be repaired by the administration of the adrenal steroids most closely concerned with carbohydrate metabolism, whereas we came to the somewhat opposing view that the defect was best remedied by those hormones primarily concerned with electrolyte metabolism (Folley and Cowie, 1944; Cowie and Folley, 1947b, c). The reasons for these differing observations are not yet entirely clear. Virtually complete restoration of milk secretion was subsequently obtained in our strain of rat by the combined administration of desoxycorticosterone acetate (DCA) and cortisone, or with the halogenated steroids, 9a-chlorocortisol and 9afluorocortisol (Cowie, 1952; Cowie and Tindal, 1955; Cowie and Tindal, unpublished; see also Table 10.1). It would therefore seem that both glucocorticoid and mineralocorticoid activity was necessary to maintain the intensity of milk secretion at its normal level. The interesting observation was made by Flux (1955» and later confirmed by Cowie and Tindal (unpublished) that the ovaries contribute to the maintenance of lactation after adrenalectomy, a contribution which could be simulated in the adrenalectomized-ovariectomized rat by the administration of 3 mg. progesterone daily. The differences in the size of the ovarian contribution may partly accoimt for the apparent differences in various strains of rat of the relative importance of mineralo- and glucocorticoids in sustaining milk secretion after adrenalectomy. The only other species in which the maintenance of lactation after adrenalectomy has been studied is the goat in which, as in the rat, lactation can be maintained with cortisone and desoxycorticosterone, the latter being apparently the more critical steroid (Cowie and Tindal. 1958; Figs. 10.12a, b).

MAMM.\RY GLAND AND LACTATION

613

There have been several studies on the effects of corticoids and adrenocortieotrophin on lactation in the intact animal. ACTH and the corticoids depress lactation in the intact cow (Fig. 10.10) (Cotes, Crichton, Folley and Young, 1949; Flux, Folley and Rowland, 1954; Shaw, Chung and Bunding, 1955; Shaw, 1955), whereas in the rat ACTH and cortisone have been reported as exhibiting galactopoietic effects (Meites, private communication; Johnson and Meites, 1958). With larger doses of cortisone, however, an inhibition of milk secretion in the rat has been reported (MercierParot, 1955).

The main function of the cortical steroids in lactation is still uncertain. They may act in a "supporting" or "permissive" manner (see Ingle, 1954), maintaining the alveolar cells in a state responsive to the galacto])oictic complex, or they may act by maintaining the necessary levels of milk precursors in the blood.

Biochemical studies are, however, Ix'ginning to add to our information on the role of the corticoids in lactation. In the rat, adrenalectomy prevents the increase in liver and mammary gland arginase which occurs during normal lactation and it has been suggested that this depression of arginase activity interferes with deamination of amino acids, and thereby inhibits any increase in gluconeogenesis from protein and thus starves the mammary gland of nonnitrogenous milk precursors (Folley and Greenbaum, 1947, 1948). As there is little arginase in the mammary gland of other species {e.g., rabbit, cow, goat, sheep), this mechanism may not have general validity (for further discussion see Folley, 1956). Other biochemical studies have suggested that the steroids of the adrenal cortex may be concerned in mammary lipogenesis, but the results so far have been conflicting and no firm conclusions can as yet be drawn (see Folley, 1956).

C. OVARIAN HORMONES

There is no evidence that ovariectomy has any deleterious effect on lactation (Kuramitsu and Loeb, 1921; de Jongh, 1932; Folley and Kon, 1938; Flux, 1955); neither is there evidence for the belief, once

TABLE 10.1

Replacement therapy in lactating rats

adrenalectomized on the fourth

day of lactation

(From A. T. Cowie and S. J. Folley,

J. Endocrinol., 5, 9-13, 1947.)

Treatment

Number of Litters

Number

of Pups

per

Litter

Litter-growth

Index* gm. + S.E.

Control

Adrenalectomy

Adrenalectomy + cortisone + DC A (tablet implantsf)

8

9

7

8 8 8

15.6 + 0.5

7.5 ± 0.6 14.9 ± 0.6

(Above results from Cowie, 1952)

Control

6

8

14.5 ± 0.8

Adrenalectomy

6

8

6.2 ± 0.4

Adrenalectomy + chloro

5

8

13.1 ± 0.5

cortisol (100 Mg per

day)

(Above results from Cowie and Tindal, 1955)

Control

8

12

17.7 ± 0.8

Adrenalectomy

8

12

7.5 ± 0.5

Adrenalectomy + ovari

5

12

3.6 ± 0.5

ectomy

Adrenalectomy + ovari

7

12

14.5 ± 0.7

ectomy + fiuorocorti

sol (200 Mg per day)

(Above results from Cowie and Tindal, unpublished)

• The litter-growth index is defined as the mean

daily gain in weight per litter over the 5-day period from the 6th to the 11th days.

t 2 X 11 mg. tablets cortisone giving mean daily absorption of 850 ^ig., and 1 X 50 mg. tablet DCA giving mean daily absorption of 360 ng.

widely held, that ovariectomy increases and prolongs lactation in the nonpregnant cow (see Richter, 1936).

Although the integrity of the ovary is not essential for the maintenance of lactation, there can be no doubt that ovarian hormones, in certain circumstances, profoundly influence milk secretion. Estrogens have long been regarded as possessing the power to inhibit lactation, a concept on which Nelson based his theory of the mechanism of lactation initiation (see page 606 1 . Some workers, however, have expressed doubts that the effect is primarily on milk secretion, and have suggested that in ex

614

PHYSIOLOGY OF GONADS

periments on laboratory animals the apparent failure in milk secretion could be a secondary effect due to either a toxic action of the estrogen causing an anorexia in the mother, interference with milk ejection, or disturbance of maternal behavior or to toxic effects on the young, whose growth rate serves as a measure of lactational performance, through estrogens being excreted in milk. The evidence to date shows that in

the intact rat estrogens even in very low doses inhibit milk secretion, their action depending on the presence of the ovary ; the ovarian factor concerned appears to be progesterone, estrogen and progesterone acting locally on the mammary gland and rendering it refractory to the lactogenic complex. In the ovariectomized rat much larger doses of estrogen are necessary to inhibit lactation, and the evidence is not entirely

Body

Goat 478

weight ^^L

.) 45 L

Plasma Na (m-equiv./l.) ^^^^

Plasma K (m-equiv./l

Milk K 40 (m-equiv./l.) 30

Milk Na ,

(m-equiv./l.)

Solids-notfat (%)

Yield of solids-notfat (g) Fat (%)

Milk yield (kg)

Goat died-*

5 15 25 4 14 24 Mgr. Apr.

Fig. 10.12i4. Effect of replaconi(>nt therapy with (losoxycoiticostcM-oiu c-ortisone aoetate (CA) on milk yield, milk composition, and concent

(DCA) and tion of Na and K in milk and blood plasma of the goat after adrenalectomy. Duration of replacement therapy (pellet implantation) indicated by horizontal lines; the names of steroids and their mean daily absorption rates are given adjacent to the lines. Note in Figure 12.4 the considerable maintenance of milk vield with DCA alone. See also Figure 12/?. (From A. T. Cowic and J. S. Tindal. J. Endocrinol., 16, 403-414, 1958.)

MAMMARY GLAND AND LACTATION

6L

Goat 515

Body 5Q _ weight —

(kg) 40 150 Plasma Na ^ ^. / /I \ ^40 —

(m-equiv./l) —

130

Plasma K (m-equiv./l)

Milk K (m-equiv./l.)

Milk Na (m-equiv./l.)

Solids-not- ^ H

fat {%) 7 U

Yield of 200 solids-not- —

fat (g) 100 Fat (- ^

Fat yield

Milk yield (kg)

13 23 2 12 22 2 12 22 Oct. Nov Dec.

Fig. 12B.

11 21 31 10 20

Jan. Feb

conclusive that there is a true inhibition of milk secretion (see Cowie, 1960). In the cow estrogen in sufficient doses depresses milk yield, but its mode of action has not been fully elucidated. In women, estrogens are used clinically to suppress unwanted lactation, but as the suckling stimulus is also removed about the same time, the role of the estrogen is difficult to assess (see Meites and Turner, 1942a).

It has been well established that progesterone by itself has no effect on milk secretion (see Folley, 1952a), save in the adrenalectomized animal (see page 612), and so it would appear that the physiologic inhibition of lactation is effected Ijy estrogen and progesterone acting synergistically as first demonstrated by Fauvet (1941b) and confirmed by others including Masson (1948), Walker and Matthews (1949),

GIG

PHYSIOLOGY OF GONADS

Cowie, FoUey, Malpress and Richarcl.son (1952J,, and Meites and Sgouris (1954). There is clear evidence that the estrogenprogesterone combination acts at least partly on the mammary parenchyma (Desclin, 1952; Meites and Sgouris, 1953) but the mechanism of the action is unknown. The hormonal interplay and complex endocrine interactions in the process of lactation inhibition with estrogen has recently been discussed at length by von Berswordt-Wallrabe (1958).

Lactogenic effects of estrogens have already been mentioned; these have been demonstrated most strikingly in cows and goats, in which milk secretion has been induced in udders being developed by exogenous estrogen. These experiments have been reviewed in some detail by Folley and Malpress (1948b) and Folley (1956).^ It is generally assumed that estrogens act by

stimulating the production of lactogenic and galactopoietic factors by the anterior pituitary. In experiments on the ovariectomized goat we have shown (Cowie, Folley, Malpress and Richardson, 1952; Benson, Cowie, Cox, Flux and Folley, 1955) that it is possible to select a daily dose of estrogen which will induce mammary growth but relatively little secretion in the sense that the udder does not become tense and distended as will happen when a lower dose of estrogen is given — an observation we may quote in support of the "double-threshold" theory of estrogen action. The lactogenic effect of the lower dose of estrogen could be abolished, however, by administering progesterone simultaneously with the estrogen (Fig. 10.13), an observation in accord with those of other workers on the rabbit and rat (see above).

In 1936 one of us (Folley, 1936) reported

Fig. 10.13. Photographs of goat uddois dovelopcd by daily injections of hoxoostiol (HX) with and without progesterone (PG). The hibels indicate the daily dose in mg. of each substance. (Results from A. T. Cowie, S. J. Folley, F. H. Malpre.ss and K. C. Ricliardson, J. Endocrinol., 8, 64-88, 1952.)

MAMMARY GLAND AND LACTATION

GK

that certain dose levels of estrogen in the lactating cow produced long-lasting changes in milk composition characterized by increases in the percentages of fat and nonfatty solids. This was regarded as an example of galactopoiesis and was termed the "enrichment" effect. The effect, however, w^as somewhat erratic and it has recently been re-investigated by Hiitton (1958) who confirmed and extended the earlier observations. Hutton found that galactopoietic responses (Figs. 10.14 and 10.15) were obtained only within a restricted dose range, the limits of which were affected by the stage of pregnancy and the breed of the cow. Hutton further concluded that in the normal cow changes in milk composition and yield associated with advancing pregnancy were probably determined by the progressive rise of blood estrogen levels.

D. THYROID HORMONES

Studies on the effect of removal of the thyroids on milk secretion have been reviewed by one of us (Folley, 1952a) ; the evidence strongly suggests that the thyroid glands are not essential for milk secretion, but in their absence the intensity and duration of lactation is reduced. Histologic and cytologic studies of the thyroid of the lactating cat suggest that there is a considerable outpouring of the thyroid secretion in the early stages of lactation (Racadot, 1957), and Grosvenor and Turner (1958b) have reported that the thyroid secretion rate is higher in lactating than in nonlactating rats.

Since the last edition of this l)ook, a great volume of experimental results has been published on the use of thyroid-active materials for increasing the milk yield of cows. These experiments have been extensively reviewed by Blaxter (1952) and Meites (1960) and we need here only touch on the salient points.

In the early studies i^reparations of dried thyroid gland were fed to cows or injections of DL-thyroxine were given, but the use on a large scale of thyroid-active materials for increasing the milk yield of cows only became feasible when it was shown that certain iodinated proteins exhibited thyroidlike activitv when given in the feed. Al

9-9 97

o 9-3 ^ 9-1

8-9

• Guernsey

Shorthorn

8-5

•^U^ri

I L

20 40 60 80 100

Oestradiol monobenzoite (mg)

Fig. 10.14. Effect of graded doses of estradiol benzoate on percentage of nonfatty solids in milk from cows of three breeds. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.)

Oestradiol monobenzoate (mg) (arith. scale)

10 20 30 40 50

6-25 12-5 250 500

Oestradiol monobenzoate (mg) (log scale)

Fig. 10.15. Effect of graded doses of estradiol benzoate on fat content of cows' milk. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.)

though these materials were readily made and were economical for large-scale use, they possessed several disadvantages. Their activity was difficult to assay and standardize, they were frequently unpalatable, and their administration entailed a considerable intake of iodine which could be undesirable. Nevertheless, a large number of experiments were carried out all over the world with this type of material. In 1949, however, a new and improved method for the synthesis of L-thyroxine was developed (Chalmers, Dickson, Elks and Hems, 1949) and thyroxine became available in large quantities. It was then shown jjy Bailey, Bartlett and Folley (1949) that this material was ealac

618

PHYSIOLOGY OF GONADS

,

/" \^

Cont-rol.

A<' / " "^ - ^*

— • DO m§.

.^\ / V- -' v;.

100 m|.

^..^-Av / V

150mg.

^^^4?^^/ . V

,.-•*.. \ vv / .^-r \ \

• — •• tva --^-^ y \ \ \

•••\-'^\ \ x- ^ .. \ ^

\. *-^ '• •■*— . \ \

• ■*•—., \ \ \ \

.... -.... "•N-:w<r:Viy: y^

Sl-art of hrcAhnc.ih \\ y' i'

hrc iXhuciil' \\ //

\v/y

\ V /

\ /

\ /

\/

V

10

50

50

Dau5

Fig. 10.16. Effect of L-thyroxine given in the feed on the milk yield of groups of cows (the indicated dose levels were fed daily). (From G. L. Bailey, S. Bartlett and S. J. Folley, Nature, London, 163, 800. 1949.)

topoietic when ]ed to lactating cows in daily doses of about 100 mg. (Fig. 10.16). It had, moreover, none of the drawbacks of the iodinated proteins, its purity could be checked chemically, it was odorless and tasteless. AVith the introduction of synthetic thyroxine, iodinated proteins have become obsolete as galactopoietic agents.

The more recently isolated 3:5:3-triiodo-L-thyronine, reported to be 5 to 7 times more active than thyroxine in various biologic tests in small animals and also in man, has little or no effect on the milk yield when fed to cows, but is somewhat more active than thyroxine in promoting galactopoiesis when administered subcutaneously, which suggests that the material is inactivated in the gut, probably in the rumen f Bartlett, Burt, Folley and Rowland, 1954).

The extensive experiments on galactopoiesis in dairy cattle with thyroxine and thyroid-active substances have made it possible to reach reasonably firm conclusions as to the practical value of the procedure. There is great variability in the response to treatment; in general a better response is ol)taincd during the decline of lactation than at the peak and end of lactation. The use of thyroid-active substances

in animals undergoing their first, second, or third lactation is of doubtful benefit because the boost in yield is largely cancelled out by a shortening of the lactation period. Short-term administration at suitable times can result in considerable galactopoiesis, but this is frequently followed by marked falls in yield when the administration of thyroid-active material ends. The administration of thyroid-active materials to dairy cows, if carried out with due care, has no ill effects on the health and reproductive abilities of the cows (see Leech and Bailey, 1953) , but because of the rather small net gain in yield (about 3 per cent) the practical application of the procedure seems to be limited.

The mode of action of thyroxine and thyroid-active substances on milk secretion is uncertain. It is tmlikely that it is a specific effect on the alveolar cells; rather is it probably related to the effects of the thyroid hormone on the general metabolic rate.

E. PARATHYROm HORMONE

The early studies on the influence of the parathyroid glands on milk secretion indicated, as might be expected from their

MAMMARY GLAND AND LACTATION

()19

role in calcium metabolism, that the parathyroids were important in the maintenance of secretion (see review by Folley, 1952a). Indeed in the rat, we demonstrated that the severe impairment of milk secretion previously observed in "thyroidectomized" rats was due not to the removal of the thyroids, but to the simultaneous ablation of the l)arathyroids (Cowie and Folley, 1945). This observation has since been confirmed and extended by Munson and his colleagues (Munson, 1955) who demonstrated an influence on the calcium-concentrating mechanism of the mammary glands. Within 24 hours of parathyroidectomy the concentration of calcium in the milk of the lactating rat was increased markedly despite a greatly depressed level of calcium in the serum; there was also a decrease in water content of the milk, but this did not entirely account for the increase in calcium content since the calcium content expressed as mg. per gm. milk solids was significantly higher after parathyroidectomy (Toverud and Munson, 1956). Further studies in this field are awaited with interest.

F. INSULIN

Early experiments (see review by Folley, 1952a) indicated that the endocrine pancreas might influence mammary function in two ways; indirectly by way of the general intermediary metabolism by which the supply of milk precursors may be regulated, and directly through its role in the carbohydrate metabolism of the mammary gland itself.

Most recent studies have been concerned with the effect of insulin on mammary tissue in vitro. Mammary gland slices from lactating rats actively synthesize fat from small molecules, glucose, and glucose plus acetate, but not from acetate alone (Folley and French, 1950). The addition of insulin to the incubation medium very markedly increases the R.Q. (see Table 10.2) and glucose uptake of the tissue slices and experiments with isotopes show that the rate of fat synthesis is increased (Balmain, Folley and Glascock, 1952). Mammary gland slices from lactating sheep, on the other hand, can utilize acetate alone but not glucose alone for fat synthesis (Folley and French, 1950) and sheep tissue is not re

TABLE 10.2

Effect of different substrates and of insulin on the

respiratory quotient (R.Q.) of lactating mammary

gland slices from various species

(From S. J. Follev and M. L. McNaught, Brit.

M. BulL, 14, 207-211, 1958.)

Respiratory Quotients

Anlrml

Substrate

Without insulin

With insulin

Mouse

Glucose

1.90

2.14

Glucose + acetate

1.46

2.14

Rat

Glucose

1.57

1.80

Acetate

0.82

Glucose + acetate

1.53

2.03

Guinea pig

Glucose

1.17

Rabbit

Glucose

1.30

_

Acetate

0.92

Glucose -t- acetate

1.24

1.67

Sheep

Glucose Acetate

0.88 1.09

1.09

Glucose + acetate

1.52

1.50

Goat

Glucose

0.86

Acetate

1.17

—

Cow

Glucose

0.84

_

Acetate

1.12

—

sponsive to insulin in vitro. This clear-cut species difference is interesting and underlines the need for further study. It is of passing interest to note that the response in vitro of rat mammary tissue to insulin has been made the basis of a highly specific in vitro bio-assay for insulin (Fig. 10.17) (Balmain, Cox, Folley and McNaught, 1954; McNaught, 1958)!

Further references and discussion on the role of insulin in mammary function and lipogenesis will be found in the reviews by Folley (1956), and Folley and McNaught (1958, 1960).

IV. Removal of Milk from the

Mammary Glands: Physiology

of Suckling and Milking

A. MILK-EJECTION REFLEX

Since the second edition of this book, there have been major advances in our knowledge of the physiology of milk removal. In the mammary gland the greater

620

PHYSIOLOGY OF GONADS

22

rs 2-5//g/ml.

20

yT

■~^

yO

i 18

- Cf

>

-o

■;;16

- i:/^

c

u=

«14

/ J3 as^g/mi.

8 12

Z' j^p^ £) 0-Vg/ml.

— 10

y rf^ ,-fP

1 3 8

si r^^ r-f^ y^ Control

o °

M J

A y^ ^cr ,^y^

4J

z

Pr( .iif^ jy^^

4

^ M^ ^r^

2

L_l 1 1 1 1 \ 1 \ 1 1 \ 1

15 30

60 90 120

Time (min)

150

Fig. 10.17. Effect of various concentrations of insulin on the respiratory metabolism of slices of rat mammarj' glands. (From J. H. Balmain, C. P. Cox, S J. Folley and M. L. McNaught, J. Endocrinol., 11, 269-276, 1954.)

portion of the milk secreted by the alveohir cells in the intervals between suckling or milking remains within the alveoli and the fine ducts. Only a small portion passes into the larger ducts and cisterns or sinuses from which it can be immediately removed by suckling, milking, or cannulation; its removal requires no maternal participation and has been termed passive withdrawal (see Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951, and page 612). The larger portion of the milk in the alveoli and fine ducts becomes available only with the active participation of the mother and requires the reflex contraction of special cells (see page 623) surrounding the alveoli in response to the milking or suckling stimulus to eject the milk from the alveoli and fine ducts into the cistern and sinuses of the gland. The occurrence of this reflex has long been known, although its true nature has only recently been generally recognized.^

-H. K. Waller {Clinical Slujlits un Lnrfallon, London: Heinemann, 1938), and later one of us (S. J. Folley, Physiology and Biochemistry of Lactation, London and Edinburgh: Oliver & Boyd, 1956) have drawn attention to the fact that the theme of the "milk-ejection reflex" was the inspiration of a paiming by II Tintoretto entitled "The Origin of the Milky Way" which hangs in the

111 the past it has been termed the "draught" in lactating women (see Isbister, 1954) and the "let-down" of milk in the cow. The latter term is particularly misleading since it implies the release of some restraint, whereas there is, in fact, an active and forceful expulsion of milk from the alveoli and we have, therefore, urged that this term be no longer used in scientific literature and that it be replaced by the term "milk ejection" (Folley, 1947; Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951), a term, incidentally, which was used by Gaines in 1915 in his classical researches on the phenomenon (see below j.

The true nature of the milk removal process was for many years not recognized, probably because it was assumed that the mammary gland could not contain all the milk obtainable at a milking, and this assumption made it necessary to postulate a very active secretion of milk during suckling or milking. Even as late as 1926 two phases of milk secretion were described in the cow ; the first phase was one of slow secretion occurring between milkings, the second phase was one of very active secretion occurring in response to the milking stimulus when a volume of milk about equal to that produced in the first phase was secreted in a matter of a few minutes (Zietzschmann, 1926). That some physiologic mechanism

National Gallery, London. Both authors point out tliat the picture shows evidence of a considerable intuiti^■e understanding of the physiologic nature of the milk-ejection reflex. Thus, it illustrates, first, that the application of the suckling stimulus causes a considerable increase in intranianiinai >• jiressure resulting, in this instance, in a sjnni cii' milk from the nipples, and second, that ihv Muklmg stimulus applied to one nipple gives rise to a systemic rather than a localized effect, for the milk is forcibly ejected from the suckled and unsuckled breasts ahke. The same theme was also treatetl by Rubens in a picture called "The Birth of the Milky Way" which can be seen in the Prado Museum, Madrid. This picture differs from Tintoretto's in one important detail, the stream of milk coming only from one breast.

The forcible ejection of milk from the nipple has doubtless been the subject of many statues. An example known to the authors is the fountain in the Sfiuare at Palos Verdes, near Los Angeles, California. The center piece of this fountain has a nude female torso at each of its four corners from whose nipples spurt streams of water.

MAMMARY GLAND AND LACTATION

621

was involved in the discharge of preformed milk from the mammary gland had, however, been recognized. Schafer (1898) considered that milk discharge was aided by contraction of plain muscle w^ithin the gland and pressure on the alveoli produced by vasodilation.

The first full investigation of the physiology of milk removal was that by Gaines in 1915. Unfortunately, his remarkably accurate observations and perspicacious conclusions aroused little general interest and were almost wholly overlooked for more than quarter of a century. It is now of interest to recall the more important of Gaines' observations. First, he made a clear distinction between milk ejection and milk secretion — "Milk secretion, in the sense of the formation of the milk constituents, is one thing; the ejection of the milk from the gland after it is formed is quite another thing. The one is probably continuous; the other, certainly discontinuous." Secondly, he concluded that "Nursing, milking and the insertion of a cannula in the teat, excite a reflex contraction of the gland musculature and expression of milk. There is a latent period of 35 to 65 seconds. . . . Removal of milk from the gland is dependent on this reflex, and it may be completely inhibited l)y anaesthesia. The conduction in the reflex arc is dependent upon the psychic condition of the mother." He also observed that the increased flow of milk following the latent period after stimulation was associated wath a steep rise in pressure within the gland cistern and that the reflex could be conditioned. Thirdly, with reference to the gland capacity, he reported that "the indication is that practically the entire quantity of milk obtained at any one time is present as such in the udder at the beginning of milking." Lastl3^ he confirmed earlier observations that injections of posterior pituitary extract caused a flow of milk in the lactating animal and he postulated that "pituitrin has a muscular action on the active mammary gland causing a constriction of the milk ducts and alveoli with a consequent expression of milk. This action holds, also, on the excised gland in the absence of any true secretory action." Gaines regarded the milk-ejection reflex as a

l)urely neural arc although he emphasized that the effect was "very similar to that produced by pituitrin." All that is required to bring these views of milk ejection in line with present day concepts is to recognize that the reflex arc is neurohormonal in character, the efferent component of which is a hormone released from the neurohypophysis. When Gaines was carrying out these experiments hardly anything was known of neuro-endocrine relationships and there was no background of knowledge to lead anyone to conceive that the effects of the posterior pituitary extract might represent a physiologic rather than a pharmacologic effect. In 1930 Turner and Slaughter hinted at a possible physiologic role of the posterior pituitary in milk ejection and, as we have noted (page 610), Gomez (1939) used posterior pituitary extract in replacement therapy given to hypophysectomized lactating rats. It was not until 1941, however, that the role of the posterior pituitary in milk ejection was seriously postulated by Ely and Petersen (1941) who, having shown in the cow that milk ejection occurred in the mammary gland to which all efferent nerve fibers had been cut, suggested that the reflex was neurohormonal, the hormonal component being derived from the posterior pituitary, and being, in all likelihood, oxytocin. The neurohormonal theory of Ely and Petersen and the subsequent work of Petersen and his colleagues (see reviews by Petersen, 1948; and Harris, 1958), unlike the earlier work of Gaines, aroused wide interest and its practical applications permitted rationalization of milking techniques in the cowshed thereby improving milk yields. Despite the attractiveness of the concept, however, a further 10 years were to elapse before unequivocal evidence of the correctness of the theory was forthcoming and this evidence we shall now briefly review.

B. ROLE OF THE NEUROHYPOPHYSIS

The first reliable indication that the suckling or milking stimulus does in fact cause an outpouring of neurohypophyseal hormones were the observations that inhibition of diuresis occurred following the application of the milking or suckling stimulus (Cross, 1950; Peeters and Cous

622

PHYSIOLOGY OF GONADS

sens, 1950; Kalliala and Karvoncn, 1951; Kalliala, Karvonen and Leppanen, 1952). It was also shown that electrical stimulation of the nerve paths to the posterior pituitary resulted in milk ejection (Cross and Harris, 1950, 1952; Andersson, 1951a, b, c; Popovich, 1958 », and that when lesions were placed in these tracts the milk-ejection reflex was abolished (Cross and Harris, 1952) .

Further evidence was adduced when it was found that removal of the posterior pituitary immediately abolished the milkejection reflex in the lactating rat, and that it was necessary to inject such animals several times a day with oxytocin if their litters were to be reared (Cowie, quoted by Folley, 1952b). Earlier workers had claimed that the posterior lolie was not essential for lactation (Smith, 1932; Houssay, 1935), but an explanation of these discordant conclusions was provided when it was shown that the impairment of the reflex after removal of the posterior lobe is not permanent and that the reflex re-establishes itself after some weeks, presumably because the remaining portions of the neurohypophysis take over the functions of the posterior lobe (Benson and Cowie, 1956). That the neurohypophysis participates in milk ejection would now appear to be beyond question.

The discovery of the role of the neurohypophyseal hormones in milk ejection has provided an explanation of some longstanding clinical observations on what has been termed the natural "sympathy" between the uterus and the breasts. Thus the beneficial effects of the suckling stimulus and the occurrence of the "draught" {i.e., milk ejection) in causing uterine contraction after parturition were emphasized over a century ago by both Smith (1844) and Patcrson (1844). 0})servations have also been made on the I'cciprocal process of stimuli arising from the reproductive organs apparently causing milk ejection. In domestic animals two such examples were mentioned by Martiny (1871). According to Herodotus, the Scythians milk their mares thus: "They take l)lowpipes of bone, very like flutes, and put them into the genitals of the mares and blow with their mouths, others milk. And they say that the I'cason why thoy do so is this, that when the marc's \-cins ai'c filled

with air, the udder cometh down" (translation by Powell, 1949). Kolbe (1727) described a similar procedure of blowing air into the vagina used by the Hottentots when milking cows which were normally suckled by calves and in which, presumably, milk ejection did not occur in response to hand nnlking. A drawing depicting this procedure from Kolbe's book was recently published in the Ciba Zeitschrift (No. 84^ 1957) along with a photograph of African natives still using the method!-^

In 1839, Busch described the occurrence of milk ejection, the milk actually spurting from the nipple, in a lactating woman during coitus. A satisfactory explanation of these curious observations is now forthcoming. Harris (1947) suggested that coitus might cause the liberation of oxytocin from the neurohypophysis and, within the next few years it was demonstrated that stimulation of the reproductive organs evoked milk ejection in the cow (Hays and VanDemark. 1953) and reports confirmatory of Busch's long forgotten observations also appeared (Harris and Pickles, 1953; Campliell and Petersen, 1953).^

C. MILK-EJECTIOX HORMONE

There is much circumstantial evidence to confirm the belief that the milk-ejection hormone is oxytocin (see Cowie and Policy. 1957). Attemi)ts, however, to demonstrate oxytocin in the blood after application of the milking stimulus have given rather inconclusive results. Early claims that the hormone could be demonstrated in blood are

^ A similar drawing, also apparently from Kolbe '.•< book, has been used in the campaign for clean milk production! Heineman (1919) discussing sanitary l^recautions in the cowshed says of the picture "another picture shows a nude Hottentot milking a cow while another one is liolding the tail of the cow to prevent its dropping into the open pail. This ])icture might well serve as a model to some modern producers who do not take such precautions and calmly lift the tail out of the milk with their hands wlicn it hnjipens to switch into the pail."

' W(- h;i\(' hi'cii able to find only one painting illustrating this plienomenon. It is a picture by a contemporary French painter, Andre Masson, entitled "Le Viol" and painted in 1939. It illustrates in Masson 's personal idiom the act of rape and it is interesting to note that a stream of milk is depicted as being I'orcibly (\iected from one breast of the

MAMMARY GLAND AND LACTATION

623

of doiil)tful validity, because the milk-ejection effect observed may have been due to 5-hydroxytryptamine (see Linzell, 1955), and more recent attempts to assay the level of oxytocin in the blood have not been entirely satisfactory or conclusive. There seem to be other polypeptide substances in blood which possess oxytocic activity, although the thiogly collate inactivation test indicates that these are different from oxytocin (Robertson and Hawker, 1957), and no marked changes in the blood oxytocic activity associated with suckling or milking have been detected (Hawker and Roberts, 1957; Hawker, 1958). However, it would seem doubtful whether the present assay techniques are sufficiently sensitive and specific to detect changes in blood oxytocin of the magnitude likely to be associated with milking or suckling. In the lactating cow the intravenous injection of 0.05 to 2.0 I.U. oxytocin will cause milk ejection (Bilek and .Tanovsk>% 1956; Donker, 1958), in the goat 0.01 to 1 I.U. (Cowie, cited by Folley, 1952b; Denamur and Martinet, 1953), in the sow 0.2 to 1.0 I.U. (Braude, 1954; Whittlestone, 1954; Cross, Goodwin and Silver, 1958) in the rabbit 0.05 I.U. (Cross, 1955b) , and in the lactating woman 0.01 I.U. (Beller, Krumholz and Zeininger, 1958) . If these (loses give any indication of the quantity of endogenous oxytocin released, then the concentration in the peripheral blood is likely to be very small ; indeed Cross, Goodwin and Silver (1958) calculated that a threshold dose (10 mU.) of oxytocin in the sow w^ould give a plasma concentration of about 1 (U,U. per ml, and until it can be shown that the assay techniques are sufficiently sensitive to detect the changes in oxytocin concentration produced by intravenous injections of "physiologic" doses of oxytocin, no great reliance can be placed on the results of assays.

Attempts have been made to demonstrate alterations in the hormone content of the neural lobe following the suckling or milking stimulus. In the goat and cow no detectable changes have been reported, but in the smaller species (dog, cat, rat, guinea pig) decreases have been described (see Cowie and Folley, 1957). It is likely that in many species the amount released is small

relative to the total hormone content of the gland and within the limits of error of the

assay.

D. EFFECTOR CONTRACTILE MECHANISM OF THE MAMMARY GLAND

In the last 10 years considerable research has been devoted to a study of the effector contractile tissue in the mammary gland; this work has recently been reviewed in some detail (see Folley, 1956) and only the salient features need be mentioned here.

Although earlier histologists had from time to time figured myoepithelial or "basket" cells in close association with the mammary alveoli, the morphology and distribution of the cells remained vague until Richardson (1949) published a detailed and illuminating description (Fig. 10.18). His beautiful observations have since been confirmed and supplemented by Linzell (1952) and Silver (1954). Richardson also disposed of the oft repeated view that smooth-muscle fibers around the alveoli played an iml)ortant role in milk ejection. From a study of the general orientation of the myoepithelial cells and the precise relationship between these cells and the folds in the secretory epithelium from contracted glands, Richardson considered it reasonable to regard the myoepithelium as the contractile tissue in the mammary gland which responds to oxytocin causing contraction of the alveoli and widening of the ducts. The evidence adduced by Richardson, although good, was nevertheless circumstantial, and it was desirable that attempts be made to visualize the contraction of the myoepithelial cells in response to oxj^tocin. In this connection it is of interest to recall that Gaines (1915) reported that when a drop of pituitrin was placed on the cut surface of the mammary gland from a lactating guinea pig, minute white dots appeared within a few seconds beneath the pituitrin and slowly swelled to tiny milky rivulets streaming beautifully through the clear liquid. Much later the local effects of posterior pituitary extract on the mammary gland were studied by Zaks (1951) in the living mouse, when it was reported that it caused contraction of the alveoli and expansion of the ducts. These observations were considerablv extended bv Linzell

624

PHYSIOLOGY OF GONADS

Fig. 10.18. Surface view of contracted alveoli (of goat) showing myoepithelial cells. (Courtesy of K. C. Richardson.)

Fig. 10.19. Recording of pressure changes witliin a galactophore of a forcibly restrained lactating rabbit. The litter was allowed to suckle the noncannulated mammary glands but obtained only 8 gm. milk, there being only a slight rise in the milk pressure probably associated with a slight contraction of the myoepithelium in response to mechanical stimulation. When 5 mU. oxytocin were injected (5P) there was a rapid milk ejection response which could be inhibited by injecting 1 yug. adrenaline (lA) just before the oxytocin. After a few minutes 5 mU. oxytocin were again effective and the litter obtained 44 gm. milk when they were allowed to suckle. A more complete milk ejection respon.so was obtained with 50 mU. oxytocin (50P) and the young obtained a further 59 gm. milk. Anesthesia did not enhance the milk-ejection response to 50 mU. oxytocin. During emotional inhibition of milk ejection the mammary gland thus remains responsive to oxytocin. (From B. A. Cross, J. Endocrinol., 12, 29-37, 1955.)

(19ooi who studied the local effects of liighly purified oxytocin and vasopressin and a number of other drugs on the mammnry gland, and confirmed that oxytocin and vasopressin produced alveolar contraction and widening of the ducts. Although in these experiments the myoepithelial cells themselves could not be visualized, nevertheless the effects observed leave little doubt that the effector mechanism was the niyoei)ithelium.

The myoepithelium is responsive to stimuli other than those arising from the presence of neurohypophyseal hormones in the blood inasmuch as partial milk ejection may occur in response to local mechanical stimulation of the mammary gland (Cross, 1954; Yokoyama, 1956; see also Fig. 10.191. These observations may explain the recent reports by Tverskoi (1958) and Denamuiand Martinet (1959a, b) that milk yields can be maintained in goats in the absence of the milk-ejection reflex.

E. INHIBITION OF MILK EJECTION

(laines (1915) stressed that the conduction in the milk-ejection reflex pathway was dei)endent on the psychic condition of the

MAMMARY GLAND AND LACTATION

625

mother. Many years later Ely and Petersen (1941) confirmed this and, having shown that injections of adrenaline blocked the milk-ejection reflex, postulated that the increased blood level of adrenaline in emotionally disturbed cows interfered with the action of oxytocin. In the last few years, the nature of the inhibitory mechanisms has been more fully investigated. Braude and Mitchell (1952) showed in the sow that adrenaline exerts at least part of its inhibitory effect at the level of the mammary gland and that, whereas the injection of adrenaline before the injection of oxytocin blocked milk ejection, less inhibition occurred if both were given together. Cross (1953, 1955a) confirmed these observations in the rabbit and demonstrated that electrical stimulation of the posterior hypothalamus (sympathetic centers) inhibited the milk-ejection response to injected oxytocin, an effect which was abolished after adrenalectomy. Cross concluded from his experiments that any central stimulation causing sympathetico-adrenal activity inhibits the milk-ejection response and that the effect appears to depend on a constriction of the mammary blood vessels resulting from the release of adrenaline and excitation of the sympathetic fibers to the mammary glands. Whereas such a mechanism could account for the emotional disturbance of the reflex. Cross was careful to point out that there was no direct proof that this was so and he later demonstrated (Cross, 1955b) that in rabbits in which emotional inhibition of milk ejection was present, milk ejection could be effected by the injection of oxytocin (Fig. 10.19). In such cases there was clearly no peripheral inhibitory effect of milk ejection. Cross concluded that the main factor in emotional disturbance of the milkejection reflex is a partial or complete inhibition of oxytocin release from the posterior pituitary gland. At present nothing is known of the nature of this central inhibitory mechanism.^

^ A curious form of the suckling stimulus is illustrated in carvings which siumount the main door of the church of Sainte Croix in Bordeaux. The carvings illustrate penances prescribed for wrong doers who have committed one of the seven deadly sins. The penance for indulgence in the sin of luxiu y is the application to the breasts of serpents or toads.

Inhibition of the milk ejection reflex may also occur when the mammary gland becomes engorged with secretion to such an extent that the capillary circulation is so reduced that oxytocin can no longer reach the myoepithelium (Cross and Silver, 1956; Cross, Goodwin and Silver, 1958).

F. NEURAL PATHWAYS OF THE MILK-EJECTION REFLEX

Interpretation of some of the earlier studies on neural pathways is difficult because investigators did not realize that, although the milk ejection reflex normally occurs in response to the suckling stimulus, it can become conditioned and can then occur in response to visual or auditory stimuli associated with the act of nursing. In such cases an apparent lack of effect on milk ejection of section of nerves or nerve tracts would not necessarily imply that the nerves normally carrying the stimuli arising from the suckling had not been cut. Studies on the effects of hemisection of the spinal cord in a few goats led Tsakhaev (1953) to the conclusion that the apparent pathway used by the milk-ejection stimulus was uncrossed. More recently pathways within the spinal cord have been investigated by Eayrs and Baddeley (1956) who found inter alia that lactation in the rat was inhibited by lesions to the lateral funiculi, and by section of the dorsal roots of nerves supplying the segments in which the suckled nipples were situated. With few exceptions hemisection of the spinal cord abolished lactation when the only nipples available for suckling were on the same side as the lesion, but not when the contralateral nipples were available. It was concluded that the pathway used by the suckling stimulus enters the central nervous system by the dorsal routes and ascends the cord deep in the lateral funiculus of the same side. Inasmuch as in these experiments lactation was assessed from the growth curve of the pups, it is not always clear whether the failure of lactation was due to a cessation of milk secretion or to loss of the milk-ejection reflex. It was noted, however, that injections of oxytocin in some

It may be questioned whether this unusual form of the suckling stimulus would not inhibit rather than evoke the milk-ejection reflex.

626

PHYSIOLOGY OF GONADS

cases restored lactation for up to 2 days after it had ceased as a result of lesions of the cord which would suggest a primary interference with milk ejection. In the goat, Andersson (1951b) considered that stimuli may reach the hypothalamus by way of the medial lemniscus in the medulla, but little definite information is available concerning the pathways used by the stimuli to reach the hypothalamus and there is here scope for further investigations. (For further discussion see review by Cross, 1960.) From the hyopthalamus there is little doubt that the route to the posterior lobe is by way of the hypothalamo-hypophyseal tract which receives nerve fibers from the cells in the hypothalamic nuclei, and in the main from the paraventricular and supra-optic nuclei. It was generally assumed that the posterior lobe hormones were secreted in the posterior lobe from the pituicytes in response to stimuli passing down the hypothalamo-hypophyseal tract. In the last decade, however, much evidence has come to light which suggests that the so-called posterior lobe hormones are in fact elaborated in the cells of the hypothalamic nuclei and are then transported down the axones as a neurosecretion and stored in the posterior lobe (see Scharrer and Scharrer, 1954).

Before leaving the neural pathways of the milk-ejection reflex, brief reference must be made to the recent discovery by Soviet physiologists that there is also a purely nervous reflex (segmental in nature) involved in the ejection of milk. It is said that within a few seconds of the application of the milking stimulus, reflex contraction of the smooth muscle in the mammary ducts occurs, causing a flow of milk from the ducts into the cistern. This reflex contraction of the smooth muscle is also believed to occur in response to stimuli arising within the gland between milkings thus aiding the redistribution of milk in the udder. This purely nervous reflex is stated to occur some 30 to 60 seconds before the reflex ejection of milk from the alveoli by oxytocin (for further details sec review by Baryshnikov, 1957). The conditioned reflexes associated with suckling and milking have been the subject of numerous investigations l)y Grachev (see Grachev,

1953, 1958) ; these and other Russian researches into the motor apparatus of the udder have been fully reviewed by Zaks (1958).

G. MECHANISM OF SUCKLING

In the past, various theories have been put forward as to how the suckling obtains milk from its mother's mammary gland. In the human infant some considered that the lips formed an airtight seal around the nipple and areola thus allowing the child to suck, whereas others believed that compression of the lacteal sinuses between the gums aided the expulsion of the milk (see Ardran, Kemp and Lind, 1958a, b for review) . In the calf the act of suckling was studied by Krzywanek and Briiggemann (1930) who described how the base of the teat was pinched off between upper and lower jaws and the teat compressed from its base towards its tip by a stripping action of the tongue. Smith and Petersen (1945) on the other hand, concluded that the calf wrapped its tongue round the teat and obtained milk by suction.

Much misunderstanding about the nature of the act of suckling has arisen because the occurrence of milk ejection was overlooked or its significance was not appreciated. As a result, the idea became prevalent that success or failure in obtaining milk could be reckoned solely in terms of the power behind the baby's suction. This erroneous concept was vigorously attacked by Waller (1938), who pointed out that once the "draught" had occurred the milk at times flowed so freely from the breast that the baby had to break off and turn its head to avoid choking. A similar observation had been made by Sir Astley Cooper in 1840 who in describing the "draught" in nursing women wrote, "If the nipple be not immediately caught by the child, the milk escapes from it, and the child when it receives the nipple is almost choked l)y the rapid and abundant flow of the fluid; if it lets go its hold, the milk spurts into the infant's eyes." An even earlier comment was made by Soranus, a writer on paediatrics in the cai'ly half of the second century A.D., that it was unwise to allow the infant to fall asleep at the breast since the milk some

MAMMARY GLAND AND LACTATION

627

times flowed without suckling and the infant choked. It must thus be emphasized that once milk ejection has occurred the milk in the gland cisterns or sinuses is under considerable pressure and the suckling has merely to overcome the resistance of the sphincters in the nipple or teat to obtain the milk.

Recently the use of cineradiograjihy has allowed a more accurate analysis of the mechanism of suckling. Studies by Ardran, Kemp and Lind (1958b) have shown that the human infant sucks the nipple to the back of the mouth and forms a "teat" from the mother's breast; when the jaw is raised this teat is compressed between the upper gum and the tip of the tongue resting on the lower gum, the tongue is then applied to the lower surface of the "teat" from before backwards pressing it against the hard palate. Suction may assist the flow of milk so expressed from the nipple, but is only of secondary importance. Studies by Ardran, Cowie and Kemp (1957, 1958) in the goat have extended these observations, because it was possible in this species to follow the withdrawal, from the udder, of milk made radiopaque with barium sulfate. As with the infant, the neck of the teat was obliterated between the tongue and the palate of the kid and the contents of the teat sinus were displaced into the mouth cavity by a suitable movement of the tongue; while the first mouthful w^as being displaced into the pharynx, the jaw and tongue were lowered to allow the refilling of the teat sinus. The normal method of obtaining milk is, therefore, for the suckling to occlude the neck of the teat and then to expel the contents of the teat sinus by exerting positive pressure on the teat (120 mm. Hg in the goat), so forcing the contents through the teat canal or nipple orifices into the mouth cavity, a process which may be aided by negative pressure created at the tip of the teat. Human infants, goat kids, and calves can obtain milk through rubber teats by suction alone provided the orifice is large enough (see Krzywanek and Briiggemann, 1930; Martyugin, 1944; Ardran, Kemp and Lind, 1958a) , but this procedure occurs only w^hen the structure of the rubber teat is such that the suckling is unable to ol)literate the

neck of the teat and cannot, therefore, strip the contents of the teat by positive pressure.

V. Relation between the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection

We have seen that the suckling or milking stimulus is responsible for initiating the reflex concerned wath the maintenance of milk secretion and also the milk-ejection reflex; the question now arises as to what extent their arcs share common paths. It would seem logical to assume that a common path to the hypothalamus exists and parts of this, as we have seen, have been partially elucidated. Although the hypothalamo-hypophyseal nerve tracts provide an obvious link between hypothalamus and the posterior lobe, the connections between the hypothalamus and anterior pituitary are still a matter of some controversy. The possible avenues of communication to the anterior lobe are neural and vascular and these may be subdivided into central and peripheral neural connections and into portal and systemic vascular connections. The various experimental findings relating to these routes have recently been critically discussed by Sayers, Redgate and Royce (1958), and by Greep and Everett in their chapters in this book, and it is clear that at present no definite conclusions can be reached concerning their relative importance. So far as the specific question of maintenance of milk secretion is concerned, the experiments of Harris and Jacobsohn (1952), which showed that pituitary grafts maintained lactation when implanted adjacent to the median eminence in hypophysectomized rats, were consistent with the existence of a hormonal transmitter, passing by w^ay of the hypophyseal portal system. On the other hand, transplantation studies by Desclin (1950, 1956) and Everett ( 1954, 1956) have revealed that in the rat the anterior lobe can spontaneously secrete prolactin in situations remote from the median eminence, and Donovan and van der Werff ten Bosch (1957) have reported that milk secretion continued in rabbits in wiiich the pituitary portal vessels had been completely destroyed, although there was, however, an inferred change in milk composition. Evidence has recentlv been obtained

628

PHYSIOLOGY OF GONADS

which has confirmed that pituitary tissue grafted under the kidney capsule in rats apparently secretes prolactin and will give slight maintenance of milk secretion in hypophysectomized animals, this maintenance being considerably enhanced if ACTH or STH is also administered (Cowie, Tindal and Benson, 1960). It would thus seem that the cells of the anterior lobe have the ability when isolated from the hypophyseal portal system to secrete prolactin, but the experiments cited above allow no conclusions to be drawn regarding the route by which the galactopoietic function of the pituitary is normally controlled.

Recent reports that bilateral cervical sympathectomy in the lactating goat causes a fall in the milk yield suggest that the galactopoietic functions of the anterior lobe may be influenced by the sympathetic nervous system (Tsakhaev, 1959; Tverskoy, 1960) . Declines in milk yield also occur after section of the pituitary stalk in the goat, but it is not clear in such cases whether the effects are due to the interruption of nervous or vascular pathways within the stalk (Tsakhaev, 1959; Tverskoy, 1960). In these studies on stalk section the cut ends of the pituitary stalk were not separated by a plastic plate, so some restoration of the hyl^ophyseal portal system may have occurred. Further experiments on the effects of section of the pituitary stalk on lactation in which restoration of the hypophyseal portal is prevented by the insertion of a plate are being conducted in our laboratory and also in the Soviet Union. Another possible mode of communication between hypothalamus and anterior pituitary has been investigated by Benson and Folley (1956, 1957a, b) who have suggested that the oxytocin released from the neurohypophysis in response to the suckling stimulus may directly act on the cells of the anterior lobe and stimulate the release of the galactopoietic complex. The careful anatomic researches of Landsmeer (1951), Daniel and Prichard (1956, 1957, 1958) and Jewell (1956) have demonstrated in several species the existence of direct vascular connections from the neurohylK)physis to the anterior lobe so that the neurohypophyseal hormones liberated into the blood stream would in fact be carried

direct to the anterior pituitary cells in very high concentrations. Clearly such a concept would provide a simple explanation of how the hormonal integration, coordination, and maintenance of mammary function is achieved. It has already been noted (see page 607) that a connection between milk ejection and the onset of copious lactation has been suggested. There is considerable evidence that oxytocin is liberated during parturition in sufficient quantities to cause contraction of the alveoli and milk ejection (see Harris, 1955; Cross, 1958; Cross, Goodwin and Silver, 1958) ; if, therefore, oxytocin can release the lactogenic and galatopoietic complexes from the anterior pituitary, a simple explanation of the mechanism triggering off the onset of copious milk secretion, before the application of the milking stimulus, is available.

We must now consider what experimental evidence there is to support this rather attractive theory. First, Benson and Folley (1956, 1957a, b) demonstrated that regular injections of oxytocin can retard mammary regression after weaning in a similar fashion to injections of prolactin (see page 610), and they have shown that the presence of the pituitary is essential for oxytocin to elicit this effect. Synthetic oxytocin proved equally effective, thus discounting the possibility of a contaminant in natural oxytocin being concerned (Fig. 10.20) . These experiments have so far only been carried out in rats, but they strongly suggest that oxytocin can elicit the secretion of prolactin. In agreement with this concept are several observations that regular injections of oxytocin have galactopoietic effects in lactating cows and that oxytocin has luteotrophic effects in rats (see review by Benson, Cowie and Tindal, 1958) . There is, moreover, some evidence that the suckling stimulus may cause the release of vasopressin or the antidiuretic hormone (ADH) from the neurohypoi)hysis (see page 621), and it has been shown that ADH or some material closely associated with it may cause the secretion of ACTH from the anterior lobe (see review by Benson, Cowie and Tindal, 1958) ; so there are some grounds for supposing that the hormones of the posterior lobe evoke the secretion of several components of the

MAMMARY GLAND AND LACTATION

Fig. 10.20. Sections from abdominal mammary gland of rats from wliuli Ur- pups were removed on the fourth day of lactation and which received thereafter for 9 daj^s: A. LO I.U. synthetic oxytocin three times daily. B. Saline daily. Note the maintenance of gland structure in A. (Courtesy of Dr. G. K. Benson.)

galactopoietic complex from the anterior lobe. It was hoped to gain further evidence on this point by studies on hypophysectomized rats bearing pituitary homografts under the kidney capsule (see Benson, Cowie, Folley and Tindal, 1959) . As already noted, such grafts secrete prolactin and will give a slight maintenance of milk secretion, but these grafts will not maintain normal milk secretion even when such animals are injected with oxytocin and ADH (Cowie, Tindal and Benson, 1960). It must, therefore, be assumed that if these posterior pituitary hormones are responsible for the release of the galactopoietic complex, some other hypothalamic factor is also necessary to maintain the anterior lobe in a responsive condition. Everett (1956) suggested that the hypothalamus by way of its neurovascular connections with the anterior lobe, normally exerts a partial inhibitory effect on prolactin secretion. It may thus be that when the anterior lobe is removed from hypothalamic influence, the synthetic activities of its cells are centered on prolactin

production to the detriment of the other components of the galactopoietic complex, so that these are no longer available for release in response to neurohypophyseal hormones. There is need, however, for experimentation in other species.

The theory that the release of the galactopoietic complex is effected by the hormones of the posterior lobe secreted in response to the suckling stimulus is attractive in that it appears to afford a simple explanation of the hormonal integration of mammary function, but it must be pointed out that the observations on the maintenance of mammary structure after weaning by injections of oxytocin do not prove that prolactin or the galactopoietic complex is released in response to oxytocin under normal conditions of milking or suckling, and more research, particularly in species other than the rat, is necessary. Grosvenor and Turner (1958a) injected oxytocin into anesthetized lactating rats and, on the basis of assays of the pituitary content of prolactin, considered that oxytocin caused no significant release of

630

PHYSIOLOGY OF GONADS

prolactin. They had previously shown that there was an immediate fall in the pituitary content of prolactin after nursing (Grosvcnor and Turner, 1957b) and therefore concluded that their findings were contrary to the hypothesis that oxytocin is a hormonal link in the discharge of prolactin. This, however, cannot be regarded as conclusive because of the difficulties of relating pituitary content of a hormone to blood levels of the hormone and also the difficulty of determining the physiologic dose of oxytocin, for if the oxytocin is carried directly from the neurohypophysis into the anterior lobe, then the concentration in the blood reaching the anterior lobe may be relatively great (see also Cowie and Folley, 1957).

Other theories of the reflex maintenance of milk secretion have been put forward. In 1953 Tverskoi, observing that repeated injections of oxytocin were galactopoietic in the goat, suggested that alveolar contraction stimulated sensory nerve endings in the alveolar walls which reflcxly caused the release of prolactin. It is obvious that his observations could be explained on the basis of the Benson-Folley theory of direct pituitary stimulation by oxytocin. This possibility was indeed considered by Tverskoi. but rejected on the grounds that oxytocin did not affect the prolactin content of the pituitary (Meites and Turner, 1948). In 1957 Tverskoi found it necessary to revise his theory, having found that full lactation could be maintained in the goat after complete and repeated denervation of the udder provided oxytocin was regularly given to evoke milk ejection. He then suggested that alveolar contraction stimulates the synthetic activities of the mammary epithelium causing an uptake of prolactin from the blood, the fall in the blood prolactin level then stimulating the further production of prolactin by the anterior lobe. Although these latter observations of Tverskoi might again be explained on the basis of direct pituitary stimulation by exogenous oxytocin, more recent studies on goats have cast doubts on the validity of such an explanation. Tverskoi (1958) and Denannir and Martinet (1959a, b, 1960) have shown that lactating goats will continue to lactate, giving nonnal or onlv niodcratelv reduced

milk yields after section of all nervous connections between the udder and brain (cord section, radicotomy, bilateral sympathectomy) and without their receiving oxytocin and in the absence of conditioned milkejection reflexes. It has already been noted that milk ejection in such animals may result from mechanical stimulation of the myoepithelial cells by udder massage (see page 624) , but the release of the galactopoietic complex from the anterior pituitary would seem in these goats to have been independent of neurohormonal reflex activities. AVhether in such animals the release is spontaneous or dependent on the level of hormones in the blood as suggested by Tverskoi (1957) is a matter for further research.

VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection

Various attempts have been made to investigate the mechanism controlling release of anterior pituitary hormones by the use of dibenamine, atropine, and other drugs. In reviewing such experiments, Harris (1955) concluded that there was no convincing evidence of the participation of adrenergic, cholinergic, or histaminergic agents in the control of gonadotrophic and adrenocorticotrophic hormone release. Recently Grosvenor and Turner (1957a) reported that various ergot alkaloids, dibenamine, and atropine blocked milk ejection in the rat; the ergot alkaloids doing so within 10 minutes of administration, the atropine and dibenamine within 2 to 4 hours. Inasmuch as milk ejection occurred in response to exogenous oxytocin, it was concluded that these drugs acted centrally, and the presence of adrenergic and cholinergic links in the neurohormone arc was postulated to be responsible for the discharge of oxytocin. Later, on the basis of assays of jntuitary prolactin after nursing in druginjected lactating rats, it was suggested that cholinergic and adrenergic links are iinohcd in the reflex resi)onsible for prolactin release (Grosvenor and Turner, 1958a). Ergot alkaloids, however, administered in our laboratory to lactating rats had no significant effect on the lactational per

MAMMARY GLAND AND LACTATION

631

fonnance as judged by the growth of the litters in comparison with the growth of litters of pair-fed control rats, showing that apparent inhibitory effects of the alkaloids on lactation were due to depressed food intake of the mothers (Tindal, 1956a). Inasmuch as growth of the litter depends on efficient milk secretion and milk ejection, Tindal's observations seem to throw doubt on the importance of the adrenergic link in these reflexes. On the other hand, IVIeites (1959) has reported that adrenaline and acetylcholine can induce or maintain mammary development and milk secretion in suitably prepared rats, observations which could be interpreted as supporting the presence of adrenergic and cholinergic links as postulated by Grosvenor and Turner (1958a).

There have been clinical reports of women developing galactorrhoea after treatment with trancjuilizing drugs {e.g., Sulman and Winnik, 1956; Marshall and Leiberman, 1956; Piatt and Sears, 19561 and interesting observations have recently ap

peared on the lactogenic effects of reserpine in animals. Milk secretion has been initiated both in virgin rabbits after suitable estrogen priming and in the pseudopregnant rabbit by reserpine (Sawyer, 1957; Meites, 1957a). On the other hand, in our laboratory Tindal (1956b, 1958) had been unable to detect any mammogenic or lactogenic effects with chlorpromazine or reserpine in rabbits (Dutch breed), rats, or goats, nor did reserpine stimulate the crop-sac when injected into pigeons. Recently, using New Zealand White rabbits, Tindal (1960) has induced milk secretion with reserpine. The reason for these contradictory results is not entirely clear, although breed differences in the response would appear to exist in the rabbit. In our laboratory, Benson (1958) has shown that reserpine is strikingly active in retarding mammary involution in the lactating rat after weaning, the effect being of such a magnitude as has so far only been equalled by a combination of prolactin and STH (Fig. 10.21). It has been tentatively suggested that the tranquilizing drugs may

^^:f/

mm\"^>m.-Wi

■w^

.•^^:j^-^ f4kr 1"

Fig. 10.2L Sections from the abdominal mammary gland of rats from whichthe pujis were removed on the fourth day of lactation and which received thereafter for 9 days: A 100 fj.g. reserpine daily. B. Sahne dailJ^ Note the retardation of involution effected by reserpine. (Courtesy of Dr. G. K. Benson.)

632

PHYSIOLOGY OF GONADS

remove .some hypothalamic restraining mechanism on the release of jn'olactin and probably of other anterior-pituitary hormones (Sulman and Winnik, 1956; Benson, Cowie and Tindal, 1958), an effect which, if confirmed, may throw light on the behavior of pituitary transplants in sites remote from the median eminence.

VII. Conclusion

Any reader familiar with the chajiter on the mammary gland in the previous edition of this book cannot fail to note the main directions in which the subject has advanced in the intervening two decades. These reflect, as they are bound to do, the road taken by the science of endocrinology itself, a road leading to greater biochemical understanding on the one hand and to ever closer rapprochement with neurophysiology on the other.

The mammary gland offers unique opportunities of studying the biochemical mechanisms of hormone action because it is an organ with quite exceptional synthetic capabilities, an organ which is perhaps the most comprehensive hormone target in the mammalian body. Biochemists are entering this promising field in increasing numbers and we may expect to reap the fruits of their labors in the future.

VIII. References

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SthicivER, p., a.vd Grueter, F. 1928. Action du lobe anterieur de I'hypophyse sur la montee laiteuse. Comp. rend. Soc. biol.. 99, 1978-1980.

Stricker. p.. and Grueter, F. 1929. Recherches

MAMMARY GLAND AND LACTATION

641

experimentales sur les fonctions du lobe anterieur de I'hypophyse: influence des extraits du lobe anterieur sur I'appareil genital de la lapine et sur la montee laiteuse. Presse med., 37, 1268-127L

SuLMAN, F. G., AND WiNNiK, H. Z. 1956. Hormonal effects of chlorpromazine. Lancet, 270, 161-162.

Sykes, J. F., AND Wrenn, T. R. 1950. Hormonal development of the mammary gland of dairy heifers. J. Dairy Sc, 33, 194-204.

Sykes, J. F., and Wrenn, T. R. 1951. Hormonal development of mammary tissue in dairy heifers. J. Dairy Sc, 34, 1174-1179.

TiNDAL, J. S. 1956a. The effect of ergotamine and dihydroergotamine on lactation in the rat. J. Endocrinol., 14, 268-274.

TiND.^L, J. S. 1956b. National Institute for Research in Dairying, Report 1956, pp. 56-57.

TiNDAL, J. S. 1958. National Institute for Research in Dairying, Report 1958, pp. 48-49.

TiNDAL, J. S. 1960. A breed difference in the lactogenic response of the rabbit to reserpine. J. Endocrinol., 20, 78-81.

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TovERUD, S. v., AND MuNSON, P. L. 1956. The influence of the parathyroids on the calcium concentration of milk. Ann. New York Acad. Sc, 64, 336.

Trentin, J. J., AND Turner, C. W. 1947. Effect of adrenalectomy on the mannnary gland of the castrated and estrogen treated castrated male rat. Endocrinology, 41, 127-134.

Trentin, J. J., and Turner, C. W. 1948. The experimental development of the mammary gland with special reference to the intei-action of the pituitary and ovarian hormones. Res. Bull. Missouri Agric Exper. Sta., No. 418.

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PHYSIOLOGY OF GONADS

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11

SOME PROBLEMS OF THE METABOLISM AND

MECHANISM OF ACTION OF STEROID

SEX HORMONES

Claude A. Villee, Ph.D.

ASSOCIATE PROFESSOR OF BIOLOGICAL CHEMISTRY, HARVARD UNIVERSITY

I. Introduction 643

II. The Biosynthesis op Steroids 643

A. Cholesterol 644

B. Progesterone 644

C. Androgens 645

D. Estrogens 647

E. Biosynthesis of Other Steroids 647

F. Interconversions of Steroids 647

G. Catabolism of Steroids (548

H. Transport, Conjugation, and Excretion 650

III. Effects of Sex Hormones on Inter mediary Metabolism 650

A. Estrogens 652

B. Androgens 659

C. Progesterone 660

IV. References 661

I. Intro<luction

The chemical structure of the sex hormones, their isohition from biologic materials, and many of their chemical properties were fully described in the previous edition of Sex and Internal Secretions (W. M. Allen, 1939; Doisy, 1939; Koch, 1939). The major steroid sex hormones were isolated and identified 20 to 30 years ago. Estrone, in fact, was crystallized from pregnancy urine by Doisy, Veler and Thayer (1929) before the true structure of the steroid nucleus was known. The isolation, identification, and chemical synthesis of estradiol, progesterone, and testosterone were accomplished during the 1930's. Additional substances with androgenic, estrogenic, or progestational activity have subsequently been isolated from urine or from tissues but these are probably metabolites of the major

sex steroids. The steroids are now routinely synthesized from cholesterol or from plant sterols. It would be possible to carry out the total synthesis of steroids from simple precursors but this is not commercially practicable.

The two decades since the previous edition have been marked by major advances in our understanding of the intermediary metabolism of steroids — the synthesis of cholesterol from two-carbon units, the conversion of cholesterol to pregnenolone and progesterone, and the derivation of corticoids, androgens, and estrogens from progesterone. These advances were made possible by the development of vastly improved methods for the isolation and identification of steroids: chromatography on paper or columns, counter-current distribution, labeling with radioactive or heavy isotopes, infrared spectroscopy, and so on. There have been concomitant increases in the information regarding the sites and mechanisms of action of these biologically important substances and the means by which they stimulate or inhibit the growth and activity of particular tissues of the body. The following discussion will attempt to present a general picture of these two fields and not an exhaustive citation of the tremendous body of relevant literature.

II. The Biosynthesis of Steroids

When the steroid hormones were first discovered it was generally believed that each

643

644

PHYSIOLOGY OF GONADS

endocrine gland made its characteristic steroid by some unique biosynthetic mechanism, one that was independent of those in other glands. However, there is now abundant evidence that the biosynthetic paths in the several steroid-secreting glands have many features which are similar or identical.

It is now well established that progesterone is not simply a female sex hormone produced by the corpus luteum, but a common precursor of adrenal glucocorticoids such as Cortisol and adrenal mineralocorticoids such as aldosterone, androgens, and estrogens. The adrenal cortex, ovary, testis, and placenta have in common many enzymes for the biosynthesis of steroids. Androgenic tumors of the human ovary, for example, have been shown to produce testosterone and its metabolites. The transplantation of an ovary into a castrate male mouse will result in the maintenance of the male secondary sex characters, which suggests that the normal ovary can also synthesize androgens.

A. CHOLESTEROL

The early work of Bloch (1951), Rilling, Tchen and Bloch (1958), and of Popjak (1950) showed that labeled acetate is converted to labeled cholesterol. The pattern of the labeling present in the cholesterol synthesized from acetate-1-C^^ or acetate-2-C^ as precursor led to speculations as to how the steroid nucleus is assembled. Further work (Langdon and Bloch, 1953) revealed that squalene and certain branched-chain, unsaturated fatty acids are intermediates in this synthesis. The current hypothesis, which is supported by a wealth of experimental evidence, states that two moles of acetyl coenzyme A condense to form acetoacetyl coenzyme A, which condenses with a third molecule of acetyl coenzyme A to form yS-hydroxy-^-methyl glutaric acyl coenzyme A (Fig. 11.1). The coenzyme A group is removed and the hydroxymethyl glutaric acid is reduced to mevalonic acid. Mevalonic acid, 3-hydroxy-3-methylpentano-5-lactone, is metabolized to a 5-carbon isoprenoid compound and three moles of these condense to form a 1 5-carbon hydrocarbon. The headto-head condensation of two molecules of this 15-carbon compound yields the 30

carbon equalene. This is metabolized, by way of lanosterol and the loss of three methyl groups, to cholesterol, which seems to be the common precursor of all of the steroid hormones (Tchen and Bloch, 1955; Clayton and Bloch, 1956).

The question of whether cholesterol is an obligate intermediate in the synthesis of steroid hormones has not been definitely answered. There is clear evidence that cholesterol is converted to steroids without first being degraded to small units and subsequently reassembled. Werbin and LeRoy (1954) administered cholesterol labeled with both carbon-14 and tritium (H^) to human subjects and isolated from their urine tetrahydrocortisone, tetrahydrocortisol, androsterone, etiocholanolone and 11ketoetiocholanolone. These substances, known to be metabolites of steroid hormones, were labeled with both C^^ and H^ and the labeled atoms were present in the ratio expected if they were derived directly from cholesterol. Experiments by Dorfman and his colleagues (Caspi, Rosenfeld and Dorfman, 1956) also provide evidence for the synthesis of steroids via cholesterol. Cortisol and 11-desoxycortisol were isolated from calf adrenals perfused with acetate-1C^^ and from a patient with an adrenal tumor to whom acetate- 1-C^^ had been administered. It is known that cholesterol synthesized from acetate-l-C^'* is labeled in carbon 20 but not in carbon 21. The Cortisol and 11-desoxycortisol also proved to be labeled in carbon 20 but not in carbon 21. This evidence does not, of course, exclude biosynthetic paths for the steroids other than one by way of cholesterol, but it does suggest that cholesterol is at least an important precursor of them. Direct evidence that cholesterol is synthesized from squalene in man is provided by the experiments of Eidinoff, Knoll, Marano, Kvamme, Rosenfeld and Hcllman (1958), who prepared tritiated squalene and administered it orally to human subjects. They found that the cholesterol of the blood achieved maximal specific aeti\'ity in 7 to 21 liours.

B. PROCiE.STERONE

Cholesterol undergoes an oxidative cleavage of its side chain to yield isocaproic acid and pregnenolone (Fig. 11.2). The lat

CH3C0-SC0A

STEROID SEX HORMONES

SCoA

645

CH^CO-SCoA

Acetyl CoA

CH3COCH2CO~SCoA

Acetoacetyl CoA

Sesquiterpene (15C)

COOH

► HO— C— CHI ^

CO-' SCO A

pOH-p methylglutaric acyl Co A

CHg

C-CHI

CH II CHo

Isoprene unit (5C)

CHO

I

CHo I ^

HO-C-CH^ 1 3

CHo I 2 COOH

Mevalonic acid

Squalene Lanosterol Cholesterol

(30C) (30C) (27C)

Fig. 11.1. Biogenesis of cholesterol.

ter is dehydrogenated in ring A by the enzyme 3-^-ol dehydrogenase and a spontaneous shift of the double bond from the A5 , 6 to the A4 , 5 position results in progesterone. Progesterone undergoes successive hydroxylation reactions, which require molecular oxygen and reduced triphosphopyridine nucleotide (TPNH), at carbons 17, 21, and 11. These hydroxylations yield, in succession, 17-a-hydroxy progesterone, Reichstein's compound S (ll-desoxy-17-hydroxycorticosterone), and Cortisol (17-a-hydroxvcorticosterone) .

C. ANDROGENS

17-a-Hydroxy progesterone is also the immediate precursor of androgens and estrogens. Oxidative cleavage of its side chain yields A-4-androstenedione, which undergoes reduction to testosterone (Fig. 11.2). A-4-Androstenedione may be hydroxylated at carbon 11 to yield ll-/3-hydroxy-A-4androstenedione, which is an androgen isolated from human urine. It has also been found as a metabolite of certain androgenic tumors of the adrenal cortex.

04 G

PHYSIOLOGY OF GONADS

CH

Y^ .

socaproic C

=0

c=o

r^

0^-OH

^r^

HO

HO

J

HO

Cholesterol

Pregnenolone

17-Hydr

oxy

Dehydroepi

preg

nenolone

androsterone

CH3

CH3

f3

H-C-OH 1

, f

c=o

1

1

c=o

1

^ ■

-x^V

t^

^XP""°"

r^^

^^<Y

„ijj

- \^Xaj

JJ

A -3- Ketopregnene

Progest

erone

17

-Hydroxy

20-oc-ol

y

y

progesterone

CH^OH

X

CH OH

/

c=o

1

c=o

"

Desoxy— corticosterone

CH2OH c=o

1 7- Hydroxy desoxycorticosterone (Reichstein's "S")

CH^OH HCO C=0

.JOJ

CH^OH

c=o

A -androstenedione

OH

o ' -- o

Corticosterone 18-aldo- 11- desoxy- Cortisol Testosterone

corticosterone (Kendall's

Cmpd. "F")

HCO C=0

19 -Hydroxy- ^■^■ androstenedione

.;^

Aldosterone

HO

Estradiol Fig. 11.2. Biosynthetic paths from cholesterol.

Estrone

STEROID SEX HORMONES

647

D. ESTROGENS

A-4-Androstenedione and testosterone are precursors of the estrogens. Baggett, Engel, Savard and Dorfman (1956) demonstrated the conversion of testosterone to estradiol17/? by slices of human ovary. Ryan (1958) found that the enzymes to carry out this conversion are also present in the human placenta, located in the microsomal fraction of placental homogenates. Homogenates of stallion testis convert labeled testosterone to labeled estradiol and estrone. Slices of human adrenal cortical carcinoma also have been shown to convert testosterone to estradiol and estrone, and Nathanson, Engel and Kelley (1951) found an increased urinary excretion of estradiol, estrone, and estriol following the administration of adrenocorticotrophic hormone to castrate women. Thus it seems that ovary, testis, placenta, and adrenal cortex have a similar biosynthetic mechanism for the production of estrogens and androgens. The first step in the conversion of testosterone or A-4-androstenedione to estrogens is the hydroxylation at carbon 19, again by an enzymatic process which requires molecular oxygen and TPNH. Meyer (1955) first isolated and characterized 19-hydroxy-A-4-androstene3,17-dione from a perfused calf adrenal. When this was incubated with dog placenta it was converted to estrone. The steps in the conversion of the 19-hydroxy-A-4-androstenedione to estrone appear to be the introduction of a second double bond into ring A, the elimination of carbon 19 as formaldehyde, and rearrangement to yield a phenolic ring A. The requirements for the aromatization of ring A by a microsomal fraction of human placenta were studied by Ryan (1958). West, Damast, Sarro and Pearson (1956) found that the administration of testosterone to castrated, adrenalectomized women resulted in an increased excretion of estrogen. This suggests that tissues other than adrenals and gonads, presumably the liver, can carry out this same series of reactions.

E. BIOSYNTHESIS OF OTHER STEROIDS

To complete the picture of the interrelations of the biosyntheses of steroids, it should be noted that other evidence shows

that progesterone is hydroxylated at carbon 21 to yield desoxycorticosterone and this is subsequently hydroxylated at carbon 11 to yield corticosterone. Desoxycorticosterone may undergo hydroxylation at carbon 18 and at carbon 11 to yield aldosterone, the most potent salt-retaining hormone known (Fig. 11.2).

Dehydroepiandrosterone is an androgen found in the urine of both men and women. Its rate of excretion is not decreased on castration and it seems to be synthesized only by the adrenal cortex. It has been postulated that pregnenolone is converted to 17-hydroxy pregnenolone and that this, by cleavage of the side chain between carbon 17 and carbon 20, would yield dehydroepiandrosterone.

F. INTERCONVERSIONS OF STEROIDS

The interconversion of estrone and estradiol has been shown to occur in a number of human tissues. A diphosphopyridine nucleotide-linked enzyme, estradiol- 17/3 dehydrogenase, which carries out this reaction has been prepared from human placenta and its properties have been described by Langer ancl Engel (1956). The mode of formation of estriol and its isomer, 16-epiestriol, is as yet unknown.

There are three major types of reactions which occur in the interconversions of the steroids: dehydrogenation, "hydroxylation," and the oxidative cleavage of the side chain. An example of a dehydrogenation reaction is the conversion of pregnenolone to progesterone by the enzyme 3-/3-ol dehydrogenase, which requires diphosphopyridine nucleotide (DPN) as hydrogen acceptor. This important enzyme, which is involved in the synthesis of progesterone and hence in the synthesis of all of the steroid hormones, is found in the adrenal cortex, ovary, testis, and placenta. Other dehydrogenation reactions in which DPN is the usual hydrogen acceptor are the readily reversible conversions of A-4-androstenedione ^ testosterone, estrone ^ estradiol, and progesterone

^ A-4-3-ketopregnene-20-a-ol. This latter

substance, and the enzymes producing it from progesterone, have been found by Zander (1958) in the human corpus luteum and placenta.

The oxidative reactions leading to the

PHYSIOLOGY OF GONADS

introduction of an OH group on the steroid nucleus are usually called "hydroxylations." Specific hydroxylases for the introduction of an OH group at carbons 11, 16, 17, 21, 18, and 19 have been demonstrated. All of these require molecular oxygen and a reduced pyridine nucleotide, usually TPNH. The ll-/3-hydroxylase of the adrenal cortex has been shown to be located in the mitochondria (Hayano and Dorfman, 1953) . Experiments with this enzyme system, utilizing oxygen 18, showed that the oxygen atoms are derived from gaseous oxygen and not from the oxygen in the water molecules (Hayano, Lindberg, Dorfman, Hancock and Doering, 1955). Thus this hydroxylation reaction also involves the reduction of molecular oxygen.

The oxidative cleavage of the side chains of the steroid molecule appears to involve similar hydroxylation reactions. The experiments of Solomon, Levitan and Lieberman (1956) indicate that the conversion of cholesterol to pregnenolone involves one and possibly two of these hydroxylation reactions, with the introduction of OH groups at carbons 20 and 22 before the splitting off of the isocaproic acid.

In summary, this newer knowledge of the biosynthetic paths of steroids has revealed that the differences between the several steroid-secreting glands are largely quantitative rather than qualitative. The testis, for example, produces progesterone and estrogens in addition to testosterone. The change from the secretion of estradiol by the follicle to the secretion of progesterone by the corpus luteum can be understood as a relative loss of activity of an enzyme in the path between progesterone and estradiol. If, for example, the enzyme for the 17-hydroxylation of progesterone became inactive as the follicle cells are changed into the corpus luteum, progesterone rather than estradiol would subsequently be produced.

Knowledge of these pathways also provides an explanation for certain abnormal changes in the functioning of the glands. Bongiovnnni (1953) and Jailer (1953) showed that the adrenogenital syndrome results from a loss of an enzyme or enzymes for the hydroxylation reactions at carbons 21 and 11 of progesterone, which results in an impairment in the production of Cortisol.

The pituitary, with little or no Cortisol to inhibit the secretion of adrenocorticotrophic hormone (ACTH), produces an excess of this hormone which stimulates the adrenal to produce more steroids. There is an excretion of the metabolites of progesterone and 17-hydroxy progesterone, pregnanediol and pregnanetriol respectively, but some of the 17-hydroxy progesterone is converted to androgens and is secreted in increased amount.

G. CATABOLISM OF STEROmS

Many of the steroid hormones are known to act on the pituitary to suppress its secretion of the appropriate trophic hormone, ACTH, the follicle-stimulating hormone (FSH), or luteinizing hormone (LH). It would seem that the maintenance of the proper feedback mechanism between steroid-secreting gland and pituitary requires that the steroids be continuously inactivated and catabolized. The catabolic reactions of the steroids are in general reductive in nature and involve the reduction of ketonic groups and the hydrogenation of double bonds. The reduction of a ketonic group to an OH group can lead to the production of two different stereoisomers. If the OH group projects from the steroid nucleus on the same side as the angular methyl groups at carbon 18 and carbon 19, i.e., above the plane of the four rings, it is said to have the yS-configuration and is represented by a heavy line. If the OH projects on the opposite side of the steroid nucleus, below the plane of the four rings, it is said to have the a-configuration and is represented by a dotted line. Although both isomers are possible, usually one is formed to a much greater extent than the other.

The first catabolic step is usually the reduction of the A4-3-ketone group of ring A, usually to 3aOH compounds with the hydrogen at carbon 5 attached in the /3configuration. The 5/3-configuration represents the CIS configuration of rings A and B. The elimination of the A4-3-ketone group greatly decreases the biologic activity of the steroid and increases somewhat its solubility in water. This reductive process occurs largely in the liver. Progesterone is converted by reduction of its A4-3-ketone group to pregnane-3a:20a-diol, and 17-hy

STEROID SEX HORMONES 649

EXCRETORY PRODUCTS

c=o

Progesterone

1 3

HCOH

HO H

Pregnanediol

CH,

-OH

17 Hydroxy progesterone

CH,

^ H-C-OH

■OH

HO H

Pregnanetriol

OH

Testosterone

Androsterone

HO

/

androstenedione

HO H

Etiocholanolone

Dehydroepiandrosterone

Fk;. 11.3. Excretory products of progesterone and androgen

(Iroxy progesterone is converted to pregnane3a:17a:20a-triol (Fig. 11.3). Testosterone and dehydrocpiandroesterone are both converted to A4-androstenedione and the reduction of its A4-3-ketone group results in a mixture of androsterone (3a,5a-configuration) and ctiochohmolone (3a,5/?-configuration ) .

The catabohsm of estradiol is not completely known. Estradiol, estrone, and estriol are found in the urine but they account for less than half of an administered dose of labeled estradiol. The /3-isomer, 16-epiestriol, and two other phenolic steroids, 16-ahydroxy estrone and 2-methoxyestrone, have recently been isolated from normal

650

PHYSIOLOGY OF GONADS

urine and are known to be estrogen metabolites (Marrian and Bauld, 1955).

H. TRANSPORT, CONJUGATION, AND EXCRETION

Steroids circulate in the blood in part as free steroids and in part conjugated with sulfate or glucuronic acid (c/. review by Roberts and Szego, 1953b j . The steroids are generally conjugated by the hydroxy 1 group at carbon 3 with inorganic sulfate or with glucuronic acid. In addition, either the conjugated or nonconjugated forms may be bound to certain of the plasma proteins such as the ^-globulins (Levedahl and Bernstein, 1954) . There is evidence of specific binding of certain steroids with particular proteins, e.g., the binding of Cortisol to "transcortin" (Daughaday, 1956). Between 50 and 80 per cent of the estrogens in the blood are present closely bound to plasma proteins. A similar large fraction of the other steroid hormones is bound to plasma proteins ; presumably this prevents the hormone from being filtered out of the blood as it passes through the glomerulus of the kidney. The steroids excreted in the urine are largely in the conjugated form, as sulfates or glucuronides.

The liver plays a prime role in the catabolism of the steroids. It is the major site of the reductive inactivation of the steroids and their conjugation with sulfate or glucuronic acid. These conjugated forms are more water-soluble and the conjugation probably promotes their excretion in the urine. Rather large amounts of certain steroids, notably estrogens, are found in the bile of certain species. These estrogens are free, not conjugated; the amount of estrogens present in the bile suggests that this is an important pathway by which they are excreted. It has been suggested that the bacteria of the gastrointestinal tract may degrade the steroids excreted in the bile and further that there is an "enterohepatic circulation" of steroids with reabsorption from the gut, transport in the portal system to the liver, and further degradation within the liver cells.

III. Effects of Sex Hormones on Intermediary Metabolism

The literature concerning the effects of hormones on intermediary metabolism is

voluminous and contains a number of contradictions, some of which are real and some, perhaps, are only apparent contradictions. Evidence that a hormone acts at one site does not necessarily contradict other evidence that that hormone may act on a different metabolic reaction. From the following discussion it should become evident that there may be more than one site of action, and more than one mechanism of action, of any given hormone.

The hormones are so different in their chemical structure, proteins, peptides, amino acids, and steroids, that it would seem unlikely, a priori, that they could all influence the cellular machinery by comparable means. The basic elements of an enzyme system are the protein enzyme, its cofactors and activators, and the substrates and products. A hormone might alter the over-all rate of an enzyme system by altering the amount or activity of the protein enzyme, or by altering the availability to the enzyme system of some cofactor or substrate molecule. Some of the mechanisms of hormone action which have been proposed are these. (1) The hormone may alter the rate at which enzyme molecules are produced de novo by the cell. (2) The hormone may alter the activity of a preformed enzyme molecule, i.e., it may convert an inactive form of the enzyme to an active form. (3) The hormone may alter the permeability of the cell membrane or the membrane around one of the subcellular structures within the cell and thus make substrate or cofactor more readily available to the enzyme. Or, (4) the hormone may serve as a coenzyme in the system, that is, it may be involved in some direct fashion as a partner in the reaction mediated by the enzyme. Each of these theories has been advanced to explain the mode of action of the sex hormones.

The problem of the hormonal control of metabolism has been investigated at a variety of biologic levels. The earliest experiments were done by injecting a hormone into an intact animal and subsequently measuring the amount of certain constituents of the blood, urine, or of some tissue. There are several difficulties with such experiments. All of the homeostatic mechanisms of the animal operate to keep condi

STEROID SEX HORMONES

651

tions constant and to minimize the effects of the injected hormone. In addition, there is a maze of interactions, some synergistic and some antagonistic, between the different hormones both in the endocrine gland and in the target organs, so that the true effect of the substance injected may be veiled. Our growing understanding of the interconversions of the steroid hormones warns us that an androgen, for example, may be rapidly converted into an estrogen, and the metabolic effects observed on the administration of an androgen may, at least in part, result from the estrogens produced from the injected androgen.

To eliminate some of the confusing effects of these homeostatic mechanisms some investigators remove the liver, kidneys, and other viscera before injecting the hormone under investigation. Such eviscerated preparations have been used by Levine and his colleagues in their investigations of the mode of action of insulin (c/. Levine and Goldstein, 1955).

Other investigators have incubated slices of liver, kidney, muscle, endocrine glands, or other tissues in glass vessels in a chemically defined medium and at constant temperature. Such experiments have the advantage that metabolism can be studied more directly, oxygen consumption and carbon dioxide production can be measured manonietrically, and aliquots of the incubation medium can be withdrawn for chemical and radiochemical analyses. The amounts of substrate, cofactors, and hormone present can be regulated and the interfering effects of other hormones and of other tissues are eliminated. Theoretically, working with a simpler system such as this should lead to greater insight into the physiologic and chemical events that occur when a hormone is added or deleted. The chief disadvantage of this experimental system is that it is difficult to prove that the conditions of the experiment are "physiologic." With tissue slices there is the possibility that the cut edges of the cells may introduce a sizeable artifact. Kipnis and Cori (1957) found that the rat diaphragm, as it is usually prepared for experiments in vitro, has an abnormally large extracellular space and is more permeable to certain pentoses than is the intact diaphragm.

It has been postulated that a hormone may influence the metabolism of a particular cell by altering the permeability of the cell membrane or of the membrane around one of the subcellular particles. Experiments with tissue homogenates, in which the cell membrane has been ruptured and removed, provide evidence bearing on such theories. If an identical hormone effect can be obtained in a cell-free system, and if suitable microscopic controls show that the system is indeed cell-free, the permeability theory may be ruled out.

Ideally the hormone effect should be studied in a completely defined system, with a single crystalline enzyme, known concentration of substrates and cofactors, and with known concentration of the pure hormone. Colowick, Cori and Slein (1947) reported that hexokinase extracted from diabetic muscle has a lower rate of activity than hexokinase from normal muscle and that it could be raised to the normal rate by the addition of insulin in vitro. The reality of this effect has been confirmed by some investigators and denied by others who were unable to repeat the observations. Cori has suggested that the decreased rate of hexokinase activity in the diabetic results from a labile inhibitor substance produced by the pituitary. Krahl and Bornstein (1954) have evidence that this inhibitor is a lipoprotein which is readily inactivated by oxidation.

The two hormones whose effects can be demonstrated reproducibly in an in vitro system at concentrations in the range which obtains in the tissues are epinephrine (or glucagon) and estradiol (and other estrogens) . Epinephrine or glucagon stimulates the reactivation of liver phosphorylase by increasing the concentration of adenosine3'-5'-monophosphate (Haynes, Sutherland and Rail, 1960), and estrogens stimulate an enzyme system found in endometrium, placenta, ventral i)rostate of the rat, and mammary gland. The estrogen-stimulable enzyme was originally described as a DPNlinked isocitric dehydrogenase, but the estrogen-sensitive enzyme now appears to be a transhydrogenase which transfers hydrogens from TPN to DPN (Talalay and Williams-x\shman, 1958; Yillee and Hngerman, 1958).

052

PHYSIOLOGY OF GONADS

The various tissues of the body respond in quite different degrees to the several hormones. This difference in response is especially marked with the sex hormones. Those tissues which respond dramatically to the administration of a hormone are termed the "target organs" of that hormone. Just what, at the cellular level, differentiates a target organ from the other tissues of the body is not known exactly but there is evidence that each kind of tissue is characterized by a certain pattern of enzymes. The pattern of enzymes is established, by means as yet unknown, in the course of embryonic differentiation. The enzyme glucose 6-phosphatase, which hydrolyzes glucose 6-phosphate and releases free glucose and inorganic phosphate, is present in liver but absent from skeletal muscle. Even though a given reaction in two different tissues may be mediated by what appears to be the same enzyme, the enzymes may be different and subject to different degrees of hormonal control. Henion and Sutherland (1957) showed that the phosphorylase of liver responds to glucagon but the phosphorylase of heart muscle does not. Further, the two enzymes are immunologically distinct. An antiserum to purified liver phosphorylase will not react with heart phosphorylase to form an inactive antigenantibody precipitate, but it does react in this manner with liver phosphorylase. Further, perhaps more subtle, differences between comparable enzymes from different tissues have appeared when lactic dehydrogenases from liver, heart, skeletal muscle, and other sources were tested for their rates of reaction with the several analogues of the pyridine nucleotides now available (Kaplan. Ciotti, Hamolsky and Bicbcr, 1960). p]xtension of this technique may reveal differences in response to added hormones.

In addition to these differences in the response to a hormone of the tissues of a single animal, there may be differences in the response of the comparable tissues of different species to a given dose of hormone. Estrone, estriol, and other estrogens have different potencies relative to estradiol in different species of mammals. There are slight differences in the amino acid sequences of the insulins and vasopressins from flifferent species and quite marked

differences in the chemical structure (Li and Papkoff, 1956) and physiologic activity (Knobil, Morse, Wolf and Greep, 1958) of the pituitar}^ growth hormones of cattle and swine, on the one hand, and of primates, on the other.

A. ESTROGENS

The amount or activity of certain enzymes in the target organs of estrogens has been found to vary with the amount of estrogen present. Examples of this phenomenon are /^-glucuronidase (Odell and Fishman, 1950) , fibrinolysin (Page, Glendening and Parkinson, 1951), and alkaline glycerophosphatase (Jones, Wade, and Goldberg, 1953). Kochakian (1947) reported that the amount of arginase in the rat kidney increased after the injection of estrogens. Enzyme activity is increased by other hormones as well; for example, progesterone has been found to increase the activity of phosphorylase (Zondek and Hestrin, 1947) and of adenosine triphosphatase (Jones, Wade, and Goldberg, 1952).

In most experiments the amount of enzyme present has been inferred from its activity, measured chemically or histochemically under conditions in which the amount of enzyme is rate-limiting. This does not enable one to distinguish between an actual increase in the number of molecules of enzyme present in the cell and an increase in the activity of the enzyme molecules without change in their number. A few enzymes can be measured by some other property, such as absorption at a specific wavelength, by which the actual amount of enzyme can be estimated (see review by Knox, Auerbach, and Lin, 1956). Knox and Auerbach (1955) found that the activity of the enzyme tryptophan peroxidase-oxidase (TPO) of the liver was decreased in adrenalectomized animals and increased by the administration of cortisone. Knox had shown previously that th(> administration of the substrate of the enzyme, tryptophan, would lead to an increase in the activity of the enzyme which was maximal in 6 to 10 hours. Evidence that the increased activity of enzyme following the administration of cortisone represents the synthesis of new protein molecules is supplied by experi

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653

ments in which it was found that the increase in enzyme activity is inhibited by ethionine and this inhibition is reversed by methionine. The amino acid analogue ethionine is known to inhibit protein synthesis and this inhibition of protein synthesis is overcome by methionine.

The injection of estrogen into the immature or castrate rodent produces a striking uptake of water by the uterus followed by a marked increase in its dry weight (Astwood, 1938). Holden (1939) postulated that the imbibition of water results from vasodilatation and from changes in the permeability of the blood vessels of the uterus. There is clear evidence (Mueller, 1957) that the subsequent increase in dry weight is due to an increased rate of synthesis of proteins and nucleic acids. The sex hormones and other steroids could be pictured as reacting with the protein or lipoprotein membrane around the cell or around some subcellular structure like a surface-wetting agent and in this way inducing a change in the permeability of the membrane. This might then increase the rate of entry of substances and thus alter the rate of metabolism within the cell. This theory could hardly account for the many notable specific relationships between steroid structure and biologic activity. Spaziani and Szego (1958) postulated that estrogens induce the release of histamine in the uterus and the histamine then alters the permeability of the blood vessels and produces the imbibition of water secondarily.

The uterus of the ovariectomized rat is remarkably responsive to estrogens and has been widely used as a test system. After ovariectomy, the content of ribonucleic acid of the uterus decreases to a low level and then is rapidly restored after injection of estradiol (Telfer, 1953). A single injection of 5 to 10 yu,g. of estradiol brings about (1) the hyperemia and water imbibition described previously; (2) an increased rate of over-all metabolism as reflected in increased utilization of oxygen (David, 1931; Khayyal and Scott, 1931; Kerly, 1937; MacLeod and Reynolds, 1938; Walaas, Walaas and Loken, 1952a; Roberts and Szego, 1953a) ; (3) an increased rate of glycolysis (Kerly, 1937; Carroll, 1942; Stuermer and Stein, 1952; Walaas, Walaas

and Loken, 1952b; Roberts and Szego, 1953a) ; (4) an increased rate of utilization of phosphorus (Grauer, Strickler, Wolken and Cutuly, 1950; Walaas and Walaas, 1950) ; and (5) tissue hypertrophy as reflected in increased dry weight (Astwood, 1938), increased content of ribonucleic acid and protein (Astwood, 1938; Telfer, 1953; Mueller, 1957), and finally, after about 72 hours, an increased content of desoxyribonucleic acid (Mueller, 1957).

An important series of experiments by Mueller and his colleagues revealed that estrogens injected in vivo affect the metabolism of the uterus which can be detected by subsequent incubation of the uterus in vitro with labeled substrate molecules. Mueller (1953) first showed that pretreatment with estradiol increases the rate of incorporation of glycine-2-C^'* into uterine protein. He then found that estrogen stimulation increases that rate of incorporation into protein of all other amino acids tested: alanine, serine, lysine, and tryptophan. The peak of stimulation occurred about 20 hours after the injection of estradiol. In further studies (Mueller and Herranen, 1956) it was found that estrogen increases the rate of incorporation of glycine-2-C^^ and formate-2-C^'* into protein, lipid, and the purine bases, adenine and guanine, of nucleic acids. A stimulation of cholesterol synthesis in the mouse uterus 20 hours after administration of estradiol was shown by Emmelot and Bos (1954).

In more detailed studies of the effects of estrogens on the metabolism of "one-carbon units" Herranen and Mueller (1956) found that the incorporation of serine-3-C^'* into adenine and guanine was stimulated by pretreatment with estradiol. The incorporation was greatly decreased when unlabeled formate was added to the reaction mixture to trap the one-carbon intermediate. In contrast, the incorporation of C^^02 into uridine and thymine by the surviving uterine segment was not increased by pretreatment with estradiol in vivo (Mueller, 1957).

To delineate further the site of estrogen effect on one-carbon metabohsm, Herranen and Mueller (1957) studied the effect of estrogen pretreatment on serine aldolase, the enzyme which catalyzes the equilibrium

654

PHYSIOLOGY OF GONADS

between serine and glycine plus an active one-carbon unit. They found that serine aldolase activity, measured in homogenates of rat uteri, increased 18 hours after pretreatment in vivo with estradiol. It seemed that the estrogen-induced increase in the activity of this enzyme might explain at least part of the increased rate of onecarbon metabolism following estrogen injection. They found, however, that incubation of uterine segments in tissue culture medium (Eagle, 1955) for 18 hours produced a marked increase in both the activity of serine aldolase and the incorporation of glycine-2-C^'* into protein. The addition of estradiol to Eagle's medium did not produce a greater increase than the control to which no estradiol was added. Uterine segments taken from rats pretreated with estradiol for 18 hours, with their glycine-incorporating system activated by hormonal stimulation, showed very little further stimulation on being incubated in Eagle's medium for 18 hours. With a shorter period of i^retreatment with estradiol, greater stimulation occurred on subsequent incubation in tissue culture fluid. These experiments suggest that the hormone and the incubation in tissue culture medium are affecting the same process, one which has a limited capacity to respond. When comparable experiments were performed with other labeled amino acids as substrates, similar results were obtained.

Mueller's work gave evidence that a considerable number of enzyme systems in the uterus are accelerated by the administration of estradiol — not only the enzymes for the incorporation of serine, glycine, and formate into adenine and guanine, but also the enzymes involved in the synthesis of fatty acids and cholesterol and indejX'ndent enzymes for the activation of amino acids by the formation of adenosine monoiihosphate (AMP) derivatives. The initial step in protein synthesis has been shown to be the activation of the carboxyl grou]) of the amino acid with transfer of energy from ATP, the formation of AMP -"amino acid, and the release of jiyrophosphate (Hoagland, Keller and Zamecnick, 1956). This reversible step was studied with homogenates of uterine tissue, P^--labeled ]n'rni)liosi)liate, and a variety of amino

acids (Mueller, Herranen and Jervell, 1958). Seven of the amino acids tested, leucine, tryptophan, valine, tryosine, methionine, glycine, and isoleucine, stimulated the exchange of P^^ between pyrophosphate and ATP. Pretreatment of the uteri by estradiol injected in vivo increased the activity of these three enzymes. The activating effect of mixtures of these amino acids was the sum of their individual effects, from which it was inferred that a specific enzyme is involved in the activation of each amino acid. Since estrogen stimulated the exchange reaction with each of these seven amino acids, Mueller concluded that the hormone must affect the amount of each of the amino acid-activating enzvmes in the soluble fraction of the cell.

Mueller (1957) postulated that estrogens increase the rate of many enzyme systems both by activating preformed enzyme molecules and by increasing the rate of de novo synthesis of enzyme molecules, possibly by removing membranous barriers covering the templates for enzyme synthesis. To explain why estrogens affect these enzymes in the target organs, but not comparable enzymes in other tissues, one would have to assume that embryonic differentiation results in the formation of enzymes in different tissues which, although catalyzing the same reaction, have different properties such as their responsiveness to hormonal stimulation.

As an alternative hypothesis, estrogen might affect some reaction which provides a substance required for all of these enzyme reactions. The carboxyl group of amino acids must be activated by ATP before the amino acid can be incorporated into proteins; the synthesis of both purines and pyrimidines requires ATP for the activation of the carboxyl group of certain precursors and for several other steps; the synthesis of cholesterol requires ATP for the conversion of mevalonic acid to squalene; and the synthesis of fatty acids is also an energy-requiring process. Thus if (>strogens acted in some way to increase the amount of biologically useful energy, in the form of ATP or of energy-rich thioesters such as acetyl coenzyme A, it would increase the rate of synthesis of all of these compo

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nents of the cell. This would occur, of course, only if the supply of ATP, rather than the amount of enzyme, substrate, or some other cofactor, were the rate-limiting factor in the synthetic processes.

When purified estrogens became available, they were tested for their effects on tissues in vitro. Estrogens added in vitro increased the utilization of oxygen by the rat uterus (Khayyal and Scott, 1931) and the rat pituitary (Victor and Andersen, 1937). The addition of estradiol- 17^ at a level of 1 fxg. per ml. of incubation medium increased the rate of utilization of oxygen and of pyruvic acid by slices of human endometrium and increased the rate at which labeled glucose and pyruvate were oxidized to C^-^Os (Hagerman and Villee, 1952, 1953a, 1953b) . In experiments with slices of human placenta similar results were obtained and it was found that estradiol increased the rate of conversion of both pyruvate-2-C^'* and acetate-l-Ci4 to C^^Os (Villee and Hagerman, 1953) . From this and other evidence it was inferred that the estrogen acted at some point in the oxidative pathway common to pyruvate and acetate, i.e., in the tricarboxylic acid cycle.

Homogenates of placenta also respond to estradiol added in vitro. With citric acid as substrate, the utilization of citric acid and oxygen and the production of a-ketoglutaric acid were increased 50 per cent by the addition of estradiol to a final concentralion of 1 fjig. per ml. (Villee and Hagerman, 1953). The homogenates were separated by differential ultracentrifugation into nuclear, mitochondrial, microsomal, and nonparticulate fractions. The estrogen-stimulable system was shown to be in the nonparticulate fraction, the material which is not sedimented by centrifugating at 57,000 X g for 60 minutes (Villee, 1955). Experiments with citric, as-aconitic, isocitric, oxalosuccinic, and a-ketoglutaric acids as substrates and with fluorocitric and transaconitic acids as inhibitors localized the estrogen-sensitive system at the oxidation of isocitric to oxalosuccinic acid, which then undergoes spontaneous decarboxylation to a-ketoglutaric acid (Villee and Gordon, 1955). Further investigations using the enzymes of the nonparticulate fraction of the human placenta revealed that, in ad

dition to isocitric acid as substrate, only DPN and a divalent cation such as Mg+ + or Mn++ were required (Villee, 1955; Gordon and Villee, 1955; Villee and Gordon, 1956). The estrogen-sensitive reaction was formulated as a DPN-linked isocitric dehydrogenase:

Isocitrate + DPN* -^ a-ketoglutarate

+ CO2 + DPXH + H*

It was found that the effect of the hormone on the enzyme can be measured by the increased rate of disappearance of citric acid, the increased rate of appearance of a-ketoglutaric acid, or by the increased rate of reduction of DPN, measured spectrophotometrically by the optical density at 340 m/x. As little as 0.001 /xg. estradiol per ml. (4 X 10~^ m) produced a measurable increase in the rate of the reaction, and there was a graded response to increasing concentrations of estrogen. The dose-response curve is typically sigmoid. This system has been used to assay the estrogen content of extracts of urine (Gordon and Villee, 1956) and of tissues (Hagerman, Wellington and Villee, 1957; Loring and Villee, 1957).

Attempts to isolate and purify the estrogen-sensitive enzyme were not very successful. By a combination of low temperature alcohol fractionation and elution from calcium phosphate gel a 20-fold purification was obtained (Hagerman and Villee, 1957). However, as the enzyme was purified it was found that an additional cofactor was required. Either uridine triphosphate (UTP) or ATP added to the system greatly increased the magnitude of the estrogen effect and, subsequently, adenosine diphosphate (ADP) was recovered from the incubation medium and identified by paper chromatography (Villee and Hagerman, 1957). Talalay and Williams-Ashman (1958) confirmed our observations and showed that the additional cofactor was triphosphopyridine nucleotide (TPN) which was required in minute amounts. This finding was confirmed by Villee and Hagerman (1958) and the estrogen-sensitive enzyme system of the placenta is now believed to be a transhydrogenase which catalyzes the transfer of hydrogen ions and electrons

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PHYSIOLOGY OF GONADS

fromTPNHtoDPN:

TPXH + DPN^ -> DPNH + TPN^

The transhydrogenation system can be coupled to glucose 6-phosphate dehydrogenase as well as to isocitric dehydrogenase (Talalay and Williams-Ashman, 1958; Villee and Hagerman, 1958) and presumably can be coupled to any TPNH-generating system.

If the estrogen-stimulable transhydrogenation reaction were readily reversible, an enzyme such as lactic dehydrogenase which requires DPN should be stimulated by estrogen if supplied with substrate amounts of TPN, catalytic amounts of DPN, and a preparation from the placenta containing the transhydrogenase. Experiments to test this prediction were made using lactic dehydrogenase and alcohol dehydrogenase of both yeast and liver (Villee, 1958a). It was not possible to demonstrate an estrogen stimulation of either enzyme system in either the forward or the reverse direction. The stimulation of the lactic dehydrogenase-DPN oxidase system of the rat uterus by estrogens administered in vivo reported by Bever, Velardo and Hisaw (1956) might be explained by the stimulation of a transhydrogenase, but it has not yet been possible to demonstrate a coupling of this transhydrogenase and lactic dehydrogenase.

The stimulating effect of a number of steroids has been tested with a system in which the transhydrogenation reaction is coupled to isocitric dehydrogenase (Villee and Gordon, 1956; Hollander, Nolan and Hollander, 1958). Estrone, equilin, equilenin, and 6-ketoestradiol have activities essentially the same as that of estradiol17 j3. Samples of 1 -methyl estrone and 2methoxy-estrone had one-half the activitj of estradiol. Estriol is only weakly estrogenic in this system; 33 fig. estriol are less active than 0.1 fig. estradiol- 17/3 (Villee, 1957a). The activities of estriol and 16epiestriol are similar, whereas 16-oxoestradiol is more active than either, with about 10 per cent as much activitv as csti'adiol17/3.

Certain analogues of stilbestrol have been shown to be anti-estrogens in vivo. When applied topically to the vagina of the rat, they prevent the cornification normally in

duced by the administration of estrogen (Barany, Morsing, Muller, Stallberg, and Stenhagen, 1955). One of these, 1,3-di-phydroxyphenylpropane, was found to be strongly anti-estrogenic in the placental system in vitro: it prevented the acceleration of the transhydrogenase-isocitric dehydrogenase system normally produced by estradiol- 17/3 (Villee and Hagerman, 1957). The inhibitory power declines as the length of the carbon chain connecting the two phenolic rings is increased and 1 , 10-di-phydroxyphenyldecane had no inhibitory action. Similar inhibitions of the estradiolsensitive system were observed with stilbestrol, estradiol-17a, and a smaller antiestrogenic effect was found with estriol (Villee, 1957a). The inhibition induced by these compounds can be overcome by adding increased amounts of estradiol-17^. When stilbestrol is added alone at low concentration, 10~' M, it has a stimulatory effect equal to that of estradiol-17^ (Glass, Loring, Spencer and Villee, 1961).

The quantitative relations between the amounts of stimulator and inhibitor suggest that this inhibition is a competitive one. It was postulated that this phenomenon involves a competition between the steroids for specific binding sites on the estrogensensitive enzyme (Villee, 1957b; Hagerman and Villee, 1957). When added alone, estriol and stilbestrol are estrogenic and increase the rate of the estrogen-sensitive enzyme. In the presence of both estradiol and estriol, the total enzyme activity observed is the sum of that due to the enzyme combined with a potent activator, estradiol- 17^, and that due to the enzyme combined with a weak activator, estriol. When the concentration of estriol is increased, some of the estradiol is displaced from the enzyme and the total activity of the enzyme system is decreased.

Two hypotheses have been proposed for the mechanism of action of estrogens on the enzyme system of the placenta. One states that the estrogen combines with an inactive form of the enzyme and converts it to an active form (Hagerman and Villee, 1957). When this theory was formulated the evidence indicated that the estrogen acted on a specific DPN-linked isocitric dehydrogenase. The theory is equally applicable if the

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657

estrogen-sensitive enzyme is a transhydrogenase, as the evidence now indicates. The results of kinetic studies with the coupled isocitric dehydrogenase-transhydrogenase system are consistent with this theory (Gordon and Villee, 1955; Villee, 1957b; Hagerman and Villee, 1957). Apparent binding constants for the enzyme-hormone complex (Gordon and Villee, 1955j and for enzyme-inhibitor complexes have been calculated (Hagerman and Villee, 1957).

The observation that estradiol and estrone, which differ in structure only by a pair of hydrogen atoms, are equally effective in stimulating the reaction suggested that the steroid might be acting in some way as a hydrogen carrier from substrate to pyridine nucleotide (Gordon and Villee, 1956). Talalay and Williams-Ashman (1958) suggested that the estrogens act as coenzymes in the transhydrogenation reaction and postulated that the reactions were:

Estrone + TPNH + H*

— Estradiol + TPN^

Estradiol + DPN+

— Estrone + DPNH + H*

Sum : TPNH

H*

- DPN^ — TPN^ + H^

DPNH

This formulation implies that the estrogen-sensitive transhydrogenation reaction is catalyzed by the estradiol-17y3 dehydrogenase characterized by Langer and Engel (1956). This enzyme was shown by Langer (1957) to use either DPN or TPN as hydrogen acceptor but it reacts more rapidly with DPN. Ryan and Engel (1953) showed that this enzyme is present in rat liver, and in human adrenal, ileum, and liver. However, no estrogen-stimulable enzyme is demonstrable in rat or human liver (Villee, 1955). The nonparticulate fraction obtained by high speed centrifugation of homogenized rabbit liver rapidly converts estradiol to estrone if DPN is present as hydrogen acceptor, but does not contain any estrogenstimulable transhydrogenation system.

It will not be possible to choose between these two hypotheses until either the estrogen-sensitive transhydrogenase and the estradiol dehydrogenase have been separated or there is conclusive proof of their

identity. Talalay, Williams-Ashman and Hurlock (1958) reported a 100-fold purification of the dehydrogenase without separation of the transhydrogenase activity and found that both activities were inhibited identically by sulfhydryl inhibitors. In contrast, Hagerman and Villee (1958) obtained partial separation of the two activities by the usual techniques of protein fractionation, and reported that a 50 per cent inhibition of transhydrogenase is obtained with p-chloromercurisulfonic acid at a concentration of 10~^ m whereas 10"^ m p-chloromercurisulfonic acid is required for a 50 per cent inhibition of the dehydrogenase. The evidence that these two activities are mediated by separate and distinct proteins has been summarized by Villee, Hagerman and Joel (1960).

The transhydrogenase present in the mitochondrial membranes of heart muscle was shown by Ball and Cooper (1957) to be inhibited by 4 X 10"^ m thyroxine. The estrogen-sensitive transhydrogenase of the placenta is also inhibited by thyroxine (Villee, 1958b). The degree of inhibition is a function of the concentration of the thyroxine and the inhibition can be overcome by increased amounts of estrogen. Suitable control experiments show that thyroxine at this concentration does not inhibit the glucose 6-phosphate dehydrogenase or isocitric dehydrogenase used as TPNH-generating systems to couple with the transhydrogenase. Triiodothyronine also inhibits the estrogen-sensitive transhydrogenase but tyrosine, diiodotyrosine and thyronine do not. The thyroxine does not seem to be inhibiting by binding the divalent cation, Mn + + or ]Mg+ + , required for activity, for the inhibition is not overcome by increasing the concentration of the cation 10-fold.

In the intact animal estrogens stimulate the growth of the tissues of certain target organs. The estrogen-sensitive enzyme has been shown to be present in many of the target organs of estrogens: in human endometrium, myometrium, placenta, mammary gland, and mammary carcinoma, in rat ventral prostate gland and uterus, and in mammotrophic-dependent transplantable tumors of the rat and mouse pituitary. In contrast, it is not demonstrable in comparable preparations from liver, heart, lung, brain, or

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PHYSIOLOGY OF GONADS

kidney. The growth of any tissue involves the utilization of energy, derived in large part from the oxidation of substrates, for the synthesis of new chemical bonds and for the reduction of substances involved in the synthesis of compounds such as fatty acids, cholesterol, purines, and pyrimidines.

The physiologic responses to estrogen action, such as water imbibition and protein and nucleic acid synthesis, are processes not directly dependent on the activity of transhydrogenase. However, all of these processes are endergonic, and one way of increasing their rate would be to increase the supply of biologically available energy by speeding up the Krebs tricarboxylic acid cycle and the flow of electrons through the electron transmitter system. Much of the oxidation of substrates by the cell produces TPNH, whereas the major fraction of the biologically useful energy of the cell comes from the oxidation of DPNH in the electron transmitter system of the cytochromes. Hormonal control of the rat of transfer of hydrogens from TPN to DPN could, at least in theory, influence the over-all rate of metabolism in the cell and secondarily influence the amount of energy available for synthetic processes. Direct evidence of this was shown in our early experiments in which the oxygen consumption of tissue slices of target organs was increased by the addition of estradiol (Hagerman and Villee, 1952; Villee and Hagerman, 1953).

This theory assumes that the supply of energy is rate-limiting for synthetic processes in these target tissues and that the activation of the estrogen-sensitive enzyme does produce a significant increase in the supply of energy. The addition of estradiol in vitro produces a significant increase in the total amount of isocitric acid dehydrogenated by the placenta (Villee, Loring and Sarner, 1958) . Slices of endometrium to which no estradiol was added in vitro utilized oxygen and metabolized substrates to carbon dioxide at rates which paralleled the levels of estradiol in the blood and urine of the patient from whom the endometrium was obtained (Hagerman and Villee, esses in these target tissues and that the 1953b). Estradiol increases the rate of synthesis of ATP by liomogenates of human

placenta (Villee, Joel, Loring and Spencer, 1960).

The reductive steps in the biosynthesis of steroids, fatty acids, purines, serine, and other substances generally require TPNH rather than DPNH as hydrogen donor. The cell ordinarily contains most of its TPN in the reduced state and most its DPN in the oxidized state (Glock and McLean, 1955). If the amount of TPN+ is ratelimiting, a transhydrogenase, by oxidizing TPN and reducing DPN, would permit further oxidation of substrates such as isocitric acid and glucose 6-phosphate, which require TPN+ as hydrogen acceptor and which are key reactions in the Krebs tricarboxylic acid cycle and the hexose monophosphate shunt, respectively. Furthermore, the experiments of Kaplan, Schwartz, Freeh and Ciotti (1956) indicate that less biologically useful energy, as ATP, is obtained when TPNH is oxidized by TPNH cytochrome c reductase than when DPNH is oxidized by DPNH cytochrome c reductase. Thus, a transhydrogenase, by transferring hydrogens from TPNH to DPN before oxidation in the cytochrome system, could increase the energy yield from a given amount of TPNH produced by isocitrate or glucose 6-phosphate oxidation. The increased amount of biologically useful energy could be used for growth, for protein and nucleic acid synthesis, for the imbibition of water, and for the other physiologic effects of estrogens.

Estrogen stimulation of the transhydrogenation reaction would tend to decrease rather than increase the amount of TPNH in the cell. Thus the estrogen-induced stimulation of the synthesis of steroids, fatty acids, proteins, and purines in the uterus can be explained more reasonably as due to an increased supply of energy rather than to an increased supply of TPNH.

The theory that estrogens stimulate transhydrogenation by acting as coenzymes which are rapidly and reversibly oxidized and reduced does not explain the pronounced estrogenic activity in vivo of stilbestrol, 17a-ethinyl estradiol, or bfsdehydrodoisynolic acid, for these substances do not contain groups that could be readily oxidized or reduced. The exact mechanism

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(359

of action of estrogens at the biochemical level remains to be elucidated, but the data available permit the formulation of a detailed working hypothesis. The notable effects of estrogens and androgens on behavior (see chapter by Young) are presumably due to some direct or indirect effect of the hormone on the central nervous system. The explanation of these phenomena in physiologic and biochemical terms remains for future investigations to provide.

B. ANDROGENS

Although there is a considerable body of literature regarding the responses at the biologic level to administered androgens and progesterone, much less is known about the site and mechanism of action of these hormones than is known about the estrogens. The review by Roberts and Szego (1953b) deals especially with the synergistic and antagonistic interactions of the several steroidal sex hormones.

The rapid growth of the capon comb following the administration of testosterone has been shown to involve a pronounced increase in the amount of mucopolysaccharide present, as measured by the content of glucosamine (Ludwig and Boas, 1950; Schiller, Benditt and Dorfman, 1952). It is not known whether the androgen acts by increasing the amount or activity of one of the enzymes involved in the synthesis of polysaccharides or whether it increases the amount or availability of some requisite cofactor. Many of the other biologic effects of androgens do not seem to involve mucopolysaccharide synthesis and the relation of these observations to the other roles of androgens remains to he determined.

Mann and Parsons (1947) found that castration of rabbits resulted in a decreased concentration of fructose in the semen. Within 2 to 3 weeks after castration the amount of fructose in the semen dropped to zero, but rapidly returned to normal following the subcutaneous implantation of a pellet of testosterone. Fructose reappeared in the semen of the castrate rat 10 hours after the injection of 10 mg. of testosterone (Rudolph and Samuels, 1949). The coagulating gland of the rat, even when trans

planted to a new site in the body, also responds by producing fructose when the host is injected with testosterone. The amount of citric acid and ergothioneine in the semen is also decreased by castration and increased by the implantation of testosterone pellets (Mann, 1955). The experiments of Hers (1956) demonstrate that fructose is produced in the seminal vesicle by the reduction of glucose to sorbitol and the subsequent oxidation of sorbitol to fructose. The reduction of glucose requires TPNH as hydrogen donor and the oxidation of sorbitol requires DPN as hydrogen acceptor. The sum of these two reactions provides for the transfer of hydrogens from TPNH to DPN. If androgens act as cofactors which are reversibly oxidized and reduced, and thus transfer hydrogens from TPNH to DPN as postulated by Talalay and Williams-Ashman (1958), one would expect that an increased amount of androgen, by providing a competing system for hydrogen transfer, would decrease rather than increase the production of fructose. The marked increases in the citric acid and ergothioneine content of semen are not readily explained by this postulated site of action of androgens.

An increase in the activity of /3-glucuronidase in the kidney has been reported following the administration of androgens (Fishman, 1951). This might be interpreted as an arlaptive increase in enzyme induced by the increased concentration of substrate, or by a direct effect of the steroid on the synthesis of the enzyme.

The respiration of slices of prostate gland of the dog is decreased by castration or by the administration of stilbestrol (Barron and Huggins, 1944). The decrease in respiration occurs with either glucose or pyruvate as substrate. The seminal vesicle of the rat responds similarly to castration. Rudolph and Samuels (1949) found that respiration of slices of seminal vesicle is decreased by castration and restored to normal values within 10 hours after the injection of testosterone. Experiments by Dr. Phillip Corfman in our laboratory with slices of prostate gland from patients with benign prostatic hypertrophy showed that oxygen utilization was reduced 50 per cent by estradiol added

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PHYSIOLOGY OF GONADS

in vitro at a level of 1 /xg. per ml. Respiration of slices of the ventral prostate gland of the rat is decreased by castration and increased by administered testosterone (Nyden and Williams-Ashman, 1953). These workers showed that lipogenesis from acetate-l-C^* in the prostate is also significantly diminished by castration and restored to normal by administered testosterone.

The succinic dehydrogenase of the liver has been found to be increased by castration and decreased by the administration of testosterone (Kalman, 1952; Rindani, 1958), the enzyme is also inhibited by testosterone added in vitro (Kalman, 1952). In contrast, Davis, Meyer and McShan (1949) found that the succinic dehydrogenase of the prostate and seminal vesicles is decreased by castration and increased by the administration of testosterone.

An interesting example of an androgen effect on a specific target organ is the decreased size of the levator ani and other perineal muscles of the rat following castration. The administration of androgen stimulates the growth of these muscles and increases their glycogen content (Leonard, 1952). However, their succinoxidase activity is unaffected by castration or by the administration of testosterone. Courrier and Marois (1952) reported that the growth of these muscles stimulated by androgen is inhibited by cortisone. The remarkable responsiveness of these muscles to androgens in vivo gave promise that slices or homogenates of this tissue incubated with androgens might yield clues as to the mode of action of the male sex hormones. Homogenates of perineal and masseter muscles of the rat responded to androgens administered in vivo with increased oxygen consumption and ATP production iLoring, Spencer and Villee, 1961). The experiments suggested that the activity of DPNH-cytochromo r reductase in these tissues is controlled by aiKh'ogeiis.

C. PROGESTERONE

Attempts to clarify the biochemical basis of the role of progesterone have been hampered by the requirement, in most instances, for a previous stimulation of the tissue by estrogen. The work of Wade and Jones

(1956a, b) demonstrated an interesting effect of progesterone added in vitro on several aspects of metabolism in rat liver mitochondria. Progesterone, but not estradiol, testosterone, 17a-hydroxyprogesterone, or any of several other steroids tested, stimulated the adenosine triphosphatase activity of rat liver mitochondria. This stimulation is not the result of an increased permeability of the mitochondrial membrane induced by progesterone, for the stimulatory effect is also demonstrable with mitochondria that have been repeatedly frozen and thawed to break the membranes. Other experiments showed that ATP was the only substrate effective in this system ; progesterone did not activate the release of inorganic phosphate from AMP, ADP, or glycerophosphate.

In other experiments with rat liver mitochondria (Wade and Jones, 1956b), progesterone at a higher concentration (6 X lO"'* m) was found to inhibit the utilization of oxygen with one of the tricarboxylic acids or with DPNH as substrate. This inhibition is less specific and occurred with estradiol, testosterone, pregnanediol, and 17a-hydroxy progesterone, as well as with progesterone. The inhibition of respiration by high concentrations of steroids in vitro has been reported many times and with several different tissues; it seems to be relatively unspecific. Wade and Jones were able to show that progesterone inhibits the reduction of cytochrome c but accelerates the oxidation of ascorbic acid. They concluded that progesterone may perhaps uncouple oxidation from phosphorylation in a manner similar to that postulated for dinitrophenol. The site of action of this uncoupling appears to be in the oxidation-reduction path between DPNH and cytochrome c. Mueller (1953) found that progesterone added in vitro decreases the incorporation of glycine-2-C^'* into the protein of strips of rat uterus, thus counteracting the stimulatory effect of estradiol administered in vivo.

Zander (1958) reported that A4-3-ketopregnene-20-a-ol and A4-3-ketopregnene20-^-ol arc effective gestational hormones in the mouse, rabbit, and man, although somewhat less active in general than is progesterone. An enzyme in rat ovary which converts progesterone to pregnene-20-a-ol, and also catalyzes the reverse reaction, was

STEROID SEX HORMONES

661

described by Wiest (1956). The conversion occurred when slices of ovary were incubated with DPN. Wiest postulated that the progesterone-pregnene-20-a-ol system might play a role in hydrogen transfer, in a manner analogous to that postulated by Talalay and Williams-Ashman (1958) for estrone-estradiol- 17^, but his subsequent experiments ruled out this possibility, for he was unable to demonstrate any progesterone-stimulable transhydrogenation reaction.

The nature of the effect of progesterone and of estrogens on myometrium has been investigated extensively by Csapo. Csapo and Corner (1952, 1953) found that ovariectomy decreased the maximal tension of the myometrium and decreased its content of actomyosin. The administration of estradiol to the ovariectomized rabbit over a period of 7 days restored both the actomyosin content and the maximal tension of the myometrium to normal. The concentration of ATP and of creatine phosphate in the myometrium is decreased by ovariectomy but is restored by only 2 days of estrogen treatment. This suggests that the effect on intermediary metabolism occurs before the effect on protein {i.e., actomyosin) synthesis. Csapo (1956a) concluded that estrogen is a limiting substance in the synthesis of the contractile proteins of myometrium, but he could not differentiate between an effect of estrogen on some particular biosynthetic reaction and an effect of estrogen on some fundamental reaction which favors synthesis in general. He was unable to demonstrate any comparable effect of progesterone on the contractile actomyosin-ATP system of the myometrium.

Other observations provide an explanation for the well known effect of progesterone in decreasing the contractile activity of myometrium, not by any effect on the contractile system itself, but in some previous step in the excitation process. Under the domination of progesterone the myometrial cells have a decreased intracellular concentration of potassium ions and an increased concentration of sodium ions (Horvath, 1954). The change in ionic gradient across the cell membrane is believed to be responsible for the altered resting potential and the partial depolarization of the cell mem

brane which results in decreased conductivity and decreased pharmacologic reactivity of the myometrial cell. The means by which progesterone produces the changes in ionic gradients is as yet unknown. Csapo postulates that the hormone might decrease the rate of metabolism which in turn would lessen the rate of the "sodium pump" of the cell membrane. The contractile elements, the actomyosin-ATP system, are capable of full contraction but, because of the partial block in the mechanism of excitation and of propagation of impulses (Csapo, 1956b), the muscle cells cannot operate effectively; the contractile activity remains localized. Csapo (1956a) showed that the progesterone block is quickly reversible and disappears if progesterone is withdrawn for 24 hours. He concluded that the progesterone block is necessary for the continuation of pregnancy and that its withdrawal is responsible for the onset of labor.

]\Iost investigators who have speculated about the mode of action of steroids — whether they believe the effect is by activating an enzyme, by altering the permeability of a membrane, or by serving as a coenzyme in a given reaction— have emphasized the physical binding of the steroid to a protein as an essential part of the mechanism of action or a preliminary step to that action. They have in this way explained the specificities, synergisms, and antagonisms of the several steroids in terms of the formation of specific steroid-protein complexes. The differences between different target organs, e.g., those that respond to androgens and those that respond to estrogens, can be attributed to differences in the distribution of the specific proteins involved in these binding reactions. Viewed in this light, the problem of the mode of action of sex hormones becomes one aspect of the larger problem of the biochemical basis of embryonic differentiation of tissues.

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ViLLEE, C. A., Loring, J. M.. and Sarner, A. 1958.

Isocitric dehydrogenases of the placenta. Fed Proc, 17, 328.

Wade, R., and Jones, H. W., Jr. 1956a. Effect of progesterone on mitochondrial adenosinetriphosphatase. J. Biol. Chem., 220, 547-551.

Wade, R., and Jones, H. W., Jr. 1956b. Effect of progesterone on oxidative phosphorylation. J. Biol. Chem.. 220, 553-562.

Walaas, O., and Walaas, E. 1950. The metabolism of uterine muscle studied with radioactive phosphorus P^". Acta physiol. scandinav 21, 18-26.

Wal-aas, O.. Wal.aas, E., .\nd Loken, F. 1952a. The effect of estradiol monobenzoate on the metaboli-sin of rat uterine muscle. Acta endocrinol., 10,201-211.

Wal.'^as, 0., Walaas, E., and Loken, F. 1952b. The effect of estradiol monobenzoate on the metabolism of the rat endometrium. Acta endocrinol., 11, 61-66.

Werbin, H. and LeRoy, G. V. 1954. Cholesterol: a precursor of tetrahydrocortisone in man. J. Am. Chem. Soc, 76, 5260-5261.

West. C. D., Damast, B. L., Sarro, S. D., and Pearson, 0. H. 1956. Conversion of testosterone to estrogens in castrated, adrenalectomized human females. J. Biol. Chem., 218, 409-418.

WiEST, W. G. 1956. The metabolism of progesterone to A4-pregnen-20a-ol-3-one in eviscerated female rats. J. Biol. Chem., 221, 461467.

Zander, J. 1958. Gestagens in human pregnancy. In Proceedings Conference on Endocrinology of Reproduction, C. W. Lloyd, Ed. New York: Academic Press, Inc.

Zondek, B., and Hestrin, S. 1947. Phosphorylase activity in human endometrium. Am. J. Obst. ct Gvnec, 54, 173-175.

NUTRITIONAL EFFECTS ON ENDOCRINE SECRETIONS

James H. Leathern, Ph.D.

PROFESSOR OF ZOOLOGY, RUTGERS, THE STATE UNIVERSITY, NEW BRUNSWICK, NEW JERSEY

I. Introduction' 666 I, Introduction

II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t ,

Studies 668 Despite the accumulation of many data

A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology

production ()68 during the past 20 years and the long es B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in C. Diabetef^Mellitus, Nutrition, and ^f^f on fertility and fecundity, knowl Reproduction ()72 edge bearing on nutrition and the endocrine

D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad E. Diet and the Liver 673 vanced comparatively slowly. However, re III. Hypophysis and Diet 674 markable advances have been made in each

A. Inanition 674 speciality SO that nutritional-endocrine

^roein. .. w problems should continue to be a fruitful

C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii

D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to

IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to

A. Testis 677 the coherence one would prefer to present

1. Inanition 677 now.

2. Protein 678 'y\^q endocrinologist appreciates the deli 4 Vitamins (i8() ^^^^ balance which exists between the hy B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi siveness of Male Reproductive Tis- lar interdependence exists between nutrition

sues to Hormones 681 and the endocrine glands, including those

1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does

2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -•

V. Female Reproductive System 683 nutrition influence synthesis and release of

A. Ovaries 683 hormones, but hormones in turn, through

1. Inanition 683 their regulation of the metabolism of pro 2. Protein 684 teins, carbohydrates, and fats, influence nu 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create

5 Vitamins 685 endocrine imbalance, and endocrine imbal B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac siveness of Female Pe])ro<luct ive tors. It follows, therefore, that, in any conTissues to Hormones 687 sideration of this interrelationship, one must

1 . Ovary ... 687 consider not only undernutrition and lack of

2. uterus and vagina 688 -n c j \ l ^ i ^ cc i. r

3. Mammary gland 689 specific foods, but also possible effects of

C. Pregnancy. . 689 antithyroid substanccs in foods, antimetab VI. Concluding Remarks 693 olites, and overnutrition, especially for the

VII.Pkferencks (;!)4 child (Forbes, 1957).

666

NUTRITIONAL EFFECTS

667

Our understanding of the means by which hormones exert their effects is relatively slight, as is our knowledge of the biochemical mechanisms by w^hich supplements and deficiencies of vitamins and amino acids influence hormone action. Nevertheless, support for the statement that modifications of nutrition influence endocrine gland secretions or hormone action on distant target organs or tissues is provided by an enumeration of a few basic cell components requiring proteins, lipids, and vitamins. (1) Proteins combine with lipids to form lipoproteins which are essential features of the internal and external cellular membranes and interfaces. Hormones, as well as nutrition, influence cell membranes and therefore cell transport is affected. It is well known that hormones influence electrolyte and carbohydrate transfer and recently an endocrine control of amino acid transport was demonstrated (Noall, Riggs, Walker and Christensen, 1957). The effect of modifications of nutrition on the capacity of hormones to influence cell transport must await study. (2) Enzymes are proteins with chemically active surfaces and often include nonprotein groups such as vitamins. Nutritional and hormonal changes cause alterations in enzyme concentrations (Knox, Auerbach and Lin, 1956). Vitamin, mineral, and fat deficiencies favor a decrease in enzymes, whereas protein deficiencies have varied effects (Van Pilsum, Speyer and Samuels, 1957). Enzyme changes caused by hormones appear to be a consequence of metabolic adaptations. The importance of a nutritional base on which a hormone can express an effect on the enzymes of the reproductive organs can only be determined after further data have been obtained. (3) Proteins combined with nucleic acids become nucleoproteins, some of which are organized in the cytoplasm and may be templates for cellular protein synthesis. Other nucleoproteins are contained in the nucleus. Nutrition and hormones influence tissue nucleoproteins but studies involving the reproductive organs are few. How^ever, one possible cause of human infertility is low desoxyribose nucleic acid in the sperm ("Weir and Leuchtenberger, 1957).

Proteins are characteristic components of

tissues and hypophyseal hormones are protein in nature; also the major portion of gonadal dry weight is protein. Such being the case, it is important to appreciate that the protein composition of the body is in a dynamic state and that proteins from the tissues and from the diet contribute to a common metabolic pool of nitrogen. This metabolic pool contains amino acids which may be withdrawn for rebuilding tissue protein and for the formation of new protein for growth. Obviously, the character of the metabolic pool of nitrogen reflects dietary protein level and quality. A food protein which is deficient in one or more amino acids will restrict tissue protein synthesis. Hormones also influence the metabolic pool by affecting appetite as well as absorption, utilization, and excretion of foods, and thus hormones could accentuate the effect of a poor diet, or create demands beyond those normally met by an adequate diet. In addition a study of the tissues and organs of the body reveals that contributions to the metabolic pool are not uniform, thus one tissue or organ may be maintained at the expense of another. In protein deprivation in adults the liver quickly contributes increased amounts of nitrogen to the metabolic pool whereas the testis does not. On the other hand, protein contributions to the nitrogen pool by the hypophysis, and the amino acid withdrawals needed for hormone synthesis, are unknown. Data suggesting that the addition of specific nutrients to diets improves hypophyseal hormone synthesis have been presented (Leathem, 1958a).

In 1939 jNIason rightfully emphasized the need for vitamins in reproduction. Since then, much additional knowledge has been obtained. Vitamins of the B complex have been more clearly identified and a better understanding of their function has been gained. Thiamine is important for carbohydrate metabolism, pyridoxine for fat metabolism, and the conversion of tryptophan to nicotinic acid, and vitamin B12 may be involved in protein synthesis. In addition, vitamins have been found to serve as coenzymes, and folic acid to be important for estrogen action on the uterus. Tliosr. and

668

PHYSIOLOGY OF GONADS

many other findings prompt a survey of the relationships of vitamins to reproduction.

It is the intention of the author to review enough of the evidence which interrelates nutrition and reproduction to create an awareness of the problems in the area. Reviews dealing with the general subject of hormonal-nutritional interrelationships have been presented (Hertz, 1948; Samuels, 1948; Ershoff, 1952; Zubiran and GomezMont, 1953; Meites, Feng and Wilwerth, 1957; Leathem, 1958a,). Other reviews have related reproduction to nutrition with emphasis on laboratory (Mason, 1939; Guilbert, 1942; Lutwak-Mann, 1958) and farm animals (Reid, 1949; Asdell, 1949), and on protein nutrition (Leathem, 1959b, c). An encyclopedic survey of the biology of human nutrition has been made by Keys, Brozec, Hernschel, Michelsen and Taylor (1950).

II. Nature of Problems in Nutritional Studies

A. THYEOID GLAND, NUTRITION, AND REPRODUCTION

Normal development of the reproductive organs and their proper functioning in

TABLE 12.1 Ovarian response to chorionic gonadrotrophin,

as modified by thiouracil and diet

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Ovarian Weight

Cholesterol

Total

Free

18 per cent casein .

18 tier cent +

I'hiouracil

per cent casein . . per cent + thiouracil

18 per cent gelatin . . .

mg.

87 342

59 133

59 186 27.5

% 0.41

0.20

0.47

0.22

0.66 0.26 1.56

0.18 0.12 0.23 0.15

0.22

18 per cent -f thio uracil

0.18 18

Chorionic gonadotrophin = 10 I. U. X 20 days.

adults are dependent not only on the endocrine glands composing the hypophysealgonadal axis, but on others as well. The importance of the thyroid, although not the same for all species, is readily apparent from the effects of prolonged hypo- and hyperthyroid states on reproduction. Many of these effects have been enumerated elsewhere (chapters by Albert, by Young on the ovary, and by Zarrow) , but others also are important. Thus steroid production may be altered in hypothyroid animals; certainly its metabolism is influenced. Myxedema is associated with a profound change in androgen metabolism. Endogenous production of androsterone is very low and subnormal amounts of administered testosterone are converted to androsterone. Triiodothyronine corrects this defect (Hellman, Bradlow, Zumoff, Fukushima and Gallagher, 1959). The gonads of male and female offspring of cretin rats are subnormal. The testes may contain a few spermatocytes but no spermatozoa, and Leydig cells seem to secrete little or no androgen. The ovaries may contain a few small follicles with antra, but corpora lutea are absent and ovarian lipid and cholesterol concentrations are very low. Nevertheless, the gonads are competent to respond to administered gonadotrophin with a marked increase in weight. However, administration of chorionic gonadotrophin to the hypothyroid rat stimulated follicular cyst formation rather than folliculogenesis and corpora lutea formation (Leathem, 1958b), but, for even this aberrant development, dietary protein was required (Leathem, 1959b) (Table 12.1). The relationship between hypothyroidism and ovarian function may provide a clue to a possible origin of ovarian cysts, long known to be a common cause of infertility and associated reproductive disorders. Clinical cases of untreated myxedema exhibit ovarian cysts, and rats made hypothyroid for eight months, had a higher percentage of cystic ovaries than did euthyroid rats (Janes, 1944).

The reproductive system of the adult male is less affected than that of the immature male by a decrease in thyroid function, just as the testis of the adult is less likely to reflect a change in protein nutrition which is sufficient to alter the immature rat

NUTRITIONAL EFFECTS

669

testis. On the other hand, the lack of demonstrable thyroid dysfunction in the adult male does not exclude the possibility of an effect of thyroid hormone on reproduction. The conversion of thyroxine to triiodothyronine may be hindered (Morton, 1958) . Thyroxine was found to decrease the number of active cells in the semen and to reduce motility, whereas triiodothyronine increased the number of active spermatozoa (Farris and Colton, 1958; Reed, Browning and O'Donnell, 1958j. Small dosages of thyroxine stimulated spermatogenesis in the mouse, rabbit, and ram (Maqsood, 1952) and were beneficial in normal guinea pigs and rats (Richter and Winter, 1947; Young, Rayner, Peterson and Brown, 1952).

In many species reproduction occurs despite hypothyroidism, but fecundity may be subnormal (Peterson, Webster, Rayner and Young, 1952). Feeding an antithyroid drug, thiouracil, to female rats may or may not prevent pregnancy, but it reduces the number of young per litter. Thiouracil feeding continued through lactation will decrease litter size (Leathem, 1959b). Hypothyroid guinea pigs gave birth to some live young, but the percentage approached normal only when thyroxine was administered (Hoar, Goy and Young, 1957). Pregnant euthyroid animals responded to thyroxine by delivering more living young than normal control pigs (Peterson, Webster, Ravner and Young, 1952) . The extent to which such effects are consequences of the reduction in appetite, metabolism, and absorption of food from the gut which are associated with hypothyroidism has not been determined.

Hyperthyroidism, on the other hand, will increase the appetite and enhance absorption of food from the gut, as the increased metabolism requires more calories, minerals, vitamins, choline, and methionine. In adult rats hyperthyroidism induces a marked loss in body fat and accelerates protein catabolism. The two effects, if unchecked, result in loss of body weight and death. In immature males hyperthyroidism slows gain in body weight, retards testis growth and maturation, and abolishes androgen secretion (Table 12.2). Altering dietary protein in adult animals failed to modify the thyroid hormone effects, thereby suggesting

TABLE 12.2

Effects of diet and thyroid {0.2 'per

cent) on immature rats

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Testis Weight

Seminal

(Casein) X 30 Days

Actual

Relative

Vesicles

20 per cent

20 per cent + thyroid

6 per cent

6 per cent + thyroid

per cent

per cent -|thyroid

mg. 1G94

1090

825

245

140

95

mg./lOOgm.

1035

881 1232

650

346

261

mg. 88

9 16

7 7 6

that the metabolic demands of other tissues were making increased withdrawals from the metabolic pool of nitrogen and thus hindering testis growth. In euthyroid rats, a 6 per cent protein diet will permit testis growth in the absence of a gain in body weight, but hyperthyroidism prevents this preferential effect. Although Moore (1939) considered the effect of thyroid hormone on reproduction as possibly due to general body emaciation, the testis seems to be less responsive than the body as a whole. Adult rats fed 0.2 per cent desiccated thyroid exhibited no correlation between loss of body weight, change in testis weight, or protein composition of testis at two levels of casein and lactalbumin (Leathem, 1959b) . The testes were seemingly not influenced by the metabolic nitrogen changes which caused a loss in carcass nitrogen and an associated increase in kidney and heart nitrogen.

The mechanism of thyroid hormone action on reproduction is far from clear. As we have noted, a part of its action may be through the regulation of nutritional processes. Thyroid function is influenced by the biologic value of the dietary protein (Leathem, 1958a) and the specifie amino acids fed (Samuels, 1953). In turn, an altered thyroid function will influence the nitrogen contributions to the metabolic pool by reducing appetite and absorption from

670

PHYSIOLOGY OF GONADS

the gut, and by changing the contributions of nitrogen to the metabolic pool made by the body tissues. Hypothyroidism interferes with the refilling of body protein stores; consequently, protein needs of the reproductive organs may not be fully met (Leathern, 1953). It is consistent with this opinion that testis recovery from protein deprivation in hypothyroid rats was aided by thyroxine treatment (Horn, 1955).

Conversion of carotene to vitamin A may be prevented by hypothyroidism, suggesting that a subnormal amount of this vitamin may contribute to fetal loss. An increased intake of B vitamins might be required as hypothyroidism aggravates a vitamin B12 deficiency, and increased intake of B vitamins enhances the capacity of young rats to withstand large doses of thiouracil (Meites, 1953). Although a reduced metabolic rate might seemingly reduce vitamin requirements, more efficient metabolic activities, in the absence of hormonal stimuli, seem to occur when vitamin intake is increased (Meites, Feng and Wilwerth, 1957).

Reproduction is influenced by effects which are the opposite of a number of those just cited, i.e., effects of malnutrition on thyroid function. The need for iodine in the prevention of goiter is well known. However, certain foods prevent the utilization of iodine in the synthesis of thyroid hormone. The foods containing an antithyroid or goitrogenic agent, as tested in man, include rutabaga, cabbage, brussels sprouts, cauliflower, turnip, rape, kale, and to a lesser extent peach, pear, strawberry, spinach, and carrot (Greer and Astwood, 1948). A potent goitrogen isolated from rutabaga is L-5-vinyl-2-thiooxazolidine ((ireer, 1950, 1956). Reduced food intake will decrease the thyroid gland response to goitrogens (Gomez-Mont, Paschkis and Cantarow, 1947; Meites and Agrawala, 1949), the uptake of I^^^ (Meites, 1953), and the level of thyrotrophic hormone in laboratory animals (D'Angelo, 1951). The thyroid changes associated with malnutrition in man arc uncertain (Zubiran and Gomez-Mont, 1953). However, a decreased functioning of the gland in anorexia nervosa, followed by an increased functioning on refeeding (Poiioff, Lasche, Nodine,

Schneeberg and \'ieillard, 1954), is suggestive of a direct nutritional need.

Changes in the thyrotrophic potency of the rat hypophysis have been observed in various vitamin deficiencies. Thiamine deficiency may increase thyroid function, but vitamin A deficiency may have the opposite effect (Ershoff, 1952). The difficulties inherent in the assay of thyroid-stimulating hormone (TSH) , even by current methods, prevent one from drawing definite conclusions from the available data.

Immature animals given thyroxine are retarded in growth and do not survive. However, increasing dietary thiamine, pyridoxine, or vitamin B12 improves the ability of the young rat to withstand large dosages of thyroid substances (Meites, 1952), as does methionine (Boldt, Harper and Elvehjem, 1958). Consideration must also be given to the need for nutritional factors which play a minor role in normal metabolic states but increase in importance in stress. Thus yeast and whole liver contain antithyrotoxic substances (Drill, 1943; Ershoff, 1952; Overby, Frost and Fredrickson, 1959).

Excessive thyroid hormone will prevent maturation of the ovary and in adult rats will cause ovarian atrophy with a cessation of estrous cycles. Addition of yeast to the diet permitted estrous cycles to continue (Drill, 1943), but gonadal inhibition in the immature animal was not ]3revented. However, whole liver or its w^ater-insoluble fraction counteracted the gonadal inhibition induced by hyperthyrodism in immature rats (Ershoff, 1952). Biochemical mechanisms by which these dietary supplements can benefit rats given excessive quantities of hormone are unknown.

B. .\DREXAL GLAND, NUTRITION, AND REPRODUCTION

The problem of the relationship between adrenal steroid secretions and the reproductive system is one that still requires clarification. Furthermore, the possible influences of nutrition can only be inferred from the effects of adrenal steroids on the major metabolic systems of the body.

In the female there is a close relationshiji between the adrenal and the estrous and

NUTRITIONAL EFFECTS

iul

menstrual cycles (Zuckerman, 1953; chapter by Young on the ovary). The ovary would seem to require cortical steroids for the normal functioning of its own metabolic processes and for those which it influences peripherally. The Addisonian patient may show ovarian follicular atresia and a loss of secondary sex characteristics, and the untreated adrenalectomized rat exhibits a decrease in ovarian size and has irregular cycles (Chester Jones, 1957). The decline in size of the ovary after adrenalectomy is due to impaired sensitivity to folliclestimulating hormone (FSH) rather than to a decreased production of FSH, and the ovarian response is corrected by cortisone (Mandl, 1954).

Reproductive potential is not necessarily lost when there is adrenal insufficiency, but pregnancy is not well tolerated by women with Addison's disease. Furthermore, adrenalectomy in rats at the time of mating or 4 to 6 days after mating resulted in abortion. Improved pregnancy maintenance was obtained in adrenalectomized rats given saline or cortisone acetate (Davis and Plotz, 1954) , whereas desoxycorticosterone acetate alone extended the pregnancy period beyond normal time (Houssay, 1945). Essentially normal pregnancies were obtained in adrenalectomized rats given both cortisone acetate and desoxycorticosterone acetate (Cupps, 1955). Substitution of cortisone acetate for adrenal secretions may be incomplete because the adrenal hormone in the normal rat is primarily corticosterone and because cortisone enhances the excretion of certain amino acids and vitamins. It would be interesting to test a diet with a high vitamin content on the capacity of an adrenalectomized rat to maintain pregnancy, because improved survival of operated rats is obtained by giving vitamin Bio (Meites, 1953) or large doses of pantothenic acid, biotin, ascorbic acid, or folic acid (Ralli and Dumm, 1952; Dumm and Ralli, 1953).

An adrenal influence over protein metabolism is well known, but protein nutrition, in turn, can influence cortical steroid effectiveness. In fact, an extension of the life span of adrenalectomized rats is not obtained with adrenal steroids if the diet

lacks protein (Leathern, 1958a). A low protein diet alone will not improve survival after adrenalectomy, but better survival is obtained when the rats are given saline. When the low protein diet was supplemented with methionine, a definite improvement in life span was observed and the possibility that cortisone exerts its effect by drawing on the carcass for methionine was suggested (Aschkenasy, 1955a, b).

Reducing dietary casein to 2 per cent seriously endangers pregnancy in the rat, but the addition of progesterone permits 80 per cent of the pregnancies to be maintained. However, removal of the adrenal glands counteracts the protective action of the progesterone, only 10 per cent of the pregnancies continuing to term. Addition of methionine to the low casein diet improved pregnancy maintenance, but 1 mg. cortisone acetate plus progesterone provided the best results (Aschkenasy-Lelu and Aschkenasy, 1957; Aschkenasy and Aschkenasy-Lelu, 1957). These data emphasize the importance of nutrition in obtaining an anticipated hormone action. Further investigation might be directed toward the study of whole proteins other than casein, for the biologic value of proteins differs from normal when tested in adrenalectomized rats (Leathem, 1958a) .

As Albert has noted in his chapter, adrenalectomy has little or no effect on the testis. Gaunt and Parkins (1933) found no degenerative changes in the testes of adult rats dying of adrenal insufficiency, although an increase in the testis : body-weight ratios was noted in rats fed 18 per cent and 4 per cent protein (Aschkenasy, 1955c). If adrenalectomized rats are kept on a maintenance dosage of cortisone acetate for 20 days and fed dietary proteins of different biologic values, one finds that testis-composition of protein, lipid, and glycogen varies in the same manner as in the normal rat (Wolf and Leathem, 1955) (Table 12.3).

When the adrenal glands are intact, the influence of diet on their functional capacity and indeed on the hypophyseal- adrenal axis must be considered. Zubiran and Gomez-]\Iont (1953) showed that patients exhibiting gonadal changes associated with chronic malnutrition also exhibit adrenal

672

PHYSIOLOGY OF GOXADS

TABLE 12.3

Nutritional effects on the testes of cortisonemaintained adrenalectomized rats (From R. C. Wolf and J. H. Leathern, Endocrinology, 57, 286, 1955.)

Testes Composition

(per cent)

Treatment

Diet

Testes

Protein

Lipid

Glycogen

mg.

Corti

20 per cent

703

68.5

30.4

0.13

sone

casein

ace

tate

Control

1211

70.5

29.4

0.15

Corti

20 per cent

808

61.0

31.8

0.17

sone

wheat

ace

ghiten

tate

Control

816

62.3

32.2

0.17

Corti

20 per cent

1014

64.3

31.3

0.17

sone

peanut

ace

flour

tate

Control

699

68.5

31.3

0.12

hypofimction. Several clinical tests permitted the evaluation of subnormal adrenal function which, however, did not reach Addisonian levels. Malnutrition not onlyreduced hypophyseal adrenocorticotrophic hormone (ACTH), but also prevented an incomplete response by the adrenal glands to injected ACTH. In laboratory rodents, anterior hypophyseal function is also influenced by dietary protein and vitamin levels (Ershoff, 1952). The importance of dietary protein in the hypophyseal-adrenal system has recently been re-emphasized (Leathem, 1957; Goth, Nadashi and Slader, 1958). Furtiiermore, adrenal cortical function is affected by vitamin deficiencies (Morgan, 1951), from which it appears that pantothenic acid is essential for cortical hormone elaboration (Eisenstcin, 1957). Administration of excessive amounts of cortical steroids can induce morphologic changes which have been compared to inanition (Baker, 1952). Not only is nitrogen loss enhanced, but hyperglycemia can also be induced which, therefore, increases the need for thiamine. Cortical steroids influence the metabolism of various vitamins

(Draper and Johnson, 1953; Dhyse, Fisher, Tullner and Hertz, 1953; Aceto, Li Moli and Panebianco, 1956; Ginoulhiac and Nani, 1956). H a vitamin deficiency already exists, administration of cortisone will aggravate the condition (Meites, Feng and Wilwerth, 1957). Nevertheless, drastic effects of therapeutic doses of cortisone on reproductive function do not occur. In rare cases loss of libido has been reported in the male, but mori)hologic changes in the testis were not observed (JVladdock, Chase and Nelson, 1953). Cortisone has little if any effect on the weight of the rat testis (Moore, 1953; Aschkenasy, 1957) and does not influence testis cholesterol (Migeon, 1952). In the female, menstrual disturbances have been noted in association with cortisone therapy, with the occurrence of hot flashes (Ward, Slocumb, Policy, Lowman and Hench, 1951). However, cortisone corrected disturbances during the follicular phase, possibly by increasing FSH release (Jones, Howard and Langford, 1953). Cortisone also increased the number of follicles in the ovary of the rat (Moore, 1953), but not in the rabbit. Cortisone administration did not prevent the enhanced ovarian response to chorionic gonadotrophin seen in hypothyroid rats (Leathem, 1958b) and had little effect on mice in parabiosis ( Noumura, 1956).

In pregnant female rabbits resorption and stunting of fetuses occurred during treatment with large doses of cortisone (Courrier and Collonge, 1951). Similar effects were noted in mice (LcRoy and Domm, 1951 ; Robson and Sharaf, 1951).

Some of the metabolic derangements of human toxemia of pregnancy have been correlated with accelerated secretion of adrenal steroids creating a steroid imbalance (see chapter by Zarrow). Cortisone is reported to have a beneficial effect on some cases (Moore, Jessop, 'Donovan, Barry, Quinn and Drury, 1951). Protein inade({uacies may also be etiologic in toxemia and further (>xamination of the possibility sliould he made.

C. DIABETES MELLITUS, NUTRITION, AND REPRODUCTION

(llycosuria can be induced experimentally by starvation, overfeeding, and shifting

NUTRITIONAL EFFECTS

673

diet^ from one of high fat content to one i; which is isocaloric but high in carbohydrate (Ingle, 1948). Force feeding a high carbohydrate diet will eventually kill a rat despite insulin administration aimed at controlling glycosuria (Ingle and Nezamis, 1947). In man excessive eating leading to obesity increases insulin demand and, in many diabetics of middle age, obesity precedes the onset of diabetes. With our present knowledge we must conclude that overfeeding is wrong when glycosuria exists and that vitamin B supplements may be of value in diabetes (Meites, Feng and Wilwerth, 1957; Salvesen, 1957).

In man urinary 17-ketosteroids and androgen levels are subnormal in diabetes (Horstmann, 1950), and in the diabetic rat pituitary gonadotrophins are reduced (Shipley and Danley, 1947), but testis hyaluronidase does not change (Moore, 1948) . When hyperglycemia exists in rats, semen ■] carbohydrates increase (Mann and Lutwak-Mann, 1951).

Hypoglycemia influences the male reproductive organs. In rats tolbutamide or insulin produce lesions of the germinal epithelium which can be prevented by \' simultaneous administration of glucose. When 2 to 5 hypoglycemic comas are induced, such testis injuries increase progressively in number and frequency, and only a partial return to normal is observed a month later (Mancini, Izquierdo, Heinrich, Penhos and Gerschenfeld, 1959).

It is well known that the incidence of infertility in the pre-insulin era was high in young diabetic women. Fertility is also reduced in diabetic experimental animals, and rat estrous cycles are prolonged (Davis, Fugo and Lawrence, 1947) . Insulin is corrective (Sinden and Longwell, 1949; Ferret, Lindan and Morgans, 1950). Pregnancy in women with uncontrolled diabetes may terminate in abortion or stillbirth, possibly l)ecause toxemia of pregnancy is high (Pedersen. 1952). In rats pancreatectomy performed the 8th to 12th day of pregnancy increased the incidence of stillbirths (Hultciuist, 1950). In another experiment almost one- fourth of 163 animals with diabetes induced by alloxan on the 10th to 12th day of pregnancy died before parturition and

about 25 ])er cent of the survivors aborted (Angcrvall, 1959).

D. STERILE-OBESE SYNDROME

A sterile-obese syndrome in one colony of mice has been shown to be a recessive monogenic trait (Ingalls, Dickie and Snell, 1950). Obesity was transmitted to subsequent generations by way of ovaries that were transplanted from obese donors to nonobese recipients (Hummel, 1957). Obesity was transmitted by obese females receiving hormonal therapy and mated to obese males kept on restricted food intake (Smithberg and Runner, 1957). In addition to the investigations of the hereditary nature of the sterile-obese syndrome, the physiologic basis for the sterility has been studied in reference to the presence of germ cells, viability of ova and sperm, integrity of the ovary, and response of the uterus to estrogen (Drasher, Dickie and Lane, 1955). The data indicate that sterility in some obese males can be prevented by food restriction and that sterility in certain obese females can be corrected.

E. DIET AND THE LIVER

The concentration of hormones which reaches the target organs in the blood is the result of the rate of their production, metabolism, and excretion. How hypophyseal hormones are destroyed is not clear, but current data make it apparent that pituitary hormones have a short half-life in the circulatory system. Exerting a major control over circulating estrogen levels is the liver, with its steroid-inactivating systems. Zondek (1934) initially demonstrated that the liver could inactivate estrogens and this finding has had repeated confirmation (Cantarow, Paschkis, Rakoff and Hansen, 1943; De:\Ieio, Rakoff, Cantarow and Paschkis, 1948; Vanderlinde and Westerfield, 1950). Other steroids are also inactivated by the liver with several enzyme systems being involved; the relative concentration of these enzymes varies among species of vertebrates (Samuels, 1949).

The liver is a labile organ which readib.' responds to nutritional modifications; the induced liver changes alter the steroid-inactivating systems of this organ. Thus, inanition (Drill and Pfeiffer, 1946; Jailer,

G74

PHYSIOLOGY OF GONADS

1948) , vitamin B complex deficiency (Segaloff and Segaloff, 1944; Biskind, 1946), and protein restriction (Jailer and Seaman, 1950) all influence the capacity of the liver to detoxify steroids. Reduced protein intake is a primary factor in decreasing the effectiveness of the steroid-inactivating system (Jailer and Seaman, 1950; Vanderlinde and Westerfield, 1950). Rats fed an 8 per cent casein diet lose their capacity to inactivate estrone within 10 days. However, ascorbic acid alone or in combination with glutathione restored the estrone-inactivating system (Vasington, Parker, Headley and Vanderlinde, 1958).

Failure of steroids to be inactivated will influence the hypophyseal-gonadal axis. In turn the excess of estrogen will decrease gonadotrophin production by the hypophysis and thus reduce steroid production by the gonad. In addition nutritional modifications influence hypophyseal and possibly gonadal secretory capacity directly. Conceivably, nutritional alterations could modify the amount of steroid secreted or interfere with complete steroid synthesis by a gland.

Fatty infiltration of the liver and a general increase in fat deposition occur in fed and fasted rats after the injection of certain pituitary extracts and adrenal steroids and after the feeding of specific diets. Impaired estrogen inactivation has been associated with a fatty liver, but Szego and Barnes (1943) believe that the major influence is inanition. In fact, estrogens, especially ethinyl estradiol, interfere with fatty infiltration of the liver induced by a low protein diet (Gyorgy, Rose and Shipley, 1947) or by a choline-deficient diet (Emerson, Zamecnik and Nathanson, 1951). Stilbestrol, however, did not prevent the increase in liver fat induced by a protein-free diet (Glasser, 1957). Estrogens that are effective in preventing fatty infiltration may act by sparing methionine or choline or by inhibiting growth hormone (Flagge, Marasso and Zimmerman, 1958).

Ethionino, the antimetabolite of nu'thioniiK', Avill induce a fatty liver and inhibit hepatic protein synthesis in female, but not in male rats (Farber and Segaloff, 1955; Farber and Corban, 1958). Pretreatment of females with testosterone prevents

the ethionine effect, but this blockage of ethionine action need not be related to androgenic or progestational properties of steroids (Ranney and Drill, 1957).

III. Hypophysis and Diet

Studies involving acute and chronic starvation have shown that gonadal hypofunction during inanition is primarily due to diminished levels of circulating gonadotrophins. Because of the similarity to changes following hypophysectomy, the endocrine response to inanition has been referred to as "pseudohypophysectomy."

A. INANITION

The hypophysis has been implicated in human reproduction disturbances associated with undernutrition. Hypophyseal atrophy and a decrease in urinary gonadotrophins have been observed in chronic malnutrition (Klinefelter, Albright and Griswold, 1943; Zubiran and Gomez-]\Iont, 1953) and anorexia nervosa (Perloff, Lasche, Nodine, Schneeberg and Vieillard, 1954). Refeeding has restored urinary gonadotrophin levels in some cases, but hypoj^hyseal damage may result from severe food restriction at puberty (VoUmer, 1943; Samuels, 1948).

The influence of inanition on the reproductive organs of lal)oratory rodents is well recognized but the cft'ects on the hypophysis cannot be presented conclusively. In support of prior investigations, Mulinos and Pomerantz (1941a, b) in rats and Giroud and Desclaux (1945) in guinea pigs observed a hypophyseal atrophy following chronic underfeeding as well as a decrease in cell numbers and mitoses. In fact, refeeding after chronic starvation resulted in only a partial recovery of hypophyseal weight (Quimby, 1948). Nevertheless, complete starvation did not influence relative gland weight in female rats (Meites and Reed, 1949), and cytologic evidence (periodic acid-Schiff (PAS) test) of an estimated 3- fold increase in gonadoti'ophin content was claimed following chronic starvation (Pearse and Rinaldini, 1950). Assays of hypophyseal gonadotrophin content in chronically starved rats of both sexes have l)een reported as decreased (Mason and Wolfe, 1930; Werner,

NUTRITIONAL EFFECTS

675

1939), unchanged (,]\Iarrian and Parkes, 1929; Pomerantz and Mulinos, 1939; Maddock and Heller, 1947; Meites and Reed, 1949; Blivaiss, Hanson, Rosenzweig and McNeil, 1954), or increased (Rinaldini, 1949; Vanderlinde and Westerfield, 1950). An increase in pituitary gonadotrophin was evident when hormone content was related to milligrams of tissue (Meites and Reed, 1949). Thus, the hormone release mechanism may fail in starvation, and eventually gonadotrophin production will be reduced to a minimum (]\laddock and Heller, 1947).

Gonadectomy of fully fed rats is followed by an increase in hypophyseal gonadotrophin content. Chronic starvation, however, prevented the anticipated changes in the pituitary gland following gonadectomy in 8 of 12 female rats (Werner, 1939). On the other hand, if adult female rats were subjected to 14 days of reduced feeding 1 month after ovariectomy, no change in the elevated gonadotrophin levels was noted (Meites and Reed, 1949). In contrast, Gomez-Mont (1959) observed above normal urinary gonadotrophins in many menopausal and postmenopausal women despite undernutrition.

It is apparent that uniformity of opinion as to how starvation influences hypophyseal gonadotrophin content has not been attained. Several explanations can be given for the discrepancies. (1) There have been unfortunate variations in experimental design. IVIaddock and Heller (1947) starved rats for 12 days, whereas Rinaldini (1949) used a low calorie diet of bread and milk for 30 days. Other variations in feeding have included feeding one-half the intake required for growth (Mulinos and Pomerantz, 1941b I, regimens of full, one-half, oneciuarter, and no feeding for 7 and 14 days (Meites and Reed, 1949), and feeding inadequate amounts of a standard rat diet for 1 to 4 months (Werner, 1939). (2) Hypophyseal implants and anterior pituitary extracts should not be compared, for variable gonadotrophin production may follow implantation procedures, depending on whether necrosis or growth occurs (Maddock and Heller, 1947). (3) There has been an insufficient standardization of experimental materials. The assay animal has usually been the immature female rat, but

occasionally the immature mouse has been used, and Rinaldini (1949) used the hypophysectomized rat.

B. PROTEIN

The need for specific food elements by the hypophysis warrants consideration, for the hormones secreted by this gland are protein in nature and the amino acids for protein synthesis must be drawn from body sources. However, dietary protein levels can vary from 15 per cent to 30 per cent without influencing hypophyseal gonadotrophin content in rats (Weatherby and Reece, 1941), but diets containing 80 per cent to 90 per cent of casein increased hypophyseal gonadotrophin (TuchmannDuplessis and Aschkenasy-Lelu, 1948). Removal of protein from the diet will decrease hypophyseal gonadotrophin content in adult male rats in comparison with pair-fed and ad libitum-ied controls, but the decrease may or may not be significant in a 30-day period; luteinizing hormone (LH) seemed to be initially reduced. Extension of the period of protein depletion another 2 months resulted in a significant lowering of hy]iophyseal gonadotrophin levels (Table 12.4) (Leathern, 1958a). On the other hand, an increased FSH with no decrease in LH activity was observed in the hypophyses of adult female rats following 30 to 35 days of protein depletion (Srebnik and Nelson, 1957). The available data indicate that not only may a sex difference exist, but also that species may differ; restitution of gonadotrophin in the discharged rabbit pituitary was not influenced by in TABLE 12.4

Influence of a protein-free diet on hypophyseal

gonadotrophin content

(From J. H. Leathern, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on PFD*

Xo. of Rats

Anterior Pituitary Weight

Recipient Ovarv Weight

nig.

mg.

9

8.3

74

30

9

7.3

54

50

7

7.0

33

90

17

6.0

23

L^ntreated recipient ovarian weight = 15.4 mg.

• PFD = Protein-free diet.

67G

PHYSIOLOGY OF GONADS

adequate dietary protein (Friedman and Friedman, 1940).

When anterior pituitar}^ glands of 60gm. male rats were extracted and administered to immature female recipients, ovarian weight increased from 13.0 to 37.3 mg. After feeding 20 per cent casein or fox chow ad libitum for 14 days, the hypophyses of male rats contained almost twice as much gonadotrophin per milligram of tissue as did the hypophyses of the initial controls. Removal of protein from the diet for 14 days, however, reduced hypophyseal gonadotrophin concentration below the level of the initial controls (Leathem and Fisher, 1959).

Data on the hypophyseal hormone content as influenced by specific amino acid deficiencies have not come to the author's attention. Cytologically, however, Scott (1956) noted that an isoleucine-deficient diet depleted the pituitary gonadotrophic cells of their PAS-positive material and reduced the size of acidophilic cells. Omission of threonine, histidine, or tryptophan invoked similar effects. The changes probably represent the interference of a single amino acid deficiency with protein metabolism rather than specific effects attributable to the lack of amino acid itself. Excessive amino acid provided by injecting leucine, methionine, valine, tyrosine, or glycine caused release of gonadotrophin (Goth, Lengyel, Bencze, Saveley and Majsay, 1955).

Administration of 0.1 mg. stilbestrol for 20 days to adult male rats eliminated detectable hypophyseal gonadotrophins. Hormone levels returned during the postinjection period provided the diet contained adequate protein, whereas a protein-free diet markedly hindered the recovery of hypophyseal gonadotrophins. The gonadotrophin content of the pituitary gland correlated well with the recovery of the reproductive system, indicating that gonadotrophin production was subnormal on ]irotein-free feeding (Leathem, 1958a).

C. CAHBOHYDR.\TE .\ND FAT

Reproduction does not appear to be influenced by carbohydrates per se and hypophyseal alterations have not been noted.

Fat-deficient diets, however, do influence reproduction and the hypophysis exhibits cellular changes. Pituitary glands of female rats fed a fat-free diet contain a subnormal number of acidophiles and an increased number of basophiles (Panos and Finerty, 1953) . In male rats the feeding oi a fat-free diet increased hypophyseal basophiles, followed progessively by more castration changes (Finerty, Klein and Panos, 1957; Panos, Klein and Finerty, 1959).

D. VITAMINS

Despite the many investigations relating reproduction to vitamin requirements, relatively few have involved hypophyseal hormone estimations. Thus in 1955, Wooten, Nelson, Simpson and Evans reported the first definitive study which related pyridoxine deficiency to hypophyseal gonadotrophin content. Using the hypophysectomized rat for assay, pituitary glands from Bo-deficient rats were shown to have a 10-fold increase in FSH per milligram of tissue and a slightly increased LH content. Earlier studies had revealed that vitamin Bi-free diets decreased pituitary gonadotrophins in male rats (Evans and Simpson, 1930) and a similar effect of the folic acid antagonist, aminopterin, in the monkey was found later (Salhanick, Hisaw and Zarrow, 1952).

Male rats deficient in vitamin A exhibited a 43 per cent increase, and castrated vitamin A-deficient rats a 100 per cent increase in hypophyseal gonadotrophin potency over the normal controls (Mason and Wolfe, 1930). The increase of gonadotrophin was more marked in vitamin A-deficient male than in vitamin A-deficient female rats. Associated with the increase in hormone level was a significant increase in basophile cells (Sutton and Brief, 1939; Hodgson, Hall, Sweetman, Wiseman and Converse, 1946; Erb, Andrews, Hauge and King, 1947).

A re\-iew of the literature up to 1944 permitted Mason to suggest that the anterior hypophysis was not the instigator of reproductive disturbances in vitamin E deficiency. Nevertheless, Griesbach, Bell and Livingston (1957), in an analysis of the pituitary gland during progessive stages of

NUTRITIONAL EFFECTS

677

tocopherol deprivation, observed cytologic changes in the hypophysis which preceded testis changes. The "peripheral or FSH gonadotrophes" increased in number, size, and activity. The LH cells exhibited a hyperplasia of lesser extent, but possibly sufficient to increase LH in circulation and to cause hypertrophy of the male accessory glands. Gonadotrophic hormone content of pituitary glands from vitamin E-deficient rats may be decreased (Rowlands and Singer, 1936), unchanged (Biddulph and Meyer, 1941), or increased to a level between normal and that of the castrate, when the adult male rats were examined after 22 weeks on a deficient diet (Nelson, 1933; Drummond, Noble and Wright, 1939). Using hypophysectomized male rats as assay animals, evidence was obtained that FSH was increased in the pituitary glands of vitamin E-deficient male and female rats (P'an, Van Dyke, Kaunitz and Slanetz, 1949).

IV. Male Reproductive System

A. TESTIS

The two basic functions of the male gonads are to produce gametes and secrete steroids. Spermatogenic activity can be estimated from testis morphology and examination of semen samples. Androgen secretion can be estimated from urinary steroid levels, accessorj^ gland weight, and from analyses of accessory sex gland secretions, i.e., fructose and citric acid. In normal maturation in the rabbit, rat, boar, and bull, androgen secretion precedes spermatogenesis (Lutwak-Mann, 1958). On this basis it would appear that well fed young bulls may come into semen production 2 to 3 months sooner than poorly fed animals (Brat ton, 1957).

1. Inanition

Complete starvation will pre^•ent maturation of immature animals. Furthermore, marked undernutrition in 700 boys, 7 to 16 years of age, was associated with genital infantilism in 37 per cent and cryptorchidism in 27 per cent (Stephens, 1941). Restriction of food intake to one-half of the normal in maturing bull calves had a

marked delaying effect on the onset of seminal vesicle secretion, but a lesser delaying effect on spermatogenesis (Davies, Mann and Rowson, 1957) . Limiting the food intake to one-third of the normal did not prevent the immature rat testis from forming spermatozoa at the same time as their controls (Talbert and Hamilton, 1955). When testis maturation was prevented by inanition, a rapid growth and maturation occurred on refeeding (Ball, Barnes and Visscher, 1947; Quimby, 1948) but Schultze (1955) observed that full body size was not attained.

The reproductive organs of the adult are more resistant to changes imposed by diet than are those of the immature animal. Thus, Mann and Walton (1953) found that 23 weeks of underfeeding produced little change in sperm density and motility in mature animals although seminal vesicle function was reduced. Li the male rat testis hypofunction follows partial or complete starvation (]\Iason and Wolfe, 1930; Mulinos and Pomerantz, 1941a; Escudero, Herraiz and Mussmano, 1948), but there is no reduction in testicular nitrogen (Addis, Poo and Lew, 1936). Loss of Leydig cell function precedes cessation of spermatogenesis (Moore and Samuels, 1931) and is evident by the atrophy of the accessory sexual organs (^lulinos and Pomerantz, 1941a) and by an alteration in accessory gland secretion (Pazos and Huggins, 1945; Lutwak-]\Iann and Mann, 1950). Evidence of a tubular effect is provided by the lack of motile sperm (Reid, 1949). Severe dietary restriction is associated with the absence of spermatozoa in the seminiferous tubules and epididymis (Mason, 1933; Menze, 1941).

The human male suffering from chronic malnutrition exhibits hypogonadism. The testes atrophy and exhibit a decrease in size of the seminiferous tubules; basement membrane thickening and small Leydig cells are seen. These individuals excrete significantly subnormal amounts of 17-ketosteroids (Zubiran and Gomez-Mont, 19531. Acute starvation may also decrease urinary 17-ketosteroid and androgen levels as much as 50 per cent, with recovery evident on refeeding (Perloff, Lasche, Nodine, Schneeberg and Vieillard. 1954).

6/

PHYSIOLOGY OF GONADS

TABLE 12.5

Effect of diet on the testes of immature rats

(From J. H. Leathern, in Re-productive Phijsiology

and Protein Nutrition, Rutgers University

Press, New Brunswick, N. J., 1959.)

Sperm

Initial control . . . 20 per cent X 30

days

6 per cent X 30

days

3 per cent X 30

days

per cent X 30

days

per cent + 5 per cent liver. ,

per cent + 5 per cent yeast

G5 per cent X 30 davs

No. of Rats

Testis

Weight

mg.

mg./lOOgm.

10

329

825

10

1694

1035

16

824

890

12

380

930

10

140

346

10

112

291

10

119

296

10

1747

1040

100

50

100

2. Protein

The minimal amount of dietary protein which will support reproduction, lactation, and growth is 16.7 per cent (Goettsch, 1949) . Thus, it is not surprising that maturation of testes and accessory sex organs was prevented in immature rats (Horn, 1955) and mice (Leathern and DeFeo, 1952) when they were fed a protein-free diet for 15 to 30 days after weaning. Furthermore, supplements of 5 per cent liver to the casein-free diet had no effect. After a month, the testes, averaging 329 mg., decreased to 140 mg. in rats fed per cent casein, but the weight increased to an average of 1694 mg. and 1747 mg. in rats fed 20 per cent and 65 per cent casein, respectively (Table 12.5). Following protein depletion, there was a decrease in tubular ribonucleic acid and an increase in lipid. Accumulation of gonadal lipid in the inactive testis may be an abnormal assimilation of a degenerative nature or simply nonutilization. A diet containing 6 per cent casein permitted the formation of spermatozoa in some animals (Guilbert and Goss, 1932). When the 6 per cent casein diet was fed to immature animals for 30 days 50 per cent of the rats exhibited some spermatozoa; in addition, testis weight increased slightly and seminal

vesicle weight doubled, but body weight was not improved (Horn, 1955). Thus, as we noted earlier, the reproductive system may gain special consideration for protein allotments when supplies are limited.

Gain in testis weight in immature male rats and the biochemical composition of the immature testis are influenced by the nutritive value of the protein fed. The testes of normal immature rats contain 85 per cent water, 10.5 per cent protein, 4.5 per cent lipid, and detectable glycogen (Wolf and Leathern, 1955). Proteins of lower nutritive value (wheat gluten, peanut flour, gelatin ) may permit some increase in testis weight, but testis protein concentration decreased, percentage of water increased, and lipid and glycogen remained unchanged (Table 12.6). The enzyme ^fi-glucuronidase, which has frequently been associated with growth processes, exhibited no change in concentration as the testis matured or was jirevented from maturing by a protein-free diet (Leathem and Fisher, 1959). Not only are the weight and composition of the testis influenced by feeding proteins of varied biologic value, but the release of androgen is more markedly altered. When a 22-day-old male rat was fed a 20 per cent casein diet for 30 days, the seminal vesicle and ventral prostate weights increased 9- to 10-fold in comparison with initial control weight. Sub TABLE 12.6

Niiiritioiial effects on testis-coin position

in immature rats

(From R. C. Wolf and J. H. Leathem,

Endocrinology, 57, 286, 1955.)

20 pel' cent casein

20 per cent wheat gluten

20 per cent i)eanut flour

20 i)er cent gelatin

5 per cent casein

Fox chow

Initial control . .

No. of Rats

Final Body Weight

gm.

7

128

8

82

8

81

5

53

5

61

8

115

7

61

72.3j30.4 04.634.0

1468 1017

1257 66.1 2101

28.8

0.11 0.18 0.11 0.10

684,62.4 29.2 0.26 1515'70.3 30.3 0.15 273 72. 7:30. 10.19

NUTRITIONAL EFFECTS

679

stitution of wheat gluten and peanut flour for casein, limited the increase in the weight of the seminal vesicles to less than 100 per cent. In fact, seminal vesicle weight as related to body weight did not increase in animals fed 20 per cent wheat gluten.

The withholding of dietary protein from an immature rat for 30 days, during which time maturation occurs in the fully fed animal, did not impose a permanent damage. Refeeding of protein permitted the rapid recovery of testis weight and the appearance of spermatoza, 70 per cent of all animals having recovered in 30 days when fully fed, whereas only 25 per cent recovered when 6 per cent casein was fed. Recovery of androgen secretion was somewhat slower than that of the tubules as estimated by seminal vesicle weight.

Variations in protein quality are a reflection of amino acid patterns, and amino acid deficiencies interfere with testis maturation (Scott, 1956; Pomeranze, Piliero, Medeci and Plachta, 1959). Alterations in food intake which follow amino acid deficiencies have required forced feeding or pair-fed controls, but it is clear from what w^as found in the controls that the gonadal changes were not entirely due to inanition (Ershoff, 1952).

If the diet is varied so that caloric intake per gram is reduced to half while retaining the dietary casein level at 20 per cent, immature rat testis growth is prevented. The effect is unlike that obtained with this level of protein in the presence of adequate calories. Furthermore, the caloric restriction may increase testis glycogen (Leathem, 1959c).

Protein anabolic levels are higher in the tissues of young growing animals and the body is more dependent on dietary protein level and quality for maintenance of the metabolic nitrogen pool than in adult animals. On the other hand, body protein reserves in adult animals permit internal shifts of nitrogen to the metabolic pool and to tissues when dietary sources are reduced or endocrine imbalances are imposed. Thus, Cole, Guilbert and Goss (1932) fed a low protein diet to adult male rats for 60 to 90 days before the sperm disappeared, but the animals would not mate. Amount of semen and sperm produced by sheep have been re

TABLE 12.7

Arlult rat testes and seminal vesicles

after protein depletion

(From J. H. Leatliem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on

No. of

Testis

H2O

Protein

Total

Seminal

PFD*

Rats

Weight

Protein

Vesical

nig.

%

%dry

gm.

mg.

Control

9

2852

85.9

66.7

0.28

1276

30

9

2600

86.0

66.1

0.24

689

50

7

2398

85.4

64.1

0.22

320

90

25

1429

85.7

69.6

0.13

168

• PFD = Protein-free diet.

lated to the dietary protein level (Popoff and Okultilschew, 1936). Removal of protein from the diet for 30 days had little effect on the adult rat testis weight, spermatogenesis, or nitrogen content (Leathem, 1954). However, seminal vesicle w^eight was reduced 50 per cent (Aschkenasy, 1954). Prolonged protein depletion was required before the testis exhibited a loss in protein and a reduction in size. A loss of spermatozoa was not observed consistently, although some testes were completely atrophic (Table 12.7). Accessory organ weight decrease reflected the disappearance of androgen (Leathem, 1958a). Interstitial cell atrophy has also been noted in rats fed a low vegetable protein (cassava) diet (Adams, Fernand and Schnieden, 1958).

Sterility may or may not be induced with diets containing 65 per cent protein (Reid. 1949; Leathem, 1959c) but a 15 to 18 per cent dietary level of a poor protein such as maize or gelatin will decrease sperm motility and increase the number of abnormal sperm. The influence of proteins having different nutritional values in support of the growth of testes from the level to which they were depressed by stilbestrol indicated that casein, lactalbumin, and wheat gluten are equally competent to support testis growth whereas gelatin is deficient. Whole proteins may have several amino acid deficiencies, but the administration of amino acid antagonists may help to identify important individual amino acids. As an example, ethionine causes severe seminiferous tubule atrophy and Leydig cell hypoplasia (Kaufman, Klavins and Kinney, 1956; Goldberg, Pfau and Ungar, 1959). Studies in man have indicated a sharp reduction in

680

PHYSIOLOGY OF GONADS

spermatozoa after 9 days on an argininedeficient diet (Holt, Albanese, Shettles, Kajdi and Wangerin, 1942).

Adequate dietary protein cannot maintain reproductive function if the diet is calorie deficient. Thus, a decrease in seminal vesicle weight could be related to a decrease in dietary calories while protein levels were constant (Rosenthal and Allison, 1956). However, the accessory gland weight loss imposed by caloric restriction could be slowed by increasing the dietary protein (Rivero-Fontan, Paschkis, West and Cantarow, 1952).

Alterations in testis function imposed by inadequate protein are corrected when protein is returned to the diet at normal levels (Aschkenasy and Dray, 1953). Nevertheless, the nutritional state of the animal as a factor influencing recovery has been demonstrated with stilbestrol-treated adult male rats. While being fed an 18 per cent casein diet, adult male rats were injected with 0.1 mg. stilbestrol daily for 20 days. Testis weight decreased from 2848 to 842 mg., spermatogenesis was abolished, and testis water and protein content were significantly reduced. Despite a reduction in food intake, pair-fed controls exhibited no effect on reproductive organs. When a protein-free diet was substituted for the normal diet during the administration of stilbestrol, atrophy of the reproductive system was observed. Cessation of hormone administration was followed by a rapid return of testicular function toward normal when 18 per cent casein was fed both during the injection period and the recovery period. Within 30 days spermatogenesis and testicular composition were fully recovered. However, when 18 per cent casein was fed in the postinjection period to rats that had received a proteinfree diet while being given stilbestrol, recovery was clearly slow. After a month, spermatozoa were observed in only 30 per cent of the testes and testis weight was subnormal. Despite the seeming similarity of response by the two nutritional groups during the injection period, the postinjection recovery on identical dietary intake revealed marked differences in rate of recoverv (Leathem, 1958a).

3. Fat

Linoleic, linolenic, and arachidonic acids are designated as essentially fatty acids, but the physiologic role of these substances is not clearly understood. Nevertheless, the male reproductive organs are influenced by dietary essential fatty acid levels. High fat diets may enhance testicular weight (Kaunitz, Slanetz, Johnson and Guilmain, 1956) whereas removal of fat from the diet resulted in a degeneration of the seminiferous tubules as evidenced by intracellular vacuolation and a reduction in spermatids and spermatozoa (Panos and Finerty, 1954). After 5 months of feeding a fatfree diet, the rat testis may be devoid of sperm (Evans, Lepkovsky and Murphy, 1934). Testis degeneration occurred despite dietary supplements of vitamins A and E and in animals whose health appeared quite normal (Ferrando, Jacques, Mabboux and Prieur, 1955; Ferrando, Jacques, Mabboux and SoUogoub, 1955).

Weanling rats fed 14 per cent arachis (peanut) oil for 15 weeks exhibited a marked impairment of spermatogenesis (Aaes-Jorgensen, Funch and Dam, 1956) and 28 per cent arachis oil induced testicular damage of such an order that 15 weeks of feeding ethyl linoleate did not restore fertility (Aaes-Jorgensen, Funch and Dam, 1957).

4. Vitamins

Testicular dysfunction as judged by failure of sperm formation or atrophy of the secondary sex organs has been observed in deprivations of thiamine, riboflavin, pyridoxine, calcium pantothenate, biotin, and vitamins A and E. One must distinguish, however, between effects of inanition associated w^ith a vitamin deficiency and a specific vitamin effect (Skelton, 1950) ; one must also consider species differences (Biskind, 1946).

There is no question but that vitamin E deficiency in the rat results in a specific and irreversible damage to the testis. Tubular damage may proceed to the point where only Sertoli cells remain and yet the interstitial cells are not influenced (Mason, 1939). Similar changes followed vitamin E deficiency in the guinea pig (Pappenheimer and SchogolefT, 1944; Curto, 1954; Ingel

NUTRITIONAL EFFECTS

681

man-Siindberg, 1954) , hamster (Mason and Mauer, 1957), and bird (Herrick, Eide and Snow, 1952; Lowe, Morton, Cunningham and Vernon, 1957). However, little or no effect of an absence of vitamin E was noted in the rabbit (Mackenzie, 1942) and mouse (Bryan and Mason, 1941), or in live stock (Blaxter and Brown, 1952), or man (Lutwak-Mann, 1958), although vitamin E is present in human testes (Dju, Mason and Filer, 1958). Treatment of low-fertility farm animals with wheat germ oil or tocopherol or the use of this vitamin clinically have provided only inconclusive results (Beckmann, 1955) . Although some positive effects have been reported in man, the results may be due in part at least to the sparing action of tocopherol toward vitamin A.

Vitamin A deficiency influences the testis but changes are closely associated with the degree of inanition. In the rat, a vitamin deficiency sufficient to cause ocular lesions did not prevent sperm formation, but a deficiency of such proportions as to cause a body weight loss did cause atrophy of the germinal epithelium (Reid, 1949). Vitamin A deficiency will induce sterility in mice (McCarthy and Cerecedo, 1952). A gross vitamin deficiency in bulls before expected breeding age prevented breeding ; adult bulls may exhibit a lower quality semen but they remain fertile (Reid, 1949). Vitamin A deficiency induces metaplastic keratinization of the epithelium lining the male accessory sex organs (Follis, 1948) and thus may influence semen.

Testis damage induced by vitamin A deficiency can be reversed, but vitamin A therapy in man for oligospermia not due to vitamin lack was without effect (Home and Maddock, 1952) .

Age of the animal and dosage are factors which influence the results obtained in male rats with administered vitamin A. Immature male rats given 250 I.U. of vitamin A per gram of body weight daily exhibited a loss of spermatocytes, an effect which was accentuated by tocopherol (Maddock, Cohen and Wolbach, 1953). Little or no effect of similar treatment was observed in adult rats. The liver is the major storage depot for vitamin A and the fact that the male rat liver is more quickly depleted and less capa

ble of storage than is the liver of the female should be considered in any attempted correlation of the vitamin and hormone levels (Booth, 1952).

Other vitamin deficiencies have been shown to influence the testis. A lack of thiamine had little effect on testis weight, but did influence the Leydig cells and prevented growth of the accessory sex organs (Pecora and Highman, 1953). A chronic lack of ascorbic acid will cause a degeneration of both Leydig cells and seminiferous tubules. The effects of vitamin deficiency on the testis has been distinguished from those due to inanition and have been related to changes in carbohydrate metabolism (Mukherjee and Banerjee, 1954; Kocen and Cavazos, 1958) . The importance of ascorbic acid in the testis as related to function is not evident, but concentrations of this vitamin are maximal at 1 week of age (Coste, Delbarre and Lacronique, 1953).

Serious anatomic and functional impairments of testes were noted in pantothenic acid deficiency (Barboriak, Krehl, Cowgill and Whedon, 1957), and development of the rat testis and seminal vesicles was retarded by a biotin deficiency (Bishop and Kosarick, 1951; Katsh, Kosarick and Alpern, 1955), but the animals did not exhibit marked alterations in other endocrine organs (Delost and Terroine, 1954). Testosterone hastened the development of vitamin deficiency and enhanced the severity of biotin deficiency in both sexes, thereby suggesting a hormone-vitamin relationship (Okey, Pencharz and Lepkovsky, 1950). On the other hand, testosterone had no effect on the tolerance of mice for aminopterin, but castration increased the tolerance (Goldin, Greenspan, Goldberg and Schoenberg 1950).

B. INFLUENCE OF NUTRITION ON THE EE SPONSIVENESS OF MALE REPRODUCTIVE

TISSUES TO HORMONES

1. Testis

a. Inanition. The testes of birds on limited food intake were more responsive to hypophyseal gonadotrophin than fully fed birds (Byerly and Burrows, 1938; Breneman, 1940). In the rat several investigators have shown that the testis will respond to gonadotrophin despite inanition (Moore and Sara

682

PHYSIOLOGY OF GONADS

TABLE 12.8

Influence of diet and pregnant mare serum (PMS)

on testes and seminal vesicles of

immature male mice

(From V. J. DeFeo and J. H. Leathern,

unpublished.)

Diet (Per cent Protein X Days Fed)

per cent X 10 per cent X 10 per cent X 20 per cent X 20

l.u.

3

3

stage of Spermatogenesis

4 1

1 5 5

Spermatids

Seminal Vesicles

mg.

2.7 4.5

2.7 3.5

uels, 1931; Funk and Funk, 1939; Meites, 1953), with a stimulation of Leydig cells, an increase in testis size, and, in 40 days, a return of spermatozoa. Underfed males injected with gonadotrophin sired litters (Mulinos and Pomerantz, 1941a, b). Improved nutrition aided by unknown liver factors enhanced the response to androgen in severe human oligospermia (Glass and Russell, 1952).

b. Protein. Feeding a protein-deficient diet to adult male rats for 60 to 90 days did not prevent stimulation of the testes and seminal vesicles after pregnant mare's serum (PMS) administration (Cole, Guilbert and Goss, 1932). As we have noted, immature animals are prevented from maturing when diets lack protein. Nevertheless, a gonadal response to injected gonadotrophin was obtained in immature mice fed a protein-free diet for 13 days; tubules and Leydig cells were stimulated and androgen was secreted (Table 12.8). Refeeding alone permitted a recovery of spermatogenesis which was not hastened by concomitant PMS (Leathem, 1959c).

The maintenance of testis weight and spermatogenic activity with testosterone propionate in hypophysectomized adult male rats is well known, but these studies have involved adequate nutrition. If hypophysectomized rats were fed a protein-free diet and injected with 0.25 mg. testosterone propionate daily, testis weight and spermatogenesis were less well maintained than in rats fed protein. Testis protein concentra

tion was also reduced. These data suggest that influences of nutrition on the testis can be direct and are not entirely mediated through hypophyeal gonadotrophin changes (Leathem, 1959b).

c. Fat. The rat fed for 20 weeks on a fatfree diet exhibits a degeneration of the seminiferous epithelium within the first weeks which progresses rapidly thereafter. Chorionic gonadotrophin or rat pituitary extract started during the 20th week failed to counteract the tubular degeneration, but testosterone propionate proved effective (Finerty, Klein and Panos, 1957). The result shows that the ineffectiveness of the gonadotrophins could not be due to the failure of androgen release (Greenberg and Ershoff, 1951).

d. Vitamins. Gonadotrophins failed to promote spermatogenesis in vitamin A(Mason, 1939) or vitamin E- (]Mason, 1933; Geller, 1933; Drummond, Noble and Wright, 1939) deficient rats, but in another experiment the atrophic accessory sex organs of vitamin A-depleted rats were stimulated (Mayer and Goodard, 1951). Lack of vitamin A favored an enhanced response to PMS when the ratios of seminal vesicle weight to body weight were computed (Meites, 1953). The failure of gonadotrophins to stimulate testis tubules suggests a specific effect of avitaminosis A and E (Mason, 1933) on the responsiveness of the germinal epithelium.

Subnormal responses of rats to PMS, as measured by relative seminal vesicle weight, were obtained when there were individual vitamin B deficiencies, but the influence was due largely to inanition (Drill and Burrill, 1944; Meites, 1953). Nevertheless, sufficient response to chorionic gonadotrophin was obtained so that fructose and citric acid levels were restored to normal. Such an effect was not observed to follow dietary correction unless an unlimited food intake was allowed (Lutwak-Mann. 1958).

2. Sc

il W.siclfx and Prosfate

a. Inanition. Although the accessory reproductive organs resppnd to direct stimulation despite an inadequate food intake (Mooi'c and Samuels. 19311, tlio increase

NUTRITIONAL EFFECTS

683

in weight may be subnormal in mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a, Grayhack and Scott, 1952), or above normal in chickens (Breneman, 1940). Complete deprivation of food reduced the quantity of prostatic fluid in the dog, but exogenous androgen restored the volume, increased acid phosphatase, and induced tissue growth (Pazos and Huggins, 1945) .

6. Protein. The response of the seminal vesicles to androgen was investigated in immature rats, using weight and /5-glucuronidase as end points. Castration and 10 days on a protein-free diet preceded the 72-hour response to 0.25 mg. testosterone propionate. The lack of protein did not prevent a normal weight increase, and enzyme concentration was unchanged. If an 18 per cent diet was fed during the 3-day period that the androgen was acting, no improvement in weight response was noted, but enzyme concentration increased 100 per cent. Thus, when protein stores are depleted, the androgen response may be incomplete in the absence of dietary protein (Leathern, 1959c). Nevertheless, varied protein levels do not influence seminal vesicle weight-response when caloric intake is reduced (RiveroFontan, Paschkis, West and Cantarow, 1952).

c. Vitamins. Vitamin deficiencies do not prevent the seminal vesicles from responding to androgen. In fact, in vitamin B deficiency, testosterone restored fructose and citric acid levels to normal despite the need for thiamine in carbohydrate metabolism (Lutwak-Mann and Mann, 1950). In the male, unlike the female, the effects of folic acid deficiency in reducing responsiveness to administered androgen were largely due to inanition in both mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a) , and vitamin A deficiency which leads to virtual castration does not prevent an essentially normal response of the accessory glands to testosterone propionate (Mayer and Truant, 1949). Restricting the caloric intake of vitamin Adeficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient animals (Mason, 1939).

V. Female Reproductive System

A. OVARIES

1. Inanition

Mammalian species generally exhibit a delay in sexual maturation when food intake is subnormal before puberty, and ovarian atrophy with associated changes in cycles if inanition is imposed on adults. In human beings a decrease in fertility and a greater incidence of menstrual irregularities were induced by war famine (Zimmer, Weill and Dubois, 1944). Ovarian atrophy with associated amenorrhea and sterility were invoked by chronic undernutrition (Stephens, 1941). The ovarian morpliologic changes were similar to those of aging. Urinary estrogens were subnormal in 22 of 25 patients exhibiting amenorrhea associated with limited food intake (Zubiran and (_lomcz-Mont, 1953).

The nutritional requirements of jM'imates other than man have been studied in female baboons. The intake of vitamins and other essential nutrients was found to be of the same order as that recommended for man. Caloric intake varied with the menstrual cycle, being least during the follicular phase and maximal during the 2 to 7 days preceding menstruation (Gilbert and Gillman, 1956). Various diets were also studied to assess their importance in maintaining the normal menstrual rhythm. The feeding of (a) maize alone, (b) assorted vegetables and fruit, or (c) maize, skimmed milk, and fat led to menstrual irregularities or to amenorrhea. The mechanism regulating ovulation was the first to be deranged. The addition of various vitamins or of animal protein did not correct the menstrual disorders. However, inclusion of ox liver in the diet did maintain the menstrual rhythmicity, but the beneficial effect could not be attributed to its protein content (Gillman and Gilbert, 1956 ) .

In lower mammals that have been studied, inanition will hinder vaginal opening, and delay puberty and ovarian maturation and functioning. In adult rats and mice ostrous cycles are interrupted and the reprorUictive system becomes atrophic when body weight loss exceeds 15 per cent. The ovaries be

684

PHYSIOLOGY OF GONADS

come smaller, ovulation fails, and large vesicular follicles decrease in number with an increase in atresia, but primary follicles show a compensatory increase (Marrian and Parkes, 1929; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Guilbert, 1942; Bratton, 1957). The ovarian interstitial cells mav be markedly altered or absent (Huseby and Ball, 1945; Rinaldini, 1949) and the ovary may exhibit excessive luteinization (Arvy, Aschkenasy, AschkenasyLelu and Gabe, 1946) or regressing corpora lutea (Rinaldini, 1949) . However, the ovarian changes induced by inanition may be reversed by refeeding, with a return to reproductive capacity (Ball, Barnes and Visscher, 1947; Schultze, 1955). The effect of feed-level on the reproductive capacity of the ewe has been reported (El-Skukh, Nulet, Pope and Casida, 1955), but one must realize that high planes of nutrition may adversely influence fertility (Asdell, 1949). Nevertheless, additional protein and calcium added to an adequate diet extended the reproductive life span (Sherman, Pearson, Bal, McCarthy and Lanford, 1956).

2. Protein

The availability of just protein has an important influence on the female reproductive system. In immature rats ovarian maturation was prevented by feeding diets containing per cent to 1.5 per cent protein (Ryabinina, 1952) and low protein diets decreased the number of ova but without altering their ribonucleic acid (RNA) or glycogen content (Ishida, 1957). Refeeding 18 per cent protein for only 3 days was marked by the appearance of vesicular follicles and the release of estrogen in mice previously fed a protein-free diet (Leathem, 1958a). In experiments involving the opposite extreme, in which 90 per cent protein diets were used, a retardation of ovarian growth, and a delay in follicular maturation, in vaginal opening, and in the initiation of estrous cycles were noted (Aschkenasy-Lelu and Tuchmann-Duplessis, 1947; TuchniannDuplcssis and Aschkenasy-Lelu, 1948).

Adult female rats fed a protein-free diet for 30 days exhibited ovaries weighing 22 mg. compared with ovaries weighing 56 mg. from i)air-fed controls fed 18 per cent casein. Ovarian glycogen, ascorbic acid, and cho

lesterol were all influenced by protein deprivation and anestrum accompanied the ovarian changes. Furthermore, uterine weight and gl3^cogen decreased in rats fed protein-free diets (Leathem, 1959b).

In adult rats the feeding of 3.5 per cent to 5 per cent levels of protein (GuillDert and Gross, 1932) was followed by irregularity of the cycles or by their cessation. The cycles became normal when 20 to 30 per cent protein was fed (Aschkenasy-Lelu and Aschkenasy, 1947). However, abnormally high levels of casein (90 per cent) induced prolonged periods of constant estrus (Tuchmann-Duplessis and AschkenasyLelu, 1947). Nevertheless, not all species responded to protein depletion in the same manner. For example, the rabbit exhibited estrus and ovulation despite a 25 per cent body weight loss imposed by to 2 per cent protein diets (Friedman and Friedman, 1940).

Despite a normal level of protein in the diet, inadequate calories will interfere with reproductive function and induce ovarian atrophy (Escudero, Herraiz and Mussmano, 1948; Rivero-Fontan, Paschkis, West and Cantarow, 1952). Furthermore, the effects of 15 per cent and 56 per cent protein levels on estrous cycles could not be distinguished when calories were reduced 50 per cent (Lee, King and Visscher, 1952). Returning mice to full feeding after months of caloric deficiency resulted in a sharp increase in reproductive performance well above that expected for the age of the animal (Visscher, King and Lee, 1952). This type of rebound phenomenon has not been explained.

Reproductive failure assigned to dietary protein may be a reflection of protein quality as well as level. Specific amino acid deficiencies lead to cessation of estrus (White and AVhite, 1942; Berg and Rohse, 1947) and thus feeding gelatin or wheat as the protein source and at an 18 per cent level was quickly followed by an anestrum (Leathem, 1959b). Supplementation of the wheat diet with lysine corrected the reproductive abnormalities (Courrier and Raynaud, 1932), but neither lysine (Pearson, Hart and Bohstedt, 1937) nor cystine (Pearson, 1936) added to a low casein diet was beneficial. Control of food intake must be considered in studies involving amino

NUTRITIONAL EFFECTS

685

acids, for a deficiency or an excess can create an imbalance and alter appetite. Opportunity to study the amino acids in reproduction is now possible because of the work of Greenstein, Birnbaum, Winitz and Otey (1957) and Schultze (1956) , who maintained rats for two or more generations on synthetic diets containing amino acids as the only source of protein. Similarly, the amino acid needs for egg-laying in hens has been reported (Fisher, 1957). Tissue culture methods also permit the study of the nutritional requirements of embryonic gonadal tissue, the success of avian gonadal tissue in culture being judged by survival, growth, and differentiation. In experiments in which this technique was used it was found that a medium made up of amino acids as the basic nitrogen source can maintain gonadal explants very successfully, even though the choice of amino acids does not exactly correspond to the 10 essential amino acids recommended for postnatal growth (StengerHaffen and Wolff, 1957).

3. Carbohydrate

The absence of dietary carbohydrate does not appreciably affect the regularity of estrous cycles in rats provided the caloric need is met. However, the substitution of 20 per cent sucrose for corn starch induced precocious sexual maturity which was followed by sterility (Whitnah and Bogart, 1956). The ovaries contained corpora lutea, but the excessive luteinization of unruptured follicles suggested a hypophyseal disturbance. Substitution of 20 per cent lactose for corn starch had no effect. Increased amounts of lactose retarded the gain in body weight and blocked ovarian maturation, possibly because the animal could not hydrolyze adequate amounts of the disaccharide. Addition of whole liver powder to the diet counteracted the depressing action of 45 per cent lactose on the ovary (Ershoff, 1949).

4. Fat

There seems to be little doubt that dietary fat is reciuired for normal cyclic activity, successful pregnancy, and lactation, and that the requirements for essential fatty acids are lower in females than in males (Deuel, 1956).

Conception, fetal development, and par

turition can take place in animals fed a diet deficient in fatty acids (Deuel, Martin and Alfin-Slater, 1954) , despite a reduction in total carcass arachidonic acid (Kummerow. Pan and Hickman, 1952). Earlier reports indicated that a deficiency of essential fatty acids caused irregular ovulation and impaired reproduction (Burr and Burr, 1930; Maeder, 1937). The large pale ovaries lead Sherman (1941) to relate essential fatty acid deficiency to carotene metabolism. In this regard the removal of essential fatty acids from an adequate diet supplemented with vitamin A and E lead to anestrum and sterility while maintaining good health (Ferrando, Jacques, Mabboux and Prieur, 1955). Perhaps the differences in opinion regarding the effects of fatty acid deficiency can be related to the duration of the experimental period. Panos and Finerty (1953) found that growing rats placed on a fat-free diet exhibited a normal time for vaginal opening, normal ovarian weight, follicles, and corpora lutea, although interstitial cells were atrophic. However, regular estrous cycles were noted for only 20 weeks, thereafter 60 per cent of the animals exhibited irregular cycles.

A decrease in reproductive function may be invoked by adding 14 per cent arachis oil to the diet (Aaes-Jorgensen, Funch and Dam, 1956) . Increasing dietary fat by adding rape oil did not influence ovarian function but did cause the accumulation of ovarian and adrenal cholesterol (Carroll and Noble, 1952).

Essential fatty acid deficiency is associated with underdevelopment and atrophic changes of the uterine mucosa. Adding fat to a stock diet enhanced uterine weight in young animals at a more rapid pace than body weight (Umberger and Gass, 1958) .

5. Vitamins

Carotenoid pigments are present in the gonads of many vertebrates and marine invertebrates, and, in mammals, are particularly prominent in the corpus luteum. However, no progress has been made in determining either the importance of the carotenoids in the ovary or of the factors controlling their concentrations. It is well known that vitamin A deficiency induces

686

PHYSIOLOGY OF GONADS

a characteristic keratinizing metaplasia of the uterus and vagina, but estrous cycles continue despite the vaginal mucosal changes. Furthermore, ovulation occurs regularly until advanced stages of deficiency appear. The estrous cycle becomes irregular in cattle fed for a long period of time on fodder deficient in carotene. The corpora lutea fail to regress at the normal rate and ovarian follicles become atretic and cystic ( Jaskowski, Watkowski, Dobrowolska and Domanski, cited by Lutwak-Mann, 1958). The alterations in reproductive organs associated with a lack of vitamin A may be due in part to a vitamin E deficiency since the latter enhances the rate at which liver stores of vitamin A are depleted.

Definite effects of hypervitaminosis A have been observed on reproduction. Masin (1950) noted that estrus in female rats could be prolonged by administration of 37,000 I.U. of vitamin A daily. The implications, however, have not been studied. The effect of hypervitaminosis A may actually induce secondary hypovitaminoses. The displacement of vitamin K by excess A is almost certain and similar relationships appear to exist with vitamin D (Nieman and Klein Obbink, 1954).

The failure of vitamin E-deficient female rats to become pregnant is apparently due to disturbances of the implantation process rather than to the failure of ovulation. There is no direct proof of ovarian dysfunction (Blandau, Kaunitz and Slanetz, 1949). However, the ovary of the rat deficient in vitamin E may have more connective tissue and pigment, and Kaunitz (1955) showed by ovarian transplantation that some nonspecific ovarian dysfunction appears to exist (cited by Cheng, 1959 (. Vitamin E is essential for birds, but there is little evidence for a dependency in most mammals; sheep, cows, goats, and pigs have been studied. Treatment of low-fertility farm animals with tocopherol has not provided conclusive data favoring its use (Lutwak-Mann, 1958), nor has the treatment of human females been rewarded with any indication that vitamin E might be helpful in cases of abnormal cycles and habitual abortion (Beckmann, 1955).

No specific reproductive disturbances in man, the rhesus monkey, or the guinea pig

have been associated with vitamin C deficiency (Mason, 1939). Nevertheless, the high ascorbic acid content of ovarian and luteal tissue and of the adrenal cortex suggests a physiologic role in association with steroid synthesis. (3varian ascorbic acid varies with the estrous cycle, dropping sharply in the proestrum (Coste, Delbarre and Lacronique, 1953), and decreasing in resjionse to gonadotrophin (Hokfelt, 1950; Parlow, 1958). Virtually no ascorbic acid is present in bovine follicular fluid (LutwakMann, 1954) or in rat ovarian cyst fluid (Blye and Leathem, 1959). Uterine ascorbic acid decreased in immature mice treated with estrogen, but remained unchanged in rats following thiouracil administration (Leathem, 1959a). Its role in the uterus awaits elucidation.

Delayed sexual maturation and ovarian atrophy have been described when there are deficiencies of thiamine, riboflavin, pyridoxine, pantothenic acid, biotin, and B12 (Ershoff, 1952; Ullrey, Becker, Terrill and Notzold, 1955). However, as we noted when deficiencies of the vitamins were being considered, much of the impairment of reproductive function can be related to inanition rather than to a vitamin deficiency (Drill and Burrill, 1944). Pyridoxine deficiency, although not affecting structure (Morris, Dunn and Wagner, 1953) , markedly reduces the sensitivity of the ovary to administered gonadotrophin (Wooten, Nelson, Simpson and Evans, 1958) .

Bird, frog, and fish eggs contain considerable quantities of vitamins. In fact, the daily human requirements for vitamins may be contained in a hen's egg and thus it is not surprising that hatchability is decreased l)y virtually any vitamin deficiency. Lutwak-Mann (1958) has provided an excellent survey of these data with numerous references to studies of frogs and fishes. Nearly all the B vitamins are present in fish roe and the pantothenic acid concentration in cod ovaries {Gadus morrhua) exceeds most otlicr natural sources. The amount of the latter varies with the reproductive cycle, d(>creasing to its lowest level before spawning. Riboflavin and vitamin B12 , on the other h;ui(l, do not change (Braekkan, 1955).

NUTRITIONAL EFFECTS

687

B. INFLUENCE OF NUTRITION ON THE RESPONSIVENESS OF FEMALE REPRODUCTIVE TISSUES TO HORMONES

1. Ovary

a. Inanition. Marrian and Parkes (1929) were the first to show that the quiescent ovary of the underfed rat can respond to injections of anterior pituitary as evidenced by ovulation and estrous smears. Subsequently the ovaries of underfed birds, rats, and guinea pigs were found to be responsive to serum gonadotrophin (Werner, 1939; Stephens and Allen, 1941; Mulinos and Pomerantz, 1941b; Hosoda, Kaneko, Mogi and Abe, 1956). A low calorie bread-andmilk diet for 30 days did not prevent ovarian response to rat anterior pituitary or to chorionic gonadotrophin. In these animals an increase in ovarian weight with repair of interstitial tissue, as well as folhcle stimulation and corpus luteum formation, were observed (Rinaldini, 1949). Rats from which food had been withdrawn for 12 days could respond to castrated rat pituitary extract with an increase in ovarian and uterine weight (Maddock and Heller, 1947). Nevertheless, differences in the time and degree of responsiveness to administered gonadotrophin were noted in rabbits. Animals on a high plane of nutrition responded to gonadotrophin at 12 weeks, whereas rabbits on a low plane of nutrition responded at 20 weeks and fewer eggs were shed (Adams, 1953).

b. Protein. Protein or amino acid deficiencies in the rat do not prevent a response to administered gonadotrophin (Cole, Guilbert and Goss, 1932; Courrier and Raynaud, 1932) . However, the degree and type of gonadal response is influenced by the diet. Thus, immature female mice fed to 6 per cent casein for 13 days exhibited only follicular growth in response to pregnant mare serum, whereas the ovarian response in mice fed 18 per cent casein was suggestive of follicle-stimulating and strongly luteinizing actions (Table 12.9). Furthermore, the ovarian response was significantly less after 20 days of nonprotein feeding than after 10 days of depletion (Leathem, 1958a). Ovarian stimulation by a gonadotrophin involves tissue protein synthesis and thus the type of whole protein fed

could influence the responses. Yamamoto and Chow (1950) fed casein, lactalbumin, soybean, and wheat gluten at 20 per cent levels and noted that the response to gonadotrophin as estimated by tissue nitrogen was related to the nutritive value of the protein. The ovarian weight response to chorionic gonadotrophin was less in rats fed 20 per cent gelatin than those fed 20 per cent casein (Leathem, 1959b). Inasmuch as the hypophysis may influence the gonadal response to injected hormone despite the diet, hypophysectomized rats fed a protein-free diet for 5 weeks and hyophysectomized rats on a complete diet were tested for response to gonadotrophins. The response to FSH was not influenced by diet, but the protein-depleted rats were twice as sensitive to interstitial cell-stimulating hormone (ICSH), human chorionic gonadotrophin (HCG), and PMS as the normal rats (Srebnik, Nelson and Simpson, 1958). Protein-depleted, normal mice were twice as sensitive to PMS as fully fed mice (Leathem and Defeo, 1952 1 .

c. Vitamins. In the female vitamin B deficiencies do not prevent ovarian responses to gonadotrophin (Figge and Allen, 1942), but the number of studies is limited. Be deficiency in DBA mice was associated with an increased sensitivity of the ovary to gonadotrophins (Morris, Dunn and Wagner, 1953), whereas pyridoxine deficiency in the rat decreased ovarian sensitivity, especially to FSH (Wooten, Nelson, Simp TABLE 12.9

Influence of dietary protein and pregnant mare

serum {PMS) on the mouse ovary

(From J. H. Leathem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Diet fPer cent Protein X Days Fed)

c-5,

•g-s

1*

k

r

1 11

c 1

I.U.

mg.

mg.

per cent X 23

1.2

4.8

per cent X 2.3

3

2.8

13

10.8

per cent X 13

1.4

4.9

per cent X 13

3

4.4

16

1

15.1

6 per cent X 13

3.2

6

7.7

6 per cent X 13

3

5.6

12

1

31.9

18 per cent X 13

5.0

10

2

51.6

18 per cent X 13

3

8.0

7

4

51.3

088

PHYSIOLOGY OF GONADS

son and Evans, 1958 j. Administration of vitamin C concomitant witli gonadotropliin has been claimed to enhance ovarian response (DiCio and Schteingart, 1942), but in another study the addition of ascorbic acid inhibited the hiteinizing and ovulating action of the gonadotrophin (Desaive, 1956).

Whether induced by vitamin deficiency or by inanition, the anestrum in rats which follows 2 to 3 weeks' feeding of a vitamin B-deficient diet has been explored as a method for the assay of gonadotrophin. Pugsley (1957) has shown that there is considerable convenience of method and a satisfactory precision of response for the assay of HCG and pregnant mare serum.

2. Uterus and Vagina

a. Inanition. Limited food intake does not prevent an increase in uterine weight after estrogen. Testosterone propionate will markedly increase uterine growth despite a 50 per cent reduction in food intake (Leathem, Nocenti and Granitsas, 1956). Furthermore, dietary manipulations involving caloric and protein levels did not prevent the uteri of spayed rats from responding to estrogen (Vanderlinde and Westerfield, 1950). More specific biochemical and physiologic responses must be measured because starvation for 4-day periods clearly interferes with deciduoma formation (DeFeo and Rothchild, 1953). A start in the direction of studying tissuecomposition changes has been made by measuring glycogen. However, no changes were noted in uterine glycogen in fasting rats (Walaas, 1952), and estrogen promoted glycogen deposition in the uteri of starved rats as well as in the uteri of fully fed rats (Bo and Atkinson, 1953).

b. Fat. Interest in the hormone content of fat from the tissues of animals treated with estrogen for the purpose of increasing body weight has raised the question of tissue hormone content. If estrogen was to be detected in tissues, an increase in dietary fat was necessary. However, the increase in dietary fat decreased the uterine response to stilbcstrol (I'mberger and Gass, 1958), thus complicating the assay.

c. Vitainins. Stimulation of the uterus by estrogen does not require tliianiinc, ribo

flavin, pyridoxine, or pantothenic acid. On the other hand, a deficiency of nicotinic acid appears to enhance the response to low doses of estrogen (Kline and Dorfman, 1951a, b). However, Bio appears to be needed for optimal oviduct response (Kline, 1955) and is required for methyl group synthesis from various one-carbon precursors including serine and glycine (Johnson, 1958).

Response of the bird oviduct to stilbestrol requires folic acid (Hertz, 1945, 1948). It was shown subsequently that stilbestrol and estrone effects in frogs, rats, and the rhesus monkey also require folic acid. A folic acid deficiency can be induced by feeding aminopterin. In this way the estrogen effects can be prevented. Aminopterin also prevents the action of progesterone in deciduoma formation, from which it may be inferred that folic acid is necessary for deciduoma formation in the rat. Increased steroid or folic acid levels can reverse the antagonist's effect (Velardo and Hisaw, 1953).

The mechanism of folic acid action is not clear. It may function in fundamental metabolic reactions linked with nucleic acid synthesis. Brown (1953) showed that desoxyribonucleic acid could be substituted for folic acid in the bird. In the rat aminopterin interferes with the increase in uterine nucleic acids, and with nitrogen and P-^- uptake by nucleic acids following estrogen. Folic acid has been implicated in the metabolism of several amino acids (Davis, Meyer and McShan, 1956).

Rats ovariectomized at weaning and maintained on a vitamin E-free diet for 6 weeks to 10 months responded to estradiol in the same manner as rats supplemented with tocopherol. This finding suggests that an intimate physiologic relationship between estradiol and vitamin E is not very probable (Kaunitz, Slanetz and Atkinson, 1949). Nevertheless, vitamin E has been re]:»orted to act synergistically with ovarian hormones in dc^ciduoma formation (Kehl, Douard and Lanfranchi. 1951 ) and to influence nucleic acid turnover (Dinning. Simc and Day, 1956).

A vitamin-hormone interrelationship is apparent when estrogen and vitamin A are considered. Vitamin A-deficient female rats present evidence of a metaplastic uterine

NUTRITIONAL EFFECTS

689

epithelium in 11 to 13 weeks, but similar changes failed to develop in ovariectomized rats. Vitamin A-deficient castrated rats quickly developed symptoms of metaplasia when estrogen alone was administered, but no adverse effect followed the administration of estrogen combined with vitamin A (Bo, 1955, 1956). The vagina is different. Its epithelium becomes cornified in vitamin A-deficient normal and castrated rats. The cornification is histologically indistinguishable from that occurring in the estrous rat and can be prevented by vitamin A. In fact, vitamin A will quantitatively inhibit the effect of estrogen on the vaginal mucosa when both are applied locally (Kahn, 1954). Conversion of ^-carotene to vitamin A is influenced by tocopherol, vitamin Bi2 , insulin, and thyroid, with evidence for and against a similar action by cortisone (Lowe and Morton, 1956; Rice and Bo, 1958). An additional vitamin-hormone relationship is suggested by the augmentation of progesterone action in rabbits given vitamin Do .

3. Mammary Gland

Inanition prevents mammary growth, but feeding above recommended requirements for maintenance and growth from birth to the first parturition also seems to interfere with mammary growth. Furthermore, steroid stimulation of the mammary gland is influenced by nutritional factors. Using the male mouse, Trentin and Turner (1941) showed that as food intake decreased, the amount of estradiol required to produce a minimal duct growth w^as proportionately increased. In the immature male rat a limited food intake prevented the growth of the mammary gland exhibited by fully fed controls. Nevertheless, the gland was competent to respond to estrogen (Reece, 1950) . Inasmuch as the glands of force-fed hypophysectomized rats did not respond to estrogen (Samuels, Reinecke and Peterson, 1941; Ahren, 1959), one can assume that, despite inanition, a hypophyseal factor was present to permit the response of the mammary gland to estrogen. However, inanition (IMeites and Reed, 1949) , but not vitamin deficiencies (Reece, Turner, Hathaway and Davis, 1937), did reduce the content of hypophyseal lactogen in the rat.

Growth of the mammary gland duct in the male rat in response to estradiol requires a minimum of 6 per cent casein. Protein levels of 3 per cent and per cent failed to support growth of the duct (Reece, 1959) .

C. PREGNANCY

The human male after attaining adulthood is confronted with the problem of maintaining the body tissues built up during the growth period. However, in the human female it has been estimated that the replacement of menstrual losses may require the synthesis of tissue equivalent to 100 per cent of her body weight (Flodin, 19531. In the event of pregnancy and in all viviparous species, the female is presented with even more formidable demands and a limitation of nutritional needs can lead to loss of the embryo or fetus. The role of nutrition at this point in reproduction has always received considerable attention and is complicated by the circumstance that many food substances influence pregnancy (Jackson, 1959). However, in many instances there is no evidence that fetal loss or malformation induced by nutritional modifications has been the consequence of an endocrine imbalance and thus limitation of the immense literature is permissible.

During the first 15 days of pregnancy, a rat may gain 50 gm. Since the fetuses and placentas are small, most of the gain is maternal and is associated with an' increase in food intake of as much as 100 calories per kilogram of body weight (Morrison, 1956). During the first 2 weeks of pregnancy, marked storage of fat and water occurs in the maternal body and the animal's positive nitrogen balance is above normal. Liver fat also increases (Shipley, Chudzik, Curtiss and Price, 1953). The increased food intake in early pregnancy may therefore provide a reserve for late fetal growth, as food intake may decline to 65 per cent of the general pregnancy level during the last 7 days (Morrison, 1956). During this last week, fetal growth is rapid. The rapid growth has been related to (1) greater demands of the fetus, (2) greater amounts of food in the maternal blood, and (31 greater permeability of the placenta. Certainly the anabolic potential of fetal tissues is high and the mother can lose weight while the

690

PHYSIOLOGY OF GONADS

fetuses gain. But it is also important to recall that there is a shift in protein, because its distribution in organs of pregnant rats differs from that in nonpregnant animals (Poo, Lew and Addis, 1939). Other changes in the maternal organism were enumerated b}^ Newton (1952) and by Souders and Morgan (1957).

A measure of nitrogen balance during pregnancy, rather than weight of young at birth, has been suggested as a means of determining a diet adequate for reproduction (Pike, Suder and Ross, 1954). After the 15th day, a retention of body protein increases, blood amino nitrogen and amino acids decrease, and urea formation decreases. These metabolic activities suggest an increase in growth hormone although the levels of this hormone have not been estimated (Beaton, Ryu and McHenry, 1955). Placental secretions have also been associated with the active anabolic state of the second half of pregnancy, because removal of the fetuses in the rat did not change the anabolic activity, whereas removal of the placentas was followed by a return to normal (Bourdel, 1957). A sharp increase in liver ribose nucleic acid has been observed during late pregnancy in mice and rats and the effect attributed to a placental secretion or to estrogen. Species differences also influence the results because only a modest change in liver RNA was observed in guinea pigs and no change occurred in cats (Campbell and Kostcrlitz, 1953; Campbell, Innes and Kosterlitz, 1953a, b).

Clinical observations have related both

TABLE 12.10

Nutrition and pregnancy in rats

(From J. H. Leathern, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Calories/kg. Body Weight

Fetuses, Day 20

No.

Average weight

18 per cent casein

18 per cent casein

6 per cent casein

per cent casein

18 per cent gelatin

18 per cent gelatin

200 100

250 200 200 100

8 6

gm. 6.1

3.5

toxemia of pregnancy (Pequignot, 1956) and prematurity to inadequate nutrition (Jeans, Smith and Stearns, 1955). The potential role of protein deprivation in the pathogenesis of the toxemia of pregnancy prompted studies in sheep and rats. In sheep nutritionally induced toxemia simulates the spontaneous toxemia (Parry and Taylor, 1956), but only certain aspects of toxemia were observed in the pregnant rat subjected to low protein diets. When rats were fed 5 per cent casein and mated, fluid retention was observed (Shipley, Chudzik, Curtiss and Price, 1953) and pregnancy was completed in only 48 per cent of the animals Curtiss, 1953). Gain in body weight in the adult rat and gain in fetal weight were subnormal as the result of a low protein feeding during pregnancy.

Complete removal of protein from the diet beginning at the time of mating did not prevent implantation but did induce an 86 to 100 per cent embryonic loss. The effect was not related solely to food intake (Nelson and Evans, 1953) , as we will see in what follows when the relationship between protein deficiency and the supply of estrogen and progesterone is described. Limiting protein deprivation to the first 9 to 10 days of pregnancy will also terminate a pregnancy, but when the protein was removed from the diet during only the last week of pregnancy, the maternal weight decreased without an effect on fetal or placental weight (Campbell and Kosterlitz, 1953). As would be anticipated, a successful pregnancy requires protein of good nutritional quality and the caloric intake must be adequate. Thus, an 18 per cent gelatin diet failed to maintain pregnancy when 200 calories per kilogram were fed, whereas a similar level of casein was adequate (Table 12.10). However, reducing caloric intake to 100 calories despite an otherwise adequate protein ration influenced the number and size of fetuses (Leathern, 1959b). Additional proteins should be studied and related to biochemical changes in pregnancy and to the need for specific amino acids; for example, elimination of methionine or tryptophan from the diet may or may not be followed by resorption (Sims, 1951; Kemeny, Handel, Kertesz and Sos, 1953; Albanese, Randall and Holt, 1943). Excretion of 10 amino acids was in

NUTRITIONAL EFFECTS

691

creased during normal human pregnancy (Miller, Ruttinger and Macey, 1954).

That a relationship exists, between the dietary requirements just described to the endocrine substances which participate in the control of pregnancy, is suggested by the fact that the deleterious effects of a proteinfree diet on pregnancy in rats have been counteracted by the administration of estrone and progesterone. Pregnancy was maintained in 30 per cent, 60 to 80 per cent, and per cent of protein-deficient animals by daily dosages of 0.5 /xg., 1 to 3 fig., and 6 jug. estrone, respectively. On the other hand, injection of 4 to 8 mg. progesterone alone maintained pregnancy in 70 per cent of the animals (Nelson and Evans, 1955) , and an injection of 4 mg. progesterone with 0.5 //.g. estrone provided complete replacement therapy (Nelson and Evans, 1954). Food intake did not increase. The results suggest that reproductive failure in the absence of dietary protein was due initially to lack of progesterone and secondarily to estrogen, the estrogen possibly serving as an indirect stimulation for luteotrophin secretion and release. It is well known that hypophysectomy or ovariectomy shortly after breeding will terminate a pregnancy and that replacement therapy requires both ovarian hormones. Thus, the protein-deficient state differs somewhat from the state following hypophysectomy or ovariectomy, but the factors involved are not known.

Pregnancy alters nutritional and metabolic conditions in such a way that labile protein stores of the liver and other parts of the body are influenced, but similar effects are imposed by a transplanted tumor, especially when it reaches 10 per cent of the body weight. ' Thus, transplantation of a tumor into a pregnant animal would place the fetuses in competition with the tumor for the amino acids of the metabolic pool. Under these circumstances will the pregnancy be maintained? An answer to the question may not yet be given. Nevertheless, Bly, Drevets and Migliarese (1955) observed various degrees of fetal damage in pregnant rats bearing the Walker 256 tumor, and 43 per cent fetal loss was obtained with a small hepatoma (Paschkis and Cantarow, 1958).

Essential fatty acid deficiency, at least in the initial stages, does not interfere with

development of the fetuses or parturition in the rat, but the pups may be born dead or they do not survive more than a few days (Kummerow, Pan and Hickman, 1952). A more pronounced deficiency has induced atrophic changes in the decidua, resorption of fetuses, and prolonged gestation. Death of the fetuses appears to be secondary to placental injury. Hormonal involvement, if any, when there is fatty acid deficiency and pregnancy seems not to have been investigated.

Pregnancy and lactation are major factors influencing vitamin requirements. It is not surprising, therefore, that vitamin deficiencies influence the course of a pregnancy. The subject has recently been reviewed by Lutwak-jMann (1958).

A deficiency of vitamin A does not noticeably affect early fetal development, but later in gestation placental degeneration occurs with hemorrhage and abortion. When the deficiency is moderate the pregnancy is not interrupted, but the fetuses are damaged (Warkany and Schraffenberger, 1944; Wilson, Roth and Warkany, 1953; Giroud and Martinet, 1959). In calves and pigs the abnormalities are associated with the eyes and palate (Guilbert, 1942) ; in birds skeletal abnormalities are seen (Asmundson and Kratzer, 1952). The use of hormones in an effort to counteract the effects seems to have been attempted only in the rabbit where 12.5 mg. progesterone improved reproduction impaired by vitamin A lack (Hays and Kendall, 1956). Vitamin A excess also proves highly detrimental to pregnancy, as resorption and malformations occur. Administration of excessive vitamin A on days 11 to 13 of pregnancy induced cleft palate in 90 per cent of the embryos (Giroud and Martinet, 1955) . In another experiment the effect of excessive vitamin A was augmented by cortisone (Woollam and Millen, 1957).

Vitamin E deficiency has long been known to influence pregnancy in rodents and fetal death appears to precede placental damage and involution of the corpora lutea. Gross observations of the abnormal embryos have been reported (Cheng, Chang and Bairnson, 1957). Estrogen, progesterone, and lactogen were not effective in attempts at corrective therapy (Ershoff, 1943), but estrone and progesterone markedly reduced the in

692

PHYSIOLOGY OF GONADS

cidence of congenital malformations associated with vitamin E lack (Cheng, 1959). In the test of a possible converse relationship, estradiol-induced abortion in guinea pigs was not prevented by vitamin E (Ingelman-Sundberg, 1958) .

Fat-soluble vitamins incorporated in the diet may be destroyed by oxidation of the unsaturated fatty acids. To stabilize the vitamins, the addition of diphenyl-p-phenylenediamine (DPPD) to the diet has proven successful, but recent studies show that DPPD has an adverse effect on reproduction and thus its use in rat rations is contraindicated (Draper, Goodyear, Barbee and Johnson, 1956).

Vitamin-hormone relationships in pregnancy have been studied with regard to thiamine, pyridoxine, pantothenic acid, and folic acid. Thiamine deficiency induced stillbirths, subnormal birth weights, resorption of fetuses, and loss of weight in the mother. However, as in the case of protein deficiency, pregnancy could be maintained with 0.5 fjLg. estrone and 4 mg. progesterone (Nelson and Evans, 1955). Estrone alone had some favorable effect on the maintenance of pregnancy in thiamine-deficient animals, but it was less effective in protein-deficient animals.

Fetal death and resorptions as well as serum protein and nonprotein nitrogen (NPN) changes similar to those reported for toxemia of pregnancy (Ross and Pike, 1956; Pike and Kirksey, 1959) were induced by a diet deficient in vitamin Be . Administration of 1 fxg. estrone and 4 mg. progesterone maintained pregnancy in 90 per cent of vitamin Be-deficient rats (Nelson, Lyons and Evans, 1951). However, the pyridoxinedeficient rat required both steroids to remain pregnant and in this regard resembled the hypophysectomized animal (Nelson, Lyoas and Evans, 1953). Nevertheless, a hypophyseal hormone combination which was adequate for the maintenance of pregnancy in the fully fed hypophysectomized rat (Lyons, 1951) was only partially successful when there was a deficiency of pyridoxine. An ovarian defect is suggested.

The folic acid antagonist, 4-aminopteroylglutamic acid, will rapidly induce the death of early implanted embryos in mice.

rats, and man (Thiersch, 1954j . Removal of folic acid from the diet or the addition of x-methyl folic acid will induce malformations when low doses are given and resorptions when high doses are given. Furthermore, this effect is obtained even when the folic acid deficiency is delayed until day 9 of a rat pregnancy or maintained for only a 36-hour period. A deficiency of pantothenic acid will also induce fetal resorption. The vitamin is required for hatching eggs (Gillis, Heuser and Norris, 1942). In animals deficient in folic acid or in pantothenic acid, estrone and progesterone replacement therapy did not prevent fetal loss, suggesting that the hormones cannot act (Nelson and Evans, 1956). In the above mentioned deficiencies replacement of the vitamin is effective. However, vitamins other than those specifically deleted may provide replacement, thus ascorbic acid seems to have a sparing action on calcium pantothenate (Everson, Northrop, Chung and Getty, 1954) .

Pregnancy can be interrupted by altering vitamins other than those discussed above, but the hormonal aspects have not been explored. Thus, the lack of choline, riboflavin, and Bi2 will induce fetal abnormalities and interrupt gestation (Giroud, Levy, Lefebvres and Dupuis, 1952; Dryden, Hartman and Gary, 1952; Jones, Brown, Richardson and Sinclair, 1955; Newberne and O'Dell, 1958) . Choline lack is detrimental to the placenta (Dubnov, 1958), riboflavin deficiency may impair carbohydrate use (Nelson, Arnrich and Morgan, 1957) and/or induce electrolyte disturbances (Diamant and Guggenheim, 1957) , and Bjo spares choline and may be concerned with nucleic acid synthesis (Johnson, 1958). Excessive amounts of Bio are not harmful. It is interesting to note that uterine secretions and rabbit blastocyst fluid are rich in vitamin B]2 (Lutwak-Mann, 1956), but its presence in such large amounts has not been explained.

An additional substance, lithospermin, extracted from the plant, Lathijrus odoratus, is related to hormone functioning; it is antigonadotrophic when eaten by nonpregnant animals and man. The feeding of this substance to prciiiiant rats terminated the pregnancies about the 17th day. Treatment with estrogen and progesterone was preventive (Walker and Wirtschafter, 1956). It is assumed, therefore, that lithospermin interfered with the production of these hormones. A repetition of the experiment on a species in which the hypophysis and ovaries are dispensable during much of pregnancy would be of interest.

In retrospect it has been found that a deficiency in protein and the vitamins thiamine, pyridoxine, pantothenic acid, and folic acid individually can interrupt a pregnancy. Furthermore, a combination of estrone and progesterone which is adequate to maintain pregnancy after hypophysectomy and ovariectomy, is equally effective in protein or thiamine deficiency. This suggests that the basic physiologic alteration is a deprivation of ovarian hormones. However, protein- and thiamine-deficiency states differ from each other as shown by the response to estrogen alone (thiamine deficiency is less responsive), and these states differ from hypophysectomy in which estrone alone has no effect. A pyridoxine deficiency seems to involve both ovary and hypophysis, for neither steroids nor pituitary hormones were more than partially successful in maintaining pregnancy in rats. Lastly, pantothenic acid and folic acid deficiencies may not create a steroid deficiency. What is involved is not known; many possibilities exist. Pantothenic acid, for example, participates in many chemical reactions. Furthermore, it is known that thiamine is essential for carbohydrate metabolism but not for fat metabolism whereas pyridoxine is involved in fat metabolism and in the conversion of tryptophan to nicotinic acid. It is clear, though, that much ground must be covered before the formulation of fruitful hypotheses may be anticipated.

VI. Concluding Remarks

The development, composition, and normal functioning of the reproductive system is dependent on adequate nutrition. However, the requirements are many and only gradually are data being acquired which are pertinent to the elucidation of the nutritional-gonadal relationship.

The demands for nutrient substances is not always the same. During pregnancy and lactation there is a need for supplemental feeding. A similar need exists in birds and in the many cold-blooded vertebrates in which reproduction is seasonal. Atypical endocrine states create imbalances and a need for nutrient materials which vary, unpredictably, we must acknowledge, until the numerous interrelationships have been clarified.

At many points where determination of cause and effect are possible, an indirect action of dietary factors on reproduction is indicated. No other conclusion seems possible in view of the many instances in which the effect of dietary deficiencies can be counteracted by the administration of a hormone or combination of hormones. The direct action is not immediately apparent; it probably is on the processes by which metabolic homeostasis is maintained, and is in the nature of a lowering of the responsiveness to the stimuli which normally trigger these processes into action. The processes may be those by which pituitary and gonadal hormones are produced or they may be the mechanisms by which these hormones produce their effects on the genital tracts and on the numerous other tissues on which they are known to act.

Because of the many interrelationships, some of which are antagonistic and some supportive, determination of the role of specific dietary substances is not easy. For those who work with laboratory species, the problem is further complicated by the many strain differences. For everyone, the problem is complicated by the many species differences which are the result of an evolution toward carnivorous, herbivorous, or omnivorous diets, to say nothing of the countless specific preferences within each group.

Finally, it is something of a paradox in our culture that much of our effort has been devoted to investigations of the effects of deficiencies and undernutrition rather than to the effects of excesses and overnutrition. Much evidence supports the view that in the aggregate the latter are fully as deleterious as the former, but the means by which this result is achieved are largely unknown.

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