Book - Sex and internal secretions (1961) 5

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

Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex

Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction

The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions

Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation

Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds

Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior

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SECTION C Physiology of the Gonads and Accessory Organs

The Mammalian Testis

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

Professor of Physiology, Mayo Foundation, and Head of the Endocrinology Laboratory, Mayo Clinic, Rochester, Minnesota

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 metabolism 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 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 Moore'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 importance of gene factors and of the fetal 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.

The 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, 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) .

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.)

Some interesting details on the relative 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 histologic 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 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 temperature 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

own body heat. The application of water

Fig. 5 2 Dcx rlopmental stages in tho monkey testis

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

? To^lls of .1 96-(lav fetus Tubules 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.

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.)

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.

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 28. Adult testis, age 11 years, 6 months, 20 days. 26. Seminiferous tubule lumen lined by young 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.)

(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 degenerates 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 fertility 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 castration 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 different 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 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.

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.)

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 rete and running 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).

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.)

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 generatic 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 temperature 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.

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.)

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 environmental 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 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.

VII. 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) perineural, 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 run to the walls of the tubules and enter the membrana propria to reach the Sertoli cells.

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 (b) 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.)

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.

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

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.)

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 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.

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.)

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

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.)

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 testosterone 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.

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.)

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

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).

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.)

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

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.)

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

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.)

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 infrared 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).

Fig. 5.16. Frequency 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.)

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

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 (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 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.

McCullagh 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 (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).

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

Some recent studies on compensatory 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 pituitary into the testis of guinea pigs has resisted in pronounced stimulation and hypertrophy 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 individual 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.

Neither 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 atrophy. 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 concept 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 suppression 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 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 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 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.

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).

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 effect 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.

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.)

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 repaired 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, it is obvious that androgen initiated spermatogenesis.

Fig. .').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.)

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 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.

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.)

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 Meensel 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.

Uncertainty 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 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) reported that the development 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 proceeded 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. Miscellaneous 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, pregneninolone 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 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 shortly thereafter, may 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 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 administered 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 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 atrophy 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. Non-neoplastic 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 differentiation of the Sertoli 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 normal preepubertally 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 that normal male dogs, after a brief olfactory 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 tumors 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).

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 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 be 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 endocrinologists. 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.

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