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

SECTION C Physiology of the Gonads and Accessory Organs

The Mammalian Ovary

William C. Young, Ph.D.

Professor Of Anatomy, University Of Kansas, Lawrence, Kansas

I. Introduction 449

II. FOLLICULOGENESIS 451

A. Growth of Primary and Small Ve sicular Follicles 451

B. Growth of Vesicular Follicles 455

C. Preovulatory Swelling 455

1). Ovulation . ." 456

E. Folliculogenesis in Pregnancy and

Lactation 457

III. Corpus Luteum 459

IV. Follicular Atresia 4(11

V. Hormones of the Ovary 4(i4

A. Sites of Origin 466

B. Amounts of Hormone Produced. . . . 471 VI. Age of the Animal and Ovarian

Functioning 476

VII. Other Endocrine Glands and the

Ovaries 478

A. Thyroid 478

B. Adrenal Cortex 480

VIII. Concluding Remarks 483

IX. References 484

I. Introduction

Despite the impetus given to the study of ovarian structure and physiology by the work of Edgar Allen and Edward A. Doisy in the 1920's, knowledge of the mammalian ovary has hardly progressed beyond a descriptive phase. This cannot be attributed to lack of effort, although it must be realized that the ovary as an object of investigation has not held its own with the hypophysis, the thyroid, the adrenal, and the testis. Nor lias there been any failure to apply new techniques to the many problems of ovarian structure and physiology. Histochemical and cytochemical techniques were seized ujion for what they might contribute to the prol)lem of the site of hormone production,^ and,

' Demp.sey and Bassett, 1943; Dempsey. 1948; Claesson, 1954; Claesson and Hillarp, 1947a-c ; Claesson, Diczfalusy. Hillarp and Hogberg, 1948; Claesson, Hillarp, Hogberg, and Hokfelt. 1949:

in at least one series of studies (Zachariae, 1957, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958), to the mechanism of ovulation. Methods for obtaining blood from the ovarian vein have been devised (Paschkis and Rakoff, 1950; Rakoff and Cantarow, 1950; Xeher and Zarrow, 1954; Edgar and Ronaldson, 1958) and refined techniques for the assay of secreted estrogens and progesterone have been developed (Reynolds and Ginsburg, 1942; Hooker and Forbes, 1947; Emmens, 1950a, b; Haslewood, 1950; Wolstenholme, 1952; Zander and Simmer, 1954; Brown, 1955; Loraine, 1958; Sommerville and Deshpande, 1958j . The collection of follicles and corpora lutea timed more accurately with respect to the moment of ovulation has become possible, and distinction between the normal and the pathologic has become clearer (Deane, 1952). Recently, the electron microscope has been found to have a place, in an investigation of the finer structure of the cells of the corpus luteum (Lever, 1956), in the unraveling of the jirocesses whereby the zona pellucida is formed around the developing oocyte (Chiquoine, 1959; Odor, 1959), and in studies of ovarian oocytes and unfertilized tubal ova (Odor, 1960; Odor and Renninger, 1960) (see Figs. 14.6 to 14.8). As Villee has indicated in his chapter, great strides have been taken toward an understanding of the metabolic pathways in estrogen and progesterone synthesis and degradation.

Two factors may have contributed to the

McKay and Robinson, 1947; Meyer and McShan, 1950; Barker, 1951; Rockenschaub, 1951; White, Hertig, Rock and Adams, 1951 ; Deane, 1952; Nishizuka, 1954; Ford and Hirschman, 1955; Noach and \an Rees, 1958.

449

450

PHYSIOLOGY OF GONADS

disappointment that has been expressed. First, the purification and synthesis of the hormones in the 1930's (Allen, 1939; Doisy, 1939) and the later successful development of synthetic estrogens and gestagens ( Solmssen, 1945; Dodds, 1955; Rock, Garcia and Pincus, 1956; St. Whitelock, 1958) provided a means whereby much of ovarian physiology could be studied out of context with the processes by which this organ functions. Specifically, there are many effector actions of ovarian hormones, many interrelationships with other hormones and with each other, many problems of tissue responsiveness, and many questions bearing on processes of ovarian hormone metabolism, all of which can be studied in ovariectomized animals.

Secondly, there were many practical reasons why chemists should have striven to synthesize estrogenic substances and gestagens which are suitable for replacement therapy. Once prepared, these synthetic substitutes are of interest, but their development and therapeutic application may well have diverted attention from studies of the ovary.

If there is disappointment with the progress that has been recorded, we would direct attention to substantial accomplishments which should stand us in good stead in the future. Among these are the numerous careful descriptions of the growth and maturation of ovarian follicles and the meticulous accounts of corpus luteum formation, structure, and involution. In a general way it has become clear that in many species estrogen and progesterone are produced while the follicles are maturing, and that, during the functional life of the corpus luteum, progesterone and estrogen are secreted. Estimates of the amounts produced have been numerous and of more than ordinary interest. In addition, they probably represent steps toward the determination of additional important information : the day-to-day rate of production correlated with the growth of the follicles and the development of the corpora lutea, and, in species in which variable numbers of follicles and corpora lutea develop, steps in an effort to ascertain whether, for example, 10 follicles in an individual produce more hormone than 5. This knowledge, if we possessed it, might

contribute significantly to current theories of gonadal-pituitary relationships because thresholds are involved in the regulatory processes <see chapters by Everett and Greep ) .

It could be disappointing that, on the basis of evidence which is largely circumstantial and inferential, almost every tissue component of the ovary, membrana granulosa, theca interna, and interstitial cells, has been claimed to be the source of estrogen and progesterone. But it is encouraging that information has been obtained which prompts us to recognize that it may be futile and unrealistic to attempt to identify specific cell types as the sources of hormones in the ovary. Current thought, stimulated by the discovery that testicular cells, placental tissue, and occasionally the adrenal cortex are sources of estrogen and progesterone, and that ovarian tissue produces androgens, is leaning toward the view that the several tissues involved in steroid hormone biosynthesis may be subject to metabolic aberrations which change their hormone production either in rate or in kind. The a])i)roach to the problem now seems to be through enzymatic biochemistry rather than through gross or finer morphology. Examples of this approach which are suggestive for further work on the ovaries are jirovided by the numerous studies by Samuels and his associates (Samuels, Helmreich, Lasater and Reich, 1951 ; Huseby, Samuels and Helmreich, 1954; Beyer and Samuels, 1956; Samuels and Helmreich, 1956; Slaunwhite and Samuels, 1956).

It is known in a general way that follicular maturation, ovulation, and corpus luteum formation are controlled by gonadotroi)hic hormones from the pituitary. However, as Greep emphasizes in his chapter, the specific gonadotrophic hormones have not yet been isolated and identified, nor have their specific roles in ovarian physiology b(»en demonstrated. To be sure, ovulation has been repoited following the injection of allegedly purified pituitary gonadotrophins into hypojihysectomized rats (Velardo, 1900), but until stages normally seen ill the jn'ocess of folliculogenesis and ()\-ulati()ii can he rejn'oduced consistently by the use of pituitary gonadotroI)hins, and until target organ responses simi

MAMMALIAN OVARY

451

lar to those in intact cycling animals are being evoked, no satisfactory conceptualization of ovarian functioning will be possible.

Chorionic gonadotrophins are not without practical value in the stimulation of ovulation (Cole and Miller, 1933; Folley and Malpress, 1944; Folley, Greenbaum and Roy, 1949; Marden, 1951; Umbaugh, 1951; Robinson, 1954; and others); nevertheless, their contribution to ovarian physiology may be limited. Bradbury has pointed out in a personal communication that the human placental hormone (HCG) is exotic for laboratory animals. It has practically no effect on the ovaries of guinea pigs or field mice. In rats its biologic effects arc finite different from those of pituitary luteinizing hormone (LH) or interstitial cellstimulating hormone (ICSH) (Selye, Collip and Thomson, 1935; Evans, Simpson, Tolksdorf and Jensen, 19391. HCG is so ineffective in the hypophysectomized rat that it has been assumed, in the case of the intact animal, either that it acts through the pituitary or that it requires the presence of the pituitary to be effective (Aschheim, Fortes and Mayer, 1939; Noble, Rowlands, Warwick and Williams, 1939) . If HCG and other chorionic gonadotrojihins are exotic, as such results would indicate, the extrapolation of effects which have followed their use to the normal functioning of the ovary could be seriously misleading.

What we have written is intended to set the tone for what follows. Many of the solid accomplishments of the past two decades will be recounted, but the areas of uncertainty and the difficulties which slowed down the progress of the twenties and thirties will be enumerated in the hope that the curiosity of a new generation will be aroused and guide us into a period of even more productive eft'ort.

II. Folliculogenesis

A. GROWTH OF PRIMARY AND SM.\LL VESICULAR FOLLICLES

In mammals, by the time of birth, oogonia have completed their proliferative activity and become primary oocytes. The serosal surface of the ovary is covered by a layer of cells known as the germinal epithelium. There has been and still is considerable

speculation whether adult germinal epithelium contains, or gives rise to, any germ cells (Sneider, 1940; Mandl and Zuckerman, 1950, 1951a-d, 1952b; Zuckerman, 1951; Green and Zuckerman, 1951). The subject is reviewed extensively by Brambell ( 1956 ) and in the chapter by Blandau. Careful studies of human ovaries have been made by Block (1951a, b, 1952, 1953). From all this material, it is clear that a satisfactory answer has not been given. The latter may be awaiting the development of a fresh approach and until then another review of the many conflicting reports and opinions would be repetitious.

]\Iore relevant to the present review is the relationship between ovarian estrogens, on the one hand, and germ cell proliferation and follicle development, on the other. It has been claimed that exogenous estrogen stimulates mitotic activity in the germinal epithelium in mice, rats, and the minnow, Phoximis laevis L. (Bullough, 1942a, 1943; Stein and Allen, 1942; de Wit, 1953; von Burkl, Kellner, Lindner and Springer, 1954) . Bullough (1943) suggested that new cycles of oogenesis are intiated by estrogen in the follicular fluid of large and rupturing follicles. Mandl and Zuckerman (1950) and Dornfeld and Berrian (1951) were less certain. The former expressed the belief that the direct effect of estrogenic stimulation cannot be measured by comparing the total number of oocytes in the ovary. The latter, after finding that isotonic saline, gelatine, or agar injected into the perovarian capsules of immature rats elicited mitoses in the germinal epithelium, concluded that the reaction was in response to injury rather than to the substance injected. Notwithstanding the precautionary notes sounded by ]\Iandl and Zuckerman and by Dornfeld and Berrian, the number of reports of increased mitotic activity near the site of ovulation or when estrogen is placed in contact with the germinal epithelium remains impressive. Particularly because of the analogy with the androgenic control of spermatogenesis pointed out by Bullough ( 1942b) and others, the possibility should be tested further.

As the oocyte starts to grow, the flat investing cells proliferate and form the membrana granulosa. By the time the rat oocyte has completed its growth it has acciuired

452

PHYSIOLOGY OF GONADS

^l!:

I--|(..7.1. Munlry l,yi„,pliy>r,-ini,iiz<Ml 1 y,-uv | hvm, ,u^ly . I'., Hide- :nv ,n ) ,i n^i .—in . ' ~::,-i>s (jl (le\f'lu]iiiieiil. C<nic('iitr;itiun ul' uorylcs m ihc corlcx n'S('inl)lfs llial in iVlal or jusciiilc o\aiie8. No evidence of estrogen production in this animal. (Courtesy of Dr. Ernest Knobil.)

foiii' layers of granulosa cells (Mandl and Zuokerman, 1952a). The increase in size of the growing follicle is relatively constant until the stage of antrum formation (Paesi, 1949a ». Follicles grow and develop to this stage in rats and guinea pigs even after hypophysectomy (Dempsey, 1937; Paesi, 1949b). It is thus apparent that the gonadotrophic hormones of the pituitary are not essential for the early growth of ovarian follicles (Fig. 7.1). The rate of this early follicular growth may, however, be accelerated in the presence of certain, but perhaps not all, gonadotrophic hormones (Pencharz, 1940; Sim])son, Evans, Fraenkel-Conrat and Li, 1941 ; Gaarenstroom and de Jongh, 1946; Payne and Hellbaum, 1955).

As the granulosa of the growing follicle ])roliferates, the surrounding tissue differentiates into theca interna. Dubreuil (1942, 1948, 1950) postulated that the granulosa j)roduces an inductor substance which causes the differentiation of the theca interna. Hisaw (1947), in a review of the literature bearing on this point, also suggested that there must be organizers within

the developing gramdosa cells which stimulate tlifferentiation of the theca interna. Furthermore, this autonomous process continues until the follicle reaches a stage of development at which it becomes responsive to gonadotrophic hormones. Hisaw called this the stage of "comjietency."

Somewhere in this process estrogens seem to have a role, or, if the ojjinions expressed by Bullough (1943) and the other investigators whose work has just been cited can be confirmed, perhaps there is a continued stimulation by these substances. If large doses of estrogen are administered to imnuitui'c or to liypopliyscctomi/ed immature rats, many follicles (k'\x'lop to the early antrum stage within 72 hours. The theca interna differentiates ai'ound these estrogenstimulated follicles. When immature rats ai'e giv(m large doses of estrogen there is a <le(iiiil(' incicasc in ox'arian weight and in the iiuiiihei' of medium-sized follicles ( ]ig. 7.2 1 ; small amounts, on the othei' hand, ai'e iiihihitoiy (Paesi, 1952). Increased giowth of large follicles, or at least a retai'dation of their degeneration, has

MAMMALIAN OVARY

453

y^

Fig 7.2. Intact immature lat given estrogen for 3 days. Active proliferation of granulosa in many follicles. Decreased incidence of atresia and hypertrophy of theca. (Courtesy of Dr. J. T. Bradburv.)

Ijeen reported in hypuphyscctoiuizcd I'uts given stilbc-^trol (Pencharz, 1940; Williams, 1944; Desclin, 1949; Payne and Hellbaum, 1955; Ingram, 1959 1. Histologically, when estrogen is given, there is a marked increase in mitotic activity of the granulosa cells and a decrease in atresia (Williams, 1945a; de Wit, 1953; Payne and Hellbaum, 1955; Payne, Hellbaum and Owens, 1956; Williams, 1956). The fact that estrogen stimulates the follicle and protects it against atresia suggests that the granulosa is not a significant source of estrogen. On the other hand, if estrogen is jiroduced by the theca interna, it could exert a localized stimulatory action on the membrana granulosa (Corner, 1938; Bullough, 1942b, c, 1943). The differentiation and development of the theca interna is nicely timed for a localized jiroduction of estrogen around each Graafian follicle.

Estrogen not only stimulates the granulosa to proliferate, but also renders the follicles more responsive to exogenous gonadotrophins. Williams (1945b) found that the ovary in the stilbestrol-treated hypol^hysectomized rat was more responsive to small doses of pregnant mare serum (PMS) than was the ovarv in the intact

immatui'e rat. Payne and Runser (1958) found that stilbestrol augmented the response of hypophysectomized immature rat ovaries to exogenous pituitary extracts. In Bradbury's experience at Iowa 48 hours of stilbestrol pretreatment rendered the ovaries of both intact immature and hypophysectomized rats more responsive to Armour's LH (Lot No. R377242H), but not to Armour's follicle-stimulating hormone (FSH) {Ah 1027) . Furthermore, increasing the dosage of stilbestrol from 0.02 mg. to 0.2 mg. markedly increased the response of the ovaries to a given dose of LH. He suggested that the possibility should be exi)lored that the local (})erifollicular) concentration of estrogen determines the resjionsiveness of the maturing follicles. Thoughtful discussions of the subject are given by Paesi (1952) and by Bradbury (1961). The former suggested that 2 or 3 types of estrogen action may be involved in the stimulation of the ovary which is seen wdien estrogen is administered. Bradbury applied estradiol or stilbestrol to one ovary of the immature rat, leaving the other ovary untreated. The various unilateral responses — increase in weight, formation of corpora lutea, greater ■ reactivity to gonadotrophins — demonstrated

454

PHYSIOLOGY OF GONADS

.Sj^cr^r

•■/>:

ikm

Fig. 7.O. Imuiuturu li.ypophy.sectomized rat treated with Arniuui - i>il. (iiauulo.sa ha^ proliferated and follicles have developed antra. The theca is diffeientiated but the interstitium is deficient. (Couitesy of Dr. R. M. Melampy.)

clearly the local stimulating effect of estrogens with the ovary, as well as the systemic effect by way of the pituitary.

If estrogen administration to immature or to hypophysectomized immature rats is continued 7 to 10 days, the granulosa of the stimulated follicles degenerates. This atretic process differs from natural atresia in that it seems to start peripherally rather than centrally. The oocytes do not fragment or give off polar bodies as frecjuently as do oocytes in normally atretic follicles. It seems that the stimulatory effect of estrogen on the granulosa is very temporary. Its din-ation, however, is long enough to be coni])atible with the noi'inal pi'ocess of maturation and ovuhitiou.

Before concluding the subject, a certain amount of back-tracking may be dcsii'able. One of the first suggestions to be made by Edgar Allen (1922; see also the biogi'aphic;il sketch in this book) was tliat the ovum is

the dynamic center of the follicle. If the suggestion is placed in the context that has since been developed, the sequence of events would l)e an inductive influence of the oocyte on the membrana granulosa, a continued inductive influence (oocyte or membrana granulosa?) on the surrounding connective tissue cells until the theca interna is formed,- and then the secretion of estro 'Resuhs obtained by Genther (1931), Schmidt (1936), Humphreys and Zuckerman (1954). and Wcstman (1958) suggest that a similar functional iclationship exists between the granulosa and interstitial cells. According to Genther, x-ray-iujured o\aries composed of interstitial cells produced estrogen only if a growing follicle was present. The in\oluted condition of the uteri in rabbits in wliich all oocytes and f()llicl(>s iiad been destroyed by x-rays led Humphreys and Zuckerman to conclude that the ovaries of these animals were not producing estrogen. The results reported by Westman suggested tliat interstitial cell function continues only lor .1 limited jieriod after x-ray-induced degeneration of the granulosa cells. The results from an ingenious investigation by Ingram (1957) ar(

MAMMALIAN OVARY

455

gen wliich feeds back to stimulate further growth of the meinbrana granulosa and follicle.

Attractive as such a hypothesis is, it has at least one weakness. If gonadotrophic extract rich in FSH is administered to immature rats for three days, there is a generalized stimulation of granulosa tissue (Fig. 7.3). Small follicles increase in size, medium sized follicles develop an antrum, and Graafian follicles become large and vesicular (Parkes, 1943), but ovulation is uncommon. At autopsy the ovaries are pale and edematous. The ovaries are markedly increased in size from numerous follicles becoming vesicular. Histologically there is little stimulation of the theca interna. Gaarenstroom and de Jongh (1946) recognized this ovarian response when they suggested that FSH be designated as Ge (gonadotrophin e])ithelial). This tissue response offers evidence that FSH is primarily f'oncerned with growth and proliferation of the granulosa cells, but there is no explanation to account for the failure of these granulosa cells to stimulate the differentiation of the theca interna and the eventual secretion of estrogens.

During all of follicular growth, the presence of estrogen has come to l^e assumed and its production by the growing follicle is thought to begin with the appearance of the theca interna (see below). On the other hand, the amount produced and the rate of production are unknown. The amount must increase with the growth of the follicles. Gillman and Gilbert (1946) found during their investigation of perineal turgescence in the baboon that, once the perineum reaches maximal turgescence, additional estrogen is required to maintain it. They concluded that in normal animals, during the second part of the phase of turgescence, there must be an increased output of ovarian estrogen. Direct studies have yielded little

similarly suggestive. Autografts of ovarian medulla without cortical tissue or oocytes, and autografts of cortical tissue were transplanted to various sites in .sexually mature rabbits. The grafts of cortical tissu(> p(>rsisti'(l after the medullary grafts had disappeared. Ingram concluded that medullary tissue containing interstitial tissue but no follicles cannot survive.

information. Foi'd and Hirschman (1955) estimated alkaline phosphatase activity in the ovary of the rat, but the concentrations in the theca interna and ovarian tissue as a whole were relatively constant during the phases of the cycle.

B. GROWTH OF VESICULAR FOLLICLES

The growth of the follicle which is dependent on stimulation by hypophyseal gonadotrophins has been described for a number of animals and, for a few (cow, sow, ewe, guinea pig, rat) plotted with respect to the time of the preceding ovulation (McKenzie, 1926; Hammond, 1927; Grant, 1934; Myers, Young and Dempsey, 1936; Boling, Blandau, Soderwall and Young, 1941 ; von Burkl and Kellner, 1956). Data of the latter sort are especially valuable for the baselines they provide for experimental studies of the factors affecting the pituitary-gonadal relationships. Deviations in the shape of the curve of follicular growth, and disparities in the size of the growing follicles and in the size and structure of the corpora lutea, are clear indicators of abnormalities in function which have been too little used.

C. PREOVULATORY SWELLING

Without exception in the animals listed above, and probably in the horse, goat, and bat, if we may judge from the data presented by Hammond and Wodzicki (1941), Wimsatt (1944), and Harrison (1946, 1948b), a linear period of growth during most of the diestrum is followed by a positive acceleration (preovulatory swelling) ; shortly before estrus and ovulation. The point at which this acceleration occurs is the point in the development of the follicle where physiologic evidence for the production of progesterone by the unruptured follicle was first found (Dempsey, Hertz and Young, 1936; Astwood, 1939). As we will see later, however, the ^'moment" the preovulatory swelling begins is not necessarily the point in time when the first progesterone is produced.

In most sjiecies in which the course of the preovulatory swelling has been followed, it is a 10- to 12-hour process (Hammond, 1927; Grant, 1934; Myers, Young and

456

PHYSIOLOGY OF GONADS

Dempsey, 1936; Doling, Blandau, Soderwall and Young, 1941 ; Rowlands and Williams, 1943; Rowlands, 1944), although in the cat and ferret the process is triggered by mating and extends over 25 to 30 hours. The preovulatory swelling can be initiated by injecting gonadotrophins of the LH or ICSH type, but they are effective only on well matured follicles (Hisaw, 1947; Talbert, Meyer and McShan, 1951). The younger follicles are not stimulated and, on the contrary, they may show an accelerated atresia. It could be postulated that the follicules with well developed theca interna were "competent" and that stimulated theca interna produced estrogen which favored the development of these follicles. The smaller follicles were "incompetent" in the absence of a thecal investment and became atretic. If HCG is injected into immature rats, the theca interna around the vesicular follicles hypertro])hies within 24 hours and these follicles enlarge rapidly. The vasodilation of the theca blood vessels is grossly evident within a few hours (Kupperman, McShan and Meyer, 1948; Sturgis and Politou, 1951; Odcblad, Nati, Selin and Westin, 1956).

Explanation has been sought for the nature of the changes within the follicle which lead to the accelerated enlargement culminating in ovulation. Studies of the staining qualities of such follicles reveal that the metachromatic polysaccharides of the granulosa (hyaluronic acid and chondroitin sulfuric acid) become progressively depolymerized and orthochromatic. This hydrolysis of the mucopolysaccharides gives rise to an increased osmolarity which may be the major factor in the preovulatory swelling of the follicle (Harter, 1948; Catchpole, Gersh and Pan, 1950; Odeblad, 1954; Zachariae, 1958; Zachariae and Jensen, 1958; Jensen and Zachariae, 1958). Accompanying the swelling is a dispersal of the cells of the cumulus oophorus. This may be a cons(^quence of the breakdown of the intcrcclhihii' substance in the stinudated niciubraiia gi'aiiulosa.

The time r('(|uirc(l for follicular growth and maturation fi'oin the stage when its further development is dependent on pituitary gonadotrophin stimulation to ovulation is related to the length of the cycle and

therefore varies greatly from species to species. Somewhat less than 4 to 5 days are required in the rat (Boling, Blandau, Soderwall and Young, 1941 ) , somewhat less than 16 days in the guinea pig (Myers, Young and Dempsey, 1936), somewhat less than 21 days in the cow (Hammond, 1927), and ])resumably comparable intervals in other species. Vermande-Van Eck (1956) estimated that in the rhesus monkey the average time required for the growth of a mature follicle from the large follicle without an antrum is 4 to 6 weeks; 11 days are estimated to lie necessary for the complete development of a follicle in the rabbit (Desaive, 1948) . Ovulation occurred earlier than normal when the corpora lutea from the ])receding cycle were removed, but the rate of follicular development was not altered (Dem])sey, 1937). Presumptive evidence exists, however, that the rate of growth may be slower in pubescent chimpanzees (Young and Yerkes, 1943), baboons (Gillman and Gilbert, 1946), and guinea jngs (Ford and Young, 1953).

D. OVULATION

Ovulation, under normal circumstances, ))robably is explosive (Hill, Allen and Kramer, 1935, in the rabbit; Blandau, 1955, in the rat). In 1 of 2 human i)atients Doyle (1951) saw a gush of follicular fluid at the time of ovulation. In 163 ovulations timed l)y Blandau the interval between the rupture of the stigma and the escape of the ovum was 72 seconds when most of the folliculai' fluid escai)ed in advance of the ovum, and 216 seconds when the cumulus oophorus preceded the follicular fluid. The slower, steady, continuous flow of the liquor folliculi which has been described by Walton and Hammond (1928) in the cow, Markee and Hinsey (1936) in the rabbit, and by Doyle (1951) in one human sul)ject could be an artifact of the procedures used in watching the jM'Ocess.

The mechanisiu heading up to formation of the stigma and rupture of the follicle is unknown. Claesson (1947), using the submicroscoi)ical differences which can be ol)served in ])olarized light, distinguished smooth muscle from connective tissue cells and rep()rte(l that no bundles of smooth muscle or isolated cells were found in the

MAMMALIAN OVARY

457

theca externa in ovaries from the cow, pig, rabbit, and guinea pig. The earlier contradictory results he reviewed were attributed to the nonspecificity of the older staining methods. A possible clue to the mechanism of ovulation wliich does not seem to have been explored was given by the observations of Boling, Blandau, Soderwall and Young ( 1941 ) when they were studying follicular growth in the rat. Immediately before ovulation, but at no other time, a large pocket at the base of the cumulus, and described as an invagination of the granulosa, is a constant feature of follicular structure (Fig. 9 in their article). No guess w^as made as to its significance.

In all the spontaneously ovulating infrahuman mammals that have been studied, except the dog (Evans and Cole, 1931 ) , possibly other Canidae, and the mouse (Snell, Fekete, Hummel and Law, 1940) in which it takes place early in estrus, ovulation occurs toward the end of heat (see reviews in Young, 1941; Dukes, 1943; and more recent articles on the chimpanzee, rhesus monkey, baboon, cow, and mare by Young and Ycrkes, 1943; van Wagenen, 1945, 1947; Gillman and Gilbert, 1946; Cordiez, 1949; and Trum, 1950; respectively ) . Only in the human female in which cyclic waxing ^nd waning of sexual desire is not easily detected does uncertainty exist.

Since an early period, when emphasis was given to the opinion that ovulation occurs about midway in the intermenstrual interval (Knaus, 1935; Hartman, 1936; Farris, 1948), much evidence has been produced indicating that it may occur at other times as well, even during menstruation (Teacher, 1935; Rubenstein, 1939; Sevitt, 1946; Bergman, 1949; Stieve, 1952; and many others). If we may judge from what has been found in the chimpanzee (Young and Yerkes, 1943), baboon (Gillman and Gilbert, 1946), ihcsus monkey (Rossman and Bartclmez, 1946), and man (Bergman, 1949; Buxton, 1950), irregularities in the length of the preovulatory and postovulatory phases of the cycle complicate the problem and could account for some of the confusion. In the chimjianzee, baboon, and human female, in which the irregularities can be located wdth respect to the time of ovulation, age influences the length of both phases, and fol

lowing pregnancies there are similar irregularities. In the baboon, environmental stresses result in temporary or even prolonged inhibition of ovarian activity. There is no reason for believing that the same factors have less effect on folliculogenesis in the human female; irregularities in adolescence (Engle and Shelesnyak, 1934) and following pregnancy (Sharman, 1950, 1951) are common and there are many reports of psychic effects (see reviews by Kelley, 1942; Kelley, Daniels, Poe, Easser and Monroe, 1954; Kroger and Freed, 1950; Randall and McElin, 1951; Bos and Cleghorn, 1958). In all cases follicular growth is interrupted and amenorrhea follows. But if the estimates are correct that the average fertile woman ovulates normally about 85 per cent of the time (Farris, 1952), or that perfectly healthy women may have 3 or 4 anovulatory cycles a year (de Allende, 1956; also see table in Bergman, 1949), there must also be cases in which much of follicular growth is normal, or at least adequate to stimulate growth changes in the uterus, but ovulation does not occur. As if the complications noted above are not enough, the reviews of the methods used in determining the time of ovulation (D'Amour, 1934; Cohen and Hankin, 1960) and the critical study of Buxton and Engle (1950) in which an attempt was made to correlate basal body temperature, the condition of the endometrium, and the stage of folliculogenesis in the ovary, suggest either that a really sensitive indicator of the time of ovulation has not been found, or if one exists, that it has not been used in a study sufficiently systematic to reveal the true situation in the human female. The problem is one of the many that is with us very much as it was 20 years ago.

E. FOLLICULOCJENESIS IN PREGNANCY AND LACTATION

Before leaving the subject of follicular growth, its course in pregnancy and lactation should be reviewed. Information has been obtained from many species, but in most cases it is not complete and a considerable amount of conjecture is necessary. What is certain is that pregnancy affects the process of folliculogensis in many ways ; each must be the reflection of a different in

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PHYSIOLOGY OF GONADS

terrelationsliip betwen pituitary, gonads, and placenta. In the mare, and presumably other species in which multiple ovulations occur early in pregnancy, the involvement of chorionic gonadotrophins, pituitary gonadotrophins, and estrogen of placental origin has been suggested (Rowlands, 1949). When folliculogenesis is inhibited just before the stage of the preovulatory swelling, as it is in many pregnant animals (see below), the nervous system may be involved. An unusually significant investigation in which the threshold of stimulation to ovulation in the rabbit was correlated with threshold changes in cerebral activity has recently been completed (Kawakami and Sawyer, 1959). It was demonstrated that pregnancy or prolonged treatment with progesterone maintains the electroencephalogram (EEGj after-reaction threshold to low frequency stimulation of hypothalamic or rhinencephalic nuclei at an elevated level. At this level, gonadotrophin release does not occur in response to coitus or other ovulatory stimuli. The discovery of this fact has provided a basis for understanding the various ovarian conditions associated with pregnancy and lactation, or at least those in which follicular development proceeds to the point of preovulatory swelling and then stops. It may be that some other mechanism of inhibition accounts for the more severe retardation of folliculogenesis in si)ecies in which this occurs.

The European hares, Lepus tiniidus L., and L. ciiniculus L., are reported as mating during pregnancy with the occurrence of superfetation (Lienhart, 1940). Pregnancy, therefore, has little or no effect on any stage of folliculogenesis in these species. The domestic rabbit appears to he somewhat more affected and perhaps more variable. Claesson, Hillarj), Hogberg and ll()kfeh (1949) state that the ovaiics of pii'gnant rab})its are composed almost entirely of interstitial gland, except for the corpoi'a lutea, but, according to Hannnond and Marshall (1925) and Dawson (1946), mature follicles ai'e present and pregnant animals will occasionally mate. However, if we may assume that the reaction of i)regnant animals is similar to that of ])seudopregnant animals (Makepeace, Weinstein and Friedman, 1938), pituitary gonadoti'opliin is not

released and ovulation does not occur. From examination of the ovaries and from the fact that fertile matings can occur within a very few hours after parturition (Dempsey, 1937; Boling, Blandau, Wilson and Young, 1939; Blandau and Soderwall, 1941), it is clear that follicular development in the pregnant guinea pig and rat proceeds to a point just short of the preovulatory swelling. According to Nelson (1929) and Swezy and Evans (1930), cycles of oogenesis occur in laboratory rats, and, although the follicles may form small corpora lutea (Swezy and Evans), ordinarily they do not rupture. The musk-rat. Ondatra zibethica, and the African bat, Xycteris luteola, must display an advanced follicular development during pregnancy because there is evidence of postl)artum estrus (Warwick, 1940; Matthews, 1941, respectively). Brown and Luther (1951) state that postpartum estrus occurs within 3 days after farrowing in the sow, if the young pigs are removed. We assume, from this latter statement and from the rel^ort that estrus and service may occur during pregnancy in this species (Perry and Pomeroy, 1956), that large follicles are present in the ovaries of the pregnant sow.

Heat ])eriods in the jjregnant ewe are associated with follicular growth, but ovulation does not occur, and late in pregnancy follicle size decreases significantly (Williams, Garrigus, Norton and Nalbandov, 1956 ) . The first heat after {parturition was an average of 23.9 days later, range 1 to 61 days. According to Harrison (1948b), widespread atretic changes can be seen in all the follicles in the goat, beginning the 40th day of |)regnancy. By the (iOth day. no healthy follicles can be found.

Hammond (1927) was of the opinion that during jircgnancy in the cow, follicles develop to the size at which the jireovulatory swelling begins, but Dukes (1943), citing a study by Weber, wrote that cows come into heat 3 to 7 weeks after parturition. Support for this \-iew comes from the report l)y Hafez ( 1954) that the average interval to the postpartum esti'us in another bovine, the Egyptian buffalo, is 43.8 days, range 16 to 76 days. The iclatixcly long postpartum inter\al in these two species is presumptive evidence that follicles are relatively small at the end of pregnancy in bovines.

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Between the 40th and 150th day of pregnancy in the mare the ovaries contain numerous actively growing follicles and several functional corpora lutea (Cole, Howell and Hart, 1931; Rowlands, 1949). However, from the 150th day until the late stages, there is a regression of all the corpora lutea and an absence of large follicles. In the late stages only minute vestiges of corpora lutea and small follicles remain. If the latter is true, follicular growth must be rapid after parturition, because the first heat following foaling was between the 7th and 10th days in 77 per cent of the many mares Trum (1950) studied. In the African elephant, Loxodonta africana, there is also a replacement of the corpora lutea (one plus several accessory corpora lutea j about midway through pregnancy (Perry, 1953). Some are formed following ovulation and some not. They persist until term when they involute rapidly. During the late stages of pregnancy no follicles with antra are founcl. Dawson ( 1946) wrote that the domestic cat does not possess mature follicles at the time of parturition. In nonlactating animals the proestrous level is reached the 4th week after parturition.

Presumptive evidence exists that the follicles in the parturitive chimpanzee are small (Young and Yerkes, 1943 ) . In the human female the appearance of the first ovulatory cycle after pregnancy is irregular (Sharman, 1950, 1951 ; AlcKeown, Gibson and Dougray, 1954). According to Sharman, it may occur about 6 weeks after delivery in nonlactating women. This suggests that follicles are small at the end of ju-egnancy in the human female.

Inhibitory effects of lactation on follicular development are indicated by the substance of many of the reports cited above (Dawson; Dukes; Perry; Schwartz; Sharman; Williams, Garrigus, Norton and Nalbandov) and by much other information. As would be expected, the intra- and interspecies variations are great. Studies in progress at Iowa (Bradbury, personal communication) are revealing that some women experience an atrophy of the vaginal epithelium during the second and third month of lactation. The atrophy is indicative of a lack of ovarian estrogen and suggests that follicular development is not normal. Observations that are

similarly suggestive have been made in other species. The absence of estrogen in significant ciuantities during lactation in the mouse (Atkinson and Leathem, 1946) and guinea pig (Rowlands, 1956) is believed to be the reflection of a delay in the resumption of follicular growth and ovulation. Mother rats and mice may copulate and conceive within 24 hours after delivering a litter of young. AVhile the mother is nursing the newborn litter, the fertilized eggs of the new pregnancy develop into blastocysts, but these blastocysts fail to implant in the uterus at the usual time (Talmadge, Buchanan, Kraintz, Lazo-Wasem and Zarrow, 1954; Whitten, 1955; Cochrane and Meyer, 1957). This delay in implantation is apparently due to a lack of estrogen, because an injection of estrogen will result in implantation of the blastocysts. The suppression of estrous cycles during lactation in the mouse and rat is influenced in part by the size of the litter. A litter of 8 to 10 young will inhibit cycles, whereas cycles are displayed if the litter is reduced to 2 or 3 young (Parkes, 1926a; Hain, 1935). The cottontail rabbit [Sijlvilagus floridamis) seems to be a species in which ovarian follicular development is little if any affected by lactation, for Schwartz ( 1942 ) stated that suckling does not prevent ovulation after coitus, at least in the early stages of lactation.

III. Corpus Liiteuni

The formation of the corpus luteum has been described for many species ( see reviews in Corner, 1945; Harrison, 1948a; Brambell, 1956). In general, after rupture of the follicle and discharge of the ovum, the granulosa is invaded by blood vessels from the theca interna (Bassett, 1943). They form a rich network among the enlarging granulosa lutein cells. The extent and nature of the contribution from the theca interna varies from species to species, but, as Corner states, the origin of the major part of the epithelioid cells of the corpus luteum from the granulosa may now be considered a fact.

Whereas there may be a fairly uniform pattern of development and control of ovarian follicles in mammalian forms, there are diverse mechanisms for the formation and maintenance of corpoi'a lutea; consequently

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specific examples must be presented in order to avoid the dangers of generalization.

In the rabbit copulation triggers a neurohumoral mechanism which releases gonadotrophin from the pituitary which subsequently induces ovulation in 10 to 12 hours. The ruptured follicles form corpora lutea which have a functional span of about 28 days if pregnancy ensues but only 14 days if the mating is infertile. Crystals of estrogen implanted into a corpus luteum of a rabbit will cause its persistence while other corj^ora lutea regress (Hammond and Robson, 1951 ). This suggests that either estrogen makes the corpus luteum more sensitive to pituitary maintenance (Hammond, 1956) or estrogen protects the corpus luteum from luteolytic action. In the cat, copulation induces ovulation about 25 hours after mating; the corpora lutea function for 36 days after an infertile mating, but gestation lasts 62 to 64 days. The ferret ovulates about 30 hours after copulation and the corpora lutea are functional for 42 days, w^hether the mating is fertile or infertile (Brambell, 1956 1.

In the unmated rat and mouse ovulation is spontaneous, but the resulting corpora lutea are nonfunctional and begin to regress within 2 days. After copulation the corpora lutea persist for 18 days if impregnation has occurred, but for only 12 days after an infertile mating. Copulation probably results in the release of enough additional gonadotrophin (LH or LTH) to activate the corpora lutea. In rats and mice the pituitary hormone, prolactin, is luteotrophic (LTH) (Desclin, 1949; Everett, 1956). These species have functional corpora lutea throughout lactation— actually two sets, that of pregnancy and that of the postpartum ovulation. Using the rat and taking weight and levels of ovarian enzymes as measures of activity, these corpora lutea were studied by Meyer and McShan and their associates and the results summarized in a ic\'i('\v (]\Ieyer and McShan, 1950). They found that "the weight of the corpora lutea of pregnancy increased greatly during the latt(>r half and that the amount of enzymes per corjius luteum was also greater. With some caution, they concluded that these corpora lutea are more highly functional during this phase of pregnancy than during the first half.

Not only copulation, but also injection of

estrogen at estrus is followed by the formation of functional corpora lutea. The estrogen maintenance of corpora lutea in rabbits and mice is offset by hypophysectomy (Hohn and Robson, 1949); presumably, therefore, maintenance is mediated through the anterior })ituitary. Reece and Turner (1937) showed that estrogen stimulates the rat i^ituitary to produce prolactin so the latter may be the luteotrophic agent in this s]:)ecies. Moore and Nalbandov (1955) found that prolactin is luteotrophic in sheep. To date this is the only species other than the rat and mouse in which prolactin has been shown to have luteotrophic activity.

In the guinea pig, monkey, man, and many other species, ovulation and the formation of functional corpora lutea are spontaneous. Copulation is not known to have any neurohumoral influence in these species. The corjiora lutea of the human female function for 2 to 3 months in pregnancy and for only 12 to 14 days in. an infertile cycle. Bergman ( 1949 ) states that the duration of the luteal i^hase is limited to a maximum of 16 days. In the rhesus monkey the functional life has been estimated to be about 13.5 days in the normal cycle, and approximately 30 days when pregnancy intervenes (Hisaw, 1944) . In the bitch, ovulation is spontaneous and the corpora lutea remain functional for 6 weeks irrespective of mating or pregnancy. In the lactating African elephant the corpora lutea degenerate soon after parturition (Perry, 1953) ; in the lactating domestic cat they not only persist, l)ut they become rejuvenated" (Dawson, 1946).

As a general statement, it can be said that the functional span of the corpora lutea is cithei' adequate to permit implantation or it is prolonged by copulation (as in rats and mice) so that imjilantation can occur. But inasmuch as imj:)lantation occurs in many species, including man, about the sixth day after ovulation and fertilization, the margin of safety is not great and a delay in the secretion of chorionic gonadotrophin by the tro])hoblast must reduce the chances of a successful pregnancy.

Ill some species, e.f/., rats, mice, rabbits, an ill felt ih' mating prolongs the life of the corpora hitea. This prolonged interval of functional luteal activity is known as pseudoj)regnancy. As Everett has noted in his

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chapter, in pseudopregnancy the hormonal aspects of pregnancy are duplicated, but no fetal tissues are present. In the pseudopregnant bitch, for example, the hormonal aspects of pregnancy are so nearly duplicated that lactation begins at the time a normal gestation would have terminated.

The duration of pseudopregnancy in different species offers evidence of adaptive or evolutionary mechanisms to control the duration of corpus luteum function, mechanisms that must be endogenous to the uterus. Rats and mice have a pseudopregnancy of 12 days duration after a sterile mating, cervical stimulation, or injection of estrogen at estrus. There is no comparable condition in guinea pigs, monkeys, or man. However, if rabbits, rats, or guinea pigs are hysterectomized, any subsequent corpora lutea will function for a time equivalent to the duration of gestation in each species (Chu, Lee and You, 1946; Bradbury, Brown and Gray, 1950), although Velardo, Olsen, Hisaw and Dawson (1953) stated that, in the rat, hysterectomy has no effect on the length of pseudopregnancy. Hysterectomy in the cow and sow will prolong the life of the corpus luteum (Melampy, personal communication). Experimental distention of the uterus l)y beads has resulted in alteration of the length of the estrous cycle in ewes (Nall)andov, Moore and Norton, 1955). The only explanation which seems to account for these results is that there is a luteolytic agent in the uterus (probably in the endometrium) of some polyestrous species which shortens the life of the corpora lutea in nonpregnant animals. In pregnancy, or when massive deciduomas are present, if Velardo, Olsen, Hisaw and Dawson are correct, the conversion of endometrium to decidual tissue may cause it to lose its luteolytic ability. In future studies on the duration of the functional span of cor]5ora lutea, the possibility of luteotrophic and luteolytic mechanisms should be considered. On the other hand, a fresh start may be advisable. Fewproblems in reproductive and clinical endocrinology (Marx, 1935) seem to have been as resistant to clarification.

In unmated females of species not having a spontaneous "pseudopregnancy," the corlius luteum involutes shortly after its formation. The rat, in which 4 to 8 corpora lutea

are formed in each ovary at intervals of 4 to 5 days, has recognizable involuting corpora lutea from the two preceding cycles, but no remnants of older ones. The early stages of involution of the corpus luteum have been described (Brewer, 1942; Boling, 1942; Dawson, 1946; Duke, 1949; Moss, Wrenn and Sykes, 1954; Corner, Jr., 1956; Rowlands, 1956; Dickie, Atkinson and Fekete, 1957). The timetables of cellular changes given by Brewer and by Corner, Jr. are of interest for the comparison they permit with physiologic estimates of the duration of secretory activity by the human corpus luteum. On day 7 the corpus luteum seems to have reached its peak of activity, as judged by the vacuolation of its cells in Bouin's or Zenker's fluid-fixed and hematoxylin and eosin-stained preparations. Corpora lutea of days 9 to 12 show evidence of i)rogressive secretory exhaustion.

The later stages of cori:)ora lutea degeneration have not received the same careful attention. In women the corpus luteum undergoes a slow hyaline degeneration and the corpora albicantia persist as old scars for months or years. They may be present in ovaries 15 to 20 years after the menopause. Whether the final stage of degeneration is a process of lysis, phagocytosis, or transformation into connective tissue has not been studied.

IV. Follicular Atresia

It was long ago estimated that the infantile liuman ovary contains about 400,000 oocytes (Fig. 7.4). In the 30 years of reproductive life about 400 ova may mature and ovulate. On this basis about 1 oocyte in 1000 achieves ovulation; the other 999 are lost through a degenerative process known as atresia. The problem is not different in any other species. Whether it is monotocous or polytocous, there is always an enormous wastage of oocytes in each cycle of folliculogenesis. Atresia may have its onset at any stage of follicular growth or maturation and oocytes may degenerate before they have acquired a distinct membrana granulosa (Mandl and Zuckerman. 1950; de Wit, 1953; Payne, Hellbaum and Owens, 1956; Williams, 1956). In advanced stages of follicular development the granulosa cells may show pycnotic changes before any degenera

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PHYSIOLOGY OF GONADS

Fig. 7.4. Ovary from 28-moiith-old child. Many primordial follicles just beneath the tunica albuginea. (Courtesy of Dr. J. T. Bradbury.)

Fill. 7.5. Iimuaturc rat h grow to medium size. Many become atretic and leave stitial tissue. (Courtesy of Dr. R. M. Melampy.)

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Fig. 7.6. Intact immature rat given progesterone for 3 days. The various-sized follicles in this area are in early stages of atresia. The pycnotic granulosa cells are dispersing into the follicular fluid and the oocyte in the small follicle at the upper left is denuded. (Courtesv of Dr. J. T.Bradburv.)

tive clianges are evident in the oocyte. When vesicular follicles become atretic the granulosa disintegrates and the cells disperse into the liquor folliculi (Knigge and Leathern, 19.56 ( . If the follicle has developed a distinct theca interna, the theca regresses after the granulosa has disintegrated. In rats the atretic follicles leave no recognizable histologic remnants. In some species the theca regresses back to ovarian interstitial tissue (Dawson and AlcCabe, 1951; Williams, 1956). In the ovary of the human female and rhesus monkey the atretic follicle leaves a scar (corpus atreticum) in which the meml)rana propria persists for months as a folded hyaline membrane within the loose fibrous remnant of the theca.

The cause of follicular atresia is not known. Immediately after hypophysectomy there is a wave of atresia in the ovary of the immature rat (Fig. 7.5) and rabbit (Foster, Foster and Hisaw, 1937). In adult rats the postovulatory wave of follicular atresia has

l)een attrilnited to an action of the corjiora lutea (Atkinson and Leathem, 1946). Injections of androgen or of progesterone increase the incidence of atr(>sia in rat ovaries (Fig. 7.6) (Paesi, 1949b; Barraclough, 1955; Payne, Hellbaum and Owens, 1956). Further study is necessary to determine whether the atresia is due to a direct effect of androgen or progesterone on the follicles, or whether the postovulatory decline in gonadotrophins, like hypophysectomy, withdraws a supi:)orting influence and permits the follicles to degenerate. The supporting influence may be estrogenic because injections of estrogen at the time of hypophysectomy will prevent, or at least delay, the expected follicular atresia (Fig. 7.7). Some months after hypophysectomy the number of remaining oocytes is greater than in the ovaries of normal litter-mate sisters (Ingram, 1953). This suggests that vegetating oocytes are less liable to undergo

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PHYSIOLOGY OF GONADS

It:

'^l&^h

■ ■;%.%,

Fig. 7.7. iiniuaiKi. li.\ pupliy.sectomized rat treated with estrogen (dit'thylstilbe.Ntrol). Many follicles have developed to a size appropriate for antrum formation. The interstitium i.s atrophic but the theca is differentiated. One follicle is obviously atretic. (Courtesy of Dr. J. T. Bnidburv.)

atresia than those which have entered the growth phase.

Hisaw (1947) discussed the problem and marshaled a number of facts in support of the idea that atresia is due to a defective differentiation of the theca interna, with a ^resulting deficiency of estrogen which is considered necessary for growth and differentiation of the granulosa. Ultimately the ])ituitary is involved because the success which has been achieved in the j^roduction of sui)erovulation reveals that the number of follicles maturing and ovulating is a measure of the amount of gonadotrophic hormone. But it is eciually true that there is an optimal dosage and time beyond which defects appear in the form of cystic follicles and premature luteinization (see review by Hisaw and more recently Zarrow, Caldw(>ll, Hafez and Piiicus, 19581.

The disintegration of the discus proligerus of the nuiture follicle and the dissolution of the granulosa in an atretic follicle have led several investigators to suggest that the

stimulus to ovulation and/or atresia is identical (Harman and Kirgis, 1938; Dawson and McCabe, 1951 ; Moricard and Gothie, 1953; Williams, 1956). Moricard and Gothic found that intrafollicular injection of HCG or P]\1S caused first polar body formation within 4 liours. Control injections of estrogen or scrum were ineffective. Dempsey (1939) noted that maturation spindles were present in ne:iily all the oocytes in medium and large follicles which had undergone atresia shortly after ovulation had been induced by luteinizing hormone. Inasmuch as the tubal egg ntay give off the second polar body about the time the corona radiata is lost, it is interesting to speculate on the significance of the fact that the eggs in atretic follicles may also give off polar bodies just as they are denuded of granulosa (Fig. 7.8).

V. Hormones of the Ovary

The hoi'mones of the ovary are the estrogens, pi-ogesterone. androgen, and relaxin. The first three are the o\'ai"ian steroid hor

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Vu,. 7.S. Aticiic lolliric in immature rat gi\en progesterone for 3 day: and second metaphase si>indle. (Courtesy of Dr. J. T. Biadbury.)

mones and are among the most important hormones participating in the regulation of reproductive physiology. In the present hook, as in editions 1 and 2, the discussions of their many actions consititute one of the central themes.

Relaxin is a protein rather than a steroid hormone and has always been considered more or less apart from the latter. The steps in proving its existence were reviewed by Hisaw and Zarrow in 1950, and the present status of the subject is discussed in the chapter by Zarrow. Unlike the other ovarian hormones, its clinical value is still uncertain (Swann and Schumacher, 1958; Stone, 1959).

Ovarian androgens present a perplexing problem. Evidence for their production is abundant (Guyenot and Naville-Trolliet. 1936; Hill, 1937a, b; Deanesly, 1938a; Bradbury and Gaensbauer, 1939; Greene and Burrill, 1939; Chamorro, 1943; Price, 1944; Katsh, 1950; Desclin, 1955; Johnson, 1958). l)ut the extent to which they are produced l)y the ovaries of normal females, and the nature of their action in normal females are

uncertain (Parkes, 1950). It has been postulated that they are produced during the normal cycle in the rat. Payne, Hellbaum and Owens (1956) suggested that the interstitial androgens produced at estrus are responsible for the postovulatory atresia of the partially developed follicles. The production of ovarian androgens has been demonstrated most effectively under abnormal conditions of stimulation. The newborn rat ovary will produce androgens when stimulated by human chorionic gonadotrophin (Bradbury and Gaensbauer, 1939). The treated infant rat shows a marked enlargement of the clitoris and may even develop the cartilage anlage of the os penis. Female guinea pigs are masculinized by injections of HOG (Guyenot and Naville-Trolliet, 1936). Ovaries transplanted to the ear of the castrate male mouse will produce enough androgen to maintain the prostate and seminal vesicle (Hill, 1937a, b). Ovarian transl^lants into the seminal vesicle may exhibit a localized androgenic stimulation of that tissue (Katsh, 1950). Ovaries of a rat in

4(36

PHYSIOLOGY OF GONADS

parabiosi;? with a castrate partner become hypertrophied and produce enougli androgen to stimulate prostatic tissue (Jolmson, 1958) ; associated with the condition is an unusual thecal and interstitial tissue hypertrophy. The masculinizing features of the Stein-Leventhal syndrome ( polycystic ovary) have been attributed to the presence of androgens, having their source perhaps in the hilar cells of the ovaries (Lisser and Traut, 1954) , but the action of other steroids with masculinizing properties also has been suggested (Fischer and Riley, 1952). The latter possibility might well be checked in any case when the production of androgens by the ovary is suspected.

Estrogen and progesterone are the ovarian hormones whose action in the female has been studied the most extensively. The steps which led to their extraction and chemical identification were described by Doisy and by Willard Allen in the 1932 and 1939 editions and will not be repeated here. Attention, however, will be directed to the isolation and identification of several naturally occurring gestagens in addition to progesterone (Davis and Plotz, 1957; Zander, Forbes, von Miinstermann and Neher, 1958). These are metabolic degradation products which still retain progestational activity.

Now as in the period 1932 to 1939, there are many unsolved problems, but it is equally true that much of interest and value has been learned. Not the least of these contributions has been the clarification of the l)athways of their biosynthesis (see chapter by Villeej . In the sections which follow particular attention will be given to the problem of their origin, to the rate of production, the manner of transport and storage, and to their "half-life."

A. CELLULAR ORTOIX

As Villee points out in his chajiter, estrogen and progesterone are apparently derived from cholesterol by a series of chemical changes. Within the ovary and in the corpora lutea of rats, there is a definite reduction in the concentration of ascorbic acid and cholesterol after gonadotrophin administration. These changes have been considered (ividence for tiie activation of hormone synthesis (Everett, 1947; Miller and Everett, 1948; Levin and Jailer, 1948; Aldman,

Claesson, Hillarp and Odeblad, 1949; Claesson, Hillarp, Hogberg and Hokfelt, 1949; Noach and van Rees, 1958).

Efforts to identify the cell types in which these processes take place have not been altogether successful. We have noted, for example, that several tissues such as testis,, placenta, and occasionally the adrenal cortex can produce estrogen. These are tissues,, then, which produce more than one hormone. Gardner emphasized this during a discussion of Parkes' (1950) review of androgenic activity of the ovary, when he called attention to Dr. Furth's observations that some ovarian tumors possess potentialities for bisexual hormone production. About the same time, Shippel (1950) postulated that thecal cells may be a source of estrogen and androgen and that the type of hormone produced may depend on particular stresses or stimuli. On the other hand, many investigators,, and particularly those interested in the apjilication of histocheraical procedures,^ have l^roceeded under the assumption that there are tissues of the ovary in which one hormone is iH'oduced predominantly. They found, for example, that the reactions of follicular granulosa and theca cells are strikingly different (Demi)sey and Bassett, 1943; Dempsey, 1948; Shij^pel, 1950). To be sure, the results w^iich have been obtained have not led to agreement with respect to details, but there is much evidence from the reactions which have been described, as well as from the older morphologic studies, that theca interna, interstitial, and luteal cells and, perhaps to a lesser extent, granulosa cells, are active in steroid hormone synthesis. What is less certain than the fact that these cells, and consequently the ovaries, produce estrogen, progesterone, and androgen is their relative role compared with that of other tissues. Presumably it is major; nevertheless, evidence for the extra-ovarian origin of estrogenic substances is provided by the occurrence of cyclic vaginal activity in ovariectomized animals (Kostitch and Telebakovitch, 1929; Mandl, 1951; Veziris,

"Dempsey and Bassett, 1943; Dempsey, 1948; Claesson and Hillarp, 1947a-c ; Claesson, Diczfahisy, Hillarp and Hogl)erg, 1948; McKay and Robinson. 1947: Sliii)iH>l. 1950: Barker. 1951: Rockenscliauh. 1951: Wliite, Heitis, Rock and Adams. 1951: Deane. 1«)52: Fuiulue-lm, 1954: Xisliizuka. 1954.

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1951) and by the high titer of estrogens in the urine from ovariectomized rats on a high fat diet (Ferret, 1950).

Corner, as long ago as 1938, in a consideration of the subject, emphasized that there is only circumstantial evidence that the ovary is the major site of estrogen production. Attempts to extract estradiol or any other estrogen from ovarian tissue had yielded very small amounts. MacCorquodale, Thayer and Doisy (1936) processed 4 tons of hog ovaries and recovered about 6 mg. estradiol from each ton. They estimated that the concentration in liquor folliculi was of the order of 1 part in 15,000,000 and that about 0.1 of this concentration is in the rest of the ovarian tissue. There are much better sources from which "ovarian hormone" can be extracted than from the ovary, i.e., placentas, pregnancy urine, the urine of the stallion or boar. The adrenal is also a source and there may be other tissues as well, for Bulbrook and Greenwood (1957) reported that urinary estrogen continued to be excreted after oophorectomy and adrenalectomy of a breast-cancer patient.

Whatever the relationships are quantitatively between the follicles and the other estrogen-secreting tissues, the evidence that the follicles are a major source of estrogenic substances remains impressive. This is also true of the corpus luteum. The human corpus luteum produces as much, or more, estrogen than was produced during the follicular phase. In the rat and mouse, estrogen production during the luteal phase must be low because more than 1 part of estrogen nullifies the action of 1000 parts of progesterone in these species ( Velardo and Hisaw, 1951). By contrast, ratios as high as 100 parts of estrogen to 1000 parts of progesterone enhance the progestational reactions in women (Long and Bradbury, 1951).

The different tissues of the ovary — membrana granulosa, theca interna, and interstitial cells — have been studied in efforts to ascertain whether they are sites of the production of specific hormones. Inasmuch as unruptured follicles and corpora lutea secrete both hormones, the two structures must be considered. It has long been known that corpora lutea secrete estrogen as well as progesterone. Evidence supporting the conclusion that jirogesterone is secreted by

the preovulatory follicle is more recent, but it comes from many sources. The possibility was first suggested following the discoveries that the beginning of mating behavior (Dempsey, Hertz and Young, 1936) and the decrease in tissue uterine fluid (Astwood, 1939) , which depend on the presence of small amounts of progesterone, coincide with the beginning of the preovulatory swelling. More recently, progesterone has been found in the follicular fluid from sows, cows, and the human female, and, in small quantities in blood plasma of the rabbit, human female, and rhesus monkey during the follicular phase of the cycle (Duy vene de Wit, 1942 ; Forbes, 1950, 1953; Bryans, 1951; Kaufmann, 1952; Edgar, 1953b; Buchholz, Dibbelt and Schild, 1954; Zander, 1954).

Earlier in this section it was noted that there is much evidence from both histochemical and the older morphologic studies that theca interna, interstitial tissue, and luteal cells, and to a lesser extent, granulosa cells secrete the ovarian steroid hormones. Many who have used histochemical methods still feel that, even though these methods demonstrate steroids and their precursors, the reactions are not sufficiently specific for identification of the individual hormones. Others, however, have been more confident, and when the evidence they have presented is combined with that reported in some of the more conventional morphologic studies the following summarization of opinion seems justified. Granulosa cells of the follicles and granulosa lutein cells of the corpora lutea contain progesterone or a precursor and secrete this hormone (Nishizuka, 1954; Green, 1955). Cells of the theca interna, theca lutein cells, and interstitial cells are believed to secrete estrogen and possibly androgen (Corner, 1938; Deanesly, 1938a; Pfeifter and Hooker, 1942; Hernandez, 1943; Claesson and Hillarp, 1947a, b; Rockenschaub, 1951 ; Aron and Aron, 1952; Furuhjelm, 1954; Nishizuka, 1954; Fetzer, Hillebrecht, Muschke and Tonutti, 1955; Johnson, 1958). The interstitial cells have been the object of much study and will be given especial attention.

Interstitial tissue or cells in the ovary is not as clear a concept as it is in the teste.-. In the latter interstitial or Leydig cells are derivatives of connective tissue elements

468

PHYSIOLOGY OF GONADS

irf^'t'H-'^

. 7.'.). Aii< ^lioii.-. ral)l)U. J^aigc lulliclf^ lUulcifioinK ati< Ma Iiilcr^l il luui i,> \i>ilil<- only as the narrow wedge of granular tissue extending from the cortex into the intrafollit-ular septum. (Courtesy of Dr. J. T. Bradbiuy.)

and can (ledift'erentiate to form connective tissue cells (Esaki, 1928; Williams, 1950). The role of Leydig cells as secretors of testicular androgen or a precursor is not (juestioned. In the ovary it is also presumed that undifferentiated connective tissue elements exist, indeed much of the stroma must be composed of such cells. It is believed rather generally, although unequivocal proof has not been given, that the theca interna is derived from connective tissue elements and that, as a component of the Graafian follicle, it secretes estrogen and possibly androgen Hoc. cit.). After ovulation and corpus luteura formation, and after atresia in the case of follicles not rui)turing, the cells of the theca interna may pcrhajjs I'csuinc their place as connective tissue cells or they may become interstitial cells (Mossman, 1937; Dawson and McCabe, 1955; Rennels, 1951 ; Nishizuka, 1954; Williams, 1956). The |)rominence of interstitial tissue varies from species to species and also with stages of the

rei)roductive cycle. Whether it is functional in i^roducing hormones has been controversial, but most contemporary investigators seem to feel that internal secretory capacity has been demonstrated. In all the work that has been done, supporting evidence is varied; in some cases it is circumstantial, but in others it is quite substantial.

Interstitial tissue is deficient in the anestrous rabbit, and even though there may be considerable follicular development, there seems to be little or no estrogen production (Claesson and Hillarp, 1947a) (Fig. 7.9).^ The liypei'ti'opliied iiitei'stitium of the estroiis i;il)l)it (Fig. 7.10) undergoes further de\-el()pnicnt (hii'ing prc'gnancy and seems almost as luteinized as th(^ corpora lutea

' Rogr('ssi\-e changes in tlic rciirodiictixe tract and accessory structures following ovariectomy of the anestrous opossimi were taken to indicate that these parts receive estrogenic stimidation of ovarian origin during th(^ anestnun (Morgan, 1946; Risman. 1946).

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469

Kid, 7,10. l'(isto\nl.-,l,,iy stigma is evident. Note tli of Dr. J. T. Bradburv.)

epithelioid natiii

of the hypertiopliied mterstitiuin. (Courtesy

(Fig. 7.11). Grossly the ovary in the anestrous rabbit is translucent whereas the estrous ovaries and the ovaries during pregnancy have a chalky white opacity due to the development of the interestitium. After hypophysectomy the interstitial cells of the rat ovary exhibit a deficiency condition and the nuclear appearance has suggested the name "wheel cells." If pituitary ICSH is administered, the deficiency cells are restored to normal (Fig. 7.12). Hyperplastic ovarian interstitium in older women has been considered a probable source of estrogen in some cases and of androgen in others. The stimulation of interstitium by injected gonadotrophins may be associated with the formation of estrogens and/or androgens (Bradbury and Gaensbauer, 1939; Marx and Bradbury, 1940). Some rats displayed a permanent estrus; others, during a period of androgenic function, were masculinized. During this period, the theca and interstitium were not luteinized in many cases and it was concluded from the responses of accessory organs that these small

immature cells had secreted male hormone and perhaps female hormone, too. In rats with fully luteinized theca and interstitium and the pronounced estrous symptoms, it was considered that the androgenic effect was no longer apparent. Information obtained recently, however, suggests that the permanent estrus, when it was shown, may have been a consequence of an androgenic effect. Cystic follicles which might have stimulated a permanent estrus had a vagina been present, were found in many adult guinea pigs which had received androgen prenatally (Tedford and Young, 1960).

Without necessarily excluding the possibility that the heterotypical hormone is also jiroduced, many articles contain suggestions that interstitial tissue has specific estrogenic or androgenic activity. There is the report that an ovarian interstitial-cell tumor was producing estrogens (Plate, 1957). The observation that estrogen continues to be secreted by ovaries in which the follicles have been destroyed by x-rays was reported by Parkes (1926b, i927a,^b), Brambell and

470

PHYSIOLOGY OF GONADS

V J ' *

k •'

6^i;>ir/

^ V

^,

Fi(. 7 11 ()\,ii\ liMiii jir. ^uaiii i.ililni l.amc luu uiiz. d < . IK m liiu. ih.niiiii ( uipu- liiii iim. Hvi)f'itioi)lue(l intPi-titium. Pninoidial t'olhcles in cortex. (Couitc^y of Di. J. T. Bra(U)iuy.)

Parkes (1927), Genther (1931), Schmidt (1936), Mandl and Zuckerman (1956a, b), and others. This conclusion w^ould seem to be .strengthened by the recent report that there is no intensification of the secretion of gonadotrophins by x-rayed rats in which there was an apparent destruction of the ova and folhcles (AVestman, 1958). Evidence of an entirely different sort for the secretion of estrogen by interstitial tissue has been i)resented by Ingram (1957). Autografts of medullary tissue containing interstitial tissue but no follicles were made in rabbits. Five animals from which this tissue was recovered had uteri which were not as atroi)hic as the uteri of spayed animals. He noted, however, that in the absence of the follicular apparatus the capacity to secrete estrogen is soon lost. As we have seen,- Ingram is one of several investigators who have related the functioning of interstitial tissue to granulosa elements.

Histochemical staining procedures for cholesterol indicated to Dempsey (1948) that the theca interna is a possible source of estrogen. The results obtained during a more extensive utilization of histochemical reactions in studies of the ovaries of nonpregnant, psoudopregnant, and pregnant rabbits, and in the ovaries of rats and guinea pigs were consistent with the conclusion that a li])i(l i)recursor of estrogenic substances is ]:)resent in interstitial tissue (Claesson, 1954; Claesson and Hillarji, 1947a, b; Claesson, Diczfalusy, Hillarp and Hclgberg, 1948).

Rennels (19511, on the basis of histochemical reactions in the oxaries of innnature rats, advanced the liypothesis that interstitial tissue has a dual origin. There is a primary type present between 10 and 18 (lays aftei- bii'th wliicli is dosc^ly associated with gi'anulosa ()Ut<j;i()wtlis and ingrowing cords of cells from the germinal epithelium. A secondary type is formed later from the

MAMMALIAN OVARY

471

-i:-* :^-"!r

t:^-^-!..

4"

jP^-'-^i'

Fig. 7.12. Immature hypoi)liyspftomized rat alter 3 days treatment with Armour's IC8H. The interstitium is restored and is mildly hyperplastic. Nearly all of the vesicular follicles are atretic. The theca blends into the interstitimn. Two of the oocytes contain maturation spindles. (Courtesy of Dr. R. M. Melampy.)

theca interna of atretic follicles. He presented no evidence for the production of estrogen by the latter tissue, but expressed the opinion that Claesson and Hillarp (1947b) had done so. Under the assumption that estrogen rather than androgen is produced by o^'aries of untreated rats during the early juvenile period (10th to 18th day), he interpreted the presence of histochemically reactive materials in the iirimary interstitial tissue as an indicator of estrogenic activity. To Huseby, Samuels and Helmreich (1954), the steroid-3/?-ol dehydrogenase activity in interstitial cell tumors having androgenic activity, suggested a relationship between the presence of this enzyme and the production of the androgen.

B. AMOUNTS OF HORMONE PRODUCED

Dependable estimates of the rate of estrogen and progesterone production would provide investigators of reproductive physiology with interesting and valuable information. It must be recognized, however, that there are many pitfalls and outright difficid

ties; consetiuently the estimates which have been made must be regarded as tentative. Furthermore, they are of limited value. The amounts produced probably deviate greatly from the quantities which are effective in meeting the threshold requirements of the tissues the ovarian hormones stimulate. This latter information would make the greater contribution to an understanding of the functioning of these substances in the regulation of rei^roductive processes. Most efforts to estimate the rate of secretion of estrogen have involved measurements of the amount of hormone given subcutaneously that will restore normal structure or function in ovariectomized animals. As Corner (1940) emphasized, estimates obtained in this manner are based on the assumption that a hormone injected once daily in an oil solution is utilized by the body as efficiently as a hormone produced by an animal's own ovaries. Gillman (1942), Barahona, Bruzzone and Lip.schutz (1950), and Zondek (1954) are among the many who have directed attention to the fact that the amount of an esiro

472

PHYSIOLOGY OF GONADS

gen required to e^'okc one response often is different from that required for a second response. For example, the amount of estradiol necessary to produce perineal enlargement in the baboon is less than that required to produce withdrawal bleeding. In the human female, the order of sensitivity of three tissues to estrogen is uterine cervix, vaginal mucosa, endometrium (Zondek, 1954). Theoretically, therefore, if the response of tissues is to be used in estimating the amount of hormone produced, the investigator should follow the response having the highest threshold value. Gillman also called attention to another complication. The minimal amount of estrogen necessary to produce a response does not necessarily approximate the amount being produced, because, in the instance he cites, larger amounts do not produce a larger perineum.

Another comi^licating circumstance is the occurrence of inherent" cycles (in some cases the length of a reproductive cycle) of responsiveness which have been demonstrated in ovariectomized females receiving constant amounts of estrogen from exogenous sources. Many such animals have displayed cyclic vaginal changes (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941a), uterine l)leeding (Zuckerman, 1940-41 ), and running activity (Young and Fish, 1945). This unknown factor must be taken into consideration in any attempt to estimate the rate of estrogen production. Existence of this factor gives emphasis to the importance of direct determinations, wiien they can be made, either from follicular fluid or from freshly drawn blood. Corresponding considerations would hardly be tliough of as applying to progesterone. Most, if not all, of its actions are synergistic or potentiating. Presumably, therefore, they are directly dependent, not so much on any changes in the inherent responsiveness of the tissues as on the extent to which the tissues have been conditioned or primed by the estrogen.

A part of the picture which must be brought into context with the problem of estrogen secretion comes from a review of the temporal factors in a normal cyclic animal. In animals with short estrous cycles (4 or 5 days in the rat, mouse, and hamster) . and in

species in which the cycles are longer as in the guinea pig it has generally been assumed that estrogen is produced maximally at the time of estrus. This is an assumption based on the simultaneous occurrence of the cornified vaginal smear and the display of estrous behavior. When one considers that 48 to 72 hours are necessary for vaginal epithelium to proliferate and then degenerate into cornified cells, it is obvious that the estrogen which starts these changes must be elaborated 2 or 3 days before estrus and therefore before the size of the follicles is maximal. Zondek (1940) demonstrated that if an immature rat was injected with HCG the ovaries could be removed 27 hours later and the rat would exhibit vaginal cornification in 84 to 96 hours. This emphasizes that the estrogen which caused the cornified smear had been elaborated 2 or 3 days before vaginal estrus (Zondek and Sklow, 1942; Green, 1956). The dilation of the uterus with fluid late in the proestrum (Astwood, 1939) is also evidence that the efi^ective estrogen had been elaborated 24 to 30 hours earlier.

Problems of assay are involved in any attempt to estimate secreted estrogen and are discussed briefly by Emmens (1950a, b). They are more serious in the case of the estrogens than in the case of progesterone. The bioassay of estrogens is usually based on vaginal cytology or change in uterine weight. The vaginal cytology or smear method is essentially the original method of Allen and Doisy(kahnt and Doisy, 1928; Allen, 1932). Ovariectomized rats or mice are given subcutaneous injections of the substance being tested for estrogenic i)otency. Smears are made of the vaginal contents 24, 48, 60, and 72 hours later. If the smear reveals the presence of cornified cells 60 to 72 hours after the first injection, the tested substance is judged to be estrogenic. By using groups of animals at each of several dose levels, the minimal eft'ectiA-e dose can be judged. The smallest amount of substance which will jiroduce cornified smears in 50 to 70 ]ier cent of the test grouj) is usually designated as the rat or mouse unit. Intravaginal tests, introduced by Berger (1935) and by Lyons and Templeton (1936), and refined, especially by Biggei's ( 1953) and by Biggers and Claringbold (1954, 1955), are 200 times more

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sensitive than tliose in which the estrogen is administered subcutaneously. When the mean number of arrested mitoses was used as the measure of estrogenic activity, the sensitivity was increased another ten times ( Martin and Claringbold, 1958).

Immature rats or mice can also be used foi' the assay of estrogens. The establishment of vaginal patency and mucified or cornificd vaginal smear denote estrogen effects. Vaginal patency per se, however, is not specific for estrogen because androgen will also induce precocious vaginal patency (Rubinstein, Abarbanel and Nader, 1938; Marx and Bradbury, 1940). Injection of estrogen into immature animals causes a rapid increase in the weight of the uterus, due to water imbibition. Astwood (1938) found that the immature rat uterus increases in weight as early as 6 hours after an injection of estrogen; however, the optimal response was at 30 hours. Just above threshold levels graded doses produce graded increases in uterine weight. This makes it possible to plot a dose-response curve so that closer approximations of potency can be achieved. Some authors have used ovariectoraized animals for the uterine response method. This requires a prior operation and subsequent adhesions may make it difficult to strip out the uterus cleanly at the end of the test.

Whatever the test, each estrogen derivative or synthetic estrogen has an optimal assay interval for maximal effect depending in part on its solubility, rate of absorption, and utilization (Hisaw, 1959). For this reason a standard assay may not be an accurate indicator of the estrogenic potency of several comijounds. This factor plus some competitive antagonism make this method impractical for the assay of mixtures of estrogens (Merrill, 1958). Whatever their faults, the bioassay methods in general are very sensitive and will detect estrogens in 0.1 to 1.0 ixg. quantities.

The chemical assay methods for estrogen are rather involved and have usually been relial)le only in milligram quantities. Fluorometric methods were tried and generally discarded because frequently small amounts of contaminants were strongly fluorescent; consequently fluorescence was being obtained in the absence of biologic activity

(Bitman, Wrenn and Sykes, 1958) . Recently paper chromatographic methods have permitted sufficient purification and isolation to make identification and quantification of estrogens in microgram quantities (Brown, 1955; Smith, 1960; Svendsen, 1960).

The most common bioassay methods for progesterone are still the Corner-Allen and the Clauberg tests which utilize the rabbit. The animal is primed with estrogen and then given progesterone after an appropriate interval. A portion of the uterus is removed and examined histologically for the degree of glandular develojiment in the endometrium. The test is relatively insensitive since it requires about 1 mg. progesterone per rabbit. McGinty, Anderson and McCullough (1939) increased the sensitivity of the test to 0.5 to 5.0 /xg. by injecting the progesterone into the lumen of an isolated segment of the rabbit uterus. The histologic response of the endometrium in this isolated segment was then judged.

Hooker and Forbes (1947) adapted the McGinty intra-uterine technique to the uterus of the ovariectomized mouse. The end result is judged histologically by the characteristics of the endometrial stromal nuclei of the isolated uterine segment. The sensitivity is of the order of 0.3 fxg. per ml. and the method has been used widely. This advantage of the Hooker-Forbes technique is that it is sensitive enough to detect gestagens in l)lood plasma and liquor folliculi. Disadvantages are that the test is not specific for progesterone, and that certain gestagens such as 17-a-hydroxyprogesterone which is devoid of progestational activity in some species (rabbit, guinea pig, man) are very active in the mouse test (Zarrow, Neher, Lazo-Wasem and Salhanick, 1957; Short, 1960).

There are spectrophotometric techniques for jn'ogesterone assay (Reynolds and Ginsburg, 1942; Zander and Simmer, 1954; Short, 1958; Sommerville and Deshpande, 1958 ) . These methods have the advantage of instrumental precision, but require rather tedious initial chemical purification. However, they have proved of especial value in comparative studies of the blood levels of progesterone in sheep with active and inactive ovaries, in studies of the progester

474

PHYSIOLOGY OF GONADS

one content of unruptured follicles in the ovaries of cows and sows, and in determinations of the cjuantities of progesterone secreted by 1 or 2 corpora lutea in sheep ( Edgar, 1953a, b; Edgar and Ronaldson, 19581.

After allowance was made for these considerations tentative estimates were given: rhesus monkey, 200 I.U. or 20 y estrone daily; human' female, 3000 I.U. or 300 y estrone daily (Corner, 1940); baboon {Papio porcarius) , somewhat more than 0.04 mg. estradiol benzoate daily (Gillman, 1942) ; guinea pig, less than the equivalent of 1.8 fjig. estradiol daily (Barahona, Bruzzone and Lipschutz, 1950). The variation in the responsiveness of individual animals was recognized by all the investigators. Two important variables were not considered, the cyclic growth of follicles, and the number of developing follicles. Presumptive evidence that the amount of secreted estrogen increases as the follicle enlarges was provided by the demonstration that more and more estrogen is required to maintain perineal turgescence (Gillman and Gilbert, 1946). The number of developing follicles may vary greatly within a species, in the guinea pig, for example, from 1 to at least 6. The fact that two corpora lutea in the ewe do not produce more progesterone than one (Edgar and Ronaldson, 1958) could prepare us for a corresponding finding with respect to estrogen.

The problem of estimating the rate of progesterone secretion is l)eset by many of the difficulties that confront an investigator attempting to estimate the rate of estrogen production, but one circumstance especially has facilitated progress by those especially interested in progesterone. It is that the amount of excreted free prcgnanediol or excreted sodium pregnanediol glucuronidate is about 1/7 the amount of injected progesterone (Trolle, 1955a, b) ; from determinations of cither of tlic former, therefore, the amount of the latter can be estimated with what is believed to be a reasonable degree of accuracy. The pioneer attempt of Corner (1937) to calculate the amount of progesterone secreted by the rabbit, sow, and human female resulted in estimates (60 mg. during the luteal phase of the cycle in the latter) wliicli are much lower than those made more recentlv lObei

and Weber, 1951, 200 mg.; Kaufmann, 1952, 200 mg.; Trolle, 1955a, b, 260 to 440 mg.). It now seems that the lower estimate made by Corner can be attributed to the uncontrolled loss of sodium pregnanediol glucuronidate during storage, owing to bacterial hydrolysis (Trolle, 1955a). The rise in the 24-hour values after ovulation, the attainment of a peak the 7th and 8th days, and the decline between then and menstruation, are shown nicely in Trolle's (1955a) study. His data provide an excellent confirmation of the estimates based on structural changes within the cell (Brewer, 1942; Corner, .Jr., 1956). The amount of free pregnanediol excreted during the cycle and therefore the amount of secreted progesterone varied from woman to woman, and in the same woman there was variation from one cycle to another.

Data obtained by Duncan, Bowerman, Hearn and Alelampy (1960) from their chromatographic study have provided the basis of an estimate that the following average amounts of jn-ogesterone are present in the luteal tissue from swine: 23 /Ag. on day 4 of the cycle; 213 /^g. on day 8; 335 yug. on day 12; 311 [xg. on day 16; /xg. on day 18. From day 16 to day 102 of pregnancy, the amount rose from 477 [xg. on day 16 to 578 fxg. on day 48 and then decreased to a low of 120 ixg. on day 102.

Edgar and Ronaldson (1958) made direct measurements of the progesterone in lilood collected from the ovarian vein in ewes. The assay method consisted of extraction of 20 ml. of blood by, and partition between, organic solvents, final separation by chromotographic i)artition on filter paper, and subsequent estimation of the hormone by ultraviolet absorjjtion spectroscopy. They reported that there is great variability from animal to animal. The concentration in yearling sheep was not lower than that in older animals. Because of the liypothesis advanced by Young and Yerkes ( 1943) that the amount of secreted progesterone is low in adolescent chimpanzees, an extension of the Edgar and Ronaldson jirocedures to |)iimates would be of interest. In this connection, Edgar and Ronaldson postulated what has been brought out as a generalization in so many studies of the steroid hormones. The absolute amount of progesterone

MAMMALIAN OVARY

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circulating in the fluids of the body may be less important than the minimal amount. The ewes secreting less than the minimum may be unable to maintain pregnancy, whereas those secreting more may simply have surpluses which are of little significance. The same principle may be extended to the human female (Davis, Plotz, Lupu and Ejarque, 1960 ».

An observation new to the reviewer could l)e important. When two corpora lutea were present in one ovary the concentration of I)rogesterone was in the same range as that for the ewes with one corpus luteum (Edgar and Ronaldson, 1958). In another ewe there were 2 corpora lutea in one ovary and 1 in the other, but the concentrations in the blood from the 2 ovarian veins were almost the same.

A facet of the problem of the rate of production of ovarian estrogen and progesterone which has become apparent is that endogenously and exogenously administered estrogen (Rakoff, Cantarow, Paschkis, Hansen and Walkling, 1944; Pearlman, 1957) and progesterone (Haskins, 1950; Zander, 1954; Rappaport, Goldstein and Haskins, 1957; Davis and Plotz, 1957; Plotz and Davis, 1957; Pearlman, 1957; Cohen, 1959) disai)i:)ear from the blood very cjuickly, in the human female and in such laboratory mammals as the dog, rabbit, and mouse. Zander, for example, injected 200 mg. of progesterone intravenously into menopausal and ovariectomized women. The concentration of this hormone in the blood was 1.44 /xg. per ml. after 3.5 minutes and 0.116 yu,g. per ml. after 2 hours; 24 hours after the injection progesterone could not be found by the method he employed. The data obtained by the other investigators were similar.^ Using some of these data obtained from reports in the literature and from his own studies, Pearlman (1957) divided the total amount of circulating hormone (M) by the

■'To a cpitain extent, and possibly to a considerable extent, the rapid disappearance of progesterone from the blood is explained by its storage in the fat tissue of the body (Davis and Plotz, 1957; Davis, Plotz, Lupu and Ejarque, 1960). Following intramuscular injection of C"-4-progesterone, and assuming an even distribution of radioactivity in the fat of the body, about 17.7 per cent, 33.7 per cent, and 19.6 per cent of the administered dose was present 12, 24, and 48 hours, respectively, after the administration of the labeled hormone.

endogenous production rate (r) as a means of obtaining the turnover time iT), i.e., the time refjuired for a complete replacement of the circulating hormone by a fresh supply from the endocrine gland. His method was not free from criticism by discussants; nevertheless, informative estimates were made. The turnover time of the various estrogens was calculated to be about 6 minutes or less, that of progesterone, about 3.3 minutes.

Not unrelated to the i)roblem of the amounts of hormone produced is the sul)ject of plasma (and erythrocyte) binding of the ovarian hormones. Especial attention was given the subject by Rakoff, Paschkis and Cantarow ( 1943 ) who reported that as much as 50 per cent of the total estrogen content of the serum of women is present in a combined or conjugated (bound) form, and that almost all of the estrogens of pregnancy are bound to the protein fractions of the serum. Shortly thereafter, Szego and Roberts (1946) reported that two-thirds of the total estrogen in the blood in human l)lasma is normally associated with jn-otein constituents, and in a subsequent series of publications (Roberts and Szego, 1946, 1947; Szego, 1953, 1957; and others) that the liver is the site of the formation of the protein-estrogen complex or estroprotein. The nature of the complex soon become controversial and has not yet been resolved (Eik-Nes, Schellman, Lumry and Samuels, 1954; Antoniades, McArthur, Pennell, Ingersoll, Ulfelder and Oncley, 1957; Sandberg, Slaunwhit(> and Antoniades, 1957; Daughaday, 1959). In the jiresent context, however, othei- considei'ations are more important.

Protein-liinding is not confined to the estrogens and their metabolites, but other steroidal hormones, progesterone, testosterone, and corticosteroids, are also present in the blood in a bound-state. In studies of the binding relationships of serum albumin, the link to the esti'ogens was found to be strongest, that to the corticosteroids relatively weak, and that to ])rogesterone and testosterone intermediate (Sandberg, Slaunwhite and Antoniades, 1957; Slaunwhite and Sandberg, 1958; Daughaday, 1959). The i;lationships in the case of other components of i^rotein mixtures have been sliown to be

47()

PHYSIOLOGY OF GONADS

different, but they are ai)parently eriually specific (Daughaclay, 1959; Slaunwhite and Sandberg, 1959). A considerable specificity of the binding sites may be involved ( Sandberg, Slaunwhite and Antoniades, 19571. Daughaday (1959) states that separate binding sites may exist for each of the steroid hormones studied, and Szego (1957) suggested that a competition for these sites may be the basis for antagonisms which are known to exist in many steroid interactions (Courrier, 1950; Hisaw and Velardo, 1951; Roberts and Szego, 1953; Velardo, 1959; Velardo and Hisaw, 1951 ; Zarrow and Neher, 1953).

The most important consideration has to do with the significance of protein binding for the steroid hormones, and, in the present chapter, the significance for estrogen and progesterone. Roberts and Szego (1946, 1947) and Szego (1957) proposed that formation of the estrogen-protein complex is necessary for the transport and activity of endogenous and exogenous estrogens. Riegel and Mueller (1954), on the other hand, found that the protein-estrogen complex they used had only a slight, if any, estrogenic activity, and Daughaday (1958, 1959) expressed the opinion that the unbound steroid hormones of the plasma are probably the biologically significant moieties. He suggested that the degree of protein binding imposes a major restraint on the passage of hydrocortisone (and presumably other steroids) through the cajiillary membranes, but pointed out that this view has not yet been established. He then asked, in the event that the steroid-protein complex does not function in the transi)ort of hormones from the vascular component to the cell, is it likely that the presence of a steroid-protein complex stabilizes the pliysiologically significant concentration of unbound steroid very much as buffer salts stabilize the small concentration of hydrogen ion? In this way, he continued, the organism would l)c protected against the rapid changes in concentration which characterize an unbuffered system.

At tiic ]ir(>sent stage in this controversial sul).iect, any hypothesis with res])ect to the significance of the protein binding of steroid hormones must be tentative. It would seem, however, that whatever emerges will have

validity only if it is compatible with the cyclic waxing and waning of reproductive phenomena. If the unbound, rather than the bound fractions, are the active fractions, the functioning of the ovarian steroid hormones must dei)end on the presence of unbound fractions, in some way made available at cyclic intervals to the tissues on which these hormones act. It would seem, too, that the significance of the increased capacity for l)inding in i)rcgnancy (Rakoff, Paschkis and Cantarow, 1943; Baylis, Browne, Round and Steinbeck, 1955; Daughaday, 1959; Slaunwhite and Sandberg, 1959) should be a ])art of the picture. Tentatively, this greater binding capacity on the part of the ])regnant adult, coupled with an inability of the developing fetuses to bind androgens, might account for the failure of the adult to be affected by the presence of androgen at a time when the genital tracts and neural tissues of the female fetuses she is carrying are undergoing profound modifications (Phoenix, Goy, Gerall and Young, 1959; Diamond, 1960).

VI. Age of the Animal and Ovarian Functioninij

The position of the ovary is such — at one and the same time being dependent on the pituitary, possessing its own varying capacity to function, and having an effectiveness which is limited by the responsiveness of the tissues on which its hormones act — that no simple consideration of the relationshii) between the age of the animal and ovarian functioning can be given. An investigation, therefore, should be planned accordingly and we find experiments in which the amount of gonadotrophic stimulation was varied when age was constant, and experiments in which age was the variable and the amount of gonadotrophin the constant. If hypo- or hyper-responsiveness of the tissues is suspected, the point can be checked by the use of spayed animals given variable amounts of ()\-arian hormones. When information of these sorts is brought together, a fairly accurate account of the relationship between age of the animal and ovarian activity can l)e ])repared.

The results fi'oni many studies have revealed that the ovaries in both inunature and senescent females are potentially able

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to secrete hormones, both estrogen and progesterone, in amounts which are in excess of those secreted by untreated animals. The secretion of these hormones was elevated in rats, mice, and hamsters by the implantation of whole pituitaries (Smith and Engle, 1927 » or by the administration of chorionic gonadotrophin (Price and Ortiz, 1944; Ortiz, 1947; Green, 1955). The reactivation of senile ovaries was first demonstrated by Zondek and Aschheim (1927) following the insertion of hypophyseal imjilants, and later by numerous other investigators listed in the review of the subject l)y Tlmng, Boot and Miihlbock (1956). In more recent experiments an enhanced secretion of estrogen and progesterone followed the injection of old hamsters with chorionic gonadotrojihin (Peczenik, 1942; Ortiz, 1955).

In women fertility may be lost before the menopause (Engle, 1955). Studies in progress at Iowa (Bradbury, personal communication) show that urinary gonadotroi')hins may be elevated before the menopause and the last ovarian cycles are achieved in the presence of excessive amounts of pituitary gonadotrophin. As a rule the human ovary is devoid of oocytes and produces relatively little estrogen at the time of the menopause. Frequently, however, there is enough residual ovarian activity (estrogen production) to maintain the vaginal epithelium for 10 to 15 years after the menopause. These observations on women suggest that as the supply of oocytes l)ecomes depleted, less estrogen is produced and more gonadotrophin is released to stimulate the aging ovary. This secjuence is in harmony with the concepts of Dubreuil (1942) and Hisaw (1947), because with fewer areas of granulosa there would be fewer centers of organizer to bring al)out the differentiation of thecal tissue competent to produce estrogen.

Ovarian stromal hyperplasia has been found in association with endometrial hyperplasia after the menopause (Morris and Scully, 1958). Sherman and Woolf (1959) suggested that the postmenopausal ovary may produce abnormal sexogens which bring about an endometrial proliferation and ultimately adenocarcinoma of the endomi'trium. Their urinarv l)ioassay studies

indicate that the patients were excreting ICSH-type gonadotrophin. The observation has been made at Iowa that a few postmenopausal women with endometrial carcinoma were maintaining an estrogenic vaginal epithelium when they were ovariectomized at ages varying from 65 to 70 years. Subsequently the gonadotrophin excretion increased to the ciuantities usually seen after the menopause. In these unusual cases the aging ovaries produce estrogen, or possibly estrogen and androgen, in quantities sufficient to suppress the usual excess production of gonadotrophins.

The responsiveness or sensitivity of the ovary to gonadotrophic stimulation is not constant throughout the life of an individual. If we may judge from the studies of Corey (1928) and Selye, Collip and Thomson (1935) on newborn and 10- to 15-day-old rats, Moore and Morgan (19431 on young opossums, and Price and Ortiz (1944) and Ortiz (1947) on rats and hamsters, the prepubertal period is characterized by very rapid and great increases in responsiveness to gonadotrophic stimulation. Species differences are great. The opossum ovaries do not respond to gonadotrophic stimulation until about 100 days of age (Moore and Morgan), whereas responsiveness was first detected in the rat ovary at 4 to 10 days (Price and Ortiz) and in the hamster ovary by the 10th day (Ortiz). Such data, coupled with the appearance of the ovaries at birth, would seem to exclude the possibility of gonadotrophic stimulation during the prenatal period, and perhaps the capacity for being stimulated as well.

Certain other species are different and present problems. There is an extensive follicular development and luteinization in the fetal ovaries of the giraffe which is the basis for the suggestion that the ovaries of this species are responsive to gonadotrophin before birth (Amoroso, 1955). Such a conclusion is predicated on the assumption either that serum gonadotrophin crosses the placental membrane or that the fetal pituitary secretes gonadotrophin. Neither hypothesis has been proved. Evidence exists that the ovaries of the horse and seal are strongly stimulated before birth (Cole, Hart, Lyons and Catchpole, 1933; Amoroso,

47!

PHYSIOLOGY OF GONADS

Harrison, Harrison-Matthews and Rowlands, 1951; Amoroso and Rowlands, 1951), but an unusual structural condition is found in these ovaries. No vesicular follicles are present and the ovaries, which are larger than those of the adult, are composed mostly of interstitial tissue which is enclosed by a thin cortex containing short chains of germ cells and a few oocytes surrounded by a single layer of epithelial cells. Comparable information does not exist for the seal, but in the horse the development of this condition is reached during the estrogenic phase and after the gonad-stimulating hormone is no longer detectable in the blood of the pregnant adult. As a result, and cjuite apart from the belief that serum gonadotrophin does not cross the placenta (Amoroso and Rowlands, 1955 j, the massive interstitial tissue liyperplasia is thought to have been stimukited by estrogenic rather than by gonadotrophic action.

The immediate jiostpubertal jieriod and middle age are periods of relative stability. The period of old age has been too little studied and is in need of attention. In old mice the ovaries are reported to become unresponsive to exogenous gonadotrophin (Green, 1957). Ortiz (1955), on the other hand, stated that although a certain degree of ovarian sensitivity is lost in old hamsters, there is a surprising degree of responsiveness present until death, not only after the animal is no longer fertile, but even in animals with ovaries almost completely atrophic.

In young animals and in old animals there are irregularities of ovarian function and irregularities in the character of the cycles which probably can be related to imbalances in the pituitary-ovarian relationship. In polytocous sjK'cies, fewer follicles ovulate in young animals (Young, Dempsey, JMyei-s and Hagquist, 1938; Ford and Young, 1953, the guinea pig; Perry, 1954, the domestic pig; Ingram, Mandl and Zuckerman, 1958. the mouse and rat), and in old animals (Perry; Ingram, Mandl and Zuckerman). These statements of fact, iiowcx-cr. do not reveal what is presumed to be more important. In young and old animals the nature of the irregularities, particularly those of ovarian function, seem to differ. Evidence collecte(l fi'om rhesus monkeys (Hartman,

1932) and chimpanzees (Young and Yerkes, 1943) suggests follicular growth without ovulation and luteinization, or in the guinea pig a sluggishness of follicular growth which is followed by ovulation and the formation of functional corpora lutea (Ford and Young, 1953) . In old animals there may also be abnormalities of follicular growth, but as the numerous reports are read, the impression is given that abnormalities of luteinization are more prominent (Deanesly, 1938b; Wolfe, 1943; Wolfe and Wright, 1943; Loci), 1948; Thung, Boot and Miihlbock, 1956; Dickie, Atkinson and Fekete, 1957; Green, 1957). Additional investigation will be necessary before we can be sure that the pituitary-gonadal imbalance in young animals differs from that in old animals. On the whole, the possibility seems to have received little attention, but its importance justifies more careful study.

VII. Other Endocrine Glands and the Ovaries

A. THYROID

The relationship of the thyroid to the functioning of the ovaries was one of the first subjects of modern endocrinologic investigation. Notwithstanding, disappointment must be expressed that after more than 50 years of effort, little more than cautious generalization is possible. This admission is not a confession of defeat; to be sure, there is an unfortunate number of uncertainties, but we have come to know what is necessary in the way of experimental design and techniques to enable us to proceed with the confidence that a gratifying clarification can be achieved. The greatest obstacle could be, not the lack of means, but rather the failure to use the means which are alnmdantly at liand for more coordinated eft'orts than many which lia\e cliaracterized this field in the past.

The general l)elief that the thyroid is invoh-ed in reproductive function is grounded in two categories of observations. The first includes those demonstrating that ovarian lioi'inones exert an action on the thyroid. 'I'lieic lia\-e been many I't'ports that in the human female the thyi'oid enlarges at puberty, at menstruation, and during pregnancy (Gamier, 1921; Marine, 1935; Neu

MAMMALIAN OVARY

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mann, 1937; and others). Modern counterparts are the reports of the increase during pregnancy in the concentration of serum precipitable iodine (Heinemann, Johnson and Man, 1948; Dowling, Freinkel and Ingbar, 1956a; Tanaka and Starr, 1959), in serum thyroxine (Danowski, Gow, Mateer, Everhart, Johnson and Greenman, 1950), and in the accumulation of radioiodine (Pochin, 1952). Some conflicting reports should be noted. There was said to be no consistent alteration in the concentration of serum precipitable iodine in oophorectomized women (Stoddard, Engstrom, Hovis, Servis and Watts, 1957), and Pochin (1952) found no detectable variation in P^^ uptake during the menstrual cycle in 5 women he studied. Comparable observations have been made on laboratory mammals (Greer, 1952; Soliman and Reineke, 1954; Soliman and Badawi, 1956; Feldman, 1956a) and the baboon, Papio ursinus (Van Zyl, 1957), except that Brown-Grant (1956) could not agree from his findings in the rat and rabbit that the level of gonadal function exerts any striking influence on thyroid activity in the normal experimental animal.

In man (Engstrom, Markardt and Liebman, 1952; Engstrom and Alarkardt, 1954; Bowling, Freinkel and Ingbar, 1956b) and in laboratory mammals (chiefly the rat) (Money, Kraintz, Eager, Kirschner and Rawson, 1951; Feldman, 1956a; Feldman and Danowski, 1956) the enhancement of thyroid activity is attributed to the level of circulating estrogen, whether it be endogenous or exogenous in origin. On the other hand, many who have worked with laboratory mammals have not found evidence of augmented thyroid activity, and not infreciuently decreases were reported (see Paschkis, Cantarow and Peacock, 1948; and the numerous articles cited by Farbman, 1944; and Feldman, 1956a). The conflicting results may perhaps be accounted for by the circumstance that the response of the thyroid seems to be related to the duration of the estrogen treatment and to the estrogen that was used. Decreases in thyroid activity have been reported when the estrogen treatment was prolonged (Feldman, 1956a), and Money and his associates showed clearly that estrone and some other

components increased the collection of P-^^ by the thyroid of rats whereas estradiol, estriol, and diethylstilbestrol decreased the collection. ]\Iany attempts have been made to ascertain the nature of the mechanism whereby the effective estrogenic substances exert their action on the thyroid (Noach, 1955a, b; Feldman, 1956b; Dowling, Freinkel and Ingbar, 1956a, b; Bogdanove and Horn, 1958). but they are so varied and speculative that they will not be reviewed here.

The second category of observations related to the thyroid and ovarian functioning includes those in which there is evidence of action of thyroid hormone on the ovary. Reviews of this work are contained in the articles by Peterson, Webster, Rayner and Young (1952), Hoar, Goy and Young (1957), and Parrott, Johnston and Durbin (1960) and most of their citations of work done on the relationship of the thyroid to the ovary will not be repeated here. As they point out, many investigators have reported that thyroidectomy is followed by ovarian degeneration, arrested folliculogenesis, and failure of ovulation. Irregularity of the reproductive cycles was common and much of this in the guinea pig could be attributed to retarded and sporadic follicular development (Hoar, Goy and Young, loc cit.) . The latter investigators gave especial attention to the condition of the ovaries in their hypothyroid guinea pigs. In 10 pairs from thyroidectomized animals (oxygen consumption and heart rate were depressed) follicular development was good in the sense that the follicles appeared healthy, but a generation of corpora lutea was missing in four. This absence of corpora lutea, which is not seen in normal adult guinea pigs, was believed to be a consequence of the involution of the older generation during the longer than normal interval between ovulations. It is considered significant in terms of the functional capacity of such ovaries, that although the percentage of sterile matings was higher than in the controls, that, in the course of the two studies at Kansas, 29 of 38 matings were fertile. This experience may perhaps account for the many reports (cited in tlu papers from the Kansas laboratory) that thvroidectomv or treatment with antithv

480

PHYSIOLOGY OF GONADS

roid drugs have no, or at the most rehitively little, effect on the ovary. To these, several additional reports should be mentioned. In thyroid-deficient female mice, fertility and litter frequency were affected only to the extent that the estrous cycles were prolonged (Bruce and Sloviter, 1957). In the rabbit, thyroidectomy did not interI'upt or alter the periodicity of follicular development, but it did eliminate the final stages (Desaive, 1948). Parrott, Johnston and Durl)in (1960) express the opinion that the long i)hysiologic life of thyroid hormone may account for many of the contradictions in the reports of the relationship between thyroid deficiency and reproduction.

Except as it is speculative, an unexplained action of thyroidectomy or the administration of goitrogenic drugs is the augmentation of the ovarian response to gonadotrophins and to anterior pituitary im])lants (citations in Peterson, Webster, Rayner and Young, 1952; and see in addition Janes, 1954; Janes and Bradbury, 1952; Kar and Sur, 1953). Thyroid substances, on the other hand, were inhibitory. Of alternative hyjiotheses, Janes favored the suggestion that during the period of propylthiouracil treatment, provided it was short rather than long, there was an accumulation of gonadotrophin in the blood and the ovarian response varied for some unknown reason according to the concentration of this latter substance in the body fluids. To Kar and Sur (1953) direct involvement of the hypophysis could be eliminated; instead a direct role of the thyroid seemed more plausible. They postulated that the absence of thyroid hormone reduced the utilization of gonadotrophic hormones by the ovary.

The reported effects of the hyi)erthyroid state or of administered thyroid hormone on the ovary are equally conflicting. The ovaries are described as being atrophic or exhibiting incomplete folliculogenesis, or as being essentially normal or even hypertrophied (citations in Peterson, Webster, Rayner and Young, loc. cit., Hoar, Gov and Young, loc. n't.). Irregular cycles are said to have occuitcmI in the rat and mouse. but no irregularity was detected in guinea pigs given thyroxine.

A tentative explanation can be given for

the many divergent reports of the relationship between the thyroid and the ovary, divergencies which are found in the clinical literature as well as in laboratory studies. In doing so, we will recall that there is abundant evidence that the ovary is a locus of action of thyroid hormone. The action may not be directly trophic, as is that of the pituitary, but it is assumed to be su])l)ortive, jiossil)ly directly so. or jiossiljly indirectly through regulation of the general metabolic level. Whatever its nature, there must be great interspecies and even intraspecies variation in the need of the ovary for such action. In addition, within a species there appears to be a wide range of tolerance, for Peterson, Webster, Rayner and Young (1952) found in their study, in which the thyroid state was estimated from measurements of oxygen consumption and heart rate, that reproduction occurred in females in which oxygen consumption ranged from an average of 50.0 to 93.5 cc. per 100 gr. i)er hr. (52.9 in the controls), and heait rate from 238 to 316 beats per minute (272 in the controls). In females that failed to rejiroduce, the lowest values were lower than in the animals which did reproduce; nevertheless, there was much overlai)i)ing, for in this group oxygen consumption ranged from an average of 46.7 to 94.1 cc. per 100 gr. l)er hr., and heart rate from 202 to 330 beats j)er minute. Within sucli a framework, there are bound to be more divergent results than when normal functioning depends on nioi'e narrowly circumscribed conditions, and the failure to replicate a result does not have the same significance. As a part of the investigation of sucli a problem, more and better correlated infoi'niation is re(|uiicd, and this could be the most pressing ne('(l in the field of oxarian (and icproducti\-cl functioning and the thAM'oid.

B. ADRENAL CORTEX

Tlu^ adrenal cortex elaborates its steroid )nn()iu's in a biosyiU lictic scciuencc \H'ry

siiiiilai' to that in the o\aiy. In fact, i)rogcstci'onc is an intermediate substance in the synthesis of glucocorticoids. Estrogen has been found in extracts of adrenal cortical tissue, but whether it represents a degradation pi'oduct within the adrenal or an artifact resulting from the chemical i)i'o

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cedurcs is not clear. Occasionally adrenal tumors produce physiologically significant amounts of estrogen, but normally the adrenal production of estrogen, if any, is not of physiologic significance. The atrophy of the female genital tract after bilateral ovariectomy suggests strongly that this is so.

The major hormone of the adrenal cortex, hydrocortisone or corticosterone, depending on the species, has a profound effect on protein and carbohydrate metabolism. An overproduction, as manifested by Cushing's disease, results in a wasting of body protein and other metabolic disturbances which by nonspecific influences tend to reduce gonadal function. Similarly a loss of adrenal function (Addison's disease) leads to anemia, electrolyte imbalance, and hypoglycemia. There is usually a decrease in ovarian function but some Addisonian patients have conceived and carried their pregnancies with only sodium and fluid supplements.

There is a hereditarv metabolic defect

of the human adrenal which renders it defective in i)roducing hydrocortisone. This is the adrenogenital syndrome. In these jiatients the adrenals produce excessive amounts of intermediate products which are excreted in the urine. Some of these compounds are androgenic 17-ketosteroids and may cause virilization (Bradbury, 1958). These androgens tend to inhibit the gonadotrophic activity of the pituitary and leave the ovaries unstimulated and infantile (Fig. 7.13). Replacement therapy with corticoids reduces the adrenocorticotrophic hormone ( ACTH) activity of the pituitary and then the adrenal production of androgen ceases. This then permits the pituitary to stimulate normal cyclic activity in the ovaries (Fig. 7.14). The adrenogenital syndrome thus has a profound effect on ovarian function which is specific through its production of androgen. More complete descriptions of the condition and of the rationale of treatment have been prepared by Wilkins ( 1949) ,

CHOLESTEROL

PREGNENOLONE

PROGESTERONE

HYDROXYPROGESTERONE

Fig. 7.13. Scliematic representation of the interaction of the adrenal and the ovary in the adrenogenital syndrome. The process of hormone biosynthesis is defective in the adrenal (BLOCK) and the degraded by-products (17-ketosteroids) being androgenic suppress the formation of gonadotrophins (GTH). (Courtesy of Dr. J. T. Bradbury.)

482

PHYSIOLOGY OF GONADS

CHOLESTEROL

CORTISOL

CHOLESTEROL

GONAD

\

ANDROGEN ESTROGEN

PREGNENOLONE

PREGNENOLONE

ADHeNAL

^

\

17-HYDROXYPROGESTERONE

PROGESTERONE

17-HYDROXYPR06ESTER0NE

Fit. 7 14. Tieatnieiit witli (oiti>one (or other glucocorticoid) reduces ACTH production and adrenal hormone .synthesis subside-^. This permits the normal pituitaiy-gonadal interactions to be established. (Courtesy of Dr. J. T. Bradbury.)

A\'ilkins, Crigler, Silverman, Gardner and Migeon (1952), and Bradbury (1958). Milder categories of what is believed to be adrenal cortical hyperplasia have also been described and are responsive to treatment with cortisone. They are characterized by amenorrhea or oligomenorrhea, hirsutism, slightly elevated 17-ketosteroid excretion values, and difficulty in becoming pregnant (Jones, Howard and Langford, 1953; Jefferies, AYeir, Weir and Prouty, 1958; Jefferies, I960). Indications arc that these abnormalities, like those typical of the adrenogenital syndrome, affect the ovary, not directly, but rather by the creation of a pituitary gonadotrophic-ovarian imbalance.

Some evidence exists for more direct relationships between the adrenal cortex and the ovary. These may involve actions of ovai-iaii liormones on the adrenal, and ac

tions of adrenal cortical hormones on the ovary. In general, however, the relationships are tenuous or at least not sharply defined. It is evident from the review by Parkes (1945) that sexual dimorphism in adrenal cortical structure has been demonstrated in a number of species, notably the mouse and rat and possibly the guinea pig, but it has not been detected in a number of other species. The effects of gonadcctomy and the injection of hormones, particularly estrogens, into gonadectomized animals are less clear, but they are suggestive of an action on the adrenal, however ill defined and variable it seems to be. A seasonal hypertrophy of the adrenal has been reported as occurring in the mole, Talpa europaea, (Kolmer, 1918) and the ground squirrel, Citellus tridecemlineatus (Mitchill), (Foster, 1934), as has enlargement at the time of estrus in the rat (Andersen and Kennedy,

MAMMALIAN OVARY

483

1932; Bourne and Ziickennan, 1941bj. More recently, a significantly higher excretion of 17-hydroxy corticosteroids has been found during the second and third weeks, and therefore during the luteal phase, of the menstrual cycle (Maengwyn-Davies and Weiner, 1955).

Whether a causal relationship exists between these indications of a fluctuating activity within the adrenal cortex, and seasonal and cyclic changes within the ovaries remains to be determined. Little information exists. Bourne and Zuckerman {loc. cit.) described the changes in the adrenals of ovariectomized rats injected with estrone and concluded that the changes are inde|)endent of the gonads. Foster's observation that the active appearance of the adrenal can be seen during pregnancy as well as during estrus suggests, but does not prove, that there is a hormonal regulation in the ground squirrel which is dependent on reproductive processes.

Data with respect to possible direct effects of adrenal cortical secretions on the ovary ai'e ambiguous. Cortisone acetate administered to rabbits 5 to 33 days in daily doses of 5 to 20 mg. did not inhibit the ovulation which occurs after mating or after the injection of copper acetate (De Costa and Abelman, 1953). The ability of the ovary of the rat to respond after adrenalectomy was tested by the administration of gonadotrophic extracts (Brolin and Lindl)ack, 1951). They found that the ovaries could be stimulated to increase the weight of the uterus without the cooperation of the adrenals and considered that this result does not support the view that there is a direct relationship between adrenal corticoids and the biosynthesis of ovarian (also testicular) hormones. In other experiments (Payne, 1951; Smith. 1955), adrenalectomy abolished (Payne) or interfered significantly (Smith) with the ovarian hyperemia response to injections of HCG and pituitary extract (Antuitrin T) , the response utilized by Farris (1946) as a test for early pregnancy. Cortisone and hydrocortisone were partially effective in restoring the response in adrenalectomized animals. According to Payne, isocortisone acetate and compound A acetate were also effective, but in larger doses. No report of the use of corticosterone

(comi)ound B) is gi\'en; replacement therapy with this hormone would have been more physiologic because it is the natural corticoid of rats. It was concluded that the hyperemia response is more nearly normal in animals with normal adrenal function; Payne believes that the response is mediated through this gland. Despite what seem to be clear-cut results which have been confirmed, it is felt that additional closely controlled exi^eriments must be done in order to show whether these adrenal hormones affect the ovary directly or whether most of the effects are nonspecific metabolic alterations.

VIII. Concluding Remarks

The avenue followed by investigators interested in the functioning of the mammalian ovary has long carried a two-way traffic. In addition, there has been movement into the out of many side streets. No understanding of the pattern of the traffic in such a situation is possible and no satisfactory regulation can be achieved unless something is known about the nature, origin, and destination of the vehicles composing the traffic. Equally important, this information cannot l)e obtained by standing on one spot. This analogy contains much that is relevant for what has been attempted in this book. The problem of the ovary has been approached from the vantage point of forces and substances originating in the pituitary and the environment which act centripetally on it (Creep, Everett), and from the vantage j^oint of many of the tissues and organs on which the hormones we associate with it exert their action (the Hisaws, Cowie and Folley, Zarrow, Young in his chapter on mating behavior). In this chapter and that prepared l)y Dr. Villce positions have been taken near the ovary and attemj^ts made to bring together much of the information gathered by investigators who were in a sense looking right at it.

Whether our perspective is developed from a familiarity with all the material which has been brought together or whether it is restricted by the narrower treatment given here, it is obvious that the unsolved problems outnumber by far any that have been solved, if indeed there are such. Wc have learned much about the functioning

484

PHYSIOLOGY OF GONADS

of the o\'ary, but there is little we can explain. As we indicated earlier, a part of this failure can be ascribed to the lack of gonadotrophic preparations which either singly or in combination will evoke changes identical with those in untreated normal animals, but this chapter alone contains an enumeration of many other problems solution of which does not depend on this particular advance. The disappointment we express may be a reflection of what seems to be the modus operandi in science. The extent of our application to unsolved problems is very unequal, but more often than not it can be traced to an investigator's success in achieving a "breakthrough"*' as Edgar Allen, Doisy, Smith and Englc, Willard Allen and Corner, Hisaw and other colleagues did in the twenties. At such a time, enthusiasm is intense and there follows a period of gratifying accomplishment, but obstacles are encountered and often interest lags, until another breakthrougli occurs. In the meantime, effort may have been diverted by discoveries elsewhere and the area of investigation which attracted so many is neglected and suffers. Ovarian physiology should not remain in this state for long. There is no tissue of the body in which the changes are as conspicuous and as dramatic as those in the ovary and there is no tissue which presents more variable aspects. Many of the stages in the cycle of ovarian structure and functioning are related to changes elsewhere in the body — changes in growth, in motility, in secretion, and in beliavior. All these changes, including those within the ovaries, offer excellent end points for continued quantitative and qualitative studies.

IX. References

Aldman, B., Claesson, L., Hillarp, N.-A., and OdeBLAD, E. 1949. Studies on the storage mechanism of oestrogen-precursors. Acta enclocrinol., 2, 24-32.

Allen, E. 1922. The oestrous cycle in the mouse. Am. J. Anal., 30, 297-371.

Allen, E. 1932. The ovarian foUirulai- lioriiioiic. theehn; animal reactions. In Sex and hih iinil Secretions, 1st ed., E. Allen, Ed., pp. 392-t,S0. Baltimore: The Williams & Wilkins Company.

Allen, W. M. 1939. Biochemistry of the corpus

" The word was not a part of the language of science at that time and prolial)ly was never used l)y them

luteum hormone, progesterone. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 901-928. Baltimore: The Williams & Wilkins Company.

Amoroso. E. C. 1955. Endocrinologv of pregnancy. Brit. Med. Bull., 11, 117-125.

Amoroso, E. C, Harrlsox, R. J., Harrlsox-M.\tthews, L., and Rowlands, I. W. 1951. ReIjroductive organs of near-term and new-born seals. Nature, London, 168, 771-772.

Amoro.so, E. C, and Rowlands, I. W. 1951. Hoimonal effects in the pregnant mare and foetal foal. J. Endocrinol., 7, 1-liii.

Andersen, D. H., and Kennedy. H. S. 1932. Studies on the physiology of reproduction. IV. Changes in the adrenal gland of the female rat associated with the oestrous cycle. J. Physiol., 76, 247-260.

Antonl\des, H. N., McArthur, J. W., Pennell, R. B., Ingersoll, F. M., Ulfelder, H., and Oncley, J. L. 1957. Distribution of infused estrone in human plasma. Am. J. Physiol., 189, 455-459.

Aron, M., and Aron, C. 1952. La glande thecale de lovaire de cobaye. Arch. Anat. Histol. et Embryol., 34, 27-41.

ASCHHEIM,, S., PORTES, L., AND M.\YER, M. 1939.

Les hormones gonadotropes. Etude critique de quelques points relatifs a leur role dans la physiologie et la pathologie des fonctions de I'ovaire. Ann. endocrinoL, 1, 42-54.

AsTwooD, E. B. 1938. A six-hour assay for the quantitative determination of estrogen. Endocrinology, 23, 25-31.

AsTW^ooD, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Am. J. Physiol., 126, 162-170.

Atkinson, W. B., and Leathem, J. H. 1946. The day to day level of estrogen and progestin during lactation in the mouse. Anat. Rec, 95, 147-155.

Barahona, M., Bruzzone, S., and Lipschutz, a. 1950. On the control of follicular development in intrasplenic ovarian grafts by minute (|uantities of oestrogen. Endocrinologv, 46, 407-413.

Barker, W. L. 1951. A cytochemical study of lipids in sows' ovaries during the estrous cycle. Endocrinology, 48, 772-785.

Barr.aclough, C. a. 1955. Influence of age on the response of prcweaning female mice to testosterone i)ro])i()nate. Am. J. Anat., 95, 493521.

Bassett, D. L. 1943. The changes in the va.scular pattern of the ovary of the albino lat during the estrous cycle. Am. J. Anat., 73, 251291.

Bavlis, H. I., Browne, J. C, Round, B. P., .and Steinbeck, A. W. 1955. Plasma 17-hydroxycorticosteroids in pregnancy. Lancet, 1, 62-67.

Beroer, M. 1935. Besonders hohe Wirk.'^amkeit des Follikelhormons bei vaginaler Instillation. Klin. Wchnschr., 14, 1601-1602.

Behcman, p. 1949. Sexual cycle, time of ovulation, and time of optimal fertility in women.

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485

Acta obst. et gvnec. scandinav., Supi)l. 29, 4, 1-139.

Beyer, K. F., AND Samuels, L. T. 1956. Distrilmtion of steroid-3^-ol-dehydrogenase in cellular structures of the adrenal gland. J. Biol. Chem., 219, 69-76.

BiGGERS, J. D. 1953. The characteristics of the dose-response line in Allen-Doisy tests obtained by the intravaginal administration of oestrone in distilled water and in 50 i)cr cent aciueous glycerol. J. Endocrinol., 9, 145-154.

BiGGERS, J. D., AND Clarinobold, P. J. 1954. Optimum conditions for the local (intravaginal) action of oestrogens. Australian J. Biol., 7, 118-139.

BiGGERS, J. D., AND Clakixgbold, P. J. 1955. Mitotic activity in the vaginal epithelium of the mouse following local oestrogenic stimulation. J. Anat., 89, 124-131.

BiTM.\N, J., Wrenn, T. R., and Sykes, J. F. 1958. Estrogen determination in blood and body fluids of cattle. In Reproduction and Infertility, III, Symposium, F. X. Gassner, Ed., pp. 141-156. New York: Pergamon Press.

Bl.\ndau, R. J. 1955. Ovulation in the living albino rat. Fertil. & Steril., 6, 391-404.

Blandau, R. J., AND Soderwall, A. L. 1941. Postparturitional heat and the time of ovulation in the albino rat. Data on parturition. Anat. Rec, 81, 419-430.

Block, E. 1951a. Quantitatixc morphological investigations of the follicular system in women. Methods of (luantitative determinations. Acta Anat., 12, 267-285.

Block, E. 1951b. Quantitative morphological investigations of the follicular system in women. Variations in the different phases of the sexual cycle. Acta endocrinol., 8, 33-54.

Block, E. 1952. Quantative morphological investigations of the follicular system in women. Variations at different ages. Acta anat., 14, 108-123.

Block, E. 1953. A cjuantitative morphological investigation of the follicular system in newborn female infants. Acta anat., 17, 201-206.

BoGDANOVE, E. M., and Horn, E. H. 1958. Thyroid iodide concentration in estradiol-treated hvpophysectomized rats. Endocrinology, 62, 97-99.

BoLiNG, J. L. 1942. Growth and regression of corpora lutea during the normal estrous cycle of the rat. Anat. Rec, 82, 131-145.

BoLiNG, J. L., Blandau, R. J., Wilson, J. G., and Young, W. C. 1939. Post-parturitional heat responses of newborn and adult guinea pigs. Data on parturition. Proc. Soc. Exper. Biol. & Med., 42, 128-132.

BoLiNG, J. L., Blandau, R. J., Soderwall, A. L., and Young, W. C. 1941. Growth of the Graafian follicle and the time of ovulation in the albino rat. Anat. Rec, 79, 313-331.

Bos, C., AND Cleghorn, R. a. 1958. P.sychogenic .sterility. Fertil. & Steril., 9, 84-98.

Bourne, G., and Zuckerman, S. 1941a. The influence of the adrenals on cyclical changes

in the accessory reproductive organs of female rats. J. Endocrinol., 2, 268-282.

Bourne, G., and Zuckerman, S. 1941b. Changes in the adrenals in relation to the normal and artificial threshold oestrous cycle in the rat. J. Endocrinol., 2, 283-310.

Bradbury, J. T. 1947. Ovarian influence on the response of the anterior pituitary to estrogens. Endocrinology, 41, 501-513.

Bradbury, J. T. 1958. Adrenogenital syndrome and adrenal hyperplasia. Clin. Obst. & Gynec, 1, 257-270.

Br.\dbury, J. T. 1961. Direct action of estrogen on the ovary of the immature rat. Endocrinology, 68, 115-120.

Bradbury, J. T., and G.aensbauer, F. 1939. Masculinization of the female rat by gonadotrophic extracts. Proc. Soc. Exper. Biol. & Med., 41, 128-131.

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Stoddard, F. J., Engstrom, W. W., Hovis. W. F., Servis, L. T., and Watts, A. D. 1957. Effect in women of bilateral oophorectomy on circulating thvroid hormone. J. Clin. Endocrinol.. 17, 561-564.

Stone. M. L. 1959. Effects of relaxin in the human. In Recent Progress in Endocrinology of Reproduction, pp. 429-439. New York: Academic Press, Inc.

Sturgis, S. H., and Politou, V. C. 1951. Intravenous assay of prciiarations causing ovarian hyperemia in the iminaluic rat. Endocrinologv 48, 435-442.

Svendsen, R. 1960. A double isotopic derivative method for the quantitative determination of oestrone and 17/:i-()(>stradiol in jilasma. Acta endocrinol.. 35, 161-187.

SwANN, R. O., AND Schumaciier. G. F. 1958. Relaxin ; its use in normal ol)stetrics. J. Iowa State M. A.. 48, 599-601.

Swezv. O., and Evans. H. M. 1930. 0\ arian

MAMMALIAN OVARY

495

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SzEGo, C. M. 1953. The influence of the Hver upon estrogen-protein binding in vitro. Endocrinology, 52, 669-678.

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Van Zyl, A. 1957. Serum protein-bound iodine and serimi lipid changes in the baboon (Papio ursinus). I. During the menstrual cycle. J. Endocrinol., 14, 309-316.

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Velardo, J. T., and Hisaw, F. L. 1951. Quantitative inhibition of progesterone by estrogens in de\-Glopment of deciduomata. Endocrinology, 49, 530-537.

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Vermande-Van Eck, G. J. 1956. Neo-ovogenesis in the adult monkey. Consequences of atresia of ovocytes. Anat. Rec, 125, 207-224.

Veziris, C. D. 1951. Cycle vaginal chez les femmes castrees ou menopausees. Ann. endocrinol., 12,915-921.

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Warwick, T. 1940. A contribution to the ecology of the musk-rat (Ondatra zibethica) in the British Isles. Proc. Zool. Soc. London, ser. A, 110, 165-201.

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496

PHYSIOLOGY OF GONADS

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Zacharl\e, F. 1958. Studies on the mechanism of ovulation. Permeability of the blood-li(|uor barrier. Acta endocrinol., 27, 339-342.

Zachariae, F.. and Jensen, C. E. 1958. Studies on the mechanism of ovulation. Histochemical and physicochemical investigations on genuine follicular fluids. Acta endocrinol., 27, 343-355.

Zander, J. 1954. Progesterone in human Ijlood and tissues. Nature, London, 174, 406-407.

Zander, J., Forbes, T. R., vox Munstermann, A. M., AND Neher, R. 1958. Two naturally occurring metabolites of progesterone — isolation, identification, biologic activity, and concentration in human tissues. J. Clin. Endocrinol., 18, 337-353.

Zander, J., and Simmer, H. 1954. Die chemische Bestimmimg von Progesteron in organislien Substraten. Klin. Wchnschr., 32, 529-540.

Zarrow, M. X., AND Neher, G. M. 1953. Studies on the validity of the Hooker-Forbes test for the determination of progesterone in untreated blood. J. Clin. Endocrinol., 8, 203-209.

Zarrow, M. X., Neher, G. M.. Lazo-Wasem, E. A., AND Salhanick, H. A. 1957. Biological activity of certain progesterone-like compounds as determined by the Hooker-Forbes bioassav. J. Chn. Endocrinol., 17, 658-666.

Zarrow, M. X., Caldwell, A. L., H.\fez, E. S. E., AND PiNCus, G. 1958. Superovulation in the immature rat as a possible assav for LH and HCG. Endocrinology, 63, 748-758.

Zondek, B. 1940. On the mechanism of action of gonadotrophin from pregnancy urine. J. Endocrinol., 2, 12-20.

Zondek, B. 1954. Some problems related to ovarian function and to pregnancy. Recent Progr. Hormone Res., 10, 395-423.

Zondek, B., and Ascheim, S. 1927. Hypophysen^•OI•del•lappen und Ovarium. Beziehungen der cndokrinen Driisen zur Ovarialfunktion. Arch. Gynak., 130, 1-45.

Zondek, B., and Sklow, J. 1942. Further investigations on the mechanisms of oestrone production in the ovary. J. Endocrinol., 3, 1-4.

Zuckerman, S. 1940-41. Periodic uterine bleeding in spayed rhesus monkeys injected thiily with a constant threshold dose of oestron(>. J. Endocrinol., 2, 263-267.

Zuckerman, S. 1951. The number of oocytes in the mature ovary. Recent Progr. Hormone Res.. 6, 63-109.

The Mammalian Female Reproductive Cycle and its Controlling Mechanisms

John W. Everett, Ph.D.

Professor Of Anatomy, Duke University Durham, North Carolina

I. Introduction 497

II. Cycles Spontaneously Interrcpted. 498

III. PiTUITARY-OVARIAN DORMANCY 499

A. The Ovary in Anestrum 500

B. The Hypophysis 500

C. Relationship of the Anestrum to the

Seasons 501

IV. Attainment of Maturity. Emergence OF Full Ovarian Function. . 502 V. Follicular Cycles. Growth and

Atresia 504

A. Correlation of Ovarian Secretion

with the Follicular Cycle 507

B. Cyclic Manifestations after Ovari ectomy or Hypophysectomy 509

C. Cyclic Manifestations in the Ab sence of Ovarian Follicles 509

D. Hypothalamus and Gonadotrophin

Secretion. General Considerations 510

VI. Follicle Maturation and Ovulation 513

A. Time of Ovulation 513

B. Ovarian Steroids and Ovulation. . . 514

1. Estrogens 514

2. Gestagens 517

C. Role of the Nervous System in Ovu lation " 520

1. The hypophyseal portal veins and

the chemotransmitter hypothesis ' 523

2. Central depressants and ovula tion 526

3. The central nervous system as a

timing mechanism for ovulation 520

D. Persistent Follicle 529

VII. The Luteal Phase 530

1. Luteotrophic substances 530

2. "Nonfunctional" corpora lutea. . 531 A. Pseudopregnancy 532

1. Duration of pseudopregnancy. .. . 533

2. Neural factors in pseudopreg nancy 534

B. Luteolytic Mechanisms 537

C. Effect of the Uterus on Luteal Func tion 538

VIII. Concluding Comments 540

IX. References 541

I. Introduction

The chain of events that constitutes the female reproductive process is characteristically repeated from time to time with considerable regularity during the adult life of an individual, and is therefore a cycle. In the broad sense, this sequence begins with ovogenesis and terminates when the progeny require no further shelter and nurture. In mammals this has become a highly complex process, involving profound maternal adjustments synchronized with successive stages in development of the ovum, fetus, and offspring. The complete mammalian cycle comprises a sequence of stages which may be identified as follows: (II follicle growth, including growth of the ovocyte; (2) ovulation, a progressive process including preovulatory maturation of follicles and ova, and the structural change of ruptured follicles to corpora lutea; (3) progravidity; (4) gravidity; (5) parturition; and (6) postpartum nurture, including lactation, protection, and training. Although it is obvious that this full sequence is often realized, it may nevertheless be retarded or frankly interrupted at almost any point.

In advanced human societies economic and social factors have diminished the number of complete cycles to such degree that they are rarities in the lifetime of an in

497

498

PHYSIOLOGY OF GONADS

dividual and infertile ("menstmal") cycles are the rule. Inasmuch as corresponding factors operate among domesticated animals, the expression "female reproductive cycle" commonly refers to those truly abortive cycles that succeed one another in the absence of insemination. The term is used in that restricted sense in this chapter.

With even that restriction, the female cycle is actually a multiplicity of interlocking cycles, in which the rhythmic interplay between hypophysis and ovary is fundamental. Attention must therefore be focused on the physiology of the ovary and on the hormonal and neural mechanisms that integrate hypophysis and ovary as a functional system. Cyclic alterations in sex accessories and other nongonadal tissues are considered mainly as indicators. The "menstrual cycle," being strictly a uterine cycle, comes in this category, together with changes in behavior.

No attempt is made to present an exhaustive description of the varied adaptive modifications of the ovarian cycle among the several mammalian orders. The reader may consult works of the late F. H. A. Marshall whose full bibliography is given by Parkes (1949 1. Asdell's Patterns of Mammalian Reproduction (1946» is anotlu'i' A-alual)lo source.

II. Cycles Spontaneously Interrupted

Cycles in the natural state are only imperfectly known, from random and often erratic sampling. One may safely assume that, as a rule, under optimal conditions they are complete, fertile cycles. There are, then, relatively few subhuman species in wdiich the characteristics of incomplete cycles have been studied. These species are necessarily the very ones that have been amenable to some form of human restraint.

Segregation of the sexes or any other interference with insemination should be regarded as a first experimental approach to understanding the complete cycle. Such factors unriuestionably operate in nature on occasion. Controlled changes of environmental conditions afford another approach in which natural factors are simulated.

The statement was made earlier that the complete cycle may conceivably be interrupted at almost any point. It has been

learned that in different species segregated females interrupt their cycles at different stages and that usually the point of interruption is species-characteristic. These facts have been of great service to the study of reproduction, first, by arousing the curiosity of the investigator and, second, by supplying a variety of ready made conditions individually appropriate for particular experimental studies.

Examples of mammalian cycles are schematically diagrammed in Figure 8.1. It is customary to state that the usual, or standard, infertile cycle is like that in primates or the guinea pig. The follicular phase culminates in spontaneous ovulation, after which corpora lutea are organized and become spontaneously functional for a period of time that is usually considerably shorter than in pregnancy.

In a few animals (rat, mouse, hamster) the cycle terminates shortly after ovulation before the corpora lutea become fully functional. Such corpora lutea are said to be inactive, in the sense that they cannot produce a decidual response to uterine trauma (Long and Evans, 1922). Sterile mating or analogous stimulation induces a luteal phase which corresponds to that of the "standard" mammal. This phenomenon is not entirely limited to the small rodents, having been described in the European hedgehog (Deanesly, 1934).

To this writer's knowledge there have not been described any mammalian species in which it is the rule that in isolated females the process of ovulation begins (follicle maturation, prelutein changes in granulosa, secretion of secondary liquor folliculi, and so on) without proceeding to eventual rupture of the follicles. Many cases could l3e cited, however, in which this has occurred "abnormally." Characteristically, some degree of luteinization occurs in the wall of such a follicle and a lutein cyst is formed.

On the other hand, there are numerous species (reflex ovulators) in which the preovulatory maturation of follicles and ovulation nearly always fail in the absence of the male. The known species in which this is true are widely distributed among the mammalian orders and are often closely iclated to other species in which spontaneous ovulation is usual. The domestic rabbit

MAMIMALIAN REPRODUCTIVE CYCLE

499

GUINEA PIG

PRIMATE

Fig. 8.1. Diagrams of cycles of representative, familiar mammals. , the follicular phase,

highly schematized and inaccurate in detail ; , atresia ; i , ovulation ; • , fully active

corpora lutea; O, corpora lutea regressing or otherwise not fully active. When sterile mating or equivalent stimulation (SM) is introduced, the cycles of the rat, rabbit and cat become directly comparable with those of the other species.

furnishes the classic exaini)le of reflex ovulation. Other reflex ovulators are the domestic cat (Greulich, 1934), the ferret (Hammond and Walton, 1934), mink (Hansson, 1947), marten (Pearson and Enders, 1944), the 13-lined ground squirrel (Foster, 1934), and the mole shrew (Pearson, 1944). To this list have been added the muskrat (Miegel, 1952) and a field mouse, Microtus californicus (Greenwald, 1956). Even among the marsupials, the female Didelphijs azarae is said not to form corpora lutea in the absence of the male (Martinez-Esteve, 1937). A few of these species display nearly constant estrus (rabbit, ferret), competent follicles being present most of the time in the isolated female during the breeding season.

Among even the spontaneous ovulators the cycle may sometimes not progress beyond the follicular phase. Thus, at the approach of puberty, waves of advanced follicle development and secretion of estrogen may take place without, however, leading to ovulation or corpus luteum forma

tion. The first cycles of primates are often anovulatory ones. In the adult macacjue, at least in some colonies, such cycles are characteristic during the summer months (Hartman, 1932) . A somewhat comparable seasonal effect has been reported in girls soon after the menarche (Engle and Shelesnyak, 1934). Menstrual cycles without ovulation have frecjuently been recognized in adult women in recent years, bearing no evident relationship to seasonal factors (Lopez Colombo de Allende, 1956). Anovulatory cycles were described in the mouse by Allen (1923) and have been noted occasionally in other species, but without clear measure of their incidence.

III. Pituitary-Ovarian Doriiianey

Varying levels of pituitary-ovarian dormancy are expressed in different ways from species to species or even from habitat to habitat within a given species. A general similarity exists between the anestrum of seasonal breeders and the prepubertal state. In fact, in animals that have a distinct sea

500

PHYSIOLOGY OF GONADS

son, puberty occurs at the very time when older females are emerging from anestrum. Whereas anestrum is often correlated with season of the year, there are exceptions, notably among dogs, in which the correlation is ill defined (Engle, 1946).

In its shortest form ovarian quiescence lasts for only a few days, probably often without being recognized, between the end of one cycle and the active follicular phase of the next. In the chimpanzee it is thought to be the chief factor in the irregularity of length of the cycle (Young and Yerkes, 1943). Rossman and Bartelmez (1946) described a comparable occurrence in monkeys. At the other extreme, anestrum may occupy the major part of the year in monestrous animals that have a very limited breeding season.

A. THE OVARY IN ANESTRUM

Generally speaking, depression of ovarian function is most extreme in greatly prolonged periods of quiescence. In the ferret, Hammond and Marshall (1930) reported that in the anestrous ovary follicles can hardly be recognized with the naked eye, because they remain small and deeply placed. The largest follicles at the "end of the season" averaged 460 /x in diameter whereas a "long time after" the average was only 240 jx, increasing again to 720 yu, at the api^roach of a new season. By contrast, the largest follicles of animals in full heat ranged between 1220 and 1440 /x. Follicle atresia abounds in the anestrous ovary of the 13-lined ground squirrel (Johnson, Foster and Coco, 1933). In sheep, however, follicles of large size may be present at any time during anestrum (Kammlade, Welch, Nalbandov and Norton, 1952).

Some moderate degree of secretory activity of the ovary is indicated even at the depth of i^rolonged seasonal anestrum (13lincd ground squirrel, Moore, Simmons, Wells, Zalesky and Nelson, 1934; ferret, Hill and Parkes, 1933; opossum, Risman, 1946). Although at this time uterus, vagina, and vulva are small, ovariectomy or hypol^hysectomy causes a further reduction. On the other hand, these structures are readily stimulated by injection of estrogens.

It may be said that low-grade follicular cycles proceed throughout the anestrous

interval, but whether there is any synchronization of one follicle with another is unknown. Some insight into this problem is furnished by study of (1) the transition from anestrum to the breeding season, and (2) the closely analogous phenomena of adolescence. In the report by Hammond and Marshall, it was shown that in ferrets during anestrum and proestrum there is a progressive increase in size of the vulva which directly parallels the diameter of the largest follicles. The absence of overt cyclic change is not surprising in view of the fact that estrus is continuous in this species. In polyestrous animals, on the other hand, it might be expected that during anestrum follicle growth and accompanying estrogen secretion are cyclic, at least at the approach of puberty or of "the season." Important information on this question has been obtained from some of the primates, notably the macaque (Allen, 1927; Hartman, 1932) and the chimpanzee (Zuckerman and Fulton, 1934; Schultz and Snyder, 1935).

Slight transitory reddening of the skin of the perineum ("sex skin") of the monkey may occur at intervals for several months preceding the onset of menses, accompanied by moderate desquamation of vaginal epithelium. During the long intervals of amenorrhea that some individuals exhibit during the summer, there is a tendency toward cyclic vaginal desciuamation (Fig. 8.2). The sex skin of the chimpanzee may begin to swell more than a year before the first menstruation. During the ensuing months the swelling may be irregularly cyclic or continuous. Thus, one may judge that lowgrade follicular cycles, accompanied by periodic increases in estrogen secretion, may succeed one another during seasonal or ])repubertal anestrum, but that in certain cases these cycles may overlap to such degree that rather continuous estrogen secretion takes place.

B. THE HYPOPHYSIS

The secretory activity of the anestrous ovary is apparently adequate to prevent "castration" changes in the adenohypophysis, for as shown by Moore, Simmons, \^'ells, Zalesky and Nelson (1934) removal of the ovary of anestrous ground squirrels I'esults in hypertrophy of the hypophysis,

MAMMALIAN REPRODUCTIVE CYCLE

501

z

28 days

1

28 days A

28 days

< "

3 s UJ . Q

III Menses

"^ v-^^/^

A>

^ Mensesllll

21 ' 15 JUNE JULY

' 15 AUGUST

15 SEPTEMBER

15 OCTOBER

Fig. 8.2. Vaginal cycles during seasonal amenorrhea in a monkey. (A portion of the record of monkey ^38 from C. G. Hartman, Contr. EmbryoL, Carnegie Inst. Washington, 13, Fig. 26, p. 121, 1932.)

increased gonadotrophin content thereof, and increased numbers of basophile cells. Warwick (1946) reported a highly significant increase of pituitary potency in spayed anestrous ewes. This is closely analogous to the results of ovariectomy in immature animals (Hohlweg, 1934). As measured by ovarian activity, gonadotrophin secretion (release) may be greatly diminished during profound anestrum. The actual hypophyseal content of gonadotrophin seems to be markedly reduced during anestrum in some species (Moore, Simmons, Wells, Zalesky and Nelson, 1934), but possibly not in others. Cole and Miller (1935) and Warwick (1946) reported that there is no seasonal variation in sheep. A study by Kammlade, Welch, Nalbandov and Norton (1952) indicates that the average content is somewhat higher during anestrum than it is in cycling ewes. The major factor in this difference, however, seems to be that during the cycle the potency of the pituitary drops during estrus and the early luteal phase.

Somewhat similarly the potency of the immature rat hypophysis has been stated to be as high as that of the sexually active adult (Clark, 1935). The fact that the ovaries of the immature female or of the anestrous adult can be stimulated by injection of gonadotrophin indicates that gonadotrophin content of, the hypophysis in these cases is not a fair measure of liberation of the hormone into the blood stream. Therefore, it seems justifiable to assume, as Robinson (1951) did in the interpretation of anestrum in the ewe, that, in spite of the possible absence of seasonal assay variation.

there is, nevertheless, a depression of hypophyseal gonadotrophin release during anestrum. We may further assume that it is not completely depressed, for the ovary remains slightly active. Ovary and hypophysis are evidently in a state of equilibrium at a relatively low level of function. It seems likely that this state of affairs is brought about by the central nervous system, inasmuch as the seasonal depression in some species is closely dependent on the daily ratio of light to darkness.

C. RELATIONSHIP OF THE ANESTRUM TO THE SEASONS

This relationship is so varied among different species that many interesting questions are raised. In many cases the midpoint of anestrum coincides approximately with the shortest days of the year (Fig. 8.3). There are other examples, however, largely among the Artiodactyla, in which it coincides with the longest days. Sheep are notable examples (Robinson, 1951). Others, like the European common hare, experience a short anestrum during the time of rapidly decreasing daylight (Asdell, 1946). The Russian yak, on the other hand, is said to experience anestrum from December to May (i.e., while day length is increasing). A general explanation of these varied adaptive manifestations is elusive. There is reason to believe that although illumination, or the light/darkness ratio, (Kirkpatrick and Leopold, 1952; Hammond, Jr., 1953) has a rather direct and primary effect in some cases, its role is more or less indirect in others where such things as temperature, humidity, availability of food and water assume major importance (Marshall, 1942).

502

PHYSIOLOriY OF GONADS

NDJFMAMJJASON

INSECTIVORA

MOLE SHREW

COMMON SHREW CARNIVORA

BROWN BEAR (EUR.)

FERRET

COYOTE

WILD CAT (EUR.)

BAD3ER (AMER.) LAGOMORPHA

HARE (ENG.)

COTTONTAIL (N . ENG.) RODENTIA

13-L. GROUND SQUIRREL

WOODCHUCK

GRAY SQUIRREL

DORMOUSE

FIELD MOUSE (EUR. )

MUSKRAT (MARYLAND) (IOWA)

PORCUPINE ARTIODACTYLA

LLAMA

ROE DEER

MULE DEER

GIRAFFE

SHEEP (HAMP )

BIGHORN

GOAT

YAK (RUSS. )

INDIAN ANTELOPE PERISSODACT YLA

HORSE

Fig. 8.3. Some representative seasonal breeders. Solid bars indicate breeding seasons (according to Asdell, 1946); blank intervals, periods of anestrum. Months of the year represented by letters at top of chart ; winter and summer solstaces marked by wavy lines. Southern hemisphere seasons converted to corresponding ones of the northern hemisphere. End of season for the Bighorn is vmcertain.

The complexity of the i)rol)lem is well illustrated by the 13-lined ground squirrel whose breeding season, like that of a multitude of small rodents, comes in the spring. Moore, Simmons, Wells, Zalesky and Nelson (1934) reported that increasing illumination, elevated temperature, and feeding all failed to bring the females into estrus out of season. If, however, hibernation was first induced by low temperature and darkness, premature estrus would follow. The conclusion was reached that hibernation itself is a necessary prerequisite. Ovarian development actually begins, under natural conditions, in early January in the midst of hibernation. Females exix'i-imentally maintained "continually for several months in cold and darkness, with more or less normal hibernation, [exhibit] sexual development at any time of the year, and periods of estrum have thus been . . . maintained for many months. ..." The impres

sion is given tliat the conditions favoring hibernation also favor sexual development to such extent that breeding potentiality continues for a few months after emergence, in spite of elevated temperatures and long periods of illumination. In another rodent, Peromyscus leucopus, however, the length of daily illumination is of paramount importance. Temperature changes (4 to 25°C. ) have no effect on rejiroduction when lighting is adequate (Whitaker, 1940). Whereas a similar primaiy dependence on lighting can be shown in a number of other species from several orders, it is unwise to generalize that this is usually true.

IV. Attainment of Maturity. Emergence of Full Ovarian Function

Ahhough ('merg(>nce of the ovary from the state of quiescence is gradual, there is usually some outward sign that allows the observer to say that puberty has ari'ived or the breeding season has begun. In ])ri

MAMMALIAN REPRODUCTIVE CYCLE

503

mates the accepted sign is the first menstruation ; in rats it is the opening of the vagina ; in many animals it is the swelling and reddening of the genitalia heralding the initial l^roestrum. In other eases, e.g., sheep, the only clear indication may be the behavior of the female toward the male. From these facts it is readily apparent that any one sign is employed simply because it happens to be accessible to easy observation. Yet the increasing output of estrogen, whether steady or cyclic, affects many parts of the organism at the same time. Furthermore, in any one individual the threshold for exl)ression of a given sign may be relatively liigh with respect to that of some other manifestation. Thus, in Hartman's monkeys (1932), some were noted in which desquamation of vaginal epithelium occurred in wave-like manner for a long time before menstruation. In others "menstrual" bleeding occurred with regularity while the uterus remained very small and A'aginal desquamation was negligible.

Hartman summarized the step-wise manner of maturation of ovary and accessory organs of the monkey during adolescence or following amenorrheic episodes somewhat as follows. The color of the sex skin may be the first to appear. A slight menstrual flow usually takes place before desquamation of vaginal epithelium becomes measurable. "More rarely there may be one or more low desquamation cycles before a bleeding is recorded. Whole cycles marked liy jieriodic bleeding and some vaginal desquamation may occur before there is any noticeable increase in size of the ovaries and uterus. These organs increase also in a saltatory manner, hence the term 'staircase' phenomenon for the process. Finally, the endocrines effect the acme of the reproductive process — ovulation."

Individual variation in the degree of abruptness with which the first ovulation is achieved is well illustrated in a study of puljertal guinea pigs by Ford and Young (1953). In most cases the first period of vaginal opening was much longer than in subsequent cycles. Whatever the duration, ovulation was more closely related to the end than to the beginning of the period, as indicated by histologic study of ovaries.

Even ovulation and corpus luteum for

mation do not signify that full power of reproduction has arrived. For example, the first cycle of the adolescent rat may culminate in ovulation without sexual receptivity (Blandau and Money, 1943). In the ewe, an ovulation without overt signs of heat may at times take place during the anestrum, especially just before and just after the breeding season (McKenzie and Terrill, 1936). The phenomenon is occasional in ewes during the season and has also been described in cattle (Hammond, 1946). In fact, the full manifestation of estrus in sheep seems to require the presence of a "waning" corpus luteum (Robinson, 1951). In sheep the transition from seasonal or prepubertal anestrum to the breeding season may involve relatively minor changes in hypophyseal activity. Even in the immature rat both the hypophysis and the ovary are capable of far greater secretory function than they normally display. In the equilibrium that prevails, the ovary appears to hold the upper hand by reason of a low hypophyseal threshold at which estrogen suppresses gonadotrophin secretion in the immature individual (Hohlweg and Dohrn, 1932; Byrnes and Meyer, 1951b) and a low ovarian threshold at which gonadotrophin stimulates estrogen secretion. Byrnes and Meyer (1951a) reported that suppression of hypophyseal gonadotrophin content in immature rats can be accomplished with doses of estrogen much smaller than those that affect uterine growth. It is also known that the immature ovary can be induced experimentally to secrete estrogen by injection of amounts of gonadotrophin that are too small to produce significant increase of ovarian weight or follicle development (Levin and Tyndale, 1937; Moon and Li, 1952). When a gonadectomized immature rat is united in parabiosis (Kallas, 1929, 1930 » with a normal or hypophysectomized female littermate, precocious puberty is induced in the latter animal because insufficient estrogen passes to the first partner to inhibit gonadotrophin secretion (see Finerty, 1952). The somewhat analogous experiment of transplanting ovaries to the spleen produces ovarian hypertrophy in much the same way. Here again, it is thought that the hypophysis becomes hyperactive because the amount of estrogen

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PHYSIOLOGY OF GONADS

reaching the ghmd is greatly diminished, through inactivation bj^ the liver (Biskind, 1941).

Although it is true that estrogens have a suppressing action on gonadotrophin secretion, it has become increasingly evident that they can also stimulate hypophyseal function in certain ways, as Engle pro{)osed in 1931. Thus short-term injection of estrogen into intact immature rats and mice will invoke precocious puberty not only by stimulating the sex accessories, but also by increasing gonadotrophin secretion and thus causing ovarian growth and even ovulation. Frank, Kingery and Gustavson (1925j reported that after such treatment regular cycles continued after treatment was withdrawn. Lane (1935) found that when 22day-old female rats were injected daily with estrogen there was an early increase in number of ovarian follicles, including vesicular stages. After the first 10 days the nonvesicular follicles became depressed although vesicular follicles were retained. This was interpreted to mean that for a short time estrogen actually stimulates the follicle-stimulating hormone (FSH) l)ut eventually suppresses it, although luteinizing hormone (LH) secretion remains elevated. Hohlweg (1934) had already demonstrated that when somewhat older prepubertal rats are given single, rather large injections of estrogen, ovulation and corpus luteum formation are induced within a few days (p. 514). Obviously LH secretion is greatly increased.

Various bits of evidence implicate the nervous system in the processes leading to puberty and to the onset of estrus in seasonal breeders. This will be discussed in the following section with respect to the general (juestion of the relationship of the hypothalamus to gonadotrophin secretion.

V. Follicular Cycles. Growth and Atresia

Attention will be focused here on the dynamic pattern of follicle development throughout the cycle, the extent to which this i)attern depends on hyi)o[)hyseal conti'ol, and the functional changes in the o\aiy associated with estrus in preparation foi' the more specialized events that lead to ovuhition and corpus luteum formation.

Production of primordial follicles and the early growth stages have been said to be independent of the hypophysis (Smith, 1939; Hisaw, 1947). This view derives from the fact that following hypophysectomy the ovaries retain large numbers of healthy proliferating follicles below the stage of antrum formation. There are, however, several indications that these developmental stages may be accelerated by gonadotrophic stimulation. It was briefly reported by Simpson and van Wagenen (1953) that administration of purified FSH to immature monkeys caused not only a 10- to 20-fold 'increase of ovarian weight, but also stimulation of granulosa in follicles of all sizes. Indirect evidence comes from the fact that follicle atresia generally becomes maximal late in estrus or metestrum, when depressed FSH might be expected on theoretical grounds. Harrison (1948) reported tliat in ovaries of goats killed on the third or fourth days of estrus healthy primary ovocytes are rare. Some few, however, presumably remain. Myers, Young and Dempsey (1936) stated that in the estrous guinea pig there are few nonatretic follicles aside from those destined for ovulation. However, small numbers of normal ai)pearing nonvesicular follicles were found.

There seems to be general agreement that, very quickly after this catastrophic elimination of follicles, renewed growth promptly ensues. Whether or not the wave of atresia represents a depression of FSH secretion, no one would deny that the new growth reflects this type of gonadotrophic stimulation. Characteristically the population of small and medium follicles is restored early in the luteal phase of the polyestrous cycle. This is clearly indicated for the guinea pig ovary (Myers, Young and Dempsey, 1936) when the data are converted from average volumes to average diameters (Fig. 8.4). Beginning on the fourth day after estrus, when the largest follicles are approximately 300 fx in diameter and when theca interna and antra have formed, rapid growth of granulosa, theca, and antra continues for several days. This is confirmed by counts of mitotic figures obtained by the colchicine technique (Schmidt, 1942), indicating greatest mitotic activity in theca and granulosa of follicles between 300 fi and 600 fx in diameter. By the

MAMMALIAN REPRODUCTIVE CYCLE

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4 8 12

DAYS AFTER BEGINNING OF ESTRUS

Fig. 8.4. A schematic repiesentation of the folhcuhir cycle in the guinea pig. The heavj^ sohd curve represents the diameters of the largest follicles, recalculated from the data of Myers, Young and Dempsey (1936). The arrow point indicates ovulation. The other solid curves and broken lines represent impressionistically the growth and atresia, respectivelj^ of other groups of follicles that are not ordinarily destined for ovulation.

11th or 12th day the largest follicles (ca. 800 /x) are "competent," i.e., capable of being ovulated (Dempsey, Hertz and Young, 1936; Dempsey, 1937). While the largest follicles are developing to this stage, multitudes of others begin to grow, being carried on to various stages of development before regression sets in.

This pattern of the follicular cycle seems to be generally true among mammals that have been carefully studied, when allowance is made for the fact that from one species to another the characteristic maxima of follicle diameter are extremely variable (shrew, 350 fjL-, rat, 900 /*; cow, 19,000 /x; mare 70,000 /x; Asdell, 1946). In ovulatory cycles of polyestrous animals the greater part of follicle growth is accomplished while the luteal phase of the preceding cycle is in progress. In successive anovulatory cycles like those of the cat the patterns of the follicular cycles are probably much the same (Evans and Swezy, 1931 » . In the rabbit and ferret, where more or less constant estrus char

acterizes the isolated females in season, there is probably considerable telescoping of successive waves of follicle growth such that as one set of follicles begins to undergo atresia another set is ready to take its place (Hill and White, 1933). The difference between cat cycles and rabbit cycles seems to be chiefly one of degree. The writer has seen both types represented in persistent-estrous rats, among litter mates of inbred strains (Everett, 1939, and unpublished).

At the end of the luteal phase of the cycle in polyestrous animals there are already present several competent follicles among an extensive population of smaller ones. For example, the guinea pig corpus luteum usually shows signs of regression on day 13 of the cycle. It has been proved that ovulation can be induced as early as day 12 by injection of LH (Dempsey, 1937), several days earlier than it would normalh^ occur (Fig. 8.5). In the human and monkey it is possible that the "preferred" follicles are recognizable by their larger size during

506

PHYSIOLOGY OF GONADS

1000

750

500

Volume (lO^cu.//)

250

Normal Cycle

Cycle After Removal of Corpora Luteo

Progesterone Treated or Pregnant

'Corpora Luteo Removed and Oestrin Injected

Fig. 8.5. The guinea pig follieular cycle and some of its experimental modifications. (After E. W. Dempsey, Am. J. Physiol., 120, 126-132, 1937.)

or soon after menstruation (Allen, Pratt, Newell and Bland, 1930; Hartman, 1932). In many mammals competent follicles may be present much earlier. Ablation of corpora lutea soon after ovulation in sheep (McKenzie and Terrill, 1936) and cattle (Hammond, Jr., and Bhattacharya, 1944) is followed in 2 to 4 days by another ovulation, much sooner than in the guinea pig (Fig. 8.5). Removal of the primate corpus luteum, at the other extreme, produces no such immediate response, judging from the details of three cases among Hartman's (1932) protocols (#40, #41, and #99). Whereas the next ovulations took place earlier than expectation, the intervals between unilateral ovariectomy and ovulation were 16, 14, and 22 days, respectively.

From detailed investigations in the rat, only the earlier stages of follicle growth may properly be regarded as pure FSH effects (Lane, 1935). Lane and Greep (1935) found that addition of Lli to FSH causes a marked increase in the proportion of vesicular follicles to follicles without antra. The use of more highly purified materials ((irecp, van Dyke and Chow, 1942; Fraenkel-Conrat, Li and Simpson, 1943) has amply confirmed the necessity for combination of the two gonadotrophins to yield maximal follicle growth and estrogen secretion ill rats. Morphologic evidence indicates that

LH acts selectively on thecal tissue and, therefore, on the interstitial tissue derived therefrom. Inasmuch as thecal tissue is the presumptive major source of ovarian estrogen (see below), it follows, perhaps, as Hisaw (1947) suggested that "the theca interna through the action of LH acquires competence to respond to FSH" (by secreting estrogen) .

Convincing evidence that thecal tissue and its derivatives are the principal sources of ovarian estrogen was assembled by Corner (1938). The status of this question remains essentially the same today. Few endocrinologists, however, would assume that no other ovarian cells have this capacity (see discussion in the chapter on the ovary). Nevertheless, there is a direct correlation in time between the marked rise in estrogen secretion as the follicular jihase of the cycle advances, on the one hand, and the organization of tlicca interna of the largest follicles into organs of obvious endocrine character, on the other. "When especially ])rominent the theca interna is referred to as the "thecal gland" (Mossman, 1937; Stafford, Collins and ]\Iossman (1942).

Thecal tissue from the multitudes of atretic follicles should not be neglected as a possible additional source of estrogen. From the standpoint of chronologic relations to the cycle tiiis (iiiestioii has hardly

MAMMALIAN REPRODUCTIVE CYCLE

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been touched. Pointing up our ignorance, Sturgis (1949) in a careful study of atresia of large follicles in the monkey ovary, speculated that their hypertrophied thecal tissue may serve the useful purpose of estrogen secretion during the interim between follicle rupture and organization of the corpus luteum.

We are in need of ciuantitative appraisals not only of the total numbers of healthy and atretic follicles of all categories present in representative species at progressive stages of the cycle, as in the work on the rat by Mandl and Zuckerman (1952), but also of the respective volumes of theca, granulosa, interstitial tissue, and corpora lutea. Lane and Davis (1939) determined in rat ovaries at four stages of the cycle the respective total volumes of theca, granulosa, and antra in all healthy follicles, as well as the separate mitotic indices of theca and granulosa. Such differential information on multiplication of cells and increase of antral volume is important. Although the latter accounts for a major part of the increase in volume of the larger follicles, it represents a function quite apart from protoplasmic growth per se.

There is now considerable evidence that estrogen itself exerts a growth -promoting influence on the follicle and, furthermore, sensitizes it to gonadotrophic stimulation. Details may be found in papers by Pencharz (1940), Williams (1940, 1944, 1945a, b), Simpson, Evans, Fraenkel-Conrat and Li (1941) , Gaarenstroom and de Jongh (1946) , and Desclin (1949a,) . Although it seems that these effects have not been elicited by physiologic doses, the possibility remains that estrogen operates within the confines of the ovary as a mediator of some of the effects of the gonadotrophins. In the neighborhood of cells that produce it the estrogen concentration is probably far above that which would be considered physiologic for the remainder of the body.

A. CORRELATION OF OVARIAN SECRETION W^ITH THE FOLLICULAR CYCLE

Knowledge of the secretory output of the ovary during the cycle is almost entirely indirect and derives chiefly from (1) substitution experiments carried out in a vari

ety of si)ecies, and (2) assays of urine, mainly human but occasionally from other forms. Satisfactory assays of blood estrogen have been very limited and chemical analysis of the steroid content of ovarian venous blood is in only its preliminary stages.

The early substitution experiments are chiefly of historic interest (Allen, Danforth and Doisy, 1939). In great measure these investigations constitute crucial steps in proof that the ovary secretes steroid hormones which are fundamentally responsible for the manifestations of estrus. Conversely, then, these manifestations might be considered to reflect an increase of estrogen secretion and their absence a relative decrease. It has been learned, however, that the action of estrogen in certain instances may be greatly modified by progesterone, androgens, and certain adrenocortical steroids (notably desoxycorticosterone). Androgens are known to be secreted in the female by the adrenal cortex (Dorfman and van Wagenen, 1941 ; Gassner, 1952) and by the ovaries (Hill, 1937a, b; Parkes, 1950; Deanesly, 1938; Burrill and Greene, 1941; Pfeiffer and Hooker, 1942; Alloiteau, 1952). Progesterone secretion is probably not confined to the luteal phase of the cycle (see p. 519j. Evidence for its secretion during follicle maturation is considerable and its possible production even earlier cannot be excluded. These considerations make it unwise, therefore, to regard phenomena such as vaginal cornification, turgescence of vulva and sex skin, uterine growth, as direct ciuantitative measures of estrogen output. This point may be illustrated by certain observations made in chim])anzees by Fish, Young and Dorfman (1941) and illustrated in Figure 8.6. Assays of urinary estrogens during the cycle exhibited two peaks, only the first of which coincided with the swelling of sex skin. The second peak of estrogen excretion was unaccompanied by swelling, presumably because of the coordinate increase of progesterone secretion. Had swelling been the only guide only the first peak would have been apparent.

Assays of urinary estrogen in primates have often shown double peaks such as illustrated for the chimpanzee. PedersenBjergaard and Pederson-Bjergaard (1948i.

508

PHYSIOLOGY OF GONADS

I.U. '00

I.U.

( »ott

Androgens Curve of genital swelling

25 27 29

Day of cycle

Fig. 8.6. E.strogen and androgen excretion by a female chimpanzee, Mamo. , total

estrogens; , estradiol; -•-•, estrone; , estriol. Menstruation indicated by solid

areas on base line. (From W. R. Fish, W. C. Young and R. I. Dorfman, Endocrinology, 28, 588,1941.)

studying one woman for 2 years, found single peaks at midinterval in 8 cycles and double peaks in 12 cycles. On the average the first peak was reached on day 12 and the second on day 21. Similar double peaks were noted in blood estrogen assays in a large group of normal young women (Markee and Berg, 1944). An additional lesser rise was observed during menstruation.

None of the available assays of urinary or blood estrogen can be accepted as an absolute measure of the rate of hormone production. Urinary assays have certain advantages, in spite of the fact that probably only a variable fraction of the ovarian product is measured. Intrinsically they are measures of rate, whereas assays of blood estrogen measure concentration alone at the moment of bleeding. Attempts have been made to measure estrogens in ovarian venous blood, but with little success because of the extreme dilution (Rakoff and Cantarow, 1950). We may hope that development of sufficiently sensitive methods of detection will soon allow systematic evaluation of ovarian output by such direct means. Tracer techniciucs have shown (Werthessen, Schwenk and Baker, 1953) in perfused ovaries of the sow that C^^-acetate enters into the synthesis of estrone and /^-estradiol.

Several years ago Corner (1940) esti

mated, from the known amounts of injected estrone required to maintain the normal status of sex skin and endometrium in castrates that the ovaries of an adult rhesus monkey secrete the equivalent of about 20 fig. estrone daily. On a weight basis the estrone equivalent secreted by the ovaries of a woman would then be on the order of 300 /Ag. per day. Actual substitution data from castrated women gave an estimate of the same order of magnitude (420 ;u,g. per day). Whatever the rate of secretion may be at different times, it would seem a 'priori that effects on extra-ovarian tissues should be more directly related to amount of estrogen in circulation. The assays of human bloodestrogen in normal women by Markee and Berg (1944) and in gynecologic patients by Fluhmann (1934), although differing in absolute values, agree in indicating that the variation of blood estrogen concentration from one stage of the cycle to another may be relatively small. If this is true, then it nuist be supposed that cyclic changes in the accessory organs are brought about by relatively moderate changes in circulating estrogen. In support of this view Markee (1948) demonstrated in the macaque that a mere 50 per cent reduction in the daily dose of estrogen can invoke menstruation if the change is abrupt.

MAMMALIAN REPRODUCTIVE CYCLE

509

B. CYCLIC MANIFESTATIONS AFTER OVARIECTOMY OR HYPOPHYSECTOMY

Residual cyclic changes in the vagina liave been reported in ovariectomized mice (Kostitch and Telebakovitch, 1929) and rats (Mandl, 1951). The periodicity is very nearly that of the normal cycles, at least in the latter species. Vaginal cycles of similar duration with more extreme estrous changes are found in ovariectomized rats receiving daily injection of threshold doses of estrogens (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941). The same was remarked in mice by Emmens (1939) and a report by Veziris (1951) indicates that vaginal periodicity may obtain in castrated or menopausal women receiving estrogen. Although sucli events have been called threshold cycles," the term may simply express the fact that they are most easily recognized when estrogen is given at threshold level. Hartman (1944), employing a modified Shorr stain for vaginal smears, found that castrated rats given large amounts of estrogen daily (5 to 100 fig. estradiol dipropionate) displayed complete cornification at 4- to 5-day intervals. During the time intervening there was admixture of Shorr cells, smaller epithelial cells, and leukocytes.

Analogous phenomena have been recognized in the endometrium of castrated monkeys (Zuckerman, 1937, 1941) injected daily for as long as 1 year with threshold doses of estrone (10 fig.). Larger doses prevent cyclic bleeding (see Hisaw, 1942). From the report of Veziris (1951) it may be judged that threshold endometrial cycles also occur in women and that the vaginal and endometrial cycles are synchronized in considerable extent.

Full explanation of these phenomena is not at hand. From the standpoint of the present discussion certain considerations are especially noteworthy. (1) Vaginal "threshold cycles" have been obtained in castrated rats in the absence of either hypophysis or adrenals (Bourne and Zuckerman, 1941 ; del Castillo and di Paola, 1942) . The former authors encountered the phenomenon in two rats from which both the hypophysis and adrenals had been removed. It is important

to remember, however, that the pars tuberalis remains in situ after the usual hypophysectomy procedure, that accessory adrenocortical tissue is frequent in rats, and that gonadal rests might remain unrecognized. (2) The reported lengths of vaginal and endometrial cycles agree favorably with the cycle lengths in intact individuals of the respective species. The degree of conformity between vaginal and uterine cycles indicated by Veziris {loc. cit.) suggests some sort of integrating mechanism. Much more information is required, however, before one may reject the alternative view that rhythmic activity is an innate characteristic of these organs.

C. CYCLIC MANIFESTATIONS IN THE ABSENCE OF OVARIAN FOLLICLES

Many years ago Parkes (1926a, b) and Brambell, Parkes and Fielding (1927a, b) reported vaginal and uterine cycles in mice in which the entire follicular apparatus had been destroyed by x-radiation. Schmidt (1936) described the phenomenon in the guinea pig, noting that, although most of her estrous animals had one or more large atretic or cystic follicles, as she had earlier reported (Genther, 1931), a few animals exiiil)ited periodic vaginal opening of short duration and correlated proestrous vaginal smears, in the absence of follicles. Her assays of urinary estrogen were negative in these animals, unlike the positive assays in those in which one or more follicles were demonstrable. Attempts by several workers (Drips and Ford, 1932; Levine and Witschi, 1933; Mandel, 1935) to reproduce in rats the results that Parkes and Brambell had found in mice, were unsuccessful, a fact indicating no estrogenic activity in ovaries completely lacking follicles and ova. Parkes (1952) more recently returned to this problem, reporting vaginal cycles and "fully functional" uteri in castrated rats bearing grafts of ovaries in which all organized follicles and ovocytes had been destroyed by deep freezing. These were true estrous cycles, in the sense that the animals would mate.

Many questions are posed by these observations. The fundamental one seems to be whether these cycles express periodicity of hypophyseal gonadotrophin secretion.

510

PHYSIOLOGY OF GONADS

The answer may be long in coming. Meanwhile, one would like to know whether castration changes are visible in the hypophysis and whether constant estrus may be invoked by exposure to continuous light or by postnatal treatment of the host with androgen or other steroids (see p. 529 1 .

D. HYPOTHALAMUS AND GONADOTROPHIN SECRETION. GENERAL CONSIDERATIONS

Experimental studies, ostensibly addressed to the general problem of neural control of gonadotropin secretion, have in fact often been concerned with the special problems of reflex ovulation <p. 520) or of provoked pseudopregnancy (p. 532). Whereas substantial information is now available w^ith respect to these special phenomena, particularly ovulation, information is limited about control of the day-to-day secretion of gonadotrophin that in the female is responsible for follicle stimulation and estrogen secretion (Benoit and Assenmacher, 1955; Harris, 1955). However, evidence in regard to induction of precocious puberty and early onset of estrus in seasonal breeders leaves no doubt that the nervous system is in some manner a regulator of follicle-stimulating activitv of the i)ars distalis.

Numerous reports associate precocious l)uberty with lesions in the hypothalamus (Weinberger and Grant, 1941; Bauer, 1954; Harris, 1955). Donovan and van der Werff ten Bosch (1956) reported off-season estrus in ferrets and precocious puberty in rats following retrochiasmatic lesions in the hyjiotlialamus. Exposure of immature rats to continuous light causes the vagina to open prematurely (Fiske, 1941). When 22-dayold female rats were given electrical stimulation of the cervix uteri daily for 10 days (Swingle, Seay, Perlmutt, Collins, Fedor and Barlow, 1951), a large proportion exhibited significant increase in uterine weight beyond that found in control animals, without change in ovarian weight. In fact, 7 of 50 rats ovulated or at least formed "several well-developed corpora lutca." Somewhat similarly, according to Aron and AronBrunetierc (1953), mechanical stimulation of the vagina or the adjacent segment of the uterus in innnatur(> guinea pigs regularly

provoked follicle growth and estrogen secretion. In gregarious birds the development of ovulable follicles requires that other individuals of the species be present. In the pigeon, even the mirror image of the female constitutes a sufficient stimulus (Matthews, 1939).

Studies by Flerko and his associates (1954-1957) present consistent evidence that restricted bilateral lesions in the region of the paraventricular nuclei serve to liberate the hypophysis from inhibitory effects of estrogen and androgen. This work is in agreement with that of Donovan and van der Werff ten Bosch in that somewhat similarly located lesions brought on precocious puberty. As noted elsewhere, gonadectomy in immature rats quickly results in hypersecretion of gonadotrophin.

Transplantation of the hypophysis to sites remote from the hypothalamus has produced divergent results. At the present writing, the chief divergence seems to rest between the sexes. In male guinea pigs and rats several workers have reported maintenance of male reproductive tracts by intra-ocular transplants of hypophyses (May, 1937; Schweizer, Charipper and Kleinberg, 1940; Cutuly, 1941a; Courrier, 1956; Goldberg and Knobil, 1957). Quite to the contrary, however, there has at best been only equivocal evidence of maintenance of female tracts, a matter of sex difference which needs full investigation. JNIay's (1937) report of 2 fertile female rats is unacceptable because of inadequate controls. Schweizer, Charipper and Haterius (1937) found in several hypophysectomized guinea pigs that intra-ocular pituitary grafts produced constant estrus and significant follicle stimulation, accompanied by uterine and mammary gland develoi)inent. Although the search for pituitary remnants in the sella turcica was reported negative, the histologic check was limited to scrapings from the sella floor. Other authors, notably Phelps, Ellison and Burch (1939), Westman and Jacobsohn (1940), Harris and Jacobsohn (1952), and Elverett ( 1956a) obtained in female rats little or no evidence of gonadotrophin secretion from apparently healthy, well vascularized grafts. The respective sites were intraniusculai', intra-ocular, in the sub

MAMMALIAN REPRODUCTIVE CYCLE

511

arachnoid space under the temporal lobe of the brain, and beneath the renal capsule — all distant from the hypothalamus.

Transplantation of the pars distalis into sites close to the hypothalamus, on the other hand, is characteristically followed by maintenance of the female reproductive tract and essentially normal sex functions. Greep (1936) found that re-implantation of hypophyses into the (presumably) emptied capsule was frequently followed in both male and female rats by return of virtually normal reproductive powers. Females exhibited cycles and even went through successful pregnancy and lactation. The result observed in male rats was confirmed by Cutuly (1941a). The obvious difficulty of establishing completeness of hypophysectomy has been the only criticism of these instructive experiments. This fault has been eliminated by an improved procedure devised by Harris and Jacobsohn (1952). Hypophysectomy was performed by the parapharyngeal route, after which the tissue to l)e grafted was introduced by a transtemporal approach to a site immediately beneath the median eminence. This permitted later histologic search for remnants of the original gland in its capsule. In many cases, including all in which the graft comprised several hypophyses from the animal's own newborn young, entirely normal gonadotrophic function was recorded. This included resumption of regular estrous cycles, typically during the 2nd or 3rd postoperative week. Several of the rats became pregnant and delivered viable litters. In marked contrast, none of the grafts that were placed under the temporal lobe gave any indication of gonadotrophin secretion, although they were as well preserved and richly vascularized as the others. Explanation of the difference seems to be that grafts under the median eminence acquire blood supply from regenerated hypophyseal portal veins and iience a neurovascular linkage with the hypothalamus. The importance of this relationship has been amply confirmed by Nikitovitch-Winer and Everett (1957, 1958d) in studies described below.

In lieu of significant numbers of nerve fibers entering the pars distalis (see Rasmussen, 1938; Harris. 1948a I, the hypophyseal

portal veins afford the most likely means by which the gland is brought under hypothalamic control. Recently it was demonstrated in rats and monkeys that these vessels have the power of rapid regeneration after simple stalk-section (Harris, 1949, 1950a, b). This fact at once gives a ready explanation of many of the discordant results of stalk-section experiments reported in the past. Harris (1950b) explored in rats the efficacy of various materials as barriers to regeneration, with the result that numerous examples of partial regeneration were produced. Degree of recovery of gonadotropliic activity by the hypophysis was strikingly correlated with degree of anatomic vascular recovery. Restoration of normal ovarian function after simple interruption of the stalk, as reported in the guinea pig by Dempsey (1939), in rats by Dempsey and Uotila (1940) , and in the human by Dandy (1940), is thus explained by the assumption that portal vein regeneration had taken place. On the other hand, Westman and Jacobsohn (1937-1938), who always inserted a barrier of metal foil between the median eminence and hypophyseal capsule, consistently found ovarian atrophy, as did Harris when portal vein regeneration was completely obstructed. Attempting to prove that the portal vessels are not essential in regulating the hypophysis, Thompson and Zuckerman (1954) stated that increased illumination induced estrus in two ferrets after stalk-section and in the absence of demonstrable regeneration of portal vessels. Donovan and Harris (1954), however, examining the histologic sections prepared from 1 of the 2 animals, found many such vessels that were uninfected. In their own experimental series, an estrous response to light was always associated with regeneration of the portal veins.

Greep and Barrnett (1951) rightly emphasized the prime importance of a good vascular supply for recovery of function by the pars distalis after either transplantation or stalk-section. They pointed to the extensive central infarction and scarring that characteristically followed stalk-section by their technique, an obvious factor contributing to hypopituitarism. Harris (1950a I, however, reported good function from sev

512

PHYSIOLOGY OF GONADS

eral hypophyses in which there was pronounced central necrosis in company with well regenerated portal vessels. A study by Nikitovitch-Winer and Everett (1957, 1958b) established beyond doubt that qualitative functional losses after stalk-sectron or transplantation of the pars distalis result, not from impaired blood supply per se, but from the loss of the intimate neurovascular relationship with the hypothalamus. Hypo])hyseal autografts, after first being placed under the kidney capsule for several weeks with the usual atrophy of the ovarian follicular apparatus and interstitial tissue, were later retransplanted to a site immediately under the median eminence. In the definitive series of 14 such experiments, 13 rats resumed estrous cycles spontaneously 8 to 68 days after retransplantation ; 7 were fertile and carried litters to term. A correlated study (Nikitovitch-Winer and Everett, 1959) demonstrated clearly that on the occasion of each of these successive transplantations there was massive necrosis of the interior of the glandular mass, leaving but a thin shell from which the functional tissue of the graft was reconstituted. In spite of this double insult some special influence of the hypothalamus brought about renewed function in a surprising number of cases. Together with the restoration of gonadotrophic activity there was significant improvement in thyroid-stimulating hormone (TSH) and adrenocorticotrophic hormone (ACTH) secretion. The considerable net loss of hypophyseal parenchyma resulting from the two operations was reflected only quantitatively in the effects on the various target organs. Ovarian weights, numbers of follicles and corpora lutea, adrenal weights and extent of adrenal hypertroi)liy after unilateral adrenalectomy, and thyroid uptake of P^^ were all intermediate between those of the normal female rat and control animals in which the graft remained on the kidney or was retransplanted under the temporal lobe of the brain.

Regulation of pars distalis secretion by means of the stalk vessels may conceivably be carried out either by regulation of blood How or by transmission of chemical mediatoi-s from the proximal capillary plexus in the median eminence to the pars distalis. An

experiment describetl by Swingle, Seay, Perlmutt, Collins, Fedor and Barlow (1951) suggested that a mediator subject to Dibenamine blockade might be involved in precocious puberty. Although significant uterine enlargement was produced in immature rats by daily stimulation of the cervix uteri for 10 days, no such effects were observed in similar rats given Dibenamine daily by stomach tube. Unfortunately, there were no controls for the possible effect of Dibenamine in nonstimulated or gonadotrophininjected animals.

Fluhmann (1952) invoked precocious vaginal opening and ovarian stimulation in immature rats by injection of neostigmine. The locus of such cholinergic action is unknown. Parenthetically, Barbarossa and di Ferrante (1950) reported follicle stimulation in immature rats after injection of intermedin, an effect not found in hypophysectomized subjects. Benoit and Assenmacher (1955) proposed that, in the drake, gonad-stimulating activity is governed by an agent contained in neurosecretory substance, which is demonstrable in abundance in the retrochiasmatic region of the median eminence. Capillaries there drain selectively into an anterior set of portal venules. Oxytocin has been suggested as a possible mediator for gonadotrophin secretion (Shibusawa, Saito, Fukuda, Kawai, Yamada and Tomizawa, 1955; Armstrong and Hansel, 1958). There is much interest as this is being written (1958) in the jiossibility that vasopressin, oxytocin, or other agents associated with neurosecretory substances of the neui-ohyjioiihysis are responsible for control of production and release of the various trophic hormones of the pars distalis. As an alternative or even a supplement to neurochemical regulation, a vasomotor mechanism cannot be denied (Green, 1951), for conceivably only a slight shift in blood flow through the jiars distalis might tip the balance of hormone production one way oi- anothei-. Thus the matter stands: whereas it is apjiarent that the hypothalamus intervenes in follicle growth and estrogen secretion, how it does so is little more than speculati\'e.

MAMMALIAN REPRODUCTIVE CYCLE

51)5

VI. Follicle Maturation and Ovulation

A variety of evidence indicates discontinuity between growth of large follicles, on the one hand, and their preovulatory maturation, on the other. Such is clearly the case among "reflex ovulators." Evidence that the same is true for spontaneous ovulators will be outlined below. Follicle maturation, ovulation, and structural transformation of the follicles to corpora lutea seem to represent successive stages in a distinct physiologic process, superimposed on the follicle growth cycle and brought about by a relatively abrupt increase in circulating gonadotrophin (theoretically LH). Since there is evidence (p. 519) that progesterone secretion may become detectable as this process begins, there might be justification for regarding it as merely the first portion of the luteal phase. However, the fact that luteinization ( i.e., the organization per se of luteal tissue) does not necessarily lead to functional cor|)ora lutea warrants treatment of the ovulation-luteinization phase as a distinct phenomenon.

Although it is customary to state that the hypopliysis invokes ovulation by release of LH, there is considerable question about the auxiliary roles played by other gonadotrophic hormones (Hisaw, 1947). Inasmuch as the time of release has been known in only the reflex ovulators, one might look to them for information. However, the available data (Hill, 1934) pertain only to the ovulating i)otency of the total gonadotrophin content of the hypophysis at various times after coitus. Substitution experiments are unsatisfactory because the presence of competent follicles implies the presence of l)oth FSH and a small amount of LH. The substitution of even the purest hormone preparations immediately after hypophysectomy leads to equivocal results inasmuch as it must be assumed that some FSH and LH of intrinsic origin remain in circulation. Talbert, Meyer and McShan (1951) determined that in rats, when hypophysectomy is performed at the onset of proestrum, the follicles remain capable of responding to injected LH for about 6 hours. Morphologic signs of follicle deterioration do not appear until nuich later. Adding to the uncertainty

is the fact that relatively pure preparations of either FSH or LH will ovulate an estrous rabbit (Greep, van Dyke and Chow, 1942). On the other hand, until the recent use of species-specific gonadotrophins (van Wagenen and Simpson, 1957), the primate ovary was notoriously difficult to ovulate therapeutically. Until effluent blood from the hypophysis can be assayed, there is little likelihood that the gonadotrophin complex that is normally responsible for ovulation can be known. Thus, whereas the expression, LH-release, will be employed occasionally to refer to the release of gonadotrophin that in^•okes ovulation, the term is used purely for convenience and brevity, and should be ai^propriately qualified by the reader.

A. TIME OF OVUL.\TION

The time of ovulation with respect to other events of the cycle is relatively easy to determine in reflex ovulators, but in spontaneous ovulators has proven to be more elusive. In the former, laparotomy at various intervals after the stimulus enables exact measure to be made of the time required to accomplish ovulation. For most of the spontaneous ovulators, save the few in which the ripening follicles can be palpated as in monkeys and cattle, it has been necessary to attempt to correlate ovulation with some easily detectable external sign. Inasmuch as the ovulation stimulus to the hyj)oiihysis in these animals is probably invoked by ovarian hormones and these are equally responsible for phenomena such as vaginal cornification and behavioral estrus, a considerable degree of correlation might be expected between ovulation and a given change in the vaginal smear or onset of estrous behavior. The predictability of the relationship, however, must depend in great measure on the degree of correlation among thresholds of response in the various tissues concerned. In the primates that have no sharply limited period of sex desire the i:)roblem is even more troublesome. When reference is made to the date of the last menstruation, prediction is erratic because of the variable occurrence of postmenstrual quiescence (Rossman and Bartelmez, 1943; Young and Yerkes, 1943). Consequently, attempts must be made to find indicato:

514

PHYSIOLOGY OF GONADS

such as basal body temperature fluctuations which may bear some intrinsically closer relationship to the event in question. (See Hartman, 1936, and Buxton and Engle, 1950, for discussion of this very practical ]iroblera. )

Among mammals generally, si)ontaneous ovulation takes place sometime during estrus (Asdell, 1946) . It is found during early estrus in the opossum, red fox, dog, mouse and hamster. In the rat some authors have placed it early (Young, Boling and Blandau, 1941) and others late (Long and Evans, 19221 with respect to vaginal estrus. In the writer's colony both relations hold, in 4-day and 5-day cycles, respectively. Ovulation in late estrus is reported for the cotton rat, bank vole, guinea pig, pig, horse, and ass. Sheep usually ovulate shortly before the end of heat, sometimes a few hours afterward. As stated earlier, ovulation may even occur in guinea pigs, rats, sheep, and cattle without overt estrus. The cow usually ovulates several hours after the end of heat. The marsupial cat is said to ovulate 5 days afterward (Hill and O'Donoghue, 1913). The extreme is represented by certain bats (Asdell, 1946) which copulate in autumn and ovulate in the spring after a prolonged state of subestrus. These variations probably express several factors.

Among reflex ovulators there is considerable interspecies variation in the interval between the stimulus that invokes release of gonadotroj^hin from the hypophysis and the eventual rupture of the Graafian follicles (rabbit, ca. 10 hours; ferret, ca. 30 hours; cat, 24 to 54 hours; 13-lined ground sciuirrcl, 8 to 12 hours; mink, 36 to 50 hours) . Among spontaneous ovulators the comparable interval is clearly defined for only the rat, 10 to 12 hours (Everett, Sawyer and Markce, 1949) . In the cow the data obtained by Hansel and Trimberger (1951) and Hough, Bearden and Hansel (1955) i)lace the outside limit at about 30 hours. Here again, threshold differentials among tlic various tissues of the individual are piobably of gicat importance. 'I'hus in one species the threshold foi' gonadoti'ophin release may be lower than that foi' estrous behavior with the result tliat by the time the latter makes its ai)pearance the former has already transpired and

ovulation will shortly take place. The rat, for example, releases LH during the afternoon, begins to show estrous behavior around 8 p.m., and ovulates around 2 a.m. (Everett, 1948, 1956b). In other species these time relationships may be reversed. In the cow, activation of the hypophysis apparently occurs several hours after the onset of estrus (Hansel and Trimberger, 1951). The cow remains in heat 10 to 18 hours and ovulates 13!/2 to 151/2 hours after going out of heat (Asdell, 1946). The early termination of estrus apparently reflects a refractory state which sets in after estrogen activity has continued for a time, for castrates receiving continued estrogen therapy remain in estrus for similarly brief periods. In the mare, ovulation is delayed until a few hours before the end of estrous periods that may extend for 5 to 10 days or longer. This suggests a relative refractoriness of the LHrelease mechanism in this animal. Such a state of affairs approaches that in persistent estrus or in the anovulatory cycle.

B. OVARIAN STEROmS AND OVULATION

1. Estrogens

Chronic administration of estrogen to the intact animal eventually produces ovarian atrophy by suppression of gonadotrophin secretion. However, some moderate basic level of continuous estrogen secretion must be compatible with normal function of the hypophyseal-ovarian system; witness the fact that blood estrogen assays in normal women (]Markee and Berg, 1944) indicate only a 2-fold increase at midinterval above a base value of considerable magnitude.

Induction of corpus luteum formation by injected estrogen was first demonstrated by Hohlweg (1934) in prepubertal rats^ and the phenomenon has been repeatedly observed by other woi'kers (Desclin, 1935; Mazer, Israel and Aljjcrs, 1936; Westman and Jacobsolm, 1938b; Herold and Effkemann, 1938; Price and Ortiz, 1944; Cole, 1946). The fact that the effect was not obtained in rats younger than 30 to 36 days l>y Piice and Ortiz, whereas Cole observed it in the age-range of 21 to 28 days, demon ' Tlic eH'cct was later ol)1aiii(^(l witli androgens (Holilwpg, 1937; Salmon, 1938; Xathanson, Fianspen and Sweenev, 1938).

MAMMALIAN REPRODUCTIVE CYCl.E

515

B

^,

^ >

1 2

3 4 5

e

A

12 3 4 5

Fig. 8.7. Experimental modifications of the 5-day cycle in rats. Two units of the ordinate represent full vaginal estrus. Time in days on abscissa, each unit 24 hours (midnight to midnight). X, ovulation time; -p, progesterone, usually 1 to 2 mg.; e, estradiol benzoate, standard do.se 50 /xg. (From J. W. Everett, Endocrinology, 43, 393, 1948.)

strates the existence of strain differences in the age factor. This probably explains the absence of luteinization in the experience of Lane (1935) and Merckel and Nelson (1940). Hohlweg and Chamorro (1937) demonstrated the importance of the hypophysis in the response. When hypophysectomy was performed 2 days after injection of estrogen no corpora liitea developed, but hypophysectomy on the 4th day did not interfere with corpus luteum formation. The effect could be produced in 50-gm. rats with as little as 4 |U,g. estradiol benzoate. Westman and Jacobsohn (1938b) reported that transsection of the hypophyseal stalk less than 2Vt days after injection prevented the reaction, but after that time the operation did not interfere. Bradbury (1947) assayed the gonadotrophin content of hypophyses of normal and castrated immature rats (30 to 32 days old at autopsy) 2 to 5 days after injection of estrogen or other steroids. These rats were apparently too young to form corpora lutea in response to the treatment, l)ut marked interstitial-cell stimulation, indicative of LH (ICSH) activity, was observed as early as 96 hours. In the intact animals significant loss of potency occurred 72 to 96 hours after injection, in agreement with the hypophysectomy data of Hohlweg and Chamorro (1937). In castrated rats, however, there was no loss of potency, thus suggesting that some ovarian factor in addition to estrogen is essential for stimulation of the hypophysis. It is unfortunate that the study was confined to animals too young to give the full response of luteinization.

Induction of ovulation in adult animals by estrogen was first reported by Hammond, Jr., Hammond and Parkes (1942) and by Hammond, Jr. (1945) in the anestrous ewe. Whereas the s])ontaneous occurrence of occult ovulation was approximately 5 per cent, injection of stilbestrol was followed by corpus luteum formation in 4 of 11 ewes, with recovery of ova in 3. Injection of stilbestrol di-n-butyrate was followed by corpus luteum formation in 5 of 6 ewes and ova were recovered in 3. The finding was confirmed by Casida (1946) who stated that in cycling ewes ovulation can be invoked by injection of diethylstilbestrol on the 4th day of the cycle, but not at other times. In 1947 Everett reported the induction of ovulation in pregnant rats within 40 hours after injection of estradiol benzoate (as little as 2 or 3 /xg.) or implantation of estradiol crystals or pellets. The response was not obtained in animals autopsied 24 hours after treatment nor in other animals hypophysectomized at 24 hours and autopsied the following day. In other studies with adult rats it was demonstrated (Everett, 1948) that in 5-day cyclic rats the injection of estrogen at mid-diestrum will regularly induce ovulation 24 hours earlier than exi:)ected (Figs. 8.7D, 8.8F|. Persistent-estrous rats were refractory to estrogen in this respect.- Nevertheless, when such animals were made pseudopregnant by daily injection of progesterone,

"The tendency toward refractoriness of similar animals with respect to induction of estrous 1) ha\'ior had earlier been reported by Boling, Blandau, Rundlett and Young (1941).

516

PHYSIOLOGY OF GONADS

B

12 3 4

T%

^'

2 3 4 5

1 P

2 3 4 5 e v^^ / y

r-U^'f*^ 1 tA 1

12 3 4 5

Fig. 8.8. Experimental modification of the 4-day cycle in rats. Same key as in Figure 8.7. Progesterone dosage 1.5 mg. per day. Artificial 5-day cycles in D, E, and F indicated by dotted lines and numbering. (From J. W. Everett, Endocrinology, 43, 395, 1948.)

B

e NO OVULATION

j~~r~r~r

Fig. 8.9. Experiment with persistent-estrous rats. Units of ordinate and abscissa have same meaning as in Figure 8.7. A. Secjuence of "progesterone cycles." Each dose of progesterone (p) is 1.0 mg. Ovulation (x) in about 70 per cent of the cycles. B. Progesterone cycle followed by unsuccessful attempt to induce ovulation by estrogen during the second c-ycle. C . Pseudopregnancy maintained by daily iiijoctinn of 1.5 mg. ]irogosteronr. Ovulation induced by estrogen in several such cases. (From .1. \V. Everett, EiKhxTinolojiy. 43, ;5i»9, 194S.)

ovulation and corpus luteuni loiniatioii were induced by estrogen (Fig. 8.9 1.

Early attempts to induce luteinization in the guinea pig with estrogen were unsuccessful ( Dempsey, 1937; see Fig. 8.5), but iiioiv recently Lipschutz, Iglesias, Bruzzone, 11 uniercz and Penaranda (1948) have shown by the use of intrasplenic ovarian autografts

that luteinization is a reguhir feature in experiments in which estrogen is administered systcniically. Interestingly enough the implantation of estrogen jiellets in or near the ox'ariaii grafts had tlic coiitrai'y effect of pi'cvcnliiig luteinization.

it was early I'cportcd that rabbits fail to ovulate in response to estrogen injection

MAMMALIAN REPRODUCTIVE CYCLE

517

(Bachman, 1936; Mazer, Israel and Alpers, 1936 ». Hisaw (1947j inferred that this is generally true for reflex ovulators. Nevertheless, it was found by Klein and Mayer ( 1946) and Klein (1947) that when pseudol^regnant or pregnant rabbits were treated with estrogen and then mated, new ovulation resulted and new corpora lutea were formed, events that do not otherwise occur. The phenomenon was further explored by Sawyer (1949). Whereas untreated rabbits, unlike cats, do not ovulate in response to mechanical stimulation of the vagina, treatment with estrogen on the preceding 2 days results in a positive response to this stimulus. In fact, his later observations (1959) indicate that estrogen priming for a longer period (4 days) occasionally results in "spontaneous" ovulation, especially during the winter and spring.

In the anestrous cat, in the response to mechanical stimulation of the vagina, estrogen facilitates the ovulation of follicles primed with equine gonadotrophin (Sawyer and Everett, 1953).

Induction of ovulation by estrogen in primates remains to be demonstrated. It is of interest in this connection that Funnell, Keaty and Hellbaum (1951) observed in menopausal women an increased excretion of LH during estrogen therapy, in contrast to FSH excretion at other times. The general experience has been that injection of estrogen during the early part of the cycle significantly postpones the next expected ovulation and menstruation (monkey, Ball and Hartman, 1939; baboon, Gillman, 1942; human, Sturgis and ^leigs, 1942; Brown, Bradbury and Jennings, 1948). Gillman reported that a single injection of estrogen precipitates widespread atresia of vesicular follicles. Brown and Bradbury (1947) reported IH-eliminary data that in 4 of 6 women there was increased gonadotrophin excretion during the 24 hours following estrogen administration. They proposed that delay of ovulation by estrogen given early in the primate cycle may be the result of premature discharge of gonadotrophin before the Graafian follicle is competent. Sturgis and Meigs had suggested, on the contrary, that the estrogen suppresses hypophyseal function. D'Amour (1940), finding in urinary assays that the initial peak of estrogen ex

cretion preceded the peak excretion of urinary gonadotrophin, postulated that the increase of estrogen stimulates the gonadotrophin release that is responsible for ovulation. O. W. Smith (1944) proposed that not estrogen itself, but some metabolite resulting from inactivation by the liver, is responsible for LH release. This interesting hypothesis has not been substantiated.

3. Gestagens

Suppression of estrus and ovulation by functional corpora lutea, suggested by Beard (1898), was experimentally demonstrated in the guinea pig by Loeb (1911). It is now well established in several species that removal of the corpora lutea results in early resumption of estrus and ovulation (see p. 506), and that administration of progesterone suppresses these events. There is considerable evidence favoring the view that the primary effect is to selectively suppress the secretion of LH. Dempsey (1937) noted that in guinea pigs receiving daily injection of progesterone (50 /^g.) all stages of follicle development proceeded except the maturation enlargement that heralds LH release (Fig. 8.5). Astwood and Fevold (1939) and Cutuly (1941b) found similar results in rats. Essentially the same phenomenon has been noted in sheep by Dutt and Casida (1948). Bradbury (1947) reported that in immature rats the injection of progesterone at the time of estrogen injection prevented the release of gonadotrophin (LH?) which otherwise followed estrogen injection by 72 to 96 hours. In ovariectomized guinea pigs containing intrasplenic autografts, preparations in which luteinization can be induced by estrogen (see above) , the simultaneous administration of gestagens prevented this action (Lipschutz, Iglesias, Bruzzone, Humerez and Pefiaranda, 1948; Iglesias, Lipschutz and Guillermo, 1950; Mardones, Bruzzone, Iglesias and Lipschutz, 1951). Mardones and co-w^orkers also made the interesting observation that among several steroids having progestational activity, "antiluteinizing activity is not concomitant with, or subordinated to" the former function. Proportionately very large amounts of ethinyl testosterone and ethinyl-A-'^-androstenediol exhibited very little antiluteinizing activity. There is evi

518

PHYSIOLOGY OF GONADS

denceinmice (Solve,, 1939) that siippreijsion of FSH secretion may occur when as much as 1 mg. of progesterone is injected daily. Alloiteau ( 1954 ) believes that this also occurs in the rat, although Cutuly (1941b) found only slight evidence of FSH suppression when as much as 6 mg. progesterone were given daily for several weeks.

So much emphasis has been placed on the suppressing effect of progesterone that its facilitating actions were recognized only in recent years. The first indication that progesterone can promote ovulation and corpus luteum formation in mammals was encountered in a study of persistent-estrous rats (Everett, 1940a, b). Daily injection of 0.25 to 1.0 mg. caused the prompt interrujition of the state of persistent follicle and the resumption of outwardly normal cycles. Corjiora lutea were formed in approximately 70 per cent of these cycles.^ The effect was obtained not only in older rats in which persistent estrus had developed spontaneously, but also in young rats in which the condition had been induced by continuous illumination. The dose level employed is below that required to suppress cycles in normal rats (1.5 mg. daily; Phillips, 1937). Subsequently, it was found (Everett, 1943) that the daily injection could be avoided if a single "interrupting" dose was given, followed by a single injection during proestrum or early estrus of each recurrent cycle (Fig. 8.9.4). The histologic appearance of the ovaries reverted toward the normal after a succession of "i)rogesterone cycles" and, significantly, the interstitial-cell nuclei were "repaired." Attempts in normal rats to invoke o\'u]ati()n earliei' than the expected time were sticcessful in the 5-day cycle (Figs. S.7B, 8.8^). Injection of from 0.5 mg. to 2 mg. on the "third day of diestrum" regularly (4 mg. occasionally) invoked ovulation (luring the coming night (Everett, 1944a, 1948) unless the treatment was given too late in the dai/ (EA'erett and Sawyer, 1949; see discussion on ]). 526 I'egarding the diurnal rhythm and ovulation). Attempts to advance ovulation in the 4-day cycle were unsuccessful, possibly because the follicles were not competent or the animals'

Marvin (1947) described a similar rosull willi desoxycorticosterone acetate.

intrinsic estrogen levels were not elevated sufficiently early.

Ovulation induced by progesterone has been reported in several species. A direct action on the excised ovary of the toad Xeno-pns was early demonstrated by Zwarenstein (1937) but such action is apparently not found in higher vertebrates. In the domestic hen injection of progesterone can invoke ovulation several hours ahead of schedule (Fraps and Dury, 1943; Rothchild and Fraps, 1949). Pfeiffer (1950) observed new corpora lutea in 10 rhesus monkeys treated with progesterone during presumptive anovulatory cycles of the summer months. Similar attempts have been made in women (Rothchild and Koh, 1951); although there were said to be definite indications of induced ovulation, the evidence is equivocal. On the other hand, a rei~)ort (Hansel and Trimberger, 1952) states that in heifers the injection of small doses of progesterone (5 to 10 mg. ) at the beginning of estrus significantly advances ovulation time. This is in contrast with the inhibitory effect of larger doses (50 mg.) beginning before the onset of estrus (Ulberg, Christian and Casida, 1951). Even in the rabbit (Sawyer, Everett and Markee, 1950), spontaneous ovulation was noted in 4 of 10 animals after combined estrogen and progesterone injection.

From certain of the foregoing statements it may be inferred that whether suppression or stimulation follows administration of ju-ogestcrone depends critically on the time of injection, on the amount given, on the status of the ovary, and probal)ly on a l)riming action of estrogen. A significant illustration of the critical nature of the time factor in rats is given by the experiments represented in Figure 8.8r and E. If after the first injection of 1.5 mg. progesterone on the first day of diesti'uni, a second injection follows in. about 24 lioui's. the imjiending estrus and o\-ulation are retarded an additional 24 hours. However, if the second injection is given 48 hours after the first, the impending estrus and ovulation are advanced. Evidence^ of the synergistic action of estrogen and progesterone in evoking oA'ulation is given by the ex]ieriments represented in Figuiv 8.9/> and (\ Sawver (1952)

MAMMALIAN REPRODUCTIVE CYCLE

519

investigated the synergism in rabl^ts. Employing estrogen-primed animals, he found that ovulation was facilitated when progesterone was injected less than 4 hours before either mating, mechanical stimulation of the vagina, or intravenous injection of copper acetate. Inhibition was obtained when progesterone was injected 24 hours before such stimulation, thus confirming the often-cited observations of ]Makepeace, Weinstein and Friedman (1937 » and Friedman (1941) that progesterone inhiijits ovulation in rabbits.

Preovulatory secretion of gestagens now seems likely. Morphologic luteal changes in preovulatory follicles are considered in the chapter on the ovary. A variety of evidence in primates indicates that progestational clianges in the endometrium begin before ovulation (Bartelmez, Corner and Hartman, 1951). Several workers have reported tiie excretion of small amounts of pregnanediol during the follicular phase of the human cycle (Wilson, Randall and Osterberg, 1939; Smith, Smith and Schiller, 1943; Davis and Fugo, 1948). Determination of plasma progesterone in women by the Hooker-Forbes test indicates the presence of significant amounts a day or two before a major rise in basal body temperature (Forbes, 1950). In monkeys a small quantity (ca. 0.5 to 1.0 ixg. per ml.) was detected l)etween the 4th and 9th days, rising in the 10- to 15-day period to concentrations of 2 to 6 jxg. per ml. (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). In both species a transient decline seems to intervene before the marked rise to still higher concentrations during the luteal phase. In the rat, Constantinides (1947) studied the stromal nuclei of the endometrium at different stages of the cycle and found that by the Hooker-Forbes criteria there is evidence of progesterone secretion during proestrum. Astwood (1939) on the basis of water content of rat uteri concluded that progesterone secretion begins with proestrum. In the rabl)it, Forbes (1953) assayed peripheral blood at various intervals after mating or gonadotrophin injection. Although no progesterone was detectable in controls, significant amounts appeared an average of 97 minutes after mating and 66 minutes after gonado

trophin injection. As much as 2.5 /xg. ])er ml. was found during the first 8 to 10 hours, although marked fluctuations were noted from time to time in the blood of individual animals. Verly (1951) reported that soon after mating the urine of rabbits contains significant amounts of pregnanediol.

It has become customary to state that the gestagen that appears during the follicular phase of the cycle is probably formed by the maturing follicle itself. Indeed, assays of fluid from Graafian follicles and cysts have indicated the presence of the hormone (Duyvene de Wit, 1942; Hooker and Forbes, 1947; Edgar, 1952, 1953). However, if it is to take part in the release of ovulating hormone gestagen must be secreted earlier than preovulatory maturation. For this also there is some evidence. Two reports cited above indicate that in monkeys, at least, there is a detectable amount present in the blood during the early follicular phase. The known fact that a waning corpus luteum favors the experimental induction of estrus and/or ovulation in sheep and cattle (Hammond, Jr., 1945; Robinson, 1951 ; Alarden, 1952) is suggestive. Although Hammond, Jr., Hammond and Parkes (1942) tested this possibility by progesterone sul)stitution with negative results, the amount given may have been too small, as Robinson suggested. A waning corpus luteum in the rat favors ovulation, as disclosed in persistent-estrous animals in which pseudopregnancy had been induced (Everett, 1939). Each of three pseudopregnancies was followed b}' a short cycle before the animals returned to persistent estrus.

In the course of studies growing out of this observation evidence was advanced (Everett, 1945) which indicated that corpora lutea of the normal rat are not wholly inactive during the short cycle. Transient depletion of cholesterol was observed in such corpora lutea during the proestrum that followed their formation. This implies a transient increase of luteotrophin secretion. Significantly this occurs before the release of LH. It is this writer's opinion that gestagen from such sources is not essential for the induction of ovulation but that it does facilitate the action of estrogen.

520

PHYSIOLOGY OF GONADS

C. ROLE OF THE NERVOUS SYSTEM IN OVULATION

Historically, the fact of neural control of reflex ovulation has been recognized in the ral)bit for many years. The comparable role of the nervous system in spontaneous ovulation, on the other hand, has more recently become apparent. It now seems justifiable and useful to postulate in the hypothalamus of reflex ovulators and spontaneous ovulators alike the existence of a mechanism that is peculiarly concerned with release of ovulating hormone. Whether it consists in a discrete anatomic entity is immaterial for the present.

The suggestion has been made that the outstanding difference between reflex and sjiontaneous ovulation may be in the kinds of afferent impulses that most readily excite the hypothalamus (Sawyer, Everett and Markee, 1949). The difference is not absolute, for spontaneous ovulation has been induced in rabbits by estrogen-progesterone injection (see p. 519) and reflex ovulation has been demonstrated under special circumstances in rats (Dempsey and Searles, 1943; Everett, 1952a) and cattle (Marion, Smith, Wiley and Barrett, 1950). The random distribution of reflex ovulators and spontaneous ovulators among mammalian orders becomes more understandable if one assumes that dual controls are widely represented and that special adaptations favor one or the other in given species.

The ovulation reflex in rabbits is apparently initiated by afferent impulses of multiple origin, among them not only impulses from the genitalia, but also propriocejUive impulses from muscles that are utilized in coitus. Brooks (1937, 1938) found that, although the sacral segments of the spinal cord and the abdominal sympathetic chains could be eliminated without jM'eventing ovulation, the luml)ai' cord must remain. Only by paralysis of the lower half of the body so that the female could not take pai't in coitus was ovulation pi'cvcntfMl. The neocortex could be removed, together with the olfactory bulbs, labyrinths, auditory apparatus and eyes without impairing the ovulation response. Even after complete decortication, ovulation followecl coitus in 1 out of 3 j'abbits. It must l)e admitted, how

ever, that, although various parts of the nervous system may thus be eliminated without changing the end result, some of them may normally play a considerable role. With little cjuestion, direct stimuli from the genitalia play a part in the natural reflex. Under certain experimental conditions detectable electrical activity in the rabbit rhinencephalon is associated with the induction of ovulation (Sawyer, 1955). Electrical stimulation of the amygdala will induce ovulation in rabbits and cats (Koikegami, Yamada and Usei, 1954; Shealy and Peele, 1957 ) .

In rats, Davis (1939) found that removal of the neocortex had no effect on the estrous cycle and ovulation. Removal of portions or of the entire sympathetic chains of rats likewise did not interfere with ovulation (Bacq, 1932). Bunn and Everett (1957) reported ovulation in constantestrous rats after electrical stimulation of the amygdala.

The importance of the dorsal thalamus is unknown. The reticular activating system has been implicated as a component of the ovulation mechanism (Sawyer, 1958), but the manner of its contribution is not clear. There is little cjuestion, on the other hand, of the indispensability of the hypothalamus and its neurovascular connection to the adenoliyi:)ophysis through the median eminence and the hypophyseal portal veins.

Although the observation by ]\Iarshall and Verney (1936) that ovulation can be induced by passing an electric current through the heads of estrous rabbits hardl}^ limited the effect to the hypothalamus itself, it was later shown that more localized electrical stimuli api)lied to certain hypothalamic regions are consistently effective (preoptic area, Haterius, 1937; Christian. 1956; posterior hypothalamus or tuber cinereum, Harris, 1937, 19481); tuber cinereum or adjacent hypothalamic areas, Markee, Sawyer anirHollinshead. 1946; medial hypothalamus fi'om ])i-eo])tic area to mammillai'V bodies. Kui'otsiu Kurachi and Ban, 195o"; Kuiotsu, Kurachi, Tabaya>hi and Ban, 1952).

Ahliougli liypotliahimic lesions. l)oth natural and expeiimental. hax'c frecjuently been reported to interfere with normal

MAMMALIAN REPRODUCTIVE CYCLE

sex function (.see Harris. 1948a, 1955, for references), the majority of these reports do not api^ly to the question at issue — control of ovulation. When ovarian atrophy occurs, as it frequently did in these cases, it reflects a profound depression of gonadotroi)hin secretion and absence of competent follicles. However, Dey, Fisher, Berry and Ranson (1940) and Dey (1941, 1943) 'found in guinea pigs that gross bilateral electrolytic lesions placed in the rostral hypothalamus resulted in persistent follicles with continuous estrogen secretion. Similar results were obtained in rats by Hillarp (1949) when small bilateral electrolytic lesions were placed in the anterior hypothalamic area near the paraventricular nuclei or between this region and the median eminence. Greer (1953) reported continuous estrus in rats after placing certain small lesions in the ventromedial nucleus, provided they were bilateral. There was no correlation with obesity. There are at least four significant points in common among these several ablation experiments. ( 1 ) The effective lesions were rost rally placed and either were limited to or included the medial group of nuclei. (2) The tuber cinereum, median eminence, and stalk connection to the hypophysis were intact. (3) Although development of competent follicles was not evidently impaired, estrogen secretion became continuous instead of cyclic. (4) The proper impetus for release of ovulating hormone from the hypophysis was absent. It would be most instructive to learn whether ovulation can be invoked in such animals by reflex stimulation or by direct electrical stimulation of the tuber. AUoiteau and Courvoisier (1953) reported that rats in constant estrus as a result of hypothalamic lesions did not undergo pseudopregnancy after stimulation of the cervix uteri. This observation, confirmed by Greer (1953), could be construed as indirect evidence of failure of reflex ovulation, for Greer regularly obtained pseudopregnancy by cervical stimulation, once corpora lutea had been formed by other means.

Other findings by Greer are important because of their bearing on the location and character of a presumptive ovulation center. Althougli the onset of persistent estrus after

making the lesions was sometimes almost immediate (following a brief anestrous interval), in other cases it was preceded by several apparently normal cycles. In any event, once the condition had become established, the daily injection of small amounts of progesterone brought about the recurrence of cycles and corpus luteum formation. In about half of the cases these cycles continued for awhile after withdrawal of treatment, whereas in the remainder there was a prompt return to persistent estrus. Essentially the same results were reported by AUoiteau (1954), and the observations suggest that the areas involved in such lesions may be of only secondary importance.

The use of radioactive phosphorus for estimating energy exchange in tissues affords a different approach to the problem of neural control of ovulation (Borell, Westman and Orstrom, 1947, 1948). This method has the virtue that the experimental subject remains undamaged until injection of P'*compounds 30 minutes before the end of the experiment. In rabbits there is a marked increase in phosphorus turn-over in the tuber cinereum within 2 minutes post coitum, and continuing for about an hour thereafter (Table 8.1). The adenohypoi)hysis shows increasing activity during the first 10 minutes which reaches a peak at about 1 hour and then regresses somewhat, although it remains relatively high for 24 hours. Response of the ovary to gonadotroi:)hin release is marked by a rapid rise during the second half-hour and another pronounced increase near the time of ovulation. In rats, at various stages of the estrous cycle, phosphorus exchange in both tuber cinereum and adenohypophysis is maximal during proestrum. In the ovary high values were reported during diestrum and proestrum, somewhat lower values during estrus and metestrum.

Possibly correlated with the above information is the observation (Gitsch, 1952b) that in rats the acetylcholine (ACh) content of the tuberal region becomes elevated during proestrum and estrus. It is said that ACh synthesis requires high energy phosphate (see Bain, 1952). Further investigation by Gitsch (1952a) and Gitscl: and Reitinger (1953) revealed that ACh

TABLE 8.1 Sequence of events in rabbit ovulation

Time Post Coitum Central Nervous System

Hypophysis

Ovary

Circulating Blood

<30 sec.

<2 mill.

10 mill. 30 mill.

60 mill.

75-90 mill.

13^-2 hrs. 3-5 hrs.

6-7 hrs.

7-8 hrs. 9-11 hrs.

Barbiturate-sensitive and atropinesensitive mechanisms^

t Phosphorylation in tuber cinereum^

t Phosphorylation

in tuber ciner reumt Phosphoryhition

in tuber cine reum^

t Phosphorylation in tuber cinereum

t Phosphoryhition^

Release of LH ca. 20 per cent. 6 Hypophysectomy prevents ovulation^' 12

Release of LH nowsufficient for ovulation."' 12 Phosphorylation at peak^

I Phosphor\iatioii

i Animal may be bled and transfused without prevent ! ing ovulationi2

folliculi. in egg

f Liquor Tetrad.' nuclei*

Cholesterol depletion in interstitial gland. ^ Egg nucleus migrates, membrane dissolves.** • " Prominent corona*

Liquor folliculi increasingly viscous*

First polar hotly'*

Marked .swelling of follicles. Thecal hypertrophy,! ■ i° hyperemia

OvuL.^Tion." t Phosphorylation2

Bleeding and transfusion now prevent ovulationi2

Progesterone detectable ^

Increased estrogen (endometrial hj-peremia)'

' Asdell, 1946.

2 Borell, Westman and Orstrom, 1947.

3 Claesson and Hillarp, 1947a. " Fee and Parkes, 1929.

'^ Forbes, 1953.

« Hill, 1934.

^ Sawyer, Markee and Hollinshcad, 1947.

Pincus and Enzmann, 1935.

^ Sawyer and associates, 1947, 1949, 1950.

1" Walton and Hammond, 192S.

" Waterman, 1943.

'2 Weslmaii and .lacohsohn, 1936.

522

MAMMALIAN REPRODUCTIVE CYCLE

523

in the rat hypothalamus is increased also by administration of estrogen or by castration, conditions that similarly increase lihosphorus exchange (Borell and Westman, 1949). The ACh content is depressed during pregnancy or when the rat has been injected with progesterone. It is also lowered by Pentothal anesthesia, a matter of interest in relation to the fact that the barbiturates suppress ovulation (see p. 526).

The location and measurement of activity in discrete nuclei and pathways are largely in the future, although a beginning has been made in the rabbit, cat, rat, and mouse. Sawyer (1955) found in rabbits, after the combined administration of pentobarbital intravenously and histamine by way of the 3rd ventricle, that there was associated with induction of ovulation a characteristic change in intrinsic electrical activity of the rhinencephalon, extending into the preoptic area. If the olfactory tracts were cut, however, this activity could not be elicited and ovulation failed. According to Porter, Cavanaugh and Sawyer (1954), vaginal stimulation of estrous cats caused altered electrical activity in two hypothalamic regions:

(1 ) in the lateral hypothalamic area at the anterior tuberal level during stimulation and for 15 to 45 seconds afterward; and

(2) in the anterior hypothalamic area near the medial forebrain bundle, where response was delayed as much as 5 n:iinutes after stimulation. According to a i)reliminary account (Critchlow and Sawyer, 1955) in curarized, proestrous rats, there were i)eriods lasting approximately 20 minutes in the midafternoon, during which altered electrical activity appeared differentially in the preoptic area or anterior hypothalamus.

Another approach to localization has been described by Hertl (1952, 1955). On the pro])Osition that increased function of particular cells is reflected by increased volume of their nuclei, cell nuclear volumes were measured in hypothalamic nuclei of female mice at different stages of the estrous cycle. During proestrum and estrus there was said to be a functional edema in hypothalamic nucleus 20 of Griinthal (possibly the pars posterior of the ventromedial nucleus of Krieg) and to lesser extent in nucleus 16 (Nucl. arcuatus).

1. The Ilypophi/seal Portal Veins and the Chemotransmitter Hypothesis

As noted elsewhere, hypothalamic control of the jiars distalis is probably mediated by the hypophyseal portal circulation. Evidence for this has been especially convincing with respect to control of ovulation, although indications are that other phases of the cycle are also regulated by this means. Pertinent data from numerous transplantation and stalk-section experiments may be summarized by the following statement. Aside from a questionable grafting experiment (2 rats) reported by May (1937), in no case has ovulation or luteinization been reported in the absence of vascular linkage of the pars distalis with the median eminence; on the other hand, ovulatory cycles have often been cjuickly restored when the gland has been revascularized by the portal vessels (see especially, Harris, 1950a; Harris and Jacobsohn, 1952; Nikitovitch-Winer and Everett, 1957, 1958b).

Although the importance of local vasomotor regulation in the stalk vessels remains to be evaluated (Green, 1951), there is extensive support for the hypothesis that ovulatory release of gonadotrophin is invoked by a chemotransmitter (Harris, 1948a, 1955). If one accei)ts the prevailing opinion that nerve fibers entering the pars distalis are too few to account for its secretomotor control and that the flow of blood in the hyi^ophyseal portal vessels is toward the gland (Wislocki and King, 1936; Green, 1947; Green and Harris, 1947, 1949; Barrnett and Greep, 1951 ; Landsmeer, 1951 ; ]McConnell, 1953; Xuereb, Prichard and Daniel, 1954; Worthington, 1955), the plausibility of the chemotransmitter hypothesis becomes inescapable.^

Evidence that the transmitter may l)e

^ For a dissenting view, see Zuckerman (1952). Reference should also be made to the hypothesis formulated by Spatz (1951) and associates (see Nowakowski, 1950, 1952). They postulated that a descending pathway in the spinal cord is the connecting link between hypothalamus and ovaries. With respect to ovulation, this is clearly denied by the fact that local stimulation of the hypotlialamus provokes ovulation in rabbits in which the thoracic spinal cord has been transsected (Christian, Markee and Markee, 1955).

524

PHYSIOLOGY OF GONADS

adrenergic was presented by Markee, Sawyer and Hollinshead (1948), who provoked ovulation in rabbits by instilling epinephrine directly into the pars distalis. Detailed experiments supporting this w^ere fully reviewed by Markee, Everett and Sawyer (1952). In discussion following that paper, Sawyer reported the induction of ovulation in rabbits by the injection into the third ventricle of either epinephrine or norepinephrine, and suggested that the latter is "more closely related to the natural mediator than is epinephrine." Donovan and Harris (1956), from studies in which the rabbit hypophysis was slowdy infused in situ with solutions of epinephrine or norepinephrine, concluded that neither substance is the agent in question, and that the positive results of Markee, Sawyer and Hollinshead (1948) were the effects of low pH and not of the drugs per se. Proof of the negative is elusive, however, and one must note that Donovan and Harris did not meet the conditions of timing and drug concentration that obtained in the earlier work.

Intravenous injection of Dibenamine or its congener, SKF-501,-^ will usually prevent ovulation in rabl)its when injection is completed within 1 minute after coitus (Sawyer and associates, 1947-50). On the other hand, when injection is delayed until 3 minutes or later, ovulation is unaffected. The nonadrenergic hydrolysis product of Dibenamine, 2-dibenzylaminoethanol, does not have the blocking action, although its central excitatory powers are much like those of the parent substance. The failure of blockade by Dibenamine, if injection is withheld for 3 minutes, demonstrates that the drug does not interfere with the actual discharge of ovulating hormone into the l)lood stream, for that process recjuires about an hour (Fee and Parkes, 1929; Westman and Jacobsohn, 1936). The Dibenamine-sensitive mechanism thus serves as a trigger, the gland being adequately stimulated within 1 01' 2 minutes post coitum. This estin^ate is in remarkal)le agreement with the earlier mentioned obscMA'ations on

•'■' Dibenamine is iV,iV-dibenzyl-/:i-cliloioetliy lamine. SKF-501 is A'-(9-fluorenyl)-.V-ethyl-/i-chlor()ethylamine hydrochlorifle. Banthine is /i-dictliylaminuc'tliyl-.\anthene-9-cai'l)Oxvlak' niclliohroiniilc

l)hosphorus exchange in the tuber cinereum and hypoi)hysis (p. 521, and Table 8.1 1.

A mechanism that is subject to blockade by atropine or Banthine^ evidently precedes the Dibenamine-sensitive process — temjDorally if not anatomically. To accomplish blockade in rabbits, these anticholinergic drugs must be injected intravenously within about 30 seconds after coitus (Sawyer and associates, 1949-1951). It should be recalled that Foster, Haney and Hisaw (1934) reported failure of ovulation in several rabbits treated with small amounts of atropine before mating. ]\Iakepeace (1938), however, was unable to confirm the effect with somewhat larger doses and the former observation was forgotten.

A seemingly crucial experiment devised by Sawyer gives conclusive evidence that the atropine-sensitive process is antecedent to the Dibenamine-sensitive one. It was based on two facts: (1) intravenous injection of nearly lethal doses of epinephrine does not induce ovulation in estrous rabbits, and (2) atropine protects rabbits against fatal pulmonary edema after injection of large amounts of epinephrine. In rabbits protected by atropine in dosage that was also sufficient to block the ovulation reflex, the injection of twice-lethal doses of epinephrine caused ovulation or significant degrees of follicle maturation in 5 of 7 cases. These effects were not found in rabbits protected by Dibenamine. Supporting evidence was adduced by Christian (1956i who found that atropine would not prevent ovulation in response to electi'ical stimulation of the medial preoptic area or adjacent parts of the hypothalanuis, whereas in a significant number of such rabliits ovulation was blocked by SKF-501.

Extension of the blocking experiments to the rat, as an example of a spontaneous ovulator, disclosed that in this species also ovulation can be blocked by Dibenamine, SKF501, atropine, and Banthine, when the injections ai'c appropriately timed with respect to the stage- of the cycle and time of day (Sawyer, Everett and Markee, 1949; Everett, Sawyer and Markee, 1949; Everett and Sawyer, 1949, 1950, 1953: see \). 526). Furthermore, blockade of ])oth estrogenimhiccd and pi'ogcstci-oiic-induced ovuhition

MAMMALIAN REPRODUCTIVE CYCLE

525

was aceomi)lished with cither Dibonamine or atropine. Neither agent, however, prevented ovulation after injection of sheep liypophyseal LH. A report by Hansel and Trimberger (1951) stated that in cattle a significant delay of ovulation (as great as 72 hours) followed atropine administration. In control experiments the simultaneous injection of atropine and human chorionic gonadotrophin was followed by ovulation slightly earlier than the normally expected time. Treatments were begun 1 to 5 hours after the onset of estrus. This work was confirmed and extended by Hough, Beardon and Hansel (1955). In the hen, blockade of ovulation, normal or induced by progesterone, has been reported after administration of Dibenamine, Dibenzyline, SKF-501 or atropine (Zarrow and Bastian, 1953; van Tienhoven, 1955). According to van Tienhoven, the drugs did not interfere with the ovulating action of extrinsic gonadotrophin.

It is important that the same drugs will block ovulation in lioth rabbits and rats (Table 8.2) . Of ec^ual significance is the fact that several agents that are ineffective in rabbits are also ineffective in rats (notably 2-dibenzylaminoethanol, the imidazoline adrenolytic drugs, and the ganglion blocking agents). These considerations are interjireted to mean that spontaneous ovulation is invoked by neurohumoral mechanisms that are very like those in the reflex ovulation of rabbits.

The suggestion that the l)locking effects might result from nonspecific stress, causing the hypophysis to be so actively secreting ACTH that gonadotroiihin secretion is interfered with (Dordoni and Timiras, 1952), is clearly denied by several facts. ( 1 ) In the rabbit studies, none of the various agents prevented ovulation when injected more than a minute post coitum. (2) In one study (Sawyer, Markee and Everett, 1950b) ovulation was actually induced by the intravenous injection of "lethal" doses of epinephrine when the animals WTre protected by atropine. (3) In rats ovulation is unaffected by massive intravenous doses of either the imidazoline drugs or 2-dibenzylamionethanol in amounts known to be stressing (Sawyer and Parkerson, 1953).

TABLE 8.2 Pharmacologic Agents and Blockade of Ovulation

Antiadrenergics

/3-Haloalkylamines

Dibenamine

SKF-501

Dibenzyline

ImidazoHnes

Priscoline

Regitine

Yohimbine

Anticholinergics

Atropine

Banthine

Antihistaminics

Neo-antergan

Ganglion blockers

Tetraethvlammonium. .

SC-1950/

Barbiturates

Nembutal

Dial

Ipral

Amvtal

Barbital

Phenobarl)ital

Prominal

Others

Morphine

Procaine, locally near tuber

Procaine, systemically .

Chlorpromazine

Reserpine

Ether

2,4-Dimtrophenol

Rabbit

Rat

Cow

Bi

01

0^

01

■?4. 5

Bi

BI

B6

Bi

01

Bi

Bi Bi Bi Bi B' Bi 8

B9

BIO

Bii

01

B12 B13 B14 B16

B3

I Sawyer and associates, 1947-1951; Everett and associates, 1949-1950; Christian, 1956; and see present text.

^ van Tienhoven, 1955; van Tienhoven, Nalbandov and Norton, 1954.

3 Zarrow and Bastian, 1953.

•• Fugo and Gross, 1942.

5 Sulman and Black, 1945.

^Hansel and Triml)erger, 1951; Hough, Beardon and Hansel, 1955.

' Fraps and Case, 1953.

Doring and Goz, 1952.

» Westman, 1947.

1" Barraclough and Sawyer, 1955.

II Westman and Jacol)8ohn, 1942. 1- Barraclough, 1956.

13 Barraclough, 1955.

" Unpublished. Temporary, during deep anesthesia.

1^ Unpublished. EDso : 25 mg. per kg. subcutaneously.

Key: B = Blockade.

= No blockade.

1 = Ovulation induced by the drug.

526

PHYSIOLOGY OF GONADS

Nor is it influenced by severe trauma, heat, cold, or formalin injection coincident with the known "critical period" (Everett, unpublished).

2. Central Depressants and Ovulation

Reported evidence of a blocking action of barbiturates on ovulation traces back to experiments by Westman (1947) who injected female rats with Prominal twice daily for 3 weeks. Approximately 30 per cent of these rats experienced prolonged vaginal estrus and had ovaries containing only follicles at the end of the experiment. Almost identical results were reported by Doring and Goz (1952) when rats were treated daily with phenobarbital. The agreement is not unexpected in view of the fact that in the body Prominal is quickly demethylated to phenobarbital. It was shown by Everett and Sawyer (1950) that when administration of barbiturates to rats is critically timed with respect to stage of the cycle and the time of day, blockade of ovulation can be accomplished at will in shortterm experiments (Fig. 8.10). Chronic administration introduces considerable uncertainty for reasons that are not yet clear (Everett, 1952b). In the rabbit, the rapid intravenous injection of pentobarbital or Pentothal, within as short a time as 12 seconds after coitus, generally failed to block ovulation (Sawyer, Everett and Markee, 1950) . However, it was later shown that barbiturate anesthesia will prevent the ovulation that is otherwise caused in the estrogen-primed rabbit by mechanical stimulation of the vagina, the anesthesia t)eing induced in advance of the stimulation (unjuiblished i.

Other central dcpi-es^ants reported to block ovulation in the rat are morj^hine, reserj^ine, chlorpromazine (Barraclough and Sawyer, 1955; Barraclough, 1955, 1956), and even meprobamate acting synergistically with an anticholinergic drug (Gitscli, 1958). Special interest attaches to the mori)hine work, in that anicnoi'i lica and sterility often a('C()in|)any moiphinc addiction in the human female. Related studies (Sawyer, Critchlow and Barraclough, 1955), in which recordings were made of electi'ical acti\ity in various regions in the bi-ain.

demonstrated in rats that morphine acts nmch like the barbiturates in depressing activity in the reticular activating system. The effect was also shown by atropine, in doses that would block ovulation. The inference is that all three agents block by striking at the same central elements of the LH-release apparatus.

An interesting peculiarity of domestic hens with respect to barbiturates was encountered by Fraps and Case (1953), who noted that pentobarbital induces ovulation jirematurely, and that pentobarbital and progesterone supplement each other in this capacity. Although these developments may represent pharmacologic curiosities limited to the bird, the possibility should be seriously considered that similar effects may occur in other animals. In fact, pentobarbital in rabbits facilitates the release of hypophyseal gonadotrophin in response to intraventricular injection of histamine, seemingly by an effect in the rhinencephalon (Sawyer^ 1955).

3. The Central Xervous System as a Timing Mechanism for Oi'ulation

In the rat and the hen and probably many other species the pro-ovulatory excitation of the hypophysis is dejiendent in large measure on time of day.

In the rat, the blocking agents have served to delimit a critical period on the day of proestrum, before which ovulation can be blocked and after which it will occur in sjiite of injection of the blocking agent. Under controlled illumination for 14 hours daily, this critical period extends from about 2 P.M. to 4 P.M. Administration of either atroi^ine or i^entobarbital at 2 p.m. consistently blocks ovulation (Fig. 8.10), whereas injections later in the period are progressively less eff"ecti\-e ( l']\-erett and Sawyer, 1950, 1953; Everett, 1956b). Such l)redictability of the hour of pituitary activation is, in itself, evidence of a relationship between this event and diunial physiologic I'liythnis.

Furthei' e\-i(lence is seen in the seciuelae of pentobarbital injection (Fig. 8.11). Repetition at 2 P.M. on successive days results in a follicular cycle and prolonged vaginal estrus with eventual atresia of all

MAMMALIAN REPRODUCTIVE CYCLE <- — 24hr.— >

527

DAY NIGHT

Fig. 8.10. Deinoni^lration of 24-lioiir penodu-ity in the luteinizing hormone-release apparatus of female rats (Vanderbilt strain, 4-day cycle, controlled lighting: 14 hours per day). Schematic representations of the normal cycle (A) and of characteristic results of different regimes of Nembutal treatment (B to F). Vaginal stages indicated by Roman numerals over each time scale; symbols above these show the corresponding follicle and corpus luteum stages. The device marked l prolonged the functional life of the coi'pus luteuni in nonpregnant women. Lyon rejjorted such prolongation using lactogen alone. The Squibl) lactogen was also used by Moore and Nalbandov (1955) in prolonging the luteal phase of the cycle in the ewe. As in the human experiments, howe\-er. one would like to know whether lactogen is capable of initial stimul.'iiioii of secretory activity of corpora lutea and of maintaining their function in the ab

~~MAMMALIAN REPRODUCTIVE CYCLE~~

~~531~~

~~sence of the hypophysis. There is no evidence for or against the lactogenic liormone in this capacity, except in rats.~~

Estrogens have direct hiteotrophic action in the rabbit (Robson, 1937, 1938, 1947). The effect does not depend on the hypophysis and has been produced by impLantation of estrogen crystals within corpora liitea (Hammond, Jr., and Robson, 1951; Hammond, Jr., 1952). Westman (1934) had earlier shown that operative reduction of ovarian stroma in pseudopregnant rabbits results in corpus luteum regression and that this can be prevented by administration of estrogen. Corpora lutea induced by gonadotrophin injection or by mating, as the case may be, require the presence of the hypophysis for their continued function ( Smith and White, 1931; Westman and Jacobsohn, 1936). Theoretically, then, in rabbits the hypophysis liberates FSH and LH which act on the interstitial tissue to cause estrogen secretion. This in turn stimulates the corpora lutea to secrete progesterone.

The effect of estrogen on the corpora lutea of rats is largely indirect and requires the presence of the hypophysis. Massive dosage with estrogen beginning soon after ovulation results in the enlargement of the corpora lutea and the production of sufficient amounts of progesterone to mucify the vaginal mucosa (Selye, Collip and Thomson, 1935; Wolfe, 1935; Desclin, 1935; Merckel and Nelson, 1940). In fact, a single injection of 50 /Ag. estradiol bcnzoate on the day after ovulation is sufficient to cause pseudopregnancy. These effects are now judged to be the result of induced liberation of hypophyseal luteotrophin. vSimilar effects have been reported after administration of androgens (McKeown and Zuckerman, 1937; Wolfe and Hamilton, 1937; Freed, Greenhill and Soskin, 1938; Laqueur and Fluhmann, 1942).

Desclin (1949b) stated that in hypophysectomized rats the administration of estrogen augments the hiteotrophic action of lactogen, producing functional corpora lutea in the presence of subthreshold doses of the latter hormone. A physiologic synergism of the two substances has thus been indicated. Mayer (1951) suggested that this may explain the stimulation of corpora lutea of lactation which follows estrogen treat

ment in this species. Greep and Chester Jones (1950) postulated that estrogen favors corpus luteum function in the rat by causing the luteal cells to produce cholesterol as a precursor of progesterone. Their actual data, however, indicate that the increase of visible cholesterol after estrogen treatment was confined to the interstitial tissue.

Factors responsible for cholesterol storage and mobilization in corpora lutea of the rat were analyzed by Everett (1947). In hypophysectomized rats in which corpora lutea were maintained by lactogen the injection of pituitary LH induced the storage of cholesterol, but this effect did not occur in hypophysectomized rats in the absence of lactogen. It could be induced during pregnancy or pseudopregnancy by estrogen if the hypophysis remained in place. Addition of an excess of lactogen prevented cholesterol storage. Lactogen thus tends to deplete cholesterol content of rat luteal tissue as ACTH tends to deplete adrenocortical cholesterol.

2. XonfunctionaV' Corpora Lutea

In the short cycles of the rat, mouse, hamster, and so on, the corpora lutea are commonly said to be nonfunctional. The meaning of this statement, of course, is that they are incapable of supporting a decidual reaction (Long and Evans, 1922), or of lireventing ovulation. They need not be totally inactive, however, to fail to cause these manifestations. Whereas daily injection of 1.5 mg. or more of progesterone into intact female rats will simulate pseudopregnancy and indefinitely delay ovulation (Selye, Browne and Collii^, 1936; Phillips, 1937), smaller amounts of 1.0 rag. or less are compatible with the short cycle (Lahr and Riddle, 1936; Phillips, 1937; Everett, 1940a, b; and unpublished). In the absence of estrogen in castrated females, daily injection of as little as 0.25 rag. progesterone will support deciduomata (Velardo and Hisaw, 1951). Very small amounts of estrogen augment this action of progesterone (Rothchild, Meyer and Spielman, 1940) but somewhat larger amounts are inhibitory unless the progesterone dose is proportionately increased (Velardo and Hisaw, 1951). In the intact animal the progestational effects of

~~53^~~

~~PHYSIOLOGY OF GONADS~~

~~less than 1.0 mg. progesterone would be inhibited by the periodic rise in estrogen secretion.~~

Evidence that some rats, but not all, actually experience low-grade corpus luteum activity during the short cycle was furnished by Everett (1945). In the comparison of ovaries from females of two strains of rats, it was noted in the supposedly normal Vanderbilt strain that on the two days immediately following ovulation the corpora lutea of the next youngest generation contained a great quantity of cholesterol, giving a strong Schultz reaction. By contrast, comparable corpora lutea of the DA strain were usually free of visible lipid in Sudan preparations or the Schultz test. Administration of small amounts of lactogen (luteotrophin I during the cycle preceding the current one, amounts inadequate to cause l)seudopregnancy, resulted in the rich deposition of cholesterol in these otherwise lipidfree corpora lutea. The conclusion was reached that the corpora lutea of the Vanderbilt rat must be slightly active during the short cycle and those of the DA rat less so, if at all. This would easily explain the relative indifference of the Vanderbilt rat to continuous light and the ease with which persistent cstrus could be induced in the DA rat by such treatment (Everett, 1942a, b). In fact, the low dosages of lactogen mentioned substituted for progesterone treatment in maintaining regular cycles in persistent-estrous rats of the DA strain (Everett, 1944b). Significantly, the treatment was effective in only those animals in which a set of corpora lutea had been induced by other means at the beginning of the experiment.

To be correlated with the above indications of low-grade function during the short cycle, is the finding that corpora lutea of the Vanderl)ilt rat retain full responsiveness to luteotrophin throughout most of the diestrous interval (Nikitovitch-Winer and Everett, 1958a). Responsiveness diminishes near the onset of i:)roestrum. Once the rat has entered proestrum these older corpora lutea are not capable of sustained function. The loss is not a function of time per se, but of stage of the cycle.

~~.\. I'.^KTI)OPRE(;\.\XrY~~

~~The terms coi-pu,^ hitcuni of ()^•ulation and corpus luteum of pscudojji'cgnancy are com~~

monly u.sed to differentiate the luteal bodies occurring during the normal cycles from those found during some unusually long period of luteal activity. However, the terms deny the fundamental similarity of the luteal phase in the cycles of such animals as the guinea pig and the luteal phase induced by sterile mating or its equivalent in animals like the rat. In the unmated bitch the spontaneous luteal phase of the cycle is commonly called pseudopregnancy, yet it is equally common to say that the guinea pig does not experience pseudopregnancy. The truth is that the luteal phase of the canine cycle is simply longer than the luteal phase in the guinea pig and may be marked by a period of lactation near its close. In the present discussion, the expression pseudopregnancy will be equivalent to saying the luteal phase of the infertile cycle. Under experimental conditions it will refer to any period of sustained luteal function similar to that of the normal progestational state. Wherever appropriate, the distinction will be made between a pseudopregnancy that is spontaneous and one that is induced.

In most of the familiar animals that ovulate spontanously corpus luteum function also begins spontaneously and continues for at least several days after ovulation. With respect to the rabbit, cat, and ferret, it is often said that pseudopregnancy is invoked by sterile copulation, whereas strictly speaking it is only ovulation and corpus luteum formation which are invoked. The pseudopregnancy then follows automatically. This interpretation seems appropriate, inasmuch as in all three species the formation of corpora lutea by l)rief treatment with hypophyseal or chorionic gonadotrophin is followed by long periods of progesterone secretion which can hardly be the direct effect of the injected substances (Hill and Parkes, 1930a, b; Foster and Hisaw, 1935; van Dyke and Li, 1938). Quite different is the pseudopregnancy of the rat, mouse, and hauistei', in \\liich progestational activity is invoked by stinudation of the cervix uteri, l^verett (1952a) described the ex])eriiuental dissociation in rats of the o^•^llati()n and luteotr()])hic mechanisms. respectively. When o\-uhition is blocked (by pentobarbital) in the cycle dui'ing which controlled uiatinu occurs, pseudopi-egnancy begins

~~MAMMALIAN REPRODUCTIVE CYCLE~~

~~533~~

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Fig. 8.12. Experimental dissociation in rats of the ovulation mechanism and that causing pseudopregnancy. A. Control cycles for comparison with B and C . Points on base line represent diestrum, on ascending lines proestrum, on highest level full vaginal estrum. X, ovulation. B. Blockade with Nembutal {'NB) on day of proestrum and following day (see Fig. 8.11E'). Ovulation during third night. C. Same basic procedure as B, but with copulation during first night (M). Ovulation usually failing in this cycle (contrast with B) . Corpora lutea formed after spontaneous ovulation in second cycle regularly become functional without further stimulation : the wavy line represents pseudopregnancy. The early copulation has introduced some change in the animal such that this pseudopregnancy "spontaneously" follows ovulation as in the standard mammalian cycle. (From J. W. Everett, Ciba Foundation Collofiuia Endocrinol.. 4, 172. 1952.)

"spontaneously" a]ter the next cyclic estrus (Fig. 8.12). Dissociation of the two mechanisms is expressed in another way by certain Mustelidae, e.g., the mink and marten. Ovulation in these forms is invoked by mating, whereas corpus luteum activation awaits appropriate environmental conditions, i.e., temperature and length of daily illumination (Pearson and Enders, 1944; Hansson, 1947). In the mink, during the period of relative luteal inactivity that follows mating early in the season, recurrent estrus continues. If reraating takes place at an interval of 6 days or more, new ovulations are induced (Hansson, 1947). Matings late in the season are immediately followed by luteal activity. The pseudopregnant cycles of a representative series of mammals are much alike when conditions appropriate to the respective species are applied (Fig. 8.1).

~~1. Duration of Psciidopregndncn~~

The length of time that corpora lutea remain functional in the pseudopregnant cycle is thought to be relatively uniform in the great majority of mammals, usually about 10 to 15 days. Rarely it is shorter,

e.g., the hamster, 7 days, although usually 9 to 10 days (Asdell, 1946). At the other extreme, the corpora lutea remain functional for periods corresponding to the duration of pregnancy, as in the ferret, 5 to 6 weeks. In fact, corpus luteum function lasting over a month is usual in the other two carnivores for which information is at hand: cat, 30 to 44 days (Foster and Hisaw, 1935) ; and dog, 30 days or more (Evans and Cole, 1931).

These figures are only approximations, however, as the criteria on which they are based differ. In the rat, in which pseudopregnancy is said to last 12 to 14 days, its termination is taken to be the onset of the next estrus, whereas the corpora lutea must have undergone a decline of activity 2 or 3 days earlier (Everett, 1948). The decline is probably not abrupt, inasmuch as the vaginal smear during the next estrus is very strongly mucified and, as mentioned earlier (p. 519), enough progesterone seems to be secreted by the waning corpora lutea to facilitate ovulation. Morphologic criteria are often employed as indicators of corpus luteum regression: characteristically, fatty vacuolation of luteal cells, decrease in size of the individual cells or of the entire corpus

~~534~~

~~PHYSIOLOGY OF GONADS~~

luteuni. and changes in the smusoidal pattern suggesting reduced circulation. Such changes are first observed in the guinea pig corpora lutea on about the 13th day of the cycle. One has the choice of taking this date as the end of the pseudopregnant phase or, alternatively, the date on which the first indications of estrus are noted. Either choice is arbitrary, but the former seems preferable as it suggests that progesterone secretion is diminishing and probably is no longer sufficient to maintain progestational changes in the uterus. In fact, regression of the endometrium sets in about a day earlier than frank degenerative changes in the corpora lutea. In the cat, according to van Dyke and l.i (1938) the corpora lutea 20 days after ovulation no longer secrete enough progesterone to cause motor effects of epinephrine in the myometrium, the so-called "epinephrine-reversal" effect, yet by histologic criteria corpus luteum regression is not apparent until 28 days or later (Liche, 1939; Foster and Hisaw, 1935). In the bitch the uterus begins regression 20 to 30 days after heat, l)ut the corpora lutea are said to remain in good condition for a longer time (see Asdell, 1946, for references). Regression is so gradual that anestrum is not reached until about 85 days. In the primates the beginning of menstruation offers a means of delimiting the luteal phase, inasmuch as menstruation in the ovulatory cycle reflects a marked reduction in corpus luteum function. Nevertheless, this reduction probably occurs a few days before bleeding begins. The i^eak of pregnanediol excretion in women (Venning and Browne, 1937) and of plasma progesterone concentration in women and monkeys (Forbes, 1950; Bryans. 1951 ) is passed about midway between ovuhition and menstruation.

~~2. Xciivdl Factors in Pseudoprciindiicji~~

The importance of the nervous system in control of pseudopregnancy is well recognized in only the few species re])resented by the rat. .\ neural effect in the mink and similar Mustelidae is implied by the relation of hiteal function to daily illumination, as mentioned earlier. Beyond that fact, however, no information is available. Att(>ntion will therefore be directed largely to the rat.

~~Not only sterile mating, but se\'eral other~~

procedures involving neural stimulation will cause rats to become pseudopregnant. Stimulation of the cervix by mechanical means (Long and Evans, 1922) or electric shock (Shelesnyak, 1931) have become standard methods. In fact, Greep and Hisaw (1938) obtained pseudopregnancies after electrical stimulation during early diestrum, several days before ovulation. Pseudopregnancy is also invoked by continuous stimulation of the nipples for several days (Selye and McKeown, 1934). According to Harris (1936) electric shock through the head is effective. His negative results with "spinal shock" are difficult to explain. From the description of position of the electrode it seems doubtful that the current passed through the cord itself, yet the sacral plexus must have been stimulated.

Al)dominal sympathectomy or superior cervical ganglionectomy are said to diminish the numbers of animals responding to electrical or mechanical stimulation of the cervix (Vogt, 1931; Haterius, 1933; Friedgood and Bevin, 1941). On the other hand, there is no diminution of response to sterile copulation, which shows that the sympathetic chains are not essential. Ball (1934) emphasized the quantitative aspects of the problem, noting that partial resection of the uterus or excision of the cervix diminished the response to sterile copulation, but only when "single-plug" matings were allowed. Multiple plugs gave pseudopregnancy in 100 per cent of the animals. It may be assumed that Vogt's (1933) negative results after hysterectomy resulted from single-jilug copulations. Kollar (1953) re-opened the cjuestion and found that pelvic nerve resection usually pi-evented the response to mating. It is not clear, howevei', whether multiple copulations wnv the rule, although it seems that the I'outiiie procedure was to leave the male with the female overnight. His contention was that cervicectomy fails to abolish the resjionse completely because the vagina remains sensitive.

Anesthesia with ether, nitrous oxide, or ethylene ( Mcyei', Leonard and Hisaw, 1929) diniiiiished the ficciuency of response to inecliaiiical stimulation of tlu> (•er\-ix. The statement was made, although without v\\(len(c, that spinal anesthesia |)re\-ents pseudopreiinanev. Aeeoi'ding to A'ogt (1933),

~~MAMMALIAN REPRODUCTIVP] CYCLE~~

~~535~~

~~local anesthesia of the vagina and cervix by cocaine or procaine prevented the response to sterile copulation in 23 of 35 rats.~~

Removal of neocortex (Davis, 1939) did not interfere with the pseudopregnancy response to electrical stimulation of the cervix, although there was slight impairment of the response to mechanical stimulation or sterile mating (single-plug?).

These results taken together have been construed to mean that induction of pseudopregnancy in rats involves a reflex similar to the ovulation reflex in rabbits. Certain considerations, however, raise the possibility that it may not be a "trigger" stimulus to the hypophysis as long believed (Everett, 1952a). In the first place, it seems doubtful that a trigger stimulus would result in continuation of a new pattern of secretion (luteotrophin) for as long as 10 to 12 days. Furthermore, as noted above, cervical stimulation during the diestrum preceding ovulation may induce pseudopregnancy (Greep and Hisaw, 1938). Similarly, copulation during a cycle in which ovulation is blocked by pentobarbital results in a pseudoi)regnancv that begins after the next estrus (Fig. 8.12)^.

We turn now to experiments concerned directly with the hypothalamo-pituitary system and pseudopregnancy. Westman and Jacobsohn (1938c) cut the pituitary stalks of estrous female rats. Barriers of metal foil were inserted to prevent regeneration of nerve fibers assumed to innervate the adenohypophysis. Regeneration of blood vessels must have been equally impossible. Controls were simply hypophysectomized. Two to 5 hours after the operations electrical stimulation of the cervix was administered to all animals. Pseudopregnancies were demonstrated by deciduomas in traumatized uteri of all the stalk-sectioned animals but not in the completely hypophysectomized rats. Desclin (1950) reported the maintenance of pseudopregnancy in estrogen-treated rats in which the only remaining hypophyseal tissue was in the form of grafts in the kidney. Whereas in hypophysectomized controls the estrogen treatment (stilbestrol pellets) produced cornification of the vagina and no enlargement of corpora lutea, the engrafted-estrogenized rats developed mucified vaginas and enlarged corpora lutea as

~~in intact rats similarly treated with estrogen. Desclin concluded that the grafted hypophysis is able to respond to estrogen by liberating luteotrophin.~~

It is now apparent, however, that neither cervical stimulation nor estrogen treatment is needed to invoke pseudopregnancy when the gland is isolated from the hypothalamus (Everett, 1954, 1956a; Nikitovitch-Winer and Everett, 1958a; Sanders and Rennels, 1957; Desclin, 1956a, b). When autografts of anterior hypophysis were made to the renal capsule or near the common carotid artery on the day after ovulation in adult cyclic rats, corpus luteum function was invoked and maintained without any stimulus other than the operative procedures themselves. In short-term experiments in which the uteri were traumatized 4 days after the transplantation large deciduomas were regularly found at 8 days in the proven absence of residual hypophyseal tissue at the original site (Everett, 1954). Hypophysectomized controls were negative. In longterm experiments, continuing luteal function was demonstrated for as long as 3 months. Here the test for luteal function was vaginal mucification in the presence of massive amounts of estrogen administered during the final week of the experiment (Everett, 1956a). Controls in which the grafts or the ovaries were removed at the beginning of such estrogen treatment responded with full vaginal cornification. Follicular apparatus and interstitial tissue of the ovaries atrophied promptly after the grafting operations, whereas corpora lutea forming at that time were maintained for the long periods without histologic sign of deterioration. In later work, the decidual reaction was used as the test for luteal function, positive reactions being elicited as late as 2 months after the transplantation. It was discovered that function of the graft is not influenced by stage of the cycle at which transplantation is carried out and that grafts in the anterior chamber of the eye secrete luteotrophin like those on the kidney (Nikitovitch-Winer and Everett, 1958a). Transsection of the pituitary stalk is sufficient in itself to provoke pseudopregnancy. If an effective barrier to vascular regeneration is inserted, the pseudopregnancy will heorve permanent, but otherwise it will l:i •■ i;!*'

~~536~~

~~PHYSIOLOGY OF GONADS~~

usual length of time (Xikitovitch-Winer, 1957 j. In fact, there is reason to suspect that even a transient impairment of circulation in the median eminence-hypophj'seal linkage can be a sufficient impetus to pseudopregnancy. The experiments of Taubenhaus and Soskin ( 1941 ) in which application of an acetylcholine-prostigmine mixture to the exposed hypophysis was followed by pseudopregnancy, may well be explained in some such way.

It thus seems that in the rat the deprivation of, or interference with, the normal connection of the pars distalis with the median eminence facilitates secretion of luteotrophin and at the same time eliminates luteolytic mechanisms. It is significant that transplants of pars distalis into the pituitary capsule or to the immediate vicinity of the median eminence resume cyclic function (see page 512). The hypothalamus may have an inhibitory effect on luteotrophic activity of the pars distalis during the short cycle in this species. Greep and Chester Jones (1950) made the pertinent suggestion in attempting to explain the induction of pseudopregnancy by estrogen treatment, that the fundamental action of estrogen here is the suppression of FSH and LH, after which luteotrophic secretion may "proceed apace."

There is necessarily some uncertainty concerning the amount of luteotrophin secreted by the pars distalis when dissociated from the brain. Three sets of facts indicate that the output is larger than that in the cycling animal. (1) Sufficient gestagen is secreted by the engrafted animal to maintain a ])regnancy (Everett, 1956c; Meyer, I'rasad and Cochrane, 1958) when the pituitary is trans])lanted on the day after mating. (2) After stalk-transsection, in which there is less initial destruction of glandular parenchyma than in transplantation experiments, the corpora lutea enlarge to a diameter like that usually found in late pregnancy rather than remaining like those of pseudopregnancy (Nikitovitch-Winer, 1957). (3) A single homotransplant of pars distalis placed subcutaneously in an otherwise normal female mouse will maintain a se(iueiicc of pseudopregnancies that override the short cycles expected of the animal's own hypophysis (Miihlbock and Boot, 19591. This is also true of rats (Nikitovitch

Winer, uni)ublislie(l). To avoid the conclusion that in such preparations the grafted gland is secreting luteotrophin at an increased rate, one must assume that the outl)ut of this hormone from the intact gland is only slightly below threshold and that the graft adds just enough to make the total output effective. To explain the maintenance of pregnancy one might assume that the luteotrophin output and the resulting gestagen secretion are no greater than during the normal short cycle and that the formation of deciduomas takes place because of the deficiency of estrogen. The results of stalksection, however, cannot easily be explained away. The weight of evidence, then, is in favor of increased luteotrophin secretion by hypophyses isolated from the brain by severance of the stalk or transplantation.

Under certain experimental conditions it has seemed that to establish pseudopregnancy all that is necessary is to block out the forthcoming estrus and ovulation. Thus, in cycling rats, when the hypophysis is transplanted to the kidney as late as 60 hours after ovulation, the current diestrum transforms into a permanent pseudopregnancy supported by the existing set of corpora lutea (Nikitovitch-Winer and Everett, 1958a ». Similarly, injections of chlorpromazine (Barraclough, 1957) or Pathilon (ditsch and Everett, 1958) begun during diestrum, may transform it into a pseudopregnancy by blocking out the expected estrus.

The Miihlhock-Boot experiment mentioned above furnishes an instructive model of the standard mammalian cycle, in which both ovulation and pseudopregnancy are spontaneous events. Given the extra pituitary tissue producing luteotrophin at a presumably constant rate, with the output of the normal ghmd fluctuating (juantitatively and (jualitatively, the mouse or rat undergoes one ])seudopregnancy after another. Possibly, in animals that normally liave a spontaneou.- hiteal phase, thei'c is a considei'ahle poUion ot' the pars distalis which I'unctions somewhat indepeiidentl}- of the hyputhalaiiius. with a continuous output of luteotrophin as from the grafted gland in the Mvihlb()ck-P)Oot pi-epaiation. The portion nioic (Hrectly under control of the me(lian eminence I zona tuberaHs'.'l would

~~MAMMALIAN REPRODUCTIVE CYCLE~~

~~537~~

then act like the intact hypoiihysis of the Miihlbock-Boot mouse. Such a view, unfortunately, continues to set apart species in which the luteal phase is not spontaneous, by suggesting that only in them are special neui'al nieclianisnis involved.

~~B. LUTEOLYTIC MECHANISMS~~

By luteolysis we shall refer to corpus luteum regression in any of its manifestations. Supposedly the initial change is functional, after which overt cytologic and histologic changes appear, leading eventually to the total loss of glandular tissue. Very likely the initial stages are occult and only gradually reach recognizable proportions. Mention was made earlier of the fact that in women and monkeys the peak of gestagen secretion is about midway between ovulation and menstruation. In rats indirect evidence from progesterone injection experiments leads to the deduction that toward the close of a pseudoi)regnancy gestagen secretion must drop below the estrus-suppressing level several days before estrous changes appear in the vaginal smear (see Fig. 8.8). A more al)rupt drop is reported for the ewe l)etween the 16th and 17th (last) days of the cycle (Edgar and Ronaldson, 1958).

Long-term experiments with pituitary autotransplants indicate that at least in the rat the life span of the corpus luteum is not limited by intrinsic factors. Some agent (s) of extra-ovarian origin must, therefore, be res]5onsible for at least the initial luteolytic changes. Various bits of information suggest that the agent is associated with, if not identical with, FSH and/or LH. Greep (1938) noted that after hypophysectomy in rats the daily injection of LH over a period of 10 days caused the corpora lutea to regress more rapidly than otherwise. Greep, van Dyke and Chow (1942) later were unable to repeat this with a more highly purified LH C'metakentrin") , a fact suggesting that the earlier material was effective because of impurity. During the short cycle of the rat, luteolysis is interrupted by translilantation of the pars distalis (Everett, 1954; Nikitovitch-Winer and Everett, 1958a). Whatever regressive changes are in progress at that moment are evidently suspended forthwith. They are first appar

ent during the third day of diestrum and become increasingly pronounced during proestrum and estrus. In this connection, it should be recalled that during late diestrum and proestrum patches of cells undergoing fatty necrosis are first recognizable histologically (Boling, 1942; Everett, 1945).

Why is it that, in the face of a continuing supply of luteotrophin in the MiihlbockBoot preparation, or in intact animals injected daily with lactogen (Lahr and Riddle, 1936; Aschheim, 1954), luteolysis sets in during the 2nd week? The question obviously cannot be answered from present knowledge. Nevertheless, it is clear that the pseudopregnancy that transpires when a significant i)ortion of hypophyseal tissue remains in normal relation to the hypothalamus is far from the steady state of that which becomes established by total removal of the pars distalis to an extracranial site. It is also apparent that the onset of luteolysis may be postponed by such means as hysterectomy or production of artificial deciduomas (see p. 538). Furthermore, during lactation-pseudoi)regnancies in rats, Canivenc and Mayer (1953) prolonged luteal function to 34 days by substituting successive new litters of suckling young. This technique should prove especially valuable in experimental analysis of both luteotrophic and luteolytic mechanisms.

Benson and Folley (1956) suggested that lactogen secretion is activated by oxytocin, inasmuch as its injection prevented the normal inv'olution of the mammary glands after withdrawal of the litters from lactating rats. This observation has been confirmed by McCann, Mack and Gale (1958), who also noted the interruption of lactation by lesions of the sui)raoi)tico-hyiiophyseal tract. Selye and ]\lcKeown (1934) long ago found that pseudopregnancy could be induced in cycling rats by the introduction of a suckling litter. Although all this is consistent with the above-mentioned observation by Canivenc and Alayer, other workers have observed luteolytic effects of gonadotrophinfree oxytocin administered to cycling dairy heifers (Armstrong and Hansel, 1958). Furthermore, Grosvenor and Turner (1958), after first noting that the administration of Dibenamine, atropine, or pentobarbital to rats prevented the expected drop in assay

~~588~~

~~PHYSIOLOGY OF GONADS~~

able pituitary lactogen at nursing, found no decrease when pentobarbitalized mothers were injected with physiologic doses of oxytocin intravenously. When the dosage was increased 30 to 60 times there was apparently some moderate discharge, but the authors regard it as insignificant.

~~C. EFFECT OF THE UTERUS ON LUTEAL FUNCTION~~

This subject has been reviewed by Bradbury, Brown and Gray (1950). In three species (Fig. 8.13) hysterectomy results in significant prolongation of the functional life-span of corpora lutea (guinea pig, Loeb, 1927; rabbit, Asdell and Hammond, 1933; rat, Bradbury, 1937). In each case the period of luteal function approximates that of normal pregnancy. The fact that corpora lutea in the pseudopregnant ferret normally function as long as in the pregnant animal may be a clue to the noncffect of hysterectomy in that species (Deanesly and Parkes,

19331. Ahhough Burford and Diddle (1936) rej^orted that in monkeys total hysterectomy was followed by vaginal cycles of normal length, examination of their protocols shows that during the several postoperative months just 1 corpus luteum was produced among all 5 animals. The experiment thus seems inconclusive. Impairment of pelvic circulation seems to be a common factor complicating the results of hysterectomy in women and may have been one cause of the failure of luteinization in these monkeys.

An interpretation given by Loeb (1927) and Bradbury, Brown and Gray (1950) for the prolongation of luteal function by hysterectomy is that in species in which the effect is demonstrable the uterus secretes a specific substance which abbreviates the life of the corpus luteum. Hechter, Fraenkel, Lev and Soskin (1940) found in rats that grafts of estrous uteri shortened the pseudopregnancies of hysterectomized animals to normal length. Implantation of similar tis

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~~MAMMALIAN REPRODUCTn'E CYCLE~~

~~539~~

SIR' which had been killed by freezing had no such effect, nor did successful grafts of uteri from diestrous donors. Bradbury, Brown and Gray (1950) found that partially hysterectomized rats in which the remaining uterine tissue was continuous with the cervix, hence properly drained, experienced pseudopregnancies of normal length. However, when the continuity was interrupted the uterine remnants became greatly distended, the endometrium was destroyed, and the animals had prolonged pseudopregnancies. Possibly the endometrium is the source of the hypothetical "luteolytic" substance.*'

Under other circumstances the endometrium of rats has a luteotrophic rather than luteolytic effect, for when deciduomas are induced by trauma the pseudopregnancies of otherwise normal animals are lengthened to 22 days or more (Ershoff and Duell, 1943; Velardo, Dawson, Olsen and Hisaw, 1953). This is not true in mice, however, and Kamell and Atkinson (1948) suggest that the I'eason may lie in the shorter life-span of the decidual tissue in that species. Loeb (1927) reported that deciduomas in cyclic guinea pigs ])rolonged the luteal phase, i.e., delayed the next ovulation from 3 to 7 days, which is far less than the prolongation after hysterectomy.

~~As an alternative to the concept of conI rol of the corpus luteum by humoral agents~~

" The denial by Velardo, Olsen, Hisaw and Dawson (1953) that hysterectomy in rats prolongs l)seiidopregnancy is based on operations performed later in the luteal phase than those of Bradbury, Brown and Gray and of Hechter, Fraenkel, Lev and Soskin. Whereas the latter workers had operated in the range from the 4th to the 7th day.^^ of p.seudopregnancy and many of Bradbury's cases lacked uteri when they entered pseudopregnancy, Velardo and associates excised the uteri on the 9th day. It seems possible that this difference in time may be crucial, for by the 9th day of a 12-day pseudopregnancy the corpora lutea must be on the verge of regression, if that in'ocess has not already been initiated. After maintaining pseudopregnancy in estrogenized, liypophysectomized rats b}^ means of lactogen, its withdrawal is followed about 3 days later by estrous smears (Nelson and Bichette, 1943; Nelson, 1946). A slightly longer delay occurs in nonestrogenized, intact rats at the end of a lactogeninduced pseudopregnancy (Everett, unpublished) or after withdrawal of progesterone treatment (Fig. 8C).

formed in the uterus, Loeb considered the jjossibility that neural mechanisms are involved. The idea was not acceptable, he felt, because in partial hysterectomies the result was not detennined by the locus of the part removed. The finding by Hechter, Fraenkel, Lev and Soskin (1940) that grafts of uterine tissue in hysterectomized rats return the duration of pseudopregnancy to normal, is significant evidence pointing in the same direction.

A third hypothesis was advanced by Heckel (1942) who found in rabbits that the extent of prolongation of luteal function by subtotal hysterectomy is roughly proportional to the amount of uterine tissue removed. The suggestion was offered that removal of the uterus has an estrogensparing effect. The greater amount of estrogen thus available to the corpora lutea prolongs their life, according to this view.

Later investigations by Moore and Nalbandov (1953) revive the possibility that the uterus influences the ovary by way of the nervous system. In sheep the implantation of a plastic bead in utero during the early luteal phase shortened the cycle by several days. Successive cycles tended to be unusually short. When the uterine segments containing the beads were denervated, however, the cycles were essentially normal. Other work from the same laboratory (Huston and Nalbandov, 1953) which indirectly may bear on this problem, indicates that the presence of a mechanical irritant (a thread) in the oviduct of the domestic fowl tends to block ovulation. The blockade may extend for as long as 20 days if the thread is placed in the isthmus (van Tienhoven, 1953). The ovaries remain functional, producing large follicles which may be ovulated at will by injection of LH. The authors feel that this phenomenon, like the effect of the bead in the sheep uterus, involves a neural mechanism, Init crucial information is lacking. It may be significant that stimulation of the ovaries was found in some hens, in place of blockade.

We may hope that as more information becomes available the assortment of facts given in these paragraphs will fit into a rational system. Not until this is realized can we hope to understand the regulation of the luteal phase.

~~540~~

~~PHYSIOLOGY OF COXADS~~

~~VIII. Concluding Comments~~

From what has been written here it is readily apparent that present knowledge of the mechanisms controlling the reproductive cycle is extremely spotty. The number of assumptions necessary to knit the various items of factual information into an orderly pattern is disturbing. In spite of a voluminous literature which has grown during the last 60 years, we are really only a few steps ahead of our predecessors at the turn of the century in terms of fundamental understanding. A brief recounting of some of these steps may be desirable.

The first three decades saw the gradual development of proof that the ovary is a gland of internal secretion as well as the producer of eggs, governing the uterus and other accessory organs by secretion of hormones into the blood stream. For a while it seemed that the events of the reproductive cycle could be neatly explained, with the ovary in the capacity of controlling agent. Yet there were indications that the ovary itself is not independent. As early as 19091910 (Aschner; Crowe, Gushing and Honuins) it was noted that destruction of the hypophysis is accompanied by atrophy of the gonads and reproductive tract. In 1927 the separate investigations of Smith and Engle and of Zondek and Aschheim demonstrated conclusively that function of the ovary depends vitally on the anterior hyjiophysis. Promptly it was learned that the hypophyseal secretion of gonadotrophic hormone is in turn modified by estrogens. In the early 1930's the "push-pull" hypothesis of pituitary-ovarian interaction was separately stated by Brouha and Simonnet and by Moore (see Moore and Price, 1932). Modified in detail as new facts appeared, this hypothesis is held to the present day in some quarters as a simple exi)lanation of how the cycle comes about in polyestrous, continuous breeders in which ovulation takes l)lacc spontaneously. Much of the investigation of pituitary-ovarian i)hysiology during the 1930's was performed within the framework of this hypothesis.

~~For seasonal breeders and reflex ovulators, however, the assumption was necessary that special controlling mechanisms are superimjiosed. It bccaiiie iccognizcd also that in~~

some vaguely defined manner even the human cycle is subject to intervention of the nervous system. The possible importance of the hypothalamus was debated at some length in the twenties. In 1932 the existence of a sex center there was proposed by Hohlweg and Junkmann. In an attempt to explain the coital excitation of the rabbit hypophysis which causes the liberation of gonadotrophin, Hinsey and Markee (1933) suggested diffusion of a chemical substance from the posterior lobe to the anterior lobe. Hinsey (1937) later elaborated on this possibility and mentioned the hypophyseal portal vessels as a plausible route by which the substance might travel. We have seen the later history of these ideas.

The "Sexualcentrum" of Hohlweg and Junkmann was proposed as a mediator of the effects of estrogen on the anterior hypophysis of rats. Westman and Jacobsohn (1936-1940), on the other hand, believed that through its stalk connection with the hypophysis the rat hypothalamus governs gonadotrophin synthesis, not release. The latter they regarded as a direct effect of estrogen on the gland. These views did not afford a common basis for spontaneous and refiex ovulation.

Schweizer, Chari])i)er and Haterius ( 1937) offered the first surmise of similarity, after finding that guinea pigs bearing intraocular pituitary grafts developed persistent estrus and large follicles that failed to go through maturation changes. Their feeling was that the normal connection of hypophysis with hypothalamus may be necessary for cyclic liberation of LH. Almost concurrently, Dempsey (1937) expressed a similar view as one alternative explanation of his experimental results with the guinea i)ig cycle. Suggesting cautiously that release of luteinizer may be brought about l)y a "rhythmic discharge" from the central nervous system, he went on to mention the "possibility that a high level of oestrin is necessary but not directly responsible for the release of luteinizer" (italics added). From this it is only a short transition to certain concepts set forth in the present exposition.

~~Accoi'ding to current views: (1) Reflex ovulation and spontaneous ovulation alike are go\-enicd by a liypothalamo-pituitary~~

~~MAMMALIAN REPRODUCTIVE CYCLE~~

~~541~~

apparatus whose final link to the pituitaiy is neurohumoral by way of the hypophyseal portal veins and whose activity precipitates release of LH. (2) The apparatus includes a hypothalamic center or centers whose excitation depends on estrogen-progesterone levels and afferent impulses of various kinds. (3) The sensitivity of the center (s) is influenced not only by the sex steroids, but by other poorly understood factors that vary from species to species and from time to time in individuals, e.g., the diurnal rhythm in rats. Here in bare outline is a plausible hypothesis that may be generally applied to the events immediately relating to ovulation.

Satisfactory hypotheses respecting other phases of the cycle must await future developments. The extent and manner of intervention of the nervous system in the follicular and luteal phases remain unsettled. Although the hypophyseal hormones concerned in ovarian follicle development have been characterized, their exact chemical descrijition has not been accomplished. The rate of their output at different stages of the cycle is largely a matter of conjecture. Structural changes that they jiroduce in the ovary are well known, but in chemical terms only the end products of ovarian activity are well recognized, and these probably incompletely. The fact that the estrogens, in turn, have a regulating effect on follicle-stimulating activity of the hypophysis is known, but the mechanisms by which this effect is accomplished are uncertain. The hypophyseal hormones that maintain the luteal phase are recognized with any certainty in only three species and there is a wide difference between rabbits, on the one hand, and rats and mice, on the other. For mammals generally, the luteotrophic factors have not been identified. Whether the hypothalamus is actively concerned in maintenance of the luteal i)hase in the majority of mammals is unknown. The morphologic effects of luteotrojihic stimulation on corpora lutea are well recognized, but here again the chemical mechanisms leading to the end products are obscure. The action of the corpus luteum hormone in regulation of the cycle is partially known, including the well established fact that its continual jiresence in large amount will suiijiress the estrogenic and

ovulatory phases. Yet, one cannot say whether this effect is accomplished by direct action on the hypophysis or by indirect action through the central nervous system. Nor can one state how the hypophysisgonad equilibrium of the luteal phase is interrupted in the absence of a conceptus. With respect to the ovulation mechanism itself, the hypothesis outlined above requires verification in additional species. Assuming its validity, many details remain to be studied, e.g., the neural pathways and nuclei involved, identification of neurochemical activators of the pars distalis and their sources and loci of action, the precise nature of mechanisms whereby the gonadal steroids excite or suppress, the cellular mechanisms by which ovulating hormone is released into circulation by the hypoi^hysis, and the cytochemical effects within the ovary. All too evidently an encompassing theory of the female reproductive cycle is far from realization.

~~IX. References~~

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~~BuNN, J. P., .AND Everett, J. W. 1957. Ovulation in persistent-estrous rats after electrical stimulation of the brain. Proc. Soc. Exper. Biol. & Med., 96, 369-371.~~

~~BURFORD. T. H., AND DiDDLE, A. W. 1936. Effect of total hysterectomy upon the o\ary of Macacus rhesus. Surg. Gynec. & Obst., 62," 701-707.~~

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C.ANivENc, R., AND M.AYER, G. 1953. Les corps gestatifs de la rate sont-ils prolonges par la prolactine? Etude de I'aecouchement et de I'ablation des cornes gray ides au cours de la lactation prolongee. Compt. rend. Soc. bioL, 147, 2025-2027.

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~~DoHKMAN, R. I.. AND VAN W.\GENEN, G. 1941. The~~

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~~DuYVENE DE WiT, J. J. 1942. Presence of progesterone and 17-ketosteroids in the follicular fluid of the pig, cow, and human being. Acta brev. neeri., 12, 23-25.~~

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~~Engle, E. T. 1946. No seasonal breeding cycle in dogs. J. Mammal., 27, 79-81.~~

~~Engle, E. T., and Shelesnyak, M. C. 1934. First menstruation and subsetiuent menstrual cycles of pubertal girls. Human Biol., 6, 431-453.~~

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Evans, H. M., .\nd Long, J. A. 1921. On the association of continued cornification of the vaginal mucosa with the presence of large vesicles in the ovary and the absence of corpus luteum formation. Anat. Rec, 21, 60-61.

~~Evans, H. M., and Cole, H. H. 1931. An introduction to the stud}' of the oestrous cycle in the dog. Mem. Univ. California, 9, 67-117.~~

~~Evans, H. M., and Swezy, 0. 1931. Ovogenesis and the normal follicular cycle in adult mammalia. Mem. Univ. California, 9, 119-225.~~

~~Evaxs, H. M., Si-mpson, M. E., Lyons, W. R., and Turpeinen, K. 1941. Anterior pituitary hormones which favor the production of traumatic uterine placentomata. Endocrinologv, 28, 933-945.~~

~~Everett, J. W. 1939. Spontaneous persistent estrus in a strain of albino rats. Endocrinology, 25, 123-127.~~

~~Everett, J. W. 1940a. The restoration of cyclic e.strus and ovulation in persistent-estrous rats bv progesterone. Anat. Rec, 76, No. 2, Suppl., 21.~~

~~Everett, J. W. 1940b. The restoration of o\ulatory cycles and corpus luteum formation in persistent-estrous rats by progesterone. Endocrinology, 27, 681-686.~~

~~Everett, J. W. 1942a. Certain functional interrelationships between spontaneous persistent~~

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~~Mandl, A. M., and Zuckerjl\x, S. 1952. Cyclical changes in the number of medium and large folhcles in the adult rat ovary. J. Endocrinol., 8, 341-346.~~

~~Marden, W. G. R. 1952. The hormone control of ovulation in the calf. Endocrinology, 50, 456-461.~~

~~Mardones, E., Bruzzone, S., Iglesias, R., and LiPSCHUTz, A. 1951. The non-concomitancy of the progestational and antiluteinizing actions of steroids. Endocrinology, 49, 817-825.~~

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~~Markee, J. E., Sawyer, C. H., and Hollinshead, W. H. 1946. Activation of the anterior hypophysis by electrical stimulation in the rabbit. Endocrinology, 38, 345-357.~~

~~Markee, J. E., Sawyer, C. H., and Hollinshead, W. H. 1948. Adrenergic control of the release of luteinizing hormone from the hypophvsis of the rabbit. Recent Progr. Hormone Res., 2, 117-131.~~

Markee, J. E., Everett, J. W., and Sawyer, C. H. 1952. The relationship of the nervous system to the release of gonadotroph in and the regulation of the sex cvc'le. Recent Progr. Hormone Res., 7, 139-163. '

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~~Martinez-Estkve, p. 1937. Le cycle sexual vagi~~

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Westman, A., and Jacobsohn, D. 1937a. Uber Oestrinwirkung auf die Corpus luteum-Funktion. I. Acta obst. gynec. scandinav., 17, 1-12.
Westman, A., .\nd Jacobsohn, D. 1937b. Uber Oestrinwirkung auf die Corpus luteum-Funktion. II. Acta obst. gynec. scandinav., 17, 13-23.
W^estman, a., and Jacobsohn, D. 1937c. Experimentelle Untersuchungen iiber die Bedeutung des Hypophysen-Zwischenhirnsystems fiir die Produktion gonadotroper Hormone des Hypophysenvorderlappens. Acta obst. gynec. scandinav., 17, 235-265.
Westman, A., and Jacobsohn, D. 1938a. Endokrinologische Untersuchungen an Ratten mit durchtrenntem Hypophysenstiel. I. Hypophysenveranderungen nach Kastration untl nach Oestrinbehandlungen. Acta obst. gynec. scandinav., 18, 99-108.
Westman, A., and Jacob.sohn, D. 1938b. Endokrinologische Untersuchungen an Ratten mit durchtrenntem Hypophysenstiel. III. Uber die luteinizierende Wirkung des Follikelhormons. Acta obst. gvnec. scandinav., 18, 115123.
Westman, A., and Jacobsohn, D. 1938c. Endokrinologische Untersuchimgen an Ratten mit durchtrenntem Hy])<)i>h\ x ii-t iil. VI. Produktion und Abgabe (1< : iiini.ulotropcn Hormone. Acta path, et niii-niliKil. scandinav., 15, 445-453.
Westman, A., and Jacobsohn, D. 1940. Endokrinologische Unteisucliungen an Kaninchen mit durchtrenntcMi Hypophyse.stic^l. Acta obst. gynec. scandinav., 20, 392-433.
Westman, A., and Jacobsohn, D. 1942. Die Wirkung transorbital an das Tuber cinereum injizierten Novocains auf die Ovulation. Acta ob.st. gynec. scandinav., 22, 16-23.
Whitaker, W. L. 1940. Some effects of artificial ilhunin.ition on reproduction in the whiteI'ootcd mouse, Peromyscus leucopns Novel>uracrnsis. J. Exper. Zool., 83, 33-60.
Williams. P. C. 1940. Effect of stilboestrol on

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555

the ovaries of hyi>ophyseftomized rats. Nature, London, 145, 388-389.
WiLLi.AMS, P. C. 1944. 0\'arian stimulation by oestrogens: effects in immature hypophysectomized rats. Proc. Roy. Soc. London, ser. B, 132, 189-199.
Williams, P. C. 1945a. Ovarian stimulation by oestrogens. II. Stimulation in the absence of the hypophysis, uterus, and adrenal glands. J. Endocrinol., 4, 125-126.
WiLLi.AMS, P. C. 1945b. Studies of the biological action of serum gonadotrophin. II. Ovarian response after hypophysectomy and oestrogen treatment. J. Endocrinol., 4, 131-136.
WiLsox, R. B., R.AXD.ALL, L. M., and Osterberg, A. E. 1939. Studies on pregnandiol. Am. J. Obst. & Gynec, 37, 59-74.
WiSLocKi, G. B., AND King, L. S. 1936. The permeability of the h.vpophysis and hypothalamus to vital dyes, with a study of the hypophyseal vascular supply. Am. J. Anat., 58, 421-472.
Wolfe, J. M. 1935. Reaction of ovaries of mature female rats to injection of oestrin. Proc. Soc. Exper. Biol. & Med., 32, 757-759.
Wolfe, J. M., and H.a.milton, J. B. 1937. Action of male sex hormone with and without estrin in the female rat. Proc. Soc. Exper. Biol. & Med., 37, 189-193.
WoRTHiNGTON, W. C, Jr. 1955. Some observations on the hypophyseal portal system in the living mouse. Bull. Johns Hopkins Hosp., 97, 343-357.
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Young, W. C, Boling, J. L., and Blandau, R. J. 1941. The vaginal smear picture, sexual receptivity, and time of ovulation in the albino rat. Anat. Rec, 80, 37-45.
Young, W. C, and Yerkes, R. M. 1943. Factors influencing the reproductive cycle in the chimpanzee; the period of adolescent sterility and related problems. Endocrinology, 33, 121-154.
Z.ARRow, M. X., and Bastian, J. W. 1953. Blockade of ovulation in the hen with adrenolytic and parasympatholytic drugs. Proc. Soc. Exper. Biol. & Med., 84, 457-459.
Zondek, B., and Aschheim, S. 1927. Hypophysenvorderlappen und Ovarium. Beziehungen der endokrinen Driisen zur Ovarialfunktion. Arch. Gynak., 130, 1^5.
ZucKERMAN, S. 1937. The menstrual cycle of primates. X. The oestrone threshold of the uterus of the rhesus monkey. Proc. Rov. Soc. London, ser. B, 123, 441-471.
ZucKERMAN, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with a constant threshold dose of oestrone. J. Endocrinol., 2, 263-267.
ZucKERMAN, S. 1952. The influence of environmental changes on the anterior pituitary. Ciba Foundation CoUociuia Endocrinol., 4, 213-228.
ZucKERMAN, S., AND FuLTON, J. F. 1934. The
menstrual cycle of the primates. VII. The sexual skin of the chimpanzee. J. Anat., 69, 38-46. ZwARENSTEiN, H. 1937. Experimental induction of ovulation with progesterone. Nature, London, 139, 112-113.

9

ACTION OF ESTROGEN AND PROGESTERONE
ON THE REPRODUCTIVE TRACT OF
LOWER PRIMATES
Frederick L. Hisaw, Ph.D.
THE BIOLOGICAL LAB0RAT0RIF:S, HARVARD TNIVERSITY, CAMBRIDGE, MASSACHUSETTS
and ^
Frederick L. Hisaw, Jr., Ph.D.
DEPARTMENT OF ZOOLOGY, OREGON STATE COLLEGE, CORVALLIS, OREGON

I. Introduction 556
II. Ovarian Hormones and Growth of
THE Genital Tract 558
III. Effects of Progesterone on the
Uterus 565
IV. Synergism between Estrogen and
Progesterone 567
V. Experimentally Produced Implantation Reactions 571
VI. The Cervix Uteri 572
VII. The Vagina 575
VIII. Sexual Skin 576
IX. Menstruation 578
X. The Mechanism of Menstru.^tion. . 583 XI. References 586
I. Introduction
Cyclic menstruation is the most characteristic feature of primate reproduction, and distinguishes it from the estrous cycle of lower mammals. This cardinal primate event is heralded by the bloody uterine effluent emanating from the vagina, whereas in estrus the dominant characteristic is a sudden modification in behavior featuring an intense mating drive. However, the internal secretions that regulate the various events in the menstrual and estrous cycles are the same, and this similarity is fundamentally more significant than the key descriptive differences just mentioned. Estrus comes at the peak of the growth phase of the cycle and is associated with ovulation. In

contrast, menstruation occurs in the cycle midway between times of ovulation and is not accompanied by an increase in sexual activity. From earliest times menstruation has been recognized as degenerative: the characteristic odor, and the necrotic changes in the lining of the uterus, part of which is cast off at this time, sustain this interpretation. Therefore, menstruation is at the opposite phase of the cycle from estrus. It is such an obvious event that menstrual cycles are dated from the onset of bleeding. Menstruation is not analogous to the proestrous bleeding in the dog or cow nor to the slight bleeding of primates at midpoint between menstrual periods (Hartman, 1929). The study of menstruation was at first almost entirely the province of the clinician and the material for investigation limited to w^omen. Hitschmann and Adler (1907), Meyer (1911), Schroder (1914). Novak and Te Linde (1924), and Bartelmez (1933) are among many of the earlier investigators who contributed descriptions of the cyclic changes in the human endometrium. The physiology of the menstrual cycle and attendant morphologic changes have continued to be an area of active research interest in science and medicine. Among the many more recent contributors are Bartelmez

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(1937), Latz and Reiner (1942), Haman (1942), Knaus (1950), Mazer and Israel (1951), and Crossen (1953).
The earlier concepts regarding the menstrual cycle were based primarily on the changes occurring in the human endometrium and for convenience of description the cycle was divided into four stages or periods. The first of these was the period of active menstruation, and the length of the cycle was dated from its onset. Most authors agreed that menses began by leaking of blood from superficial vessels to form lakes under the surface epithelium and that there was some sloughing of tissue after the beginning of bleeding. There was considerable disagreement as to the amount of destruction and loss of tissue; estimates of various authors ranged from very little to almost complete denudation of the surface. Bartelmez (1933) emphasized both the wide individual variability of the amount of tissue lost and differences in the stage of development of the endometria at the time of menstruation.
The second period immediately following menstruation began with regeneration of the surface epithelium, which started sometimes before menstrual bleeding had ceased and was completed in a very short time. This lieriod included the 5 to 7 days after cessation of menses, during which the endometrium grew in thickness. Frequent mitoses were recognized, especially in the glands which lengthened but remained straight and tubular.
The third ("interval") period, lasting 6 to 10 days, was characterized by a somewhat thickened endometrium, still with straight glands and showing little evidence of secretory activity. At first this was considered a quiescent period as indicated by the term "interval." However, as will be shown later, such a characterization was not justified from the physiologic viewpoint.
The fourth period, called the premenstrual period, included the 10 days or 2 weeks before menstruation. During this phase the glands continued to increase in size and became distended, coiled, or even sacculated. The glandular cells increased in height, and there was evidence of glycogen mobilization and secretion. Next, the epithelium became "frayed out" along the

outer borders, then decreased in height, indicating secretory depletion. Decidual cells appeared in the stroma at this time. The endometrium was much thickened and extremely hyperemic. At the height of this period the endometrium was approximately 5 mm. in thickness, as compared with V2 mm. toward the end of menses. The term premenstrual was usually applied to this phase but today the term progestational would seem preferable.
During and after these descriptions of the changes in the human endometrium, many attempts were made to locate the time of ovulation in the menstrual cycle. When it was found, as will be discussed later, that ovulation occurred approximately midway between two menses, and was preceded by follicular growth and followed by development of a corpus luteum, it became customary to refer to the two halves of the menstrual cycle as the follicular phase and the luteal phase. One advantage of this descriptive terminology was the emphasis it placed on the homology of the two phases of the menstrual cycle in primates with the follicular and luteal phases of the estrous cycles of lower mammals.
A theory to explain menstruation, widely adopted in 1920, was formulated from this morphologic evidence. The essentials were that menstruation occurs because the lining of the uterus, prepared for implantation of the ovum, degenerates if fertilization of the egg does not occur. This required that ovulation and corpus luteum formation precede the i^remenstrual changes in the endometrium. Subsequent research disclosed that menstrual cycles frequently occur in which ovulation does not take place and bleeding results from the breakdown of an "interval" rather than a progestational endometrium.
The discovery of anovulatory cycles not only brought about a revision of ideas regarding an explanation for menstruation but also raised questions as to what constituted a normal menstrual cycle. The length of the cycle and the amount and duration of bleeding are approximately the same regardless of whether or not ovulation has taken place. The gross features of menstruation under these two conditions are indistinguishable one from the other. However, the biologic purpose of the menstrual cycle

558

PHYSIOLOGY OF GONADS

is reproduction wliicli obviously cannot l)0 fulfilled unless an ovum is made available for fertilization. Therefore, in this sense it seems quite clear that anovulatory cycles should be considered incomplete and abnormal.
The investigation of changes taking place in the uterine endometrium at various periods of the menstrual cycle in women was confronted with many difficulties, the chief one being that of obtaining normal tissue representative of specific times of the cycle. The entire uterus and both ovaries are essential for proper evaluations and it was rarely possible to meet these requirements. The material for such studies came from autopsies and surgery and tissues usually had suffered postmortem changes or the surgical condition was one involving serious pelvic disease. There have been, however, a goodly number of instances in which these difficulties were adequately overcome (Stieve, 1926, 1942, 1943, 1944; Allen, Pratt, Newell and Bland, 1930) and the clinic will continue to make important contributions (Rock and Hertig, 1942; Hertig and Rock, 1944), but quite early the need became obvious for a suitable primate that could be used as an experimental animal for research on the different aspects of the physiology of reproduction.
Since the initial observations by Corner (1923) on the menstrual cycles of captive rhesus monkeys iMacaca mulatta) , more has been learned about the physiology of reproduction of this animal than any other primate. Monkeys of this species thrive under laboratory conditions, which has made it possible to devise accurately controlled experiments on normal healthy animals and obtain reliable information on the menstrual cycle, gestation, fetal development, and the interaction of hormones concerned with regulating reproductive processes.
Other features that make the rhesus monkey such an attractive animal for these purposes are the many morphologic and physiologic attributes that are strikingly like those of the human being. Tiic modal length of their menstrual cycles is 28 days but there is wide variation (Corner, 1923; Hartman, 1932; Zuckcrman, 1937a). From an analysis of 1000 cycles recorded for some 80 females of different ages, Zuckerman

(1937a) found an average cycle length of 33.5 ± 0.6 days, and the mode 28 days with an over-all range of 9 to 200 days. Ovulation occtu's api:)roximately midway between two menstrual periods, most between the 11th and 14th days (Hartman, 1932, 1944; van Wagenen, 1945, 1947), and although these animals breed at all seasons of the year many cycles are anovulatory, especially during the hot summer months (Eckstein and Zuckerman, 1956). A method developed by Hartman for detecting the exact time of ovulation by palpation of the ovaries in the unanesthetized animal greatly facilitated the timing of events of the menstrual cycle. This procedure also made it possible to determine the age of corpora lutea with great accuracy (Corner, 1942, 1945) and correlate their develojiment and involution with corresponding changes in the endometrium (Bartelmez, 1951 ) and, in ju-egnancy, with the exact age of developing embryos ( Wislocki and Streeter, 1938; Heuser and Streeter, 1941).
The primary purpose of the present discussion is to review the results of experimental investigations of physiologic processes occurring in the female reproductive tract of lower primates during the menstrual cycle, and particularly those processes that are under hormonal control. The brief introductory presentation of basic observations could be greatly extended and we take up the discussion of endocrine problems knowing that we must return often to the work of these authors and that of others to be cited, as conclusions based on experimental data take on meaning only in terms of normal function.
II. Ovarian Hormones and Growth of the Genital Tract
The changes that are repeated in different parts of the reproductive tract with each menstrual cycle are produced by ovarian hormones, estrogens, and progesterone. The dominant hormone of the follicular phase is estradiol-17/y, which is secreted by the Graafian follicle, and in the tissues is readily transformed in i)art to estrone, an estrogenic metabolite. Progesterone, secreted by the corinis luteum, is jirimarily a hormone of the luteal phase of the cycle. However, small amounts of progesterone may appear

ESTROGEX AND PROGESTERONE

559

ill the blood of monkeys as early as the 7th (lay and attain a concentration of 1 fxg. per ml. of serum at ovulation, whereas a maximal concentration of 10 /xg. per ml. is reached at approximately the middle of the luteal phase (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). Also, some estrogen is present during the luteal phase, probably secreted by the corpus luteum (estrogens can be obtained from luteal tissue) or it may be partially derived from developing follicles. It is unlikely that estrogen is ever entirely absent during a normal menstrual cycle or that the presence of progesterone is completely restricted to the luteal phase.
The dependence of the reproductive tract on ovarian hormones is strikingly demonstrated by the profound atrophy that follows surgical removal of the ovaries. A progressive decrease in size of the Fallopian tubes, uterus, cervix, and vagina takes place, and usually, the involution of the uterine endometrium involves tissue loss and bleeding, commonly referred to as post castrational bleeding. Dramatic as these effects may seem, it is equally dramatic to find that these atrophic structures can be restored entirely to their original condition by the administration of ovarian hormones. Therefore, it is seen at the beginning that investigations dealing with the physiology of the female reproductive tract of primates in large measure involve a study of the independent and combined actions of estrogens and progesterone on the activities of the various structures concerned.
Much can be learned about the action of ovarian hormones by observing the changes they produce in the gross appearance of the atrophied reproductive tract of castrated animals. Daily injections of an estrogen in doses equivalent to 1000 I.U. or more for 10 days or a fortnight will restore the uterus, cervix, and vagina to a condition comparal)le with that found in a normal monkey at the close of the follicular phase of a menstrual cycle (Fig. 9.1A). If a similar castrated monkey is given 1 or 2 mg. of progesterone daily for the same length of time, very little if any change in size of the reproductive organs results. However, if first the normal condition is restored by giving estrogen and then is followed by the progesterone treatment, the size of the uterus

is maintained but that of the cervix and vagina decreases to an extent approaching that in a castrated animal (Fig. 9.1B). Such experiments show that an estrogen promotes growth of the reproductive tract whereas progesterone is comparatively ineffective when given alone. Yet progesterone can maintain the size of the uterus when administered following an estrogen treatment but it does not prevent involution of the cervix and vagina.
An additional feature of the growth-stimulating action of the ovarian hormones is brought out when estrogen and progesterone are administered concurrently. If, after repair of the reproductive tract of a castrated animal has been accomplished by a series of injections of estrogen, both estrogen (1000 I.U.) and progesterone (1 or 2 mg.) are given daily for 20 days, it will be found that the uterus is larger than when either hormone is given alone for a similar length of time, whereas the cervix and vagina have involuted and are approximately the size found in animals given only progesterone. Thus it can be demonstrated that a synergistic effect on growth of the uterus occurs when the two hormones are given simultane

FiG. 9.1. Reproductive tracts of three adolescent monkeys which were castrated and given estrogen daily for approximately 3 weeks. A shows the condition at the conclusion of the estrogen treatment, B the condition following the injection of progesterone for an additional three weeks, and C the effects of continuing the treatment with both estrogen and progesterone for a like period.

560

PHYSIOLOGY OF GONADS

ously whereas in the cervix and vagina the growth -stimulating action of estrogen is inhibited by progesterone (Fig. 9.1C). This presents a most interesting situation in which the combined actions of two hormones on three closely associated structures of the reproductive tract join in a synergistic effort in promoting the growth of the uterus although progesterone prevents estrogen from affecting growth of the cervix and vagina.
These changes in gross morphology in response to the ovarian hormones are reflected in the histology of the responding tissues. Also, the character of the response differs depending upon the physiologic nature of the tissue concerned; therefore, for the sake of clarity each will be discussed separately. The first of these to be considered is the uterus and particularly growth of the endometrium.

It has been reported (Hisaw, 1935, 1950) that growth of the endometrium, as induced by estrogen, is limited. That is, a dosage of estrogen capable of maintaining the endometrium of a castrated animal for an indefinite period without the occurrence of bleeding, stimulates rapid growth for approximately the first 2 weeks. Within this time a maximal thickness of the endometrium is attained which remains constant or may become less during the course of treatment (Fig. 9.2). Engle and Smith (1935) made similar observations. They found that the endometria of castrated monkeys receiving estrogen for 100 days or longer were thinner than endometria of animals on estrogen for a much shorter time. Also, on prolonged treatment, the stroma of the endometrium becomes dense and the lumen small whereas the size of the uterus remains about the same. In fact, they state that in

I'll,. (1.2. I'll n Ml lour i-a.-^tra(c>d monkeys wliicli were givi^i 10 /ug. estradiol daily for 10 to 78 days. A was given estrogen for 10 days, B for 30 days, C for 60 days, and D for 78 days. Depression in the endometrium of anterior wall of D is the result of a biopsy taken a year previously. (From F. L. Hisaw, in A Si/niposium on Steroid Hormones, University of Wisconsin Press, 1950.)

ESTROGEN AND PROGESTERONE

561

four experimental animals the only well developed fundus was found in the animal on the shortest treatment, i.e., 60 days.
The mitotic activity in the epithelium of the glands and surface mucosa also indicates a limited effect of estrogen. This can be demonstrated to best advantage in the endometria of castrated monkeys that have been on estrogen for different lengths of time and have received an injection of colchicine 8 hours before their uteri were removed. A comparison of the number of cells in mitosis per square centimeter of surface mucosa at 10, 30, 45, and 60 days is shown in Figure 9.3. From this it can be seen that mitotic activity approaches that in a castrated animal. Although the five points used in drawing the curve are quite inadequate for an accurate analysis of the mitotic response in the epithelial components of the endometrium, they do show that cell division is most rapid soon after the beginning of an estrogen treatment and subseciuently declines.
The loss of responsiveness of the endometrium to estrogen seems related more to the length of treatment than to dosage of hormone. An endometrium of normal thickness can be produced in 2 or 3 weeks at a dosage level of estrogen that will not maintain the growth induced for longer than about 40 days without bleeding (Hisaw, 1935; Engle and Smith, 1935; Zuckerman, 1937b). The response to a low dosage of estrogen that will prevent bleeding during the course of treatment (about 10 /xg. estradiol-17/8 daily) is one of rapid endometrial growth at first, as has been described, followed by a thinning of the endometrium. The refractoriness of the endometrium to estrogen becomes so pronounced after about 100 days of treatment that very few cell divisions are seen in the epithelium of the glands and surface mucosa. The general morphology of the endometrium retains the characteristic appearance of the follicular phase of the menstrual cycle except that the stroma is usually more dense and the cells of the glandular epithelium have large deposits of glycogen between the nucleus and the basement membrane. However, metabolically such endometria are surprisingly inactive. Although they are dependent on the presence of estrogen and may bleed within about

Mitotic Response of Uterine Lpithelium TO looo I. u. Estrogen PER D«y.

Fig. 9.3. The number of mitoses per square centimeter of surface epithelium of the endometrium in a castrated monkey and in four castrated animals given 10 /xg. estradiol daily for 10, 30, 45, and 60 days, respectively. One-tenth of actual number of mitoses is shown on the ordinate. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)
48 hours if the treatment is stopped, the activity of their oxidative enzymes and the ratio of nucleoproteins (RNA:DNA) are about the same as in the involuted endometria of castrated animals.
The effects that accompany moderate estrogenic stimulation become exaggerated in several respects when large doses of estrogen are given for an extended period. The disparity between the area of myometrium and endometrium becomes greater as the treatment progresses (Fig. 9.4). Kaiser (1947) described the destruction of the spiraled arterioles of the endometrium in monkeys given large doses of estrogen and Hartman, Geschickter and Speert (1941) reported the reduction of the reproductive tract to the size of that of a juvenile animal by the end of 18 months during which injections of large doses of estrogen were supplemented by subcutaneous implantation of estrogen pellets. These observations not only show that the endometrium becomes unresponsive to estrogen when the treatment is prolonged but that large doses produce injurious effects.

562

PHYSIOLOGY OF GONADS

Fig. 9.4. .4. i'v'< — ' ri:,,ii of the uterus of a castrated monkey which had received 1.0 mg. estradiol daily for 35 days. Compare with B which shows the effects of 1/10 this dosage (100 fig. daily) when gi\en for 185 days.
The limited response of the endometrium to estrogen is in some respects surprising in view of its remarkable growth potentialities and regenerative capacity. These qualities were dramatically demonstrated by Hartman (1944) who dissected out as carefully as possible all of the endometrium from the uterus of a monkey and wiped the uterine cavity with a rough swab and yet the undetected endometrial fragments that remained were capable of restoring the entire structure. Also, considering the enormous increase in size of the uterus during gestation, it is even more difficult to account for the rather sharp limitation of growtli under the influence of estrogen.
The increase in tonus of the uterine musculature, a known effect of estrogen, has been considered as possibly exercising a restrictive influence on growth of the endo

metrium. An attempt has been made to remove this containing influence the muscle may have by making an incision through the anterior wall of the uterus (Hisaw, 1950) . A castrated monkey was given 10 ixg. estradiol daily for 21 days at which time the operation was performed and the treatment continued with 30 ^g. estradiol daily for 40 days. The uterus was laid open by a sagittal incision from fundus to cervix and most of the endometrium was removed from the anterior wall. This caused gaping of the incised uterus and exposure of the endometrium on the posterior wall. The incision was not closed and after hemorrhage was completely controlled the uterus was returned to the abdomen.
Examination of the uterus at the conclusion of the experiment showed no indications that endometrial growth had been enhanced. The muscularis had reunited and only a few small bits of endometrium were found in the incision (Fig. 9.5). It seemed probable that the purpose of the experiment had been defeated by rapid repair of the uterus. Therefore, a similar experiment was done in which the musculature of the incised uterus was held open by suturing a wire loop into the incision. Yet the incision closed and no unusual growth of the endometrium was detected (Fig. 9.6).
Observations under these conditions are necessarily limited to those made on the uterus when it is removed at the conclusion of an experiment and comparisons must be made between uteri of different animals. Obviously, it would be more desirable if the response of an individual endometrium could be followed during the course of treatment. It is possible to meet most of these requirements under conditions afforded by utero-abdominal fistulae, exteriorized uteri, and endometrial implants in the anterior chamber of the eye. In continuing our discussion we first shall present information obtained by such techni(iues that have a bearing on the response of the endometrium to estrogen.
The surgical procedure used by Hisaw ( 19501 for preparing utero-abdominal fistulae foi' studies of the exj^erimental induction of endometrial growth by estrogen and progesterone was a modification of that used by van Wagenen and Morse (1940) for ob

ESTROGEN AND PROGESTERONE

563

Figs. 9.5 and 9.6
The uteri of these two castrated monkeys were opened from fundus to cer\'ix by an incision through the anterior wall while the animals were receiving estrogen. Part of the endometrium of the anterior wall was removed and the incision in the myometrium was not closed.
Fig. 9.5. Estradiol, 10 fig., was given daily for 7 days; the uterus was opened and the animal continued on 30 /xg. estradiol daily for 40 days. (From F. L. Hisaw, in A Sy7nposiii7n on Steroid Hormones, University of Wisconsin Press, 1950.)
Fig. 9.6. Estradiol, 10 ^g., was given daily for 7 days; the uterus was opened and the treatment continued at a dosage of 30 /ig. estradiol daily for 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

serving changes in the endometrium (luring the normal menstrual cycle. This procedure makes frequent inspections possible either by hand lens or dissecting microscope, of most of the upper part of the endometrium on the anterior and posterior walls of the uterus. The elliptic slit formed by the endometrium of the two opposing walls can be located easily, the two sides pressed apart by any small smooth instrument, and the surface of the endometrium examined. Changes in thickness of the endometrium cannot be ascertained without resorting to biopsies but it is free to grow out of the opened uterus if it is so inclined. However, in such preparations the growth produced in the endometrium by daily injections of 10 fjig. estradiol for periods of 2 or 3 weeks is not sufficient to show any tendency whatever to grow out through the fistular opening or obstruct examination of the walls of the uterus. The limited growth observed in these experiments is in agreement with that obtained with estrogen on intact and incised uteri.
The cervix uteri of the rhesus monkey is sufficiently long to make it possible to bring the entire fundus to the exterior through a midal)dominal incision. Advantage of this

was taken in an attempt to exteriorize the uterus and maintain it outside the body for long enough periods to make it possible to study the growth responses of the endometrium (Hisaw, 1950). These preparations did not i)rove satisfactory in all respects but they did contribute a number of interesting observations.
The operational i^rocedure used in these experiments involved dividing the uterus transversely from fundus to cervix so that the anterior wall was deflected downward and the posterior wall upward (Fig. 9.7). The endometrium of the exteriorized uterus is difficult to maintain but with proper care it seems to retain its normal condition for at least the first few days after the uterus is opened. Small localized areas of ischemia can be seen to come and go, probably action of the coiled arteries, and there is a periodic general blanching of the endometrium associated with rhythmic contractions of the muscularis. This, however, does not seem true of the whole endometrium. A zone surrounding the internal os of the cervix tends to retain its blood-red color even during strong contractions of the uterus and the growth reactions of the endometrium in this area are of particular interest.

564

PHYSIOLOGY OF GONADS

Fig. 9.7. Exteriorized uteii. The uteri were divided transversely from fundus to cervix. The anterior half is seen deflected to the right and the posterior half to the left. A and B are of the same uterus taken 13 days after exteriorization showing the "blush" and "blanch" reaction of uterine contractions. It can be seen that during blanching the endometrium of the cervix does not become ischemic. C. Uterus 18 days after exteriorization, showing response to estrogen. Ridges formed by growth of the endometrium surrounding the internal OS of the cervix can be seen at the upper edge of the photograph. D, taken 83 days after exteriorization, shows response produced by a series of injections of 10 ^ig. estradiol and 1 mg. progesterone daily. The transverse ridge is formed by the two opposed lips of endometrium derived from the area surrounding the internal os of the cervix. Growth when the two hormones are given is greater than when only estrogen is injected. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

The endometrium on the exposed anterior and posterior halves of the uterus underwent deterioration despite the best of care that could be given, but that surrounding the internal OS of the cervix survived and retained its capacity to grow, in one animal, for as long as 9 months. When estrogen was given this endometrium grew rapidly and within a few days stood out as large elliptic lips surrounding the internal os (Fig. 9.7). Within 2 to 3 weeks the lips appeared to reach their full size and further growth was slow or absent. When estrogen treatments were discontinued the endometrial lips underwent bleeding within a few days and were entirely lost. At no time were activities observed that could be ascribed to coiled arterioles, nor did ischemia occur during involution previous to bleeding. It seems that the response of this tissue to estrogen is like that found in other experiments but the absence of ischemia preceding bleeding is ex

ceptional. The endometrium on the anterior and posterior walls of uterine fistulae invariably showed ischemia for several hours before active bleeding following the withdrawal of estrogen.
Markee (1940) approached the problem of endometrial growth in monkeys by studying the changes that occur in bits of endometrial tissue transplanted to the anterior chamber of the eye. Such transplants retain in large measure the normal morphology of endometrial tissue and changes in their cyclic growth parallel those going on simultaneously in the uterus. So much so that if the animal has an ovulatory cycle, the ocular implants show conditions characteristic of both the follicular and luteal phases, but if ovulation fails to occur then the luteal phase is omitted. Also, the morphologic events taking place at menstruation can be seen and recorded, since the transplants regress and bleed at each menstrual period.

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These ingenious experiments will be referred to often in the course of our discussion but at present the response of endometrial transplants in the eye to estrogen is of primary interest.
Monkeys having ocular transplants were given 200 to 300 R.U. of estrone daily for about 1 to 3 months. The transplants did not grow to a certain size and then remain stationary, but instead periods of rapid growth were interrupted by periods of regression which usually involved a marked decrease in size, and if regression was extensive and rapid, bleeding ensued. It also was found that these episodes of regression in the transplants were usually accompanied by a decrease in the size of the uterus.
Comparisons between the results of these experiments and those we have discussed previously may be misleading since it seems that only 1 of the 5 animals (no. 295) used was castrated. Also, the dosage of estrogen was not sufficient to maintain the endometrium of the uterus for an indefinite period without bleeding and this also was reflected in the transplants. It seems questionable that the growth capacity of endometrial transplants in the eye can be determined unless sufficient estrogen is given to prevent bleeding in the uterus. Therefore, it would seem that these experiments contribute less to an analysis of the effects of estrogen on endometrial growth than they do to an understanding of the events that precede and accompany menstruation.
In summary, it seems clear that the outstanding effect of estrogen on the uterus of the monkey is one of growth (Allen, 1927, 1928). The involuted uterus of a castrated animal can be restored to its normal size in 2 or 3 weeks by daily injections of adequate amounts of estrogen. At this time there is an increase in vascularity, a clear-cut hyperemia as seen in rodents. There also is secretion of luminal fluid (Sturgis, 1942) but this does not distend the uterus as in the mouse and rat. This is accompanied by an increase in tissue fluid, especially in epithelial tissues (surface epithelium and glands) , and in the connective tissue of the stroma. Glycogen may be present at the basal ends of epithelial cells beneath the nuclei (Overholser and Nelson, 1936) but it apparently is not readily released under the action of estrogen

alone (Lendrum and Hisaw, 1936; Engle and Smith, 1938) . The glands of the endometrium maintain a straight tubular structure with some branching near the muscle layers. The condition produced experimentally in the monkey's uterus by short term treatments with estrogen is equivalent to that present in the normal animal at midcycle, or even a few days later if ovulation does not occur.
If, however, an estrogen treatment is continued for several months conditions develop in the uterus that are not found during the follicular phase of a normal menstrual cycle. When the daily dose of estrogen is small menstruation occurs at intervals during the treatment (Zuckerman, 1937b) and probably marks periods of endometrial regression as observed by Markee (1940) in eye transplants, but if the dosage is increased by a sufficient amount (about 10 /xg. estradiol- 17/3 daily) injections may be continued for a year or longer without bleeding. Although the size of the uterus remains within the range of normal variation as the injections are continued, the myometrium tends to increase in thickness and the endometrium becomes thinner, a condition not corrected by further increases in dosage or by prolonging the treatment. The cause responsible for the limited response of the endometrium under these conditions is not known but apparently is not a restrictive influence of the myometrium as similar responses are given when the endometrium is exposed by incising the uterus, in abdominal fistulae, and in exteriorized uteri.
III. Effects of Progesterone on the Uterus
It has been mentioned that a menstrual cycle, in which ovulation occurs, can be conveniently divided into a follicular and a luteal phase. The follicular phase extends from menstruation to ovulation and the luteal phase from ovulation to the following menstruation. It has been shown in the previous discussion that the endometrial modifications characteristic of the follicular phase of the cycle can be duplicated in a castrated monkey by the injection of estrogen. Likewise, the progestational condition characteristic of the luteal phase can be developed by giving progesterone. In fact, all

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PHYSIOLOGY OF GONADS

the morphologic and physiologic features that are known for anovulatory and ovulatory cycles can be reproduced in castrated monkeys by estrogen and progesterone.
If one designs an experiment to simulate the normal cycle in a castrated monkey then estrogen should be given first to develop the conditions of the follicular phase followed by progesterone for the progestational development of the luteal phase. Experience has shown that this is the most effective procedure for the production of a progestational endometrium. Progesterone, as compared with estrogen, is a weak growth jH'omoter and although it can produce progestational changes in the atrophic endometrium of a castrated monkey when given in large doses, its action is greatly facilitated when preceded by estrogen. The first experiments in which progesterone was used for this purpose were planned on this principle (Hisaw, Meyer and Fevold, 1930; Hisaw, 1935; Engle, Smith and Shelesnyak, 1935).
The first noticeable effect of progesterone is an elongation of the epithelial cells of the surface membrane and necks of the glands. When the treatment is continued, this effect progresses down the gland towards the base. This change is followed closely by a rearrangement of the nuclei which is more pronounced in the glands than in the surface epithelium. The nuclei under the influence of estrogen in doses which reproduce the conditions of the follicular phase of a normal cvcle, are situated niostlv in the basal

Fig. 9.8. Uterus of a castratefl monkey which was given 2 mg. progesterone daily tor 113 days. The endometrium is thin but bleeding occiu\s when such treatment is stopped. The myometrimn is soft and pliable and ilir l)lood vessels are cnlarsed and have thick wails.

half of the cells, some of them touching the basement membrane. The nuclei retreat from the basement membrane when progesterone is given leaving a conspicuous clear zone. This zone is produced by intracellular deposits of glycogen. These early changes usually appear before pronounced spiraling and dilation of the glands.
Secretion begins in response to estrogenic stimulation and increases greatly as progestational changes are established. It appears first in the necks of the glands and progresses basalward. The surface epithelium takes a less conspicuous part in secretion and is usually reduced to a thin membrane when injections of progesterone are continued until a fully developed progestational endometrium is established. This progressive action of progesterone is such that it is possible to find all conditions in a single gland from active secretion and fraying in the neck region through primary swelling to an unmodified condition at the base.
When treatment is continued for 25 to 30 days at doses of about 2.0 mg. daily, the glands enter a state that has been called "secretory exhaustion" (Hisaw, 1935). This condition also is seen first in the necks of the glands and progresses toward the base. The glandular epithelium decreases in thickness, and active secretion, as judged by fraying of the cells, is absent. The glands may become narrow and straight and the endometrium may resemble that in castration atrophy. These involutionary changes become even more pronounced if the treatment is continued for several months or a year (Fig. 9.8). The endometrium by this time is extremely thin. The glands are straight, short, and narrow, and the stroma very dense. The myometrium is thick in proportion to the endometrium and the uterine blood vessels are large and have greatly thickened walls. Such uteri tend to be somewhat smaller than normal and are soft and pHable.
Thus, it is seen that when growth is produced in the endonietiiuni of a castrated monkey by giving estrogen and then continued on injections of progesterone, there follows a sequential development of all stages of the luteal phase of a normal menstiual cycle terminating in secretory exhaustion. However, this condition cannot

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be maintained by continuing the progesterone treatment, and involutionary processes set in and the endometrium is reduced to a thin structure. Yet, such degenerate endometria are dependent upon progesterone and will bleed within about 48 hours if the injections are stopped. It also was found that after discontinuence of progesterone daily injections of 10 /i.g. estradiol may not prevent bleeding.
IV. Synergism between Estrogen and Progesterone
There is considerable evidence that in primates progesterone under normal conditions rarely if ever produces its effects in the absence of estrogen. Large quantities of estrogen are present in human corpora lutea (Allen, Pratt, Newell and Bland, 1930) and during pregnancy the placenta secretes estrogens as well as progesterone (Diczfalusy, 1953) . This apparently is a common feature of primates, as indicated by the excretion of estrogens in the urine of pregnant chimpanzees and rhesus monkeys (Allen, Diddle, Burford and Elder, 1936; Fish, Young and Dorfman, 1941 ; Dorfman and van Wagenen, 1941). Also, correlated with this is the observation that estrogen and progesterone when given concurrently produce a greater effect on the uterus of castrated monkeys than either alone (Hisaw, Greep and Fevold, 1937; Engle, 1937; Hisaw and Greep, 1938; Engle and Smith, 1938) and that an ineffective dose of progesterone is greatly potentiated by estrogen. This synergistic effect of the two hormones on the uterus of monkeys is quite different from their action on the uteri of laboratory rodents and rabbits. In these animals the effects of progesterone can be inhibited quite easily by a surprisingly small dose of estrogen (see chapter 7).
The synergism between estrogen and progesterone in the promotion of endometrial growth can be demonstrated to best advantage under the conditions of some of the physiologic preparations that have been discussed. For instance, it was shown (Fig. 9.5) that growth of the endometrium under the influence of estrogen was not enhanced by relieving muscle tension by a midline incision through the anterior wall of the uterus. Now, if a similar operation is per

FiG. 9.9. Uterus of a castrated monkey that received 10 fig. estradiol and 1 mg. progesterone daily for 18 days, at which time the uterus was opened from fundus to cervix and most of the endometrium of the anterior wall removed. The incision was not closed and the treatment was continued for an additional 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)
formed on the uterus of a monkey that is receiving 10 /tg. estradiol daily and the treatment continued with the addition of a daily dose of 1 mg. progesterone, there usually follows a rapid growth of endometrial tissue out through the incision until by about 3 weeks a mass is formed which approximates the size of the entire uterus (Fig. 9.9). If this experiment is repeated and the same dosage of progesterone is given without estrogen, there is no outgrowth of the endometrium (Fig. 9.10).
A similar synergistic action can be seen in utero-abdominal fistulae. We have mentioned that estrogen does not cause excessive growth of the endometrium under these conditions. However, endometria that have reached their maximal response to estrogen will show a resumption of growth if 1 or 2 mg. progesterone are added daily to the treatment. By the 4th or 5th day lobes of blood-red endometrium begin to protrude

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PHYSIOLOGY OF GONADS

Fig. 9.10. Uteru.s of a castrated monkey which was given 1 mg. progesteione (lail>' for 18 days following an estrogen treatment. The uterus was opened as described for Figure 9.9, and the injections of progesterone continued for 20 days. (From F. L. Hisaw, in A Syiyiposium on Steroid Hormones, University of Wisconsin Press, 1950.)
through the opening of the fistula. Within a few days tongue-like processes of endometrial tissue are thrust out of the opening with each uterine contraction and are entirely or i^artially withdrawn at each relaxation.
Such outgrowths are difficult to protect from mechanical injury and consequent tissue loss so it is not possible to determine accurately how much endometrium is produced in a given time. In one experiment an animal was kept on 10 fxg. estradiol and 2 mg. progesterone daily for 98 days and it was found that the endometrium continued to grow, but the rate seemed considerably slow^er toward the conclusion of the treatment than at the beginning. How long an endometrium would continue to grow under these conditions was not determined, but it is obvious that much more endometrial tissue was produced by the treatment than is ever found at one time in the uterus of a monkey during a normal menstrual cycle. This takes on added significance when it is compared with the endometrial response in the intact uterus of an animal given the same dosage of estrogen and progesterone for a similar length of time.
The progestational development of the endometrium, when both hormones are given, passes through the same stages as those following the injection of only progesterone; i.e., presecretory swelling of the glandular epithelium, active secretion, and

secretory exhaustion. The endometrium, however, is considerably thicker than when a comparable dose of progesterone is given alone, and secretory exhaustion may not be so pronounced by the 30th day (Fig. 9.11). The glandular epithelium in the necks of the glands may be reduced to a thin membrane scarcely thicker than the nuclei whereas some secretion is usually present in the dilated basal parts of the glands. Also dilation of the glands in the basalis is more pronounced following a 30-day estrogen-])rogesterone treatment than when the same amount of progesterone is given separately.
Secretory exhaustion appears to be the initial indication of an involutionary process that ensues when an estrogen-progesterone treatment is continued for a long time (Hisaw, 1950). When a combination of the two hormones, known to be capable of producing a large uterus with a thick, fully develojied, progestational endometrium within al)out 20 days, is given for 100 days, an astonishingly different endometrium results (Fig. 9.12). It is thin, the stroma is dense and the narrow straight glands are reduced to cords of cells in the basal area. The condition is one suggesting inactivity and atrophy.
When such dosages of estrogen and i)rogesterone are given to castrated monkeys for 200 days or a year further changes in the endometrium occur. By 200 days the epithelium of the surface mucosa and glands

Fk;. 9.11, .\ late i)r()ges1ati()iial condition produced in the endometrium of a castrated monkey by giving 10 (ig. estradiol daily for 18 days followed by 10 /xg. estradiol and 2 mg. progesterone daily for 31 davs.

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Fig. 9.12. The endometnuin of a castrated monkey that had received 10 /xg. estradiol and 1 mg. progesterone daily for 99 days.

is lost except for small glandular vestiges along the musciilaris at the base of the endometrium. There are no glands, coiled arteries, or large blood vessels in what one might yet call the functionalis. All that remains is a modified stroma that resembles decidual tissue (Fig. 9.13.4 and B). It is also of interest that these endometria will menstruate if the treatment is discontinued and in most if the injections of progesterone are stopped and estrogen continued, but not if estrogen is stopped and progesterone continued.
Even though in such experiments the endometrium has been under the influence of both estrogen and progesterone for a year and has undergone extremely abnormal modification, it yet is capable of responding to estrogen in a more or less characteristic way when progesterone is stopped and in

jections of estrogen continued. Apparently within about three weeks the modified endometrium is replaced, under the influence of estrogen, by one that has few glands which tend to be cystic, a mesenchymatous stroma, and no coiled arteries (Fig. 9.14).
Under similar circumstances, if estrogen is stopped and jjrogesterone is continued, the modified endometrium is lost without bleeding and there is almost no repair of the endometrium even after a period of 3 weeks. There seems to be an incompatability between the epithelial outgrowths from the mouths of the glands and the underlying stroma of the denuded surface. Consequently the epithelium crumbles away and epithelization of the raw surface is not accomplished (Fig. 9.15j. How long this condition could continue has not been determined.

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Fk;. 9.13. The endometrium shown m .4 is (h;i( from a castraled mdiik.N wlml, l,a,l received 10 /xg- estradiol and 2 mg. progesterone daily for 200 days. In B, jiart of ilie endometrium of a snndai animal given the same treatment for 312 days is shown at a higher magnification. The endometrium is almost entirely a modified stroma in which glandular epithelium and coiled arteries are absent. Only vestiges of glands are present in the basal area next to the myometrium.
One of the most interesting aspects of these observations is that these effects were jiroduced by dosages of estrogen and progesterone that are very probably within the range of normal physiology. From this it appears that although growth of the endometrium is greater when the two hormones are given together, due to their synergistic interaction, this does not prevent involutionary changes from setting in when the treatment is continued for a period of weeks or months. In fact, greater damage to the endometrium occurs under the simultaneous action of the two hormones than when either is given alone. Also, increasing the dose intensifies the damaging action of both estrogen and progesterone, so much so that very large doses will almost completely destroy the endometrium.
The myometrium, however, shows a different response to these treatments. Estrogen stimulates myometrial growth, which is

Fir;. 9.14. Uterus of a castrated monkey which was given 10 ixg. of estradiol and 2 mg. progesterone daily for 307 days at which time the injections of progesterone were stopped and estrogen continued for 20 days. Bleeding occurred the second day following discontinuance of progesterone. The absence of coiled arteries and the presence of cystic glands and a mesenchymetous stroma characterize the endometrium.

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Fig. 9.15. Uterus of a castrated monkey which was given 10 yug. estradiol and 2 mg. progesterone daily for 275 days at which time estrogen was stopped and progesterone was continued for 21 days. A shows the thin endometrium and dense stroma whereas B shows failure of formation of a surface epithelium following the loss of the modified functionalis presumably present at the conclusion of treatment with both hormono.^^ (see Fig. 9.13).

intensified both by cln-onic treatment and high dosage, and seems to be equally effective when it is given alone or in combination with progesterone. Progesterone also promotes growth of the muscularis but seems less effective than estrogen and differs from it by causing pronounced thickening of the walls of the arcuate blood vessels. These vascular changes extend to the coiled arteries of the endometrium, which are also affected by high dosages of estrogen. It seems remarkable that estrogen is capable of preventing the action of progesterone on the myometrial blood vessels and correcting such effects after they are produced and yet at the same time it assists in the destruction of the coiled arteries in the endometrium.
V. Experimentally Produced Implantation Reactions
Progestational endometria of the normal menstrual cycle or those produced in castrated monkeys by progesterone, if mechanically traumatized, will develop endometrial proliferations which seem identical with those found at normal implantation sites of fertilized ova (Figs. 9.16 and 9.171 (Hisaw,

1935; Hisaw, Creep and Fevold, 1937; Wislocki and Streeter, 1938; Rossman, 1940). The proliferated cells originate from the surface and glandular epithelium and grow into the surrounding stroma. The reaction spreads from the point of injury and within a few days may involve the entire inner l)ortion of the endometrium bordering the lumen. The implantation plaques on the 3rd or 4th day present a fairly homogeneous appearance but soon thereafter certain cells attain the proportions of giant cells and many are multinucleated.
The development of the plaques is most rapid during the first week, by the end of which cell division is found only in the basal half of the proliferation and evidence of regression is seen in the superficial portion adjoining the uterine lumen. After 10 days degenerative and phagocytic processes are the dominant features and by 24 days the ut'prus contains few or no ]iroliferation cells. Wislocki and Streeter ( 1938,1 found that implantation plaques during pregnancy and those experimentally induced underwent ajjl^roximately the same development arid subsequent degeneration except for modifications produced by the invading troplio

PHYSIOLOGY OF GONADS

~~^a.~~

~~^^--.^~~

tx: ^ .

Fig. 9.16. An area of the normal implantation site of a developing ovum. (From Carnegie Institution, No. C467.)

Fig. 9.17. An experimentally induced implantation reaction in a castrated monkey showing condition 6 days after mechanical traumatization of the endometrium.

blast. Rossman (1940j made an extensive morphologic study of these epithelial proliferations and concluded that they should be regarded as typical metaplasias \vith an embryotrophic function.
VI. The Cervix Uteri
The cervix uteri of the rhesus monkey is remarkable for its size and complexity. It forms a large segment that is set off from the fundus by a conspicuous constriction at the level of the internal os (Fig. 9.1). A sagittal section (Fig. 9.18) shows the cervical canal not straight but thrown into several sharp turns by colliculi that extend from its walls into the lumen. The largest of these projects from the midventral wall. The functional advantage of such tortuosity of the cervical canal is not obvious but since the cervix probably serves as a barrier between the bacterial flora of the vagina and the corpus uteri, this may be a useful adaptation.
The physiology of the cervix has received much less attention than has been given the uterus. This is regrettable in view of the consideration it must receive in practical obstetrics and gynecology, as well as the possibility that physiologically the monkey cervix may be homologous with that of the human regardless of morphologic difTerences. Recent observations indicate that this is indeed quite probable.

Fig. 9.18. Sagittal section of the cervix from a normal monkey. The vagina and the external os of the cervix are shown at the left and the entrance to the fundus is at the right.

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Fig. 9.19. Sagittal section of the cervix of a pregnant monkey showing conditions present just previous to parturition on the 154th day of gestation. The dominant features are dilation of the cervical canal and reduction of the cervical lips (shown at the left) and the coUiculi. (From Carnegie Institution, No. C713.)

Hamilton (1949) made a detailed study of the changes in the cervix of rhesus monkeys during the menstrual cycle, paying particular attention to alterations that took place in the cells of the surface epithelium of the endocervical canal and the cervical glands. It was found that heights of the cells showed consistent increases and decreases during the cycle. The peaks came on the 3rd, 13th to 15th, and 22nd days, the greatest of these being the 14th day which is approximately the time of ovulation. It also was observed that, following a peak, secretion was associated with the decline.
Attention was called b3^ Hamilton to the rather close correlation between the fluctuations in height of the cervical epithelium in monkeys and the fluctuations observed by Markee and Berg (1944) in the blood estrogens of the human menstrual cycle. It was concluded that, if similar changes in estrogen levels also occur in monkeys, one would be justified in concluding that the increase in cell height in the cervical mucosa was due to the action of estrogen and the sudden periodic drops in blood estrogen caused secretion and consequent regression. However, it is not clear how this could account for the abundant secretion of the cervical glands in

the presence of high levels of estrogen during late pregnancy (Fig. 9.19).
Much has been learned regarding the physiology of the primate cervix from experiments on castrated monkeys. The cervical mucosa is very responsive to estrogen and castration atrophy can be repaired and a normal condition maintained by daily injections of small doses. Cervical secretion may become abundant when an estrogen treatment is prolonged and especially if large doses are injected. However, the amount of secretion induced by estrogen never equals that of the last half of pregnancy, and it usually subsides if the injections are continued for several months.
Under conditions of chronic treatments with estrogen metaplastic aberrations invariably appear in the epithelium of the endocervix. This reaction was first reported in monkeys by Overholser and Allen ( 1933, 1935) and has been confirmed by many investigators (Engle and Smith, 1935; Hisaw and Lendrum, 1936; Zuckerman, 1937c (. Similar lesions may be found in the cervix uteri of women (Fluhmann, 1954). They seem especially prone to occur under conditions characterized by excessive production of estrogen, such as hyperplasia of the

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PHYSIOLOGY OF GONADS

endometrium (Hellman, Rosenthal, Kistner and Gordon, 1954) and granulosa-cell tumors of the ovary. Various degrees of metaplasia may occur in the cervix during pregnancy both in the mother and newborn but Fluhmann (1954) did not find it as frequently as in nonpregnant women.
This reaction to estrogen as seen in the cervix of castrated monkeys is initiated by growth of small undifferentiated cells below the columnar mucous cells of the secretory epithelium. Fluhmann (1954) suggests that these cells are really undifferentiated cells of the cervical mucosa which have the potentiality of becoming columnar or squamous or simply undergoing multiplication and remaining as indifferent or reserve cells. These cells accumulate, in response to estrogen, to form aggregates of several cells in thickness and, although this may occur in any area of the endocervix, it is generally more pronounced below the base of the glands. As this process proceeds the columnar mucous cells are pushed outward and are finally desquamated thus exposing the underlying metaplastic cells to the lumen of the gland (Fig. 9.20).

Fig. 9.20. Al.iaph

in a castrated monkey that li.-id rci-civcd 1 nig. estriol daily for 48 days.

The cells of these lesions undergo a characteristic differentiation. When first formed they are small, cuboidal, and have spherical nuclei with dense chromatin. As they increase in number those in the center of the cellular mass become larger and acquire an eosinophilic cytoplasm. Such collections, as seen at the base of the cervical glands, may grow in height and form cone-shaped masses with the apexes protruding through the mucous epithelium into the lumen or they may remain as more or less compact structures. This difference in growth seems to have a general relation to the dosage of estrogen. Large doses cause more rapid growth and cone formation with the loss of cells from the apex either singly or in groups, whereas small doses produce slower growth and desquamated cells are seldom seen in the lumen. However, regardless of the rate of growth, the cells at the base of the lesion remain undifferentiated and continue as the principal area of cell proliferation.
Pearl formation is occasionally seen and may be quite common in animals on low dosages of estrogen. Under strong estrogenic stimulation and consequently rapid growth, these structures apparently are desquamated before they are completely formed. However, very early stages are frequently seen and may even be present in small clumps of metaplastic cells, but they are more commonly found in the larger collections at the base of the glands. Their appearance is initiated by swelling and disintegration of one or more adjacent cells that form a center around which epidermidization takes place. Further development does not proceed under the influence of esti'ogen, beyond the formation of a small central cavity.
The most conspicuous difference between the metaplastic growths produced by estrogen and true cancer of the cervix in the monkey (Hisaw and Hisaw, Jr., 1958) is that the former remain noninvasive even when the treatment is continued well over a year. They also involute when the treatiiiciit is discontinued and they do not appeal' when progesterone is given simultaneously with estrogen. When the injections of progesterone are started after metaplastic growths have been formed in response to estrogen, further growth is inhibited and

ESTROGEN AND PROGESTERONE

the keratinized cells of the lesion become vacuolated and are lost.
In contrast with the effects of estrogen on the cervix, the modifications that occur as pregnancy advances are remarkable. The cervix becomes a soft thin-walled structure, the glands increase in number, and their lumina become greatly enlarged, pressing the stroma into thin partitions between them, and the amount of mucus secreted is enormous (Fig. 9.19). Attempts at duplicating these changes in castrated animals by hormone therapy have been only partially successful. Estrogen produces a solid thickwalled cervix that tends to be larger than normal, an effect that is especially noticeable in young animals. Progesterone does not promote cervical growth and repair of the glands unless large doses are given and even then there is little if any secretion. The best results were obtained when both estrogen and progesterone were given and especially so when relaxin was added to the treatment (see chapter by Zarrow).
VII. The Vagina
The general features of the vaginal smear of rhesus monkeys have been described by several investigators (Allen, 1927; Hartman, 1932; Westman, 1932) and a detailed study of the cellular components at different times of the menstrual cycle has been made by Lopez Columbo de Allende, Shorr and Hartman (1945). The changes in the vagina of a monkey are in most respects like those found for the human being (Papanicolaou, Traut and Marchetti, 1948; Lopez Columbo de Allende and Orias, 1950). Epithelial growth and desquamation of cornified cells continue at all stages of the cycle but at various rates. The epithelium is thinnest at menstruation and gradually increases in thickness during the follicular phase, reaching a maximum at ovulation. At this time there is a well developed basal area in which numerous mitoses can be seen and from which many papillae or bulbs" extend into the underlying stroma. Above this is an intermediate zone, an interepithelial zone of cornification (so called Dierk's layer), and a heavily cornified outer zone (Fig. 9.21).
Cellular proliferation is less rapid during the luteal phase and apparently cells are

desquamated more rapidly than they are replaced. Consequently there is a decrease in the thickness of the epithelium in the luteal phase which may include an almost complete loss of the cornified zone (Davis and Hartman, 1935). The effects are probably due to progesterone because similar changes are seen following the introduction of progesterone into a treatment in which estrogen is being given.
The vaginal epithelium of a castrated monkey is remarkably sensitive to estrogen. A small daily dose of 5 to 10 /xg. estradiol will stimulate growth of an atrophic epithelium of 4 to 8 cells in thickness to one of 60 or even 80 layers thick within 3 weeks. One of the first things that is noticed as the vaginal epithelium thickens is the numerous mitotic figures in the stratum germinativum followed by a marked increase in the number of epithelial papillae along the basement membrane. This condition of rapid growth, cornification, and loss of cells into the vaginal lumen is typical of the follicular phase of the menstrual cycle and can be maintained indefinitely.

li(.. U.21. the vaginal epithelium of a castuUMJ monkej' showing growth antl cornification induced by estrogen.

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PHYSIOLOGY OF GONADS

Progesterone, in contrast with estrogen, does not produce rapid growth of the vaginal epithelium but at the same time it is not without an effect. The vaginal epithelium, weeks or months after castration, has relatively few papillae projecting from its basal border into the underlying stroma. When progesterone is given, this condition is changed but not in a spectacular way. There is very slow growth without cornification. The epithelium remains thin but the papillae become more numerous. These are mostly small epithelial buds which tend to remain solid but may show enlargement of the cells in their centers.
When estrogen and progesterone are given concurrently, the effects of estrogen on the vaginal mucosa are modified. If an estrogen treatment has continued for a sufficient time to produce full cornification and then progesterone is added, the first indication of an inhibition of estrogen is a decrease in mitotic activity. This is followed by a continuation of cornification and loss of cells faster than they are replaced ; consequently, most of the functionalis is lost and the epithelium becomes thinner. There is also a noticeable decrease in the intensity of cornification, which in the monkey is never as pronounced as in rodents, and under these conditions is quite incomplete, each cell retaining a conspicuous nucleus. Partly cornified cells may be present for several weeks

.-•^ss;^

4* ' V ^ §

Fig. 9.22. ^^•tgi^al epithelium of a pregnant monkey showing condition on the 154th day of gestation. (From Carnegie Institution, No. C713.)

when both estrogen and progesterone are given, but eventually they almost entirely disappear and the epithelium attains a condition resembling that of late pregnancy.
The inhibitory effect of progesterone on the action of estrogen is shown perhaps even better when a castrated monkey having a fully involuted reproductive tract is first given progesterone for a few days and then (>strogen is added to the treatment, or when injections of the two hormones are started at the same time. In such experiments estrogen has little effect on the vaginal mucosa even in doses that would produce marked cornification if given alone. These observations show that a fully cornified vaginal epithelium cannot be produced or maintained by estrogen when an effective dosage of progesterone is included in the treatment (Hisaw, Greep and Fevold, 1937).
Estrogens and progesterone are the dominant hormones of gestation and their simultaneous action is reflected by the changes in the vaginal epithelium. The fully cornified vagina, present at the time of ovulation, is gradually modified as pregnancy progresses into a condition strikingly like that seen in experiments when estrogen and progesterone are given concurrently. In late pregnancy the most striking feature of the thin, uncornified epithelium is the presence of numerous epithelial buds extending deeply into the underlying stroma. They may branch and rebranch and along their course there is conspicuous enlargement of the more centrally situated cells among which cavities ai^pear, enlarge, and join each other (Fig. 9.22). It seems quite probable that this process may be of considerable importance in increasing the diameter of the vagina.
VIII. Sexual Skin
A so-called sexual skin is jiresent in most catarrhine monkeys, is not found in platyriliine monkeys, and among the anthropoids occurs regularly only in the chimpanzee I l^]ckstein and Zuckcrman, 1956). Changes in the sexual skin during the menstrual cycle have been observed most extensively in the monkey (Macaca), the baboon (Papio), and the chimpanzee (Pan). The sexual skin of t!ie baboon and chimpanzee undergo jiro

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nounced swelling during the follicular phase of the cycle. A maximal size is attained by the middle of the cycle followed by a rapid regression and loss of edema which at least in the baboon is associated with a marked increase in the output of urine (Gillman, 1937a; Krohn and Zuckerman, 1937). The subsidence of the sexual skin begins approximately at the time of ovulation and remains in the reduced condition throughout the luteal phase, followed by a subsequent initiation of swelling during or soon after menstruation (Zuckerman, 1930, 1937e; Zuckerman and Parkes, 1932; Gillman and Gilbert, 1946; Young and Yerkes, 1943; Nissen and Yerkes, 1943).
A w^ell developed sexual skin is present in the monkey {Macaca mulatta) only during adolescence. With the appearance of the menstrual cycles the sexual skin undergoes a process of maturation into the adult condition in which cyclic changes in edema are absent and the most noticeable feature is a vivid red color. Such coloration is due to vascular engorgement rather than pigment (Collings, 1926) and involves the perineum, the buttocks, and may extend for various distances down the legs and over the symphysis pubis. The development and maturation of the sexual skin have been described in considerable detail by several investigators (Hartman, 1932; Zuckerman, van Wagenen and Gardiner, 1938) .
The sexual skin has been of considerable interest both as to the nature of its responsiveness to ovarian hormones and the manner in which its grossly visible changes during the menstrual cycle parallel events occurring in the reproductive tract. The sudden loss of edema at the conclusion of the follicular phase not only signals ovulation but also raises the question as to whether the loss of tissue fluid is due to a decrease in estrogen or is the direct effect of progesterone. The importance of this becomes obvious when it is considered that a similar process also goes on simultaneously in the endometrium and raises the question again as to the respective roles played by estrogen and progesterone in endometrial growth and menstruation.
That the development and edema of the sexual skin of adolescent rhesus monkeys depend on the ovaries was first demon

strated by Allen ( 1927 ) . Involution and loss of color follow castration, and the normal condition can be restored by the injection of estrogen. Also, when estrogen treatment is continued for several weeks maturation of the sexual skin occurs and a condition characteristic of that in the adult is established (Zuckerman, van Wagenen and Gardiner, 1938). The genital area loses its edema and develops a brilliant red color which is retained as long as estrogen is administered. Once this mature condition is established the response of the sexual skin to subsequent estrogen treatments is limited to a change in color.
Similar experiments have been performed on the chacma baboon, Papio porcarius (Parkes and Zuckerman, 1931; Gillman, 1937b, 1938, 1940a). The large sexual skin of these animals is very responsive to estrogen and development equal to that of the follicular phase of the menstrual cycle can be readily induced by daily injections for about 2 weeks. However, the perineal swelling of the baboon differs from the sexual skin of the genital area of the rhesus monkey in that it does not "mature" under the influence of estrogen.
When large doses of estrogen are given to a rhesus monkey a generalized edema of the skin occurs beyond the genital area. This first appears as deeply indented swellings along the sartorii from groin to knee, and next appears at the base of the tail and spreads gradually upward until it involves the entire dorsal portion of the trunk. At the same time, the skin of the face, scalp, and supraorbital ridges becomes swollen and finally the edema may extend out on the arms and down the legs to the ankles (Bachman, Collip and Selye, 1935; Hartman, Geschickter and Speert, 1941). A daily dose of 500 /xg. or more of estriol or estradiol w^ll produce this condition within 2 to 3 weeks and, when the treatment is continued for an extended period the effect tends to subside.
Progesterone has a strong inhibitory action on the effects produced by estrogen on both the genital and extragenital sexual skin of the monkey. If daily injections of progesterone are added to the treatmeiu after full development of the sexual skin has been induced by estrogen, there is a

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noticeable loss of edema by the 4th or 5th day followed by rapid involution and reduction of the turgid folds of skin to loose, flabby wrinkles within about 10 days. When estrogen and progesterone are given concurrently to a castrated monkey from the beginning of treatment edema does not appear but the sexual skin regains its normal color. In fact, progesterone alone, like estrogen, can restore the color to the sexual skin of castrated adult monkeys (Hisaw, Greep and Fevold, 1937; Hisaw, 1942).
The interaction of estrogen and i)rogesterone on the sexual skin of rhesus monkeys can best be demonstrated by the reaction of the skin of the sexual area in adolescent animals. The most striking effect and probably the most important is the sequence of events initiated by a single dose of progesterone when given to an animal on continuous estrogen treatment. Under such treatment a full response of the sexual skin is obtained by the end of 20 days. If at this time 1 mg. progesterone is given in a single dose and the estrogen treatment continued uninterruptedly, the first indication of an effect of the luteal hormone is a slight loss of edema and color of the sexual skin on the 4th or 5th day thereafter. The sexual skin is markedly reduced by the 8th day, almost gone by the 9th, and at the end of about a fortnight regains its ability to respond to estrogen as shown by a return of color and swelling. However, the most remarkable eventuation of such treatment is menstruation which usually begins on about the 10th day (Hisaw, 1942).
Involution of the sexual skin and menstruation following a single injection of progesterone also have been produced in the baboon by Gillman (1940a). He found that 5 mg. progesterone, when given on the 8th day of a normal menstrual cycle, would cause an appreciable loss of edema of the swollen perineal sexual skin by the day after injection. This was followed by a progressive involution of the perineum until the 13th day and swelling was re-initiated by the end of the 15th day. Reduction of the sexual skin at this dosage of progesterone was not associated with menstruation. However, when the dose was increased to 20 mg. both deturgescence of the sexual skin and menstruation occurred. These effects pro

duced by progesterone in the presence of endogenous estrogen have much in common with those described above as occurring in castrated monkeys on continuous estrogen treatments.
IX. Menstruation
An experimental ai^proach to the physiology of menstruation dates from the observations of Allen (1927) that uterine bleeding would occur in castrated monkeys following the discontinuance of an estrogen treatment. He suggested that normal menstruation is due to a fluctuation in estrogen secretion and proposed the "estrogen-withdrawal" theory to account for the observed facts. This concept led to an extensive investigation of the effects of estrogens on the endometrium and of conditions that modify their action. It was soon found that in both castrated monkeys and human beings there was a quantitative relationship between the dosage of estrogen given and the maintenance of the endometrium. Bleeding occurred during treatment when the daily dose of estrogen was small, but with larger doses a point was reached at which the injections could be continued for months or even years without bleeding (Werner and Collier, 1933; Zuckerman, 1937b, d).
Estrogen also will inliihit i)ostop('rative bleeding which usually follows total castration, provided the ovaries are removed before or soon after ovulation (Hartman. 1934). With the advent of a corpus luteum and development of a progestational endometrium it becomes progressively more difficult, following castration, to prevent menstruation by injecting estrogen. Similar results are obtained when estrogen is given during a normal menstrual cycle. Small doses may not prevent the onset of menstruation, but if continued, subsequent menstrual periods are delayed (Corner, 1935). Large doses when given during the luteal phase of the cycle do not disturb the normal menstrual rhythm, but may do so if the treatment is started during the follicular phase (Zuckerman, 1935. 1936a).
Progesterone, in contrast with estrogen, will prevent menstruation from an endometrium representative of any stage of the normal cycle. It will delay onset of the next menses even when the treatment is started only

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a few days before the expected menstruation (Corner, 1935; Corner and Allen, 1936) . Also, the bleeding that invariably follows the discontinuance of a long treatment with estrogen can be inhibited indefinitely by giving progesterone (Hisaw, 1935; Engle, Smith and Shelesnvak, 1935; Zuckerman, 1936b).
An impression held by many of the earlier investigators was that progesterone could not produce its effects on the primate endometrium unless it w^as preceded by the action of estrogen. It is true, of course, that progesterone is a comparatively weak growth promoter and its effects can be demonstrated to best advantage on an endometrium that has been developed by estrogen. However, Hisaw, Greep and Fevold (1937) produced a progestational endometrium in a monkey that had been castrated 242 days previously by giving synthetic progesterone. Also, the endometrium of this animal was found capable of forming a decidual plaque upon traumatization. Soon afterwards Hartman and Speert (1941) observed menstruation following the withdrawal of progesterone in castrated monkeys that had not been given estrogen and more recently similar results have been reported by Eckstein ( 1950) . At the same time it has l)een found that progesterone will induce menstruation in women suffering from amenorrhea and also that uterine bleeding can l)e jirecipitated l)y similar treatment (hiring the follicular j^hase of the cycle (Zondek and Rozin, 1938; Rakoff, 1946).
These observations have been confirmed and extended by Krohn (1951; 1955) who finds that menstrual bleeding can be induced in monkeys wdth secondary amenorrhea by the injection of 5 daily doses of progesterone. Progesterone (5 mg. daily for 5 days) also precipitates uterine bleeding in castrated monkeys at intervals of about 8 days provided the treatment is started innnediately a menstrual bleeding has been induced either by removel of the ovaries or withdrawal of estrogen. The most interesting aspect of these observations is that the number of short 8-day cycles that can be obtained in this way in a castrated animal seems to be related to the size of the initial dose of estrogen used to induce withdrawal bleeding. This also applies to pro

gesterone-withdrawal bleeding, so the effect does not depend upon the particular hormone used to obtain the bleeding. It also is of interest that such conditioning of the endometrium to subsequent responses to the 5-day treatments with progesterone may last for several months on a continuous regime. It is surprising that such a series of responses cannot be initiated unless the first injection of progesterone is given within 6 days following the initial withdrawal bleeding. These observations have much in common with those of Phelps (1947) who also studied the influence of previous treatment on experimental menstruation in monkeys.
There seems to be a quantitative relationship between the dosage of progesterone given in combination with estrogen and the ability of estrogen to prevent bleeding after the injections of progesterone are stopped. It has been mentioned that once a fully developed i^rogestational reaction has been produc(Hl l)y progesterone, it is extremely difficult, if not impossible, to inhibit menstruation by giving estrogen following the withdrawal of progesterone. However, Hisaw and Greep (1938) found that progestational endometria produced ijy small doses of estrogen plus api^roximately 0.5 mg. progesterone daily for 18 to 21 days did not bleed following progesterone withdrawal when continued on 10 to 20 times the original dosage of estrogen. In fact, such endometria were brought back to a condition typical for the action of estrogen and again transformed into a presecretory progestational state without the intervention of bleeding. Similar observations were made previously by Zuckerman (1936a, 1937d).
These experimental results give grounds for some doubt as to the adequacy of the estrogen-withdrawal theory to account fully for menstruation. Not only can progesterone bring about menstruation without the intervention of estrogen but other steroid hormones are capable of pi'oducing similar effects. Desoxycorticosterone in large doses can inhibit estrogen-withdrawal bleeding in castrated monkeys (Zuckerman, 1939, 1951 ) and induce phases of uterine bleeding in rapid succession in normal monkeys (Krohn, 1951). So too can testosterone prevent estrogen-withdrawal bleeding (Hart

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PHYSIOLOGY OF GONADS

man, 1937; Engle and Smith, 1939; Duncan, Allen and Hamilton, 1941) and inhibit progesterone-withdrawal bleeding as well (Engle and Smith, 1939). Testosterone also will precipitate bleeding during an estrogen treatment (Hisaw, 1943) and in normal monkeys if given early in the cycle (Krohn, 1951). Just what specific action these compounds have in common that enables them to produce these effects or whether there are different modes of action that lead to the same results is not known, but, before mentioning certain possibilities, it may be helpful to consider information regarding the influence of estrogen-progesterone interactions on menstruation.
Among the most significant observations regarding primary causes of menstruation are a few indications that there may be an intrinsic difference in the ways in which estrogen and progesterone produce their effects on the endometrium. One of the first indications of this was the discovery that a short series of injections of progesterone during treatment with estrogen will precipitate menstruation (Corner, 1937; Zuckerman, 1937d; Hisaw and Greep, 1938). This can be demonstrated by giving a castrated monkey a maintenance dose of estrogen daily for 2 or 3 weeks, then adding a daily injection of progesterone for 5 to 10 days and continuing the estrogen treatment. As a rule bleeding appears within 2 or 3 days after stopping progesterone. The most interesting point brought out by such experiments is that bleeding can occur under these conditions in the presence of an otherwise maintenance dosage of estrogen.
Perhaps the most surprising as well as most important fact brought out by subsequent experiments was the small amount of progesterone required to bring about bleeding under these conditions. It was found that only a single injection of 1 mg. was required for animals on chronic treatment with a maintenance dose of estradiol (1000 LIT.) and some bled when 0.5 mg. progesterone was given (Hisaw, 1942). The sequence of events following the injection of progesterone can be seen to best advantage in an adolescent monkey whose sexual skin also respond" to the estrogen treatment. When the 1 mg. ]irogesterone is given on the 20th day of estrogen treatment the edema of th(^

sexual skin will have attained its maximal development. The first indication of an effect of progesterone is a slight loss of edema and color of the sexual skin which appears on the 4th or 5th day and by the 9th or 10th day the edema is almost gone and the sexual skin is pale. Blood apjiears in the vaginal lavage between the 7th and 10th days, of about 70 per cent of the animals on this dosage. The sexual skin may remain markedly reduced and pale until about the 15th day after which both color and edema rapidly return. These effects can also be seen when 1 mg. progesterone is given for a series of days. However, neither the time of appearance nor loss of edema of the sexual skin is significantly hastened, and if the injections extend over no more than 5 days the time between the first injection and bleeding remains approximately the same.
Similar observations have been made by Gillman and Smyth (1939) on the South African baboon iPapio porcarius). They found that 3 mg. or more of progesterone when given in a single injection during the follicular phase of the cycle would cause the relatively enormous perineal swellings to pass rapidly through deturgescence and reach a flabby resting condition within 5 to 7 days, and after a delay of about 24 hours once again begin to swell. As much as 10 or 15 mg. in a single dose caused perineal deturgescence without bleeding, whereas 20 mg. in a single dose or a total of 15 mg. if divided into 2 or 3 injections and given at 3 or 4 day intervals, produced both deturgescence and bleeding (Gillman, 1940b). The l)aboon diff"ers from the monkey in that larger doses of progesterone are required to produce the effects and the sexual skin does not mature" on repeated treatments and lose its responsiveness; otherwise the basic physiology of the reaction in both animals seems to be the same.
The most important fact l)rought out by these experiments is that the effects of a single injection of progesterone can continue in the presence of estrogen for as long as 10 to 15 days. It is highly imi^robable that progesterone lingers in the body for so long a time (Zarrow, Shoger and Lazo-Wasem, 1954). In general it is considered the most ephemeral of the sex steroids and is probablv inactivated within at least a few hours

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after it is administered. It seems more lilvely that progesterone modifies the sexual skin in a way that renders it unresponsive to estrogen and that about a fortnight is required to recover the original condition.
This takes on added significance when the possibility is considered that effects similar to those seen in the sexual skin might also be going on simultaneously in the uterine endometrium. An appreciable dehydration of the endometrium occurs just previous to menstruation (van Dyke and Ch'en, 1936) and a loss of interstitial fluid before bleeding has been observed in endometrial implants in the eyes of monkeys and described in detail by Markee (1940) . This was shown by periodic regression in size and compactness of the grafts which resulted in a decrease in area of 25 to more than 75 per cent. Because cyclic changes in endometrial grafts in the eye are correlated with events of the menstrual cycle there is reason to believe that similar reactions were going on in the endometrium of the uterus.
Endometrial regression, as described by Markee, did not always lead to menstruation although it invariably preceded, accompanied, and followed menstrual bleeding. Menstruation occurred only when regression was rapid and extensive. This was seen in the endometrial grafts in the eye during a normal menstrual cycle at the time of involution of a corpus luteum and during an anovulatory cycle soon after the involution of a large follicle. It also begins soon after the last of a series of injections of estrogen or i^'ogesterone. A slow decrease in size of the ocular grafts, without concomitant bleeding, can be induced in castrated monkeys by gradual withdrawal of estrogen, and when estrogen is given in amounts that are inadequate for maintaining the endometrium for an extended period the "break through" bleeding that eventually ensues is preceded by a rapid and extensive endometrial regression. Because this reaction also occurs w^hen menstruation is induced by such an unusual procedure as spinal transection (Markee, Davis and Hinsey, 1936), it probably is a phenomenon that always precedes menstruation.
It seems from these observations that the changes in the endometrium preceding menstruation are initiated by a sudden with

drawal of a stimulus on which the endometrium at the time relies for the maintenance of a particular physiologic condition, and bleeding and tissue loss are incidents that occur during the readjustment necessary for the return to an inactive state. What this involves is only partly known, but an understanding of the initial changes in the endometrium that usher in menstruation most certainly holds the explanation of the real cause. This has been a perennial subject for discussion and many suggestions and theories have been set forth in an extensive literature to account for various aspects of menstruation. Among the more recent general discussions are those by Zuckerman (1949, 1951), Corner (19511, and Zondek ( 1954 ) .
The estrogen- withdrawal or estrogen-deprivation theory proposed by Edgar Allen has received more attention than any other. From what has been mentioned earlier it is clear that this theory can account for uterine bleeding subsequent to the discontinuance of a series of estrogen injections and also perhaps menstruation at the conclusion of an anovulatory cycle. However, it is not so obvious as to how this theory can explain the occurrence of menstruation at the close of the luteal phase of a normal cycle. Estrogen in large doses will not inhibit such bleeding, but it is postponed if progesterone is given. It is equally difficult to see how this theory is helpful in accounting for the fact that a small dose of progesterone will precipitate bleeding in the presence of a maintenance dosage of estrogen. As little as 2 /xg. progesterone will induce bleeding when applied topically to the endometrial lips of an exteriorized uterus (Fig. 9.7) in a monkey that is receiving 10 fig. estradiol daily (Hisaw, 1950).
Uterine bleeding precipitated by administering progesterone during an estrogen treatment has been explained on the grounds that progesterone in some way interferes with the action of estrogen on the endometrium. Therefore, it is assumed that an animal receiving both estrogen and progesterone is in a sense "deprived" of estrogen. That is, when the two hormones are given simultaneously, progesterone itself is capable of maintaining the endometrium without bleeding; but when it is stopped, the

582

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suggestion is that the animal is physiologically deprived of estrogen and literally deprived of progesterone (Corner, 1951). Although this view is in descriptive agreement with the observed facts the idea of the inhibitory effect of progesterone does not take into consideration the synergistic interaction of the two hormones on the endometrium.
The physiologic function of progesterone is the conversion of an estrogen-endometrium into a progestational endometrium suitable for receiving and nourishing a developing blastocyst. Such an endometrium is adapted for this specific reproductive function and accordingly its physiologic nature must be quite different from that of the follicular phase of the cycle. Indeed, it is known that these two structures (follicular and luteal phase endometrial are morphologically and biochemically unlike in a number of respects. This gradual transformation, following ovulation, occurs as progesterone becomes the dominant hormone, and consequently, as this proceeds, the endometrium progressively loses competence to respond to estrogen. However, this does not imply that estrogen is without effect in the general economy of the progestational endometrium. It has been shown in a number of ways that the action of progesterone on the primate endometrium is greatly facilitated by the presence of estrogen. In fact, it seems probable that rarely if ever does progesterone perform its function in the absence of estrogen (Hisaw, 1959; chapter by Zarrow).
After consideration of the endometrial specializations brought about by ]irogesterone, it seems rather jwintless to hark back to the follicular phase and inject the past recoi'd of accomjilishments and prerogatives that estrogen had at that time into the explanation of an entirely different hormonal situation. It seems more in keeping with the facts to state outright that menstruation following the involution of a corpus luteum or the discontinuance of progesterone, even though estrogen is present, is due to a decrease or absence of progesterone.
It also has become less certain that menstruation at the conclusion of an anovulatory cycle is really an estrogen-withdrawal bleeding. This is possible, of couisc, but at

the same time the exceedingly small amount of progesterone required to induce bleeding in the presence of estrogen makes it difficult to be sure what the situation might be. Even a negative test for progesterone in the blood, by our present methods, does not necessarily indicate the absence of a physiologically effective amount of progesterone. Zarrow, Shoger and Lazo-Wasem (1954) found that in rabbits an intramuscular injection of 40 mg. progesterone was required to produce an appreciable concentration of the hormone in the blood as determined by the Hooker-Forbes method. Yet, 0.2 mg. progesterone daily for 5 days will produce a progestational reaction in the uterus equivalent to that of the 5th day of normal pseudopregnancy. In monkeys 0.5 mg. daily when given with 10 fxg. estradiol is an adequate dosage of progesterone to induce unquestionable progestational changes in the endometrium and much less will cause bleeding. These observations indicate that the minimal effective concentration of progesterone in the blood may be less than is possible to detect by our present methods.
This also seems to hold for the human being. Estimates of secretion and metabolism of progesterone in the human being have been based primarily on the recovery of its excretory product sodium pregnanediol glucuronidate in the urine. It seems obvious that such determinations must be only general approximations because only about 20 per cent of the progesterone secreted or injected can be accounted for by the pregnanediol in the urine. Also, it is generally known that a physiologically effective dosage of progesterone does not necessarily lead to the excretion of pregnanediol (Hamblen, Cuylcr, Powell, Ashley and Baptist, 1939; Seegar, 1940). In other words, the threshold dose of progesterone for endometrial stimulation is l)elow that at which the hormone is excreted as pregnanediol. In fact, it has been suggested by some investigators that there is no quantitative relationship between the l)rogesterone present in the blood and the pregnanediol excreted in the urine (Buxton, 1940; Sommerville and Marrian, 1950; Kaufmann, Westphal and Zander, 1951).
These findings and the wide variation in the amount of prc'gnancdiol excreted during

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58.3

a menstrual cycle (Venning and Browne, 1937) suggest that, even in the absence of ovulation, sufficient progesterone may be present to influence menstruation. There also is the possibility of progestational hormone from some extra-ovarian source, such as the suprarenal cortex. This was suggested by Zuckerman (1937b, 1941 j as a possible explanation for periodic bleeding in monkeys on a constant submaintenance dose of estrogen. This thought becomes more plausible in view of the fact that progesterone is one of the precursors in the metabolic synthesis of androgens, estrogens, and adrenal cortical steroids (Dorfman, 1956). Also, it has been shown that desoxycorticosterone acetate is converted to progesterone in vivo (Zarrow, Hisaw and Bryans, 1950). Therefore, progesterone is not restricted to ovarian luteal function but instead is of rather general occurrence in the body and the likelihood is that small amounts are a constant constituent of the blood.
Also, the amount of progesterone from extra-ovarian sources may fluctuate, as suggested by Zuckerman (1949), and consequently disturb the normal menstrual rhythm and probably cause bleeding in monkeys on a continuous submaintenance dose of estrogen. However, as to the latter, there is an alternative explanation. Castrated monkeys on a continuous treatment of 10 fxg. of estradiol daily do not show "break-through" bleeding, and a synergistic effect on growth of the uterus is seen when 0.5 mg. or more of progesterone daily is introduced into the treatment. However, the simultaneous administration of 0.25 mg. or even 0.125 mg. progesterone daily in similar exj^eriments results in bleeding between a!)out the 10th to 16th day of the combination treatment. Thus, a dosage of progesterone less than that required for synergism or prevention of bleeding when given alone, modifies the endometrium so that it can no longer be maintained by 10 fxg. estradiol daily (Hisaw, Jr., unpublished). When it is considered that the endometrium becomes increasingly dependent on estrogen during a chronic treatment, even after maximal growth is attained (Hisaw, 1942), it seems plausible that the effectiveness of a dosage of estrogen only slightly alcove the thresh

old for bleeding may be decreased sufficiently by the endogenous progesterone from extra-ovarian sources to precipitate bleeding.
Although it is obvious that the normal menstrual cycle is primarily under the control of the ovarian estrogens and progesterone, it is also equally clear that menstruation is not due to a specific hormonal action. Experimental evidence indicates that any natural or synthetic compound having the capacity for promoting growth or sustaining an existing metabolic state in the endometrium is also capable of inducing withdrawal bleeding. However, this does not imply that all compounds capable of inducing menstruation do so by the same biochemical action; in fact, there is considerable evidence that this is not so (see chapter by Villee). Yet in each instance a series of events is set in motion that leads up to active bleeding.
X. The Mechanism of Menstruation
The immediate cause and mechanism of menstruation has continued to be a topic of special interest for many years and the subject of frequent general discussions. A generalization in keeping with our present knowledge is that no gross morphologic feature of the endometrium is distinctive of menstruation. A menstruating endometrium may be representative of any stage of the follicular or luteal phase of the cycle. The most frequently discussed hypothesis regarding the mechanism of menstruation is that proposed by Markee (1940, 1946) which is based on direct observations of vascular changes in endometrial grafts in the anterior chamber of the eye of monkeys (see p. 564). The changes observed in the endometrium shortly before bleeding are, briefly, as follows. (1) There is extensive and rapid regression of the endometrium due to loss of ground substance from the stroma (Fig. 9.23). (2) The rapid regression brings about a disproportion between the length of the coiled arteries and thickness of the endometrium with the formation of additional coils. (3) The increased coiling of the arteries retards the circulation of blood through them and their branches. This stasis begins 1 to 3 davs before the onset of the

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Repair

Fig. 9.23. A diagram indicating correlated changes in ovary and endometrium during an ovulatory cycle of rhesus monkey. Thickness of endometrium, density of stroma, gland form, and three types of arteries are indicated. There is a gradual rise in thickness up to the time of ovulation, and a brief decline followed by development of the luteal or progestational phase with accumulation of secretion in the glands due to relaxation of the myometrium. This is followed by loss of ground substance from the stroma, which is the primary factor in the premenstrual regression of the ischemic phase. This is a prelude to extravasation and shedding of tissue. Incidentally, secretion is extruded and glands collapse. There is further regression throughout the phase of menstruation. More than the basal zone (coarse stipple) survives menstruation. During repair, thickening of the endometrium is associated with increase in ground substance in the stroma and growth in the glands. (From G. W. Bartelmez, 1957, Am. J. Obst. & Gynec, 74, 931-955, 1957, with some modification of description.)

flow, and is associated with leukocytosis in the endometrium. (4) The portion of the coiled arteries located adjacent to the muscularis constricts 4 to 24 hours before the onset of the flow. This vasoconstriction persists throughout the menstrual period except when individual coiled arteries relax and blood circulates through them for a few minutes. Markee postulated that the immediate cause of menstruation under these conditions was the injurious effect of anoxemia upon the tissues of the endometrium l)rought about by mechanical compression and constriction of the coiled arteries. Therefore, the coiled arteries and their modifications become the central feature upon which the theory is based.
Although this offers an explanation for many of the facts, it falls short in that now it is known that menstruation can occur in the absence of coiled arteries. Kaiser (1947) showed that no spiral arteries are present in

the endometrium of three species of South American monkeys known to menstruate. He also found that the coiled vessels of the endometrium could be destroyed almost completely by giving large doses of estrogen and yet bleeding followed estrogen withdrawal.
Several experimental conditions under which the coiled vessels of the endometrium are destroyed have been mentioned in the present discussion and in each instance bleeding invariably followed withdrawal of the supporting stimulus. The extremely atrophic endometrium present at the conclusion of a prolonged treatment with progesterone (Fig. 9.8) will bleed when the injections are stopped, and if estrogen injections are started immediately thereafter the endometrium that develops is normal with the exception of the absence of coiled arteries; even so, it also will bleed when the treatment is stopped. Even a more

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58c

drastic destruction of endometrial structures occurs when both estrogen and progesterone are given for several months. Not only are the coiled arteries destroyed but also the glands and the luminal epithelium. All that remains is a modified stroma penetrated by a few small blood and lymph vessels and scattered glandular rudiments along the myometrium (Fig. 9.13). Yet, in spite of this, bleeding follows discontinuance of the treatment.
These observations prove conclusively that the spiral arteries of the endometrium do not hold the solution to the menstrual process. However, the descriptive account by Markee of the events that take place in the endometrium during the cycle remains one of the major contributions to our knowledge of the primate endometrium. Phelps (1946) also made a very careful study of the vascular changes in intraocular endometrial transplants in ovariectomized monkeys receiving estrogen and progesterone, and concluded that the primary function of the coiled arteries is concerned with vascularization of the implantation site of a developing embryo.
There also is reason for doul^ting that ischemia is a determining factor in the menstrual process. That constriction of the endometrial vessels does occur is well established, but that tissue destruction and bleeding are consequences of prolonged anoxemia may be questioned. The endometrium around the internal cervical os as seen in incised exteriorized uteri (Fig. 9.7) contains very few coiled arteries and does not take part in the periodic blushing and blanching of the fundus, but instead remains blood-red even during menstruation. Also, certain tongues of endometrium in a uterine fistula may become crowded by their neighbors to an extent of being partly or completely deprived of blood, yet they do not bleed even though their unfavorable situation leads to deterioration within a few days.
Emmel, Worthington and Allen (1941) attempted to induce menstruation in monkeys by operative ischemia. Circulation to the fundus of the uterus was interrupted by means of a tourniquet for periods of 1 to 8V4 hours, and in two instances for 19 hours. This procedure did not precijiitate uterine

bleeding nor did it hasten the onset of an expected bleeding following estrogen withdrawal. In fact, when the uterus was deprived of blood for periods longer than 3 hours impairment of the bleeding response to estrogen withdrawal was observed, and 19 hours of ischemia caused atrophy of the uterus without bleeding.
It also has been reported that a toxic substance formed in the endometrium is responsible for menstruation. This menstrual toxin is supposed to be present in the endometrium just previous to and during menstruation, and to be a substance resembling or identical with necrosin, a material found in pleural exudate following an inflammatory reaction (Smith and Smith, 1951). Zondek (1953) reports that menstrual blood, when obtained under relatively sterile conditions, is no more toxic to experimental animals than sterile tissue extracts. He also found that death of animals given injections of menstrual blood was due to bacteremia, an effect that could be prevented by giving antibiotics. Nor was he able to demonstrate a toxic substance in the premenstrual or menstrual endometrium. It might be mentioned in this connection that endometrial tissue destroyed by experimental ischemia in the experiments by Emmel, Worthington and Allen (1941), obviously did not influence menstruation nor did involuting endometrial tissue in uterine fistulae (p. 564). Therefore, the presence of a specific toxin that may induce menstruation has not been conclusively demonstrated.
Regardless of the specific cause of menstruation, the evidence shows that it can occur in the absence of coiled arteries, endometrial glands, or surface mucosa, and is unrelated to the thickness of the endometrium. This statement is based on conditions that have been experimentally induced in the monkey and they strongly indicate that menstruation, whatever the cause, is a stromal phenomenon. This view seems to be in agreement with the observations reported by Bartelmez in his elegant studies of the morphology of the endometrium of both monkeys and the human being. He emphasizes changes taking place in the connective tissue elements of the stroma and points out that much less tissue is lost at menstruation

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than i.< commonly thought (Bartehnez, 1957). The reduction in thickness is clue primariiy to loss of ground substance from the stroma, and conversely, the outstanding feature of repair is the increase in stromal ground substance (Fig. 9.23). ^Mitoses are rarely seen in the stroma during repair and arc not abundant enough in any phase according to Bartelmez to account for the observed increase in thickness of the endometrium. Our present knowledge indicates that an explanation of menstruation may be found in the metabolic effects induced in the stromal connective tissue of the endometrium by a sudden withdrawal of a supporting hormonal stimulus.
XI. References
Allen, E. 1927. The menstrual CA'cle in the monkey, Macacus rhesus: observations on normal animals, the effects of removal of the ovaries and the effects of injections of ovarian and placental extracts into the .spayed animals. Contr. Embrvol., Carnegie Inst. Washington, 19, 1-44.
Allen. E. 1928. Further experiments with an ovarian hormone in the ovariectomized adult monkey, Macacus rhesus, especially the degenerative phase of the experimental menstrual cycle. Am. J. Anat., 42, 467^87.
Allen, E., Diddle, A. W., Burford, T. H., .and Elder, J. H. 1936. Analyses of urine of the chimpanzee for estrogenic content during various stages of the menstrual cycle. Endocrinology, 20, 546-549.
Allen, E., Pr.\tt, J. P., Xewell, Q. U., .and Bl.and, L. J. 1930. Human tubal ova ; related early corpora lutea and uterine tubes. Contr. Embryol., Carnegie Inst. Washington, 22, 45-76.
B.ACHMAN, C, CoLLip, J. B., .\ND Selye, H. 1935. The effects of prolonged estriol administration upon the sex skin of Macaca mulatta. Proc. Roy. Soc, London, .■^er. B., 117, 16-21.
B.artel.mez, G. W. 1933. Histologic studies on the menstruating mucous membrane of the human uterus. Contr. Embryol.. Carnegie Inst. Washington, 24, 141-186.
B.artel.mez, G. W. 1937. ^Menstruation. Plnsiol. Rev.. 17, 28-72.
B.ARTELMEZ, G. W. 1951. Cyclic changes in the endometrium of the rhesus monkey {Macacus mulaltn). Contr. Embryol., Carnegie Inst. Washington. 34, 101-144.
B.ARTELMEz, G. W. 1957. The pha.scs of the menstrual cycle and their interpretation in terms of the pregnancv cycle. Am. J. Obst. & Gvnec, 74, 931-955.
BRY.AN.S, F. E. 1951. Progesterone of the lilood in the menstrual cycle of the monkey. Endocrinology, 48, 733-740.
BrxTo.N, C. L. 1940. Pregnanediol determina

tions as an aid in clinical diagnosis. Am. J. Obst. & Gynec, 40, 202-211.
CoLLiNGS, M. R. 1926. A study of the cutaneous reddening and swelling about the genitalia of the monkev, Macacus rhesus. Anat. Rec, 33, 271-287.
Corner, G. W. 1923. Ovulation and menstruation in Macacus rhesus. Contr. Embrj^oL, Carnegie Inst. Washington, 15, 73-101.
Corner, G. W. 1935. Influence of the ovarian hormones, estrin and progestin, upon the menstrual cvcle of the monkev. Am. J. Phj'siol., 113,238-250.
Corner, G. W. 1937. Experimental menstruation. Science, 85, 437-438.
Corner, G. W. 1942. The fate of the corpora lutea and the nature of the corpora aberrantia in the rhesus monkey. Contr. Embryol., Carnegie Inst. Washington, 30, 87-96.
Corner, G. W. 1945. Development, organization and breakdown of the corpus luteum in the rhesus monkey. Contr. Embrj'ol., Carnegie Inst. Washington, 31, 119-146.
Corner, G. W. 1951. Our knowledge of the menstrual cycle, 1910-1950. Lancet, 1, 919-923.
Corner, G. W., and Allen, W. M. 1936. Inhibition of menstruation by crj-stalline progesterone. Proc. Soc. Exper. Biol. & Med.. 34, 723724.
Crossex, R. J. 1953. Diseases of Women. St. Louis: C. V. Mosby Company.
Davls, M. E., and H.art^lan, C. G" 1935. Changes in vaginal epithelium during pregnancy in relation to the vaginal cvcle. J. A. M. A., 104, 279-285.
Diczfall'.sy, E. 1953. Chorionic gonadotrophin and estrogens in the human placenta. Acta endocrinol., Suppl. 12, 87-167.
DoRFMAN, R. I. 1956. Metabolism of androgens, estrogens and corticoids. Am. J. Med., 21, 679687.
DoRFMAN, R. I., AND VAN W.AGENEN, G. 1941. The
sex hormone excretion of adult female and pregnant monkevs. Surg. Gvnec. & Obst., 73, 545-548.
Duncan, P. A., Allen. E., and Ha.milton. J. B. 1941. The action of testosterone proprionate on experimental men.struation in the monkev. Endocrinology. 28, 107-111.
Eckstein, P. 1950. The induction of progesterone withdrawal bleeding in spayed monkeys. J. Endocrinol., 6, 405-411.
EcK.STEiN, P., AND ZucKERMAN, S. 1956. In Marshall's Physiology of Reproducliori. A. S. Parkes, Ed. Vol. 1, ]\ 334. London: Longmans Green & Company.
EmMEL, V. M., WORTHINGTON, R. V., AND AlLEN. E.
1941. Attempts to induce menstruation by operative ischemia in monkey's. Endocrinologv, 29, 330-335.
Engle. E. T. 1937. Problems of experimental menstruation. Cold Spring Harbor Svmposia Quant. Biol., 5, 111-114.
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ESTROGEN AND PROGESTERONE

587

reference to tlio r('i\ ix uteri. Auat. Rec, 6, 471-483.
Encle, E. T., Smith, P. E., and Shelesnyak, M. C. 1935. The role of estrin and progestin in experimental menstruation. Am. J. Obst. & Gynec. 29, 787-797.
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Fish, W. R., Young, W. C, and Dorfman, R. I. 1941. Excretion of estrogenic and androgenic substances by female and male chimpanzees with known mating behavior records. Endocrinology, 28, 585-592.
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GiLLMAN, J., AND GILBERT, C. 1946. The reproductive cycle of the chacma baboon (Papio itrsiDiis) with special reference to the problems of menstrual iriegularities as assessed by the behaviour of the sex skin. South African J. M Sc, Biol. Suppl., 11, 1-54.
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Hartman, C. G. 1932. Studies in the reproduction of the monkey, Macacus (Pithecus) rhe.S-//.S, with special reference to menstruation and piegnancy. Contr. Embryol., Carnegie Inst. Washington, 23, 1-16.
H.ARTMAN, C. G. 1934. Some attempts to influence the menstrual cvcle in the monkev. Am. J. Obst. & Gynec, 27, 564-570.
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H.ARTMAN, C. G. 1944. Regeneration of the monkey uterus after surgical removal of the endometrium and accidental endometriosis. Western J. Surg. Obst. & Gynec, 52, 87-102.
Hartman, C. G., .and Speert, H. 1941. Action of progesterone on the genital organs of the unjirimed Rhesus monkev. Endocrinology, 29, 639-648.
H.ARTMAN, C. G., GeSCHICKTER, G. F., AND SpEERT, H.
1941. Effects of continuous estrogen administration in verv large doses. Anat. Rec, Suppl. 2, 79, 31.
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Hertig, a. T., and Rock, J. 1944. On the development of the early human ovum, with special reference to the trophoblast of the previllous stage ; a description of 7 normal and 5 pathologic human ova. Am. J. Obst. & Gvnec, 47, 149-184.
Heuser, C. H., and Streeter, G. L. 1941. Development of the macaque embryo. Contr. Embryol., Carnegie Inst. Washington, 29, 17-55.
Hisaw, F. L. 1935. The physiology of menstruation in Macacus rhesus monkevs. Am. J. Obst. & Gynec, 29, 638-659.
HiSAW% F. L. 1942. The interaction of the ovarian hormones in experimental menstruation. Endocrinology, 30, 301-308.
HiSAW, F. L. 1943. Androgens and experimental menstruation in the monkey (Macaca viulatta). Endocrinology, 33, 39-47.
HisAW, F. L. 1950. Factors influencing endometrial growth in monkeys (Macaca mulatta). In A Symposium on Steroid Hormones, E. S. Gordon, Ed., pp. 259-276. Madison: University of Wisconsin Press.
Hisaw, F. L. 1959. Endocrine adaptations of the mammalian estrous cycle and gestation. In Columbia University Symposium on Comparative Endocrinology, pp. 533-552.
Hisaw, F. L., Creep, R. O., and Fevold, H. L. 1937. Effects of progesterone on the female genital tract after castration atrophy. Proc Soc. Exper. Biol. & Med., 36, 840-842."
His.\w, F. L.. AND Creep, R. O. 1938. The inhibition of uterine bleeding with estradiol and

588

PHYSIOLOGY OF GONADS

progesterone and associated endometrial modifications. Endocrinology, 23, 1-14. His.\w, F. L., .^ND His.wv, F. L., Jr. 1958. Spontaneous carcinoma of the cervix uteii in a monkey (Macaca mulatto). Cancer, 11, 810816. HiSAW, F. L., .4ND Lendrum, F. C. 1936. Squamous metaplasia in the cervical glands of the monkey following oestrin administration. Endocrinology, 20, 228-229. HiSAW, F. L., Meyer, R. K., axd Fevold, H. L. 1930. Production of a premenstrual endometrium in castrated monkeys by ovarian hormones. Proc. Soc. Exper. Biol. & Med., 27, 400-403. HiTSCHMANN, F., AND Adler, L. 1907. Die Lehre von der Endometritus. Ztschr. Geburtsh. u. GynJik., 60, 63-86. Kaiser, I. H. 1947. Absence of coiled arterioles in the endometrium of menstruating New World monkeys. Anat. Rec, 99, 353-363. Kaufmann, C, Westphal, U., and Zander, J. 1951. Untersuchungen liber die biologische Bedeutang der Ausscheidungsprodukte des Gelbkcirperhormons. Arch. Gynak., 179, 247-299. Knaus, H. 1950. Die Physiologie der Zeugung
des Menschen. Wien: Wilhelm Maudrich. Krohn, p. L. 1951. The induction of menstrual bleeding in amenorrhoeic and normal monkeys by progesterone. J. Endocrinol., 7, 310-317. Krohn, P. L. 1955. The induction of cyclic uterine bleeding in normal and spayed rhesus monkeys by progesterone. J. Endocrinol., 12, 6985. Krohn, P. L., and Zuckerman, S. 1937. Water metabolism in relation to the menstrual cycle. J. Physiol., 88, 369-387. Latz, L. J., and Reiner, E. 1942. Further studies on the sterile and fertile periods in women. Am. J. Obst. & Gynec, 43, 74-79. Lendrum, F. C., .and Hisavv^ F. L. 1936. Cytology of the monkey endometrium under influence of follicidar and corpus luteum hormones. Proc. Soc. Exper. Biol. & Med., 34, 394-396. Lopez Columbo de Allende, I., and Orias, O. 1950. Cytology of the Human Vagina. New York: Paul B. Hoeber, Inc. Lopez Colu.mbo de Allende, I., Shorr, E., and Hartman, C. G. 1945. A comparative study of the vaginal smear cycle of the rhesus monkey and the human. Contr. Embryol., Carnegie Inst. Washington, 31, 1-26. M.arkee, J. E. 1940. Menstruation in intraocular endometi'ial tr;msplants in the I'hesus monkey. Contr. Embrvol., Carnegie Inst. Washington, 28, 219-308. Markee, J. E. 1946. Morphologic and endocrine basis foi' menstrual bleeding. In Progress in Gynecology, Meigs and Sturgis, Eds. Vol. II, pp. 37-47. New York: (hune and Stiattdii. Markee, J. E., and Berg, B. 1944. Cyclic fluctuations in blood estrogen as a possible cause of menstruation. Stanford Med. Bull., 2, 55-60. Markee, J. E., D.wis, J. H., and Hinsf.y, J. C. 1936. Uterine bleeding in spinal iii()nk(>vs. Anat. Rec, 64, 231-245.

Mazer, C, and Israel, S. L. 1951. Diagnosis and Treatment of Menstrual Disorders and Sterility. New York: Paul B. Hoeber, Inc.
Meyer, R. 1911. Uber Corpus luteum-Bildung beim Menschen. Arch. Gynak., 93, 354-404.
Nissen, H. W., and Yerkes, R. M. 1943. Reproduction in the chimpanzee: report on 49 births. Anat. Rec, 86, 567-578.
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OvERHOLSER, M. D., AND Allen, E. 1933. Ovaiiau hormone and traumatic stimulation of monkey's cervix to a condition resembling early cancer. Proc Soc. Exper. Biol. & Med., 30, 1322-1326.
OvERHOLSER, M. D., AND Allen, E. 1935. Atypical growth induced in cervical epithelium of monkey by prolonged injections of ovarian hormone combined with chronic trauma. Surg. Gynec. & Obst., 60, 129-136.
OvERHOLSER, M.D.,. AND Nelson, W. 0. 1936. Migration of nuclei in uterine epithelium. A monkey following prolonged estrin injections. Proc Soc Exper. Biol. & Med., 34, 839-841.
P.APANicoL.Aou, G. N., Traut, H. F., and M.archetti, A. A. 1948. The Epithelia of Woman/s Reproductive Organs. New York: Commonwealth Fund.
Parkes, a. S., and Zuckerm.an, S. 1931. The menstrual cycle of the Primates. II. Some effects of oestrin on baboons and macaques. J. Anat., 65, 272-276.
Phelps, D. 1946. Endometrial vascular reactions and the mechanism of nidation. Am. J. Anat., 79, 167-197.
Phelps, D. H. 1947. The factor of previous treatment in experimental menstruation. J. Clin. Endocrinol., 7, 611-623.
Rakoff, a. E. 1946. Studies on high dosage progesterone therapy of amenorrhea. Am. J. Obst. & Gynec, 51, 480-491.
Rock, J., and Hertig, A. 1942. Some aspects of early human development. Am. J. Obst. & Gynec, 44, 973-983. RossM.AN, I. 1940. The decidual reaction in the rhesus monkey {Macaca mulatta). I. The epithelial proliferation. Am. J. Anat., 66, 277365. Schroder, R. 1914. Uber das Verhalten der Uterusschleimhaut um die Zeit der Menstruation. Monatsschr. Geburtsh. u. Gynak., 39, 3-21. Seeg.ar, E. G. 1940. The histologic effect of progesterone on hyperplastic endometria. Am. J. Obst. & Gynec, 39, 469-476. S.mith, O. W., and S.mith, G. V. 1951. Endocrinology and related phenomena of the human menstrual cvcle. Recent Progr. Hormone Res., 7, 209-253.
So.MMKKVILLK, I. V ., AND MaRRIAN, G. F. 1950.
I'rinary excretion of prcgnanediol in human subjects following the administration of progesterone and of pregnane-3a:20a-diol. I3iochem. J., 46, 285-289. Stieve, H. 1926. Di(> regelmassigen Verliinderungen der Muskulatur und des Bindegewebs in

ESTROGEN AND PROGESTERONE

589

der meuschlichen Gebarmutter in ihier Abhangigkeit von der Follikelreife und der Aiisbildung eines gelben Korpers, nebst Beschreibung eines menschlichen Eies im Zustand der ersten Reifteilung. Ztschr. mikroskop.-anat. Forsch, 6, 351-397.
Stieve, H. 1942. Der Einfluss von Angst und psychischer Erregung auf Bau und Funktion der weiblit'hen Geschlechtsorgane. Zeutralbl. Gynak., 66, 1698-1708.
Stieve, H. 1943. Weitere Tatsachen zur Kliirung der Frage: Wann wird das Ei aus dem Eierstock ausgestossen? Zentralbl. Gvnak., 67, 5877.
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Sturgis, S. H. 1942. Method for obtaining uterine fluid from the monkey : effect of pilocarpine, atropine, physiologic salt solution and adrenalin. Endocrinology, 31, 664-672.
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Venning, E. H., and Browne, J. S. L. 1937. Studies on corpus luteum function. I. The vn-inary excretion of sodium pregnandiol glucuronidate in the human men.^trual cvcle. Endocrinologv, 21,711-721.
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Zarrow, M. X., Shoger, R. L., and Lazo-Wasem, E. A. 1954. The rate of disappearance of exogenous progesterone from the blood. J. Clin. Endocrinol., 14, 645-652.

Zondek, B. 1953. Does menstrual blood contain a specific toxin? Am. J. Obst. & Gvnec, 65, 1065-1068.
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Zuckerman, S. 1930. The menstrual cycle of the Primates. I. General nature and homology. Proc. Zool. Soc, London, 1930, 691-754.
Zuckerman, S. 1935. The menstrual cycles in the Primates. VIII. The estrin-vvithdrawal theory of menstruation. IX. The effect of estrin on the denervated sexual skin. Proc. Rov. Soc, London, ser. B., 118, 13-33.
Zuckerman, S. 1936a. Inhibition and induction of uterine bleeding bv means of estrone. Lancet, 2, 9-13.
Zuckerman, S. 1936b. The interrelation of estrone and progestin in the menstrual cvcle. J. PhvsioL, 86, 31-33.
Zuckerman, S. 1937a. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc, London, ser. A., 1937, 315-329.
Zuckerman, S. 1937b. The menstrual cycle of the Primates. X. The oestrone threshold of the uterus of the rhesus monkey. XL The part played by oestrogenic hormone in the menstrual cycle. Proc. Roy. Soc, London, ser. B., 123,441-471.
ZucKER.MAN, S. 1937c Effects of prolonged oestrin-stimulation on the cervix uteri. Lancet, 1, 435-437.
Zuckerman, S. 1937d. Further observations on endocrine interaction in the menstrual cvcle. J. Physiol., 89, 49-51.
Zuckerman, S. 1937e. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc London, ser. A., 1937, 315-329.
Zuckerman, S. 1939. The effect of sex hormones, cortin, and vasopressin on water-retention in the reproductive organs of monkeys. J. Endocrinol., 1, 147-155.
Zuckerman, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with constant threshold dose of oestrone. J. Endocrinol., 2, 263-267.
Zuckerman, S. 1949. The menstrual cvcle. Lancet, 2, 176.
Zuckerman, S. 1951. The hormonal basis of uterine bleeding. Acta endocrinol., 7, 378-388.
Zuckerman, S., and P.arkes, A. S. 1932. The menstrual cycle of the primates. V. The cycle of the baboon. Proc Zool. Soc. London, 1932, 139-191.
Zuckerman, S., van W.agenen, G., and Gardiner, R. H. 1938. The sexual skin of the rhesus monkey. Proc Zool. Soc, London, ser. A., 108, 385-401.

10

THE MAMMARY GLAND AND LACTATION
A. T. Cowie and S. J. FoUeij
NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING, SHINFIELD, READING, ENGLAND

I. Introduction
I. Introduction 590
II. Development of the Mammary
Gland 591
A. Histogenesis 591
B. Normal Postnatal Development . 593
1. Methods of assessing mammary
development 593
2. Mammary development in the
nonpregnant female 594
3. Mammary growth in the male . . 595
4. Mammary development during
pregnancy 596
5. Mammary involution 598
C. Experimental Analysis of Hormonal
Influences 598
1. Ovarian hormones in the animal
with intact pituitary 598
2. Anterior pituitary hormones. . . 601
3. Metabolic hormones (corticoids,
insulin, and thyroid hormones) 604 III. Endocrine Influences in Milk Secretion 606
A. Anterior Pituitary Hormones 606
1. Initiation of secretion (laeto genesis) 606
2. Maintenance of milk secretion —
galactopoiesis 609
3. Suckling stimulus and the main tenance of lactation 611
B. Hormones of the Adrenal Corte.x . . 612
C. Ovarian Hormones 613
D. Thyroid Hormones 617
E. Parathyroid Hormone 618
F. Insulin 619
IV. Removal of Milk from the Mammary
Glands: Physiology of Suckling AND Milking 619
A. Milk-Ejection Reflex 619
B. Role of the Neurohypophysis 621
C. Milk-Ejection Hormone 622
D. Effector Contractile Mechanism of
the Mammary Gland 623
E. Inhibition of Milk Ejection 624

F. Neural Pathways of the Milk-Ejec tion Reflex 625
G. Mechanism of Suckling 626
V. Relation between the Reflexes
Concerned in the Maintenance of Milk Secretion and Milk Ejection 627 VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance OF Milk Secretion and
Milk E.tection 630
VII. Conclusion 632
VIII. References 632
This account of the hormonal control of the mammary gland is in no way intended as an exhaustive treatment of mammary gland physiology, but rather an attempted synthesis of current knowledge which it is hoped will be of interest as an exposition of the authors' conception of the present status of the subject. Since the publication of the second edition of this book, the emphasis in the field under review has tended to shift towards the development of quantitative techniques for assessing the degree of mammary development, towards attempts at a ])enetration into the interactions of hormones with the biochemical mechanisms of the mammary epithelial cells, and towards an increasing preoccupation with the interplay of nervous and endocrine influences in certain phases of lactation. The reader's acquaintance with the classical foundations of the subject as described in the second edition of this book (Turner, 1939) and in other subsequent reviews (Follcy, 1940; Petersen, 1944, 1948; Folley and Malpress, 1948a, b; Mayer and Klein. 1948, 1949; Follev, 1952a, ]9r)6; Dabelow. 1957) will

590

MAMMARY GLAXD AND LACTATION

591

therefore be assumed and used as a point of departure for the present account which can most profitably be concerned mainly with developments which have occurred since the last edition was published. Reference will freciuently be made to these reviews in which authority will be found for the many ex cathedra statements that will be made, but original sources will be cited wherever appropriate.^
As an aid to logical treatment of the subject the scheme of classification proposed by Cowie, Folley, Cross, Harris, Jacobsohn and Richardson (1951) will be followed in this chapter. Besides introducing a system of terminology in respect of the physiology of suckling or milking, these writers have put forward a classification scheme which is an extension of one previously proposed by one of the present authors (Folley, 1947). This scheme considers the phenomenon of lactation as divisible into a number of phases as follows:
[ [Milk synthesis
I Milk secretion ■! Passage of milk from I I the alveolar cells
Lactation<J [Passive withdrawal of
ij milk
JThe milk-ejection re[ Hex

Milk removal

I
As is logical and customary, discussion of lactation itself will be preceded by consideration of mammary development.
II. Development of the Mammary Gland
A. HISTOGENESIS
References to the earlier work on the histogenesis of the mammary gland in various species will be found in Turner ( 1939,
^ Within the last 10 years there have been several symposia devoted to the problems of the physiology of lactation. The proceedings of these symposia have been published: Mecanisme physiologie de la secretion lactee. Strasbourg, 1950, Colloqvies Internationaux du Centre National de la Recherche Scientificiue. XXXII, 1951, Paris; Svmposium sur la physiologie de la lactation, Montreal, 1953, Rev. Canad. Biol., 13, No. 4. 1954; .Symposium sur la physiologie de la lactation, Brussels, 1956, Ann. endocrinol. 17, 519; A Discussion on the Physiology and Biochemistry of Lactation. London. 1958, Proc. Roy. Soc, .ser. B, 149, 301.

1952,) and Folley (1952a). There have also been studies on the opossum (Plagge, 1942) , the mouse and certain wild rodents (Raynaud, 1949b), the rhesus monkey (Speert, 1948), and man (Williams and Stewart, 1945; Tholen, 1949; Hughes, 1950).
A question which in the last decade has been receiving attention is whether the prenatal differentiation and development of the mammary primordium is hormonally controlled. According to Balinsky (1950a, b), the mitotic index of the mammary bud in the embryo of the mouse and rabbit is lower than that of the surrounding epidermis and he concludes that differentiation of the bud is due not to cellular proliferation (growth) but to a process of aggregation ("morphogenetic movement") of epidermal cells. This author also reports that for some time after its formation, the mammary bud is cjuiescent as regards growth, thus exhibiting negative allometry compared with the whole embryo, until the sprouting of the primary duct initiates a phase of positive allometry. The cjuestion is, what is the stimulus responsible for the onset of this allometric phase? Is the growth and ramification of the duct primordium, like that of the adult duct system, due to the action of estrogen emanating from the fetal gonad or from the mother?
Hardy (1950) has shown that dift'erentiation and growth of the mammary bud of the mouse could proceed in explants from the ventral body wall of the embryo, cultured in vitro, even when no primordia were present at the time of explantation (10-day embryo). Primary and then secondary mammary ducts and a streak canal differentiated and a developmental stage similar to that in the 7-day-old mouse could be reached. Balinsky (1950b) was also able to observe the formation and growth of mammary buds in approximately their normal locations in a minority of cases in which body-wall explants of 10-day mouse embryos were cultivated in vitro. Discounting the rather remote possibility that the effects were due to minute amounts of sex hormones present in the culture media, these observations indicate that hormonal influences are not necessary for the prenatal stages of mammary develo]iment, and in accord with

592

PHYSIOLOGY OF GONADS

this Balinsky ( 1950b j found that addition of estrogens or mouse pituitary extract to the culture medium had no effect on the growth of the mammary rudiment in vitro.
On the other hand, extensive studies by Raynaud (1947c, 1949b) of the sex difference in the histogenesis of the mammary gland in the mouse, first described by Turner and Gomez (1933), indicate that the mammary rudiment is sensitive to the influence of exogenous gonadal steroids during the prenatal stages. The mammary bud in the strain of mouse studied by Raynaud shows no sex differences in development until the 15th to 16th day at which time the genital tract, hitherto indifferent, begins to differentiate. Coincident with this the mammary bud in the male becomes surrounded by a condensation of special mesenchymal cells the action of which constricts the bud at its junction with the epidermis from which it ultimately becomes completely detached (Fig. 10.1). The inguinal glands seem particularly susceptible to this influence because they exhibit this effect earlier than the thoracic glands and in some strains the second inguinal bud in the male tends to disappear completely. Sex differences in the prenatal development of the mammary rudiment in certain species of wild mouse were also described by Raynaud (1949b).
The fact that, after x-ray desti'uction of the gonad in the 13-day male mouse embryo, the mammary bud remains attached to the epidermis and the duct primordia ramify in a manner similar to the primordia in the female shows that this phenomenon of detachment of the mammary bud is due to the action of the fetal testis (Raynaud and Frilley, 1947, 1949). That the masculinizing action of the fetal testis seems to be due to the hormonal secretion of a substance having the same effect as testosterone is suggested by the fact that injection of testosterone into the pregnant mother causes the mammary buds in the female embryo to undergo the male type of development (Fig. 10.1). Here again the inguinal glands seem most sensitive because sufficiently high doses in many cases cause complete disappearance of the primordia of the second inguinal glands (Raynaud, 1947a. 1949a).

On the other hand, destruction of the fetal gonad in the female has no effect on the development of the mammary bud (Raynaud and Frilley, 1947, 1949), yet the lattW is not completely indifferent to the action of estrogen because high doses of estrogen administered to the mother, or lower doses injected early into the embryo itself inhibit the growth of the mammary bud (Raynaud. 1947b, 1952; Raynaud and Raynaud, 1956, 1957), an effect reminiscent of the well known action of excessive doses of estrogen on the adult mammary duct system (for reference see Folley, 1952a) . In pouch young of the opossum, on the other hand, Plagge (1942) found that estrogen treatment stimulated growth of the mammary duct primordia. Similarly in the fetal male mouse low doses of estrogen stimulate growth of the mammary bud (Raynaud, 1947d), but this may be an indirect effect ascribable to estrogen's antagonizing the inhibitory action of the fetal testis.
The problem of the histogenesis of the teat has also come under experimental attack. Raynaud and Frilley (1949) showed that the formation of the epithelial hood," the circular invagination of the epidermis surrounding the mammary bud which constitutes the teat anlage in the mouse, is not hormonally determined since its appearance was not prevented by the irradiation of the fetal ovary at the 13th day of life. In the male mouse the epithelial hood does not normally appear and the male is born without teats. This is undoubtedly due to the action of the fetal testis inasmuch as the teat anlagen develop in the male embryos whose testes are irradiated at 13 days (Raynaud and Frilley, 1949).
The foregoing observations jioint to an ahormonal type of development for the teat and mammary bud in the female fetus, at least in the mouse, although the mammary bud is specifically susceptible to the action of excess exogenous estrogen which can inliibit its development without affecting that of other skin gland ])rimordia. The mammary hud is a'so sus('ei)tible to the action of anch'ogen which in the normal male fetus not only dii-ects its development along charact(M-istic lines, but also suppresses the formation of the teat.

MAMMARY GLAND AND LACTATION

593

PwokcTiL del

Fig. 101. Sex difference in the development of the mammaiy bud of the fetal mouse and effect of androgen on the histogenesis of the female mammary bud. A. First inguinal gland of female fetus (15 days, 17 hours). B. First inguinal gland of male fetus (15 days, 17 hours). C. Second inguinal gland of female fetus (15 days, 16 hours) from a mother receiving testosterone propionate. D. First inguinal gland of female fetus from the same litter as that in C. (From A. Ravnaud, Ann. endocrinol., 8, 248-253, 1947.)

For further information on the morphogenesis of the mammary ghmd, the reader is referred to the recent detailed accounts by Dabelow (1957) and Raynaud (1960).
B. NORM.\L POSTNATAL DEVELOPMENT
1. Methods of Assessing Mammary Development
In the last two decades the increasing availability of the ovarian hormones in pure form and the prospect of the large scale practical application of fundamental knowledge of the hormonal control of the mammary gland to the artificial stimulation of udder growth and lactation in the cow, have together effected a demand for greater accuracy in studying and assessing the degree of mammary development. Various quantitative and objective procedures have now been evolved which allow results of developmental studies to be subjected to statistical investigation. These methods have been re

viewed recently (Folley, 1956) and we need but mention them briefly.
In those species in which, save in late pregnancy, the mammae are more or less flat sheets of tissue, the classical wholemount preparations have been the basis for several quantitative studies. From such preparations the area covered by the duct systems can be measured by suitable means (e.g., as in our studies on the rat mammary gland; Cowie and Folley, 1947d), thus providing an accurate measure of duct extension. Such measurements, however, give no information on the morphologic changes within this area and so a semiquantitative scoring system to assess the degree of duct complexity has been used in conjunction with the measurements of area (see Cowie and Folley, 1947d) . More reliable and objective techniciues for measuring duct complexity were later developed in our laboratory by Silver (1953a) and Flux (1954a). Species such as the guinea pig in which the gland,

594

PHYSIOLOGY OF GONADS

even when immature, is three-dimensional demand other methods. For such cases a precise but rather tedious method has been described by Benson, Cowie, Cox and Goldzveig (1957) which involves the determination of the volume of glandular tissue from area measurements of serial sections of the gland in conjunction with semiquantitative scoring procedures for assessing the morphologic characteristics of the tissue.
Particularly applicable to the lactating gland is the procedure developed by Richardson (see Cowie, Folley, Malpress and Richardson, 1952; Richardson, 1953) for assessing the total internal surface area of the mammary alveoli. It is of interest to note in passing that this technique is based on that developed by Short (1950) for measuring the surface area of the alveoli in the lung, the similarity in the geometry of the two organs allowing ready transference of the method from one to the other.
At present these quantitative procedures have the disadvantage of being slow and time consuming, and it seems likely that their further development will involve the use of electronic scanning methods to speed up the examination of the tissues. Of recent introduction are some biochemical procedures for assessing changes in mammary development. The desoxyribonucleic acid (DNA) content of any particular type of cell is said to be remarkably constant (see Vendrely, 1955, for review) and the amount of DNA in a tissue has been used as a reference standard directly related to the number of cells present in a tissue and to provide an estimate of the number of cells formed during the developmental phases of a gland or tissue (see Leslie, 1955, for review). Studies on DNA changes which occur in the mammary gland during pregnancy and lactation have been made in the rat by Kirkham and Turner (1953), Grecnbaum and Slater (1957a), Griffith and Turner (1957), and Shimizu (1957). It should be noted, however, that some authorities have doubts as to the constancy under all conditions of the DNA content of a cell (see Brachet, 1957) and results obtained by this technique should be interpreted with some caution (see also Griffith and Turner, 1957). Other chemical methods for assessing mammary development include (a) the determination

of the iron content of the gland, based on the observation that iron retention occurs in the epithelium of the mammary glands of mice (Rawlinson and Pierce, 1950) ; (b) whole-mount autoradiographs using P^(Lundahl, Meites and Wolterink, 1950) ; and (c) determination of the total content of alkaline phosphatase in the mammary gland (Huggins and Mainzer, 1957, 1958).
In view of the relative rapidity of the biochemical methods it seems likely that they will be used increasingly in the future.
A technique of clinical interest allowing the qualitative assessment of changes in mammary structure in the breast of pregnant and lactating women is the radiographic method described by Ingleby, Moore and Gershon-Cohen (1957).
To those seeking information of the microscopic anatomy of the human mammary gland we would recommend the excellent and beautifully illustrated review by Dabelow (1957), and new facts on the cytologic changes occurring during milk secretion will be found in the electron microscopic study of the rat mammary gland by Bargmann and Knoop (1959), and of the mouse mammary gland by Hollmann (1959).
Having briefly outlined the various quantitative methods of assessing mammary development we will now consider recent studies on normal mammary growth.
2. Mammary Development in the X on pregnant Female
It has been the general belief that until puberty the mammary ducts show little growth, but more precise studies in which the rate of increase in mammary gland area has been related to the increase in body size have now shown that in the monkey, rat, and mouse a phase of ra])id duct growth is initiated before puberty.
The first use of this procrdure, relative gi'owth analysis (for terminology see Huxley and Teissier, 1936), for the quantitative investigation of mammary duct growth was made by Folley, Guthkelch and Zuckerman (1939), who showed that over a wide range of body weights, the breast in the nonpregnant female rhesus monkey grows faster than the body as a whole. Subsequently, more detailed studies of the dynamics of mammary growth using relative growth

MAMMARY GLAXD AND LACTATION

595

olO.

rCMALC RATS
ACt$ : i - lOO DAYS

C 22 NO DAY

LOC„ CBOOY WtlCHT C>

Fig. 10.2. Relative mammary gland growth in the female hooded Norway Cowie. J. Endocrinol.. 6, 145-157, 1949.)

(From A.T.

analysis were made in the rat by Cowie (1949) and Silver (1953a, b) and in the mouse by Flux (1954a, b), and their results will now be summarized. In the rat the total mammary area increased isometrically with the body surface (a = 1.1 as compared with the theoretic value of 1.0) until the 21st to 23rd day when a phase of allometry (a = 3.0) set in. The onset of the allometric phase could be prevented by ovariectomy on the 22nd day (see Fig. 10.2). Since estrous cycles do not begin until the 35th to 42nd day in this strain of rat, it is clear that the rapid extension of the mammary ducts began well before puberty. In the immature male rat the increase of mammary area on body surface was slightly but significantly allometric; this was not altered by castration at the 22nd day. Earlier ovariectomy, i.e., when the pups were 10 days old, was followed by a phase of slightly allometric growth of the mammary glands in the fe

males (a = 1.5). With regard to the female mouse (CHI strain) a i)hase of marked allometry in mammary duct growth set in about the 24th day (a = 5.2) which could also be prevented by prior ovariectomy.
It is clear that the presence of the ovary is essential for the change from isometry to allometry, but the nature of the mechanisms governing the change is still uncertain (for further discussion, see Folley, 1956).
3. Mammary Growth in the Male
The testes have apparently little effect on mammary duct extension in the rat inasmuch as the gland in the male grows isometrically or nearly so and its specific growth rate is unaffected by castration. Castration at 21 days, however, does prevent for a time development of the lobules of alveoli, first described by Turner and Schultze (1931 ) , which are characteristic of the mammary gland in the male rat. Eventually.

596

PHY,SI(3L0GY OF GONADS

however, some alveoli do develop in the mammae of immaturely castrated male rats (Cowie and Folley, 1947d; Cowie, 1949; Ahren and Etienne, 1957) and it has been ])Ostulated that these arise from the enhanced production by the adrenal cortex of mammogenic steroids (androgens or progesterone) due to the hormone imbalance brought about by gonadectomy (see Folley, 1956 L
In a recent study, Ahren and Etienne (1957) have shown that the ducts and alveoli in the mammary gland of the male rat are remarkable in that their epithelial lining is unusually thick, being composed of several layers of cells. It had been previously noted by van Wagenen and Folley (1939) and Folley, Guthkelch and Zuckerman (1939) that testosterone caused a thickening of the mammary duct epithelium in the monkey and sometimes papillomatous outgrowths of epithelium into the lumen of the duct. It would thus seem that, although the hormone of the testis is capable of eliciting alveolar development, these alveoli and ducts differ from those occurring in the female in the nature of their epithelium. It w^as further observed by Ahren and Etienne (1957) that in the castrated male rat the alveoli, which eventually developed, had a simple epithelial lining somewhat similar to that seen in the normal female rat, suggesting that, if the adrenals are responsible, the mammogenic steroid is more likely to be progesterone than an androgen.
A study of considerable clinical interest is that of Pfaltz (1949) on the developmental changes in the mammary gland in the human male. The greatest development reached was at the 20th year; by the 40th year there occurred an atrophy first of the l)arenchyma and later of the connective tissue. In the second half of the fifth decade there was renewed growth of the parenchyma and connective tissues. The hormonal background of these changes and the possible relationship with prostatic hyjiertrophy are discussed by Pfaltz. (Further details of the microscopic anatomy of the mammary gland of the human male may be found in the studies by Graumann, 1952, 1953, and Dabclow, 1957.)

4- Mammary Development during Pregnancy
It has been customary to divide mammary changes during pregnancy into two phases, a phase of growth and a secretory phase. In the former there occurs hyperplasia of the mammary parenchyma whereas, in the latter, the continued increase in gland size is due to cell hypertrophy and the distension of the alveoli with secretion (see Folley, 1952a j . Although it was realized that these two phases merged gradually, recent studies have confirmed earh^ reports {e.g., those of Cole, 1933; Jeffers, 1935) that a wave of cell division occurs in the mammary gland towards the end of parturition or at the beginning of lactation. Al'tman (1945) described a doubling in number of cells per alveolus, in the mammary gland of the cow at parturition, but the statistical significance of his findings is difficult to assess. More recently, how^ever, Greenbaum and Slater (1957a) found that the DNA content of the rat mammary gland doubled between the end of pregnancy and the 3rd day of lactation, a finding which they interpret as resulting in the main from hyperplasia of the gland cells. Likewise in the mouse mammary gland, Lewin (1957) observed between parturition and the 4th day of lactation a great increase both in the DNA content of the mammary gland and in the total cell count. Studies on the factors controlling this wave of cell division are awaited with interest. Also associated with the onset of copious milk secretion is a considerable increase in cell volume and coincident ally the mitochondria elongate and may increase in diameter (Howe, Richardson and Birbeck, 1956). Cross, Goodwin and Silver (1958) have followed the histologic changes in the mammary glands of the sow, by means of a biopsy technique, at the end of pregnancy, during parturition, and at weaning. At the end of pregnancy there was a ])i'()gr('ssi\-c' distension of the alveoli, the existing hyaline eosinoi)hilic secretion within the alveoli was gradually replaced by a basophilic material, and fat globules appeared. At i)arturition the alveoli were contracted and their walls appeared folded (Fig. 10.3).

MAMMARY GLAND AND LACTATION

597

Fig. 10.3. Sections of biopsy specimens from the mammary gland of a sow before and din-ing parturition. A. Six days before parturition: the mammary alveoh are small and contain a nongranular eosinophilic secretion. B. Two days before parturition: alveoli have increased in size and fat globules are conspicuous. C. Fifteen hours before parturition: alveoli are now distended with secretion which consists of an outer zone of eosinophilic material and fat globules, and a central zone of basophilic granular secretion. D. During parturition: alveoli contracted with folded epithelium and sparse secretion. (From B. A. Cross, R. F. W. Goodwm and L A. Silver, J. Endocrinol., 17, 63-74, 1958.)

598

PHYSIOLOGY OF GONADS

5. Mam /nary Involution
The involutionary changes which occur in the mammary gland after weaning in various species were described in the previous edition of this book (Turner, 1939) and in a later review by Folley (1952a). Since that time, a few further studies have appeared.
There is evidence that the course of the histologic changes in the regressing mammary gland may differ according to whether the young are weaned after lactation has reached its peak and is declining, or whether they are removed soon after parturition, when the effects of engorgement with milk seem to be more marked (see, for example, Williams, 1942, for the mouse). In rats whose young were weaned soon after parturition Silver (1956) was able to re-establish lactation provided suckling was resumed within 4 or 5 days; after that time irreversible changes in the capillary blood supply to the alveoli had set in. A further point arises from a study on the cow by Mosimann (1949) which indicates that the course of the regressive changes in a gland which has undergone one lactation only may differ from those seen in glands from muciparous animals. Oshima and Goto (1955) have used quantitative histometric methods in a study of the involuting rat mammary gland ; the values which they obtained for the percentage parenchyma 7 to 10 days after removal of the young agree quite well with tiiose reported by Benson and Folley ( 1957b) for rats weaned at the 4th day and killed 9 days later.
The biochemical changes occurring in mammary tissue during involution arc of some interest and have been studied in our laboratory by McNaught (1956, 1957). She studied mammary slices taken from rats whose young were removed at the 10th day and also slices from suckled glands, the escajie of milk from which was prevented by ligation of the galactophores, the other glands in the same animals remaining intact and serving as controls. Her results, some of whichare summarized in Figure 10.4, suggest that functional changes which may be taken as indicative of involution (decrease in oxygen up-take, respiratory quotient (R.Q.), and glucose up-take; increase in lactic acid prcxUiction ) are seen as early as

8 to 12 hours after weaning. Continued suckling without removal of milk retards the onset of these changes, but only for some hours. Injections of oxytocin into the rats after weaning (see page 607) did not retard these biochemical changes. Essentially simihii' results were independently reported by Ota and Yokoyama (1958) and Mizuno and Chikamune (i958).
C. EXPERIMENTAL ANALYSIS OF HORMONAL INFLUENCES
1. Ovarian Hortnones in the Animal with Intact Pituitary
We shall see later (page 602) that the mammogenic effects of the ovarian hormones are largely dependent on the integrity of the a'nterior pituitary and thus to analyze accurately the role of hormones in mammary development it is necessary to use hypophysectomized animals. Information of considerable academic and practical importance has been obtained, however, from studies in the animal with intact pituitary and these we shall now consider.
Early studies involving hormone administration pointed to the conclusion that estrogens were in general resi)onsible for the growth of the mammary (hicts, whereas progesterone was necessary for complete lobulealveolar growth (see reviews, l)y Turner, 1939; Folley and Malpress, 1948a; Folley, 1952a). The foundation for i^liis general statement is now more sure, for as a result of experimental studies over the last 10 years, what seemed to be exceptions to this generalization have been shown to be otherwise. In some species (mouse, rat, guinea \)ig, and monkey) it is true that progesterone alone, if given in sufficiently large doses, will evoke duct and alveolar development in the ovariectomized animal, but this is probably a pharmacologic rather than a physiologic effect. There are great differences in the response of the mammary ducts to estrogen and on this basis it has become usual to divide species into three broad categories (see FoUey, 1956). It is, however, necessary to add the warning that in the estrogentre.'ited spayed animal progesterone from the a(h'eiial eoiiex may synergize with the exogenous estrogen (see Folley, 1940; Trentin and 1'ui'iier, 1947; Hohn, 1957) and it mav

MAMMARY GLAND AND LACTATION

599

O2 Uptake

G\

ucose

uptake

Lactic acid production.

s 12 ■Hours
Fig. 10.4. Oxygen uptake, respiratory quotient, glucose uptake, and lactic acid production of mammary gland slices from lactating rats killed at various times after weaning (A — A) and from rats in which svickling was maintained, but in which the galactophores of certain
glands were ligatured (• •) to prevent the escape of milk, the nonligatured glands
(O O) acting as controls. (Courtesy of Dr. M. L. McNaught.)

be that the I'eal basis for the categories is to be found largely in differences in endogenous progesterone production by the adrenal cortex.
The first category comprises those in which estrogens, in what are believed to be physiologic doses, evoke primarily and mainly duct growth; alveoli may appear, but only if high doses are given and the administration is prolonged. Examples of this class are the mouse, rat, rabbit, and cat. Silver (1953a), using the relative-growth technique, has obtained information on the

levels of estrogen necessary for normal mammary duct growth in the nonpregnant rat. In the young ovariectomized rat, the normal mammary growth rate was best imitated by injecting 0.1 ;u,g. estradiol dipropionate every second day (from 21 days of age) and increasing the dose step- wise with body weight. In the ovariectomized mouse, Flux (1954a) found it necessary to give 0.055 /jLg. estrone daily to attain mammarv duct growth comparable with that obser\-( . i in intact mice.
In the second category are those s]:»ecies

(JOO

PHYSIOLOGY OI-' GONADS

in which estrogen in physiologic doses causes growth of the ducts and the lobule-alveoL^r system, the classical example being the guinea pig in which functional mammae can be developed after gonadectomy in either sex by estrogen alone. A recent study by Hohn (1957), however, strongly suggests that progesterone from the adrenal cortex participates in the effect. The earlier view, moreover, that complete mammary growth can be evoked in the gonadectomized guinea l)ig by estrogen alone (Turner and Gomez. 1934; Nelson, 1937.) does not find support in the recent study of Benson, Cowie, Cox and Goldzveig (1957), who, using both subjective and objective methods of assessing the degree of mammary development, found that over a wide dose range of estrone, further development of the mammary gland was obtained when jirogesterone was also administered; essentially similar conclusions have been reached by Smith and Richterich (1958).
Also in this second category are cattle and goats in which, however, the male mammary gland is not equipotential with that of the female. The early studies on these species have been reviewed at length by FoUey and Malpress (1948a) and Folley (1952a, 1956). Briefly it may be said that these studies clearly showed that estrogen alone induced extensive growth of lobule-alveolar tissue of which the functional capacity was considerable although the milk yields in general were less than those expected from similar animals after parturition. The response to estrogen treatment was, moreover, very erratic. It was generally believed that the deficiencies of this treatment could be made good if progesterone were also administered, a view supported by the observations of Mixner and Turner (1943) that the mammary gland of goats treated with estrogens, when examined histologically, showed the i)resence of cystic alv(>oli, an abnormality which tended to disappear when jirogestcrone was also administered.
When progesterone became more readily available, an extensive study of the role of estrogen and progesterone in mammary development in the goat was carried out (Cowie, Folley, ^lalpress and Richai'dson.

1952; Benson, Cowie, Cox, Flux and Folley, 1955). The mammary tissue was examined histologically and the procedure devised by Richardson (see page 594) used to estimate the area and "porosity" of the alveolar epithelium. The udders grown in immaturely ovariectomized virgin goats by combined treatment with estrogens and progesterone in various proportions and at different absolute dose levels were compared with udders resulting from treatment with estrogen alone. As in the earlier observations of Mixner and Turner (1943) , histologic abnormalities were noted, the more widespread being a marked deficiency of total epithelial surface, associated with the presence of cystic alveoli, in the udders of the estrogen-treated animals. The addition of progesterone prevented the appearance of many of these abnormalities and increased the surface area of the secretory epithelium. JMoreover, when estrogen and progesterone were given in a suitable ratio and absolute level the milk yields obtained were remarkably uniform as between different animals and the glandular tissue was virtually free from abnormalities.
Studies in the cow have been less extensive, but there is evidence that both estrogen and progesterone are necessary for complete normal mammary development (Sykes and Wrenn, 1950, 1951; Reineke, INIeites, Cairy and Huffman, 1952; Flux and Folley, cited by Folley, 1956; Meites, 1960).
The case for the inclusion of the monkey in the present category has been strengthened by the excellent monograph of Speert ( 1948) who has had access to more extensive material than many of the earlier workers whose results are reviewed by him (see also Folley, 1952a). The sum total of available evidence now justifies the conclusion that estrogen alone will cause virtually complete growth of the duct and lobule-alveolar systems of the monkey breast. Extensive lobulealveolar development in the monkey breast in response to estrogen is shown in Figure 10.5. The synergistic effect of estrogen and jirogesterone on the monkey breast has not yet been adequately studied, but from available evidence it does not seem to be very dramatic. If it is permissible to argue from pi'iinates to man. it seems jiossible that coidd

MAMMARY GLAND AND LACTATION

601

Fig. 10.5. Wliole mounts of breast of an ovariectomized immature female rhesus monkey before (left) and after (right) e.strogen treatment. (From H. Speert, Contr. Embrvol., Carnegie Inst. Washington, 32, 9-65, 1948.)

the necessary experiments be done the human breast would show a considerable growth response to estrogen alone.
Finally, in the third category are those species in which estrogen in physiologic doses causes little or no mammary growth. The bitch and probably the ferret seem to belong to this class (see Folley, 1956).
There has been considerable discussion in the past regarding the ratio of progesterone to estrogen optimal for mammary growth. Only recently, however, has this question been fully investigated in any species. Benson, Cowie, Cox and Goldzveig (1957) have shown that in the guinea pig the absolute quantities of progesterone and estrogen are the crucial factors in controlling mammary growth; altering the dose levels but maintaining the ratio gave entirely different growth responses. In view of the varying ability of the different estrogens to stimulate mammary duct growth (Reece, 1950) it is essential in discussing ratios to take into consideration the nature of the estrogen used, a fact not always recognized in the past.
2. Anterior Pituitary Hormones
Soon after the discovery by Strieker and Grueter (1928, 1929) of the lactogenic effects of anterior iiituitarv extracts, it was

shown that anterior i)ituitary extracts had a mammogenic effect in the ovariectomized animal and that the ovarian steroids had little or no mammogenic effect in hypophysectomized animals. C. W. Turner and his colleagues postulated that mammogenic activity of the anterior pituitary was due to specific factors which they termed "mammogens"; other workers, in particular W. R. Lyons, believed the mammogenic effect was due to prolactin. The theory of specific mammogens has been fully reviewed in the past (Trentin and Turner, 1948; Folley and Malpress, 1948a) and we do not propose to discuss it further for there is now little evidence to support it. Damm and Turner ( 1958) , while recently seeking new evidence for the existence of a specific pituitary mammogen, concur in the view expressed by Folley and Malpress (1948a) that final proof of the existence of a specific mammogen will depend on the development of l)etter assay techniques and the characterization or isolation of the active principle.
The mammogenic effects of prolactin were observed in the rabbit by Lyons (1942) who injected small quantities of prolactin directly into the galactophores of the suitably prepared mammary gland. IV'Iilk secretion occurred but Lyons also noted that the l)rolactin caused active growth of the alveo

602

PHYSIOLOGY OF CIOXADS

lar epithelium. Recently, Mizuno, lida and Naito (1955) and Mizuno and Naito (19561 have confirmed Lyons' observations on the mammogenic effect of intracluct injections of prolactin in the rabbit both by histologic and biochemical means (DNA estimations) and there seems little doubt that the prolactin is capable of exerting a direct effect on the growth of the mammary parenchyma, at least in the rabbit whose pituitary is intact.
In the last 18 years much information on the role of the anterior pituitary in mammary growth has been obtained by Lyons and his colleagues in studies on hypophysectomized, hypophysectomized-ovariectomized, and hypophysectomized-ovariectomized-adrenalectomized (triply operated) rats of the Long-Evans strain. In 1943 Lyons showed that in the hypophysectomized-ovariectomized rat, estrogen + progesterone + prolactin induced lobulealveolar development, but the degree of development was less than that obtained in the ovariectomized rat with intact pituitary receiving estrogen and progesterone. When supplies of purified anterior-pituitary hormones became available the experiments were extended (Lyons, Li and Johnson, 1952) and it was shown that if somatotrophin (STH) was added to the hormone combination of estrogen -f progesterone + prolactin, the degree of lobulealveolar development obtained in the hypophysectomized-ovariectomized rat was much enhanced. The omission of prolactin from the hormonal tetrad prevented lobulealveolar development from occurring. In the hypophysectomized-ovariectomized-adrenalectomized rat the above hormonal tetrad could also evoke lobule-alveolar development, provided the animals were given saline to drink (Lyons, Li, Cole and Johnson, 1953). In yet more recent experiments Lyons, Li and Johnson (1958) observed that somatotrophin has a direct stimulatory effect on duct growth, but in the hypophysectomized-ovariectomized rat, the presence of estrogen is also necessary to evoke normal duct development (Fig. 10.6a, b, c) ; Likewise, in the triply operated rat, STH plus estrogen is mammogenic, but the presence of a corticoid is r('([ui]'ed to o])tain full duct de

velopment (Fig. 10.6r/). Lyons and his colleagues were able to build up the mammary glands of triply operated rats from the state of bare regressed ducts to full prolactational lobule-alveolar development by giving estrogen + STH + corticoids for a period of 10 days to obtain duct proliferation followed by a further treatment (for 10 to 20 days) with estrone + progesterone -I- STH -I- prolactin + corticoid to induce lobulealveolar development. Alilk secretion could then be induced by a third course of treatment lasting about 6 days in which only prolactin and corticoids were given (Fig. 10. 6e, /). Essentially similar results have been obtained in studies with the hooded Norway rat (Cowie and Lyons, 1959).
Studies on mammogenesis in the hypophysectomized mouse have revealed some differences in the response of the mammary gland of this species in comparison with that of the rat and indications of strain differences within the species. The mammary gland of the hypophysectomized male weanling mouse of the Strong A2G strain shows no response to the ovarian steroids alone, to prolactin, or to STH alone, but it responds with vigorous duct proliferation to combinations of estrogen + progesterone + prolactin, or of estrogen 4- progesterone + STH (Hadfield, 1957; Hadfield and Young, 1958). In the hypophysectomized male mouse of the CHI strain slight duct growth occurs in response to estrogen + jirogesterone and this is much enhanced when STH is also given; the further addition of prolactin then results in alveolar development (Flux, 1958). Extensive studies in triply operated mice of the C3H 'HeCrgl strain have been reported by Nandi (1958a, b). In this strain some duct growth was observed in triply operated animals in response to steroids alone (estrogen -I- progesterone + corticoids), but normal duct develojmient was believed to be due to the action of estrogen + STH + corticoids, a conclusion in agreement with Lyons' observations in the rat. Extensive lobuleahcohii' development could be induced by a number of hormone coml)inations, one of the most effective being estrogen + progesterone + corticoids + prolactin + STH, milk secretion occurring when the ovarian

MAMMARY C5LAND AND LACTATION

603

Fig. 10.6. Typical areas of whole mounts of the abdominal mammary gland of rat.s after the following treatments: A. Untreated rat on day 31, 14 days after hypophysectomy. The gland has regressed to a bare duct system. B. Rat hypophysectomized and ovariectomized on day 30 and injected daily with 2 mg. somatotrophin (STH) for 7 days. Note the presence of end clubs, r. Rat treated as in B but which received, in addition to the STH, 1 ^g. estrone. Note profuse eiid-rhil' ] iroliferatiou. D. Rat li.\|M)]ili\s(>ctomized on day 30. ovariectomized and adri'nali^ctoinized on day 60, and injected daily from days 60 to 69 with 1 mg. STH + 0.1 mg. DCA + 1 fig. estrone. Note again the profuse number of end buds indicative of duct proliferation. E. Same treatment as in D followed by 10 days treatment with 5 mg. prolactin + 2 mg. STH + 1 /xg. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate. Note excellent lobule-alveolar growth. F. Same treatment as in D followed by 20 days treatment with 5 mg. prolactin + 2 mg. STH + 1 fig. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate; thereafter given 0.1 mg. prolactin locally over this gland and 0.1 mg. DCA + 0.1 mg. prednisolone acetate systemically for 6 days. Note fully developed lobules with ah'eoli filled with milk. (All glands at the same magnification.) (From W. R. Lyons. C. H. Li and R. E. Johnson, Recent Progr. Hormone Res., 14, 219-254, 1958.)

604

PHYSIOLOGY OF GONADS

steroids were withdrawn, while the })rohictin, STH, and Cortisol were continued. A further interesting observation made by Nandi is that in the C3H/HeCrgl mouse STH can replace prolactin in the stimulation of all phases of mammary development and in the induction of milk secretion; enhanced effects were obtained, however, when prolactin and STH were given together. Nandi also considers that progesterone plays a greater role in duct development in the mouse than in the rat.
The above experiments clearly indicate that both in the triply operated rat and mouse, it is possible to build up the mammary gland to the full prolactational state by injecting the known ovarian, adrenal cortical, and anterior pituitary hormones. There would thus seem to be no necessity to postulate the existence of other unidentified pituitary mammogens. It must be recognized, however, that in normal pregnancy the placenta may be an important source of mammogenic hormones. The placenta of the rat contains a substance or substances possessing luteotrophic, mammogenic, lactogenic, and crop-sac stimulating properties, but it is uncertain whether this material is identical with pituitary prolactin (Averill, Ray and Lyons, 1950; Canivenc, 1952; Canivenc and Mayer, 1953; Ray, Averill, Lyons and Johnson, 1955). There is also some evidence of the presence of a somatotrophin-like principle in rat placenta (Ray, Averill, Lyons and Johnson, 19551.
3. Metabolic Hormones {Corticoids, Insulin, and Thyroid Hormones)
We have already noted that Lyons and his colleagues were able to obtain full duct development in the triply operated rat only when corticoids were given. Early studies of the role of the adrenals in mammary development have given conflicting and uncertain results (see review by Folley, 1952a). Recent studies have not entirely clarified the position. Flux (1954b) tested a number of 11 -oxygenated corticoids, and found that not only were they devoid of mammogenic activity in the ovariectomized virgin mouse, but that they inhibited the gi'owth-promoting effects of estrogen on the mammary ducts, whereas 11-desoxycorticosterone acted synergistically with estro

gen in promoting duct growth. In subsequent studies it was shown that injections of adrenocorticotrophin (ACTH) into intact female mice did not influence mammary growth (Flux and ]\lunford, 1957), but that Cortisol acetate in low doses (12.5 /^g. l)er day) stimulated mammary development in ovariectomized and in ovariectomized estrone-treated mice, whereas at higher levels (25 and 50 ftg. per day) it was without effect (Munford, 1957). In the virgin rat, on the other hand, glucocorticoids are said to stimulate mammary growth and to induce milk secretion (Selye, 1954; Johnson and Meites, 1955). Some light on these conflicting results has been shed by the studies of Ahren and Jacobsohn (1957) who investigated the effects of cortisone on the mammary glands of ovariectomized and of ovariectomized-hypophysectomized rats, both in the presence and absence of exogenous ovarian hormones. In the hypophysectomized animals, cortisone promoted enlargement and proliferation of the epithelial cells lining the duct walls, but normal growth and differentiation did not occur, nor did the addition of estrogen and progesterone appreciably alter these effects ; in rats with intact pituitaries, however, cortisone stimulated secretion but not mammary growth, whereas the addition of estrogen and progesterone promoted both growth and al)undant secretion. Ahren and Jacobsohn concluded that "the effect elicited by cortisone in the mammary gland should be analysed with due regard to the endocrine state of the animal both as to its effects on the structures of the mammary gland and to the consequences resulting from an eventual upset of the general metabolic equilibrium." They consider that in circumstances optimal for mammary gland growth and maintenance of homeostasis the predominant actions of cortisone are enhancement of alveolar growth and stimulation of secretion, whereas under conditions ill which the metabolic actions of cortisone are not efficiently counteracted, gland growth is either inhibited or an abnormal development of certain iiianimaiy cells may be e^■()ked.
That the general metabolic milieu may indeed profoundly influence the response of the iiuuiimarv gland to hormones has

MAMMARY GLAND AND LACTATION

605

been emiiha.-^ized by the recent experiments of Jacobsohn and her colleagues. Following on the work of Salter and Best (1953) who showed that hypophysectomized rats could be made to resume body growth by the injections of long-acting insulin, Jacobsohn and her colleagues (Ahren and Jacobsohn, 1956; Ahren and Etienne, 1958; Ahren, 1959) found that treatment with estrogen and progesterone would stimulate considerable mammary duct growth in hypophysectomized-gonadectomized rats when given with suitable doses of long-acting insulin (Fig. 10.7). This growth-supporting effect of insulin could be nullified if cortisone was also administered (Ahren and Jacobsohn, 1957) but could be enhanced by giving thyroxine (Jacobsohn, 1959).
The thyroid would thus appear to be another endocrine gland whose hormones affect

mammary growth intlirectly by altering the metabolic environment. Studies in this field, reviewed by Folley (1952a, 1956), indicate that in the rat some degree of hypothyroidism enhances alveolar development wdiereas in the mouse, hypothyroidism seems to inhibit mammary development. Chen, Johnson, Lyons, Li and Cole (1955) have shown that mammary growth can be induced in hypophysectomized - adrenalectomized-thyroidectomized rats by giving estrone, progesterone, prolactin, STH, and Cortisol, no replacement of the thyroid hormones being necessary.
These investigations on the effect of the metabolic environment on mammary development seem to ])e opening up new avenues of approach to the advancement of our understanding of the mechanisms of mammary growth and we would recommend.

0-5 cm.

Fig. 10.7. Whole mount preparation of .second thoracic mammary gland of : ^. Ovariectomized rats injected with estrone and progesterone. B. Hypophysectomized-ovariectomized rat injected with estrone and progesterone. C. Hypophysectomizcd-o\ariectomized rat. D. Hypophysectomized-ovariectomized rat injected with estrone, progesterone, and insulin. (From K. Ahren and D. Jacobsohn, Acta physiol. scandinav., 37, 190-203, 1956.)

GOG

PHYSIOLOGY OF GONADS

to those seeking further information about this important new fiekl, the recent review by Jacobsohn (19581.
III. Endocrine Influences in Milk Secretion
A. ANTERIOR PITUITARY HORMONES
1. Initiation of Secretion iLactogenesis)
The early experiments leading to the view that the anterior pituitary was not only necessary for the initiation of milk secretion, but in fact i)rovided a positive lactogenic stimulus, are now well known and the reader is referred to the reviews by Folley (1952a, 1956) and Lyons (1958) for further particulars. That pituitary prolactin can evoke milk secretion in the suitably de\-eloped mammary gland of the rabbit with intact pituitary has been amply confirmed, and the original experiments of Lyons (1942) involving the intraduct injection of prolactin have been successfully repeated by Meites and Turner (1947) and

Fk;. 10.8. Liictation.'il lespon.scs in pseudoincgnant rabbit to different doses of prolactin injeclcd intraductallv. (Fiom T. R. Bradley and P. M. Clarke, J. Endo.ninol., 14, 28-36, 1956.)

Bradley and Clarke (1956) (Fig. 10.8). However, endogenous pituitary hormones may have participated in the response in such experiments and in the last 20 years there has been considerable discussion as to whether prolactin should be regarded as the lactogenic hormone or as a component of a lactogenic complex. This whole question has been fully discussed in recent years (see Folley, 1952a, 1956) and it now seems reasonably certain that lactogenesis is a response to the co-operative action of more than one anterior pituitary hormone, that is, to a lactogenic hormone complex of which prolactin is an important component, as first suggested by Folley and Young (1941 ) . The recent reports by Nandi (1958a, b) that STH -I- Cortisol can induce milk secretion in triply operated mice with suitably developed glands is further strong evidence against regarding prolactin as the lactogenic hormone.
Secretory activity is evident in the mammary gland during the second half of pregnancy, but abundant milk secretion does not set in until parturition or shortly thereafter. The nature of the mechanism controlling the initiation of abundant secretion has been the subject of speculation for many years. The earlier theories w^ere discussed l)y Turner ( 1939 ) in the second edition of this book, and included the theory put forward by Nelson with reference to the guinea pig, that the high levels of blood estrogen in late pregnancy suppressed the secretion or release of prolactin from the pituitary and had also a direct inhibitory cttcct on the mammary parenchyma, the fall in the levels of estrogen occurring at parturition then allowing the anterior pituitary to exert its full lactogenic effect. This concept proved inadequate to exjilain observations in other species and it was later extended by Folley and Malpress (1948b) to embrace the concept of two thresholds for oi:)posing influences of estrogen upon jiituitary lactogenic function, a lower threshold for stimulation and a higher one for inhibition. Subsequent observations on the inhibitory role of progesterone, in the pix'sence of estrogen, on milk secretion, however, necessitated further modification of the theorv. Before discussing these modifica

MAMMARY GLAND AND LACTATION

607

tions it is convenient to refer to the ingenious theory put forward by Meites and Turner (1942a, b; 1948) which was based on their extensive investigation of the prolactin content of the pituitary in various physiologic and experimental states. According to Meites and Turner, estrogen elicits the secretion of prolactin from the anterior pituitary thereby causing lactogenesis, whereas progesterone is an inhibitory agent, operative in pregnancy, inhibiting or over-riding the lactogenic action of estrogen. The induction of lactation was thus ascribed to a fall in the body level of progesterone relative to that of estrogen heheved to occur at the time of parturition. Subsequent studies in the rabbit by jVIeites and Sgouris (1953, 1954) revealed that combinations of estrogen and progesterone could inhibit, at the mammary gland level, the lactogenic effects of exogenous prolactin. This effect was, however, relative and by increasing the prolactin or decreasing the steroids, lactogenesis ensued. Inasmuch as the theory of Meites and Turner did not take into account the eventuality that estrogen and progesterone act at the level of the mammary gland, Meites ( 1954) modified the con('ei)t, postulating that milk secretion was held in check during pregnancy first by the combined effect of estrogen and progesterone which make the mammary gland refractory to prolactin and, secondly, by a low rate of prolactin secretion. The role of progesterone in over-riding the stimulatory effect of estrogen on the pituitary he now considered to be of only minor importance. Meites also explained the continuance of lactation in pregnant animals by postulating that the initial level of prolactin was sufficiently high as a result of the suckling stimulus to overcome the inhibitory action of the ovarian hormones on the mammary gland. One of us (Folley, 1954, 1956) put forward a tentative theory, combining various features of previous hypotheses, which seemed capable of harmonizing most of the known facts regarding the initiation of milk secretion. In this it was emphasized that measurements of the prolactin content of the pituitary were not necessarily indicative of the rate of prolactin release (a recent study bv Grosvenor and Turner (1958c)

lends further support to this contention) and were best considered as largely irrelevant; low circulating levels of estrogen activate the lactogenic function of the anterior pituitary whereas higher levels tend to inhibit lactation even in the absence of the ovary; lactogenic doses of estrogen may be deprived of their lactogenic action by suitable doses of progesterone, the combination then acting as a potent inhibitor of lactation, this being the influence operating in pregnancy; at parturition the relative fall in the progesterone to estrogen ratio removes the inhibition which is replaced by the positive lactogenic effect of estrogen acting unopposed.
It was observed by Gaines in 1915 that although a colostral secretion accumulated in the mammary gland during pregnancy, the initiation of copious secretion was associated with functioning of the contractile mechanisms in the udder responsible for milk ejection; later Petersen (1944) also suggested that the suckling or milking stimulus might be partly responsible for the onset of lactation. Recent studies have provided evidence that this may well be so, and these will be considered later when discussing the role of the suckling and milking stimulus in the maintenance of milk secretion (see page 611).
During the past decade a fair amount of information has been obtained about the biochemical changes which occur in mammary tissue near the time of parturition, and which are almost certainly related to lactogenesis. The earlier work has been reviewed in some detail by one of us (Folley, 1956) and need only be referred to briefly here.
Folley and French (1949), studying rat mammary gland slices incubated in media containing glucose, showed that — QOo increased from a value of about 1.3 in late pregnancy to a value of about 4.4 at day 1 of lactation, and thereafter increased still further. At the same time the R.Q. which was below unity (approximately 0.83) at the end of pregnancy, increased to unity soon after parturition, and by day 8 had reached a value of 1.62 at approximately which level it remained for the rest of the lactation period. In accord with the

G08

PHYSIOLOGY OF GONADS

increased respiratory activity of the tissue about the time of parturition in the rat mammary gland, Moore and Nelson (1952) reported increases in the content of certain respiratory enzymes, succinic oxidase and cytochrome oxidase, in the guinea pig mammary gland at about this time. Greenbaum and Slater (1957b) made similar observations about mammary gland succinic oxidase in the rat. Recent work is beginning to throw light on the metabolic pathways involved in this increase in respiratory activity. Thus McLean (1958a) has adduced evidence indicating an increase in the activity of the pentose phosphate pathway in the rat mammary gland at about the time of parturition. Mammary gland slices taken from rats at various stages of the lactation cycle were incubated in media containing either glucose 1-C^^ or glucose 6-C^-^, and the amount of radioactivity appearing in the respiratory CO2 was determined. The results given in Figure 10.9 show that although the recovery of C^^'Oo from C-6 was relatively unaffected by the initiation of lactation, the C^^Oo originating from C-1 began a striking increase at the time of parturition (see also Glock, McLean and Whitehead, 1956, and Glock and McLean, 1958, from which Figure 10.9 was taken).

pregnancy

in\'oliition

Imc;. 1().<», The relative amounts of C'Oi; formed fioin iiiilucosc 1-C'^ and glucose 6-C" by rat niani maiy gland slices. O O, C'^Oi formed from
glucose 1-C^'. • • . C^'Oi! formed from glucose
6-C". (From G. E. Glock and P. McLean, Proc. Roy. Soc, London, ser. B, 149, 354-362, 1958.)

Despite the well known pitfalls which surround the interpretation of C-1: C-6 quotients in experiments such as these, it seems clear that lactation is associated with an increase in the metabolism of glucose by the pentose phosphate cycle, whereas the proportion going by the Embden-Meyerhof jmthway would appear to be relatively unaffected. These conclusions are supported by the fact that the levels in rat mammary tissue of two enzymes concerned in this pathway of glucose breakdown, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, show very striking increases at the time of parturition (Glock and McLean, 1954; McLean, 1958a). Other enzymes concerned in glucose breakdown whose activities in mammary tissue begin to increase at parturition are hexokinase and phosphoglucose isomerase (]\IcLean, 1958a). In connection with the glucose metabolism of rat mammary tissue it may be noted that addition of insulin to the incubation medium markedly increases the — QOo and R.Q. of rat mammary slices metabolizing glucose or glucose plus acetate (see page 619), and that this tissue only becomes sensitive to insulin just after parturition (Balmain and Folley, 1951). It is interesting to speculate which of the two above-mentioned pathways of glucose breakdown in mammary tissue resjjonds to the action of insulin. According to Abraham, Cady and Chaikoff (1957) addition of insulin in vitro increased the production by lactating rat mammary slices of C^'^Oo from glucose l-C^'*, but not from glucose 6-C^'*, which might indicate that insulin stimulates preferentially the pentose phosphate pathway. Against this, insulin increased the incorporation of both these carbon atoms (and also the 3:4 carbon atoms of glucose) into fatty acids of the slices to about the same extent. McLean (1959) believes that the stimulatory effect of insulin on the pentose jihosphate pathway in the lactating rat mammary gland is secondary to its stimulating effect on lipogenesis. The latter l)rocess generates the oxidized form of tril)hosphopyridine nucleotide (TPN) which is needed for the first two steps of the pentose phosphate cycle.
The inci-casc in the R.Q. of mammary

MAMMARY GLAXD AND LACTATION

009

tissue beginning at parturition observed by Folley and French (1949) was interi:)reted as indicating that this tissue assumes the power of effecting net fatty acid synthesis from ghicose at this time. Much subsequent evidence confirming this idea has been reviewed by Folley (1956). It only rt'mains to add that Ringler, Becker and Nelson (1954), Lauryssens, Peelers and Donck (1956), and Read and Moore (1958) ha^-e shown that the amount of coenzyme A in mammary tissue undergoes an increase at parturition. Moreover, the recent findings of McLean (1958b), who showed that the levels of pyridine nucleotides in the mammary gland of the rat begin to increase at parturition, reaching a high level by the end of lactation, may be significant in this connection. McLean found that although the increase in the tissue levels of diphosl^hopyridine nucleotide was almost entirely due to an increase in the oxidized form (DPN), in the case of TPN it was the reduced form (TPNH) which increased. The latter might well be used for reductive syntheses such as lipogenesis.
The rate of synthesis of milk constituents other than fat must also begin to increase at parturition, and Greenbaum and Greenwood (1954) showed that an increase in the levels of glutamic aspartic transaminase and of glutamic dehydrogenase in rat mammary tissue occurs at this time. The authors believe these enzymes are concerned in the provision of substrates for the synthesis of milk protein. It is significant in connection with milk protein synthesis that the mammary gland ribonucleic acid (RNA) in the rat undergoes a marked rise at parturition (Greenbaum and Slater, 1957a).
The above - mentioned biochemical changes in mammary tissue which occur at al)out the time of parturition are almost certainly closely related to the effect on this tissue of members of the anterior pituitary lactogenic complex, and particularly prolactin. Attempts have been made to elicit the characteristic respiratory changes, described above, in mammary slices in vitro by addition of prolactin and adrenal glucocorticoids to the incubation medium (see Folley, 1956). So far, however, definitive results luive not been obtained and it is doubt

ful whether any biochemical changes in lactating mammary gland slices in vitro have been demonstrated which could with certainty be ascribed to the action of prolactin (in this connection see also Bradley and Mitchell. 1957).
2. Maintenance of Milk Secretion — Galactopoiesis
It is well known that the removal of the pituitary of a lactating animal will end milk secretion (for references see Folley, 1952a). The cessation of milk secretion has been generally ascribed to the loss of the anterior lobe, but when the importance of the neurohypophysis in milk ejection became established (see page 621), it was clear that in the hypophysectomized animal it was necessary to distinguish between a failure in milk secretion and a failure in milk ejection, since either would lead to failure of lactation. It has now been shown in the rat that adequate oxytocin therapy ensuring the occurrence of milk ejection after hypophysectomy will not restore lactation (Cowie, 1957) and it may thus be concluded that the integrity of the anterior lobe is essential for the maintenance of milk secretion. The effect of hypophysectomy on milk secretion is dramatic, because in the rat, milk secretion virtually ceases within a day of the operation and biochemical changes in the metabolic activity of the mammary tissue can be detected within 4 to 8 hours (Bradley and Cowie, 1956). It is of interest to note that these metabolic changes are similar to those observed during mammary involution (see page 598).
Since the second edition of this book, there have been surprisingly few studies on replacement therapy in hypophysectomized lactating animals. In such studies we would stress the need for rigorous methods of assessing the efficacy of treatment. In the past the presence of milk in the gland as revealed by macroscopic or microscopic examination has been regarded as an indication of successful replacement. This, however, gives no measure of the degree of maintenance of lactation and some measure of the daily milk yield of such animals should be obtained (see also Cowie, 1957).

GIO

PHYSIOLOGY OF GONADS

It is abo now obvious that oxytocin may have to be injected to ensure milk ejection; under certain circumstances, however, the neurohypophyseal tissue remaining after the removal of the posterior lol^e may be capable of releasing oxytocin and permitting milk ejection (see Benson and Cowie, 1956; Bintarningsih, Lyons, Johnson and Li. 1957, 1958).
The earliest report on the maintenance of lactation after hypophysectomy is that of Gomez (1939, 1940), who found that hypophysectomized lactating rats could rear their litters if given anterior-pituitary extract, adrenal cortical extracts, glucose, and posterior pituitary extract. These experiments are difficult to assess because they are reported only in abstract, but the use of posterior pituitary extract at a time when the role of oxytocin in milk ejection was not generally recognized is worthy of note. Recently, slight maintenance of milk secretion in hypophysectomized rats has been obtained with prolactin alone, and greater maintenance when adrenocorticotrophic hormone ( ACTH I or STH was administered with prolactin (Cowie, 1957). Similar studies were reported by Bintarningsih, Lyons, Johnson and Li (1957, 1958) (see also Lvons, Li and Johnson, 1958) in which

I «
c
-^ 4
-0
I 1

£

Z -6

z

J

E

~~^ 2 ^^,~~

TV
2 ^

Fig. 10.10. Effect on the luilk yield of the cow of injected hormones of the anterior pituitary. (From the results of P. M. Cotes, J. A. Crichton, S. J. Folley and F. G. Young, Nature, London. 164, 992-993, 1919.)

considerable maintenance of milk secretion was obtained in hypophysectomized rats with prolactin and certain corticoids. Of related interest is the observation by Elias (1957) that Cortisol and prolactin can induce secretory activity in explants of mouse mammary gland growing on a synthetic medium. (Tissue culture techniques have been little exploited in mammary studies and further developments in this field may be expected.)
The evidence to date suggests that, in the rat, prolactin is an essential component of the hormone complex involved in the maintenance of lactation with ACTH and STH also participating, but further studies are recjuired to determine the most favorable balance of these factors.
Preliminary studies on the maintenance of lactation in the goat after hypophysectomy suggest that both prolactin and STH are important in the initiation and maintenance of milk secretioii (Cowie and Tindal, 1960). Our knowledge of the process in other species is derived from studies on the effect of exogenous anterior pituitary hormones on established lactation in intact animals— galactopoietic effects (for reference see Folley, 1952a, 1956). In the cow, considerable increase in milk yield can be obtained by injecting STH (Cotes, Crichton, Folley and Young, 1949), whereas prolactin has a negligible galactopoietic effect (Fig. 10.10; for discussion see also Folley, 1955). Recently the precise relationship between the dose of STH (ox) and the lactational response in the cow was established in our laboratory by Hutton (1957) who observed a highly significant linear relationship between log doses of STH (single injection) and the increase in milk yield obtained (Fig. 10.11 ) ; increases in fat yield relative to the yield of nonfatty solids also occurred. In the lactating rat, on the other hand, STH has no galactopoietic effect (Meites, 1957b; Cowie, Cox and Naito, 1957), whereas prolactin has (Johnson and Meites, 1958). Such studies must be interpreted with caution as endogenous pituitary hormones were present ; nevertheless, it seems reasonable to conclude that STH is likely to be an impoi'tant factor in the maintenance of lactation in the row.

MAMMARY GLAND AND LACTATION

611

mq qro\Om hormone (onthmeTTc scale)

fa-25 12-5 25-0 50-0

100-0

200-0

S-«^0

'Zoo-o

Fig. 10. IL Effect of graded doses of growth hormone on milk yield of row. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 16, 115-125, 1957.)

C)ther hormones of the anterior pituitary in all probability influence milk secretion through their target glands and these will be dealt with later.
3. Suckling Stimulus and the Maintenance of Lactation
It has been long believed that regular milking is an important factor in maintaining lactation and that if milk is allowed to accumulate in the gland, as occurs at weaning, atrophy of the alveolar epithelium and glandular involution occur. Evidence in support of this concept was obtained in studies showing that ligature or occlusion of

the main ducts of some of the mammae of a lactating animal resulted in atrophy of the glands concerned although the other glands were suckled normally (Kuramitsu and Loeb, 1921; Hammond and Marshall, 1925; Fauvet, 1941a). Studies by Selye and his colleagues, however, revealed that such occluded glands did not atrophy as quickly as did glands of animals in which the suckling stimulus was no longer maintained (Selye, 1934; Selye, Collip and Thomson, 1934) and it was postulated that the suckling stimulus evoked from the anterior pituitary the secretion of prolactin which maintained the secretory activity of the gland. This theory has been widely accepted

012

PHYSIOLOGY OF GONADS

although it has been suggested that a complex of hormones rather than prolactin alone is released (Folley, 1947). Williams (1945) showed that prolactin could in fact maintain the integrity of the mammary gland in the unsuckled mouse thus mimicking the effects of the suckhng stimulus; other supporting evidence has been reviewed by Folley (1952a). Recent studies in goats, however, have shown that milk secretion may continue more or less at the normal level after complete denervation of the udder (Tverskoi, 1958; Denamur and Martinet, 1959a, b, 1960) and it may be that in some species the suckling or milking stimulus is loss important in the maintenance of milk secretion.
Milk secretion is essentially a continuous process whereas the suckling or milking stimulus is intermittent ; indeed the milking stimulus may be of remarkably brief duration (in the cow about 10 minutes in all per 24 hours) and it is therefore likely that the stimulus triggers off the release of sufficient galactopoietic complex to maintain mammary function for some hours. Grosvenor and Turner (1957b) reported that suckling causes a rapid drop in the prolactin content of the pituitary in the rat, and that the prenursing level of prolactin in the pituitary is not fully regained some 9 hours later. It is difficult, however, to relate pituitary levels of prolactin to the rate of its secretion into the circulation and, although these observations are interesting, further advances are unlikely until a method of assay for blood prolactin becomes available and the "half-life" of prolactin in circulation is known.
The experiments of Gregoire (1947) on the maintenance of involution of the thymus during nursing suggests that the suckling stimulus releases ACTH which, as we have seen, is galactopoietic in the rat; thus, so far as the rat is concerned, there would appear to be good evidence that the suckling stimulus releases at least two known important components of the galactopoietic complex.
The milking and suckling stimulus is also responsible for eliciting the milk-ejection reflex and the relation between the two reflexes will be discussed later in this chapter (sec ])age 619 1.

B. HORMONES OF THE ADRENAL CORTEX
Adrenalectomy results in a marked inhibition of milk secretion and the early experiments in this field were reviewed by Turner in 1939. Since then, however, purified adrenal steroids have become available enabling further analysis to be made of the role of the adrenal cortex in lactation.
Gaunt, Eversole and Kendall (1942) considered that in the rat the defect in milk secretion after adrenalectomy could be repaired by the administration of the adrenal steroids most closely concerned with carbohydrate metabolism, whereas we came to the somewhat opposing view that the defect was best remedied by those hormones primarily concerned with electrolyte metabolism (Folley and Cowie, 1944; Cowie and Folley, 1947b, c). The reasons for these differing observations are not yet entirely clear. Virtually complete restoration of milk secretion was subsequently obtained in our strain of rat by the combined administration of desoxycorticosterone acetate (DCA) and cortisone, or with the halogenated steroids, 9a-chlorocortisol and 9afluorocortisol (Cowie, 1952; Cowie and Tindal, 1955; Cowie and Tindal, unpublished; see also Table 10.1). It would therefore seem that both glucocorticoid and mineralocorticoid activity was necessary to maintain the intensity of milk secretion at its normal level. The interesting observation was made by Flux (1955» and later confirmed by Cowie and Tindal (unpublished) that the ovaries contribute to the maintenance of lactation after adrenalectomy, a contribution which could be simulated in the adrenalectomized-ovariectomized rat by the administration of 3 mg. progesterone daily. The differences in the size of the ovarian contribution may partly accoimt for the apparent differences in various strains of rat of the relative importance of mineralo- and glucocorticoids in sustaining milk secretion after adrenalectomy. The only other species in which the maintenance of lactation after adrenalectomy has been studied is the goat in which, as in the rat, lactation can be maintained with cortisone and desoxycorticosterone, the latter being apparently the more critical steroid (Cowie and Tindal. 1958; Figs. 10.12a, b).

MAMM.\RY GLAND AND LACTATION

613

There have been several studies on the effects of corticoids and adrenocortieotrophin on lactation in the intact animal. ACTH and the corticoids depress lactation in the intact cow (Fig. 10.10) (Cotes, Crichton, Folley and Young, 1949; Flux, Folley and Rowland, 1954; Shaw, Chung and Bunding, 1955; Shaw, 1955), whereas in the rat ACTH and cortisone have been reported as exhibiting galactopoietic effects (Meites, private communication; Johnson and Meites, 1958). With larger doses of cortisone, however, an inhibition of milk secretion in the rat has been reported (MercierParot, 1955).
The main function of the cortical steroids in lactation is still uncertain. They may act in a "supporting" or "permissive" manner (see Ingle, 1954), maintaining the alveolar cells in a state responsive to the galacto])oictic complex, or they may act by maintaining the necessary levels of milk precursors in the blood.
Biochemical studies are, however, Ix'ginning to add to our information on the role of the corticoids in lactation. In the rat, adrenalectomy prevents the increase in liver and mammary gland arginase which occurs during normal lactation and it has been suggested that this depression of arginase activity interferes with deamination of amino acids, and thereby inhibits any increase in gluconeogenesis from protein and thus starves the mammary gland of nonnitrogenous milk precursors (Folley and Greenbaum, 1947, 1948). As there is little arginase in the mammary gland of other species {e.g., rabbit, cow, goat, sheep), this mechanism may not have general validity (for further discussion see Folley, 1956). Other biochemical studies have suggested that the steroids of the adrenal cortex may be concerned in mammary lipogenesis, but the results so far have been conflicting and no firm conclusions can as yet be drawn (see Folley, 1956).
C. OVARIAN HORMONES
There is no evidence that ovariectomy has any deleterious effect on lactation (Kuramitsu and Loeb, 1921; de Jongh, 1932; Folley and Kon, 1938; Flux, 1955); neither is there evidence for the belief, once

TABLE 10.1
Replacement therapy in lactating rats
adrenalectomized on the fourth
day of lactation
(From A. T. Cowie and S. J. Folley,
J. Endocrinol., 5, 9-13, 1947.)

Treatment

Number of Litters

Number
of Pups
per
Litter

Litter-growth
Index* gm. + S.E.

Control
Adrenalectomy
Adrenalectomy + cortisone + DC A (tablet implantsf)

8
9
7

8 8 8

15.6 + 0.5
7.5 ± 0.6 14.9 ± 0.6

(Above results from Cowie, 1952)

Control

6

8

14.5 ± 0.8

Adrenalectomy

6

8

6.2 ± 0.4

Adrenalectomy + chloro
5

8

13.1 ± 0.5

cortisol (100 Mg per

day)

(Above results from Cowie and Tindal, 1955)

Control

8

12

17.7 ± 0.8

Adrenalectomy

8

12

7.5 ± 0.5

Adrenalectomy + ovari
5

12

3.6 ± 0.5

ectomy

Adrenalectomy + ovari
7

12

14.5 ± 0.7

ectomy + fiuorocorti

sol (200 Mg per day)

(Above results from Cowie and Tindal, unpublished)
~~The litter-growth index is defined as the mean~~
daily gain in weight per litter over the 5-day period from the 6th to the 11th days.
t 2 X 11 mg. tablets cortisone giving mean daily absorption of 850 ^ig., and 1 X 50 mg. tablet DCA giving mean daily absorption of 360 ng.
widely held, that ovariectomy increases and prolongs lactation in the nonpregnant cow (see Richter, 1936).
Although the integrity of the ovary is not essential for the maintenance of lactation, there can be no doubt that ovarian hormones, in certain circumstances, profoundly influence milk secretion. Estrogens have long been regarded as possessing the power to inhibit lactation, a concept on which Nelson based his theory of the mechanism of lactation initiation (see page 606 1 . Some workers, however, have expressed doubts that the effect is primarily on milk secretion, and have suggested that in ex

614

PHYSIOLOGY OF GONADS

periments on laboratory animals the apparent failure in milk secretion could be a secondary effect due to either a toxic action of the estrogen causing an anorexia in the mother, interference with milk ejection, or disturbance of maternal behavior or to toxic effects on the young, whose growth rate serves as a measure of lactational performance, through estrogens being excreted in milk. The evidence to date shows that in

the intact rat estrogens even in very low doses inhibit milk secretion, their action depending on the presence of the ovary ; the ovarian factor concerned appears to be progesterone, estrogen and progesterone acting locally on the mammary gland and rendering it refractory to the lactogenic complex. In the ovariectomized rat much larger doses of estrogen are necessary to inhibit lactation, and the evidence is not entirely

Body

Goat 478

weight ^^L
~~.) 45 L~~
Plasma Na (m-equiv./l.) ^^^^
Plasma K (m-equiv./l
Milk K 40 (m-equiv./l.) 30

Milk Na ,
(m-equiv./l.)

Solids-notfat (%)
Yield of solids-notfat (g) Fat (%)

Milk yield (kg)

Goat died-*
5 15 25 4 14 24 Mgr. Apr.

Fig. 10.12i4. Effect of replaconi(>nt therapy with (losoxycoiticostcM-oiu c-ortisone aoetate (CA) on milk yield, milk composition, and concent

(DCA) and tion of Na and K in milk and blood plasma of the goat after adrenalectomy. Duration of replacement therapy (pellet implantation) indicated by horizontal lines; the names of steroids and their mean daily absorption rates are given adjacent to the lines. Note in Figure 12.4 the considerable maintenance of milk vield with DCA alone. See also Figure 12/?. (From A. T. Cowic and J. S. Tindal. J. Endocrinol., 16, 403-414, 1958.)

MAMMARY GLAND AND LACTATION

6L

Goat 515

Body 5Q _ weight —
(kg) 40 150 Plasma Na ^ ^. / /I \ ^40 —
(m-equiv./l) —
130

Plasma K (m-equiv./l)

Milk K (m-equiv./l.)

Milk Na (m-equiv./l.)
Solids-not- ^ H
fat {%) 7 U
Yield of 200 solids-not- —
fat (g) 100 Fat (- ^

Fat yield

Milk yield (kg)

13 23 2 12 22 2 12 22 Oct. Nov Dec.
Fig. 12B.

11 21 31 10 20
Jan. Feb

conclusive that there is a true inhibition of milk secretion (see Cowie, 1960). In the cow estrogen in sufficient doses depresses milk yield, but its mode of action has not been fully elucidated. In women, estrogens are used clinically to suppress unwanted lactation, but as the suckling stimulus is also removed about the same time, the role of the estrogen is difficult to assess (see Meites and Turner, 1942a).

It has been well established that progesterone by itself has no effect on milk secretion (see Folley, 1952a), save in the adrenalectomized animal (see page 612), and so it would appear that the physiologic inhibition of lactation is effected Ijy estrogen and progesterone acting synergistically as first demonstrated by Fauvet (1941b) and confirmed by others including Masson (1948), Walker and Matthews (1949),

GIG

PHYSIOLOGY OF GONADS

Cowie, FoUey, Malpress and Richarcl.son (1952J,, and Meites and Sgouris (1954). There is clear evidence that the estrogenprogesterone combination acts at least partly on the mammary parenchyma (Desclin, 1952; Meites and Sgouris, 1953) but the mechanism of the action is unknown. The hormonal interplay and complex endocrine interactions in the process of lactation inhibition with estrogen has recently been discussed at length by von Berswordt-Wallrabe (1958).
Lactogenic effects of estrogens have already been mentioned; these have been demonstrated most strikingly in cows and goats, in which milk secretion has been induced in udders being developed by exogenous estrogen. These experiments have been reviewed in some detail by Folley and Malpress (1948b) and Folley (1956).^ It is generally assumed that estrogens act by

stimulating the production of lactogenic and galactopoietic factors by the anterior pituitary. In experiments on the ovariectomized goat we have shown (Cowie, Folley, Malpress and Richardson, 1952; Benson, Cowie, Cox, Flux and Folley, 1955) that it is possible to select a daily dose of estrogen which will induce mammary growth but relatively little secretion in the sense that the udder does not become tense and distended as will happen when a lower dose of estrogen is given — an observation we may quote in support of the "double-threshold" theory of estrogen action. The lactogenic effect of the lower dose of estrogen could be abolished, however, by administering progesterone simultaneously with the estrogen (Fig. 10.13), an observation in accord with those of other workers on the rabbit and rat (see above).
In 1936 one of us (Folley, 1936) reported

Fig. 10.13. Photographs of goat uddois dovelopcd by daily injections of hoxoostiol (HX) with and without progesterone (PG). The hibels indicate the daily dose in mg. of each substance. (Results from A. T. Cowie, S. J. Folley, F. H. Malpre.ss and K. C. Ricliardson, J. Endocrinol., 8, 64-88, 1952.)

MAMMARY GLAND AND LACTATION

GK

that certain dose levels of estrogen in the lactating cow produced long-lasting changes in milk composition characterized by increases in the percentages of fat and nonfatty solids. This was regarded as an example of galactopoiesis and was termed the "enrichment" effect. The effect, however, w^as somewhat erratic and it has recently been re-investigated by Hiitton (1958) who confirmed and extended the earlier observations. Hutton found that galactopoietic responses (Figs. 10.14 and 10.15) were obtained only within a restricted dose range, the limits of which were affected by the stage of pregnancy and the breed of the cow. Hutton further concluded that in the normal cow changes in milk composition and yield associated with advancing pregnancy were probably determined by the progressive rise of blood estrogen levels.
D. THYROID HORMONES
Studies on the effect of removal of the thyroids on milk secretion have been reviewed by one of us (Folley, 1952a) ; the evidence strongly suggests that the thyroid glands are not essential for milk secretion, but in their absence the intensity and duration of lactation is reduced. Histologic and cytologic studies of the thyroid of the lactating cat suggest that there is a considerable outpouring of the thyroid secretion in the early stages of lactation (Racadot, 1957), and Grosvenor and Turner (1958b) have reported that the thyroid secretion rate is higher in lactating than in nonlactating rats.
Since the last edition of this l)ook, a great volume of experimental results has been published on the use of thyroid-active materials for increasing the milk yield of cows. These experiments have been extensively reviewed by Blaxter (1952) and Meites (1960) and we need here only touch on the salient points.
In the early studies i^reparations of dried thyroid gland were fed to cows or injections of DL-thyroxine were given, but the use on a large scale of thyroid-active materials for increasing the milk yield of cows only became feasible when it was shown that certain iodinated proteins exhibited thyroidlike activitv when given in the feed. Al

9-9 97
o 9-3 ^ 9-1

8-9

• Guernsey

Shorthorn

8-5

•^U^ri
I L

20 40 60 80 100
Oestradiol monobenzoite (mg)
Fig. 10.14. Effect of graded doses of estradiol benzoate on percentage of nonfatty solids in milk from cows of three breeds. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.)
Oestradiol monobenzoate (mg) (arith. scale)
10 20 30 40 50

6-25 12-5 250 500
Oestradiol monobenzoate (mg) (log scale)
Fig. 10.15. Effect of graded doses of estradiol benzoate on fat content of cows' milk. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.)
though these materials were readily made and were economical for large-scale use, they possessed several disadvantages. Their activity was difficult to assay and standardize, they were frequently unpalatable, and their administration entailed a considerable intake of iodine which could be undesirable. Nevertheless, a large number of experiments were carried out all over the world with this type of material. In 1949, however, a new and improved method for the synthesis of L-thyroxine was developed (Chalmers, Dickson, Elks and Hems, 1949) and thyroxine became available in large quantities. It was then shown jjy Bailey, Bartlett and Folley (1949) that this material was ealac

618

PHYSIOLOGY OF GONADS

,

/" \^

Cont-rol.

A<' / " "^ - ^*

— • DO m§.

.^\ / V- -' v;.

100 m|.

^..^-Av / V

150mg.

^^^4?^^/ . V

,.-•*.. \ vv / .^-r \ \

• — •• tva --^-^ y \ \ \

•••\-'^\ \ x- ^ .. \ ^

\. *-^ '• •■*— . \ \

~~■*•—., \ \ \ \~~

.... -.... "•N-:w<r:Viy: y^

Sl-art of hrcAhnc.ih \\ y' i'

hrc iXhuciil' \\ //

\v/y

\ V /

\ /

\ /

\/

V

10

50

50

Dau5

Fig. 10.16. Effect of L-thyroxine given in the feed on the milk yield of groups of cows (the indicated dose levels were fed daily). (From G. L. Bailey, S. Bartlett and S. J. Folley, Nature, London, 163, 800. 1949.)

topoietic when ]ed to lactating cows in daily doses of about 100 mg. (Fig. 10.16). It had, moreover, none of the drawbacks of the iodinated proteins, its purity could be checked chemically, it was odorless and tasteless. AVith the introduction of synthetic thyroxine, iodinated proteins have become obsolete as galactopoietic agents.
The more recently isolated 3:5:3-triiodo-L-thyronine, reported to be 5 to 7 times more active than thyroxine in various biologic tests in small animals and also in man, has little or no effect on the milk yield when fed to cows, but is somewhat more active than thyroxine in promoting galactopoiesis when administered subcutaneously, which suggests that the material is inactivated in the gut, probably in the rumen f Bartlett, Burt, Folley and Rowland, 1954).
The extensive experiments on galactopoiesis in dairy cattle with thyroxine and thyroid-active substances have made it possible to reach reasonably firm conclusions as to the practical value of the procedure. There is great variability in the response to treatment; in general a better response is ol)taincd during the decline of lactation than at the peak and end of lactation. The use of thyroid-active substances

in animals undergoing their first, second, or third lactation is of doubtful benefit because the boost in yield is largely cancelled out by a shortening of the lactation period. Short-term administration at suitable times can result in considerable galactopoiesis, but this is frequently followed by marked falls in yield when the administration of thyroid-active material ends. The administration of thyroid-active materials to dairy cows, if carried out with due care, has no ill effects on the health and reproductive abilities of the cows (see Leech and Bailey, 1953) , but because of the rather small net gain in yield (about 3 per cent) the practical application of the procedure seems to be limited.
The mode of action of thyroxine and thyroid-active substances on milk secretion is uncertain. It is tmlikely that it is a specific effect on the alveolar cells; rather is it probably related to the effects of the thyroid hormone on the general metabolic rate.
E. PARATHYROm HORMONE
The early studies on the influence of the parathyroid glands on milk secretion indicated, as might be expected from their

MAMMARY GLAND AND LACTATION

()19

role in calcium metabolism, that the parathyroids were important in the maintenance of secretion (see review by Folley, 1952a). Indeed in the rat, we demonstrated that the severe impairment of milk secretion previously observed in "thyroidectomized" rats was due not to the removal of the thyroids, but to the simultaneous ablation of the l)arathyroids (Cowie and Folley, 1945). This observation has since been confirmed and extended by Munson and his colleagues (Munson, 1955) who demonstrated an influence on the calcium-concentrating mechanism of the mammary glands. Within 24 hours of parathyroidectomy the concentration of calcium in the milk of the lactating rat was increased markedly despite a greatly depressed level of calcium in the serum; there was also a decrease in water content of the milk, but this did not entirely account for the increase in calcium content since the calcium content expressed as mg. per gm. milk solids was significantly higher after parathyroidectomy (Toverud and Munson, 1956). Further studies in this field are awaited with interest.
F. INSULIN
Early experiments (see review by Folley, 1952a) indicated that the endocrine pancreas might influence mammary function in two ways; indirectly by way of the general intermediary metabolism by which the supply of milk precursors may be regulated, and directly through its role in the carbohydrate metabolism of the mammary gland itself.
Most recent studies have been concerned with the effect of insulin on mammary tissue in vitro. Mammary gland slices from lactating rats actively synthesize fat from small molecules, glucose, and glucose plus acetate, but not from acetate alone (Folley and French, 1950). The addition of insulin to the incubation medium very markedly increases the R.Q. (see Table 10.2) and glucose uptake of the tissue slices and experiments with isotopes show that the rate of fat synthesis is increased (Balmain, Folley and Glascock, 1952). Mammary gland slices from lactating sheep, on the other hand, can utilize acetate alone but not glucose alone for fat synthesis (Folley and French, 1950) and sheep tissue is not re

TABLE 10.2
Effect of different substrates and of insulin on the
respiratory quotient (R.Q.) of lactating mammary
gland slices from various species
(From S. J. Follev and M. L. McNaught, Brit.
M. BulL, 14, 207-211, 1958.)

Respiratory Quotients

Anlrml

Substrate

Without insulin

With insulin

Mouse

Glucose

1.90

2.14

Glucose + acetate

1.46

2.14

Rat

Glucose

1.57

1.80

Acetate

0.82

Glucose + acetate

1.53

2.03

Guinea pig

Glucose

1.17

Rabbit

Glucose

1.30

_

Acetate

0.92

Glucose -t- acetate

1.24

1.67

Sheep

Glucose Acetate

0.88 1.09

1.09

Glucose + acetate

1.52

1.50

Goat

Glucose

0.86

Acetate

1.17

—

Cow
Glucose

0.84

_

Acetate

1.12

—

sponsive to insulin in vitro. This clear-cut species difference is interesting and underlines the need for further study. It is of passing interest to note that the response in vitro of rat mammary tissue to insulin has been made the basis of a highly specific in vitro bio-assay for insulin (Fig. 10.17) (Balmain, Cox, Folley and McNaught, 1954; McNaught, 1958)!
Further references and discussion on the role of insulin in mammary function and lipogenesis will be found in the reviews by Folley (1956), and Folley and McNaught (1958, 1960).
IV. Removal of Milk from the
Mammary Glands: Physiology
of Suckling and Milking
A. MILK-EJECTION REFLEX
Since the second edition of this book, there have been major advances in our knowledge of the physiology of milk removal. In the mammary gland the greater

620

PHYSIOLOGY OF GONADS

22

rs 2-5//g/ml.

20

yT

■~^

yO

i 18

- Cf

>

-o

■;;16

- i:/^

c

u=

«14

/ J3 as^g/mi.

8 12

Z' j^p^ £) 0-Vg/ml.

— 10

y rf^ ,-fP

1 3 8

si r^^ r-f^ y^ Control

o °

M J

A y^ ^cr ,^y^

4J

z

Pr( .iif^ jy^^

4

^ M^ ^r^

2

L_l 1 1 1 1 \ 1 \ 1 1 \ 1

15 30

60 90 120
Time (min)

150

Fig. 10.17. Effect of various concentrations of insulin on the respiratory metabolism of slices of rat mammarj' glands. (From J. H. Balmain, C. P. Cox, S J. Folley and M. L. McNaught, J. Endocrinol., 11, 269-276, 1954.)
portion of the milk secreted by the alveohir cells in the intervals between suckling or milking remains within the alveoli and the fine ducts. Only a small portion passes into the larger ducts and cisterns or sinuses from which it can be immediately removed by suckling, milking, or cannulation; its removal requires no maternal participation and has been termed passive withdrawal (see Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951, and page 612). The larger portion of the milk in the alveoli and fine ducts becomes available only with the active participation of the mother and requires the reflex contraction of special cells (see page 623) surrounding the alveoli in response to the milking or suckling stimulus to eject the milk from the alveoli and fine ducts into the cistern and sinuses of the gland. The occurrence of this reflex has long been known, although its true nature has only recently been generally recognized.^
-H. K. Waller {Clinical Slujlits un Lnrfallon, London: Heinemann, 1938), and later one of us (S. J. Folley, Physiology and Biochemistry of Lactation, London and Edinburgh: Oliver & Boyd, 1956) have drawn attention to the fact that the theme of the "milk-ejection reflex" was the inspiration of a paiming by II Tintoretto entitled "The Origin of the Milky Way" which hangs in the

111 the past it has been termed the "draught" in lactating women (see Isbister, 1954) and the "let-down" of milk in the cow. The latter term is particularly misleading since it implies the release of some restraint, whereas there is, in fact, an active and forceful expulsion of milk from the alveoli and we have, therefore, urged that this term be no longer used in scientific literature and that it be replaced by the term "milk ejection" (Folley, 1947; Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951), a term, incidentally, which was used by Gaines in 1915 in his classical researches on the phenomenon (see below j.
The true nature of the milk removal process was for many years not recognized, probably because it was assumed that the mammary gland could not contain all the milk obtainable at a milking, and this assumption made it necessary to postulate a very active secretion of milk during suckling or milking. Even as late as 1926 two phases of milk secretion were described in the cow ; the first phase was one of slow secretion occurring between milkings, the second phase was one of very active secretion occurring in response to the milking stimulus when a volume of milk about equal to that produced in the first phase was secreted in a matter of a few minutes (Zietzschmann, 1926). That some physiologic mechanism
National Gallery, London. Both authors point out tliat the picture shows evidence of a considerable intuiti^■e understanding of the physiologic nature of the milk-ejection reflex. Thus, it illustrates, first, that the application of the suckling stimulus causes a considerable increase in intranianiinai >• jiressure resulting, in this instance, in a sjnni cii' milk from the nipples, and second, that ihv Muklmg stimulus applied to one nipple gives rise to a systemic rather than a localized effect, for the milk is forcibly ejected from the suckled and unsuckled breasts ahke. The same theme was also treatetl by Rubens in a picture called "The Birth of the Milky Way" which can be seen in the Prado Museum, Madrid. This picture differs from Tintoretto's in one important detail, the stream of milk coming only from one breast.
The forcible ejection of milk from the nipple has doubtless been the subject of many statues. An example known to the authors is the fountain in the Sfiuare at Palos Verdes, near Los Angeles, California. The center piece of this fountain has a nude female torso at each of its four corners from whose nipples spurt streams of water.

MAMMARY GLAND AND LACTATION

621

was involved in the discharge of preformed milk from the mammary gland had, however, been recognized. Schafer (1898) considered that milk discharge was aided by contraction of plain muscle w^ithin the gland and pressure on the alveoli produced by vasodilation.
The first full investigation of the physiology of milk removal was that by Gaines in 1915. Unfortunately, his remarkably accurate observations and perspicacious conclusions aroused little general interest and were almost wholly overlooked for more than quarter of a century. It is now of interest to recall the more important of Gaines' observations. First, he made a clear distinction between milk ejection and milk secretion — "Milk secretion, in the sense of the formation of the milk constituents, is one thing; the ejection of the milk from the gland after it is formed is quite another thing. The one is probably continuous; the other, certainly discontinuous." Secondly, he concluded that "Nursing, milking and the insertion of a cannula in the teat, excite a reflex contraction of the gland musculature and expression of milk. There is a latent period of 35 to 65 seconds. . . . Removal of milk from the gland is dependent on this reflex, and it may be completely inhibited l)y anaesthesia. The conduction in the reflex arc is dependent upon the psychic condition of the mother." He also observed that the increased flow of milk following the latent period after stimulation was associated wath a steep rise in pressure within the gland cistern and that the reflex could be conditioned. Thirdly, with reference to the gland capacity, he reported that "the indication is that practically the entire quantity of milk obtained at any one time is present as such in the udder at the beginning of milking." Lastl3^ he confirmed earlier observations that injections of posterior pituitary extract caused a flow of milk in the lactating animal and he postulated that "pituitrin has a muscular action on the active mammary gland causing a constriction of the milk ducts and alveoli with a consequent expression of milk. This action holds, also, on the excised gland in the absence of any true secretory action." Gaines regarded the milk-ejection reflex as a

l)urely neural arc although he emphasized that the effect was "very similar to that produced by pituitrin." All that is required to bring these views of milk ejection in line with present day concepts is to recognize that the reflex arc is neurohormonal in character, the efferent component of which is a hormone released from the neurohypophysis. When Gaines was carrying out these experiments hardly anything was known of neuro-endocrine relationships and there was no background of knowledge to lead anyone to conceive that the effects of the posterior pituitary extract might represent a physiologic rather than a pharmacologic effect. In 1930 Turner and Slaughter hinted at a possible physiologic role of the posterior pituitary in milk ejection and, as we have noted (page 610), Gomez (1939) used posterior pituitary extract in replacement therapy given to hypophysectomized lactating rats. It was not until 1941, however, that the role of the posterior pituitary in milk ejection was seriously postulated by Ely and Petersen (1941) who, having shown in the cow that milk ejection occurred in the mammary gland to which all efferent nerve fibers had been cut, suggested that the reflex was neurohormonal, the hormonal component being derived from the posterior pituitary, and being, in all likelihood, oxytocin. The neurohormonal theory of Ely and Petersen and the subsequent work of Petersen and his colleagues (see reviews by Petersen, 1948; and Harris, 1958), unlike the earlier work of Gaines, aroused wide interest and its practical applications permitted rationalization of milking techniques in the cowshed thereby improving milk yields. Despite the attractiveness of the concept, however, a further 10 years were to elapse before unequivocal evidence of the correctness of the theory was forthcoming and this evidence we shall now briefly review.
B. ROLE OF THE NEUROHYPOPHYSIS
The first reliable indication that the suckling or milking stimulus does in fact cause an outpouring of neurohypophyseal hormones were the observations that inhibition of diuresis occurred following the application of the milking or suckling stimulus (Cross, 1950; Peeters and Cous

622

PHYSIOLOGY OF GONADS

sens, 1950; Kalliala and Karvoncn, 1951; Kalliala, Karvonen and Leppanen, 1952). It was also shown that electrical stimulation of the nerve paths to the posterior pituitary resulted in milk ejection (Cross and Harris, 1950, 1952; Andersson, 1951a, b, c; Popovich, 1958 », and that when lesions were placed in these tracts the milk-ejection reflex was abolished (Cross and Harris, 1952) .
Further evidence was adduced when it was found that removal of the posterior pituitary immediately abolished the milkejection reflex in the lactating rat, and that it was necessary to inject such animals several times a day with oxytocin if their litters were to be reared (Cowie, quoted by Folley, 1952b). Earlier workers had claimed that the posterior lolie was not essential for lactation (Smith, 1932; Houssay, 1935), but an explanation of these discordant conclusions was provided when it was shown that the impairment of the reflex after removal of the posterior lobe is not permanent and that the reflex re-establishes itself after some weeks, presumably because the remaining portions of the neurohypophysis take over the functions of the posterior lobe (Benson and Cowie, 1956). That the neurohypophysis participates in milk ejection would now appear to be beyond question.
The discovery of the role of the neurohypophyseal hormones in milk ejection has provided an explanation of some longstanding clinical observations on what has been termed the natural "sympathy" between the uterus and the breasts. Thus the beneficial effects of the suckling stimulus and the occurrence of the "draught" {i.e., milk ejection) in causing uterine contraction after parturition were emphasized over a century ago by both Smith (1844) and Patcrson (1844). 0})servations have also been made on the I'cciprocal process of stimuli arising from the reproductive organs apparently causing milk ejection. In domestic animals two such examples were mentioned by Martiny (1871). According to Herodotus, the Scythians milk their mares thus: "They take l)lowpipes of bone, very like flutes, and put them into the genitals of the mares and blow with their mouths, others milk. And they say that the I'cason why thoy do so is this, that when the marc's \-cins ai'c filled

with air, the udder cometh down" (translation by Powell, 1949). Kolbe (1727) described a similar procedure of blowing air into the vagina used by the Hottentots when milking cows which were normally suckled by calves and in which, presumably, milk ejection did not occur in response to hand nnlking. A drawing depicting this procedure from Kolbe's book was recently published in the Ciba Zeitschrift (No. 84^ 1957) along with a photograph of African natives still using the method!-^
In 1839, Busch described the occurrence of milk ejection, the milk actually spurting from the nipple, in a lactating woman during coitus. A satisfactory explanation of these curious observations is now forthcoming. Harris (1947) suggested that coitus might cause the liberation of oxytocin from the neurohypophysis and, within the next few years it was demonstrated that stimulation of the reproductive organs evoked milk ejection in the cow (Hays and VanDemark. 1953) and reports confirmatory of Busch's long forgotten observations also appeared (Harris and Pickles, 1953; Campliell and Petersen, 1953).^
C. MILK-EJECTIOX HORMONE
There is much circumstantial evidence to confirm the belief that the milk-ejection hormone is oxytocin (see Cowie and Policy. 1957). Attemi)ts, however, to demonstrate oxytocin in the blood after application of the milking stimulus have given rather inconclusive results. Early claims that the hormone could be demonstrated in blood are
^ A similar drawing, also apparently from Kolbe '.•< book, has been used in the campaign for clean milk production! Heineman (1919) discussing sanitary l^recautions in the cowshed says of the picture "another picture shows a nude Hottentot milking a cow while another one is liolding the tail of the cow to prevent its dropping into the open pail. This ])icture might well serve as a model to some modern producers who do not take such precautions and calmly lift the tail out of the milk with their hands wlicn it hnjipens to switch into the pail."
' W(- h;i\(' hi'cii able to find only one painting illustrating this plienomenon. It is a picture by a contemporary French painter, Andre Masson, entitled "Le Viol" and painted in 1939. It illustrates in Masson 's personal idiom the act of rape and it is interesting to note that a stream of milk is depicted as being I'orcibly (\iected from one breast of the

MAMMARY GLAND AND LACTATION

623

of doiil)tful validity, because the milk-ejection effect observed may have been due to 5-hydroxytryptamine (see Linzell, 1955), and more recent attempts to assay the level of oxytocin in the blood have not been entirely satisfactory or conclusive. There seem to be other polypeptide substances in blood which possess oxytocic activity, although the thiogly collate inactivation test indicates that these are different from oxytocin (Robertson and Hawker, 1957), and no marked changes in the blood oxytocic activity associated with suckling or milking have been detected (Hawker and Roberts, 1957; Hawker, 1958). However, it would seem doubtful whether the present assay techniques are sufficiently sensitive and specific to detect changes in blood oxytocin of the magnitude likely to be associated with milking or suckling. In the lactating cow the intravenous injection of 0.05 to 2.0 I.U. oxytocin will cause milk ejection (Bilek and .Tanovsk>% 1956; Donker, 1958), in the goat 0.01 to 1 I.U. (Cowie, cited by Folley, 1952b; Denamur and Martinet, 1953), in the sow 0.2 to 1.0 I.U. (Braude, 1954; Whittlestone, 1954; Cross, Goodwin and Silver, 1958) in the rabbit 0.05 I.U. (Cross, 1955b) , and in the lactating woman 0.01 I.U. (Beller, Krumholz and Zeininger, 1958) . If these (loses give any indication of the quantity of endogenous oxytocin released, then the concentration in the peripheral blood is likely to be very small ; indeed Cross, Goodwin and Silver (1958) calculated that a threshold dose (10 mU.) of oxytocin in the sow w^ould give a plasma concentration of about 1 (U,U. per ml, and until it can be shown that the assay techniques are sufficiently sensitive to detect the changes in oxytocin concentration produced by intravenous injections of "physiologic" doses of oxytocin, no great reliance can be placed on the results of assays.
Attempts have been made to demonstrate alterations in the hormone content of the neural lobe following the suckling or milking stimulus. In the goat and cow no detectable changes have been reported, but in the smaller species (dog, cat, rat, guinea pig) decreases have been described (see Cowie and Folley, 1957). It is likely that in many species the amount released is small

relative to the total hormone content of the gland and within the limits of error of the
assay.
D. EFFECTOR CONTRACTILE MECHANISM OF THE MAMMARY GLAND
In the last 10 years considerable research has been devoted to a study of the effector contractile tissue in the mammary gland; this work has recently been reviewed in some detail (see Folley, 1956) and only the salient features need be mentioned here.
Although earlier histologists had from time to time figured myoepithelial or "basket" cells in close association with the mammary alveoli, the morphology and distribution of the cells remained vague until Richardson (1949) published a detailed and illuminating description (Fig. 10.18). His beautiful observations have since been confirmed and supplemented by Linzell (1952) and Silver (1954). Richardson also disposed of the oft repeated view that smooth-muscle fibers around the alveoli played an iml)ortant role in milk ejection. From a study of the general orientation of the myoepithelial cells and the precise relationship between these cells and the folds in the secretory epithelium from contracted glands, Richardson considered it reasonable to regard the myoepithelium as the contractile tissue in the mammary gland which responds to oxytocin causing contraction of the alveoli and widening of the ducts. The evidence adduced by Richardson, although good, was nevertheless circumstantial, and it was desirable that attempts be made to visualize the contraction of the myoepithelial cells in response to oxj^tocin. In this connection it is of interest to recall that Gaines (1915) reported that when a drop of pituitrin was placed on the cut surface of the mammary gland from a lactating guinea pig, minute white dots appeared within a few seconds beneath the pituitrin and slowly swelled to tiny milky rivulets streaming beautifully through the clear liquid. Much later the local effects of posterior pituitary extract on the mammary gland were studied by Zaks (1951) in the living mouse, when it was reported that it caused contraction of the alveoli and expansion of the ducts. These observations were considerablv extended bv Linzell

624

PHYSIOLOGY OF GONADS

Fig. 10.18. Surface view of contracted alveoli (of goat) showing myoepithelial cells. (Courtesy of K. C. Richardson.)

Fig. 10.19. Recording of pressure changes witliin a galactophore of a forcibly restrained lactating rabbit. The litter was allowed to suckle the noncannulated mammary glands but obtained only 8 gm. milk, there being only a slight rise in the milk pressure probably associated with a slight contraction of the myoepithelium in response to mechanical stimulation. When 5 mU. oxytocin were injected (5P) there was a rapid milk ejection response which could be inhibited by injecting 1 yug. adrenaline (lA) just before the oxytocin. After a few minutes 5 mU. oxytocin were again effective and the litter obtained 44 gm. milk when they were allowed to suckle. A more complete milk ejection respon.so was obtained with 50 mU. oxytocin (50P) and the young obtained a further 59 gm. milk. Anesthesia did not enhance the milk-ejection response to 50 mU. oxytocin. During emotional inhibition of milk ejection the mammary gland thus remains responsive to oxytocin. (From B. A. Cross, J. Endocrinol., 12, 29-37, 1955.)

(19ooi who studied the local effects of liighly purified oxytocin and vasopressin and a number of other drugs on the mammnry gland, and confirmed that oxytocin and vasopressin produced alveolar contraction and widening of the ducts. Although in these experiments the myoepithelial cells themselves could not be visualized, nevertheless the effects observed leave little doubt that the effector mechanism was the niyoei)ithelium.
The myoepithelium is responsive to stimuli other than those arising from the presence of neurohypophyseal hormones in the blood inasmuch as partial milk ejection may occur in response to local mechanical stimulation of the mammary gland (Cross, 1954; Yokoyama, 1956; see also Fig. 10.191. These observations may explain the recent reports by Tverskoi (1958) and Denamuiand Martinet (1959a, b) that milk yields can be maintained in goats in the absence of the milk-ejection reflex.
E. INHIBITION OF MILK EJECTION
(laines (1915) stressed that the conduction in the milk-ejection reflex pathway was dei)endent on the psychic condition of the

MAMMARY GLAND AND LACTATION

625

mother. Many years later Ely and Petersen (1941) confirmed this and, having shown that injections of adrenaline blocked the milk-ejection reflex, postulated that the increased blood level of adrenaline in emotionally disturbed cows interfered with the action of oxytocin. In the last few years, the nature of the inhibitory mechanisms has been more fully investigated. Braude and Mitchell (1952) showed in the sow that adrenaline exerts at least part of its inhibitory effect at the level of the mammary gland and that, whereas the injection of adrenaline before the injection of oxytocin blocked milk ejection, less inhibition occurred if both were given together. Cross (1953, 1955a) confirmed these observations in the rabbit and demonstrated that electrical stimulation of the posterior hypothalamus (sympathetic centers) inhibited the milk-ejection response to injected oxytocin, an effect which was abolished after adrenalectomy. Cross concluded from his experiments that any central stimulation causing sympathetico-adrenal activity inhibits the milk-ejection response and that the effect appears to depend on a constriction of the mammary blood vessels resulting from the release of adrenaline and excitation of the sympathetic fibers to the mammary glands. Whereas such a mechanism could account for the emotional disturbance of the reflex. Cross was careful to point out that there was no direct proof that this was so and he later demonstrated (Cross, 1955b) that in rabbits in which emotional inhibition of milk ejection was present, milk ejection could be effected by the injection of oxytocin (Fig. 10.19). In such cases there was clearly no peripheral inhibitory effect of milk ejection. Cross concluded that the main factor in emotional disturbance of the milkejection reflex is a partial or complete inhibition of oxytocin release from the posterior pituitary gland. At present nothing is known of the nature of this central inhibitory mechanism.^
^ A curious form of the suckling stimulus is illustrated in carvings which siumount the main door of the church of Sainte Croix in Bordeaux. The carvings illustrate penances prescribed for wrong doers who have committed one of the seven deadly sins. The penance for indulgence in the sin of luxiu y is the application to the breasts of serpents or toads.

Inhibition of the milk ejection reflex may also occur when the mammary gland becomes engorged with secretion to such an extent that the capillary circulation is so reduced that oxytocin can no longer reach the myoepithelium (Cross and Silver, 1956; Cross, Goodwin and Silver, 1958).
F. NEURAL PATHWAYS OF THE MILK-EJECTION REFLEX
Interpretation of some of the earlier studies on neural pathways is difficult because investigators did not realize that, although the milk ejection reflex normally occurs in response to the suckling stimulus, it can become conditioned and can then occur in response to visual or auditory stimuli associated with the act of nursing. In such cases an apparent lack of effect on milk ejection of section of nerves or nerve tracts would not necessarily imply that the nerves normally carrying the stimuli arising from the suckling had not been cut. Studies on the effects of hemisection of the spinal cord in a few goats led Tsakhaev (1953) to the conclusion that the apparent pathway used by the milk-ejection stimulus was uncrossed. More recently pathways within the spinal cord have been investigated by Eayrs and Baddeley (1956) who found inter alia that lactation in the rat was inhibited by lesions to the lateral funiculi, and by section of the dorsal roots of nerves supplying the segments in which the suckled nipples were situated. With few exceptions hemisection of the spinal cord abolished lactation when the only nipples available for suckling were on the same side as the lesion, but not when the contralateral nipples were available. It was concluded that the pathway used by the suckling stimulus enters the central nervous system by the dorsal routes and ascends the cord deep in the lateral funiculus of the same side. Inasmuch as in these experiments lactation was assessed from the growth curve of the pups, it is not always clear whether the failure of lactation was due to a cessation of milk secretion or to loss of the milk-ejection reflex. It was noted, however, that injections of oxytocin in some
It may be questioned whether this unusual form of the suckling stimulus would not inhibit rather than evoke the milk-ejection reflex.

626

PHYSIOLOGY OF GONADS

cases restored lactation for up to 2 days after it had ceased as a result of lesions of the cord which would suggest a primary interference with milk ejection. In the goat, Andersson (1951b) considered that stimuli may reach the hypothalamus by way of the medial lemniscus in the medulla, but little definite information is available concerning the pathways used by the stimuli to reach the hypothalamus and there is here scope for further investigations. (For further discussion see review by Cross, 1960.) From the hyopthalamus there is little doubt that the route to the posterior lobe is by way of the hypothalamo-hypophyseal tract which receives nerve fibers from the cells in the hypothalamic nuclei, and in the main from the paraventricular and supra-optic nuclei. It was generally assumed that the posterior lobe hormones were secreted in the posterior lobe from the pituicytes in response to stimuli passing down the hypothalamo-hypophyseal tract. In the last decade, however, much evidence has come to light which suggests that the so-called posterior lobe hormones are in fact elaborated in the cells of the hypothalamic nuclei and are then transported down the axones as a neurosecretion and stored in the posterior lobe (see Scharrer and Scharrer, 1954).
Before leaving the neural pathways of the milk-ejection reflex, brief reference must be made to the recent discovery by Soviet physiologists that there is also a purely nervous reflex (segmental in nature) involved in the ejection of milk. It is said that within a few seconds of the application of the milking stimulus, reflex contraction of the smooth muscle in the mammary ducts occurs, causing a flow of milk from the ducts into the cistern. This reflex contraction of the smooth muscle is also believed to occur in response to stimuli arising within the gland between milkings thus aiding the redistribution of milk in the udder. This purely nervous reflex is stated to occur some 30 to 60 seconds before the reflex ejection of milk from the alveoli by oxytocin (for further details sec review by Baryshnikov, 1957). The conditioned reflexes associated with suckling and milking have been the subject of numerous investigations l)y Grachev (see Grachev,

1953, 1958) ; these and other Russian researches into the motor apparatus of the udder have been fully reviewed by Zaks (1958).
G. MECHANISM OF SUCKLING
In the past, various theories have been put forward as to how the suckling obtains milk from its mother's mammary gland. In the human infant some considered that the lips formed an airtight seal around the nipple and areola thus allowing the child to suck, whereas others believed that compression of the lacteal sinuses between the gums aided the expulsion of the milk (see Ardran, Kemp and Lind, 1958a, b for review) . In the calf the act of suckling was studied by Krzywanek and Briiggemann (1930) who described how the base of the teat was pinched off between upper and lower jaws and the teat compressed from its base towards its tip by a stripping action of the tongue. Smith and Petersen (1945) on the other hand, concluded that the calf wrapped its tongue round the teat and obtained milk by suction.
Much misunderstanding about the nature of the act of suckling has arisen because the occurrence of milk ejection was overlooked or its significance was not appreciated. As a result, the idea became prevalent that success or failure in obtaining milk could be reckoned solely in terms of the power behind the baby's suction. This erroneous concept was vigorously attacked by Waller (1938), who pointed out that once the "draught" had occurred the milk at times flowed so freely from the breast that the baby had to break off and turn its head to avoid choking. A similar observation had been made by Sir Astley Cooper in 1840 who in describing the "draught" in nursing women wrote, "If the nipple be not immediately caught by the child, the milk escapes from it, and the child when it receives the nipple is almost choked l)y the rapid and abundant flow of the fluid; if it lets go its hold, the milk spurts into the infant's eyes." An even earlier comment was made by Soranus, a writer on paediatrics in the cai'ly half of the second century A.D., that it was unwise to allow the infant to fall asleep at the breast since the milk some

MAMMARY GLAND AND LACTATION

627

times flowed without suckling and the infant choked. It must thus be emphasized that once milk ejection has occurred the milk in the gland cisterns or sinuses is under considerable pressure and the suckling has merely to overcome the resistance of the sphincters in the nipple or teat to obtain the milk.
Recently the use of cineradiograjihy has allowed a more accurate analysis of the mechanism of suckling. Studies by Ardran, Kemp and Lind (1958b) have shown that the human infant sucks the nipple to the back of the mouth and forms a "teat" from the mother's breast; when the jaw is raised this teat is compressed between the upper gum and the tip of the tongue resting on the lower gum, the tongue is then applied to the lower surface of the "teat" from before backwards pressing it against the hard palate. Suction may assist the flow of milk so expressed from the nipple, but is only of secondary importance. Studies by Ardran, Cowie and Kemp (1957, 1958) in the goat have extended these observations, because it was possible in this species to follow the withdrawal, from the udder, of milk made radiopaque with barium sulfate. As with the infant, the neck of the teat was obliterated between the tongue and the palate of the kid and the contents of the teat sinus were displaced into the mouth cavity by a suitable movement of the tongue; while the first mouthful w^as being displaced into the pharynx, the jaw and tongue were lowered to allow the refilling of the teat sinus. The normal method of obtaining milk is, therefore, for the suckling to occlude the neck of the teat and then to expel the contents of the teat sinus by exerting positive pressure on the teat (120 mm. Hg in the goat), so forcing the contents through the teat canal or nipple orifices into the mouth cavity, a process which may be aided by negative pressure created at the tip of the teat. Human infants, goat kids, and calves can obtain milk through rubber teats by suction alone provided the orifice is large enough (see Krzywanek and Briiggemann, 1930; Martyugin, 1944; Ardran, Kemp and Lind, 1958a) , but this procedure occurs only w^hen the structure of the rubber teat is such that the suckling is unable to ol)literate the

neck of the teat and cannot, therefore, strip the contents of the teat by positive pressure.
V. Relation between the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection
We have seen that the suckling or milking stimulus is responsible for initiating the reflex concerned wath the maintenance of milk secretion and also the milk-ejection reflex; the question now arises as to what extent their arcs share common paths. It would seem logical to assume that a common path to the hypothalamus exists and parts of this, as we have seen, have been partially elucidated. Although the hypothalamo-hypophyseal nerve tracts provide an obvious link between hypothalamus and the posterior lobe, the connections between the hypothalamus and anterior pituitary are still a matter of some controversy. The possible avenues of communication to the anterior lobe are neural and vascular and these may be subdivided into central and peripheral neural connections and into portal and systemic vascular connections. The various experimental findings relating to these routes have recently been critically discussed by Sayers, Redgate and Royce (1958), and by Greep and Everett in their chapters in this book, and it is clear that at present no definite conclusions can be reached concerning their relative importance. So far as the specific question of maintenance of milk secretion is concerned, the experiments of Harris and Jacobsohn (1952), which showed that pituitary grafts maintained lactation when implanted adjacent to the median eminence in hypophysectomized rats, were consistent with the existence of a hormonal transmitter, passing by w^ay of the hypophyseal portal system. On the other hand, transplantation studies by Desclin (1950, 1956) and Everett ( 1954, 1956) have revealed that in the rat the anterior lobe can spontaneously secrete prolactin in situations remote from the median eminence, and Donovan and van der Werff ten Bosch (1957) have reported that milk secretion continued in rabbits in wiiich the pituitary portal vessels had been completely destroyed, although there was, however, an inferred change in milk composition. Evidence has recentlv been obtained

628

PHYSIOLOGY OF GONADS

which has confirmed that pituitary tissue grafted under the kidney capsule in rats apparently secretes prolactin and will give slight maintenance of milk secretion in hypophysectomized animals, this maintenance being considerably enhanced if ACTH or STH is also administered (Cowie, Tindal and Benson, 1960). It would thus seem that the cells of the anterior lobe have the ability when isolated from the hypophyseal portal system to secrete prolactin, but the experiments cited above allow no conclusions to be drawn regarding the route by which the galactopoietic function of the pituitary is normally controlled.
Recent reports that bilateral cervical sympathectomy in the lactating goat causes a fall in the milk yield suggest that the galactopoietic functions of the anterior lobe may be influenced by the sympathetic nervous system (Tsakhaev, 1959; Tverskoy, 1960) . Declines in milk yield also occur after section of the pituitary stalk in the goat, but it is not clear in such cases whether the effects are due to the interruption of nervous or vascular pathways within the stalk (Tsakhaev, 1959; Tverskoy, 1960). In these studies on stalk section the cut ends of the pituitary stalk were not separated by a plastic plate, so some restoration of the hyl^ophyseal portal system may have occurred. Further experiments on the effects of section of the pituitary stalk on lactation in which restoration of the hypophyseal portal is prevented by the insertion of a plate are being conducted in our laboratory and also in the Soviet Union. Another possible mode of communication between hypothalamus and anterior pituitary has been investigated by Benson and Folley (1956, 1957a, b) who have suggested that the oxytocin released from the neurohypophysis in response to the suckling stimulus may directly act on the cells of the anterior lobe and stimulate the release of the galactopoietic complex. The careful anatomic researches of Landsmeer (1951), Daniel and Prichard (1956, 1957, 1958) and Jewell (1956) have demonstrated in several species the existence of direct vascular connections from the neurohylK)physis to the anterior lobe so that the neurohypophyseal hormones liberated into the blood stream would in fact be carried

direct to the anterior pituitary cells in very high concentrations. Clearly such a concept would provide a simple explanation of how the hormonal integration, coordination, and maintenance of mammary function is achieved. It has already been noted (see page 607) that a connection between milk ejection and the onset of copious lactation has been suggested. There is considerable evidence that oxytocin is liberated during parturition in sufficient quantities to cause contraction of the alveoli and milk ejection (see Harris, 1955; Cross, 1958; Cross, Goodwin and Silver, 1958) ; if, therefore, oxytocin can release the lactogenic and galatopoietic complexes from the anterior pituitary, a simple explanation of the mechanism triggering off the onset of copious milk secretion, before the application of the milking stimulus, is available.
We must now consider what experimental evidence there is to support this rather attractive theory. First, Benson and Folley (1956, 1957a, b) demonstrated that regular injections of oxytocin can retard mammary regression after weaning in a similar fashion to injections of prolactin (see page 610), and they have shown that the presence of the pituitary is essential for oxytocin to elicit this effect. Synthetic oxytocin proved equally effective, thus discounting the possibility of a contaminant in natural oxytocin being concerned (Fig. 10.20) . These experiments have so far only been carried out in rats, but they strongly suggest that oxytocin can elicit the secretion of prolactin. In agreement with this concept are several observations that regular injections of oxytocin have galactopoietic effects in lactating cows and that oxytocin has luteotrophic effects in rats (see review by Benson, Cowie and Tindal, 1958) . There is, moreover, some evidence that the suckling stimulus may cause the release of vasopressin or the antidiuretic hormone (ADH) from the neurohypoi)hysis (see page 621), and it has been shown that ADH or some material closely associated with it may cause the secretion of ACTH from the anterior lobe (see review by Benson, Cowie and Tindal, 1958) ; so there are some grounds for supposing that the hormones of the posterior lobe evoke the secretion of several components of the

MAMMARY GLAND AND LACTATION

Fig. 10.20. Sections from abdominal mammary gland of rats from wliuli Ur- pups were removed on the fourth day of lactation and which received thereafter for 9 daj^s: A. LO I.U. synthetic oxytocin three times daily. B. Saline daily. Note the maintenance of gland structure in A. (Courtesy of Dr. G. K. Benson.)

galactopoietic complex from the anterior lobe. It was hoped to gain further evidence on this point by studies on hypophysectomized rats bearing pituitary homografts under the kidney capsule (see Benson, Cowie, Folley and Tindal, 1959) . As already noted, such grafts secrete prolactin and will give a slight maintenance of milk secretion, but these grafts will not maintain normal milk secretion even when such animals are injected with oxytocin and ADH (Cowie, Tindal and Benson, 1960). It must, therefore, be assumed that if these posterior pituitary hormones are responsible for the release of the galactopoietic complex, some other hypothalamic factor is also necessary to maintain the anterior lobe in a responsive condition. Everett (1956) suggested that the hypothalamus by way of its neurovascular connections with the anterior lobe, normally exerts a partial inhibitory effect on prolactin secretion. It may thus be that when the anterior lobe is removed from hypothalamic influence, the synthetic activities of its cells are centered on prolactin

production to the detriment of the other components of the galactopoietic complex, so that these are no longer available for release in response to neurohypophyseal hormones. There is need, however, for experimentation in other species.
The theory that the release of the galactopoietic complex is effected by the hormones of the posterior lobe secreted in response to the suckling stimulus is attractive in that it appears to afford a simple explanation of the hormonal integration of mammary function, but it must be pointed out that the observations on the maintenance of mammary structure after weaning by injections of oxytocin do not prove that prolactin or the galactopoietic complex is released in response to oxytocin under normal conditions of milking or suckling, and more research, particularly in species other than the rat, is necessary. Grosvenor and Turner (1958a) injected oxytocin into anesthetized lactating rats and, on the basis of assays of the pituitary content of prolactin, considered that oxytocin caused no significant release of

630

PHYSIOLOGY OF GONADS

prolactin. They had previously shown that there was an immediate fall in the pituitary content of prolactin after nursing (Grosvcnor and Turner, 1957b) and therefore concluded that their findings were contrary to the hypothesis that oxytocin is a hormonal link in the discharge of prolactin. This, however, cannot be regarded as conclusive because of the difficulties of relating pituitary content of a hormone to blood levels of the hormone and also the difficulty of determining the physiologic dose of oxytocin, for if the oxytocin is carried directly from the neurohypophysis into the anterior lobe, then the concentration in the blood reaching the anterior lobe may be relatively great (see also Cowie and Folley, 1957).
Other theories of the reflex maintenance of milk secretion have been put forward. In 1953 Tverskoi, observing that repeated injections of oxytocin were galactopoietic in the goat, suggested that alveolar contraction stimulated sensory nerve endings in the alveolar walls which reflcxly caused the release of prolactin. It is obvious that his observations could be explained on the basis of the Benson-Folley theory of direct pituitary stimulation by oxytocin. This possibility was indeed considered by Tverskoi. but rejected on the grounds that oxytocin did not affect the prolactin content of the pituitary (Meites and Turner, 1948). In 1957 Tverskoi found it necessary to revise his theory, having found that full lactation could be maintained in the goat after complete and repeated denervation of the udder provided oxytocin was regularly given to evoke milk ejection. He then suggested that alveolar contraction stimulates the synthetic activities of the mammary epithelium causing an uptake of prolactin from the blood, the fall in the blood prolactin level then stimulating the further production of prolactin by the anterior lobe. Although these latter observations of Tverskoi might again be explained on the basis of direct pituitary stimulation by exogenous oxytocin, more recent studies on goats have cast doubts on the validity of such an explanation. Tverskoi (1958) and Denannir and Martinet (1959a, b, 1960) have shown that lactating goats will continue to lactate, giving nonnal or onlv niodcratelv reduced

milk yields after section of all nervous connections between the udder and brain (cord section, radicotomy, bilateral sympathectomy) and without their receiving oxytocin and in the absence of conditioned milkejection reflexes. It has already been noted that milk ejection in such animals may result from mechanical stimulation of the myoepithelial cells by udder massage (see page 624) , but the release of the galactopoietic complex from the anterior pituitary would seem in these goats to have been independent of neurohormonal reflex activities. AVhether in such animals the release is spontaneous or dependent on the level of hormones in the blood as suggested by Tverskoi (1957) is a matter for further research.
VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection
Various attempts have been made to investigate the mechanism controlling release of anterior pituitary hormones by the use of dibenamine, atropine, and other drugs. In reviewing such experiments, Harris (1955) concluded that there was no convincing evidence of the participation of adrenergic, cholinergic, or histaminergic agents in the control of gonadotrophic and adrenocorticotrophic hormone release. Recently Grosvenor and Turner (1957a) reported that various ergot alkaloids, dibenamine, and atropine blocked milk ejection in the rat; the ergot alkaloids doing so within 10 minutes of administration, the atropine and dibenamine within 2 to 4 hours. Inasmuch as milk ejection occurred in response to exogenous oxytocin, it was concluded that these drugs acted centrally, and the presence of adrenergic and cholinergic links in the neurohormone arc was postulated to be responsible for the discharge of oxytocin. Later, on the basis of assays of jntuitary prolactin after nursing in druginjected lactating rats, it was suggested that cholinergic and adrenergic links are iinohcd in the reflex resi)onsible for prolactin release (Grosvenor and Turner, 1958a). Ergot alkaloids, however, administered in our laboratory to lactating rats had no significant effect on the lactational per

MAMMARY GLAND AND LACTATION

631

fonnance as judged by the growth of the litters in comparison with the growth of litters of pair-fed control rats, showing that apparent inhibitory effects of the alkaloids on lactation were due to depressed food intake of the mothers (Tindal, 1956a). Inasmuch as growth of the litter depends on efficient milk secretion and milk ejection, Tindal's observations seem to throw doubt on the importance of the adrenergic link in these reflexes. On the other hand, IVIeites (1959) has reported that adrenaline and acetylcholine can induce or maintain mammary development and milk secretion in suitably prepared rats, observations which could be interpreted as supporting the presence of adrenergic and cholinergic links as postulated by Grosvenor and Turner (1958a).
There have been clinical reports of women developing galactorrhoea after treatment with trancjuilizing drugs {e.g., Sulman and Winnik, 1956; Marshall and Leiberman, 1956; Piatt and Sears, 19561 and interesting observations have recently ap

peared on the lactogenic effects of reserpine in animals. Milk secretion has been initiated both in virgin rabbits after suitable estrogen priming and in the pseudopregnant rabbit by reserpine (Sawyer, 1957; Meites, 1957a). On the other hand, in our laboratory Tindal (1956b, 1958) had been unable to detect any mammogenic or lactogenic effects with chlorpromazine or reserpine in rabbits (Dutch breed), rats, or goats, nor did reserpine stimulate the crop-sac when injected into pigeons. Recently, using New Zealand White rabbits, Tindal (1960) has induced milk secretion with reserpine. The reason for these contradictory results is not entirely clear, although breed differences in the response would appear to exist in the rabbit. In our laboratory, Benson (1958) has shown that reserpine is strikingly active in retarding mammary involution in the lactating rat after weaning, the effect being of such a magnitude as has so far only been equalled by a combination of prolactin and STH (Fig. 10.21). It has been tentatively suggested that the tranquilizing drugs may

^^:f/

mm\"^>m.-Wi

■w^

.•^^:j^-^ f4kr 1"

Fig. 10.2L Sections from the abdominal mammary gland of rats from whichthe pujis were removed on the fourth day of lactation and which received thereafter for 9 days: A 100 fj.g. reserpine daily. B. Sahne dailJ^ Note the retardation of involution effected by reserpine. (Courtesy of Dr. G. K. Benson.)

632

PHYSIOLOGY OF GONADS

remove .some hypothalamic restraining mechanism on the release of jn'olactin and probably of other anterior-pituitary hormones (Sulman and Winnik, 1956; Benson, Cowie and Tindal, 1958), an effect which, if confirmed, may throw light on the behavior of pituitary transplants in sites remote from the median eminence.
VII. Conclusion
Any reader familiar with the chajiter on the mammary gland in the previous edition of this book cannot fail to note the main directions in which the subject has advanced in the intervening two decades. These reflect, as they are bound to do, the road taken by the science of endocrinology itself, a road leading to greater biochemical understanding on the one hand and to ever closer rapprochement with neurophysiology on the other.
The mammary gland offers unique opportunities of studying the biochemical mechanisms of hormone action because it is an organ with quite exceptional synthetic capabilities, an organ which is perhaps the most comprehensive hormone target in the mammalian body. Biochemists are entering this promising field in increasing numbers and we may expect to reap the fruits of their labors in the future.
VIII. References
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SthicivER, p., a.vd Grueter, F. 1928. Action du lobe anterieur de I'hypophyse sur la montee laiteuse. Comp. rend. Soc. biol.. 99, 1978-1980.
Stricker. p.. and Grueter, F. 1929. Recherches

MAMMARY GLAND AND LACTATION

641

experimentales sur les fonctions du lobe anterieur de I'hypophyse: influence des extraits du lobe anterieur sur I'appareil genital de la lapine et sur la montee laiteuse. Presse med., 37, 1268-127L
SuLMAN, F. G., AND WiNNiK, H. Z. 1956. Hormonal effects of chlorpromazine. Lancet, 270, 161-162.
Sykes, J. F., AND Wrenn, T. R. 1950. Hormonal development of the mammary gland of dairy heifers. J. Dairy Sc, 33, 194-204.
Sykes, J. F., and Wrenn, T. R. 1951. Hormonal development of mammary tissue in dairy heifers. J. Dairy Sc, 34, 1174-1179.
TiNDAL, J. S. 1956a. The effect of ergotamine and dihydroergotamine on lactation in the rat. J. Endocrinol., 14, 268-274.
TiND.^L, J. S. 1956b. National Institute for Research in Dairying, Report 1956, pp. 56-57.
TiNDAL, J. S. 1958. National Institute for Research in Dairying, Report 1958, pp. 48-49.
TiNDAL, J. S. 1960. A breed difference in the lactogenic response of the rabbit to reserpine. J. Endocrinol., 20, 78-81.
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TovERUD, S. v., AND MuNSON, P. L. 1956. The influence of the parathyroids on the calcium concentration of milk. Ann. New York Acad. Sc, 64, 336.
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642

PHYSIOLOGY OF GONADS

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11

SOME PROBLEMS OF THE METABOLISM AND
MECHANISM OF ACTION OF STEROID
SEX HORMONES
Claude A. Villee, Ph.D.
ASSOCIATE PROFESSOR OF BIOLOGICAL CHEMISTRY, HARVARD UNIVERSITY

I. Introduction 643
II. The Biosynthesis op Steroids 643
A. Cholesterol 644
B. Progesterone 644
C. Androgens 645
D. Estrogens 647
E. Biosynthesis of Other Steroids 647
F. Interconversions of Steroids 647
G. Catabolism of Steroids (548
H. Transport, Conjugation, and Excretion 650
III. Effects of Sex Hormones on Inter mediary Metabolism 650
A. Estrogens 652
B. Androgens 659
C. Progesterone 660
IV. References 661
I. Intro<luction
The chemical structure of the sex hormones, their isohition from biologic materials, and many of their chemical properties were fully described in the previous edition of Sex and Internal Secretions (W. M. Allen, 1939; Doisy, 1939; Koch, 1939). The major steroid sex hormones were isolated and identified 20 to 30 years ago. Estrone, in fact, was crystallized from pregnancy urine by Doisy, Veler and Thayer (1929) before the true structure of the steroid nucleus was known. The isolation, identification, and chemical synthesis of estradiol, progesterone, and testosterone were accomplished during the 1930's. Additional substances with androgenic, estrogenic, or progestational activity have subsequently been isolated from urine or from tissues but these are probably metabolites of the major

sex steroids. The steroids are now routinely synthesized from cholesterol or from plant sterols. It would be possible to carry out the total synthesis of steroids from simple precursors but this is not commercially practicable.
The two decades since the previous edition have been marked by major advances in our understanding of the intermediary metabolism of steroids — the synthesis of cholesterol from two-carbon units, the conversion of cholesterol to pregnenolone and progesterone, and the derivation of corticoids, androgens, and estrogens from progesterone. These advances were made possible by the development of vastly improved methods for the isolation and identification of steroids: chromatography on paper or columns, counter-current distribution, labeling with radioactive or heavy isotopes, infrared spectroscopy, and so on. There have been concomitant increases in the information regarding the sites and mechanisms of action of these biologically important substances and the means by which they stimulate or inhibit the growth and activity of particular tissues of the body. The following discussion will attempt to present a general picture of these two fields and not an exhaustive citation of the tremendous body of relevant literature.
II. The Biosynthesis of Steroids
When the steroid hormones were first discovered it was generally believed that each

643

644

PHYSIOLOGY OF GONADS

endocrine gland made its characteristic steroid by some unique biosynthetic mechanism, one that was independent of those in other glands. However, there is now abundant evidence that the biosynthetic paths in the several steroid-secreting glands have many features which are similar or identical.
It is now well established that progesterone is not simply a female sex hormone produced by the corpus luteum, but a common precursor of adrenal glucocorticoids such as Cortisol and adrenal mineralocorticoids such as aldosterone, androgens, and estrogens. The adrenal cortex, ovary, testis, and placenta have in common many enzymes for the biosynthesis of steroids. Androgenic tumors of the human ovary, for example, have been shown to produce testosterone and its metabolites. The transplantation of an ovary into a castrate male mouse will result in the maintenance of the male secondary sex characters, which suggests that the normal ovary can also synthesize androgens.
A. CHOLESTEROL
The early work of Bloch (1951), Rilling, Tchen and Bloch (1958), and of Popjak (1950) showed that labeled acetate is converted to labeled cholesterol. The pattern of the labeling present in the cholesterol synthesized from acetate-1-C^^ or acetate-2-C^ as precursor led to speculations as to how the steroid nucleus is assembled. Further work (Langdon and Bloch, 1953) revealed that squalene and certain branched-chain, unsaturated fatty acids are intermediates in this synthesis. The current hypothesis, which is supported by a wealth of experimental evidence, states that two moles of acetyl coenzyme A condense to form acetoacetyl coenzyme A, which condenses with a third molecule of acetyl coenzyme A to form yS-hydroxy-^-methyl glutaric acyl coenzyme A (Fig. 11.1). The coenzyme A group is removed and the hydroxymethyl glutaric acid is reduced to mevalonic acid. Mevalonic acid, 3-hydroxy-3-methylpentano-5-lactone, is metabolized to a 5-carbon isoprenoid compound and three moles of these condense to form a 1 5-carbon hydrocarbon. The headto-head condensation of two molecules of this 15-carbon compound yields the 30

carbon equalene. This is metabolized, by way of lanosterol and the loss of three methyl groups, to cholesterol, which seems to be the common precursor of all of the steroid hormones (Tchen and Bloch, 1955; Clayton and Bloch, 1956).
The question of whether cholesterol is an obligate intermediate in the synthesis of steroid hormones has not been definitely answered. There is clear evidence that cholesterol is converted to steroids without first being degraded to small units and subsequently reassembled. Werbin and LeRoy (1954) administered cholesterol labeled with both carbon-14 and tritium (H^) to human subjects and isolated from their urine tetrahydrocortisone, tetrahydrocortisol, androsterone, etiocholanolone and 11ketoetiocholanolone. These substances, known to be metabolites of steroid hormones, were labeled with both C^^ and H^ and the labeled atoms were present in the ratio expected if they were derived directly from cholesterol. Experiments by Dorfman and his colleagues (Caspi, Rosenfeld and Dorfman, 1956) also provide evidence for the synthesis of steroids via cholesterol. Cortisol and 11-desoxycortisol were isolated from calf adrenals perfused with acetate-1C^^ and from a patient with an adrenal tumor to whom acetate- 1-C^^ had been administered. It is known that cholesterol synthesized from acetate-l-C^'* is labeled in carbon 20 but not in carbon 21. The Cortisol and 11-desoxycortisol also proved to be labeled in carbon 20 but not in carbon 21. This evidence does not, of course, exclude biosynthetic paths for the steroids other than one by way of cholesterol, but it does suggest that cholesterol is at least an important precursor of them. Direct evidence that cholesterol is synthesized from squalene in man is provided by the experiments of Eidinoff, Knoll, Marano, Kvamme, Rosenfeld and Hcllman (1958), who prepared tritiated squalene and administered it orally to human subjects. They found that the cholesterol of the blood achieved maximal specific aeti\'ity in 7 to 21 liours.
B. PROCiE.STERONE
Cholesterol undergoes an oxidative cleavage of its side chain to yield isocaproic acid and pregnenolone (Fig. 11.2). The lat

CH3C0-SC0A

STEROID SEX HORMONES
SCoA

645

CH^CO-SCoA

Acetyl CoA

CH3COCH2CO~SCoA

Acetoacetyl CoA

Sesquiterpene (15C)

COOH
► HO— C— CHI ^
CO-' SCO A
pOH-p methylglutaric acyl Co A

CHg
C-CHI
CH II CHo

Isoprene unit (5C)

CHO
I
CHo I ^
HO-C-CH^ 1 3
CHo I 2 COOH
Mevalonic acid

Squalene Lanosterol Cholesterol
(30C) (30C) (27C)
Fig. 11.1. Biogenesis of cholesterol.

ter is dehydrogenated in ring A by the enzyme 3-^-ol dehydrogenase and a spontaneous shift of the double bond from the A5 , 6 to the A4 , 5 position results in progesterone. Progesterone undergoes successive hydroxylation reactions, which require molecular oxygen and reduced triphosphopyridine nucleotide (TPNH), at carbons 17, 21, and 11. These hydroxylations yield, in succession, 17-a-hydroxy progesterone, Reichstein's compound S (ll-desoxy-17-hydroxycorticosterone), and Cortisol (17-a-hydroxvcorticosterone) .

C. ANDROGENS
17-a-Hydroxy progesterone is also the immediate precursor of androgens and estrogens. Oxidative cleavage of its side chain yields A-4-androstenedione, which undergoes reduction to testosterone (Fig. 11.2). A-4-Androstenedione may be hydroxylated at carbon 11 to yield ll-/3-hydroxy-A-4androstenedione, which is an androgen isolated from human urine. It has also been found as a metabolite of certain androgenic tumors of the adrenal cortex.

04 G

PHYSIOLOGY OF GONADS

CH

Y^ .

socaproic C

=0

c=o

r^

0^-OH

^r^

HO

HO

J

HO

Cholesterol

Pregnenolone

17-Hydr

oxy
Dehydroepi

preg

nenolone

androsterone

CH3

CH3

f3

H-C-OH 1

, f

c=o
1

1
c=o
1

^ ■

-x^V

t^

^XP""°"

r^^

^^<Y

„ijj

- \^Xaj

JJ

A -3- Ketopregnene

Progest

erone

17

-Hydroxy
20-oc-ol

y

y

progesterone

CH^OH

X

CH OH

/

c=o

1
c=o

"

Desoxy— corticosterone

CH2OH c=o

1 7- Hydroxy desoxycorticosterone (Reichstein's "S")

CH^OH HCO C=0

.JOJ

CH^OH
c=o

A -androstenedione

OH

o ' -- o
Corticosterone 18-aldo- 11- desoxy- Cortisol Testosterone
corticosterone (Kendall's
Cmpd. "F")

HCO C=0

19 -Hydroxy- ^■^■ androstenedione

.;^

Aldosterone

HO
Estradiol Fig. 11.2. Biosynthetic paths from cholesterol.

Estrone

STEROID SEX HORMONES

647

D. ESTROGENS
A-4-Androstenedione and testosterone are precursors of the estrogens. Baggett, Engel, Savard and Dorfman (1956) demonstrated the conversion of testosterone to estradiol17/? by slices of human ovary. Ryan (1958) found that the enzymes to carry out this conversion are also present in the human placenta, located in the microsomal fraction of placental homogenates. Homogenates of stallion testis convert labeled testosterone to labeled estradiol and estrone. Slices of human adrenal cortical carcinoma also have been shown to convert testosterone to estradiol and estrone, and Nathanson, Engel and Kelley (1951) found an increased urinary excretion of estradiol, estrone, and estriol following the administration of adrenocorticotrophic hormone to castrate women. Thus it seems that ovary, testis, placenta, and adrenal cortex have a similar biosynthetic mechanism for the production of estrogens and androgens. The first step in the conversion of testosterone or A-4-androstenedione to estrogens is the hydroxylation at carbon 19, again by an enzymatic process which requires molecular oxygen and TPNH. Meyer (1955) first isolated and characterized 19-hydroxy-A-4-androstene3,17-dione from a perfused calf adrenal. When this was incubated with dog placenta it was converted to estrone. The steps in the conversion of the 19-hydroxy-A-4-androstenedione to estrone appear to be the introduction of a second double bond into ring A, the elimination of carbon 19 as formaldehyde, and rearrangement to yield a phenolic ring A. The requirements for the aromatization of ring A by a microsomal fraction of human placenta were studied by Ryan (1958). West, Damast, Sarro and Pearson (1956) found that the administration of testosterone to castrated, adrenalectomized women resulted in an increased excretion of estrogen. This suggests that tissues other than adrenals and gonads, presumably the liver, can carry out this same series of reactions.
E. BIOSYNTHESIS OF OTHER STEROIDS
To complete the picture of the interrelations of the biosyntheses of steroids, it should be noted that other evidence shows

that progesterone is hydroxylated at carbon 21 to yield desoxycorticosterone and this is subsequently hydroxylated at carbon 11 to yield corticosterone. Desoxycorticosterone may undergo hydroxylation at carbon 18 and at carbon 11 to yield aldosterone, the most potent salt-retaining hormone known (Fig. 11.2).
Dehydroepiandrosterone is an androgen found in the urine of both men and women. Its rate of excretion is not decreased on castration and it seems to be synthesized only by the adrenal cortex. It has been postulated that pregnenolone is converted to 17-hydroxy pregnenolone and that this, by cleavage of the side chain between carbon 17 and carbon 20, would yield dehydroepiandrosterone.
F. INTERCONVERSIONS OF STEROIDS
The interconversion of estrone and estradiol has been shown to occur in a number of human tissues. A diphosphopyridine nucleotide-linked enzyme, estradiol- 17/3 dehydrogenase, which carries out this reaction has been prepared from human placenta and its properties have been described by Langer ancl Engel (1956). The mode of formation of estriol and its isomer, 16-epiestriol, is as yet unknown.
There are three major types of reactions which occur in the interconversions of the steroids: dehydrogenation, "hydroxylation," and the oxidative cleavage of the side chain. An example of a dehydrogenation reaction is the conversion of pregnenolone to progesterone by the enzyme 3-/3-ol dehydrogenase, which requires diphosphopyridine nucleotide (DPN) as hydrogen acceptor. This important enzyme, which is involved in the synthesis of progesterone and hence in the synthesis of all of the steroid hormones, is found in the adrenal cortex, ovary, testis, and placenta. Other dehydrogenation reactions in which DPN is the usual hydrogen acceptor are the readily reversible conversions of A-4-androstenedione ^ testosterone, estrone ^ estradiol, and progesterone
~~^ A-4-3-ketopregnene-20-a-ol. This latter~~
substance, and the enzymes producing it from progesterone, have been found by Zander (1958) in the human corpus luteum and placenta.
The oxidative reactions leading to the

PHYSIOLOGY OF GONADS

introduction of an OH group on the steroid nucleus are usually called "hydroxylations." Specific hydroxylases for the introduction of an OH group at carbons 11, 16, 17, 21, 18, and 19 have been demonstrated. All of these require molecular oxygen and a reduced pyridine nucleotide, usually TPNH. The ll-/3-hydroxylase of the adrenal cortex has been shown to be located in the mitochondria (Hayano and Dorfman, 1953) . Experiments with this enzyme system, utilizing oxygen 18, showed that the oxygen atoms are derived from gaseous oxygen and not from the oxygen in the water molecules (Hayano, Lindberg, Dorfman, Hancock and Doering, 1955). Thus this hydroxylation reaction also involves the reduction of molecular oxygen.
The oxidative cleavage of the side chains of the steroid molecule appears to involve similar hydroxylation reactions. The experiments of Solomon, Levitan and Lieberman (1956) indicate that the conversion of cholesterol to pregnenolone involves one and possibly two of these hydroxylation reactions, with the introduction of OH groups at carbons 20 and 22 before the splitting off of the isocaproic acid.
In summary, this newer knowledge of the biosynthetic paths of steroids has revealed that the differences between the several steroid-secreting glands are largely quantitative rather than qualitative. The testis, for example, produces progesterone and estrogens in addition to testosterone. The change from the secretion of estradiol by the follicle to the secretion of progesterone by the corpus luteum can be understood as a relative loss of activity of an enzyme in the path between progesterone and estradiol. If, for example, the enzyme for the 17-hydroxylation of progesterone became inactive as the follicle cells are changed into the corpus luteum, progesterone rather than estradiol would subsequently be produced.
Knowledge of these pathways also provides an explanation for certain abnormal changes in the functioning of the glands. Bongiovnnni (1953) and Jailer (1953) showed that the adrenogenital syndrome results from a loss of an enzyme or enzymes for the hydroxylation reactions at carbons 21 and 11 of progesterone, which results in an impairment in the production of Cortisol.

The pituitary, with little or no Cortisol to inhibit the secretion of adrenocorticotrophic hormone (ACTH), produces an excess of this hormone which stimulates the adrenal to produce more steroids. There is an excretion of the metabolites of progesterone and 17-hydroxy progesterone, pregnanediol and pregnanetriol respectively, but some of the 17-hydroxy progesterone is converted to androgens and is secreted in increased amount.
G. CATABOLISM OF STEROmS
Many of the steroid hormones are known to act on the pituitary to suppress its secretion of the appropriate trophic hormone, ACTH, the follicle-stimulating hormone (FSH), or luteinizing hormone (LH). It would seem that the maintenance of the proper feedback mechanism between steroid-secreting gland and pituitary requires that the steroids be continuously inactivated and catabolized. The catabolic reactions of the steroids are in general reductive in nature and involve the reduction of ketonic groups and the hydrogenation of double bonds. The reduction of a ketonic group to an OH group can lead to the production of two different stereoisomers. If the OH group projects from the steroid nucleus on the same side as the angular methyl groups at carbon 18 and carbon 19, i.e., above the plane of the four rings, it is said to have the yS-configuration and is represented by a heavy line. If the OH projects on the opposite side of the steroid nucleus, below the plane of the four rings, it is said to have the a-configuration and is represented by a dotted line. Although both isomers are possible, usually one is formed to a much greater extent than the other.
The first catabolic step is usually the reduction of the A4-3-ketone group of ring A, usually to 3aOH compounds with the hydrogen at carbon 5 attached in the /3configuration. The 5/3-configuration represents the CIS configuration of rings A and B. The elimination of the A4-3-ketone group greatly decreases the biologic activity of the steroid and increases somewhat its solubility in water. This reductive process occurs largely in the liver. Progesterone is converted by reduction of its A4-3-ketone group to pregnane-3a:20a-diol, and 17-hy

STEROID SEX HORMONES 649
EXCRETORY PRODUCTS

c=o

Progesterone

1 3
HCOH

HO H
Pregnanediol

CH,

-OH

17 Hydroxy progesterone

CH,

^ H-C-OH
■OH

HO H
Pregnanetriol

OH

Testosterone

Androsterone

HO

/

androstenedione

HO H
Etiocholanolone

Dehydroepiandrosterone
Fk;. 11.3. Excretory products of progesterone and androgen

(Iroxy progesterone is converted to pregnane3a:17a:20a-triol (Fig. 11.3). Testosterone and dehydrocpiandroesterone are both converted to A4-androstenedione and the reduction of its A4-3-ketone group results in a mixture of androsterone (3a,5a-configuration) and ctiochohmolone (3a,5/?-configuration ) .

The catabohsm of estradiol is not completely known. Estradiol, estrone, and estriol are found in the urine but they account for less than half of an administered dose of labeled estradiol. The /3-isomer, 16-epiestriol, and two other phenolic steroids, 16-ahydroxy estrone and 2-methoxyestrone, have recently been isolated from normal

650

PHYSIOLOGY OF GONADS

urine and are known to be estrogen metabolites (Marrian and Bauld, 1955).
H. TRANSPORT, CONJUGATION, AND EXCRETION
Steroids circulate in the blood in part as free steroids and in part conjugated with sulfate or glucuronic acid (c/. review by Roberts and Szego, 1953b j . The steroids are generally conjugated by the hydroxy 1 group at carbon 3 with inorganic sulfate or with glucuronic acid. In addition, either the conjugated or nonconjugated forms may be bound to certain of the plasma proteins such as the ^-globulins (Levedahl and Bernstein, 1954) . There is evidence of specific binding of certain steroids with particular proteins, e.g., the binding of Cortisol to "transcortin" (Daughaday, 1956). Between 50 and 80 per cent of the estrogens in the blood are present closely bound to plasma proteins. A similar large fraction of the other steroid hormones is bound to plasma proteins ; presumably this prevents the hormone from being filtered out of the blood as it passes through the glomerulus of the kidney. The steroids excreted in the urine are largely in the conjugated form, as sulfates or glucuronides.
The liver plays a prime role in the catabolism of the steroids. It is the major site of the reductive inactivation of the steroids and their conjugation with sulfate or glucuronic acid. These conjugated forms are more water-soluble and the conjugation probably promotes their excretion in the urine. Rather large amounts of certain steroids, notably estrogens, are found in the bile of certain species. These estrogens are free, not conjugated; the amount of estrogens present in the bile suggests that this is an important pathway by which they are excreted. It has been suggested that the bacteria of the gastrointestinal tract may degrade the steroids excreted in the bile and further that there is an "enterohepatic circulation" of steroids with reabsorption from the gut, transport in the portal system to the liver, and further degradation within the liver cells.
III. Effects of Sex Hormones on Intermediary Metabolism
The literature concerning the effects of hormones on intermediary metabolism is

voluminous and contains a number of contradictions, some of which are real and some, perhaps, are only apparent contradictions. Evidence that a hormone acts at one site does not necessarily contradict other evidence that that hormone may act on a different metabolic reaction. From the following discussion it should become evident that there may be more than one site of action, and more than one mechanism of action, of any given hormone.
The hormones are so different in their chemical structure, proteins, peptides, amino acids, and steroids, that it would seem unlikely, a priori, that they could all influence the cellular machinery by comparable means. The basic elements of an enzyme system are the protein enzyme, its cofactors and activators, and the substrates and products. A hormone might alter the over-all rate of an enzyme system by altering the amount or activity of the protein enzyme, or by altering the availability to the enzyme system of some cofactor or substrate molecule. Some of the mechanisms of hormone action which have been proposed are these. (1) The hormone may alter the rate at which enzyme molecules are produced de novo by the cell. (2) The hormone may alter the activity of a preformed enzyme molecule, i.e., it may convert an inactive form of the enzyme to an active form. (3) The hormone may alter the permeability of the cell membrane or the membrane around one of the subcellular structures within the cell and thus make substrate or cofactor more readily available to the enzyme. Or, (4) the hormone may serve as a coenzyme in the system, that is, it may be involved in some direct fashion as a partner in the reaction mediated by the enzyme. Each of these theories has been advanced to explain the mode of action of the sex hormones.
The problem of the hormonal control of metabolism has been investigated at a variety of biologic levels. The earliest experiments were done by injecting a hormone into an intact animal and subsequently measuring the amount of certain constituents of the blood, urine, or of some tissue. There are several difficulties with such experiments. All of the homeostatic mechanisms of the animal operate to keep condi

STEROID SEX HORMONES

651

tions constant and to minimize the effects of the injected hormone. In addition, there is a maze of interactions, some synergistic and some antagonistic, between the different hormones both in the endocrine gland and in the target organs, so that the true effect of the substance injected may be veiled. Our growing understanding of the interconversions of the steroid hormones warns us that an androgen, for example, may be rapidly converted into an estrogen, and the metabolic effects observed on the administration of an androgen may, at least in part, result from the estrogens produced from the injected androgen.
To eliminate some of the confusing effects of these homeostatic mechanisms some investigators remove the liver, kidneys, and other viscera before injecting the hormone under investigation. Such eviscerated preparations have been used by Levine and his colleagues in their investigations of the mode of action of insulin (c/. Levine and Goldstein, 1955).
Other investigators have incubated slices of liver, kidney, muscle, endocrine glands, or other tissues in glass vessels in a chemically defined medium and at constant temperature. Such experiments have the advantage that metabolism can be studied more directly, oxygen consumption and carbon dioxide production can be measured manonietrically, and aliquots of the incubation medium can be withdrawn for chemical and radiochemical analyses. The amounts of substrate, cofactors, and hormone present can be regulated and the interfering effects of other hormones and of other tissues are eliminated. Theoretically, working with a simpler system such as this should lead to greater insight into the physiologic and chemical events that occur when a hormone is added or deleted. The chief disadvantage of this experimental system is that it is difficult to prove that the conditions of the experiment are "physiologic." With tissue slices there is the possibility that the cut edges of the cells may introduce a sizeable artifact. Kipnis and Cori (1957) found that the rat diaphragm, as it is usually prepared for experiments in vitro, has an abnormally large extracellular space and is more permeable to certain pentoses than is the intact diaphragm.

It has been postulated that a hormone may influence the metabolism of a particular cell by altering the permeability of the cell membrane or of the membrane around one of the subcellular particles. Experiments with tissue homogenates, in which the cell membrane has been ruptured and removed, provide evidence bearing on such theories. If an identical hormone effect can be obtained in a cell-free system, and if suitable microscopic controls show that the system is indeed cell-free, the permeability theory may be ruled out.
Ideally the hormone effect should be studied in a completely defined system, with a single crystalline enzyme, known concentration of substrates and cofactors, and with known concentration of the pure hormone. Colowick, Cori and Slein (1947) reported that hexokinase extracted from diabetic muscle has a lower rate of activity than hexokinase from normal muscle and that it could be raised to the normal rate by the addition of insulin in vitro. The reality of this effect has been confirmed by some investigators and denied by others who were unable to repeat the observations. Cori has suggested that the decreased rate of hexokinase activity in the diabetic results from a labile inhibitor substance produced by the pituitary. Krahl and Bornstein (1954) have evidence that this inhibitor is a lipoprotein which is readily inactivated by oxidation.
The two hormones whose effects can be demonstrated reproducibly in an in vitro system at concentrations in the range which obtains in the tissues are epinephrine (or glucagon) and estradiol (and other estrogens) . Epinephrine or glucagon stimulates the reactivation of liver phosphorylase by increasing the concentration of adenosine3'-5'-monophosphate (Haynes, Sutherland and Rail, 1960), and estrogens stimulate an enzyme system found in endometrium, placenta, ventral i)rostate of the rat, and mammary gland. The estrogen-stimulable enzyme was originally described as a DPNlinked isocitric dehydrogenase, but the estrogen-sensitive enzyme now appears to be a transhydrogenase which transfers hydrogens from TPN to DPN (Talalay and Williams-x\shman, 1958; Yillee and Hngerman, 1958).

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The various tissues of the body respond in quite different degrees to the several hormones. This difference in response is especially marked with the sex hormones. Those tissues which respond dramatically to the administration of a hormone are termed the "target organs" of that hormone. Just what, at the cellular level, differentiates a target organ from the other tissues of the body is not known exactly but there is evidence that each kind of tissue is characterized by a certain pattern of enzymes. The pattern of enzymes is established, by means as yet unknown, in the course of embryonic differentiation. The enzyme glucose 6-phosphatase, which hydrolyzes glucose 6-phosphate and releases free glucose and inorganic phosphate, is present in liver but absent from skeletal muscle. Even though a given reaction in two different tissues may be mediated by what appears to be the same enzyme, the enzymes may be different and subject to different degrees of hormonal control. Henion and Sutherland (1957) showed that the phosphorylase of liver responds to glucagon but the phosphorylase of heart muscle does not. Further, the two enzymes are immunologically distinct. An antiserum to purified liver phosphorylase will not react with heart phosphorylase to form an inactive antigenantibody precipitate, but it does react in this manner with liver phosphorylase. Further, perhaps more subtle, differences between comparable enzymes from different tissues have appeared when lactic dehydrogenases from liver, heart, skeletal muscle, and other sources were tested for their rates of reaction with the several analogues of the pyridine nucleotides now available (Kaplan. Ciotti, Hamolsky and Bicbcr, 1960). p]xtension of this technique may reveal differences in response to added hormones.
In addition to these differences in the response to a hormone of the tissues of a single animal, there may be differences in the response of the comparable tissues of different species to a given dose of hormone. Estrone, estriol, and other estrogens have different potencies relative to estradiol in different species of mammals. There are slight differences in the amino acid sequences of the insulins and vasopressins from flifferent species and quite marked

differences in the chemical structure (Li and Papkoff, 1956) and physiologic activity (Knobil, Morse, Wolf and Greep, 1958) of the pituitar}^ growth hormones of cattle and swine, on the one hand, and of primates, on the other.
A. ESTROGENS
The amount or activity of certain enzymes in the target organs of estrogens has been found to vary with the amount of estrogen present. Examples of this phenomenon are /^-glucuronidase (Odell and Fishman, 1950) , fibrinolysin (Page, Glendening and Parkinson, 1951), and alkaline glycerophosphatase (Jones, Wade, and Goldberg, 1953). Kochakian (1947) reported that the amount of arginase in the rat kidney increased after the injection of estrogens. Enzyme activity is increased by other hormones as well; for example, progesterone has been found to increase the activity of phosphorylase (Zondek and Hestrin, 1947) and of adenosine triphosphatase (Jones, Wade, and Goldberg, 1952).
In most experiments the amount of enzyme present has been inferred from its activity, measured chemically or histochemically under conditions in which the amount of enzyme is rate-limiting. This does not enable one to distinguish between an actual increase in the number of molecules of enzyme present in the cell and an increase in the activity of the enzyme molecules without change in their number. A few enzymes can be measured by some other property, such as absorption at a specific wavelength, by which the actual amount of enzyme can be estimated (see review by Knox, Auerbach, and Lin, 1956). Knox and Auerbach (1955) found that the activity of the enzyme tryptophan peroxidase-oxidase (TPO) of the liver was decreased in adrenalectomized animals and increased by the administration of cortisone. Knox had shown previously that th(> administration of the substrate of the enzyme, tryptophan, would lead to an increase in the activity of the enzyme which was maximal in 6 to 10 hours. Evidence that the increased activity of enzyme following the administration of cortisone represents the synthesis of new protein molecules is supplied by experi

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ments in which it was found that the increase in enzyme activity is inhibited by ethionine and this inhibition is reversed by methionine. The amino acid analogue ethionine is known to inhibit protein synthesis and this inhibition of protein synthesis is overcome by methionine.
The injection of estrogen into the immature or castrate rodent produces a striking uptake of water by the uterus followed by a marked increase in its dry weight (Astwood, 1938). Holden (1939) postulated that the imbibition of water results from vasodilatation and from changes in the permeability of the blood vessels of the uterus. There is clear evidence (Mueller, 1957) that the subsequent increase in dry weight is due to an increased rate of synthesis of proteins and nucleic acids. The sex hormones and other steroids could be pictured as reacting with the protein or lipoprotein membrane around the cell or around some subcellular structure like a surface-wetting agent and in this way inducing a change in the permeability of the membrane. This might then increase the rate of entry of substances and thus alter the rate of metabolism within the cell. This theory could hardly account for the many notable specific relationships between steroid structure and biologic activity. Spaziani and Szego (1958) postulated that estrogens induce the release of histamine in the uterus and the histamine then alters the permeability of the blood vessels and produces the imbibition of water secondarily.
The uterus of the ovariectomized rat is remarkably responsive to estrogens and has been widely used as a test system. After ovariectomy, the content of ribonucleic acid of the uterus decreases to a low level and then is rapidly restored after injection of estradiol (Telfer, 1953). A single injection of 5 to 10 yu,g. of estradiol brings about (1) the hyperemia and water imbibition described previously; (2) an increased rate of over-all metabolism as reflected in increased utilization of oxygen (David, 1931; Khayyal and Scott, 1931; Kerly, 1937; MacLeod and Reynolds, 1938; Walaas, Walaas and Loken, 1952a; Roberts and Szego, 1953a) ; (3) an increased rate of glycolysis (Kerly, 1937; Carroll, 1942; Stuermer and Stein, 1952; Walaas, Walaas

and Loken, 1952b; Roberts and Szego, 1953a) ; (4) an increased rate of utilization of phosphorus (Grauer, Strickler, Wolken and Cutuly, 1950; Walaas and Walaas, 1950) ; and (5) tissue hypertrophy as reflected in increased dry weight (Astwood, 1938), increased content of ribonucleic acid and protein (Astwood, 1938; Telfer, 1953; Mueller, 1957), and finally, after about 72 hours, an increased content of desoxyribonucleic acid (Mueller, 1957).
An important series of experiments by Mueller and his colleagues revealed that estrogens injected in vivo affect the metabolism of the uterus which can be detected by subsequent incubation of the uterus in vitro with labeled substrate molecules. Mueller (1953) first showed that pretreatment with estradiol increases the rate of incorporation of glycine-2-C^'* into uterine protein. He then found that estrogen stimulation increases that rate of incorporation into protein of all other amino acids tested: alanine, serine, lysine, and tryptophan. The peak of stimulation occurred about 20 hours after the injection of estradiol. In further studies (Mueller and Herranen, 1956) it was found that estrogen increases the rate of incorporation of glycine-2-C^^ and formate-2-C^'* into protein, lipid, and the purine bases, adenine and guanine, of nucleic acids. A stimulation of cholesterol synthesis in the mouse uterus 20 hours after administration of estradiol was shown by Emmelot and Bos (1954).
In more detailed studies of the effects of estrogens on the metabolism of "one-carbon units" Herranen and Mueller (1956) found that the incorporation of serine-3-C^'* into adenine and guanine was stimulated by pretreatment with estradiol. The incorporation was greatly decreased when unlabeled formate was added to the reaction mixture to trap the one-carbon intermediate. In contrast, the incorporation of C^^02 into uridine and thymine by the surviving uterine segment was not increased by pretreatment with estradiol in vivo (Mueller, 1957).
To delineate further the site of estrogen effect on one-carbon metabohsm, Herranen and Mueller (1957) studied the effect of estrogen pretreatment on serine aldolase, the enzyme which catalyzes the equilibrium

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PHYSIOLOGY OF GONADS

between serine and glycine plus an active one-carbon unit. They found that serine aldolase activity, measured in homogenates of rat uteri, increased 18 hours after pretreatment in vivo with estradiol. It seemed that the estrogen-induced increase in the activity of this enzyme might explain at least part of the increased rate of onecarbon metabolism following estrogen injection. They found, however, that incubation of uterine segments in tissue culture medium (Eagle, 1955) for 18 hours produced a marked increase in both the activity of serine aldolase and the incorporation of glycine-2-C^'* into protein. The addition of estradiol to Eagle's medium did not produce a greater increase than the control to which no estradiol was added. Uterine segments taken from rats pretreated with estradiol for 18 hours, with their glycine-incorporating system activated by hormonal stimulation, showed very little further stimulation on being incubated in Eagle's medium for 18 hours. With a shorter period of i^retreatment with estradiol, greater stimulation occurred on subsequent incubation in tissue culture fluid. These experiments suggest that the hormone and the incubation in tissue culture medium are affecting the same process, one which has a limited capacity to respond. When comparable experiments were performed with other labeled amino acids as substrates, similar results were obtained.
Mueller's work gave evidence that a considerable number of enzyme systems in the uterus are accelerated by the administration of estradiol — not only the enzymes for the incorporation of serine, glycine, and formate into adenine and guanine, but also the enzymes involved in the synthesis of fatty acids and cholesterol and indejX'ndent enzymes for the activation of amino acids by the formation of adenosine monoiihosphate (AMP) derivatives. The initial step in protein synthesis has been shown to be the activation of the carboxyl grou]) of the amino acid with transfer of energy from ATP, the formation of AMP -"amino acid, and the release of jiyrophosphate (Hoagland, Keller and Zamecnick, 1956). This reversible step was studied with homogenates of uterine tissue, P^--labeled ]n'rni)liosi)liate, and a variety of amino

acids (Mueller, Herranen and Jervell, 1958). Seven of the amino acids tested, leucine, tryptophan, valine, tryosine, methionine, glycine, and isoleucine, stimulated the exchange of P^^ between pyrophosphate and ATP. Pretreatment of the uteri by estradiol injected in vivo increased the activity of these three enzymes. The activating effect of mixtures of these amino acids was the sum of their individual effects, from which it was inferred that a specific enzyme is involved in the activation of each amino acid. Since estrogen stimulated the exchange reaction with each of these seven amino acids, Mueller concluded that the hormone must affect the amount of each of the amino acid-activating enzvmes in the soluble fraction of the cell.
Mueller (1957) postulated that estrogens increase the rate of many enzyme systems both by activating preformed enzyme molecules and by increasing the rate of de novo synthesis of enzyme molecules, possibly by removing membranous barriers covering the templates for enzyme synthesis. To explain why estrogens affect these enzymes in the target organs, but not comparable enzymes in other tissues, one would have to assume that embryonic differentiation results in the formation of enzymes in different tissues which, although catalyzing the same reaction, have different properties such as their responsiveness to hormonal stimulation.
As an alternative hypothesis, estrogen might affect some reaction which provides a substance required for all of these enzyme reactions. The carboxyl group of amino acids must be activated by ATP before the amino acid can be incorporated into proteins; the synthesis of both purines and pyrimidines requires ATP for the activation of the carboxyl group of certain precursors and for several other steps; the synthesis of cholesterol requires ATP for the conversion of mevalonic acid to squalene; and the synthesis of fatty acids is also an energy-requiring process. Thus if (>strogens acted in some way to increase the amount of biologically useful energy, in the form of ATP or of energy-rich thioesters such as acetyl coenzyme A, it would increase the rate of synthesis of all of these compo

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nents of the cell. This would occur, of course, only if the supply of ATP, rather than the amount of enzyme, substrate, or some other cofactor, were the rate-limiting factor in the synthetic processes.
When purified estrogens became available, they were tested for their effects on tissues in vitro. Estrogens added in vitro increased the utilization of oxygen by the rat uterus (Khayyal and Scott, 1931) and the rat pituitary (Victor and Andersen, 1937). The addition of estradiol- 17^ at a level of 1 fxg. per ml. of incubation medium increased the rate of utilization of oxygen and of pyruvic acid by slices of human endometrium and increased the rate at which labeled glucose and pyruvate were oxidized to C^-^Os (Hagerman and Villee, 1952, 1953a, 1953b) . In experiments with slices of human placenta similar results were obtained and it was found that estradiol increased the rate of conversion of both pyruvate-2-C^'* and acetate-l-Ci4 to C^^Os (Villee and Hagerman, 1953) . From this and other evidence it was inferred that the estrogen acted at some point in the oxidative pathway common to pyruvate and acetate, i.e., in the tricarboxylic acid cycle.
Homogenates of placenta also respond to estradiol added in vitro. With citric acid as substrate, the utilization of citric acid and oxygen and the production of a-ketoglutaric acid were increased 50 per cent by the addition of estradiol to a final concentralion of 1 fjig. per ml. (Villee and Hagerman, 1953). The homogenates were separated by differential ultracentrifugation into nuclear, mitochondrial, microsomal, and nonparticulate fractions. The estrogen-stimulable system was shown to be in the nonparticulate fraction, the material which is not sedimented by centrifugating at 57,000 X g for 60 minutes (Villee, 1955). Experiments with citric, as-aconitic, isocitric, oxalosuccinic, and a-ketoglutaric acids as substrates and with fluorocitric and transaconitic acids as inhibitors localized the estrogen-sensitive system at the oxidation of isocitric to oxalosuccinic acid, which then undergoes spontaneous decarboxylation to a-ketoglutaric acid (Villee and Gordon, 1955). Further investigations using the enzymes of the nonparticulate fraction of the human placenta revealed that, in ad

dition to isocitric acid as substrate, only DPN and a divalent cation such as Mg+ + or Mn++ were required (Villee, 1955; Gordon and Villee, 1955; Villee and Gordon, 1956). The estrogen-sensitive reaction was formulated as a DPN-linked isocitric dehydrogenase:
Isocitrate + DPN* -^ a-ketoglutarate
+ CO2 + DPXH + H*
It was found that the effect of the hormone on the enzyme can be measured by the increased rate of disappearance of citric acid, the increased rate of appearance of a-ketoglutaric acid, or by the increased rate of reduction of DPN, measured spectrophotometrically by the optical density at 340 m/x. As little as 0.001 /xg. estradiol per ml. (4 X 10~^ m) produced a measurable increase in the rate of the reaction, and there was a graded response to increasing concentrations of estrogen. The dose-response curve is typically sigmoid. This system has been used to assay the estrogen content of extracts of urine (Gordon and Villee, 1956) and of tissues (Hagerman, Wellington and Villee, 1957; Loring and Villee, 1957).
Attempts to isolate and purify the estrogen-sensitive enzyme were not very successful. By a combination of low temperature alcohol fractionation and elution from calcium phosphate gel a 20-fold purification was obtained (Hagerman and Villee, 1957). However, as the enzyme was purified it was found that an additional cofactor was required. Either uridine triphosphate (UTP) or ATP added to the system greatly increased the magnitude of the estrogen effect and, subsequently, adenosine diphosphate (ADP) was recovered from the incubation medium and identified by paper chromatography (Villee and Hagerman, 1957). Talalay and Williams-Ashman (1958) confirmed our observations and showed that the additional cofactor was triphosphopyridine nucleotide (TPN) which was required in minute amounts. This finding was confirmed by Villee and Hagerman (1958) and the estrogen-sensitive enzyme system of the placenta is now believed to be a transhydrogenase which catalyzes the transfer of hydrogen ions and electrons

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PHYSIOLOGY OF GONADS

fromTPNHtoDPN:
TPXH + DPN^ -> DPNH + TPN^
The transhydrogenation system can be coupled to glucose 6-phosphate dehydrogenase as well as to isocitric dehydrogenase (Talalay and Williams-Ashman, 1958; Villee and Hagerman, 1958) and presumably can be coupled to any TPNH-generating system.
If the estrogen-stimulable transhydrogenation reaction were readily reversible, an enzyme such as lactic dehydrogenase which requires DPN should be stimulated by estrogen if supplied with substrate amounts of TPN, catalytic amounts of DPN, and a preparation from the placenta containing the transhydrogenase. Experiments to test this prediction were made using lactic dehydrogenase and alcohol dehydrogenase of both yeast and liver (Villee, 1958a). It was not possible to demonstrate an estrogen stimulation of either enzyme system in either the forward or the reverse direction. The stimulation of the lactic dehydrogenase-DPN oxidase system of the rat uterus by estrogens administered in vivo reported by Bever, Velardo and Hisaw (1956) might be explained by the stimulation of a transhydrogenase, but it has not yet been possible to demonstrate a coupling of this transhydrogenase and lactic dehydrogenase.
The stimulating effect of a number of steroids has been tested with a system in which the transhydrogenation reaction is coupled to isocitric dehydrogenase (Villee and Gordon, 1956; Hollander, Nolan and Hollander, 1958). Estrone, equilin, equilenin, and 6-ketoestradiol have activities essentially the same as that of estradiol17 j3. Samples of 1 -methyl estrone and 2methoxy-estrone had one-half the activitj of estradiol. Estriol is only weakly estrogenic in this system; 33 fig. estriol are less active than 0.1 fig. estradiol- 17/3 (Villee, 1957a). The activities of estriol and 16epiestriol are similar, whereas 16-oxoestradiol is more active than either, with about 10 per cent as much activitv as csti'adiol17/3.
Certain analogues of stilbestrol have been shown to be anti-estrogens in vivo. When applied topically to the vagina of the rat, they prevent the cornification normally in

duced by the administration of estrogen (Barany, Morsing, Muller, Stallberg, and Stenhagen, 1955). One of these, 1,3-di-phydroxyphenylpropane, was found to be strongly anti-estrogenic in the placental system in vitro: it prevented the acceleration of the transhydrogenase-isocitric dehydrogenase system normally produced by estradiol- 17/3 (Villee and Hagerman, 1957). The inhibitory power declines as the length of the carbon chain connecting the two phenolic rings is increased and 1 , 10-di-phydroxyphenyldecane had no inhibitory action. Similar inhibitions of the estradiolsensitive system were observed with stilbestrol, estradiol-17a, and a smaller antiestrogenic effect was found with estriol (Villee, 1957a). The inhibition induced by these compounds can be overcome by adding increased amounts of estradiol-17^. When stilbestrol is added alone at low concentration, 10~' M, it has a stimulatory effect equal to that of estradiol-17^ (Glass, Loring, Spencer and Villee, 1961).
The quantitative relations between the amounts of stimulator and inhibitor suggest that this inhibition is a competitive one. It was postulated that this phenomenon involves a competition between the steroids for specific binding sites on the estrogensensitive enzyme (Villee, 1957b; Hagerman and Villee, 1957). When added alone, estriol and stilbestrol are estrogenic and increase the rate of the estrogen-sensitive enzyme. In the presence of both estradiol and estriol, the total enzyme activity observed is the sum of that due to the enzyme combined with a potent activator, estradiol- 17^, and that due to the enzyme combined with a weak activator, estriol. When the concentration of estriol is increased, some of the estradiol is displaced from the enzyme and the total activity of the enzyme system is decreased.
Two hypotheses have been proposed for the mechanism of action of estrogens on the enzyme system of the placenta. One states that the estrogen combines with an inactive form of the enzyme and converts it to an active form (Hagerman and Villee, 1957). When this theory was formulated the evidence indicated that the estrogen acted on a specific DPN-linked isocitric dehydrogenase. The theory is equally applicable if the

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estrogen-sensitive enzyme is a transhydrogenase, as the evidence now indicates. The results of kinetic studies with the coupled isocitric dehydrogenase-transhydrogenase system are consistent with this theory (Gordon and Villee, 1955; Villee, 1957b; Hagerman and Villee, 1957). Apparent binding constants for the enzyme-hormone complex (Gordon and Villee, 1955j and for enzyme-inhibitor complexes have been calculated (Hagerman and Villee, 1957).
The observation that estradiol and estrone, which differ in structure only by a pair of hydrogen atoms, are equally effective in stimulating the reaction suggested that the steroid might be acting in some way as a hydrogen carrier from substrate to pyridine nucleotide (Gordon and Villee, 1956). Talalay and Williams-Ashman (1958) suggested that the estrogens act as coenzymes in the transhydrogenation reaction and postulated that the reactions were:
Estrone + TPNH + H*
— Estradiol + TPN^
Estradiol + DPN+
— Estrone + DPNH + H*

Sum : TPNH

H*

- DPN^ — TPN^ + H^

DPNH
This formulation implies that the estrogen-sensitive transhydrogenation reaction is catalyzed by the estradiol-17y3 dehydrogenase characterized by Langer and Engel (1956). This enzyme was shown by Langer (1957) to use either DPN or TPN as hydrogen acceptor but it reacts more rapidly with DPN. Ryan and Engel (1953) showed that this enzyme is present in rat liver, and in human adrenal, ileum, and liver. However, no estrogen-stimulable enzyme is demonstrable in rat or human liver (Villee, 1955). The nonparticulate fraction obtained by high speed centrifugation of homogenized rabbit liver rapidly converts estradiol to estrone if DPN is present as hydrogen acceptor, but does not contain any estrogenstimulable transhydrogenation system.
It will not be possible to choose between these two hypotheses until either the estrogen-sensitive transhydrogenase and the estradiol dehydrogenase have been separated or there is conclusive proof of their

identity. Talalay, Williams-Ashman and Hurlock (1958) reported a 100-fold purification of the dehydrogenase without separation of the transhydrogenase activity and found that both activities were inhibited identically by sulfhydryl inhibitors. In contrast, Hagerman and Villee (1958) obtained partial separation of the two activities by the usual techniques of protein fractionation, and reported that a 50 per cent inhibition of transhydrogenase is obtained with p-chloromercurisulfonic acid at a concentration of 10~^ m whereas 10"^ m p-chloromercurisulfonic acid is required for a 50 per cent inhibition of the dehydrogenase. The evidence that these two activities are mediated by separate and distinct proteins has been summarized by Villee, Hagerman and Joel (1960).
The transhydrogenase present in the mitochondrial membranes of heart muscle was shown by Ball and Cooper (1957) to be inhibited by 4 X 10"^ m thyroxine. The estrogen-sensitive transhydrogenase of the placenta is also inhibited by thyroxine (Villee, 1958b). The degree of inhibition is a function of the concentration of the thyroxine and the inhibition can be overcome by increased amounts of estrogen. Suitable control experiments show that thyroxine at this concentration does not inhibit the glucose 6-phosphate dehydrogenase or isocitric dehydrogenase used as TPNH-generating systems to couple with the transhydrogenase. Triiodothyronine also inhibits the estrogen-sensitive transhydrogenase but tyrosine, diiodotyrosine and thyronine do not. The thyroxine does not seem to be inhibiting by binding the divalent cation, Mn + + or ]Mg+ + , required for activity, for the inhibition is not overcome by increasing the concentration of the cation 10-fold.
In the intact animal estrogens stimulate the growth of the tissues of certain target organs. The estrogen-sensitive enzyme has been shown to be present in many of the target organs of estrogens: in human endometrium, myometrium, placenta, mammary gland, and mammary carcinoma, in rat ventral prostate gland and uterus, and in mammotrophic-dependent transplantable tumors of the rat and mouse pituitary. In contrast, it is not demonstrable in comparable preparations from liver, heart, lung, brain, or

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kidney. The growth of any tissue involves the utilization of energy, derived in large part from the oxidation of substrates, for the synthesis of new chemical bonds and for the reduction of substances involved in the synthesis of compounds such as fatty acids, cholesterol, purines, and pyrimidines.
The physiologic responses to estrogen action, such as water imbibition and protein and nucleic acid synthesis, are processes not directly dependent on the activity of transhydrogenase. However, all of these processes are endergonic, and one way of increasing their rate would be to increase the supply of biologically available energy by speeding up the Krebs tricarboxylic acid cycle and the flow of electrons through the electron transmitter system. Much of the oxidation of substrates by the cell produces TPNH, whereas the major fraction of the biologically useful energy of the cell comes from the oxidation of DPNH in the electron transmitter system of the cytochromes. Hormonal control of the rat of transfer of hydrogens from TPN to DPN could, at least in theory, influence the over-all rate of metabolism in the cell and secondarily influence the amount of energy available for synthetic processes. Direct evidence of this was shown in our early experiments in which the oxygen consumption of tissue slices of target organs was increased by the addition of estradiol (Hagerman and Villee, 1952; Villee and Hagerman, 1953).
This theory assumes that the supply of energy is rate-limiting for synthetic processes in these target tissues and that the activation of the estrogen-sensitive enzyme does produce a significant increase in the supply of energy. The addition of estradiol in vitro produces a significant increase in the total amount of isocitric acid dehydrogenated by the placenta (Villee, Loring and Sarner, 1958) . Slices of endometrium to which no estradiol was added in vitro utilized oxygen and metabolized substrates to carbon dioxide at rates which paralleled the levels of estradiol in the blood and urine of the patient from whom the endometrium was obtained (Hagerman and Villee, esses in these target tissues and that the 1953b). Estradiol increases the rate of synthesis of ATP by liomogenates of human

placenta (Villee, Joel, Loring and Spencer, 1960).
The reductive steps in the biosynthesis of steroids, fatty acids, purines, serine, and other substances generally require TPNH rather than DPNH as hydrogen donor. The cell ordinarily contains most of its TPN in the reduced state and most its DPN in the oxidized state (Glock and McLean, 1955). If the amount of TPN+ is ratelimiting, a transhydrogenase, by oxidizing TPN and reducing DPN, would permit further oxidation of substrates such as isocitric acid and glucose 6-phosphate, which require TPN+ as hydrogen acceptor and which are key reactions in the Krebs tricarboxylic acid cycle and the hexose monophosphate shunt, respectively. Furthermore, the experiments of Kaplan, Schwartz, Freeh and Ciotti (1956) indicate that less biologically useful energy, as ATP, is obtained when TPNH is oxidized by TPNH cytochrome c reductase than when DPNH is oxidized by DPNH cytochrome c reductase. Thus, a transhydrogenase, by transferring hydrogens from TPNH to DPN before oxidation in the cytochrome system, could increase the energy yield from a given amount of TPNH produced by isocitrate or glucose 6-phosphate oxidation. The increased amount of biologically useful energy could be used for growth, for protein and nucleic acid synthesis, for the imbibition of water, and for the other physiologic effects of estrogens.
Estrogen stimulation of the transhydrogenation reaction would tend to decrease rather than increase the amount of TPNH in the cell. Thus the estrogen-induced stimulation of the synthesis of steroids, fatty acids, proteins, and purines in the uterus can be explained more reasonably as due to an increased supply of energy rather than to an increased supply of TPNH.
The theory that estrogens stimulate transhydrogenation by acting as coenzymes which are rapidly and reversibly oxidized and reduced does not explain the pronounced estrogenic activity in vivo of stilbestrol, 17a-ethinyl estradiol, or bfsdehydrodoisynolic acid, for these substances do not contain groups that could be readily oxidized or reduced. The exact mechanism

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of action of estrogens at the biochemical level remains to be elucidated, but the data available permit the formulation of a detailed working hypothesis. The notable effects of estrogens and androgens on behavior (see chapter by Young) are presumably due to some direct or indirect effect of the hormone on the central nervous system. The explanation of these phenomena in physiologic and biochemical terms remains for future investigations to provide.
B. ANDROGENS
Although there is a considerable body of literature regarding the responses at the biologic level to administered androgens and progesterone, much less is known about the site and mechanism of action of these hormones than is known about the estrogens. The review by Roberts and Szego (1953b) deals especially with the synergistic and antagonistic interactions of the several steroidal sex hormones.
The rapid growth of the capon comb following the administration of testosterone has been shown to involve a pronounced increase in the amount of mucopolysaccharide present, as measured by the content of glucosamine (Ludwig and Boas, 1950; Schiller, Benditt and Dorfman, 1952). It is not known whether the androgen acts by increasing the amount or activity of one of the enzymes involved in the synthesis of polysaccharides or whether it increases the amount or availability of some requisite cofactor. Many of the other biologic effects of androgens do not seem to involve mucopolysaccharide synthesis and the relation of these observations to the other roles of androgens remains to he determined.
Mann and Parsons (1947) found that castration of rabbits resulted in a decreased concentration of fructose in the semen. Within 2 to 3 weeks after castration the amount of fructose in the semen dropped to zero, but rapidly returned to normal following the subcutaneous implantation of a pellet of testosterone. Fructose reappeared in the semen of the castrate rat 10 hours after the injection of 10 mg. of testosterone (Rudolph and Samuels, 1949). The coagulating gland of the rat, even when trans

planted to a new site in the body, also responds by producing fructose when the host is injected with testosterone. The amount of citric acid and ergothioneine in the semen is also decreased by castration and increased by the implantation of testosterone pellets (Mann, 1955). The experiments of Hers (1956) demonstrate that fructose is produced in the seminal vesicle by the reduction of glucose to sorbitol and the subsequent oxidation of sorbitol to fructose. The reduction of glucose requires TPNH as hydrogen donor and the oxidation of sorbitol requires DPN as hydrogen acceptor. The sum of these two reactions provides for the transfer of hydrogens from TPNH to DPN. If androgens act as cofactors which are reversibly oxidized and reduced, and thus transfer hydrogens from TPNH to DPN as postulated by Talalay and Williams-Ashman (1958), one would expect that an increased amount of androgen, by providing a competing system for hydrogen transfer, would decrease rather than increase the production of fructose. The marked increases in the citric acid and ergothioneine content of semen are not readily explained by this postulated site of action of androgens.
An increase in the activity of /3-glucuronidase in the kidney has been reported following the administration of androgens (Fishman, 1951). This might be interpreted as an arlaptive increase in enzyme induced by the increased concentration of substrate, or by a direct effect of the steroid on the synthesis of the enzyme.
The respiration of slices of prostate gland of the dog is decreased by castration or by the administration of stilbestrol (Barron and Huggins, 1944). The decrease in respiration occurs with either glucose or pyruvate as substrate. The seminal vesicle of the rat responds similarly to castration. Rudolph and Samuels (1949) found that respiration of slices of seminal vesicle is decreased by castration and restored to normal values within 10 hours after the injection of testosterone. Experiments by Dr. Phillip Corfman in our laboratory with slices of prostate gland from patients with benign prostatic hypertrophy showed that oxygen utilization was reduced 50 per cent by estradiol added

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in vitro at a level of 1 /xg. per ml. Respiration of slices of the ventral prostate gland of the rat is decreased by castration and increased by administered testosterone (Nyden and Williams-Ashman, 1953). These workers showed that lipogenesis from acetate-l-C^* in the prostate is also significantly diminished by castration and restored to normal by administered testosterone.
The succinic dehydrogenase of the liver has been found to be increased by castration and decreased by the administration of testosterone (Kalman, 1952; Rindani, 1958), the enzyme is also inhibited by testosterone added in vitro (Kalman, 1952). In contrast, Davis, Meyer and McShan (1949) found that the succinic dehydrogenase of the prostate and seminal vesicles is decreased by castration and increased by the administration of testosterone.
An interesting example of an androgen effect on a specific target organ is the decreased size of the levator ani and other perineal muscles of the rat following castration. The administration of androgen stimulates the growth of these muscles and increases their glycogen content (Leonard, 1952). However, their succinoxidase activity is unaffected by castration or by the administration of testosterone. Courrier and Marois (1952) reported that the growth of these muscles stimulated by androgen is inhibited by cortisone. The remarkable responsiveness of these muscles to androgens in vivo gave promise that slices or homogenates of this tissue incubated with androgens might yield clues as to the mode of action of the male sex hormones. Homogenates of perineal and masseter muscles of the rat responded to androgens administered in vivo with increased oxygen consumption and ATP production iLoring, Spencer and Villee, 1961). The experiments suggested that the activity of DPNH-cytochromo r reductase in these tissues is controlled by aiKh'ogeiis.
C. PROGESTERONE
Attempts to clarify the biochemical basis of the role of progesterone have been hampered by the requirement, in most instances, for a previous stimulation of the tissue by estrogen. The work of Wade and Jones

(1956a, b) demonstrated an interesting effect of progesterone added in vitro on several aspects of metabolism in rat liver mitochondria. Progesterone, but not estradiol, testosterone, 17a-hydroxyprogesterone, or any of several other steroids tested, stimulated the adenosine triphosphatase activity of rat liver mitochondria. This stimulation is not the result of an increased permeability of the mitochondrial membrane induced by progesterone, for the stimulatory effect is also demonstrable with mitochondria that have been repeatedly frozen and thawed to break the membranes. Other experiments showed that ATP was the only substrate effective in this system ; progesterone did not activate the release of inorganic phosphate from AMP, ADP, or glycerophosphate.
In other experiments with rat liver mitochondria (Wade and Jones, 1956b), progesterone at a higher concentration (6 X lO"'* m) was found to inhibit the utilization of oxygen with one of the tricarboxylic acids or with DPNH as substrate. This inhibition is less specific and occurred with estradiol, testosterone, pregnanediol, and 17a-hydroxy progesterone, as well as with progesterone. The inhibition of respiration by high concentrations of steroids in vitro has been reported many times and with several different tissues; it seems to be relatively unspecific. Wade and Jones were able to show that progesterone inhibits the reduction of cytochrome c but accelerates the oxidation of ascorbic acid. They concluded that progesterone may perhaps uncouple oxidation from phosphorylation in a manner similar to that postulated for dinitrophenol. The site of action of this uncoupling appears to be in the oxidation-reduction path between DPNH and cytochrome c. Mueller (1953) found that progesterone added in vitro decreases the incorporation of glycine-2-C^'* into the protein of strips of rat uterus, thus counteracting the stimulatory effect of estradiol administered in vivo.
Zander (1958) reported that A4-3-ketopregnene-20-a-ol and A4-3-ketopregnene20-^-ol arc effective gestational hormones in the mouse, rabbit, and man, although somewhat less active in general than is progesterone. An enzyme in rat ovary which converts progesterone to pregnene-20-a-ol, and also catalyzes the reverse reaction, was

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described by Wiest (1956). The conversion occurred when slices of ovary were incubated with DPN. Wiest postulated that the progesterone-pregnene-20-a-ol system might play a role in hydrogen transfer, in a manner analogous to that postulated by Talalay and Williams-Ashman (1958) for estrone-estradiol- 17^, but his subsequent experiments ruled out this possibility, for he was unable to demonstrate any progesterone-stimulable transhydrogenation reaction.
The nature of the effect of progesterone and of estrogens on myometrium has been investigated extensively by Csapo. Csapo and Corner (1952, 1953) found that ovariectomy decreased the maximal tension of the myometrium and decreased its content of actomyosin. The administration of estradiol to the ovariectomized rabbit over a period of 7 days restored both the actomyosin content and the maximal tension of the myometrium to normal. The concentration of ATP and of creatine phosphate in the myometrium is decreased by ovariectomy but is restored by only 2 days of estrogen treatment. This suggests that the effect on intermediary metabolism occurs before the effect on protein {i.e., actomyosin) synthesis. Csapo (1956a) concluded that estrogen is a limiting substance in the synthesis of the contractile proteins of myometrium, but he could not differentiate between an effect of estrogen on some particular biosynthetic reaction and an effect of estrogen on some fundamental reaction which favors synthesis in general. He was unable to demonstrate any comparable effect of progesterone on the contractile actomyosin-ATP system of the myometrium.
Other observations provide an explanation for the well known effect of progesterone in decreasing the contractile activity of myometrium, not by any effect on the contractile system itself, but in some previous step in the excitation process. Under the domination of progesterone the myometrial cells have a decreased intracellular concentration of potassium ions and an increased concentration of sodium ions (Horvath, 1954). The change in ionic gradient across the cell membrane is believed to be responsible for the altered resting potential and the partial depolarization of the cell mem

brane which results in decreased conductivity and decreased pharmacologic reactivity of the myometrial cell. The means by which progesterone produces the changes in ionic gradients is as yet unknown. Csapo postulates that the hormone might decrease the rate of metabolism which in turn would lessen the rate of the "sodium pump" of the cell membrane. The contractile elements, the actomyosin-ATP system, are capable of full contraction but, because of the partial block in the mechanism of excitation and of propagation of impulses (Csapo, 1956b), the muscle cells cannot operate effectively; the contractile activity remains localized. Csapo (1956a) showed that the progesterone block is quickly reversible and disappears if progesterone is withdrawn for 24 hours. He concluded that the progesterone block is necessary for the continuation of pregnancy and that its withdrawal is responsible for the onset of labor.
]\Iost investigators who have speculated about the mode of action of steroids — whether they believe the effect is by activating an enzyme, by altering the permeability of a membrane, or by serving as a coenzyme in a given reaction— have emphasized the physical binding of the steroid to a protein as an essential part of the mechanism of action or a preliminary step to that action. They have in this way explained the specificities, synergisms, and antagonisms of the several steroids in terms of the formation of specific steroid-protein complexes. The differences between different target organs, e.g., those that respond to androgens and those that respond to estrogens, can be attributed to differences in the distribution of the specific proteins involved in these binding reactions. Viewed in this light, the problem of the mode of action of sex hormones becomes one aspect of the larger problem of the biochemical basis of embryonic differentiation of tissues.
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ViLLEE, C. A., Loring, J. M.. and Sarner, A. 1958.

Isocitric dehydrogenases of the placenta. Fed Proc, 17, 328.
Wade, R., and Jones, H. W., Jr. 1956a. Effect of progesterone on mitochondrial adenosinetriphosphatase. J. Biol. Chem., 220, 547-551.
Wade, R., and Jones, H. W., Jr. 1956b. Effect of progesterone on oxidative phosphorylation. J. Biol. Chem.. 220, 553-562.
Walaas, O., and Walaas, E. 1950. The metabolism of uterine muscle studied with radioactive phosphorus P^". Acta physiol. scandinav 21, 18-26.
Wal-aas, O.. Wal.aas, E., .\nd Loken, F. 1952a. The effect of estradiol monobenzoate on the metaboli-sin of rat uterine muscle. Acta endocrinol., 10,201-211.
Wal.'^as, 0., Walaas, E., and Loken, F. 1952b. The effect of estradiol monobenzoate on the metabolism of the rat endometrium. Acta endocrinol., 11, 61-66.
Werbin, H. and LeRoy, G. V. 1954. Cholesterol: a precursor of tetrahydrocortisone in man. J. Am. Chem. Soc, 76, 5260-5261.
West. C. D., Damast, B. L., Sarro, S. D., and Pearson, 0. H. 1956. Conversion of testosterone to estrogens in castrated, adrenalectomized human females. J. Biol. Chem., 218, 409-418.
WiEST, W. G. 1956. The metabolism of progesterone to A4-pregnen-20a-ol-3-one in eviscerated female rats. J. Biol. Chem., 221, 461467.
Zander, J. 1958. Gestagens in human pregnancy. In Proceedings Conference on Endocrinology of Reproduction, C. W. Lloyd, Ed. New York: Academic Press, Inc.
Zondek, B., and Hestrin, S. 1947. Phosphorylase activity in human endometrium. Am. J. Obst. ct Gvnec, 54, 173-175.

NUTRITIONAL EFFECTS ON ENDOCRINE SECRETIONS
James H. Leathern, Ph.D.
PROFESSOR OF ZOOLOGY, RUTGERS, THE STATE UNIVERSITY, NEW BRUNSWICK, NEW JERSEY

I. Introduction' 666 I, Introduction
II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t ,
Studies 668 Despite the accumulation of many data
A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology
production ()68 during the past 20 years and the long es B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in C. Diabetef^Mellitus, Nutrition, and ^f^f on fertility and fecundity, knowl Reproduction ()72 edge bearing on nutrition and the endocrine
D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad E. Diet and the Liver 673 vanced comparatively slowly. However, re III. Hypophysis and Diet 674 markable advances have been made in each
A. Inanition 674 speciality SO that nutritional-endocrine
^roein. .. w problems should continue to be a fruitful
C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii
D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to
IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to
A. Testis 677 the coherence one would prefer to present
1. Inanition 677 now.
2. Protein 678 'y\^q endocrinologist appreciates the deli 4 Vitamins (i8() ^^^^ balance which exists between the hy B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi siveness of Male Reproductive Tis- lar interdependence exists between nutrition
sues to Hormones 681 and the endocrine glands, including those
1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does
2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -•
V. Female Reproductive System 683 nutrition influence synthesis and release of
A. Ovaries 683 hormones, but hormones in turn, through
1. Inanition 683 their regulation of the metabolism of pro 2. Protein 684 teins, carbohydrates, and fats, influence nu 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create
5 Vitamins 685 endocrine imbalance, and endocrine imbal B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac siveness of Female Pe])ro<luct ive tors. It follows, therefore, that, in any conTissues to Hormones 687 sideration of this interrelationship, one must
1 . Ovary ... 687 consider not only undernutrition and lack of
2. uterus and vagina 688 -n c j \ l ^ i ^ cc i. r
3. Mammary gland 689 specific foods, but also possible effects of
C. Pregnancy. . 689 antithyroid substanccs in foods, antimetab VI. Concluding Remarks 693 olites, and overnutrition, especially for the
VII.Pkferencks (;!)4 child (Forbes, 1957).
666

NUTRITIONAL EFFECTS

667

Our understanding of the means by which hormones exert their effects is relatively slight, as is our knowledge of the biochemical mechanisms by w^hich supplements and deficiencies of vitamins and amino acids influence hormone action. Nevertheless, support for the statement that modifications of nutrition influence endocrine gland secretions or hormone action on distant target organs or tissues is provided by an enumeration of a few basic cell components requiring proteins, lipids, and vitamins. (1) Proteins combine with lipids to form lipoproteins which are essential features of the internal and external cellular membranes and interfaces. Hormones, as well as nutrition, influence cell membranes and therefore cell transport is affected. It is well known that hormones influence electrolyte and carbohydrate transfer and recently an endocrine control of amino acid transport was demonstrated (Noall, Riggs, Walker and Christensen, 1957). The effect of modifications of nutrition on the capacity of hormones to influence cell transport must await study. (2) Enzymes are proteins with chemically active surfaces and often include nonprotein groups such as vitamins. Nutritional and hormonal changes cause alterations in enzyme concentrations (Knox, Auerbach and Lin, 1956). Vitamin, mineral, and fat deficiencies favor a decrease in enzymes, whereas protein deficiencies have varied effects (Van Pilsum, Speyer and Samuels, 1957). Enzyme changes caused by hormones appear to be a consequence of metabolic adaptations. The importance of a nutritional base on which a hormone can express an effect on the enzymes of the reproductive organs can only be determined after further data have been obtained. (3) Proteins combined with nucleic acids become nucleoproteins, some of which are organized in the cytoplasm and may be templates for cellular protein synthesis. Other nucleoproteins are contained in the nucleus. Nutrition and hormones influence tissue nucleoproteins but studies involving the reproductive organs are few. How^ever, one possible cause of human infertility is low desoxyribose nucleic acid in the sperm ("Weir and Leuchtenberger, 1957).
Proteins are characteristic components of

tissues and hypophyseal hormones are protein in nature; also the major portion of gonadal dry weight is protein. Such being the case, it is important to appreciate that the protein composition of the body is in a dynamic state and that proteins from the tissues and from the diet contribute to a common metabolic pool of nitrogen. This metabolic pool contains amino acids which may be withdrawn for rebuilding tissue protein and for the formation of new protein for growth. Obviously, the character of the metabolic pool of nitrogen reflects dietary protein level and quality. A food protein which is deficient in one or more amino acids will restrict tissue protein synthesis. Hormones also influence the metabolic pool by affecting appetite as well as absorption, utilization, and excretion of foods, and thus hormones could accentuate the effect of a poor diet, or create demands beyond those normally met by an adequate diet. In addition a study of the tissues and organs of the body reveals that contributions to the metabolic pool are not uniform, thus one tissue or organ may be maintained at the expense of another. In protein deprivation in adults the liver quickly contributes increased amounts of nitrogen to the metabolic pool whereas the testis does not. On the other hand, protein contributions to the nitrogen pool by the hypophysis, and the amino acid withdrawals needed for hormone synthesis, are unknown. Data suggesting that the addition of specific nutrients to diets improves hypophyseal hormone synthesis have been presented (Leathem, 1958a).
In 1939 jNIason rightfully emphasized the need for vitamins in reproduction. Since then, much additional knowledge has been obtained. Vitamins of the B complex have been more clearly identified and a better understanding of their function has been gained. Thiamine is important for carbohydrate metabolism, pyridoxine for fat metabolism, and the conversion of tryptophan to nicotinic acid, and vitamin B12 may be involved in protein synthesis. In addition, vitamins have been found to serve as coenzymes, and folic acid to be important for estrogen action on the uterus. Tliosr. and

668

PHYSIOLOGY OF GONADS

many other findings prompt a survey of the relationships of vitamins to reproduction.
It is the intention of the author to review enough of the evidence which interrelates nutrition and reproduction to create an awareness of the problems in the area. Reviews dealing with the general subject of hormonal-nutritional interrelationships have been presented (Hertz, 1948; Samuels, 1948; Ershoff, 1952; Zubiran and GomezMont, 1953; Meites, Feng and Wilwerth, 1957; Leathem, 1958a,). Other reviews have related reproduction to nutrition with emphasis on laboratory (Mason, 1939; Guilbert, 1942; Lutwak-Mann, 1958) and farm animals (Reid, 1949; Asdell, 1949), and on protein nutrition (Leathem, 1959b, c). An encyclopedic survey of the biology of human nutrition has been made by Keys, Brozec, Hernschel, Michelsen and Taylor (1950).
II. Nature of Problems in Nutritional Studies
A. THYEOID GLAND, NUTRITION, AND REPRODUCTION
Normal development of the reproductive organs and their proper functioning in
TABLE 12.1 Ovarian response to chorionic gonadrotrophin,
as modified by thiouracil and diet
(From J. H. Leathem, in Recent Progress in
the Endocrinology of Reproduction, Academic
Press, Inc., New York, 1959.)

Diet

Ovarian Weight

Cholesterol

Total

Free

18 per cent casein .
18 tier cent +
I'hiouracil
per cent casein . . per cent + thiouracil
18 per cent gelatin . . .

mg.
87 342
59 133
59 186 27.5

% 0.41
0.20
0.47
0.22
0.66 0.26 1.56

0.18 0.12 0.23 0.15
0.22

18 per cent -f thio uracil

0.18 18

Chorionic gonadotrophin = 10 I. U. X 20 days.

adults are dependent not only on the endocrine glands composing the hypophysealgonadal axis, but on others as well. The importance of the thyroid, although not the same for all species, is readily apparent from the effects of prolonged hypo- and hyperthyroid states on reproduction. Many of these effects have been enumerated elsewhere (chapters by Albert, by Young on the ovary, and by Zarrow) , but others also are important. Thus steroid production may be altered in hypothyroid animals; certainly its metabolism is influenced. Myxedema is associated with a profound change in androgen metabolism. Endogenous production of androsterone is very low and subnormal amounts of administered testosterone are converted to androsterone. Triiodothyronine corrects this defect (Hellman, Bradlow, Zumoff, Fukushima and Gallagher, 1959). The gonads of male and female offspring of cretin rats are subnormal. The testes may contain a few spermatocytes but no spermatozoa, and Leydig cells seem to secrete little or no androgen. The ovaries may contain a few small follicles with antra, but corpora lutea are absent and ovarian lipid and cholesterol concentrations are very low. Nevertheless, the gonads are competent to respond to administered gonadotrophin with a marked increase in weight. However, administration of chorionic gonadotrophin to the hypothyroid rat stimulated follicular cyst formation rather than folliculogenesis and corpora lutea formation (Leathem, 1958b), but, for even this aberrant development, dietary protein was required (Leathem, 1959b) (Table 12.1). The relationship between hypothyroidism and ovarian function may provide a clue to a possible origin of ovarian cysts, long known to be a common cause of infertility and associated reproductive disorders. Clinical cases of untreated myxedema exhibit ovarian cysts, and rats made hypothyroid for eight months, had a higher percentage of cystic ovaries than did euthyroid rats (Janes, 1944).
The reproductive system of the adult male is less affected than that of the immature male by a decrease in thyroid function, just as the testis of the adult is less likely to reflect a change in protein nutrition which is sufficient to alter the immature rat

NUTRITIONAL EFFECTS

669

testis. On the other hand, the lack of demonstrable thyroid dysfunction in the adult male does not exclude the possibility of an effect of thyroid hormone on reproduction. The conversion of thyroxine to triiodothyronine may be hindered (Morton, 1958) . Thyroxine was found to decrease the number of active cells in the semen and to reduce motility, whereas triiodothyronine increased the number of active spermatozoa (Farris and Colton, 1958; Reed, Browning and O'Donnell, 1958j. Small dosages of thyroxine stimulated spermatogenesis in the mouse, rabbit, and ram (Maqsood, 1952) and were beneficial in normal guinea pigs and rats (Richter and Winter, 1947; Young, Rayner, Peterson and Brown, 1952).
In many species reproduction occurs despite hypothyroidism, but fecundity may be subnormal (Peterson, Webster, Rayner and Young, 1952). Feeding an antithyroid drug, thiouracil, to female rats may or may not prevent pregnancy, but it reduces the number of young per litter. Thiouracil feeding continued through lactation will decrease litter size (Leathem, 1959b). Hypothyroid guinea pigs gave birth to some live young, but the percentage approached normal only when thyroxine was administered (Hoar, Goy and Young, 1957). Pregnant euthyroid animals responded to thyroxine by delivering more living young than normal control pigs (Peterson, Webster, Ravner and Young, 1952) . The extent to which such effects are consequences of the reduction in appetite, metabolism, and absorption of food from the gut which are associated with hypothyroidism has not been determined.
Hyperthyroidism, on the other hand, will increase the appetite and enhance absorption of food from the gut, as the increased metabolism requires more calories, minerals, vitamins, choline, and methionine. In adult rats hyperthyroidism induces a marked loss in body fat and accelerates protein catabolism. The two effects, if unchecked, result in loss of body weight and death. In immature males hyperthyroidism slows gain in body weight, retards testis growth and maturation, and abolishes androgen secretion (Table 12.2). Altering dietary protein in adult animals failed to modify the thyroid hormone effects, thereby suggesting

TABLE 12.2
Effects of diet and thyroid {0.2 'per
cent) on immature rats
(From J. H. Leathem, in Recent Progress in
the Endocrinology of Reproduction, Academic
Press, Inc., New York, 1959.)

Diet

Testis Weight

Seminal

(Casein) X 30 Days

Actual

Relative

Vesicles

20 per cent
20 per cent + thyroid
6 per cent
6 per cent + thyroid
per cent
per cent -|thyroid

mg. 1G94
1090
825
245
140
95

mg./lOOgm.
1035
881 1232
650
346
261

mg. 88
9 16
7 7 6

that the metabolic demands of other tissues were making increased withdrawals from the metabolic pool of nitrogen and thus hindering testis growth. In euthyroid rats, a 6 per cent protein diet will permit testis growth in the absence of a gain in body weight, but hyperthyroidism prevents this preferential effect. Although Moore (1939) considered the effect of thyroid hormone on reproduction as possibly due to general body emaciation, the testis seems to be less responsive than the body as a whole. Adult rats fed 0.2 per cent desiccated thyroid exhibited no correlation between loss of body weight, change in testis weight, or protein composition of testis at two levels of casein and lactalbumin (Leathem, 1959b) . The testes were seemingly not influenced by the metabolic nitrogen changes which caused a loss in carcass nitrogen and an associated increase in kidney and heart nitrogen.
The mechanism of thyroid hormone action on reproduction is far from clear. As we have noted, a part of its action may be through the regulation of nutritional processes. Thyroid function is influenced by the biologic value of the dietary protein (Leathem, 1958a) and the specifie amino acids fed (Samuels, 1953). In turn, an altered thyroid function will influence the nitrogen contributions to the metabolic pool by reducing appetite and absorption from

670

PHYSIOLOGY OF GONADS

the gut, and by changing the contributions of nitrogen to the metabolic pool made by the body tissues. Hypothyroidism interferes with the refilling of body protein stores; consequently, protein needs of the reproductive organs may not be fully met (Leathern, 1953). It is consistent with this opinion that testis recovery from protein deprivation in hypothyroid rats was aided by thyroxine treatment (Horn, 1955).
Conversion of carotene to vitamin A may be prevented by hypothyroidism, suggesting that a subnormal amount of this vitamin may contribute to fetal loss. An increased intake of B vitamins might be required as hypothyroidism aggravates a vitamin B12 deficiency, and increased intake of B vitamins enhances the capacity of young rats to withstand large doses of thiouracil (Meites, 1953). Although a reduced metabolic rate might seemingly reduce vitamin requirements, more efficient metabolic activities, in the absence of hormonal stimuli, seem to occur when vitamin intake is increased (Meites, Feng and Wilwerth, 1957).
Reproduction is influenced by effects which are the opposite of a number of those just cited, i.e., effects of malnutrition on thyroid function. The need for iodine in the prevention of goiter is well known. However, certain foods prevent the utilization of iodine in the synthesis of thyroid hormone. The foods containing an antithyroid or goitrogenic agent, as tested in man, include rutabaga, cabbage, brussels sprouts, cauliflower, turnip, rape, kale, and to a lesser extent peach, pear, strawberry, spinach, and carrot (Greer and Astwood, 1948). A potent goitrogen isolated from rutabaga is L-5-vinyl-2-thiooxazolidine ((ireer, 1950, 1956). Reduced food intake will decrease the thyroid gland response to goitrogens (Gomez-Mont, Paschkis and Cantarow, 1947; Meites and Agrawala, 1949), the uptake of I^^^ (Meites, 1953), and the level of thyrotrophic hormone in laboratory animals (D'Angelo, 1951). The thyroid changes associated with malnutrition in man arc uncertain (Zubiran and Gomez-Mont, 1953). However, a decreased functioning of the gland in anorexia nervosa, followed by an increased functioning on refeeding (Poiioff, Lasche, Nodine,

Schneeberg and \'ieillard, 1954), is suggestive of a direct nutritional need.
Changes in the thyrotrophic potency of the rat hypophysis have been observed in various vitamin deficiencies. Thiamine deficiency may increase thyroid function, but vitamin A deficiency may have the opposite effect (Ershoff, 1952). The difficulties inherent in the assay of thyroid-stimulating hormone (TSH) , even by current methods, prevent one from drawing definite conclusions from the available data.
Immature animals given thyroxine are retarded in growth and do not survive. However, increasing dietary thiamine, pyridoxine, or vitamin B12 improves the ability of the young rat to withstand large dosages of thyroid substances (Meites, 1952), as does methionine (Boldt, Harper and Elvehjem, 1958). Consideration must also be given to the need for nutritional factors which play a minor role in normal metabolic states but increase in importance in stress. Thus yeast and whole liver contain antithyrotoxic substances (Drill, 1943; Ershoff, 1952; Overby, Frost and Fredrickson, 1959).
Excessive thyroid hormone will prevent maturation of the ovary and in adult rats will cause ovarian atrophy with a cessation of estrous cycles. Addition of yeast to the diet permitted estrous cycles to continue (Drill, 1943), but gonadal inhibition in the immature animal was not ]3revented. However, whole liver or its w^ater-insoluble fraction counteracted the gonadal inhibition induced by hyperthyrodism in immature rats (Ershoff, 1952). Biochemical mechanisms by which these dietary supplements can benefit rats given excessive quantities of hormone are unknown.
B. .\DREXAL GLAND, NUTRITION, AND REPRODUCTION
The problem of the relationship between adrenal steroid secretions and the reproductive system is one that still requires clarification. Furthermore, the possible influences of nutrition can only be inferred from the effects of adrenal steroids on the major metabolic systems of the body.
In the female there is a close relationshiji between the adrenal and the estrous and

NUTRITIONAL EFFECTS

iul

menstrual cycles (Zuckerman, 1953; chapter by Young on the ovary). The ovary would seem to require cortical steroids for the normal functioning of its own metabolic processes and for those which it influences peripherally. The Addisonian patient may show ovarian follicular atresia and a loss of secondary sex characteristics, and the untreated adrenalectomized rat exhibits a decrease in ovarian size and has irregular cycles (Chester Jones, 1957). The decline in size of the ovary after adrenalectomy is due to impaired sensitivity to folliclestimulating hormone (FSH) rather than to a decreased production of FSH, and the ovarian response is corrected by cortisone (Mandl, 1954).
Reproductive potential is not necessarily lost when there is adrenal insufficiency, but pregnancy is not well tolerated by women with Addison's disease. Furthermore, adrenalectomy in rats at the time of mating or 4 to 6 days after mating resulted in abortion. Improved pregnancy maintenance was obtained in adrenalectomized rats given saline or cortisone acetate (Davis and Plotz, 1954) , whereas desoxycorticosterone acetate alone extended the pregnancy period beyond normal time (Houssay, 1945). Essentially normal pregnancies were obtained in adrenalectomized rats given both cortisone acetate and desoxycorticosterone acetate (Cupps, 1955). Substitution of cortisone acetate for adrenal secretions may be incomplete because the adrenal hormone in the normal rat is primarily corticosterone and because cortisone enhances the excretion of certain amino acids and vitamins. It would be interesting to test a diet with a high vitamin content on the capacity of an adrenalectomized rat to maintain pregnancy, because improved survival of operated rats is obtained by giving vitamin Bio (Meites, 1953) or large doses of pantothenic acid, biotin, ascorbic acid, or folic acid (Ralli and Dumm, 1952; Dumm and Ralli, 1953).
An adrenal influence over protein metabolism is well known, but protein nutrition, in turn, can influence cortical steroid effectiveness. In fact, an extension of the life span of adrenalectomized rats is not obtained with adrenal steroids if the diet

lacks protein (Leathern, 1958a). A low protein diet alone will not improve survival after adrenalectomy, but better survival is obtained when the rats are given saline. When the low protein diet was supplemented with methionine, a definite improvement in life span was observed and the possibility that cortisone exerts its effect by drawing on the carcass for methionine was suggested (Aschkenasy, 1955a, b).
Reducing dietary casein to 2 per cent seriously endangers pregnancy in the rat, but the addition of progesterone permits 80 per cent of the pregnancies to be maintained. However, removal of the adrenal glands counteracts the protective action of the progesterone, only 10 per cent of the pregnancies continuing to term. Addition of methionine to the low casein diet improved pregnancy maintenance, but 1 mg. cortisone acetate plus progesterone provided the best results (Aschkenasy-Lelu and Aschkenasy, 1957; Aschkenasy and Aschkenasy-Lelu, 1957). These data emphasize the importance of nutrition in obtaining an anticipated hormone action. Further investigation might be directed toward the study of whole proteins other than casein, for the biologic value of proteins differs from normal when tested in adrenalectomized rats (Leathem, 1958a) .
As Albert has noted in his chapter, adrenalectomy has little or no effect on the testis. Gaunt and Parkins (1933) found no degenerative changes in the testes of adult rats dying of adrenal insufficiency, although an increase in the testis : body-weight ratios was noted in rats fed 18 per cent and 4 per cent protein (Aschkenasy, 1955c). If adrenalectomized rats are kept on a maintenance dosage of cortisone acetate for 20 days and fed dietary proteins of different biologic values, one finds that testis-composition of protein, lipid, and glycogen varies in the same manner as in the normal rat (Wolf and Leathem, 1955) (Table 12.3).
When the adrenal glands are intact, the influence of diet on their functional capacity and indeed on the hypophyseal- adrenal axis must be considered. Zubiran and Gomez-]\Iont (1953) showed that patients exhibiting gonadal changes associated with chronic malnutrition also exhibit adrenal

672

PHYSIOLOGY OF GOXADS

TABLE 12.3
Nutritional effects on the testes of cortisonemaintained adrenalectomized rats (From R. C. Wolf and J. H. Leathern, Endocrinology, 57, 286, 1955.)

Testes Composition

(per cent)

Treatment

Diet

Testes

Protein

Lipid

Glycogen

mg.

Corti
20 per cent

703

68.5

30.4

0.13

sone

casein

ace

tate

Control

1211

70.5

29.4

0.15

Corti
20 per cent

808

61.0

31.8

0.17

sone

wheat

ace
ghiten

tate

Control

816

62.3

32.2

0.17

Corti
20 per cent

1014

64.3

31.3

0.17

sone

peanut

ace
flour

tate

Control

699

68.5

31.3

0.12

hypofimction. Several clinical tests permitted the evaluation of subnormal adrenal function which, however, did not reach Addisonian levels. Malnutrition not onlyreduced hypophyseal adrenocorticotrophic hormone (ACTH), but also prevented an incomplete response by the adrenal glands to injected ACTH. In laboratory rodents, anterior hypophyseal function is also influenced by dietary protein and vitamin levels (Ershoff, 1952). The importance of dietary protein in the hypophyseal-adrenal system has recently been re-emphasized (Leathem, 1957; Goth, Nadashi and Slader, 1958). Furtiiermore, adrenal cortical function is affected by vitamin deficiencies (Morgan, 1951), from which it appears that pantothenic acid is essential for cortical hormone elaboration (Eisenstcin, 1957). Administration of excessive amounts of cortical steroids can induce morphologic changes which have been compared to inanition (Baker, 1952). Not only is nitrogen loss enhanced, but hyperglycemia can also be induced which, therefore, increases the need for thiamine. Cortical steroids influence the metabolism of various vitamins

(Draper and Johnson, 1953; Dhyse, Fisher, Tullner and Hertz, 1953; Aceto, Li Moli and Panebianco, 1956; Ginoulhiac and Nani, 1956). H a vitamin deficiency already exists, administration of cortisone will aggravate the condition (Meites, Feng and Wilwerth, 1957). Nevertheless, drastic effects of therapeutic doses of cortisone on reproductive function do not occur. In rare cases loss of libido has been reported in the male, but mori)hologic changes in the testis were not observed (JVladdock, Chase and Nelson, 1953). Cortisone has little if any effect on the weight of the rat testis (Moore, 1953; Aschkenasy, 1957) and does not influence testis cholesterol (Migeon, 1952). In the female, menstrual disturbances have been noted in association with cortisone therapy, with the occurrence of hot flashes (Ward, Slocumb, Policy, Lowman and Hench, 1951). However, cortisone corrected disturbances during the follicular phase, possibly by increasing FSH release (Jones, Howard and Langford, 1953). Cortisone also increased the number of follicles in the ovary of the rat (Moore, 1953), but not in the rabbit. Cortisone administration did not prevent the enhanced ovarian response to chorionic gonadotrophin seen in hypothyroid rats (Leathem, 1958b) and had little effect on mice in parabiosis ( Noumura, 1956).
In pregnant female rabbits resorption and stunting of fetuses occurred during treatment with large doses of cortisone (Courrier and Collonge, 1951). Similar effects were noted in mice (LcRoy and Domm, 1951 ; Robson and Sharaf, 1951).
Some of the metabolic derangements of human toxemia of pregnancy have been correlated with accelerated secretion of adrenal steroids creating a steroid imbalance (see chapter by Zarrow). Cortisone is reported to have a beneficial effect on some cases (Moore, Jessop, 'Donovan, Barry, Quinn and Drury, 1951). Protein inade({uacies may also be etiologic in toxemia and further (>xamination of the possibility sliould he made.
C. DIABETES MELLITUS, NUTRITION, AND REPRODUCTION
(llycosuria can be induced experimentally by starvation, overfeeding, and shifting

NUTRITIONAL EFFECTS

673

diet^ from one of high fat content to one i; which is isocaloric but high in carbohydrate (Ingle, 1948). Force feeding a high carbohydrate diet will eventually kill a rat despite insulin administration aimed at controlling glycosuria (Ingle and Nezamis, 1947). In man excessive eating leading to obesity increases insulin demand and, in many diabetics of middle age, obesity precedes the onset of diabetes. With our present knowledge we must conclude that overfeeding is wrong when glycosuria exists and that vitamin B supplements may be of value in diabetes (Meites, Feng and Wilwerth, 1957; Salvesen, 1957).
In man urinary 17-ketosteroids and androgen levels are subnormal in diabetes (Horstmann, 1950), and in the diabetic rat pituitary gonadotrophins are reduced (Shipley and Danley, 1947), but testis hyaluronidase does not change (Moore, 1948) . When hyperglycemia exists in rats, semen ■] carbohydrates increase (Mann and Lutwak-Mann, 1951).
Hypoglycemia influences the male reproductive organs. In rats tolbutamide or insulin produce lesions of the germinal epithelium which can be prevented by \' simultaneous administration of glucose. When 2 to 5 hypoglycemic comas are induced, such testis injuries increase progressively in number and frequency, and only a partial return to normal is observed a month later (Mancini, Izquierdo, Heinrich, Penhos and Gerschenfeld, 1959).
It is well known that the incidence of infertility in the pre-insulin era was high in young diabetic women. Fertility is also reduced in diabetic experimental animals, and rat estrous cycles are prolonged (Davis, Fugo and Lawrence, 1947) . Insulin is corrective (Sinden and Longwell, 1949; Ferret, Lindan and Morgans, 1950). Pregnancy in women with uncontrolled diabetes may terminate in abortion or stillbirth, possibly l)ecause toxemia of pregnancy is high (Pedersen. 1952). In rats pancreatectomy performed the 8th to 12th day of pregnancy increased the incidence of stillbirths (Hultciuist, 1950). In another experiment almost one- fourth of 163 animals with diabetes induced by alloxan on the 10th to 12th day of pregnancy died before parturition and

about 25 ])er cent of the survivors aborted (Angcrvall, 1959).
D. STERILE-OBESE SYNDROME
A sterile-obese syndrome in one colony of mice has been shown to be a recessive monogenic trait (Ingalls, Dickie and Snell, 1950). Obesity was transmitted to subsequent generations by way of ovaries that were transplanted from obese donors to nonobese recipients (Hummel, 1957). Obesity was transmitted by obese females receiving hormonal therapy and mated to obese males kept on restricted food intake (Smithberg and Runner, 1957). In addition to the investigations of the hereditary nature of the sterile-obese syndrome, the physiologic basis for the sterility has been studied in reference to the presence of germ cells, viability of ova and sperm, integrity of the ovary, and response of the uterus to estrogen (Drasher, Dickie and Lane, 1955). The data indicate that sterility in some obese males can be prevented by food restriction and that sterility in certain obese females can be corrected.
E. DIET AND THE LIVER
The concentration of hormones which reaches the target organs in the blood is the result of the rate of their production, metabolism, and excretion. How hypophyseal hormones are destroyed is not clear, but current data make it apparent that pituitary hormones have a short half-life in the circulatory system. Exerting a major control over circulating estrogen levels is the liver, with its steroid-inactivating systems. Zondek (1934) initially demonstrated that the liver could inactivate estrogens and this finding has had repeated confirmation (Cantarow, Paschkis, Rakoff and Hansen, 1943; De:\Ieio, Rakoff, Cantarow and Paschkis, 1948; Vanderlinde and Westerfield, 1950). Other steroids are also inactivated by the liver with several enzyme systems being involved; the relative concentration of these enzymes varies among species of vertebrates (Samuels, 1949).
The liver is a labile organ which readib.' responds to nutritional modifications; the induced liver changes alter the steroid-inactivating systems of this organ. Thus, inanition (Drill and Pfeiffer, 1946; Jailer,

G74

PHYSIOLOGY OF GONADS

1948) , vitamin B complex deficiency (Segaloff and Segaloff, 1944; Biskind, 1946), and protein restriction (Jailer and Seaman, 1950) all influence the capacity of the liver to detoxify steroids. Reduced protein intake is a primary factor in decreasing the effectiveness of the steroid-inactivating system (Jailer and Seaman, 1950; Vanderlinde and Westerfield, 1950). Rats fed an 8 per cent casein diet lose their capacity to inactivate estrone within 10 days. However, ascorbic acid alone or in combination with glutathione restored the estrone-inactivating system (Vasington, Parker, Headley and Vanderlinde, 1958).
Failure of steroids to be inactivated will influence the hypophyseal-gonadal axis. In turn the excess of estrogen will decrease gonadotrophin production by the hypophysis and thus reduce steroid production by the gonad. In addition nutritional modifications influence hypophyseal and possibly gonadal secretory capacity directly. Conceivably, nutritional alterations could modify the amount of steroid secreted or interfere with complete steroid synthesis by a gland.
Fatty infiltration of the liver and a general increase in fat deposition occur in fed and fasted rats after the injection of certain pituitary extracts and adrenal steroids and after the feeding of specific diets. Impaired estrogen inactivation has been associated with a fatty liver, but Szego and Barnes (1943) believe that the major influence is inanition. In fact, estrogens, especially ethinyl estradiol, interfere with fatty infiltration of the liver induced by a low protein diet (Gyorgy, Rose and Shipley, 1947) or by a choline-deficient diet (Emerson, Zamecnik and Nathanson, 1951). Stilbestrol, however, did not prevent the increase in liver fat induced by a protein-free diet (Glasser, 1957). Estrogens that are effective in preventing fatty infiltration may act by sparing methionine or choline or by inhibiting growth hormone (Flagge, Marasso and Zimmerman, 1958).
Ethionino, the antimetabolite of nu'thioniiK', Avill induce a fatty liver and inhibit hepatic protein synthesis in female, but not in male rats (Farber and Segaloff, 1955; Farber and Corban, 1958). Pretreatment of females with testosterone prevents

the ethionine effect, but this blockage of ethionine action need not be related to androgenic or progestational properties of steroids (Ranney and Drill, 1957).
III. Hypophysis and Diet
Studies involving acute and chronic starvation have shown that gonadal hypofunction during inanition is primarily due to diminished levels of circulating gonadotrophins. Because of the similarity to changes following hypophysectomy, the endocrine response to inanition has been referred to as "pseudohypophysectomy."
A. INANITION
The hypophysis has been implicated in human reproduction disturbances associated with undernutrition. Hypophyseal atrophy and a decrease in urinary gonadotrophins have been observed in chronic malnutrition (Klinefelter, Albright and Griswold, 1943; Zubiran and Gomez-]\Iont, 1953) and anorexia nervosa (Perloff, Lasche, Nodine, Schneeberg and Vieillard, 1954). Refeeding has restored urinary gonadotrophin levels in some cases, but hypoj^hyseal damage may result from severe food restriction at puberty (VoUmer, 1943; Samuels, 1948).
The influence of inanition on the reproductive organs of lal)oratory rodents is well recognized but the cft'ects on the hypophysis cannot be presented conclusively. In support of prior investigations, Mulinos and Pomerantz (1941a, b) in rats and Giroud and Desclaux (1945) in guinea pigs observed a hypophyseal atrophy following chronic underfeeding as well as a decrease in cell numbers and mitoses. In fact, refeeding after chronic starvation resulted in only a partial recovery of hypophyseal weight (Quimby, 1948). Nevertheless, complete starvation did not influence relative gland weight in female rats (Meites and Reed, 1949), and cytologic evidence (periodic acid-Schiff (PAS) test) of an estimated 3- fold increase in gonadoti'ophin content was claimed following chronic starvation (Pearse and Rinaldini, 1950). Assays of hypophyseal gonadotrophin content in chronically starved rats of both sexes have l)een reported as decreased (Mason and Wolfe, 1930; Werner,

NUTRITIONAL EFFECTS

675

1939), unchanged (,]\Iarrian and Parkes, 1929; Pomerantz and Mulinos, 1939; Maddock and Heller, 1947; Meites and Reed, 1949; Blivaiss, Hanson, Rosenzweig and McNeil, 1954), or increased (Rinaldini, 1949; Vanderlinde and Westerfield, 1950). An increase in pituitary gonadotrophin was evident when hormone content was related to milligrams of tissue (Meites and Reed, 1949). Thus, the hormone release mechanism may fail in starvation, and eventually gonadotrophin production will be reduced to a minimum (]\laddock and Heller, 1947).
Gonadectomy of fully fed rats is followed by an increase in hypophyseal gonadotrophin content. Chronic starvation, however, prevented the anticipated changes in the pituitary gland following gonadectomy in 8 of 12 female rats (Werner, 1939). On the other hand, if adult female rats were subjected to 14 days of reduced feeding 1 month after ovariectomy, no change in the elevated gonadotrophin levels was noted (Meites and Reed, 1949). In contrast, Gomez-Mont (1959) observed above normal urinary gonadotrophins in many menopausal and postmenopausal women despite undernutrition.
It is apparent that uniformity of opinion as to how starvation influences hypophyseal gonadotrophin content has not been attained. Several explanations can be given for the discrepancies. (1) There have been unfortunate variations in experimental design. IVIaddock and Heller (1947) starved rats for 12 days, whereas Rinaldini (1949) used a low calorie diet of bread and milk for 30 days. Other variations in feeding have included feeding one-half the intake required for growth (Mulinos and Pomerantz, 1941b I, regimens of full, one-half, oneciuarter, and no feeding for 7 and 14 days (Meites and Reed, 1949), and feeding inadequate amounts of a standard rat diet for 1 to 4 months (Werner, 1939). (2) Hypophyseal implants and anterior pituitary extracts should not be compared, for variable gonadotrophin production may follow implantation procedures, depending on whether necrosis or growth occurs (Maddock and Heller, 1947). (3) There has been an insufficient standardization of experimental materials. The assay animal has usually been the immature female rat, but

occasionally the immature mouse has been used, and Rinaldini (1949) used the hypophysectomized rat.
B. PROTEIN
The need for specific food elements by the hypophysis warrants consideration, for the hormones secreted by this gland are protein in nature and the amino acids for protein synthesis must be drawn from body sources. However, dietary protein levels can vary from 15 per cent to 30 per cent without influencing hypophyseal gonadotrophin content in rats (Weatherby and Reece, 1941), but diets containing 80 per cent to 90 per cent of casein increased hypophyseal gonadotrophin (TuchmannDuplessis and Aschkenasy-Lelu, 1948). Removal of protein from the diet will decrease hypophyseal gonadotrophin content in adult male rats in comparison with pair-fed and ad libitum-ied controls, but the decrease may or may not be significant in a 30-day period; luteinizing hormone (LH) seemed to be initially reduced. Extension of the period of protein depletion another 2 months resulted in a significant lowering of hy]iophyseal gonadotrophin levels (Table 12.4) (Leathern, 1958a). On the other hand, an increased FSH with no decrease in LH activity was observed in the hypophyses of adult female rats following 30 to 35 days of protein depletion (Srebnik and Nelson, 1957). The available data indicate that not only may a sex difference exist, but also that species may differ; restitution of gonadotrophin in the discharged rabbit pituitary was not influenced by in TABLE 12.4
Influence of a protein-free diet on hypophyseal
gonadotrophin content
(From J. H. Leathern, Recent Progr. Hormone
Res., 14, 141, 1958.)

Days on PFD*

Xo. of Rats

Anterior Pituitary Weight

Recipient Ovarv Weight

nig.

mg.

9

8.3

74

30

9

7.3

54

50

7

7.0

33

90

17

6.0

23

L^ntreated recipient ovarian weight = 15.4 mg.
~~PFD = Protein-free diet.~~

67G

PHYSIOLOGY OF GONADS

adequate dietary protein (Friedman and Friedman, 1940).
When anterior pituitar}^ glands of 60gm. male rats were extracted and administered to immature female recipients, ovarian weight increased from 13.0 to 37.3 mg. After feeding 20 per cent casein or fox chow ad libitum for 14 days, the hypophyses of male rats contained almost twice as much gonadotrophin per milligram of tissue as did the hypophyses of the initial controls. Removal of protein from the diet for 14 days, however, reduced hypophyseal gonadotrophin concentration below the level of the initial controls (Leathem and Fisher, 1959).
Data on the hypophyseal hormone content as influenced by specific amino acid deficiencies have not come to the author's attention. Cytologically, however, Scott (1956) noted that an isoleucine-deficient diet depleted the pituitary gonadotrophic cells of their PAS-positive material and reduced the size of acidophilic cells. Omission of threonine, histidine, or tryptophan invoked similar effects. The changes probably represent the interference of a single amino acid deficiency with protein metabolism rather than specific effects attributable to the lack of amino acid itself. Excessive amino acid provided by injecting leucine, methionine, valine, tyrosine, or glycine caused release of gonadotrophin (Goth, Lengyel, Bencze, Saveley and Majsay, 1955).
Administration of 0.1 mg. stilbestrol for 20 days to adult male rats eliminated detectable hypophyseal gonadotrophins. Hormone levels returned during the postinjection period provided the diet contained adequate protein, whereas a protein-free diet markedly hindered the recovery of hypophyseal gonadotrophins. The gonadotrophin content of the pituitary gland correlated well with the recovery of the reproductive system, indicating that gonadotrophin production was subnormal on ]irotein-free feeding (Leathem, 1958a).
C. CAHBOHYDR.\TE .\ND FAT
Reproduction does not appear to be influenced by carbohydrates per se and hypophyseal alterations have not been noted.

Fat-deficient diets, however, do influence reproduction and the hypophysis exhibits cellular changes. Pituitary glands of female rats fed a fat-free diet contain a subnormal number of acidophiles and an increased number of basophiles (Panos and Finerty, 1953) . In male rats the feeding oi a fat-free diet increased hypophyseal basophiles, followed progessively by more castration changes (Finerty, Klein and Panos, 1957; Panos, Klein and Finerty, 1959).
D. VITAMINS
Despite the many investigations relating reproduction to vitamin requirements, relatively few have involved hypophyseal hormone estimations. Thus in 1955, Wooten, Nelson, Simpson and Evans reported the first definitive study which related pyridoxine deficiency to hypophyseal gonadotrophin content. Using the hypophysectomized rat for assay, pituitary glands from Bo-deficient rats were shown to have a 10-fold increase in FSH per milligram of tissue and a slightly increased LH content. Earlier studies had revealed that vitamin Bi-free diets decreased pituitary gonadotrophins in male rats (Evans and Simpson, 1930) and a similar effect of the folic acid antagonist, aminopterin, in the monkey was found later (Salhanick, Hisaw and Zarrow, 1952).
Male rats deficient in vitamin A exhibited a 43 per cent increase, and castrated vitamin A-deficient rats a 100 per cent increase in hypophyseal gonadotrophin potency over the normal controls (Mason and Wolfe, 1930). The increase of gonadotrophin was more marked in vitamin A-deficient male than in vitamin A-deficient female rats. Associated with the increase in hormone level was a significant increase in basophile cells (Sutton and Brief, 1939; Hodgson, Hall, Sweetman, Wiseman and Converse, 1946; Erb, Andrews, Hauge and King, 1947).
A re\-iew of the literature up to 1944 permitted Mason to suggest that the anterior hypophysis was not the instigator of reproductive disturbances in vitamin E deficiency. Nevertheless, Griesbach, Bell and Livingston (1957), in an analysis of the pituitary gland during progessive stages of

NUTRITIONAL EFFECTS

677

tocopherol deprivation, observed cytologic changes in the hypophysis which preceded testis changes. The "peripheral or FSH gonadotrophes" increased in number, size, and activity. The LH cells exhibited a hyperplasia of lesser extent, but possibly sufficient to increase LH in circulation and to cause hypertrophy of the male accessory glands. Gonadotrophic hormone content of pituitary glands from vitamin E-deficient rats may be decreased (Rowlands and Singer, 1936), unchanged (Biddulph and Meyer, 1941), or increased to a level between normal and that of the castrate, when the adult male rats were examined after 22 weeks on a deficient diet (Nelson, 1933; Drummond, Noble and Wright, 1939). Using hypophysectomized male rats as assay animals, evidence was obtained that FSH was increased in the pituitary glands of vitamin E-deficient male and female rats (P'an, Van Dyke, Kaunitz and Slanetz, 1949).
IV. Male Reproductive System
A. TESTIS
The two basic functions of the male gonads are to produce gametes and secrete steroids. Spermatogenic activity can be estimated from testis morphology and examination of semen samples. Androgen secretion can be estimated from urinary steroid levels, accessorj^ gland weight, and from analyses of accessory sex gland secretions, i.e., fructose and citric acid. In normal maturation in the rabbit, rat, boar, and bull, androgen secretion precedes spermatogenesis (Lutwak-Mann, 1958). On this basis it would appear that well fed young bulls may come into semen production 2 to 3 months sooner than poorly fed animals (Brat ton, 1957).
1. Inanition
Complete starvation will pre^•ent maturation of immature animals. Furthermore, marked undernutrition in 700 boys, 7 to 16 years of age, was associated with genital infantilism in 37 per cent and cryptorchidism in 27 per cent (Stephens, 1941). Restriction of food intake to one-half of the normal in maturing bull calves had a

marked delaying effect on the onset of seminal vesicle secretion, but a lesser delaying effect on spermatogenesis (Davies, Mann and Rowson, 1957) . Limiting the food intake to one-third of the normal did not prevent the immature rat testis from forming spermatozoa at the same time as their controls (Talbert and Hamilton, 1955). When testis maturation was prevented by inanition, a rapid growth and maturation occurred on refeeding (Ball, Barnes and Visscher, 1947; Quimby, 1948) but Schultze (1955) observed that full body size was not attained.
The reproductive organs of the adult are more resistant to changes imposed by diet than are those of the immature animal. Thus, Mann and Walton (1953) found that 23 weeks of underfeeding produced little change in sperm density and motility in mature animals although seminal vesicle function was reduced. Li the male rat testis hypofunction follows partial or complete starvation (]\Iason and Wolfe, 1930; Mulinos and Pomerantz, 1941a; Escudero, Herraiz and Mussmano, 1948), but there is no reduction in testicular nitrogen (Addis, Poo and Lew, 1936). Loss of Leydig cell function precedes cessation of spermatogenesis (Moore and Samuels, 1931) and is evident by the atrophy of the accessory sexual organs (^lulinos and Pomerantz, 1941a) and by an alteration in accessory gland secretion (Pazos and Huggins, 1945; Lutwak-]\Iann and Mann, 1950). Evidence of a tubular effect is provided by the lack of motile sperm (Reid, 1949). Severe dietary restriction is associated with the absence of spermatozoa in the seminiferous tubules and epididymis (Mason, 1933; Menze, 1941).
The human male suffering from chronic malnutrition exhibits hypogonadism. The testes atrophy and exhibit a decrease in size of the seminiferous tubules; basement membrane thickening and small Leydig cells are seen. These individuals excrete significantly subnormal amounts of 17-ketosteroids (Zubiran and Gomez-Mont, 19531. Acute starvation may also decrease urinary 17-ketosteroid and androgen levels as much as 50 per cent, with recovery evident on refeeding (Perloff, Lasche, Nodine, Schneeberg and Vieillard. 1954).

6/

PHYSIOLOGY OF GONADS

TABLE 12.5
Effect of diet on the testes of immature rats
(From J. H. Leathern, in Re-productive Phijsiology
and Protein Nutrition, Rutgers University
Press, New Brunswick, N. J., 1959.)

Sperm

Initial control . . . 20 per cent X 30
days
6 per cent X 30
days
3 per cent X 30
days
per cent X 30
days
per cent + 5 per cent liver. ,
per cent + 5 per cent yeast
G5 per cent X 30 davs

No. of Rats

Testis

Weight

mg.

mg./lOOgm.

10

329

825

10

1694

1035

16

824

890

12

380

930

10

140

346

10

112

291

10

119

296

10

1747

1040

100
50

100

2. Protein
The minimal amount of dietary protein which will support reproduction, lactation, and growth is 16.7 per cent (Goettsch, 1949) . Thus, it is not surprising that maturation of testes and accessory sex organs was prevented in immature rats (Horn, 1955) and mice (Leathern and DeFeo, 1952) when they were fed a protein-free diet for 15 to 30 days after weaning. Furthermore, supplements of 5 per cent liver to the casein-free diet had no effect. After a month, the testes, averaging 329 mg., decreased to 140 mg. in rats fed per cent casein, but the weight increased to an average of 1694 mg. and 1747 mg. in rats fed 20 per cent and 65 per cent casein, respectively (Table 12.5). Following protein depletion, there was a decrease in tubular ribonucleic acid and an increase in lipid. Accumulation of gonadal lipid in the inactive testis may be an abnormal assimilation of a degenerative nature or simply nonutilization. A diet containing 6 per cent casein permitted the formation of spermatozoa in some animals (Guilbert and Goss, 1932). When the 6 per cent casein diet was fed to immature animals for 30 days 50 per cent of the rats exhibited some spermatozoa; in addition, testis weight increased slightly and seminal

vesicle weight doubled, but body weight was not improved (Horn, 1955). Thus, as we noted earlier, the reproductive system may gain special consideration for protein allotments when supplies are limited.
Gain in testis weight in immature male rats and the biochemical composition of the immature testis are influenced by the nutritive value of the protein fed. The testes of normal immature rats contain 85 per cent water, 10.5 per cent protein, 4.5 per cent lipid, and detectable glycogen (Wolf and Leathern, 1955). Proteins of lower nutritive value (wheat gluten, peanut flour, gelatin ) may permit some increase in testis weight, but testis protein concentration decreased, percentage of water increased, and lipid and glycogen remained unchanged (Table 12.6). The enzyme ^fi-glucuronidase, which has frequently been associated with growth processes, exhibited no change in concentration as the testis matured or was jirevented from maturing by a protein-free diet (Leathem and Fisher, 1959). Not only are the weight and composition of the testis influenced by feeding proteins of varied biologic value, but the release of androgen is more markedly altered. When a 22-day-old male rat was fed a 20 per cent casein diet for 30 days, the seminal vesicle and ventral prostate weights increased 9- to 10-fold in comparison with initial control weight. Sub TABLE 12.6
Niiiritioiial effects on testis-coin position
in immature rats
(From R. C. Wolf and J. H. Leathem,
Endocrinology, 57, 286, 1955.)

20 pel' cent casein
20 per cent wheat gluten
20 per cent i)eanut flour
20 i)er cent gelatin
5 per cent casein
Fox chow
Initial control . .

No. of Rats

Final Body Weight

gm.

7

128

8

82

8

81

5

53

5

61

8

115

7

61

72.3j30.4 04.634.0

1468 1017
1257 66.1 2101

28.8

0.11 0.18 0.11 0.10

684,62.4 29.2 0.26 1515'70.3 30.3 0.15 273 72. 7:30. 10.19

NUTRITIONAL EFFECTS

679

stitution of wheat gluten and peanut flour for casein, limited the increase in the weight of the seminal vesicles to less than 100 per cent. In fact, seminal vesicle weight as related to body weight did not increase in animals fed 20 per cent wheat gluten.
The withholding of dietary protein from an immature rat for 30 days, during which time maturation occurs in the fully fed animal, did not impose a permanent damage. Refeeding of protein permitted the rapid recovery of testis weight and the appearance of spermatoza, 70 per cent of all animals having recovered in 30 days when fully fed, whereas only 25 per cent recovered when 6 per cent casein was fed. Recovery of androgen secretion was somewhat slower than that of the tubules as estimated by seminal vesicle weight.
Variations in protein quality are a reflection of amino acid patterns, and amino acid deficiencies interfere with testis maturation (Scott, 1956; Pomeranze, Piliero, Medeci and Plachta, 1959). Alterations in food intake which follow amino acid deficiencies have required forced feeding or pair-fed controls, but it is clear from what w^as found in the controls that the gonadal changes were not entirely due to inanition (Ershoff, 1952).
If the diet is varied so that caloric intake per gram is reduced to half while retaining the dietary casein level at 20 per cent, immature rat testis growth is prevented. The effect is unlike that obtained with this level of protein in the presence of adequate calories. Furthermore, the caloric restriction may increase testis glycogen (Leathem, 1959c).
Protein anabolic levels are higher in the tissues of young growing animals and the body is more dependent on dietary protein level and quality for maintenance of the metabolic nitrogen pool than in adult animals. On the other hand, body protein reserves in adult animals permit internal shifts of nitrogen to the metabolic pool and to tissues when dietary sources are reduced or endocrine imbalances are imposed. Thus, Cole, Guilbert and Goss (1932) fed a low protein diet to adult male rats for 60 to 90 days before the sperm disappeared, but the animals would not mate. Amount of semen and sperm produced by sheep have been re

TABLE 12.7
Arlult rat testes and seminal vesicles
after protein depletion
(From J. H. Leatliem, Recent Progr. Hormone
Res., 14, 141, 1958.)

Days on

No. of

Testis

H2O

Protein

Total

Seminal

PFD*

Rats

Weight

Protein

Vesical

nig.

%

%dry

gm.

mg.

Control

9

2852

85.9

66.7

0.28

1276

30

9

2600

86.0

66.1

0.24

689

50

7

2398

85.4

64.1

0.22

320

90

25

1429

85.7

69.6

0.13

168

~~PFD = Protein-free diet.~~
lated to the dietary protein level (Popoff and Okultilschew, 1936). Removal of protein from the diet for 30 days had little effect on the adult rat testis weight, spermatogenesis, or nitrogen content (Leathem, 1954). However, seminal vesicle w^eight was reduced 50 per cent (Aschkenasy, 1954). Prolonged protein depletion was required before the testis exhibited a loss in protein and a reduction in size. A loss of spermatozoa was not observed consistently, although some testes were completely atrophic (Table 12.7). Accessory organ weight decrease reflected the disappearance of androgen (Leathem, 1958a). Interstitial cell atrophy has also been noted in rats fed a low vegetable protein (cassava) diet (Adams, Fernand and Schnieden, 1958).
Sterility may or may not be induced with diets containing 65 per cent protein (Reid. 1949; Leathem, 1959c) but a 15 to 18 per cent dietary level of a poor protein such as maize or gelatin will decrease sperm motility and increase the number of abnormal sperm. The influence of proteins having different nutritional values in support of the growth of testes from the level to which they were depressed by stilbestrol indicated that casein, lactalbumin, and wheat gluten are equally competent to support testis growth whereas gelatin is deficient. Whole proteins may have several amino acid deficiencies, but the administration of amino acid antagonists may help to identify important individual amino acids. As an example, ethionine causes severe seminiferous tubule atrophy and Leydig cell hypoplasia (Kaufman, Klavins and Kinney, 1956; Goldberg, Pfau and Ungar, 1959). Studies in man have indicated a sharp reduction in

680

PHYSIOLOGY OF GONADS

spermatozoa after 9 days on an argininedeficient diet (Holt, Albanese, Shettles, Kajdi and Wangerin, 1942).
Adequate dietary protein cannot maintain reproductive function if the diet is calorie deficient. Thus, a decrease in seminal vesicle weight could be related to a decrease in dietary calories while protein levels were constant (Rosenthal and Allison, 1956). However, the accessory gland weight loss imposed by caloric restriction could be slowed by increasing the dietary protein (Rivero-Fontan, Paschkis, West and Cantarow, 1952).
Alterations in testis function imposed by inadequate protein are corrected when protein is returned to the diet at normal levels (Aschkenasy and Dray, 1953). Nevertheless, the nutritional state of the animal as a factor influencing recovery has been demonstrated with stilbestrol-treated adult male rats. While being fed an 18 per cent casein diet, adult male rats were injected with 0.1 mg. stilbestrol daily for 20 days. Testis weight decreased from 2848 to 842 mg., spermatogenesis was abolished, and testis water and protein content were significantly reduced. Despite a reduction in food intake, pair-fed controls exhibited no effect on reproductive organs. When a protein-free diet was substituted for the normal diet during the administration of stilbestrol, atrophy of the reproductive system was observed. Cessation of hormone administration was followed by a rapid return of testicular function toward normal when 18 per cent casein was fed both during the injection period and the recovery period. Within 30 days spermatogenesis and testicular composition were fully recovered. However, when 18 per cent casein was fed in the postinjection period to rats that had received a proteinfree diet while being given stilbestrol, recovery was clearly slow. After a month, spermatozoa were observed in only 30 per cent of the testes and testis weight was subnormal. Despite the seeming similarity of response by the two nutritional groups during the injection period, the postinjection recovery on identical dietary intake revealed marked differences in rate of recoverv (Leathem, 1958a).

3. Fat
Linoleic, linolenic, and arachidonic acids are designated as essentially fatty acids, but the physiologic role of these substances is not clearly understood. Nevertheless, the male reproductive organs are influenced by dietary essential fatty acid levels. High fat diets may enhance testicular weight (Kaunitz, Slanetz, Johnson and Guilmain, 1956) whereas removal of fat from the diet resulted in a degeneration of the seminiferous tubules as evidenced by intracellular vacuolation and a reduction in spermatids and spermatozoa (Panos and Finerty, 1954). After 5 months of feeding a fatfree diet, the rat testis may be devoid of sperm (Evans, Lepkovsky and Murphy, 1934). Testis degeneration occurred despite dietary supplements of vitamins A and E and in animals whose health appeared quite normal (Ferrando, Jacques, Mabboux and Prieur, 1955; Ferrando, Jacques, Mabboux and SoUogoub, 1955).
Weanling rats fed 14 per cent arachis (peanut) oil for 15 weeks exhibited a marked impairment of spermatogenesis (Aaes-Jorgensen, Funch and Dam, 1956) and 28 per cent arachis oil induced testicular damage of such an order that 15 weeks of feeding ethyl linoleate did not restore fertility (Aaes-Jorgensen, Funch and Dam, 1957).
4. Vitamins
Testicular dysfunction as judged by failure of sperm formation or atrophy of the secondary sex organs has been observed in deprivations of thiamine, riboflavin, pyridoxine, calcium pantothenate, biotin, and vitamins A and E. One must distinguish, however, between effects of inanition associated w^ith a vitamin deficiency and a specific vitamin effect (Skelton, 1950) ; one must also consider species differences (Biskind, 1946).
There is no question but that vitamin E deficiency in the rat results in a specific and irreversible damage to the testis. Tubular damage may proceed to the point where only Sertoli cells remain and yet the interstitial cells are not influenced (Mason, 1939). Similar changes followed vitamin E deficiency in the guinea pig (Pappenheimer and SchogolefT, 1944; Curto, 1954; Ingel

NUTRITIONAL EFFECTS

681

man-Siindberg, 1954) , hamster (Mason and Mauer, 1957), and bird (Herrick, Eide and Snow, 1952; Lowe, Morton, Cunningham and Vernon, 1957). However, little or no effect of an absence of vitamin E was noted in the rabbit (Mackenzie, 1942) and mouse (Bryan and Mason, 1941), or in live stock (Blaxter and Brown, 1952), or man (Lutwak-Mann, 1958), although vitamin E is present in human testes (Dju, Mason and Filer, 1958). Treatment of low-fertility farm animals with wheat germ oil or tocopherol or the use of this vitamin clinically have provided only inconclusive results (Beckmann, 1955) . Although some positive effects have been reported in man, the results may be due in part at least to the sparing action of tocopherol toward vitamin A.
Vitamin A deficiency influences the testis but changes are closely associated with the degree of inanition. In the rat, a vitamin deficiency sufficient to cause ocular lesions did not prevent sperm formation, but a deficiency of such proportions as to cause a body weight loss did cause atrophy of the germinal epithelium (Reid, 1949). Vitamin A deficiency will induce sterility in mice (McCarthy and Cerecedo, 1952). A gross vitamin deficiency in bulls before expected breeding age prevented breeding ; adult bulls may exhibit a lower quality semen but they remain fertile (Reid, 1949). Vitamin A deficiency induces metaplastic keratinization of the epithelium lining the male accessory sex organs (Follis, 1948) and thus may influence semen.
Testis damage induced by vitamin A deficiency can be reversed, but vitamin A therapy in man for oligospermia not due to vitamin lack was without effect (Home and Maddock, 1952) .
Age of the animal and dosage are factors which influence the results obtained in male rats with administered vitamin A. Immature male rats given 250 I.U. of vitamin A per gram of body weight daily exhibited a loss of spermatocytes, an effect which was accentuated by tocopherol (Maddock, Cohen and Wolbach, 1953). Little or no effect of similar treatment was observed in adult rats. The liver is the major storage depot for vitamin A and the fact that the male rat liver is more quickly depleted and less capa

ble of storage than is the liver of the female should be considered in any attempted correlation of the vitamin and hormone levels (Booth, 1952).
Other vitamin deficiencies have been shown to influence the testis. A lack of thiamine had little effect on testis weight, but did influence the Leydig cells and prevented growth of the accessory sex organs (Pecora and Highman, 1953). A chronic lack of ascorbic acid will cause a degeneration of both Leydig cells and seminiferous tubules. The effects of vitamin deficiency on the testis has been distinguished from those due to inanition and have been related to changes in carbohydrate metabolism (Mukherjee and Banerjee, 1954; Kocen and Cavazos, 1958) . The importance of ascorbic acid in the testis as related to function is not evident, but concentrations of this vitamin are maximal at 1 week of age (Coste, Delbarre and Lacronique, 1953).
Serious anatomic and functional impairments of testes were noted in pantothenic acid deficiency (Barboriak, Krehl, Cowgill and Whedon, 1957), and development of the rat testis and seminal vesicles was retarded by a biotin deficiency (Bishop and Kosarick, 1951; Katsh, Kosarick and Alpern, 1955), but the animals did not exhibit marked alterations in other endocrine organs (Delost and Terroine, 1954). Testosterone hastened the development of vitamin deficiency and enhanced the severity of biotin deficiency in both sexes, thereby suggesting a hormone-vitamin relationship (Okey, Pencharz and Lepkovsky, 1950). On the other hand, testosterone had no effect on the tolerance of mice for aminopterin, but castration increased the tolerance (Goldin, Greenspan, Goldberg and Schoenberg 1950).
B. INFLUENCE OF NUTRITION ON THE EE SPONSIVENESS OF MALE REPRODUCTIVE
TISSUES TO HORMONES
1. Testis
a. Inanition. The testes of birds on limited food intake were more responsive to hypophyseal gonadotrophin than fully fed birds (Byerly and Burrows, 1938; Breneman, 1940). In the rat several investigators have shown that the testis will respond to gonadotrophin despite inanition (Moore and Sara

682

PHYSIOLOGY OF GONADS

TABLE 12.8
Influence of diet and pregnant mare serum (PMS)
on testes and seminal vesicles of
immature male mice
(From V. J. DeFeo and J. H. Leathern,
unpublished.)

Diet (Per cent Protein X Days Fed)

per cent X 10 per cent X 10 per cent X 20 per cent X 20

l.u.
3
3

stage of Spermatogenesis

4 1
1 5 5

Spermatids

Seminal Vesicles

mg.
2.7 4.5
2.7 3.5

uels, 1931; Funk and Funk, 1939; Meites, 1953), with a stimulation of Leydig cells, an increase in testis size, and, in 40 days, a return of spermatozoa. Underfed males injected with gonadotrophin sired litters (Mulinos and Pomerantz, 1941a, b). Improved nutrition aided by unknown liver factors enhanced the response to androgen in severe human oligospermia (Glass and Russell, 1952).
b. Protein. Feeding a protein-deficient diet to adult male rats for 60 to 90 days did not prevent stimulation of the testes and seminal vesicles after pregnant mare's serum (PMS) administration (Cole, Guilbert and Goss, 1932). As we have noted, immature animals are prevented from maturing when diets lack protein. Nevertheless, a gonadal response to injected gonadotrophin was obtained in immature mice fed a protein-free diet for 13 days; tubules and Leydig cells were stimulated and androgen was secreted (Table 12.8). Refeeding alone permitted a recovery of spermatogenesis which was not hastened by concomitant PMS (Leathem, 1959c).
The maintenance of testis weight and spermatogenic activity with testosterone propionate in hypophysectomized adult male rats is well known, but these studies have involved adequate nutrition. If hypophysectomized rats were fed a protein-free diet and injected with 0.25 mg. testosterone propionate daily, testis weight and spermatogenesis were less well maintained than in rats fed protein. Testis protein concentra

tion was also reduced. These data suggest that influences of nutrition on the testis can be direct and are not entirely mediated through hypophyeal gonadotrophin changes (Leathem, 1959b).
c. Fat. The rat fed for 20 weeks on a fatfree diet exhibits a degeneration of the seminiferous epithelium within the first weeks which progresses rapidly thereafter. Chorionic gonadotrophin or rat pituitary extract started during the 20th week failed to counteract the tubular degeneration, but testosterone propionate proved effective (Finerty, Klein and Panos, 1957). The result shows that the ineffectiveness of the gonadotrophins could not be due to the failure of androgen release (Greenberg and Ershoff, 1951).
d. Vitamins. Gonadotrophins failed to promote spermatogenesis in vitamin A(Mason, 1939) or vitamin E- (]Mason, 1933; Geller, 1933; Drummond, Noble and Wright, 1939) deficient rats, but in another experiment the atrophic accessory sex organs of vitamin A-depleted rats were stimulated (Mayer and Goodard, 1951). Lack of vitamin A favored an enhanced response to PMS when the ratios of seminal vesicle weight to body weight were computed (Meites, 1953). The failure of gonadotrophins to stimulate testis tubules suggests a specific effect of avitaminosis A and E (Mason, 1933) on the responsiveness of the germinal epithelium.
Subnormal responses of rats to PMS, as measured by relative seminal vesicle weight, were obtained when there were individual vitamin B deficiencies, but the influence was due largely to inanition (Drill and Burrill, 1944; Meites, 1953). Nevertheless, sufficient response to chorionic gonadotrophin was obtained so that fructose and citric acid levels were restored to normal. Such an effect was not observed to follow dietary correction unless an unlimited food intake was allowed (Lutwak-Mann. 1958).

2. Sc

il W.siclfx and Prosfate

a. Inanition. Although the accessory reproductive organs resppnd to direct stimulation despite an inadequate food intake (Mooi'c and Samuels. 19311, tlio increase

NUTRITIONAL EFFECTS

683

in weight may be subnormal in mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a, Grayhack and Scott, 1952), or above normal in chickens (Breneman, 1940). Complete deprivation of food reduced the quantity of prostatic fluid in the dog, but exogenous androgen restored the volume, increased acid phosphatase, and induced tissue growth (Pazos and Huggins, 1945) .
6. Protein. The response of the seminal vesicles to androgen was investigated in immature rats, using weight and /5-glucuronidase as end points. Castration and 10 days on a protein-free diet preceded the 72-hour response to 0.25 mg. testosterone propionate. The lack of protein did not prevent a normal weight increase, and enzyme concentration was unchanged. If an 18 per cent diet was fed during the 3-day period that the androgen was acting, no improvement in weight response was noted, but enzyme concentration increased 100 per cent. Thus, when protein stores are depleted, the androgen response may be incomplete in the absence of dietary protein (Leathern, 1959c). Nevertheless, varied protein levels do not influence seminal vesicle weight-response when caloric intake is reduced (RiveroFontan, Paschkis, West and Cantarow, 1952).
c. Vitamins. Vitamin deficiencies do not prevent the seminal vesicles from responding to androgen. In fact, in vitamin B deficiency, testosterone restored fructose and citric acid levels to normal despite the need for thiamine in carbohydrate metabolism (Lutwak-Mann and Mann, 1950). In the male, unlike the female, the effects of folic acid deficiency in reducing responsiveness to administered androgen were largely due to inanition in both mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a) , and vitamin A deficiency which leads to virtual castration does not prevent an essentially normal response of the accessory glands to testosterone propionate (Mayer and Truant, 1949). Restricting the caloric intake of vitamin Adeficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient animals (Mason, 1939).

V. Female Reproductive System
A. OVARIES
1. Inanition
Mammalian species generally exhibit a delay in sexual maturation when food intake is subnormal before puberty, and ovarian atrophy with associated changes in cycles if inanition is imposed on adults. In human beings a decrease in fertility and a greater incidence of menstrual irregularities were induced by war famine (Zimmer, Weill and Dubois, 1944). Ovarian atrophy with associated amenorrhea and sterility were invoked by chronic undernutrition (Stephens, 1941). The ovarian morpliologic changes were similar to those of aging. Urinary estrogens were subnormal in 22 of 25 patients exhibiting amenorrhea associated with limited food intake (Zubiran and (_lomcz-Mont, 1953).
The nutritional requirements of jM'imates other than man have been studied in female baboons. The intake of vitamins and other essential nutrients was found to be of the same order as that recommended for man. Caloric intake varied with the menstrual cycle, being least during the follicular phase and maximal during the 2 to 7 days preceding menstruation (Gilbert and Gillman, 1956). Various diets were also studied to assess their importance in maintaining the normal menstrual rhythm. The feeding of (a) maize alone, (b) assorted vegetables and fruit, or (c) maize, skimmed milk, and fat led to menstrual irregularities or to amenorrhea. The mechanism regulating ovulation was the first to be deranged. The addition of various vitamins or of animal protein did not correct the menstrual disorders. However, inclusion of ox liver in the diet did maintain the menstrual rhythmicity, but the beneficial effect could not be attributed to its protein content (Gillman and Gilbert, 1956 ) .
In lower mammals that have been studied, inanition will hinder vaginal opening, and delay puberty and ovarian maturation and functioning. In adult rats and mice ostrous cycles are interrupted and the reprorUictive system becomes atrophic when body weight loss exceeds 15 per cent. The ovaries be

684

PHYSIOLOGY OF GONADS

come smaller, ovulation fails, and large vesicular follicles decrease in number with an increase in atresia, but primary follicles show a compensatory increase (Marrian and Parkes, 1929; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Guilbert, 1942; Bratton, 1957). The ovarian interstitial cells mav be markedly altered or absent (Huseby and Ball, 1945; Rinaldini, 1949) and the ovary may exhibit excessive luteinization (Arvy, Aschkenasy, AschkenasyLelu and Gabe, 1946) or regressing corpora lutea (Rinaldini, 1949) . However, the ovarian changes induced by inanition may be reversed by refeeding, with a return to reproductive capacity (Ball, Barnes and Visscher, 1947; Schultze, 1955). The effect of feed-level on the reproductive capacity of the ewe has been reported (El-Skukh, Nulet, Pope and Casida, 1955), but one must realize that high planes of nutrition may adversely influence fertility (Asdell, 1949). Nevertheless, additional protein and calcium added to an adequate diet extended the reproductive life span (Sherman, Pearson, Bal, McCarthy and Lanford, 1956).
2. Protein
The availability of just protein has an important influence on the female reproductive system. In immature rats ovarian maturation was prevented by feeding diets containing per cent to 1.5 per cent protein (Ryabinina, 1952) and low protein diets decreased the number of ova but without altering their ribonucleic acid (RNA) or glycogen content (Ishida, 1957). Refeeding 18 per cent protein for only 3 days was marked by the appearance of vesicular follicles and the release of estrogen in mice previously fed a protein-free diet (Leathem, 1958a). In experiments involving the opposite extreme, in which 90 per cent protein diets were used, a retardation of ovarian growth, and a delay in follicular maturation, in vaginal opening, and in the initiation of estrous cycles were noted (Aschkenasy-Lelu and Tuchmann-Duplessis, 1947; TuchniannDuplcssis and Aschkenasy-Lelu, 1948).
Adult female rats fed a protein-free diet for 30 days exhibited ovaries weighing 22 mg. compared with ovaries weighing 56 mg. from i)air-fed controls fed 18 per cent casein. Ovarian glycogen, ascorbic acid, and cho

lesterol were all influenced by protein deprivation and anestrum accompanied the ovarian changes. Furthermore, uterine weight and gl3^cogen decreased in rats fed protein-free diets (Leathem, 1959b).
In adult rats the feeding of 3.5 per cent to 5 per cent levels of protein (GuillDert and Gross, 1932) was followed by irregularity of the cycles or by their cessation. The cycles became normal when 20 to 30 per cent protein was fed (Aschkenasy-Lelu and Aschkenasy, 1947). However, abnormally high levels of casein (90 per cent) induced prolonged periods of constant estrus (Tuchmann-Duplessis and AschkenasyLelu, 1947). Nevertheless, not all species responded to protein depletion in the same manner. For example, the rabbit exhibited estrus and ovulation despite a 25 per cent body weight loss imposed by to 2 per cent protein diets (Friedman and Friedman, 1940).
Despite a normal level of protein in the diet, inadequate calories will interfere with reproductive function and induce ovarian atrophy (Escudero, Herraiz and Mussmano, 1948; Rivero-Fontan, Paschkis, West and Cantarow, 1952). Furthermore, the effects of 15 per cent and 56 per cent protein levels on estrous cycles could not be distinguished when calories were reduced 50 per cent (Lee, King and Visscher, 1952). Returning mice to full feeding after months of caloric deficiency resulted in a sharp increase in reproductive performance well above that expected for the age of the animal (Visscher, King and Lee, 1952). This type of rebound phenomenon has not been explained.
Reproductive failure assigned to dietary protein may be a reflection of protein quality as well as level. Specific amino acid deficiencies lead to cessation of estrus (White and AVhite, 1942; Berg and Rohse, 1947) and thus feeding gelatin or wheat as the protein source and at an 18 per cent level was quickly followed by an anestrum (Leathem, 1959b). Supplementation of the wheat diet with lysine corrected the reproductive abnormalities (Courrier and Raynaud, 1932), but neither lysine (Pearson, Hart and Bohstedt, 1937) nor cystine (Pearson, 1936) added to a low casein diet was beneficial. Control of food intake must be considered in studies involving amino

NUTRITIONAL EFFECTS

685

acids, for a deficiency or an excess can create an imbalance and alter appetite. Opportunity to study the amino acids in reproduction is now possible because of the work of Greenstein, Birnbaum, Winitz and Otey (1957) and Schultze (1956) , who maintained rats for two or more generations on synthetic diets containing amino acids as the only source of protein. Similarly, the amino acid needs for egg-laying in hens has been reported (Fisher, 1957). Tissue culture methods also permit the study of the nutritional requirements of embryonic gonadal tissue, the success of avian gonadal tissue in culture being judged by survival, growth, and differentiation. In experiments in which this technique was used it was found that a medium made up of amino acids as the basic nitrogen source can maintain gonadal explants very successfully, even though the choice of amino acids does not exactly correspond to the 10 essential amino acids recommended for postnatal growth (StengerHaffen and Wolff, 1957).
3. Carbohydrate
The absence of dietary carbohydrate does not appreciably affect the regularity of estrous cycles in rats provided the caloric need is met. However, the substitution of 20 per cent sucrose for corn starch induced precocious sexual maturity which was followed by sterility (Whitnah and Bogart, 1956). The ovaries contained corpora lutea, but the excessive luteinization of unruptured follicles suggested a hypophyseal disturbance. Substitution of 20 per cent lactose for corn starch had no effect. Increased amounts of lactose retarded the gain in body weight and blocked ovarian maturation, possibly because the animal could not hydrolyze adequate amounts of the disaccharide. Addition of whole liver powder to the diet counteracted the depressing action of 45 per cent lactose on the ovary (Ershoff, 1949).
4. Fat
There seems to be little doubt that dietary fat is reciuired for normal cyclic activity, successful pregnancy, and lactation, and that the requirements for essential fatty acids are lower in females than in males (Deuel, 1956).
Conception, fetal development, and par

turition can take place in animals fed a diet deficient in fatty acids (Deuel, Martin and Alfin-Slater, 1954) , despite a reduction in total carcass arachidonic acid (Kummerow. Pan and Hickman, 1952). Earlier reports indicated that a deficiency of essential fatty acids caused irregular ovulation and impaired reproduction (Burr and Burr, 1930; Maeder, 1937). The large pale ovaries lead Sherman (1941) to relate essential fatty acid deficiency to carotene metabolism. In this regard the removal of essential fatty acids from an adequate diet supplemented with vitamin A and E lead to anestrum and sterility while maintaining good health (Ferrando, Jacques, Mabboux and Prieur, 1955). Perhaps the differences in opinion regarding the effects of fatty acid deficiency can be related to the duration of the experimental period. Panos and Finerty (1953) found that growing rats placed on a fat-free diet exhibited a normal time for vaginal opening, normal ovarian weight, follicles, and corpora lutea, although interstitial cells were atrophic. However, regular estrous cycles were noted for only 20 weeks, thereafter 60 per cent of the animals exhibited irregular cycles.
A decrease in reproductive function may be invoked by adding 14 per cent arachis oil to the diet (Aaes-Jorgensen, Funch and Dam, 1956) . Increasing dietary fat by adding rape oil did not influence ovarian function but did cause the accumulation of ovarian and adrenal cholesterol (Carroll and Noble, 1952).
Essential fatty acid deficiency is associated with underdevelopment and atrophic changes of the uterine mucosa. Adding fat to a stock diet enhanced uterine weight in young animals at a more rapid pace than body weight (Umberger and Gass, 1958) .
5. Vitamins
Carotenoid pigments are present in the gonads of many vertebrates and marine invertebrates, and, in mammals, are particularly prominent in the corpus luteum. However, no progress has been made in determining either the importance of the carotenoids in the ovary or of the factors controlling their concentrations. It is well known that vitamin A deficiency induces

686

PHYSIOLOGY OF GONADS

a characteristic keratinizing metaplasia of the uterus and vagina, but estrous cycles continue despite the vaginal mucosal changes. Furthermore, ovulation occurs regularly until advanced stages of deficiency appear. The estrous cycle becomes irregular in cattle fed for a long period of time on fodder deficient in carotene. The corpora lutea fail to regress at the normal rate and ovarian follicles become atretic and cystic ( Jaskowski, Watkowski, Dobrowolska and Domanski, cited by Lutwak-Mann, 1958). The alterations in reproductive organs associated with a lack of vitamin A may be due in part to a vitamin E deficiency since the latter enhances the rate at which liver stores of vitamin A are depleted.
Definite effects of hypervitaminosis A have been observed on reproduction. Masin (1950) noted that estrus in female rats could be prolonged by administration of 37,000 I.U. of vitamin A daily. The implications, however, have not been studied. The effect of hypervitaminosis A may actually induce secondary hypovitaminoses. The displacement of vitamin K by excess A is almost certain and similar relationships appear to exist with vitamin D (Nieman and Klein Obbink, 1954).
The failure of vitamin E-deficient female rats to become pregnant is apparently due to disturbances of the implantation process rather than to the failure of ovulation. There is no direct proof of ovarian dysfunction (Blandau, Kaunitz and Slanetz, 1949). However, the ovary of the rat deficient in vitamin E may have more connective tissue and pigment, and Kaunitz (1955) showed by ovarian transplantation that some nonspecific ovarian dysfunction appears to exist (cited by Cheng, 1959 (. Vitamin E is essential for birds, but there is little evidence for a dependency in most mammals; sheep, cows, goats, and pigs have been studied. Treatment of low-fertility farm animals with tocopherol has not provided conclusive data favoring its use (Lutwak-Mann, 1958), nor has the treatment of human females been rewarded with any indication that vitamin E might be helpful in cases of abnormal cycles and habitual abortion (Beckmann, 1955).
No specific reproductive disturbances in man, the rhesus monkey, or the guinea pig

have been associated with vitamin C deficiency (Mason, 1939). Nevertheless, the high ascorbic acid content of ovarian and luteal tissue and of the adrenal cortex suggests a physiologic role in association with steroid synthesis. (3varian ascorbic acid varies with the estrous cycle, dropping sharply in the proestrum (Coste, Delbarre and Lacronique, 1953), and decreasing in resjionse to gonadotrophin (Hokfelt, 1950; Parlow, 1958). Virtually no ascorbic acid is present in bovine follicular fluid (LutwakMann, 1954) or in rat ovarian cyst fluid (Blye and Leathem, 1959). Uterine ascorbic acid decreased in immature mice treated with estrogen, but remained unchanged in rats following thiouracil administration (Leathem, 1959a). Its role in the uterus awaits elucidation.
Delayed sexual maturation and ovarian atrophy have been described when there are deficiencies of thiamine, riboflavin, pyridoxine, pantothenic acid, biotin, and B12 (Ershoff, 1952; Ullrey, Becker, Terrill and Notzold, 1955). However, as we noted when deficiencies of the vitamins were being considered, much of the impairment of reproductive function can be related to inanition rather than to a vitamin deficiency (Drill and Burrill, 1944). Pyridoxine deficiency, although not affecting structure (Morris, Dunn and Wagner, 1953) , markedly reduces the sensitivity of the ovary to administered gonadotrophin (Wooten, Nelson, Simpson and Evans, 1958) .
Bird, frog, and fish eggs contain considerable quantities of vitamins. In fact, the daily human requirements for vitamins may be contained in a hen's egg and thus it is not surprising that hatchability is decreased l)y virtually any vitamin deficiency. Lutwak-Mann (1958) has provided an excellent survey of these data with numerous references to studies of frogs and fishes. Nearly all the B vitamins are present in fish roe and the pantothenic acid concentration in cod ovaries {Gadus morrhua) exceeds most otlicr natural sources. The amount of the latter varies with the reproductive cycle, d(>creasing to its lowest level before spawning. Riboflavin and vitamin B12 , on the other h;ui(l, do not change (Braekkan, 1955).

NUTRITIONAL EFFECTS

687

B. INFLUENCE OF NUTRITION ON THE RESPONSIVENESS OF FEMALE REPRODUCTIVE TISSUES TO HORMONES
1. Ovary
a. Inanition. Marrian and Parkes (1929) were the first to show that the quiescent ovary of the underfed rat can respond to injections of anterior pituitary as evidenced by ovulation and estrous smears. Subsequently the ovaries of underfed birds, rats, and guinea pigs were found to be responsive to serum gonadotrophin (Werner, 1939; Stephens and Allen, 1941; Mulinos and Pomerantz, 1941b; Hosoda, Kaneko, Mogi and Abe, 1956). A low calorie bread-andmilk diet for 30 days did not prevent ovarian response to rat anterior pituitary or to chorionic gonadotrophin. In these animals an increase in ovarian weight with repair of interstitial tissue, as well as folhcle stimulation and corpus luteum formation, were observed (Rinaldini, 1949). Rats from which food had been withdrawn for 12 days could respond to castrated rat pituitary extract with an increase in ovarian and uterine weight (Maddock and Heller, 1947). Nevertheless, differences in the time and degree of responsiveness to administered gonadotrophin were noted in rabbits. Animals on a high plane of nutrition responded to gonadotrophin at 12 weeks, whereas rabbits on a low plane of nutrition responded at 20 weeks and fewer eggs were shed (Adams, 1953).
b. Protein. Protein or amino acid deficiencies in the rat do not prevent a response to administered gonadotrophin (Cole, Guilbert and Goss, 1932; Courrier and Raynaud, 1932) . However, the degree and type of gonadal response is influenced by the diet. Thus, immature female mice fed to 6 per cent casein for 13 days exhibited only follicular growth in response to pregnant mare serum, whereas the ovarian response in mice fed 18 per cent casein was suggestive of follicle-stimulating and strongly luteinizing actions (Table 12.9). Furthermore, the ovarian response was significantly less after 20 days of nonprotein feeding than after 10 days of depletion (Leathem, 1958a). Ovarian stimulation by a gonadotrophin involves tissue protein synthesis and thus the type of whole protein fed

could influence the responses. Yamamoto and Chow (1950) fed casein, lactalbumin, soybean, and wheat gluten at 20 per cent levels and noted that the response to gonadotrophin as estimated by tissue nitrogen was related to the nutritive value of the protein. The ovarian weight response to chorionic gonadotrophin was less in rats fed 20 per cent gelatin than those fed 20 per cent casein (Leathem, 1959b). Inasmuch as the hypophysis may influence the gonadal response to injected hormone despite the diet, hypophysectomized rats fed a protein-free diet for 5 weeks and hyophysectomized rats on a complete diet were tested for response to gonadotrophins. The response to FSH was not influenced by diet, but the protein-depleted rats were twice as sensitive to interstitial cell-stimulating hormone (ICSH), human chorionic gonadotrophin (HCG), and PMS as the normal rats (Srebnik, Nelson and Simpson, 1958). Protein-depleted, normal mice were twice as sensitive to PMS as fully fed mice (Leathem and Defeo, 1952 1 .
c. Vitamins. In the female vitamin B deficiencies do not prevent ovarian responses to gonadotrophin (Figge and Allen, 1942), but the number of studies is limited. Be deficiency in DBA mice was associated with an increased sensitivity of the ovary to gonadotrophins (Morris, Dunn and Wagner, 1953), whereas pyridoxine deficiency in the rat decreased ovarian sensitivity, especially to FSH (Wooten, Nelson, Simp TABLE 12.9
Influence of dietary protein and pregnant mare
serum {PMS) on the mouse ovary
(From J. H. Leathem, Recent Progr. Hormone
Res., 14, 141, 1958.)

Diet fPer cent Protein X Days Fed)

c-5,
•g-s
1*

k
r

1 11

c 1

I.U.

mg.

mg.

per cent X 23

1.2

4.8

per cent X 2.3

3

2.8

13

10.8

per cent X 13

1.4

4.9

per cent X 13

3

4.4

16

1

15.1

6 per cent X 13

3.2

6

7.7

6 per cent X 13

3

5.6

12

1

31.9

18 per cent X 13

5.0

10

2

51.6

18 per cent X 13

3

8.0

7

4

51.3

088

PHYSIOLOGY OF GONADS

son and Evans, 1958 j. Administration of vitamin C concomitant witli gonadotropliin has been claimed to enhance ovarian response (DiCio and Schteingart, 1942), but in another study the addition of ascorbic acid inhibited the hiteinizing and ovulating action of the gonadotrophin (Desaive, 1956).
Whether induced by vitamin deficiency or by inanition, the anestrum in rats which follows 2 to 3 weeks' feeding of a vitamin B-deficient diet has been explored as a method for the assay of gonadotrophin. Pugsley (1957) has shown that there is considerable convenience of method and a satisfactory precision of response for the assay of HCG and pregnant mare serum.
2. Uterus and Vagina
a. Inanition. Limited food intake does not prevent an increase in uterine weight after estrogen. Testosterone propionate will markedly increase uterine growth despite a 50 per cent reduction in food intake (Leathem, Nocenti and Granitsas, 1956). Furthermore, dietary manipulations involving caloric and protein levels did not prevent the uteri of spayed rats from responding to estrogen (Vanderlinde and Westerfield, 1950). More specific biochemical and physiologic responses must be measured because starvation for 4-day periods clearly interferes with deciduoma formation (DeFeo and Rothchild, 1953). A start in the direction of studying tissuecomposition changes has been made by measuring glycogen. However, no changes were noted in uterine glycogen in fasting rats (Walaas, 1952), and estrogen promoted glycogen deposition in the uteri of starved rats as well as in the uteri of fully fed rats (Bo and Atkinson, 1953).
b. Fat. Interest in the hormone content of fat from the tissues of animals treated with estrogen for the purpose of increasing body weight has raised the question of tissue hormone content. If estrogen was to be detected in tissues, an increase in dietary fat was necessary. However, the increase in dietary fat decreased the uterine response to stilbcstrol (I'mberger and Gass, 1958), thus complicating the assay.
c. Vitainins. Stimulation of the uterus by estrogen does not require tliianiinc, ribo

flavin, pyridoxine, or pantothenic acid. On the other hand, a deficiency of nicotinic acid appears to enhance the response to low doses of estrogen (Kline and Dorfman, 1951a, b). However, Bio appears to be needed for optimal oviduct response (Kline, 1955) and is required for methyl group synthesis from various one-carbon precursors including serine and glycine (Johnson, 1958).
Response of the bird oviduct to stilbestrol requires folic acid (Hertz, 1945, 1948). It was shown subsequently that stilbestrol and estrone effects in frogs, rats, and the rhesus monkey also require folic acid. A folic acid deficiency can be induced by feeding aminopterin. In this way the estrogen effects can be prevented. Aminopterin also prevents the action of progesterone in deciduoma formation, from which it may be inferred that folic acid is necessary for deciduoma formation in the rat. Increased steroid or folic acid levels can reverse the antagonist's effect (Velardo and Hisaw, 1953).
The mechanism of folic acid action is not clear. It may function in fundamental metabolic reactions linked with nucleic acid synthesis. Brown (1953) showed that desoxyribonucleic acid could be substituted for folic acid in the bird. In the rat aminopterin interferes with the increase in uterine nucleic acids, and with nitrogen and P-^- uptake by nucleic acids following estrogen. Folic acid has been implicated in the metabolism of several amino acids (Davis, Meyer and McShan, 1956).
Rats ovariectomized at weaning and maintained on a vitamin E-free diet for 6 weeks to 10 months responded to estradiol in the same manner as rats supplemented with tocopherol. This finding suggests that an intimate physiologic relationship between estradiol and vitamin E is not very probable (Kaunitz, Slanetz and Atkinson, 1949). Nevertheless, vitamin E has been re]:»orted to act synergistically with ovarian hormones in dc^ciduoma formation (Kehl, Douard and Lanfranchi. 1951 ) and to influence nucleic acid turnover (Dinning. Simc and Day, 1956).
A vitamin-hormone interrelationship is apparent when estrogen and vitamin A are considered. Vitamin A-deficient female rats present evidence of a metaplastic uterine

NUTRITIONAL EFFECTS

689

epithelium in 11 to 13 weeks, but similar changes failed to develop in ovariectomized rats. Vitamin A-deficient castrated rats quickly developed symptoms of metaplasia when estrogen alone was administered, but no adverse effect followed the administration of estrogen combined with vitamin A (Bo, 1955, 1956). The vagina is different. Its epithelium becomes cornified in vitamin A-deficient normal and castrated rats. The cornification is histologically indistinguishable from that occurring in the estrous rat and can be prevented by vitamin A. In fact, vitamin A will quantitatively inhibit the effect of estrogen on the vaginal mucosa when both are applied locally (Kahn, 1954). Conversion of ^-carotene to vitamin A is influenced by tocopherol, vitamin Bi2 , insulin, and thyroid, with evidence for and against a similar action by cortisone (Lowe and Morton, 1956; Rice and Bo, 1958). An additional vitamin-hormone relationship is suggested by the augmentation of progesterone action in rabbits given vitamin Do .
3. Mammary Gland
Inanition prevents mammary growth, but feeding above recommended requirements for maintenance and growth from birth to the first parturition also seems to interfere with mammary growth. Furthermore, steroid stimulation of the mammary gland is influenced by nutritional factors. Using the male mouse, Trentin and Turner (1941) showed that as food intake decreased, the amount of estradiol required to produce a minimal duct growth w^as proportionately increased. In the immature male rat a limited food intake prevented the growth of the mammary gland exhibited by fully fed controls. Nevertheless, the gland was competent to respond to estrogen (Reece, 1950) . Inasmuch as the glands of force-fed hypophysectomized rats did not respond to estrogen (Samuels, Reinecke and Peterson, 1941; Ahren, 1959), one can assume that, despite inanition, a hypophyseal factor was present to permit the response of the mammary gland to estrogen. However, inanition (IMeites and Reed, 1949) , but not vitamin deficiencies (Reece, Turner, Hathaway and Davis, 1937), did reduce the content of hypophyseal lactogen in the rat.

Growth of the mammary gland duct in the male rat in response to estradiol requires a minimum of 6 per cent casein. Protein levels of 3 per cent and per cent failed to support growth of the duct (Reece, 1959) .
C. PREGNANCY
The human male after attaining adulthood is confronted with the problem of maintaining the body tissues built up during the growth period. However, in the human female it has been estimated that the replacement of menstrual losses may require the synthesis of tissue equivalent to 100 per cent of her body weight (Flodin, 19531. In the event of pregnancy and in all viviparous species, the female is presented with even more formidable demands and a limitation of nutritional needs can lead to loss of the embryo or fetus. The role of nutrition at this point in reproduction has always received considerable attention and is complicated by the circumstance that many food substances influence pregnancy (Jackson, 1959). However, in many instances there is no evidence that fetal loss or malformation induced by nutritional modifications has been the consequence of an endocrine imbalance and thus limitation of the immense literature is permissible.
During the first 15 days of pregnancy, a rat may gain 50 gm. Since the fetuses and placentas are small, most of the gain is maternal and is associated with an' increase in food intake of as much as 100 calories per kilogram of body weight (Morrison, 1956). During the first 2 weeks of pregnancy, marked storage of fat and water occurs in the maternal body and the animal's positive nitrogen balance is above normal. Liver fat also increases (Shipley, Chudzik, Curtiss and Price, 1953). The increased food intake in early pregnancy may therefore provide a reserve for late fetal growth, as food intake may decline to 65 per cent of the general pregnancy level during the last 7 days (Morrison, 1956). During this last week, fetal growth is rapid. The rapid growth has been related to (1) greater demands of the fetus, (2) greater amounts of food in the maternal blood, and (31 greater permeability of the placenta. Certainly the anabolic potential of fetal tissues is high and the mother can lose weight while the

690

PHYSIOLOGY OF GONADS

fetuses gain. But it is also important to recall that there is a shift in protein, because its distribution in organs of pregnant rats differs from that in nonpregnant animals (Poo, Lew and Addis, 1939). Other changes in the maternal organism were enumerated b}^ Newton (1952) and by Souders and Morgan (1957).
A measure of nitrogen balance during pregnancy, rather than weight of young at birth, has been suggested as a means of determining a diet adequate for reproduction (Pike, Suder and Ross, 1954). After the 15th day, a retention of body protein increases, blood amino nitrogen and amino acids decrease, and urea formation decreases. These metabolic activities suggest an increase in growth hormone although the levels of this hormone have not been estimated (Beaton, Ryu and McHenry, 1955). Placental secretions have also been associated with the active anabolic state of the second half of pregnancy, because removal of the fetuses in the rat did not change the anabolic activity, whereas removal of the placentas was followed by a return to normal (Bourdel, 1957). A sharp increase in liver ribose nucleic acid has been observed during late pregnancy in mice and rats and the effect attributed to a placental secretion or to estrogen. Species differences also influence the results because only a modest change in liver RNA was observed in guinea pigs and no change occurred in cats (Campbell and Kostcrlitz, 1953; Campbell, Innes and Kosterlitz, 1953a, b).
Clinical observations have related both
TABLE 12.10
Nutrition and pregnancy in rats
(From J. H. Leathern, in Recent Progress in
the Endocrinology of Reproduction, Academic
Press, Inc., New York, 1959.)

Calories/kg. Body Weight

Fetuses, Day 20

No.

Average weight

18 per cent casein
18 per cent casein
6 per cent casein
per cent casein
18 per cent gelatin
18 per cent gelatin

200 100
250 200 200 100

8 6

gm. 6.1
3.5

toxemia of pregnancy (Pequignot, 1956) and prematurity to inadequate nutrition (Jeans, Smith and Stearns, 1955). The potential role of protein deprivation in the pathogenesis of the toxemia of pregnancy prompted studies in sheep and rats. In sheep nutritionally induced toxemia simulates the spontaneous toxemia (Parry and Taylor, 1956), but only certain aspects of toxemia were observed in the pregnant rat subjected to low protein diets. When rats were fed 5 per cent casein and mated, fluid retention was observed (Shipley, Chudzik, Curtiss and Price, 1953) and pregnancy was completed in only 48 per cent of the animals Curtiss, 1953). Gain in body weight in the adult rat and gain in fetal weight were subnormal as the result of a low protein feeding during pregnancy.
Complete removal of protein from the diet beginning at the time of mating did not prevent implantation but did induce an 86 to 100 per cent embryonic loss. The effect was not related solely to food intake (Nelson and Evans, 1953) , as we will see in what follows when the relationship between protein deficiency and the supply of estrogen and progesterone is described. Limiting protein deprivation to the first 9 to 10 days of pregnancy will also terminate a pregnancy, but when the protein was removed from the diet during only the last week of pregnancy, the maternal weight decreased without an effect on fetal or placental weight (Campbell and Kosterlitz, 1953). As would be anticipated, a successful pregnancy requires protein of good nutritional quality and the caloric intake must be adequate. Thus, an 18 per cent gelatin diet failed to maintain pregnancy when 200 calories per kilogram were fed, whereas a similar level of casein was adequate (Table 12.10). However, reducing caloric intake to 100 calories despite an otherwise adequate protein ration influenced the number and size of fetuses (Leathern, 1959b). Additional proteins should be studied and related to biochemical changes in pregnancy and to the need for specific amino acids; for example, elimination of methionine or tryptophan from the diet may or may not be followed by resorption (Sims, 1951; Kemeny, Handel, Kertesz and Sos, 1953; Albanese, Randall and Holt, 1943). Excretion of 10 amino acids was in

NUTRITIONAL EFFECTS

691

creased during normal human pregnancy (Miller, Ruttinger and Macey, 1954).
That a relationship exists, between the dietary requirements just described to the endocrine substances which participate in the control of pregnancy, is suggested by the fact that the deleterious effects of a proteinfree diet on pregnancy in rats have been counteracted by the administration of estrone and progesterone. Pregnancy was maintained in 30 per cent, 60 to 80 per cent, and per cent of protein-deficient animals by daily dosages of 0.5 /xg., 1 to 3 fig., and 6 jug. estrone, respectively. On the other hand, injection of 4 to 8 mg. progesterone alone maintained pregnancy in 70 per cent of the animals (Nelson and Evans, 1955) , and an injection of 4 mg. progesterone with 0.5 //.g. estrone provided complete replacement therapy (Nelson and Evans, 1954). Food intake did not increase. The results suggest that reproductive failure in the absence of dietary protein was due initially to lack of progesterone and secondarily to estrogen, the estrogen possibly serving as an indirect stimulation for luteotrophin secretion and release. It is well known that hypophysectomy or ovariectomy shortly after breeding will terminate a pregnancy and that replacement therapy requires both ovarian hormones. Thus, the protein-deficient state differs somewhat from the state following hypophysectomy or ovariectomy, but the factors involved are not known.
Pregnancy alters nutritional and metabolic conditions in such a way that labile protein stores of the liver and other parts of the body are influenced, but similar effects are imposed by a transplanted tumor, especially when it reaches 10 per cent of the body weight. ' Thus, transplantation of a tumor into a pregnant animal would place the fetuses in competition with the tumor for the amino acids of the metabolic pool. Under these circumstances will the pregnancy be maintained? An answer to the question may not yet be given. Nevertheless, Bly, Drevets and Migliarese (1955) observed various degrees of fetal damage in pregnant rats bearing the Walker 256 tumor, and 43 per cent fetal loss was obtained with a small hepatoma (Paschkis and Cantarow, 1958).
Essential fatty acid deficiency, at least in the initial stages, does not interfere with

development of the fetuses or parturition in the rat, but the pups may be born dead or they do not survive more than a few days (Kummerow, Pan and Hickman, 1952). A more pronounced deficiency has induced atrophic changes in the decidua, resorption of fetuses, and prolonged gestation. Death of the fetuses appears to be secondary to placental injury. Hormonal involvement, if any, when there is fatty acid deficiency and pregnancy seems not to have been investigated.
Pregnancy and lactation are major factors influencing vitamin requirements. It is not surprising, therefore, that vitamin deficiencies influence the course of a pregnancy. The subject has recently been reviewed by Lutwak-jMann (1958).
A deficiency of vitamin A does not noticeably affect early fetal development, but later in gestation placental degeneration occurs with hemorrhage and abortion. When the deficiency is moderate the pregnancy is not interrupted, but the fetuses are damaged (Warkany and Schraffenberger, 1944; Wilson, Roth and Warkany, 1953; Giroud and Martinet, 1959). In calves and pigs the abnormalities are associated with the eyes and palate (Guilbert, 1942) ; in birds skeletal abnormalities are seen (Asmundson and Kratzer, 1952). The use of hormones in an effort to counteract the effects seems to have been attempted only in the rabbit where 12.5 mg. progesterone improved reproduction impaired by vitamin A lack (Hays and Kendall, 1956). Vitamin A excess also proves highly detrimental to pregnancy, as resorption and malformations occur. Administration of excessive vitamin A on days 11 to 13 of pregnancy induced cleft palate in 90 per cent of the embryos (Giroud and Martinet, 1955) . In another experiment the effect of excessive vitamin A was augmented by cortisone (Woollam and Millen, 1957).
Vitamin E deficiency has long been known to influence pregnancy in rodents and fetal death appears to precede placental damage and involution of the corpora lutea. Gross observations of the abnormal embryos have been reported (Cheng, Chang and Bairnson, 1957). Estrogen, progesterone, and lactogen were not effective in attempts at corrective therapy (Ershoff, 1943), but estrone and progesterone markedly reduced the in

692

PHYSIOLOGY OF GONADS

cidence of congenital malformations associated with vitamin E lack (Cheng, 1959). In the test of a possible converse relationship, estradiol-induced abortion in guinea pigs was not prevented by vitamin E (Ingelman-Sundberg, 1958) .
Fat-soluble vitamins incorporated in the diet may be destroyed by oxidation of the unsaturated fatty acids. To stabilize the vitamins, the addition of diphenyl-p-phenylenediamine (DPPD) to the diet has proven successful, but recent studies show that DPPD has an adverse effect on reproduction and thus its use in rat rations is contraindicated (Draper, Goodyear, Barbee and Johnson, 1956).
Vitamin-hormone relationships in pregnancy have been studied with regard to thiamine, pyridoxine, pantothenic acid, and folic acid. Thiamine deficiency induced stillbirths, subnormal birth weights, resorption of fetuses, and loss of weight in the mother. However, as in the case of protein deficiency, pregnancy could be maintained with 0.5 fjLg. estrone and 4 mg. progesterone (Nelson and Evans, 1955). Estrone alone had some favorable effect on the maintenance of pregnancy in thiamine-deficient animals, but it was less effective in protein-deficient animals.
Fetal death and resorptions as well as serum protein and nonprotein nitrogen (NPN) changes similar to those reported for toxemia of pregnancy (Ross and Pike, 1956; Pike and Kirksey, 1959) were induced by a diet deficient in vitamin Be . Administration of 1 fxg. estrone and 4 mg. progesterone maintained pregnancy in 90 per cent of vitamin Be-deficient rats (Nelson, Lyons and Evans, 1951). However, the pyridoxinedeficient rat required both steroids to remain pregnant and in this regard resembled the hypophysectomized animal (Nelson, Lyoas and Evans, 1953). Nevertheless, a hypophyseal hormone combination which was adequate for the maintenance of pregnancy in the fully fed hypophysectomized rat (Lyons, 1951) was only partially successful when there was a deficiency of pyridoxine. An ovarian defect is suggested.
The folic acid antagonist, 4-aminopteroylglutamic acid, will rapidly induce the death of early implanted embryos in mice.

rats, and man (Thiersch, 1954j . Removal of folic acid from the diet or the addition of x-methyl folic acid will induce malformations when low doses are given and resorptions when high doses are given. Furthermore, this effect is obtained even when the folic acid deficiency is delayed until day 9 of a rat pregnancy or maintained for only a 36-hour period. A deficiency of pantothenic acid will also induce fetal resorption. The vitamin is required for hatching eggs (Gillis, Heuser and Norris, 1942). In animals deficient in folic acid or in pantothenic acid, estrone and progesterone replacement therapy did not prevent fetal loss, suggesting that the hormones cannot act (Nelson and Evans, 1956). In the above mentioned deficiencies replacement of the vitamin is effective. However, vitamins other than those specifically deleted may provide replacement, thus ascorbic acid seems to have a sparing action on calcium pantothenate (Everson, Northrop, Chung and Getty, 1954) .
Pregnancy can be interrupted by altering vitamins other than those discussed above, but the hormonal aspects have not been explored. Thus, the lack of choline, riboflavin, and Bi2 will induce fetal abnormalities and interrupt gestation (Giroud, Levy, Lefebvres and Dupuis, 1952; Dryden, Hartman and Gary, 1952; Jones, Brown, Richardson and Sinclair, 1955; Newberne and O'Dell, 1958) . Choline lack is detrimental to the placenta (Dubnov, 1958), riboflavin deficiency may impair carbohydrate use (Nelson, Arnrich and Morgan, 1957) and/or induce electrolyte disturbances (Diamant and Guggenheim, 1957) , and Bjo spares choline and may be concerned with nucleic acid synthesis (Johnson, 1958). Excessive amounts of Bio are not harmful. It is interesting to note that uterine secretions and rabbit blastocyst fluid are rich in vitamin B]2 (Lutwak-Mann, 1956), but its presence in such large amounts has not been explained.
An additional substance, lithospermin, extracted from the plant, Lathijrus odoratus, is related to hormone functioning; it is antigonadotrophic when eaten by nonpregnant animals and man. The feeding of this substance to prciiiiant rats terminated the pregnancies about the 17th day. Treatment with estrogen and progesterone was preventive (Walker and Wirtschafter, 1956). It is assumed, therefore, that lithospermin interfered with the production of these hormones. A repetition of the experiment on a species in which the hypophysis and ovaries are dispensable during much of pregnancy would be of interest.
In retrospect it has been found that a deficiency in protein and the vitamins thiamine, pyridoxine, pantothenic acid, and folic acid individually can interrupt a pregnancy. Furthermore, a combination of estrone and progesterone which is adequate to maintain pregnancy after hypophysectomy and ovariectomy, is equally effective in protein or thiamine deficiency. This suggests that the basic physiologic alteration is a deprivation of ovarian hormones. However, protein- and thiamine-deficiency states differ from each other as shown by the response to estrogen alone (thiamine deficiency is less responsive), and these states differ from hypophysectomy in which estrone alone has no effect. A pyridoxine deficiency seems to involve both ovary and hypophysis, for neither steroids nor pituitary hormones were more than partially successful in maintaining pregnancy in rats. Lastly, pantothenic acid and folic acid deficiencies may not create a steroid deficiency. What is involved is not known; many possibilities exist. Pantothenic acid, for example, participates in many chemical reactions. Furthermore, it is known that thiamine is essential for carbohydrate metabolism but not for fat metabolism whereas pyridoxine is involved in fat metabolism and in the conversion of tryptophan to nicotinic acid. It is clear, though, that much ground must be covered before the formulation of fruitful hypotheses may be anticipated.
VI. Concluding Remarks
The development, composition, and normal functioning of the reproductive system is dependent on adequate nutrition. However, the requirements are many and only gradually are data being acquired which are pertinent to the elucidation of the nutritional-gonadal relationship.

The demands for nutrient substances is not always the same. During pregnancy and lactation there is a need for supplemental feeding. A similar need exists in birds and in the many cold-blooded vertebrates in which reproduction is seasonal. Atypical endocrine states create imbalances and a need for nutrient materials which vary, unpredictably, we must acknowledge, until the numerous interrelationships have been clarified.
At many points where determination of cause and effect are possible, an indirect action of dietary factors on reproduction is indicated. No other conclusion seems possible in view of the many instances in which the effect of dietary deficiencies can be counteracted by the administration of a hormone or combination of hormones. The direct action is not immediately apparent; it probably is on the processes by which metabolic homeostasis is maintained, and is in the nature of a lowering of the responsiveness to the stimuli which normally trigger these processes into action. The processes may be those by which pituitary and gonadal hormones are produced or they may be the mechanisms by which these hormones produce their effects on the genital tracts and on the numerous other tissues on which they are known to act.
Because of the many interrelationships, some of which are antagonistic and some supportive, determination of the role of specific dietary substances is not easy. For those who work with laboratory species, the problem is further complicated by the many strain differences. For everyone, the problem is complicated by the many species differences which are the result of an evolution toward carnivorous, herbivorous, or omnivorous diets, to say nothing of the countless specific preferences within each group.
Finally, it is something of a paradox in our culture that much of our effort has been devoted to investigations of the effects of deficiencies and undernutrition rather than to the effects of excesses and overnutrition. Much evidence supports the view that in the aggregate the latter are fully as deleterious as the former, but the means by which this result is achieved are largely unknown.

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Book - Sex and internal secretions (1961) 7

The Mammalian Ovary

The Mammalian Female Reproductive Cycle and its Controlling Mechanisms