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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
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, 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.
' Dempsey and Bassett, 1943; Dempsey. 1948; Claesson, 1954; Claesson and Hillarp, 1947a-c ; Claesson, Diczfalusy. Hillarp and Hogberg, 1948; Claesson, Hillarp, Hogberg, and Hokfelt. 1949:
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.
Two factors may have contributed to the 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 similar 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 effort.
II. Folliculogenesis
A. Growth of Primary and Small 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.
More 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 acquired
Fig..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 "competency."
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
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.)
been 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 hypophysectomized rat was more responsive to small doses of pregnant mare serum (PMS) than was the ovarv in the intact immature 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 clearly the local stimulating effect of estrogens with the ovary, as well as the systemic effect by way of the pituitary.
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.)
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 desirable. One of the first suggestions to be made by Edgar Allen (1922; see also the biographical 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( gen which 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 information. Ford 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.
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 tissue persisting after the medullary grafts had disappeared. Ingram concluded that medullary tissue containing interstitial tissue but no follicles cannot survive.
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 species 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 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 consequence of the breakdown of the intcrcclhihii' substance in the stinudated niciubraiia granulosa.
The time r('(|uirc(l for follicular growth and maturation from 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 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 was 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 following 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. Folliculogenesis 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 interrelationhip 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 pseudopregnant animals (Makepeace, Weinstein and Friedman, 1938), pituitary gonadotropliin 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.
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 Luteum
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 corpora lutea; consequently 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 corpora 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, whether the mating is fertile or infertile (Brambell, 1956).
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 (Meyer 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 pituitary. Reece and Turner (1937) showed that estrogen stimulates the rat pituitary 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 phase 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, but 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.
In some species, e.g., 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 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 corpus 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 corpora 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 degenerative 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.
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.)
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.)
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 been 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 atresia than those which have entered the growth phase.
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.)
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 ovarian steroid hormones 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.
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.)
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 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 Origin
As Villee points out in his chapter, 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. 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 progesterone 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
Fig. 7.09. 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 dedifferentiate 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 prominence of interstitial tissue varies from species to species and also with stages of the reproductive 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).
Fig, 7.10. l'(isto\nl.-,l,,iy stigma is evident. Note tli (Courtesy of Dr. J. T. Bradbury.)
epithelioid natiii
of the hypertiopliied mterstitiuin.
(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 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.
Fig.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.)
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).
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.)
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 days after birth which is dosc^ly associated with granulosa ()Ut<j;i()wtlis and ingrowing cords of cells from the germinal epithelium. A secondary type is formed later from the 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 ovaries 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 difficulties; 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 Lipschutz (1950), and Zondek (1954) are among the many who have directed attention to the fact that the amount of an estrogen 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 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 progesterone 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 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 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.
- To a certain 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 percent, and 19.6 per cent of the administered dose was present 12, 24, and 48 hours, respectively, after the administration of the labeled hormone.
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 relationships in the case of other components of i^rotein mixtures have been sliown to be 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 the present 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 Functioning
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 ovarian 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 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, 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. There have been many reports that in the human female the thyoid enlarges at puberty, at menstruation, and during pregnancy (Gamier, 1921; Marine, 1935; Neumann, 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. Many 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 antithyroid 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
The adrenal cortex elaborates its steroid )nn()iu's in a biosynthetic 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 procedurcs 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.)
CHOLESTEROL
CORTISOL
CHOLESTEROL
GONAD
ANDROGEN
ESTROGEN
PREGNENOLONE
PREGNENOLONE
ADHeNAL
17-HYDROXYPROGESTERONE
PROGESTERONE
PROGESTERONE
17-HYDROXYPR06ESTER0NE
Fig. 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 actions 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, 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 (compound 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. We have learned much about the functioning of the ovary, 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.
" The word was not a part of the language of science at that time and probably was never used by them
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Reference: Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
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