Book - Sex and internal secretions (1961) 8
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SECTION C Physiology of the Gonads and Accessory Organs
The Mammalian Female Reproductive Cycle and its Controlling Mechanisms
John W. Everett, Ph.D.
Professor Of Anatomy, Duke University Durham, North Carolina
I. Introduction
The chain of events that constitutes the female reproductive process is characteristically repeated from time to time with considerable regularity during the adult life of an individual, and is therefore a cycle. In the broad sense, this sequence begins with ovogenesis and terminates when the progeny require no further shelter and nurture. In mammals this has become a highly complex process, involving profound maternal adjustments synchronized with successive stages in development of the ovum, fetus, and offspring. The complete mammalian cycle comprises a sequence of stages which may be identified as follows: (II follicle growth, including growth of the ovocyte; (2) ovulation, a progressive process including preovulatory maturation of follicles and ova, and the structural change of ruptured follicles to corpora lutea; (3) progravidity; (4) gravidity; (5) parturition; and (6) postpartum nurture, including lactation, protection, and training. Although it is obvious that this full sequence is often realized, it may nevertheless be retarded or frankly interrupted at almost any point.
In advanced human societies economic and social factors have diminished the number of complete cycles to such degree that they are rarities in the lifetime of an individual and infertile ("menstmal") cycles are the rule. Inasmuch as corresponding factors operate among domesticated animals, the expression "female reproductive cycle" commonly refers to those truly abortive cycles that succeed one another in the absence of insemination. The term is used in that restricted sense in this chapter.
With even that restriction, the female cycle is actually a multiplicity of interlocking cycles, in which the rhythmic interplay between hypophysis and ovary is fundamental. Attention must therefore be focused on the physiology of the ovary and on the hormonal and neural mechanisms that integrate hypophysis and ovary as a functional system. Cyclic alterations in sex accessories and other nongonadal tissues are considered mainly as indicators. The "menstrual cycle," being strictly a uterine cycle, comes in this category, together with changes in behavior.
No attempt is made to present an exhaustive description of the varied adaptive modifications of the ovarian cycle among the several mammalian orders. The reader may consult works of the late F. H. A. Marshall whose full bibliography is given by Parkes (1949 1. Asdell's Patterns of Mammalian Reproduction (1946» is anotlu'i' A-alual)lo source.
II. Cycles Spontaneously Interrupted
Cycles in the natural state are only imperfectly known, from random and often erratic sampling. One may safely assume that, as a rule, under optimal conditions they are complete, fertile cycles. There are, then, relatively few subhuman species in wdiich the characteristics of incomplete cycles have been studied. These species are necessarily the very ones that have been amenable to some form of human restraint.
Segregation of the sexes or any other interference with insemination should be regarded as a first experimental approach to understanding the complete cycle. Such factors unriuestionably operate in nature on occasion. Controlled changes of environmental conditions afford another approach in which natural factors are simulated.
The statement was made earlier that the complete cycle may conceivably be interrupted at almost any point. It has been learned that in different species segregated females interrupt their cycles at different stages and that usually the point of interruption is species-characteristic. These facts have been of great service to the study of reproduction, first, by arousing the curiosity of the investigator and, second, by supplying a variety of ready made conditions individually appropriate for particular experimental studies.
Examples of mammalian cycles are schematically diagrammed in Figure 8.1. It is customary to state that the usual, or standard, infertile cycle is like that in primates or the guinea pig. The follicular phase culminates in spontaneous ovulation, after which corpora lutea are organized and become spontaneously functional for a period of time that is usually considerably shorter than in pregnancy.
In a few animals (rat, mouse, hamster) the cycle terminates shortly after ovulation before the corpora lutea become fully functional. Such corpora lutea are said to be inactive, in the sense that they cannot produce a decidual response to uterine trauma (Long and Evans, 1922). Sterile mating or analogous stimulation induces a luteal phase which corresponds to that of the "standard" mammal. This phenomenon is not entirely limited to the small rodents, having been described in the European hedgehog (Deanesly, 1934).
To this writer's knowledge there have not been described any mammalian species in which it is the rule that in isolated females the process of ovulation begins (follicle maturation, prelutein changes in granulosa, secretion of secondary liquor folliculi, and so on) without proceeding to eventual rupture of the follicles. Many cases could l3e cited, however, in which this has occurred "abnormally." Characteristically, some degree of luteinization occurs in the wall of such a follicle and a lutein cyst is formed.
On the other hand, there are numerous species (reflex ovulators) in which the preovulatory maturation of follicles and ovulation nearly always fail in the absence of the male. The known species in which this is true are widely distributed among the mammalian orders and are often closely related to other species in which spontaneous ovulation is usual. The domestic rabbit
Fig. 8.1. Diagrams of cycles of representative, familiar mammals. , the follicular phase, highly schematized and inaccurate in detail ; , atresia ; i , ovulation ; • , fully active corpora lutea; O, corpora lutea regressing or otherwise not fully active. When sterile mating or equivalent stimulation (SM) is introduced, the cycles of the rat, rabbit and cat become directly comparable with those of the other species.
furnishes the classic exaini)le of reflex ovulation. Other reflex ovulators are the domestic cat (Greulich, 1934), the ferret (Hammond and Walton, 1934), mink (Hansson, 1947), marten (Pearson and Enders, 1944), the 13-lined ground squirrel (Foster, 1934), and the mole shrew (Pearson, 1944). To this list have been added the muskrat (Miegel, 1952) and a field mouse, Microtus californicus (Greenwald, 1956). Even among the marsupials, the female Didelphijs azarae is said not to form corpora lutea in the absence of the male (Martinez-Esteve, 1937). A few of these species display nearly constant estrus (rabbit, ferret), competent follicles being present most of the time in the isolated female during the breeding season.
Among even the spontaneous ovulators the cycle may sometimes not progress beyond the follicular phase. Thus, at the approach of puberty, waves of advanced follicle development and secretion of estrogen may take place without, however, leading to ovulation or corpus luteum formation. The first cycles of primates are often anovulatory ones. In the adult macacjue, at least in some colonies, such cycles are characteristic during the summer months (Hartman, 1932) . A somewhat comparable seasonal effect has been reported in girls soon after the menarche (Engle and Shelesnyak, 1934). Menstrual cycles without ovulation have frecjuently been recognized in adult women in recent years, bearing no evident relationship to seasonal factors (Lopez Colombo de Allende, 1956). Anovulatory cycles were described in the mouse by Allen (1923) and have been noted occasionally in other species, but without clear measure of their incidence.
III. Pituitary-Ovarian Dormancy
Varying levels of pituitary-ovarian dormancy are expressed in different ways from species to species or even from habitat to habitat within a given species. A general similarity exists between the anestrum of seasonal breeders and the prepubertal state. In fact, in animals that have a distinct season, puberty occurs at the very time when older females are emerging from anestrum. Whereas anestrum is often correlated with season of the year, there are exceptions, notably among dogs, in which the correlation is ill defined (Engle, 1946).
In its shortest form ovarian quiescence lasts for only a few days, probably often without being recognized, between the end of one cycle and the active follicular phase of the next. In the chimpanzee it is thought to be the chief factor in the irregularity of length of the cycle (Young and Yerkes, 1943). Rossman and Bartelmez (1946) described a comparable occurrence in monkeys. At the other extreme, anestrum may occupy the major part of the year in monestrous animals that have a very limited breeding season.
A. The Ovary in Anestrum
Generally speaking, depression of ovarian function is most extreme in greatly prolonged periods of quiescence. In the ferret, Hammond and Marshall (1930) reported that in the anestrous ovary follicles can hardly be recognized with the naked eye, because they remain small and deeply placed. The largest follicles at the "end of the season" averaged 460 /x in diameter whereas a "long time after" the average was only 240 jx, increasing again to 720 yu, at the api^roach of a new season. By contrast, the largest follicles of animals in full heat ranged between 1220 and 1440 /x. Follicle atresia abounds in the anestrous ovary of the 13-lined ground squirrel (Johnson, Foster and Coco, 1933). In sheep, however, follicles of large size may be present at any time during anestrum (Kammlade, Welch, Nalbandov and Norton, 1952).
Some moderate degree of secretory activity of the ovary is indicated even at the depth of i^rolonged seasonal anestrum (13lincd ground squirrel, Moore, Simmons, Wells, Zalesky and Nelson, 1934; ferret, Hill and Parkes, 1933; opossum, Risman, 1946). Although at this time uterus, vagina, and vulva are small, ovariectomy or hypol^hysectomy causes a further reduction. On the other hand, these structures are readily stimulated by injection of estrogens.
It may be said that low-grade follicular cycles proceed throughout the anestrous interval, but whether there is any synchronization of one follicle with another is unknown. Some insight into this problem is furnished by study of (1) the transition from anestrum to the breeding season, and (2) the closely analogous phenomena of adolescence. In the report by Hammond and Marshall, it was shown that in ferrets during anestrum and proestrum there is a progressive increase in size of the vulva which directly parallels the diameter of the largest follicles. The absence of overt cyclic change is not surprising in view of the fact that estrus is continuous in this species. In polyestrous animals, on the other hand, it might be expected that during anestrum follicle growth and accompanying estrogen secretion are cyclic, at least at the approach of puberty or of "the season." Important information on this question has been obtained from some of the primates, notably the macaque (Allen, 1927; Hartman, 1932) and the chimpanzee (Zuckerman and Fulton, 1934; Schultz and Snyder, 1935).
Slight transitory reddening of the skin of the perineum ("sex skin") of the monkey may occur at intervals for several months preceding the onset of menses, accompanied by moderate desquamation of vaginal epithelium. During the long intervals of amenorrhea that some individuals exhibit during the summer, there is a tendency toward cyclic vaginal desciuamation (Fig. 8.2). The sex skin of the chimpanzee may begin to swell more than a year before the first menstruation. During the ensuing months the swelling may be irregularly cyclic or continuous. Thus, one may judge that lowgrade follicular cycles, accompanied by periodic increases in estrogen secretion, may succeed one another during seasonal or prepubertal anestrum, but that in certain cases these cycles may overlap to such degree that rather continuous estrogen secretion takes place.
B. The Hypophysis
The secretory activity of the anestrous ovary is apparently adequate to prevent "castration" changes in the adenohypophysis, for as shown by Moore, Simmons, \^'ells, Zalesky and Nelson (1934) removal of the ovary of anestrous ground squirrels results in hypertrophy of the hypophysis,
Fig. 8.2. Vaginal cycles during seasonal amenorrhea in a monkey. (A portion of the record of monkey ^38 from C. G. Hartman, Contr. EmbryoL, Carnegie Inst. Washington, 13, Fig. 26, p. 121, 1932.)
increased gonadotrophin content thereof, and increased numbers of basophile cells. Warwick (1946) reported a highly significant increase of pituitary potency in spayed anestrous ewes. This is closely analogous to the results of ovariectomy in immature animals (Hohlweg, 1934). As measured by ovarian activity, gonadotrophin secretion (release) may be greatly diminished during profound anestrum. The actual hypophyseal content of gonadotrophin seems to be markedly reduced during anestrum in some species (Moore, Simmons, Wells, Zalesky and Nelson, 1934), but possibly not in others. Cole and Miller (1935) and Warwick (1946) reported that there is no seasonal variation in sheep. A study by Kammlade, Welch, Nalbandov and Norton (1952) indicates that the average content is somewhat higher during anestrum than it is in cycling ewes. The major factor in this difference, however, seems to be that during the cycle the potency of the pituitary drops during estrus and the early luteal phase.
Somewhat similarly the potency of the immature rat hypophysis has been stated to be as high as that of the sexually active adult (Clark, 1935). The fact that the ovaries of the immature female or of the anestrous adult can be stimulated by injection of gonadotrophin indicates that gonadotrophin content of, the hypophysis in these cases is not a fair measure of liberation of the hormone into the blood stream. Therefore, it seems justifiable to assume, as Robinson (1951) did in the interpretation of anestrum in the ewe, that, in spite of the possible absence of seasonal assay variation.
there is, nevertheless, a depression of hypophyseal gonadotrophin release during anestrum. We may further assume that it is not completely depressed, for the ovary remains slightly active. Ovary and hypophysis are evidently in a state of equilibrium at a relatively low level of function. It seems likely that this state of affairs is brought about by the central nervous system, inasmuch as the seasonal depression in some species is closely dependent on the daily ratio of light to darkness.
C. Relationship of the Anestrum to the Seasons
This relationship is so varied among different species that many interesting questions are raised. In many cases the midpoint of anestrum coincides approximately with the shortest days of the year (Fig. 8.3). There are other examples, however, largely among the Artiodactyla, in which it coincides with the longest days. Sheep are notable examples (Robinson, 1951). Others, like the European common hare, experience a short anestrum during the time of rapidly decreasing daylight (Asdell, 1946). The Russian yak, on the other hand, is said to experience anestrum from December to May (i.e., while day length is increasing). A general explanation of these varied adaptive manifestations is elusive. There is reason to believe that although illumination, or the light/darkness ratio, (Kirkpatrick and Leopold, 1952; Hammond, Jr., 1953) has a rather direct and primary effect in some cases, its role is more or less indirect in others where such things as temperature, humidity, availability of food and water assume major importance (Marshall, 1942).
Fig. 8.3. Some representative seasonal breeders. Solid bars indicate breeding seasons (according to Asdell, 1946); blank intervals, periods of anestrum. Months of the year represented by letters at top of chart ; winter and summer solstaces marked by wavy lines. Southern hemisphere seasons converted to corresponding ones of the northern hemisphere. End of season for the Bighorn is vmcertain.
The complexity of the i)rol)lem is well illustrated by the 13-lined ground squirrel whose breeding season, like that of a multitude of small rodents, comes in the spring. Moore, Simmons, Wells, Zalesky and Nelson (1934) reported that increasing illumination, elevated temperature, and feeding all failed to bring the females into estrus out of season. If, however, hibernation was first induced by low temperature and darkness, premature estrus would follow. The conclusion was reached that hibernation itself is a necessary prerequisite. Ovarian development actually begins, under natural conditions, in early January in the midst of hibernation. Females exix'i-imentally maintained "continually for several months in cold and darkness, with more or less normal hibernation, [exhibit] sexual development at any time of the year, and periods of estrum have thus been . . . maintained for many months. ..." The impression is given tliat the conditions favoring hibernation also favor sexual development to such extent that breeding potentiality continues for a few months after emergence, in spite of elevated temperatures and long periods of illumination. In another rodent, Peromyscus leucopus, however, the length of daily illumination is of paramount importance. Temperature changes (4 to 25°C. ) have no effect on rejiroduction when lighting is adequate (Whitaker, 1940). Whereas a similar primaiy dependence on lighting can be shown in a number of other species from several orders, it is unwise to generalize that this is usually true.
IV. Attainment of Maturity. Emergence of Full Ovarian Function
Ahhough ('merg(>nce of the ovary from the state of quiescence is gradual, there is usually some outward sign that allows the observer to say that puberty has ari'ived or the breeding season has begun. In primates the accepted sign is the first menstruation ; in rats it is the opening of the vagina ; in many animals it is the swelling and reddening of the genitalia heralding the initial proestrum. In other eases, e.g., sheep, the only clear indication may be the behavior of the female toward the male. From these facts it is readily apparent that any one sign is employed simply because it happens to be accessible to easy observation. Yet the increasing output of estrogen, whether steady or cyclic, affects many parts of the organism at the same time. Furthermore, in any one individual the threshold for exl)ression of a given sign may be relatively liigh with respect to that of some other manifestation. Thus, in Hartman's monkeys (1932), some were noted in which desquamation of vaginal epithelium occurred in wave-like manner for a long time before menstruation. In others "menstrual" bleeding occurred with regularity while the uterus remained very small and A'aginal desquamation was negligible.
Hartman summarized the step-wise manner of maturation of ovary and accessory organs of the monkey during adolescence or following amenorrheic episodes somewhat as follows. The color of the sex skin may be the first to appear. A slight menstrual flow usually takes place before desquamation of vaginal epithelium becomes measurable. "More rarely there may be one or more low desquamation cycles before a bleeding is recorded. Whole cycles marked liy jieriodic bleeding and some vaginal desquamation may occur before there is any noticeable increase in size of the ovaries and uterus. These organs increase also in a saltatory manner, hence the term 'staircase' phenomenon for the process. Finally, the endocrines effect the acme of the reproductive process — ovulation."
Individual variation in the degree of abruptness with which the first ovulation is achieved is well illustrated in a study of puljertal guinea pigs by Ford and Young (1953). In most cases the first period of vaginal opening was much longer than in subsequent cycles. Whatever the duration, ovulation was more closely related to the end than to the beginning of the period, as indicated by histologic study of ovaries.
Even ovulation and corpus luteum formation do not signify that full power of reproduction has arrived. For example, the first cycle of the adolescent rat may culminate in ovulation without sexual receptivity (Blandau and Money, 1943). In the ewe, an ovulation without overt signs of heat may at times take place during the anestrum, especially just before and just after the breeding season (McKenzie and Terrill, 1936). The phenomenon is occasional in ewes during the season and has also been described in cattle (Hammond, 1946). In fact, the full manifestation of estrus in sheep seems to require the presence of a "waning" corpus luteum (Robinson, 1951). In sheep the transition from seasonal or prepubertal anestrum to the breeding season may involve relatively minor changes in hypophyseal activity. Even in the immature rat both the hypophysis and the ovary are capable of far greater secretory function than they normally display. In the equilibrium that prevails, the ovary appears to hold the upper hand by reason of a low hypophyseal threshold at which estrogen suppresses gonadotrophin secretion in the immature individual (Hohlweg and Dohrn, 1932; Byrnes and Meyer, 1951b) and a low ovarian threshold at which gonadotrophin stimulates estrogen secretion. Byrnes and Meyer (1951a) reported that suppression of hypophyseal gonadotrophin content in immature rats can be accomplished with doses of estrogen much smaller than those that affect uterine growth. It is also known that the immature ovary can be induced experimentally to secrete estrogen by injection of amounts of gonadotrophin that are too small to produce significant increase of ovarian weight or follicle development (Levin and Tyndale, 1937; Moon and Li, 1952). When a gonadectomized immature rat is united in parabiosis (Kallas, 1929, 1930 » with a normal or hypophysectomized female littermate, precocious puberty is induced in the latter animal because insufficient estrogen passes to the first partner to inhibit gonadotrophin secretion (see Finerty, 1952). The somewhat analogous experiment of transplanting ovaries to the spleen produces ovarian hypertrophy in much the same way. Here again, it is thought that the hypophysis becomes hyperactive because the amount of estrogen reaching the ghmd is greatly diminished, through inactivation bj^ the liver (Biskind, 1941).
Although it is true that estrogens have a suppressing action on gonadotrophin secretion, it has become increasingly evident that they can also stimulate hypophyseal function in certain ways, as Engle pro{)osed in 1931. Thus short-term injection of estrogen into intact immature rats and mice will invoke precocious puberty not only by stimulating the sex accessories, but also by increasing gonadotrophin secretion and thus causing ovarian growth and even ovulation. Frank, Kingery and Gustavson (1925j reported that after such treatment regular cycles continued after treatment was withdrawn. Lane (1935) found that when 22day-old female rats were injected daily with estrogen there was an early increase in number of ovarian follicles, including vesicular stages. After the first 10 days the nonvesicular follicles became depressed although vesicular follicles were retained. This was interpreted to mean that for a short time estrogen actually stimulates the follicle-stimulating hormone (FSH) l)ut eventually suppresses it, although luteinizing hormone (LH) secretion remains elevated. Hohlweg (1934) had already demonstrated that when somewhat older prepubertal rats are given single, rather large injections of estrogen, ovulation and corpus luteum formation are induced within a few days (p. 514). Obviously LH secretion is greatly increased.
Various bits of evidence implicate the nervous system in the processes leading to puberty and to the onset of estrus in seasonal breeders. This will be discussed in the following section with respect to the general (juestion of the relationship of the hypothalamus to gonadotrophin secretion.
V. Follicular Cycles. Growth and Atresia
Attention will be focused here on the dynamic pattern of follicle development throughout the cycle, the extent to which this i)attern depends on hyi)o[)hyseal conti'ol, and the functional changes in the o\aiy associated with estrus in preparation foi' the more specialized events that lead to ovuhition and corpus luteum formation.
Production of primordial follicles and the early growth stages have been said to be independent of the hypophysis (Smith, 1939; Hisaw, 1947). This view derives from the fact that following hypophysectomy the ovaries retain large numbers of healthy proliferating follicles below the stage of antrum formation. There are, however, several indications that these developmental stages may be accelerated by gonadotrophic stimulation. It was briefly reported by Simpson and van Wagenen (1953) that administration of purified FSH to immature monkeys caused not only a 10- to 20-fold 'increase of ovarian weight, but also stimulation of granulosa in follicles of all sizes. Indirect evidence comes from the fact that follicle atresia generally becomes maximal late in estrus or metestrum, when depressed FSH might be expected on theoretical grounds. Harrison (1948) reported tliat in ovaries of goats killed on the third or fourth days of estrus healthy primary ovocytes are rare. Some few, however, presumably remain. Myers, Young and Dempsey (1936) stated that in the estrous guinea pig there are few nonatretic follicles aside from those destined for ovulation. However, small numbers of normal ai)pearing nonvesicular follicles were found.
There seems to be general agreement that, very quickly after this catastrophic elimination of follicles, renewed growth promptly ensues. Whether or not the wave of atresia represents a depression of FSH secretion, no one would deny that the new growth reflects this type of gonadotrophic stimulation. Characteristically the population of small and medium follicles is restored early in the luteal phase of the polyestrous cycle. This is clearly indicated for the guinea pig ovary (Myers, Young and Dempsey, 1936) when the data are converted from average volumes to average diameters (Fig. 8.4). Beginning on the fourth day after estrus, when the largest follicles are approximately 300 fx in diameter and when theca interna and antra have formed, rapid growth of granulosa, theca, and antra continues for several days. This is confirmed by counts of mitotic figures obtained by the colchicine technique (Schmidt, 1942), indicating greatest mitotic activity in theca and granulosa of follicles between 300 fi and 600 fx in diameter. By the
Fig. 8.4. A schematic repiesentation of the folhcuhir cycle in the guinea pig. The heavj^
sohd curve represents the diameters of the largest follicles, recalculated from the data of
Myers, Young and Dempsey (1936). The arrow point indicates ovulation. The other solid
curves and broken lines represent impressionistically the growth and atresia, respectivelj^
of other groups of follicles that are not ordinarily destined for ovulation.
11th or 12th day the largest follicles (ca. 800 /x) are "competent," i.e., capable of being ovulated (Dempsey, Hertz and Young, 1936; Dempsey, 1937). While the largest follicles are developing to this stage, multitudes of others begin to grow, being carried on to various stages of development before regression sets in.
This pattern of the follicular cycle seems to be generally true among mammals that have been carefully studied, when allowance is made for the fact that from one species to another the characteristic maxima of follicle diameter are extremely variable (shrew, 350 fjL-, rat, 900 /*; cow, 19,000 /x; mare 70,000 /x; Asdell, 1946). In ovulatory cycles of polyestrous animals the greater part of follicle growth is accomplished while the luteal phase of the preceding cycle is in progress. In successive anovulatory cycles like those of the cat the patterns of the follicular cycles are probably much the same (Evans and Swezy, 1931 » . In the rabbit and ferret, where more or less constant estrus characterizes the isolated females in season, there is probably considerable telescoping of successive waves of follicle growth such that as one set of follicles begins to undergo atresia another set is ready to take its place (Hill and White, 1933). The difference between cat cycles and rabbit cycles seems to be chiefly one of degree. The writer has seen both types represented in persistent-estrous rats, among litter mates of inbred strains (Everett, 1939, and unpublished).
At the end of the luteal phase of the
cycle in polyestrous animals there are already present several competent follicles
among an extensive population of smaller
ones. For example, the guinea pig corpus
luteum usually shows signs of regression on
day 13 of the cycle. It has been proved that
ovulation can be induced as early as day
12 by injection of LH (Dempsey, 1937),
several days earlier than it would normalh^
occur (Fig. 8.5). In the human and monkey
it is possible that the "preferred" follicles
are recognizable by their larger size during
Fig. 8.5. The guinea pig follieular cycle and some of its experimental modifications. (After
E. W. Dempsey, Am. J. Physiol., 120, 126-132, 1937.)
or soon after menstruation (Allen, Pratt, Newell and Bland, 1930; Hartman, 1932). In many mammals competent follicles may be present much earlier. Ablation of corpora lutea soon after ovulation in sheep (McKenzie and Terrill, 1936) and cattle (Hammond, Jr., and Bhattacharya, 1944) is followed in 2 to 4 days by another ovulation, much sooner than in the guinea pig (Fig. 8.5). Removal of the primate corpus luteum, at the other extreme, produces no such immediate response, judging from the details of three cases among Hartman's (1932) protocols (#40, #41, and #99). Whereas the next ovulations took place earlier than expectation, the intervals between unilateral ovariectomy and ovulation were 16, 14, and 22 days, respectively.
From detailed investigations in the rat, only the earlier stages of follicle growth may properly be regarded as pure FSH effects (Lane, 1935). Lane and Greep (1935) found that addition of Lli to FSH causes a marked increase in the proportion of vesicular follicles to follicles without antra. The use of more highly purified materials ((irecp, van Dyke and Chow, 1942; Fraenkel-Conrat, Li and Simpson, 1943) has amply confirmed the necessity for combination of the two gonadotrophins to yield maximal follicle growth and estrogen secretion ill rats. Morphologic evidence indicates that
LH acts selectively on thecal tissue and, therefore, on the interstitial tissue derived therefrom. Inasmuch as thecal tissue is the presumptive major source of ovarian estrogen (see below), it follows, perhaps, as Hisaw (1947) suggested that "the theca interna through the action of LH acquires competence to respond to FSH" (by secreting estrogen) .
Convincing evidence that thecal tissue and its derivatives are the principal sources of ovarian estrogen was assembled by Corner (1938). The status of this question remains essentially the same today. Few endocrinologists, however, would assume that no other ovarian cells have this capacity (see discussion in the chapter on the ovary). Nevertheless, there is a direct correlation in time between the marked rise in estrogen secretion as the follicular jihase of the cycle advances, on the one hand, and the organization of tlicca interna of the largest follicles into organs of obvious endocrine character, on the other. "When especially ])rominent the theca interna is referred to as the "thecal gland" (Mossman, 1937; Stafford, Collins and ]\Iossman (1942).
Thecal tissue from the multitudes of atretic follicles should not be neglected as a possible additional source of estrogen. From the standpoint of chronologic relations to the cycle this (iiiestioii has hardly been touched. Pointing up our ignorance, Sturgis (1949) in a careful study of atresia of large follicles in the monkey ovary, speculated that their hypertrophied thecal tissue may serve the useful purpose of estrogen secretion during the interim between follicle rupture and organization of the corpus luteum.
We are in need of ciuantitative appraisals not only of the total numbers of healthy and atretic follicles of all categories present in representative species at progressive stages of the cycle, as in the work on the rat by Mandl and Zuckerman (1952), but also of the respective volumes of theca, granulosa, interstitial tissue, and corpora lutea. Lane and Davis (1939) determined in rat ovaries at four stages of the cycle the respective total volumes of theca, granulosa, and antra in all healthy follicles, as well as the separate mitotic indices of theca and granulosa. Such differential information on multiplication of cells and increase of antral volume is important. Although the latter accounts for a major part of the increase in volume of the larger follicles, it represents a function quite apart from protoplasmic growth per se.
There is now considerable evidence that estrogen itself exerts a growth -promoting influence on the follicle and, furthermore, sensitizes it to gonadotrophic stimulation. Details may be found in papers by Pencharz (1940), Williams (1940, 1944, 1945a, b), Simpson, Evans, Fraenkel-Conrat and Li (1941) , Gaarenstroom and de Jongh (1946) , and Desclin (1949a,) . Although it seems that these effects have not been elicited by physiologic doses, the possibility remains that estrogen operates within the confines of the ovary as a mediator of some of the effects of the gonadotrophins. In the neighborhood of cells that produce it the estrogen concentration is probably far above that which would be considered physiologic for the remainder of the body.
A. Correlation of Ovarian Secretion with the Follicular Cycle
Knowledge of the secretory output of the ovary during the cycle is almost entirely indirect and derives chiefly from (1) substitution experiments carried out in a vari
ety of si)ecies, and (2) assays of urine,
mainly human but occasionally from other
forms. Satisfactory assays of blood estrogen
have been very limited and chemical analysis of the steroid content of ovarian venous
blood is in only its preliminary stages.
The early substitution experiments are chiefly of historic interest (Allen, Danforth and Doisy, 1939). In great measure these investigations constitute crucial steps in proof that the ovary secretes steroid hormones which are fundamentally responsible for the manifestations of estrus. Conversely, then, these manifestations might be considered to reflect an increase of estrogen secretion and their absence a relative decrease. It has been learned, however, that the action of estrogen in certain instances may be greatly modified by progesterone, androgens, and certain adrenocortical steroids (notably desoxycorticosterone). Androgens are known to be secreted in the female by the adrenal cortex (Dorfman and van Wagenen, 1941 ; Gassner, 1952) and by the ovaries (Hill, 1937a, b; Parkes, 1950; Deanesly, 1938; Burrill and Greene, 1941; Pfeiffer and Hooker, 1942; Alloiteau, 1952). Progesterone secretion is probably not confined to the luteal phase of the cycle (see p. 519j. Evidence for its secretion during follicle maturation is considerable and its possible production even earlier cannot be excluded. These considerations make it unwise, therefore, to regard phenomena such as vaginal cornification, turgescence of vulva and sex skin, uterine growth, as direct ciuantitative measures of estrogen output. This point may be illustrated by certain observations made in chim])anzees by Fish, Young and Dorfman (1941) and illustrated in Figure 8.6. Assays of urinary estrogens during the cycle exhibited two peaks, only the first of which coincided with the swelling of sex skin. The second peak of estrogen excretion was unaccompanied by swelling, presumably because of the coordinate increase of progesterone secretion. Had swelling been the only guide only the first peak would have been apparent.
Assays of urinary estrogen in primates have often shown double peaks such as illustrated for the chimpanzee. PedersenBjergaard and Pederson-Bjergaard (1948i.
Fig. 8.6. E.strogen and androgen excretion by a female chimpanzee, Mamo. , total estrogens; , estradiol; -•-•, estrone; , estriol. Menstruation indicated by solid areas on base line. (From W. R. Fish, W. C. Young and R. I. Dorfman, Endocrinology, 28,
588,1941.)
studying one woman for 2 years, found single peaks at midinterval in 8 cycles and double peaks in 12 cycles. On the average the first peak was reached on day 12 and the second on day 21. Similar double peaks were noted in blood estrogen assays in a large group of normal young women (Markee and Berg, 1944). An additional lesser rise was observed during menstruation.
None of the available assays of urinary or blood estrogen can be accepted as an absolute measure of the rate of hormone production. Urinary assays have certain advantages, in spite of the fact that probably only a variable fraction of the ovarian product is measured. Intrinsically they are measures of rate, whereas assays of blood estrogen measure concentration alone at the moment of bleeding. Attempts have been made to measure estrogens in ovarian venous blood, but with little success because of the extreme dilution (Rakoff and Cantarow, 1950). We may hope that development of sufficiently sensitive methods of detection will soon allow systematic evaluation of ovarian output by such direct means. Tracer techniciucs have shown (Werthessen, Schwenk and Baker, 1953) in perfused ovaries of the sow that C^^-acetate enters into the synthesis of estrone and /^-estradiol.
Several years ago Corner (1940) estimated, from the known amounts of injected estrone required to maintain the normal status of sex skin and endometrium in castrates that the ovaries of an adult rhesus monkey secrete the equivalent of about 20 fig. estrone daily. On a weight basis the estrone equivalent secreted by the ovaries of a woman would then be on the order of 300 /Ag. per day. Actual substitution data from castrated women gave an estimate of the same order of magnitude (420 ;u,g. per day). Whatever the rate of secretion may be at different times, it would seem a 'priori that effects on extra-ovarian tissues should be more directly related to amount of estrogen in circulation. The assays of human bloodestrogen in normal women by Markee and Berg (1944) and in gynecologic patients by Fluhmann (1934), although differing in absolute values, agree in indicating that the variation of blood estrogen concentration from one stage of the cycle to another may be relatively small. If this is true, then it nuist be supposed that cyclic changes in the accessory organs are brought about by relatively moderate changes in circulating estrogen. In support of this view Markee (1948) demonstrated in the macaque that a mere 50 per cent reduction in the daily dose of estrogen can invoke menstruation if the change is abrupt.
B. Cyclic Manifestations After Ovariectomy Or Hypophysectomy
Residual cyclic changes in the vagina liave been reported in ovariectomized mice (Kostitch and Telebakovitch, 1929) and rats (Mandl, 1951). The periodicity is very nearly that of the normal cycles, at least in the latter species. Vaginal cycles of similar duration with more extreme estrous changes are found in ovariectomized rats receiving daily injection of threshold doses of estrogens (del Castillo and Calatroni, 1930; Bourne and Zuckerman, 1941). The same was remarked in mice by Emmens (1939) and a report by Veziris (1951) indicates that vaginal periodicity may obtain in castrated or menopausal women receiving estrogen. Although sucli events have been called threshold cycles," the term may simply express the fact that they are most easily recognized when estrogen is given at threshold level. Hartman (1944), employing a modified Shorr stain for vaginal smears, found that castrated rats given large amounts of estrogen daily (5 to 100 fig. estradiol dipropionate) displayed complete cornification at 4- to 5-day intervals. During the time intervening there was admixture of Shorr cells, smaller epithelial cells, and leukocytes.
Analogous phenomena have been recognized in the endometrium of castrated monkeys (Zuckerman, 1937, 1941) injected daily for as long as 1 year with threshold doses of estrone (10 fig.). Larger doses prevent cyclic bleeding (see Hisaw, 1942). From the report of Veziris (1951) it may be judged that threshold endometrial cycles also occur in women and that the vaginal and endometrial cycles are synchronized in considerable extent.
Full explanation of these phenomena is not at hand. From the standpoint of the present discussion certain considerations are especially noteworthy. (1) Vaginal "threshold cycles" have been obtained in castrated rats in the absence of either hypophysis or adrenals (Bourne and Zuckerman, 1941 ; del Castillo and di Paola, 1942) . The former authors encountered the phenomenon in two rats from which both the hypophysis and adrenals had been removed. It is important to remember, however, that the pars tuberalis remains in situ after the usual hypophysectomy procedure, that accessory adrenocortical tissue is frequent in rats, and that gonadal rests might remain unrecognized. (2) The reported lengths of vaginal and endometrial cycles agree favorably with the cycle lengths in intact individuals of the respective species. The degree of conformity between vaginal and uterine cycles indicated by Veziris {loc. cit.) suggests some sort of integrating mechanism. Much more information is required, however, before one may reject the alternative view that rhythmic activity is an innate characteristic of these organs.
C. Cyclic Manifestations in the Absence of Ovarian Follicles
Many years ago Parkes (1926a, b) and Brambell, Parkes and Fielding (1927a, b) reported vaginal and uterine cycles in mice in which the entire follicular apparatus had been destroyed by x-radiation. Schmidt (1936) described the phenomenon in the guinea pig, noting that, although most of her estrous animals had one or more large atretic or cystic follicles, as she had earlier reported (Genther, 1931), a few animals exiiil)ited periodic vaginal opening of short duration and correlated proestrous vaginal smears, in the absence of follicles. Her assays of urinary estrogen were negative in these animals, unlike the positive assays in those in which one or more follicles were demonstrable. Attempts by several workers (Drips and Ford, 1932; Levine and Witschi, 1933; Mandel, 1935) to reproduce in rats the results that Parkes and Brambell had found in mice, were unsuccessful, a fact indicating no estrogenic activity in ovaries completely lacking follicles and ova. Parkes (1952) more recently returned to this problem, reporting vaginal cycles and "fully functional" uteri in castrated rats bearing grafts of ovaries in which all organized follicles and ovocytes had been destroyed by deep freezing. These were true estrous cycles, in the sense that the animals would mate.
Many questions are posed by these observations. The fundamental one seems to be whether these cycles express periodicity of hypophyseal gonadotrophin secretion.
The answer may be long in coming. Meanwhile, one would like to know whether castration changes are visible in the hypophysis and whether constant estrus may be invoked by exposure to continuous light or by postnatal treatment of the host with androgen or other steroids (see p. 529 1 .
D. Hypothalamus and Gonadotrophin Secretion. General Considerations
Experimental studies, ostensibly addressed to the general problem of neural control of gonadotropin secretion, have in fact often been concerned with the special problems of reflex ovulation <p. 520) or of provoked pseudopregnancy (p. 532). Whereas substantial information is now available w^ith respect to these special phenomena, particularly ovulation, information is limited about control of the day-to-day secretion of gonadotrophin that in the female is responsible for follicle stimulation and estrogen secretion (Benoit and Assenmacher, 1955; Harris, 1955). However, evidence in regard to induction of precocious puberty and early onset of estrus in seasonal breeders leaves no doubt that the nervous system is in some manner a regulator of follicle-stimulating activitv of the i)ars distalis.
Numerous reports associate precocious l)uberty with lesions in the hypothalamus (Weinberger and Grant, 1941; Bauer, 1954; Harris, 1955). Donovan and van der Werff ten Bosch (1956) reported off-season estrus in ferrets and precocious puberty in rats following retrochiasmatic lesions in the hyjiotlialamus. Exposure of immature rats to continuous light causes the vagina to open prematurely (Fiske, 1941). When 22-dayold female rats were given electrical stimulation of the cervix uteri daily for 10 days (Swingle, Seay, Perlmutt, Collins, Fedor and Barlow, 1951), a large proportion exhibited significant increase in uterine weight beyond that found in control animals, without change in ovarian weight. In fact, 7 of 50 rats ovulated or at least formed "several well-developed corpora lutca." Somewhat similarly, according to Aron and AronBrunetierc (1953), mechanical stimulation of the vagina or the adjacent segment of the uterus in immature guinea pigs regularly provoked follicle growth and estrogen secretion. In gregarious birds the development of ovulable follicles requires that other individuals of the species be present. In the pigeon, even the mirror image of the female constitutes a sufficient stimulus (Matthews, 1939).
Studies by Flerko and his associates (1954-1957) present consistent evidence that restricted bilateral lesions in the region of the paraventricular nuclei serve to liberate the hypophysis from inhibitory effects of estrogen and androgen. This work is in agreement with that of Donovan and van der Werff ten Bosch in that somewhat similarly located lesions brought on precocious puberty. As noted elsewhere, gonadectomy in immature rats quickly results in hypersecretion of gonadotrophin.
Transplantation of the hypophysis to sites remote from the hypothalamus has produced divergent results. At the present writing, the chief divergence seems to rest between the sexes. In male guinea pigs and rats several workers have reported maintenance of male reproductive tracts by intra-ocular transplants of hypophyses (May, 1937; Schweizer, Charipper and Kleinberg, 1940; Cutuly, 1941a; Courrier, 1956; Goldberg and Knobil, 1957). Quite to the contrary, however, there has at best been only equivocal evidence of maintenance of female tracts, a matter of sex difference which needs full investigation. JNIay's (1937) report of 2 fertile female rats is unacceptable because of inadequate controls. Schweizer, Charipper and Haterius (1937) found in several hypophysectomized guinea pigs that intra-ocular pituitary grafts produced constant estrus and significant follicle stimulation, accompanied by uterine and mammary gland develoi)inent. Although the search for pituitary remnants in the sella turcica was reported negative, the histologic check was limited to scrapings from the sella floor. Other authors, notably Phelps, Ellison and Burch (1939), Westman and Jacobsohn (1940), Harris and Jacobsohn (1952), and Elverett ( 1956a) obtained in female rats little or no evidence of gonadotrophin secretion from apparently healthy, well vascularized grafts. The respective sites were intraniusculai', intra-ocular, in the subarachnoid space under the temporal lobe of the brain, and beneath the renal capsule — all distant from the hypothalamus.
Transplantation of the pars distalis into sites close to the hypothalamus, on the other hand, is characteristically followed by maintenance of the female reproductive tract and essentially normal sex functions. Greep (1936) found that re-implantation of hypophyses into the (presumably) emptied capsule was frequently followed in both male and female rats by return of virtually normal reproductive powers. Females exhibited cycles and even went through successful pregnancy and lactation. The result observed in male rats was confirmed by Cutuly (1941a). The obvious difficulty of establishing completeness of hypophysectomy has been the only criticism of these instructive experiments. This fault has been eliminated by an improved procedure devised by Harris and Jacobsohn (1952). Hypophysectomy was performed by the parapharyngeal route, after which the tissue to l)e grafted was introduced by a transtemporal approach to a site immediately beneath the median eminence. This permitted later histologic search for remnants of the original gland in its capsule. In many cases, including all in which the graft comprised several hypophyses from the animal's own newborn young, entirely normal gonadotrophic function was recorded. This included resumption of regular estrous cycles, typically during the 2nd or 3rd postoperative week. Several of the rats became pregnant and delivered viable litters. In marked contrast, none of the grafts that were placed under the temporal lobe gave any indication of gonadotrophin secretion, although they were as well preserved and richly vascularized as the others. Explanation of the difference seems to be that grafts under the median eminence acquire blood supply from regenerated hypophyseal portal veins and iience a neurovascular linkage with the hypothalamus. The importance of this relationship has been amply confirmed by Nikitovitch-Winer and Everett (1957, 1958d) in studies described below.
In lieu of significant numbers of nerve fibers entering the pars distalis (see Rasmussen, 1938; Harris. 1948a I, the hypophyseal portal veins afford the most likely means by which the gland is brought under hypothalamic control. Recently it was demonstrated in rats and monkeys that these vessels have the power of rapid regeneration after simple stalk-section (Harris, 1949, 1950a, b). This fact at once gives a ready explanation of many of the discordant results of stalk-section experiments reported in the past. Harris (1950b) explored in rats the efficacy of various materials as barriers to regeneration, with the result that numerous examples of partial regeneration were produced. Degree of recovery of gonadotropliic activity by the hypophysis was strikingly correlated with degree of anatomic vascular recovery. Restoration of normal ovarian function after simple interruption of the stalk, as reported in the guinea pig by Dempsey (1939), in rats by Dempsey and Uotila (1940) , and in the human by Dandy (1940), is thus explained by the assumption that portal vein regeneration had taken place. On the other hand, Westman and Jacobsohn (1937-1938), who always inserted a barrier of metal foil between the median eminence and hypophyseal capsule, consistently found ovarian atrophy, as did Harris when portal vein regeneration was completely obstructed. Attempting to prove that the portal vessels are not essential in regulating the hypophysis, Thompson and Zuckerman (1954) stated that increased illumination induced estrus in two ferrets after stalk-section and in the absence of demonstrable regeneration of portal vessels. Donovan and Harris (1954), however, examining the histologic sections prepared from 1 of the 2 animals, found many such vessels that were uninfected. In their own experimental series, an estrous response to light was always associated with regeneration of the portal veins.
Greep and Barrnett (1951) rightly emphasized the prime importance of a good vascular supply for recovery of function by the pars distalis after either transplantation or stalk-section. They pointed to the extensive central infarction and scarring that characteristically followed stalk-section by their technique, an obvious factor contributing to hypopituitarism. Harris (1950a I, however, reported good function from several hypophyses in which there was pronounced central necrosis in company with well regenerated portal vessels. A study by Nikitovitch-Winer and Everett (1957, 1958b) established beyond doubt that qualitative functional losses after stalk-sectron or transplantation of the pars distalis result, not from impaired blood supply per se, but from the loss of the intimate neurovascular relationship with the hypothalamus. Hypo])hyseal autografts, after first being placed under the kidney capsule for several weeks with the usual atrophy of the ovarian follicular apparatus and interstitial tissue, were later retransplanted to a site immediately under the median eminence. In the definitive series of 14 such experiments, 13 rats resumed estrous cycles spontaneously 8 to 68 days after retransplantation ; 7 were fertile and carried litters to term. A correlated study (Nikitovitch-Winer and Everett, 1959) demonstrated clearly that on the occasion of each of these successive transplantations there was massive necrosis of the interior of the glandular mass, leaving but a thin shell from which the functional tissue of the graft was reconstituted. In spite of this double insult some special influence of the hypothalamus brought about renewed function in a surprising number of cases. Together with the restoration of gonadotrophic activity there was significant improvement in thyroid-stimulating hormone (TSH) and adrenocorticotrophic hormone (ACTH) secretion. The considerable net loss of hypophyseal parenchyma resulting from the two operations was reflected only quantitatively in the effects on the various target organs. Ovarian weights, numbers of follicles and corpora lutea, adrenal weights and extent of adrenal hypertroi)liy after unilateral adrenalectomy, and thyroid uptake of P^^ were all intermediate between those of the normal female rat and control animals in which the graft remained on the kidney or was retransplanted under the temporal lobe of the brain.
Regulation of pars distalis secretion by means of the stalk vessels may conceivably be carried out either by regulation of blood How or by transmission of chemical mediatoi-s from the proximal capillary plexus in the median eminence to the pars distalis. An experiment describetl by Swingle, Seay, Perlmutt, Collins, Fedor and Barlow (1951) suggested that a mediator subject to Dibenamine blockade might be involved in precocious puberty. Although significant uterine enlargement was produced in immature rats by daily stimulation of the cervix uteri for 10 days, no such effects were observed in similar rats given Dibenamine daily by stomach tube. Unfortunately, there were no controls for the possible effect of Dibenamine in nonstimulated or gonadotrophininjected animals.
Fluhmann (1952) invoked precocious vaginal opening and ovarian stimulation in immature rats by injection of neostigmine. The locus of such cholinergic action is unknown. Parenthetically, Barbarossa and di Ferrante (1950) reported follicle stimulation in immature rats after injection of intermedin, an effect not found in hypophysectomized subjects. Benoit and Assenmacher (1955) proposed that, in the drake, gonad-stimulating activity is governed by an agent contained in neurosecretory substance, which is demonstrable in abundance in the retrochiasmatic region of the median eminence. Capillaries there drain selectively into an anterior set of portal venules. Oxytocin has been suggested as a possible mediator for gonadotrophin secretion (Shibusawa, Saito, Fukuda, Kawai, Yamada and Tomizawa, 1955; Armstrong and Hansel, 1958). There is much interest as this is being written (1958) in the jiossibility that vasopressin, oxytocin, or other agents associated with neurosecretory substances of the neui-ohyjioiihysis are responsible for control of production and release of the various trophic hormones of the pars distalis. As an alternative or even a supplement to neurochemical regulation, a vasomotor mechanism cannot be denied (Green, 1951), for conceivably only a slight shift in blood flow through the jiars distalis might tip the balance of hormone production one way oi- anothei-. Thus the matter stands: whereas it is apjiarent that the hypothalamus intervenes in follicle growth and estrogen secretion, how it does so is little more than speculative.
VI. Follicle Maturation and Ovulation
A variety of evidence indicates discontinuity between growth of large follicles, on the one hand, and their preovulatory maturation, on the other. Such is clearly the case among "reflex ovulators." Evidence that the same is true for spontaneous ovulators will be outlined below. Follicle maturation, ovulation, and structural transformation of the follicles to corpora lutea seem to represent successive stages in a distinct physiologic process, superimposed on the follicle growth cycle and brought about by a relatively abrupt increase in circulating gonadotrophin (theoretically LH). Since there is evidence (p. 519) that progesterone secretion may become detectable as this process begins, there might be justification for regarding it as merely the first portion of the luteal phase. However, the fact that luteinization ( i.e., the organization per se of luteal tissue) does not necessarily lead to functional cor|)ora lutea warrants treatment of the ovulation-luteinization phase as a distinct phenomenon.
Although it is customary to state that the hypopliysis invokes ovulation by release of LH, there is considerable question about the auxiliary roles played by other gonadotrophic hormones (Hisaw, 1947). Inasmuch as the time of release has been known in only the reflex ovulators, one might look to them for information. However, the available data (Hill, 1934) pertain only to the ovulating i)otency of the total gonadotrophin content of the hypophysis at various times after coitus. Substitution experiments are unsatisfactory because the presence of competent follicles implies the presence of l)oth FSH and a small amount of LH. The substitution of even the purest hormone preparations immediately after hypophysectomy leads to equivocal results inasmuch as it must be assumed that some FSH and LH of intrinsic origin remain in circulation. Talbert, Meyer and McShan (1951) determined that in rats, when hypophysectomy is performed at the onset of proestrum, the follicles remain capable of responding to injected LH for about 6 hours. Morphologic signs of follicle deterioration do not appear until nuich later. Adding to the uncertainty is the fact that relatively pure preparations of either FSH or LH will ovulate an estrous rabbit (Greep, van Dyke and Chow, 1942). On the other hand, until the recent use of species-specific gonadotrophins (van Wagenen and Simpson, 1957), the primate ovary was notoriously difficult to ovulate therapeutically. Until effluent blood from the hypophysis can be assayed, there is little likelihood that the gonadotrophin complex that is normally responsible for ovulation can be known. Thus, whereas the expression, LH-release, will be employed occasionally to refer to the release of gonadotrophin that invokes ovulation, the term is used purely for convenience and brevity, and should be appropriately qualified by the reader.
A. Time Of Ovulation
The time of ovulation with respect to other events of the cycle is relatively easy to determine in reflex ovulators, but in spontaneous ovulators has proven to be more elusive. In the former, laparotomy at various intervals after the stimulus enables exact measure to be made of the time required to accomplish ovulation. For most of the spontaneous ovulators, save the few in which the ripening follicles can be palpated as in monkeys and cattle, it has been necessary to attempt to correlate ovulation with some easily detectable external sign. Inasmuch as the ovulation stimulus to the hyj)oiihysis in these animals is probably invoked by ovarian hormones and these are equally responsible for phenomena such as vaginal cornification and behavioral estrus, a considerable degree of correlation might be expected between ovulation and a given change in the vaginal smear or onset of estrous behavior. The predictability of the relationship, however, must depend in great measure on the degree of correlation among thresholds of response in the various tissues concerned. In the primates that have no sharply limited period of sex desire the i:)roblem is even more troublesome. When reference is made to the date of the last menstruation, prediction is erratic because of the variable occurrence of postmenstrual quiescence (Rossman and Bartelmez, 1943; Young and Yerkes, 1943). Consequently, attempts must be made to find indicato: such as basal body temperature fluctuations which may bear some intrinsically closer relationship to the event in question. (See Hartman, 1936, and Buxton and Engle, 1950, for discussion of this very practical ]iroblera. )
Among mammals generally, si)ontaneous ovulation takes place sometime during estrus (Asdell, 1946) . It is found during early estrus in the opossum, red fox, dog, mouse and hamster. In the rat some authors have placed it early (Young, Boling and Blandau, 1941) and others late (Long and Evans, 19221 with respect to vaginal estrus. In the writer's colony both relations hold, in 4-day and 5-day cycles, respectively. Ovulation in late estrus is reported for the cotton rat, bank vole, guinea pig, pig, horse, and ass. Sheep usually ovulate shortly before the end of heat, sometimes a few hours afterward. As stated earlier, ovulation may even occur in guinea pigs, rats, sheep, and cattle without overt estrus. The cow usually ovulates several hours after the end of heat. The marsupial cat is said to ovulate 5 days afterward (Hill and O'Donoghue, 1913). The extreme is represented by certain bats (Asdell, 1946) which copulate in autumn and ovulate in the spring after a prolonged state of subestrus. These variations probably express several factors.
Among reflex ovulators there is considerable interspecies variation in the interval between the stimulus that invokes release of gonadotroj^hin from the hypophysis and the eventual rupture of the Graafian follicles (rabbit, ca. 10 hours; ferret, ca. 30 hours; cat, 24 to 54 hours; 13-lined ground sciuirrcl, 8 to 12 hours; mink, 36 to 50 hours) . Among spontaneous ovulators the comparable interval is clearly defined for only the rat, 10 to 12 hours (Everett, Sawyer and Markce, 1949) . In the cow the data obtained by Hansel and Trimberger (1951) and Hough, Bearden and Hansel (1955) i)lace the outside limit at about 30 hours. Here again, threshold differentials among tlic various tissues of the individual are piobably of gicat importance. 'I'hus in one species the threshold foi' gonadoti'ophin release may be lower than that foi' estrous behavior with the result tliat by the time the latter makes its ai)pearance the former has already transpired and ovulation will shortly take place. The rat, for example, releases LH during the afternoon, begins to show estrous behavior around 8 p.m., and ovulates around 2 a.m. (Everett, 1948, 1956b). In other species these time relationships may be reversed. In the cow, activation of the hypophysis apparently occurs several hours after the onset of estrus (Hansel and Trimberger, 1951). The cow remains in heat 10 to 18 hours and ovulates 13!/2 to 151/2 hours after going out of heat (Asdell, 1946). The early termination of estrus apparently reflects a refractory state which sets in after estrogen activity has continued for a time, for castrates receiving continued estrogen therapy remain in estrus for similarly brief periods. In the mare, ovulation is delayed until a few hours before the end of estrous periods that may extend for 5 to 10 days or longer. This suggests a relative refractoriness of the LHrelease mechanism in this animal. Such a state of affairs approaches that in persistent estrus or in the anovulatory cycle.
B. Ovarian Steroms and Ovulation
1. Estrogens
Chronic administration of estrogen to the intact animal eventually produces ovarian atrophy by suppression of gonadotrophin secretion. However, some moderate basic level of continuous estrogen secretion must be compatible with normal function of the hypophyseal-ovarian system; witness the fact that blood estrogen assays in normal women (]Markee and Berg, 1944) indicate only a 2-fold increase at midinterval above a base value of considerable magnitude.
Induction of corpus luteum formation by injected estrogen was first demonstrated by Hohlweg (1934) in prepubertal rats^ and the phenomenon has been repeatedly observed by other woi'kers (Desclin, 1935; Mazer, Israel and Aljjcrs, 1936; Westman and Jacobsolm, 1938b; Herold and Effkemann, 1938; Price and Ortiz, 1944; Cole, 1946). The fact that the effect was not obtained in rats younger than 30 to 36 days l>y Piice and Ortiz, whereas Cole observed it in the age-range of 21 to 28 days, demonstrates the existence of strain differences in the age factor. This probably explains the absence of luteinization in the experience of Lane (1935) and Merckel and Nelson (1940). Hohlweg and Chamorro (1937) demonstrated the importance of the hypophysis in the response. When hypophysectomy was performed 2 days after injection of estrogen no corpora liitea developed, but hypophysectomy on the 4th day did not interfere with corpus luteum formation. The effect could be produced in 50-gm. rats with as little as 4 |U,g. estradiol benzoate. Westman and Jacobsohn (1938b) reported that transsection of the hypophyseal stalk less than 2Vt days after injection prevented the reaction, but after that time the operation did not interfere. Bradbury (1947) assayed the gonadotrophin content of hypophyses of normal and castrated immature rats (30 to 32 days old at autopsy) 2 to 5 days after injection of estrogen or other steroids. These rats were apparently too young to form corpora lutea in response to the treatment, l)ut marked interstitial-cell stimulation, indicative of LH (ICSH) activity, was observed as early as 96 hours. In the intact animals significant loss of potency occurred 72 to 96 hours after injection, in agreement with the hypophysectomy data of Hohlweg and Chamorro (1937). In castrated rats, however, there was no loss of potency, thus suggesting that some ovarian factor in addition to estrogen is essential for stimulation of the hypophysis. It is unfortunate that the study was confined to animals too young to give the full response of luteinization.
- The effect was later obtained with androgens (Holilwpg, 1937; Salmon, 1938; Xathanson, Fianspen and Sweenev, 1938).
Fig. 8.7. Experimental modifications of the 5-day cycle in rats. Two units of the ordinate
represent full vaginal estrus. Time in days on abscissa, each unit 24 hours (midnight to midnight). X, ovulation time; -p, progesterone, usually 1 to 2 mg.; e, estradiol benzoate, standard
do.se 50 /xg. (From J. W. Everett, Endocrinology, 43, 393, 1948.)
Induction of ovulation in adult animals
by estrogen was first reported by Hammond,
Jr., Hammond and Parkes (1942) and by
Hammond, Jr. (1945) in the anestrous ewe.
Whereas the s])ontaneous occurrence of occult ovulation was approximately 5 per
cent, injection of stilbestrol was followed by
corpus luteum formation in 4 of 11 ewes,
with recovery of ova in 3. Injection of stilbestrol di-n-butyrate was followed by corpus luteum formation in 5 of 6 ewes and ova
were recovered in 3. The finding was confirmed by Casida (1946) who stated that in
cycling ewes ovulation can be invoked by
injection of diethylstilbestrol on the 4th day
of the cycle, but not at other times. In 1947
Everett reported the induction of ovulation
in pregnant rats within 40 hours after injection of estradiol benzoate (as little as 2 or
3 /xg.) or implantation of estradiol crystals
or pellets. The response was not obtained in
animals autopsied 24 hours after treatment
nor in other animals hypophysectomized at
24 hours and autopsied the following day. In
other studies with adult rats it was demonstrated (Everett, 1948) that in 5-day cyclic
rats the injection of estrogen at mid-diestrum will regularly induce ovulation 24
hours earlier than exi:)ected (Figs. 8.7D,
8.8F|. Persistent-estrous rats were refractory to estrogen in this respect.- Nevertheless, when such animals were made pseudopregnant by daily injection of progesterone,
"The tendency toward refractoriness of similar animals with respect to induction of estrous 1) ha\'ior had earlier been reported by Boling, Blandau, Rundlett and Young (1941).
Fig. 8.8. Experimental modification of the 4-day cycle in rats. Same key as in Figure 8.7. Progesterone dosage 1.5 mg. per day. Artificial 5-day cycles in D, E, and F indicated by dotted lines and numbering. (From J. W. Everett, Endocrinology, 43, 395, 1948.)
Fig. 8.9. Experiment with persistent-estrous rats. Units of ordinate and abscissa have same
meaning as in Figure 8.7. A. Secjuence of "progesterone cycles." Each dose of progesterone
(p) is 1.0 mg. Ovulation (x) in about 70 per cent of the cycles. B. Progesterone cycle followed
by unsuccessful attempt to induce ovulation by estrogen during the second c-ycle. C . Pseudopregnancy maintained by daily iiijoctinn of 1.5 mg. ]irogosteronr. Ovulation induced by
estrogen in several such cases. (From .1. \V. Everett, EiKhxTinolojiy. 43, ;5i»9, 194S.)
ovulation and corpus luteuni loiniatioii were induced by estrogen (Fig. 8.9 1.
Early attempts to induce luteinization in the guinea pig with estrogen were unsuccessful ( Dempsey, 1937; see Fig. 8.5), but iiioiv recently Lipschutz, Iglesias, Bruzzone, 11 uniercz and Penaranda (1948) have shown by the use of intrasplenic ovarian autografts that luteinization is a reguhir feature in experiments in which estrogen is administered systcniically. Interestingly enough the implantation of estrogen jiellets in or near the ox'ariaii grafts had the coiitrai'y effect of pi'cvcnliiig luteinization.
it was early I'cportcd that rabbits fail to ovulate in response to estrogen injection
(Bachman, 1936; Mazer, Israel and Alpers,
1936 ». Hisaw (1947j inferred that this is
generally true for reflex ovulators. Nevertheless, it was found by Klein and Mayer
( 1946) and Klein (1947) that when pseudol^regnant or pregnant rabbits were treated
with estrogen and then mated, new ovulation resulted and new corpora lutea were
formed, events that do not otherwise occur.
The phenomenon was further explored by
Sawyer (1949). Whereas untreated rabbits,
unlike cats, do not ovulate in response to
mechanical stimulation of the vagina, treatment with estrogen on the preceding 2 days
results in a positive response to this stimulus. In fact, his later observations (1959)
indicate that estrogen priming for a longer
period (4 days) occasionally results in
"spontaneous" ovulation, especially during
the winter and spring.
In the anestrous cat, in the response to mechanical stimulation of the vagina, estrogen facilitates the ovulation of follicles primed with equine gonadotrophin (Sawyer and Everett, 1953).
Induction of ovulation by estrogen in primates remains to be demonstrated. It is of interest in this connection that Funnell, Keaty and Hellbaum (1951) observed in menopausal women an increased excretion of LH during estrogen therapy, in contrast to FSH excretion at other times. The general experience has been that injection of estrogen during the early part of the cycle significantly postpones the next expected ovulation and menstruation (monkey, Ball and Hartman, 1939; baboon, Gillman, 1942; human, Sturgis and ^leigs, 1942; Brown, Bradbury and Jennings, 1948). Gillman reported that a single injection of estrogen precipitates widespread atresia of vesicular follicles. Brown and Bradbury (1947) reported IH-eliminary data that in 4 of 6 women there was increased gonadotrophin excretion during the 24 hours following estrogen administration. They proposed that delay of ovulation by estrogen given early in the primate cycle may be the result of premature discharge of gonadotrophin before the Graafian follicle is competent. Sturgis and Meigs had suggested, on the contrary, that the estrogen suppresses hypophyseal function. D'Amour (1940), finding in urinary assays that the initial peak of estrogen ex
cretion preceded the peak excretion of urinary gonadotrophin, postulated that the
increase of estrogen stimulates the gonadotrophin release that is responsible for ovulation. O. W. Smith (1944) proposed that
not estrogen itself, but some metabolite resulting from inactivation by the liver, is
responsible for LH release. This interesting
hypothesis has not been substantiated.
3. Gestagens
Suppression of estrus and ovulation by functional corpora lutea, suggested by Beard (1898), was experimentally demonstrated in the guinea pig by Loeb (1911). It is now well established in several species that removal of the corpora lutea results in early resumption of estrus and ovulation (see p. 506), and that administration of progesterone suppresses these events. There is considerable evidence favoring the view that the primary effect is to selectively suppress the secretion of LH. Dempsey (1937) noted that in guinea pigs receiving daily injection of progesterone (50 /^g.) all stages of follicle development proceeded except the maturation enlargement that heralds LH release (Fig. 8.5). Astwood and Fevold (1939) and Cutuly (1941b) found similar results in rats. Essentially the same phenomenon has been noted in sheep by Dutt and Casida (1948). Bradbury (1947) reported that in immature rats the injection of progesterone at the time of estrogen injection prevented the release of gonadotrophin (LH?) which otherwise followed estrogen injection by 72 to 96 hours. In ovariectomized guinea pigs containing intrasplenic autografts, preparations in which luteinization can be induced by estrogen (see above) , the simultaneous administration of gestagens prevented this action (Lipschutz, Iglesias, Bruzzone, Humerez and Pefiaranda, 1948; Iglesias, Lipschutz and Guillermo, 1950; Mardones, Bruzzone, Iglesias and Lipschutz, 1951). Mardones and co-w^orkers also made the interesting observation that among several steroids having progestational activity, "antiluteinizing activity is not concomitant with, or subordinated to" the former function. Proportionately very large amounts of ethinyl testosterone and ethinyl-A-'^-androstenediol exhibited very little antiluteinizing activity. There is evidence in mice (Solve,, 1939) that suppression of FSH secretion may occur when as much as 1 mg. of progesterone is injected daily. Alloiteau ( 1954 ) believes that this also occurs in the rat, although Cutuly (1941b) found only slight evidence of FSH suppression when as much as 6 mg. progesterone were given daily for several weeks.
So much emphasis has been placed on the suppressing effect of progesterone that its facilitating actions were recognized only in recent years. The first indication that progesterone can promote ovulation and corpus luteum formation in mammals was encountered in a study of persistent-estrous rats (Everett, 1940a, b). Daily injection of 0.25 to 1.0 mg. caused the prompt interrujition of the state of persistent follicle and the resumption of outwardly normal cycles. Corjiora lutea were formed in approximately 70 per cent of these cycles.^ The effect was obtained not only in older rats in which persistent estrus had developed spontaneously, but also in young rats in which the condition had been induced by continuous illumination. The dose level employed is below that required to suppress cycles in normal rats (1.5 mg. daily; Phillips, 1937). Subsequently, it was found (Everett, 1943) that the daily injection could be avoided if a single "interrupting" dose was given, followed by a single injection during proestrum or early estrus of each recurrent cycle (Fig. 8.9.4). The histologic appearance of the ovaries reverted toward the normal after a succession of "i)rogesterone cycles" and, significantly, the interstitial-cell nuclei were "repaired." Attempts in normal rats to invoke o\'u]ati()n earliei' than the expected time were sticcessful in the 5-day cycle (Figs. S.7B, 8.8^). Injection of from 0.5 mg. to 2 mg. on the "third day of diestrum" regularly (4 mg. occasionally) invoked ovulation (luring the coming night (Everett, 1944a, 1948) unless the treatment was given too late in the dai/ (EA'erett and Sawyer, 1949; see discussion on ]). 526 I'egarding the diurnal rhythm and ovulation). Attempts to advance ovulation in the 4-day cycle were unsuccessful, possibly because the follicles were not competent or the animals'
Marvin (1947) described a similar rosull willi desoxycorticosterone acetate.
intrinsic estrogen levels were not elevated sufficiently early.
Ovulation induced by progesterone has been reported in several species. A direct action on the excised ovary of the toad Xeno-pns was early demonstrated by Zwarenstein (1937) but such action is apparently not found in higher vertebrates. In the domestic hen injection of progesterone can invoke ovulation several hours ahead of schedule (Fraps and Dury, 1943; Rothchild and Fraps, 1949). Pfeiffer (1950) observed new corpora lutea in 10 rhesus monkeys treated with progesterone during presumptive anovulatory cycles of the summer months. Similar attempts have been made in women (Rothchild and Koh, 1951); although there were said to be definite indications of induced ovulation, the evidence is equivocal. On the other hand, a rei~)ort (Hansel and Trimberger, 1952) states that in heifers the injection of small doses of progesterone (5 to 10 mg. ) at the beginning of estrus significantly advances ovulation time. This is in contrast with the inhibitory effect of larger doses (50 mg.) beginning before the onset of estrus (Ulberg, Christian and Casida, 1951). Even in the rabbit (Sawyer, Everett and Markee, 1950), spontaneous ovulation was noted in 4 of 10 animals after combined estrogen and progesterone injection.
From certain of the foregoing statements it may be inferred that whether suppression or stimulation follows administration of ju-ogestcrone depends critically on the time of injection, on the amount given, on the status of the ovary, and probal)ly on a l)riming action of estrogen. A significant illustration of the critical nature of the time factor in rats is given by the experiments represented in Figure 8.8r and E. If after the first injection of 1.5 mg. progesterone on the first day of diesti'uni, a second injection follows in. about 24 lioui's. the imjiending estrus and o\-ulation are retarded an additional 24 hours. However, if the second injection is given 48 hours after the first, the impending estrus and ovulation are advanced. Evidence^ of the synergistic action of estrogen and progesterone in evoking ovulation is given by the ex]ieriments represented in Figuiv 8.9/> and (\ Sawver (1952) investigated the synergism in rabl^ts. Employing estrogen-primed animals, he found that ovulation was facilitated when progesterone was injected less than 4 hours before either mating, mechanical stimulation of the vagina, or intravenous injection of copper acetate. Inhibition was obtained when progesterone was injected 24 hours before such stimulation, thus confirming the often-cited observations of ]Makepeace, Weinstein and Friedman (1937 » and Friedman (1941) that progesterone inhiijits ovulation in rabbits.
Preovulatory secretion of gestagens now seems likely. Morphologic luteal changes in preovulatory follicles are considered in the chapter on the ovary. A variety of evidence in primates indicates that progestational clianges in the endometrium begin before ovulation (Bartelmez, Corner and Hartman, 1951). Several workers have reported tiie excretion of small amounts of pregnanediol during the follicular phase of the human cycle (Wilson, Randall and Osterberg, 1939; Smith, Smith and Schiller, 1943; Davis and Fugo, 1948). Determination of plasma progesterone in women by the Hooker-Forbes test indicates the presence of significant amounts a day or two before a major rise in basal body temperature (Forbes, 1950). In monkeys a small quantity (ca. 0.5 to 1.0 ixg. per ml.) was detected l)etween the 4th and 9th days, rising in the 10- to 15-day period to concentrations of 2 to 6 jxg. per ml. (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). In both species a transient decline seems to intervene before the marked rise to still higher concentrations during the luteal phase. In the rat, Constantinides (1947) studied the stromal nuclei of the endometrium at different stages of the cycle and found that by the Hooker-Forbes criteria there is evidence of progesterone secretion during proestrum. Astwood (1939) on the basis of water content of rat uteri concluded that progesterone secretion begins with proestrum. In the rabl)it, Forbes (1953) assayed peripheral blood at various intervals after mating or gonadotrophin injection. Although no progesterone was detectable in controls, significant amounts appeared an average of 97 minutes after mating and 66 minutes after gonadotrophin injection. As much as 2.5 /xg. ])er ml. was found during the first 8 to 10 hours, although marked fluctuations were noted from time to time in the blood of individual animals. Verly (1951) reported that soon after mating the urine of rabbits contains significant amounts of pregnanediol.
It has become customary to state that the gestagen that appears during the follicular phase of the cycle is probably formed by the maturing follicle itself. Indeed, assays of fluid from Graafian follicles and cysts have indicated the presence of the hormone (Duyvene de Wit, 1942; Hooker and Forbes, 1947; Edgar, 1952, 1953). However, if it is to take part in the release of ovulating hormone gestagen must be secreted earlier than preovulatory maturation. For this also there is some evidence. Two reports cited above indicate that in monkeys, at least, there is a detectable amount present in the blood during the early follicular phase. The known fact that a waning corpus luteum favors the experimental induction of estrus and/or ovulation in sheep and cattle (Hammond, Jr., 1945; Robinson, 1951 ; Alarden, 1952) is suggestive. Although Hammond, Jr., Hammond and Parkes (1942) tested this possibility by progesterone sul)stitution with negative results, the amount given may have been too small, as Robinson suggested. A waning corpus luteum in the rat favors ovulation, as disclosed in persistent-estrous animals in which pseudopregnancy had been induced (Everett, 1939). Each of three pseudopregnancies was followed b}' a short cycle before the animals returned to persistent estrus.
In the course of studies growing out of this observation evidence was advanced (Everett, 1945) which indicated that corpora lutea of the normal rat are not wholly inactive during the short cycle. Transient depletion of cholesterol was observed in such corpora lutea during the proestrum that followed their formation. This implies a transient increase of luteotrophin secretion. Significantly this occurs before the release of LH. It is this writer's opinion that gestagen from such sources is not essential for the induction of ovulation but that it does facilitate the action of estrogen.
C. Role of the Nervous System in Ovulation
Historically, the fact of neural control of reflex ovulation has been recognized in the ral)bit for many years. The comparable role of the nervous system in spontaneous ovulation, on the other hand, has more recently become apparent. It now seems justifiable and useful to postulate in the hypothalamus of reflex ovulators and spontaneous ovulators alike the existence of a mechanism that is peculiarly concerned with release of ovulating hormone. Whether it consists in a discrete anatomic entity is immaterial for the present.
The suggestion has been made that the outstanding difference between reflex and sjiontaneous ovulation may be in the kinds of afferent impulses that most readily excite the hypothalamus (Sawyer, Everett and Markee, 1949). The difference is not absolute, for spontaneous ovulation has been induced in rabbits by estrogen-progesterone injection (see p. 519) and reflex ovulation has been demonstrated under special circumstances in rats (Dempsey and Searles, 1943; Everett, 1952a) and cattle (Marion, Smith, Wiley and Barrett, 1950). The random distribution of reflex ovulators and spontaneous ovulators among mammalian orders becomes more understandable if one assumes that dual controls are widely represented and that special adaptations favor one or the other in given species.
The ovulation reflex in rabbits is apparently initiated by afferent impulses of multiple origin, among them not only impulses from the genitalia, but also propriocejUive impulses from muscles that are utilized in coitus. Brooks (1937, 1938) found that, although the sacral segments of the spinal cord and the abdominal sympathetic chains could be eliminated without jM'eventing ovulation, the luml)ai' cord must remain. Only by paralysis of the lower half of the body so that the female could not take pai't in coitus was ovulation pi'cvcntfMl. The neocortex could be removed, together with the olfactory bulbs, labyrinths, auditory apparatus and eyes without impairing the ovulation response. Even after complete decortication, ovulation followecl coitus in 1 out of 3 j'abbits. It must be admitted, however, that, although various parts of the nervous system may thus be eliminated without changing the end result, some of them may normally play a considerable role. With little cjuestion, direct stimuli from the genitalia play a part in the natural reflex. Under certain experimental conditions detectable electrical activity in the rabbit rhinencephalon is associated with the induction of ovulation (Sawyer, 1955). Electrical stimulation of the amygdala will induce ovulation in rabbits and cats (Koikegami, Yamada and Usei, 1954; Shealy and Peele, 1957 ) .
In rats, Davis (1939) found that removal of the neocortex had no effect on the estrous cycle and ovulation. Removal of portions or of the entire sympathetic chains of rats likewise did not interfere with ovulation (Bacq, 1932). Bunn and Everett (1957) reported ovulation in constantestrous rats after electrical stimulation of the amygdala.
The importance of the dorsal thalamus is unknown. The reticular activating system has been implicated as a component of the ovulation mechanism (Sawyer, 1958), but the manner of its contribution is not clear. There is little cjuestion, on the other hand, of the indispensability of the hypothalamus and its neurovascular connection to the adenoliyi:)ophysis through the median eminence and the hypophyseal portal veins.
Although the observation by ]\Iarshall and Verney (1936) that ovulation can be induced by passing an electric current through the heads of estrous rabbits hardl}^ limited the effect to the hypothalamus itself, it was later shown that more localized electrical stimuli api)lied to certain hypothalamic regions are consistently effective (preoptic area, Haterius, 1937; Christian. 1956; posterior hypothalamus or tuber cinereum, Harris, 1937, 19481); tuber cinereum or adjacent hypothalamic areas, Markee, Sawyer anirHollinshead. 1946; medial hypothalamus fi'om ])i-eo])tic area to mammillai'V bodies. Kui'otsiu Kurachi and Ban, 195o"; Kuiotsu, Kurachi, Tabaya>hi and Ban, 1952).
Ahliougli liypotliahimic lesions. l)oth natural and expeiimental. hax'c frecjuently been reported to interfere with normal sex function (.see Harris. 1948a, 1955, for references), the majority of these reports do not api^ly to the question at issue — control of ovulation. When ovarian atrophy occurs, as it frequently did in these cases, it reflects a profound depression of gonadotroi)hin secretion and absence of competent follicles. However, Dey, Fisher, Berry and Ranson (1940) and Dey (1941, 1943) 'found in guinea pigs that gross bilateral electrolytic lesions placed in the rostral hypothalamus resulted in persistent follicles with continuous estrogen secretion. Similar results were obtained in rats by Hillarp (1949) when small bilateral electrolytic lesions were placed in the anterior hypothalamic area near the paraventricular nuclei or between this region and the median eminence. Greer (1953) reported continuous estrus in rats after placing certain small lesions in the ventromedial nucleus, provided they were bilateral. There was no correlation with obesity. There are at least four significant points in common among these several ablation experiments. ( 1 ) The effective lesions were rost rally placed and either were limited to or included the medial group of nuclei. (2) The tuber cinereum, median eminence, and stalk connection to the hypophysis were intact. (3) Although development of competent follicles was not evidently impaired, estrogen secretion became continuous instead of cyclic. (4) The proper impetus for release of ovulating hormone from the hypophysis was absent. It would be most instructive to learn whether ovulation can be invoked in such animals by reflex stimulation or by direct electrical stimulation of the tuber. AUoiteau and Courvoisier (1953) reported that rats in constant estrus as a result of hypothalamic lesions did not undergo pseudopregnancy after stimulation of the cervix uteri. This observation, confirmed by Greer (1953), could be construed as indirect evidence of failure of reflex ovulation, for Greer regularly obtained pseudopregnancy by cervical stimulation, once corpora lutea had been formed by other means.
Other findings by Greer are important because of their bearing on the location and character of a presumptive ovulation center. Althougli the onset of persistent estrus after making the lesions was sometimes almost immediate (following a brief anestrous interval), in other cases it was preceded by several apparently normal cycles. In any event, once the condition had become established, the daily injection of small amounts of progesterone brought about the recurrence of cycles and corpus luteum formation. In about half of the cases these cycles continued for awhile after withdrawal of treatment, whereas in the remainder there was a prompt return to persistent estrus. Essentially the same results were reported by AUoiteau (1954), and the observations suggest that the areas involved in such lesions may be of only secondary importance.
The use of radioactive phosphorus for estimating energy exchange in tissues affords a different approach to the problem of neural control of ovulation (Borell, Westman and Orstrom, 1947, 1948). This method has the virtue that the experimental subject remains undamaged until injection of P'*compounds 30 minutes before the end of the experiment. In rabbits there is a marked increase in phosphorus turn-over in the tuber cinereum within 2 minutes post coitum, and continuing for about an hour thereafter (Table 8.1). The adenohypoi)hysis shows increasing activity during the first 10 minutes which reaches a peak at about 1 hour and then regresses somewhat, although it remains relatively high for 24 hours. Response of the ovary to gonadotroi:)hin release is marked by a rapid rise during the second half-hour and another pronounced increase near the time of ovulation. In rats, at various stages of the estrous cycle, phosphorus exchange in both tuber cinereum and adenohypophysis is maximal during proestrum. In the ovary high values were reported during diestrum and proestrum, somewhat lower values during estrus and metestrum.
Possibly correlated with the above information is the observation (Gitsch, 1952b) that in rats the acetylcholine (ACh) content of the tuberal region becomes elevated during proestrum and estrus. It is said that ACh synthesis requires high energy phosphate (see Bain, 1952). Further investigation by Gitsch (1952a) and Gitscl: and Reitinger (1953) revealed that ACh
TABLE 8.1 Sequence of events in rabbit ovulation
Time Post Coitum Central Nervous System
Hypophysis
Ovary
Circulating Blood
<30 sec.
<2 mill.
10 mill. 30 mill.
60 mill.
75-90 mill.
13^-2 hrs. 3-5 hrs.
6-7 hrs.
7-8 hrs. 9-11 hrs.
Barbiturate-sensitive and atropinesensitive mechanisms^
t Phosphorylation in tuber cinereum^
t Phosphorylation
in tuber ciner reumt Phosphoryhition
in tuber cine reum^
t Phosphorylation in tuber cinereum
t Phosphoryhition^
Release of LH ca. 20 per cent. 6 Hypophysectomy prevents ovulation^' 12
Release of LH nowsufficient for ovulation."' 12 Phosphorylation at peak^
I Phosphor\iatioii
i Animal may be bled and transfused without prevent ! ing ovulationi2
folliculi. in egg
f Liquor Tetrad.' nuclei*
Cholesterol depletion in interstitial gland. ^ Egg nucleus migrates, membrane dissolves.** • " Prominent corona*
Liquor folliculi increasingly viscous*
First polar hotly'*
Marked .swelling of follicles. Thecal hypertrophy,! ■ i° hyperemia
OvuL.^Tion." t Phosphorylation2
Bleeding and transfusion now prevent ovulationi2
Progesterone detectable ^
Increased estrogen (endometrial hj-peremia)'
' Asdell, 1946.
2 Borell, Westman and Orstrom, 1947.
3 Claesson and Hillarp, 1947a. " Fee and Parkes, 1929.
'^ Forbes, 1953.
« Hill, 1934.
^ Sawyer, Markee and Hollinshcad, 1947.
- Pincus and Enzmann, 1935.
^ Sawyer and associates, 1947, 1949, 1950.
1" Walton and Hammond, 192S.
" Waterman, 1943.
'2 Weslmaii and .lacohsohn, 1936.
in the rat hypothalamus is increased also by administration of estrogen or by castration, conditions that similarly increase lihosphorus exchange (Borell and Westman, 1949). The ACh content is depressed during pregnancy or when the rat has been injected with progesterone. It is also lowered by Pentothal anesthesia, a matter of interest in relation to the fact that the barbiturates suppress ovulation (see p. 526).
The location and measurement of activity in discrete nuclei and pathways are largely in the future, although a beginning has been made in the rabbit, cat, rat, and mouse. Sawyer (1955) found in rabbits, after the combined administration of pentobarbital intravenously and histamine by way of the 3rd ventricle, that there was associated with induction of ovulation a characteristic change in intrinsic electrical activity of the rhinencephalon, extending into the preoptic area. If the olfactory tracts were cut, however, this activity could not be elicited and ovulation failed. According to Porter, Cavanaugh and Sawyer (1954), vaginal stimulation of estrous cats caused altered electrical activity in two hypothalamic regions:
(1 ) in the lateral hypothalamic area at the anterior tuberal level during stimulation and for 15 to 45 seconds afterward; and
(2) in the anterior hypothalamic area near the medial forebrain bundle, where response was delayed as much as 5 n:iinutes after stimulation. According to a i)reliminary account (Critchlow and Sawyer, 1955) in curarized, proestrous rats, there were i)eriods lasting approximately 20 minutes in the midafternoon, during which altered electrical activity appeared differentially in the preoptic area or anterior hypothalamus.
Another approach to localization has been described by Hertl (1952, 1955). On the pro])Osition that increased function of particular cells is reflected by increased volume of their nuclei, cell nuclear volumes were measured in hypothalamic nuclei of female mice at different stages of the estrous cycle. During proestrum and estrus there was said to be a functional edema in hypothalamic nucleus 20 of Griinthal (possibly the pars posterior of the ventromedial nucleus of Krieg) and to lesser extent in nucleus 16 (Nucl. arcuatus).
1. The Hypophyseal Portal Veins and the Chemotransmitter Hypothesis
As noted elsewhere, hypothalamic control of the jiars distalis is probably mediated by the hypophyseal portal circulation. Evidence for this has been especially convincing with respect to control of ovulation, although indications are that other phases of the cycle are also regulated by this means. Pertinent data from numerous transplantation and stalk-section experiments may be summarized by the following statement. Aside from a questionable grafting experiment (2 rats) reported by May (1937), in no case has ovulation or luteinization been reported in the absence of vascular linkage of the pars distalis with the median eminence; on the other hand, ovulatory cycles have often been cjuickly restored when the gland has been revascularized by the portal vessels (see especially, Harris, 1950a; Harris and Jacobsohn, 1952; Nikitovitch-Winer and Everett, 1957, 1958b).
Although the importance of local vasomotor regulation in the stalk vessels remains to be evaluated (Green, 1951), there is extensive support for the hypothesis that ovulatory release of gonadotrophin is invoked by a chemotransmitter (Harris, 1948a, 1955). If one accei)ts the prevailing opinion that nerve fibers entering the pars distalis are too few to account for its secretomotor control and that the flow of blood in the hyi^ophyseal portal vessels is toward the gland (Wislocki and King, 1936; Green, 1947; Green and Harris, 1947, 1949; Barrnett and Greep, 1951 ; Landsmeer, 1951 ; ]McConnell, 1953; Xuereb, Prichard and Daniel, 1954; Worthington, 1955), the plausibility of the chemotransmitter hypothesis becomes inescapable.^
Evidence that the transmitter may be
^ For a dissenting view, see Zuckerman (1952). Reference should also be made to the hypothesis formulated by Spatz (1951) and associates (see Nowakowski, 1950, 1952). They postulated that a descending pathway in the spinal cord is the connecting link between hypothalamus and ovaries. With respect to ovulation, this is clearly denied by the fact that local stimulation of the hypotlialamus provokes ovulation in rabbits in which the thoracic spinal cord has been transsected (Christian, Markee and Markee, 1955).
adrenergic was presented by Markee, Sawyer and Hollinshead (1948), who provoked ovulation in rabbits by instilling epinephrine directly into the pars distalis. Detailed experiments supporting this w^ere fully reviewed by Markee, Everett and Sawyer (1952). In discussion following that paper, Sawyer reported the induction of ovulation in rabbits by the injection into the third ventricle of either epinephrine or norepinephrine, and suggested that the latter is "more closely related to the natural mediator than is epinephrine." Donovan and Harris (1956), from studies in which the rabbit hypophysis was slowdy infused in situ with solutions of epinephrine or norepinephrine, concluded that neither substance is the agent in question, and that the positive results of Markee, Sawyer and Hollinshead (1948) were the effects of low pH and not of the drugs per se. Proof of the negative is elusive, however, and one must note that Donovan and Harris did not meet the conditions of timing and drug concentration that obtained in the earlier work.
Intravenous injection of Dibenamine or its congener, SKF-501,-^ will usually prevent ovulation in rabl)its when injection is completed within 1 minute after coitus (Sawyer and associates, 1947-50). On the other hand, when injection is delayed until 3 minutes or later, ovulation is unaffected. The nonadrenergic hydrolysis product of Dibenamine, 2-dibenzylaminoethanol, does not have the blocking action, although its central excitatory powers are much like those of the parent substance. The failure of blockade by Dibenamine, if injection is withheld for 3 minutes, demonstrates that the drug does not interfere with the actual discharge of ovulating hormone into the l)lood stream, for that process recjuires about an hour (Fee and Parkes, 1929; Westman and Jacobsohn, 1936). The Dibenamine-sensitive mechanism thus serves as a trigger, the gland being adequately stimulated within 1 01' 2 minutes post coitum. This estin^ate is in remarkal)le agreement with the earlier mentioned obscMA'ations on
•'■' Dibenamine is iV,iV-dibenzyl-/:i-cliloioetliy lamine. SKF-501 is A'-(9-fluorenyl)-.V-ethyl-/i-chlor()ethylamine hydrochlorifle. Banthine is /i-dictliylaminuc'tliyl-.\anthene-9-cai'l)Oxvlak' niclliohroiniilc
phosphorus exchange in the tuber cinereum and hypoi)hysis (p. 521, and Table 8.1 1.
A mechanism that is subject to blockade by atropine or Banthine^ evidently precedes the Dibenamine-sensitive process — temjDorally if not anatomically. To accomplish blockade in rabbits, these anticholinergic drugs must be injected intravenously within about 30 seconds after coitus (Sawyer and associates, 1949-1951). It should be recalled that Foster, Haney and Hisaw (1934) reported failure of ovulation in several rabbits treated with small amounts of atropine before mating. ]\Iakepeace (1938), however, was unable to confirm the effect with somewhat larger doses and the former observation was forgotten.
A seemingly crucial experiment devised by Sawyer gives conclusive evidence that the atropine-sensitive process is antecedent to the Dibenamine-sensitive one. It was based on two facts: (1) intravenous injection of nearly lethal doses of epinephrine does not induce ovulation in estrous rabbits, and (2) atropine protects rabbits against fatal pulmonary edema after injection of large amounts of epinephrine. In rabbits protected by atropine in dosage that was also sufficient to block the ovulation reflex, the injection of twice-lethal doses of epinephrine caused ovulation or significant degrees of follicle maturation in 5 of 7 cases. These effects were not found in rabbits protected by Dibenamine. Supporting evidence was adduced by Christian (1956i who found that atropine would not prevent ovulation in response to electi'ical stimulation of the medial preoptic area or adjacent parts of the hypothalanuis, whereas in a significant number of such rabliits ovulation was blocked by SKF-501.
Extension of the blocking experiments to the rat, as an example of a spontaneous ovulator, disclosed that in this species also ovulation can be blocked by Dibenamine, SKF501, atropine, and Banthine, when the injections ai'c appropriately timed with respect to the stage- of the cycle and time of day (Sawyer, Everett and Markee, 1949; Everett, Sawyer and Markee, 1949; Everett and Sawyer, 1949, 1950, 1953: see \). 526). Furthermore, blockade of ])oth estrogenimhiccd and pi'ogcstci-oiic-induced ovuhition was aceomi)lished with cither Dibonamine or atropine. Neither agent, however, prevented ovulation after injection of sheep liypophyseal LH. A report by Hansel and Trimberger (1951) stated that in cattle a significant delay of ovulation (as great as 72 hours) followed atropine administration. In control experiments the simultaneous injection of atropine and human chorionic gonadotrophin was followed by ovulation slightly earlier than the normally expected time. Treatments were begun 1 to 5 hours after the onset of estrus. This work was confirmed and extended by Hough, Beardon and Hansel (1955). In the hen, blockade of ovulation, normal or induced by progesterone, has been reported after administration of Dibenamine, Dibenzyline, SKF-501 or atropine (Zarrow and Bastian, 1953; van Tienhoven, 1955). According to van Tienhoven, the drugs did not interfere with the ovulating action of extrinsic gonadotrophin.
It is important that the same drugs will block ovulation in lioth rabbits and rats (Table 8.2) . Of ec^ual significance is the fact that several agents that are ineffective in rabbits are also ineffective in rats (notably 2-dibenzylaminoethanol, the imidazoline adrenolytic drugs, and the ganglion blocking agents). These considerations are interjireted to mean that spontaneous ovulation is invoked by neurohumoral mechanisms that are very like those in the reflex ovulation of rabbits.
The suggestion that the l)locking effects might result from nonspecific stress, causing the hypophysis to be so actively secreting ACTH that gonadotroiihin secretion is interfered with (Dordoni and Timiras, 1952), is clearly denied by several facts. ( 1 ) In the rabbit studies, none of the various agents prevented ovulation when injected more than a minute post coitum. (2) In one study (Sawyer, Markee and Everett, 1950b) ovulation was actually induced by the intravenous injection of "lethal" doses of epinephrine when the animals WTre protected by atropine. (3) In rats ovulation is unaffected by massive intravenous doses of either the imidazoline drugs or 2-dibenzylamionethanol in amounts known to be stressing (Sawyer and Parkerson, 1953).
TABLE 8.2 Pharmacologic Agents and Blockade of Ovulation
Antiadrenergics
/3-Haloalkylamines
Dibenamine
SKF-501
Dibenzyline
ImidazoHnes
Priscoline
Regitine
Yohimbine
Anticholinergics
Atropine
Banthine
Antihistaminics
Neo-antergan
Ganglion blockers
Tetraethvlammonium. .
SC-1950/
Barbiturates
Nembutal
Dial
Ipral
Amvtal
Barbital
Phenobarl)ital
Prominal
Others
Morphine
Procaine, locally near tuber
Procaine, systemically .
Chlorpromazine
Reserpine
Ether
2,4-Dimtrophenol
Rabbit
Rat
Cow
Bi
Bi
Bi
Bi
01
01
0^
01
■?4. 5
Bi
BI
B6
Bi
Bi
01
01
01
01
Bi
Bi
Bi
Bi
Bi
B'
Bi 8
B9
BIO
Bii
01
B12
B13
B14
B16
B3
I Sawyer and associates, 1947-1951; Everett and associates, 1949-1950; Christian, 1956; and see present text.
^ van Tienhoven, 1955; van Tienhoven, Nalbandov and Norton, 1954.
3 Zarrow and Bastian, 1953.
•• Fugo and Gross, 1942.
5 Sulman and Black, 1945.
^Hansel and Triml)erger, 1951; Hough, Beardon and Hansel, 1955.
' Fraps and Case, 1953.
- Doring and Goz, 1952.
» Westman, 1947.
1" Barraclough and Sawyer, 1955.
II Westman and Jacol)8ohn, 1942. 1- Barraclough, 1956.
13 Barraclough, 1955.
" Unpublished. Temporary, during deep anesthesia.
1^ Unpublished. EDso : 25 mg. per kg. subcutaneously.
Key: B = Blockade.
= No blockade.
1 = Ovulation induced by the drug.
Nor is it influenced by severe trauma, heat,
cold, or formalin injection coincident with
the known "critical period" (Everett, unpublished).
2. Central Depressants and Ovulation
Reported evidence of a blocking action of barbiturates on ovulation traces back to experiments by Westman (1947) who injected female rats with Prominal twice daily for 3 weeks. Approximately 30 per cent of these rats experienced prolonged vaginal estrus and had ovaries containing only follicles at the end of the experiment. Almost identical results were reported by Doring and Goz (1952) when rats were treated daily with phenobarbital. The agreement is not unexpected in view of the fact that in the body Prominal is quickly demethylated to phenobarbital. It was shown by Everett and Sawyer (1950) that when administration of barbiturates to rats is critically timed with respect to stage of the cycle and the time of day, blockade of ovulation can be accomplished at will in shortterm experiments (Fig. 8.10). Chronic administration introduces considerable uncertainty for reasons that are not yet clear (Everett, 1952b). In the rabbit, the rapid intravenous injection of pentobarbital or Pentothal, within as short a time as 12 seconds after coitus, generally failed to block ovulation (Sawyer, Everett and Markee, 1950) . However, it was later shown that barbiturate anesthesia will prevent the ovulation that is otherwise caused in the estrogen-primed rabbit by mechanical stimulation of the vagina, the anesthesia t)eing induced in advance of the stimulation (unjuiblished i.
Other central dcpi-es^ants reported to block ovulation in the rat are morj^hine, reserj^ine, chlorpromazine (Barraclough and Sawyer, 1955; Barraclough, 1955, 1956), and even meprobamate acting synergistically with an anticholinergic drug (Gitscli, 1958). Special interest attaches to the mori)hine work, in that anicnoi'i lica and sterility often a('C()in|)any moiphinc addiction in the human female. Related studies (Sawyer, Critchlow and Barraclough, 1955), in which recordings were made of electi'ical acti\ity in various regions in the brain.
demonstrated in rats that morphine acts nmch like the barbiturates in depressing activity in the reticular activating system. The effect was also shown by atropine, in doses that would block ovulation. The inference is that all three agents block by striking at the same central elements of the LH-release apparatus.
An interesting peculiarity of domestic hens with respect to barbiturates was encountered by Fraps and Case (1953), who noted that pentobarbital induces ovulation jirematurely, and that pentobarbital and progesterone supplement each other in this capacity. Although these developments may represent pharmacologic curiosities limited to the bird, the possibility should be seriously considered that similar effects may occur in other animals. In fact, pentobarbital in rabbits facilitates the release of hypophyseal gonadotrophin in response to intraventricular injection of histamine, seemingly by an effect in the rhinencephalon (Sawyer^ 1955).
3. The Central Nervous System as a Timing Mechanism for Ovulation
In the rat and the hen and probably many other species the pro-ovulatory excitation of the hypophysis is dejiendent in large measure on time of day.
In the rat, the blocking agents have served to delimit a critical period on the day of proestrum, before which ovulation can be blocked and after which it will occur in sjiite of injection of the blocking agent. Under controlled illumination for 14 hours daily, this critical period extends from about 2 P.M. to 4 P.M. Administration of either atroi^ine or i^entobarbital at 2 p.m. consistently blocks ovulation (Fig. 8.10), whereas injections later in the period are progressively less eff"ecti\-e ( l']\-erett and Sawyer, 1950, 1953; Everett, 1956b). Such l)redictability of the hour of pituitary activation is, in itself, evidence of a relationship between this event and diunial physiologic I'liythnis.
Furthei' e\-i(lence is seen in the seciuelae of pentobarbital injection (Fig. 8.11). Repetition at 2 P.M. on successive days results in a follicular cycle and prolonged vaginal estrus with eventual atresia of all
Fig. 8.10. Deinoni^lration of 24-lioiir penodu-ity in the luteinizing hormone-release apparatus of female rats (Vanderbilt strain, 4-day cycle, controlled lighting: 14 hours per day).
Schematic representations of the normal cycle (A) and of characteristic results of different
regimes of Nembutal treatment (B to F). Vaginal stages indicated by Roman numerals over
each time scale; symbols above these show the corresponding follicle and corpus luteum
stages. The device marked l prolonged the functional
life of the coi'pus luteuni in nonpregnant
women. Lyon rejjorted such prolongation
using lactogen alone. The Squibl) lactogen
was also used by Moore and Nalbandov
(1955) in prolonging the luteal phase of the
cycle in the ewe. As in the human experiments, howe\-er. one would like to know
whether lactogen is capable of initial stimul.'iiioii of secretory activity of corpora lutea
and of maintaining their function in the absence of the hypophysis. There is no evidence for or against the lactogenic liormone
in this capacity, except in rats.
Estrogens have direct hiteotrophic action
in the rabbit (Robson, 1937, 1938, 1947).
The effect does not depend on the hypophysis and has been produced by impLantation
of estrogen crystals within corpora liitea
(Hammond, Jr., and Robson, 1951; Hammond, Jr., 1952). Westman (1934) had earlier shown that operative reduction of
ovarian stroma in pseudopregnant rabbits
results in corpus luteum regression and that
this can be prevented by administration of
estrogen. Corpora lutea induced by gonadotrophin injection or by mating, as the case
may be, require the presence of the hypophysis for their continued function ( Smith and
White, 1931; Westman and Jacobsohn,
1936). Theoretically, then, in rabbits the
hypophysis liberates FSH and LH which act
on the interstitial tissue to cause estrogen
secretion. This in turn stimulates the corpora lutea to secrete progesterone.
The effect of estrogen on the corpora lutea
of rats is largely indirect and requires the
presence of the hypophysis. Massive dosage
with estrogen beginning soon after ovulation results in the enlargement of the corpora lutea and the production of sufficient
amounts of progesterone to mucify the vaginal mucosa (Selye, Collip and Thomson,
1935; Wolfe, 1935; Desclin, 1935; Merckel
and Nelson, 1940). In fact, a single injection of 50 /Ag. estradiol bcnzoate on the day
after ovulation is sufficient to cause pseudopregnancy. These effects are now judged to
be the result of induced liberation of hypophyseal luteotrophin. vSimilar effects have
been reported after administration of androgens (McKeown and Zuckerman, 1937;
Wolfe and Hamilton, 1937; Freed, Greenhill
and Soskin, 1938; Laqueur and Fluhmann,
1942).
Desclin (1949b) stated that in hypophysectomized rats the administration of estrogen augments the hiteotrophic action of
lactogen, producing functional corpora lutea in the presence of subthreshold doses of
the latter hormone. A physiologic synergism
of the two substances has thus been indicated. Mayer (1951) suggested that this
may explain the stimulation of corpora lutea
of lactation which follows estrogen treatment in this species. Greep and Chester
Jones (1950) postulated that estrogen favors corpus luteum function in the rat by
causing the luteal cells to produce cholesterol as a precursor of progesterone. Their
actual data, however, indicate that the increase of visible cholesterol after estrogen
treatment was confined to the interstitial
tissue.
Factors responsible for cholesterol storage
and mobilization in corpora lutea of the rat
were analyzed by Everett (1947). In hypophysectomized rats in which corpora lutea
were maintained by lactogen the injection
of pituitary LH induced the storage of
cholesterol, but this effect did not occur in
hypophysectomized rats in the absence of
lactogen. It could be induced during pregnancy or pseudopregnancy by estrogen if
the hypophysis remained in place. Addition
of an excess of lactogen prevented cholesterol storage. Lactogen thus tends to deplete
cholesterol content of rat luteal tissue as
ACTH tends to deplete adrenocortical cholesterol.
2. XonfunctionaV' Corpora Lutea
In the short cycles of the rat, mouse,
hamster, and so on, the corpora lutea are
commonly said to be nonfunctional. The
meaning of this statement, of course, is that
they are incapable of supporting a decidual
reaction (Long and Evans, 1922), or of lireventing ovulation. They need not be totally
inactive, however, to fail to cause these
manifestations. Whereas daily injection of
1.5 mg. or more of progesterone into intact
female rats will simulate pseudopregnancy
and indefinitely delay ovulation (Selye,
Browne and Collii^, 1936; Phillips, 1937),
smaller amounts of 1.0 rag. or less are compatible with the short cycle (Lahr and
Riddle, 1936; Phillips, 1937; Everett, 1940a,
b; and unpublished). In the absence of estrogen in castrated females, daily injection
of as little as 0.25 rag. progesterone will support deciduomata (Velardo and Hisaw,
1951). Very small amounts of estrogen augment this action of progesterone (Rothchild,
Meyer and Spielman, 1940) but somewhat
larger amounts are inhibitory unless the
progesterone dose is proportionately increased (Velardo and Hisaw, 1951). In the
intact animal the progestational effects of less than 1.0 mg. progesterone would be inhibited by the periodic rise in estrogen secretion.
Evidence that some rats, but not all, actually experience low-grade corpus luteum
activity during the short cycle was furnished
by Everett (1945). In the comparison of
ovaries from females of two strains of rats,
it was noted in the supposedly normal Vanderbilt strain that on the two days immediately following ovulation the corpora
lutea of the next youngest generation contained a great quantity of cholesterol, giving a strong Schultz reaction. By contrast,
comparable corpora lutea of the DA strain
were usually free of visible lipid in Sudan
preparations or the Schultz test. Administration of small amounts of lactogen (luteotrophin I during the cycle preceding the
current one, amounts inadequate to cause
l)seudopregnancy, resulted in the rich deposition of cholesterol in these otherwise lipidfree corpora lutea. The conclusion was
reached that the corpora lutea of the Vanderbilt rat must be slightly active during the
short cycle and those of the DA rat less so, if
at all. This would easily explain the relative
indifference of the Vanderbilt rat to continuous light and the ease with which persistent
cstrus could be induced in the DA rat by
such treatment (Everett, 1942a, b). In fact,
the low dosages of lactogen mentioned substituted for progesterone treatment in maintaining regular cycles in persistent-estrous
rats of the DA strain (Everett, 1944b). Significantly, the treatment was effective in
only those animals in which a set of corpora
lutea had been induced by other means at
the beginning of the experiment.
To be correlated with the above indications of low-grade function during the short
cycle, is the finding that corpora lutea of
the Vanderl)ilt rat retain full responsiveness
to luteotrophin throughout most of the diestrous interval (Nikitovitch-Winer and
Everett, 1958a). Responsiveness diminishes
near the onset of i:)roestrum. Once the rat
has entered proestrum these older corpora
lutea are not capable of sustained function.
The loss is not a function of time per se, but
of stage of the cycle.
.\. I'.^KTI)OPRE(;\.\XrY
The terms coi-pu,^ hitcuni of ()^•ulation and
corpus luteum of pscudojji'cgnancy are com
monly u.sed to differentiate the luteal bodies
occurring during the normal cycles from
those found during some unusually long
period of luteal activity. However, the
terms deny the fundamental similarity of
the luteal phase in the cycles of such animals as the guinea pig and the luteal phase
induced by sterile mating or its equivalent
in animals like the rat. In the unmated bitch
the spontaneous luteal phase of the cycle is
commonly called pseudopregnancy, yet it
is equally common to say that the guinea
pig does not experience pseudopregnancy.
The truth is that the luteal phase of the
canine cycle is simply longer than the luteal
phase in the guinea pig and may be marked
by a period of lactation near its close. In
the present discussion, the expression pseudopregnancy will be equivalent to saying
the luteal phase of the infertile cycle. Under
experimental conditions it will refer to any
period of sustained luteal function similar
to that of the normal progestational state.
Wherever appropriate, the distinction will
be made between a pseudopregnancy that is
spontaneous and one that is induced.
In most of the familiar animals that ovulate spontanously corpus luteum function
also begins spontaneously and continues for
at least several days after ovulation. With
respect to the rabbit, cat, and ferret, it is
often said that pseudopregnancy is invoked
by sterile copulation, whereas strictly speaking it is only ovulation and corpus luteum
formation which are invoked. The pseudopregnancy then follows automatically. This
interpretation seems appropriate, inasmuch
as in all three species the formation of
corpora lutea by l)rief treatment with hypophyseal or chorionic gonadotrophin is followed by long periods of progesterone secretion which can hardly be the direct effect
of the injected substances (Hill and Parkes,
1930a, b; Foster and Hisaw, 1935; van
Dyke and Li, 1938). Quite different is the
pseudopregnancy of the rat, mouse, and
hauistei', in \\liich progestational activity is
invoked by stinudation of the cervix uteri,
l^verett (1952a) described the ex])eriiuental
dissociation in rats of the o^•^llati()n and
luteotr()])hic mechanisms. respectively.
When o\-uhition is blocked (by pentobarbital) in the cycle dui'ing which controlled
uiatinu occurs, pseudopi-egnancy begins
Fig. 8.12. Experimental dissociation in rats of the ovulation mechanism and that causing
pseudopregnancy. A. Control cycles for comparison with B and C . Points on base line
represent diestrum, on ascending lines proestrum, on highest level full vaginal estrum. X,
ovulation. B. Blockade with Nembutal {'NB) on day of proestrum and following day (see
Fig. 8.11E'). Ovulation during third night. C. Same basic procedure as B, but with copulation
during first night (M). Ovulation usually failing in this cycle (contrast with B) . Corpora
lutea formed after spontaneous ovulation in second cycle regularly become functional without further stimulation : the wavy line represents pseudopregnancy. The early copulation has
introduced some change in the animal such that this pseudopregnancy "spontaneously"
follows ovulation as in the standard mammalian cycle. (From J. W. Everett, Ciba Foundation Collofiuia Endocrinol.. 4, 172. 1952.)
"spontaneously" a]ter the next cyclic estrus
(Fig. 8.12). Dissociation of the two mechanisms is expressed in another way by certain Mustelidae, e.g., the mink and marten.
Ovulation in these forms is invoked by
mating, whereas corpus luteum activation
awaits appropriate environmental conditions, i.e., temperature and length of daily
illumination (Pearson and Enders, 1944;
Hansson, 1947). In the mink, during the period of relative luteal inactivity that follows mating early in the season, recurrent
estrus continues. If reraating takes place at
an interval of 6 days or more, new ovulations are induced (Hansson, 1947). Matings
late in the season are immediately followed
by luteal activity. The pseudopregnant cycles of a representative series of mammals
are much alike when conditions appropriate to the respective species are applied
(Fig. 8.1).
1. Duration of Psciidopregndncn
The length of time that corpora lutea remain functional in the pseudopregnant cycle is thought to be relatively uniform in the great majority of mammals, usually about 10 to 15 days. Rarely it is shorter,
e.g., the hamster, 7 days, although usually 9 to 10 days (Asdell, 1946). At the other extreme, the corpora lutea remain functional for periods corresponding to the duration of pregnancy, as in the ferret, 5 to 6 weeks. In fact, corpus luteum function lasting over a month is usual in the other two carnivores for which information is at hand: cat, 30 to 44 days (Foster and Hisaw, 1935) ; and dog, 30 days or more (Evans and Cole, 1931).
These figures are only approximations, however, as the criteria on which they are based differ. In the rat, in which pseudopregnancy is said to last 12 to 14 days, its termination is taken to be the onset of the next estrus, whereas the corpora lutea must have undergone a decline of activity 2 or 3 days earlier (Everett, 1948). The decline is probably not abrupt, inasmuch as the vaginal smear during the next estrus is very strongly mucified and, as mentioned earlier (p. 519), enough progesterone seems to be secreted by the waning corpora lutea to facilitate ovulation. Morphologic criteria are often employed as indicators of corpus luteum regression: characteristically, fatty vacuolation of luteal cells, decrease in size of the individual cells or of the entire corpus luteum. and changes in the smusoidal pattern suggesting reduced circulation. Such changes are first observed in the guinea pig corpora lutea on about the 13th day of the cycle. One has the choice of taking this date as the end of the pseudopregnant phase or, alternatively, the date on which the first indications of estrus are noted. Either choice is arbitrary, but the former seems preferable as it suggests that progesterone secretion is diminishing and probably is no longer sufficient to maintain progestational changes in the uterus. In fact, regression of the endometrium sets in about a day earlier than frank degenerative changes in the corpora lutea. In the cat, according to van Dyke and l.i (1938) the corpora lutea 20 days after ovulation no longer secrete enough progesterone to cause motor effects of epinephrine in the myometrium, the so-called "epinephrine-reversal" effect, yet by histologic criteria corpus luteum regression is not apparent until 28 days or later (Liche, 1939; Foster and Hisaw, 1935). In the bitch the uterus begins regression 20 to 30 days after heat, l)ut the corpora lutea are said to remain in good condition for a longer time (see Asdell, 1946, for references). Regression is so gradual that anestrum is not reached until about 85 days. In the primates the beginning of menstruation offers a means of delimiting the luteal phase, inasmuch as menstruation in the ovulatory cycle reflects a marked reduction in corpus luteum function. Nevertheless, this reduction probably occurs a few days before bleeding begins. The i^eak of pregnanediol excretion in women (Venning and Browne, 1937) and of plasma progesterone concentration in women and monkeys (Forbes, 1950; Bryans. 1951 ) is passed about midway between ovuhition and menstruation.
2. Xciivdl Factors in Pseudopregnancy
The importance of the nervous system in control of pseudopregnancy is well recognized in only the few species re])resented by the rat. .\ neural effect in the mink and similar Mustelidae is implied by the relation of hiteal function to daily illumination, as mentioned earlier. Beyond that fact, however, no information is available. Att(>ntion will therefore be directed largely to the rat.
Not only sterile mating, but se\'eral other
procedures involving neural stimulation will cause rats to become pseudopregnant. Stimulation of the cervix by mechanical means (Long and Evans, 1922) or electric shock (Shelesnyak, 1931) have become standard methods. In fact, Greep and Hisaw (1938) obtained pseudopregnancies after electrical stimulation during early diestrum, several days before ovulation. Pseudopregnancy is also invoked by continuous stimulation of the nipples for several days (Selye and McKeown, 1934). According to Harris (1936) electric shock through the head is effective. His negative results with "spinal shock" are difficult to explain. From the description of position of the electrode it seems doubtful that the current passed through the cord itself, yet the sacral plexus must have been stimulated.
Al)dominal sympathectomy or superior cervical ganglionectomy are said to diminish the numbers of animals responding to electrical or mechanical stimulation of the cervix (Vogt, 1931; Haterius, 1933; Friedgood and Bevin, 1941). On the other hand, there is no diminution of response to sterile copulation, which shows that the sympathetic chains are not essential. Ball (1934) emphasized the quantitative aspects of the problem, noting that partial resection of the uterus or excision of the cervix diminished the response to sterile copulation, but only when "single-plug" matings were allowed. Multiple plugs gave pseudopregnancy in 100 per cent of the animals. It may be assumed that Vogt's (1933) negative results after hysterectomy resulted from single-jilug copulations. Kollar (1953) re-opened the cjuestion and found that pelvic nerve resection usually pi-evented the response to mating. It is not clear, howevei', whether multiple copulations wnv the rule, although it seems that the I'outiiie procedure was to leave the male with the female overnight. His contention was that cervicectomy fails to abolish the resjionse completely because the vagina remains sensitive.
Anesthesia with ether, nitrous oxide, or ethylene ( Mcyei', Leonard and Hisaw, 1929) diniiiiished the ficciuency of response to inecliaiiical stimulation of tlu> (•er\-ix. The statement was made, although without v\\(len(c, that spinal anesthesia |)re\-ents pseudopreiinanev. Aeeoi'ding to A'ogt (1933),
local anesthesia of the vagina and cervix by cocaine or procaine prevented the response to sterile copulation in 23 of 35 rats.
Removal of neocortex (Davis, 1939) did not interfere with the pseudopregnancy response to electrical stimulation of the cervix, although there was slight impairment of the response to mechanical stimulation or sterile mating (single-plug?).
These results taken together have been construed to mean that induction of pseudopregnancy in rats involves a reflex similar to the ovulation reflex in rabbits. Certain considerations, however, raise the possibility that it may not be a "trigger" stimulus to the hypophysis as long believed (Everett, 1952a). In the first place, it seems doubtful that a trigger stimulus would result in continuation of a new pattern of secretion (luteotrophin) for as long as 10 to 12 days. Furthermore, as noted above, cervical stimulation during the diestrum preceding ovulation may induce pseudopregnancy (Greep and Hisaw, 1938). Similarly, copulation during a cycle in which ovulation is blocked by pentobarbital results in a pseudoi)regnancv that begins after the next estrus (Fig. 8.12)^.
We turn now to experiments concerned directly with the hypothalamo-pituitary system and pseudopregnancy. Westman and Jacobsohn (1938c) cut the pituitary stalks of estrous female rats. Barriers of metal foil were inserted to prevent regeneration of nerve fibers assumed to innervate the adenohypophysis. Regeneration of blood vessels must have been equally impossible. Controls were simply hypophysectomized. Two to 5 hours after the operations electrical stimulation of the cervix was administered to all animals. Pseudopregnancies were demonstrated by deciduomas in traumatized uteri of all the stalk-sectioned animals but not in the completely hypophysectomized rats. Desclin (1950) reported the maintenance of pseudopregnancy in estrogen-treated rats in which the only remaining hypophyseal tissue was in the form of grafts in the kidney. Whereas in hypophysectomized controls the estrogen treatment (stilbestrol pellets) produced cornification of the vagina and no enlargement of corpora lutea, the engrafted-estrogenized rats developed mucified vaginas and enlarged corpora lutea as in intact rats similarly treated with estrogen. Desclin concluded that the grafted hypophysis is able to respond to estrogen by liberating luteotrophin.
It is now apparent, however, that neither cervical stimulation nor estrogen treatment is needed to invoke pseudopregnancy when the gland is isolated from the hypothalamus (Everett, 1954, 1956a; Nikitovitch-Winer and Everett, 1958a; Sanders and Rennels, 1957; Desclin, 1956a, b). When autografts of anterior hypophysis were made to the renal capsule or near the common carotid artery on the day after ovulation in adult cyclic rats, corpus luteum function was invoked and maintained without any stimulus other than the operative procedures themselves. In short-term experiments in which the uteri were traumatized 4 days after the transplantation large deciduomas were regularly found at 8 days in the proven absence of residual hypophyseal tissue at the original site (Everett, 1954). Hypophysectomized controls were negative. In longterm experiments, continuing luteal function was demonstrated for as long as 3 months. Here the test for luteal function was vaginal mucification in the presence of massive amounts of estrogen administered during the final week of the experiment (Everett, 1956a). Controls in which the grafts or the ovaries were removed at the beginning of such estrogen treatment responded with full vaginal cornification. Follicular apparatus and interstitial tissue of the ovaries atrophied promptly after the grafting operations, whereas corpora lutea forming at that time were maintained for the long periods without histologic sign of deterioration. In later work, the decidual reaction was used as the test for luteal function, positive reactions being elicited as late as 2 months after the transplantation. It was discovered that function of the graft is not influenced by stage of the cycle at which transplantation is carried out and that grafts in the anterior chamber of the eye secrete luteotrophin like those on the kidney (Nikitovitch-Winer and Everett, 1958a). Transsection of the pituitary stalk is sufficient in itself to provoke pseudopregnancy. If an effective barrier to vascular regeneration is inserted, the pseudopregnancy will heorve permanent, but otherwise it will l:i •■ i;!* usual length of time (Xikitovitch-Winer, 1957 j. In fact, there is reason to suspect that even a transient impairment of circulation in the median eminence-hypophj'seal linkage can be a sufficient impetus to pseudopregnancy. The experiments of Taubenhaus and Soskin ( 1941 ) in which application of an acetylcholine-prostigmine mixture to the exposed hypophysis was followed by pseudopregnancy, may well be explained in some such way.
It thus seems that in the rat the deprivation of, or interference with, the normal connection of the pars distalis with the median eminence facilitates secretion of luteotrophin and at the same time eliminates luteolytic mechanisms. It is significant that transplants of pars distalis into the pituitary capsule or to the immediate vicinity of the median eminence resume cyclic function (see page 512). The hypothalamus may have an inhibitory effect on luteotrophic activity of the pars distalis during the short cycle in this species. Greep and Chester Jones (1950) made the pertinent suggestion in attempting to explain the induction of pseudopregnancy by estrogen treatment, that the fundamental action of estrogen here is the suppression of FSH and LH, after which luteotrophic secretion may "proceed apace."
There is necessarily some uncertainty concerning the amount of luteotrophin secreted by the pars distalis when dissociated from the brain. Three sets of facts indicate that the output is larger than that in the cycling animal. (1) Sufficient gestagen is secreted by the engrafted animal to maintain a ])regnancy (Everett, 1956c; Meyer, I'rasad and Cochrane, 1958) when the pituitary is trans])lanted on the day after mating. (2) After stalk-transsection, in which there is less initial destruction of glandular parenchyma than in transplantation experiments, the corpora lutea enlarge to a diameter like that usually found in late pregnancy rather than remaining like those of pseudopregnancy (Nikitovitch-Winer, 1957). (3) A single homotransplant of pars distalis placed subcutaneously in an otherwise normal female mouse will maintain a se(iueiicc of pseudopregnancies that override the short cycles expected of the animal's own hypophysis (Miihlbock and Boot, 19591. This is also true of rats (Nikitovitch Winer, uni)ublislie(l). To avoid the conclusion that in such preparations the grafted gland is secreting luteotrophin at an increased rate, one must assume that the outl)ut of this hormone from the intact gland is only slightly below threshold and that the graft adds just enough to make the total output effective. To explain the maintenance of pregnancy one might assume that the luteotrophin output and the resulting gestagen secretion are no greater than during the normal short cycle and that the formation of deciduomas takes place because of the deficiency of estrogen. The results of stalksection, however, cannot easily be explained away. The weight of evidence, then, is in favor of increased luteotrophin secretion by hypophyses isolated from the brain by severance of the stalk or transplantation.
Under certain experimental conditions it has seemed that to establish pseudopregnancy all that is necessary is to block out the forthcoming estrus and ovulation. Thus, in cycling rats, when the hypophysis is transplanted to the kidney as late as 60 hours after ovulation, the current diestrum transforms into a permanent pseudopregnancy supported by the existing set of corpora lutea (Nikitovitch-Winer and Everett, 1958a ». Similarly, injections of chlorpromazine (Barraclough, 1957) or Pathilon (ditsch and Everett, 1958) begun during diestrum, may transform it into a pseudopregnancy by blocking out the expected estrus.
The Miihlhock-Boot experiment mentioned above furnishes an instructive model of the standard mammalian cycle, in which both ovulation and pseudopregnancy are spontaneous events. Given the extra pituitary tissue producing luteotrophin at a presumably constant rate, with the output of the normal ghmd fluctuating (juantitatively and (jualitatively, the mouse or rat undergoes one ])seudopregnancy after another. Possibly, in animals that normally liave a spontaneou.- hiteal phase, thei'c is a considei'ahle poUion ot' the pars distalis which I'unctions somewhat indepeiidentl}- of the hyputhalaiiius. with a continuous output of luteotrophin as from the grafted gland in the Mvihlb()ck-P)Oot pi-epaiation. The portion nioic (Hrectly under control of the me(lian eminence I zona tuberaHs'.'l would then act like the intact hypoiihysis of the Miihlbock-Boot mouse. Such a view, unfortunately, continues to set apart species in which the luteal phase is not spontaneous, by suggesting that only in them are special neui'al nieclianisnis involved.
B. Luteolytic Mechanisms
By luteolysis we shall refer to corpus luteum regression in any of its manifestations. Supposedly the initial change is functional, after which overt cytologic and histologic changes appear, leading eventually to the total loss of glandular tissue. Very likely the initial stages are occult and only gradually reach recognizable proportions. Mention was made earlier of the fact that in women and monkeys the peak of gestagen secretion is about midway between ovulation and menstruation. In rats indirect evidence from progesterone injection experiments leads to the deduction that toward the close of a pseudoi)regnancy gestagen secretion must drop below the estrus-suppressing level several days before estrous changes appear in the vaginal smear (see Fig. 8.8). A more al)rupt drop is reported for the ewe l)etween the 16th and 17th (last) days of the cycle (Edgar and Ronaldson, 1958).
Long-term experiments with pituitary autotransplants indicate that at least in the rat the life span of the corpus luteum is not limited by intrinsic factors. Some agent (s) of extra-ovarian origin must, therefore, be res]5onsible for at least the initial luteolytic changes. Various bits of information suggest that the agent is associated with, if not identical with, FSH and/or LH. Greep (1938) noted that after hypophysectomy in rats the daily injection of LH over a period of 10 days caused the corpora lutea to regress more rapidly than otherwise. Greep, van Dyke and Chow (1942) later were unable to repeat this with a more highly purified LH C'metakentrin") , a fact suggesting that the earlier material was effective because of impurity. During the short cycle of the rat, luteolysis is interrupted by translilantation of the pars distalis (Everett, 1954; Nikitovitch-Winer and Everett, 1958a). Whatever regressive changes are in progress at that moment are evidently suspended forthwith. They are first apparent during the third day of diestrum and become increasingly pronounced during proestrum and estrus. In this connection, it should be recalled that during late diestrum and proestrum patches of cells undergoing fatty necrosis are first recognizable histologically (Boling, 1942; Everett, 1945).
Why is it that, in the face of a continuing supply of luteotrophin in the MiihlbockBoot preparation, or in intact animals injected daily with lactogen (Lahr and Riddle, 1936; Aschheim, 1954), luteolysis sets in during the 2nd week? The question obviously cannot be answered from present knowledge. Nevertheless, it is clear that the pseudopregnancy that transpires when a significant i)ortion of hypophyseal tissue remains in normal relation to the hypothalamus is far from the steady state of that which becomes established by total removal of the pars distalis to an extracranial site. It is also apparent that the onset of luteolysis may be postponed by such means as hysterectomy or production of artificial deciduomas (see p. 538). Furthermore, during lactation-pseudoi)regnancies in rats, Canivenc and Mayer (1953) prolonged luteal function to 34 days by substituting successive new litters of suckling young. This technique should prove especially valuable in experimental analysis of both luteotrophic and luteolytic mechanisms.
Benson and Folley (1956) suggested that lactogen secretion is activated by oxytocin, inasmuch as its injection prevented the normal inv'olution of the mammary glands after withdrawal of the litters from lactating rats. This observation has been confirmed by McCann, Mack and Gale (1958), who also noted the interruption of lactation by lesions of the sui)raoi)tico-hyiiophyseal tract. Selye and ]\lcKeown (1934) long ago found that pseudopregnancy could be induced in cycling rats by the introduction of a suckling litter. Although all this is consistent with the above-mentioned observation by Canivenc and Alayer, other workers have observed luteolytic effects of gonadotrophinfree oxytocin administered to cycling dairy heifers (Armstrong and Hansel, 1958). Furthermore, Grosvenor and Turner (1958), after first noting that the administration of Dibenamine, atropine, or pentobarbital to rats prevented the expected drop in assay able pituitary lactogen at nursing, found no decrease when pentobarbitalized mothers were injected with physiologic doses of oxytocin intravenously. When the dosage was increased 30 to 60 times there was apparently some moderate discharge, but the authors regard it as insignificant.
C. Effect of the Uterus on Luteal Function
This subject has been reviewed by Bradbury, Brown and Gray (1950). In three species (Fig. 8.13) hysterectomy results in significant prolongation of the functional life-span of corpora lutea (guinea pig, Loeb, 1927; rabbit, Asdell and Hammond, 1933; rat, Bradbury, 1937). In each case the period of luteal function approximates that of normal pregnancy. The fact that corpora lutea in the pseudopregnant ferret normally function as long as in the pregnant animal may be a clue to the noncffect of hysterectomy in that species (Deanesly and Parkes, 1933). Ahhough Burford and Diddle (1936) rej^orted that in monkeys total hysterectomy was followed by vaginal cycles of normal length, examination of their protocols shows that during the several postoperative months just 1 corpus luteum was produced among all 5 animals. The experiment thus seems inconclusive. Impairment of pelvic circulation seems to be a common factor complicating the results of hysterectomy in women and may have been one cause of the failure of luteinization in these monkeys.
An interpretation given by Loeb (1927) and Bradbury, Brown and Gray (1950) for the prolongation of luteal function by hysterectomy is that in species in which the effect is demonstrable the uterus secretes a specific substance which abbreviates the life of the corpus luteum. Hechter, Fraenkel, Lev and Soskin (1940) found in rats that grafts of estrous uteri shortened the pseudopregnancies of hysterectomized animals to normal length. Implantation of similar tis
Fig. 8.13. Reliilive duiation.s of p.seiulopicgnaiicy in normal and hysterectomizeil animals of four species in relation to the duration of j^ostation characteristic of each. Gestation plotted as a common unit of time. (After J. T. Bradbury, W. E. Brown and L. A. Gray, Recent Proj,n-. Hormone Res., 5, 151-194, 1950.)
SIR' which had been killed by freezing had
no such effect, nor did successful grafts of
uteri from diestrous donors. Bradbury,
Brown and Gray (1950) found that partially hysterectomized rats in which the remaining uterine tissue was continuous with
the cervix, hence properly drained, experienced pseudopregnancies of normal length.
However, when the continuity was interrupted the uterine remnants became greatly
distended, the endometrium was destroyed,
and the animals had prolonged pseudopregnancies. Possibly the endometrium is the
source of the hypothetical "luteolytic" substance.*'
Under other circumstances the endometrium of rats has a luteotrophic rather than luteolytic effect, for when deciduomas are induced by trauma the pseudopregnancies of otherwise normal animals are lengthened to 22 days or more (Ershoff and Duell, 1943; Velardo, Dawson, Olsen and Hisaw, 1953). This is not true in mice, however, and Kamell and Atkinson (1948) suggest that the I'eason may lie in the shorter life-span of the decidual tissue in that species. Loeb (1927) reported that deciduomas in cyclic guinea pigs ])rolonged the luteal phase, i.e., delayed the next ovulation from 3 to 7 days, which is far less than the prolongation after hysterectomy.
As an alternative to the concept of conI rol of the corpus luteum by humoral agents
" The denial by Velardo, Olsen, Hisaw and Dawson (1953) that hysterectomy in rats prolongs l)seiidopregnancy is based on operations performed later in the luteal phase than those of Bradbury, Brown and Gray and of Hechter, Fraenkel, Lev and Soskin. Whereas the latter workers had operated in the range from the 4th to the 7th day.^^ of p.seudopregnancy and many of Bradbury's cases lacked uteri when they entered pseudopregnancy, Velardo and associates excised the uteri on the 9th day. It seems possible that this difference in time may be crucial, for by the 9th day of a 12-day pseudopregnancy the corpora lutea must be on the verge of regression, if that in'ocess has not already been initiated. After maintaining pseudopregnancy in estrogenized, liypophysectomized rats b}^ means of lactogen, its withdrawal is followed about 3 days later by estrous smears (Nelson and Bichette, 1943; Nelson, 1946). A slightly longer delay occurs in nonestrogenized, intact rats at the end of a lactogeninduced pseudopregnancy (Everett, unpublished) or after withdrawal of progesterone treatment (Fig. 8C).
formed in the uterus, Loeb considered the jjossibility that neural mechanisms are involved. The idea was not acceptable, he felt, because in partial hysterectomies the result was not detennined by the locus of the part removed. The finding by Hechter, Fraenkel, Lev and Soskin (1940) that grafts of uterine tissue in hysterectomized rats return the duration of pseudopregnancy to normal, is significant evidence pointing in the same direction.
A third hypothesis was advanced by Heckel (1942) who found in rabbits that the extent of prolongation of luteal function by subtotal hysterectomy is roughly proportional to the amount of uterine tissue removed. The suggestion was offered that removal of the uterus has an estrogensparing effect. The greater amount of estrogen thus available to the corpora lutea prolongs their life, according to this view.
Later investigations by Moore and Nalbandov (1953) revive the possibility that the uterus influences the ovary by way of the nervous system. In sheep the implantation of a plastic bead in utero during the early luteal phase shortened the cycle by several days. Successive cycles tended to be unusually short. When the uterine segments containing the beads were denervated, however, the cycles were essentially normal. Other work from the same laboratory (Huston and Nalbandov, 1953) which indirectly may bear on this problem, indicates that the presence of a mechanical irritant (a thread) in the oviduct of the domestic fowl tends to block ovulation. The blockade may extend for as long as 20 days if the thread is placed in the isthmus (van Tienhoven, 1953). The ovaries remain functional, producing large follicles which may be ovulated at will by injection of LH. The authors feel that this phenomenon, like the effect of the bead in the sheep uterus, involves a neural mechanism, Init crucial information is lacking. It may be significant that stimulation of the ovaries was found in some hens, in place of blockade.
We may hope that as more information becomes available the assortment of facts given in these paragraphs will fit into a rational system. Not until this is realized can we hope to understand the regulation of the luteal phase.
VIII. Concluding Comments
From what has been written here it is readily apparent that present knowledge of the mechanisms controlling the reproductive cycle is extremely spotty. The number of assumptions necessary to knit the various items of factual information into an orderly pattern is disturbing. In spite of a voluminous literature which has grown during the last 60 years, we are really only a few steps ahead of our predecessors at the turn of the century in terms of fundamental understanding. A brief recounting of some of these steps may be desirable.
The first three decades saw the gradual development of proof that the ovary is a gland of internal secretion as well as the producer of eggs, governing the uterus and other accessory organs by secretion of hormones into the blood stream. For a while it seemed that the events of the reproductive cycle could be neatly explained, with the ovary in the capacity of controlling agent. Yet there were indications that the ovary itself is not independent. As early as 19091910 (Aschner; Crowe, Gushing and Honuins) it was noted that destruction of the hypophysis is accompanied by atrophy of the gonads and reproductive tract. In 1927 the separate investigations of Smith and Engle and of Zondek and Aschheim demonstrated conclusively that function of the ovary depends vitally on the anterior hyjiophysis. Promptly it was learned that the hypophyseal secretion of gonadotrophic hormone is in turn modified by estrogens. In the early 1930's the "push-pull" hypothesis of pituitary-ovarian interaction was separately stated by Brouha and Simonnet and by Moore (see Moore and Price, 1932). Modified in detail as new facts appeared, this hypothesis is held to the present day in some quarters as a simple exi)lanation of how the cycle comes about in polyestrous, continuous breeders in which ovulation takes l)lacc spontaneously. Much of the investigation of pituitary-ovarian i)hysiology during the 1930's was performed within the framework of this hypothesis.
For seasonal breeders and reflex ovulators, however, the assumption was necessary that special controlling mechanisms are superimjiosed. It bccaiiie iccognizcd also that in some vaguely defined manner even the human cycle is subject to intervention of the nervous system. The possible importance of the hypothalamus was debated at some length in the twenties. In 1932 the existence of a sex center there was proposed by Hohlweg and Junkmann. In an attempt to explain the coital excitation of the rabbit hypophysis which causes the liberation of gonadotrophin, Hinsey and Markee (1933) suggested diffusion of a chemical substance from the posterior lobe to the anterior lobe. Hinsey (1937) later elaborated on this possibility and mentioned the hypophyseal portal vessels as a plausible route by which the substance might travel. We have seen the later history of these ideas.
The "Sexualcentrum" of Hohlweg and Junkmann was proposed as a mediator of the effects of estrogen on the anterior hypophysis of rats. Westman and Jacobsohn (1936-1940), on the other hand, believed that through its stalk connection with the hypophysis the rat hypothalamus governs gonadotrophin synthesis, not release. The latter they regarded as a direct effect of estrogen on the gland. These views did not afford a common basis for spontaneous and refiex ovulation.
Schweizer, Chari])i)er and Haterius ( 1937) offered the first surmise of similarity, after finding that guinea pigs bearing intraocular pituitary grafts developed persistent estrus and large follicles that failed to go through maturation changes. Their feeling was that the normal connection of hypophysis with hypothalamus may be necessary for cyclic liberation of LH. Almost concurrently, Dempsey (1937) expressed a similar view as one alternative explanation of his experimental results with the guinea i)ig cycle. Suggesting cautiously that release of luteinizer may be brought about l)y a "rhythmic discharge" from the central nervous system, he went on to mention the "possibility that a high level of oestrin is necessary but not directly responsible for the release of luteinizer" (italics added). From this it is only a short transition to certain concepts set forth in the present exposition.
According to current views: (1) Reflex ovulation and spontaneous ovulation alike are governed by a hypothalamo-pituitary apparatus whose final link to the pituitaiy is neurohumoral by way of the hypophyseal portal veins and whose activity precipitates release of LH. (2) The apparatus includes a hypothalamic center or centers whose excitation depends on estrogen-progesterone levels and afferent impulses of various kinds. (3) The sensitivity of the center (s) is influenced not only by the sex steroids, but by other poorly understood factors that vary from species to species and from time to time in individuals, e.g., the diurnal rhythm in rats. Here in bare outline is a plausible hypothesis that may be generally applied to the events immediately relating to ovulation.
Satisfactory hypotheses respecting other phases of the cycle must await future developments. The extent and manner of intervention of the nervous system in the follicular and luteal phases remain unsettled. Although the hypophyseal hormones concerned in ovarian follicle development have been characterized, their exact chemical descrijition has not been accomplished. The rate of their output at different stages of the cycle is largely a matter of conjecture. Structural changes that they jiroduce in the ovary are well known, but in chemical terms only the end products of ovarian activity are well recognized, and these probably incompletely. The fact that the estrogens, in turn, have a regulating effect on follicle-stimulating activity of the hypophysis is known, but the mechanisms by which this effect is accomplished are uncertain. The hypophyseal hormones that maintain the luteal phase are recognized with any certainty in only three species and there is a wide difference between rabbits, on the one hand, and rats and mice, on the other. For mammals generally, the luteotrophic factors have not been identified. Whether the hypothalamus is actively concerned in maintenance of the luteal i)hase in the majority of mammals is unknown. The morphologic effects of luteotrojihic stimulation on corpora lutea are well recognized, but here again the chemical mechanisms leading to the end products are obscure. The action of the corpus luteum hormone in regulation of the cycle is partially known, including the well established fact that its continual jiresence in large amount will suiijiress the estrogenic and ovulatory phases. Yet, one cannot say whether this effect is accomplished by direct action on the hypophysis or by indirect action through the central nervous system. Nor can one state how the hypophysisgonad equilibrium of the luteal phase is interrupted in the absence of a conceptus. With respect to the ovulation mechanism itself, the hypothesis outlined above requires verification in additional species. Assuming its validity, many details remain to be studied, e.g., the neural pathways and nuclei involved, identification of neurochemical activators of the pars distalis and their sources and loci of action, the precise nature of mechanisms whereby the gonadal steroids excite or suppress, the cellular mechanisms by which ovulating hormone is released into circulation by the hypoi^hysis, and the cytochemical effects within the ovary. All too evidently an encompassing theory of the female reproductive cycle is far from realization.
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