Recent Progress in Hormone Research: Proceedings of the 1982 Laurentian Hormone Conference

Zor

PREFACE

As this the thirty-ninth volume of Recent Progress in Hormone Research, representing the proceedings of the 1982 Laurentian Hormone Conference, goes to press it seems not amiss, perhaps even edifying, to take a backward glance. The forerunner of this Conference, the Hormone Conference of the American Association for the Advancement of Science, met for the first time in 1943 at Gibson Island. There an unanticipated segregation problem arose. In 1944, through the kindly intercession of the Montreal Physiological Society, the Conference met at Mt. Tremblant Lodge in the Laurentian Mountains some ninety miles northwest of Montreal. This setting for both formal and informal scientific exchange proved to be propitious. At a return meeting in 1945 it was unanimously voted to call the assembly the Laurentian Hormone Conference and to publish the papers and discussions.

In inaugurating this series of annual publications the editor, Gregory Pincus, expressed the hope that the publication of critical evaluations and work-in-progress by leading authorities will be valuable not only as records of knowledge and accomplishment but as excitements to research. The spirit of inquiry dies without criticism and discussion, and it is largely the purpose of these Conferences to nourish that spirit. What was then a hope became an enduring reality from which there has been no deviation. As with Volume 1, this volume will attest that in the field of hormone research the spirit of inquiry remains very much alive and that the Laurentian Hormone Conference from which this series derives remains, as in Olympia, a gathering where the best is put to test. It has been my good fortune to attend all but four of these Conferences, including the one at Gibson Island, and to edit the last ten volumes of this series. On the basis of that perspective it is clear that hormone research has been keeping pace with the momentous advances in science and technology and continues to offer challenging problems of great intrigue and biomedical importance. To my knowledge the record of progress in hormone research as depicted in these volumes remains unmatched elsewhere. Volume 39 is in keeping with that proud tradition.

Publication of the hour-long discussions following each paper is a valuable feature of Recent Progress in Hormone Research. Persons chairing these discussion sessions are carefully chosen for their relevant expertise and nimbleness in the arena of issues and answers. For their services in this capacity we are indebted to Drs. Ernest J. Peck, Alexander D. Kenny, Murray Saffran, Gerald D. Aurbach, John D. Baxter, Iones Kourides, Joseph Lamer, and Maria New.

It is again my pleasure to thank our executive secretary, Martha Wright, for efficient and dedicated attention to every aspect of the arrangements for the 1982 Conference and the assemblage of all the copy for this volume. To Lucy Felicissimo and Linda Carsagnini we extend our gratitude for transcribing the tapes with skill and alacrity, and, as always, the expertise and care lavished on the production of this volume by Academic Press is deeply appreciated.

Roy O. Greep

The Ovarian Triad of the Primate Menstrual Cycle¹

ARNOLD L. GOODMAN and GARY D. HODGEN,     Department of Physiology and Biophysics, University of Alabama in Birmingham, Birmingham, Alabama Pregnancy Research Branch, National Institute of Child, Health and Human Development National Institutes of Health, Bethesda, Maryland

Publisher Summary

This chapter reviews the primate ovarian cycle and the regulation of its endocrine and gametogenic activities, based on the studies in rhesus and cynomolgus monkeys. Unlike adult male mammals, which continuously produce new sperm, female mammals are born with their lifetime supply of eggs, enclosed in primordial follicles. This pool of primordial follicles serves as a non-replenished, progressively depleted stockpile from which growing follicles and oocytes are continuously withdrawn. Each adult mammalian female ovulates a species-characteristic number of ova each week, month, season, or year—the frequency again being a species characteristic. Thus, litter size and annual fecundity rate of a species are proximately determined by the physiology of the female through the size of the ovulatory quota, frequency of the ovarian cycle, and the duration of pregnancy and lactation. Typically ovulation of a single, fertilizable ovum in each menstrual cycle completes a course of oogenesis that begins during fetal development.

I Introduction

To be offered so coveted a forum to present our findings on the workings of the primate ovary leaves us flattered by the opportunity and awed by the prospect of addressing such a distinguished audience. This good moment derives much from the contributions of co-workers—the energetic efforts of a cohort of imaginative postdoctoral fellows and the unrelenting labors of a talented and devoted technical staff. Speaking for all of them, we are pleased that our efforts will become a part of the legacy of The Gregory Pincus Memorial Lectures.

While not a strict chronicle, this presentation will review our efforts aimed toward an improved understanding of the primate ovarian cycle and the regulation of its endocrine and gametogenic activities, based on studies in rhesus and cynomolgus monkeys. We will concentrate on a few fundamental, unifying issues. These studies, along with the findings of others, have enabled us to decipher and construct a clearer image of the workings of the primate ovary. However, as you will see, what emerges is far from a portrait in vivid color; rather, it is more a sketch in chiaroscuro.

II The Ovarian Triad

As we began our work in the early 1970s, we were fortunate to have a rather firm foundation on which to design our own studies on the regulation of follicle growth. In particular, we had the advantage of drawing on knowledge of the rhesus monkey menstrual cycle, provided by the classical studies of Hartman, Hisaw, Corner, Allen, van Wagenen, and Knobil, among many others. Detailed characterizations of the human cycle by Ross, Gemzell, Vande Wiele, Lunenfeld, Yen and others were also highly instructive. Without their solid contributions, our opportunities would have been more constrained and our attempts less focused.

Since the fundamental themes of folliculogenesis appear to transcend species differences, we considered them then, and again here, as axiomatic for primates, as well. Unlike adult male mammals, which continuously produce new sperm, female mammals are born with their lifetime supply of eggs, enclosed in primordial follicles. This pool of primordial follicles serves as a nonreplenished, progressively depleted stockpile (or genetic bank) from which growing follicles (and oocytes) are continuously withdrawn. As an adult, each mammalian female ovulates a species-characteristic number of ova (the ovulatory quota) each week, month, season, or year—the frequency again being a species characteristic. Thus, litter size and annual fecundity rate of a species are proximately determined by the physiology of the female through the size of the ovulatory quota, frequency of the ovarian cycle, and the duration of pregnancy and lactation. The process of folliculogenesis may be seen as providing three products essential for successful reproduction: (1) the surge of estradiol secretion to trigger the release of the preovulatory LH surge, (2) the species-characteristic number of fertilizable haploid ova, and (3) the luteal body(ies) and secretion of progesterone necessary to prepare the uterus for implantation and to maintain at least the early stages of pregnancy. Thus, this ovarian triad—the ovulatory follicle, oocyte, and corpus luteum—must each function properly for successful reproduction; impairment of follicle growth and secretion, oocyte maturation, or luteal function will preclude natural fertility.

III Regulation of Folliculogenesis

A SOME QUESTIONS

At first, the questions we posed were not new; indeed, they were much the same as asked by earlier workers (Hisaw, 1947; Young, 1961). Later, as our understanding improved, we began to ask more precise questions. Some we have answered; others, including many important ones, remain unanswered.

We accept as dogma that typically ovulation of a single, fertilizable ovum each menstrual cycle completes a course of oogenesis that began during fetal development. But, how is it that, from among the thousands of follicles present from birth, only a relative few are recruited each cycle to grow, at the same time others remain at rest? How is it that, from the host of follicles maturing in each ovary, (typically) only a single follicle escapes atresia and is selected to ovulate each cycle? Since the vast majority of follicles falls victim to atresia (>99%), we recognized that understanding the selection of the follicle destined to ovulate is fraught with the inherent difficulty of studying the rare exception rather than the predominant rule. As a consequence, we have tried to be assiduous in distinguishing follicle growth that culminates in ovulation (gametogenic follicle growth) from that ending in atresia (see illustrated overview of the life cycle of the primate ovary—Fig. 1a and b).

FIG. 1 (a) Pie chart depicting the fate of follicles as a function of age and as fraction of the total present from birth. (b) Hypothetical curve depicting progressive depletion of viable follicles from the ovary from fetal-life through menopause.

The latter stages of oogenesis in adults (i.e., folliculogenesis) are known to depend, to a large degree, on a complex interplay of hormones from the hypothalamus, pituitary, and ovary. However, even though much more is understood today of these endocrine relationships (diZerega and Hodgen, 1981a; Knobil, 1980; Richards, 1979; Jones, 1978), what determines the fate of an individual follicle remains largely unknown. Is it primarily extraovarian factors that regulate follicle growth, or do intraovarian factors play important governing roles, as well (Franchimont and Channing, 1981; Jagiello and Vogel, 1981)? When is the follicle destined to ovulate selected? Is it only events in the current cycle that determine the outcome, or do events in the preceding cycle(s) also influence this process.

This last question leads immediately to another intriguing aspect of folliculogenesis in monotocous primates, including women and macaques: how just a single follicle matures to ovulation on only one ovary each cycle, even though both ovaries are perfused by a common systemic circulation. Since in higher primates both ovaries are functional (i.e., one has not remained rudimentary), the maturation of a single follicle with the potential to ovulate brings with it the obvious concomitant of one active and one quiescent ovary each cycle. Is this ovarian asymmetry and side-to-side partition of ovulation between cycles regulated or random? Do the ovaries communicate, and, if so, how? Which comes first, so to speak, the follicle or the ovary? That is, is one ovary in a given cycle somehow more suited than the other to sponsor follicle development (as a consequence of events in the preceding cycle?), or is ovarian asymmetry merely a consequence of follicle selection?

B A NASCENT HYPOTHESIS

As we attempt to answer some of these questions, we will also present evidence in support of an emerging hypothesis of the regulation of follicle growth and selection during the primate ovarian cycle. This hypothesis suggests that many remaining answers, and doubtless new questions as well, will be found within the ovary.

We hypothesize (1) that the precise regulation of follicle growth and selection is accomplished primarily by specific ovarian factors that act directly on the ovaries, and (2) that gonadotropins, at tonic levels, are merely permissive to folliculogenesis. We envisage a two-tier ovarian mechanism (Fig. 2). At the first tier, specific ovarian factors govern the progressive winnowing of the cohort of developing follicles down to the size of the species characteristic ovulatory quota each cycle. Some factors may act within the ovary of origin as intraovarian regulators; other ovarian factors may be secreted and circulate to the opposite ovary, to act as extraovarian signals (but of ovarian origin). Together, they regulate the culling-out or inhibit the maturation of supernumerary growing follicles. This first tier of the proposed ovarian mechanism, which precisely regulates follicle selection (cohort size), is operative, however, only when circulating gonadotropins are above minimal tonic levels and near the tonic set-point. At the second tier, ovarian hormones (steroidal and nonsteroidal) inhibit gonadotropin secretion in a negative feedback fashion to constrain circulating gonadotropin levels to an appropriate range around the tonic set-point. If gonadotropin levels are too far below this tonic set-point, then folliculogenesis will be arrested as a result of inadequate stimulation. Contrariwise, if circulating gonadotropin levels are too far above the tonic set-point, then first tier ovarian mechanisms, ordinarily at work to regulate the size of the ovulatory quota, are impaired or inactivated; in such instances superovulation occurs. That is, we propose that the emergence of multiple follicles on both ovaries after administration of exogenous gonadotropins to monkeys or women is not only the result of augmenting the availability of gonadotropins, per se, but is also an indirect result of overriding first tier ovarian mechanisms of follicle selection.

FIG. 2 A two tier mechanism involving both intra- and extraovarian regulation of folliculogenesis is proposed. Ovarian (follicular) factors acting directly on the ovaries, against a background of permissive gonadotropic support, are hypothesized to be the primary regulators of follicle selection and dominance.

Clearly, as exploited by several well-known bioassays for gonadotropins, both FSH and LH can have graded, dose-dependent effects on the ovary. However, in the physiological setting of the menstrual cycle, we find it more useful to consider that the folliculogenic actions of gonadotropins (principally FSH) are permissive at tonic levels and that the steroidogenic actions of gonadotropins (principally LH) are graded. If FSH at tonic levels is actually permissive to folliculogenesis, then graded effects observed may be attributable to supraphysiological (supratonic) levels. Graded actions of gonadotropins on steroidogenesis [and perhaps on inhibin secretion(s) as well, see below] are necessary for the second tier of the ovarian mechanism to constrain circulating gonadotropins near the tonic set-point, so that first tier mechanisms of follicle selection are effective. Evidence that these two activities (tiers) are dissociable, in some circumstances, is presented below. More direct evidence for this hypothesis must come from future studies.

C TOWARD A MORE CONSISTENT TERMINOLOGY

Before considering how we arrived at this hypothesis, we shall explain some important terms, with the hope of avoiding misunderstandings later on. These terms are not new yet, although in wide use, they have not been employed with uniform precision. While even our definitions remain lacking, they are, nonetheless, useful in drawing important distinctions.

That the ovary performs dual roles as an organ of reproduction and a gland of internal secretion is well known. To distinguish the regulation of these ovarian activities, we shall refer to the gametogenic activity as folliculogenesis and to the secretory activity as hormonogenesis (Fig. 3). Extra- or intraovarian factors that directly influence the ovary’s gametogenic role have a folliculogenic action or elicit a folliculogenic response. While some may balk at the introduction of a term like hormonogenesis, it is used here because we find current nomenclature inadequate. Since, in our scheme, some ovarian secretions may act locally within the ovary in a paracrine (or perhaps even autocrine) manner, they are not, in the strictest sense, endocrine. In addition, since some ovarian hormones secreted into the circulation may be nonsteroidal (inhibins), steroidogenesis is too restrictive. Consequently, we will use the term hormonogenesis to encompass all nature and manner of such ovarian secretions.

FIG. 3 Hypothetical scheme depicting the relationships between the regulation of the ovary’s gametogenic and secretory activities by extraovarian and intraovarian factors.

During each cycle primordial follicles depart the resting pool and begin a well-characterized pattern of growth and development (Brambell, 1956; Harrison and Weir, 1977). Groups of (quasi)synchronously growing follicles are called cohorts. In the same or some subsequent cycle,² a few (or only one) members of one cohort continue to develop and escape atresia,³ until they become preovulatory Graafian follicles, ultimately providing the species-characteristic ovulatory quota of eggs. Schwartz (1974) has aptly termed this pattern the trajectory of follicle growth. Extending the trajectory metaphor into our hypothesis outlined above, gonadotropins may be seen as providing the thrust and ovarian factors the guidance along the trajectory, not unlike some surface launched missle (Fig. 4). Clearly, without continued thrust the trajectory will be limited; with thrust in excess of the guidance system’s design the accuracy and precision of the course are compromised.

FIG. 4 Proposed relationship between gonadotropins and ovarian factors in regulating maturation or atresia along the so-called trajectory of follicle growth.

We shall use the term recruitment to indicate that a follicle has entered on this growth trajectory. Thus, under this definition, recruitment includes the entry of primordial follicles onto the trajectory, without excluding the reentry of more mature follicles which may have been transiently at rest. Pedersen’s (1970) studies in mice have generally been interpreted to mean that, once a follicle leaves the resting primordial pool, it must continue to mature or succumb to atresia, i.e., it does not again rest. Whether or not this is true for primates is unknown, hence the broader definition used here. Since follicles at various preantral stages of development were observed in ovaries of hypophysectomized rats and rabbits (Hertz and Hisaw, 1934), recruitment of primordial follicles is not wholly dependent on gonadotropins, but may be only enhanced by these hormones (Lunenfeld et al., 1976). Growing follicles are vulnerable to atresia and may depart the trajectory at any point. Thus, while an obligatory step, recruitment does not guarantee ovulation. That recruitment is a necessary, but not a sufficient, condition for ovulation is particularly important when interpreting results of experiments employing exogenous gonadotropins to stimulate follicle development, as discussed below.

The term selection will be used here to indicate the final winnowing of the cohort (via atresia of excess follicles) down to a size equal to the species-characteristic ovulatory quota. That is, when the number of healthy follicles (i.e., with ovulatory potential) in the cohort equals the size of the ovulatory quota, then selection is complete. Implicit in this definition is the notion that the cohort may be the regulated variable rather than the fate of an individual follicle. That is, which follicles, in particular, are culled from the cohort may be due to a random process that continues until cohort size matches the ovulatory quota, in contrast to a deterministic process in which specific follicles are individually chosen according to some unknown criteria. The character (stochastic vs deterministic) of the selection mechanism remains uncertain. What is certain, however, is that the process operates in primates with great precision; a spontaneous multiple ovulation is extremely atypical.⁴ Like recruitment, selection does not guarantee ovulation, but, given its greater temporal proximity to ovulation, selection may, with high probability, be expected to be followed by ovulation in a typical cycle. Evidence will be presented that selection is begun and is completed only during the cycle in which ovulation occurs. In contrast, the time of recruitment, and thus the total length (duration) of the trajectory are unknown. Based on findings discussed below, the duration of the trajectory in macaques and women appears to be not less than about 2 weeks.

Clearly, the ovulatory quota in higher primates is generally unity; hence, although actual cohort size as a function of day-of-cycle is unknown, the possibility of a cohort of one even as early as recruitment is not excluded. Even if this were true, cohort remains a meaningful construct, and it is still useful to consider recruitment and selection as distinct processes.

Although anticipating facts not yet in evidence, so to speak, the term dominance is introduced here to limit our lexicography to this section. As we shall demonstrate below, the follicle destined to ovulate plays a key role in regulating the size of the ovulatory quota, at least in monkeys. That is, the follicle selected for ovulation is functionally (not merely morphologically) dominant; it inhibits the development of other competing follicles on both ovaries. As a necessary corollary, the dominant follicle (i.e., the sole follicle destined to ovulate) somehow continues to thrive in a milieu it, itself, has made inhospitable for others. Whether this capacity to thrive under these circumstances results from a unique ability of the dominant follicle which is newly acquired or from a preexisting ability originally shared by the entire cohort, but which is retained only by the dominant follicle, is unclear. That is, does the survival of the dominant follicle depend on a process of acquisition or retention of metabolic properties to resist atresia? Underpinning this issue is how the dominant follicle actually exerts its eminence. How is it spared from the very inhibition it imposes on others? As one mechanism, we hypothesize that the dominant follicle secretes a substance we call selectron, which acts directly on the ovaries to inhibit the development of potentially competing follicles. The motivation for this hypothesis is developed in more detail below. As we will show, the selected follicle becomes dominant about a week before ovulation. Consequently, it must maintain its dominance during the week before ovulation. Unresolved is whether the mechanism(s) by which the follicle attains dominance is the same as the mechanism(s) by which the follicle maintains dominance. Unresolved as well is the precise temporal relationship between selection and dominance.

D SELECTION OF THE DOMINANT FOLLICLE

1 The Role of the Ovary’s Cyclic Structures

In one of our first studies (Goodman et al., 1977b) we learned that the ovary’s cyclic structures: the follicle destined to ovulate and its successor, the corpus luteum, play important roles in the regulation of gametogenic follicle growth. Since the outcome of our initial experiment provided the rationale for a series of subsequent studies, its key findings are reviewed in detail. If logical flaws exist, they may be traced to this first effort.

After we cauterized the largest visible follicle (days 8–12), the cycle in progress was halted (Fig. 5). Estradiol levels, which had been increasing progressively, fell promptly to early follicular phase values. Surges of LH and FSH did not occur at midcycle; instead, gonadotropins were maintained at tonic levels for an average of ∼12.5 days before the next preovulatory gonadotropin surges were observed (i.e., about 6 weeks after surges in the previous cycle). These surges, preceded by typical rises in estradiol levels, were followed, in turn, by typical luteal phase patterns of progesterone secretion. As confirmed at laparotomy, only a single follicle ovulated in each monkey; in almost all monkeys the new corpus luteum was contralateral to the side of cautery. In a control group, cautery of the ovarian surface remote from the largest visible follicle, in contrast, did not deter the progressive rise in estradiol levels, delay the midcycle surges of LH and FSH, or diminish progesterone secretion during the ensuing luteal phase.

FIG. 5 Hormonal patterns before and after cautery of the largest visible follicle in rhesus monkeys. Important time intervals are marked at top. (From Goodman et al., 1977b, with permission.)

In another group of monkeys (Fig. 6), after we excised the corpus luteum and cauterized the resulting crater, the cycle in progress was halted during the midluteal phase (days 16–19). Progesterone levels dropped to follicular phase levels in 1–2 days and the onset of menses was premature, appearing 3–4 days after luteal excision. The midluteal phase rise in estrogens was also terminated. As in the follicle cautery group, gonadotropins were maintained at tonic levels until the next preovulatory surges ∼12.5 days after luteectomy, or, equivalently, about 18 days after the preceding surges. These surges of FSH and LH were preceded by a typical progressive increase in circulating estradiol levels and were succeeded, in turn, by a typical luteal phase pattern of progesterone secretion. Again, only a single follicle ovulated, in every instance on the contralateral ovary.

FIG. 6 Hormonal patterns before and after luteectomy in rhesus monkeys. Important time intervals are shown at top. (From Goodman et al., 1977b, with permission.)

That follicle cautery had delayed, and luteectomy had advanced, the expected times of the next preovulatory gonadotropin surges was consistent with assignment of a zeitgeber role to the ovary in the monkey menstrual cycle (Knobil, 1973, 1974). Thus, the time-keeping function of the primate ovary appeared to be subserved by the activities of the ovary’s cyclic structures. More than this, these kinds of findings provided new insights into the regulation of follicle growth and selection in this primate. Not all these inferences are obvious, however, without some explanation.

That cautery of the largest visible follicle halted the cycle in progress, i.e., interrupted the progressive rise in estradiol levels and thereby prevented the expected midcycle surges of LH and FSH, led us to conclude that we had, in fact, destroyed the follicle destined to ovulate that cycle. Moreover, given the lengthy (see below) 2 week delay to the next ovulation, it appeared that no other follicle of the cohort, if any, was competent to serve as a surrogate for the cauterized follicle to achieve a timely, midcycle ovulation. Instead, the next follicle to ovulate was likely derived from another cohort. This, in turn, meant that the selection of the follicle destined to ovulate had already occurred by the time of cautery, i.e., by day 8 of the cycle.

The use of follicle cautery and luteectomy was pioneered by workers studying the ovarian cycle in domestic animals (Dufour et al., 1971). However, the outcome of these kinds of experiments in monkeys versus sheep, swine, or cattle is significantly different. In the domestic species, cautery of the current crop of follicles is rapidly compensated. Removal of the cyclic structures is followed by ovulation within about 2 days in sheep, about 6 days in pigs, and 2 to 3 days in cows, in contrast to the 2 week interval observed in monkeys. In women, too, the interval from ablation of the dominant follicle or corpus luteum to the next ovulation is a fortnight (Nilsson et al., 1982). Thus, since the overall time-course of follicle growth presumably exceeds the brief interval from ablation to ovulation, growth of new follicles in domestic species ordinarily occurs in the presence of the prevailing cyclic structures. In higher primates, in contrast, new gametogenic follicle growth appears to be inhibited in the presence of the cyclic structure and proceeds only after its removal. Consequently, in monkeys and women, the follicle destined to ovulate is not merely morphologically dominant, it is functionally dominant, as well.

Thus, we concluded that the dominant follicle, itself, plays a key role in regulating the size of the ovulatory quota in this primate by inhibiting the development of any competing follicles. Inhibition of gametogenic follicle growth is continued by its successor, the corpus luteum. The next round of new gametogenic follicle growth occurs only after the influence of the cyclic structure is removed either artificially by experimental intervention or naturally after the demise of the corpus luteum.

We were also struck by the fact that the interval from either follicle cautery or luteectomy to the next preovulatory LH surge was about 12.5 days (i.e., about the length of a typical follicular phase after adding the 36 to 40 hours for follicular rupture). The similarity of the duration of these intervals was very significant. Based on studies in rats, Schwartz (1974) had hypothesized that the role of the preovulatory FSH elevations was not to ripen the current crop of follicles but rather to recruit new follicles for the next cycle. If this were true in monkeys, then one would predict a priori that the time-course of new gametogenic follicle growth after interuption of the cycle in the follicular phase—before the FSH surge occurred—would differ from the time-course of new gametogenic follicle growth after luteectomy—after the midcycle FSH surge had already occurred. Clearly, this was not the case. Consequently, we concluded that the preovulatory surge of FSH is not necessary for follicle recruitment in monkeys. In addition, since, in this first study at least, gonadotropin levels were apparently maintained after the ablations, follicle recruitment and the subsequent emergence of a single follicle occurred without an attendant increment in circulating gonadotropins. Conversely, then, the inhibition of follicle growth in the presence of the cyclic structure was presumably not due to a decrement or limitation in gonadotropins. Instead, it appeared as if removal of these cyclic structures produced an unbolting (Parkes, 1966) or disinhibition of gametogenic follicle growth at the level of the ovaries. Moreover, the steroid milieu (estrogen-dominated vs progesterone-dominated) prevailing at ablation seemed without differential effect, as well. Relationships between tonic FSH levels and follicle recruitment and selection are discussed in more detail later on. The key point here, however, is that the midcycle surge of FSH is not an important determinant of follicle growth in monkeys.

That gametogenic follicle growth is held in abeyance during the luteal phase in primates was already appreciated by earlier workers, based on morphological findings (Block, 1951; Koering, 1969). The mechanism for this inhibition was unknown. In comparing follicle growth in sheep and primates Baird and co-workers (1975) hypothesized that the reason follicles mature during the luteal phase in sheep, but not in primates, was due to the difference in luteal estradiol secretion. The primate corpus luteum secretes estrogens; the ovine corpus luteum does not. They reasoned that luteal estradiol along with progesterone exerted a stronger negative feedback on gonadotropin secretion and thereby indirectly inhibited follicle growth in the primate luteal phase. However, when we examined this issue in monkeys, we could not confirm their hypothesis.

To learn how gametogenic follicle growth may be inhibited during the primate luteal phase, we (Goodman and Hodgen, 1977) luteectomized monkeys around day 18 of the menstrual cycle and immediately implanted silastic capsules containing crystalline progesterone for 10 days to approximate the duration of an intact cycle (Fig. 7). These capsules maintained serum progesterone at luteal phase levels (∼4 ng/ml) and prevented the premature onset of menses seen after luteectomy alone. Circulating estradiol, however, declined to early follicular phase levels (≤ 50 pg/ml). Tonic gonadotropin levels appeared to be maintained after luteectomy, during progesterone treatment, and after capsule removal. Menses began 3 to 4 days after progesterone withdrawal and the next preovulatory go nadotropin surges occurred, on average, 12.5 days after capsule removal, or, equivalently, ∼22.5 days after luteectomy. Thus, replacement with progesterone alone for 10 days was sufficient to delay the next ovulation after luteectomy by 10 days. Consequently, progesterone appears to be the principal luteal hormone inhibiting gametogenic follicle growth in this primate. In a related context, in addition to its well-known ability to block an estradiol-induced LH surge, progesterone has a more profound antifertility action, namely, inhibiting the growth of the follicle that would be the source of the estradiol surge.

FIG. 7 Effects of systemic progesterone replacement on time of next ovulation after luteectomy in rhesus monkeys. (From Goodman and Hodgen, 1977, with permission.)

Since we observed no changes in gonadotropin levels before and after luteectomy and capsule insertion and withdrawal, we hypothesized that progesterone may exert its inhibition at the level of the ovaries. However, we were unable to inhibit follicle growth by implanting progesterone-impregnated silastic wafers into either ovary after luteectomy. These findings were recently confirmed (Nass et al., 1981). Significantly, however, the side of the next ovulation after systemic progesterone replacement or bilateral ovarian surgery was randomized (see below).

We recently considered the possibility that (exogenous) progesterone’s action was mediated by peripheral conversion to 17α-hydroxyprogesterone, which is secreted by both the corpus luteum and dominant follicle in women and monkeys (Goodman and Hodgen, 1982b). This hypothesis appeared attractive for two reasons. First, it might explain the failure of intraovarian implants of progesterone to inhibit follicle growth. Second, it might account for the seemingly similar kind of inhibition of follicle growth exerted by the dominant follicle during the follicular phase when progesterone levels are not elevated. Suffice it to say that whatever may be progesterone’s mode of inhibition, it appears not to be mediated by peripheral conversion to 17α-hydroxyprogesterone or, presumably, other steroids along the so-called delta-4 pathway. We shall return to the issue of progesterone’s inhibition of gametogenic follicle growth in a later section.

2 The (Un)importance of Previous Ovarian Status

As mentioned above, after surgical removal of the cyclic structure the next ovulation was almost always on the contralateral ovary. Was this merely an experimental artifact or did it indicate a residual inhibitory effect of the ablated cyclic structure? To address this and other issues, we performed the following experiments (Goodman and Hodgen, 1979a,b) whose design is depicted in Figs. 8a and b. Since rhesus monkeys were not available in sufficient supply, in this study we employed cynomolgus monkeys, another macaque (M. fascicularis) whose reproductive physiology differs little from the rhesus (Goodman et al., 1977a).

FIG. 8 (a and b) Experimental design employed to investigate interaction between ovaries. (From Goodman and Hodgen, 1979a,b, with permission.)

When the cyclic structure (i.e., dominant follicle or corpus luteum) alone was removed, we observed the same kind of responses described earlier, namely, the current cycle was interrupted and the next ovulation of a single follicle occurred about 2 weeks later on the contralateral ovary (Fig. 9a and b). These same responses were also observed when the entire ovary bearing the structure was removed (Fig. 10a and b). Thus, apparently, the activity of the ovary bearing the cyclic structure can be accounted for virtually exclusively by the cyclic structure itself. That is, removal of other portions of that ovary was without discernible additive effects. In the third group, we removed the cyclic structure and the entire contralateral ovary in order to force new follicle growth back to the ipsilateral ovary. Despite this maneuver, responses stereotypical of the removal of the cyclic structure alone were observed (Fig. 11a and b).

FIG. 9 (a and b) Effects of ablation of the cyclic structure in the follicular (a) and luteal phase (b) in cynomolgus monkeys. (From Goodman and Hodgen, 1979a,b, with permission.)

FIG. 9b

FIG. 10 (a and b) Effects of ablating the entire ovary bearing the cyclic structure in the follicular (a) and luteal phase (b) in cynomolgus monkeys. (From Goodman and Hodgen, 1979a,b, with permission.)

FIG. 10b

FIG. 11 (a and b) Effects of ablating the cyclic structure and the entire contralateral ovary in the follicular (a) and luteal phase (b) to constrain new follicle growth to the ipsilateral ovary. (From Goodman and Hodgen, 1979a,b, with permission.)

FIG. 11b

This last outcome provided two important clues. First, it meant that new gametogenic follicle growth could resume on the operated ovary with the typical time-course to ovulation; thus, any residual inhibitory effects of the cyclic structure, or trauma attending its removal, were not of major significance. More importantly, it suggested that selection of the next dominant follicle did not occur until after the cyclic structure was removed. That is, since a monkey could not have known which ovary would be removed, she could not have queued up the next one ahead of time. This observation seems inconsistent with the hypothesis that follicles are selected to ovulate in some predetermined fashion (Edwards et al., 1977).

In the fourth group, removal of the ovary contralateral to the cyclic structure during the mid-follicular or mid-luteal phase was without discernible effect (Fig. 12a and b). In each case the cycle continued along its typical course; circulating levels of gonadotropins and ovarian steroids were unaffected. Consequently, the contralateral ovary appeared to be virtually quiescent after the emergence of the dominant follicle and throughout much of the luteal phase. Thus, the influence of the cyclic structure is not limited to its own ovary but extends to the other, as well, to inhibit its gametogenic and endocrine activities. Discounting trauma attending ablation, the previous status of the ovary did not, by itself, seem to influence subsequent gametogenic follicle growth.

FIG. 12 (a and b) Effects of ablating only the contralateral ovary during the follicular (a) or luteal phase (b) in cynomolgus monkeys. (From Goodman and Hodgen, 1979a,b, with permission.)

FIG. 12b

This last inference was tested in another but related experiment, namely, in chronically hemiovariectomized rhesus monkeys in which gametogenic follicle growth is repetitively constrained to a single ovary. Such monkeys display seemingly normal ovulatory menstrual cycles (Goodman and Hodgen, 1979c). When we (Goodman et al., 1979) luteectomized chronically hemiovariectomized monkeys, we observed the same, stereotypical folliculogenic response described for two-ovary monkeys—a single follicle ovulated about 2 weeks later (Fig. 13). The same kind of folliculogenic response was observed in monkeys with two ovaries even though the contralateral ovary also underwent wedge-resection as a surgical control (Fig. 14). In this luteectomized wedge-resected group, however, about half of the new ovulations occurred back on the luteectomized ovary. That is, bilateral trauma of the ovaries led to a randomization of the side of the next ovulation as observed previously (Goodman et al., 1979), without affecting the time-course of follicle growth.

FIG. 13 Effects of luteectomy on FSH secretion and follicle growth in chronically hemiovariectomized rhesus monkeys. (From Goodman et al., 1979, with permission.)

FIG. 14 Effects of luteectomy and concurrent wedge-resection of contralateral ovary. (From Goodman et al., 1979, with permission.)

The hormonal response to luteectomy in one-ovary monkeys, however, was not stereotypical; after luteectomy, FSH levels increased 2- to 4-fold and did not return to baseline before about 7 days. Consequently, timely new gametogenic follicle growth proceeded, with the maintenance of the ovulatory quota at unity, even though FSH levels were markedly elevated; this indicated that the ability of the remaining ovary to inhibit FSH secretion was somehow temporarily impaired (about 1 week). That is, new follicle growth and feedback inhibition were temporally dissociated. In monkeys with two ovaries, in contrast, luteectomy was followed by either no change in FSH levels or only a comparatively small (<50%), highly transient increase. Thus, the contralateral ovary makes a major contribution to the negative feedback regulation of FSH immediately after luteectomy. However, as indicated above, when the contralateral ovary was subjected to wedge-resection, only about half of the new ovulations occurred contralateral to luteectomy. Curiously, then, we are forced to conclude that, even though the contralateral ovary contributes to the negative feedback of FSH secretion, this activity is not sufficient to ensure that it is the site of the next ovulation. That is, in this circumstance, gametogenic follicle growth and negative feedback on FSH secretion were spatially dissociated in half the luteectomized wedge-resected monkeys.

From these kinds of results we concluded that, during the cycle, previous ovarian status, by itself, does not appear to be an important determinant of the side of the next ovulation. This same conclusion was reached by Clark et al. (1978) based on serial laparoscopic observations of rhesus monkeys across several cycles. The frequency of a new corpus luteum on the left or right ovary from cycle to cycle occurred as a binomial distribution with L = R = 0.5. Thus, the left-to-right alternation of ovulation in consecutive cycles seems as random as heads-after-tails at the flip of an honest coin.

In other physiological conditions, however, previous ovarian status may influence follicle growth. Ovulation was delayed after luteectomyinduced abortion in fertile cycles (Goodman and Hodgen, 1979d). This delay may be due to an extraluteal antifolliculogenic effect of chorionic gonadotropin (Goodman and Hodgen, 1982a) and may be mediated by differences in intraovarian progesterone levels (diZerega et al., 1982a).

3 Ovarian Asymmetry

Having considered the importance of previous ovarian status, we return now to the question: When is the dominant follicle selected? From the findings discussed so far we can answer with the interval estimate: after the demise of the corpus luteum in the preceding cycle and before day 8 of the cycle in which ovulation occurs. That is, the onset of ovarian asymmetry appears to be the result of events in the current cycle rather than a reflection of events in the preceding cycle. Do the times of follicle selection and onset of ovarian asymmetry coincide? In this section we bring together evidence that provides a narrower estimate of the time of follicle selection, the attainment of dominance, and the onset of ovarian asymmetry.

a Follicular Aspects.

As described above, when the ovary bearing the dominant follicle was removed, the cycle was interrupted and ovulation was delayed for about 2 weeks; ablation of the entire contralateral ovary, in contrast, had no apparent effect. Thus, when the dominant follicle is clearly recognizable in situ, the ovaries are functionally asymmetrical, as well. Conversely, then, we reasoned that if ovarian asymmetry were demonstrable shortly after luteectomy or luteolysis, before the dominant follicle was visible, such asymmetry would signal a latent dominant follicle and thereby refine estimates of when the dominant follicle is selected (Goodman et al., 1982).

To obtain a more precise estimate of the time of follicle selection it was important to employ a procedure that (1) provided a clear starting time-point and (2) synchronized subsequent follicle growth within each experimental group. Luteectomy is such a procedure. To recapitulate, luteectomy in rhesus monkeys is typically followed by the next LH surge in about 12.5 days. Only a single follicle ovulates after luteectomy, as in intact cycles, and this is almost always (>90%) on the opposite ovary. While the last outcome is likely an experimental artifact, since trauma of the opposite ovary randomized the side of the next ovulation (Goodman et al., 1979), it was purposely exploited for its advantage in localizing (predicting) the next side of ovulation. Moreover, in addition to synchronizing subsequent follicle growth among monkeys, luteectomy necessarily ensures that the preceding cycle was ovulatory. Thus, based on these considerations, the time and place of the next ovulation were highly predictable; ovulation would occur about 2 weeks after luteectomy, most likely on the opposite ovary. Accordingly, the procedures of luteectomy and hemiovariectomy were combined into a single study to detect the onset of ovarian asymmetry and thereby reveal a latent dominant follicle.

In the first part of the study, intact rhesus monkeys were luteectomized (day 16–19) and assigned at random to one of two groups. In one group the contralateral ovary was removed 4 days after luteectomy, in the other group the luteectomized (ipsilateral) ovary was removed instead. Since the next ovulation almost always occurs on the contralateral ovary after luteectomy alone, we predicted that removal of this ovary 4 days later—if the next dominant follicle were already selected—would delay the next LH surge by about 4 days over the common reference interval of 12.5 days. [The choice of 4 days was influenced by the variance around the mean interval from luteectomy to the next LH surge (SEM ≅ 1); we reckoned that 4 days was the shortest span which would produce a detectable delay of statistical significance.] Alternatively, no delay was expected if gametogenic follicle growth had yet to resume and/or the next follicle destined to ovulate had yet to be selected. The former prediction was seemingly borne out. The interval from luteectomy to the next LH surge was extended to 17.0 ± 1.5 days (p < 0.01), which corresponds to an interval of 13.0 ± 1.5 days from contralateral hemiovariectomy to the LH surge.

Since the next ovulation after luteectomy seldom (frequency <10%) occurs on the ipsilateral ovary, we predicted that removing this ovary 4 days after luteectomy would have no effect. Curiously, however, this intervention, like its preceding counterpart, also delayed the next LH surge by about 4 days. The interval from luteectomy to the next LH surge was 12.7 ± 1.6 days, thereby extending the luteectomy–LH surge interval to nearly 17 days (p < 0.01).

An overt vesicular follicle was not discernible at hemiovariectomy on day 4 in either group and removal of either ovary at this time produced comparable delays in the next LH surge without differentially affecting tonic gonadotropins levels. Thus, morphological symmetry was reflected in functional symmetry. However, since removal of either ovary engendered a 4-day delay in ovulation, gametogenic follicle growth had apparently resumed promptly after luteal ablation, otherwise, intervention on day 4 presumably would not have delayed ovulation by 4 days.

What was unexpected in these findings was not functional symmetry, per se, but rather that ablation of the ipsilateral ovary had any effect, at all. As discussed above, removal of the entire ovary containing the corpus luteum produced no effect different from luteectomy alone, suggesting that the ipsilateral ovary remained quiescent (vis-à-vis folliculogenesis) after luteal ablation. As mentioned below, laparotomy and collection of ovarian venous blood around this time do not delay the next LH surge (diZerega et al., 1981a). Consequently, the ipsilateral ovary appears to participate in the ovarian cycle for some time after luteectomy in some, as yet inexplicable, fashion. Since this outcome was at variance with our original predictions, we extended the study in an effort to clarify the issue.

In the second portion of the study, hemiovariectomy was performed 8 days after luteectomy—a time when previous evidence indicated that the next dominant follicle was already selected. As expected, ablation of the ipsilateral ovary on day 8 did not significantly delay the next LH surge beyond the 12.5 day reference interval after luteectomy alone; the next LH surge occurred about 5 days after ipsilateral hemiovariectomy or, equivalently, ∼13 days after luteectomy. In contrast, removal of the contralateral ovary 8 days after luteal ablation significantly prolonged the luteectomy-LH surge interval to nearly 27 days, roughly twice the interval after luteectomy alone. (The protracted 19-day interval from contralateral hemiovariectomy to the next LH surge was an unexpected departure from the 12.5-day reference interval. The extension may represent combined effects of luteectomy and the subsequent inhibition of follicles on the ipsilateral ovary by the ablated-with-the-contralateral-ovary, new dominant follicle.) We do not consider the protracted difference of significance with respect to the typical cycle. The key finding is that removal of the contralateral ovary, which contained a morphologically distinct dominant follicle, significantly delayed the next ovulation.

Thus, when a dominant follicle is clearly discernible, functional ovarian asymmetry can be demonstrated. Since hemiovariectomy on day 4 (before a dominant follicle was discernible) delayed the next ovulation, the next follicle destined to ovulate had apparently begun to develop, but it had yet to attain dominance over the opposite ovary. Consequently, we cannot be sure whether or not selection was completed by day 4. By day 8, however, unambiguous dominance had been attained and selection could be verified.

In other words, when follicle dominance is demonstrable morphologically or functionally, selection of the follicle destined to ovulate is already a fait accompli. Selection, then, appears not to be an instantaneous event, but rather may be a progressive process whose culmination is signaled by the attainment of dominance by a single follicle. If selection is a progressive process, rather than a discrete event, one may never be able to describe its occurrence more precisely than by an interval (rather than a point) estimate.

What emerges from all this is that selection and dominance are conceptually distinct, but physiologically intertwined activities. Attainment of functional dominance is an integral component of the selection process; the detection of dominance is a sufficient condition for adducing selection (if dominance, then selection). Whether the attainment of dominance is a necessary condition for selection is as yet unresolved (if not dominance, then not selection?). That is, the precise temporal relationship between selection and dominance is unclear. Which comes first, dominance or selection? Does one follicle select itself by first (somehow) attaining dominance over other potential competitors; alternatively, is a single follicle first selected by some other mechanism and afterward ensures its destiny by exerting dominance as some kind of a safety mechanism against multiple ovulations? In either case, the selected follicle, which has attained dominance, must thereafter maintain dominance until ovulation is triggered about a week later. Possible mechanisms of dominance are considered in a later section.

Relating these findings to the menstrual cycle, we conclude (1) that the attainment of unambiguous functional and morphological dominance signals the completion of a follicle selection process that begins (or is perhaps, held in abeyance until) promptly after luteolysis, and (2) that the follicle destined to ovulate attains dominance 4–8 days after the demise of the corpus luteum. Since menses begins 2–4 days after luteolysis, dominance is attained, and hence, selection completed, between the second and sixth day of the cycle.

That a new dominant follicle has not yet emerged by day 4 after luteectomy is supported by our histological data from these ovaries (Koering et al., 1982). However, even though the ipsilateral and contralateral ovaries appeared functionally symmetrical on day 4, morphological asymmetry was already present as divergent follicle distributions. Day 4 contralateral ovaries contained significantly more healthy follicles 0.5–1.0 mm in diameter than did ipsilateral ovaries, even though no obvious dominant follicle was detectable in either group. Moreover, the ipsilateral ovary appears to assume its quiescent status soon after day 4, since the distribution of follicles in day 8 ipsilateral ovaries was not different from day 4, whereas in day 8 contralateral ovaries a presumptive dominant follicle was almost always identifiable.

Other evidence that the dominant follicle is emerging during this interval (i.e., day 2–6 of the cycle) comes from studies of follicle labeling using biologically active fluorescein-tagged hCG (Fig. 15). On days 9 and 11 of the cycle, only the largest follicle among those present on both ovaries displayed a unique pattern of thecal fluorescence. On day 7 this pattern was observed circumscribing only one follicle, even though this follicle was otherwise indistinguishable by size from other follicles in either ovary (diZerega and Hodgen, 1980). Before day 7 no unique pattern of fluorescence was discernible around any single follicle. Autoradiography has also been used to detect specific uptake of radiolabeled hCG by the dominant follicle (Zeleznik et al., 1981).

FIG. 15 Ovarian uptake of biologically active fluorescein-tagged hCG on day 7 of the menstrual cycle in rhesus monkeys. DF, Dominant follicle. (From diZerega and Hodgen, 1980, with permission.)

Using a specially designed laparoscope, which permitted transillumination of the ovary in rhesus monkeys, Clark et al. (1978) were able to distinguish one follicle during the first week of the cycle. However, frequently its identity as the dominant follicle required retrospective confirmation.

b Hormonal Aspects.

Clearly, the gametogenic and hormonal activities of the ovary are closely linked. As described above, ablation of the ovary containing the cyclic structure had profound effects not only on the time-course of the cycle but on circulating ovarian hormone levels as well (Fig. 10a and b). In contrast, removal of the contralateral (quiescent) ovary was without discernible effects (Fig. 12a and b).

How early in the cycle does ovarian endocrine activity become asymmetrical, as indicated by steroid hormone levels in ovarian venous blood? Will differences in hormone secretion between ovaries reliably anticipate the side of the next dominant follicle or merely reflect its presence after it is already discernible by other means? To answer these questions, ovarian venous blood was drawn from monkeys at various times after the onset of menses (diZerega et al., 1980) or after luteectomy (diZerega et al., 1981a). Levels of estradiol in ovarian venous serum were significantly different between ovaries as early as days 5 to 7 of the cycle and by 5 days after luteectomy (diZerega and Hodgen, 1981a). Not surprisingly, the degree of asymmetry increased with progression to ovulation (Fig. 16). Without exception, the ovary secreting more estradiol early on was later found to contain a fresh corpus luteum. Only in monkeys in which the cycle proved to be anovulatory was no difference observed. Clearcut differences in androstenedione and progesterone levels developed only in the last 3 days before the LH surge in intact monkeys. In luteectomized monkeys, however, progesterone levels were initially higher in the blood draining the luteectomized ovary, but this difference disappeared within 3 to 5 days. The potential significance of early differences in progesterone secretion is considered in a later section. The important point here is that divergence of estrogen secretion between ovaries provides the earliest hormonal index we have so far in revealing the emergence of the dominant follicle. Indeed, the onset of this asymmetry by days 5 to 7 of the cycle corresponds well with follicular indices of ovarian asymmetry and follicle selection. That the dominant follicle is the source of the differential estradiol secretion at this early stage is uncertain. Later in the cycle, the dominant follicle is doubtless the preponderant source of circulating estradiol.

FIG. 16 Relationship between ovarian vein steroid levels throughout the follicular phase and side of current ovulation (ovary with CL). (From diZerega et al., 1980, with