Recent Progress in Hormone Research: Proceedings of the 1977 Laurentian Hormone Conference

Yoshinaga

PREFACE

Contained in this volume are the proceedings of the 1977 Laurentian Hormone Conference held at Mont Tremblant, Quebec, Canada August 28 through September 2. The program covered a diversity of topics that ranged in technical intricacy from an overview of twentieth century research in reproductive endocrinology to to the X-ray crystallography, conformation, transport, and metabolism of thyroid hormones. Adding to the intellectual fare were reports of recent progress in many of the most highly active fields of endocrine research. Leading authorities detailed their findings concerning hypothalamo-pituitary relationships; the structure and biology of relaxin; pineal regulatory mechanisms; ultrastructural and autoradiographic analysis of the biosynthesis, secretion, and binding of insulin; isolation of a new molecular form of growth hormone from plasma; the action of calcemic hormones on the formation and adsorption of bone; and the isolation of newly identified genera of short-lived prostaglandins, the endoperoxides and thromboxanes. These in-depth reports were enriched by the comments, criticisms, and queries from the assembled audience of experts in endocrinology and related medical disciplines.

It is a pleasure to acknowledge our indebtedness to the following chairpersons for their skillful guidance of the lively and stimulating discussion sessions: Drs. Abraham White, Henry G. Friesen, Roger G. Ungar, Urban J. Lewis, Frederic C. Bartter, Paul L. Munson, Muriel Feigelson, Jack Oppenheimer, and Valerie Galton. Our gratitude goes also to Lucy Felicissimo and Linda Passalalpi for their on-location verbatim transcriptions of the recorded discussions. The Conferees wish to thank the management and staff of the Mont Tremblant Lodge for their courteous service and the ever-present ambience of pleasantries and goodwill. As editor of this volume I wish to acknowledge my grateful appreciation of the invaluable assistance of Miss Martha Wright and extend my thanks to the staff of Academic Press for their careful and dedicated attention to the production of this the thirty-fourth volume of Recent Progress in Hormone Research.

Roy O. Greep

Reproductive Endocrinology: Concepts and Perspectives, an Overview¹

ROY O. GREEP,     Harvard Medical School, Boston, Massachusetts

Publisher Summary

This chapter provides an overview of concepts and perspectives of reproductive endocrinology. For the purpose of contraception by the rhythm method, there is urgent need for an accurate, rapid, convenient, and low-cost method of detecting ovulation in women. All present methods are imprecise and have a high failure rate. What causes the human corpus luteum to cease producing progesterone and allow menstruation to ensue has to be determined. There is need also for a new discipline representing an amalgamation of neurophysiology, neuropharmacology, and endocrinology to explore the no-man’s-land that exists between purely neural phenomena as modified by drugs and the manifestations of hormonal action. Progress in reproductive endocrinology over the remainder of this century will almost certainly have far greater consequences for humanity than the much talked about genetic engineering. Although different species have adopted a variety of mechanisms for the purpose of reproduction, it is evident that the basic unity of nature prevails as a dominant feature.

I Introduction

First let me aver with all the force at my command that this was not my idea but something perpetrated by the Program Committee on whose shoulders must rest a fair share of the responsibility. To those who might well ask: Why did you agree?–I would have to admit to being temporarily blinded by flattery. The agony that followed once the euphoria had worn off can be imagined only by those who have faced this audience before. That I was asked to give an overview of the field of reproductive endocrinology can be ascribed only to my antiquity. A presentation of this nature marks a sharp and perhaps dangerous break with tradition at the Laurentian Hormone Conference where in-depth presentations have been a hallmark from the beginning. Happily, there is no way I can lose, for if I fail then no precedent will have been set for presentations of this type.

In a more serious vein, I do deeply appreciate the opportunity and the honor of presenting this Gregory Pincus Memorial Lecture of the Laurentian Hormone Conference, of which Pincus was the founding father. A few years ago I also had the privilege of presenting the Gregory Pincus Memorial Lecture at the Worcester Foundation for Experimental Biology, of which Pincus was a cofounder. There I spoke on Science, Politics, and Society, which, to my mind, epitomized the life of Gregory Pincus. He was a distinguished scientist, a master in the art of persuasion for the purpose of accomplishing worthy objectives, and a benefactor of human society. While it may appear that my cup runneth over with Pincus lectures, so does my admiration and respect for that striking figure known to many as Goody.

This is the tenth anniversary of the death of Gregory Pincus, and, while his place in history is assured, I am not satisfied that the better world which he helped build has paid proper tribute to his leadership. Credit for the development of the Pill now being utilized by some 60 million women to control their fertility must, of course, be shared by his several collaborators, Chang, Rock, and Celso-Garcia, but it was Pincus’ phenomenal capacity for daring leadership that sparked a worldwide revolution in contraceptive practice. Few developments in history have brought about a more marked and rapid change in human society than did the introduction of the pill. The liberation of millions of women from the adversities of excessive childbearing and illegal abortions is monumental, as is the impact the pill has had on the health, welfare, and happiness of millions of people in all parts of the world.

It is widely assumed that untimely death robbed Pincus of a Nobel Prize for his much heralded achievements in science and in medicine. The intervening years have also witnessed some chipping away at his just rewards. The toppling of giants and disparaging the achievements of the past has been a popular activity and a mark of sophistication ever since David encountered Goliath. A fellowship program in Pincus’ honor has been established by the Searle Company at the Harvard School of Public Health, but I still feel that a truly fitting memorial to this man has not yet been created. It is not enough that his work will stand as an enduring monument. What is needed is for humanity to show its appreciation. A multimillion dollar international memorial fund for the promotion of study and research in the reproductive sciences throughout the world would be in keeping with Pincus’ achievements, his internationalism, and his tireless pursuit of human betterment through research.

II Historical Perspective

In looking back over the history of reproductive endocrinology, one is impressed by its erratic upbringing. Its unheralded and almost abortive birth in 1849 was followed by a prolonged and barely viable infancy lasting nearly a half century. In the first quarter of the present century, this field experienced in the teens appropriately a stimulation of growth, but puberty was delayed until the early twenties. The pubescent period was followed by a burst of youthful vigor and pioneering exuberance that lasted up to the beginning of the Second World War, which brought work in this field to a very low ebb. The fifties witnessed a slow but complete recovery and set the stage for the modern period of absolutely spectacular growth and development.

Speaking now in more precise terms, Berthold’s 1849 discoveries were disputed and largely ignored through the remainder of the nineteenth century. There were some studies in the final decade, but they were mainly confined to observations of sexual behavior and the cyclicity of heat and mating periods in wild and domestic animals. The study of reproduction was then shunned by the elite in both science and medicine and suffered tainted respectability because it dealt with subject matter long shielded by Victorian and Puritan prudery. Application of the experimental method as a means of learning about the reproductive system in male and female mammals was not introduced until the turn of the century. At that time the gametogenic functions of the ovaries and testes were well known, but there was no definitive knowledge concerning their secretory functions. Circumstantial evidence abounded, but no reproductive hormone had been identified. From a study of seasonal breeders Walter Heape, writing in 1905, concluded that the stimulus which affects the generative system during the onset of the breeding season is due to a special substance in the blood, a generative ferment thought by him to be nutritive in nature and induced by the warming spring climate and abundance of new food. He was actually hot on the trail. His prophetic lines are worth noting: There is strong evidence that the increased activity of the generative glands (the gonads), consequent on the presence of this ‘ferment’ results in the secretion of material which exercises a profound effect upon the rest of the generative system and possibly upon other organs; for this secretion I will suggest the term ‘gonadin’. Heape’s generative ferment became, of course, the gonadotropins, as we know them, and his gonadin was the ethereal forerunner of the ovarian estrogens.

At this point in history, the effects of removing the ovary on the cyclic appearance of heat, menses, and pregnancy came into conflict. Some claimed that ovariectomy abolished these phenomena, other found no change. It turned out that those claiming no effect were being misled by poor surgery and the then unsuspected fact that ovariectomy does not interrupt an established pregnancy in some of the mammals being studied, such as guinea pigs, monkeys, and humans. By 1910, and through the work of a first generation of mainly English, French, and German investigators, especially Marshall, Heape, Hammond, Born, Fraenkel, Prenant, and Ancel and Bouin plus Leo Loeb in the United States, growth of the uterus and the establishment of pregnancy and pseudopregnancy were known to be dependent upon some factor or factors emanating from the ovary. Extracts, mainly aqueous, of fresh and dried ovaries were prepared by a host of investigators, but with only an occasional hint of activity.

The accomplishments during the early part of this century were elementary by any standard. There was no knowledge base on which to build. The important consequence of these early explorations was to stimulate interest on the part of a new generation of mainly American and Canadian clinicians and basic scientists. They had little more than thyroid powder in their effective armamentarium but were intent on gaining insight as to the common disorders of the endocrine system. It was this same group that organized the Society for the Study of Internal Secretions in 1917, later termed the Endocrine Society. That era also marked a shift in this field’s center of gravity from the European continent to North America, due largely to the devastation and heavy loss of life inflicted by the First World War. Prior to that event, nearly all of our aspiring young investigators sought their training with the masters abroad. Afterward, the flow of trainees was more and more in the reverse direction.

The year 1917 also marks a discovery that was to have a far-reaching impact on reproductive research. This was Stockard and Papanicolaou’s (1917) observation that sloughed cells in the vaginal smear of guinea pigs correlated with cyclic morphologic changes in the ovaries and with estrous behavior. This soon led to the classic description of the estrous cycle in rats by Long and Evans (1922). This new technique greatly facilitated research by making possible precise monitoring of the internal ovarian cycle by means of an external index. The vaginal smear remains today one of the most commonly used techniques in research on reproduction. When I joined the Hisaw laboratory in 1930, learning to smear rats was second only to the requirement that I register as a graduate student.

Nothing could have better set the stage for the opening of meaningful research in reproductive endocrinology than the vaginal smear. Although by 1923 it was virtually certain that the ovary was the source of an estrus-inducing hormone, the final proof came with Allen and Doisy’s demonstration that bovine follicular fluid induced vaginal estrus and enlargement of the uterus in immature rats. They found too that the smear could be used as a quantitative assay of estrogenic activity. With this necessary tool, and a source of raw material, the first race was on to isolate a hormone, and reproductive endocrinology entered a new era. The amount of follicular fluid available proved inadequate, but luckily another source became available with the 1927 discovery by Aschheim and Zondek that human pregnancy urine contained an abundance of a similar activity. From this source, Doisy’s group isolated estrone in 1929, and a year later Marrian did the same for estriol.

This was the first instance of public excitement over a glandular development since 1889 when the elderly Brown-Séquard claimed to have rejuvenated himself with an extract of dog testes. His report was premature; Doisy’s was not. Science had brought forth what was hailed as the female sex hormone and shown that it could be obtained in quantity for clinical use. This new glandular product was already known to induce growth of the female reproductive organs and mammary glands and to bring about estrus and mating behavior in animals. Its news value lay in what might be in store for people. The excitement of the moment touched off a search for new sources of this hormone, including the blood and urine of many pregnant and nonpregnant animals and a variety of substances ranging from the pussywillow to petroleum.

It was as a participant in this heady exploration of a newly opened field during the late 1920s that my own career in endocrinology sprouted a small bud. My qualifications were those not of a steroid biochemist but of a college zoology major. I was asked by one of my professors, who was almost as green to endocrine research as I, to assist in a project that involved the collection of urine from the dairy barn located at the edge of the campus. Owing to the professor’s tight teaching schedule, we had to transport open tubs of this golden effluvia to the chemistry laboratory at intermission time, with all my classmates rushing across campus. Needless to say they gave us a wide birth and some querulous glances. The raw material had also to be stockpiled because the professor was free to work only on weekends. What I remember most vividly from this less than propitious introduction to research was the odor. The starting material was bad enough with all windows open, but when we got to the stage of evaporating down our concentrate, something of a more permeating nature came off. As I would wind my way home to the shower, I sensed a popularity akin to that of a skunk at a ladies’ tea party. Our extracts proved not to have the slightest trace of activity for the simple reason that, as we learned later, none was present to start with. As a final blow, our unpublished negative findings were immediately confirmed in print by Hisaw and Meyer (1929).

At the risk of sinning on the side of omissions, I am going to list in capsular form what I consider to be the major breakthroughs or milestones in reproductive endocrinology from the 1920s up to the inception of the modern period in the early 1960s. First, let me make clear that by major breakthroughs I mean those advances of observational, conceptual, or methodical nature that opened doors and led rather quickly to widespread stimulation of research.

Heading the list must be the already mentioned methodological description of the vaginal estrous cycle in the rat by Long and Evans in 1922 and the discovery of the ovarian follicular hormone by Allen and Doisy a year later.

The mid-1920s witnessed a veritable explosion of endocrine discoveries relating to reproductive physiology. First came the essentially simultaneous discoveries by Zondek and Aschheim (1926) and by Smith (1926) that daily implants of rat anterior pituitary glands induced precocious ovarian enlargement and sexual maturation. Their revolutionizing conclusion that ovarian secretion is under the control of the pituitary was placed beyond doubt by Smith (1927), who found that involution and total loss of gonadal function followed surgical removal of the pituitary gland. Considering that the thyroid, adrenal, and body growth were similarly affected, and that all deficiencies were repaired by replacement therapy, plus the impact this had on future research, Smith’s contributions must stand as the single most important set of observations ever made in the history of endocrine research. The mid-1920s also witnessed the discovery of chorionic gonadotropin in the urine of pregnant women by Aschheim and Zondek (1927), of relaxin in the blood of pregnant rabbits by Hisaw (1926), and of a potent androgen in a lipid extract of bull testes by McGee (1927). Active luteal extracts prepared by Hisaw et al. (1928) and Corner and Allen (1929) made possible the immediate elucidation of all the major physiological functions of the corpus luteum, such as inhibition of ovulation, secretory endometria, progestational proliferation, implantation, and maintenance of pregnancy.

The 1930s were also fruitful, opening as they did with the discovery of two more gonadotropins, one in the blood of pregnant mares by Cole and Hart (1930) and another in the urine of postmenopausal women by Zondek (1930). In 1931, light dawned on the enigma as to how the pituitary could control both the follicular and the luteal phases of ovarian function. This came with the preliminary evidence by Fevold, Hisaw, and Leonard (1931) that the pituitary produces not one but two gonadotropins–a follicle-stimulating and a luteinizing hormone. This new concept survived a hostile reception and imparted an enduring stimulus to research on the gonadotropins.

The suppressive action of estrogen on pituitary gonadotropin content and secretion was first demonstrated by Meyer et al. in 1930, but it remained for Moore and Price to put together in 1932 the first explanation of the estrous cycle based on reciprocal endocrine interactions between the pituitary and the ovary. Their concept won general acceptance for many years. Simply stated, the belief was that estrogen from maturing follicles inhibited FSH and stimulated the secretion of LH resulting in ovulation. LH was then, in turn, inhibited by the luteal hormone progesterone, allowing a new cycle to be initiated by the action of FSH. Although the hypothalamic influence had later to be superimposed on the Moore–Price concept, much of its essence still stands. Their paper provided an enormous stimulus to research and conceptual thinking and remains one of the truly great classics in the history of reproductive endocrinology.

The year 1932 was a banner year for classics, as it also included Hartman’s delineation of the ovulatory period at midcycle in the primate menstrual cycle, an observation that gave birth to the rhythm method of birth control. This method more commonly known as the safe period enjoyed considerable popularity until it became evident that the difference between theory and practice was parenthood.

The concept of neural regulation of pituitary gonadotropic functions arose piecemeal over a period of nearly two decades extending from the late 1920s to the late 1940s. Early work by Rowan (1926), Bissonnette (1932), and Marshall (1936) indicating that environmental factors, such as food, heat, light, and changing seasons, influence reproductive processes led Marshall and Verney (1936) to try passing a diffuse electric current through the head of rabbits to see if this would lead to ovulation. It did.

The next problem was to localize the site of effective stimulation. Haterius and Deryshire (1937) and Harris (1937) used electrical probes and obtained ovulation following stimulation of the hypothalamus, but a controversy ensued as to whether direct stimulation of the pituitary also caused ovulation. Since the anterior pituitary was known not to have secretomotor innervation, this issue became a matter of considerable importance. It was not until 9 years later that the pituitary was conclusively ruled out as a site of effective electrical stimulation by Markee, Sawyer, and Hollinshead (1946). The prior positive results were due to spread of the electrical stimulus from the pituitary to the hypothalamus.

Taking a myriad of related observations into account, Harris in 1948 formulated the neurovascular concept of neural regulation of the anterior pituitary. No other concept has so completely altered the course of endocrine research. It added an important new dimension to the mechanism regulating gonadal function and established a new specialty area, neuroendocrinology. It has served to provide a comprehensive explanation of all the evidence gathered before and after its formulation. That concept is now a basic component of reproductive endocrinology.

The role of adrenergic and cholinergic components in the ovulatory process, as first delineated in 1947 by Sawyer, Markee, and colleagues (Sawyer et al., 1947) initiated broad-scale studies of the brain monoamines in relation to reproductive functions. This has now become one of the most active fronts in reproductive research.

Since gonadal function is controlled by the pituitary and is cyclic in females and acyclic in males, question arose early as to whether sexuality of the pituitary is determined genetically or by other factors. In 1936, Pfeiffer proposed that the sex of the pituitary was permanently determined at puberty depending on whether it was exposed to male or female hormones. By removing gonads and transplanting them between sexes at birth, he found that the male pituitary could be made to function cyclically and conversely the female pituitary could be made to function in an acyclic manner. In the same year I transferred pituitaries between sexes and found that the pituitary had no fixed sexuality but would function either as male or female depending on the sex of the adult host (Greep, 1936). This left open the question as to what determined the functional characteristics of the pituitary. Twenty-five years later the missing link was discovered by Barraclough and Gorski (1961), who found that it is not the pituitary but the hypothalamus that is sexually bipotential. Actually all rats are born with a female type of hypothalamus and will retain that characteristic unless exposed to male sex hormones during the first few days of life. In newborn males the presence of testicular androgens causes the hypothalamus to differentiate as male and function in an acyclic manner. Similarly when newborn females are experimentally exposed to androgen, their hypothalamus also differentiates as male, yielding the familiar androgenized female. Since these animals remain in constant estrus, fail to ovulate, and are sterile, they have become a valuable model for neuroendocrine studies.

A surprise development came in 1941 when Astwood found that the essential stimulus to luteal function in the rat is the lactogenic hormone prolactin. Why this holds in only two or three other mammals remains a mystery. Some current evidence suggests that the luteotropic role of prolactin may not be so narrowly confined.

Research on the gonadotropins was greatly facilitated by the introduction of the ventral prostate assay for LH in 1941 (Greep et al., 1941), the Steelman–Pohley assay for FSH in 1953, and the ovarian ascorbic acid depletion assay in 1961 (Parlow, 1961).

The most helpful breakthrough in elucidating the neuroendocrine mechanism controlling ovulation came with the discovery of the so-called critical period in 1949 by Everett, Sawyer, and Markee. They found that injections of dibenamine or atropine given before 2:00 P.M., but not later than 4:00 P.M. on the afternoon of proestrus, would block ovulation.

The 1950s were short on breakthroughs, but the ones that did come bore fruit in abundance. The tip-off to the possibility of developing an orally active contraceptive agent came in 1953 with the discovery by Pincus and Chang that ovulation in mated rabbits could be inhibited by the oral administration of synthetic progestins. The rest is history and known throughout the world.

Then came a study by McArthur and associates in 1958 that revealed for the first time an elevated output of LH in the urine of women at or near the time of ovulation. Out of this was born during the next decade the cluster of spectacular midcycle surges, pulses, and peaks in nearly every known female reproductive hormone—a virtual fireworks that builds to a flurry of follicular ovulatory explosions. To pursue this analogy a bit further, it might even be said that the succeeding 1960s ended in a bang through the works of Abraham, Knobil, Midgley, Odell, Parlow, Ross, and Taymore whose studies of the preovulatory events were made possible by the advent of a new and highly sensitive radioimmunoassay—a gift from Berson and Yalow. Squire and Li (1959) brought the 1950s to a close by winning a 29-year marathon effort that ended with isolation of the luteinizing hormone, rechristened by them the interstitial-cell-stimulating hormone.

The sixth decade marked the opening of what may rightly be called the modern period in the history of research on reproduction. It was a notable decade in terms of scientific advances. The opening year was to mark the unequivocal demonstration by McCann et al. (1960) of the presence of an LH releasing factor in extracts of the hypothalamus. This triggered an explosion in neuroendocrine research and led, a decade hence, to the isolation and synthesis of porcine LHRH by Schally’s group and ovine LHRH by Guillemin’s group. Everett (1961) announced his finding that the stimulus resulting in the preovulatory LH surge was initiated in the preoptic area. It is now well established that in rats the acute, as opposed to the toxic, discharge of gonadotropins is under the control of the preoptic area.

I have already alluded to a few of the notable advances made during the modern era. They are much too numerous and too well known to review here. This has been by far the most productive period in the history of this field, topped perhaps by the elucidation of the chemical structure of the gonadotropins and their subunits and several hypothalamic releasing hormones. This plethora of progress, like the boxes of Cracker Jacks that my parents used to bring from town, has yielded some surprises, such as the positive-feedback mechanism, the pulsatile patterns of secretion, and the casting of a halo over the Sertoli cell.

The study of the male reproductive system having been much neglected, the major advances on the way to current enlightment are few and mainly of recent vintage. The ancient custom of removing the testicles from man and beasts had such consistent and unfailing results as to have left little motive for questioning why. Finally, in 1849., Berthold did. The birth of endocrinology came with his observation that the transplantation of testes into long-term castrated cocks restored the comb and wattles and, as Berthold put it, a friendly interest in hens. The next major discovery did not come until 78 years later, when Smith (1927) found that both the sperm-forming and secretory functions of the testes were controlled by the anterior pituitary. Over the ensuing years, a controversy developed as to whether the testicular secretion came from the seminiferous tubules or the Leydig cells. The argument was settled in 1936 when the Wisconsin group (Greep et al., 1936) were able to show that in hypophysectomized immature rats LH restored the Leydig cells to a functional state while FSH failed. In a preceding paper, Walsh, Cuyler, and McCullagh (1934) made the almost heretical observation that androgens could maintain spermatogenesis in the complete absence of the pituitary gland. This provided a ready explanation of the later observation (Greep and Fevold, 1937) that in hypophysectomized adult male rats, the LH-induced secretion of androgen by the Leydig cells maintained the entire male reproductive system in a normal functional state. Most discoveries that mark a significant step forward open doors and stimulate research; these certainly did not. Henceforth, male reproductive endocrinology lapsed in a diapause that lasted for nearly 30 years. Now and then a faint exhortation from the wilderness would question the role of FSH. Since testosterone could maintain gametogenesis, some felt that perhaps the male had no need for FSH. Then in 1961, Woods and Simpson brought this field to its feet with a report that highly purified FSH had no effect on the regressed germinal epithelium of hypophysectomized rats except at doses high enough for the effect to be attributed to LH contamination. For most workers this served to deny a physiologic role for FSH. I was not among them. FSH was known to be present in the male rat pituitary, to circulate in the blood stream, and to stimulate sperm formation in reptiles, birds, and most mammals, including the subhuman primates and man.

After 1961, male research lapsed back into stagnation until late in the 1960s, when Means and Hall (1967, 1969) opened a new front with their demonstration that FSH increased protein synthesis in the testes of immature rats. This did open doors. The attack was joined by a regiment of biochemically trained investigators including Ritzén, French, Mancini, Fritz, Frick, Vernon, the Steinbergers, Hansson, Dorrington, and Armstrong, among others. The attack centered almost exclusively on FSH and the Sertoli cell. This work is still in progress and is too well known to warrant detailed attention here. It is also too new to be evaluated definitively.

The current and radically altered view of testis physiology is that FSH and androgen from the Leydig cells acting in concert stimulate the Sertoli cells to synthesize and secrete androgen-binding protein. This androphilic protein appears to serve the purpose of maintaining a high local concentration of androgen necessary for the generation of sperm. This, however, has not been proved. FSH has been shown to bind to the Sertoli cells and to induce a series of biochemical events, including adenyl cyclase activation, cyclic AMP accumulation, protein kinase activation, and the synthesis of proteins and RNA. It is now virtually certain that the Sertoli cell, once the lowliest of all cells on the testis totem pole, is the target cell of FSH. These findings do not preclude the possibility that FSH may exert some direct action on the germinal tissue. Means has found that FSH reduces the number of spermatogonia undergoing involution and Steinberger has produced evidence that FSH is necessary for completion of the first wave of spermatogenesis at the time of puberty. Lately, Dym, Madhwa Raj, and Chemes (1977) found that antisera to highly purified FSH severely restricts the growth of the testes in immature rats. Obviously the role of FSH in spermatogenesis has not yet been delineated. The most striking thing that has happened to male reproductive endocrinology over the past few years is that it has become the glamor area of reproductive research.

Although biologists led the way in identifying the activity of reproductive hormones, what raised reproductive endocrinology to the level of a quantitative science was the work of an early generation of steroid biochemists including Willard Allen, J. S. L. Brown, Collip, Butenandt, David, DeJongh, Dingemanse, Doisy, Fels, Fernholz, Hirschman, Inhiffen, Kober, Koch, Laqueur, Marker, Marrian, MacCorquodale, Ruschig, Ruzicka, Schwenk, Slotta, Thayer, Veler, Westerfeld, Westphal, Wettstein, and Wintersteiner. They were aided by the fact that organic chemistry was then in a much more advanced state than protein chemistry. Once these workers knew that the hormones existed and could be obtained in quantity from raw material such as tank cars of urine or tons of pig ovaries, the steroids succumbed to chemical characterization in domino fashion. This dazzling conquest was almost in the nature of a turkey shoot compared to the difficulties experienced in bagging the protein hormones. The steroid hormones and many of their metabolites became entities of known structure within the first decade of active endocrine research, the structure of the protein and peptide hormones was revealed only during the last decade. For the steroids it usually took only 4–6 years from a potent extract to pure substance of known structure. For the proteins it generally took 30 years from active extract to isolation and at least another 10 years to determine the structure. Relaxin, which holds the record for elusiveness, was identified in blood and luteal tissue over half a century ago, and, despite increasing effort, the structure is only now a fait accompli. Again, once the structure of one protein hormone, LH, had been determined, that of LHRH, FSH, prolactin, and relaxin was achieved in rapid order, aided by major advances in protein chemistry. These spectacular achievements were accomplished by a small group of outstanding investigators comprised of Amoss, Bahl, Burgus, Butt, Canfield, Ekblad, Guillemin, Hartree, Kammerman, Li, Liao, Liu, Morgan, Papkoff, Parlow, Pierce, Reichert, Sairam, Saxena, Schally, Schwabe, Shome, Vale, and Ward.

III The Overall Record of Accomplishments

At the forefronts of research in this field, as in others, investigators, are constantly coming to grips with the unknown. Their overriding emphasis on what remains to be understood fosters the impression that reproductive science may be a vast wasteland of ignorance. To counterbalance that intimation, I wish to reflect for a moment on the record. It has been my privilege to witness nearly the entire span of meaningful research on reproduction, and it is easy for me to appreciate how far this field has advanced. At the time of the isolation of several hormones in the 1920s, glandular products, in the forms of potions and patent medicines, were the order of the day, as was the practice of organotherapy. Physicians were thereby prescribing consumption of animal organs, including glands in the fresh or dried state, in the belief that this would compensate for deficiencies of the same organs in the human body. An ethical advertisement that appeared in a 1921 issue of Endocrinology (Fig. 1) depicts the status of general and reproductive endocrinology at that time and underscores the phenomenal transition of this field from a pseudoscience to a science that has become the hope of mankind. The rise of reproductive endocrinology is a proud and glorious record of accomplishment. Over these past 50 years, I have seen one difficult problem after another completely resolved. No one in the 1930s could rightly have expected to live to see the complete structure of the gonadotropins with their concatenation of over 200 amino acids linked together in a special order. I am mindful too of how pleased Hisaw would have been to view, as we shall, the structure of relaxin, in which he had consuming interest.

FIG. 1 Advertisement in a 1921 issue of Endocrinology.

Reflect also, if you will, on the heritage that would be left to biology and medicine if all reproductive research were brought to an immediate halt. Out of our accumulated knowledge and experience, normal reproductive function including every step from mating behavior to birth of live young could be carried out solely by the action of exogenous hormones of known structure, function, and potency. Women with longstanding sterility amenable to treatment would be enabled to conceive and bear offspring. Other disorders of the human reproductive system would be treated on a rational basis, although more effectively in women than in men. Consider too that upward of 70 million women throughout the world are now using the pill or the IUD to plan and regulate their childbearing in accordance with their wishes and to the benefit of their well being. All of the 500-odd million women in the world today who are at risk of pregnancy could do the same. It is no fault of reproductive science that millions of women are denied the right to control their fertility.

IV Problem Areas for Future Research

Lest it appear that there is little of importance yet to be learned in this field, let me identify only a few of the areas where our knowledge is woefully deficient. For the purpose of contraception by the rhythm method, there is urgent need of an accurate, rapid, convenient and low-cost method of detecting ovulation in women. All present methods are imprecise and have a high failure rate. By the same token, there is need to determine what causes the human corpus luteum to cease producing progesterone and allow menstruation to ensue. To date, there are clues as to the nature of the luteolytic agent in some animals, but none for the human. It is a virtual certainty that, if the luteolytic stimulus were identified, an end-of-the-month pill could be developed that would greatly shorten the long period of chemical contraception necessitated by the present combination pill.

The area of greatest ignorance and the one that is under most vigorous attack lies at the subcellular and molecular level. This includes everything subsumed under mechanism of hormone action, such as hormone binding, isolation of receptors, hormone-receptor interaction, hormonal control of gene action, hormone-induced biosynthesis of another hormone by a target cell, and intracellular packaging, transport, and release of hormones and other secretory products. Inroads, some superficial, some deep, are being carved out in these difficult and challenging areas by highly skilled investigators using the most sophisticated tools of modern science.

Other challenging problems in critical need of solution include: the nature of the mechanism that brings about the changeover from negative to positive feedback action; the mechanism whereby in each cycle one or more follicles are selected for development; the role, if any, of follicular atresia in ovarian physiology; what controls the transport of ova through the oviduct; what initiates parturition in the human female; what is the mechanism whereby hyperprolactinemia inhibits ovulation; what is the function, if any, of the recently discovered androphilic protein in the testis and epididymis; and, last, there is the powder-keg problem: Is abnormal sexual behavior in humans, such as homosexuality and lesbianism, determined by hormones during sexual differentiation of the brain?

All of these latter problems and many more are being pursued to some extent, but only a few are being investigated on a major scale. Viewing the field as a whole, this represents an imbalance in research effort. The imbalance is further exaggerated by a preponderance of interest in certain fashionable areas—especially neuroendocrinology and mechanism of hormone action. There would be no gain in achieving balance at the expense of one area over another. What is needed is for young people to realize that they should not choose to work in these areas of high technology unless they have the necessary capabilities. They should also keep in mind some poetic advice that the opportunities for self-fulfillment are often best in the ways less-traveled by. Put more pointedly, it is easy to get lost by running with the herd. Having noted how young recruits in science tend to flock to research areas having the prominent names and a firm grip on the purse strings, I sometimes feel that they should reflect for a moment on that lovely lady of mythology Lorelei and what happened to those boatmen on the Rhine who were enchanted by her luringly dulcet tones.

Where there are heights, there must also be valleys. In recent years the going for reproductive endocrinology has all been up and away, but I sense that the ascent has slowed. It is not that new heights are not there, only uncertainty as to which ones to tackle next. Some are more important than others, and some are not amenable to conquest with the equipment presently available. It will be difficult to maintain the pace of progress set over the past 10 years. Already, I sense that the next breakthrough is overdue.

On the matter of new frontiers, I feel that there is a treasure-house of fundamental knowledge to be gained from comparative studies, and by that I mean the phylogenetic evolution of the hormones and of the uses to which they have been put. There should be no apologies for the study of the reproductive endocrinology of fishes, amphibians, reptiles, birds, or lower mammals, including the marsupials. Nothing could provide a more substantial base to the fundamental science of reproduction.

I would also like to see more high-powered work on structure–function relationships of the reproductive hormones by persons highly trained in biophysical chemistry. In the same vein, there is need to exploit the use of the electron microprobe in elemental analysis and ionic movements in subcellular compartments and fluids associated with the reproductive system, including gametes and early embryoes. This instrument, first used in the study of metals, has only recently been put to use in the study of biological materials and promises to do for physiology what the electron microscope did for anatomy. Immunoreproduction, another very promising area, continues to limp along despite its demonstrated value as a means of neutralizing the activity of any given hormone in an in vivo system. What this particular field needs is some bolstering of expertise in immunology, as most of the work has been done by persons whose interest and expertise lies primarily in endocrinology.

There is need also for a new discipline representing an amalgamation of neurophysiology, neuropharmacology, and endocrinology to explore the no-man’s-land that exists between purely neural phenomena as modified by drugs and the manifestations of hormonal action. This type of interdisciplinary expertise is needed if we are ever to understand such matters as: psychogenic amenorrhea; why crowding inhibits ovarian function in many mammals; why the odor of a strange male inhibits early pregnancy in mice; and how suckling results in an increased secretion of oxytocin and prolactin.

Progress in reproductive endocrinology over the remainder of this century will almost certainly have far greater consequences for humanity than the much talked about genetic engineering. Some will question whether this intense effort in basic research will merely add to the pool of fundamental knowledge or will also find application in solving man’s most pressing problem, the control of human fertility. The only assured answer is that basic knowledge will be augmented. Although the practical benefits of research cannot be predicted, none can be had in its absence. From the remarkable gains made in knowledge and insight, especially over the past 15 years, this field is poised as never before for even more rapid advancement. With the quality and volume of expertise now available, the potential for payoffs in practical application is more than sufficient to justify the large expenditures in funds and human endeavor that will be required.

V Funding

Although reproductive research has experienced an acceleration in pace of progress and a buildup in manpower and financial support over the past decade (Fig. 2), the field is still far short of the intensive effort necessary to meet society’s needs and expectations. If that expanded effort is to be realized, reproductive research much recruit more of the topmost talent among the life sciences. Pari passu the opportunities for rewarding careers, the facilities for research, and the opportunities for effective communications at the national and international level must all be increased. This will require a priori a sharp and sustained rise in the level of financial support. It is an absolute certainty that none of these improvements can or will be effected without the necessary funds being available beforehand.

FIG. 2 Worldwide expenditure for research on reproduction. Current vs constant U.S. dollars (1970=100).

To appreciate better the handicaps in support that have plagued this field from the beginning, and continue to hamper progress, a few comments on the history of funding seem in order.

The study of reproduction, having been beset by historic social taboos, could not and did not keep pace with research on other major bodily systems or organs. Government support of research in this politically sensitive area was especially slow in coming, and support for any research bearing on birth control was forbidden up to 1960. Except for the past 10 years, the funding of reproductive research has been in a hand-to-mouth situation. From the 1920s through the 1940s the bulk of support came from university departmental budgets with some outside support from the National Research Council (NRC), the U.S. Department of Agriculture, and especially the pharmaceutical industry.

Early grants from the NRC were in terms of a few hundred dollars to a maximum of around $5000 per year. From 1922 through 1945, the average annual support in the United States by all private agencies was only $69,000, an amount that might now suffice for the support of a single investigator. From 1946 through 1960, the average annual support climbed to a mere $325,000 from private sources, and government support was next to negligible. This, mind you, included the 1950s, when governmental support of research in both the medical and the natural sciences was on a sharply upward course.

Beginning with the creation of the National Institutes of Health (NIH) and the National Science Foundation (NSF) at mid-century, support for health-oriented research became available from NIH and support for research in the natural sciences became available from the NSF. Through the next dozen years, reproductive research found itself in the role of an outcast. It was not sufficiently disease oriented to qualify for NIH support, and it was too medical to qualify for support by NSF. Thus, while medical research soared, reproductive studies had to be content with crumbs dropped from Uncle Sam’s bounteous table.

By the late 1950s, President Eisenhower had made his famous but ill-considered pronouncement that he could not imagine anything further from the concern of government than the control of human fertility. But by this time, public attitudes were changing; the baby boom had reached its peak and there was mounting concern over the burgeoning growth of the human population. Within a year public pressure had not only forced the President to reverse his stand, but created a demand that the government take some action. As a result, the National Institute of Child Health and Human Development was brought into being in 1963, but again the emphasis was limited to human fetal development and the well child with only token support for reproduction. A 1963 Department of Health, Education, and Welfare report declared that research on birth and population control is not an objective toward which NIH has a planned effort. In 1964, out of a total budget of 34 million dollars the NICHD spent only 2.7 million on reproductive research. Finally in 1968, public and political pressure forced creation of the Center for Population Research, with the sole objective of stimulating and supporting research on reproduction and contraceptive development. Thus, after what a western farmer would have termed a long dry spell, the drought was broken. Between 1969 and 1973, NIH expenditures for research in this area jumped from 6 to 40 million dollars, but the shower was short-lived. Support in real dollars leveled off in 1973 and has since been declining owing to inflation and the rising costs of conducting research.

What kept reproductive research afloat until it was rescued by the Center for Population Research was action on the part of private philanthropy. In 1952, the Population Council, with the backing of the Ford, Rockefeller, and Scaife Foundations, initiated support of research and training to the tune of around one million dollars annually. Then in 1960, the Ford Foundation undertook direct support of research in reproductive biology and contraceptive development with yearly expenditures reaching 10 million dollars or more. The Rockefeller Foundation also contributed additional support, especially for recruitment of talent from related disciplines. From 1960 to 1965, while the transition was being made from seed monies to substantial support by government, a full 50% of the support for research in this area came from private philanthropy. Now, with governmental support languishing, support from the private foundations is also being reduced. What lies ahead is unclear, but the handwritings on the walls of the White House are not encouraging. What has become absolutely certain is that support for a research program, adequate to the needs of the nation and the world, can be met only by government, and that is where the future responsibility will rest.

The 1976 survey of research progress and funding in the reproductive sciences and contraceptive development sponsored by the Ford Foundation documented evidence that the current level of support should be tripled immediately, reaching one-half billion dollars in 1980 (Greep et al., 1976). Over the past dozen years, population-related research has been declared a high priority area by three presidents and several acts of congress, but comparable support has not been forthcoming. Two other areas given high priority status, cancer and heart and lung research, received 33% and 15%, respectively, of the 1975 NIH research budget, but only 2% was allotted to reproductive research. Had research on reproduction been given 10%, which seems a minimum figure for a high-priority item, it would have received 260 million dollars instead of the paltry 52 million (a personal communication from Dr. Donald Whedon indicates that the present allotment for 1977 may reach 3%.) Considering that the future of mankind depends not on the conquest of cancer but on the control of human reproduction, the allotment of 360 million dollars to reproductive research as proposed by the Ford Report does not seem at all unreasonable compared to the current annual allotments of around 700 million to cancer research.

This long delay followed by a period of expediency in the funding of research on reproduction has left its mark on the field. This is evident in an inadequate number of highly qualified investigators. Within the general field of endocrinology, reproduction has not competed favorably with its sister science areas, such as adrenal and thyroid research, in attracting top research talent. This is especially true in the clinical area and is most severe in respect to the endocrinology of the human male. One needs little more than the fingers of one hand to count the clinicians in this country who are actively advancing research on the male. That shortage is not peculiar to the United States. WHO has recently taken note of the deficient state of knowledge concerning male reproductive biology and the scarcity of investigators interested in the problems, particularly clinicians. There can also be no doubt that the large amounts of money available over the past several years for cancer and heart and lung research have attracted talent away from the field of reproduction.

VI Constraints

Why has reproductive endocrinology not fared better in attracting an adequate number of outstanding investigators? The answer is simple. There is within the vauntedly broad-minded scientific community a residium of bias against being identified professionally with reproductive matters. No Nobel Prize has ever been awarded in this area despite the undeniable fact that several deserving candidates have been available over the past 50 years. The extremely limited number of reproductive endocrinologists who have become members of the National Academy of Sciences tells the same story. Candidates who elect to work in this field cannot be unaware of its handicaps in public and scientific esteem. Large gains in image have come about, but stigma still casts a shadow on the field. No other branch of science has ever had to contend with a handicap of this nature.

The baby boom of the 1950s and the alarming growth of world population brought a precipitate rush to establish fellowship and training programs and a flood of applicants. Most of these training programs lacked hard-nosed external quality control, and, although they were not designed to turn out highly skilled investigators, a few such did emerge. Most of these programs were informally structured and offered only one or two, or at most three, years of training. Others were largely service oriented and offered a minimum of actual experience in modern-day research. These hastily implemented programs were largely discontinued when it became evident that the needs of both United States and foreign trainees could be better met by other means.

The lesson to be learned here is that mass production methods are not suited to augmenting the supply of high-caliber scientific manpower. Skill in research is not taught but is acquired through independent experience in an ambience of excellence, as is well illustrated by the experience of the Astwood laboratory (see Recent Progress in Hormone Research, Vol. 33, p. xviii). As a stepping-stone to a successful career in science, the competitive research fellowship is without peer. There is great need today for the training of an additional supply of highly qualified young investigators.

While I am at pains of laundering the field’s linen, let me mention some minor points that need attention. First, there is an undue incidence of continued fixation on problem areas that are no longer productive. There is a large difference between panning for gold where gold exists and sifting the sands of an abandoned claim. Success in this young and rapidly developing field requires some versatility and periodic reevaluation of one’s research efforts in terms of scientific pay dirt. What was good enough for a Ph.D. thesis may become about as rewarding as for a dog to be chasing its own tail. Second, of the many old and new journals devoted in whole or in part to the publication of papers pertaining to reproduction, all have increased their standards over the years, some more than others. In a losing effort at keeping abreast of the ever-growing volume of literature, I often come across a paper that strikes me as little more than a minor variation on a theme, and with little relevance to problem-solving. These may be grist for the grant mill, but they are otherwise on a par with a miscarriage. Third, there is the distressingly high incidence of reinvention of the wheel. With the introduction of every new model test system and with each new and improved assay or analytical procedure, the findings of yesteryears are reoutfitted in fashionable garb and entered in the journalistic parade of virginal findings. Apparently the disguise works, as these entries continue to slip by the editorial watchdogs. This duplication of effort brings to mind the Queen’s fitting admonition that in Wonderland it takes all the running you can do to keep in the same place. If you want to get somewhere else you must run at least twice as fast as that! The same holds in science.

I am also concerned that the free expression of hypotheses and the exchange of ideas is being stifled by the tight grant situation. These items have become the investigators private capital. In earlier times, the publication of working hypotheses enjoyed priority status. Those that panned out won the jackpot of glory, the others were forgotten. Investigators can no longer risk being wrong. Back in the 1930s there was a dictum, attributed to Herbert Evans, that being right 35% of the time was sufficient to assure a successful career in research.

VII Animal Models

The gathering of information applicable to man is an important objective of much of the experimentation conducted on laboratory and domestic animals. Few of the necessary experimental procedures can be carried out on humans for obvious reasons. The animals most commonly used as models include rats, rabbits, hamsters, sheep, pigs, cows, and subhuman primates, mostly rhesus monkeys. Opinion is divided as to which animal species serves best as a prototype for humans. The bulk of the experimental work has been carried out on rats, but that practice has been severely criticized on the basis that the estrous cycle of the rat has no luteal phase and the rat is virtually alone in requiring prolactin for luteal function. While many of the basic features of reproductive endocrinology in monkeys are similar to or identical with those in women, they also differ in some important aspects. The pulsatile pattern of LH secretion as originally observed only in ovariectomized female monkeys (Dierschke et al., 1970) is seen in sexually mature women. Also, the circulating level of progesterone during pregnancy is low in the monkey and high in women.

Most investigators become closely wedded to a given species, so that direct comparison of responses in different species is seldom possible. The maintenance of more than one or two species for experimentation is generally impractical for economic reasons and also because the investigator must be thoroughly familiar with every aspect of the species under study including all the extraneous influences, such as food, photoperiod, handling, and environmental stresses, that can alter the experimental results. Many investigators hold some bias as to the merits of the species of their choice and tend to generalize about the significance of results based on a single species. The concluding statements in articles and abstracts often leave the unwarranted impression that the findings are of general significance. As Tweedledee said: If it was so it might be; and if it were so, it would be; but as it isn’t, it ain’t. That’s logic.

In connection with the recent Review Project sponsored by the Ford Foundation, Schwartz, Dierschke, McCormack, and Waltz (1977) made a compilation in tabular form of the existing data on the response elicited in rats, sheep, monkeys, and humans by 43 different experimental situations involving steroid feedback mechanisms. The impressive gist of their findings is the fundamental similarity of the induced responses in these four species. Important differences in the response to a given stimulus were noted in only 6 of the 43 test situations examined and where significant differences existed, the sheep or rat sometimes turned out to be more satisfactory than the monkey in predictive value for the human.

Although different species have adopted a variety of mechanisms for the purpose of reproduction, it is evident that the basic unity of nature prevails as a dominant feature. It should also be evident from the remarkable progress already achieved that the animal models now in use have been serving the field admirably well. That they will also continue to do so in the future should not sidetrack the search for even better models, keeping in mind Alexander Pope’s dictum of many years ago that: The proper study of mankind is man. This still stands, but moral and legal restriction have greatly limited use of humans as experimental subjects, especially in technologically advanced nations. It is a disconcerting fact that these legal restrictions vary widely among different countries of the