Recognition that sustained pituitary gonadotropin secretion requires pulsatile GnRH stimulation: a Pittsburgh Saga

The origin of the saga can be traced to Knobil’s uneasiness with using data derived from studies of the 4–5 day estrous cycle of the rat to explain to medical students the physiological control of the 28-day human menstrual cycle: a discomfort that contributed to his decision in the mid-1960s to submit the regulation of the menstrual cycle of the rhesus monkey to systematic study. Establishing such a nonhuman primate model first required precise descriptions of the time courses of the circulating concentrations of the ovarian steroids (estradiol and progesterone) and the pituitary gonadotropins throughout the entire 28-day menstrual cycle of the rhesus macaque. In the case of progesterone, the task was relatively easy, as the use of competitive protein binding for measuring small quantities of blood steroids had been established by Beverly Murphy in Montreal in the early 1960s. Using corticosteroid-binding protein from dog as the binding protein for progesterone, the Knobil laboratory, with Jimmy Neill as the lead investigator, provided in 1967 a precise description of plasma progesterone throughout the menstrual cycle of the rhesus monkey (). Parenthetically, at the time of publication of this paper, I was struggling with my Ph.D. project in the laboratory of Richard Michael in London, where I was attempting to develop a method to indirectly measure ovarian progesterone secretion in the monkey by quantitating the 24 h urinary excretion of the steroid’s metabolites. Needless to say, Jimmy Neill’s paper was a wake-up call for me and later led me to seek a postdoctoral position in the Knobil laboratory in Pittsburgh. Estradiol in nonpregnant monkeys was present in only picogram concentrations, and the measurement of this circulating steroid required a specific antiserum with high affinity for the hormone: in the late 60s, steroid antibodies were not commercially available and were typically generated inhouse. Using such an antiserum (provided by Michel Ferin and Raymond Vande Wiele at Columbia University), Julane Hotchkiss led the development of a sensitive and specific radioimmunoassay (RIA) for measurement of circulating estradiol and, in 1971, described the time course of concentrations of this plasma steroid throughout the monkey menstrual cycle ().

When it came to the pituitary gonadotropins, the failure of these hormones from the monkey to cross-react with antibodies to the cognate human glycoproteins also necessitated the development of inhouse RIAs. Instrumental in this regard was William Peckham, a talented biochemist, who used side fractions of monkey pituitary generated earlier by Knobil while extracting growth hormone to isolate highly purified preparations of monkey LH and FSH that were then employed to generate primary antibodies, trace, and standards for use in homologous, double antibody RIAs. A sensitive and specific RIA for LH was reported in 1970 (), and 3 years later, that for FSH followed (). In the 1973 Gregory Pincus Memorial Lecture at the Laurentian Hormone Conference, Knobil provided a composite figure showing the concentrations of both pituitary hormones and ovarian steroids throughout the rhesus monkey menstrual cycle (): they were remarkably similar to those described earlier for women, establishing this Old World monkey as a powerful paradigm for the human situation.

In contrast to the human female, the monkey was, of course, highly tractable to experimental manipulation, and the stage was thus set for what became a remarkable phase of linear scholarship by the Knobil laboratory that spanned close to a decade and culminated in the proposal of a compelling model for the neuroendocrine control of the primate menstrual cycle (). In the early 1970s, the concept of negative feedback regulation of gonadotropin secretion by ovarian steroids was well established, and the ability of estradiol to elicit a discharge of LH (positive feedback) that mimicked the preovulatory LH surge triggering ovulation had been demonstrated in the ewe. With this intelligence, the Knobil laboratory began to examine the feedback actions of estradiol in the monkey using physiologic replacement paradigms, which in some cases required the administration of steroids over days or weeks. As noted by Fred Karsch (FK), who joined the laboratory in 1972, chronic intravenous infusion of the hormone was fraught with difficulty. Fortunately, during his graduate student years at the University of Illinois, FK had gained experience using subcutaneously implanted steroid-filled Silastic capsules, and he advocated the exploration of the latter approach as an alternative to intravenous infusion for long-term delivery of estradiol to the monkey. FK recounts that he “searched the lab for Silastic tubing and found just one piece about one foot long. I had no idea what size to use, so I cut off a piece about one inch long, packed it with crystalline estradiol, and sealed the ends shut with Silastic glue. My first implant was a total shot in the dark”. “Low and behold, we hit paydirt. The implant elevated the plasma estradiol concentration to ∼70 pg/ml, exactly the value observed in ovary-intact monkeys during the early follicular phase of the menstrual cycle. Further, that level of estradiol was sustained for several weeks without any sign of decline over time. To add icing to the cake, the plasma concentration of LH in that monkey dropped from the high value typical of ovariectomized monkeys to low values seen in ovary-intact monkeys during the early follicular phase of the cycle. My laboratory mates thought I was a genius. Little did they know it was dumb luck!”

Karsch’s introduction of the steroid-filled Silastic capsule to the Knobil laboratory provided the key tool that served as the catalyst for an elegant series of physiological studies of the negative and positive feedback actions of estradiol on gonadotropin secretion in the monkey. These were published with Karsch as the first author in 1973 (, , ) and underlined the view that the pattern of gonadotropin secretion throughout the menstrual cycle could be accounted for simply by the negative and positive feedback actions of estradiol on the secretion of LH and FSH, and led to the notion of a “pelvic clock” governing the menstrual cycle (). The negative feedback action of estradiol was argued to be responsible for the relatively low levels of gonadotropin secretion (tonic) throughout most of the follicular phase and the luteal phase, whereas the positive feedback action of high levels of estradiol secreted by the dominant follicle was responsible for eliciting and timing the massive preovulatory gonadotropin surge at the end of the follicular phase that, in turn, triggered ovulation.

The question then became—at what sites (brain or pituitary) does estradiol exert its feedback actions on the gonadotropins, and if central neural sites are involved, where are they located? This question had been fermenting since the arrival of Lewis Krey (LK) in Pittsburgh in 1971. LK had completed his graduate studies with John (Jack) Everett at Duke University and was, therefore, steeped in concepts regarding the neural control of the ovarian cycle founded on the classic studies of the rat by Everett, Markee, and Sawyer in the 40s and 50s. Central to these ideas was the tenet that the preovulatory LH surge on the afternoon of proestrus was timed by a neural signal, which had been subsequently argued by Halasz and Gorski (in Sawyer’s Department at UCLA) to originate from an area within the rostral hypothalamus or preoptic area (). The latter view was based on Halasz and Gorski’s finding that ovulation was blocked when the neural connections between the preoptic area and medial basal hypothalamus (MBH) in the rat were interrupted surgically with a bayonet-shaped knife. On his arrival in the Knobil laboratory, LK proposed to use a “Halasz” type knife for surgical deafferentation of the MBH of the monkey: an idea that received little enthusiasm at the time. Nevertheless, over the next 2 years, LK, with the ardent support of Ron Butler, another postdoctoral fellow in the laboratory, doggedly pursued the idea: a rotating double-edged knife to “fit” the monkey MBH was designed and built, a neurosurgical procedure was developed for midline insertion and stereotaxic placement of the knife, and informal support was obtained from the Hospital’s Radiology Department to develop multiple radiograms generated from the series of sagittal roentgenograms required to confirm the accurate placement of the knife visually. Extensive postoperative maintenance protocols were written, and an initial series of animals were prepared and studied. Sadly, histological examination of the lesions revealed that all the initial deafferentations were unilateral, and, therefore, the afferent input to the MBH had not been fully interrupted. LK recounts that “I had a breakthrough in December 1973 when I went to visit family in Westchester, NY. I had read an article by Paul Maclean about deafferentation of the ventromedial nucleus on feeding behavior in rhesus monkeys, so I went to visit his lab in nearby White Plains at the Cornell Primate Facility. I described my problem, and he gave me his procedural ‘secrets’ for bilateral cuts. When I returned to Pittsburgh, I incorporated his techniques and had complete success on the next group of monkeys.” The completeness of MBH deafferentation was verified histologically and supported by concomitant findings that the GH response to vasopressin or insulin hypoglycemia was abolished, as was the diurnal variation of cortisol. On the other hand, the positive feedback action of estradiol to elicit the LH surge remained intact, and spontaneous ovulatory menstrual cycles were observed in some monkeys; findings indicating that, in contrast to the rat, the site of both the positive and negative feedback actions of estradiol in the monkey were exerted within the MBH-pituitary unit. The Krey results for the monkey were published in 1975 in an elegant and now classic series of 4 back-to-back papers in Endocrinology. The first paper describing that the positive feedback action of estradiol to elicit the preovulatory-like LH surge was not abolished by the complete surgical isolation of the MBH () caused considerable and prolonged consternation within the neuroendocrine community that had been brought up on dogma generated from the studies of the rat.

It was at about this point in the saga that I joined the Knobil laboratory as a postdoctoral fellow and, together with David Hess, who was already in Pittsburgh, became immersed in the pursuit of the answer to the question of the relative importance of feedback by estradiol at the level of the MBH versus that at the pituitary. The initial approach focused on neural sites and applied the technique of push-pull perfusion to either administer estradiol to the MBH and monitor LH secretion or to sample GnRH from the anterior pituitary under conditions of negative and positive estradiol feedback. To our chagrin, these approaches provided only equivocal results, and after 2 years of unremitting struggle, neither we nor the laboratory was any the wiser regarding the sites of estradiol feedback.

In parallel with the push-pull experiments, we were also using the stereotaxic placement of radiofrequency lesions to target specific structures within the MBH with the aim of identifying hypothalamic nuclei regulating gonadotropin secretion: a study that benefited greatly from both the expertise in the stereotaxic placement of hypothalamic probes and the considerable bank of knowledge on the neuroanatomy of the monkey hypothalamus that LK had earlier established (and subsequently willingly shared) during the deafferentation studies. A major finding of the lesion study was that discrete destruction of the arcuate nucleus, a small bilateral sausage-shaped structure immediately dorsal to the median eminence, resulted in the cessation of both LH and FSH secretion without compromising the blood supply to the pituitary, as indicated by a large but unsustained increment of LH and FSH in response to a continuous infusion of GnRH at 6.8 ug/h for 48 h (). On the other hand, much larger lesions placed dorsal to the arcuate nucleus were without a major discernable effect on gonadotropin secretion. At the time, we were greatly baffled by the finding that such small lesions involving only the arcuate nucleus had such a profound effect on LH and FSH secretion: a puzzle that remained for approximately 3 decades until the significance of kisspeptin in controlling gonadotropin secretion was revealed by human genetics, and the arcuate nucleus was shown to be densely populated with neurons expressing this neuropeptide ().

In the context of the present story, it was immediately recognized that the arcuate lesioned monkey offered a potential in vivo model to explore feedback actions of estradiol directly at the pituitary. If gonadotropin secretion could be restored in the lesioned animal by constant exogenous GnRH stimulation, any change in LH and FSH secretion that followed the administration of estradiol would have to be accounted for by the action of the steroid directly at the pituitary, as hypothalamic stimulation of the gonadotrophs in these animals had presumably been eliminated and replaced by a “clamped” input of exogenous GnRH. As mentioned above, a continuous intravenous infusion of GnRH at 6.8 ug/h elicited an acute discharge of LH and FSH lasting several hours but failed to sustain gonadotropin secretion despite the continued administration of the peptide (). A similar phenomenon had been noted earlier by LK while studying the response of the monkey to a preparation of ovine GnRH that had been provided by Roger Guillemin. Despite these ominous results, the continuous mode of GnRH administration was not initially abandoned, and we continued to examine additional rates of continuous GnRH infusion in the hope that one would be found to be effective in sustaining gonadotropin secretion. It was Yoshikatsu Nakai (YN), who joined the laboratory in early 1975 at the time of the arcuate lesion study, that became the major proponent for an intermittent mode of exogenous GnRH stimulation: he spent many hours searching for insight in the Pitt medical school library, where he came on the interesting and, at the time, little known finding that continuous exposure to certain peptide hormones can lead to inhibition of the second messenger and loss of responsiveness. However, his proposal to administer GnRH intermittently was initially turned down. YN recalls that this was probably “because my English was poor and the Chief did not have much knowledge of biochemistry.” After some cajoling, “the Chief” agreed to test intermittent delivery in one arcuate lesioned monkey: Nakai’s pulsatile regimen of GnRH at a dose of 1 ug/min for 6 min every h was agreed on. The interpulse interval was selected to mimic the approximately hourly frequency of spontaneous pulsatile LH secretion established earlier for the ovariectomized monkey, but the selection of infusion rate and duration of the pulse of the peptide was largely serendipitous. The experiment was conducted while YN and I went to the 1976 Annual Meeting of the Endocrine Society in San Francisco, and when we returned, the samples were immediately sent for assay. To our great joy, LH and FSH had risen over a period of days from undetectable values characteristic of the lesioned animal to the high levels typically seen in brain-intact ovariectomized animals, and moreover, the subsequent administration of estradiol was followed by a rapid decline in gonadotropin secretion followed by a surge of LH, indicating that both the negative and positive feedback actions of estradiol were expressed directly at the level of the pituitary. Additional experiments were performed as quickly as possible, and 2 papers describing, respectively, the positive and the negative feedback actions of estradiol at the pituitary level in arcuate lesioned monkeys in which LH and FSH secretion was restored by Nakai’s pulsatile GnRH regime were together submitted to Endocrinology in August 1977 (, ). Although it was recognized in the Material and Methods of the first of these papers () that continuous infusion of GnRH was not successful in restoring gonadotropin secretion in the lesioned animals, the phenomenon was not further discussed. However, by the time the 2 Endocrinology papers were published in April 1978, the definitive study to determine whether the failure of the pituitary to respond to continuous exposure of GnRH was attributable to the pattern of hypophysiotropic stimulation per se or the quantity of the decapeptide delivered to the pituitary, had been completed and the results submitted to Science. In the later study, led by Paul Belchetz, a clinical endocrinologist from the United Kingdom, continuous intravenous infusion of GnRH at 4 doses (0.001, 0.01, 0.1, and 1.0 ug/min) for 10 days failed, without exception, to sustain gonadotropin secretion: in marked contrast, restoration of gonadotropin secretion was achieved with surprising ease in the same animals when a pulsed infusion of GnRH at 1 ug/min for 6 min once every hour was administered (). These findings led to the proposal that the pattern of GnRH stimulation rather than the total mass of the peptide delivered to the pituitary was critical in driving LH and FSH secretion: a conclusion that was beautifully illustrated by the Belchetz experiment in which elevated gonadotropin secretion sustained by the pulsatile mode of GnRH administration was suppressed over a period of approximately 10 days after initiation of continuous GnRH administration but was again restored by reestablishing pulsatile GnRH stimulation after an additional 10 days of inhibition imposed by the continuous exposure to GnRH (Figure 1).

In retrospect, the reticence to examine pulsatile administration of GnRH is difficult to understand as the laboratory had proposed 5–6 years earlier that discharges by the hypothalamus of an “LH releasing factor” were responsible for the approximately hourly episodic discharges of LH that had been observed in ovariectomized monkeys in a study led by Dierschke et al.(). Indeed, YN remembers that “at a later lab meeting, Dr. Knobil graciously recognized that he should have placed more weight on the Dierschke paper.” The seeming belated recognition that GnRH stimulation had to be intermittent to sustain gonadotropin secretion is now viewed by YN as a “Columbus egg.”

The discovery in 1978 that sustained secretion of pituitary LH and FSH, which is necessary for maintaining adult gonadal function and fertility, required intermittent stimulation by the hypothalamic hormone, GnRH, was the result of conceptualization and empirical struggle for approximately 15 years. It was orchestrated in a systematic and linear fashion by Ernst Knobil, an erudite leader with great vision and exceptional tenacity, and executed by sequential teams of creative and industrious postdoctoral fellows. The journey, however, was not all plain sailing: the work was conducted with the rhesus monkey, an expensive and difficult experimental model, and many major methodological hurdles had to be overcome along the way; there were several sub-plots of herculean, but not always successful, lines of experimentation; and there were occasions when critical ideas were not immediately acted on. Fortunately, the hard times were interrupted by instances of serendipity, which led to quantum leaps in either conceptualization or experimental technique. For the writer of this review, his participation in the culmination of the saga was breathtaking and became the most formative phase of his development as a scientist.

The author thanks Fred Karsch (Emeritus Professor, University of Michigan), Lewis Krey (Professor of Obstetrics-Gynecology and Cell Biology, New York University School of Medicine: retired), and Yoshikatsu Nakai (Professor Emeritus, Kyoto University) for sharing their thoughts about the work they proposed and conducted in the laboratory of Ernst Knobil in Pittsburgh. The work of Fred and Lew embodies critical milestones in the evolution of the saga told above, whereas that of Yoshi underpins the final chapter of the story.