Hypothalamic Control of the Pituitary-Gonadal Axis in Higher Primates: Key Advances over the Last Two Decades

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1 Journal of Neuroendocrinology From Molecular to Translational Neurobiology REVIEW ARTICLE Journal of Neuroendocrinology 2, ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd Hypothalamic Control of the Pituitary-Gonadal Axis in Higher Primates: Key Advances over the Last Two Decades T. M. Plant Department of Cell Biology and Physiology, University of Pittsburgh, Magee-Womens Research Institute, Pittsburgh, PA, USA. Journal of Neuroendocrinology Correspondence to: Tony M. Plant, Department of Cell Biology and Physiology, University of Pittsburgh, Magee-Womens Research Institute, 24 Craft Avenue; Rm. B311, Pittsburgh, PA 15213, USA ( This review provides a brief historical background to the foundation of primate reproductive neuroendocrinology that was laid by Ernst Knobil during the late 196s and early 197s. This is followed by a discussion of studies conducted over the last two decades that I view as having contributed to the current understanding of the field of primate reproductive neuroendocrinology. The review concludes with a short summary of key questions that remain to be addressed. Key words: puberty, ovulation, primate, GnRH, kisspeptin. doi: /j x The purpose of this review is to identify some of the studies that have captured my interest over the last two decades, and which I view as having contributed to our current understanding of the neuroendocrine mechanisms that control postnatal development and function of the gonad in higher primates 1. Before embarking on this journey, it is first necessary to briefly outline where this rather circumscribed and relatively scantly studied area of neuroendocrinology stood in 1988: a recent starting point when it is recalled that the neuroendocrine control of the gonad was first appreciated, albeit dimly, at the turn of the 2th Century as a result of the work of W. Heape (1) with the rabbit, a reflex ovulator, and of F. H. A. Marshall (2), who stressed the correlation between the external environment and reproduction. Early milestones The hypothalamus became the focus of the neuro component of neuroendocrine control systems governing gonadal function as a result of lesion and stimulation studies conducted by Smith, by Marshall and Verney, and by Fevold, Hisaw and Greep in the late 192s and 193s (3 5). This was followed during the 194s by the development of the tenet championed by Geoffrey Harris that the hypothalamic control of the anterior pituitary gland, including those cells regulating gonadal function, was achieved by neurohormones released into the primary plexus of the hypophysial portal circulation (6). Final proof of Harris neurovascular hypothesis was obtained in 197 when the laboratories of Guillemin and of Schally 1 Higher primates are comprised of three families: Old World monkeys, apes and humans. published the isolation of the hypothalamic-releasing factor, thyroid-stimulating hormone-releasing factor or thyrotrophin-releasing hormone. One year later, the isolation and structure of luteinising hormone releasing factor (LRF) or follicle-stimulating hormone (FSH)- and luteinising hormone-releasing hormone (LHRH) was announced by the same groups (7, 8) 2. Although Harris died in 1971, he has come to be recognised as the father of neuroendocrinology, while Guilleman and Schally, who outlived Harris (both are alive at the time of writing), were awarded the 1977 Nobel Prize in Physiology or Medicine for their work. Primate reproductive neuroendocrinology in the late 198s The foundation for this area of neuroendocrinology was based largely on an elegant and systematic series of experiments conducted by Ernst Knobil at the University of Pittsburgh during the 196s and early 197s that provided a rational model for the control of the menstrual cycle. Borrowing from control theory, he proposed that the pattern of gonadotrophin secretion throughout the ovarian cycle of the rhesus monkey could be accounted for by the negative- and positive-feedback actions of ovarian oestradiol secretion on LH and FSH release (9). In a characteristic linear approach, he then addressed the sites of the negative and positive feedback actions of oestradiol on gonadotrophin secretion in the female monkey. Using animals in which the spontaneous hypophysiotrophic drive to the gonadotrophs was first abolished with hypothalamic 2 The decapeptide, gonadotropin-releasing hormone (GnRH), which is synonymous with LRF and LHRH, is used throughout this review to describe mammalian GnRH (GnRH-1).

2 72 T. M. Plant lesions and subsequently restored with a pulsatile infusion of synthetic GnRH, he demonstrated that invariant intermittent GnRH stimulation of the pituitary was sufficient to drive ovulatory menstrual cycles with normal follicular and luteal phases (1). Knobil concluded that a pituitary site of action could account for both the negative- and positive-feedback actions of oestradiol on gonadotrophin secretion (1). The same experimental model led to the discovery that an intermittent hypothalamic GnRH signal to the pituitary was an obligatory component of the neuroendocrine control system that governs normal gonadotrophin secretion (11). Although the concept of a hypothalamic GnRH pulse generator remains a corner stone of reproductive neuroendocrinology, Knobil s conclusions regarding the pituitary as a major site of oestradiol action in the control of gonadotrophin secretion throughout the menstrual cycle, including the periovulatory period, are now often ignored. I believe this stems from the fact that the molecular and cellular bases of oestradiol action to regulate gonadotrophin secretion are being studied primarily in rats and mice; species in which hypothalamic sites of oestradiol action are critical for the unfolding of the 4 5-day oestrous cycle. It is ironic to note that Knobil s interest in the control of the menstrual cycle of the rhesus monkey was born in the lecture hall where he found that, using data obtained primarily from studies of the laboratory rat, it was impossible to explain the regulation of ovulation in women to medical students. By the late 198s, it was also well established that in primates, as in nonprimate species, the onset of gametogenesis at the time of puberty resulted from an increase in gonadotrophin secretion triggered, in turn, by an increase in GnRH pulse generator activity at this critical stage of development (12, 13). It was also known at this time that the postnatal ontogeny of GnRH pulsatility in primates is fundamentally different from that in rodents. This is because the increase in GnRH pulsatility that triggers puberty at the end of the protracted juvenile phase of primate development reflects a resurgence in hypothalamic GnRH pulse generator activity that has been held in check since late infancy. This is in striking contrast to rodents where the postnatal development of the pubertal GnRH signal unfolds progressively and without interruption (14). The characteristic on off on pattern 3 of GnRH pulse generator activity during postnatal development in higher primates was convincingly revealed in 1975 by Grumbach and his colleagues (15), who described, in agonadal human subjects, a characteristic diphasic pattern of gonadotrophin secretion from birth to adulthood with elevated levels during infancy and puberty separated by a hiatus during the childhood and juvenile years. This feature of primate development is exemplified by the agonadal rhesus monkey in which gonadotrophin secretion is amplified because of the absence of testicular or ovarian feedback signals (Fig. 1). The qualitative preservation of the diphasic pattern of gonadotrophin secretion in agonadal monkeys and human subjects also led to the rejection of the gonadostat hypothesis to account for the onset of puberty in higher primates (16). The latter idea was replaced over time by the notion of a gonad independent central inhibition or restraint to account for the prepubertal reduction in pulsatile GnRH release (17 2), and such a neurobiological brake now represents a 3 Strictly speaking, on-diminished-on. LH (ng/ml) FSH (ng/ml) major component of contemporary models of puberty onset in primates. It was also recognised prior to the 198s that the pubertal increase in GnRH could result from either the removal of an inhibitory input or the application of a stimulatory signal to the GnRH neuronal network of the juvenile hypothalamus, or a combination of the two (21) (i.e. the notion of a brake is conceptual). In 1988, however, the neural components of the brake that is imposed on GnRH pulsatility during juvenile development (and childhood in man) were unknown. Similarly, the control system that dictates when the brake is applied and when it is released was unknown, as it remains today. In this regard, the notion of a brain clock to time the initiation of puberty, or a hypothalamic somatometer to track growth and thereby coordinate the onset of puberty with a sufficient level of somatic development, has been in the literature for several decades (22, 23). Primate reproductive neuroendocrinology: the last two decades Puberty Age (weeks) Fig. 1. The on off on pattern of gonadotrophin-releasing hormone pulse generator activity during postnatal development in agonadal male (stippled area) and female (closed data points error bars) rhesus monkeys as reflected by circulating mean luteinising hormone (LH) (top panel) and follicle-stimulating hormone (FSH) (bottom panel) concentrations from birth to weeks of age. Note, in females, the intensity and duration of the prepubertal hiatus in the secretion of FSH, and LH to a lesser extent, is truncated in comparison to males. Adapted with permission (16) from TM. Plant. Puberty in primates. In: E. Knobil, JB. Neill, eds. The Physiology of Reproduction, 2nd edn. New York, NY: Raven Press Ltd., 1994; Vol. 2, Chapter 42, Copyright Elsevier, GnRH neurones are not limiting to the onset of puberty As demonstrated by my laboratory in the late 198s (24), the network of GnRH neurones in the hypothalamus of the juvenile male ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,

3 Hypothalamic control of the primate gonad 721 monkey may be reawakened at any time and, with surprising ease, by intermittent chemical stimulation with N-methyl-D-aspartate, a glutamate receptor agonist (Fig. 2). This finding indicates that, in primates, the network of GnRH neurones, which in adulthood provides the drive to the gonadotrophs, must be viewed together with the pituitary and gonads as a nonlimiting component of the control system that governs the onset of puberty in these species. The subsequent finding that levels of GnRH and of GnRH mrna in the hypothalamus of the juvenile monkey are similar to those in the pubertal adult brain (2) is consistent with the notion of a dormant GnRH pulse generator, and suggests that the molecular substrate for the biosynthesis and secretion of GnRH is maintained in a state of suspended animation throughout the juvenile phase of development when release of the decapeptide into the hypophysial portal circulation is restrained. It should be noted here that the intensity of the prepubertal brake that is imposed on GnRH release between infancy and puberty appears to be sexually differentiated. In the female, the brake is less intense and is imposed for a shorter duration; a sex difference that is reflected in the postnatal time course of gonadotrophin secretion in the agonadal situation (Fig. 1). Whether this difference, which is presumably the result of testicular programming of the fetal hypothalamus, is solely quantitative is unknown. This, however, would seem to be the most parsimonious situation. Components of the neurobiologic brake on prepubertal GnRH pulsatility During the last 2 years, we have learnt that glutamate (see above), c amino butyric acid (GABA), neuropeptide Y (NPY) and kisspeptin are important components of the neurobiological brake that holds or releases the check on GnRH pulsatility from infancy until puberty. A major role for GABA in inducing a state of relative dormancy in the network of GnRH neurones of the prepubertal female was firmly established during the 199s by an elegant series of studies by Terasawa and her colleagues at the Wisconsin National Primate Research Centre employing the rhesus monkey. Using the demanding technique of hypothalamic perfusion, the Wisconsin group found that release of GABA into the median eminence declined as pulsatile GnRH release was increasing at the onset of puberty (25). On the other hand, acute administration to the median eminence of the GABA A receptor antagonist, bicuculline, or the antisense oligodeoxynucleotide for the mrna coding the GABA synthesising enzyme, glutamate acid decarboxylase 67 (GAD 67 ), elicited a discharge of GnRH in the juvenile animal (25, 26). Moreover, initiation of chronic repetitive administration of bicuculline into the base of the third cerebroventricle at approximately 15 months of age in premenarcheal monkeys led to precocious puberty, as indicated by a dramatically premature menarche and first ovulation (27). Because of the sex difference in the intensity of the prepubertal brake on GnRH pulsatility, analogous studies of the role of GABA in the male primate would be of considerable interest, but these have not been conducted; a state of affairs probably related to the high cost of experimentation with nonhuman primates. In the male, on the other hand, exploration of inhibitory inputs to the GnRH neuronal network during juvenile development has focused on NPY and compelling indirect evidence is available to support the view that NPY is an important inhibitory component of this neuroendocrine control system (28). 1 Weeks of NMDA treatment Post-NMDA 8 Testosterone (ng/ml) Time of day (h) LH (ng/ml) Fig. 2. Chronic intermittent stimulation of the gonadotrophin-releasing hormone (GnRH) neuronal network in the hypothalamus of juvenile male monkeys (15 16 months of age) with brief i.v. infusions of N-methyl-D-aspartate (NMDA) once every 3 h for 15 weeks results in the immediate initiation of pubertal activity of the hypothalamic-pituitary-testicular axis. After 11 weeks of stimulation, the pulsatile pattern of hormone activity in the pituitary-leydig cell axis was indistinguishable from that observed spontaneously in adults, and motile sperm were recovered in the epididymides of two animals at 16 and 22 weeks after initiation of NMDA treatment. The latency to full activation of the pituitary-testicular axis is due to a progressive upregulation of pituitary gonadotrophin synthesis, and the action of NMDA on the axis was abolished by concomitant treatment with a GnRH receptor antagonist (not shown). LH, luteinising hormone. Reprinted with permission (24) from TM. Plant et al. Puberty in monkeys is triggered by chemical stimulation of the hypothalamus. Proc Nalt Acad Sci USA 1989;86: Copyright PNAS, ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,

4 722 T. M. Plant During the last 5 years, kisspeptin has emerged as a totally unexpected and novel component of the neurobiological machinery underlying the on off on pattern of GnRH release throughout postnatal development. Kisspeptins are encoded by the KiSS-1 gene and signal at the G-protein coupled receptor, GPR54 (29). Significantly, because of the primate focus of this review, the initial evidence for the importance of this signalling pathway in regulating the hypothalamic-pituitary-gonadal axis in any species was obtained by contemporary genetic studies of human pathophysiology and the findings that were reported were incontrovertible. Two groups, one led by de Roux in Paris (3) and the other by Crowley in Boston (31), each almost simultaneously reported that several members of a large consanguineous family presenting with hypogonadotrophic hypogonadism and absent puberty carried homozygous mutations for GPR54. In one of these studies (31), it was further reported that, in an additional subject bearing a compound heterozygote mutation of GPR54, pituitary responsiveness to pulsatile GnRH administration was not compromised (interestingly, it was actually exaggerated), indicating a hypothalamic locus for the deficit associated with this genetic disorder (Fig. 3). The impact of these studies on the field of reproductive neuroendocrinology in general has been, and continues to be, enormous, and this is reflected by the extent to which the initial human findings have, and are being, reversely translated to nonprimate species serving as paradigms for investigating a variety of modulators of the hypothalamic-pituitary-gonadal axis, such as development, season, nutrition, metabolism and stress. In the field of reproductive neuroendocrinology, it is difficult to identify a precedent for such a scenario. In collaboration with the Boston group and the Ojeda laboratory at the Oregon National Primate Research Centre, my laboratory pursued the notion that kisspeptin signalling at GPR54 is a major component of the trigger for the pubertal resurgence of pulsatile GnRH release in primates. It was reasoned that, if this is indeed the case, then the following premises should apply. First, KiSS-1 and the gene coding for GPR54 should be expressed in the primate hypothalamus. Second, an increase in kisspeptin signalling should occur in association with the pubertal resurgence of pulsatile GnRH release and, third, an increase in hypothalamic kisspeptin levels in the juvenile monkey should lead to a precocious activation of a pubertal mode of pulsatile GnRH release. Using in situ hybridisation, KiSS-1 expressing neurones were found to be restricted to the region of the arcuate nucleus in the medial basal hypothalamus (MBH), whereas GPR54 mrna was more diffusely distributed in the MBH (32). More recently, we have shown that the location of kisspeptin perikarya, as revealed by immunohistochemistry, is restricted to the arcuate nucleus, and therefore consistent with the expression of KiSS-1. Parenthetically, the absence of kisspeptin perikarya in the more rostral regions of the monkey hypothalamus, such as the anteroventral periventricular nucleus, has also been reported in the human (33), and contrasts with the situation in rodents: a comparative difference that may be related to differences in gonadal feedback control systems governing the preovulatory gonadotrophin surge in primates and rodents (see above). Reverse transcription-polymerase chain reaction revealed that KiSS-1 (A) LH (IU/l) (B) LH (IU/l) (C) LH amplitude Time (h) ng/kg 75 ng/kg 7.5 ng/kg 25 ng/kg Time (h) IHH patient with GPR54 mutations: R331X, X399R Regression line Regression line, mean LH Amp for 6 IHH men, no GPR54 coding sequence abnormalities 95% confidence limits 1 LOG (1) GnRH Fig. 3. Spontaneous and induced luteinising hormone (LH) secretion in a male patient carrying a GPR54 mutation. (A) Solid circles show spontaneous moment to moment changes in circulating LH concentrations at baseline over a 24-h period. The range for normal men is shown by the crosshatched area. Arrow heads show LH pulses. (B) LH responses to brief i.v. boluses of synthetic gonadotrophin-releasing hormone (GnRH) ( ng ml) administered after the patient had received intermittent s.c. GnRH treatment for several months to heighten pituitary responsiveness to the decapeptide. (C) Dose response curve describing LH pulse amplitude versus GnRH dose (log 1 ng kg) for the patient compared to the same regression for subjects without GPR54 mutations. Reprinted with permission (31) from SB. Seminara et al. The GPR54 gene as a regulator of puberty. N Engl J Med 23;349: Copyright Massachusetts Medical Society, 23. expression was increased in association with the pubertal resurgence in pulsatile GnRH release in agonadal males and intact females, whereas an increase in GPR54 mrna levels was only observed in the females. A precocious sustained release of pulsatile GnRH from juvenile monkeys, however, could only be elicited by intermittent stimulation with kisspeptin (34) (Fig. 4). Continuous i.v. infusions of the same peptide failed to initiate a sustained release of LH, and downregulation of GPR54 became apparent at high doses of uninterrupted administration (35). Taking the foregoing considerations together with the finding that GnRH neurones express GPR54 (29), this leads to the conclu- 2 3 ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,

5 Hypothalamic control of the primate gonad 723 LH (ng/ml) Day 1 Day 2 Day 3 Time (h) Fig. 4. Luteinising hormone (LH) discharges induced precociously in agonadal juvenile male monkeys (2 24 months of age) by an intermittent i.v. infusion of gonadotrophin-releasing hormone (GnRH) that mimics a castrate adult hypophysiotrophic drive to the gonadotrophs (open arrows on day 1) and by brief infusions of kisspeptin every hour (closed arrows) initiated on day 1 immediately following termination of GnRH priming and maintained for 48 h in a crossover design (kisspeptin-closed data points; vehicle-open data points). LH responses are shown for the first three kisspeptin or vehicle pulses on day 1, three such pulses on day 2 and the last two pulses on day 3. GnRH priming (open arrows) was reinitiated after completion of kisspeptin or vehicle administration on day 3. The LH response to pulsatile kisspeptin administration was abolished by concomitant treatment with a GnRH receptor antagonist (not shown) indicating a hypothalamic site of action of kisspeptin. Reprinted with permission (34) from TM. Plant et al. Repetitive activation of hypothalamic G protein - coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin - releasing hormone discharges. Endocrinology 26:147: Copyright 26, The Endocrine Society. sion that kisspeptin signalling at GPR54 is a critically important component of the neurobiological substrate that triggers the pubertal resurgence of GnRH release. The question remains, however, as to the precise role of this neuropeptide. Do kisspeptin neurones in the arcuate nucleus constitute a pubertal clock, or do they mediate a signal from a pubertal clock located elsewhere in the brain? Do kisspeptin neurones constitute a hypothalamic somatometer that senses a circulating signal reflecting somatic development? At the cellular level, the finding that robust GnRH pulsatility was induced precociously only when the kisspeptin stimulus is applied intermittently (Fig. 4) must be reconciled with the intriguing clinical observations of pulsatile LH release with low amplitude and approximately normal frequencies in patients with inactivating mutations of GPR54 (Fig. 3) (36). The latter findings suggest that kisspeptin may not be responsible for GnRH pulse generation, but rather functions to amplify the activity of the GnRH pulse generator. Moreover, as discussed above, there is compelling evidence for the involvement of other neurotransmitters (GABA and glutamate) and neuropeptides (NPY) in the neurobiological brake holding or releasing the check on GnRH pulsatility during juvenile development. Thus, the role of kisspeptin in triggering the pubertal resurgence in pulsatile GnRH release must be integrated with that of other neural signals, and probably additional signals of glial origin in order to provide a rational model for the neurobiological bases of puberty onset: this is a major challenge for the future and requires much more than just pasting neurones glia of different phenotypes onto a wiring diagram of positive and negative inputs to GnRH neurones. Leptin: an obligatory, albeit permissive, factor for the pubertal resurgence of pulsatile GnRH release Based on unchanging plasma leptin levels throughout peripubertal development in the male monkey, my laboratory proposed, in 1997, that the role of this adipocyte hormone in primate puberty was permissive (37). The evidence that engrained this notion into our thinking, however, was provided by studies of a classical endocrine paradigm in man. In 1999, Farooqi and colleagues (38) reported that leptin replacement for 12 months to a 1-year-old prepubertal child with advanced bone age and severe obesity and congenital leptin deficiency due to homozygosity for a frameshift mutation in the LEP gene was associated with the onset of puberty. Over the last decade, this finding has been confirmed in an additional two children of pubertal age whereas, significantly, in younger children (n = 4), there has been no evidence of premature puberty following leptin replacement (38, 39; I. S. Farooqi, unpublished observations). These key clinical observations emphasising the obligatory, albeit permissive, action of leptin in the onset of puberty also provide a precedent for the view that a circulating hormone reflecting somatic status (fat mass in this case) can serve as a profound modulator of the hypothalamic GnRH pulse generator. Structural remodelling of the GnRH neuronal network and its afferent inputs That neuroendocrine systems exhibit morphological plasticity was first established for vasopressin and oxytocin neurones, where glial ensheathment and synaptic input may be modified when magnocellular nuclei are activated by either an osmotic challenge or by application of a suckling stimulus (4). Building on these fascinating findings, my laboratory demonstrated that the postnatal mediobasal hypothalamus of the monkey continues to express the embryonic form of neural cell adhesion molecule (NCAM), polysialic acid-ncam (41), a previously established marker of neuronal plasticity, and subsequently demonstrated at the ultrastructural level that synaptic innervation of GnRH perikarya is reduced in animals in which the resurgence in pulsatile GnRH release has occurred compared with juvenile monkeys in which pulsatile GnRH release has not yet escaped the prepubertal check (42). Recent application of contemporary imaging techniques to the rodent hypothalamus indicate that synaptic plasticity of the GnRH neurone across development may be most extensive at the dendritic level (43). ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,

6 724 T. M. Plant Adulthood The preovulatory GnRH surge As discussed above, studies by Knobil three decades ago had demonstrated that oestradiol induced pre-ovulatory gonadotrophin surges could be elicited in female monkeys in the absence of a discharge of GnRH, leading to the proposal that the role of the hypothalamus in the control of the menstrual cycle was permissive (1). Knobil s model was not accepted with universal enthusiasm because it was fundamentally different from those proposed to account for the ovarian cycle of the rodent, which included an obligatory discharge of hypothalamic GnRH to trigger the preovulatory LH surge. The polarising nature of the Knobil model led many to overlook the experimental data, which, of course, do not exclude the possibility of an additional action of oestradiol at the level of the hypothalamus. Thanks to the perseverance of Spies and his colleagues (44), who demonstrated, in the rhesus monkey, an unambiguous increase in GnRH release in the median eminence concomitant with the preovulatory LH surge, such an additional hypothalamic action of oestradiol in the monkey is now established. Interestingly, the situation in the human female remains a topic of debate. Although ovulatory menstrual cycles may be induced in women with deficiencies in hypothalamic GnRH with invariant pulsatile GnRH treatment (45, 46), studies by Hall using a GnRH antagonist to indirectly quantitate endogenous GnRH secretion in normal women suggest that GnRH release during the preovulatory gonadotrophin surge may actually be decreased (47). If this is indeed the case, it would have to be argued, nevertheless, that it is the result of a hypothalamic action of oestradiol or a related ovarian signal. We are therefore left to determine the relative importance of hypothalamic versus pituitary sites of the positive-feedback action of oestradiol between species, and the extent, if any, to which the neurobiology of the circadian coupled hypothalamic signal for ovulation in the rodent may be translated to the human female. Interplay of nutrition and other stressors in modulating hypothalamic GnRH pulsatility That the hypothalamic network of GnRH neurones is the primary locus of the negative impact of undernutrition on the reproductive axis was convincingly demonstrated in 1986 by the finding that a chronic intermittent GnRH infusion could restore a normal pattern of gonadotrophin secretion in castrate male monkeys maintained on a calorie deficient diet (48). This study provided the impetus for Cameron, a co-author of the 1986 paper, to subsequently initiate a systematic series of studies aimed at elucidating the nutritional control of gonadotrophin secretion in the monkey (49). During the course of these studies, Berga was beginning to explore the notion that, in many cases, stress induced amenorrhea in women was the result of exposure to a combination of psychogenic and metabolic stresses, the latter arising from both nutritional factors and exercise. At this time, these investigators were both at the Center for Research in Reproductive Physiology at the University of Pittsburgh, and a successful collaboration was initiated to systematically explore the impact of combinations of stressors on GnRH pulsatility and menstrual cyclicity using the nonhuman primate as an experimental model. Recently, these investigators have provided compelling evidence that, in the monkey, the addition of a mild psychosocial stress (moving from one cage room to another) to an exercise schedule coupled with caloric restriction results in an interruption of normal menstrual cyclicity (5). A better understanding of interactions between stressors to impair GnRH pulsatility may have direct relevance to the treatment of life style amenorrhea in women. Conclusions In writing this review, I was struck by the fact that, despite Herculean efforts by several laboratories over the last 2 years, what might be called fundamental advances or breakthroughs in our understanding of the neuroendocrine physiology of the primate hypothalamic-pituitary-gonadal axis are few and far between. Certainly, the discovery that kisspeptin-gpr54 signalling is a dominant pathway regulating GnRH release across all mammalian species is such a breakthrough. At the same time, the control of the timing of human puberty remains a mystery, the precise neuroendocrine mechanisms underlying the preovulatory gonadotrophin surge in women is far from being clarified, and the cell biology responsible for GnRH pulse generation continues to be debated. Acknowledgements I would like to thank my colleagues, Drs Melvin Grumbach, Ei Terasawa, Harold Spies, William Crowley and Stephanie Seminara for their thoughts and help while I was writing this review. I am also grateful to Dr Sadaf Farooqi for sharing with me unpublished data on her leptin deficient patients. Work from my laboratory during the last 3 years has been generously supported by the National Institutes of Health (Grants HD and 816). Received: 29 February 28, accepted 1 March 28 References 1 Heape W. Ovulation and degeneration of ova in the rabbit. Proc Roy Soc B 195; 76: Marshall FHA. Sexual periodicity and the causes which determine it. The Croonian Lecture. Philos Trans R Soc London B 1936; 226: Smith PE. The disabilities caused by hypophysectomy and their repair. The tuberal (hypothalamic) syndrome in the rat. J Am Med Assoc 1927; 88: Marshall FHA, Verney EB. The occurrence of ovulation and pseudopregnancy in the rabbit as a result of central nervous stimulation. J Physiol 1936; 86: Fevold HL, Hisaw FL, Greep R. Augmentation of the gonad stimulating action of pituitary extracts by inorganic substances, particularly copper salts. Am J Physiol 1936; 117: Harris GW. Neural Control of the Pituitary Gland. London: Edward Arnold Ltd, ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,

7 Hypothalamic control of the primate gonad Amoss M, Burgus R, Blackwell R, Vale W, Fellows R, Guillemin R. Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem Biophys Res Commun 1971; 44: Schally AV, Arimura A, Baba Y, Nair RM, Matsuo H, Redding TW, Debeljuk L, White WF. Isolation and properties of the FSH- and LHreleasing hormone. Biochem Biophys Res Commun 1971; 43: Knobil E. On the control of gonadotropin secretion in the rhesus monkey. Recent Prog Horm Res 1974; 3: Knobil E, Plant TM, Wildt L, Belchetz PE, Marshall G. Control of the rhesus monkey menstrual cycle: permissive role of the hypothalamic gonadotropin-releasing hormone. Science 198; 27: Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 1978; 22: Wildt L, Marshall G, Knobil E. Experimental induction of puberty in the infantile female rhesus monkey. Science 198; 27: Watanabe G, Terasawa E. In vivo release of luteinizing hormone releasing hormone increases with puberty in the female rhesus monkey. Endocrinology 1989; 125: Fraser MO, Plant TM. Further studies on the role of the gonads in determining the ontogeny of gonadotropin secretion in the guinea pig (Cavia porcelus). Endocrinology 1989; 125: Conte FA, Grumbach MM, Kaplan SL. A diphasic pattern of gonadotropin secretion in patients with the syndrome of gonadal dysgenesis. J Clin Endocrinol Metab 1975; 4: Plant TM. Puberty in primates. In: Knobil E, Neill JD, eds. The Physiology of Reproduction, 2nd edn. New York, NY: Raven Press, Ltd., 1994; Vol. 2, Chapter 42, Grumbach MM, Roth JC, Kaplan SL, Kelch RP. Hypothalamic-pituitary regulation of puberty: evidence and concepts derived from clinical research. In: Grumbach MM, Grave GD, Mayer FE, eds. Control of the Onset of Puberty. New York, NY: John Wiley & Sons, Inc. 1974; Chapter 6, Plant TM, Zorub DS. The role of non-gonadal restraint of gonadotropin secretion in the delay of the onset of puberty in the rhesus monkey (Macaca mulatta). J Anim Sci 1982; 55 (Suppl. 2): Tereasawa E, Nass TE, Yeoman RR, Loose MD, Schultz NJ. Hypothalamic control of puberty in the female rhesus macaque. In: Norman RL, ed. Neuroendocrine Aspects of Reproduction. New York, NY: Academic Press, 1983: Plant TM. Neurobiological bases underlying the control of the onset of puberty in the rhesus monkey: a representative higher primate. Front Neuroendocrinol 21; 22: Winter JSD, Faiman C. Gonadotropins and sex hormone patterns in puberty, clinical data. In: Grumbach MM, Grave GD, Mayer FE, eds. Control of the Onset of Puberty. New York, NY: John Wiley & Sons, Inc. 1974: Chapter 2, Grave GD. Introduction. In: Grumbach MM, Grave GD, Mayer FE, eds. Control of the Onset of Puberty. New York, NY: John Wiley & Sons, Inc., Plant TM, Fraser MO, Medhamurthy R, Gay VL. Somatogenic control of GnRH neuronal synchronization during development in primates: a speculation. In: Delemarre van de Waal HA, Plant TM, van Rees GP, Schoemaker J, eds. Control of the Onset of Puberty, III. Amsterdam: Elsevier Science Publishers BV, 1989: Plant TM, Gay VL, Marshall GR, Arslan M. Puberty in monkeys is triggered by chemical stimulation of the hypothalamus. Proc Natl Acad Sci USA 1989; 86: Mitsushima D, Hei DL, Terasawa E. c-aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty. Proc Natl Acad Sci USA 1994; 91: Mitsushima D, Marzban F, Luchansky LL, Burich AJ, Keen KL, Durning M, Golos TG, Terasawa E. Role of glutamic acid decarboxylase in the prepubertal inhibition of the luteinizing hormone releasing hormone release in female rhesus monkeys. 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The GPR54 gene as a regulator of puberty. N Engl J Med 23; 349: Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 25; 12: Rometo AM, Krajewski SJ, Voytko ML, Rance NE. Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. J Clin Endocrinol Metab 27; 92: Plant TM, Ramaswamy S, DiPietro MJ. Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology 26; 147: Seminara SB, DiPietro MJ, Ramaswamy S, Crowley WF Jr, Plant TM. Continuous human metastin infusion desensitizes GPR54-induced GnRH release monitored indirectly in the juvenile male rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology 26; 147: Tenenbaum-Rakover Y, Commenges-Ducos M, Iovane A, Aumas C, Admoni O, de Roux N. Neuroendocrine phenotype analysis in five patients with isolated hypogonadotropic hypogonadism due to a L12P inactivating mutation of GPR54. J Clin Endocrinol Metab 27; 92: Plant TM, Durrant AR. Circulating leptin does not appear to provide a signal for triggering the initiation of puberty in the male rhesus monkey (Macaca mulatta). Endocrinology 1997; 138: Farooqi IS, Jebb SA, Langmack G, Lawrence E, Cheetham CH, Prentice AM, Hughes IA, McCamish MA, O Rahilly S. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med 1999; 341: Farooqi IS. Leptin and the onset of puberty: insights from rodent and human genetics. 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8 726 T. M. Plant 41 Perera AD, Lagenaur CF, Plant TM. Postnatal expression of polysialic acid-neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta). Endocrinology 1993; 133: Perera AD, Plant TM. Ultrastructural studies of neuronal correlates of the pubertal reaugmentation of hypothalamic gonadotropin-releasing hormone (GnRH) release in the rhesus monkey (Macaca mulatta). J Comp Neurol 1997; 385: Cottrell EC, Campbell RE, Han SK, Herbison AE. Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons. Endocrinology 26; 147: Pau K-YF, Berria M, Hess DL, Spies HG. Preovulatory gonadotropinreleasing hormone surge in ovarian-intact rhesus macaques. Endocrinology 1993; 133: Crowley WF Jr, McArthur JW. Stimulation of the normal menstrual cycle in Kallman s syndrome by pulsatile administration of luteinizing hormone-releasing hormone (LHRH). J Clin Endocrinol Metab 198; 51: Leydendecker G, Wildt L, Hansmann M. Pregnancies following chronic intermittent (pulsatile) administration of Gn-RH by means of a portable pump ( Zyklomat )-a new approach to infertility in hypothalamic amenorrhea. J Clin Endocrinol Metab 198; 51: Hall JE, Taylor AE, Martin KA, Rivier J, Schoenfeld DA, Crowley WF Jr. Decreased release of gonadotropin-releasing hormone during the preovulatory midcycle luteinizing hormone surge in normal women. Proc Natl Acad Sci USA 1994; 91: Dubey AK, Cameron JL, Steiner RA, Plant TM. Inhibition of gonadotropin secretion in castrated male rhesus monkeys (Macaca mulatta) induced by dietary restriction: analogy with the prepubertal hiatus of gonadotropin release. Endocrinology 1986; 118: Cameron JL. Regulation of reproductive hormone secretion in primates by short-term changes in nutrition. Rev Reprod 1996; 1: Williams NI, Berga SL, Cameron JL. Synergism between psychosocial and metabolic stressors: impact on reproductive function in cynomolgus monkeys. Am J Physiol Metab 27; 293: E27 E276. ª 28 The Author. Journal Compilation ª 28 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 2,