Ovarian regulation of kisspeptin neurones in the arcuate nucleus of the rhesus monkey (macaca mulatta).

Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative-feedback actions of ovarian oestradiol (E2) and progesterone. Although kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in nonprimate species, this issue has been less well studied in the monkey. In the present study, we used immunohistochemistry and in situ hybridisation to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E2, progesterone and E2 + progesterone replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurones and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys but, on the other hand, in intact animals, kisspeptin cell bodies were small in size and number and only fine fibres were observed. Replacement of OVX monkeys with physiological levels of E2, either alone or with luteal phase levels of progesterone, abolished KISS1 expression in the ARC. Interestingly, progesterone replacement alone for 14 days also resulted in a significant down-regulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signalling in ARC neurones appears to play an important role in mediating the negative-feedback action of E2 on gonadotrophin secretion, and also indicate the need to study further their regulation by progesterone.

Hypothalamic control of the pituitary-gonadal axis in higher primates: key advances over the last two decades.

This review provides a brief historical background to the foundation of primate reproductive neuroendocrinology that was laid by Ernst Knobil during the late 1960s and early 1970s. 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.

Evidence that down regulation of hypothalamic KiSS-1 expression is involved in the negative feedback action of testosterone to regulate luteinising hormone secretion in the adult male rhesus monkey (Macaca mulatta).

In the male monkey, luteinising hormone (LH) secretion is regulated by a negative feedback action of testicular testosterone that is exerted indirectly at the hypothalamic level to decelerate pulsatile gonadotrophin-releasing hormone release (GnRH). The purpose of the present experiment was to investigate whether the kisspeptin-G protein-coupled receptor 54 (GPR54) signalling pathway is involved in mediating the action of testosterone to suppress GnRH release in the monkey, as has been indicated by studies of nonprimates. To this end, 12 castrated adult male rhesus monkeys were implanted with either testosterone containing or empty Silastic capsules. Testosterone treatment produced a square wave increment in circulating testosterone levels within the physiologic range. After suppression of LH and follicle-stimulating hormone secretion was established at 5-6 weeks of testosterone exposure, the animals were killed and expression of the genes encoding for kisspeptin, GPR54 and GnRH determined in the mediobasal hypothalamus and preoptic area of both treated and control animals using RNase protection assays. The suppression in pituitary gonadotrophin secretion was associated with a reduction in kisspeptin mRNA levels in the mediobasal hypothalamus, but not the preoptic area. GPR54 mRNA levels, on the other hand, were not influenced by testosterone treatment. These results are consistent with those previously reported for the rodent, and suggest that the neurobiology of the negative feedback action of testicular testosterone on LH secretion in the monkey, a representative higher primate, may be mediated by kisspeptinergic neurones upstream to the GnRH network.

Neurobiological bases underlying the control of the onset of puberty in the rhesus monkey: a representative higher primate.

The purpose of this article is to discuss our understanding of the neurobiological mechanisms that govern the timing of the onset of puberty in the rhesus monkey, a representative higher primate, and, whenever possible, to place findings obtained from studies of this macaque in perspective with those for the human situation. Specifically, the dynamics in the postnatal ontogeny of hypothalamic GnRH gene expression and release are described, and the roles of neuropeptide Y and gamma-aminobutyric acid in imposing the restraint on pulsatile GnRH release during juvenile development are examined. Finally, the hypothesis that circulating leptin provides the signal that times the reaugmentation of pulsatile GnRH release at the termination of juvenile development, and therefore triggers the onset of primate puberty, is discussed.

Changes in hypothalamic gene expression associated with the arrest of pulsatile gonadotropin-releasing hormone release during infancy in the agonadal male rhesus monkey (Macaca mulatta).

This study examined whether changes in the levels of the messenger RNAs (mRNAs) encoding the gamma-aminobutyric acid (GABA) synthesizing enzymes, glutamate decarboxylase (GAD)65 and GAD67 and transforming growth factor-alpha (TGFalpha) in the hypothalamus are correlated with the arrest of pulsatile GnRH release during infancy in the agonadal male monkey. This experiment also provided the opportunity to examine changes in hypothalamic GnRH gene expression during this critical phase of primate development. Male rhesus monkeys were castrated at 1 week of age: four were killed 4-7 weeks after orchidectomy while pulsatile GnRH release was robust as reflected by high circulating LH levels, and four were killed at 12-15 months of age after establishing that pulsatile GnRH release had been arrested. GAD65, GAD67, TGFalpha, and GnRH mRNA levels were estimated using RNase protection assays employing homologous probes and the results were expressed relative to cyclophilin mRNA levels. GnRH peptide was measured by RIA. GAD65 and GAD67 mRNA levels in the hypothalamus of juveniles were significantly greater than those in neonatal monkeys. On the other hand, hypothalamic TGFalpha and GnRH mRNA (and peptide) levels in agonadal neonate and juvenile monkeys were indistinguishable. These results indicate that the molecular concomitants associated with bringing the hypothalamic GnRH pulse generator into check in agonadal neonatal males are not a mirror image of those previously reported at the time this neuronal network is reactivated at puberty when TGFalpha and GnRH gene expression increase and GAD65 and GAD67 mRNA levels remain unchanged. Thus, the neurobiological mechanism that reactivates pulsatile GnRH release at puberty is likely to involve more than a simple reversal of that underlying inhibition of the same network in late infancy.

Neuropeptide Y: A hypothalamic brake restraining the onset of puberty in primates.

The adult reproductive axis is driven by an intermittent discharge of gonadotropin-releasing hormone (GnRH) generated by a network of hypothalamic neurons known as the GnRH pulse generator. Although this signal generator is operational in infant primates, puberty in these species is delayed by activation shortly after birth of a central neural mechanism that holds GnRH release in check during juvenile development. Here, we show that, in the male rhesus monkey, the postnatal pattern in GnRH pulse generator activity is inversely related to that in neuropeptide Y (NPY) gene and protein expression in the mediobasal hypothalamus and that central administration of an NPY Y(1) receptor antagonist to juvenile animals elicits precocious GnRH release. Cell imaging indicated that the developmentally regulated NPY neurons may be located in regions dorsal to the arcuate nucleus. These findings lead us to propose that NPY is a fundamental component of the neurobiological brake restraining the onset of puberty in primates.

Development and organization of the hypophysiotropic hypothalamus driving the pituitary-gonadal axis in the rhesus monkey.

The purpose of the present review is to describe, with particular emphasis on the rhesus monkey, the ontogeny and functional organisation of the hypothalamic GnRH pulse generator. Control of pituitary-gonadal axis in higher primates is provided by a group of some 1,000 GnRH neurons that are diffusely distributed throughout the hypothalamus. After synthesis of a prehormone and formation of the mature decapeptide, GnRH is released in the hypophysial portal circulation and stimulates FSH and LH production.

Indirect assessment of pulsatile gonadotropin-releasing hormone release in agonadal prepubertal rhesus monkeys (Macaca mulatta).

The major purpose of this study was to characterize the open-loop frequency of pulsatile GnRH release in the female rhesus monkey at an age (15-20 months) when the prepubertal restraint on the hypothalamic-pituitary axis is maximally imposed. Additionally, evidence for pulsatile GnRH release in agonadal males of comparable age was also sought. Episodic LH secretion from the pituitary was used as an indirect index of GnRH discharges. In order to maximize the sensitivity of this in situ bioassay, the responsiveness of the pituitary gonadotrophs was usually first heightened by an i.v. intermittent infusion of the synthetic peptide. Monkeys (five females, three males) were castrated between 9 and 14 months of age, implanted with indwelling venous catheters, fitted with nylon jackets and housed in specialized cages that permitted remote access to the venous circulation with minimal restraint and without interruption of the light-darkness cycle. In females, LH secretion was generally assessed at 20-day intervals during alternate nighttime (1900-0200 h) and daytime (0700-1400 h) windows. In males, LH was assessed less frequently and only at night. The mean frequency of pulsatile LH release in agonadal prepubertal females was 4 pulses/7 h during the night and 2 pulses/7 h during the day. These findings indicate that, prior to puberty in the female monkey, the GnRH pulse generator operates at a relatively slow frequency and is subjected to diurnal modulation. In males, evidence for robust pulsatile GnRH release was not observed. The striking difference in activity of the GnRH pulse generator in agonadal prepubertal male and female monkeys reinforces the view that the ontogeny of the hypothalamic drive to the pituitary-gonadal axis in higher primates, including man, is sexually differentiated.

Effect of estrogen on hypothalamic transforming growth factor alpha and gonadotropin-releasing hormone gene expression in the female rhesus monkey.

In order to study whether hypothalamic transforming growth factor alpha (TGFalpha) gene expression in the monkey is estrogen-sensitive, long-term ovariectomized rhesus macaques were implanted subcutaneously with either estradiol-containing (n = 3) or blank (n = 3) Silastic capsules. Blood samples were collected every other day while the animals were lightly sedated with ketamine hydrochloride to monitor circulating LH and estradiol concentrations. Animals were killed with a lethal dose of pentobarbital sodium after a marked suppression of LH secretion was confirmed (81 days of estradiol treatment); the preoptic area (POA), mediobasal hypothalamus (MBH) and samples of cerebral cortex were dissected out, snap-frozen in liquid nitrogen and processed for the determination of TGFalpha messenger RNA (mRNA) by ribonuclease protection assay using a cRNA probe. The opportunity was also taken to study the action of estrogen on hypothalamic GnRH mRNA levels. Although circulating estradiol concentrations of 50-150 pg/ml achieved in the steroid-treated group produced a decrease in hypothalamic GnRH mRNA levels, which was significant in the MBH, TGFalpha mRNA levels in this hypothalamic region and in the POA were not influenced by estrogen treatment. These findings indicate that TGFalpha is probably not involved in mediating the inhibitory action of estradiol on GnRH neurons. Additionally, the relevance of our results to the understanding of the neurobiological mechanisms underlying the initiation of puberty in primates is discussed.

Ultrastructural studies of neuronal correlates of the pubertal reaugmentation of hypothalamic gonadotropin-releasing hormone (GnRH) release in the rhesus monkey (Macaca mulatta).

This study tested the hypothesis that puberty in primates is triggered by a remodeling of synaptic inputs and/or glial coverage of hypothalamic gonadotropin releasing hormone (GnRH) neurons. Male rhesus monkeys were prepubertally castrated at 16 months of age and were killed and perfused either 1 month later (n = 4, juvenile group) or at 30 months of age, shortly after initiation of the pubertal increase in pulsatile GnRH release (n = 4, adult group). Hypothalami were sectioned, immunocytochemically stained for GnRH, and processed for electron microscopy. Cross-sectional profiles of 77 GnRH cells from the medial basal hypothalamus (MBH) and the region of the organum vasculosum of the lamina terminalis (OVLT) were compared between the two developmental stages. GnRH cell and nucleolus size in the two groups were the same. The percentage of GnRH perikaryal membrane occupied by synaptic density in the MBH of juveniles was significantly greater (P < 0.05) than that of adults. Differences in the percentage of GnRH perikaryal membrane occupied by synaptic density were not observed in the OVLT nor on GnRH dendrites in either brain region. Qualitative analysis, based on synaptic vesicle shape, failed to reveal developmental differences in putatively excitatory or inhibitory synapses on GnRH cells. The degree of glial ensheathment of GnRH neurons did not change significantly during the two developmental stages. These findings provide ultrastructural evidence for the view that, in primates, neuronal plasticity, and specifically a decrease in synaptic input to GnRH perikarya, may underlie the initiation of the pubertal mode of release of this neuropeptide, and therefore, the onset of puberty in these species.

Glutamate-immunoreactive neurons and their gonadotropin-releasing hormone-neuronal interactions in the monkey hypothalamus.

Glutamate (Glu) is the most prevalent excitatory neurotransmitter in the brain and has been implicated in the regulation of GnRH secretion in several mammalian species, including the monkey. To investigate the neuroanatomical basis for Glu-GnRH interactions, we performed an immunocytochemical study at both the light and electron microscopic levels on the brains of four female and five male macaques. Initially, we determined the location of Glu-immunoreactive (-ir) elements using a monoclonal antibody specific for glutaraldehyde-fixed Glu (Glu-2) and 3,3'-diaminobenzidine-4-HCl (DAB). Glu-ir was observed in the cytoplasm and to a variable degree in the nuclei of neurons in the diencephalon. Cytoplasmic staining was particularly intense in numerous neurons in the arcuate nucleus, supraoptic nucleus, and many paraventricular nucleus neurons. Short Glu-ir processes were evident in these and other hypothalamic regions and were extremely dense in the infundibular stalk and median eminence. Prior absorption of the Glu-2 antibody with a Glu-glutaraldehyde-BSA conjugate completely abolished all immunostaining in both neuronal nuclei and cytoplasm. Double label Glu-GnRH immunostaining for light microscopy was performed using Glu-2 and DAB without enhancement, and a polyclonal antibody (LR1 or LR2) with silver-enhanced DAB for Glu and GnRH, respectively. Glu-ir interactions with GnRH-ir cell bodies were not apparent, but a few Glu-ir axons seemed to contact GnRH-ir dendrites in the organum vasculosum of the lamina terminalis, medial septum, and arcuate nucleus regions. Reciprocal interactions occurred more frequently, however, in which GnRH-ir axons and dendritic fibers engaged Glu-ir cell bodies en passant, particularly toward the medial and posterior hypothalamus. For ultrastructural analyses, Glu-ir elements were stained with the Glu-2 antibody and 15 nm immunogold or DAB. Electron microscopy demonstrated that Glu-ir was associated with clear microvesicles within the neuronal cytoplasm. Glu-ir processes made classical asymmetrical synapses with one another and received asymmetrical synapses from unlabeled afferents. In sections double labeled for Glu with immunogold and for GnRH with DAB, axo-somatic interactions were not observed. However, axo-dendritic Glu-GnRH synapses were seen, which usually exhibited Glu-ir labeling of terminal vesicles and inconsistent postsynaptic densities, with GnRH-ir neurosecretory granules sometimes congregated in the apposing dendrite or spine. Surprisingly, reverse GnRH-Glu interactions were observed more frequently.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal expression of polysialic acid-neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta).

Puberty in primates is triggered by a gonad-independent reinitiation of a pulsatile mode of GnRH release. The purpose of the present study was to begin to examine the hypothesis that this neuroendocrine event is the result of structural or plastic changes within the neural network governing the activity of GnRH neurons. Specifically, we sought to determine whether polysialic acid neural cell adhesion molecule (PSA-NCAM), a plasma membrane-associated glycoprotein that has previously been proposed to be a marker for postnatal neuronal plasticity, was expressed within GnRH neuron containing areas of the rhesus monkey hypothalamus. The study employed male monkeys that were castrated prepubertally. Immunocytochemistry of hypothalamic tissue from four animals of pubertal age employing a monoclonal antibody (12F8) specific for PSA-NCAM revealed the presence of PSA-NCAM immunoreactivity within the region of the arcuate nucleus and median eminence of the medial basal hypothalamus (MBH) and in the region of the organum vasculosum of the lamina terminalis of the rostral hypothalamus, two areas in the monkey brain where GnRH neurons are concentrated. As expected, immunostaining for total NCAM using a polyclonal rabbit antibody to mouse total NCAM was uniformly distributed throughout hypothalamic sections containing the MBH. Double staining showed that some, though not all, GnRH cell bodies of the MBH were located within the PSA-NCAM-immunopositive region of the arcuate nucleus and the median eminence. The pattern of PSA-NCAM immunoreactivity in the MBH of three prepubertal monkeys was similar to that seen for the older animals. Western analysis of a membrane extract from the MBH of a monkey of pubertal age, employing antibody 12F8, identified a broad band of staining at the expected molecular weight for this adhesion molecule. A similar, but less intense, immunoreactive band was observed for the preoptic area. In contrast, an immunoblot of a membrane extract of cerebral cortex was only faintly positive for PSA-NCAM. Taken together, the foregoing findings are consistent with the notion that structural changes within the MBH may underlie the pubertal reinitiation of pulsatile GnRH release. Moreover, the presence of PSA-NCAM in the MBH of prepubertal monkeys suggests that the role, if any, of this molecule in the onset of sexual maturation in primates is permissive in nature.

Repetitive injections of L-glutamic acid, in contrast to those of N-methyl-D,L-aspartic acid, fail to elicit sustained hypothalamic GnRH release in the prepubertal male rhesus monkey (Macaca mulatta).

The purpose of the present study was to examine whether repetitive intravenous injections of L-glutamic acid (Glu), like those of N-methyl-D,L-aspartic acid (NMA), are able to elicit a sustained train of gonadotropin releasing hormone (GnRH) discharges from the hypothalamus of the prepubertal male monkey. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, the responsiveness of the gonadotroph of the prepubertal animals was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every h). Sequential intravenous injections of Glu (150 mg/kg BW) were administered at 3-hour intervals for 6 or 24 h. Although the first injection of this acidic amino acid elicited a robust discharge of GnRH, subsequent stimulation with Glu resulted in GnRH discharges with progressively decreasing magnitudes, and by the 9th injection Glu-induced GnRH release was abolished. Peak concentrations of circulating Glu following the 1st and 4th Glu injection were indistinguishable (3,959 +/- 437 vs. 4,139 +/- 72 nmol/ml, respectively). Interestingly, the failure of repetitive intravenous injections of Glu to sustain pulsatile GnRH release was not associated with a loss of responsiveness to NMA administration, nor was it accompanied by a corresponding decrement in Glu induced growth hormone (GH) discharges. As previously demonstrated, repetitive intravenous administration of NMA (2-5 mg/kg BW) every 3 h for 9 h sustained pulsatile GnRH secretion without decrement. A similar intermittent infusion of kainic acid (KA; 1 mg/kg BW every 3 h for 6 h), however, elicited a GnRH response that mimicked that observed in response to intermittent Glu treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the role of the N-methyl-D-aspartate (NMDA) receptor in the hypothalamic control of prolactin secretion.

To further examine the role of excitatory amino acids in the control of prolactin (PRL) secretion, the effects of administering a specific agonist and an antagonist of the N-methyl-D-aspartate (NMDA) receptor on plasma PRL concentrations were examined in the adult male rat. Animals of the Sprague-Dawley strain weighing 250-300 g were implanted with an indwelling cardiac catheter via the right jugular vein. Blood samples were collected through the catheter at 5 min intervals for 40 min, beginning 5 min before the iv administration of drug or the saline vehicle (V). Plasma PRL and luteinizing hormone (LH) concentrations were estimated using RIAs. Groups of animals (n = 5-7) received N-methyl-D,L-aspartate (NMA), D,L-2-amino-5-phosphonopentanoic acid (AP5), AP5 and NMA, norvaline (NOR), or V. The effects of administering the NMDA receptor antagonist alone were studied on two separate occasions. Injection of NMA (4.5 mg/rat) resulted in unambiguous PRL and LH discharges. Treatment with AP5 (9 mg/rat) 1 min prior to NMA administration completely blocked the LH releasing action of NMA, but did not significantly alter the discharge of PRL. Injection of AP5, alone, generally elicited a distinct and robust discharge of PRL, although plasma LH levels in these animals remained unchanged. NOR, an amino acid structurally related to AP5, administered at a dose (5.3 mg/animal) isomolar to that of AP5, was without effect on PRL and LH secretion, as was injection of V alone. These findings suggest that neuroexcitatory amino acids acting at the NMDA receptor may play a role in modulating the activity of neuronal systems that govern the release of both PRL releasing factor (PRF) and PRL inhibiting factor (PIF) into hypophysial portal blood.

The neurobiology of primate puberty.

In higher primates the protracted prepubertal phase of development is occasioned by a mechanism that suppresses pulsatile hypothalamic gonadotropin-releasing hormone (GnRH) secretion from late infancy until the onset of puberty and thereby guarantees, in the juvenile, the quiescence of the pituitary-gonadal axis. Studies from our laboratory have employed the rhesus monkey, a representative higher primate, as an experimental paradigm. GnRH release has been measured using luteinizing hormone secretion by the in situ pituitary as a bioassay for the hypothalamic hormone. The nature of the prepubertal brake on pulsatile GnRH release in the monkey has been probed using physiological, neuroanatomical and neuropharmacological approaches. Such studies have led to the view that the prepubertal hiatus in pulsatile GnRH release results from a withdrawal in late infancy of a synchronized frequency-facilitated afferent neural input to the GnRH network, which in all other respects appears to exhibit properties identical to those in the postpubertal animal. The mechanism timing the onset of puberty, i.e. that responsible for the reactivation of synchronous activity in the GnRH network, is posited to be under the control of a central neural time- or growth-tracking device.

Stimulation of gonadotropin secretion in prepubertal monkeys after hypothalamic excitation with aspartate and glutamate.

To test the hypothesis that aspartate (Asp) and glutamate (Glu) can be used to probe the functional integrity of the GnRH neuron, the dose-response relationships between i.v. administered, endogenously occurring amino acids (Asp and Glu) and GnRH release were determined in the prepubertal male monkey. GnRH release was assessed indirectly by monitoring the LH response by the pituitary, the sensitivity of which had been heightened by prior exposure to pulsatile GnRH. Four of these animals received an i.v. bolus of 0, 1.5, 4.8, 15, 48 and 150 mg/kg BW of each of the amino acids. Plasma gonadotropin and amino acid concentrations were measured immediately before and for 3 hours after administration of Asp and Glu. The 150 mg/kg dose of both amino acids resulted in a dramatic rise in plasma LH concentrations that peaked at 10 min after injection. At this dose plasma Asp and Glu levels increased 200-fold and 50-fold, respectively. No significant LH release was seen with any of the lower doses. These results indicate that i.v. administration of these acidic amino acids in prepubertal monkeys stimulates GnRH release. Based upon this observation, we hypothesize that Asp or Glu could be used to develop a clinical test of GnRH neuronal function.

The prepubertal hiatus in gonadotropin secretion in the male rhesus monkey (Macaca mulatta) does not appear to involve endogenous opioid peptide restraint of hypothalamic gonadotropin-releasing hormone release.

To examine the possibility that the prepubertal hiatus in gonadotropin secretion in primates is occasioned by an endogenous opioid peptide (EOP)-dependent suppression of pulsatile GnRH release, the ability of an EOP receptor antagonist, naloxone (NAL), to elicit GnRH release was examined indirectly in the rhesus monkey. For this purpose, six castrated male monkeys, aged 18-24 months, first received an intermitten iv infusion of GnRH (0.1 micrograms/min for 3 min every h) to enhance the responsiveness of the gonadotroph to endogenous GnRH. Acute and chronic blockade of EOP receptors with single bolus injections of NAL at three doses (0.2, 2.0, and 10 mg/kg BW) and a continuous infusion of the antagonist (2 mg/h for 36 h), respectively, failed to elicit significant increments in circulating concentrations of mean LH. In addition, changes in plasma LH concentrations during a chronic intermitten iv infusion of NAL (2 mg/kg BW every 6 h for up to 16 days) were unremarkable. Unequivocal discharges of LH, however, were observed in response to small doses of GnRH (0.3 micrograms/monkey) administered iv after all modes of NAL administration. Taken together, these findings fail to provide evidence for the view that in primates, EOPs underlie the hiatus in pulsatile GnRH release, which in these species is responsible for the quiescence of the pituitary-testicular axis during the greater part of prepubertal development.

Bilateral orchidectomy and concomitant testosterone replacement in the juvenile male rhesus monkey (Macaca mulatta) receiving an invariant intravenous gonadotropin-releasing hormone (GnRH) infusion results, as in the hypothalamus lesioned GnRH-driven adult male, in a selective hypersecretion of follicle-stimulating hormone.

The purpose of this experiment was to determine whether the testes of the juvenile male rhesus monkey, receiving an invariant intermittent iv infusion of GnRH, produce a specific FSH secretion-inhibiting hormone that exerts its action directly at the level of the pituitary gland. To this end, five male rhesus monkeys between 13-18 months of age were treated with a chronic intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h) for 10 weeks to elicit an adult-like pattern in the episodic activity of the pituitary-Leydig cell axis. Animals were then bilaterally orchidectomized, and on the day of castration testosterone replacement with testosterone-containing Silastic capsules that maintained circulating levels of the steroid at approximately 6 ng/ml was initiated. Sequential blood samples were collected before castration and at 4-5, 11-13, and 18-19 days thereafter. Removal of the testes resulted in a marked and selective hypersecretion of FSH, a response very similar to that observed previously in hypothalamus-lesioned GnRH-treated adult males. This finding indicates that it will be possible to substitute the juvenile male for the much larger hypothalamus-lesioned adult in future studies requiring a hypophysiotropic clamp preparation. Such a modification of this experimental model will facilitate an examination of the effects on FSH secretion of passive immunization with inhibin antisera and of administering pure inhibin peptides.

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content.

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.

Puberty in monkeys is triggered by chemical stimulation of the hypothalamus.

Gonadal quiescence prior to puberty in primates results from a diminished secretion of the pituitary gonadotropic hormones, follicle-stimulating hormone and luteinizing hormone, which, in turn, is occasioned by an interruption of pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus during this phase of development. A discharge of GnRH may be provoked from the hypothalamus of prepubertal monkeys, however, by an i.v. injection of N-methyl-D-aspartate (NMDA), an analog of the putative excitatory neurotransmitter, aspartate. Since this action of NMDA is blocked by the specific NMDA receptor antagonist, DL-2-amino-5-phosphonopentanoic acid, the release of GnRH is likely mediated by NMDA receptors located either on the GnRH neurons themselves or on afferents to the GnRH cells. We report here that prolonged intermittent NMDA stimulation of GnRH neurons within the hypothalamus of the juvenile monkey for 16-30 wk results, with surprising ease, in the onset of precocious puberty with full activation of the hypothalamic-pituitary-Leydig cell axis and initiation of spermatogenesis. These findings demonstrate that, in primates, the network of hypothalamic GnRH neurons, which in adulthood provides the drive to the gonadotropin-secreting cells of the anterior pituitary gland, must now 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.