Minireview: timely ovulation: circadian regulation of the female hypothalamo-pituitary-gonadal axis

The preovulatory surge in the secretion of LH is timed by a neuroendocrine integrative mechanism that involves ovarian estradiol levels and the endogenous circadian system. Studies in female rats and hamsters have established that the clock in the hypothalamic suprachiasmatic nucleus has a preeminent role in setting the LH surge, and anatomical, physiological, and pharmacological data are revealing the responsible connections between suprachiasmatic nucleus neurons and GnRH and estradiol-receptive areas. Recent investigations show that GnRH and pituitary cells express circadian clock genes that might play a role in the release and reception of the GnRH signal. Analysis of the circadian regulation of the LH surge may provide a model for understanding how multiple neural oscillators function within other neuroendocrine axes.     

Changes in the control of gonadotrophin secretion by neurotransmitters during sexual development in rats.

Gonadotrophin hormone releasing hormone (GnRH) is the primary messenger involved in sexual maturation and the onset of puberty. The activity of these neurons are controlled by several neurotransmitters systems. The onset of puberty implies changes from a prepubertal type of gonadotrophin secretion, characterized by a low activity of GnRH neurons, to an adult pattern of secretion with phasic and synchronous activation of GnRH neurons resulting in an increase in the amplitute and frequency of GnRH pulses. Neurotransmitter systems are involved in these changes of GnRH secretion during the onset of puberty by quantitative and qualitative modifications in the effect on GnRH secretion. Serotonin (5-HT), GABA and catecholamines (CA) have qualitative differences in the effects on GnRH and LH secretion in early prepubertal than in late prepubertal and adult female rats. The administration of 5-hydroxytryptophan a precursor of serotonin (5-HT) which increases 5-HT hypothalamic levels induces GnRH and LH release in early prepubertal female rats, these effects dissapear in late prepubertal stage having an inhibitory action in adult female rats. GABAergic system also stimulates GnRH and LH secretion in early prepubertal female rats and has an inhibitory action on this axis in late prepubertal period and in adult female rats. On the contrary the inhibition of catecholamines synthesis by alpha-methyl-p-tyrosine induced an increase of LH secretion in early prepubertal female rats and inhibitory effect in late prepubertal and adult stage. These effects indicate tha CA has an inhibitory effects on GnRH-LH secretion in early prepubertal female rats changing to an stimulatory action in the late puberty and adult rats. These qualitative modifications were observed only in female rats and are probably connected with the hypothalamic differentiation into a female type of gonadotrophin control. Opiadergic and excitatory amino acid systems have quantitative differences on GnRH-LH secretion during prepubertal and peripubertal and adult stages. Opiates has an high inhibitory tone in early prepubertal rats that is decreasing during sexual maturation to reach puberty. On the contrary EAA increases its stimulatory activity on GnRH-LH secretion during sexual maturation by increasing the hypothalamic release of aspartate and glutamate, the excitatory amino acids involved in GnRH release, and the sensibility of NMDA receptors to these amino acids. In conclusion sexual maturation and the onset of puberty in the female rats involve qualitative and quantitative modifications in the effects of neurotrasmitters system on GnRH secretion.     

Regulatory role of excitatory amino acids in reproduction

Glutamate, the major excitatory amino acid (EAA) transmitter in the central nervous system, has been implicated as a critical mediator in brain function. Glutamate and its receptors are found in all key hypothalamic areas critically involved in reproduction. Administration of glutamate and its agonists can bring about LH release in animals with a steroid background. Antagonists of the ionotropic glutamate receptors inhibited LH release and abolished the steroid-induced and the preovulatory LH surge. Both NMDA and non-NMDA receptor antagonists can also inhibit pulsatile LH release in castrated animals. The preoptic area has been implicated as a primary site of action of NMDA, while non-NMDA agonists have been suggested to act primarily at the arcuate/median eminence level. While EAAs may act directly on GnRH neurons to enhance GnRH release, the majority of evidence suggests that an indirect mechanism, involving EAA activation of nitric oxide and/or catecholamines, plays a major role in the GnRH-releasing effects of EAAs. Furthermore, there is also some evidence that the tonic inhibitory effect of opioids on GnRH may also involve, at least in part, a suppression of glutamate. Finally, EAA stimulation of GnRH/LH release is markedly attenuated in middle-aged rats, suggesting that a defect in glutamate neurotransmission may underlie the attenuated LH surge observed in aging.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

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

The purpose of the present study was to further examine the hypothesis that activation of G protein-coupled receptor 54 (GPR54) signaling at the end of the juvenile phase of primate development is responsible for initiation of gonadarche and the onset of puberty. Accordingly, we determined whether repetitive iv administration of the GPR54 receptor agonist kisspeptin-10 (2 microg as a brief 1-min infusion once every hour for 48 h) to the juvenile male rhesus monkey would prematurely elicit sustained, pulsatile release of hypothalamic GnRH, the neuroendocrine trigger for gonadarche. GnRH release was monitored indirectly by measuring LH secretion from the in situ pituitary, the GnRH responsiveness of which had been heightened before the experiment with an intermittent iv infusion of synthetic GnRH. Agonadal animals (n = 4) were employed to eliminate any confounding and secondary effects of changing feedback signals from the testis. The first brief infusion of kisspeptin-10 evoked an LH discharge that mimicked those produced by GnRH priming, and this was followed by a train of similar LH discharges in response to hourly activation of GPR54 by repetitive kisspeptin-10 administration. Concomitant treatment with a GnRH receptor antagonist, acyline, abolished kisspeptin-10-induced LH release. Repetitive kisspeptin-10 administration also provided a GnRH-dependent signal to FSH secretion. These findings are consistent with the notion that, in primates, the transition from the juvenile (attenuated GnRH release) to pubertal (robust GnRH release) state is controlled by activation of GPR54 resulting from increased expression of hypothalamic KiSS-1 and release of kisspeptin in this region of the brain.     

Disruption of GnRH pulses by anti-GnRH serum and phentolamine obliterates pulsatile LH but not FSH secretion in ovariectomized rabbits

It has been hypothesized that the secretion of gonadotropins, i.e. luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is driven by a synchronized neural network ('pulse generator'). This network, regulated in part by alpha-adrenergic activity, ultimately generates bursts of hypothalamic gonadotropin-releasing hormone (GnRH) release. In this study, we used the push-pull (PP) perfusion technique in ovariectomized rabbits to investigate three aspects of the ('GnRH/gonadotropin pulse generator') hypothesis. The objectives were to determine: (1) if plasma LH and FSH pulses occur concomitantly with mediobasal hypothalamic (MBH-) GnRH pulses, (2) changes in the patterns of pulsatile LH and FSH secretion when pulsatile MBH GnRH signals are interrupted by either local immunoneutralization of GnRH or intravenous infusion of the alpha-adrenergic antagonist phentolamine (PHEN, 4 mg/kg BW), and (3) whether third cerebroventricular (3VT-) GnRH patterns reflect neuronal GnRH release from the MBH. We found that while both plasma LH and FSH patterns were pulsatile, MBH GnRH pulses were significantly coupled only with LH pulses (94% coincidence). Both the local immunoneutralization of MBH GnRH pulses and the PHEN-induced suppression of MBH GnRH pulses obliterated the pulsatile secretion of LH, but not FSH. Neither MBH GnRH nor plasma LH or plasma FSH pulses were concurrent with 3VT GnRH pulses. However, the PP perfusion of the 3VT appeared to alter the pulsatile release of MBH GnRH and pituitary LH. The results support the hypothesis that in the absence of ovarian signals, the 'pulse generator' is maintained by tonic alpha-adrenergic input and that a 'cellular unity' of MBH GnRH release (GnRH pulses) drives the gonadotrophs to secrete LH in pulses. In contrast, the pulsatile release of FSH appears to involve additional nonovarian regulatory events to those controlling LH secretion.     

Leptin stimulates the reproductive male axis in rats during sexual maturation by acting on hypothalamic excitatory amino acids.

The purpose of the present study was to determine the effect of treatment with leptin on gonadotrophin secretion and hypothalamic GnRH, excitatory and inhibitory amino acids release, in prepubertal (15 days old) and peripubertal (30 days old) male rats. Rats of both ages received a single (ip) injection of 30 microg/kg leptin 60 minutes previous to sacrifice. Serum LH was determined, and the hypothalamus dissected and incubated in Earle's medium. GnRH and amino acids release were determined in the media. LH and GnRH were measured by RIA. Amino acids were assessed by HPLC-UV detection. In the two prepubertal stages, (prepubertal and peripubertal, 15 and 30 days of age respectively) leptin increased plasmatic LH levels (p < 0.01) and hypothalamic GnRH release (p < 0.01). Glutamate (GLU) release showed an increment in leptin-treated rats (p < 0.01) at both ages, while only the 30 days old rats showed an increment of the aspartate (ASP) release. GABA secretion was not modified by leptin treatment. In conclusion, the results demonstrated that leptin stimulates the LH-GnRH axis during sexual development in male rats, increasing the secretion of both hormones. The hypothalamic excitatory amino acid neurotransmitter system appears to be involved in this change.     

The role of histamine in the neuroendocrine regulation of pituitary hormone secretion

The neurotransmitter histamine participates in the neuroendocrine regulation of pituitary hormone secretion by an indirect action at a hypothalamic level where histaminergic neurons are abundant. The effect of histamine is caused by activation of postsynaptic H1- or H2-receptors. Histamine stimulates the secretion of ACTH, beta-endorphin (mediated by CRH and AVP), alpha-MSH (mediated by dopamine and peripheral catecholamines), and PRL (mediated by dopamine, serotonin and AVP), and participates in the stress-induced release of these hormones and possibly in the suckling- and estrogen-induced PRL release. The release of GH and TSH is predominantly inhibited by histamine; however, uncertainty exists regarding its role and the hypothalamic factors involved. Histamine increases the secretion of LH in females (mediated by GnRH), and may be involved in the mediation of the estrogen-induced LH surge. AVP and oxytocin are stimulated by histamine, probably by an effect in the supraoptic and paraventricular nuclei of the hypothalamus.     

Altered neuroendocrine regulation of gonadotropin secretion in women distance runners

We tested the hypothesis that the neuroendocrine control of gonadotropin secretion is altered in certain women distance runners with secondary amenorrhea. To this end, we quantitated the frequency and amplitude of spontaneous pulsatile LH secretion during a 24-h interval in nine such women. The ability of the pituitary gland to release LH normally was assessed by administration of graded bolus doses of GnRH during the subsequent 8 h. Compared to normally menstruating women, six of nine amenorrheic distance runners had a distinct reduction in spontaneous LH pulse frequency, with one, three, six, five, four, or two pulses per 24 h (normal, 8-15 pulses/24 h). This reduction in LH pulse frequency occurred without any significant alterations in plasma concentrations of estradiol and free testosterone or 24-h integrated serum concentrations of LH, FSH, or PRL. Moreover, in long-distance runners, the capacity of the pituitary gland to release LH was normal or accentuated in response to exogenous pulses of GnRH. In the six women athletes with diminished spontaneous LH pulsatility, acute ovarian responsiveness also was normal, since serum estradiol concentrations increased normally in response to the GnRH-induced LH pulses. Although long-distance runners had significantly lower estimated percent body fat compared to control women, specific changes in pulsatile gonadotropin release did not correlate with degree of body leanness. In summary, certain long-distance runners with secondary amenorrhea or severe oligomenorrhea have unambiguously decreased spontaneous LH pulse frequency with intact pituitary responsiveness to GnRH. This neuroendocrine disturbance may be relevant to exercise-associated amenorrhea, since pulsatile LH release is a prerequisite for cyclic ovarian function. We speculate that such alterations in pulsatile LH release in exercising women reflect an adaptive response of the hypothalamic pulse generator controlling the intermittent GnRH signal to the pituitary gland. The basis for amenorrhea in the remaining runners who have normal pulsatile properties of LH release is not known.     

Neuroendocrine aspects of polycystic ovary syndrome.

A series of investigations have emphasized the heterogeneous nature of the clinical condition known as PCOS and have delineated several factors that may contribute to the hyperandrogenemia and anovulation in this condition. Currently, it remains unclear whether intrinsic abnormalities of ovarian steroidogenesis, the effects of hyperinsulinemia in augmenting LH stimulation of ovarian androgen production, and the persistent rapid frequency of LH/GnRH secretion are primary factors in all patients. Indeed, these factors may have variable roles in different patients, all of whom present with the clinical syndrome of PCOS. A consensus has emerged that abnormalities in the neuroendocrine control of GnRH secretion exist in a significant subset of patients and lead to persistent hypersecretion of LH, which seems to be an important component of the syndrome, particularly in nonobese patients. The relative frequency of primary abnormalities in the regulation of GnRH secretion versus secondary changes reflecting altered circulating concentrations of ovarian steroid remains uncertain. No clear evidence exists for an underlying neuroendocrine abnormality of GnRH regulation in all patients. The recent data showing insensitivity of the hypothalamic GnRH pulse generator to E2 progesterone feedback have suggested potential mechanisms that may explain the abnormalities of GnRH secretion seen in adolescent girls in whom the clinical syndrome of PCOS is destined to develop. Further studies are required in adolescents to establish whether GnRH regulation is impaired during puberty or whether data in adults simply reflect the long-term effects of elevated androgens, estrogens, or other hormones on the hypothalamus. Studies in carefully delineated subgroups of patients with PCOS are needed to establish these points, with a long-term goal of providing patients with improved methods of inducing ovulation and reducing hyperandrogenemia.     

Amino acid neurotransmitter release in the preoptic area of rats during the positive feedback actions of estradiol on LH release.

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of gamma-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the luteinizing hormone and prolactin responses to multiple injections of kainate, as compared to N-methyl-D,L-aspartate, in cycling rats.

We have previously reported that repetitive iv injections of NMA [N-methyl-D,L-aspartate, the mixed analog acting on the N-methyl-D-aspartate (NMDA) receptor] can induce a consistent increase in LH and PRL secretion in cycling rats, but not in lactating rats. To further explore the use of excitatory amino acids (EAAs) as tools for understanding the regulation of the neuroendocrine reproductive axis, we have examined the effects of multiple injections of kainate, an agonist to another subclass of EAA receptor, on LH and PRL secretion in cycling rats. Recent studies suggest that kainate receptors may be more abundant than NMDA receptors in the hypothalamus. Five iv injections of kainate were administered at 50-min intervals to diestrous or estrous rats. Blood samples were collected every 10 min and assayed for LH and PRL. LH, but not PRL secretion, was stimulated by this regimen of kainate treatment. Surprisingly, the LH response to kainate, unlike NMA, decreased with repetitive injections of the drug. The response to the last pulse of kainate was approximately 30-40% of the first pulse. This decline in LH responsiveness to kainate was not due to desensitization at the level of the pituitary or to refractoriness of GnRH neurons, since further stimulation of LH release could be obtained by the administration of GnRH or NMA. The mechanisms responsible for the diminishing GnRH response to kainate remain unclear. However, we speculate that it might be due to the delayed activation of inhibitory inputs to GnRH neurons or to the desensitization of kainate receptors. On the other hand, the absence of a PRL response to kainate, in contrast to the stimulatory effect of NMA, most likely reflects differences in the distribution of kainate and NMDA receptors on dopamine neurons and neurons containing PRL-releasing factors, or on extrahypothalamic afferent neuronal populations projecting to the hypothalamus. In conclusion, the effects of systemic injections of kainate on LH and PRL secretion differed from NMA in that the LH response could not be sustained with multiple injections and PRL was unresponsive to kainate stimulation.     

Photoperiodic induction in vitro: the dynamics of gonadotropin-releasing hormone release from hypothalamic explants of the Japanese quail.

The Japanese quail is a photoperiodic animal that under certain experimental conditions can respond to a single long day with a wave of LH secretion. Such a system offers an opportunity to analyze the photoneuroendocrine changes as they occur in real time, especially as all of the neural machinery (photoreceptor, clock, and GnRH system) is believed to lie within the hypothalamus. The first detectable rise in LH occurs at about hour 23 of the long day, and this single inductive event leads to prolonged LH secretion lasting for up to 2 weeks and peaking 2-4 days after the dawn of the long day. The size of the quail's hypothalamus is such that the entire structure, including both the GnRH cell bodies and the median eminence, can be cultured for some hours, and the rates of GnRH release measured therefrom. The present experiments used hypothalamic explants from quail at different times throughout the photoperiodic response, superfused them for up to 7.5 h in vitro, and measured the dynamics of GnRH release. A significant step increase of 80% in GnRH release occurred between hours 22.5 and 23 in quail that had been exposed to a long day: an equivalent change was not found in hypothalami taken from quail maintained only under short day lengths. In explants taken from quail at the peak of LH secretion (53 h after dawn of the long day), the rates of GnRH release were double those found in control quail not exposed to the long day. Explants taken 14 days after the long day, when LH secretion had subsided fully, showed no difference in GnRH release between photo-stimulated and control quail. These results suggest that photoperiodic induction involves a timed increase in GnRH release, and the rise at hour 23 is believed to represent photoperiodic induction actually taking place within the brain in vitro. They also suggest that the wave of LH secretion triggered by the single long day is, at least in part, a neuroendocrine or neural phenomenon; this confirms earlier indirect evidence to this effect.     

A role for kisspeptins in the regulation of gonadotropin secretion in the mouse

Kisspeptins are products of the KiSS-1 gene, which bind to a G protein-coupled receptor known as GPR54. Mutations or targeted disruptions in the GPR54 gene cause hypogonadotropic hypogonadism in humans and mice, suggesting that kisspeptin signaling may be important for the regulation of gonadotropin secretion. To examine the effects of kisspeptin-54 (metastin) and kisspeptin-10 (the biologically active C-terminal decapeptide) on gonadotropin secretion in the mouse, we administered the kisspeptins directly into the lateral cerebral ventricle of the brain and demonstrated that both peptides stimulate LH secretion. Further characterization of kisspeptin-54 demonstrated that it stimulated both LH and FSH secretion, at doses as low as 1 fmol; moreover, this effect was shown to be blocked by pretreatment with acyline, a potent GnRH antagonist. To learn more about the functional anatomy of kisspeptins, we mapped the distribution of KiSS-1 mRNA in the hypothalamus. We observed that KiSS-1 mRNA is expressed in areas of the hypothalamus implicated in the neuroendocrine regulation of gonadotropin secretion, including the anteroventral periventricular nucleus, the periventricular nucleus, and the arcuate nucleus. We conclude that kisspeptin-GPR54 signaling may be part of the hypothalamic circuitry that governs the hypothalamic secretion of GnRH.     

Metastin/Kisspeptin and control of estrous cycle in rats

Estrous cyclicity is controlled by a cascade of neuroendocrine events, involving the activation of the hypothalamo-pituitary-gonadal axis. Two modes of gonadotropin-releasing hormone (GnRH) are well established to regulate the estrous cycle: one is a tonic or pulse mode of secretion which is responsible for the stimulation of follicular development and steroidogenesis; the other is a surge mode, which is solely responsible for the induction of luteinizing hormone (LH) surges, eventually leading to ovulation. Metastin/kisspeptin-GPR54 signaling has been suggested to control ovarian cyclicity through regulating the two modes of GnRH release. A population of metastin/kisspeptin neurons located in the anteroventral periventricular nucleus (AVPV) is considered to trigger GnRH surge and thus to mediate the estrogen positive feedback action on GnRH release. The other hypothalamic population of metastin/kisspeptin neurons is located in the arcuate nucleus (ARC) and could be involved in generating GnRH pulses and mediating negative feedback action of estrogen on GnRH release. GnRH neurons express mRNA for GPR54, a metastin/kisspeptin receptor, and have a close association with metastin/kisspeptin neurons at the cell body and terminal level, but the precise mechanism by which this peptide regulates the two modes of GnRH release needs to be determined. Metastin/kisspeptin, therefore, is a key hypothalamic neuropeptide, which is placed immediately upstream of GnRH neurons and relays the peripheral steroidal information to GnRH neurons to control estrous cyclicity.     

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. III. A23187, cAMP, phorbol ester and DiC8-stimulated luteinizing hormone release

Dispersed estradiol-treated rat pituitary cells were used to characterize progesterone (P) modulation of luteinizing hormone (LH) secretion in response to a variety of pharmacologic secretagogues which influence cell biochemistry. Acute (less than 3 h) and chronic (24 h) exposures to P prior to secretagogue challenge respectively enhanced and inhibited Ca2+ ionophore (A23187)-stimulated and gonadotropin-releasing hormone (GnRH)-stimulated LH release in similar quantitative fashion without any effect on concurrent prolactin release. Similar responses were also noted with cholera toxin-stimulated secretion. However, when protein kinase C activators such as phorbol esters and dioctanoylglycerol were used to trigger LH release, chronic exposure to P did not inhibit, but rather enhanced, LH release. Again, P had no effect on prolactin release. 'Washout' studies indicated that chronic treatments with P would suppress LH secretion stimulated by these compounds, but only when the steroid was cleared from the cells 4 h beforehand. These studies provide further evidence that P specifically modulates gonadotroph secretory function via mechanisms which bypass GnRH receptors. Moreover, they suggest that P exerts many different actions within the gonadotroph and question the role of protein kinase C in GnRH action.     

Gonadotropin-releasing hormone secretion from hypothalamic neurons: stimulation by insulin and potentiation by leptin

Insulin and leptin are peripheral metabolic factors signaling the body needs in energy to the central nervous system. Because energy homeostasis and reproductive function are closely related phenomena, we investigated the respective roles played by insulin and leptin in the hypothalamic control of GnRH secretion. We observed that increasing circulating insulin levels, by performing hyperinsulinemic clamp studies in male mice, was associated with a significant rise in LH secretion. This effect of insulin is likely mediated at the hypothalamic level, because it was also found to stimulate the secretion and the expression of GnRH by hypothalamic neurons in culture. Leptin was found to potentiate the effect of insulin on GnRH secretion in vitro but was devoid of any effect on its own. These data represent the first evidence of direct insulin sensing by hypothalamic neurons involved in activating the neuroendocrine gonadotrope axis. They also demonstrate that these neurons can integrate different hormonal signals to modulate net hypothalamic GnRH output. We propose that such integration is an essential mechanism for the adaptation of reproductive function to changes in the metabolic status of an individual.     

Inhibition of hypothalamic GnRH secretion in the ewe by antigonadotropic decapeptide during the estrous cycle and nonbreeding season

Previous experiments from our laboratory and others have shown that the peptide antigonadotropic decapeptide (AGD) has marked inhibitory effects on luteinizing hormone (LH) secretion in rats and ewes. The first objective of this study was to determine whether AGD inhibits LH secretion by regulating hypothalamic release of gonadotropin hormone (GnRH). AGD (200 μg in 200 μL of 0.3% bovine serum albumin [BSA] saline) or vehicle was infused into the lateral ventricle of ovariectomized (OVX) ewes with hypophyseal-portal cannulae, and GnRH secretion was monitored. The frequency of GnRH and LH pulses in AGD-treated ewes was significantly decreased (p<0.05) but did not change in the control ewes. The second objective of this investigation was to evaluate changes in hypothalamic sensitivity to AGD in the ewe during the estrous cycle and nonbreeding season. During the estrous cycle, the effects of AGD on LH secretion were assessed following ovariectomy, during the metestrous, diestrous, and proestrous phases of the estrous cycle. The response to AGD during the estrous cycle was compared to its effect during the anestrous season. LH, cortisol, and prolactin (PRL) concentrations were assayed in peripheral blood samples obtained at 10-min intervals over a 6-h period prior to injection of either vehicle (200 μL of 0.3% BSA in 0.9% saline) or AGD (200 μg in 200 μL of vehicle), and for an additional 10 h following treatment. LH pulse frequency decreased after treatment with AGD (p<0.05) at all times in OVX and intact ewes compared to vehicle-treated controls. During the anestrous season, AGD treatment was more effective in inhibiting LH pulse frequency than during the breeding season (p<0.05). Furthermore, there was a significant increase (p<0.05) in mean cortisol concentrations after AGD infusion in all AGD-treated groups compared to controls independent of season or reproductive status. PRL concentrations were also increased (p<0.05) following treatment with AGD. These results suggest that inhibition of pulsatile LH release induced by AGD is modulated by alterations in frequency of hypothalamic discharges of GnRH. Furthermore, changes in the inhibitory actions of AGD may contribute to the seasonal regulation of hypothalamic GnRH secretion in the ewe.     

Gonadotrophin regulation and clinical applications of GnRH

Gonadotrophin secretion is determined by the interplay of neural and gonadal influences. The neural influence is mediated for both LH and FSH by the decapeptide GnRH which is secreted into the hypophyseal portal vessels. LH is secreted in a pulsatile fashion apparently driven by episodic release of GnRH. Unremitting exposure of the pituitary to GnRH eventually abolishes gonadotrophin secretion. In primates, as opposed to the rat, GnRH appears to have a permissive role in the regulation of gonadotrophin secretion, priming the pituitary to secrete and show both negative and positive feedback responses to oestrogen in adult females. Striking physiological changes occur from fetal life to puberty in gonadotrophin regulation. GnRH acts on surface receptors. Chemical dissection of the GnRH molecule has disclosed a structure-activity relationship, allowing the development of both antagonist and 'superagonist' analogues. The initial stage in activation of gonadotrophs by GnRH appears to be binding to and clustering--probably dimerization--of GnRH receptors. Subsequent intracellular events are not fully clarified but grounds exist to suggest the involvement of both cyclic AMP and calcium fluxes within the cell. There is strong evidence that GnRH secretion influences the number of its own receptors in various situations in the rat. The phenomenon of pulsatile GnRH release in experimental animals survives hypothalamic deafferentation. Catecholamines are probably intimately involved in the generation of GnRH pulses--which for noradrenaline poses a paradox as all noradrenergic cell bodies lie outside the MBH. LH pulse frequency can be absent or altered in various states (e.g., Kallman's syndrome, hyperprolactinaemia and exposure to opiates--exogenous or apparently endogenous). The existence of GnRH receptors in gonadal tissue has been described but it is debatable whether this is true in man. Therapeutic uses of GnRH initially was aimed at correcting hypogonadotrophic hypogonadism. Development of GnRH superagonists demonstrated desensitization and thus their paradoxical application to the areas of contraception, precocious puberty and endocrine-dependent cancers. The development of miniaturized programmable infusion pumps has made pulsatile GnRH therapy a practical prospect. It holds considerable therapeutic promise in selected cases of hypogonadotrophic hypogonadism, especially in women.