Stimulation Of Gonadotropin-releasing Hormone Release Research Articles

Neuroendocrine mechanisms underlying estrogen positive feedback and the LH surge.

A fundamental principle in reproductive neuroendocrinology is sex steroid feedback: steroid hormones secreted by the gonads circulate back to the brain to regulate the neural circuits governing the reproductive neuroendocrine axis. These regulatory feedback loops ultimately act to modulate gonadotropin-releasing hormone (GnRH) secretion, thereby affecting gonadotropin secretion from the anterior pituitary. In females, rising estradiol (E2) during the middle of the menstrual (or estrous) cycle paradoxically “switch” from being inhibitory on GnRH secretion (“negative feedback”) to stimulating GnRH release (“positive feedback”), resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation. While upstream neural afferents of GnRH neurons, including kisspeptin neurons in the rostral hypothalamus, are proposed as critical loci of E2 feedback action, the underlying mechanisms governing the shift between E2 negative and positive feedback are still poorly understood. Indeed, the precise cell targets, neural signaling factors and receptors, hormonal pathways, and molecular mechanisms by which ovarian-derived E2 indirectly stimulates GnRH surge secretion remain incompletely known. In many species, there is also a circadian component to the LH surge, restricting its occurrence to specific times of day, but how the circadian clock interacts with endocrine signals to ultimately time LH surge generation also remains a major gap in knowledge. Here, we focus on classic and recent data from rodent models and discuss the consensus knowledge of the neural players, including kisspeptin, the suprachiasmatic nucleus, and glia, as well as endocrine players, including estradiol and progesterone, in the complex regulation and generation of E2-induced LH surges in females.

Hypothalamic Astrocyte Development and Physiology for Neuroprogesterone Induction of the Luteinizing Hormone Surge.

Neural circuits in female rats sequentially exposed to estradiol and progesterone underlie so-called estrogen positive feedback that induce the surge release of pituitary luteinizing hormone (LH) leading to ovulation and luteinization of the corpus hemorrhagicum. It is now well-established that gonadotropin releasing hormone (GnRH) neurons express neither the reproductively critical estrogen receptor-α (ERα) nor classical progesterone receptor (PGR). Estradiol from developing ovarian follicles acts on ERα-expressing kisspeptin neurons in the rostral periventricular region of the third ventricle (RP3V) to induce PGR expression, and kisspeptin release. Circulating estradiol levels that induce positive feedback also induce neuroprogesterone (neuroP) synthesis in hypothalamic astrocytes. This local neuroP acts on kisspeptin neurons that express PGR to augment kisspeptin expression and release needed to stimulate GnRH release, triggering the LH surge. In vitro and in vivo studies demonstrate that neuroP signaling in kisspeptin neurons occurs through membrane PGR activation of Src family kinase (Src). This signaling cascade has been also implicated in PGR signaling in the arcuate nucleus of the hypothalamus, suggesting that Src may be a common mode of membrane PGR signaling. Sexual maturation requires that signaling between neuroP synthesizing astrocytes, kisspeptin and GnRH neurons be established. Prior to puberty, estradiol does not facilitate the synthesis of neuroP in hypothalamic astrocytes. During pubertal development, levels of membrane ERα increase in astrocytes coincident with an increase of PKA phosphorylation needed for neuroP synthesis. Currently, it is not clear whether these developmental changes occur in existing astrocytes or are due to a new population of astrocytes born during puberty. However, strong evidence suggests that it is the former. Blocking new cell addition during puberty attenuates the LH surge. Together these results demonstrate the importance of pubertal maturation involving hypothalamic astrocytes, estradiol-induced neuroP synthesis and membrane-initiated progesterone signaling for the CNS control of ovulation and reproduction.

Role of Kisspeptin and NKB in Puberty in Nonhuman Primates: Sex Differences.

To understand the roles of kisspeptin and neurokinin B (NKB) in puberty and sex differences in their involvement, we conducted a series of experiments measuring the release of gonadotropin-releasing hormone (GnRH) and kisspeptin in the median eminence of the hypothalamus in male and female monkeys throughout sexual development. Results indicate that kisspeptin-10 and the NKB agonist, senktide, stimulated GnRH release in males and females at the prepubertal and pubertal stages, but females are much more sensitive to kisspeptin signaling than males. Moreover, throughout the progress of puberty, major remodeling of kisspeptin and NKB signaling pathways for the regulation of GnRH release takes place. In females during puberty, reciprocal pathways (i.e., kisspeptin signaling mediated through NKB neurons and NKB signaling mediated through kisspeptin neurons) are established, to provide powerful and flexible mechanisms for GnRH neurosecretory activity necessary for complex female reproductive function in adulthood. By contrast, during puberty in males, reciprocal pathways are consolidated to a simpler kisspeptin-dominant signaling pathway. Nevertheless, in primates, both kisspeptin and NKB signaling are contributing factors for the pubertal increase in GnRH release, rather than initiating puberty.

Kisspeptin and Neurokinin B Signaling Network Underlies the Pubertal Increase in GnRH Release in Female Rhesus Monkeys.

Loss-of-function or inactivating mutations in the genes coding for kisspeptin and its receptor (KISS1R) or neurokinin B (NKB) and the NKB receptor (NK3R) in humans result in a delay in or the absence of puberty. However, precise mechanisms of kisspeptin and NKB signaling in the regulation of the pubertal increase in gonadotropin-releasing hormone (GnRH) release in primates are unknown. In this study, we conducted a series of experiments infusing agonists and antagonists of kisspeptin and NKB into the stalk-median eminence, where GnRH, kisspeptin, and NKB neuroterminal fibers are concentrated, and measuring GnRH release in prepubertal and pubertal female rhesus monkeys. Results indicate that (1) similar to those previously reported for GnRH stimulation by the KISS1R agonist (i.e., human kisspeptin-10), the NK3R agonist senktide stimulated GnRH release in a dose-responsive manner in both prepubertal and pubertal monkeys; (2) the senktide-induced GnRH release was blocked in the presence of the KISS1R antagonist peptide 234 in pubertal but not prepubertal monkeys; and (3) the kisspeptin-induced GnRH release was blocked in the presence of the NK3R antagonist SB222200 in the pubertal but not prepubertal monkeys. These results are interpreted to mean that although, in prepubertal female monkeys, kisspeptin and NKB signaling to GnRH release is independent, in pubertal female monkeys, a reciprocal signaling mechanism between kisspeptin and NKB neurons is established. We speculate that this cooperative mechanism by the kisspeptin and NKB network underlies the pubertal increase in GnRH release in female monkeys.

Timeline: kisspeptins

Kisspeptins are peptides encoded by KISS1. The gene was first discovered in 1996, in Hershey, PA, USA, and was subsequently named after Hershey's chocolate kisses. The peptide product of KISS1 is a 145 aminoacid peptide that is cleaved into four shorter peptides: kisspeptin-54, kisspeptin-14, kisspeptin-13, and kisspeptin-10 (the numbers denote how many aminoacids in each). All these isoforms activate the kisspeptin receptor. Kisspeptins produced in the hypothalamus stimulate neurons in the hypothalamus that secrete gonadotropin-releasing hormone (GnRH), which subsequently stimulates release of gonadotropins and sex steroid hormones. Kisspeptin has been shown to have a crucial role in puberty. Inactivating mutations of the kisspeptin receptor or KISS1 gene lead to pubertal failure due to hypogonadotrophic hypogonadism in mice and in people. Conversely, activating mutations of the kisspeptin receptor in people lead to central precocious puberty. Exogenous kisspeptin stimulates gonadotropin release in all animal species studied and in people. This effect is mediated predominantly by stimulating GnRH release (because pretreatment with a GnRH antagonist completely blocks the ability of kisspeptin to stimulate gonadotropin release). Recent evidence also suggests that the kisspeptin system is important in the generation of GnRH pulses, which are crucial for normal fertility. In a subpopulation of neurons in the hypothalamic arcuate nucleus, kisspeptin colocalises with two other hormones known to affect GnRH release, namely neurokinin B (NKB) and dynorphin (DYN). This group of cells is often referred to as the KNDy neurons. Increasing data suggest that KNDy neurons have a key role in mediation of pulsatile GnRH release. Evidence thus far suggests that kisspeptin has exciting therapeutic potential in people—eg, acute use of kisspeptin for infertility could stimulate reproductive hormone release more naturally than by conventional methods. Data for human beings also suggest that kisspeptin might stimulate pulses of luteinising hormone, indicating its therapeutic potential for the treatment of women with anovulatory infertility. Chronic use of high doses of kisspeptin causes desensitisation and reduces secretion of gonadotropins. Hence, longacting forms of kisspeptin are being developed to treat hormone-sensitive cancers, with promising early results in people.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from −2 to −0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1μmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHβ and GnRH-R in the pituitary. In the case of the GnRH and LHβ genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

Kisspeptin/G protein-coupled receptor-54 system as an essential gatekeeper of pubertal development

Puberty is the end-point of a complex series of developmental events, defined by the dynamic interaction between genetic factors and environmental cues, ultimately leading to the attainment of reproductive capacity. Kisspeptins, products of the KISS1 gene, were originally identified as metastasis suppressor peptides with the ability to bind G protein-coupled receptors (GPR54). In 2003, loss-of-function mutations of the GPR54 gene were found in patients with hypogonadotropic hypogonadism. This finding triggered study of the role of the kisspeptin/GPR54 system as an essential gatekeeper of control of reproduction and pubertal development. Kisspeptins are very potent elicitors of gonadotropin secretion, primarily through stimulation of gonadotropin-releasing hormone release. KISS1 also functions as an essential integrator for peripheral inputs, including gonadal steroids and nutritional signals, and for controlling GnRH and gonadotropin secretion. Whether the kisspeptin/GPR54 system is the trigger for puberty onset and/or it operates as integrator and effector of up-stream regulatory factors warrants further investigation.

Inactivating KISS1 Mutation and Hypogonadotropic Hypogonadism

Gonadotropin-releasing hormone (GnRH) is the central regulator of gonadotropins, which stimulate gonadal function. Hypothalamic neurons that produce kisspeptin and neurokinin B stimulate GnRH release. Inactivating mutations in the genes encoding the human kisspeptin receptor (KISS1R, formerly called GPR54), neurokinin B (TAC3), and the neurokinin B receptor (TACR3) result in pubertal failure. However, human kisspeptin loss-of-function mutations have not been described, and contradictory findings have been reported in Kiss1-knockout mice. We describe an inactivating mutation in KISS1 in a large consanguineous family that results in failure of pubertal progression, indicating that functional kisspeptin is important for puberty and reproduction in humans. (Funded by the Scientific and Technological Research Council of Turkey [TÜBİTAK] and others.)

Central Precocious Puberty due to Hypothalamic Hamartomas Correlates with Anatomic Features but Not with Expression of GnRH, TGFα, or KISS1

Background/Aims: Hypothalamic hamartomas are the most common identifiable cause of central precocious puberty (CPP). Hamartoma characteristics proposed to be associated with CPP include specific anatomic features and expression of molecules such as gonadotropin-releasing hormone (GnRH), transforming growth factor α (TGFα), and GRM1A, which encodes the type 1 metabotropic glutamate receptor α isoform. We sought to determine whether hamartomas that cause CPP could be distinguished by anatomic features, expression of these molecules, or expression of KISS1, whose products signal through the receptor GPR54 to stimulate GnRH release. Methods: Clinical records and radiologic images were reviewed for 18 patients who underwent hamartoma resection for intractable seizures; 7 had precocious puberty. Resected tissue was examined for expression of GnRH, GnRH receptor (GnRHR), TGFα, KISS1, GPR54, and GRM1A. Results: Hypothalamic hamartomas associated with CPP were more likely to contact the infundibulum or tuber cinereum and were larger than hamartomas not associated with CPP. GnRH, TGFα, and GnRHR were expressed by all hamartomas studied. Expression of KISS1, GPR54, and GRM1A did not differ significantly between hamartomas associated and not associated with CPP. Conclusion: Anatomic features rather than expression patterns of candidate molecules distinguish hypothalamic hamartomas that are associated with CPP from those that are not.

Differential Regulation of Gonadotropin-Releasing Hormone Neuron Activity and Membrane Properties by Acutely Applied Estradiol: Dependence on Dose and Estrogen Receptor Subtype

Gonadotropin-releasing hormone (GnRH) neurons are critical to controlling fertility. In vivo, estradiol can inhibit or stimulate GnRH release depending on concentration and physiological state. We examined rapid, nongenomic effects of estradiol. Whole-cell recordings were made of GnRH neurons in brain slices from ovariectomized mice with ionotropic GABA and glutamate receptors blocked. Estradiol was bath applied and measurements completed within 15 min. Estradiol from high physiological (preovulatory) concentrations (100 pm) to 100 nm enhanced action potential firing, reduced afterhyperpolarizing potential (AHP) and increased slow afterdepolarization amplitudes (ADP), and reduced I(AHP) and enhanced I(ADP). The reduction of I(AHP) was occluded by previous blockade of calcium-activated potassium channels. These effects were mimicked by an estrogen receptor (ER) beta-specific agonist and were blocked by the classical receptor antagonist ICI182780. ERalpha or GPR30 agonists had no effect. The acute stimulatory effect of high physiological estradiol on firing rate was dependent on signaling via protein kinase A. In contrast, low physiological levels of estradiol (10 pm) did not affect intrinsic properties. Without blockade of ionotropic GABA and glutamate receptors, however, 10 pm estradiol reduced firing of GnRH neurons; this was mimicked by an ERalpha agonist. ERalpha agonists reduced the frequency of GABA transmission to GnRH neurons; GABA can excite to these cells. In contrast, ERbeta agonists increased GABA transmission and postsynaptic response. These data suggest rapid intrinsic and network modulation of GnRH neurons by estradiol is dependent on both dose and receptor subtype. In cooperation with genomic actions, nongenomic effects may play a role in feedback regulation of GnRH secretion.

Kisspeptin Can Stimulate Gonadotropin-Releasing Hormone (GnRH) Release by a Direct Action at GnRH Nerve Terminals

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.

Hypocretin/Orexin: A Molecular Link Between Sleep, Energy Regulation, and Pleasure

Hypocretin (Hcrt) is a neurotransmitter of the dorsal and lateral hypothalamus that regulates sleep, appetite, and energy consumption. Recent evidence indicates that it is also involved in pleasure/reward-seeking. Mutation of the Hcrt-receptor gene causes narcolepsy in canines, and Hcrt knockout mice exhibit narcolepsy-like symptoms. Human narcoleptics do not commonly have mutations in the ligand or receptor but do have degeneration of Hcrt-containing neurons, possibly through an autoimmune mechanism. When Hcrt neurons degenerate in mice, hypophagia and obesity are observed, symptoms that are also present in some human narcoleptics. This article reviews the recent literature with regard to the many functions of this single molecule. The authors suggest that eating habits and impulsivity may be topics worth exploring in the evaluation of narcoleptic patients.

17-Beta-Estradiol Directly Regulates the Expression of Adrenergic Receptors and Kisspeptin/GPR54 System in GT1-7 GnRH Neurons

Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17β-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) α₁A-, α₁B-, α₁D-, α₂A-, α₂C-, and β₁-AR, and showed for the first time that mRNAs for α₂B-, β₂- and β₃-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17β-estradiol, α₁B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while α₁B- and α₁D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1–10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.

Testosterone response to GnRH in a female songbird varies with stage of reproduction: implications for adult behaviour and maternal effects

Summary Despite considerable recent interest in plasma and yolk testosterone (T) in female birds, relatively little is known about environmental regulation of female T, individual variation in female T or the relationship between plasma and yolk T. In breeding females of a wild population of dark‐eyed junco (Junco hyemalis), we assessed variation in the responsiveness of the hypothalamo‐pituitary‐gonadal (HPG) axis to a challenge with gonadotropin‐releasing hormone (GnRH) by measuring circulating T before and 30 min after a standardized injection of GnRH. We asked whether response to challenge varied seasonally or with stage of reproduction and whether it was repeatable within individuals or related to T deposited in eggs. Initial and post‐challenge levels of T were measured using enzyme immunoassay. In a subset of these females, luteinising hormone (LH) was measured using radioimmunoassay (RIA). In addition, eggs were collected from nests of 15 females that had received a GnRH challenge, and yolk T was measured using RIA. During most of the breeding season, plasma T did not increase in response to GnRH. GnRH consistently caused increases in plasma T only during the 7 days before oviposition, when females were rapidly depositing yolk in eggs but had not yet begun to lay them. Among a small subset of females we found a positive correlation between the magnitude of this increase in plasma T in response to GnRH during egg development and the amount of T deposited in the yolk of eggs collected at a later time. These results suggest that ovarian response to GnRH‐induced increases in LH is greatest when females are actively depositing yolk into eggs. Factors that stimulate the release of GnRH during egg formation may result in higher levels of plasma T which could influence adult female behaviour. Further, because plasma T was correlated with later yolk T, factors that stimulate GnRH release may also lead to higher levels of yolk T potentially influencing offspring development or behaviour.

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

Orexin A, a recently discovered hypothalamic peptide, has been shown to have a stimulatory effect on release of gonadotropin-releasing hormone (GnRH) from rat hypothalamic explants in vitro. However, it is presently unclear whether in vivo this effect is mediated directly at the level of the GnRH neuron, or via multiple afferent neuronal connections. Therefore, in the present study, we investigated the direct action of orexin A on GnRH neurons using the immortalized GnRH-secreting GT1-7 hypothalamic cells. Orexin-1 receptor (OX1R) expression was detected in GT1-7 cells by RT-PCR and Western blot. Results showed that 0.1–1 nM orexin A, when administered in culture media for 4 h, can significantly stimulate GnRH mRNA expression in GT1-7 cells (p < 0.05). Administration of 1 µM OX1R antagonist, SB-334867, completely blocked the observed orexin A responses in these cells, indicating that orexin A stimulation of GnRH neurons is specifically through OX1R. Moreover, 0.1 nM orexin A stimulated GnRH release after 30–45 min. To examine possible signal transduction pathways involved in mediating these effects, a MEK inhibitor (UO-126), PKC inhibitor (calphostin C), and PKA inhibitor (H-89), were used, with each blocking orexin A-induced GnRH transcription and release from immortalized cells. Collectively, our results show that orexin A is capable of directly stimulating GnRH transcription and neuropeptide release from these immortalized hypothalamic neurons, and that the effects of orexin A appear to be mediated via the OX1R, coupled with activation of the PKC-, MAPK- and PKA-signaling pathways. It is suggested that the stimulatory effect of orexin A on GnRH transcription and release may also occur directly at the level of GnRH neurons in vivo.

The roles of kisspeptins and G protein-coupled receptor-54 in pubertal development

This review summarizes the experimental data demonstrating the fundamental role of kisspeptins and their G protein-coupled receptor GPR54 in the control of reproduction, with special emphasis on their function at puberty. Kisspeptins, products of the KiSS-1 gene, were originally identified as metastasis suppressor peptides with the ability to bind G protein-coupled receptor GPR54. In late 2003, loss-of-function mutations of the GPR54 gene were found in patients suffering from hypogonadotropic hypogonadism. This finding kicked off the analysis of the role of the KiSS-1/GPR54 system in the control of reproduction. Kisspeptins are very potent elicitors of gonadotropin secretion, primarily through stimulation of gonadotropin-releasing hormone release. Enhanced expression of KiSS-1 and GPR54 genes, as well as increased GPR54 signaling, are detected at the hypothalamus during pubertal development, and activation of GPR54 by administration of kisspeptin is sufficient to induce precocious activation of the gonadotropic axis in immature rodents and monkeys. Hypothalamic KiSS-1 also functions as an essential integrator for peripheral inputs, including gonadal steroids and nutritional signals, controlling gonadotropin-releasing hormone and gonadotropin secretion. Kisspeptins and their putative receptor, GPR54, have recently emerged as indispensable factors for pubertal development, with a key role as gatekeepers of gonadotropin-releasing hormone release neurons and, hence, of reproductive function.

Interaction Between Norepinephrine, Oxytocin, and Nitric Oxide in the Stimulation of Gonadotropin‐Releasing Hormone Release From Proestrous Rat Basal Hypothalamus Explants

In the proestrous female rat, norepinephrine, oxytocin and nitric oxide (NO) all participate in the regulation of the preovulatory gonadotropin-releasing hormone (GnRH) surge. Recent studies from our laboratory have demonstrated that oxytocin induces dose-dependent release of GnRH from proestrous basal hypothalamus explants. The present studies were undertaken to determine whether norepinephrine could also stimulate GnRH release from similar explants, to identify the receptors responsible for this effect and to investigate interactions between norepinephrine, oxytocin and NO. Norepinephrine significantly stimulated GnRH release from proestrous basal hypothalamus explants, and coadministration of the alpha(1)-adrenergic antagonist prazosin blocked this effect. Combined administration of oxytocin and norepinephrine stimulated significantly more GnRH release than either drug alone, and this stimulation was blocked by inhibition of NO synthase, or by an oxytocin receptor antagonist. NO production was measured from the same samples using a modified Griess reaction. Oxytocin, but not norepinephrine, significantly increased NO production, as did norepinephrine and oxytocin in combination. Oxytocin receptor antagonist administration attenuated the stimulation of NO production by norepinephrine/oxytocin. These results demonstrate for the first time that oxytocin and norepinephrine dramatically stimulate GnRH release from basal hypothalamus explants harvested on the afternoon of proestrus, and indicate that this involves oxytocin receptor and NO-dependent mechanisms.

Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants

Gonadotropin-releasing hormone (GnRH) is critical for the initiation and maintenance of reproduction in vertebrates. Information regarding GnRH release is abundant in mammals, but absent in poikilothermic tetrapods. In this study, we established a novel GnRH enzyme immunoassay (EIA) to measure GnRH release over time from hypothalamic explants isolated from mature field-caught and commercially-acquired male bullfrogs, Rana catesbeiana. Hypothalamic explants from rats were used as a positive control to test the sensitivity and accuracy of our EIA and to ensure our in vitro system could detect GnRH pulses. Prominent GnRH pulses were present in the majority (9/10) of rat hypothalamic explants, but absent in all (17/17) of the commercial bullfrogs and the majority (5/8) of field-caught bullfrogs. In three cases where GnRH pulses were observed in field-caught bullfrogs, there was only one pulse during the 2-h incubation period; high-frequency pulses similar to those observed in rats were not observed. Veratridine, which opens voltage-gated sodium channels, stimulated GnRH release in all explants cultured in the presence of Ca 2+, demonstrating explant viability. The levels of both spontaneous and veratridine-induced GnRH release were significantly higher in field-caught than commercial bullfrogs. This study demonstrated, for the first time, the temporal pattern of GnRH release in a poikilothermic tetrapod. Further, our results suggest the levels and patterns of GnRH output in bullfrogs are subject to the dynamic regulation by physiological and environmental cues.

Central nervous system receptors involved in mediating the inhibitory action of neuropeptide Y on luteinizing hormone secretion in the male rhesus monkey (Macaca mulatta).

An earlier finding that gonadotropin-releasing hormone (GnRH) secretion may be triggered prematurely in the juvenile male monkey by central administration of 1229U91, a Y1 receptor antagonist, contributed to our current hypothesis that neuropeptide Y (NPY) is a major component of the brake that holds pulsatile GnRH release in check during prepubertal development in primates. However, 1229U91 is also a Y4 receptor agonist, and the present study was conducted to further examine the role of the Y1 receptor in mediating the putative inhibitory action of NPY on GnRH release. Agonadal juvenile and postpubertal male monkeys were implanted with i.v. and i.c.v. cannulae to gain continuous access to the venous and cerebroventricular circulations without sedation. Luteinizing hormone (LH) secretion was measured to provide an indirect index of GnRH release. The specific Y1 antagonists, VD-11 (476 microg; n = 4) and isopropyl 3-chloro-5-[1-((6-[2-(5-ethyl-4-methyl-1,3-thiazol-2-yl)ethyl]-4-morpholin-4-ylpyridin-2-yl)amino)ethyl]phenylcarbamate (Compound A, 300 microg; n = 4), did not mimic the stimulatory action of 1229U91 on GnRH secretion in the juvenile male monkey. Additionally, neither NPY (200 microg; n = 2), a general Y receptor agonist, nor rPP (100 microg; n = 4), a Y4 agonist, mimicked the action of 1229U91 in stimulating GnRH release. Moreover, previous exposure of the hypothalamus of juvenile monkeys (n = 5) to NPY (660 microg) failed to block 1229U91-induced (200 microg) GnRH release. However, the action of NPY (364 microg) in inhibiting GnRH release postpubertally was attenuated by 1229U91 (300 microg). We conclude that, although the action of exogenous NPY to suppress GnRH release from the postpubertal hypothalamus appears to be mediated, at least in part, by the Y1 receptor, the existence of a Y1 receptor pathway inhibitory to GnRH release in the prepubertal hypothalamus remains to be substantiated.

Astrocytes and brain function: implications for reproduction.

Recent evidence suggests that astrocytes have important neuroregulatory functions in addition to their classic functions of support and segregation of neurons. These newly revealed functions include regulation of neuron communication, neurosecretion, and synaptic plasticity. Although these actions occur throughout the brain, this review will focus on astrocyte-neuron interactions in the hypothalamus, particularly with respect to their potential contribution to the regulation of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Hypothalamic astrocytes have been documented to release a variety of neuroactive factors, including transforming growth factors-alpha and -beta, insulin-like growth factor-1, prostaglandin E2, and the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnane-20-one. Each of these factors has been shown to stimulate GnRH release, and receptors for each factor have been documented on GnRH neurons. Astrocytes have also been implicated in the regulation of synaptic plasticity in key areas of the hypothalamus that control GnRH release, an effect achieved by extension and retraction of glial processes (i.e., glial ensheathment). Through this mechanism, the number of synapses on GnRH neurons and GnRH regulatory neurons can potentially be modulated, thereby influencing the activation state of GnRH neurons. The steroid hormone 17beta-estradiol, which triggers the GnRH and luteinizing hormone surge, has been shown to induce the astrocyte-regulated changes in hypothalamic synaptic plasticity, as well as enhance formation and release of the astrocyte neuroactive factors, thereby providing another potential mechanistic layer for astrocyte regulation of GnRH release. As a whole, these studies provide new insights into the diversity of astrocytes and their potential role in reproductive neuroendocrine function.