Gonadotropin-releasing Hormone Neuronal Activity Research Articles

Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice

AbstractAlthough the participation of sex hormones and sex hormone-responsive neurons in aggressive behavior has been extensively studied, the role of other systems within the hypothalamus-pituitary-gonadal (HPG) axis remains elusive. Here we assessed how the gonadotropin-releasing hormone (GnRH) and kisspeptin systems are impacted by escalated aggression in male mice. We used a combination of social isolation and aggression training (IST) to exacerbate mice’s aggressive behavior. Next, low-aggressive (group-housed, GH) and highly aggressive (IST) mice were compared regarding neuronal activity in the target populations and hormonal levels, using immunohistochemistry and ELISA, respectively. Finally, we used pharmacological and viral approaches to manipulate neuropeptide signaling and expression, subsequently evaluating its effects on behavior. IST mice exhibited enhanced aggressive behavior compared to GH controls, which was accompanied by elevated neuronal activity in GnRH neurons and arcuate nucleus kisspeptin neurons. Remarkably, IST mice presented an increased number of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV). In addition, IST mice exhibited elevated levels of luteinizing hormone (LH) in serum. Accordingly, activation and blockade of GnRH receptors (GnRHR) exacerbated and reduced aggression, respectively. Surprisingly, kisspeptin had intricate effects on aggression, i.e., viral ablation of AVPV-kisspeptin neurons impaired the training-induced rise in aggressive behavior whereas kisspeptin itself strongly reduced aggression in IST mice. Our results indicate that IST enhances aggressive behavior in male mice by exacerbating HPG-axis activity. Particularly, increased GnRH neuron activity and GnRHR signaling were found to underlie aggression whereas the relationship with kisspeptin remains puzzling.

Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

Serotonin stimulates female preoptic area kisspeptin neurons via activation of type 2 serotonin receptors in mice.

The neuroendocrine control of ovulation is orchestrated by neuronal circuits that ultimately drive the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus to trigger the preovulatory surge in luteinizing hormone (LH) secretion. While estrogen feedback signals are determinant in triggering activation of GnRH neurons, through stimulation of afferent kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3VKISS1 neurons), many neuropeptidergic and classical neurotransmitter systems have been shown to regulate the LH surge. Among these, several lines of evidence indicate that the monoamine neurotransmitter serotonin (5-HT) has an excitatory, permissive, influence over the generation of the surge, via activation of type 2 5-HT (5-HT2) receptors. The mechanisms through which this occurs, however, are not well understood. We hypothesized that 5-HT exerts its influence on the surge by stimulating RP3VKISS1 neurons in a 5-HT2 receptor-dependent manner. We tested this using kisspeptin neuron-specific calcium imaging and electrophysiology in brain slices obtained from male and female mice. We show that exogenous 5-HT reversibly increases the activity of the majority of RP3VKISS1 neurons. This effect is more prominent in females than in males, is likely mediated directly at RP3VKISS1 neurons and requires activation of 5-HT2 receptors. The functional impact of 5-HT on RP3VKISS1 neurons, however, does not significantly vary during the estrous cycle. Taken together, these data suggest that 5-HT2 receptor-mediated stimulation of RP3VKISS1 neuron activity might be involved in mediating the influence of 5-HT on the preovulatory LH surge.

FRI283 Fragile X Gene Mutations Alter GnRH Neuron And Ovarian Innervation With Consequences On Reproductive Function

Abstract Disclosure: P.A. Villa: None. N. Lainez: None. C.R. Jonak: None. S. Berlin: None. I. Ethell: None. D. Coss: None. Mutations in the Fragile X messenger ribonucleoprotein 1 gene (FMR1) cause Fragile X syndrome, the most common cause of inherited mental disability, attributed to the loss of the Fragile X messenger ribonucleoprotein (FMRP). FMR1 mutations also comprise a majority of the genetic causes of premature ovarian failure (POF) in women, mechanisms of which are unknown. We utilized the Fmr1KO mouse model to determine the role of the FMR1 gene in the reproductive system in females. Similarly to what occurs in women harboring the mutation, our data demonstrate that Fmr1KO female mice experienced premature reproductive senescence, where reproductive function stopped at p163; in contrast, wild type controls stopped reproducing at p263. Since POF in women can occur due to either an insufficient pool of primordial follicles or early depletion of follicles, we quantified primordial follicles in 3 week old females and determined there was no difference between KO and controls. We then determined that Fmr1KO females had a higher number of corpora lutea at 8 weeks of age and gave birth to larger litters, suggesting that KO females recruit more follicles in each estrus cycle. Given that follicle stimulating hormone (FSH) and luteinizing hormone (LH) are essential in ovarian function, we analyzed gonadotropin levels in Fmr1KO females and determined they have higher serum LH and FSH. Inhibin b, progesterone, and testosterone levels were also higher, suggesting that high gonadotropins do not result from the lack of feedback. Ovariectomy determined that the hypothalamus drives high LH in KO females, while increased FSH is dependent on the ovaries. Increased vasculature in the corpora lutea and higher sympathetic innervation of developing follicles may increase steroid hormone production and drive increased serum FSH levels. To investigate the hypothalamic role in increased LH, we determined that a majority of gonadotropin-releasing hormone (GnRH) neurons express FMRP, which also have a higher number of GABAergic synapses in Fmr1KO females. Given that GABA is excitatory to GnRH neurons, alterations in afferent synaptic input can affect GnRH and LH secretion. LH pulsatile analysis determined higher LH pulse frequency in both unmodified and ovariectomized females, indicating an increase in GnRH neuron activity. In summary, our results reveal hypothalamic and ovarian contributions to the reproductive phenotype of Fragile X mutation carriers that may lead to early menopause. Presentation: Friday, June 16, 2023

Review: Early and late determinants of puberty in ruminants and the role of nutrition.

This article reviews the scientific literature on puberty with a focus on ruminants and draws inference, where appropriate, from recent findings in transgenic mouse models and human pathology. Early genetic determinants of puberty have been discovered in humans suffering from hypogonadotropic hypogonadism or central precocious puberty. Transgenic mouse models selected on the basis of the causative defective genes helped in discovering the cellular and molecular mechanisms involved. Most of the genes found are involved in the development of neuroendocrine networks during embryo development and early postnatal life. Notwithstanding that the development of neuroendocrine networks takes place early in puberty, a delay or acceleration in the development of Gonadotropin Releasing Hormone (GnRH) neurons has an impact on puberty onset inducing a delay or an advance, respectively. Among the genes discovered in humans and laboratory models, only a few of them displayed polymorphisms associated with advanced sexual maturity, but also marbling, growth traits and callipygian conformation. This could be related to the fact that rather than puberty onset, most research monitored sexual maturity. Sexual maturity occurs after puberty onset and involves factors regulating the maturation of gonads and in the expression of sexual behaviour. The association with growth and metabolic traits is not surprising since nutrition is the major environmental factor that will act on late genetic determinants of puberty onset. However, a recent hypothesis emerged suggesting that it is the postnatal activation of the GnRH neuronal network that induces the acceleration of growth and weight gain. Hence, nutritional factors need the activation of GnRH neurons first before acting on late genetic determinants. Moreover, nutritional factors can also affect the epigenetic landscape of parental gamete's genome with the consequence of specific methylation of genes involved in GnRH neuron development in the embryo. Season is another important regulator of puberty onset in seasonal small ruminants and appears to involve the same mechanisms that are involved in seasonal transition in adults. The social environment is also an underestimated factor affecting puberty onset in domestic ruminants, most research studies focused on olfactory cues, but the genetic basis has not heretofore been adequately tackled by the scientific community. Additionally, there is some evidence to suggest transgenerational effects exist, in that nutritional and social cues to which parents were exposed, could affect the epigenetic landscape of parental gametes resulting in the epigenetic regulation of early genetic determinants of puberty onset in their offspring.

Acetylcholine regulation of GnRH neuronal activity: A circuit in the medial septum.

In vertebrates, gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility by regulating gonadotrophs in the anterior pituitary. While it is known that acetylcholine (ACh) influences GnRH secretion, whether the effect is direct or indirect, and the specific ACh receptor (AChR) subtype(s) involved remain unclear. Here, we determined 1) whether ACh can modulate GnRH cellular activity and 2) a source of ACh afferents contacting GnRH neurons. Calcium imaging was used to assay GnRH neuronal activity. With GABAergic and glutamatergic transmission blocked, subtype-specific AChR agonists and antagonists were applied to identify direct regulation of GnRH neurons. ACh and nicotine caused a rise in calcium that declined gradually back to baseline after 5-6min. This response was mimicked by an alpha3-specific agonist. In contrast, muscarine inhibited GnRH calcium oscillations, and blocking M2 and M4 together prevented this inhibition. Labeling for choline acetyltransferase (ChAT) and GnRH revealed ChAT fibers contacting GnRH neurons, primarily in the medial septum (MS), and in greater number in females than males. ChAT positive cells in the MS are known to express p75NGFRs. Labeling for p75NGFR, ChAT and GnRH indicated that ChAT fibers contacting GnRH cells originate from cholinergic cells within these same rostral areas. Together, these results indicate that cholinergic cells in septal areas can directly regulate GnRH neurons.

Suppression of neurotransmission on gonadotropin-releasing hormone neurons in letrozole-induced polycystic ovary syndrome: A mouse model.

Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine disorder in reproductive-age women, characterized by the accretion of small cystic follicles in the ovary associated with chronic anovulation and overproduction of androgens. Ovarian function in all mammals is controlled by gonadotropin-releasing hormone (GnRH) neurons, which are the central regulator of the hypothalamic-pituitary-gonadal (HPG) axis. However, the impact on the neurotransmitter system regulating GnRH neuronal function in the letrozole-induced PCOS mouse model remains unclear. In this study, we compared the response of various neurotransmitters and neurosteroids regulating GnRH neuronal activities between letrozole-induced PCOS and normal mice via electrophysiological techniques. Response to neurotransmitter systems like GABAergic, glutamatergic and kisspeptinergic were suppressed in letrozole-fed compared to normal mice. In addition, neurosteroids tetrahydrodeoxycorticosterone (THDOC) and 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) mediated response on GnRH neurons were significantly smaller on letrozole-fed mice compared to normal mice. Furthermore, we also found that letrozole-fed mice showed irregularity in the estrous cycle, increased body weight, and anovulation in female mice. These findings suggest that PCOS is an endocrine disorder that may directly affect the neurotransmitter system regulating GnRH neuronal activity at the hypothalamic level and impact reproductive physiology.

Epilepsy-associated increase in gonadotropin-releasing hormone neuron firing in diestrous female mice is independent of chronic seizure burden severity

Reproductive endocrine disorders are common comorbidities of temporal lobe epilepsy (TLE). Our previous studies using the intrahippocampal kainic acid (IHKA) mouse model of TLE demonstrated that many females show prolonged estrous cycles and hypothalamic gonadotropin-releasing hormone (GnRH) neurons exhibit elevated firing during diestrus. However, it is unknown whether the degree of change in GnRH neuron activity is dependent on epilepsy severity. Here, we used 24/7 in vivo electroencephalography (EEG) and in vitro electrophysiological recordings in acute brain slices to assess GnRH neuron firing in relation to chronic seizure burden in diestrous female mice at two months after IHKA injection. We found that percentage of time in seizure activity in the 24 h prior to slice preparation is an accurate proxy of overall seizure burden. Firing rates of GnRH neurons from EEG-recorded IHKA mice were increased in comparison to controls, but no relationships were found between GnRH neuron firing and seizure burden measured in vivo. The independence of GnRH neuron firing rate in relation to seizure burden was unaffected by GnRH neuron soma location or estrous cycle length. Furthermore, GnRH neuron firing rates were not yet different from control values when measured 1 month after injection, when epileptogenesis is already complete in IHKA mice. These findings indicate that the severity of epilepsy and the degree of downstream disruption to GnRH neuron activity are independent, suggesting that susceptibility to reproductive endocrine comorbidities is driven by other risk factors.

Unusual suspects: Glial cells in fertility regulation and their suspected role in polycystic ovary syndrome.

Gonadotropin‐releasing‐hormone (GnRH) neurons sitting within the hypothalamus control the production of gametes and sex steroids by the gonads, therefore ensuring survival of species. As orchestrators of reproductive function, GnRH neurons integrate information from external and internal cues. This occurs through an extensively studied neuronal network known as the “GnRH neuronal network.” However, the brain is not simply composed of neurons. Evidence suggests a role for glial cells in controlling GnRH neuron activity, secretion and fertility outcomes, although numerous questions remain. Glial cells have historically been seen as support cells for neurons. This idea has been challenged by the discovery that some neurological diseases originate from glial dysfunction. The prevalence of infertility disorders is increasing worldwide, with one in four couples being affected; therefore, it remains essential to understand the mechanisms by which the brain controls fertility. The “GnRH glial network” could be a major player in infertility disorders and represent a potential therapeutic target. In polycystic ovary syndrome (PCOS), the most common infertility disorder of reproductive aged women worldwide, the brain is considered a prime suspect. Recent studies have demonstrated pathological neuronal wiring of the “GnRH neuronal network” in PCOS‐like animal models. However, the role of the “GnRH glial network” remains to be elucidated. In this review, I aim to propose glial cells as unusual suspects in infertility disorders such as PCOS. In the first part, I state our current knowledge about the role of glia in the regulation of GnRH neurons and fertility. In the second part, based on our recent findings, I discuss how glial cells could be implicated in PCOS pathology.

The Dopamine D4 Receptor Regulates Gonadotropin-Releasing Hormone Neuron Excitability in Male Mice.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both luteinizing hormone (LH) and Follicle stimulation hormone (FSH) from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on Gαi/o protein coupling, while the D1/5R-mediated excitation required Gαs protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

Prenatal Androgen Treatment Does Not Alter the Firing Activity of Hypothalamic Arcuate Kisspeptin Neurons in Female Mice

Neuroendocrine control of reproduction is disrupted in many individuals with polycystic ovary syndrome (PCOS), who present with increased luteinizing hormone (LH), and presumably gonadotropin-releasing hormone (GnRH), release frequency, and high androgen levels. Prenatal androgenization (PNA) recapitulates these phenotypes in primates and rodents. Female offspring of mice injected with dihydrotestosterone (DHT) on gestational days 16–18 exhibit disrupted estrous cyclicity, increased LH and testosterone, and increased GnRH neuron firing rate as adults. PNA also alters the developmental trajectory of GnRH neuron firing rates, markedly blunting the prepubertal peak in firing that occurs in three-week (3wk)-old controls. GnRH neurons do not express detectable androgen receptors and are thus probably not the direct target of DHT. Rather, PNA likely alters GnRH neuronal activity by modulating upstream neurons, such as hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B (gene Tac2), and dynorphin, also known as KNDy neurons. We hypothesized PNA treatment changes firing rates of KNDy neurons in a similar age-dependent manner as GnRH neurons. We conducted targeted extracellular recordings (0.5–2 h) of Tac2-identified KNDy neurons from control and PNA mice at 3wks of age and in adulthood. About half of neurons were quiescent (<0.005 Hz). Long-term firing rates of active cells varied, suggestive of episodic activity, but were not different among groups. Short-term burst firing was also similar. We thus reject the hypothesis that PNA alters the firing rate of KNDy neurons. This does not preclude altered neurosecretory output of KNDy neurons, involvement of other neuronal populations, or in vivo networks as critical drivers of altered GnRH firing rates in PNA mice.

A role for glial fibrillary acidic protein (GFAP)-expressing cells in the regulation of gonadotropin-releasing hormone (GnRH) but not arcuate kisspeptin neuron output in male mice

GnRH neurons are the final central neural output regulating fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (KNDy neurons) are considered the main regulator of GnRH output. GnRH and KNDy neurons are surrounded by astrocytes, which can modulate neuronal activity and communicate over distances. Prostaglandin E2 (PGE2), synthesized primarily by astrocytes, increases GnRH neuron activity and downstream pituitary release of luteinizing hormone (LH). We hypothesized that glial fibrillary acidic protein (GFAP)-expressing astrocytes play a role in regulating GnRH and/or KNDy neuron activity and LH release. We used adeno-associated viruses to target designer receptors exclusively activated by designer drugs (DREADDs) to GFAP-expressing cells to activate Gq- or Gi-mediated signaling. Activating Gq signaling in the preoptic area, near GnRH neurons, but not in the arcuate, increases LH release in vivo and GnRH firing in vitro via a mechanism in part dependent upon PGE2. These data suggest that astrocytes can activate GnRH/LH release in a manner independent of KNDy neurons.

Anti-Müllerian Hormone, Growth Hormone, and Insulin-Like Growth Factor 1 Modulate the Migratory and Secretory Patterns of GnRH Neurons.

Anti-Müllerian hormone (AMH) is secreted by Sertoli or granulosa cells. Recent evidence suggests that AMH may play a role in the pathogenesis of hypogonadotropic hypogonadism (HH) and that its serum levels could help to discriminate HH from delayed puberty. Moreover, the growth hormone (GH)/insulin-like growth factor 1 (IGF1) system may be involved in the function of gonadotropin-releasing hormone (GnRH) neurons, as delayed puberty is commonly found in patients with GH deficiency (GHD) or with Laron syndrome, a genetic form of GH resistance. The comprehension of the stimuli enhancing the migration and secretory activity of GnRH neurons might shed light on the causes of delay of puberty or HH. With these premises, we aimed to better clarify the role of the AMH, GH, and IGF1 on GnRH neuron migration and GnRH secretion, by taking advantage of previously established models of immature (GN11 cell line) and mature (GT1-7 cell line) GnRH neurons. Expression of Amhr, Ghr, and Igf1r genes was confirmed in both cell lines. Cells were then incubated with increasing concentrations of AMH (1.5–150 ng/mL), GH (3–1000 ng/mL), or IGF1 (1.5–150 ng/mL). All hormones were able to support GN11 cell chemomigration. AMH, GH, and IGF1 significantly stimulated GnRH secretion by GT1-7 cells after a 90-min incubation. To the best of our knowledge, this is the first study investigating the direct effects of GH and IGF1 in GnRH neuron migration and of GH in the GnRH secreting pattern. Taken together with previous basic and clinical studies, these findings may provide explanatory mechanisms for data, suggesting that AMH and the GH-IGF1 system play a role in HH or the onset of puberty.

Time-restricted feeding improves the reproductive function of female mice via liver fibroblast growth factor 21.

BackgroundThere has been a significant increase, to epidemic levels, of obese and overweight women of reproductive age, causing impairments to reproductive health. Time‐restricted feeding (TRF) including isocaloric intake has shown to be preventive of obesity‐related disorders. However, its therapeutic ability to improve the reproductive function of female remains largely unknown.MethodsHere, we investigated the ability of TRF to improve the reproductive function in wild‐type and liver‐specific FGF21 knockout female mice. To study fertility, a continuous and a short‐term fertility test, gonadotropin releasing‐hormone (GnRH), and Kisspeptin test were performed. Immortalized GnRH neuron was used to examine the direct role of liver fibroblast growth factor 21 (FGF21) on GnRH secretion.ResultsWe found that TRF rescues female mice from bodyweight gain and glucose intolerance, as well as ovarian follicle loss and dysfunction of estrus cyclicity induced by high‐fat diet. Furthermore, the beneficial effects of the TRF regimen on the reproductive performance were also observed in mice fed both chow and high‐fat diet. However, those beneficial effects of TRF on metabolism and reproduction were absent in liver‐specific FGF21 knockout mice. In vitro, FGF21 directly acted on GnRH neurons to modulate GnRH secretion via extracellular regulated protein kinases (ERK1/2) pathway.ConclusionsOverall, time‐restricted feeding improves the reproductive function of female mice and liver FGF21 signaling plays a key role in GnRH neuron activity in female mice.

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice.

SummaryEpidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Melatonin Suppresses the Kainate Receptor-Mediated Excitation on Gonadotropin-Releasing Hormone Neurons in Female and Male Prepubertal Mice.

Melatonin, a pineal gland secretion, is an amphiphilic neurohormone involved in the biological and physiologic regulation of bodily functions. Numerous studies have shown the effects of melatonin on the release of gonadotropins and their actions at one or several levels of the hypothalamic–pituitary–gonadal axis. However, direct melatonin action on gonadotropin-releasing hormone (GnRH) neurons and its mechanism of action remain unclear. Here, plasma melatonin levels were measured and the effect of melatonin on GnRH neurons was assessed using brain slice patch clamp techniques. The plasma melatonin levels in prepubertal mice were higher than those in the adults. Melatonin itself did not change the firing activity of GnRH neurons. Interestingly, the kainate receptor-mediated responses but not the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartic acid (NMDA)-induced responses were suppressed by melatonin in both the voltage clamp and current clamp modes. The inhibitory effects of the kainate-induced response by melatonin tended to increase with higher melatonin concentrations and persisted in the presence of tetrodotoxin, a voltage-sensitive Na+ channel blocker, or luzindole, a non-selective melatonin receptor antagonist. However, the response was completely abolished by pretreatment with pertussis toxin. These results suggest that melatonin can regulate GnRH neuronal activities in prepubertal mice by partially suppressing the excitatory signaling mediated by kainate receptors through pertussis toxin-sensitive G-protein-coupled receptors.

Ovarian Androgens Maintain High GnRH Neuron Firing Rate in Adult Prenatally-Androgenized Female Mice.

Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens, and disrupted cycles, but before puberty, GnRH neuron activity is reduced in PNA mice compared with controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of green fluorescent protein (GFP)-identified GnRH neurons were made 5 to 7 days after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX + DHT). In 3-week-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In adult PNA mice, however, OVX decreased GnRH neuron firing rate, and DHT restored firing rate to sham-operated levels. In contrast to the differential effects of ovarian feedback on GnRH neuron firing rate, serum LH increased after OVX in both control and PNA mice and was not altered by DHT. Pituitary gene expression largely reflected changes expected with OVX, although in PNA but not control mice, DHT treatment increased Lhb expression. These results suggest prenatal androgen exposure programs marked changes in GnRH neuron regulation by homeostatic steroid feedback. PNA lowers GnRH neuron activity in low-steroid states (before puberty, OVX), and renders activity in adulthood dependent upon ongoing exposure to elevated ovarian androgens.

The Role of the Brain in the Pathogenesis and Physiology of Polycystic Ovary Syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder, affecting at least 10% of women of reproductive age. PCOS is typically characterized by the presence of at least two of the three cardinal features of hyperandrogenemia (high circulating androgen levels), oligo- or anovulation, and cystic ovaries. Hyperandrogenemia increases the severity of the condition and is driven by increased luteinizing hormone (LH) pulse secretion from the pituitary. Indeed, PCOS women display both elevated mean LH levels, as well as an elevated frequency of LH pulsatile secretion. The abnormally high LH pulse frequency, reflective of a hyperactive gonadotropin-releasing hormone (GnRH) neural circuit, suggests a neuroendocrine basis to either the etiology or phenotype of PCOS. Several studies in preclinical animal models of PCOS have demonstrated alterations in GnRH neurons and their upstream afferent neuronal circuits. Some rodent PCOS models have demonstrated an increase in GnRH neuron activity that correlates with an increase in stimulatory GABAergic innervation and postsynaptic currents onto GnRH neurons. Additional studies have identified robust increases in hypothalamic levels of kisspeptin, another potent stimulator of GnRH neurons. This review outlines the different brain and neuroendocrine changes in the reproductive axis observed in PCOS animal models, discusses how they might contribute to either the etiology or adult phenotype of PCOS, and considers parallel findings in PCOS women.

Modulation of Gonadotropin-Releasing Hormone Neuron Activity and Secretion in Mice by Non-peptide Neurotransmitters, Gasotransmitters, and Gliotransmitters

Gonadotropin-releasing hormone (GnRH) neuron activity and GnRH secretion are essential for fertility in mammals. Here, I review findings from mouse studies on the direct modulation of GnRH neuron activity and GnRH secretion by non-peptide neurotransmitters (GABA, glutamate, dopamine, serotonin, norepinephrine, epinephrine, histamine, ATP, adenosine, and acetylcholine), gasotransmitters (nitric oxide and carbon monoxide), and gliotransmitters (prostaglandin E2 and possibly GABA, glutamate, and ATP). These neurotransmitters, gasotransmitters, and gliotransmitters have been shown to directly modulate activity and/or GnRH secretion in GnRH neurons in vivo or ex vivo (brain slices), from postnatal through adult mice, or in embryonic or immortalized mouse GnRH neurons. However, except for GABA, nitric oxide, and prostaglandin E2, which appear to be essential for normal GnRH neuron activity, GnRH secretion, and fertility in males and/or females, the biological significance of their direct modulation of GnRH neuron activity and/or GnRH secretion in the central regulation of reproduction remains largely unknown and requires further exploration.

SUN-LB083 Functional Role of Arcuate Nucleus NPY/AgRP Neurons in the GnRH Circuit Regulating LH Secretion

Homeostatic processes like metabolism and reproduction are tightly regulated by neural circuits, and any disruption of these neural circuits causes energy imbalance and infertility, respectively. Furthermore, in conditions of energy imbalance, such as anorexia or obesity, there is also reproductive dysfunction. This causal relationship between metabolism and reproduction, strongly suggests an interdependence of these neuronal networks. In order to better understand the mechanisms underlying infertility caused by metabolic imbalance, it is important to identify the specific circuitry governing the coordination of energy balance and reproductive function. Neuropeptide Y (NPY) / Agouti related peptide (AgRP) expressing neurons in the arcuate nucleus (ARN) are likely to play an important role as they are crucial in regulating food intake and also affect gonadotropin-releasing hormone (GnRH) neuronal activity and luteinizing hormone (LH) secretion. However, the precise impact of the ARN NPY/AgRP circuit on GnRH neuron activity and fertility remains unclear. To assess this, we used the stimulatory DREADD in AgRP-Cre mice to specifically activate ARN NPY/AgRP neurons and measured pulsatile LH secretion, as a readout of GnRH neuronal activity, in conscious male and female mice. We found that activation of NPY/AgRP neurons by injection of the DREADD ligand, clozapine-N-oxide (CNO), decreased post-castration LH pulse secretion in both sexes, suggesting a slowing of GnRH pulse generation. To map the specific circuit associated with slowing of LH secretion following ARN NPY/AgRP neuron activation, region specific activation of NPY/AgRP fibres was achieved with optogenetics. Selective optogenetic stimulation of NPY/AgRP terminals surrounding GnRH neuron cell bodies in the rostral preoptic area (rPOA) was capable of decreasing GnRH neuron activity in ex vivo slices and significantly decreased LH pulsatility in conscious animals, indicating a functional ARN NPY/AgRP to GnRH neuron circuit capable of slowing pulse generation. Chemogenetic and optogenetic activation of ARN NPY/AgRP neurons was also able to significantly reduce LH pulse frequency in a prenatally androgenised (PNA) model of polycystic ovary syndrome that exhibits high LH pulse frequency, thereby demonstrating therapeutic potential. However, chronic activation of ARN NPY/AgRP neurons with CNO delivery in drinking water over 2 weeks did not ameliorate the PCOS-like reproductive pathology in PNA mice. Together, these data show that activation of ARN NPY/AgRP neurons can slow GnRH/LH pulse generation, potentially through a direct circuit to GnRH neurons in the rPOA. These findings identify a specific mechanism by which ARN NPY/AgRP neurons mediate nutritional infertility and highlight the therapeutic potential of modulating ARN NPY/AgRP activity in infertile conditions associated with high GnRH/ LH pulsatility. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.