17α-Estradiol (17αE2), a less-feminising enantiomer of 17β-estradiol, has been shown to prolong lifespan and improve metabolic health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor α (ERα) in mediating the effects of 17αE2 on metabolic health. However, the specific tissues and/or neuronal signalling pathways that 17αE2 acts through remain to be elucidated. ERα expression in glutamatergic and GABAergic neurons (principal excitatory and inhibitory neurons respectively) in the hypothalamus is essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one of these neuronal populations would attenuate the established beneficial metabolic effects of 17αE2 in male mice exposed to a high fat diet. To test this hypothesis we used two established brain specific ERα KO models, targeting either glutamatergic or GABAergic neurons (Vglut2/Vgat-ERαKO). We show that both of these ERα KO models exhibit a strong reduction in ERα expression in the arcuate nucleus of the hypothalamus, a control centre for metabolic regulation. Deletion of ERα from GABAergic neurons significantly diminished the effect of 17αE2 on body weight relative to controls, although these animals still show metabolic benefits with 17αE2 treatment. The response to 17αE2 was unaffected by ERα deletion in glutamatergic neurons. Our results support a benefit of 17αE2 treatment in protection against metabolic dysfunction, but these effects do not depend on exclusive ERα expression in glutamatergic and GABAergic neurons and persist when ERα expression is strongly reduced in the arcuate nucleus of the hypothalamus.

Abstract Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility in women of reproductive age, characterised by androgen excess, polycystic appearance of the ovary and irregular menstruation. PCOS is also frequently associated with metabolic abnormalities, including increased adiposity and insulin resistance. The origins of PCOS are unknown, however recent findings in animal models strongly implicate androgen signalling in the brain in the development of PCOS pathophysiology. Exposure to androgen excess, either acutely during prenatal development or chronically from a peripubertal timepoint, can drive the development of PCOS-like features in adulthood. Prenatally androgenized (PNA) mice exhibit the cardinal reproductive features of PCOS and increased luteinizing hormone (LH) pulse frequency. This phenotype is associated with increased GABAergic innervation of gonadotropin-releasing hormone (GnRH) neurons, postulated to drive elevated GnRH/LH release and downstream effects. Chronic exposure to di-hydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic PCOS-like features that are ameliorated by selective AR loss from the brain. Here, we aimed to determine whether chronic exposure to DHT drives a similar increase in LH pulsatility and elevated GABAergic innervation to GnRH neurons as seen following prenatal exposure to androgen excess. GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day (PND) 21 (N = 6-7/group). Serial tail tip blood sampling was used to measure pulsatile LH and fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. Chronic androgen excess from the peripubertal period resulted in acyclicity and increased body weight as expected. However, LH pulsatility was not different between DHT-treated females and controls. Similarly, the density of VGAT appositions to GnRH neurons was not different between groups. Therefore, the programmed changes in the GnRH neuronal network and hyperactive LH secretion that result from prenatal androgen excess are not affected by chronic DHT exposure initiated at 3 weeks of age. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

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. Abstracts 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.

GnRH neuron cell bodies are scattered throughout the basal forebrain but funnel their projections to the median eminence to release GnRH into the pituitary portal system to control fertility. Prior studies have shown that GnRH neurons located in the anterior hypothalamus send projections to the median eminence that have characteristics of both dendrites and axons. These unusual structures have been termed "dendrons." To address whether the dendron is unique to anterior hypothalamic GnRH neurons or is also a characteristic of more rostral GnRH neurons, we used viral vector‒mediated GnRH neuron‒specific tract-tracing coupled with CLARITY optical clearing. Individual rostral preoptic area GnRH neurons in female mice were identified to elaborate processes up to 4 mm in length that exhibited spines and projected all the way to the median eminence before branching into multiple short axons. The synaptic innervation patterns of distal GnRH neuron dendrons and their short axons in the vicinity of the median eminence were examined using electron microscopy. This revealed the presence of a high density of synaptic inputs to distal dendrons at the border of the median eminence. In contrast, no synapses were detected on any GnRH neuron axons. These studies demonstrate that GnRH neurons in the rostral preoptic area project dendrons to the edge of the median eminence, whereupon they branch into multiple short axons responsible for GnRH secretion. The dense synaptic innervation of these distal dendrons likely represents an efficient mechanism for controlling GnRH secretion required for fertility.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Polycystic ovarian syndrome (PCOS), the leading cause of female infertility, is associated with an increase in luteinizing hormone (LH) pulse frequency, implicating abnormal steroid hormone feedback to gonadotropin-releasing hormone (GnRH) neurons. This study investigated whether modifications in the synaptically connected neuronal network of GnRH neurons could account for this pathology. The PCOS phenotype was induced in mice following prenatal androgen (PNA) exposure. Serial blood sampling confirmed that PNA elicits increased LH pulse frequency and impaired progesterone negative feedback in adult females, mimicking the neuroendocrine abnormalities of the clinical syndrome. Imaging of GnRH neurons revealed greater dendritic spine density that correlated with increased putative GABAergic but not glutamatergic inputs in PNA mice. Mapping of steroid hormone receptor expression revealed that PNA mice had 59% fewer progesterone receptor-expressing cells in the arcuate nucleus of the hypothalamus (ARN). To address whether increased GABA innervation to GnRH neurons originates in the ARN, a viral-mediated Cre-lox approach was taken to trace the projections of ARN GABA neurons in vivo. Remarkably, projections from ARN GABAergic neurons heavily contacted and even bundled with GnRH neuron dendrites, and the density of fibers apposing GnRH neurons was even greater in PNA mice (56%). Additionally, this ARN GABA population showed significantly less colocalization with progesterone receptor in PNA animals compared with controls. Together, these data describe a robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome.