Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome.

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.

Systemic metabolic benefits of 17α-estradiol are not exclusively mediated by ERα in glutamatergic or GABAergic neurons.

17α-Estradiol (17αE2), a less-feminising enantiomer of 17ß-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.

Synaptic Innervation of the GnRH Neuron Distal Dendron in Female Mice.

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.

Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.

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.

Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone 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.