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.GnRH | PCOS | GABA | progesterone receptor | luteinizing hormone G onadotropin-releasing hormone (GnRH) neurons, located in the hypothalamus, control fertility by driving the secretion of the gonadotrophins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) from the pituitary gland. The pulse amplitude and frequency of LH and FSH shape the sequence of events that occur at the ovary, including follicular development, gonadal steroid synthesis, and ovulation.

Gonadal steroid hormone feedback is impaired in polycystic ovarian syndrome (PCOS), a common endocrine disorder characterized by hyperandrogenism and an associated increase in LH pulse frequency. Using a prenatal androgen (PNA)-treated mouse model of PCOS, we aimed to investigate negative and positive feedback effects of estrogens on the hypothalamic-pituitary axis regulation of LH. PNA-treated mice exhibited severely disrupted estrous cycles, hyperandrogenism, significantly reduced fertility, and altered ovarian morphology. To assess the negative feedback effects of estrogens, LH was measured before and after ovariectomy and after estradiol (E2) administration. Compared with controls, PNA-treated mice exhibited a blunted postcastration rise in LH (P < .001) and an absence of LH suppression after E2 administration. To assess E2-positive feedback, control and PNA-treated GnRH-green fluorescent protein transgenic mice were subjected to a standard ovariectomy with E2-replacement regimen, and both plasma and perfusion-fixed brains were collected at the time of the expected GnRH/LH surge. Immunocytochemistry and confocal imaging of cFos and green fluorescent protein were used to assess GnRH neuron activation and spine density. In the surged group, both control and PNA-treated mice had significantly increased LH and cFos activation in GnRH neurons (P < .05) compared with nonsurged animals. Spine density was quantified in cFos-positive and -negative GnRH neurons to examine whether there was an increase in spine density in cFos-expressing GnRH neurons of surged mice as expected. A significant increase in spine density in cFos-expressing GnRH neurons was evident in control animals; however, no significant increase was observed in the PNA-treated mice because spine density was elevated across all GnRH neurons. These data support that PNA treatment results in a PCOS-like phenotype that includes impaired E2-negative feedback. Additionally, although E2-positive feedback capability is retained in PNA mice, elevated GnRH neuron spine density may reflect altered synaptic regulation.

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.

Androgen excess is a hallmark of polycystic ovary syndrome (PCOS), a prevalent yet poorly understood endocrine disorder. Evidence from women and preclinical animal models suggests that elevated perinatal androgens can elicit PCOS onset in adulthood, implying androgen actions in both PCOS ontogeny and adult pathophysiology. Prenatally androgenized (PNA) mice exhibit a robust increase of progesterone-sensitive GABAergic inputs to gonadotropin-releasing hormone (GnRH) neurons implicated in the pathogenesis of PCOS. It is unclear when altered GABAergic wiring develops in the brain, and whether these central abnormalities are dependent upon adult androgen excess. Using GnRH-GFP-transgenic mice, we determined that increased GABA input to GnRH neurons occurs prior to androgen excess and the manifestation of reproductive impairments in PNA mice. These data suggest that brain circuit abnormalities precede the postpubertal development of PCOS traits. Despite the apparent developmental programming of circuit abnormalities, long-term blockade of androgen receptor signaling from early adulthood rescued normal GABAergic wiring onto GnRH neurons, improved ovarian morphology, and restored reproductive cycles in PNA mice. Therefore, androgen excess maintains changes in female brain wiring linked to PCOS features and the blockade of androgen receptor signaling reverses both the central and peripheral PNA-induced PCOS phenotype.