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.Gonadotropin-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. Hormones secreted from the ovary, in turn, provide critical feedback signals to GnRH neurons through a network of hormone-sensitive neurons. Gonadal hormone feedback directs both the firing of GnRH neurons and pulsatile release of the GnRH peptide (1).Polycystic ovarian syndrome (PCOS), the most common form of anovulatory infertility (2), is estimated to affect more than 100 million women worldwide (3). Most women diagnosed with PCOS exhibit increased LH pulse frequency and decreased FSH release, suggestive of rapid GnRH pulse frequency (4). High LH, in turn, contributes to increased androgen production from ovarian theca cells, whereas decreased FSH disrupts follicle maturation and ovulation. Elevated GnRH/LH secretion in women with PCOS is less responsive to exogenous estrogen and progesterone (P4) administration (5, 6), suggesting that steroid hormone negative feedback to GnRH neurons is impaired. In animal models, elevated androgens are associated with blunted P4 negative feedback in particular (7, 8).Although the origin of GnRH/LH hypersecretion in PCOS is unknown, impaired steroid hormone negative feedback may lie within the hormone-sensitive afferent neuronal network to GnRH neurons. Identifying the specific neuronal elements affected is challenging in women; however, discoveries can be made in animal models (9, 10). PCOS is most commonly modeled through exposure to androgens during critical periods of development (11). Women exposed to elevated prenatal androgens (PNAs) develop the cardinal reproductive and endocrine features of PCOS in adulthood (12, 13), and PNA treatment produces a PCOS-like phenotype in all mammalian species studied to date (11). In the mouse, PNA elicits many of the key neuroendocrine features of the syndrome, suggestive of impaired steroid hormone feedback to the GnRH pulse generator (14, 15), however, it remains to be determined directly whether P4 negative feedback and LH pulse frequency are modified.P4 modulation of GnRH neurons via classical progesterone receptors (PRs) is most likely transsynaptic, as GnRH neurons do not express PRs. Many P4-sensitive populations have been identified throughout the hypothalamus, including neurons expressing gamma-aminobutyric acid (GABA) (16), glutamate (17), and various neuropeptides (18, 19). Both endogenous GnRH neuron firing activity and GABAergic postsynaptic currents are increased in PNA mice (15, 20). However, the specific P4-sensitive neuronal phenotype that relays feedback signals to GnRH neurons is so far unknown. The aim of the present study was to characterize whether LH pulse frequency and P4 negative feedback are impaired in a mouse model of PCOS and to investigate what modifications exist in the GnRH neuronal network that may impair negative feedback.