Abstract
Polycystic ovary syndrome (PCOS), the leading cause of anovulatory infertility, is associated with a hyperactive reproductive axis. The increased frequency of pulsatile luteinising hormone (LH) secretion in women with PCOS implicates upstream changes in gonadotropin-releasing hormone (GnRH) neurons, the final output neurons of the neuronal network that controls fertility. The frequency of pulsatile GnRH/LH secretion is largely determined by negative feedback effects of ovarian steroid hormones, particularly progesterone, mediated via actions onto an afferent neuronal network. Women with PCOS are less responsive to the suppressive effects of progesterone on pulsatile GnRH/LH secretion, implicating alterations in the progesterone-sensitive afferent neuronal circuits that regulate GnRH neuron activity in the pathophysiology of PCOS.
Research using a prenatally androgenised (PNA) mouse model, which recapitulates the cardinal features of clinical PCOS, suggests that hyperactive GnRH/LH secretion and downstream reproductive dysfunction may be underpinned by enhanced excitatory signalling to GnRH neurons from arcuate nucleus (ARN) GABA neurons with diminished progesterone sensitivity. The present study used the PNA mouse model of PCOS to investigate when and how a reduction in progesterone receptor (PR) expression in ARN GABA neurons arises, and the contribution of the ARN GABA-to-GnRH circuit to PCOS pathology.
Identifying the developmental timing of changes within the ARN GABA-to-GnRH circuit provides insights into their role in PCOS pathophysiology. Here, immunolabelling of PR-expressing cells in the ARN, enabled by the robust validation of a newly-generated PR antibody, revealed normal progesterone sensitivity in PNA mice at the late-pubertal timepoint of postnatal day (PND)40. This suggests that diminished progesterone sensitivity in ARN GABA neurons in PNA mice may occur as a result of the post-pubertal hyperandrogenism that occurs by PND50. However, this finding is tempered by an inability to detect the previously reported reduction in PR expression in adulthood (PND60).
A role for post-pubertal hyperandrogenism in the development of reduced progesterone sensitivity was investigated using pharmacological blockade of androgen receptor (AR) signalling. Administration of the AR antagonist, flutamide, to PNA mice has been previously reported to rescue normal GABA-to-GnRH neuron wiring, ovarian function and reproductive cyclicity. I aimed to determine whether the beneficial effects of blocking hyperandrogenism are associated with the rescue of normal PR expression in ARN GABA neurons and a slowing of pulsatile LH secretion. Several delivery methods of flutamide were trialled and optimised for anti-androgen effects in male mice. However, despite using a range of suppliers, doses, and delivery methods, a flutamide-induced rescue of fertility could not be reproduced in current cohorts of PNA mice. Therefore, the impact of blocking hyperandrogenism on PR expression in ARN GABA neurons remains to be addressed. Potential future avenues are discussed.
Chronic activation of ARN GABA neurons in healthy mice is sufficient to drive PCOS-like neuroendocrine and reproductive dysfunction, but it is unclear whether inhibiting ARN GABA neurons can ameliorate pathology in PCOS-like PNA mice. Here, the activity of the ARN GABA neuron population was chronically reduced in PNA mice using an inhibitory chemogenetic tool, which was carefully validated using in vitro brain slice electrophysiology. While PNA mice appeared to exhibit a less severe disruption of reproductive function before inhibition than previously reported, chronically reducing the activity of ARN GABA neurons in PNA mice did not significantly impact reproductive cyclicity, pulsatile LH secretion, circulating testosterone, ovarian morphology, or putative GABAergic input to GnRH neurons.
Together, these data suggest that reduced sensitivity to the suppressive effects of progesterone in ARN GABA neurons may arise as a consequence of post-pubertal androgen excess, and targeting the hyperactivity of the ARN GABA-to-GnRH circuit does not appear to ameliorate pathology in this preclinical model of PCOS. These findings suggest that there is likely an array of emerging and yet-to-be identified changes in the GnRH neuronal network that contribute to the development and maintenance of PCOS-like pathology, and highlight the need for ongoing research into the central mechanisms that underpin PCOS pathophysiology.