Abstract
Clinical and preclinical evidence highlights the brain as a prime suspect in both the development and maintenance of polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility worldwide. Prenatally androgenized (PNA) models of PCOS exhibit excessive GABAergic wiring associated with PCOS-like reproductive deficits in adulthood, with aberrant brain wiring detected as early as postnatal day (P) 25, prior to disease onset. The mechanisms by which PNA exposure drives this aberrant brain wiring however, remains unknown. Microglia, the immune cells of the brain, are sculptors of neuronal wiring across development, mediating both the formation and removal of neuronal inputs, although whether microglia play a role in PCOS is yet to be defined. This PhD project aimed to understand whether microglia play an important role in PCOS pathogenesis.
This work used the PNA mouse model that recapitulates the cardinal features of PCOS and exhibits excessive GABAergic wiring onto gonadotropin-releasing hormone (GnRH) neurons as early as P25, prior to the emergence of PCOS-like features. Initial studies assessed whether the microglia population in the PNA brain is altered compared to controls within two regions of the hypothalamus implicated in fertility regulation, the rostral preoptic area (rPOA), where GnRH neurons reside, and the arcuate nucleus (ARN), the likely source of the aberrant GABA inputs. Using specific immunolabelling for the microglia-specific marker, Iba-1, microglia number and morphology associated with activation states were analyzed in PNA and control mice across developmental timepoints, including embryonic day 17.5, P0 and P25. At P0, fewer amoeboid microglia were observed in the rPOA of PNA mice. However, the greatest changes were observed at P25, with PNA mice exhibiting fewer total microglia, and specifically fewer “sculpting” microglia within the rPOA. These findings reveal time and location-specific changes in microglia number and morphology following prenatal androgen excess.
Based on these anatomical findings at P25, microglia-mediated refinement of GABAergic synaptic terminals was assessed at two developmental stages of peak synaptic refinement: P7 and P15. Using a reporter mouse line expressing GFP in microglia, specific immunolabelling was combined with confocal imaging and 3D reconstruction to assess microglia content of cleaved GABAergic synaptic terminals in PNA and control mice. PNA mice showed a significant reduction in the engulfment of GABAergic synaptic material at P15 which was restricted to the rPOA, suggesting that microglia are key mediators of androgen excess-induced changes in the wiring of GnRH neurons.