(351) Androgen and Glucocorticoid Receptor Involvement in Ovarian Progesterone Receptor Action During Ovulation

The process of ovulation is critical for successful fertilisation and pregnancy, and is therefore essential for female reproduction. Ovulation is driven by the transcription factor progesterone receptor (PGR), which regulates genes that are crucial for oocyte maturation and follicle rupture. PGR is part of the steroid receptor family, which also includes androgen (AR) and glucocorticoid (GR) receptors that share a common canonical DNA binding sequence (nuclear receptor response element – NRE). The roles of AR and GR in ovulation, however, remain elusive. Through qPCR, Western blot and immunofluorescence we showed that all three steroid receptors were present in granulosa cells during mid-ovulation, where PGR largely exerts influence in mediating ovulatory gene transcription. To elucidate the relationship between AR, GR and PGR in the ovary, we characterised and contrasted the chromatin binding profiles of PGR, AR and GR using ChIP-seq in granulosa cells from mice undergoing in vivo hormone stimulation for ovulation. More than half of all AR and GR chromatin binding was constitutive (i.e. unaffected by ovulatory LH-stimulus), and within close proximity (< 1 kb) to transcription start sites. In response to ovulatory hormones, PGR and ~20% of GR gained novel binding sites, whereas AR binding was largely repressed. Integration of the AR, GR and PGR cistromes showed that two-thirds of PGR binding sites were co-bound by AR and/or GR, indicating substantial interaction between all three steroid receptors. Importantly, PGR and LH-induced GR co-binding significantly affected LH- and PGR-dependent ovulatory gene induction. Interestingly, AR showed the strongest binding preference for the canonical NRE sequence, while PGR and GR favoured motifs that correspond to other transcription factor families, including AP-1, CEBP and RUNX. PGR recruited to pre-accessible, constitutively AR/GR-bound chromatin sites had a more modest contribution to ovulatory gene regulation. Unexpectedly, ATAC-seq and ChIP-seq on PGR knockout mice showed that PGR was required to enable LH-induced GR binding and chromatin accessibility to target promoters. Our results indicate a novel way through which PGR activates ovulatory genes, which involves chromatin remodelling and GR recruitment to target promoters, that distinguishes hormone response in the ovary from that in other tissue contexts. These findings shed further light on molecular mechanisms that are crucial to ovulation and female fertility.