Progesterone receptor (PGR)-mediated transcriptional networks in human ovulatory follicles: Identification of SOX9 as a novel PGR-downstream mediator [RNA-seq]

Progesterone receptor (PGR) is an essential transcription factor for successful ovulation. Clinically, PGR inhibitors have been used as emergency contraceptives due to their ability to delay or block ovulation. While much of the current understanding of PGR’s function has been derived from animal models, the specific downstream target genes and pathways PGR regulates in human ovulatory follicles remain largely unexplored. In the present study, we utilized a human granulosa cell (hGLC) model that mimics preovulatory hCG-induced increases in PGR expression and progesterone production, combined with a PGR inhibitor (RU486), to identify hCG-induced, PGR-regulated genes in human ovulatory follicles. RNA-Seq and ChIP-Seq analyses revealed numerous genes regulated by PGR in response to human chorionic gonadotropin (hCG) treatment for 12 h, which are involved in diverse biological processes, including metabolism, steroidogenesis, signaling activation, cell cycle regulation, and transcriptional control. By comparing these findings with the existing data from PgrKO mouse models, we also identified both conserved and species-specific PGR-regulated genes. Notably, SOX9 was identified as a human-specific, hCG-induced PGR-target gene in hGLCs. A further characterization study using dominant follicle samples collected before and at different times after ovulatory hCG administration from regularly cycling women demonstrated that SOX9 expression is predominantly induced in the granulosa cells of ovulatory follicles and persists in the developing corpus luteum. Functional studies showed that SOX9 knockdown by siRNA reduced progesterone production and the expression of steroidogenesis-related genes (CYP11A1 and STAR) in hGLCs. In summary, the present finding provides the first comprehensive list of preovulatory PGR-downstream genes in human granulosa cells and unveils SOX9 as a novel human-specific ovulatory transcription factor with potential roles in steroidogenesis. This foundational information not only enhances our understanding of the PGR-regulated ovulatory pathway but also provides a critical resource for developing improved strategies to manage women’s fertility.