The Molecular Mechanism of Progesterone Receptor in Regulating Gene Expression in Mouse Granulosa Cells during Ovulation

Abstract

The process of ovulation is critical for successful fertilisation and pregnancy. Of utmost importance is the progesterone receptor (PGR), which regulates various biological processes preceding pregnancy including ovulation, oviductal oocyte/embryo transportation and embryo implantation. How PGR can achieve divergent reproductive roles is still poorly understood. This thesis aims to explore the molecular mechanisms that allow for highly specialised PGR ovulatory functions through describing the PGR cistrome and transcriptome in mouse peri-ovulatory granulosa cells when PGR is highly induced and active. In addition, the relationship between PGR and other transcription factors, especially RUNX1, as well as isoform-specific actions were also determined. As PGR acts through direct binding to the PGR response element (PRE), differences in PGR chromatin targets can influence PGR actions. Characterisation of the PGR cistrome using chromatin immunoprecipitation – sequencing (ChIP-seq) showed striking distinctions in preferential PGR targets in peri-ovulatory granulosa cells compared to the uterus. Granulosa PGR favourably interacted with transcriptionally active promoters and had few mutual chromatin targets with uterine PGR. Interestingly, motif analysis of PGR peaks identified specific patterns in the degree of PRE occupancy and the enrichment of distinct non-canonical motifs, suggesting that PGR interacts with other transcription factors in a context-specific manner. Motif analysis of PGR peaks in granulosa cells implied a number of potential protein partners such as the JUN/FOS, LRH1, and RUNX families. The physical interaction of these proteins with PGR in mouse peri-ovulatory granulosa cells was confirmed through proximity ligation assay. Among these, RUNX was a granulosa-specific factor and thus potentially important in granulosa-specific PGR roles. RUNX1 displayed context-specific chromatin binding properties as shown through RUNX1 ChIP-seq of mouse foetal and adult granulosa cells before and after the LH surge. In peri-ovulatory granulosa cells, PGR/RUNX1 interaction was specifically hCG-induced, RUNX1 shared mutual targets and non-canonical binding motifs with PGR that resulted in the regulation of mutual ovulatory genes. This indicates a close interplay between PGR and RUNX1 in granulosa cells during ovulation, likely in conjunction with other modulators. The PGR-A and PGR-B isoforms play distinct roles in different biological contexts, with PGR-A being prominent in peri-ovulatory granulosa cells. To further assess the specific roles of PGR-A and PGR-B during ovulation, transcriptomes of peri-ovulatory granulosa cells from mice lacking both isoforms (PGRKO), PGR-A (AKO) or PGR-B (BKO) were obtained through RNA-seq. More than 600 differentially expressed genes were identified in PGRKO and AKO with few identified in BKO. Mutual PGR/RUNX1 direct binding was important in the regulation of these genes. PGRKO and AKO transcriptomes shared nearly half of their genes with little similarities with the BKO transcriptome. The transcriptomic data supports the key physiological roles of PGR-A in ovulation. Altogether, this study provides the first description of the PGR cistrome, interactome and transcriptome in granulosa cells. A unique cooperation between PGR, especially PGR-A, and specific transcription factors, especially RUNX1, in a mutual transcription complex leads to the specification of PGR ovulatory action in granulosa cells. Such understanding in tissue-specific PGR actions is crucial for the development of novel contraceptives targeting ovulation.