The role of nuclear progesterone receptor (PGR) in regulating gene expression, morphology and function in the ovary and oviduct during the periovulatory period.

Abstract

Ovulation requires sequential molecular events and structural remodelling in the ovarian follicle for the successful release of a mature oocyte into the oviduct. Critical to this process is progesterone receptor (PGR), a transcription factor highly yet transiently expressed in granulosa cells (GC) of preovulatory follicles and abundant in the oviduct. Progesterone receptor knockout (PRKO) mice are anovulatory, with a specific and complete defect in follicle rupture. Therefore, this model was used to examine the critical molecular and biochemical mechanisms necessary for successful ovulation. Progesterone is known to affect oviductal cells in vitro, but how PGR regulates oviductal structure and function is poorly understood. A systematic evaluation of ovarian and oviductal morphology during the periovulatory period revealed no structural defects in PRKO mice relative to heterozygous (PR+/-) littermates. However, in response to the LH surge/hCG treatment, ovulation only occurred in PR+/- ovaries, with numerous corpora lutea observed and cumulus oocyte complexes (COCs) in oviducts, while PRKO ovaries did not ovulate and showed entrapped COCs within large, luteinising follicles. Transplantation of PRKO ovaries into wild-type mice (PRWT) did not rescue the infertility phenotype. Therefore, although PGR is expressed in other tissues, ovarian PGR is essential for ovulation. Further experiments identified PGR-regulated processes at multiple levels. In whole ovaries 10 h post-hCG, inflammatory genes were disrupted in PRKO mice, including cytokines, endothelial adhesion factors, vasoconstrictors, T-cell antigens, and the prostaglandin synthase, Ptgs2. In GCs and COCs isolated 8 h post-hCG, microarray analyses identified 296 and 44 differentially expressed genes respectively between PRKO and PR+/- samples. Gene ontology analysis revealed associations with the processes of proteolysis, vascular remodelling/angiogenesis, inflammatory responses, cell adhesion, migration and invasion. The latter three processes were characterised in periovulatory COCs using in vitro assays and were shown to be transiently activated, peaking at ovulation then declining dramatically in COCs collected from the oviduct immediately post-ovulation. However, periovulatory PRKO and PR+/- COCs showed similar rates of adhesion, migration and invasion in the presence of collagen I. Upregulation of the chemokine receptor, Cxcr4, by LH/hCG via PGR in both GCs and COCs was validated by RT-PCR and immunohistochemistry. Mitochondrial membrane potential was altered in PRKO oocytes compared to PR+/- and therefore their developmental potential may be reduced. Further, a bioassay measuring retention of prostaglandin (PGE2) within the matrix of expanded COCs suggested that the matrix integrity of PRKO COCs may be compromised. Therefore, PGR in granulosa cells appears to have down-stream impacts on COCs. In oviducts, microarray analysis comparing gene expression in PRKO and PR+/- mice 8 h post-hCG, when P4 levels are high, identified 1003 PGR-regulated genes. Gene ontology analysis identified significant associations with the functions of cell adhesion, migration, invasion, chemotaxis, muscle contraction and vasoconstriction. Several genes were confirmed to be PGR-regulated by RT-PCR (Adamts1, Itga8 and Edn3) and were induced by LH/hCG. Therefore, the identification of novel gene targets for PGR regulation in the ovary and oviduct exposes several new, down-stream influences of PGR on inflammation, the COC and oviductal function, highlighting the essential role of PGR as master regulator in the ovary and oviduct during the periovulatory period.