Chris presented "SELECTIVE ABLATION OF PROGESTERONE RECEPTOR IN THE GRANULOSA CELL SHUTS DOWN OVULATION MACHINERY: PGR, A DIRECT REGULATOR OF THE EXPRESSION OF PROSTAGLANDIN SYNTHASES?" at the 2017 National Centers for Translational Research in Reproduction and Infertility (NCTRI) Research Meeting (Wednesday and Thursday, May 24th and 25th) in Natcher Auditorium at the NIH (Bethesda, MD)

Progesterone receptor (PGR), a classical nuclear receptor transcription factor, is a critical player in regulating ovulation and implantation at all levels of female reproductive axis from hypothalamus to pituitary, ovary and uterus. In the ovary, the ovulatory LH surge triggers progesterone (P4) synthesis and and induces PGR expression in the granulosa cells of preovulatory follicles. If either P4 synthesis is inhibited, PGR is antagonized or its gene (Pgr) is ablated, LH fails to induce ovulation. The ovulatory process resembles an acute inflammatory reaction with a massive influx of leukocytes to the ovary prior to ovulation, which is triggered by locally produced chemokines and cytokines. Once infiltrated into the ovary, they secrete a cohort of proteases that aid ovulatory processes. Importantly, we found that antagonism of PGR by RU486, a PGR antagonist, significantly inhibit leukocyte infiltration into the ovary (p<=0.01). We therefore hypothesized that PGR regulates the expression of pro-inflammatory genes in the granulosa cells of preovulatory ovary. To test this hypothesis, we created a novel mouse model in which Pgr gene was selectively ablated in the granulosa cells by cross-breeding a floxed Pgr mouse line and a novel transgenic mouse line (Esr2-iCre, Genesis, 2016) that expresses the Cre recombinase specifically in granulosa cells in the ovary. This double transgenic mouse (Esr2-PgrKO) was first tested for fertility and ovulatory capacity. Female Esr2-PgrKO mice were individually housed with proven males, and the numbers of litters and pups/litter were counted. No litter was produced by any of the Esr2-PgrKO mice tested (n=9), whereas normal fertility was seen in the wild type (WT) controls (6.2 pups/litter, n=9). Interestingly, the ovaries of the Esr2-PgrKO mice displayed a normal pattern of folliculogenesis and corpus luteum formation. But, the corpus lutea of the Esr2-PgrKO mice contained oocytes in them, indicating ovulatory failure. We then tested their ovulatiory capacity employing a superovulation protocol. In brief, WT and Esr2-PgrKO mice were injected with gonadotropins and the numbers of oocytes counted in the oviduct. WT littermates had a normal number of oocytes in the oviduct (25.4 oocytes/oviduct, n=6), but no oocyte was found in the Esr2-PgrKO mice (n=10), showing a complete loss of ovulatory capacity in the Esr2-PgrKO ovary. To identify genes whose expressions were afftected by the mutation, RNA-seq was conducted with the ovaries from WT (n=3) and Esr2-PgrKO mice (n=3). Among 221 genes downregulated, inflammatory genes (>70 genes) were one the most affected. Of note, the expressions of prostaglandin synthase1 (Ptgs1) and Ptgs2 were significantly lower than WT control (5.9 and 3.4 fold, respectively; p<=0.05). These findings indicate that PGR mediates the inflammatory process in the preovulatory follicle by up-regulating the expression of pro-inflmmatory genes. In silico study identified that two putative PGR binding sites in the 75kb and 55kb upstream of Ptgs2 transcription start site. A ChIP assay is underway to determine if PGR directly binds to the putative PGR binding sites and to determine the significance of the changes of Ptgs1 and Ptgs2 expression.