Progesterone Receptor (PGR)-mediated Molecular Mechanisms On Ovulation In Mice

Progesterone plays a very important role in the reproductive activity in mammals,the major physiological function of progesterone is mediated by the progesteronereceptor (Progesterone receptor, PGR). Progesterone receptor knockout mouse modelhave confirmed the importance of PGR gene for mammalian female reproductivefunction, particularly played an irreplaceable role in ovulation. In recent years,microarray experiments screened by a large number of downstream genes of PGR, buthas so far failed to find progesterone response element in the traditional sense in thepromoter regions of these downstream genes. ADAMTS-1and Cathepsin L has beenconsidered as the key signaling molecules during wall rupture mediated by PGR gene inovarian follicle, but how the two proteins is regulated by PGR gene and what it hasplayed a role in the rupture of the follicle wall remains unclear.In this experiment, we used exogenous gonadotropins (PMSG/hCG) to inducesexually immature female Kunming mice superovulation, and detected the expression ofPGR, ADAMTS-1and Cathepsin L mRNA by quantitive real-time PCR in the ovary. Wefound that the expression of ADAMTS-1mRNA significantly increased at24h afterPMSG injection, and continued to4h after hCG injection, and further significantlyincreased at12h after hCG injection which was also the ovulation moment in mice,suggesting that ADAMTS-1plays an important role not only in the process of folliculardevelopment, but also in the ovulation process. Besides, the further rise of ADAMTS-1mRNA occurred just followed the the time when the mRNA expression of PGR reachedits maximum value, which is likely to implied that the sudden increase of ADAMTS-1mRNA expression at the ovulation time was mediated by PGR. In contrast, the mRNAexpression of Cathepsin L didn’t changed during the whole process (P>0.05), showingthat Cathepsin L mRNA mainly constitutively expressed in the ovary, and exogenousgonadotropin couldn’t change the Cathepsin L mRNA levels of the entire ovary.Further, treated mice with the progesterone receptor antagonist RU486, and detectedthe ovarian mRNA expression of genes12h after hCG injection, we found that RU486could significantly inhibit the stimulation effect of exogenous gonadotropins to themRNA expression of ADAMTS-1(P <0.05), indicating that the stimulation ofexogenous gonadotropins for ADAMTS-1mRNA at ovulation time was mediated byPGR. In addition, hypoxia-inducible factor1α (Hypoxia inducible factor1, HIF-1α) andHIF-1β known as the downstream gene of PGR in mouse ovaries, exerted the role oftranscription factors in the form of heterodimers (ie, HIF-1). Moreover, it had beenfound that HIF-1can bind directly to ADAMTS-1promoter region in human vascularendothelial cells. So we naturally assumed that, PGR indirectly regulated the expressionof ADAMTS-1by HIF-1. To test this hypothesis, we selected a human breast cancer cellline (MCF-7) for study, owing to its convenience for study and the simultaneousexpression of PGR, ADAMTS-1, HIF-1α and HIF-1β gene. We treated MCF-7cells withdifferent concentrations (10nM,100nM and1μM) of exogenous progestinmedroxyprogesterone acetate (MPA), and found that with the increase of theconcentration of MPA, the stimulation on ADAMTS-1mRNA was more obvious (P <0.05), the mRNA expression of HIF-1α, HIF-1β and its typical target gene VEGFAgradually increased, but the difference was not significant (P>0.05). On this basis,treated with different concentrations of RU486(0.1μM,1μM and10μM), and foundthat as the concentration increase, inhibition of RU486for ADAMTS-1mRNAexpression was more obvious, but not for HIF-1α, HIF-1β and VEGFA, and theexpression of VEGFA increased very significantly when RU486concentration was10μM (P <0.01). These results showed that PGR could regulate the the expression ofADAMTS-1, but not for HIF-1α, HIF-1β and VEGFA. We used cobalt chloride to mimichypoxia and to increase the HIF-1α protein concentration, found cobalt chloridetreatment can stimulate the expression of ADAMTS-1mRNA and protein, indicatingthat in MCF-7cells, HIF-1can also regulate the expression of ADAMTS-1. However,treated with MPA and cobalt chloride simultaneously, HIF-1α and HIF-1β mRNA wasnot significantly changed, but there was a significant increase in VEGFA mRNA (P <0.05), while the PGR mRNA expression was significantly inhibited (P <0.05), whileADAMTS-1mRNA is also inhibited. This showed that in MCF-7cells, ADAMTS-1expression is mainly regulated by PGR, while high concentration of HIF-1α proteinwas able to inhibit the expression of PGR, and further inhibited ADAMTS-1expression.Thus, in MCF-7cells, the regulation of PGR on ADAMTS-1was not mediated byHIF-1, at least not exactly.In summary, PGR mediated the stimulation on ADAMTS-1by exogenousgonadotropins at ovulation time in mouse ovary, and also regulated the expression ofADAMTS-1in MCF-7cells, but had no significant role in the regulation on HIF-1α and HIF-1β both of which are known downstream gene of PGR in mouse ovary.