Expression And Function Of Membrane Progestin Receptor β In Pig Oocytes Matured In Vitro

Membrane progestin receptors(mPRs)were the members of a protein family isolated recently.MPRs included three isoforms,which were named mPRα,βandγrespectively.According to prior studies,they play roles in regulating the oocytes maturation of lower vertebrates.However,as far as I know,there were no any data reported about their expression and function in the procedure of in vitro maturation of mammalian oocyte.In order to detect whether the mPRβmRNA could express in pig oocyte-cumulus complexes(COCs)matured in vitro,RT-PCR and DNA sequencing were used.The PCR product with the expected size of 1447bp was obtained using pig mPRβspecial primers.The BLAST result showed that the sequence obtained in the experiment was a part of mPRβ,which confirmed the expression of mPRβmRNA in pig COCs.The dynamic rule of mPRβmRNA was also analyzed by semi-quantative RT-PCR.The COCs matured for 0h,12h, 20h,36h,44h respectively were divided into five groups.The result of semi-quantative RT-PCR showed that the mPRβexpression levels of 20h and 36h groups were significantly higher than that of 0h group(P＜0.05).There was no significant difference among the other groups.When the LH or FSH excluded from the maturation medium,the expression of mPRβwas not affected, which indicated the expression of mPRβwas not regulated by gonadotrophin.In order to analyze the expression of mPRβin the translational level,the anti- mPRβserum was prepared.Subsequent western blotting analysis indicated that mPRβprotein was not detected at 0h during IVM,however,there was a high level at 20h and thereafter decreased to a low level at 44h.There were significant differences among the three treatment groups(P＜0.05).The immunofluorescence analysis showed that mPRβwas located at the plasma membrane of cumulus cells.However,in mouse embryo fibroblasts,mPRβwas located at the endoplasm reticulum.When 10%anti-mPRβserum was added into the maturation medium,the cumulus expansion of pig COCs was significantly impaired(P＜0.05).The result of bioinformatics analysis implied that the protein might be involved in cumulus expansion through exocytosis.In conclusion,mPRβcan be expressed with a special pattern in pig COCs. The subcellular localization of mPRβis varied from tissue to tissue and the protein is related to cumulus expansion.