Estrogen And Estrogen Receptor Were Associated With Oocytes Maturation

Object:In this study, by measuring estradiol levels of serum and follicularfluid on oocyte retrieval day as well as the estrogen-β receptor mRNA of cumuluscells investigate the relationship between the estrogen concentration offollicular fluid and estrogen-β receptor mRNA expression of cumulus cells; toinvestigate the correlation of oocyte maturation and estrogen-β receptor mRNAexpression in cumulus cells by measuring the estrogen-β receptor mRNAexpression in different stages of oocytes. At the same time by observing thelocation and expression of G-protein-coupled receptor30(GPR30) in mouseoocytes with different developing stages as well as the different expressionbetween in vivo matured oocytes and in vitro matured oocytes to find out therole of GPR30on mouse oocyte maturation and differential role on oocytesmaturated in vivo and in vitro. Try to verity the feasibility of controllingoocytes development and apoptosis by regulation of GPR30expression which canbe affected by adjusting the dosage of estrogen under vitro environment,provide a basis theoretical for that oocytes maintenance、growth and maturationcontrolled by regulation of the micro-environment in vivo.Method:A total of34patients received intra-cytoplasmic sperm injection（ICSI） were enrolled in this study. Samples of blood and follicular fluid (FF)were obtained on the day of oocyte pick-up (OPU).3-6oocytes were obtained fromeach patient and total282oocytes collected from patients.Electro-chemiluminesence immunoassay (ECLIA) was used to measure blood serumand follicular fluid concentrations of E2, ER-β mRNA expression in cumulus cells was measured by Real-time PCR， Oocytes maturation was recorded and then analysistheir relevance. Cells in vitro experimental study: Mice (CD1, female:8–10weeks-old) were employed in this study. Obtained mature oocytes (MII) fromovaries stimulated by gonadotropins. Immature oocyte collected from the naturalcycling mice (without any stimulation with gonadotropins). The fully growingimmature oocytes (GV)surrounded with cumulus cells from antral follicles wereselectively collected for in vitro maturation(IVM) of culture. By maturatingin vitro (IVM), the immature oocytes (GV) develop to mature oocytes (MII) andaging oocyte (Aging). The expression of GPER from different maturation stagesof oocytes, in vivo and in vitro matured oocytes was examined byimmune-fluorescence GPR30antibody and the images were analyzed by laserscanning confocal microscope. Further confirmation was performed by Westernblots analysis of GPR30for cell fractionation.Results：(1) Concentrations of E2in blood serum were significantly lowerthan follicular fluid and there were positive correlations between them.(2)ER-βconcentrations in the FF of MII oocytes were significantly higher than inthe FF of MⅠ and GV oocytes.(3) ER-β mRNA expression in cumulus GC from MIIoocytes was significantly higher than in MⅠ and GV oocytes and showedsignificant differences（P<0.01）.(4) Experiments (immuno-fluoresence andwestern-blots) indicate that GPER is expressed on the plasma membrane as aestrogen receptor.(5) GPER protein expression changes in different stages ofmouse oocytes and there were significant differences among them (p<0.01)(6)The level of GPER expression on plasma membrane was significantly higher inoocytes matured in vivo compared to the oocytes matured in vitro (p<0.05).Conclusion:(1) Estrogen levels in follicular fluid and peripheral blood havea direct correlation. The estrogen levels in peripheral blood can indirectly reflect the level of estrogen in the follicular fluid that regards as indicatorsof predicting oocyte maturity;(2) ER-β may promote oocyte maturation throughenhancing bioavailability of E2and ER-β expression in cumulus cells waspositively correlated with oocyte maturity as well as estrogen levels infollicular fluid.(3) GPER was observed on the plasma membrane of mouse oocytesas estrogen receptor.(4) The GPR30expression was related to the oocytesmaturation.(5) The changes of expression of GPER on mouse oocytes plasmamembrane confirm oocyte membrane maturation, suggesting that those changes ofGPER may be related to the functional role of oocyte maturation.