| With the development of mammalian embryo biotechnology,the demand for high quality oocytes is increasing.The number of mature oocytes obtained in vivo is limited,so it is necessary to understand the regulatory network and mechanism of follicular development and oocyte maturation.Studies have shown that adding appropriate amounts of estradiol(E2)to the culture system in vitro can promote oocyte maturation.In estrous cycle of female animals,oogenesis always changes with follicular development.Only when the hormone and other cytokines in the follicle reach an appropriate level,the oocyte can undergo meiosis recovery and further development and maturation,in which the regulation of E2 is very important.Existing research shows that the regulation of E2 on mammalian reproduction is mediated by estrogen receptor,on the one hand,E2 regulates target gene transcription through nuclear receptor,on the other hand,G protein coupled estrogen receptor(GPER)can rapidly mediate the non-genomic effect of E2.In this study,we first investigated the distribution and expression of GPER in porcine follicles.Then,we used the model of porcine oocyte cultured in vitro to study the effect of GPER on porcine oocytes maturation by adding E2,GPER specific agonist G1 and antagonist G15,as well as the role of PI3K/AKT signaling pathway during GPER action.It provides an experimental basis for understanding the mechanism of E2 involved in the regulation of mammalian follicular development and oocyte maturation.The results of this experiment are as follows:1.The distribution and expression of GPER in porcine follicles of different sizesThe purpose of this study was to investigate the distribution and expression of G protein coupled estrogen receptor(GPER)in porcine follicles of different sizes.According to the diameter of follicles,different groups were set up.The healthy antral follicles of pigs with diameters less than 3 mm,3-5 mm and more than 5 mm were separated,and the COC,follicular fluid and follicular wall of each group were collected.The concentration of estradiol in follicular fluid was detected by ELISA,the distribution and relative expression level of GPER in ovary were detected by immunohistochemistry and WB,and the location of GPER in COCs was detected by indirect immunofluorescence combined with confocal microscopy.The results showed that the concentration of estradiol in follicular fluid increased with the development of follicles.GPER immunoreactive products were distributed in granulosa cells,theca cells,cumulus cells,vitelline membrane and oocyte cytoplasm of porcine follicles of different sizes,which granulosa cells of the>5 mm group were stained most deeply.The relative expression of GPER protein gradually increased with the development of follicles.Confocal images showed that GPER was mainly located on the membrane of cumulus cells in the COC of porcine follicles,but were stained slightly in the whole oocytes.These results showed that the distribution and expression of GPER in porcine follicles of different sizes were consistent with the development of follicles,suggesting that GPER may mediate the regulation of estrogen in porcine follicular development and oocyte maturation.2.Effect of GPER on porcine oocytes maturation in vitroIn order to investigate the effect of GPER on porcine oocytes maturation in vitro,the established model of porcine oocytes cultured in vitro was used in this experiment.The optimal concentration of GPER natural ligand E2,GPER specific agonist G1 and antagonist G15 were screened,and then five groups were set up:control group,E2 group,G1 group,G15 group and E2+G15 group.WB was used to detect the relative expression level of GPER;Then the first polar body extrusion rate and the cumulus expansion index of different groups were counted;Finally,the mRNA levels of the cumulus expansion related genes,such as Has-2,Pgr,Ptgs-2 and Ptx-31 of cumulus cells were detected by RT-qPCR,respectively.The results showed that E2 and G1 significantly increased the expression level of GPER but this effect was weakened when GPER was inhibited by G15(P<0.05).The activation of GPER promoted porcine oocytes maturation and cumulus cells expansion in vitro,however,the inhibition of GPER hindered porcine oocytes maturation and cumulus cells expansion,The mRNA levels of the cumulus expansion related genes also changed significantly.These results suggested that E2 can promote the maturation of porcine oocytes by regulating the expression of GPER.3.Activation of GPER by estradiol(E2)regulates oocyte maturation via PI3K/AKT pathwayIn order to explore the molecular mechanism of E2 promoting porcine oocyte maturation in vitro through GPER and its relationship with PI3K/AKT signaling pathway,the established model of porcine oocytes cultured in vitro was used in this experiment.The optimal concentration of PI3K specific inhibitor was screened,and then six groups were set up,including control group,E2 group,G15 group,E2+G15 group,LY294002 group and E2+LY294002 group.Firstly,the phosphorylation level of AKT protein was detected by WB to explore whether E2 could activate the PI3K/AKT signaling pathway through GPER,then the relative expression level of GPER was detected,and finally the phenotype was verified.The results showed that E2 significantly increased the relative expression of p-AKT/AKT,while G15 or LY294002 significantly decreased the expression of p-AKT/AKT,in the E2+G15 and E2+LY294002 group,the induction of E2 were significantly blocked(P<0.05).E2 significantly increased the relative expression of GPER(P<0.05).Different from G15,GPER expression was not significantly inhibited in LY294002 group(P>0.05).In the E2+LY294002 co-treatment group,the induction effect of E2 still exists.E2 significantly improved oocytes maturation and cumulus cells expansion of porcine COCs cultured in vitro(P<0.05),while G15 or LY294002 significantly decreased oocytes maturation and cumulus cells expansion(P<0.05).In the E2+G15 and E2+LY294002 group,the induction effect of E2 weakened.These results suggested that E2 could activate PI3K/AKT signaling pathway through GPER to promote porcine oocytes maturation and cumulus cells expansion in vitro. |
