Regulation Of Estrogen Membrane Receptor GPER1 On EgGF-like Factor Expression In Mouse Cumulus Cells And Its Mechanism

The signal network mediated by EGF（epidermal growth factor,EGF）-like factors plays a vital role in the maturation of mammalian oocytes.In the later stage of oocyte development,the conduction of EGF signal in the follicle induces a series of biological events such as the expansion of cumulus cells,the closure of gap junction communication,the resumption of oocyte meiosis and ovulation.Studies have found that 17β-estradiol（17β-estradiol,17β-E₂,denoted by E₂in the following text）in the oocyte maturation culture system in vitro is beneficial to the closure of gap junction communication,the expansion of cumulus cells and the meiosis resumption of oocytes,but the molecular mechanism by which estrogen plays a role in this process is still unclear.In this study,we examined the expression and localization of GPER1 in mouse follicles and cumulus-oocyte complexes（COCs）to investigate whether estrogen can induce EGF-like factor expression in mouse cumulus cells by activating GPER1,and the mechanism of estrogen-induced EGF-like factor expression in mouse cumulus cells.The research contents and results are as follows:（1）Immunofluorescence staining,reverse transcription PCR（RT-PCR）and Western blot techniques were used to detect the location and expression of GPER1 in mouse follicles and COCs.The results of immunofluorescence staining showed that GPER1 was localized in granulosa cells,cumulus cells and oocytes.RT-PCR and Western blot results showed that both m RNA and protein of GPER1 are expressed in mouse cumulus cells and oocytes.（2）ELISA and Western blot were used to detect the level of cyclic adenosine monophosphate（c AMP）and phosphorylated c AMP response element binding protein（CREB）in mouse cumulus cells after GPER1 was activated.1μM E₂or 1μM G1（GPER1agonist）were treated with COCs for different times to detect the changes in the levels of c AMP and phosphorylated CREB in cumulus cells,and set up the GPER1 inhibitor G15treatment group,the estrogen nuclear receptor inhibitor ICI182780 treatment group,and the protein kinase A（protein kinase A,PKA）specific inhibitor H89 treatment group.The results showed that after treating COCs with 1μM E₂or 1μM G1,c AMP levels in cumulus cells increased rapidly and reached a peak at 45 min after treatment;while G15 and H89 inhibited the effect of E₂on the up-regulation of c AMP levels in mouse cumulus cells,ICI182780treatment did not show an effect on the action of E₂.Consistent with this result,1μM E₂or 1μM G1 treated COCs for 45 min and 1 h,the phosphorylation level of CREB in cumulus cells was significantly increased,and G15 instead of ICI182780 inhibited the effect of E₂on CREB phosphorylation in mouse cumulus cells.In addition,we found that H89 also inhibited the up-regulation of CREB phosphorylation level in mouse cumulus cells by E₂.These findings indicate that E₂induces the increase of c AMP level in mouse cumulus cells through GPER1,and PKA mediates the c AMP-dependent CREB phosphorylation in mouse cumulus cells induced by E₂.（3）During follicle development,c AMP-dependent CREB activation can induce the transcriptional expression of EGF-like factors in granulosa cells.We used q RT-PCR technology to detect the relative expression of EGF-like factors（Areg,Ereg,Btc）m RNA in mouse cumulus cells.The results showed that treatment with 1μM E₂or 1μM G1 for 1 h,respectively,can significantly up-regulate the expression of EGF-like factor m RNA in mouse cumulus cells.Both G15 and H89 showed an inhibitory effect on the up-regulation of EGF-like factor m RNA expression in mouse cumulus cells by E₂,while ICI182780 treatment did not show an effect on the action of E₂.These findings indicate that E₂induces the expression of EGF-like factor m RNA in mouse cumulus cells through the GPER1/c AMP/PKA/CREB signaling pathway.（4）To further explore the role of GPER1 in the expression of EGF-like factors in cumulus cells induced by E₂,we cultured isolated mouse cumulus cells in vitro,used GPER1si RNA technology to interfere with the expression of GPER1 in cumulus cells,and detected the change of EGF-like factor m RNA expression induced by E₂and G1 in cumulus cells interference with GPER1.The results showed that after the GPER1 expression in cumulus cells was disturbed,neither E₂nor G1 could induce the up-regulation of EGF-like factor m RNA expression in cumulus cells.The results of this study further indicate that GPER1mediates the expression of EGF-like factors in mouse cumulus cells induced by E₂.In summary,E₂induces the expression of EGF-like factors in mouse cumulus cells through the c AMP/PKA/CREB signaling pathway,and this biological effect is mediated by GPER1.