Effect Of Estrogen Membrane Receptor GPR30 On Gap Junctional Communication Of Goat Cummulus Oocyte Complex And Its Mechanism

Mammalian follicles are mainly composed of follicular somatic cells and germ cells.There are gap junctions between granulosa cells,cumulus cells and between cumulus cells and oocytes,thereby forming a functional syncytium.During the development of follicles,oocyte produces an extracellular coat termed the zona pellucida,transzonal cytoplasmic projections?TZPs?formed between the cumulus cells and the oocyte membrane,which morphologically resemble filopodia,and in the end of the TZPs also form gap junctions;The gap junctions transport signal substances and energy for the development and maturation of oocyte and ovulation.During spontaneous or gonadotropin-induced oocyte maturation,granulosa cells gap junctional intercellular communications?GJIC?are reduced,and gap junctions between cumulus cells and oocytes are gradually closed or disappear.luteinizing hormone?LH?can act on the gap junctions of granulosa cells and attenuate the production and transmission of meiotic inhibitory substances,for instance cGMP.Estrogen can also regulate the expression of gap junction-associated proteins during the development of ovarian follicles.Nevertheless the relevant mechanisms of estrogen-regulated GJIC in cumulus-oocyte complexes?COCs?have not yet been reported.In the present study,we investigated the regulation of estrogen on goat oocyte germinal vesicle breakdown?GVBD?,as well as the effect of GJIC activity in the process of oocyte nuclear maturation of goat COCs,in order to explore the mechanism of estrogen on GJIC and resumption of meiotic maturation.The contents and results are as follows:?1?Hoechst33342 was used for goat oocyte nuclear staining to observe the chromatin morphology and detect the effect of 17?-E₂ in goat COCs.The goat COCs were treated with1?g/mL 17?-E₂ for 2,4,6 h and 8 h in vitro.It was found that 17?-E₂ treatment significantly promoted GVBD of oocytes at 4 and 6 h.Estrogen nuclear receptor?ER?inhibitor ICI182780,G receptor G30?GPR30?inhibitor G15 or agonist G1 was used to explore the signaling of estrogen on goat oocyte maturation.The results show tha the effect of 17?-estradiol on goat oocyte maturation can be inhibited by GPR30 antagonist G15 rather than ICI182780.Importantly,G1 can mimic the effect of 17?-estradiol on goat oocyte maturation.It was demonstrated that 1?g/mL 17?-E₂ promote GVBD of oocytes in goat COCs in the early stage in vitro culture,and17?-estradiol promoted oocyte nuclear maturation might be mediated by GPR30..?2?During the process of oocyte nuclear maturation,the number of TZPs and gap junction function will change dynamically.However,whether these dynamic changes are relatedtotheGPR30-mediatedsignalingpathwayneedsfurthervalidation.Immunofluorescence technique was used to detect the expression and localization of GPR30and connexin CX43 in freshly isolated goat COCs.The results showed that GPR30 and CX43expression in the cumulus cells and oocytes of goat COCs and localization on the plasma membrane.In order to verify whether TZPs play a role in 17?-E₂ promoting premature GVBD in goat oocytes,In the presence or absence of 17?-E₂ in vitro culture system,the expression of TZPs in goat COCs was detected at 0,6,24 h in vitro culture.It was found that the number of intact TZPs of all COCs was reduced significantly at 6 h and 24 h of in vitro culture?P<0.05?.The expression of TZPs in the 17?-E₂ treatment group was significantly decreased compared with the control at 24 h?P<0.05?,however,in 6h culture,there was no significant difference between the group of 17?-E₂ treatment and the control,suggesting that a decrease in the amount of TZPs does not account for 17?-E₂ induced premature nuclear maturation of oocytes.In order to further verify whether gap junctions play a role in 17?-E₂promoting premature GVBD,goat COCs were cultured in a culture system supplemented with 1?g/mL 17?-E₂,before and after cultured 2,4,6 and 8 h,lucifer yellow?LY?was injected into the cytoplasm of oocytes using a micromanipulation system to assess gap junction cummunication.Gap junctin communication were classified as open,partial or closed,according to spread of the dye into neighboring cumulus of COCs.GJCs was assrssed at the time of collection and after 2,4,6 and 8 h culture in basic medium?control?or supplemented with 1?g/mL 17?-E₂.It was showed that,at the time of collection,87.5%COCs of gap junctions were open.Gap junctional communication was significantly downregulated?P<0.05?at 4h and 6h of 17?-E₂ treatment.1?g/m L 17?-E₂ was added to the COCs culture system.or estrogen membrane receptor agonist G1,or co-administration of17?-E₂ and estrogen membrane receptor inhibitors G15,or co-administration of 17?-E₂ and estrogen nuclear receptor inhibitor ICI182780,in vitro culture of COCs.At 4 h,the COCs were transferred to phosphate buffer containing calcein-AM and the transfer of calcein from cumulus cells to oocytes was monitored to assess the effect of estrogen receptor pathway on gap junction communication activity.The results showed that the addition of 17?-E₂ or estrogen membrane agonist G1 alone could decrease the gap junction activity and significantly inhibit the transfer of calcein from cumulus cells to oocytes?P<0.05?;Supplement membrane receptor inhibitor G15 blocked the inhibitory effect of estrogen on gap junctions without affecting calcein transfer;Nevertheless,the supplement of estrogen nuclear receptor inhibitor ICI182780 did not affect the inhibitory effect of 17?-E₂ on calcein transfer.It was suggested that 17?-E₂ could down-regulate the gap junction communication between cumulus cells and oocytes in COCs,and it was mediated by estrogen receptor GPR30.?3?In order to investigate the mechanism of estrogen on the regulation pf gan junction communication between cumulus cells and oocytes in COCs,the effect of 17?-E₂ on the phosphorylation level of connexin CX43 in goat COCs was detected by Western Blotting.The results showed that there was no significant change in the phosphorylation level of CX43 Ser368 in COCs within 4 h after 17?-E₂ treatment.The phosphorylation level of CX43was significantly increased at 4 h,6 h,and 8 h after treatment with 17?-E₂ alone.The phosphorylation levels of CX43 and extracellular-signal regulated protein kinase1/2?ERK1/2?,a key molecules in the GPR30 signaling pathway,in COCs were significantly increased after treatment with 17?-E₂ or the GPR30 agonist G1 for 4h,and higher than those in the control group and co-administration of 17?-E₂ and estrogen membrane receptor inhibitors G15.On the basis of 1?g/mL 17?-E₂,borth ERK inhibitor PD98059?50?M?and AG1478?50?M?,an inhibitor of EGFR,another key molecule in the GPR30 signaling pathway,could significantly down-regulate phosphorylation levels of ERK1/2 and CX43 Ser368.?P<0.05?;while co-administration estrogen nuclear receptor inhibitor ICI182780 with 17?-E₂ the phosphorylation levels of ERK1/2 and CX43 Ser368 were not significantly different from the control group.It was demonstrated that 17?-E₂ promote the phosphorylation of CX43 in goat COCs mediated by GPR30,and the EGFR-ERK1/2 signaling pathway is involved in this process.?4?To further verify that the EGFR pathway is involved in estrogen regulation of CX43phosphorylation mediated by the GPR30 in goat COCs.The expression of EGFR ligands EGF-like factors?AREG,EREG,BTC?in goat cumulus cells were detected by qRT-PCR.The results showed that the supplement of 17?-E₂ or GPR30 agonist G1 alone for 4 h increased the expression of EGF-like mRNA in cumulus cells,and was significantly higher than that of the control and the group of co-treated with 17?-E₂ and G15?P<0.05?.The treatment of estrogen-nuclear receptor inhibitors ICI182780 did not affect 17?-E₂ induced expression of EGF-like factors in cumulus cells.It was demonstrated that 17?-E₂ promotes EGF-like factor mRNA expression mediated by GPR30.In summary,In goat COCs,17?-E₂ promotes EGF like factor expression in cumulus cells via GPR30,which in turn activates ERK1/2 signaling pathway to promote CX43phosphorylation and resumption of meiotic maturation.