Effect And Mechanism Of Estrogen Membrane Receptor GPR30 On Cumulus Expansion During Goat Oocytes Maturation

The cumulus cells are a kind of highly specific cells that around the oocyte of antral follicle and the early embryos.During contacting with the oocyte,cumulus cells maintain the communications between oocyte and cumulus cells,which have an important effect on the development and maturation of oocyte.Studies had shown that cumulus expansion was the primary morphological indicator of mature oocyte and high doses of estrogen could significantly promote the expansion of cumulus cells.Estrogen exerted a physiological role mainly through estrogen nuclear receptor,ER? and ER?,which were widely distributed in different tissues and cells.Although these two estrogen receptor subtypes mediated most of the effects of estrogen,estrogen also could rapidly regulate intracellular events,such as the production of cAMP,the migration of calcium ions and so on,these rapid estrogen effects did not involve transcriptional activation in certain cells,but directly act on the surfaces of the cells.Filardo and his colleagues found an estrogen membrane receptor G-protein coupled receptor 30?GPR30?.Their studies had shown that GPR30 could rapidly mediate these estrogen events under certain conditions.Although the GPR30 played an important role in many cells and tissues,whether in goat cumulus cells and oocyte also existed the expression of GPR30 has not yet been reported.Therefore,this study first tested the localization and expression of GPR30 in goat COCs before and after cultured 24 h in vitro.On the basis of determining the presence of GPR30 in goat COCs,the effect and mechanism of GPR30 on the expansion of cumulus cells during goat oocyte maturation were further studied using GPR30-specific agonist G1 and specific antagonist G15.The following shows the contents and results of this study:?1?The localization and expression of GPR30 in goat COCs before and after cultured 24 h were detected by immunofluorescence.The results showed that the expression of GPR30 in goat COCs was localized on the cells plasma membrane.In addition,qRT-PCR and Western Blotting showed that there was the expression of GPR30 mRNA and protein before and after in vitro cultured goat COCs 24 h,and the expression levels of GPR30 mRNA and protein were significantly increased after cultured 24 h compared to 0 h?P < 0.05?.However,the GPR30 mRNA and protein levels in mature oocytes were significantly lower than before cultured?P < 0.05?.?2?On the basis of the above studies,the effect of estrogen on oocyte maturation was studied by using G1,a specific GPR30 agonist,and specific antagonist G15.The maturation rates of oocytes in 17?-E₂ group,G1 group,control group,17?-E₂ and G15 combined treatment group were calculated according to the presence of the first polar body in the oocyte after 24 h incubation in vitro.The results suggested that 17?-E₂ group and G1 group obviously promoted the maturation of oocytes compared with the control group?P < 0.05?.The maturation rate of G1 group was lower than that of 17?-E₂ group,but there was no obvious difference between above two groups?P > 0.05?.These results suggest that G1 can mimic the promotion effect of 17?-E₂ on oocyte maturation to some extent.However,the maturation rates of oocytes in 17?-E₂ and G15 combined treatment group were obviously lower than that of 17?-E₂ group and G1 group?P < 0.05?,the maturation rates of goat oocytes were obviously higher than that of the control group?P < 0.05?,indicating that the maturation of goat oocyte was regulated by GPR30-mediated estrogen signaling pathway,at least in part,mediated by GPR30.?3?In order to study the effect of estrogen membrane receptor GPR30-mediated estrogen signaling pathway on cumulus expansion during oocyte maturation,after we cultured COCs for 24 h,according to Vanderhyden et al.?1990?reported the cumulus expansion grading and expansion index assessment methods,the cumulus expansion index in different treatments and control group were calculated.We found that 17?-E₂ can obviously increased the expansion index of goat cumulus cells compared with the control group?P < 0.05?,And G1 can also increased the cumulus expansion index.In addition,17?-E₂ and G15 were combined treatment with COCs for 24 h,the expansion index of cumulus cells was increased than that of the control group,but there was no differ from the two groups?P > 0.05?,however,compared with G1 group and 17?-E₂ group,the expansion index of cumulus cells was decreased.After 24 h incubation in vitro,we also studied the relative expression levels of cumulus expansion-related genes?HAS2,PTGS2,PTX3,TNFAIP6?in the above groups.The results of qRT-PCR showed that the expression levels of HAS2 and PTX3 mRNA in cumulus cells were significantly increased in 17?-E₂ treatment group and G1 treatment group compared with the control group?P < 0.05?.However,the expression levels of PTGS2 and TNFAIP6 mRNA were no obvious difference among the 17?-E₂ treatment group,the G1 treatment group and the 17?-E₂ and G15 co-treated group?P > 0.05?.?4?In order to further explore the mechanism of GPR30-mediated estrogen signaling pathway on the cumulus expansion during oocyte maturation,the ELISA method was used to analyze cAMP levels in goat COCs.The results showed that 17?-E₂ and G1 both could significantly increase the levels of cAMP in goat COCs compared with the control group,further suggesting that GPR30 was activated.17?-E₂ group compared with G1 group,there were obvious differences?P < 0.05?,the levels of cAMP in 17?-E₂ and G15 co-treated group was significantly reduced compared with the 17?-E₂ group and G1 group,indicating that GPR30 mediated the effect of 17?-E₂ on cAMP levels in COCs.Moreover,when compared to the control group,the co-treated group obviously increased the levels of cAMP in COCs?P < 0.05?,suggesting estrogen may also promote the increase of cAMP levels in goat COCs through other signaling pathways.According to the previous studies showed that ERK1/2 could promote the expansion of cumulus cells,we hypothesized that estrogen might promote the expansion of cumulus cells by influencing ERK1/2 phosphorylation levels.Therefore,we cultured goat COCs in 17?-E₂?1 ?g/mL?,G1?100 nM?,17?-E₂?1 ?g/mL?+G15?100 nM?and control group?no 17?-E₂?for 30 min.Western Blotting showed that the levels of ERK1/2 in the 17?-E₂ group or G1 group were obviously higher than those in the control group?P < 0.05?.However,the phosphorylation levels of ERK1/2 in G1 group was markedly lower than that of ERK1/2 in 17?-E₂ group.The addition of G15 significantly decreased the promotion effect of 17?-E₂ on the phosphorylation of ERK1/2?P < 0.05?,however,the phosphorylation levels of ERK1/2 in G15 and 17?-E₂ combined treatment group was still significantly higher than that in control group?P < 0.05?.In summary,the expression of GPR30,which was located on the cytoplasmic membrane,was present on goat cumulus cells and oocytes.17?-E₂?1 ?g/mL?promoted the maturation of goat oocytes by GPR30.GPR30 mediated-17?-E₂?1 ?g/mL?increased cAMP levels and ERK1/2 phosphorylation in goat COCs,and ultimately promoted the expansion of cumulus cells.