Effect Of FSH On E₂/GPR30-Mediated Mouse Cumulus–Oocyte Complexes Maturation In Vitro

Under normal physiological conditions,the main role of follicle stimulating hormone（FSH）in the ovaries of female mammals is to promote follicle growth and development,expression of luteinizing hormone receptor（LHR）receptor and acquisition of oocytes’ability to restore meiosis.In follicular membrane cells,luteinizing hormone（LH）is combined with LHR,promotes the synthesis and secretion of androstenedione.The synthesis of androstenedione was catalyzed by P450 aromatase from granulosa cells induced by FSH,and then transformed into17β-estradiol（E₂）by 17B-hydroxyl steroid dehydrogenase（HSD-17B）.The synthesized estrogen is mediated by estrogen nuclear receptors（ERs）and transmembrane G protein-coupled receptor（GPR30）.In other words,FSH plays an indirect physiological role by promoting estrogen synthesis and may be mediated by estrogen receptor.Studies have shown that when sheep COCs were added to mature culture medium with different concentrations of FSH,or FSH and follicle stimulating hormone receptor-binding inhibitors,the estrogen nuclear receptor ERβm RNA level and protein level will not significant chang.This indicates that the indirect physiological effects of FSH may not achieved by ERs（ERαand ERβ）.Therefore,how FSH plays an indirect physiological role through estrogen and whether oocyte maturation and cumulus expansion are mediated by estrogen membrane receptor GPR30 is a scientific problem to be explored.Therefore,mouse COCs were used as a model in this study to investigate the effect of FSH on the in vitro maturation of mouse oocytes mediated by E₂/GPR30.And the mechanism of its effect was studied.The contents and results of this research are as follows:（1）Firstly,the concentration of FSH in the process of maturation and culture of mouse COCs in vitro was screened,and the concentration gradient of 0 IU/m L,0.01 IU/m L,0.1 IU/m L,1 IU/m L,10 IU/m L FSH were set.The statistical results of CEI showed that the addition of 0.1IU/m L FSH could effectively promote the cumulus cells expansion.The ELISA method was used to detect the change of estrogen level in the medium supplemented with FSH during the in vitro maturation culture of mouse COCs.The results showed that FSH could promote the production of E₂in vitro matured mouse COCs compare with control group.Letrozole（Let）,a specific inhibitor of aromatase,was used for further quantitative study of aromatase associated with E₂production.q RT-PCR results showed that the m RNA level of HSD-17B1 and CYP51 in FSH group was significantly higher than that in control group and FSH add Let group（P<0.05）,but there was no significant difference in CYP19A1 transcription level among the three groups（P>0.05）.The results showed that the addition of 0.1 IU/ml FSH,could activate aromatase in cumulus cells and promote the production of estrogens during the in vitro maturation of mouse COCs.（2）Based on the above studies,using G15,a specific inhibitor of estrogen,to further investigate whether E₂,which is produced by FSH,is mediated by estrogen membrane receptor GPR30 in the process of in vitro maturation of mouse COCs.Immunofluorescence staining of mouse cumulus cells and COCs was first carried out.Laser scanning confocal microscopy showed that GPR30 was present on the cytoplasmic membrane of mouse cumulus cells and oocytes,but the expression of the GPR30 on the plasma membrane of the oocytes was lower compared with the cumulus cells.Then,the changes of m RNA and protein levels of GPR30 in the mature culture process were further studied.q RT-PCR results showed that GPR30 m RNA levels were lower in FSH+G15,FSH+Let or FSH+G15+Let treatment of COCs than in FSH group（P<0.05）.Western blotting analysis further showed that GPR30 protein levels and m RNA levels showed the same trend.Our results indicate that FSH activates the production of estrogen by activating the aromatase of mouse COCs,and the resulting endogenous estrogen activates GPR30,thereby indirectly regulating GPR30.（3）Based on the above findings and theories,we investigated the role of FSH-mediated GPR30 and its downstream ERK1/2 signaling pathway in cumulus expansion.We found that after 8 hours of IVM,the FSH group had more significant cumulus cells expansion than the control group and other small molecule inhibitors（G15,Let,PD98059）groups（P<0.05）.After16 hours,the cumulus cells were further expanded in the FSH group compared to the control group and other small molecule inhibitor groups（P<0.05）.The results of CEI showed that the CEI of FSH group,FSH+G15 group,FSH+PD98059 group,FSH+G15+PD98059 group,FSH+G15+Let+PD98059 group were higher than those of control group after 8 h culture（P<0.05）.However,after 16 hours of IVM,the FSH group FSH+Let group,FSH+G15 group had higher CEI than the control group（P<0.05）,but there was no significant difference in CEI between the control group and FSH+PD98059 group,FSH+PD98059+G15 group or FSH added three small molecule inhibitor groups（P>0.05）.In addition,the relative expression of genes related to cumulus expansion（HAS2,PTGS2,PTX3,TNFAIP6）in the above groups was studied after 6 hours of in vitro culture of mouse COCs.The results of q RT-PCR showed that compared with the control group and added with the inhibitor group,FSH significantly increased the m RNA expression of HAS2 and PTGS2 in COCs（P<0.05）.On the contrary,except for the control group,there was no significant difference in the expression of PTX3 and TNFAIP6 m RNA between the groups（P>0.05）,but the levels of PTX3 and TNFAIP6 m RNA in each group were significantly higher than those in the control group（P<0.05）.This indicates that FSH-induced expansion of cumulus cells in mouse COCs is regulated by GPR30-mediated estrogen signaling pathway.（4）Next,the role of FSH-mediated GPR30 and its downstream ERK1/2 signaling pathway in oocyte maturation was further studied The statistical results of the first polar body（PBI）excretion rate showed that the excretion rate of PBI in the FSH group was significantly higher than that in the control group and other small molecule inhibitors groups（P<0.05）.The m RNA levels of the genes related to oocyte maturation（GDF9,BMP15,GREM1）were quantified.The q RT-PCR results showed that the m RNA levels of GDF9 and BMP15 in the FSH group were significantly lower than those in the control group（P<0.05）,even though with the addition of different inhibitors,the inhibitory effect of FSH was not reversed.However,the expression level of GREM1 m RNA in the FSH group was significantly higher than that in the other groups（P<0.05）.It indicated that the in vitro maturation of mouse oocytes induced by FSH was regulated by GPR30-mediated estrogen signaling pathway.（5）To further explore the mechanism of action of GPR30-mediated estrogen signaling pathway on FSH-induced mouse oocyte maturation.The effect of GPR30-mediated estrogen signaling pathway on phosphorylation of ERK1/2 in mouse COCs was examined by Western blot.Our results showed that FSH treatment of COCs for 6 h was significantly higher than the phosphorylation level of ERK1/2 in the control group（P<0.05）.However,the addition of G15,Let,and PD98059 alone or in combination significantly reduced the promotion of ERK1/2phosphorylation by FSH（P<0.05）.It was shown that the addition of FSH to promote the production of COCs 17β-E₂can regulate the phosphorylation level of ERK1/2 through the estrogen membrane receptor GPR30,thereby promoting the expansion of mouse cumulus cells and the maturation of oocytes.In conclusion,during mouse COCs maturation in vitro,the mechanism of FSH promoting oocyte nuclear maturation and cumulus cell expansion maily by activating aromatase,transforming androstenedione into E₂,and then the phosphorylation of ERK1/2 was promoted by GPR30.