The Role And Mechanism Of Extragonadal FSHR In The Decidualization Of Mouse Endometrial Stromal Cells

Objectives :To investigate the localization and expression of FSHR in mouse endometrium and its role in the decidualization of pregnant mice endometrium.Methods:1.To observe the expression of FSHR in endometrial tissue of D1-D9 pregnant mice by immunohistochemistry.2.To observe whether the expression of FSHR is influenced by the decidualization via using artificial induced mice deciduation model in vivo.3.To determine the effects of steroid hormones(E2 and P4)on the expression FSHR by the ovariectomized mice.4.The expression levels of FSHR and FSH were observed in mouse uterus during artificial decidualization after endometrial stromal cells were isolated and cultured and induced in vitro.5.To observe the effects of different concentrations of recombinant FSH on the proliferation of mouse endometrial stromal cells by MTS method.6.To observe and analyze the effects of FSH on deciduization of endometrium by real-time fluorescence quantitative PCR and western blot.7.The effects of FSH on the decidualization of endometrial stromal cells were observed and analyzed by RNA interference technique.8.To observe the effects of FSH on the Erk1/2 and AKT signaling pathways in mouse endometrial stromal cells by western blot.Results:1.Immunohistochemical results showed that FSHR was mainly located in the glandular epithelium and luminal epithelial cells during gestation D1-D4 days.With the progresses of decidua(D5-D9 days of pregnancy),the expression level of FSHR at the implantation site increased significantly,and FSHR was mainly expressed in the uterine decidua surrounding the embryo.However,FSHR was mainly located in glandular epithelium and luminal epithelial cells in the inter-implantation sites.2.In the artificial induced deciduation model,sesame oil was injected into the corner of the uterus from D4.Compared with the Non-oil side,the angle of the uterus on one side of the sesame oil gradually increased with the progresses of the pregnancy time(D5-D9 days),but there was no obvious change in the shape of the horn on the Non-oil side.In the uterine tissue of induced decidualization,the expression of FSHR was increased as the prolongation of pregnant time in D5-D9 days and mainly concentrated in the decidual tissue,while FSHR in the non-oil uterine tissues was mainly expressed in the glandular epithelium and luminal epithelial cells.3.Immunohistochemical results shows that FSHR was mainly expressed in the luminal epithelium and glandular epithelium cells in the ovariectomized uteri at 12 h and 24 h after E2 treatment,and the FSHR expression almost disappeared in uterine tissue after E2 and its antagonist Tam treatment;FSHR was mainly expressed in luminal epithelium,glandular epithelial cells and uterine stromal cells of the uterine tissues at 12 h and 24 h after treated with P4,but FSHR was only expressed in luminal epithelium and glandular epithelial cells after P4 and its antagonist RU486 treatment.4.The purity of isolated endometrial stromal cells was assessed by staining for vimentin(positive)and cytokeratin(negative)at 72 h of culture.At 24 h,48h and 72 h after treatment with E2 and P4 in vitro,the immunofluorescence assay showed that the FSHR protein was clearly expressed in the cytoplasm and membrane of.stromal cells.The results of immunoblotting and real-time quantitative PCR showed that the expression levels of Cyclin D3 protein and prl8a2 mRNA increased significantly in vitro(P<0.05 or 0.01).At the same time,the expression levels of Fshb and Fshr mRNA also increased with the progresses of decidualization(P<0.01).5.MTS experimental results showed that the isolated stromal cells were incubated with different concentrations of FSH for 96 hours and 3ng/ml,10ng/ml,30ng/ml and 100ng/ml FSH significantly promoted the proliferation of stromal cells compared with control group.However,300 ng/ml,600 ng/ml,and 1000 ng/ml FSH significantly inhibited the proliferation of stromal cells(P<0.05 or 0.01).The stromal cells with 10 ng/ml FSH treatment significantly promoted the proliferation of stromal cells at 24 h,48h,72 h,and 96 h compared with 0 h in vitro(P<0.05 or 0.01).6.Western blot and real-time fluorescence quantitative PCR results showed that the expression level of cyclin D3 protein significantly increased compared with the control group at 48 h and 72 h of culture after treatment with FSH,E2+P4 and E2+P4+FSH(P<0.05 or 0.01).At the same time,the expression level of prl8a2 mRNA significantly increased in E2+P4+FSH group compared with E2+P4 group(P<0.01).7.In vitro culture of uterine stromal cells,compared to blank control group,the Fshr mRNA and FSHR protein expression levels were significantly decreased at 24 h,48h,72 h in the FSHR siRNA+FSH group after transfection with FSHR siRNA,but there was no significant change in the scramble siRNA group,suggesting that FSHR siRNA can specificity interfere FSHR mRNA expression.At the same time,the expression levels of prl8a2 mRNA and Cyclin D3 protein levels were significantly decreased(P<0.05 or 0.01),indicating that FSHR siRNA can inhibit the decidualization of endometrial stromal cells.8.Western blotting results showed that 10 ng/ml FSH significantly increased phosphorylation of AKT and Erk1/2 levels at 15 min,30 min and 60 min compared with 0 min,with the peak at 60min(P < 0.01).Conclusion:During early the pregancy,FSHR was temporally and spatially expressed at the maternal-fetal interface in pregnant mice uteri.The expression site and expression levels of FSHR were influenced by E2,P4 and endometrium decidualization,and low dose FSH can promote the proliferation and decidua of endometrial stromal cells.After the intervention of FSHR,the decidualization of endometrial stromal cells was significantly inhibited,suggesting that FSHR can promote the decidualization of endometrium.