Effect And Mechanism Of FSH On Uterine Endometrium

Part I The effects of FSH treatment on AQPs and receptive factors in human endometriumObjective:To identify whether FSHR expresses in human endometrium and Ishikawa cells, the endometrial adenocarcinoma cell line. To observe the human endometrial morphological and functional changes, as well as endometrial expression of FSHR, AQPs and endometrial receptive factors after controlled ovarian stimulation (COS) using FSH treatment.Patients and methods:We collected 15 endometrial samples in implantation window after COS, and 40 samples in implantation window of natural cycle as control group. We measured the serum FSH and E2 with chemoluminescence, detected FSHR expression in human endometrial tissue and Ishikawa cells with a few methods, including sequencing RT-PCR product, Western blotting, immunohistochemistry and immunofluorescence. Endometrial ultrastructure was investigated with electron microscopy. We observe the changes of endometrial FSHR, AQPs(AQPl, AQP2, AQP 8), endometrial receptive factors (Integrinβ3,LIF) and Claudin4, a tight-junction associated protein with Western blotting.Results:We identified FSHR mRNA and protein in endometrial tissue and Ishikawa cells, and make 100% matchup between FSHR RT-PCR product sequence and BLAST results from NCBI. Protein mainly expressed on cellular membrane and cytoplasma. The serum FSH and E2 level from COS group is much higher than those of control group. With scanning electron microscope, we observed that endometrial luminal epithelium were arranged well, with pinopode seen in the control group, in contrast, endometrial luminal epithelium was deranged, without any pinopode in the COS group. With the transmission electron microscope, apoptosis of endometrial epithelium increased. No significant difference of tight-junction was observed. The Western blotting result shows that FSHR expression was up-regulated significantly while expression of AQP1, AQP2, AQP8, Integrinβ3, LIF were down-regulated significantly in the COS group, and Claudin4 did not change much in the COS group.Conclusion:FSHR is expressed in human endometrial tissue and Ishikawa cells. Compared with control group, women of COS group exhibited elevated serum FSH and E2 level, up-regulation of FSHR expression in endometrium, down-regulation of AQP1, AQP2, AQP8, Integrinβ3 and LIF expression, as well as alternation of endometrial luminal epithelium morphology and apoptosis. FSH may regulate the morphology aspect and function of child-bearing period endometrium directly through FSHR.Part II The mechanism of FSH affecting child-bearing period endometrium receptivityObjective:To investigate the effect and mechanism of FSH on child-bearing period endometrium receptivity.Patients and methods:We set up the OS mouse model by injecting exogenous rFSH, and assessed the influence of exogenous rFSH on serum FSH/E2 level, endometrial AQPs, receptive factors, pregnancy rate and live birth rate. We constructed the mouse blastocyst-adhesion model, and assessed the effect of FSH/E2, PCMB (AQP inhibitor) pre-treatment on blastocyst adhesion and implantation. We measured AQPs, receptive factors (integrinβ3, LIF) gene expression level after FSH/E2 treatment by RT-Real time quantitative PCR based on in vitro cell experiment. Assessing expression of endometrial receptive factors and JAr adhesion rate after AQP2/AQP8siRNA interfering.Results:In the OS group, serum FSH/E2 level elevated significantly, endometrial expression of AQP2, AQP3 protein expression was down-regulated and FSHR was slightly up-regulated, the pregnant rate and live birth rate decreased drastically. The results from the mouse blastocyst adhesion model showed that in vitro FSH, FSH/E2 and PCMB treatment on endometrium cells lead to decreased blastocyst adhesion rate at 48hr and 72hr. In Ishikawa cells, E2 display a concentration-dependent up-regulation on expression of AQP2 and AQP8 mRNA, while FSH (0,1,3,10,30 IU/L) display a concentration-dependent and time-dependent down-regulation on AQP2, AQP8 and LIF mRNA, especially 10IU/L and 30IU/L at 48hr. After AQP2siRNA interfering, the expression of LIF and IntegrinB3 were up-regulated and OLFM1 was up-regulated. After AQP8siRNA interfering, the expression of LIF was down-regulated and OLFM1 was up-regulated. The JAr adhesion rate decreased after either AQP8 siRNA or AQP2siRNA interfering.Conclusion:Superphysiological FSH level disturb endometrium receptivity and embryo implantation through the down-regulation expression of endometrium AQPs and receptive factors.Part III The effects of high serum FSH on menopausal uterine endometrial atropy.Objective:To investigate the effect and mechanism of high FSH level during the post-menopause period on endometrial function and atrophy.Patients and methods:We measured serum FSH, LH and E2 dynamic level with chemoluminescence, and detect endometrial protein expression of FSHR, Ki67, AQP1, AQP2 and AQP8 with immunohistochemistry of pre-menopausal group and post-menopausal group. We assessed the effect of FSH on proliferation of human primary cultured endometrial glandular epithelium and Ishikawa cells with MTT assay, on cell cycles of Ishikawa cells with flow cytometry, and on endometrial expression of AQP2 and AQP8 mRNA with Real time quantitative PCR, and also assessed the influence of AC activator and inhibitor on FSH-associated effect. We constructed surgery castrated (OVX), drug castrated (GnRHa) animal model as well as SHAM group, measured their serum FSH and E2 level using RIA assay, and observed morphology aspect and apoptosis signs of endometrial cells via transmission electron microscope. We also detected the expression level of factors related to endometrial proliferation and apoptosis using Real time PCR and Western blotting.Results:The serum FSH level in post-menopausal women increased with aging from 41-50yrs to 61-70yrs, but 71-80 yrs group did not display a higher FSH level than 61-70yrs group, however, the serum E2 showed the exact opposite trend. Compared with FSHR, AQP2, and AQP1 protein expression of child-bearing period endometrium, those of post-menopausal endometrium did not change significantly, while expression of Ki67 decreased in post-menopausal group. AQP8 protein expression increased in endometrial interstitium, but decreased in endometrial gland in post-menopausal group. FSH displayed a concentration-dependent inhibition on the proliferation of primary endometrial gland epithelium, but not on that of Ishikawa cells. High concentration FSH (50IU/L,100IU/L,200IU/L,400IU/L) up-regulated the expression of AQP2 mRNA in Ishikawa cells as a concentration-dependent way, however, it can be partly antagonized by SQ22536, an AC inhibitor. Forskolin, an AC activator, treatment led to increased expression of AQP2 mRNA in Ishikawa cells. We constructed surgery castrated group(OVX, high FSH, low E2), drug castrated (GnRHa, low FSH,low E2) and sham group(SHAM, normal FSH and E2). The uterus of Mice in OVX group developed atrophy, and was observed plenty of apoptosis cells and few apoptosis body in the transmission electron scope. The extent of morphological and ultrastructural changes of GnRHa group lay between OVX group and SHAM group. The protein expression of C-FOS, C-JUN and Bcl-2 decreased in the GnRHa group, compared those in SHAM group. The protein expression of C-FOS, C-JUN and Bcl-2 decreased drastically while Caspase3 increased in OVX group, compared with SHAM group and GnRHa group.Conclusion:High serum FSH after menopause can up-regulate uterine endometrial AQP2 partly through AC pathway, inhibit the endometrial proliferation, and promote apoptosis of endometrial cells, and eventurally regulate the physiological atrophy of uterine endometrium, directly or coordinating with low serum E2.