Expression Of Transcription Factor FoxO1 In The Endometrium During Ordinary Menstrual Cycle And Deciduas Of Early Pregnancy And Study On The Regulation Of FoxO1

In human the transformation of the mucosal lining of the uterus from endometrium to dicidua is essential for normal implantation of the blastocyst and duration of pregnancy.Decidualization represents a process of morphological and biochemical differentiation.The period between implantation and menstruation,i.e. 7-14 days after the luteinizing hormone surge,is critical for the endometrium to make the decision whether to continue the process of decidualization or to break down and be shed at menstruation. Up to 50% of all pregnancy loss happen in this period around implantation.Little is known about the molecular basis of the decidualization.Thus,increased attention to the changes occurring during this time may be critical to understand the normal implantation and be particularly helpful for some infertility and the implantation failure seen in IVF.FoxO1 has been identified as one gene that regulates a number of decidualization specific genes,i.e. IGFBP1,PRL,DCN,TIMP3 and CNR1. Overexpressing FoxO1 caused a change in cell shape, in that the stromal fibroblasts acquired a decidual cell-like appearance. It is implied FoxO1 significantly increases in PRL promoter activity and significantly decreases in TIMP3 promoter activity.Researchs are not found abount the protein expression and cellular localization of transcription factor FoxO1 in the endometrium during ordinary menstrual cycle and deciduas of early pregnancy in detail. Using RT-Q-PCR, immunohistochemical analysis and western blot, the expression and cellular localization of transcription factor FoxO1 in the endometrium during ordinary menstrual cycle and deciduas of early pregnancy is analyzed.And the same time,the researchs about the regulation of FoxO1 in endometrium are few.The research is to determine whether FoxOl is regulated by cAMP,P,E and HCG in order to find the regulator of FoxO1 in endometrium stroma cell,and the related signaling pathway about FoxO1.Part one: Expression of transcription factor FoxO1 in the endometrium during ordinary menstrual cycle and deciduas of early pregnancyObjective:To investigate the protein expression and cellular localization of transcription factor FoxO1 in the endometrium during ordinary menstrual cycle and deciduas of early pregnancy in detail.Method: Using RT-Q-PCR, immunohistochemical analysis and western blot, the expression and cellular localization of transcription factor FoxO1 in the endometrium during ordinary menstrual cycle and deciduas of early pregnancy is analyzed.Endometrial samples were obtained from women undergoing hysterectomy for myoma of uterus and cervical intraepithelial neoplasia,including 12 cases of proliferative phase,7 cases of early secrectory phase,10 cases of mid-secrectory phase and 10 cases of late-secrectory phase.Decidua were obtained at the time of elective first-trimester pregnancy termination of pregnancies(n=12).Results: Epithelial cells showed weak FoxO1 immunostaining in cytoplasm in Proliferative phase and strong FoxO1 immunostaining in cytoplasm and nucleus in secretory phase.In contrast, we observed that in stroma the expression of FoxO1 was confined to the secretory phase of the cycle and early pregnancy,and FoxO1 immunostaining was in cytoplasm in early and mid secretory phase and mainly in nucleus in late secretory phase. Immunoreactivity for FoxO1 was found in cytoplasm of deciduas in early pregnancy. During proliferative phase endometrium exhibited the lowest level of FoxOl mRNA and protein by RT-Q-PCR and western blot. FoxO1 mRNA and protein was significantly up-regulated in the mid-and late-secretory phases,and levels of FoxO1 mRNA and protein were highest in early pregnant decidua.The expression of FoxO1 between mid and late secretory phase has no difference.Conlusion:The expression level of FoxO1 in human endometrium fluctuates with the menstrual cycle,and reaches its peak during the mid-and late-secretory phases.If pregnancies happen,the expression of FoxO1 is further increased.Part two: The regulation of FoxOl in human endometrium stroma cell and the expression of FoxO1 in vitro decidualizationObjective:To determine whether FoxOl is regulated by cAMP,P,E and HCG in order to find the regulator of FoxO1 in endometrium stroma cell and investigate the expression of FoxO1 in vitro decidualization.Method: Using RT-Q-PCR, cytoimmunohistochemical analysis and western blot,the regulation of FoxO1 by cAMP,P,E and HCG in endometrium stroma cell was studied. The expression of FoxO1 in vitro decidualization was investigated.ResuIts:Compared with the control group, the expression of FoxO1 mRNA in the P,P+E and HCG groups has no difference(P>0.05).The expression of FoxO1 protein was not detected by western blot in the P,P+E, HCG and control groups. Compared with the control group, the expression of FoxO1 mRNA and protein in the cAMP and cAMP+P groups was significantly up-regulated(P<0.05). Compared with the cAMP group, the expression of FoxO1 mRNA and protein in cAMP+P group was increased(P<0.05). In the cAMP group, the expression of FoxO1 was confined to the nucleus of endometrium stroma cell by cytoimmunohistochemical analysis.By contrast, in the cAMP+P group endometrium stroma cell showed FoxO1 immunostaining in cytoplasm.During in vitro decidualization induced by cAMP,there was the expression of FoxO1 protein.Conlusion:A regulation of FoxOl in vitro by P,P+E and HCG could not be detected.The expression of FoxO1 was regulated by cAMP.P can cooperatively stimulate the expression of FoxO1 with cAMP. During in vitro decidualization induced by cAMP,there was the expression of FoxO1 protein.Part three: cAMP-PKA signaling pathway and the expression of FoxO1Objective:To study whether cAMP-PKA signaling pathway is involved in the modulation of FoxO1.Method:We test the effet of H89,a PKA inhibitor on the expression of FoxO1 in endometrium stroma cell treated with cAMP and cAMP+P.Results:The expression of FoxO1 in endometrium stroma cell treated with cAMP and cAMP+P was decreased by H89(P<0.05).Conlusion: cAMP-PKA signaling pathway is involved in the expression of FoxO1.