Expression And Regulation Of CD82and S100A10/Annexin A2in Embryo Implantation And Relationships With Establishment Of Endometrial Receptivity

I. ObjectiveEmbryo implantation is a process that requires both temporal and spatialsynchronisation of the uterine endometrium and the embryo. A series of molecules inthe endometrium change dynamically in menstrual cycles. Especially during thewindow of implantation, expression of some molecules facilitates the process of embryoimplantation. Glycoproteins, found on cell surfaces, are involved the adhesion of cellsto other cells, and adhesion of cells to the extracellular matrix. CD82is a glycoproteinthat belongs to the transmembrane4superfamily and it can inhibit metastasis. Decidualcells at the maternal-fetal interface express a significant amount of CD82, indicating thatCD82may participate in embryo implantation.The endometrial cycle is regulated by estrogen and progesterone. Indeed,progesterone plays critical roles for the embryo implantation and is required for themaintenance of pregnancy. In humans, the receptivity of endometrium for embryoadhesion is regulated closely by progesterone. Progesterone and the progesteronereceptor are the targets of many contraceptive drugs. Mifepristone (RU486) is acommon contraceptive drug that acts as a competitive progesterone receptor antagonist.There are many contraceptive mechanisms of RU486, but the relationship betweenRU486and fucosyltransferase IV which is a marker of endometrial receptivity has notbeen reported.The S100A10is also called Annexin A2ligand. S100A10/Annexin A2tetramertransfers into cell membranes and is involved in many biological functions. It has beenreported that S100A10is highly expressed at implantation sites in mice and rhesus monkeys of early pregnancy. Annexin A2increases progressively during gestation inhuman fetomaternal interface.In this study, we research the expression and regulation of CD82, S100A10andAnnexin A2on cell surface, and evaluate their roles on cell surface during implantation.The study will promte the further elucidation of the mechanism in embryo implantaion.II. Methods1. Expression and localization of CD82, S100A10and Annexin A2in the humanendometrium were analysed by immunohistochemistry.2. The recombinant vector was transfected into cells.3. The expression of CD82and FUT4were detected by the methods of RT-PCR.4. The expression of S100A10and Annexin A2were detected by the methods ofreal-time PCR.5. The expression of CD82, FUT4, S100A10and Annexin A2were detected bythe method of Western blot6. Indirect immunofluorescence staining was used to detect CD82protein.7. Detect the percent adhesion of JAR cell adhesion to RL95-2or HEC-1A cellmonolayer.III. Results(I) CD82expression alters with human endometrial cycles and affects the uterineendometrial receptivity in vitro1. Immunohistochemistry found that the highest expression of CD82occurred inmid-and late-secretory phases which are considered the window of implantation.2. The expression of CD82on RL95-2cells was also higher than on HEC-1A cellsby RT-PCR, Western blot and indirect immunofluorescence staining.3. Progesterone induced the expression of CD82in both RL95-2and HEC-1Acells by RT-PCR and Western blot4. RT-PCR, Western blot and indirect immunofluorescence staining found that theexpression of CD82was dramatically decreased by CD82-siRNA transfection incomparison to untransfected controls. The percent adhesion of JAR cells to RL95-2cellmonolayer was inhibited by CD82-siRNA or CD82antibody. Inversely, the percentadhesion of JAR cells to HEC-1A cell monolayer was promoted by CD82expressionplasmid.5. To study the effect of CD82expression on FAK signaling pathway, tyrosinephosphorylation of FAK and pTYr was assessed by Western blot. In RL95-2cells, CD82knocking down cells expressed less pFAK and pTYr. Meanwhile, overexpressionof CD82in HEC-1A cells could increase the synthesis of pFAK and pTYr.(II) Mifepristone(RU486) decreases the expression of fucosyltransferaseIV mediated by CD82and inhibits the adhesion of embryos in vitro1. RU486inhibited the adhesion of JAR cells to RL95-2monolayer2. RT-PCR detected the inhibition of RU486on the expression profile of variousFUTs in RL95-2cells. RU486predominantly decreased FUT4mRNA expression.3. RU486inhibited the expression of FUT4in RL95-2cells and in humanendometrial tissues by RT-PCR and Western blot.4. RU486inhibited the expression of CD82in RL95-2cells by RT-PCR andWestern blot.5. CD82-siRNA inhibited the expression of FUT4in RL95-2cells. Meanwhile,overexpression of CD82increased the expression of FUT4in RL95-2cells.6. Inhibition of embryonic adhesion by RU486correlated with RU486/CD82/FUT4signaling pathway.(III) Cyclic changes of S100A10and Annexin A2expression in humanendometrium1. Immunohistochemistry and Western blot found that the highest expression ofS100A10and Annexin A2protein occurred in mid-secretory phases in the cytoplasmand cytomembrane of the luminal and glandular epithelia, scatteredly and weakly instromal cells.2. Relative mRNA quantity of S100A10and Annexin A2was analyzed byreal-time PCR. S100A10and Annexin A2mRNA was significantly up-regulated duringmid-secretory phases of the menstrual cycle compared with other stages.IV. Conclusions(I) CD82expression alters with human endometrial cycles and affects the uterineendometrial receptivity in vitro1. The highest expression of CD82occurred in the window of implantation.Meanwhile, the expression CD82on RL95-2cells is higher than which on HEC-1Acells. The results indicate that CD82may been considered as an endometrial receptivitymarker.2. Progesterone facilitates embryo implantation by up-regulating CD82-dependentembryo-endometrial adhesion. Therefore, CD82plays a role in embryo implantation.3. CD82can regulate the FAK signaling pathway in RL95-2and HEC-1A cells and participates embryo implantation.(II)Mifepristone(RU486) decreases the expression of fucosyltransferase IVmediated by CD82and inhibits the adhesion of embryos in vitro1. FUT4is regulated by progesterone recepter and participates embryoimplantation.2. CD82facilitates embryo implantation by up-regulating FUT4-dependentembryo-endometrial adhesion.3. RU486inhibits embryonic adhesion through an RU486/CD82/FUT4pathway.(III) Cyclic changes of S100A10and Annexin A2expression in humanendometrium1. The highest expression of S100A10occurred in the window of implantation.The results indicate that S100A10may been considered as an endometrial receptivitymarker.2. Annexin A2is necessary for establishment of endometrial receptivity and maybeen considered as a marker.