| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Flow cytometry demonstrated the expression of the beta_1, alpha_1, alpha_2, alpha_3, alpha_5, and alpha_6 subunits of beta_1 integrin family in cultured decidual cells. Autoradiography of irnmunoprecipitate subjects to SDS-PAGE revealed three major polypeptides with molecular weights of 130 kD (beta_1 subunit), 165 kD (alpha_2 subunit), and 210 kD (alpha_1 subunit) under reducing conditions. These data demonstrated that endometrial stromal and decidual cells expressed beta_1 integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, beta_1 integrins in human endometrium and decidua may be important in mediating the organization of ECM proteins derived from embryos during the early stage of implantation. Although the area of trophob last outgrowth in the presence of a mouse monoclonal IgGI antibody was increased during 96 h of culture, an
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ti-beta_1 antibody blocked the outgrowth of trophoblasts in a dose-dependent manner. Both the incidence and extent of trophoblastic outgrowth also were significantly reduced in the presence of antibodies against the alpha_1, alpha_2, alpha_5, and alpha6 subunits. These observations suggest that beta_1integrins on decidual cells may be involved in blastocyst development and differentiation following attachment. Double stainings for FAK and phosphotyrosine, FAK and talin, and FAK and beta_1 integrin demonstrated that FAK colocalized with integrins in cellular focal adhesions. Treatment of decidual cells with herbimycin A, a tyrosine kinase inhibitor, did not affect the rate of hatching or attachment of blastocysts. However, the outgrowth of embryos on the decidual cells was inhibited by addition of herbimycin A in a dose-dependent manner, implying that blastocyst attachment and outgrowth are mediated by different mechanisms. This study suggest that tyrosine phosphorylation of FAK on decidual cells may be important in development and differentiation following attachment. The sequence integrinbeta_1[140-164] is referred as the Arg-GIy-Asp (RGD) cross-linking region. The corresponding peptide to integrin beta_1[l40-164] (DDL ; DYPIDLYYLMDLSYSMKDDLENVKS) inhibited decidual cell attachment to FN-coated dish in a dose-dependent manner. The variant integrin peptide in which both Asp 157 and Asp 158 were replaced by Ala (AAL ; DYPIDLYYLMDLSYSMKAALENVKS), had no effect on decidual cell attachment to FN.Treatment of decidual cells with synthetic peptides did not affect the rates of hatching and attachment of blastocysts. The outgrowth of embryos on decidual cells was inhibited by addition of DDL peptide in a dose-dependent manner, but AAL peptide did not affect the outgrowth of embryo. These findings suggest that integrin beta_1[140-1641 on decidual cell may be important region in embryonic development and differentiation following attachment. Less
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