The Effects Of Epigallocatechin-3-gallate On The Development Of Kunming Mouse One-cell Embryos Pretreated With H₂O₂and On The Distribution Of Estrogen Receptor-alpha In Two-cell Embryos In Vitro

Objectives：To investigate the effects of different concentrations of H₂O₂on thedevelopment of mouse preimplantation embryos, and to observe whether the adverseeffects of H₂O₂pretreatment could be reversed by EGCG supplement, and to test theexpression levels of ERα and phosphorylated ERα （Ser167） in the2-cell embryos.Methods:1.1-cell embryos were recovered from female KM mouse, cultured in M16medium added with different final concentrations of H₂O₂for25mins, and thenremoved respectively to M16medium or EGCG+M16medium. The developmentalpotential of preimplantation embryos was observed and recorded continually.2.1-cell embryos were recovered, and cultured in M16medium and M16+EGCG.2-cell embryos were collected and tested the expression levels of ERα andphosphorylated ERα （Ser167） by laser scanning confocal microscopy at50h-posthCG.Results:1. Low concentrations （1μM,10μM） of H₂O₂improved the developmentpotential of KM mouse preimplantation embryos.While higher concentrations of（20μM,40μM） H₂O₂had a severe hazard on the preimplantation embryo development.2. The developmental blockage caused by H₂O₂pretreatment could be reversedby the supplement of EGCG （10μg/mL）, which improved the developmental ratio of4-cell and morula embryos. However, the blastocyst ratio could not be improved.3. The observation of laser scanning confocal microscopy showed a higherintensity of p-ERα（Ser167） fluorescence in the2-cell embryos cultured in10μg/mL EGCG.Conclusion:Keeping a proper concentration of ROS has an advantage to the preimplantationembryo development. High concentrations of ROS have irreversible harm to the earlyembryos, while low concentrations of ROS improves the development of early embryos.A proper concentration of EGCG reverses the hazards caused by H₂O₂pretreatment, butcould not improve blastocyst formation. So, in this situation, the function of EGCG is toclear out ROS and to sustain the proceeding of cleavage, however, EGCG can notreverse the blastocyst formation blockage caused by high concentration of H₂O₂.EGCGimproves the level of phosphorylated ERα（Ser167） level in the2-cell embryos, but theexpression and distribution of total ERα remains unchanged, implying a posttranscription modification mechanism following EGCG treatment, rather than changesin ERα protein expression level.