Effects Of Microtubule Distribution Of Aged Oocytes In Mice On The Development Of Preimplantation Embryos After In Vitro Fertilization

Objective:1.The aged oocytes not only affects the normal fertility,but also has a serious impact on the progress of assisted reproductive technology.For example,in the process of in vitro fertilization,the rate of obtained eggs decreases,the fertilization rate decreases,the quality of embryos declines,and abortion probability increases during early pregnancy,abnormal development of offspring.Therefore,the study of oocyte aging and its mechanism is great importance in improving the quality of oocytes and improving the outcome of ARTs.In this experiment,the distribution of microtubules in mouse oocytes and embryos was studied to explore the effect of microtubule distribution on embryonic development after aged oocytes IVF.2.To provide reference for improving the developmental efficiency of aged egg IVF.Method:1.In this experiment,female ICR mice aged 7-8 weeks were used as experimental animals.After promoting ovulation,oocytes were removed from oviducts and then IVF was carried out;2.The experimental aging oocyte model was formed by oxidative stress induced aging in mouse oocytes treated with hydrogen peroxide（H₂O₂）at a concentration of 10、50、100、150μmol/L,and select the optimal concentration;3.The development rate of embryos in the 2,4-cell stage and blastocysts was used as the main evaluation index of oocyte quality;4.Two groups of oocytes,2 and 4 cell stage embryos and blastocysts were immunofluorescent staining respectively.The expression and distribution ofβ-Tubulin in two groups of oocytes and embryo development stages were compared.Result:1.Compared with 10,50,150,100μmol/L H₂O₂ was the optimal concentration for inducing oocyte aging;2.Mouse oocytes were treated with 100μmol/L H₂O₂ for 3 hours,then IVF,compared with the normal group,the formation rate of 2 cells,4 cells8cells and blastocyst were all reduced（P<0.05）;3.β-Tubulin forms spindles in mouse oocytes of the normal group.β-Tubulin is unevenly distributed in mouse oocytes in the aged group,mainly around the nucleus,and spindles are not seen;4.In the 2 and 4 cell phase,β-Tubulin is distributed evenly in the cytoplasm of each blastomere in mouse embryos of the normal group,and expression is increased in the cleavage furrow and the under of the cell membrane.In the aged embryos,β-Tubulin is expressed in each blastomere evenly but its expression is reduced.5.In normal mice blastocyst period showed different distribution ofβ-Tubulin in each blastomere,In some blastomeres,the spindle formed byβ-Tubulin was clearly visible.However,in other blastomeres,β-Tubulin shows a scattered distribution.The expression ofβ-Tubulin in the blastocytic spheres of the aged mouse embryos was less than that of the normal group,and spindles are not seen.Conclusion:1.Mouse oocytes treated with 100μmol/L H₂O₂ could be used as an aging oocyte model;2.The formation rate of 2 cells,4 cells 8cells and blastocyst in the aging group were all reduced.3.The disorder ofβ-Tubulin microtubulin distribution in aged mouse oocytes hinders the formation of spindles in cleavage cells and blastocysts,and also hinders the formation of contraction rings incleavage furrow.