The Developmental Competence Of Oocytes Parthenogenetically Activated And Somatic Cell Nuclear Transplantation By An Eletric Pulse And Anisomycin Treatment

Parthenogenetic activation and somatic cell nuclear transfer plays an important role in animal breeding,production of transgenic animals,establishment of animal model,protecting the endangered animals and providing artificial organs xenotransplantation research and developmental biology research.The oocytes activation is one of the important factors on affecting the parthenogenetic activation and somatic cell nuclear transfer efficiency.And determined the parthenogenetic activation and nuclear transfer is also one of the basic factors of the subsequent development of cloned embryos.It is very important to improve the efficiency of oocyte activation.Anisomycin is a kind of protein synthesis inhibitors.It is first time used in bovine parthenogenetic activation and somatic cell nuclear transfer embryos for activate processing application.The results showed that anisomycin could significantly improve bovine parthenogenetic activation and somatic cell nuclear transfer embryos blastocyst rates.However,no researches have not yet been reported that anisomycin used in porcine parthenogenetic activation and somatic cell nuclear transfer embryo.In this study,we used the electrical activation post-processing with anisomycin in porcine parthenogenetic activation and somatic cell nuclear transfer embryos to observe the effect of anisomycin.We obtained the following results:1.Anisomycin,untreated group compared with CB group discharged the second polar body.Anisomycin and untreated groups are significant differences with CB group on the second polar body extrusion(51.9%and 51.6%vs.14.3%p<0.05).The results show that anisomycin cannot inhibit the second polar body eduction.2.There are significant differences between 1?/mL anisomycin and other concentrations in embryonic development to 2-4 cell stages(table3-2).There are no significant difference anisomycin group and control group(CB)on the number of cells in the blastocyst by hoechest 33342(table2).PA embryos treated with 0.01?g/mL,0.1 ?g/mL anisomycin had a greater rate of blastocyst formation compared with CB or embryos treated with 1?g/mL anisomycin(31.8%and 27.1%vs.0 and 16.7%,p<0.05).Under the optimal concentration of anisomycin,we evaluated the 0 h,2 h,4 h and 6 h the embryo development.The results show that the best condition is 4h(39.5%vs.20.3%,33.3%,and 29.2%,p<0.05).There are no difference on the number of cells per blastocyst in all groups.All this results indicate the optimum condition anisomycin is 0.01 ?g/mL for 2h.3.PA embryos treated with 0.01 ?g/mL anisomycin and 6-DMAP groups for 4h had a greater rate of blastocyst formation compared with untreated group(37.6%and 30.0%vs 19.9%,p<0.05).4.The level of MPF was significantly lower in the anisomycin-treated group than in the control group(361.3 pg/ml vs 254.2 pg/mL,p<0.05)5.Chromosomes of early blastocysts derived from 7 day parthenogenetically activated oocytes treated with anisomycin,6-DMAP and control group(CB).Anisomycin are significant differences with CB and 6-DMAP groups group on the chromosome abnormality(33.3%vs 60%and 50%,p<0.05).All this result shows anisomycin could reduce the rate of chromosome abnormality.6.The effect of anisomycin treatment on the expression of pluripotency-related genes(Oct4,Sox2,and Nanog)and apoptosis-related genes(Bcl-2 and Bax)in blastocysts were assessed.The mRNA expression levels of these genes did not differ between the control and anisomycin-treated groups.7.After SCNT,anisomycin-and 6-DMAP-treated groups compared with CB group.There are no significant in 2-4cell and number cells per blastocyst.Anisomycin compared with the untreated group,can significantly improve the SCNT embryos development(18.3%vs 4.2%,p<0.05).Conclusion:1)Anisomycin could be affected parthenogenetic activation embryos and somatic cell nuclear transfer embryos development by changing the activity of MPF.2)Anisomycin could significantly reduce the parthenogenetic activation blastocysts chromosome abnormality rate.3)The mRNA expression levels of these genes did not differ between the control and anisomycin-treated groups.