The Research Of DNA Methylation And Histone H3K36 Trimethylation In Mouse Oocytes And Early Embryos

The development of oocytes and early embryonic were regulated by epigenetic modifications,and the mechanism can also be revealed about the growth,maturation,fertilization,early embryo cleavage and development of oocytes by probing epigenetic modifications.The epigenetic pattern is the crucial criterion to evaluate the quality of oocytes during the process of oogenesis,meanwhile,it can affect many important biological events,for instance: affect the maturation of oocyte and the development of early embryo.The modification of histone H3K36 trimethylation plays an important role in regulating the transcriptional activity and methylation of gene,and its dynamic remodeling process is similar to DNA methylation during oocyte growth and embryonic development.The research about the two kinds of epigenetic modification patterns of DNA methylation and H3K36 trimethylation and the transcription level of related enzymes,which reveals the oogenesis,oocyte genome DNA methylation patterns and the regulation of DNA methylation enzymes in this paper.At the same time,the dynamic changes of maternal and early embryonic H3K36 trimethylation were observed during the accumulation and function of maternal regulatory genes,and the transcription level of related enzymes was studied in the assisted reproductive process to stimulate follicular development,in addition this hormone stimulation will reduce the quality of the eggs.We further studied the effect of hormones on the methylation of oocytes and the transcription level of related enzymes during egg generation,based on the study of DNA methylation pattern and gene transcription level in vivo.A large number of epigenetic modification and abnormalities of maternal modulation gene occurred though SCNT technology.However,the abnormal modification of H3K36me3 has important theoretical significance for the abnormal development of embryonic during SCNT.Therefore,we further research the transcriptional expression of H3K36 trimethylation and the cumulus cell nuclear transfer embryos during the activation of zygotic gene based on the study of the methylation of oocytes and embryos H3K36 in vivo.Finally,the single cell transcription technique was used to analyze the transcription of DNA methylation,H3K36 trimethylation and embryonic development related genes during the activation of zygotic gene.The results of this study will provide important theoretical support for the research of mammalian oocytes and early embryos,meanwhile provide new ideas for assisted reproductive technology and livestock breeding.Experimental Materials: mouse immature oocytes(hormone +/-),mature oocytes,in vivo prokaryotic embryos,in vivo 2-cells embryos,cumulus cells nuclear transfer prokaryotic and cumulus cell nuclear transfer 2-cell embryos.Experimental methods: indirect immunofluorescence,qPCR,somatic cell transplantation,single cell transcription analysis.Experimental results:1.The methylation degree of GV oocytes was significantly higher than the non-injected group after injecting of PMSG,of which the degree of NSN methylation was significantly higher than others(NSN-N 7054.16±994.23,SN-N4950.64±799.06,NSN-P 17336.75±2179.49,SN-P 9195.5±832.75,p<0.05).The transcription level of DNMT1 was significantly higher in NSN-N than in other groups,while DNMT3A and DNMT3L mRNA were significantly higher in NSN-P and the trend of expression was similar to that of methylation.2.The H3K36me3 NSN configuration of GV oocytes was significantly higher than that of SN(SN 819.10±84.89 VS NSN 3198.78±142.08,p<0.05).3.H3K36me3 is in a dynamical condition(GV 2118.36±124.81、MII726.15±45.41、合子158.25±17.3)during the oocyte maturation and early embryonic development.The transcription level of setd2 during the oogenesis was significantly reduced(p<0.05)The expression of mRNA of setd2 was significantly decreased during oocyte maturation(p<0.05).4.H3K36me3 keep a dynamical change(GV 2118.36±124.81、MII726.15±45.41、zygote 158.25±17.3、2cell 566.87±44.73,p<0.05)and reconstruct after the lowest zygote period during the oocyte maturation and early embryonic development.In addition,H3K36me3 modification is asymmetric in the zygote period,and male nucleus is not expressed.5.H3K36me3 lack of reconstruction in the prokaryotic and 2-cell phase,and maintain little difference level of modification with fertilization 2 cell during nuclear transfer(zygote 158.25±17.3、2 cell 566.87±44.73、NTC–zygote 508.13±39.83、NTC-2 cell 607.82±39.44).Conclusion: 1.There are no direct relation between H3K36 trimethylation and DNA methylation related enzyme genes in mouse oocytes and embryos,i.e.DNA methylation was not directly regulated by H3K36 trimethylation.2.Nuclear transfer affect H3K36 trimethylation,maybe,the Dnmt1、Dnmt3a、 Dnmt3 L and Elp3 are related to H3K36 trimethylation,and the Setd2、MRG15、Dnmt3b、Trim28、Tet3 as well as the Dppa3 are not related to H3K36 trimethylation during this stage.3.Hormones affect DNA methylation of oocytes though Dnmt3 s rather than DNMT1.4.Reveal the dynamic expression patterns of H3K36 trimethylation in ovums and embryos.