| Somatic cell nuclear transfer technology not only proves that highly differentiated nucleus can be reprogrammed to achieve pluripotency under certain conditions,but also demonstrates the advantages of this technology in the protection of endangered species and conservation of improved varieties.Although somatic cell cloned animals have important application value,the low birth efficiency of cloned animals severely restricted the application and development of porcine somatic cell cloning technology.There are many factors that result to the low developmental efficiency of cloned embryos produced by somatic cell nuclear transfer technology.Recent studies have shown that including cytosine DNA Methylation,chemical modification of histone modification sites,genomic imprinting,and chromatin remodeling.Epigenetic reprogramming plays an extremely important role in the development of cloned embryos.Methylation modification is a ubiquitous form in the mammalian genome,which can achieve cell differentiation by regulating the transcription and inhibition of genes during development,and making the expression of the gene tissue-specific.In addition,methylation may be involved in gene imprinting and play a key regulatory role in the early development of the embryo.However,in cloned embryos,there are still many unknown reprogramming processes for DNA methylation that need to be explored.The aim of this study was to compare the methylation dynamics of fertilized embryos and cloned embryos in pig germ cells and pigs at different stages by RRBS sequencing,and to reveal the DNA methylation reprogramming rules of pig cloned embryos before planting,and to find out the key genes of embryonic developmental abnormality,in order to improve the development efficiency of cloned pigs,expand the application of cloning technology in scientific research and production,and provide theoretical basis for this.The results showed that:1.The methylation levels of porcine germ cells,in vivo embryos and cloned embryos at different development stages(1cell,2cell,4cell,8cell,Morula and ICM at blastocyst stage)were detected by RRBS method for the first time.Through mapping the Cp G island in the process of embryonic development and methylation in the promoter region of the dynamic trend of porcine reproductive cells and embryos were studied and the distribution characteristics of cloned embryos methylation level and change of gametes and embryos in vivo analysis of pigs and the distribution of the cloned embryo methylation levels,two embryos were identified in methylation phases: the first is sperm egg,sperm to methylation;The second time is the oocyte demethylation after the zygote genome activation,and the lack of demethylation in the development of cloned embryos can also be directly reflected.In addition,by studying the methylation level of m C in pig germ cells and in vivo embryos and cloned embryos,it was found that the decrease of methylation level in embryos was mainly related to the decrease of m C(80%-100%)with high methylation degree to m C(0%-40%)with low methylation degree.2.For the first time,the methylation of Cytosine base in pig oocytes and sperm was analyzed through RRBS,and 26115 DMRs were obtained.According to the methylation degree,1059 oocyte-specific DMR and 17810 sperm-specific DMR were found,and 3852 related genes were enriched and annotation by GO analysis.Then 14,492 DMR and 47,116 DMR were found by analyzing oocytes,sperm and 1cell embryos,respectively,and 2996 and 5,557 related genes were enriched and annotated by GO analysis.After that,DMR analysis of pig embryos and cloned embryos(1cell,2cell,4cell,8cell,Morula and ICM at blastocyst stage)at different development stages was carried out,and relevant DMR was obtained successively and relevant genes were enriched and annotated by GO analysis.By studying the number and methylation degree of oocyte-specific and sperm-specific DMR in embryos and cloned embryos in vivo,the genes involved in embryonic development were found,and the reasons for the low development efficiency in cloned embryos were expounded.3.The development efficiency of cloned embryos can be significantly improved by adding DZNep and UNC0642 into the in vitro culture system of pig cloned embryos(PZM3)with the final concentrations of DZNep and UNC0642 at 10 n M and 5n M respectively and the treatment time at 24h(P<0.05).DZNep can inhibit the expression of EZH2 gene,reduce the level of H3K27me3 in early cloned pig embryos,and promote embryo development.UNC0642 can inhibit the expression of GLP/G9 a gene,reduce the level of H3K9me2 in early cloned pig embryos,and promote embryonic development,but it is not capable of regulating the level of H3K9me3 in embryos.The experiment found that the processing of DZNep and UNC0642 not only effectively corrected the abnormal histone methylation modification in cloned embryos,but also improved the quality of embryos,promoted the expression of pluripotent genes and significantly improved the development efficiency of cloned embryos.Conclusion:1.The methylation level in Cp G islands decreased rapidly after fertilization,but decreased slowly with the development after 2cells stage.The methylation level in the Cp G islands of cloned embryos decreased slowly than in-vivo embryos with the development of the embryo,which lead to the insufficient of demethylation.The methylation levels of promoter region decreased rapidly and reached a stabilized levels after sperm-egg binding.After 8 cells,the methylation level of in-vivo enbryos decreased rapidly again and the dynamics of methylation in promoter region of cloned embryo was similar as in-vivo embryos.2.Oocyte-specific and Sperm-specific DMR is not only largely absent in cloned embryos,but also the main cause of low embryonic development efficiency due to the gene expression changes caused by its high degree of methylation.3.When DZnep and UNC0642 were used as histone methylation modification drugs to clone embryos,they can significantly promote the efficiency of embryo development and improve the quality of embryos. |
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