Whole Genome Methylation Sequencing And Analysis Of Bovine In Vivo And In Vitro Blastocysts

As an efficient assisted reproduction technology,in vitro embryo production is of great significance to improve the reproductive efficiency and genetic improvement of livestock.Vitrification has the advantages of low cost,simple operation and high efficiency.It is increasingly used by the majority of scientific research workers and clinical staff.However,there are differences in cell metabolism,freezing tolerance and ultrastructure between embryos produced in vitro and in vivo,which may be related to abnormal expression of imprinted genes and abnormal epigenetic modification.Compared with fresh oocytes,vitrification affects the ability of development and fertilization of oocytes,which is related to the effect of vitrification on the expression of important developmental genes during oocyte maturation.In this study,single cell whole genome methylation sequencing technique was used to analyze the whole genome methylation level and DMR region cluster analysis of bovine in vivo blastocysts,fresh oocyte IVF blastocysts and vitrified oocytes IVF blastocysts.The GO annotation and KEGG functional enrichment of differentially methylated genes were preliminarily explored.The main results are as follows:1.The whole genome methylation of IVF blastocysts produced in vivo and fresh oocytes were sequenced and analyzed.The results of the current study show that the whole genome methylation level of blastocysts produced in vivo were higher than the IVF blastocysts of fresh oocytes.Among the 1149 DMR regions,the methylation level of 1099 DMR regions of blastocysts produced in vivo were higher than the IVF blastocysts of fresh oocytes.GO annotation of differentially methylated genes showed the presence of many genes enriched in cell growth and development,cytoskeleton,ATP binding etc.KEGG functional enrichment analysis was used to analyze the functional enrichment of differentially methylated genes.Differentially methylated genes were enriched in PI3K-Akt pathway,oocyte meiotic pathway,oocyte maturation pathway etc.Finally,the important differentially methylated genes screened were of IGF1,SVIL and CDC20.2.The whole genome methylation of fresh and vitrified oocytes IVF blastocysts was sequenced and analyzed.The results showed that the whole genome methylation level of the IVF blastocysts of fresh oocytes were higher than the IVF blastocysts of vitrified oocytes.Among the 151 DMR regions,122 DMR regions of fresh oocyte IVF blastocysts were higher than those of vitrified oocytes IVF blastocysts.GO annotation of differentially methylated genes showed that there were many genes enriched in cell growth,cell development,cytoskeleton etc.Through KEGG functional enrichment analysis,differentially methylated genes were enriched in MAPK pathway.Finally,the important differentially methylated genes screened were FAM3 D,EGFR and MAP4K4.3.The whole genome methylation of blastocysts produced in vivo and vitrified frozen oocytes IVF blastocysts were sequenced and analyzed.The results showed that the whole genome methylation level of blastocysts produced in vivo was higher than that of vitrified frozen oocytes IVF blastocysts.Among the 1572 DMR regions,1546 DMR regions of blastocysts produced in vivo were higher than the IVF blastocysts of vitrified oocytes.The differentially methylated genes were annotated by GO.The current study indicated the presence of many genes enriched in cell growth and development,ATP binding etc.By KEGG functional enrichment analysis,there were differentially methylated genes in gap junction,endoplasmic reticulum processing,oocyte meiotic division,oocyte maturation etc.Finally,the important differentially methylated genes screened were IGF,IGF1 R,IGF2BP3 and FGFR2.