Effect Of Vitrification On Methylation Pattern Of Genomic DNA In Bovine Oocytes

The cryopreservation technology of mammalian oocytes provides an egg bank for livestock embryo biotechnology,which is conducive to the long-term preservation of genetic resources of endangered and rare wild animals,and is an important way to maintain species diversity and even human fertility,has important theoretical significance and application value.So far,vitrification has been successfully used for cryopreservation of oocytes in many mammals.At the same time,the effect of vitrification on the cytological damage and epigenetics of oocytes has become a key bottleneck restricting its application potential.In this study,we used single-cell whole-genome methylation sequencing technology to analyze the genome-wide methylation level,DMR region clustering,imprinted genes and GO annotation and KEGG preliminary exploration of functional enrichment of differential methylation genes in bovine GV stage oocytes and their MII oocytes.The main results are as follows:1.Whole genome methylation sequencing and analysis of fresh and vitrified bovine GV oocytes.The results showed that vitrification did not significantly affect the methylation level of whole genome of bovine GV oocytes.A total of 140 DMRs regions were screened in this experiment,fresh GV oocytes had 76 DMR regions with higher methylation levels than frozen GV oocytes,and 64 DMR regions with lower methylation levels than frozen GV oocytes.And differentially methylated genes related to oocyte maturation?TSC2?,cytoskeleton?NUDC?,and cell viability?MAFK?were screened in DMRs.Our experiment indicated that vitrification significantly increased the methylation level of imprinted gene IGF2 R.Based on GO annotation analysis,it was found that the differential genes were mainly involved in cell development,cytoskeleton organization,cell connection,protein binding,ATP binding and other items.Based on KEGG enrichment analysis,it was found that differential genes were mainly concentrated on the pathways such as PI3K-Akt,GnRH,and oocyte meiosis.Among them,in the oocyte meiosis pathway,the significantly upregulated genes,ADCY1 and APC1 affect the oocyte meiosis and cell development process.2.Whole genome methylation sequencing and analysis of MII oocytes from fresh and vitrified bovine GV oocytes after in vitro maturation?IVM?.The results showed that the whole genome methylation level of fresh MII oocytes was higher than the MII oocytes from vitrified GV oocytes.A total of 1308 DMR regions were screened in this experiment,fresh MII oocytes had 847 DMR regions with higher methylation levels than MII oocytes from vitrified GV oocytes,and 461 DMR regions with lower methylation levels than MII oocytes from vitrified GV oocytes.And differentially methylated genes such as VWF?PDGFRL?COL8A1 were screened in DMRs.Our experiment indicated that vitrification significantly reduced the methylation level of imprinted gene SNRPN,and the methylation level of imprinted gene DGAT1 had no changed in bovine MII oocytes.Based on GO annotation analysis,it was found that the differential genes were mainly involved in cytoskeleton organization,embryo development,cytoplasm,spindle,protein binding and other items.Based on KEGG enrichment analysis,it was found that the differential genes were mainly concentrated on the pathways such as MAPK,PI3K-Akt,Wnt,apoptosis and Notch signaling pathway.Among them,in the Notch signaling pathway,genes?RBPSUH?MAML?EP300?ADAM17?NCSTN?up-regulation and genes?HDAC1₂?NOTCH1?FNG?DLL?JAGGED?down-regulation will affect oocyte maturation and growth and development,thus affecting embryonic development.In conclusion,our results showed that vitrification of bovine ooyctes at GV stage signficanlty altered their whole genome methylation levels during the IVM progress.