Effects Of Gamete Cryopreservation On DNA Methylation Pattern In Mouse In Vitro Fertilized Early Embryos

The present study is designed to investigate the effect of mouse gamete (including spermatozoa and oocyte) cryopreservation on DNA methylation pattern during in vitro fertilized early embryo development.Firstly, zygotic pronuclear formation and early embryo development were detected after fertilization with fresh and freezed sperm, showing that freezed sperm declined normal fertilization rate (P< 0.01) and delayed pronuclear formation progression (P< 0.05) and resulting early embryos acquired significantly declined cleavage rate and blastocyst formation rate compared with fresh sperm (P< 0.05). Genome-wide DNA methylation level of various stage zygotes were examined by immunofluorescent staining, showing that zygotes fertilized with freezed sperm obtained significantly increased relative DNA methylation level at late zygotic stage (P< 0.05). Moreover, relative mRNA transcript abundance of Tet3 was detected by real time quantitative PCR, indicating that relative mRNA expression of Tet3 was remarkably decreased in zygotes fertilized with freezed sperm compared with fresh sperm (P< 0.01).Secondly, expression and distribution of DNA methyltransferases in fresh and vitrified oocytes and subsequent early embryos were examined by immunofluorescent staining, showing that abnormal expression of DNA methyltransferases was observed in vitrified oocytes and subsequent early embryos. Relative mRNA transcript abundance of DNA methyltransferases was detected by real time quantitative PCR, indicating that relative mRNA expression of all three DNA methyltransferases was substantially decreased in vitrified oocytes, whereas Dnmt3b still keeps a lower expression level at blastocyst stage compared with fresh oocytes (P< 0.05).Finally, Bisulfite sequencing PCR was used to analyse the methylation status of gene promoter region of Dnmtl, Tet3, Dppa3 and Sall4, which are related to DNA demethylation in fresh and vitrified oocytes and subsequent early embryos. Relative mRNA transcript abundance of four genes was detected by real time quantitative PCR. Results showed that the methylation level of Tet3 promoter had a rise that exceeded 10% in vitrified oocytes, derivative zygotes,2-cells and blastocysts. The percentage of hypermethylated clones rose significantly in vitrified oocytes and blastocysts (P< 0.05), relative mRNA expression of Tet3 was significantly declined compared with fresh oocytes accordingly (P< 0.05). The methylation level of Dnmtl promoter experienced a more than 10% decline in vitrified oocytes, but showed only a slight change in early embryos. Surprisingly, relative mRNA expression of Dnmtl also had a significant reduction in vitrified oocytes (P<0.05), while there was no significant difference at blastocyst stage. Moreover, the hypomethylation of Dppa3 and Sall4 promoter was remained throughout entire early embryo development, as well as the high relative mRNA expression.In conclusion, sperm cryopreservation disturbed the progression of DNA demethylation relugated by TET3 in zygotic paternal genome and thus delayed the pronuclear formation in zygotes, which could have adverse effect on the early embryo development. Vitrification caused the abnormal expression of DNA methyltransferases in oocytes, which should be one of important reasons for abnormal DNA methylation pattern during early embryo development. Furthermore, abnormal promoter methylation statuses and mRNA expression of Tet3 and Dnmtl found in vitrified oocytes shoud be responsible for the abnormality of DNA demethylation during zygotic development.