| In order to testify the effect of oocyte vitrification on DNA methylation, this experiment studied the DNA methylation patterns of different methylated regions at imprined genes (H19, Peg3, Snrpn) in fresh and vitrified oocytes, the relative expression of enzymes associated with DNA methylation were also studied. Results showed that, the DNA methylation status in vitrified oocytes were similar with fresh oocytes. Compared to fresh oocytes, the relative expression levels of DNA methytransferases, Dnmtl, Dnmt3a, Dnmt3b and Dnmt3l, were significantly declined (P<0.05) in vitrified oocytes, and the relative expression level of DNA demethyltransferase, Tet3, was also significantly declined (P<0.05) in vitrified oocytes.Secondly, the DNA methylation patterns of different methylated regions at imprinted genes in blastocysts derived from fresh oocytes and vitrified oocytes in vitro were examined. Results showed that the methylation level of the H19DMR in vitrified group (30.95%) and fresh group (32.15%), had no significant difference (P>0.05), the average methylation level of Peg DMR was30.19%in vitrified grou, which was significantly lower than that of fresh group (35.38%)(P<0.05), the average methylation level of DMR at Snrpn (18.13%) was notable lower than that of the fresh group (35.48%)(P<0.05). Compared to fresh goups, both the relative expression levels of the three DNA methyltransferases (Dnmtl, Dnmt3a and Dnmt3l), and three related enzymes for DNA demethylation (Tetl, Tet2and Tet3), were not siginificantly changed in vitrified groups (P>0.05). The Dnmt3b relative expression levels were significantly lower than those in fresh frozen group groups (P<0.05).Wether the oocyte vitrified could produce transgenerational effect on DNA methylation, the DNA methylation patterns of imprinted genes and retrotransposons were examined in E13.5primordial germ cell in fetus derived from vitrified oocytes.(1) In the E13.5female primordial germ cell, average DNA methylation level at H19DMR in vitrified group (3.34%) was significantly lower than that of fresh group (16.44%)(P<0.05), and in male primordial germ cell there was no differences between two groups (P>0.05), which were in completely demethylation.(2) In the female primordial germ cell of vitrified group, the DNA methylation level of Peg3DMR was5.88%, which was significantly lower than that of fresh group (0%)(P<0.05), and in males primordial germ cells in vitrified group, the methylation level of Peg3DMR was41.97%which was notable (P<0.05) higher than that of fresh group (0%).(3) The DNA methylation status of Snrpn DMR in E13.5primordial germ cell, both females and males primordial germ cells were demethylation. There was no difference between frozen and fresh groups.(4) The DNA methylation at Line1-5’ in13.5female primordial germ cells of vitrified group was38.02%, which had no difference compared to fresh group (36.51%)(P>0.05). While in males primordial germ cells the DNA methylation level of the Group (14.03%) in vitrified group was significantly higher than the fresh group (6.25%)(P<0.05).(5) f The DNA methylation at IAP-LTR in E13.5female primordial germ cells of vitrified group (36.37%) was significantly lower than that of fresh female (48.18%), while in males primordial germ cells in vitrified group, the DNA methylation level (93.93%) was significantly higher than the fresh group (57.14%).To sum up, vitrification of mouse M Ⅱ oocytes did not affect DNA methylation of imprinted genes in oocyte, vitrification of oocytes reduced the relatived abundance of DNMTs and TETs mRNA. DNA methylation levels at imprinted genes in blastocysts derived from vitrified oocyte s were significantly lower than the fresh group. Oocytes vitrification affected the relative expression level of Dnmt3b in blastocyst, vitrification oocytes showed that will influence the late embryonic DNA methylation reprogramming. Peg3DMR and retrotransposon repeated sequences showed abnormal methylation patterns in primordial germ cells obtained from vitrified oocytes. |
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