The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming.

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.