Maternal O‐ GlcNAc Transferase Is Required for the Asymmetry of Epigenetic Modifications in Mouse Zygotes

After fertilization in mammals, there is an epigenetic asymmetry reflected by differences in DNA demethylation and histone modifications between female and male pronuclei (FPN and MPN, respectively). Based on its expression level and amount, we investigated the role of maternal O-GlcNAc transferase (OGT), a key enzyme mediating O-GlcNAcylation, in regulating this asymmetry. By using a specific small-molecule inhibitor and small interfering RNA (siRNA)-mediated knockdown of OGT during oocyte maturation in mice, we evaluated the downstream effects on epigenetic modifications and early developmental capability. OGT inhibition significantly reduced fertilization rates and led to developmental arrest at the zygote or 2-cell stage, whereas the siRNA-mediated decrease of Ogt mRNA had less or no significant effect on preimplantation development. Immunostaining analyses revealed that OGT inhibition reduced 5-hydroxymethylcytosine levels in MPN, attributed to a reduction in Tet methylcytosine dioxygenase 3. In contrast, FPN showed delayed epigenetic changes, with the loss of 5-methylcytosine protection mediated by H3K9me2. Moreover, OGT inhibition increased histone methylation levels in MPN and disrupted epigenetic and size asymmetry between FPN and MPN. These alterations suggest that maternal OGT regulates multiple layers of epigenetic reprogramming in early zygotes. Taken together, these findings suggest that maternal OGT is essential for maintaining epigenetic asymmetry between parental pronuclei, primarily by modulating DNA demethylation and histone methylation in MPN.