Superovulation Influences Methylation Reprogramming and Delays Onset of DNA Replication in Both Pronuclei of Mouse Zygotes

Although an essential component of assisted reproductive technologies, ovarian stimulation, or superovulation, may interfere with the epigenetic reprogramming machinery during early embryogenesis and gametogenesis. To investigate the possible impact of superovulation particularly on the methylation reprogramming process directly after fertilization, we performed immunofluorescence staining of pronuclear (PN) stage embryos with antibodies against 5mC and 5hmC. PN stage embryos obtained by superovulation displayed an increased incidence of abnormal methylation and hydroxymethylation patterns in both maternal and paternal pronuclear DNA. Subsequent single-cell RT-qPCR analyses of theTet1,Tet2, andTet3 genes revealed no significant expression differences between PN stage embryos from spontaneously and superovulated matings that could be causative for the abnormal methylation and hydroxymethylation patterns. To analyze the possible contribution of TET-independent replication-associated demethylation mechanisms, we then determined the 5mC and 5hmC levels of PN stage mouse embryos using immunofluorescence analyses after inhibition of DNA replication with aphidicolin. Inhibition of DNA replication had no effect on abnormal methylation and hydroxymethylation patterns that still persisted in the superovulated group. Interestingly, the onset of DNA replication, which was also analyzed in these experiments, was remarkably delayed in the superovulated group. Our findings imply an impact o...

Source: Cytogenetic and Genome Research - October 29, 2018 Category: Genetics & Stem Cells Source Type: research