DNA Methylation and Hydroxymethylation in Early Rabbit Embryos: Consequences of in vitro Culture

During the first developmental stages, the genome of the embryo is transcriptionally silent and developmental changes are under the control of maternally inherited factors. Embryonic genome activation (EGA) takes place at later stages (8/16-cell-stage in rabbit) and involves epigenetic modifications. DNA methylation at CpG dinucleotides is an epigenetic mark. CpG methylation is depleted at the early stages and reinstated at the blastocyst stage. Recent findings have shown that demethylation involves the oxidation of methylated DNA into hydroxymethylated DNA. However the role of hydroxymethylation can probably not be restricted to an intermediate in DNA demethylation. Indeed, hydroxymethylation seems involved in gene activation and maintenance of pluripotency, and could therefore be important for EGA. Several studies have suggested that in vitro conditions can have a negative impact on epigenetic reprogramming. Therefore, our aim was to investigate the impact of two culture media on methylation and hydroxymethylation in rabbit embryos. To quantify methylated and hydroxymethylated DNA, we implemented an immunofluorescence detection protocol on rabbit embryos cultured in those media until different developmental stages. Our results show that the dynamics of methylation and hydroxymethylation are different between the two culture conditions. Further investigation is needed to compare the in vitro cultured embryos to in vivo developed ones. To draw solid conclusions, it is advisable to reproduce the experiment with other species such as bovine embryos ahead of further steps to demonstrate on human embryo. Our results will be helpful for the advancement of ART which is challenged by abnormal embryonic development and unsuccessful pregnancy.