DNA methylation and hydroxymethylation in rabbit embryos: effect of in vitro culture
Résumé
DNA methylation is deeply reprogrammed in early mammalian embryos. After an initial phase of demethylation, that is thought to be linked with the embryonic genome activation (EGA) and the reprogramming to totipotency, remethylation at the blastocyst stage goes along with the first differentiation steps. The discovery of 5-hydroxymethylcytosine as an intermediate of active DNA demethylation but also as a potential player of gene activation and of pluripotency has added a new layer of complexity to methylation reprogramming in early embryos. While mouse is the most widely studied model of mammalian development, it has a very early EGA (2-cell stage) that sets it apart from other mammalian species and makes it difficult to separate reprogramming events linked with fertilization from those linked to EGA. We have thus decided to take advantage of the rabbit model which exhibits a later EGA (8/16-cell stage), and have recently reported that demethylation in rabbit embryos is delayed as compared to mouse and that methylation reaches its lowest point at the time of EGA. In addition, our study has shown that the kinetics of demethylation in in vitro cultured embryos differs from that of in vivo developed ones. In vitro culture is widely used for human Assisted Reproductive Technologies (ART), and it is of major importance to determine the impact it can have on epigenetic reprogramming. Indeed, in some cases consequences of inappropriate reprogramming during early development can be observed only much later in life, which makes them difficult to observe and evaluate. Moreover, different kinds of culture media are currently in use, and there is so far very little evidence to help deciding which is the most appropriate one. In this work, we have compared the dynamics of methylation and hydroxymethylation during preimplantation development of in vivo fertilized rabbit embryos that were collected at the 1-cell stage and subsequently cultured in vitro in 2 different media commonly used in human ART. Our first results indicate that there is indeed an effect of the culture medium used on DNA hydroxymethylation.