Dynamics of DNA-methylation in rabbit embryos during preimplantation | Dinâmica da metilação do DNA em embriões de coelho no período pré-implantação

DNA methylation in the early embryo bas been considered important for differentiai parental genome reprogramming, embryonic genome transcriptional activation (EGA) and further development. However, DNA methylation dynamics (DNA-MD) seems to differ among species and may be altered by the in vitro culture environment. The rabbit thus appears to be a pertinent madel for analyses of the epigenetic events involved in EGA regulation due to its delayed EGA as in most mammals. Moreover, in these animais, bath in vivo and in vitro developed embryos are easy to obtain. However, the variation ofDNA methylation was never quantified during the preimplantation development of in vivo-developed rabbit embryos and paternal pronucleus demethylation is still controversial in these animais. This study reported DNA-MD in in vivo-developed (IVD) and in vitro cultured (IVC) rabbit embryos from one cell to the blastocyst stage. IVC embryos were collected after in vivo fertilization and cultured in B2 medium. Imrnunostaining for 5-methyl cytosine and DNA labeling by EthD2 were performed and the embryos were analyzed by fluorescence quantification. The results showed that DNA-MD differed between the maternai and paternal pronuclei in IVD zygotes. The maternai pronucleus displayed a constant DNA methylation level over the one cell stage, resulting in a maintenance methylation throughout DNA replication. On the other hand, in the patemal pronucleus, partial demethylation occurred before replication, probably by active DNA demethylation, while a maintenance methylation took place over the S-phase. Moreover, a demethylation took place during cell cleavages in IVD and IVC rabbit embryos. Nonetheless, DNA-MD differed between the IVD and IVC groups. This study provided the frrst detailed description of DNA-MD in rabbit embryos and taken together, the extent of these in vivo results paves the way to the analysis of the consequences of different in vitro culture conditions in DNA methylation.