Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos.

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.