Genome reprogramming during the first cell cycle in in vitro produced porcine embryos

Abstract

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in <i>in vitro</i> produced porcine zygotes, whereas <i>in vitro</i> produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two <i>in vitro</i> techniques commonly used for <i>in vitro</i > embryo production (<i>in vitro</i> fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after <i>in vitro</i> fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that <i>in vitro</i> matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the <i>in vitro</i> technique for embryo production.