Impaired Active Demethylation of the Paternal Genome in Pronuclear-Stage Rat Zygotes Produced by In Vitro Fertilization or Intracytoplasmic Sperm Injection

This study was designed to investigate the effect of in vitro production systems of rat zygotes such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) on demethylation dynamics of the paternal genome. Immunostaining with anti-5-methyl-cytosine indicated that in vivo derived zygotes harvested at 20 hr post-hCG injection had no active demethylation of paternal genomes as a mean relative methylation (RM) value, total fluorescence in the paternal genome divided by that in maternal one, at 1.17. Leaving zygotes in vivo or in culture for an additional 4 or 8 hr resulted in significant decreases of RM values to 0.14-0.31. Since in vitro-derived zygotes were produced using oocytes harvested at 14 hr post-hCG injection, zygotes at 6 hr after IVF or ICSI were considered developmentally comparable to the in vivo derived zygotes harvested at 20 hr post-hCG injection, with RM values at 1.04 and 0.92, respectively. At 10 hr post-IVF and ICSI, the RM values of the in vitro derived zygotes decreased significantly, butto a lesser extent compared with in vivo derived zygotes, to 0.53 and 0.62, respectively, without further decreases at 14 hr. Treatment of IVF-derived zygotes with 5-aza-2'-deoxycytidine (5-azadC; an inhibitor for methylation) or trichostatin A (TSA; an inhibitor for deacetylation) resulted in the decreased RM values at 14 hr post-IVF. However, developmental potential of the 5-azadC- or TSA-treated IVF zygotes up to the blastocyst stage was not improved. Thus, the demethylation dynamics of the paternal genome in pronuclear-stage rat zygotes was impaired by routine protocols for in vitro embryo production such as IVF and ICSI.