110 COMPARATIVE ANALYSIS OF DNA AND HISTONE H3 METHYLATION REPROGRAMMING IN MOUSE, BOVINE, AND RABBIT ZYGOTES

Active demethylation of paternal DNA in zygotes has been documented for many mammalian species, including mouse (Mayer et al. 2000 Nature 403, 501–505), rat (Zaitseva et al. 2007 Mol. Reprod. Dev. 74, 1255–1261), pig (Fulka et al. 2006 Histochem. Cell. Biol. 126, 213–217), Homo sapiens (Fulka et al. 2004 Reproduction 128, 703–708), and cow (Beaujean et al. 2004 Curr. Biol. 14, 266–267). The generality of this concept has been challenged by reports stating that there is no detectable genome-wide paternal DNA demethylation in rabbit (Shi et al. 2004 Biol. Reprod. 71, 340–347) and ovine (Beaujean et al. 2004 Curr. Biol. 14, 266–267), whereas others report partial evidence for the opposite (Zhang et al. 2005 Mol. Reprod. Dev. 72, 530–533). In order to compare the fate of DNA and histone H3 methylation (H3/K4triMe and H3/K9diMe), we performed studies on mouse, bovine, and rabbit zygotes using specific antibodies in an indirect immunofluorescence approach. We observed a similar distribution of these epigenetic modifications in maternal and paternal pronuclei of the zygote, indicating that the overall epigenetic reprogramming activities — including the DNA demethylation of the paternal pronucleus — are conserved between mammalian species. For all 3 species we found a clear asymmetry in mouse monoclonal antimethylcytosine (MeC) antibody signal intensities between paternal and maternal pronuclei at advanced stages, which suggests the presence of paternal DNA demethylation. To obtain additional evidence for the presence of DNA demethylation activity in rabbit zygotes, we examined 1-cell embryos after somatic cell nuclear transfer (SCNT) using rabbit fetal fibroblasts as nuclear donors, and we found a strong signal reduction in SCNT embryos 4 h after activation. For the majority of SCNT 1-cell embryos, we estimated at least a 4- to 6-fold decrease of MeC signal. Along with the similarities of DNA demethylation kinetics in rabbit, mouse, and bovine zygotes, we found a conserved distribution of H3/K4triMe and H3/K9diMe signals on maternal and paternal chromatin. In all 3 species, H3/K4triMe, which predominantly demarcate open chromatin, is present in both pronuclear chromatin, whereas H3/K9diMe, a hallmark of condensed chromatin, is strongly enriched in maternal pronuclear chromatin and undetectable in paternal chromatin. Furthermore, we observed a similar asymmetric compartmentalization of paternal and maternal chromosomes in early 2-cell rabbit and bovine embryos as monitored by H3/K9diMe, which marks maternally derived chromosomes. In summary, our data suggest that mechanisms of epigenetic reprogramming are conserved in mammalian species both on the level of DNA and chromatin methylation.