127. EPIGENETIC REPROGRAMMING IN ZYGOTES INVOLVES THE GLOBAL CYTOSINE DEMETHYLATION OF BOTH THE PATERNAL AND MATERNAL GENOMES

Abstract

Epigenetic reprogramming is essential for normal development and has been held to occur in a different manner for the paternally and maternally inherited genomes. The current paradigm implicates active global demethylation of the paternal pronucleus soon after fertlization, but passive demethylation of maternally-derived genome over many cell-cycles. This parent-of-origin difference has been difficult to reconcile with other biological processes prompting us to re-examine this evidence. DNA methylation levels were examined in mouse zygotes by immunolocalization with methylcytosine specific antibodies. Zygotes were isolated from the oviduct at times after hCG and staged for pronuclei maturity (PN1-5, least to most mature) or metaphase commencement. We found methylation levels to be high in PN1-2 stage pronuclei but then progressively declined. By PN5 stage methylcytosine staining was greatly diminished. Yet, contrary to the current paradigm, demethylation generally occurred in both the male and female pronucleus. We found no methylcytosine staining in any metaphase chromosomes. The contrast of our results with those widely cited prompted us to review the methodology previously used. In previous studies zygotes that had been collected after fertilization and then cultured in vitro, or produced by IVF and then cultured were used. When we prepared zygotes by these methods we found that many PN5-stage cultured zygotes displayed relatively more demethylation of the male pronucleus than the female. When zygotes were generated by IVF this asynchrony was further exacerbated. In contrast to the zygotes collected directly from the reproductive tract, metaphase chromosomes in cultured post-syngamal zygotes commonly showed extensive methylcytosine staining. Our results show that the normal process of epigenetic reprogramming in the mouse involves global demethylation of both the paternal and maternal genomes. This was variably perturbed (particularly in the female pronucleus) by IVF and zygote culture.