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DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of spermderived pronucleus have been reported in mammals. In the first, the male pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono-and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.

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