Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.

In an effort to establish cloning technology for the rat, we tested several methods (electric stimulation, treatment with ethanol or strontium) for the parthenogenetic activation of rat oocytes. We observed marked individual differences among rats of the outbred Wistar strain in their ability to yield activatable oocytes. These differences were independent of the activation protocol and may be due to a genetic predisposition that is crucial for the parthenogenetic activation of oocytes. The activation of oocytes was dependent upon the time between superovulation of the donor animal and the collection of the embryos. Aged oocytes (derived about 24 h after superovulation) were more prone to activation by each method than were younger oocytes, and some even underwent spontaneous activation without treatment and exhibited pronuclear formation and blastocyst development. All activation methods were effective in generating parthenogenetic rat embryos, and rat parthenotes developed until implantation. However, in general, short-term (15 min) and long-term (2 h) strontium treatment was superior to stimulation by ethanol or electric pulse for parthenogenetic activation. These results will be helpful in achieving successful cloning in the rat.

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