Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage.

The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.