The methylation of cytosine residues in CpG dinucleotides, which is associated with transcriptional silencing [14], is an example of an epigenetic process that must be reprogrammed during preimplantation development. It has been shown that mammalian genomic DNA undergoes gradual demethylation during preimplantation development and subsequently becomes methylated de novo during postimplantation development (de novo methylation occurs at CpG dinucleotides on a strand of DNA whose complement is not already methylated) [15,16,17,18]. To examine the dynamics of this process in more detail, Dean and colleagues [13] used antibodies against 5-methylcytosine that mainly detect densely methylated cytosine residues within highly and interspersed repetitive sequences. Their data revealed that, in the mouse, DNA within the larger paternal pronucleus is significantly demethylated by a putative demethylase activity after fertilization but before the first S phase (Figure (Figure1).1). This replication-independent demethylation is conserved in eutherian mammals: cattle, pigs and rats all exhibit active global demethylation of the presumed paternal genome. Asymmetric behavior of the parental genomes is corroborated by the replication-independent demethylation of the paternal allele of several single-copy genes in mouse zygotes [19]. During mouse and bovine preimplantation development, the maternal genome is also demethylated, notably in a replication-dependent manner (Figure (Figure1).1). Reduction in global methylation is first observed at the 2-cell stage and proceeds through the next cell divisions, suggesting that the maternal genome is somehow protected from the active demethylase activity and instead undergoes passive demethylation [20]. Both mouse and cattle genomes are hypomethylated by the 8- to 16-cell stage. In contrast to the mouse, in which genome-wide de novo methylation begins in the inner cell mass (ICM) of the blastocyst-stage embryo, in bovine embryos this process commences at the 8- to 16-cell stage, before blastocyst formation. As a result, there is divergence in the methylation pattern between mice and cattle, with bovine blastocysts exhibiting methylation both in the ICM and in cells of the outer cell layer (trophectoderm). Similar results were reported in the mouse by Rougier and colleagues [21] and in cattle by Bourc'his et al. [22]. Differential demethylation (and topological separation) of the maternal and paternal genomes continues through to at least the 4-cell stage in the mouse, after which the two parental genomes have attained roughly the same levels of global hypomethylation [23]. Taken together, these data show that the parental genomes undergo extensive changes in global methylation during preimplantation development.

The results discussed here point to an impaired ability of clones to appropriately reprogram the somatic genome. The retention of a donor-like methylation profile in reconstituted embryos may be explained by the possible presence of the somatic form of DNA methyltransferase 1 that may be introduced by the original donor cells [26]. In the presence of this enzyme the genome-wide demethylation characteristic of preimplantation embryos may fail to be recapitulated. Impaired epigenetic reprogramming capabilities of the somatic donor DNA during the earliest stage of clone embryogenesis may be one of the factors contributing to the lethality and abnormal development of cloned animals. Continuing investigations that examine the reprogramming events during normal and clonal embryonic preimplantation development will yield further insights into the molecular mechanisms that regulate the transformation of gametic epigenetic marks into the appropriate embryonic patterns in various mammals and the extent to which cloned embryos can reprogram their somatic genome.