DNA methylation and DNA methyltransferase 1 expression during early embryonic development

Methylation of specific CpG residues in the vertebrate genome is critical for cellular differentiation and embryonic development. The regulation of DNA methylation during bovine early embryonic development is subject to much controversy and little information is available regarding the expression of DNA methyltransferase 1 (Dnmt1), the enzyme responsible for maintenance of genome-wide methylation. In our first study we identified transcripts coding for the somatic form of Dnmt1 (Dnmt1s) and to its splice variant (Dnmt1v), both ubiquitously transcribed in all samples analyzed. Levels of Dnmt1s transcripts were shown to be reduced until 8-cell stages of development when compared to immature oocytes and embryos from 16-cell until blastocyst stage. Day 16 conceptuses showed reduced levels of Dnmt1s expression which were comparable to that observed in somatic tissues. Immunocytochemical localization studies revealed that Dnmt1 is retained in the cytoplasm of oocytes and embryos until the 8-cell stage undergoing nuclear translocation at the 16-cell stage. Dnmt1 was found in the nucleus of Inner Cell Mass (ICM) cells and not trophectodermal (TE) cells of expanded blastocysts which suggest its involvement in early cell-lineage differentiation. Confocal image densitometric analysis showed that in vitro fertilized (IVF) embryos show a gradual decrease in DNA methylation levels until the 8-cell stage. After that these levels decline sharply (53%) being reestablished by the 16-cell stage. Thereafter, DNA methylation levels remained stable until the blastocyst stage when the ICM appeared undermethylated compared to the TE cell lineage. In contrast, nuclear transfer (NT) embryos displayed only a gradual loss of DNA methylation levels throughout early development. DNA methylation patterns in IVF embryos were even within the nucleus of early embryos until the 32-cell stage when patterns composed of large foci were established. Taken together, our data show that Dnmt1s transcript levels, timing and localization positively correlate with DNA methylation changes undergone by cattle pre-attachment embryos and that a novel active demethylation and de novo methylation events occur in IVF but not in NT embryos. These results allow for a better understanding of the epigenetic deregulations that lead to poor developmental capacity of embryos.