Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine

Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells.

Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 μM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4.

In comparison with untreated cells, 0.01 and 0.08 μM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 μM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 μM 5-aza-dc treated cells when compared with 0 and 0.01 μM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 μM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 μM 5-aza-dc remained unchanged.

These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.