Reconstructed embryos from terminally differentiated somatic cells revealed high levels of genomic methylation which results into inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during development of clones. Improvement in cloning competency maybe achieved through modification in epigenetic markers of donor cells.
Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-2'-dc) (0-0.01-0.08 and 0.3 µM), a DNA methyl transferase inhibitor, improved development and if this treatment can change cellular characteristics in Bovine Ear Fibroblast cells (BEFs) such as cellular morphology, growth, proliferation, cell cycle, apoptosis and epigenetic markers passage 5.
5-aza-2'-dc inhibited cell proliferation at all tested concentrations in a dose dependent manner. Additionally, 5-aza-2'-dc changed morphology of cells, especially at the highest dose. In such a way that untreated cells had significant connection to the substratum compared with treated cells, which appeared elongated and less connected. Moreover 5-aza-2'-dc induced apoptosis in a dose-dependent manner. 5-aza-2'-dc at lower concentration showed little effect on cell cycle. However 0.3 µM 5-aza-2'-dc resulted in an increased cell population at the G0/G1 stage. Increased 5-aza-2'-dc concentrations led to elevated in acetylation of H3K9 and reduction in DNA methylation. In compare to untreated cells. Treating cells with 0.01 and 0.08 µM 5-aza-2'-dc decreased blastocyst rate insignificantly (32.1% vs. 28.6% and 27.2% respectively) while it was significant for 0.3 µM treated cells (6.5%). The quality of embryo (TCN) decreased in a dose-related fashion which reaches to significance level in 0.08 and 0.3 µM 5-aza-2'-dc treated cells in compare with 0 and 0.01 µM 5-aza-2'-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 µM 5-aza-2'-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation. The epigenetic markers of embryos cloned from 0.01 µM 5-aza-2'-dc were remained unchanged.
In conclusion these results show that 0.01 µM 5-aza-2'-dc had less deleterious effects. Nonetheless this chemical is not a suitable choice for modifying nuclear reprogramming. Finally we could conclude that wide genomic hypomethylation induced by 5-aza-2'-dc have deleterious effects on developmental competency of cloned embryos.