The influence of demethylating agents on preimplantation development of mice.

Abstract

The effects of two demethylating drugs with a different mechanism of action (5-azacytidine (Aza) and L-ethionine (Eth)) on mouse preimplantation development were investigated. Preimplantation embryos were cultured for 24 h in the presence of the drug and for an additional 24 or 48 h (depending on the cleavage stage) in medium supplemented with bromodeoxyuridine to reveal sister chromatid exchanges (SCEs) and the number of cell cycles performed before harvesting. Striking differences between the two drugs were observed in their influence on proliferation of blastomeres, primary differentiation and sister chromatid differentiation (SCD), and in the pattern of DNA methylation and the frequency of SCEs per cell. At a final concentration of 1 microM Aza had no effects, whereas higher concentrations stopped development of all stages except the zygote. In contrast Eth treatments (5 mM) resulted in a severe reduction of the mean cell number per embryo in comparison with controls. Moreover both the absence of blastocyst formation and no effects on mitotic activity were detected. The most prominent effect of Eth was detected at the zygote and 4-cell stages. An unexpected decrease in SCE frequency in Eth-treated morulae and 4-cell embryos has been observed. Data are explained taking into account the different mechanisms of action of the agents.