Increased cleavage rate of human nuclear transfer embryos after 5-aza-2'-deoxycytidine treatment.

As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. The present study found that the use of cytochalasin B was not necessary for, and even harmful to, the enucleation of human zygotes. This study also decreased the DNA methylation levels in reconstructed embryos using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), in an attempt to correct the abnormalities in DNA methylation that might play an important role in the failure of embryo development. After 5-aza-dC treatment and nuclear transfer (NT-Aza group), 32.7% of reconstructed embryos developed to the 8-cell stage, which is a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that of the NT group, as determined by 5-methylcytosine immunodetection. Based on the present results, this study recommends performing the enucleation procedure without cytochalasin B treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies.