Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro.

ABSTRACT: PURPOSE: The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation. METHODS: The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR. RESULTS: Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2- and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P<0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4- and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05). CONCLUSIONS: The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.

SUBMITTER: Ju S 

PROVIDER: S-EPMC2826623 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature