42 EFFECT OF S-ADENOSYLHOMOCYSTEINE, A NON-TOXIC EPIGENETIC MODIFYING REAGENT, ON PORCINE FEMALE DONOR CELLS AND CLONED EMBRYOS

Abstract

Despite great advances in the field of cloning techniques, the efficiency of production of cloning animals is very low. Maybe the poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell or cloned embryos. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH), the reversible nontoxic inhibitor of DNA methyltransferases (DNMT), on porcine female fibroblast donor cells and in vitro development of cloned embryos. We hypothesized that SAH targeting DNA methylation could alter chromatin configuration and turn it more amenable to reprogramming. Thus, the female fibroblast donor cells were cultured in media containing respective concentrations of SAH [0 (control), 0.1, 0.5, and 1 mM) for 2 passages. One-way ANOVA was used to determine significant differences in the data and a Tukey test was done to determine statistical differences among groups. Compared with nontreated controls, the cells treated with SAH, especially 1 mM, revealed significantly (P < 0.05) reduced global DNA methylation, proved by commercial kit and immunocytochemistry analysis, and elevation of transcript levels for X chromosome-linked genes (XIST and HPRT), estimated by real-time PCR analysis compared with the control group. It was suggested that treatment with SAH in female cells could make cells into more valuable donor cells for cloning. In another trial, cloned embryos using normal donor cells were cultured in media containing 1 mM SAH for 0 (control), 12, and 24 h after activation on different time interval of DNMT inhibition, transferred to PZM5 media, and subsequently cultured for 7 days. Treatment with SAH for 12 h resulted in 13.0 ± 1.9% blastocyst production, which was significantly greater than cloned embryos treated with SAH for 24 h (11.2 ± 2.1%) and control cloned embryos (9.1 ± 1.2%). It was suggested that the appropriate DNMT inhibition might have an important role in in vitro development of porcine SCNT, and improving effects on developmental competency of cloned embryos. We concluded that SAH induced global DNA demethylation that partially reactivated the X chromosome and that a hypomethylated genome may facilitate the nuclear reprogramming process. This study was supported by IPET (no. 311011-05-1-SB010), MKE (no. 10033839-2012-21), Institute for Veterinary Science, the BK21 program, and TS Corporation.