42 DEVELOPMENTAL POTENTIAL AND REPROGRAMMING EFFICIENCY OF BOVINE EMBRYOS CLONED WITH ADULT FIBROBLASTS TREATED BY A DEMETHYLATING AGENT, S-ADENOSYL-HOMOCYSTEINE

Abstract

Animal cloning by somatic cell nuclear transfer (SCNT) has been successfully applied to several species although with low efficiency and often associated with severe abnormalities. These poor outcomes are thought to be a consequence of aberrant DNA methylation patterns that result from incomplete epigenetic reprogramming of the transplanted nucleus into recipient oocytes. Telomerase, an enzyme not expressed in most somatic cells, should be expressed in cloned embryos. Therefore its activity has been used as an index of reprogramming in SCNT embryos. The objective of this study was to investigate the DNA methylation status of donor fibroblasts treated with a non-cytotoxic transmethylation inhibitor, S-adenosyl homocysteine (SAH), and to assess the relative telomerase activity (RTA) and developmental potential of SCNT embryos derived from such cells. Adult ear skin fibroblasts were cultured in DMEM supplemented with 0, 0.5, 1.0, or 2.0 mM SAH for 144 h by daily media change prior to nuclear transfer. The SAH-treated fibroblasts were immunostained with a fluorescein isothiocyanate (FITC) conjugated 5-methylcytosine antibody and the relative fluorescence intensity (RFI) was analyzed using a fluorescence microscope equipped with an Openlab^" program (Improvision, Coventry, UK). RTA was measured in Day 8 SCNT blastocysts using the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP). Fibroblasts treated with 0.5, 1.0, and 2.0 mM SAH showed lower levels of DNA methylation compared to nontreated controls, and the values did not differ among the treatment groups. Cleavage rates did not differ between the SCNT embryos derived from 0.5 mM SAH-treated cells and nontreated control cells (92.3% vs. 91.3%, respectively). However, the rates of blastocyst development and hatching were significantly (P < 0.05) higher in SCNT embryos derived from 0.5 mM SAH treated donor cells compared to controls (60.0 and 40.0% vs. 34.3 and 26.4%, respectively). Moreover, RTA of the 0.5 mM SAH SCNT embryos was significantly (P < 0.05) increased (1.5-fold) in relation to controls. S-adenosyl homocysteine treatment induces global DNA demethylation in donor fibroblasts and enhances the blastocyst frequencies for bovine SCNT embryos that also exhibit greater telomerase activity levels. These results suggest that use of hypomethylated donor somatic cells increases the developmental potential for SCNT embryos by enhancing the nuclear reprogramming efficiency.

This work was funded by NSERC, OMAF, OCAG, and CRC.