Procaine and S-adenosyl-L-homocysteine (SAH) affect the expression of genes related to the epigenetic machinery and change the DNA methylation status of in vitro cultured bovine skin fibroblasts

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterise the effects of procaine and S-adenosyl-L-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterised the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH (P=0.0495) and by the combination of SAH and procaine (P=0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells [procaine (P=0.0116), SAH (P=0.0408), and both (P=0.0163)]. Regarding the transcriptional profile, there was a decrease in total DNMT transcript levels in cells cultivated with SAH and procaine. There was a higher level of TET3 transcripts in treated cells than in the controls. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.