Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5 nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50 nM) for 1 h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5 nM 5-aza-dC for 1 h and subsequently treated with 50 nM TSA for another 1 h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5 nM 5-aza-dC and 50 nM TSA for 1 h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5 nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.