Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer

• Epigenetic status of pig embryonic germ cells were evaluated when they were in vitro cultured at different passages.
• Expression level of genes related to DNA methylation and histone modification varied with passages of embryonic germ cells.
• Developmental competence of cloned pig embryos was affected by the passages of embryonic germ cells.

Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages. In addition, the expression level of H3K27me3 functional methylase EZH2 increased while no changes were observed on H3K27me3 demethylase JMJD3 in relation to passage number. Moreover, the expression levels of both the H3K4me3 methylase MLL1 and the H3K4me3 demethylase RBP2 were increased at high passages. By using lower passage (numbers 3–5) EG cells as donor cells, the SCNT efficiency was significantly lower compared with use of fetal fibroblast donor cells. However, similar blastocyst rates were achieved when using higher passage (numbers 9–12) EG cells as donor cells. In conclusion, the present study suggests that the epigenetic status of EG cells change with increasing passage numbers, and that higher passage number EG cells are better primed for SCNT.