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Previously, we reported that the osmolarity conditions in the satellite region were affected CpG DNA methylation status while Pre-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. This study was conducted to investigate the DNA methylation status of repeat sequences in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05 M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. The DNA methylation status of the Pre-1 sequences in blastocysts was characterized using a bisulfite-sequencing method. Intriguingly, in the present study, we found the unique DNA methylation at several non-CpG sequences at the Pre-1 sequences in all groups. The non-CpG methylation was hypermethylated in all three groups, including in vivo group (86.90% of PZM- 3; 83.87% of NaCl; 84.82% of sucrose; 90.94% of in vivo embryos). To determine whether certain non-CpG methylated sites were preferentially methylated, we also investigated the methylation degree of CpA, CpT and CpC. Excepting in vivo group, preference of methylation was CpT>CpC>CpA in all three groups investigated. These results indicate that DNA methylation of Pre-1 sequences was hypermethylated in CpG as well as non-CpG site, regardless modification of osmolarity in a culture media.
