23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

Abstract

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.