Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine

Abstract

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7 and A8 had blastocyst rates of 11.7%(a), 16.7%(ab) and 20.0%(b), respectively ((ab) p < 0.05). Adult somatic cells included kidney, mammary (large), and mammary (small) also had different blastocyst rates (ab p < 0.05) of 4.2% (a), 10.7% (ab) and 18.3% (b), respectively. For clonal donor cells and for adult somatic cell groups the donor cells with the highest blastocyst rates also had methylation profiles with the lowest similarity to the methylation profiles of the in vivo-produced blastocysts. Conversely, the donor cells with the lowest blastocyst rates had methylation profiles with the highest similarity to the methylation profiles of the in vivo-produced blastocysts. Our findings show there is an inverse correlation to the similarity of the methylation profiles of the donor cells and the in vivo-produced embryos, and to the blastocyst rates following SCNT.

Figure 1

A. Clonal cell lines cultured (A2, A7 & A8) and trypsinized (A2′, A7′ & A8′). B. Somatic donor cells from mammary (A) and kidney (B) tissues. Arrows indicate the large (L) and small (S) cells that were selected from the mammary cells.

Figure 2

Hierarchical clustering of the methylation profiles of the clonal donor cells (A2, A7, and A8,), somatic cells (kidney, (K), Mammary-Large (ML), and Mammary-Small (MS)), and in vivo-produced blastocysts. Developmental potential is negatively correlated to similarity to the in vivo-produced blastocyst methylation profile. Donor cells that with the lowest blastocyst rates after SCNT had the most similar methylation profiles while donor cells with higher blastocyst rate did not cluster with the in vivo-produced blastocysts. The blastocyst rate after SCNT is shown in parentheses below each of the donor cell types.

Figure 3

Clones with similar methylation profiles in the donor cells and the in vivo-produced blastocysts were clustered by using Self Organizing Map analysis. Hypermethylation (A) and hypomethylation (B) of the donor cells are correlated with lower blastocyst rates after SCNT. Those shown above were found to be different (P<0.01) in at least one of the biological samples of donor cells.