Epigenetic characteristics of bovine donor cells for nuclear transfer: Levels of histone acetylation

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone Hi in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone HI and acetyl histone H3 increased with cell passage (passages 5-15) in G(0)/G(1)- and G(2)/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G(0)/G(1)-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G(0)/G(1) into the S and G(2)/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.