Epigenetic Characteristics of Bovine Donor Cells for Nuclear Transfer: Levels of Histone Acetylation1

Abstract

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 H1 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 H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/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 G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

“…There is increasing evidence that successful reprogramming of a donor nucleus after transfer into a recipient cytoplast is largely dependent on its epigenetic state and can be influenced by in vitro culture conditions, including passage number, serum concentration, cell density, and physical/chemical treatment (Enright et al 2003a, Shi et al 2003a. Major epigenetic characteristics of donor cells are DNA methylation and histone modifications (Shi et al 2003b).…”

“…Following nuclear transfer (NT), the histone acetylation status of the donor nucleus, which must be reprogrammed to an embryonic one, is affected by a number of factors and can be experimentally manipulated. For example, histone acetylation levels in bovine cumulus and fibroblast cells were shown to be affected by the stage of the cell cycle, cell origin, and cell passage numbers (Enright et al 2003a). Moreover, global histone acetylation in donor cells could be increased by treatment with histone deacetylase (HDAC) inhibitor, trichostatin A (Enright et al 2003b).…”

Analysis of Permissive and Repressive Chromatin Markers in In Vitro Fertilized Bovine Embryos Just After Embryonic Genome Activation

“…In fact, the lower rates of blastocysts observed for bovine and human comparing to mouse embryos might be as well a result of extended IVC, since culture environment can affect epigenetic modifications pattern (Enright et al, 2003). This aspect may contribute to the different gene transcription profiles observed between bovine embryos produced in vitro and in vivo (Wrenzycki et al, 2004).…”

“…These and other modifications could explain the altered expression of vital developmental genes later in development. Bovine cultured cells also present a disrupted pattern of epigenetic modifications (Enright et al, 2003). In this respect, we described that it is possible to artificially increase histone acetylation during in vitro culture.…”

Establishment of customized mouse stem cell lines by sequential nuclear transfer

“…Confluent NT-ES cells at passage 10 were trypsinized and resuspended in cold "saline GM" [28]. Fixed cells in 100% ethanol overnight or for one week and to stain for DNA, cells were incubated in PBS containing 10 µg/ml of propidium iodide and 0.3 mg/ml of RNase A.…”