Effect Of Different Types And Treatments On The Level Of Histone Acetylation In The Sheep Donor Cells And Cloned Embryos

The epigenetic reprogramming of the somatic cell genome has been suggested as a key event occurring in the SCNT, if the reprogramming is not completed, the efficiency of SCNT will be reduced. Different cell type and the treatments will affect the potential of reprogramming. In this study, the fibroblast and the cumulus cells were dealed with Trichostatin A (TSA), Roscovitine (ROS), serum starvation and contact inhibition. We detected the change of acetylation, the cell cycle, the development of cloned embryos after the treatments, the level of acetylation in IVF, cumulus cell cloned embryos, fibroblast cell cloned embryos, and the fibroblast cell dealed with TSA. The results are as followaed:1. The cell morphology and the motility rate were detected, the fibroblast cells were becaming flat, but the cumulus cells'motility rate was quite lower than the fibroblast cells after dealed with TSA. After dealed with different density of TSA, the two types level of acetylation were increased obviously, the cumulus cells were higher, and 10ng/mL TSA was suitable for the two types of cells.2. The fibroblast and the cumulus cells were treated with Trichostatin A(TSA), Roscovitine(ROS), serum starvation and contact inhibition seperately, the motility rate kept at 94%. After treated with TSA, the level of acetylation that detected by the indirect immunofluorescence was increased obviously(P<0.05). The level of acetylation was also increased by treated with ROS, but lower than the TSA treatment (P<0.05), differently, the serum starvation was lower than the control. We found that the cumulus cells had a higher level of acetylation than the fibroblast cells. The cell cycle was analyzed by flow cytometry , The cell stage was stoped at the G0/G1 after dealed with TSA.3.The highest cleavage and blastula rate (P<0.05, 81.2±3.4% vs 57.3±8.9%; 28.2±6.3% vs 7.1±7.6%) were obtained after the donor cell dealed with TSA, when the donor cell was fibroblast cells. Oppositely, a low cleavage and blastula rate were founded when the donor cell was cumulus cells.4. The cloned embryos were obtained a quite higher lever of acetylation than the other treatments(P < 0.05)after dealed with TSA,the serum starvation treatment was also lower than the control,when the donor cell was fibroblast cells. However, when the donor cell was cumulus cells, the cloned embryos were not detected any discrepancy significant exept TSA; Without any treatment, a higher level of acetylation were detected in the cumulus cells cloned embryos than the fibroblast cells.5. The distribution of acetylation in either group of cloned embryos did not resemble that in IVF embryos suggested that reprogramming of this epigenetic mark was aberrant in cloned embryos and could not be corrected by treatment of donor cells with TSA.