Effect Of5-aza-dC And NaBu On Cell Growth,Cycle,Apoptosis Of Buffalo Donor Cell And The Development Of Cloned Embryos

Somatic cell nuclear transfer technique (SCNT) has been frequently associated with in vitro production of embryos, however, its low efficiency limits its application. The main reason for the low efficiency might be the incomplete reprogramming of donor cells. In order to improve the epigenetic reprogramming level and the efficiency of SCNT, donor cells or embryos are treated with DNA methyl-transferase inhibitors and histone deacetylase inhibitor. In the present study, we treated buffalo fibroblasts with DNA methyl-transferase inhibitor5-aza-dC and histone deacetylase inhibitor NaBu and assessed their morphology, growth, cell cycle and apoptosis, then introduced them to the cattle oocytes, optimizing the best way of treating donor cells of two reagents. The results are as follows:1. The effect of different concentrations of NaBu on morphology, growth, cell cycle and apoptosis of buffalo fibroblast cellsThe present experiment was conducted for optimization of the sodium butyrate (NaBu) in buffalo fibroblast cells after assessing their morphology, growth, cell cycle and apoptosis. Buffalo fibroblastic cells were treated with different concentrations of NaBu (0、0.5、1.0、2.0、2.5and3.0mmol/L) after exposure for48h. The results showed that treatment with0.5or1.0mmol/L NaBu did not affect cell morphology and resulted in unaltered early apoptosis and cell cycle phases, similarly,0.5mmol/L dosage did not change late apoptosis(P>0.05) and proliferation inhibition rate compared to control (2.75vs0,P>0.05). On the other hand, cells treated with NaBu (2.0,2.5and3.0mmol/L) increased size of the fibroblasts, a more elongated appearance and a large number of dead cells. All NaBu treatment groups (2.0,2.5and3.0mmol/L) significantly increased early apoptosis and cells in G0/G1and S phases compared to control. From these results, it can be concluded that0.5mmol/L and1.0mmol/L NaBu has little effect on the cell morphology, appearance and apoptotic rate and thus suitable as treatment of donor cells. 2. The effect of a combination of5-aza-dC and NaBu on growth, cell cycle and apoptosis of buffalo fibroblast cellsBased on our previous experimental results, we utilized0.01μmol/L5-aza-dC for72h and1.0mmol/LNaBu for48h to treat buffalo fibroblast cells and assessed their growth, cell cycle and apoptosis(P<0.05). Treatment with5-aza-dC did not affect different cell cycle phases (P>0.05), while the combined treatments of NaBu and5-aza-dC significantly increased cells in G0/G1(P<0.05); The total number of normal cells in combined treatment were significantly reduced than5-aza-dC treatment and the control group(P<0.05), but there were no significant difference with NaBu treatment (P>0.05). Combined treatment significantly increased late apoptosis compared to5-aza-dC, NaBu and control groups (P<0.05), however, there were no significant difference in early apoptosis among all experimental groups(P>0.05). The results showed that combined treatment can significantly affect cell growth, cell apoptosis and different cell cycle phases.3. The effect of5-aza-dC and NaBu on the buffalo-cattle interspecies nuclear transferIn this experiment, we used5-aza-dC, NaBu and a combination of5-aza-dC and NaBu to treat donor cells to produce the buffalo-cattle nuclear transfer embryos while the untreated group and the embryos of cattle and buffalo as controls. The development of buffalo-cattle interspecies2-cell,8-to16-cell and blastocyst was significantly lower than buffalo or cattle embryos while, treatment with NaBu (1.0mmol/L) significantly increased the growth rate of buffalo-cattle interspecies cloned embryos (8-to16-cell and blastocyst). Treatment with combination of5-aza-dC and NaBu increased the buffalo-cattle embryos(8-to16-cell) developmental rate only than the controls significantly(P<0.05), but the blastocyst developmental rate was significantly increased than5-aza-dC, NaBu and the controls(P<0.05). These results suggest that the development of interspecies nuclear transfer embryosis lower than that of cattle or buffalo nuclear transfer embryos. Furthermore,donor cells treated with NaBu and a combination of5-aza-dC and NaBu can significantly improve the developmental potential of interspecies nuclear transfer embryos.