Effects Of Trichostatin A On The Proliferation Of Buffalo Somatic Cells And Development Of Nuclear Transfer Embryos

Effects of Trichostatin A treatment on cell proliferation and expression patterns of Cx43 in buffalo fibroblasts were investigated. Cells were grown to confluence at passage 6 and subsequently treated with TSA of different concentration and treated time. The patterns of cell proliferation were investigated by the methods of MTT colorimetric assay, and also the expression patterns of Cx43 in buffalo fibroblasts were detected after TSA-treated by using real time fluorescence PCR. The results showed that the treatment of TSA could significantly inhibit cell proliferation in buffalo fibroblasts, and the effects were time- and dose-dependent; proper TSA concentration and treating time can significantly enhance the Cx43 gene expression level, and changed corresponding to the different TSA concentration and treated time. The level of mRNA for Cx43 was increased respectively 7, 9, 11-fold after treating by 0.1, 0.5, 1μmol/L TSA for 48h, was significant higher than that of the control group(P<0.05).Effect of TSA on buffalo somatic cell nuclear transfer embryos developmental potential was investigated. The reconstructed buffalo embryos were treated with TSA(0 nmol/L, 5 nmol/L, 50 nmol/L, 100 nmol/L) for 10h, there were not significant difference in cleavage rate of reconstructed embryos among the four groups(P>0.05), in group treated with 50 nmol/L TSA the blastocyte development rate was significantly higher than the control group(22.0% Vs 13.0%, P<0.05). 50 nmol/L TSA treatment for 10, 24, 36,48h resulted a significantly higher blastocyte development rate than the control group(P<0.05), but no significant difference in cleavage rate(P>0.05); also there were not significant difference among the four groups(P>0.05), however the blastocyte development rate was the highest in group with 50 nmol/L TSA treatment for 24h(26.4%). Results showed that the treatment with 50 nmol/L TSA for 24h can improve the buffalo nuclear transfer efficiency.The expression pattern of Cx43 protein in the IVF and SCNT buffalo preimplantation embryos was investigated by using immunocytochemical techniques. Results showed that Cx43 was expressed in preimplantation embryos from 2-cell to blastocyst stage, at 2-cell, 4-cell, 8-cell stage Cx43 was strongly expressed at the cell membranes of the blastomeres, but at the morula and blastocyst stage Cx43 was much less expressed, and the expression of Cx43 protein in the SCNT embryos was lower than that in IVF embryos.In conclusion: (1) TSA treatment of the buffalo fibroblasts could significantly inhibit cell proliferation, and the effects were time- and dose-dependent; (2) effect of TSA on the buffalo fibroblasts depends on TSA concentration and treating time, proper TSA concentration and treating time can significantly enhance the Cx43 gene expression level; (3) proper TSA treatment after SCNT can improve the buffalo nuclear transfer efficiency; (4) Cx43 plays an important role during the embryo development, and the low level expression of Cx43 may be the cause of the low developmental potential for buffalo SCNT embryos.