Effects Of Trichostatin A (TSA) On The Morphology, Proliferation Of Somatic Cells And The Development Of Nuclear Transfer Reconstructed Embryos In Yanbian Yellow Cattle

Recent years, histone acetylation involves in a variety of chromatin-mediated mitosis, which has a very important role in embryonic development. Researchers have been focused on the process and development of reconstructed embryos. The selection of donor cells can significantly improve the rate of blastocyst. In this experiment, histone acetylation enzyme inhibitors trichostatin A (TSA) was added to somatic cells with different concentrations and durations to test its effects on the morphology and the proliferation of somatic cells in Yanbian Yellow Cattle, and then investigate its effects on nuclear transfer reconstructed embryos. The research result shows as follows:1. The purpose of this study was to investigate the inhibitory effect of TSA on somatic cells, MTT was utilized to draw cellular growth curve, and also to study the effect of different handling time on the morphology of cells. The results shows:low concentration groups (5、10ng/ml group) of TSA had little toxic effect on those cells, the inhibitory phenomenon came outs after24h, but there was no significant difference at36h between10、25ng/ml groups (P>0.05), this might due to the appearance of drug resistance, as culturing time became longer, significant difference appeared at48h(P<0.05). However, cellular toxic effect increased in high concentration groups (25、50、100ng/ml groups), most cells were dead in100ng/ml group compared with control group, TSA had significant inhibitory effect on the growth of the somatic cells in Yanbian yellow cattle, and it showed time-dependent and concentration-dependent phenomenon.2. The effects of different TSA concentrations on the cleavage and blastocyst rates of reconstructed embryos were determined.10ng/ml and25ng/ml TSA handing groups had signification higher (P<0.05) cleavage and blastocyst rate of reconstructed embryos than those which were in control group of and (10ng/ml vs.25ng/ml) handing groups, but there was no significant difference between those two groups(73.35%vs.75.94%, P>0.05). The blastocyst rate in25ng/ml treatment group was significantly higher than the10ng/ml treated(13.88vs.17.56, P<0.05) group. These results showed the optimum TSA concentration was25ng/ml, and it can be used somatic cell cloning in Yanbian yellow cattle.3. Investigated the effect of TSA different treatment time on the nuclear transfer embryo cleavage and blastocyst rate. There was no significant difference between treatment groups at12h and24h, but cleavage and blastocyst rate in24hours processing time was higher than12hours group. From the results it can determine that24hours processing time was better than12hours. Two time group was significantly higher than control group in cleavage (70.81%,75.17%vs.66.18,P<0.05)and blastocyst rate (16.91%,17.92%vs.ll.09%,P<0.05).4. The effects of different culture methods on the blastocyst rate of reconstructed embryos with TSA treated somatic cells were also tested. The result showed that the cleavage and blastocyst rate of nuclear transfer reconstructed embryos with somatic cells which were treated with25ng/ml TSA for24hours in granulosa cell co-cultured method have slightly higher than conventional culture in Yanbian yellow cattle.