Studies On The Effect Of Scriptaid And Caffine On Sheep Somatic Cell Nuclear Transplantation

Somatic cell nuclear transplantation (SCNT) is a powerful technique which is benefit for animal breeding, extincted animal preservation and it also plays an important role for the solution of many basic biological problems. However, the overall efficiency of SCNT is very low and its low successful rate has become the main hurdle that restricted the application of the technique. Therefore, it is necessary for us to optimize the protocal of SNT technique and investigate its mechanisms fully and completely.In this study, we want to improve the histone acetylation levels of sheep nuclear transfer donor cells and nuclear transfer embryos by the histone deacetylase inhibitors - Scriptaid and examine the effect of the sheep somatic cell nuclear transfer embryos in vitro by the improvement of the histone acetylation levels. In addition, the caffeine in the development of nuclear transfer embryos in vitro was also studied.We examined the effects of treatment with caffeine on the blastocyst formation rate in sheep somatic cell nuclear transferred (SCNT) embryos derived from cumulus cell. It has been demonstrated that the rate of development to the cleaved stage ,morula stage and blastocyst stage, by applying 0mmol/L,2.5mmol/L, 5mmol/L caffeine after fusion, did not significantly enhance the developmental competence(P>0.05). Treatment with 2.5mmol/L or 5mmol/L caffeine significantly increased the cell number of blastocyst(P<0.05).We examined the effects of treatment with scriptaid on the blastocyst formation rate in cumulus cell. Scriptaid significantly enhanced the rate of development of SCNT embryos to the blastocyst stage which is 24.39%, when 0.2μmol/L scriptaid was added to the cumulus cell culture medium. The concentration increased the rate of blastocyst significantly when compared with 0μmol/L and 0.8μmol/L team(P<0.05). We examined the effects of treatment with scriptaid on the blastocyst formation rate in SCNT embryos derived from cumulus cell. Scriptaid significantly enhanced the rate of development of SCNT embryos to the blastocyst stage which is 24.31%, when 0.2μmol/L scriptaid was added to the embryo culture medium. The concentration increased the rate of blastocyst significantly when compared with 0μmol/L and 0.8μmol/L(P<0.05). However,it did not significantly enhance the developmental competence when compared with 0.4μmol/L(P>0.05).Then, we examined the effects of treatment with 0.2μmol/L scriptaid on the blastocyst formation rate in SCNT embryos derived from cumulus cell and on the blastocyst formation rate in cumulus cell.Scriptaid significantly enhanced the rate of development of SCNT embryos to the blastocyst stage(P<0.05),but the concentration induced the rate of blastocyst, when compared with the blastocyst formation rate that scriptaid was added to the embryo culture medium and the cumulus cell culture medium(P<0.05). This is most likely that Scriptaid have a certain degree of toxicity for cells and embroy.We examined the effects of the time for the embroys on the treatment of 0.2μmol/L scriptaid. 0.2μmol/L Scriptaid significantly enhanced the rate of development of SCNT embryos to the blastocyst stage which is 24.94% and 24.27% for 24h and 48h, which are significantly higher when compared with control and 96h (P<0.05),however,it did not significantly different between 24h and 28h(P>0.05).We examined that the pregnancy rate of sheep is increased with 0.2μmol/L scriptaid significantly when compared with the control(P<0.05).At last, the intensity of histone acetylation at H4K12 at nuclear transfer embryos which 0.2μmol/L Scriptaid were added to the embryo culture medium from 1-cell to 8-cells periods was significantly stronger than the control group, the H4K12ac intensity of two groups of blastocysts was not significantly different.In summary, when the 0.2μmol/L Scriptaid is added into added to the embryo culture medium or the cumulus cell culture medium separately, the rate of nuclear transfer embryonic blastocyst can be significantly improved; and the H4K12 acetylation level of experimental group of 1-cells, 2-cells, 4-cell and 8-cell nuclear transfer embryos was significantly higher then control. So, the histone acetylation level of nuclear transfer reconstructed embryos can help to improve the efficiency of in vitro development of them.