| Somatic cell nuclear transfer (SCNT) provides a wealth of raw materials for the field of livestock industry, medicine and basic research of life sciences, and brings hope to conservation of genetic resources, multiplication of elites, rescue of endangered animals, production of transgenic animals, establishment of human disease models etc. Pigs are increasingly being regarded as an ideal model animal, not only because they are farm animal which have an important economic value, but because their genetics, anatomy and physiology are very similar to human. Pigs are multiparous animal, as well as, there are mature feeding and management technology, less ethical issues, relatively modest life span, and can make up for a deficiency of mice as the animal models of human disease. Therefore the success of pig somatic cell cloning has more great significance. Despite somatic cell cloned pigs have been come out at least for ten years, the efficiency is extremely low. The fundamental reason is that reprogramming of epigenetic modification of the donor cells nuclear are not completely. Treatment of donor cells and/or cloned embryos using epigenetic modifiers including histone deacetylase inhibitors (TSA, Scriptaid, VPA, etc.) can improve the early development of pig cloned embryos. Romidepsin, (FK228), a cyclic peptide HDACi can effectively inhibit HDAC1 and HDAC2 activity, and could enhance gene transcription activity by increasing histone acetylation levels. However, as yet there is no study showing if FK228 affects reprogramming of cloned embryos. The present study was designed to explore effects on growth of donor cell and in vitro development of cloned embryos after brief treatment using FK228, in order to improving the efficiency of porcine somatic cell nuclear transfer, enriching epigenetic modification interventions, and laying the foundation for further reveal the impact and mechanisms of histone acetylation on embryonic development. Specific tests and results are as follows: Expenriment I was designed to evaluate influence of FK228 on pig fetus fibroblast cells. The present experiment was conducted to optimize FK228 treatment in preparation of donor cells for nuclear transfer. Growth characteristics including morphology, growth curve, apoptosis and chromosome numbers of pig fetus fibroblast cells after FK228 treatment were examined. Pig fetus fibroblastic cells were cultured with medium containing different concentrations of FK228 (0,0.05,0.1, lμM) after exposure for various times(24,48,72h). From these results, it can be concluded that 0.1 μM FK228 for 24h has little effect on the cell morphology, appearance and number of chromosomes, and thus suitable as treatment of donor cells.Expenriment II aims to explore the development competence of somatic nuclear transfer embryos after donor cells treated by FK228. Fusion rate, cleavage rate, blastocyst formation rate, and the total cell numbers of blastocysts were recorded in order to evaluate the in vitro development competence of SCNT embryos derived from donor cells after treatment at various concentrations (0,0.05,0.1, 1μM) of FK228 with exposure 24h. The results showed that fusion rate of embryos were higher significantly than the control group when the donor cells treated with 0.1 μM or 1μM for 24h. Cleavage rate among groups was not significant difference. Blastocyst rate appeared to be the highest at 0.1 μM FK228 for 24h group. Compared to control, the total cell numbers of blastocysts both increased at 0.1 μM or 1μM FK228. Therefore, donor cells treated with 0.1 μM FK228 for 24h may improve the developmental potential of pig nuclear transfer embryos.Expenriment III was to investigate the in vitro developmental competence of cloned embryos after treatment by FK228. Pig SCNT embryos were cultured with PZM-3 containing different concentrations of FK228(0,0.05,0.1,1,5,50, 100μM) after exposure for various times (24h or 36h). Somatic cell cloned embryos were randomly placed in these different culture groups. The results showed that no significant differences were found among groups in terms of the rate of cleavage, blastocyst formation, and the total cell numbers per blastocyst, but there was an increasing trend when concentrations of FK228 climbed up till 1μM if the exposure time is 24h. Although no significant difference exist for the cleavage rate when SCNT embryos treated with 0,5,50, and 100μM FK228 for 24h, no blastocyst formation happened in the test groups except control. We then utilized various concentrations (0, 0.05,0.1, 1μM) of FK228 to treat pig SCNT embryos exposure 36h. The result suggested that the blastocyst rate reached to be the highest in 0.1 μM FK228 group, and be the lowest at 1μM FK228 group; The total cell numbers of blastocysts were enhanced along with the concentrations of FK228(0～1μM), and were higher significantly than the control group at 0.1 μM FK228 for 36h. The results indicated that FK228 may significantly improve the blastocyst rate and total cell numbers of blastocysts with 0.1 μM FK228 for 36h, which is helpful for improving in vitro development of pig SCNT embryos.In Expenriment Ⅳ, based on our previous experimental results, we treated donor cells with 0.1 μM FK228 for 24h and cloned embryos with 0.1μM FK228 for 36h.The results showed that the cleavage rate were higher significantly in the test group than the control. But other indicators did not differ between the groups. It indicated that the combined treatment had no synergistic promotion role to the development of pig cloned embryos.In summary, the treatment of pig donor cells using 0.1 μM FK228 for 24h alone, or treatment of cloned embryos solely for 36h could promote the in vitro development of porcine SCNT embryos. However, combined treatment of donor cells and embryos with FK228 is of no use. Taken together, we demonstrated the role of FK228 on growth of donor cells and in vitro development of cloned embryos in pigs for the first time. The results provides theoretical basis and lay a solid foundation for further study of the role of FK228. |
PDF Full Text Request