| Somatic cell nuclear transfer is a major content of mammal embryo engineering, and it is useful not only for shortening the time of animal breed and increasing the availability of good species but also for the study of fertilization mechanism. There are many steps in the procedure of mammal somatic cell nuclear transfer from recipient oocyte culture, donor cell preparation, nuclear transfer, reconstructed embryo formation, reconstructed embryo activation, reconstructed embryo culture, embryo transfer to foetus birth, and each of these steps influence the efficiency of somatic cell nuclear transfer. In these steps, donor cell preparation, recipient oocyte culture and reconstructed embryo formation play important role. In our study, we investigated several factors which affected donor cell preparation, recipient oocyte culture and reconstructed embryo formation in bovine nuclear transfer. We investigated the effects of culture passages, serum starvation concentration and serum starvation time on cell cycle and apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelia cells, and we also studied the mechanism of apoptosis origined from serum starvation and the inhibit effect of different antioxidant for their apoptosis. We added various concentrations of iron or copper to culture media from which we had previously removed the respective element in order to examine the effects of iron and copper on bovine oocyte maturation and preimplantation embryo development in vitro. Furthermore, our study determined lower osmotic pressure duration optimal for breaking granulosa cell membrane, and to determine its effects on reconstructed embryos formation and reconstructed embryos developmental potential in bovine nuclear transfer by intraplasmic injection.1. Investigated the effects of culture time, serum starvation and starvation time on cell cycle of in vitro cultured bovine granulosa cells and oviduct epithelium cells. Cell cycleanalysis of propidium iodide staining was performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5% and 0.05% FCS seum starvation for 2 d, 3 d, 4 d, and 5 d. The results were as follows: bovine granulosa cells and oviduct epithelium cells of P 2, P 10 and P 25 have no significant differences in the percentages of G0/G1 phase (P＞0.05). 0.5% and 0.05% FCS in culture medium significantly increased the percentages of G0/G1 phase than their control(10% FCS)(P＜0.05), and there were no significant differences between 0.5% FCS and 0.05% FCS (P＞0.05). The cells cultured for 5 d at 0.5% FCS starvation had significant higher percentages of G0/G1 phase than the cells cultured for 2 d at 0.5% FCS starvation. In concluded that culture time of bovine granulosa cells and oviduct epithelium cells in vitro has no effect on the percentages of G0/G1 phase, and serum starvation and prolonged serum starvation time synchronized the cell cycle effectively.2. Investigated the effects of culture time, serum starvation and serum starvation time on apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelium cells and investigated its possible mechanisms. Cell apoptosis detection of Annexin V/FITC double staining were performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5%FCS and 0.05% FCS seum starvation for 2 d, 3 d, 4 d and 5 d. Caspase inhibitor z-VAD-fmk was used to determine whether serum starvation-induced apoptosis was mediated by caspase activation. The results were as follows: there were no significant differences in apoptosis rates of granulosa cells between P 2 and P 10, and the cells of P 25 had significantly higher apoptosis rates than the cells of P 2 and P 10. Apoptosis rates of 0.5%FCS and 0.05%FCS serum starvation were significant differences and significant greater than serum-fed treatment (10% FCS). The cells cultured for 4 d and 5 d at 0.5% FCS starvation had significantly higher apoptosis rates than the cells cultured for 2 d and 3 d. Culture medium with caspase inhibitor z-VAD-fmk significantly decreased the cells apoptosis rates of 0.5% and 0.05% FCS starvation than culture medium without z-VAD-fmk, and they were no significant differences in the cells apoptosis rates of 10% FCS between z-VAD-fmk and the control. In concluded that long-term curture in vitro, serum starvation and prolonged serum starvation time resulted in high apoptosis rate of bovine granulosa cells and oviduct epithelium cells, and serum starvation-induced apoptosis has the involvement of caspases.3. Investigated the effects of different antioxidant on inhibit of apoptosis origined from serum starvation, and somatic cells were bovine granulosa cells and oviduct epithelium cells. Cell apoptosis detection of Annexin V/FITC double staining was performed by flow cytometric assay. Cells of P 5 were detected in 2 d culture in 10% and 0.5% FCS culture medium with antioxidant vitamin E, selenium and tathion. The results were as follows: 10% FCS treatments with antioxidant vitamin E, selenium and tathion decreased apoptosis rates of granulosa cells compared with their control, but there were no significant differences. However, 0.5% FCS treatments with antioxidant vitamin E, selenium and tathion significantly decreased apoptosis rates of granulosa cells compared with their control. 10% FCS treatments with antioxidant decreased apoptosis rates of oviduct epithelium cells compared with their control, but there were no significant differences, and 0.5% FCS treatments with antioxidant significantly decreased apoptosis rates of the cells compared with their control. In concluded that bovine granulosa cells and oviduct epithelium cells apoptosis induced by serum starvation could be active suppression through adding antioxidant vitamin E, selenium and tathion in culture medium, and antioxidant vitamin E, selenium and tathion in culture medium could not inhibit apoptosis of the cells cultured in 10% FCS culture medium.4. This study was on the effects of iron and copper on bovine oocytes maturation, preimplantational embryos development and apoptosis in blastocysts. Iron concentrations in culture medium were 0 mg/L(control), 0.45mg/L, 0.81mg/L, 1.96mg/Land 3.26mg/L, and copper were 0mg/L(control), 0.093mg/L, 0.27mg/L, 0.46mg/Land 0.68mg/L. The variance of iron concentration (1.96mg/L) and copper concentration (0.46mg/L) in culture medium were mensurated at 22h of oocytes maturation and at 48h, 96h, 144h and 192h of zygotes culture. The results were as follows: there were no significant differences in oocytes maturation and cleavage between iron and control, but iron had higher (p＜0.05) rate of 8-cell embryos, morulae and blastocysts than the control, and 1.96mg/Lof iron increased blastocysts rate (p＜0.05). The effects of copper on oocytes maturation and cleavage was similar to iron, and 0.46 and 0.68mg/Lof copper increased the rate of morulae and blastocysts (p＜0.05). Iron or copper had a significant decrease in apoptotic blastomeres than the control (p＜0.05). At 22h of oocytes maturation and 48h of zygotes culture, decreased percentage of iron concentrations were 3.6% and 9.2%, respectively, and that of copper were 6.5 and 10.9%, respectively. At 96h, 144h and 192h of zygotes culture, decreased percentage of iron concentrations were 21.4%, 25.5% and 27.0%, respectively, and that of copper were 23.9%, 28.3% and 30.4%, respectively. In conclusion: iron or copper played an important role in the success of culture of 8-cell embryos, morulae and blastocysts, and long-term lack of iron or copper increased apoptotic blastomeres. There might has a transition of primary demand for trace element iron or copper utilized by zygotes from cytoplast to culture medium after 8-cell of in vitro zygotes culture.5. Determined lower osmotic pressure (0.075 M KCl) duration optimal for breaking granulosa cell membrane, and determined its effects on reconstructed embryos formation, reconstructed embryos developmental potential and cells apoptosis of blastocysts in bovine nuclear transfer by intracytoplasmic injection. Enucleated bovine oocytes were divided into five groups according to duration of 0.075 M KCl to granulosa cells: Os (control), 30s, 60s, 90s and 180s. Typical apoptotic nuclei was shown by staining of granulosa cells with DNA-binding fluorochrome Hoechst 33342. Different duration of 0.075 M KCl significantly increased the formation of reconstructed embryos than control. The treatment of 180s had significantly lower rate of blastocysts than other groups, and nuclei of blastocysts from 180s duration of 0.075 M KCl showed typical apoptosis morphology. These results suggested that lower osmotic pressure to bovine granulosa cells accelerated the formation of reconstructed embryos in nuclear transfer of intracytoplasmic injection, and long duration of lower osmotic pressure resulted in low blastocysts rate and high cells apoptosis rate of blastocysts. |
