Pan Xiaoyan, Wang Zhengchao, Li Zhixin, Jin Yuji, Dou Zhaohua, Guo Zhiqin, Wang Feng
Department of Histology and Embryology, Jilin Medical College, Jilin 132013, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):503-8.
In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 microg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 microg/mL) for 1 h, and the rate of cytoplast protrusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 microg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 microg/mL colchicine for 0.5 h. 3) A maturation time of 18-23 h did not affect the rates of cytoplast protrusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21-23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.
为提高绵羊体细胞核移植效率，我们采用秋水仙碱化学辅助去核法，研究秋水仙碱浓度、卵母细胞在秋水仙碱中的孵育时间以及卵母细胞成熟时间对去核率和重构胚发育的影响。结果表明：1）在秋水仙碱（0.4μg/mL）中孵育0.5 h的卵母细胞与在秋水仙碱（0.4μg/mL）中孵育1 h的卵母细胞，其胞质体突出率和去核率无显著差异，胞质体突出率可达85.4%，胞质体去核率为100%。2）用含0.2μg/mL秋水仙碱的培养基处理0.5 h的卵母细胞与用含0.4μg/mL秋水仙碱的培养基处理0.5 h的卵母细胞，其去核率无显著差异。3）18 - 23 h的成熟时间不影响化学辅助去核的胞质体突出率和去核率，而盲法去核的卵母细胞去核率随孵育时间延长而降低。4）这两种去核方法均不影响重构胚的发育率，从成熟21 - 23 h的卵母细胞获得的重构胚发育率有所提高。这些结果表明，优化的秋水仙碱化学辅助去核法可快速、有效地对绵羊卵母细胞进行去核，提高绵羊卵母细胞去核率及体外重构胚的早期发育能力。
