化学辅助去核法对绵羊卵母细胞去核率和重构胚发育率的影响

Effect of the chemically assisted enucleation on the enucleation of sheep oocytes and the development of their reconstructed embryos

摘要:

为提高绵羊体细胞核移植的效率, 本研究采用一种新的去核方法—化学辅助去核法, 对绵羊体外成熟的卵母细胞进行去核, 研究了化学诱导剂秋水仙素的处理浓度、作用时间、卵母细胞的成熟时间对去核效果及重构胚发育的影响。结果表明: 1) 卵母细胞在0.4 mg/mL的秋水仙素溶液中分别孵育0.5 h和1 h, 胞质突起率和去核率没有显著的差异, 突起率可高达85.4%, 去核率达到100%; 2) 0.2 mg/mL或0.4 mg/mL秋水仙素溶液将卵母细胞处理0.5 h, 对去核效果没有显著影响; 3) 对于体外成熟18~23 h的卵母细胞, 随着成熟时间的延长, 盲吸法的去核率降低, 但没有影响秋水仙素诱导胞质突起的比率和去核率; 4) 两种去核方法对重构胚的发育没有产生显著影响, 但成熟21~23 h卵母细胞重构胚囊胚的发育率显著高于成熟18~20 h卵母细胞重构胚囊胚的发育率。综上所述, 本试验优化了绵羊卵母细胞化学辅助的去核程序, 利用化学辅助去核法对高卵龄的绵羊卵母细胞进行去核, 提高了去核率和重构胚的体外发育率。

Abstract:

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 μg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 μg/mL) for 1 h, and the rate of cytoplast protusion 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 μg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 μg/mL colchicine for 0.5 h. 3) A maturation time of 18–23 h did not affect the rates of cytoplast protusion 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.