摘要目的:探讨小鼠卵母细胞纺锤体置换过程中，容易导致卵母细胞正常受精率低的因素及应对策略。方法:获取C57BL/6（简称C57）及ICR品系小鼠中处于第二次减数分裂中期（metaphase Ⅱ，MⅡ）的卵母细胞，进行核质置换操作观测：显微操作取出大、中、小三种不同大小的纺锤体胞体，在明场及偏振光下观察取出后0 min、10 min、20 min、30 min时的形态；对于纺锤体小胞体，分别在取出10 min、20 min、30 min后进行2%多聚甲醛固定，免疫荧光染色后观察其纺锤体及染色体的形态；对卵母细胞进行卵胞质内单精子注射（intracytoplasmic sperm injection，ICSI），随即取纺锤体小胞体，然后分别在取出后30 min（传统法置换组）、10 min（改良法1置换组）、0 min（改良法2置换组）时进行胞体融合，而只行ICSI未经纺锤体置换的卵母细胞作为对照组，14~16 h后，观察重构卵母细胞受精情况并统计分析。结果:①卵母细胞纺锤体胞体在刚取出时（0 min），胞体膜平滑完整，纺锤体呈现正常纺锤型。在取出10 min、20 min、30 min后，均可见胞体膜扭曲或拉长、形态不规则，纺锤体随之扭曲拉伸或散乱、亮度变弱。且胞体越小，变形越严重。②刚取出时（0 min）的纺锤体胞体，免疫荧光染色可见纺锤体呈现标准纺锤型，染色体规则整齐地排列于赤道板上。而随着放置时间的延长，纺锤体拉长变细或呈哑铃型，染色体散乱、排列不规则。③对于传统法置换的C57BL/6小鼠重构卵母细胞正常受精率为63.16%（24/38），与对照组［90.00%（36/40）］相比，差异有统计学意义（ P=0.002）。在改良法1置换组的正常受精率［68.00%（34/50）］显著低于对照组（ P=0.019），但与传统法置换组比较，差异无统计学意义（ P=0.260）。而在改良法2置换组的正常受精率［82.50%（33/40）］与对照组比较，差异无统计学意义（ P=0.422），但显著高于传统法置换组（ P=0.010）。 结论:小鼠卵母细胞纺锤体胞体取出后放置时间越久，形态变化越严重，纺锤体及染色体越不稳定，正常受精率越低；纺锤体胞体取出后立即融合为核质置换操作的最佳时机。

abstractsObjective:To explore the factors that tend to lead to a low normal fertilization rate in spindle transfer of mouse oocytes and its strategies.Methods:Oocytes in metaphase Ⅱ (MⅡ) of C57BL/6 and ICR strains mouse were obtained for observations of mitochondrial replacement operations. Spindle karyoplasts of three different sizes (large, medium, small) were isolated by micromanipulation, and their morphology was observed under bright field and polarized light at 0 min, 10 min, 20 min and 30 min after suction. For small spindle karyoplasts, 2% paraformaldehyde fixation was performed after 10 min, 20 min and 30 min respectively, and the morphology of spindle and chromosomes was observed after immunofluorescence staining. Intracytoplasmic sperm injection (ICSI) was performed on oocytes, followed by small spindle karyoplasts isolation, and then fusion was conducted after 30 min (traditional method), 10 min (modified method 1) and 0 min (modified method 2) respectively. ICSI-oocytes without spindle transfer were used as control group, and 14-16 h later, the fertilization of reconstructed oocytes was observed and statistically analyzed.Results:1) When spindle karyoplast was just retrieved (0 min), the karyoplast membrane was smooth and complete, and the spindle showed a normal spindle shape. After 10 min, 20 min and 30 min of suction, the karyoplast membrane was twisted or elongated with an irregular morphology, and the spindle was also twisted, stretched or scattered with a weaker brightness. And the smaller spindle karyoplast, the more serious the deformation was. 2) When the spindle was just taken out (0 min), immunofluorescence staining showed that the spindle was in a standard spindle shape, and the chromosomes were regularly and neatly arranged on the equatorial plate. With the prolongation of placement time, spindles were elongated and tapered or dumbbell-shaped, and chromosomes were scattered and irregularly arranged. 3) For the traditional method of spindle transfer, the normal fertilization rate of C57BL/6 mouse reconstructed oocytes was 63.16% (24/38), which was statistically different from that of control group ( P=0.002). The normal fertilization rate [68.00% (34/50)] in the modified method 1 group was significantly lower than that in control group [90.00% (36/40), P=0.019], but there was no statistical difference with the traditional method ( P=0.260). However, the normal fertilization rate [82.50% (33/40)] in the modified method 2 group was similar to control group ( P=0.422), and was significantly higher than that of the traditional method group ( P=0.010). Conclusion:The longer the spindle karyoplast of mouse oocytes is stayed after retrieval, the more serious the morphological changes, the more unstable the spindles and chromosomes, and the lower normal fertilization rate. Immediate fusion of spindle karyoplast after isolation is the best time for mitochondrial replacement micromanipulation.