Detection of Aging on Goat(Capra hircus) Oocytes Cultured In vitro Using Comet Assay | 彗星试验检测体外培养山羊卵母细胞的老化程度

Chinese. 为提高体外成熟山羊卵母细胞利用率，减少因卵母细胞老化而造成的质量下降，本研究通过彗星试验对体外成熟培养山羊(Capra hircus)卵母细胞的DNA受损程度进行研究。结果表明，随着成熟培养时间的延长，尤其是培养时间超过24 h以后，卵母细胞的极体逐渐退化，其DNA损伤程度明显增加，卵母细胞凋亡和老化的现象越来越严重。在体外成熟培养24 h之内，检测到极体的卵母细胞的比例随着培养时间的延长而显著增加(20 h, (45.74±2.67)%; 22 h, (66.11±1.12)%)，卵母细胞平均彗尾长度均小于100 μm，其中有极体卵母细胞彗尾长度在0~50 μm之间的比例(20 h, (77.27±2.27)%; 22 h, (40.12±1.55)%)小于无极体卵母细胞(20 h, (83.86±1.34)%; 22 h, (51.39±8.45)%)。体外成熟培养24 h时，有极体的卵母细胞比例达到最高值(80.19±2.07)%，有极体和无极体卵母细胞的平均彗尾长度基本相同((96.08±4.52) μm vs (95.25±9.28) μm)，有极体卵母细胞的彗尾分布主要以50~100 μm ((45.71±0.50)%)和100~150 μm ((35.45±2.92)%)区段为主，而无极体卵母细胞主要分布在0~50 μm ((30.92±4.02)%)和50~100 μm ((33.02±3.23)%)两个区段。当培养时间超过26 h以后，有极体的卵母细胞比例显著下降(26 h, (72.01±2.88)%; 28 h, (59.77±3.59)%)；无论是有极体或无极体的卵母细胞，其平均彗尾长度都继续增加，彗尾长度分布在150 μm以上的卵母细胞比例迅速增加。培养至30 h时，有极体的卵母细胞比例下降至(46.34±2.07)%，其平均彗尾长度为(180.11±10.33) μm，彗尾长度分布在150 μm以上的比例为(58.33±4.81)%；无极体卵母细胞平均彗尾长度为(270±17.72) μm，彗尾长度分布在150 μm以上的比例为(80.95±2.38)%，均高于有极体卵母细胞。本研究结果表明，山羊卵母细胞在体外成熟后会逐渐老化，尤其是在成熟培养时间超过24 h后，老化卵母细胞比例显著增加。本研究可使我们对卵母细胞老化的机理有了更进一步的了解和认识，有助于通过减少卵母细胞老化来提高卵母细胞的质量。

English. To improve the utilization of goat oocytes and reduce the decline in oocytes quality caused by aging, the senescence of goat(Capra hircus) oocytes cultured in vitro were studied by comet assay. The results showed that, with the prolonged culture time, especially more than 24 h, the first polar body(PBⅠ) degenerated significantly and the DNA damage increased gradually. These results suggested that apoptosis increased seriously during oocytes culture in vitro. Before 24 h of culture, the percentage of oocytes with PBⅠ increased visibly with the extended culture(20 h, (45.74±2.67)%; 22 h, (66.11±1.12)%). All the mean length of comet tail was less than 100 μm, however, among oocytes with PBⅠ, the percentage of oocytes with comet tail length distributed in 0~50 μm region (20 h, (77.27±2.27)%; 22 h, (40.12±1.55)%) was less than oocytes without PBⅠ(20 h, (83.86±1.34)%; 22 h, (51.39±8.45)%). At 24 h of culture, oocytes with PBⅠ increased to the peak rate(80.19±2.07)%. And there was no difference in the mean comet tail length for both of oocytes with PBⅠor not ((96.08±4.52) μm vs (95.25±9.28) μm). Moreover, the distribution of mean comet tail length was major in 50~100 μm((45.71±0.50)%) and 100~150 μm ((35.45±2.92)%) for oocytes with PBⅠ, while 0~50 μm ((30.92±4.02)%) and 50~100 μm ((33.02±3.23)%) were the major distribution for oocytes without PBⅠ. When the culture time extended to 26 h later, the rate of oocytes with PBⅠ decreased dramatically (26 h, (72.01±2.88)%; 28 h, (59.77±3.59)%). And the mean comet tail length increased continuely for both of oocytes with PBⅠor not, and the percentage of oocytes with more than 150 μm comet tail length increased rapidly. At 30 h of culture, only (46.34±2.07)% of PBⅠ could be observed in oocytes, and the mean comet tail length was (180.11±10.33) μm, (58.33±4.81)% of them with a more than 150 μm comet tail; but for oocytes without PBⅠ, the mean comet tail length was (270±17.72) μm, and (80.95±2.38)% of them with a more than 150 μm comet tail. This study showed that goat oocytes matured after in vitro muturation became increasingly aged along with the extended culture time, and the proportion of aging oocytes increased dramatically when cultured more than 24 h. Through this study, we can get a further more understanding on the mechanism of oocyte aging. It contributed to improve the oocyte quality by reducing the oocyte aging.