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目的探讨高龄小鼠卵母细胞线粒体、皮质颗粒、微丝、纺锤体及染色体形态和排布的变化规律。方法将雌性昆明小鼠分为年轻组(4~8周)和高龄组(48~52周)。腹腔内注射孕马血清促性腺激素(PMSG)10IU/只行促排卵,48 h后腹腔内注射人绒毛膜促性腺激素(h CG)10 IU/只,14 h后收集输卵管的卵冠丘复合物(OCCC)。脱去颗粒细胞,获取成熟卵母细胞(MⅡ期)经2%多聚甲醛固定。抗小鼠α-tubulin单克隆抗体染色卵母细胞纺锤体的微管,罗丹明标记的鬼笔环肽染色微丝,FITC结合的小扁豆凝集素染色皮质颗粒,Mito Tracker Green FM染色线粒体。激光扫描共焦显微镜下观察。结果共获取小鼠卵母细胞372枚,其中年轻组179枚,高龄组193枚。高龄组小鼠卵母细胞纺锤体异常率(71.78%±1.27%)、染色体排列异常率(65.16%±1.52%)明显高于年轻组(18.93%±1.27%,32.24%±1.10%)(P<0.01,P<0.01);高龄组小鼠卵母细胞线粒体的荧光强度(28.09±6.62)明显低于年轻组(45.07±5.90)(P<0.01);高龄组小鼠成熟皮质颗粒(Ⅲ级皮质颗粒)(51.00%±1.00%)明显低于年轻组(94.04%±0.71%)(P<0.01)。结论高龄小鼠卵母细胞中各主要细胞器、细胞骨架及染色体排列异常率明显增加,可能是导致高龄受孕率低的重要原因。
Objective To investigate the changes of morphology and arrangement of mitochondria, cortical granules, microfilaments, spindle and chromosomes in aged mouse oocytes. Methods Female Kunming mice were divided into young group (4-8 weeks) and senior group (48-52 weeks). Intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) 10IU / line ovulation induction, 48 h after intraperitoneal injection of human chorionic gonadotropin (hCG) 10 IU / only 14 h after collecting oviduct cumulus complex Things (OCCC). Remove the granulosa cells, obtain mature oocytes (M Ⅱ phase) fixed by 2% paraformaldehyde. Microtubules of anti-mouse α-tubulin monoclonal antibody-stained oocyte spindle, rhodamine-labeled phalloidin-stained microfilaments, FITC-conjugated lentil lectin-stained cortical granules, Mito Tracker Green FM-stained mitochondria. Laser scanning confocal microscopy. Results A total of 372 mouse oocytes were obtained, of which 179 were young and 193 were elderly. The abnormal rate of spindle oocyte (71.78% ± 1.27%) and chromosomal aberration (65.16% ± 1.52%) were significantly higher in advanced age group than in young group (18.93% ± 1.27%, 32.24% ± 1.10%) (P <0.01, P <0.01). The fluorescence intensity of mitochondria in aged mice (28.09 ± 6.62) was significantly lower than that in young mice (45.07 ± 5.90) (P <0.01) Cortical granules) (51.00% ± 1.00%) was significantly lower than the young group (94.04% ± 0.71%) (P <0.01). Conclusion The anomalous rate of major organelles, cytoskeleton and chromosomal abnormalities in the oocytes of the aged mice is significantly increased, which may be the important reason for the low conception rate in the elderly.