解剖学报

解剖学报 ›› 2017, Vol. 48 ›› Issue (6): 740-745.doi: 10.16098/j.issn.0529-1356.2017.06.018

• 组织学胚胎学发育生物学 • 上一篇    下一篇

影响高龄小鼠卵母细胞质量的超微结构因素

刘美菊1,2 陈新霞1,3*   

  1. 1.山东大学附属省立医院生殖医学中心，济南 250000; 2.临沂市人民医院生殖医学科，山东 临沂 276000; 3.山东大学护理学院，济南 250000
  • 收稿日期:2017-01-09 修回日期:2017-02-14 出版日期:2017-12-06 发布日期:2017-12-06
  • 通讯作者: 陈新霞 E-mail:chenxinxia@sdu.edu.cn
  • 基金资助:
    山东省医药卫生科技发展计划项目;山东省自然科学基金

Ultrastructural factors affecting the quality of oocytes in aged mice

LIU Mei-ju 1,2 CHEN Xin-xia 1,3*   

  1. 1. Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Ji’nan 250000, China;2. Department of Reproductive Medicine, Linyi People’s Hospital，Shandong Linyi 276000,China;3. School of Nursing of Shandong University, Ji’nan 250000,China
  • Received:2017-01-09 Revised:2017-02-14 Online:2017-12-06 Published:2017-12-06
  • Contact: CHEN Xin-xia E-mail:chenxinxia@sdu.edu.cn

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摘要：

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）10 IU/只行促排卵，48 h后腹腔内注射人绒毛膜促性腺激素（hCG）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%±100%）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。

关键词: 卵母细胞, 线粒体, 皮质颗粒, 纺锤体, 微丝, 染色体, 激光扫描共焦显微术, 小鼠

Abstract:

Objective To investigate the morphology and distribution of cortical granules, mitochondria, microfilaments, spindles, and chromosomes in aged mice. Methods Kunming mice were divided into a young (4-8 weeks) and a old age group (48-52 weeks). Female mice were injected intraperitoneally with 10 IU prenant mare serum gonadotropin (PMSG), and followed 48 hours later by 10 IU of human chorionic gonadotrophin (hCG). MII oocytes were collected from the oviducts 14 hours after the hCG injection into M199 medium. The cumulus cells were removed by a brief incubation. Oocyte-corona-cumulus complex(OCCC) in fallopian tube were collected by superovulation to female mice. Anti-mouse α-tubulin monoclonal antibody was used to label the spindle microtubules, phalloidin labeled actin rhodamine to label microfilaments, lentil lectin combined with FITC to label cortical granules and Mito Tracker Green FM to label mitochondria. The specimen was examined under a laser scanning confocal microscope. Results Totally 372 oocytes were obtained, 179 oocytes from the young group and 193 oocytes from aged group. The abnormal rate of spindle of elderly mouse oocytes (71.78% ± 1.27%) was significantly higher than that of young mice (18.93% ± 1.27%) (P<0.01). The elderly mouse oocytes chromosomes arranged abnormal rate (65.16% ± 1.52%) was significantly higher than that of young mice (32.24% ± 1.10%) (P<0.01). Fluorescence intensity of aged mouse oocyte mitochondria (28.09 ± 6.62) was significantly lower than that of young mice (45.07 ± 5.90) (P<0.01). The aged mouse mature cortical granules (grade III cortical granules) rate (51% ± 1%) was significantly lower than that of young mice (94.04% ± 0.71%) (P<0.01). Conclusion The abnormal rate of aged mouse oocytes’ major organelles and cytoskeleton increased significantly than that of the younger mice, which may be the main reason for the low pregnancy rate in the elderly.

Key words: Oocytes, Mitochondria, Cortical granule, spindle, Microfilament, Chromosome, Laser scanning confocal microscopy, Mouse