van den Hurk R, Spek E R, Hage W J, Fair T, Ralph J H, Schotanus K
Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht, The Netherlands.
Hum Reprod Update. 1998 Nov-Dec;4(6):833-41. doi: 10.1093/humupd/4.6.833.
Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes.
体外分离卵巢卵泡和卵母细胞成熟的技术具有巨大的生殖潜力。保持正常组织功能至关重要。本研究着重于机械分离的牛小（直径40 - 100微米）腔前卵泡和大（140 - 450微米）腔前/早期窦状卵泡的超微结构和活力。对小腔前卵泡进行了活力研究。酯酶活性、活跃线粒体和死细胞的存在作为卵母细胞和颗粒细胞活力的参数。培养1天后，所有卵泡的颗粒细胞均存活，其所有细胞均显示活跃的线粒体和/或酯酶活性，尽管17%的卵泡中存在少数（通常<5个）死亡的颗粒细胞。在卵母细胞中，分别有35%和80%具有酯酶活性和活跃的线粒体，而8%看起来已死亡。培养过程中显示酯酶活性和活跃线粒体的卵母细胞百分比下降，而含有死亡卵母细胞或死亡颗粒细胞的卵泡百分比则大幅增加。超过90%的分离小卵泡显示出较差的超微结构，尤其是其卵母细胞，这表明在本研究中存在对劣质卵泡的负面选择性分离和/或分离过程导致的卵泡损伤。对于大腔前卵泡，分布在皮质中的42%以及分离并培养1天的64%具有较差的超微结构。与小卵泡不同，培养5天后，超微结构较差的大腔前卵泡百分比降至27%，这可能是由于更好的分离和培养条件。建议使用超微结构和/或活力细胞标记物来评估体外培养卵泡的质量，尤其是其卵母细胞的质量。
