Wongsrikeao Pimprapar, Otoi Takeshige, Murakami Masako, Karja Ni Wayan Kurniani, Budiyanto Agung, Murakami Masao, Nii Masaru, Suzuki Tatsuyuki
Laboratory of Animal Reproduction, United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan.
Reprod Fertil Dev. 2004;16(8):773-80. doi: 10.1071/rd03099.
The present study was conducted to investigate the effects of the attachment of cumulus cells to oocytes and coculture with cumulus cells during maturation culture on the nuclear status and DNA fragmentation of porcine denuded oocytes (DOs). In the first experiment, cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 8, 16, 24 or 32 h after the onset of maturation culture and the DOs were then cultured in their original droplets until 42 h of culture was reached. In the second experiment, all COCs were denuded before the onset of culture and the DOs were cocultured with their removed cumulus cells. The DOs were transferred into fresh medium at 0, 8, 16, 24 or 32 h after the onset of coculture with cumulus cells and then cultured until 42 h of culture was reached. After culture, DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) method. When the DOs were returned to the same droplets after removal of the cumulus cells, the removal of the cumulus cells after 16 h of culture significantly decreased the proportion of oocytes remaining at the germinal vesicle (GV) stage. However, coculture treatment of DOs in the presence of their removed cumulus cells had no significant effects on the GV breakdown (GVBD) of oocytes. There were no significant differences in the proportion maturing to MII oocytes among the groups following removal of cumulus cells after the onset of maturation culture; however, DOs cocultured with cumulus cells until the end of maturation culture exhibited an increased maturation rate compared with DOs cocultured for 8 and 16 h. The total proportion of TUNEL-positive oocytes of oocytes remaining at the GV stage was higher than that of oocytes reaching other stages, irrespective of the removal of cumulus cells and coculture treatments. However, coculture for more than 16 h decreased the total proportion of TUNEL-positive oocytes. Our results indicate that the attachment of cumulus cells to oocytes may have a critical role for oocytes undergoing GVBD and that coculture with cumulus cells promotes the ability of oocytes to complete maturation. Moreover, coculture with cumulus cells may assist the oocyte to avoid undergoing DNA fragmentation.
本研究旨在探讨卵丘细胞与卵母细胞的附着以及在成熟培养过程中与卵丘细胞共培养对猪裸卵（DOs）核状态和DNA片段化的影响。在第一个实验中，在成熟培养开始后的0、8、16、24或32小时从卵丘 - 卵母细胞复合体（COCs）中去除卵丘细胞，然后将DOs在其原有的液滴中培养至42小时。在第二个实验中，所有COCs在培养开始前均被去除卵丘细胞，然后将DOs与其去除的卵丘细胞进行共培养。在与卵丘细胞共培养开始后的0、8、16、24或32小时将DOs转移到新鲜培养基中，然后培养至42小时。培养后，使用末端脱氧核苷酸转移酶介导的dUTP - 地高辛标记（TUNEL）方法检测卵母细胞的DNA片段化和核状态。当去除卵丘细胞后将DOs放回相同的液滴中时，培养16小时后去除卵丘细胞显著降低了处于生发泡（GV）期的卵母细胞比例。然而，DOs在其去除的卵丘细胞存在下进行共培养处理对卵母细胞的生发泡破裂（GVBD）没有显著影响。在成熟培养开始后去除卵丘细胞的各组中，成熟为MII期卵母细胞的比例没有显著差异；然而，与共培养8小时和16小时的DOs相比，与卵丘细胞共培养至成熟培养结束的DOs表现出更高的成熟率。无论去除卵丘细胞和共培养处理如何，处于GV期的卵母细胞中TUNEL阳性卵母细胞的总比例高于达到其他阶段的卵母细胞。然而，共培养超过16小时会降低TUNEL阳性卵母细胞的总比例。我们的结果表明，卵丘细胞与卵母细胞的附着可能对经历GVBD的卵母细胞具有关键作用，并且与卵丘细胞共培养可促进卵母细胞完成成熟的能力。此外，与卵丘细胞共培养可能有助于卵母细胞避免发生DNA片段化。
