Tatemoto H, Sakurai N, Muto N
School of Bioresources, Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan.
Biol Reprod. 2000 Sep;63(3):805-10. doi: 10.1095/biolreprod63.3.805.
The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P: < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P: < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.
本研究旨在探讨卵丘细胞对猪卵母细胞体外成熟过程中由次黄嘌呤-黄嘌呤氧化酶（XOD）系统产生的活性氧（ROS）所导致的卵母细胞损伤的保护作用。将卵丘-卵母细胞复合体（COCs）和裸卵（DOs）在添加了半胱氨酸、促性腺激素、10%猪卵泡液和次黄嘌呤的NCSU37中培养44小时，培养过程中分别添加或不添加XOD。分别使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法和单细胞凝胶电泳（彗星）试验分析DNA裂解和损伤情况，并测定各实验组中的半胱天冬酶-3活性和谷胱甘肽（GSH）含量。DOs暴露于ROS会导致减数分裂停滞和退化卵母细胞数量增加。这些退化的DOs发生了凋亡，表现为其生发泡内TUNEL阳性反应以及半胱天冬酶-3的激活。用XOD处理的DOs中DNA迁移长度显著长于未处理的DOs（P：<0.05）。然而，在COCs中未观察到由ROS引起的无法修复的细胞损伤，并且在用和未用XOD处理的COCs中，半胱天冬酶-3活性没有差异。与从卵泡中新鲜分离的卵母细胞相比，培养44小时后的COCs中GSH水平显著升高（P：<0.05），而无论是否用XOD处理，DOs中的GSH含量均显著降低。这些发现表明，卵丘细胞通过提高卵母细胞中的GSH含量，在保护卵母细胞免受氧化应激诱导的凋亡方面具有关键作用。
