Bilodeau J F, Chatterjee S, Sirard M A, Gagnon C
Urology Research Laboratory, Royal Victoria Hospital and McGill University, Montreal, Canada.
Mol Reprod Dev. 2000 Mar;55(3):282-8. doi: 10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7.
Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H(2)O(2)). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk-Tris-glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5 degrees C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function.
越来越多的证据表明，活性氧（ROS）的产生及其被抗氧化剂解毒在生育中起着非常重要的作用。然而，精子中抗氧化剂水平与冻融循环后生育力下降之间的关系仍知之甚少。我们评估了牛精液中过氧化氢酶、谷胱甘肽过氧化物酶（GPx）、超氧化物歧化酶（SOD）等抗氧化酶的活性以及还原型/氧化型谷胱甘肽（GSH/GSSG）的水平。使用Percoll梯度分离精子细胞，以避免来自精浆、细胞碎片和其他细胞类型的污染。我们发现牛精子对有毒的过氧化氢（H₂O₂）的代谢适应性很差。事实上，观察到GPx水平非常低且缺乏过氧化氢酶。我们还研究了在蛋黄 - Tris - 甘油稀释液（EYTG）中冻融牛精子的影响。冷冻保存显著降低了精子GSH水平78%，SOD活性降低了50%。我们还研究了GSH水平的降低是否与氧化代谢有关，发现与在有限氧气供应条件下孵育相比，新鲜精子细胞在38.5℃有氧条件下于EYTG中孵育6小时后，细胞内GSH水平的降低幅度更大。我们的结果强烈表明冻融循环过程中存在氧化应激，并且与这样一个循环中产生的ROS对精子功能有害的假设一致。
