Biotechnology and Germplasm Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, Maryland, USA.
Theriogenology. 2012 Nov;78(8):1700-8. doi: 10.1016/j.theriogenology.2012.05.002. Epub 2012 Jun 14.
Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.
活性氧 (ROS) 的形成和膜脂质过氧化已被认为是精子存活和生育能力的问题。由于已建立的化学发光测定技术的特异性和灵敏度较低，因此超氧化物 (SO)、过氧化氢 (HP) 和膜脂质过氧化的精确作用和检测一直存在问题。我们开发了用于测量猪精子中 SO、HP、膜脂质过氧化和线粒体跨膜电位的流式细胞术检测方法。这些方法足够灵敏，可以检测到精子细胞中 ROS 形成的早期变化，而这些细胞仍然具有活力。新鲜和冷冻解冻的猪精液中活精子的基础 ROS 形成和膜脂质过氧化在没有 ROS 生成剂的情况下很低，平均影响不到 4%的精子细胞。然而，在其他物种中并非如此，因为人类、牛和家禽精子的 ROS 形成、脂质过氧化、运动能力丧失和体外死亡都会大幅增加。使用甲萘醌（线粒体 SO 生成剂）和 HP 处理更深入地研究了 ROS 形成对猪精子运动能力和 ATP 含量之间关系的影响。甲萘醌或 HP 分别立即破坏运动能力，而精子 ATP 含量延迟或没有下降。总的来说，ROS 对运动能力的抑制作用表明存在与线粒体无关的机制。运动能力的降低可能是由于 ROS 诱导的 ATP 利用或鞭毛的收缩装置损伤所致。
