Sperm cryopreservation leads to various structural and functional damages, some of which induce by oxidative stress. The reactive oxygen species (ROS) generates by mitochondria and membrane NADPH oxidases (NOXs). Among the NOXs, only NOX5 has been identified in the cell membrane of human sperm. This study was designed to clarify the possible role of NOX5 on sperm cryoinjury. Forty human semen samples were washed and randomly divided into fresh and cryopreserved groups. Each group was divided into 4 subgroups containing Ham’s F10 (control), 0.1% DMSO (vehicle), 100 nM of PMA (phorbol 12-myristate 13-acetate) and 1 µM of DPI (diphenyleneiodonium), as NOX5 activator and inhibitor. The samples of cryopreserved groups were preserved in liquid nitrogen for 1 month. The sperm kinematics, membrane integrity, ROS production, apoptosis rate, mitochondrial membrane potential (MMP), intracellular ATP and calcium concentration [Ca²⁺]_i were evaluated. The percent of sperm with intact membrane and motile sperm reduced significantly after thawing (p ≤ 0.01). The ROS production (p ≤ 0.01) and the apoptotic rate increased, MMP dissipated, and the percentage of live cells with high [Ca²⁺]_i decreased significantly in the cryopreserved control group relative to the fresh control group. DPI, in contrast to PMA, improved sperm progressive motility (p ≤ 0.01), membrane integrity in fresh and cryopreserved groups and reduced the ROS amount in cryopreserved group (p ≤ 0.01). Apoptotic rate, [Ca²⁺]_i, ATP, and MMP did not change with DPI and PMA in cryopreserved groups. We conclude that NOX5 activity in fresh sperm is low, and it increases during cryopreservation. NOX5 inhibition improves the cryopreserved sperm quality.

精子冷冻保存会导致各种结构和功能损伤，其中一些是由氧化应激引起的。活性氧 (ROS) 由线粒体和膜 NADPH 氧化酶 (NOX) 产生。在 NOXs 中，只有 NOX5 存在于人类精子的细胞膜中。本研究旨在阐明 NOX5 对精子冷冻损伤的可能作用。洗涤四十份人类精液样本，随机分为新鲜组和冷冻组。每组分为 4 个亚组，包含 Ham's F10（对照）、0.1% DMSO（载体）、100 nM PMA（佛波醇 12-肉豆蔻酸酯 13-乙酸酯）和 1 μM DPI（二苯碘鎓），作为 NOX5 激活剂和抑制剂。冷冻保存组的样品在液氮中保存1个月。精子运动学、膜完整性、ROS 产生、细胞凋亡率、²⁺ ]_我被评估了。有完整的包膜并活动精子的精子的百分比解冻（后显著降低p  ≤0.01）。所述ROS产生（p  ≤0.01）和细胞凋亡率增加，MMP消散，和活细胞的高百分比的[Ca ²⁺ ]_我显著冻存相对于新鲜对照组，对照组降低。与 PMA 相比，DPI 改善了新鲜和冷冻组的精子进行性运动 ( p  ≤ 0.01)、膜完整性，并降低了冷冻组的 ROS 量 ( p  ≤ 0.01)。细胞凋亡率，[Ca ²⁺ ] _i、ATP 和 MMP 在冷冻保存组中不随 DPI 和 PMA 变化。我们得出结论，新鲜精子中的 NOX5 活性很低，并且在冷冻保存过程中会增加。NOX5 抑制提高了冷冻保存的精子质量。