Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Zand Blvd, 71348-45794, Shiraz, Iran.
Department of Immunology and Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, 71345-1798, Shiraz, Iran.
Cell Tissue Bank. 2020 Dec;21(4):675-684. doi: 10.1007/s10561-020-09845-0. Epub 2020 Jul 1.
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 [Ca] 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 [Ca] 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, [Ca], 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，其中一些损伤是由氧化应激引起的。活性氧（ROS）由线粒体和膜 NADPH 氧化酶（NOXs）产生。在 NOXs 中，只有 NOX5 被鉴定为人类精子的细胞膜上。本研究旨在阐明 NOX5 在精子冷冻损伤中的可能作用。将 40 个人类精液样本洗涤并随机分为新鲜组和冷冻组。每组分为新鲜组和冷冻组，每组分为 4 个亚组，分别含有 Ham's F10（对照）、0.1% DMSO（载体）、100 nM PMA（佛波醇 12-肉豆蔻酸 13-乙酸酯）和 1 μM DPI（二苯基碘），作为 NOX5 激活剂和抑制剂。冷冻组的样本在液氮中保存 1 个月。评估精子运动学、膜完整性、ROS 产生、凋亡率、线粒体膜电位（MMP）、细胞内 ATP 和钙离子浓度[Ca]。解冻后，完整膜精子和活动精子的百分比显著降低（p≤0.01）。与新鲜对照组相比，冷冻对照组 ROS 产生（p≤0.01）和凋亡率增加，MMP 耗散，活细胞中高[Ca]的百分比显著降低。与 PMA 相比，DPI 显著改善新鲜和冷冻组精子的前向运动（p≤0.01）和膜完整性，并降低冷冻组 ROS 量（p≤0.01）。DPI 和 PMA 在冷冻组中对凋亡率、[Ca]、ATP 和 MMP 没有影响。我们得出结论，新鲜精子中 NOX5 活性较低，冷冻过程中活性增加。NOX5 抑制可改善冷冻精子质量。
