Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima, Olsztyn, Poland.
J Anim Sci. 2018 Apr 14;96(4):1453-1465. doi: 10.1093/jas/sky063.
During semen cryopreservation, spermatozoa are exposed to physical and chemical stressors that result in their functional and structural damage. Growing evidence suggests that most cryoinjuries result from oxidative stress accompanying sperm cryopreservation. Elevated amounts of reactive oxygen species (ROS) generated during cryopreservation can react with sperm macromolecules, including proteins. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of carp spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level, and motility of spermatozoa. The spermatozoa proteins that were specifically carbonylated were identified and quantified by Western blotting, in conjunction with 2-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Cryopreservation decreased spermatozoa motility (P < 0.01) and viability (P < 0.0001) and significantly increased (P < 0.0001) the number of ROS-positive cells. We identified 25 protein spots, corresponding to 19 proteins, with increases (P < 0.05) in carbonylation level due to freezing/thawing. The identified proteins are involved in motility, metabolism, calcium-ion binding, signal transduction, protein folding, and intracellular transport. The results suggest that carbonylation of flagellar proteins can result in motility disorders and may contribute to the reduced percentage of motile spermatozoa and disturbances in movement trajectory after sperm cryopreservation. Moreover, cryopreservation may contribute to impaired cellular respiration, ATP regeneration, disturbances of Ca2+ turnover, unfolding of cytoplasmic or histone proteins, disturbances of cell signaling and intracellular transport, and reduced membrane stability. Our results contribute to the knowledge concerning cryoinjury and to further development of a modified cryopreservation procedure aimed at minimizing oxidative damage of carp sperm proteins.
在精液冷冻保存过程中，精子会暴露于物理和化学应激源中，从而导致其功能和结构受损。越来越多的证据表明，大多数冷冻损伤是由于冷冻保存过程中伴随的氧化应激引起的。冷冻保存过程中产生的大量活性氧（ROS）可以与精子大分子（包括蛋白质）反应。本研究的目的是研究冷冻保存过程中鲤鱼精子蛋白的氧化修饰（通过羰基化水平变化来衡量）。流式细胞术和计算机辅助精子分析用于评估精子活力、ROS 水平和运动性的变化。通过 Western 印迹与二维电泳（2D-oxyblot）和基质辅助激光解吸/电离飞行时间/飞行时间质谱联用，鉴定和定量特定羰基化的精子蛋白。冷冻保存降低了精子的活力（P < 0.01）和存活率（P < 0.0001），并显著增加了（P < 0.0001）ROS 阳性细胞的数量。我们鉴定出 25 个蛋白斑点，对应于 19 种蛋白质，由于冷冻/解冻导致羰基化水平增加（P < 0.05）。鉴定出的蛋白质参与运动、代谢、钙离子结合、信号转导、蛋白质折叠和细胞内运输。结果表明，鞭毛蛋白的羰基化可能导致运动障碍，并可能导致冷冻保存后的精子活力下降和运动轨迹紊乱。此外，冷冻保存可能导致细胞呼吸受损、ATP 再生减少、Ca2+ 周转紊乱、细胞质或组蛋白蛋白解折叠、细胞信号转导和细胞内运输紊乱以及膜稳定性降低。我们的研究结果有助于了解冷冻损伤，并进一步开发旨在最小化鲤鱼精子蛋白氧化损伤的改良冷冻保存程序。
