Mostek Agnieszka, Dietrich Mariola Aleksandra, Słowińska Mariola, Ciereszko Andrzej
Institute of Animal Reproduction and Food Research, Department of Gamete and Embryo Biology, Polish Academy of Sciences, Tuwima 10 Str., 10-748 Olsztyn, Poland.
Institute of Animal Reproduction and Food Research, Department of Gamete and Embryo Biology, Polish Academy of Sciences, Tuwima 10 Str., 10-748 Olsztyn, Poland.
Theriogenology. 2017 Apr 1;92:95-102. doi: 10.1016/j.theriogenology.2017.01.011. Epub 2017 Jan 10.
Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull 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. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation.
使用冷冻精液进行人工授精能够以可承受的成本大规模传播具有优良遗传特性的配子。然而，冷冻保存会对精子造成功能和结构损伤，这与活性氧（ROS）的产生、精子活力受损以及受精潜力降低有关，但人们对蛋白质变化关注较少。本研究的目的是调查冷冻保存过程引发的公牛精子蛋白质的氧化修饰（以羰基化水平变化衡量）。使用流式细胞术和计算机辅助精子分析来评估精子活力、ROS水平和运动能力的变化。采用蛋白质免疫印迹法，结合二维电泳（二维氧化印迹法）和基质辅助激光解吸/电离飞行时间/飞行时间质谱法，来鉴定和定量精子中特异性羰基化的蛋白质。冷冻保存降低了精子的运动能力和活力，但增加了ROS阳性细胞的数量。我们鉴定出11种蛋白质（roporin-1、外致密纤维蛋白2、谷胱甘肽S-转移酶、磷酸丙糖异构酶、帽蛋白β3亚型、肌动蛋白相关蛋白M1、肌动蛋白相关蛋白T2、NADH脱氢酶、异柠檬酸脱氢酶、纤毛和鞭毛相关蛋白161、磷脂酰乙醇胺结合蛋白4），它们在冷冻保存后蛋白质羰基化方面表现出差异。所鉴定的蛋白质与细胞骨架和鞭毛组织、解毒及能量代谢相关。此外，几乎所有鉴定出的羰基化蛋白质都参与了精子获能过程。我们的结果首次表明，冷冻保存通过羰基化诱导特定精子蛋白质发生氧化。我们认为，精子蛋白质的羰基化可能是氧化应激的直接结果，并可能导致参与获能的蛋白质受到干扰，或者表明冷冻保存诱导了过早的精子获能。
