Marti E, Marti J I, Muiño-Blanco T, Cebrián-Pérez J A
Department of Biochemistry and Molecular and Cell Biology, School of Veterinary Medicine, University of Zaragoza, Zaragoza, Spain.
J Androl. 2008 Jul-Aug;29(4):459-67. doi: 10.2164/jandrol.107.003459. Epub 2008 Feb 22.
In this study, certain enzymes in ram semen involved in reactive oxygen species elimination and their changes during the cryopreservation process were characterized in order to investigate the hypothesis that the antioxidant defense system is involved in the maintenance of frozen sperm quality. Glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities were quantified in ram sperm samples subjected to cooling and freezing/thawing processes. In addition, their distribution on the sperm surface and the changes due to cryoinjury were determined by indirect immunofluorescence. SOD showed the highest antioxidant activity, which was also twice as high in fresh and cooled samples as in frozen/thawed ones. Enzymatic activity of GPx and GR showed no significant change throughout the freezing process. Seminal plasma proteins (SPPs) added alone or with other compounds showed a protective effect and accounted for an increase in the sperm quality parameters and enzyme activity levels not only in the fresh sample but also after cooling and freezing/thawing. These antioxidant enzymes were distributed over several sperm regions, and we were able to define several subpopulations according to the obtained sperm immunofluorescence patterns. The sperm membrane distribution of SOD, GPx, and GR changed considerably during cryopreservation, and the type and percentage of the immunofluorescence patterns found in fresh samples were severely modified. This remodeling was strongly affected by the use of different cryoprotectants. The mixture of SPPs, oleic/linoleic acids, and vitamin E was able to partly maintain and recover the fresh enzyme distribution, particularly of SOD.
在本研究中，对参与活性氧清除的公羊精液中的某些酶及其在冷冻保存过程中的变化进行了表征，以研究抗氧化防御系统参与维持冷冻精子质量这一假说。对经过冷却和冷冻/解冻过程的公羊精子样本中的谷胱甘肽还原酶（GR）、谷胱甘肽过氧化物酶（GPx）和超氧化物歧化酶（SOD）活性进行了定量分析。此外，通过间接免疫荧光法确定了它们在精子表面的分布以及冷冻损伤引起的变化。SOD显示出最高的抗氧化活性，在新鲜和冷却样本中的活性也是冷冻/解冻样本中的两倍。在整个冷冻过程中，GPx和GR的酶活性没有显著变化。单独添加或与其他化合物一起添加的精浆蛋白（SPP）显示出保护作用，不仅在新鲜样本中，而且在冷却和冷冻/解冻后，精子质量参数和酶活性水平均有所提高。这些抗氧化酶分布在精子的几个区域，根据获得的精子免疫荧光模式，我们能够定义几个亚群。在冷冻保存过程中，SOD、GPx和GR的精子膜分布发生了显著变化，新鲜样本中发现的免疫荧光模式的类型和百分比也发生了严重改变。这种重塑受到不同冷冻保护剂使用的强烈影响。SPP、油酸/亚油酸和维生素E的混合物能够部分维持并恢复新鲜酶的分布，尤其是SOD的分布。
