Manandhar Gaurishankar, Miranda-Vizuete Antonio, Pedrajas Jose R, Krause William J, Zimmerman Shawn, Sutovsky Miriam, Sutovsky Peter
Division of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Biol Reprod. 2009 Jun;80(6):1168-77. doi: 10.1095/biolreprod.108.071738. Epub 2009 Feb 4.
Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.
过氧化物酶体增殖物激活受体γ辅激活因子2（PRDX2）是一种高效的氧化还原蛋白，可中和过氧化氢，从而保护细胞免受氧化损伤，并调节过氧化物介导的信号转导事件。PRDX2的氧化形式通过硫氧还蛋白系统恢复为还原形式。在本研究中，我们使用蛋白质组学技术和免疫细胞化学方法，研究了PRDX2在小鼠和公猪精子以及小鼠精子细胞中的存在情况。精子和精子细胞提取物在蛋白质印迹上显示出一条20 kDa的PRDX2条带。PRDX2在精子细胞中以Triton可溶形式存在，在成熟精子中以Triton不溶形式存在。用PRDX2抗体对公猪生精小管提取物进行免疫沉淀，然后通过SDS-PAGE分离。通过基质辅助激光解吸电离飞行时间（TOF）进行肽质量指纹分析，以及通过纳喷雾四极杆-四极杆TOF串联质谱进行微测序，结果显示在免疫沉淀条带中存在PRDX2离子，同时还有精子线粒体相关富含半胱氨酸蛋白、细胞核酸结合蛋白和谷胱甘肽过氧化物酶4。在小鼠精母细胞和精子细胞中，在细胞质和残余小体中观察到PRDX2的弥漫性标记。精子形成后，PRDX2的定位局限于精子尾部中段的线粒体鞘。公猪精子显示出与小鼠精子相似的PRDX2定位。顶体受损的公猪精子在顶体后鞘区域表达PRDX2。通过评估在存在或不存在谷胱甘肽消耗剂（马来酸二乙酯）或谷胱甘肽还原酶抑制剂（卡莫司汀）的情况下NADPH氧化速率，对公猪精子提取物的过氧化物酶活性进行了评估。马来酸二乙酯部分抑制过氧化物酶活性，而卡莫司汀显示出不显著的作用。这些观察结果表明，谷胱甘肽和谷胱甘肽还原酶活性仅部分有助于精子提取物的总过氧化物酶活性。虽然PRDX2在精子提取物总过氧化物酶活性中的具体作用仍然是一个悬而未决的问题，但本研究首次（据我们所知）表明PRDX2存在于哺乳动物精子中。精子提取物中的过氧化物酶活性不是由于谷胱甘肽系统，因此可能涉及PRDX2和其他过氧化物酶。
