Cabrillana María E, Uribe Pamela, Villegas Juana V, Álvarez Juan, Sánchez Raúl, Fornés Miguel W
a Laboratory of Andrology Research of Mendoza (LIAM) Institute of Histology and Embriology of Mendoza (IHEM) Histology and Embryology Area, Department of Morphology and Physiology , School of Medicine, National University of Cuyo & CCT-Mendoza , CONICET, Mendoza , Argentina.
b Research Institute (CIUDA), Medicine Faculty , Universidad del Aconcagua , Mendoza , Argentina.
Syst Biol Reprod Med. 2016 Oct;62(5):325-34. doi: 10.1080/19396368.2016.1208782. Epub 2016 Aug 3.
Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility.
DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation
analysis of variance.
过氧亚硝酸根是一种高反应性氮物种，当它大量生成时会导致亚硝化应激，这是精子功能受损的一个重要原因。已表明高水平的过氧亚硝酸根与不育男性精液质量下降相关。精子中的硫醇基团主要存在于轴丝中的酶、抗氧化分子和结构蛋白中。过氧亚硝酸根主要与含半胱氨酸蛋白质的硫醇基团发生反应。尽管众所周知过氧亚硝酸根会氧化精子中的巯基，但这种氧化的亚细胞定位仍不清楚。本研究的主要目的是确定过氧亚硝酸根诱导的亚硝化应激在硫醇基团中的亚细胞定位及其与人类精子活力的关系。为此，将健康供体的精子在体外暴露于3-吗啉代-sydnonimine（SIN-1），一种能产生过氧亚硝酸根的化合物。为了检测过氧亚硝酸根和还原型硫醇基团，分别使用了荧光探针二氢罗丹明123和单溴联苯胺（mBBr）。通过碘化丙啶染色分析精子活力。通过共聚焦显微镜监测过氧亚硝酸根的生成和硫醇氧化还原状态，而通过流式细胞术评估精子活力。使用ISAS（®）系统通过计算机辅助精子分析（CASA）分析精子活力。结果表明，人类精子暴露于过氧亚硝酸根会导致硫醇氧化增加，主要定位于精子头部和主段区域。硫醇氧化与活力丧失相关。硫醇基团对过氧亚硝酸根诱导氧化的高敏感性至少可以部分解释活性氮物种对精子活力的负面影响。
DHR：二氢罗丹明123；mBBr：单溴联苯胺；ONOO(-)：过氧亚硝酸根；RNS：活性氮物种；RFI：相对荧光强度；SIN-1：3-吗啉代-sydnonimine；CASA：计算机辅助精子分析；PARP：聚ADP核糖聚合酶；VCL：曲线速度；VSL：直线速度；VAP：平均路径速度；PRDXs：过氧化物酶；ODF：外致密纤维；ODF1：外致密纤维1；PI：碘化丙啶；DMSO：二甲基亚砜；SD：标准差；
方差分析。
