title: Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa.
abstract: 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.
  
  
  BACKGROUND
  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.
authors.affiliation: 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.
authors.forename: María E
authors.initials: ME
authors.lastname: Cabrillana
authors.affiliation: c Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR-BIOREN) , Universidad de La Frontera , Temuco , Chile.
authors.forename: Pamela
authors.initials: P
authors.lastname: Uribe
authors.affiliation: c Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR-BIOREN) , Universidad de La Frontera , Temuco , Chile.|d Department of Internal Medicine, Medicine Faculty , Universidad de La Frontera , Temuco , Chile.
authors.forename: Juana V
authors.initials: JV
authors.lastname: Villegas
authors.affiliation: e Centro ANDROGEN, La Coruña , Spain.|f Harvard Medical School , Boston , Massachusetts , USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Álvarez
authors.affiliation: c Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR-BIOREN) , Universidad de La Frontera , Temuco , Chile.|g Department of Preclinical Science, Medicine Faculty , Universidad de La Frontera , Temuco , Chile.
authors.forename: Raúl
authors.initials: R
authors.lastname: Sánchez
authors.affiliation: 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.
authors.forename: Miguel W
authors.initials: MW
authors.lastname: Fornés
mesh_terms: D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism*; D055101:Semen Analysis; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; C002385:linsidomine; D008981:Molsidomine
keywords: Human spermatozoa; nitrosative stress; oxidative stress; peroxynitrite; sperm motility; thiol groups