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.

Abbreviations: 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 ANOVA: analysis of variance

KEYWORDS:

• Human spermatozoa
• nitrosative stress
• oxidative stress
• peroxynitrite
• sperm motility
• thiol groups

Declaration of interest

This work was supported by: PIP (Proyecto de Investigación plurianual, grupo de investigación, 2012-2014) National Research Council (CONICET), Sectyp 06/J307 (Secretaria de Ciencia y Tecnología) National University of Cuyo, and CIUDA (Consejo de Investigaciones de la Universidad del Aconcagua) Aconcagua University, Argentina; DI12-0102 from Universidad de La Frontera (J.V.V.), MEC-80130007 from CONICYT (M.F.-R.S.) and 21110812 scholarships from CONICYT (P.U.), Chile. The authors report no declarations of interest.

Additional information

Notes on contributors

María E. Cabrillana

Study conception and design, data acquisition, analysis and interpretation, drafting of the article: MEC, PU; Study conception and design, drafting of the article, and revising of the article critically for important intellectual content: JVV; Data analysis and interpretation, revising of the article critically for important intellectual content: JA, RS, MWF. All authors have read and approved the final version of the manuscript and agree with the order of presentation of the authors.