title: Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.
abstract: Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: P
authors.initials: P
authors.lastname: Uribe
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Boguen
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: F
authors.initials: F
authors.lastname: Treulen
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Sánchez
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco 4781218, Chile juana.villegas@ufrontera.cl.
authors.forename: J V
authors.initials: JV
authors.lastname: Villegas
mesh_terms: D000328:Adult; D001562:Benzimidazoles; D002232:Carbocyanines; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000494:pharmacology*; D012235:Rhodamines; D055101:Semen Analysis; D013050:Spectrometry, Fluorescence; D013081:Sperm Motility / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects* / Q000502:physiology
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D012235:Rhodamines; C058319:dihydrorhodamine 123; D030421:Peroxynitrous Acid; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C002385:linsidomine; D008981:Molsidomine
keywords: infertility; mitochondrial membrane potential; peroxynitrite; reactive nitrogen species; sperm motility