title: Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.
abstract: Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Plaza Davila
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Martin Muñoz
authors.affiliation: Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Tapia
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ortega Ferrusola
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Balao da Silva C
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors*; D006019:Glycolysis / Q000187:drug effects; D006736:Horses; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D042967:Electron Transport Complex I