title: Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium.
abstract: Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase (X, 0.1 mM; XO, 0.01 U/mL) generating system for the production of O2*-, with or without the addition of superoxide dismutase (SOD, 150 U/mL) or the SOD mimetic, Tiron (0.1, 1.0 or 5.0 mM) for 1h. Changes in fluorescence of DHE were determined for the live cell population (calcein-positive cells) by flow cytometry. The DHE fluorescence increased with the X-XO incubation; this increase was inhibited by SOD or Tiron, indicating that DHE is specific for O2*- detection. In Experiment 2, spermatozoa were loaded with DHE/CAM, treated with calcium ionophore A23187 (0, 0.8, or 8.0 microM), and incubated for 15 min. Cell fluorescence was again determined by flow cytometry. Calcium ionophore A23187 increased O2*- production in a dose-dependent manner. In Experiment 3, cells were loaded with DHE/CAM, treated with NADPH (0.0, 0.25, 0.5, or 1 mM) with or without 0.5% Triton X-100, and incubated for 15 min prior to flow cytometry. Cells treated with NADPH with or without 0.5% Triton X-100 did not have O2*- levels that were significantly different from the control. In Experiment 4, spermatozoa loaded with DHE/CAM were incubated under capacitating conditions (1.2 mM dibutryl-cAMP+1.0 mM caffeine) or in control media for 3h. Although O2*- generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O2*- production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O2*- in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O2*-.
authors.affiliation: Department of Population Health and Reproduction, 1114 Tupper Hall, University of California, Davis, CA 95616, USA.
authors.forename: L
authors.initials: L
authors.lastname: Burnaugh
authors.forename: K
authors.initials: K
authors.lastname: Sabeur
authors.forename: B A
authors.initials: BA
authors.lastname: Ball
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D019411:Clinical Laboratory Techniques / Q000662:veterinary; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D005434:Flow Cytometry / Q000662:veterinary; D005453:Fluorescence; D006736:Horses / Q000502:physiology*; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D009249:NADP / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000494:pharmacology
chemical_list: D007476:Ionophores; C067883:dihydroethidium; D013481:Superoxides; D000001:Calcimycin; D009249:NADP; D014969:Xanthine Oxidase; D004996:Ethidium