Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium

Low-level production of the superoxide anion (O₂{radical dot}^-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O₂{radical dot}^- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O₂{radical dot}^- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 μM) 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{radical dot}^-, 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 1 h. 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{radical dot}^- detection. In Experiment 2, spermatozoa were loaded with DHE/CAM, treated with calcium ionophore A23187 (0, 0.8, or 8.0 μM), and incubated for 15 min. Cell fluorescence was again determined by flow cytometry. Calcium ionophore A23187 increased O₂{radical dot}^- 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{radical dot}^- 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 3 h. Although O₂{radical dot}^- generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O₂{radical dot}^- production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O₂{radical dot}^- in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O₂{radical dot}^-.