Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium

• Published in: Theriogenology 2007-02, Vol.67 (3), p.580-589
• Main Authors: Burnaugh, L., Sabeur, K., Ball, B.A.
• Format: Article
• Language: English
• Subjects: Animals; Calcimycin - pharmacology; calcium; calcium ionophore A23187; capacitation; Clinical Laboratory Techniques - veterinary; detection; Dihydroethidium; dihydroethidium fluorescence; Ethidium - analogs & derivatives; Ethidium - metabolism; Flow Cytometry - veterinary; Fluorescence; heterocyclic compounds; Horse; horses; Horses - physiology; ionophores; Ionophores - pharmacology; Male; NADP (coenzyme); NADP - pharmacology; Spermatozoa; Spermatozoa - drug effects; Spermatozoa - metabolism; Superoxide anion; superoxide dismutase; Superoxides - metabolism; xanthine; xanthine oxidase; Xanthine Oxidase - pharmacology
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2006.07.021

Low-level production of the superoxide anion (O 2 −) is an important signal transduction event in sperm function including capacitation; however, excessive production of O 2 − can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O 2 − 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 O 2 −, 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 O 2 − 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 O 2 − 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 O 2 − 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 O 2 − generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O 2 − production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O 2 − in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O 2 −.