Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium

L. Burnaugh, K. Sabeur, B. A. Ball

Research output: Contribution to journalArticlepeer-review

81 Scopus citations


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}-.

Original languageEnglish
Pages (from-to)580-589
Number of pages10
Issue number3
StatePublished - Feb 2007

Bibliographical note

Funding Information:
This research was supported by the John P. Hughes Endowment, by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California pari-mutuel fund, and contributions by private donors and by the National Research Initiative Competitive Grant no. 2002-35203-12260 from the USDA Cooperative State Research, Education, and Extension Service.


  • Dihydroethidium
  • Horse
  • Spermatozoa
  • Superoxide anion

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine


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