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*-.
Publication Date: 2006-10-12 PubMed ID: 17045638DOI: 10.1016/j.theriogenology.2006.07.021Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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The research aimed to understand the production of a molecule called superoxide anion (O2*-), and how this impacts sperm function in horses. Using a particular probe, dihydroethidium (DHE), the study was able to detect and measure the generation of this molecule during different conditions and treatment methods.
Superoxide Anion and Sperm Function
- The molecule superoxide anion (O2*-) is beneficial in low-level production as it aids in capacitation – the process that prepares sperm for fertilisation.
- However, excessive creation of O2*- can negatively affect sperm function.
DHE Probe for O2*-
- Researchers assessed DHE as a probe for detecting O2*- in equine spermatozoa (horse sperm).
- When the sperm were treated with certain substances, the DHE would react depending on the levels of the O2*- present, enabling detection and measurement.
Experiments with the Probe
- Four experiments were carried out during the study, each altering the treatment of the sperm to generate different levels of O2*- and examining the effectiveness of DHE as a probe.
- The first experiment used a combination of substances called the xanthine-xanthine oxidase (X-XO) system, producing O2*-. When superoxide dismutase (SOD) or the SOD mimetic, Tiron, were added, DHE fluorescence was inhibited, confirming its specificity to O2*- detection.
- The second experiment used calcium ionophore A23187 and demonstrated increased O2*- production in a dose-dependent manner.
- In the third experiment, cells were treated with NADPH (a form of energy in cells) and did not show a significant difference in O2*- levels.
- The fourth experiment found that spermatozoa incubated under capacitating conditions had higher O2*- production than those in control media, indicating that capacitation processes were associated with increased generation of O2*-.
Concluding Remarks
- The study concluded that DHE is indeed a useful probe for detecting O2*- in equine spermatozoa.
- The research also identified factors that increase O2*- production, including internal calcium elevation and capacitation in vitro (laboratory conditions).
Cite This Article
APA
Burnaugh L, Sabeur K, Ball BA.
(2006).
Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium.
Theriogenology, 67(3), 580-589.
https://doi.org/10.1016/j.theriogenology.2006.07.021 Publication
Researcher Affiliations
- Department of Population Health and Reproduction, 1114 Tupper Hall, University of California, Davis, CA 95616, USA.
MeSH Terms
- Animals
- Calcimycin / pharmacology
- Clinical Laboratory Techniques / veterinary
- Ethidium / analogs & derivatives
- Ethidium / metabolism
- Flow Cytometry / veterinary
- Fluorescence
- Horses / physiology
- Ionophores / pharmacology
- Male
- NADP / pharmacology
- Spermatozoa / drug effects
- Spermatozoa / metabolism
- Superoxides / metabolism
- Xanthine Oxidase / pharmacology
Citations
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