Using Fluorescence-Activated Flow Cytometry to Determine Reactive Oxygen Species Formation and Membrane Lipid Peroxidation in Viable Boar Spermatozoa

  • H. David Guthrie
  • Glenn R. Welch
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 594)

Abstract

Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa loaded with, respectively, hydroethidine (HE) or the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C11BODIPY581/591 (BODIPY). ROS was detected by red fluorescence emission from oxidization of HE and membrane lipid peroxidation was detected by green fluorescence emission from oxidation of BODIPY in individual live sperm. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeSo4/ascorbate (FeAc), because menadione and H2O2 had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H2O2; FeAc had no effect. The incidence of basal or spontaneous ROS formation and membrane lipid peroxidation were low in boar sperm (<1% of live sperm) in fresh semen or after low temperature storage; however the sperm were quite susceptible to treatment-induced ROS formation and membrane lipid peroxidation.

Key words

C11-BODIPY581/591 Lipid peroxidation Hydroethidine Flow cytometry Motility 

References

  1. 1.
    Johnson LA (1985) Fertility results using frozen boar spermatozoa: 1970–1985. In: Johnson LA, Larsson K (eds) Deep freezing of Boar semen. Swedish Univ. Agric Sci, Uppsala, pp 199–222Google Scholar
  2. 2.
    Waberski D, Weitze KF, Gleumes T, Schwarz M, Willmen T, Petzoldt R (1994) Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen. Theriogenology 42:831–840PubMedCrossRefGoogle Scholar
  3. 3.
    Roca J, Gil MA, Hernandez M, Parrilla I, Vazquez JM, Martinez EA (2004) Survival and fertility of boar spermatozoa after freeze-thawing in extender supplemented with butylated hydroxytoluene. J Androl 25:397–405PubMedGoogle Scholar
  4. 4.
    Cerolini S, Maldjian A, Surai P, Noble R (2000) Viability, susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage. Anim Reprod Sci 58:99–111PubMedCrossRefGoogle Scholar
  5. 5.
    Breininger E, Beorlegui NB, O’Flaherty CM, Beconi MT (2005) Alpha-tocopherol improves biochemical and dynamic variables in cryopreserved boar semen. Theriogenology 63:2126–2135PubMedCrossRefGoogle Scholar
  6. 6.
    Fridovich I (2003) Editorial commentary on “Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: Potential implications in intracellular fluorescence detection of superoxide” by H. Zhao et al. Free Radic Biol Med 34:1357–1358CrossRefGoogle Scholar
  7. 7.
    Bass DA, Parce JW, DeChatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 130:1910–1917PubMedGoogle Scholar
  8. 8.
    Carter WO, Narayanan PK, Robinson JP (1994) Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol 55:253–258PubMedGoogle Scholar
  9. 9.
    Guthrie HD, Welch GR (2006) Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci 84:2089–2100PubMedCrossRefGoogle Scholar
  10. 10.
    Spiteller G (2006) Peroxyl radicals: inductors of neurodegenerative and other inflammatory diseases. Their origin and how they transform cholesterol, phospholipids, plasmalogens, polyunsaturated fatty acids, sugars, and proteins into deleterious products. Free Radic Biol Med 41:362–387Google Scholar
  11. 11.
    Storey BT (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod 3:203–213PubMedCrossRefGoogle Scholar
  12. 12.
    Guthrie HD, Welch GR (2007) Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with4, 4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid. J Anim Sci 85:1402–1411PubMedCrossRefGoogle Scholar
  13. 13.
    Brouwers JF, Gadella BM (2003) In situ detection and localization of lipid peroxidation in individual bovine sperm cells. Free Radic Biol Med 35:1382–1391PubMedCrossRefGoogle Scholar
  14. 14.
    Pap EH, Drummen GP, Winter VJ, Kooij TW, Rijken P, Wirtz KW, Op den Kamp JA, Hage WJ, Post JA (1999) Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591). FEBS Lett 453:278–282Google Scholar
  15. 15.
    Drummen GPC, van Liebergen LCM, Op den Kamp JAF, Post JA (2002) C11-BODIPY581-591, an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic Biol Med 33:473–490Google Scholar
  16. 16.
    Haugland RP (2005) The handbook, 10th edn. Molecular Probes Inc., EugeneGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • H. David Guthrie
    • 1
  • Glenn R. Welch
    • 1
  1. 1.U. S. Department of Agriculture, Biotechnology and Germplasm LaboratoryAgricultural Research ServiceBeltsvilleUSA

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