Advertisement

Sperm DNA Fragmentation Analysis Using the TUNEL Assay

  • Rakesh Sharma
  • Jayson Masaki
  • Ashok Agarwal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 927)

Abstract

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling or the TUNEL assay is an important technique in the assessment of DNA damage. Semen samples are routinely assessed microscopically to assess their fertilization ability. In addition to routine semen analysis, the use of the TUNEL assay can provide information on the level of DNA damage present within a sample. This chapter provides a practical walk-through guide aimed at directing a researcher or a clinical facility interested in setting up and using TUNEL and flow cytometry or fluorescence microscopy for sperm DNA analysis.

Key words

TUNEL DNA damage Flow cytometry Chromatin 

References

  1. 1.
    Aitken RJ et al (2009) Biological and clinical significance of DNA damage in the male germ line. Int J Androl 32:46–56PubMedCrossRefGoogle Scholar
  2. 2.
    Sharma RK et al (2004) Sperm DNA damage and its clinical relevance in assessing reproductive outcome. Asian J Androl 6:139–148PubMedGoogle Scholar
  3. 3.
    Tremellen K (2008) Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 14:243–258PubMedCrossRefGoogle Scholar
  4. 4.
    Twigg J et al (1998) Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod 4:439–445PubMedCrossRefGoogle Scholar
  5. 5.
    Agarwal A, Prabakaran SA (2005) Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology. Indian J Exp Biol 43:963–974PubMedGoogle Scholar
  6. 6.
    Koppers AJ et al (2008) Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab 93:3199–3207PubMedCrossRefGoogle Scholar
  7. 7.
    Plante M et al (1994) Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertil Steril 62:387–393PubMedGoogle Scholar
  8. 8.
    Henkel R et al (2005) Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients. Fertil Steril 83:635–642PubMedCrossRefGoogle Scholar
  9. 9.
    Zini A et al (2009) Antioxidants and sperm DNA damage: a clinical perspective. J Assist Reprod Genet 26:427–432PubMedCrossRefGoogle Scholar
  10. 10.
    Makker K et al (2009) Oxidative stress & male infertility. Indian J Med Res 129:357–367PubMedGoogle Scholar
  11. 11.
    Desai N et al (2009) Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil Steril 92:1626–1631PubMedCrossRefGoogle Scholar
  12. 12.
    Wang X et al (2003) Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 80:531–535PubMedCrossRefGoogle Scholar
  13. 13.
    Mahfouz R et al (2010) Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril 94:2141–2146PubMedCrossRefGoogle Scholar
  14. 14.
    Robbins DJ, Coleman MS (1988) Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions. Nucleic Acids Res 16:2943–2957PubMedCrossRefGoogle Scholar
  15. 15.
    Robbins DJ et al (1987) Interaction of terminal transferase with single-stranded DNA. J Biol Chem 262:9494–9502PubMedGoogle Scholar
  16. 16.
    Loft S et al (2003) Oxidative DNA damage in human sperm influences time to pregnancy. Hum Reprod 18:1265–1272PubMedCrossRefGoogle Scholar
  17. 17.
    Shen HM et al (1999) Evaluation of oxidative DNA damage in human sperm and its association with male infertility. J Androl 20:718–723PubMedGoogle Scholar
  18. 18.
    Derijck A et al (2008) DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation. Hum Mol Genet 17:1922–1937PubMedCrossRefGoogle Scholar
  19. 19.
    Lewis SE et al (2008) Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med 54:111–125PubMedCrossRefGoogle Scholar
  20. 20.
    Mitchell LA et al (2011) The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl 34:2–13PubMedCrossRefGoogle Scholar
  21. 21.
    Ward WS, Coffey DS (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 44:569–574PubMedCrossRefGoogle Scholar
  22. 22.
    Sharma RK et al (2010) TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 76:1380–1386PubMedCrossRefGoogle Scholar
  23. 23.
    Duran EH et al (2002) Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 17:3122–3128PubMedCrossRefGoogle Scholar
  24. 24.
    Benchaib M et al (2003) Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 18:1023–1028PubMedCrossRefGoogle Scholar
  25. 25.
    Henkel R et al (2003) DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online 7:477–484PubMedCrossRefGoogle Scholar
  26. 26.
    Henkel R et al (2004) Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 81:965–972PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Center for Reproductive Medicine, Glickman Urological & Kidney InstituteCleveland ClinicClevelandUSA

Personalised recommendations