DNA Damage in Human and Mouse Spermatozoa after In Vitro-Irradiation Assessed by the Comet Assay

  • Grant Haines
  • Brian Marples
  • Paul Daniel
  • Ian Morris
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 444)


The comet assay is widely employed as a method to measure DNA damage in a wide variety of cell types following genotoxic insult. We have used this method in order to characterise DNA damage in spermatozoa following in vitro irradiation with 137Cs gamma rays. In contrast to somatic cells, the DNA of mammalian spermatozoa is bound by protamine molecules allowing a sixfold more highly compact structure and thus rendering conventional cell lysis protocols ineffective. Therefore, this new method uses an extensive lysis step to ensure effective removal of DNA-associated proteins allowing DNA damage to be scored reproducibly in both murine and human spermatozoa. Mouse spermatozoa collected from the vas deferens at post-mortem or human spermatozoa provided by donors were irradiated with doses of γ-rays from 0–100 Gy using a 137Cs source and then processed for both alkaline and neutral comet assays. Under neutral electrophoresis conditions, which permits the measurement of double-stranded DNA breaks, a linear increase in the amount of DNA damage measured was observed with increasing radiation dose for both murine and human spermatozoa. Similarly, using alkaline electrophoresis conditions to examine DNA single-strand breaks and alkali-labile sites, a linear relationship was also observed for murine sperm but in contrast no such relationship was apparent for human spermatozoa subjected to the same radiation treatments. Interestingly, unirradiated sperm (both human and mouse) showed extensive DNA migration from the nucleus after alkaline assay. Since it is unlikely that the DNA of normal spermatozoa contains high numbers of single-strand breaks and damage was not detected for unirradiated sperm in the neutral assay, it is more likely that this DNA migration is due to the presence of high numbers of alkali labile sites within sperm DNA and that these may be related to the highly condensed structure of spermatozoal DNA. The large radiation doses used in these experiments to produce measurable amounts of DNA damage reflects the high radioresistance of spermatozoa compared to somatic cells and this may also be related to the differences in DNA packaging and conformation. In conclusion, this work shows that the comet assay represents a new method for examining DNA damage in spermatozoa and should be evaluated for use in reproductive toxicity testing.


Comet Assay Human Sperm Comet Tail Alkaline Comet Assay Mouse Sperm 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Angelis, K.J., Veleminsky, J., Reiger, R., and Schubert, I., 1989, Repair of bleomycin-induced DNA double-strand breaks in Vicia Faba. Mutat. Res. 212: 155–157.PubMedCrossRefGoogle Scholar
  2. Balhorn, R., 1982, A model for the structure of mammalian sperm. J. Cell. Biol. 93: 298–305.PubMedCrossRefGoogle Scholar
  3. Ballachey, B.E., Hohenboken, W.D., and Evenson, D.P., 1987, Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility. Biol. Reprod. 36: 915–925.PubMedCrossRefGoogle Scholar
  4. Biggers, J.D., Whitten, K.K., and Whittingham, D.G., 1971, The culture of mouse embryos in vitro, in: Methods in Mammalian Embryology, J.C. David, ed., Freeman Press, San Francisco.Google Scholar
  5. Brandriff, B., Gordon, L., Ashworth, L., Watchmaker, G., Moore, D., Wyrobek, A.J., and Carrano, A.V., 1985, Chromosomes of human sperm: variability among normal individuals. Hum. Genet. 70: 18–24.PubMedCrossRefGoogle Scholar
  6. Brandriff, B.F., Gordon, L.A., Ashworth, L.K., and Carrano, A.V., 1988, Chromosomal aberrations induced by in vitro irradiation: comparisons between human sperm and lymphocytes. Environ. Mol. Mutagen. 12: 167–177.PubMedCrossRefGoogle Scholar
  7. Carle, G.F., Frank, M., and Olson, M.V., 1986, Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 232: 65–68.PubMedCrossRefGoogle Scholar
  8. Carlsen, E., Giwercman, A., Keiding, N., and Skakkebæk, N.E., 1992, Evidence for decreasing quality of semen during past 50 years. Br. Med. J. 305: 609–613.CrossRefGoogle Scholar
  9. Cattanach, B.M., Patrick, G., Papworth, D., Goodhead, D.T., Hacker, T., Cobb, L., and Whitehall, E., 1995, Investigation of lung tumour induction in BALB/cJ mice following paternal X-irradiation. Int. J. Radiat. Biol. 67: 607–615.PubMedCrossRefGoogle Scholar
  10. Dobrzynska, M.M., and Gajewski, A.K., 1994, Mouse dominant lethal and sperm abnormality studies with combined exposure to X-rays and mitomycin C. Mutat. Res. 306: 203–209.PubMedCrossRefGoogle Scholar
  11. Ehling, U.H., 1971, Comparison of radiation-and chemically-induced dominant lethal mutations in male mice. Mutat. Res. 11: 35–44.PubMedCrossRefGoogle Scholar
  12. Evenson, D.P., Jost, L.K., Baer, R.K., Turner, T.W., and Schrader, S.M., 1991, Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol. 5: 115–125.PubMedCrossRefGoogle Scholar
  13. Fairbairn, D.W., Olive, P.L., and O’Neill, K.L., 1995, The comet assay: a comprehensive review. Mutat. Res. 339: 37–59.PubMedCrossRefGoogle Scholar
  14. Freidenrich, S., and Hand, R., 1981, The use of agarose gel electrophoresis to measure the size of DNA molecules in crude cell lysates. Analyt. Biochem. 115: 231–235.PubMedCrossRefGoogle Scholar
  15. Hassold, T., Chiu, D., and Yamane, J.A., 1984, Parental origin of autosomal trisomies. Ann. Hum. Genet. 48: 129–144.PubMedCrossRefGoogle Scholar
  16. Hughes, C.M., Lewis, S.E.M., McKelvey-Martin, V.J., and Thompson, W., 1996, A comparison of baseline and induced DNA damage in human sperm from fertile and infertile men, using a modified comet assay. Mol. Human. Reprod. 2: 613–619.CrossRefGoogle Scholar
  17. Hughes, C.M., Lewis, S.E.M., McKelvey-Martin, V.J., and Thompson, W., 1997, Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutat. Res. 374: 261–268.PubMedCrossRefGoogle Scholar
  18. Jacobs, P., Hassold, T., Harvey, J., and May, K., 1989, The origin of sex chromosome aneuploidy. Prog. Clin. Biol. Res. 311: 135–151.PubMedGoogle Scholar
  19. Kamiguchi, Y., Tateno, H., and Mikamo, K., 1990a, Types of structural chromosome aberrations and their incidences in human spermatozoa X-irradiated in vitro. Mutat. Res. 228: 133–140.PubMedCrossRefGoogle Scholar
  20. Kamiguchi, Y., Tateno, H., and Mikamo, K., 1990b, Dose-response relationship for the induction of structural chromosome aberrations in human spermatozoa after in vitro exposure to tritium beta-rays. Mutat. Res. 228: 125–131.PubMedCrossRefGoogle Scholar
  21. Kohn, K.W., Erickson, L.C., Ewig, R.G., and Friedman, C.A., 1976, Fractionation of DNA from mammalian cells by alkaline elution. Biochem. 15: 4629–4637.CrossRefGoogle Scholar
  22. Lett, J.T., Klucis, E.S., and Sun, C., 1970, On the size of DNA in the mammalian chromosome. Biophys. J. 10: 277–292.PubMedCrossRefGoogle Scholar
  23. Lipetz, P.D., Brash, D.E., Joseph, L.B., Jewett, H.D., Lisle, D.R., Lantry, L.E., and Stephens, R.E., 1982, Determination of DNA superhelicity and extremely low level of DNA strand breaks in low numbers of nonradiolabeled cells by 4′6′-diamidino-2-phenylindol fluorescence in nucleoid gradients. Analyt. Biochem. 121: 330–348.CrossRefGoogle Scholar
  24. Martin, R.H., 1985, Chromosomal abnormalities in human sperm. Basic. Life. Sci. 36: 91–102.PubMedGoogle Scholar
  25. Martin, R.H., Rademaker, A.W., Hildebrand, K., Long-Simpson, L., Peterson, D., and Yamamoto, J., 1987, Variation in the frequency and type of sperm chromosomal abnormalities among normal men. Hum. Genet. 11: 108–114.CrossRefGoogle Scholar
  26. Martin, R.H., Rademaker, A., Hildebrand, K., Barnes, M., Arthur, K., Ringrose, T., Brown, I.S., and Douglas G, 1989, A comparison of chromosomal aberrations induced by in vivo radiotherapy in human sperm and lymphocytes. Mutat. Res. 226: 21–30.PubMedCrossRefGoogle Scholar
  27. Matsuda, Y., Yamada, T., and Tobari, I., 1985, Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation. Mutat. Res. 148: 113–117.PubMedCrossRefGoogle Scholar
  28. McKelvey-Martin, V.J., Green, M.H.L., Schmezer, P., Pool-Zobel, B.L., DeMeo, M.P., and Collins, A., 1993, The single cell gel electrophoresis assay (comet assay): a European review. Mutat. Res. 288: 47–63.PubMedCrossRefGoogle Scholar
  29. McKelvey-Martin, V.J., Melia, N., Walsk, I.K., Johnston, S.R., Hughes, C.M., Lewis, S.E.M., and Thompson, W., 1997, Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1) human bladder washings and transitional cell carcinoma of the bladder; and (2) human sperm and male infertility. Mutat. Res. 375: 93–104.PubMedCrossRefGoogle Scholar
  30. Meistrich, M.L., 1986, Critical components of testicular function and sensitivity to disruption. Biol Reprod, 34: 17–28.PubMedCrossRefGoogle Scholar
  31. Meistrich, M.L., 1993, Effects of chemotherapy and radiotherapy on spermatogenesis. Eur. Urol. 23: 136–141.PubMedGoogle Scholar
  32. Mikamo, K., Kamiguchi, Y., and Tateno, H., 1990, Spontaneous and in vitro radiation-induced chromosome aberrations in human spermatozoa: application of a new method. Prog. Clin. Biol. Res. 340B: 447–456.PubMedGoogle Scholar
  33. Ono, T., and Okada, S., 1977, Radiation-induced DNA single-strand scission and its rejoining in spermatogonia and spermatozoa of mouse. Mutat. Res. 43: 25–36.PubMedCrossRefGoogle Scholar
  34. Quinn, P., Barros, C., and Whittingham, D.G., 1982, Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fertil. 66: 161–168.PubMedCrossRefGoogle Scholar
  35. Rydberg, B., 1980, Detection of induced DNA strand breaks with improved sensitivity in human cells. Radiat. Res. 81: 492–495.PubMedCrossRefGoogle Scholar
  36. Sailer, B.L., Jost, L.K., and Evenson, D.P., 1995, Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J. Androl. 16: 80–87.PubMedGoogle Scholar
  37. Searle, A.G., and Beechey, C.V., 1974, Sperm-count, egg-fertilization and dominant lethality after X-irradiation of mice. Mutat. Res. 22: 63–72.PubMedCrossRefGoogle Scholar
  38. Sega, G.A., 1976, Molecular dosimetry of chemical mutagens: measurement of molecular dose and DNA repair in mammalian germ cells. Mutat. Res. 38: 317–326.PubMedCrossRefGoogle Scholar
  39. Sega, G.A., Sluder, A.E., McCoy, L.S., Owens, J.G., and Generoso, E.E., 1986, The use of alkaline elution procedures to measure DNA damage in spermiogenic stages of mice exposed to methyl methanesulfonate. Mutat. Res. 159: 55–63.PubMedCrossRefGoogle Scholar
  40. Sega, G.A., and Generoso, E.E., 1988, Measurement of DNA breakage in spermiogenic germ-cell stages of mice exposed to ethylene oxide using an alkaline elution procedure. Mutat. Res. 197: 93–99.PubMedCrossRefGoogle Scholar
  41. Singh, N.P., McCoy, M.T., Tice, R.R., and Schneider, E.L., 1988, A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175: 184–191.PubMedCrossRefGoogle Scholar
  42. Singh, N.P., Danner, D.B., Tice, R.R., McCoy, M.T., Collins, G.D., and Schneider, E.L., 1989, Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp. Cell. Res. 184: 461–470.PubMedCrossRefGoogle Scholar
  43. Tateno, H., Kamiguchi, Y., and Mikamo, K., 1989, Cytogenetic effects of X-and γ-rays on human sperm chromosomes. J. Radiat. Res. (Tokyo). 30: 95.Google Scholar
  44. Van Loon, A.A.W.N., Den Boer, P.J., Van der Schans, G.P., Mackenbach, P., Grootegoed, J.A., Baan, R.A., and Lohman, P.H.M., 1991, Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation. Exp. Cell. Res. 193: 303–309.PubMedCrossRefGoogle Scholar
  45. Van Loon, A.A.W.N., Groenendijk, R.H., Timmerman, A.J., Van der Schans, G.P., Lohman, P.H.M., and Baan, R.A., 1992, Quantitative detection of DNA damage in cells after exposure to ionizing radiation by means of an improved immunochemical assay. Mutat. Res. 274: 19–27.PubMedCrossRefGoogle Scholar
  46. Van Loon, A.A.W.N., Sonneveld, E., Hoogerbrugge, J., Van der Schans, G.P., Grootegoed, J.A., Lohman, P.H.M., and Baan, R.A., 1993, Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation. Mutat. Res. 294: 139–148.PubMedCrossRefGoogle Scholar
  47. Ward, W.S., and Coffey, D.S., 1991, DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod. 44: 569–574.PubMedCrossRefGoogle Scholar
  48. Wheeler, K.T., and Wierowski, J.V., 1983, DNA accessibility: a determinant of mammalian cell differentiation. Radiat. Res. 93: 312–318.PubMedCrossRefGoogle Scholar
  49. World Health Organisation, 1992, Laboratory Manual for the Examination of Semen and Sperm Cervical Mucus Interaction. University Press, Cambridge.Google Scholar

Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Grant Haines
    • 1
  • Brian Marples
    • 2
  • Paul Daniel
    • 3
  • Ian Morris
    • 1
  1. 1.School of Biological SciencesUniversity of ManchesterManchesterUK
  2. 2.Paterson Institute for Cancer ResearchChristie HospitalManchesterUK
  3. 3.Department of GeneticsWestlakes Research InstituteMoor Row, CumbriaUK

Personalised recommendations