Journal of Assisted Reproduction and Genetics

, Volume 24, Issue 12, pp 561–569 | Cite as

Mammalian sperm chromatin structure and assessment of DNA fragmentation

  • S. M. H. AndrabiEmail author


This review article illustrates the biology of mammalian sperm chromatin structure. The possible causes of DNA (deoxyribonucleic acid) fragmentation are discussed. Also available molecular techniques for assessment of mammalian sperm DNA damage are described.


Sperm chromatin DNA fragmentation Mammals 


  1. 1.
    Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005;322:33–41.PubMedGoogle Scholar
  2. 2.
    Zini A, Bielcki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation in fertile and infertile men. Fertil Steril 2001;75:674–7.PubMedGoogle Scholar
  3. 3.
    Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male fertility and comparisons with other techniques. J Androl 2002;23:25–43.PubMedGoogle Scholar
  4. 4.
    Fuentes-Mascorro G, Serrano H, Rosado A. Sperm chromatin. Arch Androl 2000;45:215–25.PubMedGoogle Scholar
  5. 5.
    Holstein A-F, Schulze W, Davidoff M. Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol 2003;1:107.PubMedGoogle Scholar
  6. 6.
    Agarwal A, Said TM. Sperm chromatin assessment. In: Gardner DK, Weissman A, Howles CM, Shoham A, editors. Textbook of assisted reproductive technology and clinical perspectives. London: Taylor & Francis; 2004. p. 93–106.Google Scholar
  7. 7.
    Akama K, Sato H, Furihata-Yamauchi M, Komatsu Y, Tobita T, Nakano M. Interaction of nucleosome core DNA with transition proteins 1 and 3 from boar late spermatid nuclei. J Biochem 1996;119:448–5.PubMedGoogle Scholar
  8. 8.
    Chevaillier P, Chirat F, Sautiere P. The amino acid sequence of the ram spermatidal protein 3—a transition protein TP3 or TP4? Eur J Biochem 1998;258:460–4.PubMedGoogle Scholar
  9. 9.
    Steger K, Klonisch T, Gavenis K, Drabent B, Doenecke D, Bergmann M. Expression of mRNA and protein of nucleoproteins during human spermiogenesis. Mol Hum Reprod 1998;4:939–45.PubMedGoogle Scholar
  10. 10.
    Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 1997;389:251–60.PubMedGoogle Scholar
  11. 11.
    Khochbin S. Histone H1 diversity: bridging regulatory signals to linker histone function. Gene 2001;271:1–12.PubMedGoogle Scholar
  12. 12.
    Arents G, Moudrianakis EN. Topography of the histone octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA. Proc Natl Acad Sci USA 1993;90:10489–93.PubMedGoogle Scholar
  13. 13.
    Govin J, Caron C, Lestrat C, Rousseaux S, Khochbin S. The role of histones in chromatin remodeling during mammalian spermiogenesis. Eur J Biochem 2004;271:3459–69.PubMedGoogle Scholar
  14. 14.
    Malik HS, Henikoff S. Phylogenomics of the nucleosome. Nat Struct Biol 2003;10:882–91.PubMedGoogle Scholar
  15. 15.
    D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival. Anim Reprod Sci 2007;101:1–17.PubMedGoogle Scholar
  16. 16.
    Marzluff WF, Gongidi P, Woods KR, Jin J, Maltais LJ. The human and mouse replication-dependent histone genes. Genomics 2002;80:487–98.PubMedGoogle Scholar
  17. 17.
    Loir M, Lanneau M. Structural function of the basic nuclear proteins in ram spermatids. J Ultrastruct Res 1984;86:262–72.PubMedGoogle Scholar
  18. 18.
    Oliva R. Protamines and male infertility. Hum Reprod 2006;12:417–35.Google Scholar
  19. 19.
    McKay DJ, Renaux BS, Dixon GH. Human sperm protamines amino-acid sequences of two forms of protamine P2. Eur J Biochem 1986;156:5–8.PubMedGoogle Scholar
  20. 20.
    Yelick PC, Balhorn R, Johnson PA, Corzett M, Mazrimas JA, Kleene KC, Hecht NB. Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not. Mol Cell Biol 1987;7:2173–9.PubMedGoogle Scholar
  21. 21.
    Sautière P, Martinage A, Bélaïche D, Arkhis A, Chevaillier P. Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines. J Biol Chem 1988;263:11059–62.PubMedGoogle Scholar
  22. 22.
    Chauvière M, Martinage A, Debarle M, Sautière P, Chevaillier P. Molecular characterization of six intermediate proteins in the processing of mouse protamine P2 precursor. Eur J Biochem 1992;204:759–65.PubMedGoogle Scholar
  23. 23.
    Green GR, Balhorn R, Poccia DL, Hecht NB. Synthesis and processing of mammalian protamines and transition proteins. Mol Reprod Dev 1994;37:255–63.PubMedGoogle Scholar
  24. 24.
    Queralt R, Oliva R. Demonstration of trans-acting factors binding to the promoter region of the testis-specific rat protamine P1 gene. Biochem Biophys Res Com 1995;208:802–12.PubMedGoogle Scholar
  25. 25.
    Wouters-Tyrou D, Martinage A, Chevaillier P, Sautiere P. Nuclear basic proteins in spermiogenesis. Biochimie 1998;80:117–28.PubMedGoogle Scholar
  26. 26.
    Banerjee S, Smallwood A. Chromatin modifi cation of imprinted H19 gene in mammalian spermatozoa. Mol Reprod Dev 1998;50:474–84.PubMedGoogle Scholar
  27. 27.
    Gusse M, Chevaillier PH. Electron microscope evidence for the presence of globular structure in different sperm chromatins. J Cell Biol 1980;87:280–84.PubMedGoogle Scholar
  28. 28.
    Oliva R, Dixon GH. Vertebrate protamine genes and the histoneto-protamine replacement reaction. Prog Nucleic Acid Res Mol Biol 1991;40:25–94.PubMedCrossRefGoogle Scholar
  29. 29.
    Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CV. Sequence-specific packaging of DNA in human sperm chromatin. Science 1987;236:962–4.PubMedGoogle Scholar
  30. 30.
    Baskaran R, Rao MRS. Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs. Biochem Biophys Res Commun 1991;179:1491–9.PubMedGoogle Scholar
  31. 31.
    Pogany GC, Corzett M, Weston S, Balhorn R. DNA and protein content of mouse sperm implication regarding sperm chromatin structure. Exp Cell Res 1981;136:127–36.PubMedGoogle Scholar
  32. 32.
    Rhim JA, Connor W, Dixon GH, Harendza CJ, Evenson DP, Palmiter RD, Brinster RL. Expression of an avian protamine in transgenic mice disrupts chromatin structure in spermatozoa. Biol Reprod 1995;52:20–32.PubMedGoogle Scholar
  33. 33.
    Koheler JK. A freeze-etching study of rabbit spermatozoa with particular reference to head structure. J Ultrastruct Res 1970;33:598–614.Google Scholar
  34. 34.
    Koheler JK, Würschmidt U, Larsen MP. Nuclear and chromatin structure in rat spermatozoa. Gamet Res 1983;8:357–70.Google Scholar
  35. 35.
    Livolant F. Cholesteric organization of DNA in the stallion sperm head. Tiss Cell 1983;16:535–55.Google Scholar
  36. 36.
    Sipski ML, Wagner TE. The total structure and organization of chromosomal fibers in eutherian sperm nuclei. Biol Reprod 1977;16:428–40.PubMedGoogle Scholar
  37. 37.
    Bench GS, Friz AM, Corzett MH, Morse DH, Balhorn R. DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry 1996;23:263–71.PubMedGoogle Scholar
  38. 38.
    Gineitis AA, Zalenskaya IA, Yau PM, Morton BE, Zalensky AO. Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment. J Cell Biol 2000;151:1591–8.PubMedGoogle Scholar
  39. 39.
    Meistrich ML, Mohapatra B, Shirley CR, Zhao M. Roles of transition nuclear proteins in spermiogenesis. Chromosoma 2003;111:483–8.PubMedGoogle Scholar
  40. 40.
    Kistler WS, Noyes C, Hsu R, Henrikson RL. The amino acid sequence of a testis-specific basic protein that is associated with spermatogenesis. J Biol Chem 1975;250:1847–53.PubMedGoogle Scholar
  41. 41.
    Schumacher JM, Artzt K, Braun RE. Spermatid perinuclear ribonucleic acid.binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice. Biol Reprod 1998;59:69–76.PubMedGoogle Scholar
  42. 42.
    Grimes SR, Platz RD, Meistrich ML, Hnilica LS. Partial characterization of a new basic nuclear protein from rat testis elongated spermatids. Biochem Biophys Res Commun 1975;67:182–9.PubMedGoogle Scholar
  43. 43.
    Engel W, Keime S, Kremling H, Hameister H, Schluter G. The genes for protamine 1 and 2 (PRM1 and PRM2) and transition protein 2 (TNP2) are closely linked in the mammalian genome. Cytogenet Cell Genet 1992;61:158–9.PubMedGoogle Scholar
  44. 44.
    Heidaran MA, Kozak CA, Kistler WS. Nucleotide sequence of the Stp-1 gene coding for rat spermatid nuclear transition protein 1 (TP1): homology with protamine P1 and assignment of the mouse Stp-1 gene to chromosome 1. Gene 1989;75:39–46.PubMedGoogle Scholar
  45. 45.
    Illison L. Spermatozoal head shape in two inbred strains of mice and their F1 and F2 progenic. Aust J Biol Sci 1969;22:947–63.Google Scholar
  46. 46.
    Ostermeier GC, Sartor-Bergfelt R, Susko-Parrish JL, Parrish JJ. Bull fertility and sperm nuclear shape. AgBiotechNet 2000;2:1–6.Google Scholar
  47. 47.
    Meistrich ML, Brock WA, Grimes SR, Platz RD, Hnilica LS. Nuclear protein transitions during spermatogenesis. Fed Proc 1978;37:2522–5.PubMedGoogle Scholar
  48. 48.
    Warrant RW, Kim SH. Helix-double helix interaction shown in the structure of a protamine transfer RNA complex and a nucleoprotamine model. Nature 1978;271:130–5.PubMedGoogle Scholar
  49. 49.
    Balhorn RA. model for the structure of chromatin in mammalian sperm. J Cell Biol 1982;93:298–305.PubMedGoogle Scholar
  50. 50.
    Kornberg RD. Chromatin structure: repeating unit of histones and DNA. Science 1974;184:868–71.PubMedGoogle Scholar
  51. 51.
    Thoma F, Koller T, Klug A. Involvement of histone H1 in the organization of the nucleosome and the salt-dependent superstructures of chromatin. J Cell Biol 1979;83:402–27.Google Scholar
  52. 52.
    Ciejek EM, Tsai MJ, O’Malley BW. Actively transcribed genes are associated with the nuclear matrix. Nature 1983;306:607–9.PubMedGoogle Scholar
  53. 53.
    Robinson SI, Small D, Idzerda R, McKnight GS, Vogelstein B. The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct. Nucleic Acids Res 1983;11:5113–30.PubMedGoogle Scholar
  54. 54.
    Jarman AP, Higgs DR. Nuclear scaffold attachment sites in the human globin gene complexes. EMBO J 1998;7:3337–44.Google Scholar
  55. 55.
    Boulikas T. Nature of DNA sequences at the attachment regions of genes to the nuclear matrix. J Cell Biochem 1998;52:14–22.Google Scholar
  56. 56.
    Bode J, Stengert-Iber M, Kay V, Schlake T, Dietz-Pfeilstetter A. Scaffold/matrix-attached regions: topological switches with multiple regulatory functions. Crit Rev Eukaryot Gene Expr 1996;6:115–38.PubMedGoogle Scholar
  57. 57.
    Singh GB, Kramer JA, Krawetz SA. Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res 1997;25:1419–25.PubMedGoogle Scholar
  58. 58.
    Walter WR, Singh GB, Krawetz SA. MARs mission update. Biochem Biophys Res Commun 1998;242:419–22.PubMedGoogle Scholar
  59. 59.
    Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod 2003;9:331–45.Google Scholar
  60. 60.
    Sailer B, Jost L, Evenson D. 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 1995;16:80–7.PubMedGoogle Scholar
  61. 61.
    Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59:1037–46.PubMedGoogle Scholar
  62. 62.
    Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993;53:1945–51.Google Scholar
  63. 63.
    de Yebra L, Ballescà JL, Vanrell JA, Corzett M, Balhorn R, Oliva R. Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Fertil Steril 1998;69:755–9.PubMedGoogle Scholar
  64. 64.
    Evenson DP, Jost LK, Corzett M, Balhorn R. Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study. J Androl 2000;21:739–46.PubMedGoogle Scholar
  65. 65.
    Page AW, Orr-Weaver TL. Stopping and starting the meiotic cell cycle. Curr Opin Genet Dev 1997;7:23–31.PubMedGoogle Scholar
  66. 66.
    Bannister LA, Schimenti JC. Homologous recombinational repair proteins in mouse meiosis. Cytogenet Genome Res 2004;107:191–200.PubMedGoogle Scholar
  67. 67.
    Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl 2006;8:11–29.PubMedGoogle Scholar
  68. 68.
    McPherson SM, Longo FJ. Chromatin structure–function alterations during mammalian spermatogenesis—DNA nicking and repair in elongating spermatids. Eur J Histochem 1993;37:109–28.PubMedGoogle Scholar
  69. 69.
    McPherson SM, Longo FJ. Endogenous nicks in elongating spermatid DNA-involvement of DNA topoisomerase-ii and protamine. Mol Biol Cell 1992;3:A102.Google Scholar
  70. 70.
    Laberge RM, Boissonneault G. On the nature and origin of DNA strand breaks in elongating spermatids. Biol Reprod 2005;73:289–96.PubMedGoogle Scholar
  71. 71.
    Erenpreiss J, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001;22:45–53.PubMedGoogle Scholar
  72. 72.
    Warren JS, Johnson KJ, Ward PA. Oxygen radicals in cell injury and cell death. Pathol Immunopathol Res 1987;6:301–15.PubMedGoogle Scholar
  73. 73.
    Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology 1996;48:835–50.PubMedGoogle Scholar
  74. 74.
    Zini A, Libman J. Sperm DNA damage: importance in the era of assisted reproduction. Curr Opin Urol 2006;16:428–34.PubMedGoogle Scholar
  75. 75.
    Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001;65:1102–13.PubMedGoogle Scholar
  76. 76.
    Aitken RJ, Fisher HM, Fulton N, Gomez E, Knox W, Lewis B, Irvine S. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol Reprod Dev 1997;47:468–82.PubMedGoogle Scholar
  77. 77.
    Banfi B, Molnar G, Maturana A, Steger K, Hegedus B, Demaurex N, Krause KH. A Ca(2)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem 2001;276:37594–601.PubMedGoogle Scholar
  78. 78.
    Oliw EH, Sprecher H. Metabolism of polyunsaturated fatty acids by an (n-6)-lipoxygenase associated with human ejaculates. Biochim Biophys Acta 1989;1002:283–91.PubMedGoogle Scholar
  79. 79.
    Aitken RJ, Ryan AL, Curry BJ, Baker MA. Multiple forms of redox activity in populations of human spermatozoa. Mol Hum Reprod 2003;9:645–61.PubMedGoogle Scholar
  80. 80.
    Morré DJ, Morré DM. Cell surface NADH oxidases (ECTONOX proteins) with roles in cancer, cellular time-keeping, growth, aging and neurodegenerative diseases. Free Radic Res 2003;37:795–808.PubMedGoogle Scholar
  81. 81.
    Baker MA, Krutskikh A, Curry BJ, McLaughlin EA, Aitken RJ. Identification of cytochrome P450-reductase as theenzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biol Reprod 2004;71:307–18.PubMedGoogle Scholar
  82. 82.
    Cummins JM, Jequier AM, Kann R. Molecular biology of human male infertility: links with ageing, mitochondrial genetics and oxidative stress? Mol Reprod Dev 1994;37:345–62.PubMedGoogle Scholar
  83. 83.
    Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998;13:1864–71.PubMedGoogle Scholar
  84. 84.
    Donnelly ET, McClure N, Lewis SE. The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis 1999;14:505–12.PubMedGoogle Scholar
  85. 85.
    Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21:33–44.PubMedGoogle Scholar
  86. 86.
    Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl 1996;17:276–87.PubMedGoogle Scholar
  87. 87.
    Sinha Hikim AP, Rajavashisth TB, Sinha Hikim I, Lue Y, Bonavera JJ, Leung A, Wang C, Swerdloff RS. Significance of apoptosis in the temporal and stage-specific loss of germ cells in the adult rat after gonadotropin deprivation. Biol Reprod 1997;57:1193–201.PubMedGoogle Scholar
  88. 88.
    Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999;4:38–47.PubMedGoogle Scholar
  89. 89.
    Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodelling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol Reprod 1997;56:1020–4.PubMedGoogle Scholar
  90. 90.
    Francavilla S, D’Abrizio P, Rucci N, Silvano G, Properzi G, Straface E, Cordeschi G, Necozione S, Gnessi L, Arizzi M, Ulisse S. Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 2000;85:2692–700.PubMedGoogle Scholar
  91. 91.
    Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281:1312–6.PubMedGoogle Scholar
  92. 92.
    Haaf T, Ward DC. Higher order nuclear structure in mammalian sperm revealed by in situ hybridisation and extended chromatin fibres. Exp Cell Res 1995;219:604–11.PubMedGoogle Scholar
  93. 93.
    Pérez-Llano B, Enciso M, García-Casado P, Sala R, Gosálvez J. Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders. Theriogenology 2006;66:2137–43.PubMedGoogle Scholar
  94. 94.
    Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;240:1131–3.Google Scholar
  95. 95.
    Host E, Lindenberg S, Ernst E, Christensen F. DNA strand breaks in human spermatozoa: a possible factor to be considered in couples suffering from unexplained fertility. Acta Obstet Gynec Scand 1999;78:622–5.PubMedGoogle Scholar
  96. 96.
    Host E, Lindenberg S, Kahn JA, Christensen F. DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynec Scand 1999;78:336–9.PubMedGoogle Scholar
  97. 97.
    Host E, Lindenberg S, Smidt-Jensen S. DNA strand breaks in human spermatozoa: correlation with fertilisation in vitro in oligozoospermic men and in men with unexplained fertility. Acta Obstet Gynec Scand 2000;79:189–93.PubMedGoogle Scholar
  98. 98.
    Host E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynec Scand 2000;79:559–63.PubMedGoogle Scholar
  99. 99.
    Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7.PubMedGoogle Scholar
  100. 100.
    Lopes S, Sun JG, Juriscova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor quality sperm samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528–32.PubMedGoogle Scholar
  101. 101.
    Ahmadi A, Ng S-C. Fertilising ability of DNA damaged spermatozoa. J Exp Zool 1999;284:696–704.PubMedGoogle Scholar
  102. 102.
    Haines GA, Hendry JH, Daniel CP, Morris ID. Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatognia. Mutat Res 2001;495:21–32.PubMedGoogle Scholar
  103. 103.
    Fraser L. Structural damage to nuclear DNA in mammalian spermatozoa: its evaluation techniques and relationship with male infertility. Pol J Vet Sci 2004;7:311–21.PubMedGoogle Scholar
  104. 104.
    Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosalvez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82:251–6.PubMedGoogle Scholar
  105. 105.
    Fernandez JL, Vazquez-Gundin F, Delgado A, Goyanes VJ, Ramiro-Diaz J, de la Torre J, Gosalvez J. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453:77–82.PubMedGoogle Scholar
  106. 106.
    Fernandez JL, Gosalvez J. Application of FISH to detect DNA damage: DNA breakage detection-FISH (DBD-FISH). Methods Mol Biol 2002;203:203–16.PubMedGoogle Scholar
  107. 107.
    Evenson DP, Higgins PJ, Grueneberg D, Ballachey BE. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 1985;6:238–53.PubMedGoogle Scholar
  108. 108.
    Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jefferies TM. Sperm chromatin changes associated with male smoking. Mutat Res 1999;423:103–11.PubMedGoogle Scholar
  109. 109.
    Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984;42:87–91.PubMedGoogle Scholar
  110. 110.
    Foresta C, De Carlo E, Mioni R, Zorzi M. Sperm nuclear chromatin heterogeneity in infertile subjects. Andrologia 1989;21:384–90.PubMedGoogle Scholar
  111. 111.
    Peluso JP, Luciano AA, Nulsen JC. The relationship between alterations in spermatozoal deoxyribonucleic acid, heparin binding sites, and semen quality. Fertil Steril 1992;57:665–70.PubMedGoogle Scholar
  112. 112.
    Hoshi K, Katayose H, Yanagida K, et al. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996;66:634–9.PubMedGoogle Scholar
  113. 113.
    Duran EH, Gurgan T, Gunalp S, Enginsu ME, Yarali H, Ayhan A. A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum Reprod 1998;13:1235–9.PubMedGoogle Scholar
  114. 114.
    Martins CF, Dode MN, Bao SN, Rumpf R. The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA. Genet Mol Res 2007;6:94–104.PubMedGoogle Scholar
  115. 115.
    Martins CF, Bao SN, Dode MN, Correa GA, Rumpf R. Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm. Theriogenology 2007;67:1307–15.PubMedGoogle Scholar
  116. 116.
    Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl 2006;27:53–9.PubMedGoogle Scholar
  117. 117.
    Anderson D, Dobrzynska MM, Basaran N. Effect of various genotoxins and reproductive toxins in lymphocytes and sperm samples in the Comet assay. Teratog Carcinog Mutagen 1997;17:29–43.PubMedGoogle Scholar
  118. 118.
    Anderson D, Basaran N, Dobrzynska MM, Basaran AA, Yu TW. Modulating effects of flavonoids on food mutagens in human blood and sperm samples in the Comet assay. Teratog Carcinog Mutagen 1997;17:45–58.PubMedGoogle Scholar
  119. 119.
    Klaude M, Eriksson S, Nygren J, Ahnstrom G. The comet assay: mechanisms and technical considerations. Mutat Res 1996;363:89–96.PubMedGoogle Scholar
  120. 120.
    Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240–7.PubMedGoogle Scholar
  121. 121.
    Singh N, Stephens R. X-ray induced DNA double-strand breaks in human sperm. Mutagenesis 1998;13:75–9.PubMedGoogle Scholar
  122. 122.
    Haines G, Marples B, Daniel P, Morris I. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the COMET assay. Adv Exp Med Biol 1998;444:79–91.PubMedGoogle Scholar
  123. 123.
    Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91.PubMedGoogle Scholar
  124. 124.
    Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24:59–66.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Animal Reproduction LaboratoryAnimal Sciences InstituteIslamabadPakistan

Personalised recommendations