Human sperm DNA integrity assessed by the Comet and ELISA assays.
DNA integrity in sperm is essential for the accurate transmission of genetic information and therefore the maintenance of good health in future generations. The ELISA and Comet assays, two techniques that detect DNA damage in cells, are compared in this study of DNA integrity in human sperm. Both techniques rely on alkaline unwinding for the release of single strands of DNA from the nucleus. The ELISA detects single strands immunochemically whereas the Comet assay measures single strands drawn out by electrophoresis, stained with ethidium bromide and quantified by image analysis. The two techniques, both modified for use with sperm, detect similar levels of baseline DNA damage along with similar dose-dependent patterns of induced damage by X-ray irradiation at 10 and 30 Gy (P < 0.05). The assays are also comparable in the detection of a significant protective effect by ascorbic acid (300 and 600 microM) and alpha-tocopherol (30 and 60 microM) on DNA integrity, both at baseline levels and following X-ray irradiation (p < 0.01). The advantages and disadvantages of each technique are discussed.
Hughes
C M
CM
Department of Obstetrics and Gynaecology, Queens University of Belfast, UK.
McKelvey-Martin
V J
VJ
Lewis
S E
SE
D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D004247:DNA / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005298:Fertility / Q000235:genetics; D006801:Humans; D007248:Infertility, Male / Q000235:genetics; D008297:Male; D013094:Spermatozoa / Q000737:chemistry* / Q000528:radiation effects; D014810:Vitamin E / Q000494:pharmacology
D000975:Antioxidants; D004277:DNA, Single-Stranded; D014810:Vitamin E; D004247:DNA; D001205:Ascorbic Acid