Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay.
BACKGROUND
In this study we present the physiological changes observed in ejaculated spermatozoa of normospermic men after exposure to hydrogen peroxide (H(2)O(2)) or gamma irradiation.


METHODS
Motility changes as well as membrane and DNA-damage were determined in spermatozoa after incubation with 25 micromol/l of H(2)O(2) during increasing intervals of time (0--60 min and after 24 h) or after irradiation of cells using alpha rays. Annexin V-binding in combination with propidium iodide was used for the assessment of membrane changes after each incubation time. TdT-mediated-dUTP nick-end labelling (TUNEL) was used to evaluate DNA damage.


RESULTS
After 1 h incubation of the spermatozoa with H(2)O(2), almost all cells were positive for Annexin-V, while no significantly increase in TUNEL positivity was observed. TUNEL results were significantly higher 24 h after incubation with H(2)O(2) (10--16.3%, P = 0.03). In the control group (cumulus cells), an increase in the percentage of TUNEL positive cells was observed after 15 min of incubation with H(2)O(2) and showed a five-fold increase after 24 h (from 8.1-72.1%, P < 0.001). TUNEL positive cells after alpha irradiation increased with the doses and post-irradiation time (from 10.8--47.2%). Interestingly, when only motile spermatozoa from irradiated samples were analysed, only 0.5% were TUNEL positive.


CONCLUSIONS
Motility may be a relevant physiological marker for DNA-intact sperm after exposure of spermatozoa to H(2)O(2) and alpha irradiation.
Ramos
L
L
Fertility Laboratory, Department of Obstetrics and Gynaecology, University Medical Centre St. Radboud, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. l.ramos@obgyn.azn.nl
Wetzels
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D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D004249:DNA Damage; D053938:DNA Fragmentation; D005720:Gamma Rays; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008297:Male; D018384:Oxidative Stress; D013081:Sperm Motility; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects
D017304:Annexin A5; D006861:Hydrogen Peroxide