Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay

• Published in: Human reproduction (Oxford) 2001-08, Vol.16 (8), p.1703-1707
• Main Authors: Ramos, Liliana, Wetzels, Alex M. M.
• Format: Article
• Language: English
• Subjects: Annexin A5 - analysis; Apoptosis; Biological and medical sciences; Birth control; DNA Damage; DNA Fragmentation; gamma radiation; Gamma Rays; Gynecology. Andrology. Obstetrics; Humans; Hydrogen Peroxide - pharmacology; ICSI; In Situ Nick-End Labeling; Male; Medical sciences; Oxidative Stress; reactive oxygen species; Sperm Motility; Spermatozoa - drug effects; Spermatozoa - physiology; Spermatozoa - radiation effects; Sterility. Assisted procreation; TUNEL
• ISSN: 0268-1161; 1460-2350; 1460-2350
• DOI: 10.1093/humrep/16.8.1703

BACKGROUND: In this study we present the physiological changes observed in ejaculated spermatozoa of normospermic men after exposure to hydrogen peroxide (H2O2) or γ irradiation. METHODS: Motility changes as well as membrane and DNA-damage were determined in spermatozoa after incubation with 25 μmol/l of H2O2 during increasing intervals of time (0–60 min and after 24 h) or after irradiation of cells using α 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 H2O2, 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 H2O2 (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 H2O2 and showed a five-fold increase after 24 h (from 8.1–72.1%, P < 0.001). TUNEL positive cells after α 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. CONCLUSION: Motility may be a relevant physiological marker for DNA-intact sperm after exposure of spermatozoa to H2O2 and α irradiation.