Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation.
The origin and the meaning of DNA fragmentation in ejaculated human spermatozoa are not yet clear, although some hypotheses have been proposed. In the present study, we used investigated sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry to investigate DNA fragmentation in spermatozoa that were selected by the swim-up procedure and incubated long-term. In addition, using flow cytometry we detected annexin V binding assay and propidium iodide staining, and we also studied membrane phosphatidylserine translocation and the loss of membrane integrity in the same sperm populations that we used in the TUNEL investigation. We found that in vitro sperm DNA fragmentation 1) occurs after ejaculation under experimental conditions without the involvement of any external factor, 2) is not affected by treatment with the nuclease inhibitor aurintricarboxylic acid, 3) is increased by treatment with the glutathione peroxidase inhibitor mercaptosuccinate, 4) correlates with basal values (ie, just after swim-up selection) of DNA fragmentation in teratozoospermic but not in normospermic semen samples, 5) develops in a sharply associated manner with the in vitro occurrence of sperm necrosis, and 6) is predicted by the basal value of annexin V binding in viable spermatozoa. These findings suggest an involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation.
Muratori
Monica
M
Department of Clinical Physiopathology, Andrology Unit, University of Florence, Florence, Italy.
Maggi
Mario
M
Spinelli
Silvia
S
Filimberti
Erminio
E
Forti
Gianni
G
Baldi
Elisabetta
E
D017304:Annexin A5 / Q000378:metabolism; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D004542:Ejaculation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005307:Fertilization in Vitro; D005434:Flow Cytometry / Q000379:methods; D016166:Free Radical Scavengers / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000037:antagonists & inhibitors; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013081:Sperm Motility; D013094:Spermatozoa / Q000378:metabolism* / Q000473:pathology*; D013870:Thiomalates / Q000494:pharmacology
D017304:Annexin A5; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D013870:Thiomalates; D001312:Aurintricarboxylic Acid; C046062:2-thiomalic acid; D005979:Glutathione Peroxidase; D003851:Deoxyribonucleases