The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology.

摘要: The purpose of this study was to evaluate the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay as a method for assessing DNA damage in human spermatozoa. conventional shown be insensitive and unresponsive fragmentation induced mouse spermatozoa on exposure Fenton reagents (H₂O₂ Fe(2+) ). However, both time- dose-dependent responses could readily detected if chromatin exposed 2 mm dithiothreitol (DTT) 45 min prior fixation. This modified version significantly enhanced TUNEL signals generated by subpopulations isolated discontinuous Percoll gradients well triggered (arachidonic acid menadione) that are known stimulate superoxide anion production DTT also improved with chromomycin A₃ (CMA₃), probe designed determine efficacy protamination, correlation observed between criterion sperm quality assay. Finally, output found highly correlated vitality. methodology therefore further refined incorporate vital stain covalently bound intracellular amine groups non-viable cells. tag remained associated during fixation processing so ultimately, integrity vitality simultaneously assessed same flow cytometry methods described article simple robust should facilitate research into causes