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

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. The conventional assay was shown to be insensitive and unresponsive to the DNA fragmentation induced in human and mouse spermatozoa on exposure to Fenton reagents (H₂O₂ and Fe²⁺). However, both time- and dose-dependent responses could be readily detected if the chromatin was exposed to 2 mm dithiothreitol (DTT) for 45 min prior to fixation. This modified version of the assay significantly enhanced the TUNEL signals generated by subpopulations of spermatozoa isolated on discontinuous Percoll gradients as well as the responses triggered by reagents (arachidonic acid and menadione) that are known to stimulate superoxide anion production by human spermatozoa. DTT exposure also improved the signals detected with chromomycin A₃ (CMA₃), a probe designed to determine the efficacy of chromatin protamination, and enhanced the correlation observed between this criterion of sperm quality and the TUNEL assay. Finally, the output of the TUNEL assay was found to be highly correlated with sperm vitality. The TUNEL methodology was therefore further refined to incorporate a vital stain that covalently bound to intracellular amine groups in non-viable cells. This tag remained associated with the spermatozoa during fixation and processing for the TUNEL assay so that ultimately, both DNA integrity and vitality could be simultaneously assessed in the same flow cytometry assay. The methods described in this article are simple and robust and should facilitate research into the causes of DNA damage in human spermatozoa.