248. Oxidative stress and DNA damage in human spermatozoa

Abstract

Unusually high levels of DNA damage in the male germ line are, unfortunately, characteristic of our species. A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the mechanisms responsible for creating this stress have not yet been elucidated. We have undertaken a detailed analysis of the ability of estrogens, electromagnetic radiation and xenobiotics including metal ions to trigger reactive oxygen species (ROS) production and/or DNA damage in human spermatozoa in vitro. This investigation was completed using a range of techniques validated for use in these highly specialised cells. DNA integrity was assessed using the Comet and TUNEL assays, oxidative DNA adducts were detected by an anti-8-oxo-dG assay and cross-linking adducts were characterised by mass spectrometry. Intracellular redox activity was monitored using dihydroethidium as the probe. Of the factors evaluated, catechol estrogens, certain transition metal ions, radio frequency electromagnetic radiation and heat were all capable of stimulating ROS production in human spermatozoa. The oxidative stress created by exposure to such factors lead to the induction of significant DNA damage. Generally, redox inert compounds including non-catechol estrogens and xenobiotics such as phthalate esters did not lead to ROS production or measurable DNA damage. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin. These findings raise fundamental questions about the importance of xenobiotics, environmental factors as well endogenous compounds in creating oxidative stress and DNA damage in the male germ line.