Estrogenic Compounds Induce Oxidative Stress in Human Spermatozoa: Implications for DNA Integrity and Fertilization Success.

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. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure to estrogenic xenobiotics. We have undertaken a detailed analysis of the ability of these compounds to trigger oxidative stress and/or DNA damage in human spermatozoa. This investigation was completed using a range of techniques validated for use in these highly specialized cells. DNA integrity was assessed using the Comet and TUNEL assays, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens (reactive metabolites of estrogen), quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity in human spermatozoa while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3- dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation and potentially preventing fertilization. These findings raise fundamental questions about the importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line.