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Submitted on December 15, 2005
Accepted on February 22, 2006
ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW 2308
* To whom correspondence should be addressed. E-mail: jaitken{at}mail.newcastle.edu.au.
Context: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved.
Objective: To chemically validate the use of dihydroethidium as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function.
Method: Dihydroethidium and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells.
Results: HPLC, mass spectrometry, NMR spectroscopy and spectrofluorimetry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from dihydroethidium. Spontaneous superoxide production by human spermatozoa was found to originate from a non-mitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage
Conclusions: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.
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