Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa.
BACKGROUND
Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear.


OBJECTIVE
The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function.


METHODS
Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C(11) in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis.


RESULTS
Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of alpha-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production.


CONCLUSIONS
We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.
Koppers
Adam J
AJ
Discipline of Biological Sciences, University of Newcastle. Callaghan, New South Wales 2308, Australia.
De Iuliis
Geoffry N
GN
Finnie
Jane M
JM
McLaughlin
Eileen A
EA
Aitken
R John
RJ
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D006801:Humans; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013081:Sperm Motility; D013094:Spermatozoa / Q000378:metabolism*
D017382:Reactive Oxygen Species; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone