Significance of mitochondrial reactive oxygen species production in male infertility

Research Doctorate - Doctor of Philosophy (PhD)Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The aetiology of this condition is thought to involve the excessive generation of reactive oxygen species (ROS) by human spermatozoa; however the cause and sub-cellular origins of this aberrant activity is unknown. The objective of this doctoral thesis was to determine the importance of sperm mitochondria in creating oxidative stress associated with defective sperm function and subsequently to investigate causes of increased mitochondrial ROS generation. In the first study, intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C11 in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. The induction of mitochondria ROS resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of α-tocopherol. Concluding that the sperm mitochondria make a significant contribution to the oxidative stress exhibited by defective human spermatozoa. In order to investigate causes of irregular mitochondrial ROS production, we demonstrated that defective human sperm populations are characterized by high cellular contents of both total and free fatty acids using gas chromatography. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation. This relationship was causal and mediated by the range of unsaturated fatty acids that are present in human spermatozoa. Direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We followed with examination of the ability of human spermatozoa to undergo apoptosis and generate mitochondrial ROS. We report that these cells are prevented from entering the apoptotic pathway as long as the PI3kinase/AKT complex is phosphorylated. If PI3 kinase phosphorylation is inhibited with wortmannin then these cells default to an intrinsic apoptotic cascade characterized by caspase activation in the cytosol, annexin V binding to the cell surface, mitochondrial ROS generation, cytoplasmic vacuolization, oxidative DNA damage and motility loss. However the physical architecture of the cell subsequently prevents endonucleases released from the mitochondria or activated in the cytosol from penetrating the sperm nucleus and, as a result DNA fragmentation does not occur although oxidative DNA adducts can clearly be detected. These results for the first time highlight the importance of mitochondrial ROS generation in human spermatozoa in the aetiology of male infertility. Subsequent studies revealed that higher intracellular concentrations of unsaturated fatty acids and the stimulation of apoptosis are both mechanisms of mitochondrial ROS generation which can result in increased lipid peroxidation, loss of sperm motility and oxidative DNA damage. These data and further investigation will aid in the diagnosis, prevention and treatment of male infertility