Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 30, 2007
Publication Date: November 1, 2008
Citation: Guthrie, H.D., Welch, G., Long, J.A. 2008. Mitochondrial function and reactive oxygen species action in relation to boar motility. Theriogenology. 70:1209-1215. Interpretive Summary: Flow cytometric assays were developed for reactive oxygen species (ROS) formation (ROS-induced oxidization of hydroethidine to ethidium), membrane lipid peroxidation (C11-BODIPY-581/591 oxidation), and mitochondrial transmembrane potential (MMP) (MMP-induced JC-1 aggregation, red fluorescence) in viable sperm during hypothermic liquid and frozen storage and to investigate the relationship between ROS, MMP, motility, and ATP production. During aerobic incubation of sperm at 37C for 30 min ROS formation and lipid peroxidation affected less than 4% of sperm; the results did not did differ among the storage treatments. However, during incubation fresh, liquid stored and frozen-thawed sperm appeared to be equally susceptible to the activity ROS generators such as xanthine/xanthine oxidase, menadione, FeSO4/ascorbate, and hydrogen peroxide. Motility was depressed by 90% during at 60 min, but viability was not affected. Of the ROS generators tested, membrane lipid peroxidation was specific for FeSO4/ascorbate; menadione and hydrogen peroxide had little or no effect. The ROS-induced oxidization of hydroethidine to ethidium was specific for menadione, xanthine/xanthine oxidase, and hydrogen peroxide; FeSO4/ascorbate had no effect. Surprisingly, while both menadione and H2O2 decreased motility, and menadione decreased MMP, neither decreased ATP production over a 60 min incubation period. Overall, the inhibitory affects of ROS on motility point to a mitochondrial independent mechanism for ROS induced motility inhibition.
Technical Abstract: Flow cytometric assays of viable boar sperm were developed to measure reactive oxygen species (ROS) formation (oxidization of hydroethidine to ethidium), membrane lipid peroxidation (oxidation of lipophilic probe C11-BODIPY581/591), and mitochondrial inner transmembrane potential (aggregation of mitochondrial probe JC-1) during hypothermic liquid storage and freeze-thawing of boar semen and to investigate the relationship between ROS, motility, mitochondrial inner transmembrane potential, and ATP production. Basal ROS formation and membrane lipid peroxidation were low in viable sperm of both fresh and frozen-thawed semen, affecting less than 4%. Sperm in fresh, liquid stored and frozen-thawed semen appeared to be equally susceptible to the activity ROS generators xanthine/xanthine oxidase, FeSO4/ascorbate, and hydrogen peroxide (H2O2). Of the ROS generators tested, FeSO4/ascorbate was specific for membrane lipid peroxidation; menadione, xanthine/xanthine oxidase, and H2O2 were specific for oxidization of hydroethidine. Menadione (30 microM) and H2O2 (300 microM) decreased (P less than 0.05) motility by 90% during 60 min of incubation. Menadione decreased (P less than 0.05) the incidence of sperm with high mitochondrial inner transmembrane potential by 95% during 60 min of the incubation, while ATP content was not decreased (P greater than 0.05) until 120 min. In contrast, H2O2 did not affect mitochondrial inner transmembrane potential or ATP at any time. The formation of ROS was not associated with any change in viability (90%) for either menadione or H2O2 through 120 min. Overall, the inhibitory affects of ROS on motility point to a mitochondrial independent mechanism. The reduction in motility may have been due to a ROS induced lesion in ATP utilization or in the contractile apparatus of the flagellum.