304 TREATMENT OF BOAR SPERM WITH RESPIRATION INHIBITOR MENADIONE: EFFECTS ON MOTILITY, REACTIVE OXYGEN SPECIES (ROS), MITOCHONDRIAL TRANSMEMBRANE POTENTIAL (MMP), AND ATP CONTENT

Abstract

Previously we found that live, fresh or thawed boar sperm show little tendency to accumulate ROS spontaneously, but live sperm accumulated ROS during a 30-min incubation with xanthine and xanthine oxidase and showed marked reduction in motility. High mitochondrial transmembrane potential (MMP) is required to drive the F0/F1 ATPase responsible for producing ATP in most cell types, and ATP is required for sperm motility. This experiment was conducted to investigate the effects of menadione (disrupter of electron transport at Complex I) on sperm motility, MMP, and ATP content. The incidence of cells with high MMP was determined by measuring the fluorescence of JC-1 aggregates bound to the inner mitochondrial membrane using flow cytometry. Computer-assisted motion analysis was conducted using the IVOS version 12 (Hamilton Thorne Research, Beverly, MA, USA), and ATP (pmoles/10⁶ sperm) was determined using the luciferin-luciferase assay. Sperm from six boars were individually Percoll washed to eliminate seminal plasma and incubated at 40 × 10⁶/mL with 0, 1, 10, or 30 µM menadione for 5, 30, 60, and 120 min at 38°C in a modified Tyrode's medium containing 0.1% polyvinyl alcohol with no bicarbonate or BSA. The formation of ROS was confirmed by measuring the red fluorescence developed by the oxidation of hydroethidine to ethidium using flow cytometry. Whereas the basal level of ethidium fluorescence in the absence of menadione was low (2% ethidium-positive cells at 5 min), 10 and 30 µM menadione increased (P < 0.05) the percentage of ethidium-positive cells to 47 and 87%, respectively, at 30 min. Sperm motility did not decrease significantly (79-83%) during the 120 min incubation with no menadione, but menadione caused a significant dose-related decrease (P < 0.05) over time, with 10 and 30 µM menadione decreasing motility to 60 and 40%, 51 and 7%, and 10 and 1% at 30, 60, and 120 min, respectively. JC-1 aggregate fluorescence intensity decreased (P < 0.05) by 45-60% in a dose-related fashion at 120 min compared to the same doses at 5 min. Sperm viability, as measured by number of propidium iodide negative cells, averaged 93.6% during the incubation and was not significantly affected by treatment. The effect of menadione on ATP content was much less than that on motility or JC-1 fluorescence intensity. Mean ATP content averaged 63 pmoles through 60 min at all menadione doses; at 120 min only 30 ¼M menadione decreased (P < 0.05) ATP to 43 pmoles, compared to all other treatments. Menadione caused an increase in ROS formation and a decline in MMP, which suggested uncoupling of the respiratory chain and oxidative phosphorylation. However, sperm ATP content was not highly correlated with motility. This suggests that ATP content was maintained by the activity of other intermediary metabolism pathways. The reduction in motility may have been due to a ROS induced lesion in ATP utilization or in the contractile apparatus of the cell.