Abstract
Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.
Highlights
The mitochondria of the spermatozoa control numerous functions and are considered to be hallmarks of sperm functionality [1, 2]
Alteration in the redox homeostasis of the cell leads to sperm senescence and death[5]; in humans, it has been reported that the mitochondria of defective sperm are the major source of reactive oxygen species (ROS) originating from electron leakage in the electron transport chain (ETC) [6]
We studied the effects of interrupting the electron flux in the mitochondria of stallion sperm at two specific points, complex I (NADH-ubiquinone oxidoreductase), and complex III
Summary
The mitochondria of the spermatozoa control numerous functions and are considered to be hallmarks of sperm functionality [1, 2]. Alteration in the redox homeostasis of the cell leads to sperm senescence and death[5]; in humans, it has been reported that the mitochondria of defective sperm are the major source of ROS originating from electron leakage in the electron transport chain (ETC) [6]. This has been assumed to be true for horses, as reviewed in [7], and recent data support this hypothesis[8]. Other effects on sperm parameters were investigated as well, the role of glycolysis in sperm function was investigated using specific inhibitors and through the incubation of stallion spermatozoa in media free of glucose and pyruvate
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