Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

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.

Olive Leaves Extract Improved Sperm Quality and Antioxidant Status in the Testis of Rat Exposed to Rotenone

“…Davila observed that exposure to ROT induced sperm death and reduce motility and membrane integrity in sperm. These events attributed to increased production of H 2 O 2 , which occurred in the present ROT (47). On the other hand, ROT caused mitochondrial damage, which resulted in apoptotic cell death by disrupting cellular energy metabolism and enhancing ROS production, inhibition of the microtubular network, and condensation/fragmentation of the cell nucleus (48).…”

Membrane Lipid Replacement: Clinical Studies Using a Natural Medicine Approach to Restoring Membrane Function and Improving Health

“…At lower glycerolphospholipid concentrations vesicle fusion with sperm resulted in enhanced motility [57], which is directly related to mitochondrial activity [58]. However, this was not seen at higher concentrations of the vesicles where membrane swelling due to incorporation of excess membrane glycerolphospholipids occurred [57].…”

“…However, this was not seen at higher concentrations of the vesicles where membrane swelling due to incorporation of excess membrane glycerolphospholipids occurred [57]. Thus it is important to maintain the compact nature of cellular membranes to retain biological activity [58]- [60]. The fusion of high, nonphysiological concentrations of glycerolphospholipids into mitochondrial membranes in vitro cannot be expected to mimic natural in vivo MLR.…”

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

“…() and Plaza Dávila et al. () who observed a sperm viability similar to that in control even using a higher rotenone concentration (10 μ m ) for 1 h; only after 3 h of incubation, rotenone at the concentration of 10 μ m induced a significant reduction in the percentage of intact sperm (Plaza Dávila et al. ).…”

“…() who observed a sperm viability similar to that in control even using a higher rotenone concentration (10 μ m ) for 1 h; only after 3 h of incubation, rotenone at the concentration of 10 μ m induced a significant reduction in the percentage of intact sperm (Plaza Dávila et al. ). In contrast to the results of those authors, we did not observe any significant decrease in the percentage of live spermatozoa with high mitochondrial membrane potential.…”