Use of specific mitochondrial complex inhibitors to investigate mitochondrial involvement on horse sperm motility and ROS production

Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1h and 3h at 37°C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.8 μM, ANTI), the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine (5 μM, CCCP), ATP synthase inhibitor oligomycin (5 μM, OLIGO), and 2 μl vehicle DMSO (control, CTL). Samples were analyzed for sperm motility and for mitochondrial membrane potential (MMP), mitochondrial integrity, mitochondrial O2•– production, and cytoplasmic H2O2. A multivariate analysis was performed on the data. CCCP caused a pronounced MMP reduction at both time points while ROT and ANTI showed the same effect at 3h. All treatments at 3h incubation significantly reduced the percentage of sperm with early changes in membrane permeability with active mitochondria. The H2O2 production of live cells was low at 1h incubation in all treatments; after 3h a slight decrease in the percentage of low-H2O2 producing cells was recorded. All treatments, except DMM, induced a significant decline in sperm motility and kinematics and modified the pattern of sperm subpopulations. The effect of DMM was evident only after 3h, increasing the percentage of slow sperm subpopulation. In conclusion, the disruption of mitochondrial integrity induces an increase of mitochondrial ROS production that could be detrimental for cell function and survivior.