Flow cytometric assessment of changes in rat sperm mitochondrial function after treatment with pentachlorophenol

The fluorophore 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) localizes to the mitochondria and is affected by membrane potential, fluorescing bright orange when the membrane potential is high and green when mitochondrial membrane potential is low. The present study used flow cytometric analysis of JC-1 staining patterns of large numbers of spermatozoa to detect chemical-induced alterations of sperm mitochondrial membrane potential. Cauda epididymal rat spermatozoa were incubated with pentachlorophenol (PCP; 0.1 microM or 1.0 microM), a known uncoupler of mitochondrial oxidative phosphorylation. Microscopic evaluation showed that the midpiece (mitochondrial location) of live, highly motile spermatozoa stained bright orange, while the midpiece of live, non-motile spermatozoa stained green. The midpiece of slightly or non-progressively motile spermatozoa stained a faint orange-green. The percentage of spermatozoa stained bright orange and the total percentage of spermatozoa stained orange (bright orange+faint orange) in the control samples of spermatozoa were significantly higher (P<0.001) than in the 0.1 microM and 1.0 microM PCP treated samples. These data indicate that sperm mitochondrial membrane potential is highly sensitive to the uncoupling effects of PCP and that JC-1 staining and flow cytometric analysis may be a sensitive assay to detect the effect of toxicants on rat sperm mitochondrial function.