Factors affecting the redox state of bovine epididymal spermatozoa.

The oxidation-reduction state of bovine epididymal spermatozoa was determined in vitro by fluorescence spectroscopy and by direct chemical analysis. Enhanced NADH fluorescence in sperm was observed with the onset of anaerobiosis in the sample cuvette. However, part of this increased fluorescence was temporary and a stable pyridine nucleotide fluorescence was not reached until 25 min after the onset of anaerobiosis. The transient was not paralleled by an equivalent increase in cellular NADH as measured by absorption spectroscopy. Hypotonic treatment of sperm, which removed the plasma membrane, liberated greater than 50% of the cellular NAD and that remaining was reduced by rotenone addition, indicating its mitochondrial location. Hypotonically treated sperm did not demonstrate a transient fluorescence above that due to the increases in NADH from anaerobiosis. Addition of pyruvate to anaerobic sperm resulted in a rapid decrease in fluorescence that corresponded to NADH oxidation coupled with the reduction of pyruvate to lactate. The duration of this oxidized state was dependent on the amount of pyruvate added. Analysis of cellular NAD under similar conditions confirmed this result. The pyridine nucleotides of hypotonically treated cells were also oxidized by pyruvate but were not reduced by added glucose as in untreated sperm. These results indicate that pyruvate reduction served to balance reducing equivalents and temporarily reoxidized the intracellular milieu of the anaerobic spermatozoon. The data also support the hypothesis that pyruvate and lactate can serve as reducing equivalent carriers between cytosol and mitochondria.