Rabbit spermatozoa, like other mammalian spermatozoa, have part of their lactate dehydrogenase in the mitochondrial matrix. This enables lactate to be utilized directly as a NAD-linked oxidizable substrate by the mitochondria, but it also enables pyruvate to act as an oxidant of intramitochondrial NADH which should inhibit 02 uptake. These reactions have been investigated in hypotonically treated rabbit epididymal sperm (HTRES) by means of 0, uptake rate determinations and fluorimetric measurements of the reduction level of intramitochondrial NAD. Pyruvate is a potent inhibitor of lactate oxidation; the inhibition is overcome by malate. Pyruvate alone is oxidized slowly but is oxidized rapidly when malate is present. Lactate alone is oxidized rapidly but malate is required for maximal 02 uptake. Fluorimetric measurements show a high reduction level for intramitochondrial NAD with lactate as substrate; this level is increased by malate. Pyruvate reoxidizes intramitochondrial NAD reduced by lactate and inhibits 0, uptake; reversal of this inhibition by malate partially restores the NAD reduction level. Malate plus pyruvate maintain a NAD reduction level sufficient for rapid 02 uptake. We conclude that malate is essential to pyruvate and lactate oxidation by rabbit sperm mitochondria and functions by 2 mechanisms: a) The NADH generated from malate dehydrogenase does not have access to LDH and is made directly available to the respiratory chain. b) The oxaloacetate from malate oxidation stimulates pyruvate dehydrogenase by reaction with the product acetyl C0A. These mechanisms appear to act in concert to prevent inhibition of respiration by pyruvate oxidation of intramitochondrial NADH catalyzed by LDH.
