CHARACTERIZATION OF THE MITOCHONDRIAL MEMBRANE POTENTIAL IN DOMESTIC CAT TESTICULAR CELLS: POSSIBLE IMPLICATIONS FOR GERM CELL FUNCTION

Abstract

In mitochondria, substrates generated in the Krebs cycle are oxidized by a sequence of cytochromes in the mitochondrial respiratory chain (MRC), located in the inner mitochondrial membrane, and which is composed by integrated units, or Complexes. Concomitantly, the MRC actively extrudes protons from the mitochondrial matrix, and, as the inner mitochondrial membrane is proton-impermeable, energy is stored as a pH and electrical gradient (delta pH and delta psi, respectively), with the matrix negatively charged. The gradient is then used to synthesize ATP in the ATP synthase. Barring sole recourse to glycolysis the mitochondrial membrane potential is thus paramount for energy production. We have isolated testicular mitochondria from neutered domestic cats (Felis catus) and monitored variations in the mitochondrial electric potential using a tetraphenylphosphonium cation (TPP+) selective electrode. The delta psi was calculated with NADH as a substrate for the entire MRC (Complexes I through IV), or succinate, which bypasses complex I and reduces complex II directly. We show that the maximum mitochondrial delta psi varies between −174 and −221mV, reaching the most negative value (and highest delta psi) around 4 years of age, thus coinciding with the fertility potential monitored during the life span of the animal. Accordingly, we found a negative correlation (r=-0,564, P<0,05) between delta psi generated by succinate oxidation in Complex II and age. When comparing two distinct age groups (cats older and younger than 12 months) our results show significant differences (P<0.001), with younger cats presenting a lower mitochondrial delta psi generated when using both Complex I and Complex II substrates. The results may be due to the higher spermatogonia-to-spermatocyte ratio during puberty and early adulthood, as it has been suggested that mitochondria from spermatogonia are less metabolically active than spermatocyte mitochondria. This is the first study on mitochondrial bioenergetics in testicular cells from a carnivore. Besides giving a basic insight into the energy production machinery of the testis, the results also generate a model to further test some of the drugs now being used to rescue fertility, such as oral administration of antioxidants and energetic substrates added to preservation mediums, the effect of which on germ cells and sperm is still poorly understood. (Supported by FCT, SFRH/BD/23643/2005). (poster)