Assessment of ram sperm mitochondrial function by quantitative determination of sperm rhodamine 123 accumulation

A simple procedure is described for determining the functional state of ram sperm mitochondria by quantitative measurement of sperm rhodamine 123 (R 123) accumulation. Sperm were incubated with 1 microgram/ml R 123, and the accumulated R 123 was measured fluorimetrically after release from washed sperm by detergent lysis. Ram sperm R 123 uptake was maximal after 30 min of incubation and responded to changes in both sperm (P < 0.01) and R 123 (P < 0.01) concentration. There was a linear relationship (r = 0.98) between R 123 uptake and the proportion of cold-shocked sperm present in a sperm sample. R 123 uptake was unaffected by 20 mM 2-deoxyglucose or by 10 mM malonate (the latter being sufficient to reduce O2 uptake; P < 0.01). R 123 accumulation in ram sperm was reduced by 6 mg/ml sodium pentobarbitone (P < 0.05), by 1 microM 2,4-dinitrophenol (P < 0.01), and by 0.05% Triton X-100 (P < 0.01). It is concluded that quantitative estimation of R 123 uptake complements oxygen uptake in detecting mitochondrial dysfunction in ram sperm. While it is largely unaffected by inhibition of glycolysis, and is less sensitive than oxygen uptake to trichloroacetic acid cycle inhibition, R 123 uptake is sensitive to factors directly reducing the mitochondrial membrane potential of ram sperm. It may therefore by useful in the evaluation of the effects of such membrane-mediated injuries as cold shock and freezing damage on ram sperm mitochondria.