Effect of various substrates on mitochondrial and cellular energy state of intact spermatozoa

Ejaculated bull spermatozoa separated from seminal plasma by repeated washing were used to study the effects of various substrates on respiration, mitochondrial energization, cellular ATP/ADP ratios, and motility. The respiratory rate in the presence of lactate, pyruvate, acetate or glycerol was about two to three times higher compared to cells oxidizing amino acids, fatty acids, or endogenous substrates. When well oxidizable substrates were present, the uncoupler FCCP produced a marked increase in respiration (two to three times at optimal FCCP concentrations). Mitochondrial energization was monitored in intact spermatozoa by spectrophotometry with the dye safranin. Lactate and pyruvate yielded more pronounced mitochondrial energization than did alanine or endogenous substrates. Cellular ATP/ADP ratios, with lactate, pyruvate, acetate or glycerol, were found to range from 6 to 10, whereas endogenous substrates and amino acids were able to establish ATP/ADP quotients as low as about 1 only. The capacity to oxidize various substrates was clearly reflected by the amount of motile cells. Motility in the presence of lactate, acetate or glycerol was seen in more than 60% of spermatozoa, while spermatozoa in the presence of alanine or butyrate showed a motility not above the endogenous level (approx. 30% motile cells). The experiments demonstrated that cellular performance is highly dependent on the energy state as made possible by the capacity of substrate utilization.