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31P NMR signals assigned to intracellular adenine nucleotides and to inorganic phosphate were detected in dense suspensions of epididymal sperm obtained from bulls or hamsters. Similar adenine nucleotide signals and an additional large resonance peak, attributable to extracellular glycerylphosphorylcholine, were observed with whole bovine cauda epididymides. Provision of the glycolytic substrate fructose to such sperm suspensions promoted apparent conversion of intracellular ADP to ATP with a concomitant decrease in cellular inorganic phosphate (Pi) content. Subsequent treatment with the methylxanthine caffeine resulted in diminution of the intracellular gamma-P-ATP signal that was consistent with the decreased ATP and ADP contents previously demonstrated by chemical analyses of cellular extracts. Alternatively, treatment with fructose followed by the membrane-selective detergent digitonin produced loss of the nucleotide NMR signals, indicating release of ATP and Pi from the sperm cytosol with subsequent hydrolysis in the extracellular medium. Comparison of intracellular Pi and ATP resonance signals with those of ATP and Pi in vitro, in media of varied pH and cation composition, allowed calculation of a cytosolic pH of 6.5-6.6 and a cytosolic Mg2+ concentration of 0.5 mM for fresh suspensions of bovine cauda epididymal sperm. Intracellular Pi of hamster epididymal sperm reported a similar cytosolic pH. Other, more acidic compartments were not detected in these experiments. However, during prolonged incubation, the pH of the bovine sperm interior slowly decreased as the extracellular medium was acidified by extensive production of lactate. Intracellular ATP was detectable until cytosolic pH declined to approximately 5.5. Rapid intracellular acidification, resulting from exchange of internal K+ for H+, was observed after treatment with carboxylic acid ionophore nigericin. This lowering of internal pH was followed by a slower return toward initial internal pH values, probably as a consequence of secondary exchange of internal protons for other external monovalent cations, rather than as a result of the operation of a cellular homeostatic mechanism. Together, these studies utilizing noninvasive NMR techniques provide evidence that within the bovine epididymis sperm utilize an unknown energy source to phosphorylate adenine nucleotides and maintain a slightly acidic cytosolic pH.

A 31P NMR Study of the Epididymis and Epididymal Sperm of the Bull and Hamster1