The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.
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