Energy metabolism and intracellular pH in boar spermatozoa

Abstract

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• 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 ppm appeared representing glycerol 3-phosphate.

Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa

“…However, this is unlikely to happen in spermatozoa under physiological conditions. Boar spermatozoa studied in vitro by 31 P NMR and confocal microscopy showed an intracellular pH of 7.2 in the head and in the principal piece of the flagellum, whereas the mid-piece (containing the mitochondria) was more acidic (Kamp et al 2003). The pH of ejaculated spermatozoa in vivo (i.e.…”

“…The pH of ejaculated spermatozoa in vivo (i.e. in the female genital tract) is not known, but might be affected by the surrounding media (Gatti et al 1993) or by sperm metabolism (Kamp et al 2003). Yet, pH is certainly not the only factor affecting PFK activity in vivo.…”

“…Moreover, the concentration of AMP in boar sperm responds to environmental conditions. Thus, hypoxia increases AMP in live spermatozoa, but this can readily be reverted when oxygen is readmitted (Kamp et al 2003). This is due to the fact that, unlike in vertebrate muscle (for review see, Krause & Wegener 1996a, 1996c, AMP is not deaminated to IMP in boar sperm and also not dephosphorylated to adenosine (Kalic et al 1997).…”

“…Inorganic phosphate (P i ) is an activator of PFK, but large fractional changes in its concentration, as would be required for significant effects, have not been observed in boar spermatozoa (Kamp et al 2003). However, they have been demonstrated in other spermatozoa (like those from sea urchins or carp, for review see Kamp et al 1996) or in somatic cells (like skeletal muscles, for review Krause & Wegener 1996c) that contain a phosphagen such as phosphocreatine.…”

A novel pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and the acrosome

“…We have proposed (Westhoff & Kamp 1997) that glycolysis in the principal piece is important to supply ATP locally for dynein-ATPases since the mitochondria are confined to the midpiece and the phosphocreatine/creatine kinase shuttle for transport of mitochondrial energy-rich phosphate is poorly developed in mammalian spermatozoa (Kamp et al 1996). Support for this hypothesis comes from the observations that lactate production by boar spermatozoa occurs even under normoxic conditions (Kamp et al 2003) and is increased if sperm are stimulated to hyperactivity (unpublished results) which is vital for fertility (Yanagimachi 1994, Stauss et al 1995, Mortimer 1997, Bedford 1998.…”