Effects of Calcium Ions on the Malate-Aspartate Shuttle in Slow-Cooled Boar Spermatozoa

BIOLOGY OF REPRODUCTION 49, 537-543 (1993) Effects of Calcium Ions on the Malate-Aspartate Shuttle in Slow-Cooled Boar Spermatozoa KAZUHIKO NISHIMURA' Nara PrefecturalLivestock Experiment Station, Ouda-cho, Uda-gun, Nara, 633-21 Japan ABSTRACT 2+ INTRODUCTION Cold shock by rapid cooling to 0°C causes an irreversible loss of sperm viability [1, 2] and alters the permeability of the plasma membrane [2-5]. By some unknown mechanism, boar spermatozoa acquire resistance to low temperature during incubation at 15 0C. This treatment is an important step in the preparation of frozen spermatozoa and is necessary for the prevention of cold shock [6]. It has been reported that high concentrations of Ca 2+ ions in the extracellular medium at 37 0C inhibit the movement of spermatozoa [7, 8]. However, the concentration of intracellular free Ca 2+ ions of spermatozoa plays a regulatory role in the control of motility [7-10] and the acrosome reaction [11]. Because Ca 2+ ions are involved in the damage to frozen and thawed spermatozoa, Ca 2+ ions are often omitted from the procedure for freezing spermatozoa [6]. It has been reported that an influx of Ca 2+ ions is induced by slow cooling [12]. Influx of Ca 2+ ions induced with ionophore activates both motility [9,10,13] and the acrosome reaction [11] in spermatozoa, and premature induction of these events is incompatible with low-temperature storage. Moreover, within the range from 25 0C to 5C, changes in temperature affect membrane fluidity in a manner that involves membrane permeability to Ca 2+ ions [2, 4, 14]. Therefore, it is important with regard to the preparation of frozen spermatozoa to study the role of Ca 2+ ions in slowcooled spermatozoa. Glucose is a major substrate for respiration in spermatozoa and generates cytosolic NADH when it is metabolized to pyruvate. Oxidation of cytosolic NADH in the mitochonAccepted April 19, 1993. Received October 23, 1992. 'Correspondence: Kazuhiko Nishimura, 637-1, Hieda-cho, Yamatokohriyama-si, Nara, 639-11 Japan. 537 drial respiratory chain is important in the production of energy [15, 16]. Cytosolic NADH cannot traverse the mitochondrial membranes, so the reducing equivalents of cytosolic NADH are transferred to the mitochondria by shuttle systems. Previous studies [17,18] have shown that the capacity of the malate-aspartate shuttle is closely related to the ratio of ATP to ADP (ATP:ADP) in boar spermatozoa, suggesting that an assessment of the capacity of the malateaspartate shuttle may be useful for the evaluation of damage to mitochondrial membranes by freezing and thawing of boar spermatozoa. The malate-aspartate shuttle of rat liver is activated by Ca2 + ions [19]. However, the role of Ca 2+ ions in the malate-aspartate shuttle of boar spermatozoa is not clear. The purpose of the present study was to examine the relationship between the capacity of the malate-aspartate shuttle and Ca 2+ ions in slow-cooled boar spermatozoa. MATERIALS AND METHODS Materials Aminooxyacetate and pyrazole were purchased from Wako Pure Chemical Ind., Osaka, Japan. The Ca2+ ionophore A23187 was purchased from Sigma Chemical Co., St. Louis, MO. Reagents for assays of metabolites were obtained from Boehringer-Mannheim, Mannheim, FRG and Sigma Chemical Co. All other chemicals were of the highest quality available. Preparationof Washed Spermatozoa Ejaculated semen was collected separately from five boars by the gloved-hand method and filtered through gauze at 37°C. Spermatozoa were separated from seminal plasma by centrifugation at 600 x g for 10 min; they were then washed three times by centrifugation at 400 x g for 2 min with The effects of intracellular free calcium (Ca ) ions on the malate-aspartate shuttle were studied in slow-cooled boar spermatozoa. The capacity of the malate-aspartate shuttle was assessed by an indirect method on the basis of accumulation of lactate relative to pyruvate when ethanol is provided as substrate. The capacity of the malate-aspartate shuttle at 37 0C was dependent on the presence of Ca2+ ions and was stimulated by an influx of Ca2 + ions induced by the combination of the Ca 2+ ionophore A23187 and 100 tAM CaC2,. When washed spermatozoa were cooled slowly to 150C, the percentage of progressive motile spermatozoa was about half that at 370 C in 100 5AMCa2+-containing medium, while the capacity of the malate-aspartate shuttle remained equal to that at 37 0C. The motility decreased further at higher concentrations of Ca2 + ions. Spermatozoa in EGTAcontaining medium barely moved at 15°C and the capacity of the malate-aspartate shuttle decreased. Even in Ca2+-containing medium, LaCI 3 caused a decrease in the capacity of the malate-aspartate shuttle at 15°C. These results suggest that an influx of a low concentration of Ca2+ ions activates the malate-aspartate shuttle at 15°C, with a subsequent increase in the proportion of cells that maintain progressive motility. 538 NISHIMURA Cytosol inner Mitochondrion modified Krebs' Ringer bicarbonate medium plus HEPES at 37 0C. The medium consisted of 155.37 mM NaCl, 4.78 mM KCl, 1.19 mM KH 2PO 4, 1.19 mM MgSO 4, 5.07 mM NaHCO 3, and 20 mM HEPES, pH 7.4. The washed spermatozoa were resuspended at 2 x 108 cells/ml in medium that contained 100 mM CaC12 or 1 mM EGTA at 370 C. The motility of the washed spermatozoa, assessed as described below, was more than 85%. Procedurefor Slow Cooling Thirty-milliliter suspensions of washed spermatozoa, in 50-ml tubes, were cooled at a rate of 0.3°C/min to 15°C in a water bath (ML-10 Cooling Bath; Taitec Co., Saitama, Japan) and held at 15°C for 1 h. The cooled spermatozoa were centrifuged at 600 x g for 5 min at 15 0C and resuspended at 2 x 108 cells/ml in each medium. For control, washed spermatozoa were held at 37°C for 2 h and resuspended at 37°C. Assessment of the Capacity of the Malate-AspartateShuttle Assessment of the capacity of the malate-aspartate shuttle was made by the method of Sugano et al. [19, 20], which determines the accumulation of lactate relative to pyruvate when ethanol is provided as substrate. Ethanol is oxidized by alcohol dehydrogenase, increasing the level of NADH and the redox state in the cytosol. The ratio of lactate to pyruvate (L:P) increases with the cytosolic redox state, so L:P increases during cellular oxidation of ethanol. Because the reducing equivalents of cytosolic NADH are transferred to mitochondria by the malate-aspartate shuttle and are ox- idized in the respiratory chain, the shuttle acts to oxidize the cytosol and decrease L:P (Fig. 1). Aminooxyacetate (AOA), an inhibitor of aspartate transaminase [21], inhibits the malate-aspartate shuttle [19, 20] and increases L:P. Therefore, the capacity of the malate-aspartate shuttle can be assessed indirectly by measurement of L:P [19, 20] during cellular oxidation of ethanol. Quantitationof Lactate, Pyruvate, ATP, and ADP and Assessment of Motility One-milliliter suspensions of washed and slow-cooled sper (...truncated)