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R. V. Hyne and Kim P. Edwards

Summary. When guinea-pig spermatozoa were suspended in a minimal culture medium (MCM-PL), 2-deoxy-d-glucose (100 μm) and 2-amino-2-deoxy-d-glucose (100 μm) were potent inhibitors of the acrosome reaction without affecting the sperm motility, whereas the N-acetyl derivative 2-acetamido-2-deoxy-d-glucose (2 mm) had no inhibitory effect. The addition of d-glucose (2 mm) partly inhibited the percentage acrosome reaction of spermatozoa suspended in Medium MCM-PL, but dl-aglycerophosphate (2 mm) and myo-inositol (2 mm) had no effect. In addition, dl-α-glycerophosphate (2 mm) did not overcome the inhibitory effect of 2-deoxy-d-glucose on the sperm acrosome reaction. The inhibitory action of 2-deoxy-d-glucose (100 μm) on the sperm acrosome reaction assessed after a 3-h incubation was irreversible and was only completely effective if the sugar was added within 30 min of the start of incubation. When spermatozoa suspended in Medium MCM-PL were treated with 2-deoxy-d-glucose (100–200 μm) for an extended incubation up to 6 h, the inhibitory effect of 2-deoxy-d-glucose was partly overcome. Spermatozoa treated with 2-deoxy-d-glucose had significantly reduced concentrations of ATP after incubation for 2 h in Ca2+ -free media, compared with the ATP concentrations of spermatozoa preincubated for 2 h in Ca2+ -free media that supported acrosome reactions. The addition of Ca2+ (5 mm) caused a rapid decrease in ATP concentrations of spermatozoa suspended in Medium MCM-PL, while the addition of the monovalent ionophore monensin (50 μm) and Ca2+ stimulated sperm acrosome reactions as well as an additional decline in the sperm ATP concentrations. However, monensin (50 μm) in the absence of Ca2 + caused only a slight decline in the sperm ATP concentrations over the 15-min incubation period. The depletion of the sperm ATP concentrations by 2-deoxy-d-glucose may retard completion of the capacitation process and the resultant acrosome reaction.

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R. V. Hyne and D. L. Garbers

Summary. The acrosome reaction was induced by jelly coat factors, nigericin, or elevated pH. When spermatozoa were preincubated for 5 min in sea water maintained at pH 7·9 in the presence of 1 mm-phenylmethylsulphonyl-flouride (PMSF), 1 mm-benzamidine, 0·1 mm-1-chloro-3-tosyl-amido-7-amino-2-heptanone (TLCK), 5 mm-diisopropylphosphofluoridate (DFP) or 5 mm-DFP that was previously hydrolysed, only DFP or its hydrolysis product(s) prevented formation of the acrosomal filament induced by jelly coat factors. When incubation with inhibitors was extended to 2 h only TLCK and its hydrolysis products inhibited the jelly-induced acrosome reaction. Only DFP significantly inhibited the acrosome reaction induced by elevated pH (9·0). Nigericin induced acrosome reactions in the presence of DFP or TLCK. These findings do not support the concept of an active role for acrosin in development of an acrosome reaction.

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R. V. Hyne, R. N. Murdoch, and B. Boettcher

Summary. The metabolism and motility of human ejaculated spermatozoa incubated in vitro with steroids were studied. Progesterone and norethynodrel depressed the respiration, glycolytic metabolism and the motility of washed spermatozoa. Lynoestrenol did not affect the respiration or glycolysis of the spermatozoa, but did inhibit motility. Oestradiol did not cause any consistent alteration of the sperm metabolism, and did not affect the motility. Progesterone and norethynodrel appear to act on the plasma membrane of human spermatozoa to increase its permeability and hence to facilitate the loss of essential cofactors required for the glycolytic and oxidative processes.

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R. V. Hyne, Ruth E. Higginson, Debbie Kohlman, and A. Lopata

Summary. Guinea-pig spermatozoa, incubated in a minimal culture medium (MCM-PL) containing 150 mM-Na+ and no added K +, capacitate within 2 h and respond to added Ca2+ with maximal percentage of acrosome reactions. When spermatozoa, initially capacitated in Medium MCM-PL, were washed and resuspended in saline-based media, the motile spermatozoa showed acrosome reactions only in response to added Ca2 + at an extracellular pH > 7·4. In contrast, resuspension of capacitated spermatozoa into isotonic sucrose or choline-based media containing no added Na+ and K+ (pH 7·8) did not support acrosome reactions in response to the addition of Ca2 +. Examination under the electron microscope showed that these spermatozoa responded to added Ca2 + with swelling or cavitation of the acrosomal matrix but fusion of the plasma membrane and acrosomal membrane did not occur. Treatment of spermatozoa with monensin, a monovalent cationic ionophore, induced rapid acrosome reactions in the presence of extracellular Ca2 + and Na+. In contrast, spermatozoa incubated for 3 h in a sucrose-based Na+-deficient medium or Medium MCM-PL containing 10 mM-KCl failed to give a significant percentage of acrosome reactions in response to the addition of Ca2 +, but the addition of ouabain (0·1 mM) prevented the K+ inhibition. Amiloride, a sodium channel blocker in various other tissues, retarded the development of acrosome reactions of spermatozoa incubated in Medium MCM-PL. Based on these data, it appears that an increase in intracellular Na+ has a functional role in acquisition of guinea-pig sperm fertilizing ability.

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D. L. Garbers, J. R. Hansbrough, E. W. Radany, R. V. Hyne, and G. S. Kopf

Summary. Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85°C, ammonium sulphate precipitation at pH 4·2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8·3 μg calmodulin were recovered per 1010 sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.