J. Cell Sci. 32, 153-164 (1978) 153 Printed in Great Britain © Company of Biologists Limited lgyS THE ACTIVATION OF PROTEOLYSIS IN THE ACROSOME REACTION OF GUINEA-PIG SPERM D. P. L. GREEN Physiological Laboratory, Cambridge CBz 3EG, England SUMMARY The divalent metal cation ionophore A23187 rapidly induces a normal acrosome reaction in a population of guinea-pig sperm suspended in calcium medium. In the course of the acrosome reaction, proacrosin, the zymogen precursor of the protease acrosin, is activated. Although the acrosome reaction causes exocytosis of the acrosomal contents, 'soluble' acrosin is not released in significant amounts until well after the sperm population as a whole has undergone an acrosome reaction. This suggests that proacrosin is stored within the acrosome in an insoluble form and that exocytosis of the acrosomal contents in the acrosome reaction is insufficient, by itself, to cause its immediate dissolution. Electron micrographs of sperm undergoing an A23i87-induced acrosome reaction in the presence of the acrosin inhibitors benzamidine, p-amino-benzamidine and phenylmethyl- sulphonyl fluoride show that the acrosome reaction proceeds normally but that dispersal of the acrosomal contents is inhibited. These morphological changes are, for the most part, below the limit of resolution of the light microscope and using light microscopy to assess whether an acrosome reaction has taken place, it can be mistakenly inferred that the reaction itself is inhibited by the acrosin inhibitors. The inhibition of the dispersal of the acrosomal contents by acrosin inhibitors suggests that acrosin activity is important in solubilizing acrosin. These experimental observations, taken with the evidence that the acrosome reaction is a response to an increase in intracellular free calcium, have been taken as the basis of a proposal for the mechanism of proacrosin activation in the acrosome reaction. INTRODUCTION The acrosomes of mammalian sperm contain a number of enzymes. One of these is acrosin, a protease which is broadly similar to trypsin; it hydrolyses the same synthetic substrates (e.g. benzoyl arginine ethyl ester (BAEE), etc.) and it is inhibited by a wide range of synthetic and naturally occurring trypsin inhibitors (e.g. benzamidine, p-aminobenzamidine, soybean trypsin inhibitor, ovomucoid, etc.). Before the acrosome reaction acrosin exists in sperm almost completely as an inactive zymogen precursor, proacrosin (Meizel & Mukerji, 1976). Proacrosin, as far as is known, can only be activated by proteolytic cleavage (cf. trypsinogen, etc.). Activation can be caused by either trypsin or acrosin. There is no direct evidence that proacrosin is activated during the acrosome reaction, although if acrosin is necessary for sperm penetration of the zona it must be. The fate of proacrosin in the acrosome reaction has been examined using the A23i87-induced reaction of guinea-pig sperm (Green, 1978 a). At the same time, the effect of trypsin inhibitors on the acrosome reaction has been studied in view of a report that some inhibitors prevent the acrosome reaction from taking place (Meizel & Lui, 1976). 154 D. P. L. Green METHODS Phenyl methyl sulphonyl fluoride (PMSF), ^-aminobenzamidine, soybean trypsin inhibitor (STI) Type I-S, lima bean trypsin inhibitor (LTI), ovomucoid and benzoyl arginine ethyl ester (BAEE) were purchased from Sigma, and benzamidine from Aldrich. The basic experimental procedures were the same as described previously (Green, 1978a). The time course for the loss of the acrosome induced by A23187 in the presence of trypsin inhibitors was estimated from inspection by light microscopy of sperm fixed at suitable intervals after addition of A23187. The acrosome reaction was induced by addition of 50 /il of the stock solution of A23187 in dimethyl sulphoxide (DMSO) to 5 ml of sperm suspension at 37 °C. Samples (0-5 ml) were pipetted into Karnovsky's fixative in which magnesium and 100 fiM EGTA were substituted for calcium. 500 sperm were examined for each time point. With the exception of PMSF, all inhibitors were dissolved directly in calcium medium. For the experiments with PMSF, 20/tl of a 25 mM stock solution in DMSO were added directly to s ml sperm suspension. In one experiment, sperm were incubated with PMSF in magnesium medium for 0-5 h and then washed 5 times in magnesium medium, 10 ml each time, before resuspension in calcium medium and addition of A23187. Acrosin activity was measured after acid extraction of sperm by following the hydrolysis of BAEE at 256 ran. The acrosome reaction was started with addition of A23187 from a stock solution in DMSO. In a typical experiment, o-s-ml samples were removed from 5 ml of sperm suspension, one sample before and the rest after addition of A23187, and pipetted into 0-5 ml of 20 mM HC1. The acid suspension was centrifuged at 14000 g,v after not less than 15 min and the supernatant assayed for acrosin activity. The assay solution contained 300 mM Tris HC1 buffer, pH 8-o, 150 mM CaCl! and 3 mM BAEE. To 0-9 ml of this solution was first added 0-9 ml of 10 mM KOH followed, after mixing, by 09 ml of acid extract. The change in absorbance at 256 nm was measured at 20 °C in a Zeiss PMQ II spectrophotometer. In one experiment the appearance of soluble acrosin discharged from sperm undergoing an A23i87-induced acrosome reaction was compared with the appearance of acid-extractable acrosin under the same conditions. Sperm in calcium medium were induced to undergo the acrosome reaction by addition of A23187 as before. At suitable time intervals, o-6 ml of sperm suspension was transferred to an Eppendorf tube and spun for 15 s at 14000 g,v. The centrifuge was rapidly stopped and 05 ml of the supernatant pipetted into a test tube containing 0-5 ml of 20 mM HC1. The whole procedure takes about 40-45 s: it was started 15 s before each time point and ended 25-30 s after. The acid solution was assayed as described previously for the acid-extracted acrosin. RESULTS An acrosome reaction is induced in guinea-pig sperm in calcium medium by the divalent cation ionophore A23187 (Green, 1976; Summers et at. 1976; Talbot et al. 1976; Green, 1978 a). The acrosome reaction results in the loss of the acrosome and a substantial change in the shape and size of the head. The time course for the loss of the acrosome is shown in Fig. 3. In the course of the acrosome reaction, the total quantity of acrosin rises (Figs, i, 2). This increase in acrosin reflects the proteolytic activation of proacrosin. Activation is dependent on the concentration of A23187 and external calcium, both in rate and extent (Figs. 1, 2): activation does not occur in magnesium medium after addition of A23187 (Fig. 1) or in magnesium medium containing o-i % Triton X-100. Extraction of sperm with acid removes acrosin and proacrosin (Meizel & Mukerji, 1976) and at the same time dissociates the acrosin inhibitor (Brown, Andani & Hartree, 1975; Schleuning, Hell & Fritz, 1976). The acrosin activity measured there- fore represents total acrosin, not free acrosin. Proteolysis in the acrosome reaction 155 The effect of acrosin inhibitors on the loss of the acrosome during the acrosome reaction is shown in Figs. 3-5. The reversible acrosin inhibitors benzamidine and p-aminobenzamidine and the irreversible acrosin inhibitor PMSF delay but do not prevent the loss of the acrosome (Figs. 3, 5), but the naturally occurring protein acrosin inhibitors, soybean trypsin inhibitor, lima bean trypsin inhibitor and ovomucoid are without effect (Fig. 4). 0-2 r Time, min Figs. 1, 2. Proacrosin activation, induced in guinea-pig sperm in calcium medium by A23187. Fig. 1. Proacrosin activation for a fixed A23187 concentration, 80 fiM, and different external calcium concentrations, inmni: 10, # ; 2, O ; and 0-4, •. Sperm in magnesium show no activation on addition of A23187, •• When sperm in magnesium medium are incubated with PMSF and then extensively washed before resuspension in calcium medium and addition of A23187, the loss of the acrosome takes place normally (Fig. 5). Electron micrographs of sperm undergoing an acrosome reaction in the presence of either p-aminobenzamidine or PMSF fixed 5 min after addition of A23187 (i.e. when Fig. 3 shows the population of sperm to have undergone almost completely an acrosome reaction in the absence of inhibitors) have the appearance shown in Figs. 6 and 7. Both micrographs show head-to-tail sections through a stack of sperm heads. They 156 D.P.L. Green are to be compared with figs. 1 and 4 of the preceding paper (Green, 1978 a). What they both show is that all the sperm have undergone an acrosome reaction, i.e. mem- brane fusion is well advanced, but the acrosomal contents have failed to disperse. As a result, all the sperm, irrespective of the sequence in which they underwent the acrosome reaction have been arrested at the same stage. 0-2 r- 0 5 10 Time, min Fig. 2. Proacrosin activation for a fixed external calcium concentration, 10 mM, and different concentrations of A23187, in/iM; 80, •; 16, •; and 3-2, O- A comparison of the rate of appearance of acid-extractable acrosin with 'soluble' acrosin is shown in Fig. 8. This shows that 'soluble' acrosin does not significantly appear in the supernatant until proacrosin activation is essentially complete. This statement errs on the side of caution because a 15-s spin at 14000 g cannot be con- sidered an adequate test of solubility. It is almost certain, therefore, that release of soluble acrosin is delayed well beyond the period of its formation from proacrosin, if it ever occurs completely at all. Proteolysis in the acrosome reaction 1OOr Time, min Figs. 3—5.