Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.     

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.     

Studies of three major proteases associated with guinea pig sperm acrosomes

The major proteases associated with guinea pig sperm were investigated by using immunological and electrophoretic techniques. Three major proteases were detected following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in gels containing 0.1% gelatin. These enzymes had molecular weights of 47,000, 34,000, and 32,000 relative to reduced protein standards and 58,500, 40,000, and 37,500 relative to unreduced standards. All three protease activities were present in acid extracts of sperm, detergent extracts of sperm, and the soluble acrosomal components of sperm released following induction of the acrosome reaction with the Ca2+-ionophore A23187. As determined by indirect immunofluorescence, an antibody to purified boar acrosin specifically cross-reacted with the acrosomes of guinea pig sperm. Decreased fluorescence was associated with sperm that had lost their acrosomes. Immunoblot analysis demonstrated that this antibody reacted with the 47,000 Mr protease but not the 32,000 and 34,000 Mr proteases. All three proteases were maximally active in the pH 7.0-8.5 region and were inhibited by classical inhibitors of acrosin activity. During a 3-hour incubation period, MgCl2 (10 mM) inhibited the activities of the 32,000 and 34,000 Mr proteases while the 47,000 Mr protease was stimulated. Although these proteases shared properties that would classify them as trypsin-like proteases, only the 47,000 Mr protease could be definitely classified as a member of the proacrosin-acrosin family based upon cross-reaction with an antibody to purified boar acrosin.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Diisopropyl fluorophosphate labeling of sperm-associated proteinases

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.     

Characterisation of an epitope shared by an acrosomal acrosin-like protein and the surface of tammar wallaby (Macropus eugenii) spermatozoa

Monoclonal antibodies (mAb) have been raised against marsupial sperm proteins to provide insights into the molecular nature of marsupial spermatozoa, and the proteins that mediate sperm maturation and interaction with the oocyte. This study reports the production of a mAb, designated WSA-1, which bound acrosomal and surface determinants on tammar wallaby spermatozoa. The acrosomal antigen was first detected in the wallaby testis; however, ejaculated spermatozoa demonstrated whole cell WSA-1 immunoreactivity as a result of binding an epididymal protein. Ultrastructural and agglutination analyses localised the WSA-1 epitope to the acrosomal matrix and the whole sperm plasmalemma. The WSA-1 mAb bound three polypeptides with relative molecular weights of 35, 31 and 15 kDa on western blots under reducing conditions. The N-terminal amino acid sequence obtained for the 35 kDa wallaby sperm polypeptide demonstrated identity with the eutherian acrosomal protein acrosin. The 31 kDa polypeptide was of epididymal origin and will be the subject of a separate study. Further studies of the WSA-1 antigens are likely to provide useful insights into the function and maturation of marsupial sperm since proacrosin has a number of putative roles in eutherian fertilisation, and epididymal proteins are thought to mediate sperm maturation and storage.     

The inhibition of acrosin by sterol sulphates

Four 3 beta-hydroxy-delta 5-steroid sulphates were found to be potent and specific inhibitors of the sperm acrosomal proteinase, acrosin. Two of these acrosin inhibitors, desmosteryl sulphate and cholesteryl sulphate, occur naturally in spermatozoa. Desmosteryl sulphate, an inhibitor of the in-vitro capacitation of hamster spermatozoa, has a Ki of 3.5 x 10(-6) M for the inhibition of acrosin. The mechanism of inhibition of sperm capacitation by sterol sulphates is probably due to their inhibition of acrosin.     

Characterization of isolated acrosomal matrices from hamster spermatozoa

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.     

Partial purification and characterization of human sperminogen

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.     

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.     

Characterization of a 23 kDa sperm-binding polypeptide of the golden hamster epididymis

A 23 kDa polypeptide has been identified on the flagellum of sperm obtained from the cauda epididymis of the golden hamster. A monospecific antiserum to the 23 kDa hamster polypeptide was prepared and used to study its distribution on sperm, in the epididymis, and in epididymal fluid. In the cauda, the polypeptide is found on the midpiece and endpiece of the sperm tail, in detergent extracts of sperm, and in epididymal luminal fluid-enriched fractions. It is not present on sperm or in luminal fluid-enriched fractions from the caput epididymis. Immunocytochemical staining of epididymal tissue has demonstrated the 23 kDa polypeptide in the Golgi region of the principal cells of the proximal cauda and on sperm in the tubules of this segment and in tubules distal to it. Antiserum to the 23 kDa golden hamster polypeptide cross-reacts with sperm from rats and Chinese hamsters, but not with sperm from rabbits, cattle, mice, and guinea pigs. The antigen is localized to the tail of sperm obtained from the cauda of the rat and from the distal caput of the Chinese hamster. Immunoblots of detergent extracts of sperm and luminal fluid-enriched fractions from these two species reveal a 26 dKa polypeptide that is immunologically related to the golden hamster polypeptide.     

Further evidence in support of a role for hamster sperm hydrolytic enzymes in the acrosome reaction

The effects of trypsin inhibitors and phospholipase inhibitors on the acrosome reaction of washed cauda epididymal sperm of golden hamsters were studied using two different incubation systems. One incubation system, a non-synchronous acrosome reaction inducing system, included the use of a highly purified BSA and a protein-free motility factor preparation from hamster adrenal gland. The other system was a relatively synchronous acrosome reaction-inducing-system utilizing the calcium ionophore A23187. Acrosome reactions were inhibited by three low molecular weight synthetic trypsin inhibitors, benzamidine, NPGB and TLCK, when they were added five minutes prior to the initial occurrence of acrosome reactions in the non-synchronous system or five minutes prior to induction of acrosome reactions by A23187 in the synchronous system. Two phospholipase A inhibitors, p-bromophenacyl bromide and mepacrine, were also effective in inhibiting hamster sperm acrosome reactions in both incubation systems. TPCK, an inhibitor of several non-trypsin-like proteases, indomethacin, a prostaglandin synthetase inhibitor, and soybean trypsin inhibitor, a large molecular weight polypeptide, did not inhibit acrosome reactions. The inhibition of those acrosome reactions induced by A23187 provides further indirect evidence that the effective inhibitors were functioning at a site within the sperm. The overall results provide: (1) further support for our earlier work suggesting the involvement of an internal trypsin-like enzyme (presumably acrosin) rather than an exogenous trypsin-like enzyme in the hamster sperm acrosome reaction and (2) the first evidence suggesting the possibility that a sperm phospholipase may also be involved in the mammalian acrosome reaction.     

Benzamidine as an inhibitor of proacrosin activation in bull sperm.

Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.     

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.