Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization.

When denuded ram spermatozoa were suspended in weakly buffered 0.25M sucrose, the acrosin remained bound to the acrosomal membranes of the sperm heads. Media containing CaCl2 caused complete solubilization of the enzyme. Effects of acrosin inhibitors on soluble and bound enzyme were studied in Tris HCl(pH 8.2) containing sucrose. Denuded spermatozoa were used as a preparation of bound acrosin. Trasylol (Kunitz basic pancreatic trypsin inhibitor) acted more strongly on bound scrosin than on soluble acrosin, but soya-bean trypsin inhibitor acted more strongly on soluble acrosin. At concentrations 0.5 - 2.0muM, the inhibitors isolated from ram acrosomes and from ram seminal plasma inhibited soluble acrosin but had negligible effects on bound acrosin. However, bound acrosin was sensitive to high concentrations of the acrosomal inhibitor. The two forms of acrosin were inhibited to about the same degree by p-aminobenzamidine and also by Tos-Lys-CH2Cl. It is proposed that membrane-bound acrosin is the form that functions in penetration of the zona pellucida, and that a role for acrosin inhibitors is suppression of an antifertility effect of soluble acrosin on mammalian eggs. This hypothesis is supported by 1) the results of work on the impaired fertilizing capacity of rabbit spermatozoa that have been treated with acrosin inhibitors, 2) the anti-fertility effects on hamster eggs of solutions of acrosin and of bovine trypsin, and 3) the results in this paper.     

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.     

Multiple forms of boar acrosin and their relationship to proenzyme activation.

Further evidence is presented that the acrosomal proteinase acrosin exists as a zymogen precursor in freshly ejaculated boar spermatozoa. Autoactivation of proacrosin to acrosin takes place optimally at slightly alkaline pH and in the presence of calcium ions. Activation is considerably accelerated by catalytic amounts of trypsin or highly purified acrosin. A significant acceleration of the activation is also achieved by porcine pancreatic and urinary kallikrein, whereas chymotrypsin, plasmin, thrombin or urokinase showed no effect. Activation can be inhibited by p-amino-benzamidine and p-nitrophenyl p'-guanidino-benzoate. Electrophoretic analysis at different stages of activation revealed that during this process various molecular forms of acrosin are produced, apparently by limited proteolysis.     

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.     

Acrosin and the acrosome in human spermatogenesis

Using the indirect immunofluorescent staining technique, the developmental patterns of (pro) acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.     

Isolation of a stable apical segment of the guinea pig sperm acrosome

The large apical segments of guinea pig sperm acrosomes were mechanically separated from the spermatozoa and subsequently isolated by density gradient centrifugation. The isolated acrosomal caps were very stable and maintained their crescent morphology when suspended in sucrose-based medium buffered at pH 5.6, with or without the acrosin inhibitor p-aminobenzamidine (pAB). Examination under the electron microscope showed that the acrosomal caps were free of plasma membrane and were bound by an outer acrosomal membrane which was discontinuous. Enzymatic analysis after lysis of the caps indicated that acrosin and hyaluronidase were present with high specific activity, while only a trace amount of acid phosphatase activity and no arylsulphatase, phospholipase A₂, or phospholipase C activities were present. Significant particulate acrosin activity, but only trace amounts of soluble acrosin activity, could be detected in the isolated acrosomal caps if assayed immediately after isolation in the absence of pAB. However, soluble acrosin activity of high specific activity was obtained after the acrosomal caps were extracted by 10% glycerol buffered at low pH (pH 3.0). The new procedures provide a means to isolate and purify guinea pig sperm apical acrosomal segments rapidly.     

Studies on the rabbit proacrosin-acrosin system

A single molecular form (Mr = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 117;21-11728) was initially converted by autoactivation into an acrosin (Mr = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities (Rm = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands (Rm values of 0.395-0.412 and 0.497-0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the Mr 34,000 form of acrosin. This property was not shown by Mr 68,000 acrosin. Initial acrosin (Mr = 68,000) was activated by divalent cations such as Ca2+ and Mg2+. The enzyme was inhibited by Zn2+, Fe2+, Hg2+, and sulfhydryl blockers such as 5,5'-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn2+ is involved in acrosomal stabilization. The initial rabbit acrosin (Mr = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

Comparative activation studies with extracted and purified human proacrosin

Proacrosin was purified from acid extracts of human spermatozoa by concanavalin A precipitation and Bio-Gel P-100 chromatography. Two molecular weight forms of proacrosin were obtained, a major one with a Mr of 70,000-71,000 and a minor one with a Mr of 47,000-53,000. In contrast to sperm extracts, the purified forms of proacrosin were free of acrosin inhibitor(s) and nonzymogen acrosin. By modulating pH, ionic strength and temperature, the activation of proacrosin in sperm extracts was compared to only the major form of purified proacrosin, since it seemed to be the source of the lower molecular weight form of proacrosin. In both preparations, proacrosin activation occurred maximally over a broad pH range (7.6-8.8 for purified proacrosin and 7.6-9.6 for extract). Additionally, an ionic strength of 0.1 and above caused a decrease in proacrosin activation in both preparations. Similarly, proacrosin was sensitive to short incubation periods at 45 degrees C and above which caused a decrease in the amount of proacrosin found in both preparations.     

Acrosin does not appear to be bound to the inner acrosomal membrane of bull spermatozoa.

Ferritin-conjugated soybean trypsin inhibitor was used for the ultrastructural localization of acrosin in bull spermatozoa following acrosomal disruption. The ferritin label was observed in the anterior segment of the acrosome in disrupted cells only. Emptied acrosomes were labeled, mostly on the external surface of their outer membrane. Labeling was also found on the material bound to detached acrosomal caps. However, at no time could the ferritin label be found on the inner acrosomal membrane. It is concluded that acrosin activity is not present on the inner acrosomal membrane but is lost from the acrosomal matrix as the acrosomal reaction proceeds.     

Loss of acrosin from bovine spermatozoa following cold shock: Protective effects of seminal plasma

Loss of the alkaline proteinase acrosin and other proteins from the acrosome of bovine spermatozoa was investigated following cold shock and/or incubation of the spermatozoa at either 5, 21, or 37 °C for 4 hr. As detected by electrophoretic analyses of the acrosomal material two bands of acrosin activity and 10 proteins were lost from the acrosome after cold shock and incubation for 4 hr at 5 or 21 °C, whereas one acrosin band and 10 protein bands were lost after cold shock and incubation at 37 °C. Only 45% of the total acrosin activity remained in the acrosome after both cold shock and 4-hr incubation at 37 °C. Egg yolk, present at levels above 15%, and seminal plasma prevented much of the loss of acrosin from the cells.     

Biochemical studies on proacrosin and acrosin from epididymal boar spermatozoa: in vitro translation of boar testicular proacrosin mRNA

An inactive form of acrosin was extracted from epididymal boar spermatozoa utilizing acid pH conditions. When subjected to activation in alkaline environment, this form turns into an enzymatically active species, which exhibits close-related electrophoretic characteristics. Both the precursor and the activated species, when incubated in the presence of thermolysin, give rise to two fastly moving acrosin molecular forms. In order to establish the nature of the true acrosin zymogen, we isolated poly(A+)-RNA from boar testicles, performed its translation in vitro in the presence of [35S]-methionine and reticulocyte lysate, immunoprecipitated the translation products with anti-boar acrosin antibody, and analyzed them by SDS-polyacrylamide gel electrophoresis and autoradiography. A single translation product of molecular weight 55,000 was detected. It is concluded that the polypeptide chain of the boar zymogen is of 55,000; increases in molecular weight are due to post-translational modifications, like glycosylation.     

Immunological approach to characterize proacrosin and various acrosin forms in boar and man by monoclonal antibodies

Three different monoclonal rat antibodies, Acr1, Acr2, and Acr3, have been established against boar proacrosin. They are shown by enzyme-linked immunosorbent and immunoblot assays to react with boar proacrosin and several different acrosin molecules derived therefrom during activation. The epitopes detected by the three antibodies are different from each other, one being highly sensitive to reduction and periodate treatment. The antibodies crossreact with various proacrosin and acrosin molecules derived from human sperm extract; they also show indirect immunofluorescent staining of the acrosomal region of ejaculated sperm from normal men but fail to react with round-headed spermatozoa.     

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.     

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.     

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH‐20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm‐zona binding in other species. Anti‐macaque PH‐20 IgG, anti‐pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD‐46, which is also located on the inner acrosomal membrane, but has no known function in sperm‐zona pellucida interaction. After labeling with anti‐acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH‐20 and CD‐46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti‐acrosin IgG nor anti‐CD‐46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti‐PH‐20 IgG (100 μg/ml) was present during sperm‐oocyte interaction. Ultrastructural observations of oocytes incubated with anti‐PH‐20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti‐PH‐20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm‐zona binding, rather than primary sperm‐zona binding or the zona‐induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

pH and protease control of acrosomal content stasis and release during the guinea pig sperm acrosome reaction

The purpose of this study was to examine how trypsin inhibitors affect the guinea pig sperm acrosome reaction in vitro. Using spermatozoa pretreated with lysophosphatidyl choline, we found that both naturally occurring high molecular weight and the smaller synthetic trypsin inhibitor p-aminobenzamidine (PAB) delayed the onset of the acrosome reaction as monitored by light microscopy. Examination with electron microscopy revealed that acrosomal matrix dispersal rather than membrane fusion was affected. Despite the morphologic delay in acrosomal content release, PAB unexpectedly permitted 96% of soluble acrosomal antigen to be released into the supernatant. In addition, total acrosin release in the presence of PAB was 74% of control, with the vast majority as latent rather than active enzyme. A morphologically intact but membrane-free target of acrosomal matrix (AM), which is sensitive to trypsin inhibitor, was partially purified using Triton-x-100 at pH 5.2. AM remained morphologically stable at pH 5.2; however, shift up to pH 7 resulted in rapid dissolution within several minutes as monitored by light and electron microscopy and light scattering. Trypsin inhibitor prevented dispersion of AM at pH 7. The results suggest that, during the acrosome reaction, one distinct region of the acrosomal contents disperses after membrane vesiculation in a pH and trypsin inhibitor-insensitive fashion while a pH sensitive trypsin-like activity (acrosin?) disperses another discrete region of acrosomal matrix.     

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.     

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.