Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

Proteolytic activity of rabbit perivitelline spermatozoa.

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 micrograms/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 micrograms/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

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.     

Localization of rabbit sperm acrosin during the acrosome reaction induced by immobilized zona matrix

To better understand the loss of the acrosomal cap on the surface of the zona pellucida and the function of the equatorial-postacrosomal region after the acrosome reaction, we have constructed an in vitro system using heat-solubilized zonae pellucidae dried onto a coverslip and incubated with capacitated spermatozoa. This system allows good optical resolution of spermatozoonzona interaction. Induction of the acrosome reaction by zonae on coverslips (30%) is comparable to the induction of the reaction reported previously for rabbit spermatozoa using solubilized zonae in solution. Antiserum to rabbit proacrosin, antiserum to a porcine 49-kDa proacrosin fragment, and antiserum to a porcine 14-kDa C-terminal acrosin fragment were utilized to monitor the acrosome reaction. Rabbit proacrosin/acrosin is not present on the surface of live, acrosome-intact, swimming spermatozoa. After contact with zona, the acrosome reaction begins and proacrosin/acrosin becomes available to bind antibody, first as a crescent in the apical region and then more posteriorly until the entire anterior acrosome is labeled. Proacrosin/acrosin remains on the equatorial and postacrosomal regions of acrosome-reacted spermatozoa and also remains associated with the acrosomal cap even after the spermatozoon is no longer associated with it. Further studies using zona-coated coverslips should lead to a more detailed understanding of the mechanism of zona penetration.     

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.     

Proacrosin/acrosin activity during spermiohistogenesis of the bull

Abstract. Acrosin and its zymogen form, proacrosin, were extracted from early and late spermatids, from ejaculated and epididymal spermatozoa ( caput, corpus , and cauda ) of the bull. Activity of proacrosin/acrosin and the time course of proacrosin activation were studied. It turned out that proacrosin/acrosin activity is first demonstrable in haploid spermatids, increases during spermiohistogenesis in the testis, and remains nearly constant in epididymal and ejaculated spermatozoa.     

Proacrosin/acrosin activity during spermiohistogenesis of the bull

Acrosin and its zymogen form, proacrosin, were extracted from early and late spermatids, from ejaculated and epididymal spermatozoa (caput, corpus, and cauda) of the bull. Activity of proacrosin/acrosin and the time course of proacrosin activation were studied. It turned out that proacrosin/acrosin activity is first demonstrable in haploid spermatids, increases during spermiohistogenesis in the testis, and remains nearly constant in epididymal and ejaculated spermatozoa.     

Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase

Ovulated opossum oocytes are surrounded by a zona pellucida, but not by cumulus cells. Opossum sperm carry at least four acrosomal hydrolases (hyaluronidase, acrosin, N-acetylhexosaminidase, and arylsulfatase); the functions of these enzymes in opossum fertilization are uncertain. To identify possible substrates for these hydrolases, the ultrastructure of opossum oocytes was examined after fixation in the presence of ruthenium red which stabilizes extracellular matrices. This oocyte is unusual in having a wide perivitelline space containing a highly structured extracellular matrix (ECM). The ECM is comprised of granules and filaments, and it resembles matrices known to contain hyaluronic acid in other systems. Hydrolases, known to be present in opossum acrosomes, were tested for their effect on the ultrastructure of the zona pellucida and matrix of the perivitelline space. Trypsin dissolved the zona pellucida and decreased the size of the granules in the perivitelline space. Streptomyces hyaluronidase, which specifically attacks hyaluronic acid, removed only matrix filaments. Arylsulfatase, N-acetylhexosaminidase, and beta-glucuronidase did not affect the zona pellucida or ECM in our assay. These observations are consistent with the ideas that (1) opossum sperm must penetrate two oocyte investments, the zona pellucida and ECM of the perivitelline space; (2) the ECM contains hyaluronic acid (filaments) and protein (granules); (3) opossum sperm acrosin may function in penetration of the zona pellucida and ECM; and (4) opossum sperm hyaluronidase may function in penetration of the ECM by degrading hyaluronic acid (filaments). Dissolution of the granules and filaments from oocyte microvilli is probably necessary to permit close apposition and fusion of the sperm and oocyte membranes. The evolutionary significance of these results is discussed.     

Proacrosin activation and acrosin release during the guinea pig acrosome reaction

The kinetics of proacrosin activation and release from guinea pig spermatozoa during the nonsynchronous acrosome reaction were studied. Epididymal spermatozoa were incubated at 37 degrees C in a defined medium (pH 7.8) containing 1.7 mM Ca2+. After 195 min, 78% of the motile spermatozoa had undergone the acrosome reaction as determined by light microscopy. Acrosin and proacrosin levels in the spermatozoa and medium were measured at the beginning of the incubation period. Most of the total acrosin activity (78%) was associated with the spermatozoa, of which greater than 90% was in the form of proacrosin. Proacrosin represented a small, stable fraction (23%) of the total acrosin in the medium; it did not activate to acrosin while in the medium. After 195 min, a decrease in sperm-associated total acrosin (42%; p less than 0.05) was accompanied by an increase in the total acrosin level in the medium (115%; P less than 0.05). No change in the relative proacrosin content (percent of total acrosin) was evident in either medium or spermatozoa. Additional experiments quantified acrosin and proacrosin during the progression of the acrosome reaction. Both the loss of sperm-associated total acrosin and the increase in total acrosin levels in the medium were highly correlated with the fraction of acrosome-reacted spermatozoa (r = 0.954 and 0.922, respectively; P less than 0.001). However, the rate of acrosin appearance in the medium was only 60% (P less than 0.001) of the rate of acrosin loss from the spermatozoa. The fractional proacrosin content of spermatozoa (94%) and medium (31%) remained unchanged during the acrosome reaction (r = 0.15 and 0.30, respectively; P greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of various conditions of semen storage on the acrosin system of human spermatozoa

Stability of the human sperm acrosin system (major components: non-zymogen acrosin, proacrosin and acrosin inhibitor) was studied under various conditions of semen storage used clinically or in the laboratory. Freezing at -196 degrees C caused a profound decrease in total acrosin content and in the amount of this enzyme present in zymogen form (proacrosin), but resulted in some increase in non-zymogen acrosin. Acrosin inhibitor did not appear to be significantly affected by this treatment. No relationship was present between the decreases in sperm motility induced by freezing to -196 degrees C and the alterations in total acrosin, proacrosin and non-zymogen acrosin. Storage of whole semen at -20 degrees C had deleterious effects on all the components of the acrosin system measured except for non-zymogen acrosin. Major decreases in the total acrosin, proacrosin and acrosin inhibitor occurred after only 1 day at -20 degrees C and continued slowly thereafter. Whole semen kept at room temperature for up to 24 h after ejaculation did not show any significant changes in the sperm acrosin system. Seminal plasma did not have a detrimental or stabilizing effect of acrosin and proacrosin when spermatozoa were kept at room temperature. However, removal of seminal plasma and re-suspension of spermatozoa in 0.9% NaCl resulted n the liberation of a significant amount of the acrosin inhibitor from the spermatozoa and the apparent activation of some of the proacrosin to 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.     

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.     

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.     

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.     

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida.

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.     

Boar proacrosin is a single-chain molecule which has the N-terminus of the acrosin A-chain (light chain)

Boar proacrosin was isolated from spermatozoa by a novel procedure under conditions preventing proenzyme activation. The spermatozoal extract was fractionated by gel filtration and reversed-phase FPLC, all in acidic solutions. Isolated proacrosin had a molecular mass of 55/53 kDa (doublet) and was devoid of amidolytic activity. Its single N-terminal sequence corresponded to that of the 23-residue acrosin A-chain and continued with that of the acrosin B-chain. Autoactivation at pH 7.8 did not influence the molecular mass. However, activated material contained two parallel N-terminal sequences, those of the A- and B-chain. Thus, activation of proacrosin is analogous to that of other serine proteinase proenzymes.     

Comparison of neutral proteinase activities in cock and ram spermatozoa and observations on a proacrosin in cock spermatozoa.

Cock spermatozoa, like trypsin, induced a rapid fall in the viscosity of gelatin solutions but ram spermatozoa and inhibitor-free ram acrosin were ineffective. The gelatin-hydrolysing activity in cock spermatozoa was solubilized at pH 8 in the presence of calcium ions but comparable extracts of ram spermatozoa were inactive. Both extracts showed acrosin activity (assayed with benzoylarginine ethyl ester). The two catalytic activities of cock spermatozoa were each susceptible to the same trypsin inhibitors and during fractionations they were not separable. We deduce that cock acrosin, and probably some other avian acrosins, have the power to degrade dissolved gelatin while ram acrosin does not. The acrosin in cock spermatozoa, unlike that in ram spermatozoa, was inactivated at pH 2-7. Acid extracts of the former contain an inactive precursor of acrosin which undergoes spontaneous re-activation in buffers, pH 8, containing calcium ions. In this respect it resembles the proacrosin of rabbit testis.     

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.     

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.