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.     

Is sperminogen a modified proacrosin? Isolation, purification, and partial characterization of low-molecular-mass boar proacrosin

Low-molecular-mass zymogen was extracted from boar spermatozoa together with proacrosin using 10% acetic acid supplemented with 10% glycerol, and was purified by the sequential use of gel filtration on Sephadex G-75 and (FPLC) reversed-phase chromatography. LMM zymogen represented approximately 5% of the latent trypsin-like activity present in the sperm extract. SDS-PAGE indicated a molecular mass of 33 kDa. The zymogen reacted with both mouse monoclonal and rabbit polyclonal antibodies to boar acrosin. Determination of the N-terminal sequence of 34 amino-acid residues revealed its identity with the known N-terminal sequence of boar proacrosin.     

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.     

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.     

Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure

We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.     

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.     

Monoclonal antibodies to bovine and human acrosin

Monoclonal antibodies to human acrosin were required for studies of immunological interference with fertilization. Since human acrosin was not available in adequate amounts, monoclonal antibodies have been raised in mice against purified bovine acrosin and screened for cross-reaction with human sperm cells. Two of these antibodies are described, B4F6 and C2E5. Data from enzyme-linked immunosorbent assays, immunoblots, immunoprecipitation, and indirect immunofluorescence on sperm cells indicate that B4F6 binds only to bovine acrosin, and that C2E5 binds both to bovine and to human acrosin at a conformationally determined epitope. The antibodies do not inhibit the hydrolysis of benzoylarginine ethyl ester by acrosin, but C2E5 did inhibit the dissolution of the hamster zona pellucida by purified human acrosin. The antibodies have also been used for affinity purification of acrosin and proacrosin.     

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.     

Identification of a zona-binding protein from boar spermatozoa as proacrosin

The initial stages of fertilization in vertebrates and invertebrates are thought to involve complementary recognition molecules on spermatozoa and eggs. In a previous work (C. R. Brown and R. Jones, 1987, Development) we described one such putative molecule (a protein of approximate molecular weight 53 kDa) in detergent extracts of boar spermatozoa that has affinity for glycoproteins from the zona pellucida of pig eggs. This molecule has now been identified as proacrosin, the zymogen form of the acrosomal protease acrosin, on the basis of its electrophoretic behavior, the ability of zona glycoproteins to recognize and bind to proacrosin on Western blots, and the cross-reactivity of specific antisera to the 53-kDa molecule and proacrosin. A role is proposed for this enzyme in binding the sperm head to the zona pellucida during the initial stages of sperm-egg interaction.     

Rabbit testis proacrosin: Immunological similarities between testis,sperm proacrosins and the initially formed testis acrosin

Anti-rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto-activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G-100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti-rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.     

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.     

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.     

cDNA cloning and functional analysis of ascidian sperm proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.     

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.     

The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.     

Proacrosin activation in the presence of a 32-kDa protein from boar spermatozoa

A 32-kDa protein was purified from acrosomal extracts of ejaculated boar spermatozoa as a complex with 55- and 53-kDa proacrosins. In the presence of the 32-kDa protein, these proacrosins were sequentially converted by autoactivation to a 49-kDa intermediate, a 43-kDa intermediate, and then a 35-kDa mature acrosin. This activation process was consistent with that in the absence of the 32-kDa protein, but differed in producing the 49-kDa form as the predominant acrosin intermediate. Thus, the 32-kDa protein may be a regulatory protein for proacrosin activation. The 49-kDa intermediate was a two-chain polypeptide with the amino-terminal sequences corresponding to those of the light and heavy chains of mature acrosin, whereas the carboxyl-terminal sequence of its heavy chain was identical with that of the 53-kDa proacrosin. These results suggest that the 49-kDa intermediate is produced from 53-kDa proacrosin during proacrosin activation by the cleavage of the peptide bond between Arg-23 and Val-24, which results in the formation of the light and heavy chains.     

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.     

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.     

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.