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PubMed1978

Studies on ram acrosin. Activation of proacrosin accompanying the isolation of acrosin from spermatozoa, and purification of the enzyme by affinity chromatography.

1. A previously described, freeze-dried, partially purified ram acrosin preparation was fractionated on a column of Sepharose linked to the acrosin inhibitor p-(p'-aminophenoxypropoxy)benzamidine. Two acrosin fractions were obtained. 2. beta-Acrosin was homogeneous, quite stable at low pH and very stable when freeze-dried. Its molecular weight is about 38000, and it contains about six sugar residues per molecule, but no sialic acid. psi-Acrosin consisted of at least three unstable forms of acrosin. 3. When the entire purification process, starting from collection of semen, was carried out as rapidly as possible, the yield of beta-acrosin was increased and very little psi-acrosin was obtained. 4. In fresh ram semen the acrosin is present as the intra-acrosomal zymogen, proacrosin. After its extraction from spermatozoa autoproteolytic reactions convert proacrosin into beta-acrosin; psi-acrosin appears to be breakdown products of beta-acrosin. 5. When beta-acrosin was passed through a column of Sepharose linked to the non-inhibitory deamidinated analogue of the inhibitor it behaved as a hydrophobic protein. This is consistent with our view that acrosin (as zymogen) occurs in spermatozoa as a membrane-bound protein. 6. Success in the isolation of pure acrosin in high yield calls for an affinity adsorbent with the appropriate subsidiary hydrophobic properties.

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PubMed1976

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.

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PubMedDate not supplied

On the teratogenesis of round-headed spermatozoa: investigations with antibodies against acrosin, an intraacrosomally located acrosin-inhibitor, and the outer acrosomal membrane.

Acrosin, the outer acrosomal membrane (OAM) and an acrosin inhibitor were studied in testicular cells and ejaculated spermatozoa of fertile men and in those of an infertile patient with exclusively round-headed spermatozoa in his ejaculates. The investigations were performed with the aid of immunohistochemical techniques using specific antibodies against the three acrosomal markers isolated from boar spermatozoa. The spermatozoa of fertile men exhibit staining for acrosin, the OAM and the acrosin inhibitor in the cap region while the round-headed spermatozoa of the patient are totally negative for the three markers, clearly supporting the conclusions of other authors that round-headed spermatozoa lack acrosomes. The lack of the acrosin system was further substantiated by the gelatin substrate film technique. In the course of normal human spermatogenesis acrosin, the OAM and the acrosin inhibitor we first demonstrable in early round spermatids, namely in identical compartments adjacent to the cell nucleus. During spermatid differentiation the staining for the three markers becomes flattened over the nucleus, resulting in a cap-like structure in testicular spermatozoa. In contrast to the ejaculated round-headed spermatozoa, the early round spermatids in the testis of the infertile patient exhibit fluorescent staining for the three markers in the region adjacent to the nuclear membrane. In the course of further spermiogenesis, the staining did not extend over the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, was translocated at various locations in the cytoplasm and was finally eliminated with the loss of the cytoplasm. These results are in accordance with the results of electron microscopically investigations on the teratogenesis of round-headed spermatozoa. Furthermore, the developmental pattern of the acrosin inhibitor during normal and abnormal spermiogenesis supports the intraacrosomal location of the acrosin inhibitor recently described by Tschesche et al. (1982).

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PubMed1978

Boar malpha-acrosin. Purification and characterization of the inital active enzyme resulting from the conversion of boar proacrosin to acrosin.

The preparation of highly purified malpha-acrosin is described. Purification was achieved by controlled activation of partially purified proacrosin, followed by gel chromatography over Sephadex G-100 at pH 3.0. The final malpha-acrosin preparation resulted in a single protein band with a molecular weight of 49,000 as determined by sodium dodecyl sulfate-disc gel electrophoresis. Disc arginine naphthylamide hydrolyzing band with a relative migration of 0.39 malpha-acrosin catalyzed the hydrolysis of synthetic substrates containing arginine and lysine, but not phenylalanine. Although calcium ions were not required for enzymatic activity, the addition of calcium chloride stimulated the activity through an increased substrate affinity and an increased maximal velocity. Polyamines stimulated the maximal velocity of the reaction, but were without effect on the substrate affinity. malpha-Acrosin was inhibited by lima bean, ovo-mucoid, and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and 1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an irreversible inhibition, while L-arginine, benzamidine, and p-aminobenzamidine were competitive inhibitors with respect to substrate. These properties of malpha-acrosin are very similar to those previously reported for mbeta-acrosin and suggest that the portion of the molecule lost during the conversion of malpha-acrosin to mbeta-acrosin contributes little to the topography of either the active site or regulatory sites of the enzyme.

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PubMed1990

Determination of active, non-zymogen acrosin, proacrosin and total acrosin in different andrological patients.

A spectrophotometric assay is described which, due to improved extraction conditions, allows quantitative determination of enzymatically active, non-zymogen acrosin, proacrosin and total acrosin activity from human sperm acrosomes. Acrosomal proteinase activity is assessed by acid extraction of the sperm pellet and the suspension medium before and after snap-freezing, followed by zymogen autoactivation. Release of acrosin from the acrosome can be used as a sensitive biochemical marker to characterize acrosomal membrane stability, severe disturbance of which may be the cause of impaired male fertility. Acrosin activities in different populations of semen specimens are reported and compared to data available in the literature. Different degrees of acrosomal membrane alterations are observed in men with oligozoospermia, tetratozoospermia and polyzoospermia. Particularly in oligozoospermia, a significant increase of active, non-zymogen acrosin points to severe acrosomal membrane alterations and, in addition, to a premature activation of proacrosin, which may impair fertilization in certain individuals. Finally, acrosin activity is shown to be significantly influenced by the time of sexual abstinence. It is concluded that determination of acrosin may be a useful indicator of the fertility potential in men.

Acetates↗
PubMed1976

Influence of boar acrosin antibodies produced in rabbit and sheep on chymotrypsinogen activation catalyzed by acrosin from boar, bull, ram, rabbkt and human.

Activation of bovine chymotrypsinogen is catalyzed with increasing velocity by human, rabbit, boar, bull and ram acrosin. Antiboar-acrosin rabbit gamma-globulins cause a significant reduction in the proenzyme activation rate induced by boar and bull acrosin, but only a weak reduction or none if ram or rabbit acrosin is the activating agent. The antiboar-acrosin gamma-globulins from sheep strongly inhibit chymotrypsinogen activation by ram, bull and boar acrosin, and significantly inhibit the human acrosin-catalyzed reaction.

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PubMed1990

Zona pellucida-binding of boar sperm acrosin is associated with the N-terminal peptide of the acrosin B-chain (heavy chain).

Recently, it has been shown that boar acrosin exhibits a carbohydrate-binding activity with a specificity to fucose, by which it can bind to the oocyte zona pellucida. By limited autoproteolysis of a high-molecular mass acrosin (55/53 kDa), designated as alpha-acrosin, a 15 kDa fragment was generated which interacts strongly with the porcine zona pellucida. Zona-binding was demonstrated on protein blots and by the solid-phase zona-binding assay utilizing biotinylated zona proteins. The zona-binding peptide was isolated by reversed-phase HPLC and analyzed for amino acid sequence. Its single N-terminal sequence corresponded to that of the acrosin B-chain (heavy chain). These data indicate that the zona-binding properties of acrosin are associated with the N-terminal peptide of the acrosin heavy chain.

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PubMed1976

Activation of chymotrypsinogen by boar acrosin and its prevention by antiboar acrosin rabbit gamma-globulins.

Activation of chymotrypsinogen by bovine trypsin or boar sperm acrosin was followed up using Nalpha-acetyl-L-tyrosine ethyl ester in a highly sensitive test system. Inhibition studies employing antiboar acrosin rabbit gamma-globulins showed the following results. 1) Whereas the acrosin-induced activation velocity was significantly depressed in the presence of the antibodies, the trypsin-catalyzed activation rate was not diminished. 2) The antibodies enhanced the acrosin-catalyzed cleavage rate of BzArgOEt significantly, but not the trypsin-catalyzed cleavage rate of this substrate. 3) Autodigestion of acrosin was considerably reduced in the presence of the antibodies. The enzymatic test system used is especially suitable to study the specificity of acrosin antibodies or their affinity to related enzymes if only small amounts of these substances are available.

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PubMed1978

Inactivation of boar acrosin by peptidyl-arginyl-chloromethanes. Comparison of the reactivity of acrosin, trypsin and thrombin.

A survey of the reactivity of 16 peptidyl-argininyl-chloromethanes with boar acrosin indicated that these compounds as a general group of reagents were highly effective in the inactivation of acrosin since at least half of the reagents tested rapidly inactivated this protease at a concentration of 0.10 micrometer or lower. For example, Dns-Glu-Gly-ArgCH2Cl inactivates acrosin by 50% in 1.8 min at a concentration of 75 nM, whereas in contrast, a 14000-fold higher concentration of Nalpha-tosyllysyl-chloromethane is required to obtain an equivalent rate of inactivation. A comparison of the reactivity of acrosin and trypsin with the peptides of arginyl-chloromethane containing different substituents in the P2 and P3 positions suggests that the secondary binding sites of these two proteases are very similar. Reagents with homoarginine, lysine and D-arginine in the P1 position have also been prepared and evaluated, but these were considerably less effective than the corresponding arginyl-chloromethanes in the inactivation of both acrosin and trypsin.

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PubMed1975

Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors.

1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-alpha-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca(2+), and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris-HCl buffer (pH8.2) activation by Ca(2+) did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca(2+) no longer exerts an activating effect and may become inhibitory. Ca(2+) is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca(2+) on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca(2+). Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca(2+). 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-alpha-benzoyl-dl-arginine 2-naphthylamide. 8. K(m) values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k(cat.) values are slightly higher than those for trypsin. Considerations of the influences of ions and dimethyl sulphoxide on the activities and kinetic constants of acrosin and trypsin suggest that conformational changes are the factors mainly responsible for the reported activations of acrosin. 9. The following conclusions are reached. (a) Acrosin plays a role in the penetration of the sperm cell into the egg without becoming detached from the acrosomal membrane. (b) The enzyme is a peripheral membrane protein which may be classed as a cathepsin. (c) The susceptibility of the activity of soluble acrosin to cations and solvents points to a flexible molecule, i.e. one lacking conformational restraints imposed by association (presumably ionic) with the acrosomal membrane.

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PubMed1976

Enhancement of sperm acrosin activity by glycerol-pretreatment - quantitative estimations.

Acetic acid treatment of spermatozoa, a method suitable for optimal extraction of the acrosomal proteinase acrosin, was used to show the influence of glycerol on sperm acrosin activity. Short-time pretreatment of semen samples with glycerol in concentrations up to 35% (v/v) caused a 1.5-2.5 fold increase in sperm acrosin activity. Higher glycerol concentrations caused a decrease in sperm acrosin activity due to leakage of the enzyme into the suspension medium. A further increase in sperm acrosin activity was observed during aging of semen in the presence of glycerol. In this case, the height of the increase depends on the glycerol concentration applied as well as incubation temperature and time. The acrosin activation pattern induced by glycerol was not influenced by proteinase (acrosin) inhibitors. On the other hand, addition of glycerol to spermatozoa from which the seminal plasma had been removed and substituted by physiological saline caused only a small increase in sperm acrosin activity. This indicates the occurrence of a seminal plasma factor which is either stimulated (activated) by glycerol or which can penetrate the membranes and subsequently activate acrosin only in the presence of glycerol. This seminal plasma factor was not consumed during activation and could be transfered to another sperm sample. However, a protecting influence of such a factor on the sperm head membranes and thus an indirect activation effect, i.e. better extractibility of acrosin, has also to be considered. The glycerol-induced rise of the acrosin activity is not caused by reversible conformational changes of the enzyme molecules: acrosin activity was not diminished if glycerol was removed from semen samples or extracts later on. The possibility that the observed increase in BAEE-splitting activity is due to a so far unknown proteinase may be excluded: the activity completely disappeared by neutralization due to formation of the acrosin-inhibitor complex and appeared again by acidification, a well known characteristic of acrosin and its inhibitors. Factors which are probably responsible for the glycerol-induced activation of acrosin-membrane effects and the activation of a precursor from of acrosin-are discussed.

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PubMed1990

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)

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PubMedDate not supplied

Human sperm acrosin. Further studies with the clinical assay and activity in a group of presumably fertile men.

The goals of this study were to determine the effect of the nonionic detergent, Triton X-100, on the recovery of acrosin in the clinical assay (Kennedy et al, 1989), since previous investigations have used higher concentrations for acrosin extraction from spermatozoa; and to establish the minimal acrosin activity in fertile men. The recovered acrosin activity was dependent on the concentration of Triton. A peak in acrosin activity was obtained at 0.01% to 0.02% Triton, the approximate critical micelle concentration (CMC; 0.015%). The bimodal effect of Triton was not due to substrate/buffer alterations, to the degree of acrosomal disruption as assessed by light and transmission electron microscopic examination, or to its effect on the kinetic properties of acrosin, as determined by spectrophotometric analysis of acid-extracted enzyme. However, Triton affected the conversion of proacrosin to acrosin, with peak activation occurring at 0.01% to 0.02% detergent. The acrosin activity of a group of presumably fertile men (as established by the production of offspring under natural conditions) varied from 18 to 42 microIU/10(6) spermatozoa, as assessed by the clinical assay containing 0.01% Triton. Furthermore, men who had initial acrosin values in the low normal range (18 to 25 microIU/10(6) sperm) were observed for 11 months. The acrosin activity of their ejaculates never fell below 17 microIU/10(6) sperm. Thus, it can be tentatively assumed that the minimal levels of acrosin for naturally fertile men are 17 to 18 microIU acrosin/10(6) sperm in this assay.

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PubMed1991

Antisperm antibody binding to human acrosin: a study of patients with unexplained infertility.

OBJECTIVE: Antisperm antibody binding to acrosin was investigated by Western Blotting. The clinical significance of this binding specificity was assessed in a 2-year clinical follow-up. DESIGN: Consecutive serum samples positive for antisperm antibodies by both enzyme-linked immunosorbent assay and immunobead testing were evaluated for acrosin-binding specificity. SETTING: The patients were followed in an outpatient setting by private infertility specialists. PATIENTS: Sixty-five consecutive infertile referral patients with positive antisperm antibody were evaluated. Clinical follow-up was obtained on 8 of 9 females with evidence of antibody binding to acrosin and 19 of 26 females with no specific binding to acrosin. INTERVENTIONS: Prednisone therapy was given during six courses of intrauterine insemination with husband's sperm. All treatment decisions were made by private physicians independent of the acrosin-binding result. MAIN OUTCOME MEASURES: Pregnancy status was obtained as part of a 2-year follow-up. RESULTS: Acrosin-binding specificity was demonstrated in 10 (15%) of the 65 patients. Two of the 8 women (25%) with antibody binding to acrosin and 6 of the 19 women (32%) with antisperm antibodies but no specific binding to acrosin delivered normal children. CONCLUSIONS: Although antibody-binding specificity to acrosin could be demonstrated, a 2-year clinical follow-up showed no difference in pregnancy rates when compared with women with antisperm antibodies showing no binding specificity to acrosin.

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PubMed1992

Sperm acrosin activity and fluorescence microscopic assessment of proacrosin/acrosin in ejaculates of infertile and fertile men.

OBJECTIVE: To compare biochemically active with immunoreactive sperm acrosin in fertile and infertile men. SETTING: This study was conducted in a tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila. PATIENTS: We evaluated the males in 40 infertile couples with no recognized cause of female infertility and 20 fertile men. INTERVENTIONS: Ejaculates were collected under standardized conditions of abstinence. MAIN OUTCOME MEASURES: Total sperm acrosin activity was measured on a spectrophotometer in washed sperm stored at -80 degrees C for 1 to 6 days. The percent of spermatozoa immunostained by an antiserum against proacrosin/acrosin by indirect immunofluorescence (IFL) was determined on methanol fixed sperm smears. RESULTS: Biochemically active acrosin was correlated to immunoreactive acrosin (P = 0.0028), and both were inversely correlated to the percent of spermatozoa with an abnormal head (P = 0.00024 for acrosin activity and P = 0.0013 for IFL). Biochemically active and immunoreactive acrosin were lower in infertile compared with fertile men (P = 0.0012 and P = 0.0009, respectively). Sixty-eight percent of ejaculates with an acrosin activity lower than the limit value observed in fertile men showed a normal sperm morphology and a normal immunoreactivity for acrosin. CONCLUSIONS: A low sperm acrosin activity in teratospermic ejaculates is because of a lack or a defect of the immunogenic and functional domains of the protein. A low sperm acrosin in infertile men with normal semen parameters results from a possible functional defect of the enzyme that is immunohistochemically detected in spermatozoa.

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PubMed1979

Acrosin of mouse spermatozoa.

Mouse spermatozoa possess a neutral proteinase, acrosin, that is to a large extent (70-80%) present in the zymogen (proacrosin) form. Acid extraction yields higher amounts of acrosin than detergent extraction. Synthetic inhibitor studies indicate that mouse acrosin has a serine and histidine at its active site and hydrolyzes the peptide bonds of lysine and arginine but of not phenylalanine. An inhibitor of acrosin is associated with mouse spermatozoa, capable of preventing the activity of at least 60% of all available acrosin. Acrosin activity is essential for fertilization because natural and synthetic inhibitors of mouse acrosin prevent the union of the gametes. Also, the relative inhibitory activity of synthetic agents toward acrosin runs approximately parallel to their antifertility activity. The percent of acrosin in the proacrosin form does not change after capacitating mouse spermatozoa in vitro.

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