Acrosin, proacrosin, and acrosin inhibitor of guinea pig spermatozoa capacitated and acrosome-reacted in vitro.
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[Methodological investigations on acrosin activity in human spermatozoa. Correlation between acrosin and sperm density].
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An acrosin-acrosin inhibitor complex in ejaculated boar sperm.
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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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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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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.
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Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems.
Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60 min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological differences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34,000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm.
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A simple, clinical assay to evaluate the acrosin activity of human spermatozoa.
Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.
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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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Protein-protein interactions controlling acrosin release and solubilization during the boar sperm acrosome reaction.
In this study we used previously characterized monoclonal antibodies to acrosin (ACR.2) and to an acrosomal matrix antigen (ACR.4) to analyze the acrosin-binding activity of a 28-kDa putative acrosin-binding protein from the acrosomal matrix. The 28-kDa protein bound proacrosin and the 49-kDa form of acrosin (alpha-acrosin) but it did not bind the 36-kDa acrosin form (beta-acrosin). The acrosin-binding activity of the 28-kDa protein was stimulated by Ca2+, inhibited by Mg2+, and removed by disulphide bond reduction. Induction of the acrosome reaction by a calcium ionophore resulted in proteolytic cleavage of the 28-kDa protein, giving rise to a 12-kDa degradation product that was the only form of ACR.4 antigen released to incubation media; the release of the ACR.4 antigen was closely correlated with that of acrosin. The release of alpha-acrosin to incubation media was accelerated in the presence of ACR.4 antibody. In a cell-free system, a limited cleavage of the purified 28-kDa protein into immunoreactive degradation products was catalyzed by acrosin but not by trypsin or chymotrypsin. The data suggest that the 28-kDa acrosomal protein helps to maintain acrosomal matrix integrity and controls the acrosin release from acrosome-reacted cells.
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Effects of substrate and separation method on acrosin amidase measurements.
The purpose of this study was to increase the accuracy and reproducibility of the acrosin amidase assay and to assess the effects of different methods of sperm isolation on total sperm acrosin activity. Specific acrosin activity was measured by the procedure described by Kennedy et al (1989) comparing the usual substrate, N-alpha-benzoyl-DL-arginine-p-nitroanalide hydrochloride (DL-BAPNA) with the L-(L-BAPNA) and D-(D-BAPNA) isomers. Activity measurements were also compared on sperm isolated by methods: (1) centrifugation through buffered Ficoll, (2) method 1 plus an additional wash in buffered Ficoll, (3) back addition of supernatant from method 1 to spermatozoa isolated by method 2, and (4) swim-up into synthetic human tubal fluid media (mHTF) and using L-BAPNA. The specific activity of acrosin was dependent on substrate concentration up to 2.1 mM DL-BAPNA and 2 mM L-BAPNA. The maximum reliable solubility of DL-BAPNA was approximately 2.1 mM in 10% dimethylsulfoxide (DMSO):90% detergent buffer. There were no solubility constraints for L-BAPNA through 6.3 mM (> 5 times Km). D-BAPNA (1 mM) was not hydrolyzed by acrosin. Mean specific acrosin activity was higher using 6.3 mM L-BAPNA (159 +/- 11.4 microIU/10(6) sperm) than with 2.1 mM DL-BAPNA (81.4 +/- 10.9 microIU/10(6) sperm; P < 0.001, n = 16). Sperm isolated by methods 2 and 4 had higher specific acrosin activity than sperm isolated by method 1 (P = 0.002). Sperm treated per method 3 had similar acrosin activity as sperm isolated by method 1 (140 +/- 14.1 vs. 149 +/- 13.8 microIU/10(6) sperm). The K(m) for acrosin, calculated through 6.3 mM L-BAPNA, was 0.6 microIU/10(6) sperm. L-BAPNA is superior to DL-BAPNA as substrate for a clinical acrosin assay, increasing the reproducibility and accuracy of the assay. Simple Ficoll separation is not completely effective at removing acrosin inhibitors and additional separation steps may be necessary to assess true acrosin activity.
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Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins.
During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind. The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site.
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Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.
A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.
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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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Boar acrosin. Association of an endogenous membrane proteinase with phospholipid membranes.
Acrosin, an enzyme required for fertilization, is an endogenous proteinase associated with membranes of the sperm acrosome. Liposomes were utilized as a model system to evaluate the mode of association between highly purified boar acrosin and phospholipid bilayer membranes. Acrosin was observed to bind to liposomes containing acidic phospholipids such as phosphatidylglycerol, cardiolipin, and phosphatidylserine. There was no apparent binding of acrosin to liposomes which consisted of nonacidic phospholipids, thus indicating that an ionic phospholipid constituent was required for binding. Increased ionic strength caused a significant reduction in acrosin-liposome association with an inverse effect on enzyme-membrane dissociation. Acrosin-liposome association and dissociation were similarly effected by increasing concentrations of divalent cations at constant ionic strength, and by reductions in pH. Equilibrium binding experiments, with anionic liposomes, suggest the presence of either multiple classes of independent binding sites, or apparent negative cooperativity, with a range in the apparent affinity constant (Ka) from 2 x 10(11) M-1 at low acrosin concentrations to 3 x 10(8) M-1 at high acrosin concentrations. The membrane-associated enzyme was accessible to concanavalin A, and to high molecular weight substrates, demonstrating that a portion of the acrosin molecule is exposed at the membrane surface. In addition, acrosin binding to liposomes had no apparent effect of hydrolysis of soluble protein or synthetic substrates. The results demonstrate that acrosin-liposome binding is due in part to electrostatic charge interactions and indicate that the enzyme has properties of an extrinsic membrane protein.
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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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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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Effect of recombinant boar beta-acrosin on sperm binding to intact zona pellucida during in vitro fertilization.
In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.
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