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.     

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.     

An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.

Ram seminal plasma, and ejaculated ram spermatozoa that have been washed with 0.25M sucrose, both contain acrosin inhibitor. The aim of this work was to determine whether the intracellular inhibitor originates from the seminal plasma. The amounts of inhibitor in ejaculated and epididymal spermatozoa were measured and compared with the amounts present in the seminal plasma of normal and vasectomized rams. One ejaculated ram spermatozoon contained 2.1 amol (2.1 X 10(-18) mol) of inhibitor and one epididymal spermatozoon contained 3.3 amol of inhibitor. (All molarities are mean values based on pooled ram semen or on single ejaculates from three vasectomized rams.) Calculations from results in earlier publications indicated that one ejaculated ram spermatozoon contains about 3 amol of acrosin; thus the inhibitor: acrosin ratio in washed ram spermatozoa is approximately 1. One ml of ram semen contains, on average, 3 X 10(9) spermatozoa and not more than 0.8 ml of seminal plasma. This number of ejaculated spermatozoa would contain 6.3 nmol of inhibitor, while the same number of epididymal spermatozoa would contain 9.9 nmol of inhibitor. These values exceed the quantities of inhibitor present in 0.8 ml of normal seminal plasma (approximately 1.6 nmol) or in 0.8 ml of seminal plasma from vasectomized rams (approximately 2.3 nmol). We conclude that seminal plasma is not a major source of the acrosin inhibitor that can be recovered from washed ejaculated ram spermatozoa.     

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.     

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.     

The inhibition of acrosin by sterol sulphates

Four 3 beta-hydroxy-delta 5-steroid sulphates were found to be potent and specific inhibitors of the sperm acrosomal proteinase, acrosin. Two of these acrosin inhibitors, desmosteryl sulphate and cholesteryl sulphate, occur naturally in spermatozoa. Desmosteryl sulphate, an inhibitor of the in-vitro capacitation of hamster spermatozoa, has a Ki of 3.5 x 10(-6) M for the inhibition of acrosin. The mechanism of inhibition of sperm capacitation by sterol sulphates is probably due to their inhibition of acrosin.     

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

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

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.     

Effect of aryl 4-guanidinobenzoates on the acrosin activity of human spermatozoa

Certain aryl 4-guanidinobenzoates (AGs; inhibitors of proteinases, including the sperm enzyme acrosin) have been shown to be more potent vaginal contraceptives in rabbits and less toxic than nonoxynol-9, the active ingredient of most marketed vaginal contraceptive formulations. To determine if these AGs can contact sperm and inhibit acrosin when mixed with the entire human ejaculate for a short period of time (roughly imitating clinical conditions), the inhibitors were added to semen at various concentrations for 2 min, after which the seminal plasma and unbound inhibitor were removed from the sperm by Ficoll centrifugation. Subsequently, the total arginine amidolytic activity of the spermatozoa was determined spectrophotometrically after a combined treatment that resulted in extraction, proacrosin activation, and reaction with substrate. Dose-response curves were prepared. All AGs studied were effective inhibitors of the amidolytic activity under these conditions, with ED50 values (the dose levels at which half of the acrosin associated with 10(6) sperm is inhibited) ranging from 10(-5) to 10(-7) M. To determine the effect on the proteolytic activity of individual spermatozoa, the experiment was repeated with 4'-acetamidophenyl 4-guanidinobenzoate (AGB), and the protease released from the sperm was measured by the gelatin-plate assay. The inhibition results were similar to those obtained by extraction of the spermatozoa and measurement of amidolytic activity. Thus, when mixed with the human ejaculate, AGs interact rapidly with spermatozoa to inhibit both their arginine amidolytic and proteolytic activity (probably due primarily or only to inhibition of acrosin) and remain bound even after removal of the seminal plasma. These data encourage further study of the compounds for contraceptive purposes.     

Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.     

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.     

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.     

Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes.

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.     

Studies on ram acrosin. Fluorimetric titratiion of operational molarity with 4-methylumbelliferyl p-guanidinobenzoate.

1. Titration in sodium barbiturate buffer of acrosin, a serine proteinase from sperm acrosomes, with the ester substrate 4-methylumbelliferyl p-guanidinobenzoate gave rise to an incomplete 'burst' of 4-methylumbelliferone. Studies of the effects on the reaction of activators of acrosin (Ca2+, water-miscible solvents) showed that titrations carried out in barbiturate buffer containing 1M-CaCl2 and diluted with 0.2 vol. of dimethylsulphoxide produced a rapid quantitative burst within 4 min at 20 degrees C. 2. The net post-burst production of 4-methylumbelliferone was neglibible because (a) the acyl-enzyme was very stable, and (b) the slow post-burst formation of 4-methylumbelliferone (turnover of acyl-enzyme) was virtually equal to the slow photolytic destruction of 4-methylumbelliferone that was liberated during the burst. 3. The standard procedure permits titrations of 20-100pmol of acrosin, i.e. amounts normally taken for conventional rate assays, and with these amounts the impurities present in crude enzme fractions did not interfere. The burst was judged to be quantitative on the basis of comparisons with titrations of acrosin with p-nitrophenyl p'-quanidinobenzoate. 4. The burst reaction of trypsin with the 4-methylumbelliferyl ester was inhibited by high Ca2+ concentrations and by dimethyl sulphoxide. 5. The association and dissociation of complexes of both acrosin and trypsin with protein-type inhibitors (Kunitz pancreatic trypsin inhibitor and a spermatozoal acrosin inhibitor) are rather slow. It is thus possible, in certain cases, to use the ester to titrate both total enzyme in an inhibitor-enzyme mixture and net enzyme, i.e. the stoicheiometric excess of enzyme over inhibitor.     

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

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

The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro

In a series of experiments the influence of the trypsin inhibitors aprotinin (Trasylol) and TLCK (N-p-tosyl-L-lysin chloromethyl ketone) on the gelatinolytic activity of acrosin and motility of rabbit spermatozoa was tested. Ejaculated, highly motile spermatozoa were washed in Brackett-Medium. 12.5 to 1000 microns Aprotinin and 50 to 1000 micrograms TLCK, respectively, were added to samples of 1 ml sperm suspension: the specimens were incubated at 37 degrees C. Increasing aprotinin concentrations reduced the gelatinolytic activity of acrosin and the sperm incubation at a concentration of 1000 micrograms Aprotinin/ml sperm. Spermatozoa in all TLCK specimens were entirely immotile 1.5 hours after incubation. The gelatinolytic activity of acrosin was obviously not inhibited at any TLCK concentration. These results suggest that, under these experimental conditions, aprotinin and TLCK may impair primarily the motility spermatozoa.     

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.     

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.     

Determination of acrosin amidase activity in equine spermatozoa

Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (microIU acrosin/10(6) sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r(2) = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 +/- 49 microU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.