Proacrosin Activation Research Articles

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.

Activation of boar proacrosin is effected by processing at both N-and C-terminal portions of the zymogen molecule

A mixture of 55 and 53 kDa boar proacrosins was autoactivated at pH 8.5 to produce a 43 kDa intermediate form and a 35 kDa mature acrosin, and each of four forms of (pro)acrosins was isolated. Analysis of the N-terminal sequences of the two proacrosins indicated the existence of a segment corresponding to the acrosin light chain at the N-terminal end of the zymogen. Two N-terminal sequences identical with those of the light and heavy chains were found in the intermediate form and mature acrosin. The proacrosins and the intermediate contained many more proline residues than the mature enzyme. These results indicate that the activation of boar acrosin zymogen is achieved by the removal of a C-terminal segment rich in proline residues and by the cleavage of the Arg 23Val 24 bond leading to the formation of the light and heavy chains.

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

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

Activation of proacrosin by a locally produced component of human follicular fluid

Components of human follicular fluid were separated on Sepharose 6B columns and the effects of different fractions on the conversion of pig proacrosin to acrosin were examined. A high-molecular-weight fraction (Mr greater than 3,000,000) of follicular fluid was a potent stimulator of this reaction. The proacrosin converting activity was absent in the corresponding fraction of blood serum. The acceleration of proacrosin activation was dependent on the concentration of material with proacrosin converting activity. The results indicate that human follicular fluid contains a high-molecular-weight component of local origin which is capable of accelerating proacrosin in a dose-dependent manner.

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.

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.

Studies on the rabbit proacrosin-acrosin system

A single molecular form ( M r = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 11721–11728) was initially converted by autoactivation into an acrosin ( M r = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities ( R m = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands ( R m values of 0.395 – 0.412 and 0.497 – 0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the M r 34,000 form of acrosin. This property was not shown by M r 68,000 acrosin. Initial acrosin ( M r = 68,000) was activated by divalent cations such as Ca 2+ and Mg 2+. The enzyme was inhibited by Zn 2+, Fe 2+, Hg 2+, and sulfhydryl blockers such as 5,5′-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn 2+ is involved in acrosomal stabilization. The initial rabbit acrosin ( M r = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

AbstractProacrosin 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.

Studies on acrosome labelling of mammalian spermatozoa by radioactive sugars.

The localization of glycoprotein synthesis and storage was studied during acrosome formation in guinea-pig using fine-structure autoradiography after (3H)-fucose incorporation. Three days after (3H)-fucose injection, labelling in spermatids was concentrated in the matrix of developing acrosomes, and it was evident that the fucosylation of acrosomal glycoproteins largely overshadowed the fucosylation of other spermatid glycoproteins. Acrosin labelling and its quantitative relation to labelling of other glycoproteins was examined in mature rabbit spermatozoa after incorporation of (14C)-fucose or (14C)-glucosamine during spermatogenesis. Cauda epididymis spermatozoa recovered 21 days after intratesticular application of (14C)-fucose or (14C)-glucosamine were analysed for acrosin specific labelling after acid extraction and gel filtration. In all the material examined, radioactivity was detected in the proacrosine fractions; radioactivity in purified proacrosin amounted to at least 2% of the total radioactivity in the epididymal sperm population. In addition to the peak with radioactive proacrosin, another radioactive peak in (14C)-glucosamine-labelled material was attributed to a glycoprotein intraacrosomal inhibitor of acrosin. It is concluded that (pro)acrosin (acrosin-inhibitor) complexes seem to contribute significantly to acrosomal glycoprotein labelling by radioactive sugars and that the distribution of these complexes may at least correspond to their cytochemically detectable component, acrosin. The superposition of the distribution of acrosin and of other acrosomal glycoproteins during acrosome reaction can be explained by the fact that the dispersal of most of the acrosomal content is linked to proacrosin activation.

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa.

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.

Proacrosin/acrosin activity during spermiohistogenesis of the bull

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

Isolation of rabbit testicular cathepsin D and its role in the activation of proacrosin

Cathepsin D was purified 900-fold with 30% recovery from rabbit testes using pepstatin bound Sepharose affinity chromatography. The enzyme is homogeneous as observed by acrylamide gel electrophoresis. The heat stable enzyme exhibits an apparent molecular weight of 42,000 with identical subunits of 20,000. Purified cathepsin D catalyses the conversion of proacrosin to acrosin.

The location of acrosin and proacrosin in ram spermatozoa.

Summary. Acrosin and its zymogen form proacrosin were located in various sperm fractions by biochemical and immunocytochemical techniques, using an anti-acrosin serum that cross-reacts strongly with proacrosin. If activation of proacrosin was prevented, the zymogen was associated almost entirely with the sperm heads, where it was confined to the anterior segment of the acrosome. Electron microscopy revealed that the acrosomal lumen of such heads remained full of matrix material, and the outer acrosomal membrane remained closely apposed to the other head structures. However, if activation had been allowed to take place, the resultant acrosin and the remaining proacrosin were associated with all sperm fractions and no specific location was observed. In these circumstances, the matrix material was almost entirely absent from the heads, and the outer acrosomal membrane, though usually still present, was only loosely attached. It is concluded that (1) neither acrosin nor proacrosin are truly membrane-bound; they behave as 'diffusible' or partly soluble proteins, although they display a non-specific affinity for cell membrane and other surfaces; (2) proacrosin is part of the acrosomal matrix material, and is not located specifically on the inner or the outer acrosomal membrane; (3) the matrix material plays an important mechanical role in stabilizing the position of the outer acrosomal membrane relative to the inner acrosomal membrane; it disperses during proacrosin activation.

Gossypol: an effective acrosin blocker.

The activation of proacrosin and the acrosin activity of boar ejaculated spermatozoa were studied in vitro over a wide range of gossypol concentrations and it was found that both activities were sensitive to gossypol inhibition. Acrosin, fruthermore, is by far the most gossypol sensitive enzyme among all enzymes reported. This antiacrosin effect of gossypol adds an extra advantage to its being used as a vaginal contraceptive.

Acrosin, proacrosin, and acrosin inhibitor of guinea pig spermatozoa capacitated and acrosome-reacted in vitro.

Guinea pig epididymal spermatozoa were either capacitated only, or capacitated and acrosomereacted in vitro. The spermatozoa were separated from the supernatant incubation media, the spermatozoa extracted, and the amount of acrosin, proacrosin, and acrosin inhibitor in the sperm extracts as well as in the supernatant solutions was measured. A dialyzable factor was present which inhibited proacrosin activation as well as acrosin activity, although the latter to a lesser degree, and was partially removed during the acrosome reaction. The total amount of acrosin (nonzymogen acrosin + proacrosin) in untreated, control spermatozoa was 3 to 5 times that in mouse and human spermatozoa. A reduction of �\�50% in the total amount of sperm acrosin occurred after induction of the acrosome reaction. Whether spermatozoa were untreated, capacitated, or acrosome-reacted, ‘\:90% of the acrosin present on the spermatozoa was in proacrosin form. The loss of acrosin activity after induction of the acrosome reaction was due to its release into the surrounding medium, and all of the released enzyme was in the nonzymogen acrosin form. The amount of acrosin released from control spermatozoa kept in buffer, which underwent the “false” acrosome reaction, was approximately the same as that released from “true” acrosomereacted spermatozoa. The total amount of acrosin released after induction of the acrosome reaction was never more than about 55% of the original amount present, even if more than 80% of the spermatozoa had undergone the acrosome reaction. Proacrosin activation apparently does not occur, or occurs only minimally, during in vitro capacitation of guinea pig spermatozoa, but approximately half is activated just before, during, or immediately after the spermatozoa show morphological changes in the acrosome, whether induced by sperm death or capacitation. Approximately the same small amount of acrosin inhibitor was released from capacitated spermatozoa as from noncapacitated spermatozoa kept in buffer that had undergone the “false” acrosome reaction. By contrast, a much higher amount of acrosin inhibitor was liberated from capacitated, ,sc:osome-reacted spermatozoa.

Stimulation of rhesus monkey (Macaca mulatta) proacrosin activation by oviduct fluid.

Pronase-resistant low molecular weight stimulators for the activation of proacrosin to acrosin were found in rhesus monkey oviduct fluid collected before, during and after ovulation, but the presence of high concentrations of acrosin inhibitors before and after ovulation partly masked the stimulation in unfractionated fluid. This low molecular weight fraction of oviduct fluid had no detectable esterase or amidase activity by itself, and the stimulating factors were sensitive to digestion by hyaluronidase and chondroitin ABC lyase and were presumed to be glycosaminoglycans. Heparin and hyaluronic acid had similar effects. The presence of soluble glycosaminoglycans at the site of fertilization suggests that they may have a role in capacitation and fertilization.

Acrosin, Proacrosin, and Acrosin Inhibitor of Human Spermatozoa: Extraction, Quantitation, and Stability

Ten different methods were evaluated to determine the treatment for obtaining maximal amounts of acrosin, proacrosin, and acrosin inhibitor (the acrosin system) from human spermatozoa by a single technique. Optimal results were obtained by extraction of the gametes with 10% glycerol at pH 2.8 for at least 12 hours. A single treatment by this technique provided approximately 95% of all available acrosin. The use of benzamidlne during extraction was essential for maximal recovery of proacrosin, i.e., to prevent zymogen activation. Benzamidlne did not affect the total amount of acrosin recovered. The levels of acrosin Inhibitor obtained did not vary with the method of treatment but were greatly influenced by the molecular pore size of the membrane used for dialysis of the extracts. An assay method for the simple determination of the components of the acrosin system is presented. Using the optimal extraction method and described assay technique, 42 pooled semen samples, each consisting of four to 14 ejaculates, were analyzed for the acrosin system. The total acrosin averaged 106 mlU/107 spermatozoa (S.D. = ±22 mlU/107 sperm). Almost all the acrosin was in the zymogen (proacrosin) form (93% ± 2%). After proacrosin activation, enough inhibitor was present to inhibit 94% ±5% of the acrosin. Acrosin and proacrosin were stable in spermatozoa kept in seminal plasma at room temperature for at least 6 hours. After solubilization, the enzymes were stable for at least one week when stored at pH 3.0,4 C, if the proacrosin was not activated to acrosin. After activation, the extracts were much less stable. Maximal conversion of proacrosin to acrosin occurred in 10 minutes when the extracts were incubated at pH 8.0 at room temperature. The presence of calcium ions severely retarded activation. The described extraction and assay techniques provide a standard method by which the acrosin system of spermatozoa can be measured, and may now be applied to the spermatozoa of infertile men to determine whether any abnormalities exist In the acrosin system of these gametes.

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.

The activation of proacrosin in spermatozoa from ram, bull and boar

Acrosin activity was estimated in fractions from washed ram, bull and boar spermatozoa that had been disrupted using a Stansted Cell Disruptor. When p-aminobenzamidine was included in the medium during disruption, all the acrosin (acrosomal proteinase, EC 3.4.21.10) was recovered as its inactive zymogen form, proacrosin. But if spermatozoa were damaged before disruption, or were disrupted in the absence of p-aminobenzamidine, considerable amounts of active acrosin were detectable. It was concluded that conversion of proacrosin to acrosin takes place in spermatozoa only after the acrosome has been ruptured. In a sucrose medium, all the proacrosin was bound to the sperm heads. Conversion to acrosin took place readily with all components in a bound state. Using ram sperm heads, the conversion was found to be relatively insensitive to pH, proceeding rapidly above pH 6.5; the rate of conversion was not affected by physiological levels of Ca 2+, Mg 2+ or Zn 2+, although elevated ionic strength caused a solubilization of the acrosin activity and some slowing of the rate. Electrophoretic analysis revealed that several active forms of acrosin were involved, but the final product was a single stable form. Final levels of the active acrosin (expressed as μmol N-α- benzoyl- l-arginine ethyl ester utilised/min per 10 9 heads) were: ram 26.2; bull, 15.9; boar, 133.8. But active site titration revealed that these different levels were not reflected in the numbers of active enzyme molecules on the sperm head; boar acrosin appears to be about three times more active towards benzoyl-arginine ethyl ester than do the acrosins from the other species.

The activation of proteolysis in the acrosome reaction of guinea-pig sperm

The divalent metal cation ionophore A23187 rapidly induces a normal acrosome reaction in a population of guinea-pig sperm suspended in calcium medium. In the course of the acrosome reaction, proacrosin, the zymogen precursor of the protease acrosin, is activated. Although the acrosome reaction causes exocytosis of the acrosomal contents, 'soluble' acrosin is not released in significant amounts until well after the sperm population as a whole has undergone an acrosome reaction. This suggests that proacrosin is stored within the acrosome in an insoluble form and that exocytosis of the acrosomal contents in the acrosome reaction is insufficient, by itself, to cause its immediate dissolution. Electron micrographs of sperm undergoing an A23187-induced acrosome reaction in the presence of the acrosin inhibitors benzamidine, p-amino-benzamidine and phenylmethylsulphonyl fluoride show that the acrosome reaction proceeds normally but that dispersal of the acrosomal contents is inhibited. These morphological changes are, for the most part, below the limit of resolution of the light microscope and using light microscopy to assess whether an acrosome reaction has taken place, it can be mistakenly inferred that the reaction itself is inhibited by the acrosin inhibitors. The inhibition of the dispersal of the acrosomal contents by acrosin inhibitors suggests that acrosin activity is important in solubilizing acrosin. These experimental observations, taken with the evidence that the acrosome reaction is a response to an increase in intracellular free calcium, have been taken as the basis of a proposal for the mechanism of proacrosin activation in the acrosome reaction.