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.     

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.     

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.     

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.     

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.     

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.     

Benzamidine as an inhibitor of proacrosin activation in bull sperm.

Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.     

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)     

Purification of bovine and human acrosin

Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but not detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mM HCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mM benzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7-10 U of acrosin activity, about 2.5 U (approximately 20 micrograms of protein) could be obtained in a pure state.     

Proacrosin/acrosin activity during spermiohistogenesis of the bull

Abstract. 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.     

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.     

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.     

Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa

Short-term liquid storage of turkey semen is of great interest in the management of turkey reproduction due to the extensive use of artificial insemination. This study examined changes in DNA fragmentation (using a comet assay), sperm motility characteristics (using computer-aided sperm analysis), and amidase activity (using a colorimetric assay) of turkey sperm stored for 24 and 48 h. In addition we found that turkey spermatozoa contain besides acrosin, additional two serine proteinases of molecular weight of 34 and 42 kDa. We found that, after 48 h of liquid storage, decreases in sperm motility characteristics and increases in amidase activity and DNA fragmentation occurred. An increase of amidase activity was found after 24h. Decreases in sperm motility and increase in DNA fragmentation were found after 48 h of storage. These data suggest that a decrease in turkey sperm quality during short-term storage is related to disturbances to the acrosome, presumably related to premature activation of acrosomal serine proteinases, and to a lesser extent a decrease in sperm motility characteristics and damage of sperm DNA.     

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.     

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

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.     

Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum)

Spermatozoa of paddlefish and sturgeon fishes (Acipenseriformes), unlike teleost fish, have an acrosome. The objectives of this study were to characterize acrosin-like activity of cryopreserved sperm of paddlefish (Polyodon spathula) and to test and compare stability of paddlefish acrosin-like activity with that of lake sturgeon and bull spermatozoa. Mean acrosin-like activity of cryopreserved paddlefish sperm was 0.372 +/- 0.067 microU/10(6) spermatozoa. This activity was 79% higher in the whole semen than in spermatozoa. Highest activity was recorded at pH 8.0 and 8.5. Triton X-100, zinc ions and 4'-acetamidophenyl 4-guanidinobenzoate (AGB) inhibited the activity. Amidase activity was also inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TLCK at concentrations of 0.1 and 1.0 mM gave a significant decrease in activity of 19 and 61%, respectively. However, TPCK significantly inhibited amidase activity (by 19%) only at concentration 1.0 mM. After acidification and 60 min incubation at 4 degrees C of sperm suspensions only 4% of the activity was retained. A similar phenomenon was observed in the case of lake sturgeon but not bull sperm. These results suggest that trypsin-like activity of Acipenserid fish resembles rather fish trypsin that mammalian one. In frozen-thawed paddlefish sperm a minute chymotrypsin-like activity was also indicated, when GPNA was used as substrate. This activity amounted to 0.0415 +/- 0.0138 microU/10(6) spermatozoa and was 18% of total amidase activity. This suggests that chymotrypsin-like activity may also be present in paddlefish spermatozoa.     

Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

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.     

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.