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.     

Effects of season and breed on sperm acrosin activity and semen quality of boars

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.     

Evaluation of the effect of butyl p-hydroxybenzoate on the proteolytic activity and membrane function of human spermatozoa

The inhibition of the proteolytic activity of acrosin in human spermatozoa by butyl p-hydroxybenzoate was assessed by the gelatin substrate film method. Compared with a typical acrosin inhibitor, TLCK, the inhibitory activity of butyl p-hydroxybenzoate to acrosin was much more effective (20 times) than that of TLCK, proving that butyl p-hydroxybenzoate was a potent acrosin inhibitor. The effect of butyl p-hydroxybenzoate on membrane function of human spermatozoa was evaluated using a sperm-tail hypoosmotic swelling test and supravital stain method. A good correlation (r = 0.92) was observed between the % spermatozoa with normal membrane function and the % live spermatozoa after treatment of the spermatozoa with butyl p-hydroxybenzoate for 1 min, indicating that the death of spermatozoa caused by butyl p-hydroxybenzoate is probably due to impairment of sperm membrane function. Both the inhibitory effect on acrosin and the adverse effect on membrane function suggest that butyl p-hydroxybenzoate could be developed as a new vaginal contraceptive.     

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.     

The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations.

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.     

Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization.

When denuded ram spermatozoa were suspended in weakly buffered 0.25M sucrose, the acrosin remained bound to the acrosomal membranes of the sperm heads. Media containing CaCl2 caused complete solubilization of the enzyme. Effects of acrosin inhibitors on soluble and bound enzyme were studied in Tris HCl(pH 8.2) containing sucrose. Denuded spermatozoa were used as a preparation of bound acrosin. Trasylol (Kunitz basic pancreatic trypsin inhibitor) acted more strongly on bound scrosin than on soluble acrosin, but soya-bean trypsin inhibitor acted more strongly on soluble acrosin. At concentrations 0.5 - 2.0muM, the inhibitors isolated from ram acrosomes and from ram seminal plasma inhibited soluble acrosin but had negligible effects on bound acrosin. However, bound acrosin was sensitive to high concentrations of the acrosomal inhibitor. The two forms of acrosin were inhibited to about the same degree by p-aminobenzamidine and also by Tos-Lys-CH2Cl. It is proposed that membrane-bound acrosin is the form that functions in penetration of the zona pellucida, and that a role for acrosin inhibitors is suppression of an antifertility effect of soluble acrosin on mammalian eggs. This hypothesis is supported by 1) the results of work on the impaired fertilizing capacity of rabbit spermatozoa that have been treated with acrosin inhibitors, 2) the anti-fertility effects on hamster eggs of solutions of acrosin and of bovine trypsin, and 3) the results in this paper.     

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.     

Distribution of a seminal plasma-associated protein kinase inhibitor in normal, oligozoospermic, and vasectomized men

Human sperm-free seminal plasma contains an inhibitor, which is protein in nature, of the histone kinase present in seminal plasma. Since protein kinase inhibitors have been observed to be present in spermatozoa, the objective of the present study was to determine whether this seminal plasma-associated enzyme inhibitor originates from the sperm, or whether it is a component of accessory secretion(s) comprising the seminal plasma. Sperm-free seminal plasma from normospermic (greater than 20 X 10(6) sperm/ml), oligozoospermic (less than or equal to 20 X 10(6) sperm/ml), and vasectomized donors was obtained, and inhibitor-enriched fractions were prepared by (NH4)2SO4 fractionation and gel filtration. Contamination of the sperm-free seminal plasma by spermatozoa or spermatozoan components was negligible as assessed by light microscopy, polyacrylamide gel electrophoresis, and measurement of the activity of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Specific (inhibitory units/mg protein) and total inhibitory activities were determined in each of the donors by constructing linear inhibition curves using various concentrations of inhibitor. The results were correlated with the initial sperm concentration. There was no apparent relationship between the amount of inhibitory activity present and the initial sperm concentration. The histone kinase inhibitor also did not appear to be associated with testicular or epididymal secretions since it was observed in the seminal plasma of vasectomized donors. It is concluded that this inhibitor of histone kinase originates from the accessory secretions comprising the human ejaculate.     

Acrosin activity is a good predictor of boar sperm freezability

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze–thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze–thawing procedures took place, i.e., in the refrigeration step at 15 °C.     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

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.     

Intra-acrosomal inhibition of boar acrosin by synthetic proteinase inhibitors

Thirteen serine proteinase inhibitors of the guanidine, monoamidine and diamidine type were tested for their ability to inhibit the proteinase acrosin present in the acrosome of ejaculated and capacitated boar spermatozoa. All compounds studied proved to be potent in-vitro inhibitors of acrosin. Inhibition constants (Ki) in the range of 1.2 x 10(-7) to 6 x 10(-8) M were found for the reversible inhibitors. The intra-acrosomal inhibition of acrosin was assessed by the gelatin substrate film method: 2 guanidinobenzoates, one monoamidine and one diamidine derivative proved to inhibit acrosin completely in intact spermatozoa. Intravenous injection of 6-amidino-2-(4-amidinophenyl)-indole had no effect on fertilization, but application of 4-nitrophenyl-4-guanidinobenzoate in a vaginal suppository gave a 50% reduction of fertilization.     

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.     

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.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

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.     

Role of fluid from seminal vesicles and coagulating glands in sperm transport into the uterus and fertility in rats.

The relationship between the quantity of seminal vesicle secretion in the ejaculate, the percentage of spermatozoa reaching the uterus and fertility was studied in rats. Different portions of seminal vesicles were removed from male rats; 15 min after coitus (day 0), the numbers of spermatozoa in the uterus and vagina were counted and the vaginal plug characteristics were noted. Fertility was evaluated by the number of fetuses on day 14. A gradual decrease in the percentage of spermatozoa in the uterus was positively related to the reduction in seminal vesicle secretion, estimated by plug weight. This decline was not caused by a delay in sperm transport to the uterine lumen and the results suggested that the spermatozoa that fail to enter the uterus in the first minutes after coitus never enter. The vaginal plug weight, which is related to the seminal vesicle weight, and the position of the plug, which must be firmly lodged into the cervical opening, seem to be the most important conditions for promoting the rapid passage of spermatozoa into the uterus. When the seminal vesicles were partially removed, the plug was not tightly lodged and formed a 'cup' filled with spermatozoa. The number of fetuses did not show a close correlation with the quantity of seminal vesicle secretion. Studies of males in which the seminal vesicles had been removed indicated that a normal number of fetuses can be obtained despite low numbers of spermatozoa reaching the uterus. Ablation of the coagulating glands showed that, when there is no vaginal plug, no spermatozoa reach the uterus and fertility is suppressed. Nevertheless, the complete removal of coagulating glands is difficult; when small portions of these glands remain, the vaginal plug is formed and then fertility is achieved.     

Inhibition of fertilization in the rabbit by anti-acrosin antibodies

Rabbit acrosin was purified from detergent extract of epididymal spermatozoa by molecular-sieve Chormatography (Sephadex G-75) and Priflavin-Sepharose affinity chromatography. Affinity Purified rabbit acrosin (Specific activity = 63.7 U Per mg Protein) exhibited one major band (M_r = 36,000) on SDS-polyacrylamide-slab gel electrophoresis. Sheep werweantibodies, Preared against acid-glycerol-stabilized acrosin, were Fractionated sequentitally with 50 and 33% ammonium sulfate. Anti-acrosin immunoglobulins were specific for the acrosome as evidenced by indirect fluorescence lebeling, and they inhibited acrosin's proteolytic activity when essayed for their effect on fertilization invivo, antiacrosin-treated sperm produced the lowest persent fertility (7.1%) followed by preimmune y-globulins(69%), Krebs-ringer hosphate-glucose-serum(96.4%) Analysis of antibody-treated sperm revealed no significiant chang in their motility or agglutination patterns. Antiacrosin antibodies, therefore, can effectivily neutrlize acrosin and inhibit fertilization when placed at the site of sperm-egg interaction in vivo.