Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells.

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10 mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20 mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

[Value of sperm acrosin activity detection in selecting the method of assisted reproduction for patients with unexplained infertility].

OBJECTIVE: To investigate the clinical significance of sperm acrosin activity detection in selecting the method of assisted reproduction for patients with unexplained infertility (UI). METHODS: This retrospective study included 49 UI couples treated by IVFET (49 cycles) after three failures in intrauterine insemination (IUI) and another 95 couples with uterine tube obstruction (UTO) treated by IVF (131 cycles). We analyzed the laboratory data, clinical outcomes and sperm acrosin activity in the two groups of patients. According to the level of sperm acrosin activity of the males, we further divided the UI patients into two subgroups, a < 36 IU/106 sperm group (20 cycles) and a ≥36 IU/106 sperm group (29 cycles), and compared the fertilization rates between the two groups. RESULTS: Compared with UI couples treated by IVFET, the UTO couples treated by IVF had a significantly lower rate of fertilization (67.0% vs 76.4%, P < 0.05) and a higher rate of remedial intracytoplasmic sperm injection (ICSI) (20.4% vs 6.1%, P < 0.05), but showed no statistically significant differences in the rates of MII oocytes, available embryos, highquality embryos, implantation, and clinical pregnancy from the latter group (P >0.05). The sperm acrosin activity was remarkably lower in the UI than in the UTO patients (36.03 vs 61.98 IU/106, P < 0.01), and so was the fertilization rate in the < 36 IU/106 than in the ≥36 IU/106 sperm subgroup (47.7% vs 80.3%, P < 0.01). CONCLUSIONS: The low fertilization rate caused by decreased sperm acrosin activity may be the main cause of infertility and the potential factor of UI. When sperm acrosin activity is < 36 IU/106 sperm, IVF plus shortterm fertilization by remedial ICSI should be preferred to IUI.

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

Isolation of the outer acrosomal membrane from bull sperm.

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.

Acrosin activity as a potential marker for sperm membrane characteristics in unexplained male infertility.

OBJECTIVE: To evaluate sperm membrane system integrity in unexplained infertile male subjects with three consecutive conception failures on IUI even though semen clinical parameters were normal. DESIGN: Prospective study. SETTING: Medical biotechnology laboratory, School of Medical Science and Technology IIT Kharagpur, India. PATIENT(S): Twenty-nine patients with unexplained infertility, 17 normal proven-fertile healthy donors, and 21 infertile males with low motility but with other semen parameters remaining normal. INTERVENTION(S): Semen samples were collected from unexplained infertile patients as well as from healthy fertile donors after abstinence of 3-5 days and were analyzed according to World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Release of 5'-nucleotidase (plasma membrane marker), lactate dehydrogenase (mitochondrial marker) and free acrosin, proacrosin, and total acrosin (acrosomal membrane marker). RESULT(S): Plasma membrane integrity and respiratory activity of sperm cells were comparable in all three groups. The proacrosin-acrosin system was adversely affected in unexplained infertile subjects despite high sperm motility. CONCLUSION(S): Total acrosin activity may be considered as a sensitive biochemical marker for clinical evaluation of unexplained infertility in males.

Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men.

In this experimental prospective study, we aimed to analyze the effect of transient scrotal hyperthermia on the male reproductive organs, from the perspective of sperm parameters, semen plasma biochemical markers, and oxidative stress, to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis. Two groups of volunteers (10 per group) received testicular warming in a 43°C water bath 10 times, for 30 min each time: group 1: 10 consecutive days; group 2: once every 3 days. Sperm parameters, epididymis and accessory sex gland function, semen plasma oxidative stress and serum sex hormones were tested before treatment and in the 16-week recovery period after treatment. At last, we found an obvious reversible decrease in sperm concentration (P = 0.005 for Group 1 and P= 0.008 for Group 2 when the minimums were compared with baseline levels, the same below), motility (P = 0.009 and 0.021, respectively), the hypoosmotic swelling test score (P = 0.007 and 0.008, respectively), total acrosin activity (P = 0.018 and 0.009, respectively), and an increase in the seminal plasma malondialdehyde concentration (P = 0.005 and 0.017, respectively). The decrease of sperm concentration was greater for Group 2 than for Group 1 (P = 0.031). We concluded that transient scrotal hyperthermia seriously, but reversibly, negatively affected the spermatogenesis, oxidative stress may be involved in this process. In addition, intermittent heat exposure more seriously suppresses the spermatogenesis compared to consecutive heat exposure. This may be indicative for clinical infertility etiology analysis and the design of contraceptive methods based on heat stress.

Brefeldin A and mannose 6-phosphate regulation of acrosomic related vesicular trafficking.

Acrosomal biogenesis represents a unique system for the molecular analysis of the various processes involved in vesicular membrane transport. To assess the basic membrane trafficking mechanisms used in spermatids, we have used two fluorescent lipid compounds that label: a) the Golgi and Golgi-derived vesicles (C5-DMB-Cer), and b) endocytic vesicles (FM4-64). Incubation of mouse testicular germ cells at 33 degrees C for 1.5 h with C5-DMB-Cer revealed that C5-DMB-Cer labeling is localized in the Golgi and acrosome of early-maid round spermatid stages, with no labeling of the acrosome in late round spermatid stages. Culturing germ cells with FM4-64 for 1.5 h at 33 degrees C, showed that FM4-64 labeling in spermatids was localized in endocytic vesicles and Golgi of early-mid round spermatid stages, whereas in a small population of late round spermatid stages, FM4-64 was also localized in the apex region of the acrosome. Incubation with brefeldin A (BFA) (5 micrograms/ml) inhibited the distribution of C5-DMB-Cer and FM4-64 to the acrosome, however, it did not affect the localization of acrosin-an acrosome-specific protein-indicating that there was no apparent acrosome disassembly in the presence of BFA. Localization of FM4-64 in late round spermatids in the presence of 2.5 mM mannose 6-phosphate was found in endocytic vesicles and the Golgi, but not the acrosome. These results show that there are at least two sources of vesicular transport to the acrosome derived from the Golgi and plasma membrane.

Dansylalanyllysylchloromethyl ketone as a fluorescent probe for localization of acrosin activity in boar and human spermatozoa.

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. These results are compatible with the hypothesis that, under the experimental conditions used, active acrosin is tightly bound to the IAM and that the "specificity site" of the acrosin-active center is probably of a highly hydrophobic nature.

The interaction of ram proacrosin and acrosin forms with antiserum raised against ram m beta-acrosin.

An antiserum was raised in rabbits to pure m beta-acrosin, the stable form of active acrosin from ram spermatozoa. By use of immunoprecipitation with Protein A-bearing Staphylococcus aureus cells, so as to react antigen with antibody and isolate the complex rapidly, it could be demonstrated that the anti-m beta-acrosin cross-reacted strongly with native proacrosin, with a degenerate proacrosin form, and with at least one form of active acrosin other than m beta-acrosin. Sperm proteins unrelated to acrosin were not recognized. It is concluded that rabbit anti-m beta-acrosin will react specifically with all the major ram acrosin forms; the antiserum can therefore be used with confidence in immunocytochemical studies to locate both proacrosin and acrosin in ram spermatozoa.

The organized distribution of acrosomal proteinase within the acrosomes of rabbit spermatozoa.

Acrosomal proteinase was found to be present in a highly organized distribution in the acrosomes of rabbit spermatozoa, using cytochemistry. This distribution consists of at least six linear loops of evenly spaced proteinase granules, which run diagonally across the flat side of the sperm head in a criss-crossing pattern. Two additional loops may be present, one encircling the tip of the spermatozoon in the region of the acrosome reaction, and the other at the posterior end of the acrosome. The results also seem to indicate that the enzyme granules are associated with the outer, and not the inner, acrosomal membrane.

Total acrosin activity correlates with fertility potential after fertilization in vitro.

OBJECTIVE: To evaluate the possible relationship between total acrosin activity in spermatozoa and fertility potential after fertilization in vitro. DESIGN: Total acrosin activity of spermatozoa was measured in 101 in vitro fertilization (IVF) cases by an observer unaware of fertilization and cleavage results. SETTING: University Hospital is a tertiary referral center offering a government supported In Vitro Fertilization Programme. PARTICIPANTS: Participants were couples undergoing IVF. INTERVENTIONS: A miniature assay measured total acrosin activity in the semen sample used for IVF. OUTCOME MEASURES: The proportion of mature oocytes fertilized, the proportion of mature oocytes transferred, and fertilization of at least one mature oocyte were considered outcomes with fertility potential. RESULTS: Total acrosin activity correlated with both the proportion of mature oocytes fertilized and the proportion of mature oocytes that were transferred as cleaving embryos. Total acrosin activity was higher in cycles when one or more mature oocyte fertilized compared with cycles with failed fertilization of all mature oocytes. CONCLUSIONS: The likelihood ratio for subnormal results indicates that measurement of total acrosin activity is a fair test of the fertilizing capacity of sperm.

Gelatin-substrate film technique for detection of acrosin in single mammalian sperm.

Sperm acrosin proteolytic activity in single sperm can be detected by a protein-free halo on a gelatin-substrate film. With current techniques, halos have variable sizes and are often absent because of unevenness of the hand-spread gelatin-substrate film. We prepared gelatin-substrate films with a coating machine. Using these films, halos were formed uniformly throughout the gelatin-substrate films in the vicinity of single mammalian sperm. The level of acrosin activity as determined by halo diameters was human greater than dog greater than squirrel monkey greater than mouse greater than rat. This simple and reproducible technique may be used to diagnose infertility due to decreased acrosin activity, as a screening method for identifying compounds with male sterility effects, and for identifying agents with developmental and/or genetic effects.

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration.

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.

Effect of abstinence on sperm acrosin, hypoosmotic swelling, and other semen variables.

OBJECTIVE: To compare the variability of sperm acrosin and hypoosmotic swelling to the more standard semen variables in relationship to controlled periods of sexual abstinence using a defined group of men. DESIGN: Ten men abstained sequentially for 1, 2, 3, 4, 5, and 10 days and produced an ejaculate after each time period. The ejaculate variables measured at each time point were sperm acrosin, hypoosmotic swelling, volume, sperm number, sperm concentration, sperm motility, sperm morphology, pH, and white blood cells (WBCs). Comparisons were performed between the values obtained at each abstinence period. RESULTS: The percentage of hypoosmotically reactive spermatozoa did not vary significantly with the abstinence period. Sperm acrosin remained similar up to 5 days of abstinence but decreased almost twofold after 10 days of abstinence. The sperm volume and concentration increased gradually with the length of abstinence, being approximately twofold higher after 10 days of abstinence than after 1 day of abstinence. The total sperm number increased about fourfold from 1 day of abstinence to 10 days of abstinence. The percent normal sperm forms tended to increase until 5 days of abstinence but decreased after 10 days of abstinence. The WBC count showed only a small increase with longer abstinence periods. The pH remained essentially the same. CONCLUSIONS: The length of abstinence affects the various semen variables differently. An abstinence period of up to 10 days does not alter the hypoosmotic swelling test results. However, the sperm acrosin values decrease after prolonged abstinence so that the abstinence period needs to be taken into consideration when performing this assay.

The occurrence and identification of round spermatids in the ejaculate of men with nonobstructive azoospermia.

OBJECTIVE: To evaluate the occurrence of round spermatids in the ejaculate of men suffering from nonobstructive azoospermia as a prerequisite for the therapeutic use of round spermatid injection into oocytes. DESIGN: Prospective study. SETTING: University-based diagnostic and research laboratory. PATIENT(S): Azoospermic men previously thought to be definitively infertile whose cases were revisited in view of the novel method of fertilization by round spermatid injection into oocytes. INTERVENTION(S): Smear preparations of cells from patients' ejaculates were stained by three different methods: Papanicolaou, fluorescein-labeled Pisum sativum agglutinin binding, and antiacrosin antiserum immunolabeling. MAIN OUTCOME MEASURE(S): Search for round spermatids in smear preparations of cells from azoospermic ejaculates. RESULT(S): Of 124 azoospermic men examined, 36 (69%) had round spermatids in the ejaculate. the quantity of round spermatids was not related to serum FSH concentration. CONCLUSION(S): Variable quantities of round spermatids can be detected in the ejaculate of most patients with nonobstructive azoospermia. High serum FSH levels do not predict the absence of spermatids.

A study of sperm acrosin in patients with unexplained infertility.

Radioimmunoassay (RIA) and the fluorometric enzyme method (FEM) were used to study sperm acrosin levels of semen obtained from 13 men of known fertility (group I), 14 male partners of unexplained infertile couples (group II), 4 men among unexplained infertile couples who fathered a child during evaluation and follow-up (group III), and 13 oligospermic males (group IV). Using analysis of variance with the Student-Neuman Keuls post hoc comparisons, we found statistically significant differences between acrosin levels of groups I and II, groups I and IV, groups II and III, and groups III and IV (P less than 0.01) for each comparison and P less than 0.05 for entire experiment). Although RIA was found to be superior to the fluorometric technique, there was excellent correlation between the two methods (r = 0.7; P less than 0.001). This study suggests an association between low sperm acrosin levels and infertility.

Is spermatozoan acrosin a predictor of fertilization and embryo quality in the human?

OBJECTIVE: To investigate whether acrosin is a more reliable criterion than conventional parameters for assessing semen samples. DESIGN: Total acrosin was estimated biochemically in semen samples obtained for routine screening for infertility and for IVF-ET procedures. SETTING: Assisted Conception Unit, St. James's University Hospital, Leeds, United Kingdom. PATIENTS: Four hundred sixty-three couples being investigated for causes of infertility, and 132 couples undergoing IVF-ET for any indication except antisperm antibodies between 1990 and 1991 were included in the study. INTERVENTIONS: Semen samples were collected as part of routine investigation. Samples from men with consistently high viscosity were collected in alpha-chymotrypsin. MAIN OUTCOME MEASURES: Total spermatozoan acrosin in motile spermatozoa and motile spermatozoan density in couples being assessed for IVF-ET and fertilization and embryo quality in those receiving treatment are considered. RESULTS: Total acrosin levels were less variable (within subject) than either total or motile spermatozoan concentration at ejaculation. Although severely oligozoospermic ejaculates had the lowest levels of total acrosin, overall, there was no significant correlation of spermatozoan concentration between total acrosin levels and percentage fertilization. CONCLUSIONS: Total spermatozoan acrosin activity correlates positively with fertilization rates but not with spermatozoan count. Motile spermatozoan density for insemination may be adjusted to achieve > 7.5 microIU acrosin per oocyte, without compromising fertilization or further embryo development to blastocysts in vitro.

The combination of testosterone undecanoate with tamoxifen citrate enhances the effects of each agent given independently on seminal parameters in men with idiopathic oligozoospermia.

OBJECTIVE: To evaluate the effects of combined tamoxifen citrate and T undecanoate treatment on seminal parameters in men with idiopathic oligozoospermia. DESIGN: Prospective randomized clinical study. SETTING: A state hospital tertiary clinic. PATIENT(S): Eighty oligozoospermic men were included in the protocol. INTERVENTION(S): Patients were randomized to receive placebo, T undecanoate (40 mg three times per day), tamoxifen citrate (10 mg two times per day), or T undecanoate plus tamoxifen citrate. RESULT(S): Tamoxifen citrate plus T undecanoate treatment produced a satisfactory improvement of total sperm number, motility, and functional sperm fraction after 3 and 6 months. Comparisons with other active treatment groups showed significantly higher increment values for motility and functional fraction, whereas aniline, acrosine, and free L-carnitine also were markedly better in the combination treatment group. CONCLUSION(S): These results indicate that the combination of tamoxifen citrate with T undecanoate not only improves significantly important seminal parameters but also compares favorably with the single treatments used. Therefore, this combination deserves a place as a first line of treatment in idiopathic oligozoospermia.

A simple, clinical assay to evaluate the acrosin activity of human spermatozoa.

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.