Effect of dialysis on the proacrosin/acrosin system and motility of turkey (Meleagris gallopavo) spermatozoa during liquid storage.

1. The effect of dialysis on the proacrosin/acrosin system and motility of turkey spermatozoa were examined after 24 and 48 h of liquid storage at 4°C. 2. Fifteen pools of semen diluted in extender were dialysed against Clemson Turkey Semen Diluent (dialysed semen) or stored in aerobic conditions (undialysed semen). Semen quality was assessed by measuring spermatozoa motility, amidase activity of spermatozoa suspension, spermatozoa extract and seminal plasma and anti-trypsin activity of seminal plasma. 3. Extracted amidase activity of dialysed semen was lower than undialysed by 28%. Higher values for speed parameters of spermatozoa were found in dialysed semen in comparison to undialysed, for example, 81.6 µm/s versus 75.0 µm/s for straight-line velocity (VSL), 114.7 µm/s versus 110.3 µm/s for curvilinear velocity (VCL) and 86.6 µm/s versus 79.8 µm/s for average path velocity (VAP). 4. It was concluded that dialysis caused lower amidase activity of spermatozoa and increased speed parameters of progressively motile turkey spermatozoa during storage. Lower extracted amidase activity of dialysed semen reflected better membrane integrity of dialysed semen and suggests that the proacrosin/acrosin system of dialysed spermatozoa is less susceptible to activation compared to undialysed semen.

Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica).

An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45  kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45  kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.

Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm.

We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation.

Assessment of released acrosin activity as a measurement of the sperm acrosome reaction.

AIM: To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction (AR). METHODS: Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization (WHO) criteria. Calcium ionophore A23187 and progesterone (P4) were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA). The percentage of sperm undergoing AR (AR%) was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. RESULTS: The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 (P < 0.001). There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining (r=0.916, P < 0.001). CONCLUSION: Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR.

Interactions between zona pellucida glycoproteins and sperm proacrosin/acrosin during fertilization.

Fertilization is one of the most specific and carefully regulated cell-cell interactions in the animal body and is determined to a large extent by compatibility between ligand and receptor molecules on the surface of each gamete. On the zona pellucida (ZP), sperm receptor activity is associated with glycoproteins ZP3 (primary receptor for acrosome-intact sperm) and ZP2 (secondary receptor for acrosome-reacted sperm) but their complementary binding proteins on sperm are less well defined. In this communication we review the evidence for proacrosin as a secondary ZP binding protein. Proacrosin/acrosin binds non-enzymically to ZP glycoproteins. Binding is a strong ionic interaction between polysulphate groups on ZP glycoproteins (probably on their carbohydrate moieties) and basic residues on the surface of proacrosin. The stereochemistry of the reactants is crucial and determines to a large extent the affinity of binding. Site-directed mutagenesis and a 3D-structural analysis of boar and ram acrosin have identified 2 clusters of basic residues potentially involved in binding. A polysulphonated anticancer drug, suramin, has been shown to bind strongly to proacrosin/acrosin and to inhibit sperm-egg binding in vitro. In the mouse model, 125I-ZP2 and 3H-suramin bind approximately 65% less effectively to acrosin 'null' sperm than to wild-type sperm. Neither ZP2 nor suramin bind to acrosome intact sperm and can, therefore, only exert their effects after exposure of the acrosomal contents. Overall, this combination of biochemical, genetic and functional data supports the hypothesis that proacrosin is a multifunctional protein with a significant role in retaining acrosome-reacted sperm on the ZP surface long enough to enable ZP penetration to begin.

The role of acrosin in sperm penetration through human cervical mucus.

Enzymes have been implicated in facilitating cervical mucus penetration by spermatozoa. One of these enzymes in the neutral proteinase acrosin, which is associated with the sperm acrosome. To determine the validity of this hypothesis, human spermatozoa were incubated with the following acrosin inhibitors: p-aminobenzamidine (AB), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and p-nitropheyl-p'-guanidino benzoate (NPGB). An in vitro slide test system was developed which allowed inhibitor-treated and control spermatozoa to be evaluated against the same human cervical mucus sample. At inhibitor concentrations far exceeding those necessary for the inhibition of human acrosin, there was no effect on spermatozoal penetration into or through the mucus. These findings indicate that, in man, acrosin activity is neither necessary nor facilitory to sperm penetration of cervical mucus. Evidence is also presented that demonstrates the superiority of the newly developed double-interface slide test, especially for comparative purposes, over the tests currently in use.

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin.

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Biochemical alterations in the proacrosin-acrosin system during epididymal maturation of the rat spermatozoa.

Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.

Potentials for fertility regulation at the tubal level.

PIP: Suitable systems to support the varied processes associated with the initiation of life are provided by the Fallopian tube. Certain contraceptive possibilities based on available information on tubal function and acting at the tubal level have been considered. Approaches which would alter sperm and/or ovum transport could be initiated through modification in ciliary or muscular action or both. A review of the constituents of oviductal fluid and its influences on capacitation, fertilization, and early development includes the role of specific components such as bicarbonate, calcium lactate, and enzyme inhibitors and possibilities of altering the environment so as to affect adversely the processes within it. Incomplete information indicates the need and importance of further investigations to evaluate and control tubal function.^ieng

Advancement in biochemical assays in andrology.

Determination of markers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variation characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability. Biochemical parameters may be used in clinical practice to evaluate the sperm fertilizing capacity (acrosin, aniline blue, ROS), to characterize male accessory sex gland secretions (fructose, alpha-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS).

The action of acrosin on the zona pellucida.

The presence of hydrolytic enzymes in and associated with the sperm head has long argued for their functioning in fertilization. Several observations led investigators to propose that the acrosomal trypsin-like enzyme, acrosin in mammals, functioned in fertilization in aiding the sperm to penetrate the zona pellucida. While many have raised significant objections to this role, the action of acrosin on its presumed physiological substrate has not been characterized in a biochemical fashion. The intent of this study was to examine the effect of sperm proteases on the innermost egg envelopes in a parallel study, with the pig, Sous scrofa and the South African clawed frog, Xenopus laevis. With the pig, a great deal of information exists concerning the boar enzyme, acrosin, but little is known about the chemical structure of the zona pellucida. The opposite situation exists in X. laevis where the vitelline envelope is well characterized chemically, but little is known about the putative sperm lysins.

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.

[Studies on male infertility--acrosome reaction of the human sperm incubated in vitro].

In 1981, P. Talbot developed a triple-stain technique to estimate the number of human sperm undergoing normal acrosome reaction in fixed smears. In this method, live and dead sperm are first differentiated using the vital stain trypan blue. Sperm are then fixed in glutaraldehyde, dried onto slides, and the postacrosomal region and acrosome are differentiated using Bismark brown and Rose Bengal. Slides are examined at 1,000x with a bright-field microscope and assessed for the percentage of sperm that have undergone the normal acrosome reaction. Using this method we examined the time course of the acrosome reaction of human sperm incubated in mBWW the influence of human serum albumin, the calcium concentration of incubation media, Ca ionophore A 23187, and trypsin on the acrosome reaction of human sperm and the difference between the percentage of sperm undergoing normal acrosome reaction of fertile and infertile males. We got the following results: The percentage of human sperm undergoing acrosome reaction increased for the first six hours of incubation. Without human serum albumin the acrosome reaction did not occur. Ca ion could be one of the triggers of the acrosome reaction. Ca ionophore A23187 and trypsin induced the acrosome reaction in vitro. Sperm from oligozoospermic males, especially poorly motile sperm, could not undergo acrosome reaction so easily as sperm from fertile males could.

[Significance of proteases of the male genital tract for reproduction]. / Die Bedeutung der Proteasen des männlichen Genitaltraktes für die Fortpflanzung

PIP: The roles of proteases in reproduction are discussed. Semen coagulation is controlled by a protease similar to fibrinogen, the ''clotting enzyme,'' a part of the prostate secretion. The proteolytic enzyme that controls semen fluidity is still unknown. Breakdown of clot fragments is accomplished by seminin. Sperm motility is a function of the Kallikrein-kinin system, but it is not yet known which protease is responsible for kinin liberation. Seminal proteases, especially seminin and acrosin, are of particular importance for the penetration of cervical mucus and the facilitation of sperm migration. Acrosin is also of importance in the penetration of the zona pellucida, and is regulated by specific protease inhibitors. A number of other proteases are known to be present in the male genital tract, but their functions are not precisely know. Specific inhibition of 1 of the proteases may be the basis of antienzymatic contraception.^ieng

Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I.

The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo (Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17 +/- 1.51) compared with that in the control group (9.50+/-0.36) at 2h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30min (33.27+/-2.62 vs. 26.71+/-1.02), 60min (24.24+/-3.45 vs. 18.77+/-2.09), and 90min (22.86+/-3.02 vs. 16.92+/-1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4h (41.12+/-6.44 vs. 43.53+/-5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I-treated group (56.73+/-3.70) compared with that in the control group (44.85+/-2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility.

Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm

We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation.