Acrosin in the spermiohistogenesis of mammals

Abstract. Using the indirect immunofluorescence staining technique, the developmental pattern of acrosin during spermatogenesis of boar, ram, rabbit, mouse, rat, and Russian hamster ( Phodopus sungorus ) was studied. Specific antibodies against purified boar acrosin raised in rabbits crossreacted with the acrosin of all species investigated thus suggesting that the antigenic determinants of the acrosin molecule cross-reacting with anti-boar acrosin antiserum have been highly conserved in mammalian evolution. During spermatogenesis acrosin was first demonstrable in haploid spermatids and increased in the course of the differentiation of the spermatids to spermatozoa. During the entire period of spermatid differentiation acrosin appeared in juxtaposition to the nucleus. In boar and ram the results obtained with the indirect immunofluorescence staining procedure were confirmed with the indirect immunoperoxidase staining method.     

Acrosin in the spermiohistogenesis of mammals

Using the indirect immunofluorescence staining technique, the developmental pattern of acrosin during spermatogenesis of boar, ram, rabbit, mouse, rat, and Russian hamster (Phodopus sungorus) was studied. Specific antibodies against purified boar acrosin raised in rabbits cross-reacted with the acrosin of all species investigated thus suggesting that the antigenic determinants of the acrosin molecule cross-reacting with anti-boar acrosin antiserum have been highly conserved in mammalian evolution. During spermatogenesis acrosin was first demonstrable in haploid spermatids and increased in the course of the differentiation of the spermatids to spermatozoa. During the entire period of spermatid differentiation acrosin appeared in juxtaposition to the nucleus. In boar and ram the results obtained with the indirect immunofluorescence staining procedure were confirmed with the indirect immunoperoxidase staining method.     

Localization of rabbit sperm acrosin during the acrosome reaction induced by immobilized zona matrix

To better understand the loss of the acrosomal cap on the surface of the zona pellucida and the function of the equatorial-postacrosomal region after the acrosome reaction, we have constructed an in vitro system using heat-solubilized zonae pellucidae dried onto a coverslip and incubated with capacitated spermatozoa. This system allows good optical resolution of spermatozoonzona interaction. Induction of the acrosome reaction by zonae on coverslips (30%) is comparable to the induction of the reaction reported previously for rabbit spermatozoa using solubilized zonae in solution. Antiserum to rabbit proacrosin, antiserum to a porcine 49-kDa proacrosin fragment, and antiserum to a porcine 14-kDa C-terminal acrosin fragment were utilized to monitor the acrosome reaction. Rabbit proacrosin/acrosin is not present on the surface of live, acrosome-intact, swimming spermatozoa. After contact with zona, the acrosome reaction begins and proacrosin/acrosin becomes available to bind antibody, first as a crescent in the apical region and then more posteriorly until the entire anterior acrosome is labeled. Proacrosin/acrosin remains on the equatorial and postacrosomal regions of acrosome-reacted spermatozoa and also remains associated with the acrosomal cap even after the spermatozoon is no longer associated with it. Further studies using zona-coated coverslips should lead to a more detailed understanding of the mechanism of zona penetration.     

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.     

Regulation of proacrosin conversion in isolated guinea pig sperm acrosomal apical segments

Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proacrosin has been identified in extracts of intact guinea spermatozoa as a major silver staining band which reacted immunologically with antibodies made against purified proacrosin from guinea pig testis. Proacrosin exhibited an approximate Mr of 50,000 and was rapidly converted to an Mr 45,000 protein following induction of the acrosome reaction with 2.0 mM CaCl2 and 1 micrograms/ml A23187. Apical segments isolated at pH 6.0 from guinea pig spermatozoa also contained a major silver staining band of Mr 50,000 which cross-reacted with antibodies to guinea pig testis proacrosin. Subcellular fractionation of spermatozoa indicated that proacrosin remained in the particulate fraction of homogenized spermatozoa and was enriched within the isolated acrosomal apical segment. When apical segments isolated at pH 6.0 were incubated at pH 7.5, proacrosin was rapidly converted to the Mr 45,000 form observed in spermatozoa undergoing the acrosome reaction. The conversion process in isolated apical segments was inhibited by leupeptin and was accelerated in the presence of calcium, magnesium, and manganese. Zinc completely inhibited the conversion of proacrosin to the Mr 45,000 protein. Neither proacrosin nor the Mr 45,000 protein were released into the supernatant fluid during the incubation of apical segments at pH 7.5. Furthermore, the proteins were resistant to solubilization by 150 mM NaCl and 1% Triton X-100 but were solubilized by treatment of apical segments with 1 M NaCl. These results provide evidence as to the identity and subcellular distribution of proacrosin in intact guinea pig sperm prior to zymogen conversion and suggest that isolated apical segments exhibit a subset of the exocytotic reactions leading to completion of the acrosome reaction.     

The phospholipid-binding protein of the reproductive tract of the bull — Effect on the removal of spermatozoal cytoplasm droplets in other species and influence of antibodies on its reactivity

The phospholipid-binding protein (PBP) isolated from bull seminal vesicle fluid removed cytoplasm droplets not only from bull, but also from ram, boar and rabbit epididymal spermatozoa. However, the presence of a protein cross-reacting with anti-PBP antisera was demonstrated by immunofluorescent staining in ram seminal vesicles and ampullae. In contrast to PBP from bull, the ram PBP-like protein did not lyse bull or ram erythrocytes. Rabbit antiserum against PBP only negligibly reduced the ability of PBP to remove cytoplasm droplets from bull epididymal spermatozoa, but it inhibited the haemolytic effect of the protein.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.     

Human acrosome biogenesis: immunodetection of proacrosin in primary spermatocytes and of its partitioning pattern during meiosis.

Proacrosin biosynthesis timing during human spermatogenesis has been studied using the monoclonal antibody 4D4 (mAb 4D4). Frozen and paraffin-embedded sections of testicular biopsies were labelled by standard indirect immunofluorescence and avidin-biotin immunoperoxidase procedures. The labelling specificity was checked by immunochemistry assays on unrelated tissues and by western blotting of testis extracts showing that only the 50-55 x 10(3) Mr proacrosin was recognized by mAb 4D4. Proacrosin was first observed in the Golgi region of midpachytene primary spermatocytes. In late pachytene primary spermatocytes, proacrosin was observed in two regions located at opposite nuclear poles. During the subsequent steps of the first meiotic division, the two bodies containing proacrosin were located: (i) on opposite sides of the equatorial plate during metaphase; (ii) along the microtubular spindle during anaphase; and (iii) close to each chromosomal aggregate during telophase. Two bodies containing proacrosin were still observed in interphasic secondary spermatocytes. The single labelled area observed in early spermatids was found to increase considerably in size during spermiogenesis. Anomalies of proacrosin scattering were observed in patients with Golgi complex partitioning failure. These data reveal proacrosin biosynthesis during diploid and haploid phases of human spermatogenesis and the proacrosin partitioning pattern during meiosis.     

Influence of boar acrosin antibodies produced in rabbit and sheep on chymotrypsinogen activation catalyzed by acrosin from boar, bull, ram, rabbkt and human.

Activation of bovine chymotrypsinogen is catalyzed with increasing velocity by human, rabbit, boar, bull and ram acrosin. Antiboar-acrosin rabbit gamma-globulins cause a significant reduction in the proenzyme activation rate induced by boar and bull acrosin, but only a weak reduction or none if ram or rabbit acrosin is the activating agent. The antiboar-acrosin gamma-globulins from sheep strongly inhibit chymotrypsinogen activation by ram, bull and boar acrosin, and significantly inhibit the human acrosin-catalyzed reaction.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.     

Boar proacrosin expressed in spermatids of transgenic mice does not reach the acrosome and disrupts spermatogenesis

Transgenic mice that express boar proacrosin were produced to examine mechanisms for targeting hydrolytic enzymes to the acrosome. A 2.3 kb transgene was constructed by ligating the cDNA for boar preproacrosin with the mouse protamine 2 promoter region. Six founder mice that incorporated the transgene were identified by polymerase chain reaction and Southern blot analysis. Northern blots indicated that the two male founders (Ac.2 and Ac.5) and male progeny from three female founders (Ac.3, Ac.4, Ac.6) expressed the transgene mRNA in testis, but not in somatic tissues. In these transgenic animals boar proacrosin was detected by immunohistochemistry in condensing spermatids, but was not localized in the acrosome. This acrosomal targeting defect of the transgene product may result from its delayed expression during the later steps of haploid differentiation. Furthermore, both male founders and all Ac.4 and Ac.6 males were infertile, as determined by multipe matings for at least 2 months. Ac.3 males were either infertile or rarely transmitted the transgene to their offspring The infertile males mated, produced copulatory plugs, and had seminal vesicle weights and testosterone levels within the normal range. However, they produced significantly fewer spermatozoa and had lower testis weights than controls. Although the mitotic and meiotic phases of spermatogenesis appeared normal by histological criteria, condensing spermatids were missing from most tubules, and multinucleated cells were present in the lumen of seminiferous tubules and in the epididymis. We hypothesize that boar proacrosin which fails to reach the acrosome is activated in these transgenic mice, and that its proteolytic activity disrupts spermatogenesis during spermatid formation. © 1996 Wiley-Liss, Inc.     

Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Failure of immunoperoxidase staining to detect acrosin in the equatorial segment of spermatozoa (1).

An immunoperoxidase staining procedure that readily demonstrated acrosin in the rostral portion of the acrosome failed to detect acrosin in the equatorial segment of spermatozoa representing the mammalian orders artiodactyla (bull and boar), lagomorpha (rabbit) and primate (human).     

Immunofluorescent localization of acrosin in mammalian spermatozoa (1).

Acrosin was detected by immunofluorescence in the spermatozoan acrosomes of artiodactyla (bull, ram and boar), perissodactyla (horse), carnivora (dog and cat), lagomorpha (rabbit) and primates (human) using anti-bovine acrosin immunoglobulins. The results indicate that the acrosin molecules of several mammalian species possess antigenic similarities.     

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.     

Purification and partial peptide sequence analysis of the boar proacrosin binding protein

Boar proacrosin binding protein has been purified and the partial peptide sequence of the CNBr‐digested proacrosin binding protein has been determined. Proacrosin binding protein was purified as a proacrosin and proacrosin binding protein complex from the acid extracts of boar spermatozoa through gel filtration. After the proacrosin binding protein was dissociated from proacrosin by freeze‐thaw method, the proacrosin binding protein was purified through gel filtration. Fractions containing the proacrosin binding protein were pooled and were concentrated by lyophilization and then subjected to CNBr digestion. Four major CNBr‐digested peptides were subjected to N‐terminal peptide sequencing. All four showed the same N‐terminus sequence. Mol. Reprod. Dev. 54:76–80, 1999. © 1999 Wiley‐Liss, Inc.