Acrosomal Matrix Research Articles

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii).

This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice.

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida.

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the 'scoop'. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated ‘scoop’-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the ‘scoop’. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

P-Aminobenzamidine-sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the egg zona pellucida in mouse.

It has been reported that a significant delay in protein dispersal from the acrosomal matrix is observed in wild-type sperm by adding p-aminobenzamidine, a trypsin/acrosin inhibitor, to the incubation medium. The pattern of this delayed release was similar to that of the acrosin-deficient mutant mouse sperm (Yamagata et al., J. Biol. Chem., 273, 10470-4, 1998). In the present study, no further delay in protein dispersal was found when the acrosin-deficient sperm were treated with p-aminobenzamidine, indicating that among the p-aminobenzamidine-sensitive protease(s) only acrosin may function to accelerate this process. Although the acrosin-deficient sperm penetrated the zona pellucida (Baba et al., J. Biol. Chem., 269, 31845-9, 1994), the addition of p-aminobenzamidine to the fertilisation medium caused a significant inhibition of fertilisation in vitro. This indicates that there is a p-aminobenzamidine-sensitive protease(s) other than acrosin participating in the zona penetration step. Indeed, we demonstrated that a non-acrosin protease with a size of 42 kDa was present in the supernatant of the acrosome-reacted sperm suspension. The enzyme was inhibited by p-aminobenzamidine, diisopropyl fluorophosphate and N alpha-tosyl-L-lysine chloromethyl ketone, and was apparently activated by acrosin.

Rearrangement of the PH‐20 protein on the surface of macaque spermatozoa following exposure to anti‐PH‐20 antibodies or binding to zona pellucida

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20 . The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction. Mol. Reprod. Dev. 50:207–220, 1998. © 1998 Wiley-Liss, Inc.

Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida.

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20. The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction.

Acrosome biogenesis in the hamster: ultrastructurally distinct matrix regions are assembled from a common precursor polypeptide.

The hamster sperm acrosome contains a stable matrix complex that binds specific hydrolases and appears to regulate their release during the acrosome reaction. This complex comprises two contiguous but ultrastructurally distinct regions that are segregated to specific sites within the acrosome. In this study, we define the temporal expression, processing, and localization of major matrix proteins of 29 kDa (AM29) and 22 kDa (AM22) during spermiogenesis and post-testicular sperm maturation in the epididymis. Peptide mapping, N-terminal microsequence analysis, immunoblotting, and immunocytochemistry were used to demonstrate that AM29 and AM22 of mature spermatozoa are structurally related and appear to arise from a common 40-kDa precursor protein expressed in round spermatids. A monoclonal antibody that recognized only the mature forms of the matrix proteins and a polyclonal antibody that recognized both the precursor and fully processed matrix proteins were prepared and used to demonstrate that the precursor protein is present in the acrosome of round spermatids and that it undergoes size processing during the terminal stages of spermiogenesis so that the mature matrix polypeptides are evident in epididymal spermatozoa. Finally, using light and electron microscopic immunocytochemistry, we demonstrated that the matrix polypeptides are excluded from the equatorial segment and are localized to both structurally distinct matrix domains of the mature acrosome. These data show that processing of the major proteins of the acrosomal matrix occurs in a temporally regulated fashion after their transport to the acrosome and that the processed products can assemble into ultrastructurally distinct matrix elements.

Time course of spontaneous in vitro sperm acrosome reaction.

The acrosome reaction (AR) is an exocytotic process essential for sperm penetration of the zona pellucida and binding to the oocyte (DeJonge, 1994). Evaluation of in vitro AR can suggest fertility potential. The purpose of this study was to determine AR as a function of time after removal of sperm from the seminal fluid using a novel test, the Acrobeads test (Fertility Technologies, Natick, Massachusetts), which uses paramagnetic beads coated with MH61, a monoclonal antibody that binds to acrosome-reacted sperm. Specimens were acquired from known fertile donors (n = 9) and in vitro fertilization (IVF) patients (n = 8) with no apparent male factor on the day of the IVF and processed by a minipercoll wash at 30 minutes after ejaculation. An aliquot of washed sperm was then divided into two portions. The first was placed with the Acrobeads (according to the manufacturer's instructions) and assessed for bead binding after 6 and 24 hours with the beads. The second aliquot of washed sperm was held at room temperature for 24 hours, then exposed to the beads, with bead binding assessed at 6 and 24 hours later (30 and 48 hours after washing). The Acrobeads score was determined by assessing the binding of MH61 beads in each of four replicates with a resulting score of 1 (lowest) to 4 (highest). The mean (+/-SD) motility was 62.0% (7.5) at 6 hours, 52.3% (6.4) at 24 hours, 55.9% (10.4) at 30 hours, and 54.7% (8.4) at 48 hours after removal from the seminal fluid. At 6 hours after washing and exposure to the beads, the score was 0.077 (0.27) with a range of 0-1; one donor specimen gave a score of 1, while all others had a score of 0. At 24 hours postremoval from the semen, donor and patient sperm were positive for the test, with a mean score of 3.6 (0.65). The mean fertilization rate for the IVF patients was 64.4% (range 33-90). When sperm were held for 24 hours prior to the test, there was little or no bead binding 6 hours later (score of 0.46 +/- 0.77) and at 24 hours later (48 hours after washing) (mean score of 0.25 +/- 0.45). These data suggest that completion of the acrosome reaction occurs by 24 hours after removal of the sperm from the seminal fluid. Since the MH61 beads bind to specific residues on the inner acrosomal membrane, these data also suggest that following the acrosome reaction at 24 hours with removal of the outer acrosomal contents and acrosomal matrix, the inner acrosomal membrane may be modified in some way that does not allow MH61 beads to bind to the sperm.

NMR studies of a viral protein that mimics the regulators of complement activation

Vaccinia virus complement control protein (VCP) is a 243-residue protein that is similar in sequence to the regulators of complement activation; its role is to defend the virus against attack by the host complement system. A fragment of this protein spanning the two complement protein (CP)-modules (residues 126 to 243) which make up the C-terminal half of VCP has been expressed in Pichia pastoris. A 15N-labelled sample was purified for the purposes of structure determination and measurements of dynamics in solution using NMR. Structures were calculated on the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence. The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; average r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons,=1.2 Å) but less similar to the fifth CP-module of fH (average r.m.s.d.=2.2 Å). In the VCP fragment, the orientation of one module with respect to the other is clearly defined by the experimental data, and T 1 measurements are consistent with only limited flexibility at the module-module interface. The r.m.s.d. over all of the 118 residues (backbone atoms) is 0.73 Å. The intermodular orientation is better defined than, and significantly different from, that observed in a CP-module pair from fH (re-calculated using the extended SA protocol). In VCP the long axis of the second module is tilted by 59(±4)° with respect to the first module (50(±13)° in the fH pair), and twisted with respect to the first module by 22(±6)° (223(±17)° in fH). The differences between the human and viral proteins may be rationalised in terms of the lack of hydrogen-bond stabilised secondary structure in the N-terminal portion of fH module 16, and the number and type of amino acid side-chains which make up the interface. A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matrix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimental data.

An antigenically related polypeptide family is a major structural constituent of a stable acrosomal matrix assembly in bovine spermatozoa.

The apical and principal segments of the bovine acrosome contain a stable matrix complex that is bound to the outer acrosomal membrane and exhibits hydrolase-binding activity. The present study was undertaken to determine whether the outer acrosomal membrane-associated matrix complex (OMC) is composed of a unique set of acrosomal proteins and to define its fate during both capacitation and the acrosome reaction. A purified OMC fraction was isolated from ejaculated spermatozoa, and one polypeptide of 32 kDa (OMC32) was purified to homogeneity and used for N-terminal sequence analysis and preparation of monospecific antisera. Immunofluorescence staining of sperm with anti-OMC32 demonstrated that the polypeptide localized specifically to the apical and principal segments of the acrosome. Immunoelectron microscopy further revealed that OMC32 was restricted to the stable matrix assembly and was not associated with the inner acrosomal membrane or the equatorial segment. Immunoblot analyses of sperm lysates and of the purified OMC fraction revealed that anti-OMC32 recognized an antigenically related family of polypeptides between 38 and 19 kDa. These polypeptides exhibited no size processing during capacitation or the acrosome reaction, and they were not released during the acrosome reaction but remained in the particulate cell subfraction, associated with the hybrid membrane complex. N-terminal sequence analysis of OMC32 indicated a structural relationship to the SP-10 polypeptide family of human and baboon spermatozoa. The potential function of the OMC complex and differences in the intraacrosomal distribution of bovine OMC32-related polypeptides from that reported for acrosomal SP-10 polypeptides in other species are discussed.

Protein involvement in the fusion between the equatorial segment of acrosome-reacted human spermatozoa and liposomes.

Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using anti-sperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step.

AM67, a Secretory Component of the Guinea Pig Sperm Acrosomal Matrix, Is Related to Mouse Sperm Protein sp56 and the Complement Component 4-binding Proteins

The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in acrosomal matrix-zona pellucida interactions during and immediately following the acrosome reaction in the mouse.

Posttesticular development of spermatozoa of the tammar wallaby (Macropus eugenii).

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.

A novel method for evaluating the acrosomal status of mammalian spermatozoa.

A novel method was developed to evaluate the acrosomal status of mammalian spermatozoa. The method is based on the ability of the lectin Pisum sativum agglutinin (PSA) to bind specifically to glycoproteins of the acrosomal matrix released during the acrosome reaction. The amount of released acrosomal content is proportional to the fraction of spermatozoa that underwent acrosome reaction. The released glycoproteins present in the supernatant separated from the cells were detected via an ELISA-like assay. The authors suggest that one of these glycoproteins might be the acrosin as identified by anti-acrosin antibodies, using Western blot analysis. The new method (demonstrated here with ram and bull spermatozoa) correlates well with the results obtained by conventional methods. Its advantages are simplicity, objectiveness, rapidity, and low cost. In addition, many samples can be processed in parallel. The method can be used in experimental as well as clinical applications.

Identification of hydrolase binding activities of the acrosomal matrix of hamster spermatozoa.

The interior of the mammalian sperm acrosome contains a structural framework, the acrosomal matrix, that may regulate both the distribution of hydrolases within the acrosome and their release during the acrosome reaction. To define the biochemical basis of this interaction, we examined the binding of two acrosomal hydrolase, proacrosin and N-acetylglucosaminidase (NAGA), to a purified acrosomal matrix fraction of hamster spermatozoa. Proacrosin-acrosin was chromatographically purified from acid extracts of hamster spermatozoa and consisted of four size variants of 50 kDa, 49 kDa, 45 kDa, and 43 kDa. Each of the four isoforms exhibited the same N-terminal amino acid sequence through 16 residues, suggesting that they may be modified by cleavage at the C-terminus. Polyclonal antiserum against the proacrosin isoforms specifically binds the acrosomal cap as shown by immunofluorescence microscopy. Neither proacrosin nor NAGA were solubilized when sperm were permeabilized with Triton X-100 under low ionic strength conditions; however, both hydrolases were releases by extraction with Triton X-100 containing 0.5 M NaCl. An acrosomal matrix fraction isolated under low ionic strength conditions retained bound proacrosin-acrosin and NAGA, and both hydrolases were released from the matrix by subsequent high-salt extraction. After high-salt treatment, the acrosomal matrix retained specific binding sites for both proacrosin and NAGA. In a blot overlay assay, a set of acrosomal matrix polypeptides between 29 kDa and 24 kDa specifically bound proacrosin. These data suggest that specific interactions between acrosomal matrix polypeptides and hydrolases represent a mechanism to sequester hydrolases within the acrosome and to regulate their release during the acrosome reaction.

Proacrosin-acrosomal matrix binding interactions in ejaculated bovine spermatozoa.

The mechanisms regulating hydrolase release during the mammalian sperm acrosome reaction are poorly understood. The present study demonstrates that specific domains of the acrosomal matrix of bovine spermatozoa function to maintain a particulate proacrosin pool and to regulate proacrosin/acrosin release. In sonicated sperm suspensions, 50-60% of the total proacrosin activity was sedimentable, and the amount of sedimentable proacrosin activity remained unchanged over time. Serial centrifugation and resuspension experiments demonstrated that the particulate proacrosin fraction resulted from an equilibrium binding of proacrosin to a stable sperm structure. To identify the proacrosin-binding structure of the acrosome, a purified sperm head fraction was isolated on sucrose density gradients. The sperm heads were extracted with Triton X-100, and a homogeneous acrosomal subfraction, the matrix complex associated with the outer acrosomal membrane (OMC), was isolated on Percoll density gradients. A centrifugation assay was then used to demonstrate that the OMC specifically binds proacrosin in a dose-dependent manner. These data demonstrate that the OMC represents a stable structural component of the acrosome that maintains a particulate proacrosin pool. We propose that the OMC regulates proacrosin release during the acrosome reaction and maintains elevated acrosin concentrations at the site of sperm-egg interaction.

Electron-microscopic localisation of thiol and disulphide groups by direct monomaleimido-nanogold labelling in the spermatozoa of a marsupial, the tammar wallaby (Macropus eugenii)

This study utilised a commercially available monomaleimido-nanogold reagent to directly label cellular thiol groups (SH) of marsupial (tammar wallaby) spermatozoa before and after reduction of disulphides (S-S) with mercaptoethylamine hydrochloride (MEA). The sperm surface, mitochondrial membranes, axoneme and tail fibres were all labelled with gold particles before MEA treatment and the label intensity was increased after S-S reduction. The acrosomal membranes and matrix of spermatozoa contained no detectable SH prior to MEA treatment. However, after moderate MEA treatment (1 mg/ml) gold label was associated with the acrosomal membrane and invaginated acrosomal membrane within the acrosomal matrix. After exposure to 5 and 10 mg/ml MEA, gold particles heavily labelled the acrosomal matrix. Thus, the acrosomal membranes and matrix of tammar wallaby spermatozoa both contain S-S cross-linked structures, and this may contribute to the unusual stability of the marsupial acrosome. Under all treatment conditions the nucleus remained unlabelled. This is consistent with early studies which indicated that cysteine was absent from the nuclear protamines. The study also demonstrated that monomaleimido-nanogold can be used to resolve SH- and S-S-rich cellular structures directly, in addition to its use to label antibodies and Fab fragments for immunochemical localisation.