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Regular Article |
a Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6142
b Department of Biology, Randolph-Macon College, Ashland, Virginia 23005
ABSTRACT
The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.
acrosome reaction, fertilization, gamete biology, male reproductive tract, sperm
INTRODUCTION
The acrosome is a membrane-bound, caplike organelle overlying the anterior tip of the sperm nucleus. The acrosome is essential for fertilization in mammals; humans and mice whose sperm lack acrosomes are infertile [1–4]. The exact function of this regulated secretory vesicle is still not clearly understood, but the acrosome and its contents have been implicated in sperm-zona pellucida adhesion and in the penetration of the zona pellucida. During the course of fertilization, the acrosome undergoes exocytosis near the time that the sperm encounters the zona. Although the actual site of acrosomal exocytosis is subject to debate, the prevailing dogma is that sperm must have completely intact acrosomes to adhere initially to the zona pellucida. However, the actual mechanism initiating acrosomal exocytosis is being intensely studied in many laboratories. One thing that is clear is that sperm must experience a period of "capacitation" before they are competent to undergo acrosomal exocytosis and fertilize the egg [5].
The acrosome has all of the characteristics attributed to other regulated secretory vesicles [6]. In the case of spermatozoa, secretion from the acrosome is accelerated by extracellular stimuli, the cumulus extracellular matrix and its components, and the zona pellucida [7]. The acrosomal secretory products are concentrated and condensed, and the acrosomal granule is stored for a long period of time as the spermatid and sperm mature in the testis and epididymis, respectively. As is found in other regulated secretory granules, the acrosome has an electron-opaque content known as a dense core. In sperm, the dense core is termed the acrosomal matrix and, like other regulated secretory granules, the membranes surrounding the acrosome can be removed by detergent treatment of spermatozoa without dissolving the condensed acrosomal matrix [8].
The existence of an acrosomal matrix allows the definition of two biochemical compartments within the acrosome: the acrosomal matrix and the soluble compartment. The working definition of a soluble compartment component is an acrosomal constituent that is not recovered as part of the matrix. Some of the soluble proteins described as constituents of the soluble compartment are hyaluronidase, dipeptidyl peptidase, and CRISP-2 (also known as autoantigen 1 and Tpx-1) [8]. The acrosomal matrix has been studied most extensively in the guinea pig and includes proacrosin, AM50 (also known as p50, apexin, Narp, and neuronal pentraxin II), AM67, and proacrosin-binding protein [9–11]. AM67 is a member of the complement 4-binding protein family but is most closely related to the mouse sperm protein sp56.
The roles of acrosomal matrix proteins are still being clarified. For example, proacrosin, the protease zymogen form of the serine protease acrosin, was initially thought to be the enzyme responsible for enabling the sperm to penetrate the zona. Recently, acrosin-null mice were produced, and these mice were fertile [12]. Subsequent experiments indicate that one action of acrosin is to facilitate the dispersal of the acrosomal matrix [13]. The mouse sperm protein sp56 was initially identified as a sperm surface-zona pellucida binding protein [14]; however, results from our laboratory demonstrated that sp56 is an internal acrosomal component and could not be detected on the plasma membrane by indirect immunofluorescence and immunoelectron microscopy [11].
In addition to having distinct biochemical compartments, the acrosome is also morphologically compartmentalized [15–18]. These different regions can be detected by transmission electron microscopy as distinct domains of varying degrees of electron density. In the guinea pig sperm acrosome, the domains are designated as M1, M2, and M3, and different acrosomal components have distinctly different distributions among the different morphological domains [8, 11, 17]. Although not widely recognized, the relatively smaller acrosomes of mouse spermatozoa also contain distinct domains that can be identified immunochemically; for instance, the 155 000 Mr protein recognized by monoclonal antibody mMC101 is specifically localized in the cortex of the anterior region of the mouse sperm acrosome [19]. The sperm of other species also contain an acrosomal matrix [18, 20–23].
The recognition of the domains within the acrosome led Hardy et al. [8] to propose that the release of acrosomal proteins from the sperm during exocytosis is dependent on their partitioning into either the soluble or matrix compartments of the acrosome. These authors proposed that soluble compartment components, such as CRISP-2, hyaluronidase, and dipeptidyl peptidase, would be free to diffuse away from the acrosome during the course of acrosomal exocytosis, whereas proteins of the matrix would be released more slowly. We have examined this issue by evaluating the release of acrosomal components from guinea pig sperm that were induced to undergo acrosomal exocytosis by treatment with the calcium ionophore A23187. Using indirect immunofluorescence, immunoelectron microscopy, and immunoblotting, we found that different acrosomal proteins display different patterns of release from the sperm, and the patterns of release were correlated with their association with biochemical (soluble versus matrix) and morphological (M1, M2, or M3 domains) compartments of the acrosome.
MATERIALS AND METHODS
Materials
Guinea pigs (retired breeding males; Charles River Laboratories, Wilmington, MA) were used in accordance with the International Guiding Principles for Biomedical Research Involving Animals, as promulgated by the Society for the Study of Reproduction, and research protocols involving animals were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Reagents used for electrophoresis were obtained from BioRad (Richmond, CA). Percoll was from Pharmacia (Uppsala, Sweden). Leupeptin, pepstatin A, and benzamidine HCl were from the Sigma Chemical Co. (St. Louis, MO). Pure nitrocellulose transfer and immobilization membranes were from Schleicher and Schuell (Keene, NH). An enhanced chemiluminescence (ECL) kit (Amersham Life Sciences, Buckinghamshire, England) was used for immunoblot analyses. Primary rabbit polyclonal antibodies to the following guinea pig sperm acrosomal antigens were previously characterized: AM67 [11], AM50 [10], proacrosin [24], and CRISP-2 [25]. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) was purchased from Zymed (South San Francisco, CA).
Ionophore-Induced Acrosomal Exocytosis
A slit was made near the junction of the corpus and cauda epididymides, and guinea pig sperm were recovered by retrograde perfusion of PBS through the distal end of the cauda epididymis. These sperm were washed twice by centrifugation for 5 min at 300 x g and were resuspended at 2 x 107 sperm/ml in CF-MCM (25 mM NaHCO3, 112 mM NaCl, 2.7 mM KCl, 1.5 mM MgCl2, 20 mM sodium lactate, 1 mM sodium pyruvate) containing 1 µg/ml ionophore A23187 and 2 mM CaCl2 [26] at 37°C. At various times after the introduction of the ionophore, aliquots of the sperm suspension were removed and a protease inhibitor cocktail was added. The suspensions (0.5 ml) were layered over 50% Percoll cushions (0.5 ml). (A 100% stock Percoll solution was formed by mixing 9 parts Percoll with 1 part 10x PBS; a 50% Percoll solution was 1 part 100% Percoll mixed with 1 part PBS.) The one-step Percoll gradient was then centrifuged to obtain a sperm pellet containing unreleased proteins and a supernatant fluid fraction containing proteins released during acrosomal exocytosis. The sperm pellet fractions were extracted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [27], and the concentrations of supernatant and pellet protein fractions were determined by bicinchoninic acid assay (Pierce, Rockford, IL) [28].
Indirect Immunofluorescence
The sperm were washed by two cycles of centrifugation for 5 min each at 300 x g and resuspended in PBS (2 x 107 sperm/ml). Spermatozoa were attached to poly-L-lysine-coated coverslips for 15 min. To induce acrosomal exocytosis, CF-MCM containing 2 mM CaCl2 and 1 µg/ml ionophore A23187 was placed on the coverslips and incubated for 0, 2.5, and 10 min. The medium was then aspirated and replaced with fixative (4% paraformaldehyde in PBS) for 15 min. After three washes in PBS, methanol (-20°C) was quickly added to permeabilize the spermatozoa. After 2 min, the coverslips were rinsed three times for 5 min each with PBS and then blocked with PBS containing 10% normal goat serum for 30 min at room temperature. The coverslips were then incubated with primary antibody (preimmune rabbit serum as a control, anti-AM50, anti-AM67, anti-proacrosin, or anti-CRISP-2) diluted in blocking buffer (1:100) for 1 h at 37°C in a humid chamber. After three washes in PBS, the coverslips were incubated in blocking buffer containing FITC-goat anti-rabbit IgG (1:50) for 1 h at 37°C in a humid chamber. After several washes in PBS, the coverslips were mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL), and the slides were examined by phase-contrast and fluorescence microscopy. Photographs were taken on a Zeiss Photomicroscope III (Carl Zeiss Inc., Thornwood, NY) with Kodak T-Max P3200 film (Kodak, Rochester, NY). All fluorescence images were photographed at equivalent exposure times.
For analysis of the progression of acrosomal exocytosis, the morphological and immunofluorescence patterns of the sperm were quantitated. For each time point (0, 2.5, and 10 min), triplicate samples of 
200 sperm were examined. The paired phase-contrast and fluorescence images of sperm were classified into four categories: 1) sperm showing apparently intact acrosomes and weak immunofluorescence signals, 2) sperm showing irregularly shaped acrosomes and strong immunofluorescence signals, 3) sperm without obvious acrosomes but displaying a specific immunofluorescence signal, and 4) sperm without obvious acrosomes and lacking a detectable immunofluorescence signal. The percentages of each class of sperm determined for each time point and each antigen were graphically expressed as the mean ± SD.
Immunoelectron Microscopy
Following a 2.5-min incubation period in ionophore A23187, sperm were fixed for 1 h at 4°C in 0.2 M cacodylate buffer (pH 7.4) containing 2% (w/v) paraformaldehyde. Sperm were then dehydrated in increasing concentrations of ethanol (60–100%) and embedded in LR Gold resin (Polysciences, Fort Washington, PA). Ultrathin sections were cut and placed on grids. Prior to immunostaining, grids were incubated for 30 min at room temperature in blocking buffer consisting of Tris-buffered saline (TBS: 20 mM Tris HCl, pH 7.5, 0.5 M NaCl) containing 10% (v/v) normal goat serum. For immunolocalization of AM67 and AM50, grids were incubated in blocking buffer containing anti-AM67 (diluted 1:100) or anti-AM50 (diluted 1:25) overnight at 4°C. Following three washes in TBS, grids were incubated in blocking buffer containing 18 nM colloidal gold conjugated to a goat anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:50 (v/v) in normal goat serum. Grids were washed again three times in TBS before being fixed and counterstained with 1% osmium tetroxide followed by 7% aqueous uranyl acetate. Sperm were observed and photographed using a Phillips 201 transmission electron microscope (Phillips, Eindhoven, The Netherlands).
Immunoblot Analysis
Guinea pig sperm proteins were separated by SDS-PAGE according to the method of Laemmli [27] and transferred to nitrocellulose by the method of Towbin et al. [29]. Blots were blocked in 5% nonfat dry milk in TBS and incubated for 3 h at room temperature with primary antibody in blocking buffer (anti-AM50, anti-AM67, anti-proacrosin, or anti-CRISP-2; 1:2000) followed by a secondary antibody in blocking buffer (goat anti-rabbit IgG-HRP; BioRad, Richmond, CA; 1:2000) for 1 h at room temperature. Blots were developed using ECL and exposed to autoradiography film (Action Scientific, Carolina Beach, NC).
Enzymography
Proteases were detected following SDS-PAGE in gels containing 0.1% gelatin. The samples were prepared in Laemmli sample buffer that lacked reducing agent (dithiothreitol) and were not heated prior to electrophoresis. Following SDS-PAGE, the gels were washed at room temperature with 2.5% Triton X-100 in 50 mM Tris HCl, pH 8.0 for 1 h followed by two or three washes over the course of 1 h with 50 mM Tris HCl, pH 8.0. After incubation at 37°C for 16 h with fresh buffer, the gels were stained with Coomassie brilliant blue R-250 to detect the clear zones of gelatin hydrolysis.
RESULTS
Acrosomal Exocytosis Is Exemplified by Transient, Intermediate Classes of Sperm
To evaluate the presence of specific acrosomal matrix proteins during the course of acrosomal exocytosis, cauda epididymal guinea pig sperm were prepared and treated with ionophore A23187. At 0, 2.5, and 10 min after ionophore addition, sperm were removed and processed for indirect immunofluorescence with antibodies directed against CRISP-2, AM50, AM67, and proacrosin. Representative pictures of sperm stained with the various antibodies during the course of acrosomal exocytosis are shown in Figures 1–4. Paired phase-contrast and fluorescence images were used to classify each sperm into one of four morphological categories. Class 1 sperm had apparently intact acrosomes by phase-contrast microscopy and strong immunofluorescence signals. Class 2 sperm had irregularly shaped acrosomes and stronger immunofluorescence signals than did class 1 sperm. Class 3 sperm did not have an obvious acrosome but did have a specific immunofluorescence signal that was generally less intense than the signals found with class 1 and class 2 sperm. Class 4 sperm lacked obvious acrosomes and did not display any detectable immunofluorescence signals. At each of the time points, the proportions of each sperm class were calculated and expressed graphically (Figs. 1–4, B). Variable staining of the flagellum was occasionally noted but was considered nonspecific.
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CRISP-2, a soluble component, was lost very quickly from the sperm acrosome and was not detected in sperm that had undergone morphologically complete acrosomal exocytosis (Fig. 1). AM50, an acrosomal matrix component, dissipated relatively slowly from the sperm acrosomal region (Fig. 2). At later times following ionophore exposure, many sperm still contained substantial amounts of AM50 in the acrosome at the apical tip of the sperm head. However, by 30 min, most of the AM50 staining was lost from the sperm heads lacking visible acrosomes. AM67, another acrosomal matrix constituent, disappeared more rapidly than AM50 after ionophore treatment, and at later time points, little AM67 label was detected at times when AM50 was still associated with the periacrosomal region (Fig. 3). Another acrosomal matrix member, proacrosin, displayed immunofluorescence patterns similar to those of AM50 but became less detectable in association with the acrosomal region at a slightly higher rate than did AM50 (Fig. 4). The similar immunofluorescence patterns for proacrosin and AM50 were not unexpected because proacrosin is present in the M2 and M3 domains of the acrosome and AM50 is present in the M3 domain [17].
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M1 Acrosomal Matrix Domain Breaks Down Prior to the Disintegration of the M2 and M3 Domains
Previously, various acrosomal proteins were demonstrated to be localized within specific morphological domains of the acrosome. For example, AM67 is restricted to the M1 domain and AM50 is confined to the M3 domain [11, 17]. The region of the dorsal concave surface of the acrosomal apical segment is where the plasma and outer acrosomal membranes first begin to fuse during acrosomal exocytosis [30, 31]. To determine whether the localization within a domain affected the release of AM67 or AM50 during acrosomal exocytosis, we used immunoelectron microscopy to examine guinea pig sperm that were undergoing acrosomal exocytosis following stimulation with ionophore A23187. At an early stage of exocytosis following the addition of A23187, AM67 was still detected in the dorsal bulge or M1 domain of sperm (Fig. 5A), although the acrosomal matrix in this region had begun to disintegrate. The M1 domain had extensively expanded and was characterized by a light, flocculent, diffuse matrix (Fig. 5B). The M3 domain showed evidence of disassembly, with the formation of clear sectors in this region. As detected by immunogold particles, AM50 remained associated with the dense matrix of this zone (Fig. 5B). In sperm at later stages of exocytosis, the acrosomal matrix had greatly expanded, but AM50 still remained associated with the acrosomal matrix, even in the expanded regions containing an anastomosing network of flocculent electron-dense matrix material (not shown). Acrosomal matrix material in the proximity of zones where the plasma and outer acrosome membranes had not fused showed the least amount of disintegration.
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Release of Some Acrosomal Components Is Correlated with Their Modification>
The alteration of the sperm head immunofluorescence patterns was correlated with the recovery of acrosomal proteins in the supernatant surrounding the sperm that were undergoing acrosomal exocytosis. To examine the released products, materials released from guinea pig sperm after A23187 treatment were compared with the proteins remaining with the sperm by immunoblotting and by protease detection (Fig. 6). Soluble proteins such as CRISP-2 were readily released from sperm and into the incubation medium subsequent to the addition of A23187 (to induce acrosomal exocytosis), whereas acrosomal matrix proteins such as AM50 remained associated with the sperm pellet for prolonged periods of time. Similar to the results from the immunofluorescence experiment (Figs. 1–4), AM67 was released more slowly (in the supernatant fluid 2.5 min after A23187 addition) than CRISP-2 but became soluble more readily than did AM50 (in the supernatant fluid 10 min after A23187 addition). In addition, the release of AM50 into the supernatant was correlated with its conversion to a lower molecular weight form known as AM50AR [17]. AM67 was apparently not processed as a consequence of acrosomal exocytosis. Proacrosin was detected in immunoblots of mature sperm but was not detected in either the pellet or supernatant of sperm undergoing acrosomal exocytosis. This immunoblotting result (loss of anti-proacrosin immunoreactivity in Western blots of sperm undergoing acrosomal exocytosis) seems at odds with the immunoblotting results (Fig. 4). We hypothesize that proacrosin is modified in the early stages of acrosomal exocytosis. The discrepancy between the immunofluorescence and immunoblotting with the anti-proacrosin antibody may result from conformation-sensitive epitopes that are recognized in fixed sperm (relatively native conformation) but that are destroyed by the denaturation caused by SDS and dithiothreitol treatment (electrophoresis conditions). However, a strong protease signal (presumably representing the activated acrosin that was no longer detectable by the antibody) was detected in the supernatant fluid 2.5 min after induction of acrosomal exocytosis. These data demonstrate that acrosomal matrix components are sequentially released during this process.
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DISCUSSION
Acrosomal Proteins Redistribute During the Course of Acrosomal Exocytosis
We consider the acrosome to be compartmentalized from biochemical and morphological standpoints. This compartmentalization and the four classes of sperm morphology categorized in this study are summarized in Figure 7. The immunofluorescence patterns of class 1 sperm reflect the distributions demonstrated by previous immunoelectron microscopic studies. CRISP-2 is present throughout domains M1, M2, and M3 [8]. Hence, its localization encompassed the complete acrosome, including the principal segment that extends down the anterior half of the sperm nucleus (Figs. 1 and 7). AM50 is present in domain M3 and proacrosin is present in domains M2 and M3 [17], so it was expected that the immunofluorescence signals should predominate in the apical region of the sperm acrosome, with some signal seen in the principal region (Figs. 2, 5, and 7). AM67 is restricted to domain M1 [11] and, accordingly in our experiments, was only found at the tip of the apical segment (Figs. 3, 5, and 7). As acrosomal exocytosis ensued, the acrosome began to become altered by expansion of the contents (Figs. 1–6 and 7). Using indirect immunofluorescence of paraformaldehyde-fixed and methanol-permeabilized sperm, the antibodies gained access to more of the acrosomal protein epitopes, and the intensities of the signals increased. CRISP-2, AM50, and proacrosin are all present in M3, which includes the principal segment [8, 17]; hence, our finding that the signal over the anterior half of the nucleus increased immensely during the course of acrosomal exocytosis was expected. Likewise, AM67, which is found only in domain M1 [11], remained restricted to the apical segment of the sperm acrosome (Figs. 2 and 5).
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Acrosomal Proteins Are Differentially Released from the Sperm During the Course of Exocytosis
These studies present additional evidence to support the concept that acrosomal proteins are differentially released during the course of exocytosis stimulated by calcium ionophore A23187. In other words, acrosomal exocytosis does not constitute a wholesale, instantaneous release of soluble components from a simple, fluid-filled sack. In essence, the rate of acrosomal protein release is correlated with the compartmentalization of the proteins. Proteins such as the soluble component CRISP-2 are readily released from the sperm once acrosomal exocytosis has been initiated. However, acrosomal matrix components such as AM67, AM50, and proacrosin remain associated with the sperm for prolonged periods of time. Each acrosomal matrix component apparently has its own characteristic pattern of release, e.g., AM67 was more readily released from the sperm than was either AM50 or proacrosin. Modifications of AM50 and proacrosin occurred, and these structural changes were correlated with their release from the sperm. These results open up the possibility that processing of specific acrosomal proteins may alter their association with the matrix. The earlier release of CRISP-2 and AM67 relative to AM50 and proacrosin is consistent with the findings of Flaherty and Olson [30]. In electron microscopic studies of lysolecithin-treated guinea pig sperm induced to undergo acrosomal exocytosis in response to the addition of calcium, the initial regions of fusion between the plasma membrane and outer acrosomal membrane were found on the anteroventral and dorsal surfaces of the apical segment of the acrosome, near the M1 domain. Similar to our biochemical and morphological assessments of the release of the M1 domain protein AM67 (Figs. 3 and 5), these investigators observed that the contents of the M1 domain (dorsal bulge) quickly disperse, and the matrix begins to cavitate soon after exocytosis has begun.
In interpreting these results, it is appropriate to consider that these experiments were performed using ionophore A23187 instead of a physiological stimulus, the zona pellucida, to stimulate acrosomal exocytosis. The ionophore method allowed us to screen large numbers of guinea pig sperm by indirect immunofluorescence for the various stages of exocytosis and to carry out biochemical analyses of the released acrosomal proteins. While we recognize that there may be discrete distinctions between this process in solution and acrosomal exocytosis occurring in the microenvironment immediate to the zona [32], we think our findings of acrosomal component compartmentalization can be generalized to acrosomal exocytosis in situ. In fact, the issue of acrosomal protein compartmentalization may be even more important when sperm undergo acrosomal exocytosis in response to the solid phase zona pellucida.
Acrosomal Exocytosis Is a Continuous Process, Not a One-Step Reaction
These results demonstrate that during the course of acrosomal exocytosis, transient intermediates are formed. This is not a new finding, but it is an important point to recognize because most models of acrosome dynamics ignore the intermediates and only take into account the acrosome-intact and acrosome-reacted states of sperm. Thus, many investigators view acrosomal dynamics as a binary process whereby sperm go from the intact state to the reacted state in a single step, i.e., reaction. However, some acrosomal components remain associated with sperm for extended periods of time following the initiation of acrosomal exocytosis (Figs. 1–6) [17, 20, 31, 33–36]. The intermediates of acrosomal exocytosis are often not detected because the properties used for assaying acrosomal status frequently do not take into account the existence of material associated with the sperm head. For example, class 3 sperm (Figs. 1–4) would be classified as acrosome reacted by phase-contrast microscopy; however, acrosomal matrix material is still associated with these sperm. Thus, class 3 truly merits categorization as an intermediate that precedes the final state (class 4), whereby negligible or no acrosomal material remains associated with the sperm head. In recognition of the fact that acrosomal dynamics is not a binary process, i.e., a one-step reaction of the nature
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Sn represent transitional intermediates (comprising class 2 and class 3 sperm), and AR represents the final state of exocytosis (class 4 sperm). The nomenclature chosen here was adapted from that used by Storey, Kopf, and colleagues; the earlier usages of B, S, and AR refer to states of capacitation and acrosomal status of mouse sperm as classified by staining with the fluorochrome chlortetracycline [37, 38]. Although the intermediates described by these authors refer to fluorescent staining patterns, our results demonstrate that intermediates of acrosomal exocytosis do exist, suggesting that the chlortetracycline patterns may be indicative of changes in acrosomal status. Presentation of Acrosomal Matrix Components Suggests a Mechanism for the Sperm to Adhere to the Zona Pellucida
Although the acrosome is acknowledged to be important for fertility, we still do not fully comprehend the role it plays in the fertilization process. However, recognizing that acrosomal exocytosis is not a binary (two state) event but is truly an analog (continuously variable) process, provides another perspective to view the function of this important organelle. Specifically, AM67 is the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56. Both proteins are present within the acrosomal matrices of the guinea pig and mouse sperm, respectively [11, 39]. Thus, having a solid-phase zona-binding protein that can be exposed on the sperm head during the course of acrosomal exocytosis provides a mechanism for the sperm to adhere to the zona pellucida via acrosomal matrix proteins such as sp56 (and possibly AM67). In essence, this would be similar to two cells adhering via their respective extracellular matrices, because both the acrosomal matrix and the zona pellucida are fundamentally extracellular matrices. In addition, several studies have indicated that proacrosin may also have a role in assisting sperm to adhere to the zona pellucida [40, 41]. Likewise, AM50 is a member of the carbohydrate-binding pentraxin protein family [10] and might also participate in the sperm-zona pellucida adhesion process.
Acrosomal Exocytosis Model of Acrosomal Dynamics Considers the Role of Intermediates in Sperm-Zona Pellucida Interactions
We are examining the hypothesis that acrosomal matrix proteins are functionally important in fertilization, acting as agents for sperm-zona pellucida adhesion and penetration. However, once sperm have adhered to the zona pellucida, a mechanism must exist to enable them to release from their point of attachment and then move through the zona pellucida without losing contact with the egg's extracellular matrix. To accomplish this, we envision a scenario whereby the loss of proteins from the periphery of the acrosomal matrix proteins permits the sperm to release from the attachment with the zona and move forward, re-establishing adhesion to the zona via the exposure of underlying zona-binding molecules of the acrosomal matrix. The underlying acrosomal matrix components that bind to zona pellucida ligands could include AM67, the homologue of the mouse zona-binding protein sp56 [11], proacrosin [40], and possibly AM50, which is structurally related to galactose-binding proteins [10]. Release of the peripheral matrix proteins could be mediated by proteolysis. For example, during the course of acrosomal exocytosis, AM50 is subsequently released from the sperm after it has been converted to the 
43 000 Mr AM50AR [17]. Processing of acrosomal matrix proteins could result from proteolytic cleavage by acrosin (the activated serine protease form of proacrosin). Further support for a role for acrosin in matrix structural dynamics comes from the studies of proacrosin-null mutant mice. Although these mice are fertile, penetration through the zona pellucida is delayed and seems to result from the retarded dissipation of acrosomal proteins [13].
In an extension of the mechanism proposed by Hardy et al. [8], we are developing an alternative paradigm for acrosomal dynamics that emphasizes the transitional intermediates of acrosomal exocytosis [42]. In contrast to the widely accepted, two-state acrosome reaction model, the alternative acrosomal exocytosis model views the release of acrosomal proteins as a continuous process, with different proteins being released from sperm at characteristic rates dependent upon the inherent properties of each protein and their interactions with other acrosomal components (Fig. 7). Soluble proteins such as CRISP-2 would be readily released from the sperm, whereas each acrosomal matrix component would remain particulate until changes in the matrix cause the component to become soluble. In the case of proteins such as AM50, proteolysis may be the mechanism for converting this protein from a matrix-bound protein (AM50) to a soluble protein (AM50AR). The proteolytic processing of the matrix could be regulated in part by the presence and gradual dispersion of protease inhibitors or protease regulators such as proacrosin-binding protein [9]. A stoichiometric measurement of the acrosin content of guinea pig sperm following A23187 induction of acrosomal exocytosis demonstrated that approximately half of the acrosin content of guinea pig sperm remained associated with the sperm a full 30 min and could only be released by acid extraction [43]. Because diffusion of proteins would occur from the outer margins of the acrosomal matrix, proteolytic processing could provide a mechanism for the controlled releasing of zona pellucida-binding proteins from the outer margins, thereby exposing fresh underlying matrix-associated molecules that could continue to participate in an another cycle of sperm adhesion to the zona. Hypothetically, this process could enable the sperm to maintain adhesion to the zona while moving through it, i.e., the sperm could use the acrosomal matrix-zona pellucida interactions to "ratchet" through the zona as the acrosomal matrix gradually disintegrates.
ACKNOWLEDGMENTS
We thank our laboratory associates and other colleagues at the Center for Research on Reproduction and Women's Health for their support and encouragement with these studies. We also gratefully acknowledge Dr. Stuart Moss for critically reviewing the manuscript prior to submission.
FOOTNOTES
1 This work was supported in part by National Institutes of Health grants HD22899 to G.L.G. and HD-07305 and HD-07792 to J.A.F. and by a grant from the Korean Research Foundation to K.S.K. 
2 Correspondence: George L. Gerton, Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, 421 Curie Blvd., 1311 BRB II/III, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6142. FAX: 215 573 7627; gerton{at}mail.med.upenn.edu
Accepted: August 15, 2000.
Received: June 21, 2000.
REFERENCES
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K. Mitra, N. Rangaraj, and S. Shivaji Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenase in Spermatozoa: CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION, AND ACTIVITY DURING SPERM CAPACITATION J. Biol. Chem., July 8, 2005; 280(27): 25743 - 25753. [Abstract] [Full Text] [PDF]
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 C. Dube, P. Leclerc, T. Baba, C. Reyes-Moreno, and J. L. Bailey The Proacrosin Binding Protein, sp32, Is Tyrosine Phosphorylated During Capacitation of Pig Sperm J Androl, July 1, 2005; 26(4): 519 - 528. [Abstract] [Full Text] [PDF]
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 D. Busso, D.J. Cohen, M. Hayashi, M. Kasahara, and P.S. Cuasnicu Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction Mol. Hum. Reprod., April 1, 2005; 11(4): 299 - 305. [Abstract] [Full Text] [PDF]
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 N. Inoue, M. Ikawa, T. Nakanishi, M. Matsumoto, M. Nomura, T. Seya, and M. Okabe Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm Mol. Cell. Biol., April 1, 2003; 23(7): 2614 - 2622. [Abstract] [Full Text] [PDF]
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 L. Oyewumi, F. Kaplan, S. Gagnon, and N. B. Sweezey Antisense Oligodeoxynucleotides Decrease LGL1 mRNA and Protein Levels and Inhibit Branching Morphogenesis in Fetal Rat Lung Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 232 - 240. [Abstract] [Full Text] [PDF]
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P. Talbot, B. D. Shur, and D. G. Myles Cell Adhesion and Fertilization: Steps in Oocyte Transport, Sperm-Zona Pellucida Interactions, and Sperm-Egg Fusion Biol Reprod, January 1, 2003; 68(1): 1 - 9. [Abstract] [Full Text] [PDF]
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J. Ramalho-Santos, G. Schatten, and R. D. Moreno Control of Membrane Fusion During Spermiogenesis and the Acrosome Reaction Biol Reprod, October 1, 2002; 67(4): 1043 - 1051. [Abstract] [Full Text] [PDF]
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 D. J. Miller, X. Shi, and H. Burkin Molecular Basis of Mammalian Gamete Binding Recent Prog. Horm. Res., January 1, 2002; 57(1): 37 - 73. [Abstract] [Full Text] [PDF]
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