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Regular Article |
a Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6142
ABSTRACT
Previously, we identified the guinea pig sperm acrosomal matrix glycoprotein AM67 and demonstrated that it is most closely related to mouse sperm sp56, initially reported to be a cell-surface protein. On the contrary, our studies demonstrated that sp56 is an intra-acrosomal component. Based upon the homology between guinea pig AM67 and mouse sp56, we hypothesized that sp56 was part of the acrosomal matrix, a structure that had yet to be demonstrated to exist in mouse sperm. In this paper, we show that sp56 first appeared in late meiotic cells and accumulated during spermiogenesis, the haploid stage of spermatogenic cell development. Using affinity-purified anti-peptide antisera, we determined that the molecular weight of sp56 in cauda epididymal sperm approximated that of guinea pig AM67 (
67 000 Mr) and that sp56 was present in a high molecular weight, disulfide-linked complex. The forms of sp56 in pachytene spermatocytes and spermatids had higher molecular weights than was found for the sperm form; the size differences were apparently due to alterations in carbohydrate side chains. The sp56 complex could not be solubilized by the nonionic detergent Triton X-100 but remained associated with the dorsal surface of the mouse sperm head, demonstrating that sp56 is a component of the mouse sperm acrosomal matrix.
acrosome reaction, fertilization, sperm, spermatid, spermatogenesis
INTRODUCTION
The acrosome is an exocytotic vesicle overlying the an;atterior region of the sperm head. The acrosome contains a variety of proteins, including several protease zymogens, protease inhibitors, zona pellucida-binding proteins, and other ligand-binding proteins [1–5]. The acrosome is considered essential for fertilization; men and mutant animals that produce sperm lacking acrosomes are infertile [6–9]. A variety of evidence suggests that the acrosomal components may participate in sperm-zona adhesion and penetration of the zona pellucida [4, 10].
Biogenesis of the acrosome begins in the late pachytene spermatocyte phase of meiosis and continues throughout the first half of spermiogenesis, the haploid phase of spermatogenic differentiation [11–13]. Initially, proacrosomal vesicles are formed in the perinuclear region near the Golgi apparatus. After meiosis is completed, these vesicles coalesce into a single granule that attaches to the round spermatid nucleus and continues to enlarge as Golgi-derived material is added. During the last half of spermiogenesis, the Golgi ceases to contribute glycoconjugates to the acrosome, and the acrosome-nucleus complex undergoes extensive morphological alterations to assume the characteristic shape for that species' sperm.
The interior of the acrosome is compartmentalized biochemically and morphologically. A given protein of the acrosomal lumen may be considered as a soluble constituent or an acrosomal matrix component depending on whether the protein is solubilized following extraction with Triton X-100 under conditions that block proteolysis [14]. Alternatively, specific components have been localized to discrete domains within the acrosome [15–18]. These findings suggest that the position or solubility of a specific acrosomal protein may govern its function during the course of acrosomal exocytosis and thereafter. For example, a component of the acrosomal matrix would be predicted to remain associated with the sperm head for a longer period of time than would a soluble protein.
During the course of our studies of the guinea pig sperm acrosomal matrix, we identified AM67, a member of the complement 4-binding protein family [19]. AM67 is most closely related to the mouse sperm protein sp56, initially thought to be a cell-surface protein [20, 21]. Our studies demonstrated that mouse sp56, like its apparent guinea pig orthologue AM67, is indeed localized within the lumen of the mouse sperm acrosome and is absent from the sperm surface unless the outer acrosomal and plasma membranes overlying the acrosome have begun fusing or have ruptured [19]. Although an acrosomal matrix has never been isolated from mouse sperm, these results suggested that such a compartment exists within the mouse sperm acrosome.
We have begun to examine the function of the acrosomal matrix in mouse sperm by first concentrating on acrosome biogenesis, using sp56 as a marker. In this paper, we demonstrate that sp56 is expressed during early spermiogenesis and, possibly, during late meiosis. We have also observed that sp56 is synthesized as a larger protein than has previously been reported. During the course of spermatid differentiation, sp56 undergoes post-translational modifications, including the modification of carbohydrate side chains. The final size of sp56 in cauda epididymal sperm approximates that of its guinea pig orthologue, AM67. Finally, we demonstrate that sp56 is part of a stable acrosomal matrix and is assembled into this structure in early spermatids.
MATERIALS AND METHODS
Materials
Adult (>12 wk old) CD-1 male mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Bovine serum albumin (BSA1, Fraction V) was from Sigma Chemical Co. (St. Louis, MO) or U.S. Biochemical Corp. (Cleveland, OH). Electrophoresis reagents were obtained from BioRad (Richmond, CA). A protease inhibitor cocktail (PIC) was constituted from tablets (cat. no. 1836153; Roche, Mannheim, Germany) and final concentrations were based upon the manufacturer's recommendation. Polyvinylidene fluoride (PVDF) membranes (Immobilon-P) were from Millipore (Marlborough, MA). An enhanced chemifluorescence (ECF) kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) was used for immunoblot analyses. Fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG was purchased from Zymed (So. San Francisco, CA). Monoclonal antibody mAb 7C5, specific for mouse sperm sp56, was purchased from QED Biologicals (La Jolla, CA). Trifluoromethanesulfonic acid (TFMS) was supplied by Sigma Chemical Co.
Antibody Production
To prepare peptide-specific polyclonal antisera against mouse sp56, two peptides (CPTPDMEKIKIVSERRDF and VYKLFLEIERLEHQKEK) were synthesized, conjugated to keyhole limpet hemocyanin, and used to immunize rabbits (Quality Controlled Biologicals, Hopkinton, MA). Figure 1 illustrates the relative positions of the two peptides within the linear amino acid sequence of mouse sp56. Antibodies (designated as anti-CPT and anti-VYK, respectively) were affinity purified on chromatographic columns conjugated to each respective peptide and characterized by immunoblotting using protein extracts from spermatozoa as the source of sp56. Specificity of the antibodies was verified by preadsorption of each antibody with its respective peptide.
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Isolation of Spermatogenic Cell Populations and Spermatozoa
Mixed populations of spermatogenic cells were obtained from mouse testes using the collagenase dissociation method [22]. Purified populations of spermatogenic cells were isolated following collagenase treatment of testes and trypsin digestion of isolated seminiferous tubules using unit gravity sedimentation velocity in a BSA gradient [22, 23]. Tubes containing each cell population (pachytene spermatocytes, >90% pure; round spermatids, >90% pure; and a condensing spermatid-residual body mixture) were selected by Nomarski interference contrast microscopy. Sperm were extruded from the caudae epididymides and vasa deferentia by retrograde perfusion with modified Krebs-Ringer solution (lacking CaCl2, NaHCO3, and BSA).
Extraction of Proteins from Spermatogenic Cells and Spermatozoa
Two methods were used to extract protein from the spermatogenic cells and sperm. For the most complete extraction of proteins, the cells were extracted with Laemmli SDS-PAGE sample buffer [24] containing PIC. To extract cellular membranes but maintain the acrosomal matrix structure, cells were extracted by a modification of the procedure of Hardy et al. [25]. Briefly, the cells were treated with 20 mM sodium acetate, pH 5.2, containing 0.15 M NaCl, 0.625% Triton X-100, 5% sucrose, and PIC. The cells were homogenized by extrusion through a 26-gauge syringe needle two times. The resulting cellular suspension was separated into a supernatant (containing soluble proteins and membrane extracts) by centrifugation (10 000 x g, 10 min). After washing once with 20 mM sodium acetate, pH 5.2, containing 0.15 M NaCl, and PIC, the pellets were then extracted with SDS sample buffer.
Electrophoretic and Immunoblot Analyses
Germ cell and sperm extracts were separated by SDS-PAGE according to the method of Laemmli [24] and transferred to a PVDF membrane by the method of Towbin et al. [26]. Blots were blocked in 5% nonfat dry milk in Tris-buffered saline and incubated overnight at 4°C or 2 h at room temperature with primary antibody diluted in blocking buffer. The concentrations of the antibodies used were 10 nM for anti-CPT and 15 nM for anti-VYK. After washing to remove the primary antibodies, the blots were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody diluted 1:2500 in blocking buffer. Blots were developed using ECF and analyzed on a Storm system (Molecular Dynamics, Sunnyvale, CA).
Chemical Deglycosylation
The TFMS was used to chemically deglycosylate spermatogenic and sperm glycoproteins [27]. Reactivials containing 300 µg of spermatogenic cell or sperm proteins were placed on ice. The TFMS (90 µl) and anisole (10 µl) were added to each sample and the mixtures were left on ice for 2 h with occasional mixing. Each reactivial was then placed in a dry ice-methanol bath and the TFMS was neutralized by the dropwise addition of pyridine:diethyl ether (1:9, v/v). The protein-containing precipitate that formed was collected by centrifugation and dissolved in cold 0.1 M NH4HCO3. After extensive dialysis with the same buffer, the sample was lyophilized, redissolved in Laemmli sample buffer, and analyzed by SDS-PAGE.
Indirect Immunofluorescence
Mouse spermatogenic cells or sperm obtained from caudae epididymides were washed twice by centrifugation for 5 min at 300 x g, resuspended in PBS, and placed onto polylysine-coated coverslips. Sperm were then treated with PBS containing 4% paraformaldehyde for 15 min at room temperature and then permeabilized with methanol (-20°C). After washing in PBS, coverslips were incubated in PBS containing 10% normal goat serum (blocking buffer) for 30 min at room temperature followed by primary antiserum (200 nM for anti-CPT and 300 nM for anti-VYK) diluted in blocking buffer for 1 h at 37°C. Following a washing step (3 x 5 min in PBS), coverslips were incubated with FITC-conjugated goat anti-rabbit IgG diluted 1:50 in blocking buffer for 1 h at 37°C, washed again, and mounted using Fluoromount G. Slides were examined using a Zeiss Photomicroscope III equipped with epifluorescence and photographed with Kodak T-Max P3200.
RESULTS
Anti-Peptide Antibodies React Specifically with a 67 000 Mr Protein from Mouse Sperm
We produced peptide-specific antibodies to be used for the analysis of sp56 by immunoblotting under reducing and nonreducing conditions and also to enable the localization of the protein within the developing acrosomes of spermatids and the mature acrosomes of epididymal sperm. As shown in Figure 2, anti-CPT and anti-VYK identified a major immunoreactive protein with a molecular weight of 67 000. Minor species of 43 000 and 31 000 Mr were also detected by both antibodies; a band representing an 18 000–20 000 Mr protein was also detected by the C-terminal peptide-specific anti-VYK. We have tried a variety of extraction conditions and have found the relative proportion of the smaller protein compared to the 67 000 Mr protein to be variable, depending upon the buffer pH, detergent used, and protease inhibitors present. The least degradation of sp56 occurred when the proteins were extracted with the Laemmli SDS-PAGE sample buffer containing PIC. As a control, each antibody was preincubated with its respective peptide prior to addition to the blots; under these conditions, the antibodies failed to bind to either protein, confirming the specificity of the antibodies (Fig. 2).
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Immunoreactivity of Reduced and Nonreduced Forms of sp56 with mAb 7C5 and Anti-Peptide Antibodies
We previously reported that guinea pig AM67 is found as a large molecular weight, disulfide-linked multimer in its native state [19]. Based upon the homology of mouse sp56 to AM67 and the C4BP family, we predicted that sp56 also forms a high molecular weight complex. Figure 3 demonstrates that, in the absence of reducing agent, anti-CPT and mAb 7C5 detected sp56 in high molecular weight (>>200 000 Mr) complexes. Interestingly, mAb 7C5 detected what appeared to be a second complex that is larger than the one detected by anti-CPT. The monoclonal antibody mAb 7C5 did not work well with reduced samples. Anti-CPT also detected a 31 000 Mr form of sp56 in the absence of reducing agent. Anti-VYK did not detect any sp56 forms under nonreducing conditions, presumably reflecting a masking of the peptide epitope recognized by this antibody. Reduction of the proteins with dithiothreitol (DTT) causes sp56 protein to run as a monomer in SDS polyacrylamide gels. When blots of the reduced proteins were also probed with anti-VYK, the major 67 000 Mr form of sp56 was readily detectable. When the blots of the reduced proteins were probed with anti-CPT, we observed the prominent 67 000 Mr form and the same minor forms of sp56 (43 000 and 31 000 Mr) described above. Because the 31 000 Mr form was negligibly detectable with the anti-VYK antibody, we believe this protein is a form of the 67 000 Mr monomer that has been truncated at the C-terminus.
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Anti-Peptide Antibodies Cross-React with AM67 of Guinea Pig Sperm
Mouse sperm protein sp56 was reported to have a monomer molecular weight of 56 000 while its AM67 orthologue in guinea pig was determined to be 67 000 Mr [19, 28]. For direct comparison, mAb 7C5, the commercially available monoclonal antibody that recognizes sp56, did not detect guinea pig AM67 (Fig. 4A). Dithiothreitol was omitted in the sample buffer for this comparison because the mAb 7C5 monoclonal antibody worked best for mouse sperm proteins using nonreducing conditions (Fig. 3). In the absence of DTT, mouse sp56 remained in a high molecular weight aggregate and 7C5 failed to cross-react with guinea pig proteins. Our antibody against guinea pig AM67 did show some cross-reactivity with a mouse protein similar in size to AM67, but one could argue that this antibody, made against electrophoretically purified AM67, may have detected carbohydrate determinants on an unrelated protein. To address this issue, we probed blots of mouse and guinea pig sperm proteins with anti-CPT and anti-VYK that we predicted might cross-react with both mouse sp56 and guinea pig AM67 because the proteins are very similar in the corresponding regions (Fig. 4B). In a side-by-side comparison, anti-CPT and anti-VYK identified similarly sized proteins in both mouse and guinea pig sperm, adding to the previously available information demonstrating that these two proteins are orthologues. Because of differences in cross-reactivity between species, protein levels of the extracts of sperm from each species were adjusted to give the optimal immunoreactive signal. This apparently reflects the fact that the proteins are not 100% identical in those regions selected for anti-peptide antibody production (Fig. 4B). The specific peptides blocked the ability of the antibodies to bind to sperm proteins from either species (Fig. 2 and data not shown).
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sp56 in Spermatocytes and Spermatids
We examined the presence of sp56 in the developing spermatogenic cells. By indirect immunofluorescence, the protein was first detectable within a cluster of small proacrosomal granules of late pachytene spermatocytes (Fig. 5). In the haploid round spermatids that result from the completion of the two reductional meiotic divisions of the spermatocytes, sp56 was found within the developing acrosomal granule of each spermatid. As the spermatids differentiated, the pattern of sp56 localization mirrored the structural alterations of the acrosome that occur during this time.
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When populations of mouse spermatogenic cells were purified and analyzed by immunoblotting for the presence of sp56, a low level of sp56 could be detected in pachytene spermatocytes (Fig. 6). The level of sp56 increased over the course of spermiogenesis, partly due to the accumulation of newly synthesized sp56 protein and also as a result of the reduction of spermatid volume that occurs during this time. We also observed that the molecular weights of sp56 decreased as a function of cellular differentiation. By immunoblotting, both antibodies detected maturation-dependent processing of sp56: bands at 86 000 and 77 000 Mr were found in pachytene spermatocytes and round spermatids, a single band of 77 000 Mr was detected in condensing spermatids, and the 67 000 Mr form was observed in cauda epididymal sperm.
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Previous work from our laboratory has demonstrated that the carbohydrate side-chains of glycoproteins of the guinea pig sperm acrosome were modified as the sperm matured. To examine this issue, we chemically deglycosylated sp56 using TFMS. This procedure is known to remove all N- and O-linked oligosaccharides from glycoproteins. As shown in Figure 7, the deglycosylated forms of sp56 from each stage of germ cell (pachytene spermatocyte, round spermatid, condensing spermatid, and epididymal sperm) all had the same molecular weight of 66 000–67 000, indicating that each form had the same polypeptide backbone. Thus, the maturation of sp56 during spermatogenesis appears to result from oligosaccharide processing.
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sp56 Is Part of the Acrosomal Matrix in Spermatogenic Cells and Sperm
As discussed above, sperm acrosomes are compartmentalized. Biochemically, components of the acrosomes can be classified as either soluble or as part of an acrosomal matrix depending upon their extractability with nonionic detergents such as Triton X-100. For this experiment, spermatogenic cell populations and epididymal sperm were extracted with 20 mM sodium acetate, pH 5.2, containing 0.15 M NaCl, 0.625% Triton X-100, 5% sucrose, and PIC. This is the same buffer used to isolate acrosomal matrices from guinea pig sperm [19]. As shown in Figure 8, sp56 was found as part of a detergent-insoluble complex in spermatogenic cells and sperm. Negligible sp56 was recovered in the supernatants of the cells extracted under these conditions.
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We next determined whether the treatment of spermatogenic cells and sperm under these conditions would release a particulate acrosomal matrix complex from these cells. As shown in Figure 9, extraction of the spermatogenic cells and sperm under these conditions did not release an acrosomal matrix particle from these cells. Instead, the particulate matrix containing sp56 remained associated with the head of sperm and the nuclei of the developing spermatids. Because the cells were exposed to the detergent extraction and the shear force generated by passing the cells through a 26-gauge needle twice, the acrosomal matrices must be quite firmly attached to the nuclei of these cells. This result is in contrast to guinea pig sperm that has a large acrosome that extends well beyond the end of the sperm nucleus; when treated under the same conditions, the acrosomal matrices of guinea pig sperm become dislodged from the remainder of the sperm and can be recovered after chromatography on a column of glass beads [25].
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DISCUSSION
Mouse Sperm Protein sp56 Is an Acrosomal Matrix Protein
These results demonstrate that mouse sperm contain a morphologically and biochemically defined acrosomal matrix. In addition, sp56 was detectable from the time when the acrosome first begins to be assembled (late meiosis) and accumulated during the times when the acrosome is undergoing extensive expansion (early spermiogenesis). The vast majority of sp56 was not recovered in the supernatant following extraction of spermatogenic cells and sperm under conditions used to release the acrosomal matrix as a particulate structure from guinea pig sperm. This indicates that sp56 is assembled into an acrosomal matrix early during acrosome biogenesis.
Properties of sp56
The sequence of a cDNA encoding sp56 indicated that this protein is a member of the complement 4B-binding protein family and has a predicted monomer polypeptide of 61 300 Mr [21]. Although Bookbinder et al. reported the size discrepancy between the calculated molecular weight and the experimentally derived value to be due to post-translational truncation of the sp56 polypeptide in spermatogenic cells, our results indicate that native sp56 monomer in sperm is larger (67 000 Mr) than previously reported (56 000 Mr) and that the sp56 polypeptide backbone of 66 000–67 000 Mr approximates the size predicted by the cDNA (61 300 Mr). Furthermore, we believe the 56 000 Mr reported by Bookbinder et al. resulted from a C-terminal truncation of sp56 when the proteins were extracted from the sperm. We tried a variety of extraction conditions including various buffers, detergents, and protease inhibitors and found that the conditions reported here worked best to preserve the higher molecular weight forms of sp56 from degradation. Also, the size of the sp56 monomer (67 000 Mr) was identical to that of its guinea pig orthologue, AM67. Our results further demonstrate the aggregation of sp56 into a large molecular weight multimer, similar to the guinea pig protein.
As demonstrated in Figure 7, sp56 did decrease in size as a function of spermatid maturation, but the changes were due to oligosaccharide processing, not truncation of the polypeptide backbone. Interestingly, although the predicted amino acid sequence of sp56 contains 11 canonical sites for asparagine-linked glycosylation [21], the amount of glycosylation appeared to be negligible in cauda epididymal sperm (Fig. 7). The significance of the carbohydrate side-chain changes is unclear at this time, but similar maturation-associated alterations have been noted for other acrosomal proteins [29].
The Acrosomal Matrix and Its Role in Acrosomal Exocytosis
Given that sp56 is found as an acrosomal matrix component in spermatogenic cells and sperm, what is its role? Although the exact function of sp56 in spermatogenesis is unknown, sp56 may have a role similar to other proteins involved in regulated secretory granule biogenesis such as the selective aggregation of regulated secretory proteins in the trans-Golgi network of secretory granules [30–32]. The association of sp56 with an insoluble acrosomal matrix could increase the fidelity of segregation of acrosomal proteins from constitutive secretory proteins by keeping it and other acrosomal proteins within the maturing acrosome while other nonessential materials are being removed from the acrosome as the acrosomal contents are condensed during late spermiogenesis [33–36].
Strong evidence indicates that mouse sp56 is a zona pellucida binding protein and might function in sperm-zona adhesion. Bleil and Wassarman first identified this protein as a ZP3-binding protein following the crosslinking of ZP3 to proteins of capacitated sperm with a photoactivatable heterobifunctional crosslinker [20]. Affinity chromatography on a column of purified ZP3 was also performed and identified a protein with a molecular weight of 56 000. Subsequently, the protein was purified and was used to determine the amino acid sequences of some peptides and to prepare monoclonal antibodies.
In our previous study, we found no surface exposure of sp56 unless the plasma membrane and outer acrosomal membrane had begun to fuse or were ruptured [19]. This result conflicted with earlier studies that concluded sp56 is restricted to the external face of the plasma membrane overlying the acrosome by using a nonconventional immunocolloidal gold-labeled surface replica technique and monoclonal antibodies to sp56 to localize this protein on capacitated sperm [28, 37]. In the studies reported here, we demonstrated that sp56 resides within the acrosomal matrix compartment of mouse sperm, i.e., sp56 remains particulate after Triton X-100 extraction. If sp56 were a peripheral membrane protein as initially proposed [21, 28, 37], it likely would have been extracted into the supernatant under these conditions.
How then could sp56 be misidentified as a cell surface molecule of capacitated sperm? We hypothesize that sperm capacitation represents a transitional state whereby the membranes overlying the sperm become modified by destabilization or initial fusion events. These initial phases of exocytosis could allow the antibody access to the intra-acrosomal components. Mouse sp56, being part of the insoluble acrosomal matrix (Fig. 9), would not readily diffuse from its acrosomal localization. Thus, some of the sp56 would be exposed to extracellular milieu and, under the conditions used for the immunocolloidal gold-labeled surface replica technique, sp56 could then appear to be localized on the plasma membrane overlying the acrosome [28, 37].
The finding that the zona pellucida-binding protein sp56 is within the acrosomal matrix and not on the sperm plasma membrane raises some interesting and important questions regarding the attachment of the sperm to the zona pellucida and its movement through the zona once initial adhesion has occurred [38]. A mechanism must exist to allow the sperm to release from points of attachment and move forward without coming off the zona pellucida. The acrosomal matrix may hold the key to this mechanism. Mutant male mice lacking a functional acrosin gene are fertile [39]. The acrosin-null sperm adhere to the zona pellucida normally, but the mutant sperm exhibit a delay in penetration of the zona pellucida due to an altered rate of protein dispersal from the acrosome [40]. In the case of sperm adhering to the zona pellucida via sp56, it is possible that modification of sp56 or some other sp56-anchoring protein by acrosin would lead to the release of this protein from the matrix. As shown in Figure 8, proteolytically derived lower molecular weight forms of sp56 (43 000 Mr and 31 000 Mr) appear to be less tenaciously bound to the Triton X-100 insoluble matrix than the intact 67 000 Mr form; this may provide a mechanism for the differential release of the sp56 forms from the acrosomal matrix of sperm during the course of acrosomal exocytosis. If the release of sp56 (or any anchoring protein) occurred from the outer periphery of the acrosomal matrix, then the intact, underlying sp56 would be exposed to reinitiate attachment to the zona [38]. Coupled with a mechanism for penetrating the zona, this could provide a way for the sperm to ratchet through the zona pellucida as the sperm move forward through this egg extracellular matrix.
ACKNOWLEDGMENTS
We thank Drs. Bayard T. Storey, Laura Díaz-Cueto, and Jin-Hyun Jun for their comments and critical evaluation of the manuscript.
FOOTNOTES
1 This work was supported in part by National Institutes of Health Grant HD22899 to G.L.G. 
2 Correspondence: George L. Gerton, Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, 421 Curie Blvd., 1311 BRB II/III, Philadelphia, PA 19104-6142. FAX: 215 573 7627; gerton{at}mail.med.upenn.edu
Accepted: August 8, 2000.
Received: June 26, 2000.
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