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W2034281339 abstract "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 M r 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 λgt11 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 calculatedM r of 59,768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have aM r 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 α-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. 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 M r 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 λgt11 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 calculatedM r of 59,768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have aM r 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 α-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. The sperm acrosome reaction has the characteristic hallmarks of regulated secretion: 1) secretory products are concentrated and condensed; 2) secretory granules are stored for long periods of time; and 3) secretion is coupled to an extracellular stimulus (in this case, the zona pellucida) (1Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar). Regulated secretion from sperm (i.e. the acrosome reaction) is obligatory for fertilization (2Kopf G.S. Gerton G.L. Wassarman P.M. Elements of Mammalian Fertilization.1. CRC Press, Inc., Boca Raton, FL1991: 153-203Google Scholar). It is presumed that the release of specific secretory components from the acrosome is involved in assisting the sperm in the penetration of the investments surrounding the egg. Furthermore, although sperm contain only one secretory vesicle, different enzymatic activities are released from sperm at various times following the acrosome reaction (3Talbot P. Franklin L.E. J. Reprod. Fertil. 1974; 39: 429-432Crossref PubMed Google Scholar, 4Green D.P. J. Cell Sci. 1978; 32: 153-164PubMed Google Scholar, 5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 6de Vries J.W.A. Willemson R. Geuze H.J. Eur. J. Biochem. 1985; 37: 81-88Google Scholar, 7Zao P.Z. Meizel S. Talbot P. J. Exp. Zool. 1985; 234: 63-74Crossref PubMed Scopus (28) Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar). The differential temporal release of secretory components occurs in other regulated secretory tissues such as the pancreas, but since cells of such tissues contain multiple secretory granules, it has been proposed that this “non-parallel secretion” is due to exocytosis from heterogeneous sources within the tissue or cell of study (9Adelson J.W. Miller P.E. Science. 1985; 228: 993-996Crossref PubMed Scopus (38) Google Scholar). In guinea pig sperm, the acrosome reaction results in the differential release of acrosomal components possibly due to their compartmentalization within soluble or insoluble phases of the acrosome (5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar). Following the exocytotic acrosome reaction, certain acrosomal proteins, such as proacrosin, remain associated with the matrix that establishes the “dense core” of the acrosome and are released at a slower rate than components, such as dipeptidyl peptidase, that are found in a more readily solubilized compartment.The results presented here and in recent papers (10Noland T.D. Olson G.E. Biol. Reprod. 1989; 40: 1057-1066Crossref PubMed Scopus (10) Google Scholar, 11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 12Westbrook-Case V.A. Winfrey V.P. Olson G.E. Biol. Reprod. 1994; 51: 1-13Crossref PubMed Scopus (43) Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar, 14Westbrook-Case V.A. Winfrey V.P. Olson G.E. Dev. Biol. 1995; 167: 338-349Crossref PubMed Scopus (32) Google Scholar) demonstrate that two of the components of the dense core (matrix) of the acrosome, AM50 and AM67, represent monomers of large homopolymeric proteins. AM50 has been shown to be a novel, testis-specific member of the pentraxin family of proteins (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar). Preliminary tryptic peptide sequencing indicated that AM67 is related to members of the complement-binding protein family (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar). In this paper, we show that AM67 is, indeed, a member of this class of proteins. Furthermore, the amino acid sequence of AM67 is most closely related to that of the mouse sperm protein sp56. This protein is a candidate for the cell-surface receptor of ZP3, a glycoprotein of the egg's extracellular matrix, the zona pellucida (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar). A standard post-embedding immunoelectron microscopic technique demonstrated that AM67 is restricted to a specific dorsal compartment (M1 domain of the apical segment) within the acrosome of guinea pig sperm, while mouse sp56 was detected only within the acrosome and not on the plasma membrane over the acrosome as was previously reported using an unconventional colloidal gold surface replica immunoelectron microscopic procedure (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar).DISCUSSIONThis study demonstrates that the guinea pig sperm acrosome contains a testis-specific protein related to mouse sperm sp56 and other members of the complement regulatory protein superfamily. This result is reminiscent of our previous reports concerning the acrosomal matrix protein AM50, which was found to be a member of the pentraxin superfamily (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar). Like AM50, AM67 was found to be a testis-specific member of a protein superfamily, was localized to a specific domain within the acrosome, and also formed high molecular weight homopolymers. Similar to C4BPα, we estimate that six to eight monomers of AM67 oligomerize to constitute the unreduced form of the protein (M r ∼380,000). Based upon the C4BPα sequence homology, AM67 might be predicted to form calcium-dependent complexes with other ligands within the acrosome or, after exocytosis, with extracellular ligands. Comparable calcium-dependent binding of AM50 to the apical segment complex (containing the acrosomal matrix) has already been demonstrated (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar).Of all the sequences found to be homologous to AM67, mouse sperm protein sp56 and the serum complement component 4-binding proteins within the complement regulatory protein superfamily ranked the highest in a BLAST search of GenBank™ sequences (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar). The significance of this is not clear at the present time; but it is noteworthy that serum C4BP has been shown to interact with serum pentraxins (C-reactive protein and serum amyloid P component) (38Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1991; 266: 12896-12901Abstract Full Text PDF PubMed Google Scholar, 39Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1990; 265: 21749-21757Abstract Full Text PDF PubMed Google Scholar, 40Garcı́a de Frutos P. Dahlbäck B. J. Immunol. 1994; 152: 2430-2436PubMed Google Scholar), and thus, AM67 might be predicted to interact with AM50. Although immunoelectron microscopy demonstrated that AM67 and AM50 are localized in distinct regions of the acrosomal matrix of acrosome-intact sperm, it is possible that these components could interact following the acrosome reaction. C4BPα and the pentraxins are known to bind to plasma membranes, and based upon the homologies to these proteins, AM67 and AM50 might also be expected to interact with components of the acrosomal membranes. If AM67 and AM50 bind to different ligands of the acrosomal membrane, aggregation of these proteins with their ligands during acrosome biogenesis may help to establish the M1 and M3 domains. Additionally, AM50 and AM67 may interact with membranes or react with each other upon their release following the acrosome reaction and the dissolution of the acrosomal matrix.AM67 is not the first member of the complement regulatory protein superfamily to be found in sperm. Additional members recently identified include another guinea pig sperm C4BP distinct from AM67 (13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar) as well as decay-accelerating factor (CD55), membrane cofactor protein (CD46), and protectin (CD59), which have been shown to be membrane-bound components of the sperm acrosomal region (41Rooney I.A. Oglesby T.J. Atkinson J.P. Immunol. Res. 1993; 12: 276-294Crossref PubMed Scopus (80) Google Scholar). Clusterin, another regulator of complement, is secreted by the Sertoli cells and the epididymal epithelium and has also been shown to be associated with sperm (42Sylvester S.R. Morales C. Oko R. Griswold M.D. Biol. Reprod. 1991; 45: 195-207Crossref PubMed Scopus (139) Google Scholar, 43Law G.L. Griswold M.D. Biol. Reprod. 1994; 50: 669-679Crossref PubMed Scopus (43) Google Scholar). The roles of these complement regulatory proteins in fertilization and reproductive immunology are debatable at this point, but several of them appear to be present in sperm-specific forms. The testis-specific expression of AM67 is also intriguing considering C4BPα proteins are generally considered to be liver-specific proteins.The very remarkable similarity of the amino acid sequence of guinea pig AM67 to that of mouse sp56 suggests that these proteins are orthologues. Although the apparent molecular weights of the native AM67 and sp56 multimeric proteins were ∼380,000 and 110,000, respectively (14Westbrook-Case V.A. Winfrey V.P. Olson G.E. Dev. Biol. 1995; 167: 338-349Crossref PubMed Scopus (32) Google Scholar, 15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar), these sizes are similar to the differences in the properties of the C4BPs from other mammalian species compared with mouse (44Barnum S.R. Immunol. Res. 1991; 10: 28-42Crossref PubMed Scopus (16) Google Scholar). Furthermore, AM67 and sp56 are soluble at low pH, a commonly used condition for the extraction of acrosomal proteins (45Meizel S. Mukerji S.K. Biol. Reprod. 1976; 14: 444-450Crossref PubMed Scopus (46) Google Scholar). However, our finding that AM67 is intracellular contrasts with previous reports characterizing mouse sp56 as a cell-surface protein (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 46Bleil J.D. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5563-5567Crossref PubMed Scopus (183) Google Scholar).To examine the discrepancy in the localizations of AM67 and sp56, we used the post-embedding immunoelectron microscopic method, but found that both AM67 and sp56 were localized to the acrosomal contents in mature guinea pig and mouse sperm, respectively, while neither was observed on the plasma membrane. It seems unlikely that there is a pool of sp56 on the mouse sperm surface that was not detected by this method since both surface and intracellular antigens should be detected equally well. This conclusion was further substantiated by results from pre-embedding immunoelectron microscopy, a standard method used to localize cell-surface antigens, showing that sp56 was associated with acrosomal material in damaged sperm, but not on the plasma membrane (data not shown). It is possible that the colloidal gold labeling of sp56 interpreted as surface labeling by the immunogold surface replica method (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar) could, in fact, be due to the exposure of acrosomal sp56 on the surface by permeabilization of the outer acrosomal and plasma membranes. The outer acrosomal membrane and the plasma membrane over the acrosome are known to be susceptible to mechanical and chemical disruption, and these membranes are also destabilized during capacitation (47Langlais J. Zollinger M. Plante L. Chapdelaine A. Bleau G. Roberts K.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7266-7270Crossref PubMed Scopus (121) Google Scholar). Furthermore, the mouse sperm utilized for the localization of sp56 by the immunogold surface replica technique were capacitated and lightly fixed with 1% paraformaldehyde, but not embedded prior to immunolabeling (36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar). Since formaldehyde fixation is reversible (48Barka T. Anderson P.J. Histochemistry: Theory, Practice, and Bibliography. Harper & Row Publishers, Inc., New York1963: 28-29Google Scholar), it appears that the “surface labeling” of sp56 may actually be attributable to the exposure of intra-acrosomal sp56 caused by the permeabilization, rupture, or partial fusion of the destabilized outer acrosomal and plasma membranes during the course of the sample manipulations.These results do not negate the results showing the ability of mouse sp56 to bind to the zona pellucida, but should cause us to reassess our concepts regarding sperm capacitation, sperm-egg recognition, and the acrosome reaction. Evidence from a variety of systems indicates that small dynamic fusion pores form in a common bilayer created by the apposing leaflets of the vesicular and plasmalemmal membranes (reviewed by Monck and Fernandez (49Monck J.R. Fernandez J.M. Curr. Opin. Cell Biol. 1996; 8: 524-533Crossref PubMed Scopus (119) Google Scholar)). These small pores are thought to occasionally close after the release of small non-quantal amounts of secretory products. In an application of this “dynamic fusion pore” hypothesis to sperm, capacitation may represent a state where the small fusion pores are dynamically opening and closing between the outer acrosomal and plasma membranes, thereby exposing acrosomal components. Thus, it might be possible that a capacitated sperm encountering an egg may actually bind to the zona via the transiently exposed acrosomal proteins, not a cell-surface molecule. Binding to the zona in this manner might “lock” the fusion pore into the static open state, driving exocytosis to completion.In consideration of the resistance of the acrosomal matrix to Triton X-100 solubilization, we propose that acrosomal matrix components such as AM67 and AM50 help form the dense core of the acrosome and establish the different compartments (M1, M2, and M3 domains) within this organelle. As proposed for the dynamic fusion pore hypothesis, the relative distribution of these proteins may position them for binding to other molecules such as the zona pellucida glycoproteins, and the affinity of AM67 and AM50 for other acrosomal components may regulate the differential release of certain secretory products following the acrosome reaction. Other acrosomal proteins such as dipeptidyl peptidase, autoantigen 1, and soluble hyaluronidase may not associate tightly with the matrix, and thus, they would be freely released following the acrosome reaction (5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 6de Vries J.W.A. Willemson R. Geuze H.J. Eur. J. Biochem. 1985; 37: 81-88Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar, 34Talbot P. DiCarlantonio G. J. Histochem. Cytochem. 1985; 33: 1169-1172Crossref PubMed Scopus (35) Google Scholar). Proteins such as proacrosin would be more firmly associated with certain components of the matrix (e.g. AM67, AM50, and proacrosin-binding protein) (50Green D.P.L. Hockaday A.R. J. Cell Sci. 1978; 32: 177-184PubMed Google Scholar, 51Green D.P.L. J. Cell Sci. 1978; 32: 153-164PubMed Google Scholar, 52Green D.P.L. J. Cell Sci. 1978; 32: 137-157PubMed Google Scholar). The gradual dissolution of the matrix may result from the dissociation of matrix components resulting from partial proteolysis following the progressive conversion of the zymogen proacrosin to enzymatically active acrosin. With the activation of proteolysis, AM50 would be processed to AM50AR by proteolysis, leading to the destabilization of the AM50 oligomers and continued dissolution of the matrix (12Westbrook-Case V.A. Winfrey V.P. Olson G.E. Biol. Reprod. 1994; 51: 1-13Crossref PubMed Scopus (43) Google Scholar). This working hypothesis opens up new avenues for examining the release of materials following acrosomal exocytosis. In studies in progress, the binding properties of AM67 and AM50 are being examined to identify ligands for these large oligomers and to determine their roles in acrosomal biogenesis and fertilization. The sperm acrosome reaction has the characteristic hallmarks of regulated secretion: 1) secretory products are concentrated and condensed; 2) secretory granules are stored for long periods of time; and 3) secretion is coupled to an extracellular stimulus (in this case, the zona pellucida) (1Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (743) Google Scholar). Regulated secretion from sperm (i.e. the acrosome reaction) is obligatory for fertilization (2Kopf G.S. Gerton G.L. Wassarman P.M. Elements of Mammalian Fertilization.1. CRC Press, Inc., Boca Raton, FL1991: 153-203Google Scholar). It is presumed that the release of specific secretory components from the acrosome is involved in assisting the sperm in the penetration of the investments surrounding the egg. Furthermore, although sperm contain only one secretory vesicle, different enzymatic activities are released from sperm at various times following the acrosome reaction (3Talbot P. Franklin L.E. J. Reprod. Fertil. 1974; 39: 429-432Crossref PubMed Google Scholar, 4Green D.P. J. Cell Sci. 1978; 32: 153-164PubMed Google Scholar, 5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 6de Vries J.W.A. Willemson R. Geuze H.J. Eur. J. Biochem. 1985; 37: 81-88Google Scholar, 7Zao P.Z. Meizel S. Talbot P. J. Exp. Zool. 1985; 234: 63-74Crossref PubMed Scopus (28) Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar). The differential temporal release of secretory components occurs in other regulated secretory tissues such as the pancreas, but since cells of such tissues contain multiple secretory granules, it has been proposed that this “non-parallel secretion” is due to exocytosis from heterogeneous sources within the tissue or cell of study (9Adelson J.W. Miller P.E. Science. 1985; 228: 993-996Crossref PubMed Scopus (38) Google Scholar). In guinea pig sperm, the acrosome reaction results in the differential release of acrosomal components possibly due to their compartmentalization within soluble or insoluble phases of the acrosome (5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar). Following the exocytotic acrosome reaction, certain acrosomal proteins, such as proacrosin, remain associated with the matrix that establishes the “dense core” of the acrosome and are released at a slower rate than components, such as dipeptidyl peptidase, that are found in a more readily solubilized compartment. The results presented here and in recent papers (10Noland T.D. Olson G.E. Biol. Reprod. 1989; 40: 1057-1066Crossref PubMed Scopus (10) Google Scholar, 11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 12Westbrook-Case V.A. Winfrey V.P. Olson G.E. Biol. Reprod. 1994; 51: 1-13Crossref PubMed Scopus (43) Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar, 14Westbrook-Case V.A. Winfrey V.P. Olson G.E. Dev. Biol. 1995; 167: 338-349Crossref PubMed Scopus (32) Google Scholar) demonstrate that two of the components of the dense core (matrix) of the acrosome, AM50 and AM67, represent monomers of large homopolymeric proteins. AM50 has been shown to be a novel, testis-specific member of the pentraxin family of proteins (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar). Preliminary tryptic peptide sequencing indicated that AM67 is related to members of the complement-binding protein family (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar). In this paper, we show that AM67 is, indeed, a member of this class of proteins. Furthermore, the amino acid sequence of AM67 is most closely related to that of the mouse sperm protein sp56. This protein is a candidate for the cell-surface receptor of ZP3, a glycoprotein of the egg's extracellular matrix, the zona pellucida (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar). A standard post-embedding immunoelectron microscopic technique demonstrated that AM67 is restricted to a specific dorsal compartment (M1 domain of the apical segment) within the acrosome of guinea pig sperm, while mouse sp56 was detected only within the acrosome and not on the plasma membrane over the acrosome as was previously reported using an unconventional colloidal gold surface replica immunoelectron microscopic procedure (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar). DISCUSSIONThis study demonstrates that the guinea pig sperm acrosome contains a testis-specific protein related to mouse sperm sp56 and other members of the complement regulatory protein superfamily. This result is reminiscent of our previous reports concerning the acrosomal matrix protein AM50, which was found to be a member of the pentraxin superfamily (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar). Like AM50, AM67 was found to be a testis-specific member of a protein superfamily, was localized to a specific domain within the acrosome, and also formed high molecular weight homopolymers. Similar to C4BPα, we estimate that six to eight monomers of AM67 oligomerize to constitute the unreduced form of the protein (M r ∼380,000). Based upon the C4BPα sequence homology, AM67 might be predicted to form calcium-dependent complexes with other ligands within the acrosome or, after exocytosis, with extracellular ligands. Comparable calcium-dependent binding of AM50 to the apical segment complex (containing the acrosomal matrix) has already been demonstrated (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar).Of all the sequences found to be homologous to AM67, mouse sperm protein sp56 and the serum complement component 4-binding proteins within the complement regulatory protein superfamily ranked the highest in a BLAST search of GenBank™ sequences (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar). The significance of this is not clear at the present time; but it is noteworthy that serum C4BP has been shown to interact with serum pentraxins (C-reactive protein and serum amyloid P component) (38Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1991; 266: 12896-12901Abstract Full Text PDF PubMed Google Scholar, 39Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1990; 265: 21749-21757Abstract Full Text PDF PubMed Google Scholar, 40Garcı́a de Frutos P. Dahlbäck B. J. Immunol. 1994; 152: 2430-2436PubMed Google Scholar), and thus, AM67 might be predicted to interact with AM50. Although immunoelectron microscopy demonstrated that AM67 and AM50 are localized in distinct regions of the acrosomal matrix of acrosome-intact sperm, it is possible that these components could interact following the acrosome reaction. C4BPα and the pentraxins are known to bind to plasma membranes, and based upon the homologies to these proteins, AM67 and AM50 might also be expected to interact with components of the acrosomal membranes. If AM67 and AM50 bind to different ligands of the acrosomal membrane, aggregation of these proteins with their ligands during acrosome biogenesis may help to establish the M1 and M3 domains. Additionally, AM50 and AM67 may interact with membranes or react with each other upon their release following the acrosome reaction and the dissolution of the acrosomal matrix.AM67 is not the first member of the complement regulatory protein superfamily to be found in sperm. Additional members recently identified include another guinea pig sperm C4BP distinct from AM67 (13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar) as well as decay-accelerating factor (CD55), membrane cofactor protein (CD46), and protectin (CD59), which have been shown to be membrane-bound components of the sperm acrosomal region (41Rooney I.A. Oglesby T.J. Atkinson J.P. Immunol. Res. 1993; 12: 276-294Crossref PubMed Scopus (80) Google Scholar). Clusterin, another regulator of complement, is secreted by the Sertoli cells and the epididymal epithelium and has also been shown to be associated with sperm (42Sylvester S.R. Morales C. Oko R. Griswold M.D. Biol. Reprod. 1991; 45: 195-207Crossref PubMed Scopus (139) Google Scholar, 43Law G.L. Griswold M.D. Biol. Reprod. 1994; 50: 669-679Crossref PubMed Scopus (43) Google Scholar). The roles of these complement regulatory proteins in fertilization and reproductive immunology are debatable at this point, but several of them appear to be present in sperm-specific forms. The testis-specific expression of AM67 is also intriguing considering C4BPα proteins are generally considered to be liver-specific proteins.The very remarkable similarity of the amino acid sequence of guinea pig AM67 to that of mouse sp56 suggests that these proteins are orthologues. Although the apparent molecular weights of the native AM67 and sp56 multimeric proteins were ∼380,000 and 110,000, respectively (14Westbrook-Case V.A. Winfrey V.P. Olson G.E. Dev. Biol. 1995; 167: 338-349Crossref PubMed Scopus (32) Google Scholar, 15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar), these sizes are similar to the differences in the properties of the C4BPs from other mammalian species compared with mouse (44Barnum S.R. Immunol. Res. 1991; 10: 28-42Crossref PubMed Scopus (16) Google Scholar). Furthermore, AM67 and sp56 are soluble at low pH, a commonly used condition for the extraction of acrosomal proteins (45Meizel S. Mukerji S.K. Biol. Reprod. 1976; 14: 444-450Crossref PubMed Scopus (46) Google Scholar). However, our finding that AM67 is intracellular contrasts with previous reports characterizing mouse sp56 as a cell-surface protein (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 46Bleil J.D. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5563-5567Crossref PubMed Scopus (183) Google Scholar).To examine the discrepancy in the localizations of AM67 and sp56, we used the post-embedding immunoelectron microscopic method, but found that both AM67 and sp56 were localized to the acrosomal contents in mature guinea pig and mouse sperm, respectively, while neither was observed on the plasma membrane. It seems unlikely that there is a pool of sp56 on the mouse sperm surface that was not detected by this method since both surface and intracellular antigens should be detected equally well. This conclusion was further substantiated by results from pre-embedding immunoelectron microscopy, a standard method used to localize cell-surface antigens, showing that sp56 was associated with acrosomal material in damaged sperm, but not on the plasma membrane (data not shown). It is possible that the colloidal gold labeling of sp56 interpreted as surface labeling by the immunogold surface replica method (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar) could, in fact, be due to the exposure of acrosomal sp56 on the surface by permeabilization of the outer acrosomal and plasma membranes. The outer acrosomal membrane and the plasma membrane over the acrosome are known to be susceptible to mechanical and chemical disruption, and these membranes are also destabilized during capacitation (47Langlais J. Zollinger M. Plante L. Chapdelaine A. Bleau G. Roberts K.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7266-7270Crossref PubMed Scopus (121) Google Scholar). Furthermore, the mouse sperm utilized for the localization of sp56 by the immunogold surface replica technique were capacitated and lightly fixed with 1% paraformaldehyde, but not embedded prior to immunolabeling (36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar). Since formaldehyde fixation is reversible (48Barka T. Anderson P.J. Histochemistry: Theory, Practice, and Bibliography. Harper & Row Publishers, Inc., New York1963: 28-29Google Scholar), it appears that the “surface labeling” of sp56 may actually be attributable to the exposure of intra-acrosomal sp56 caused by the permeabilization, rupture, or partial fusion of the destabilized outer acrosomal and plasma membranes during the course of the sample manipulations.These results do not negate the results showing the ability of mouse sp56 to bind to the zona pellucida, but should cause us to reassess our concepts regarding sperm capacitation, sperm-egg recognition, and the acrosome reaction. Evidence from a variety of systems indicates that small dynamic fusion pores form in a common bilayer created by the apposing leaflets of the vesicular and plasmalemmal membranes (reviewed by Monck and Fernandez (49Monck J.R. Fernandez J.M. Curr. Opin. Cell Biol. 1996; 8: 524-533Crossref PubMed Scopus (119) Google Scholar)). These small pores are thought to occasionally close after the release of small non-quantal amounts of secretory products. In an application of this “dynamic fusion pore” hypothesis to sperm, capacitation may represent a state where the small fusion pores are dynamically opening and closing between the outer acrosomal and plasma membranes, thereby exposing acrosomal components. Thus, it might be possible that a capacitated sperm encountering an egg may actually bind to the zona via the transiently exposed acrosomal proteins, not a cell-surface molecule. Binding to the zona in this manner might “lock” the fusion pore into the static open state, driving exocytosis to completion.In consideration of the resistance of the acrosomal matrix to Triton X-100 solubilization, we propose that acrosomal matrix components such as AM67 and AM50 help form the dense core of the acrosome and establish the different compartments (M1, M2, and M3 domains) within this organelle. As proposed for the dynamic fusion pore hypothesis, the relative distribution of these proteins may position them for binding to other molecules such as the zona pellucida glycoproteins, and the affinity of AM67 and AM50 for other acrosomal components may regulate the differential release of certain secretory products following the acrosome reaction. Other acrosomal proteins such as dipeptidyl peptidase, autoantigen 1, and soluble hyaluronidase may not associate tightly with the matrix, and thus, they would be freely released following the acrosome reaction (5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 6de Vries J.W.A. Willemson R. Geuze H.J. Eur. J. Biochem. 1985; 37: 81-88Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar, 34Talbot P. DiCarlantonio G. J. Histochem. Cytochem. 1985; 33: 1169-1172Crossref PubMed Scopus (35) Google Scholar). Proteins such as proacrosin would be more firmly associated with certain components of the matrix (e.g. AM67, AM50, and proacrosin-binding protein) (50Green D.P.L. Hockaday A.R. J. Cell Sci. 1978; 32: 177-184PubMed Google Scholar, 51Green D.P.L. J. Cell Sci. 1978; 32: 153-164PubMed Google Scholar, 52Green D.P.L. J. Cell Sci. 1978; 32: 137-157PubMed Google Scholar). The gradual dissolution of the matrix may result from the dissociation of matrix components resulting from partial proteolysis following the progressive conversion of the zymogen proacrosin to enzymatically active acrosin. With the activation of proteolysis, AM50 would be processed to AM50AR by proteolysis, leading to the destabilization of the AM50 oligomers and continued dissolution of the matrix (12Westbrook-Case V.A. Winfrey V.P. Olson G.E. Biol. Reprod. 1994; 51: 1-13Crossref PubMed Scopus (43) Google Scholar). This working hypothesis opens up new avenues for examining the release of materials following acrosomal exocytosis. In studies in progress, the binding properties of AM67 and AM50 are being examined to identify ligands for these large oligomers and to determine their roles in acrosomal biogenesis and fertilization. This study demonstrates that the guinea pig sperm acrosome contains a testis-specific protein related to mouse sperm sp56 and other members of the complement regulatory protein superfamily. This result is reminiscent of our previous reports concerning the acrosomal matrix protein AM50, which was found to be a member of the pentraxin superfamily (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar, 13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar). Like AM50, AM67 was found to be a testis-specific member of a protein superfamily, was localized to a specific domain within the acrosome, and also formed high molecular weight homopolymers. Similar to C4BPα, we estimate that six to eight monomers of AM67 oligomerize to constitute the unreduced form of the protein (M r ∼380,000). Based upon the C4BPα sequence homology, AM67 might be predicted to form calcium-dependent complexes with other ligands within the acrosome or, after exocytosis, with extracellular ligands. Comparable calcium-dependent binding of AM50 to the apical segment complex (containing the acrosomal matrix) has already been demonstrated (11Noland T.D. Friday B.B. Maulit M.T. Gerton G.L. J. Biol. Chem. 1994; 269: 32607-32614Abstract Full Text PDF PubMed Google Scholar). Of all the sequences found to be homologous to AM67, mouse sperm protein sp56 and the serum complement component 4-binding proteins within the complement regulatory protein superfamily ranked the highest in a BLAST search of GenBank™ sequences (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar). The significance of this is not clear at the present time; but it is noteworthy that serum C4BP has been shown to interact with serum pentraxins (C-reactive protein and serum amyloid P component) (38Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1991; 266: 12896-12901Abstract Full Text PDF PubMed Google Scholar, 39Schwalbe R.A. Dahlbäck B. Nelsestuen G.L. J. Biol. Chem. 1990; 265: 21749-21757Abstract Full Text PDF PubMed Google Scholar, 40Garcı́a de Frutos P. Dahlbäck B. J. Immunol. 1994; 152: 2430-2436PubMed Google Scholar), and thus, AM67 might be predicted to interact with AM50. Although immunoelectron microscopy demonstrated that AM67 and AM50 are localized in distinct regions of the acrosomal matrix of acrosome-intact sperm, it is possible that these components could interact following the acrosome reaction. C4BPα and the pentraxins are known to bind to plasma membranes, and based upon the homologies to these proteins, AM67 and AM50 might also be expected to interact with components of the acrosomal membranes. If AM67 and AM50 bind to different ligands of the acrosomal membrane, aggregation of these proteins with their ligands during acrosome biogenesis may help to establish the M1 and M3 domains. Additionally, AM50 and AM67 may interact with membranes or react with each other upon their release following the acrosome reaction and the dissolution of the acrosomal matrix. AM67 is not the first member of the complement regulatory protein superfamily to be found in sperm. Additional members recently identified include another guinea pig sperm C4BP distinct from AM67 (13Reid M. Blobel C.P. J. Biol. Chem. 1994; 269: 32615-32620Abstract Full Text PDF PubMed Google Scholar) as well as decay-accelerating factor (CD55), membrane cofactor protein (CD46), and protectin (CD59), which have been shown to be membrane-bound components of the sperm acrosomal region (41Rooney I.A. Oglesby T.J. Atkinson J.P. Immunol. Res. 1993; 12: 276-294Crossref PubMed Scopus (80) Google Scholar). Clusterin, another regulator of complement, is secreted by the Sertoli cells and the epididymal epithelium and has also been shown to be associated with sperm (42Sylvester S.R. Morales C. Oko R. Griswold M.D. Biol. Reprod. 1991; 45: 195-207Crossref PubMed Scopus (139) Google Scholar, 43Law G.L. Griswold M.D. Biol. Reprod. 1994; 50: 669-679Crossref PubMed Scopus (43) Google Scholar). The roles of these complement regulatory proteins in fertilization and reproductive immunology are debatable at this point, but several of them appear to be present in sperm-specific forms. The testis-specific expression of AM67 is also intriguing considering C4BPα proteins are generally considered to be liver-specific proteins. The very remarkable similarity of the amino acid sequence of guinea pig AM67 to that of mouse sp56 suggests that these proteins are orthologues. Although the apparent molecular weights of the native AM67 and sp56 multimeric proteins were ∼380,000 and 110,000, respectively (14Westbrook-Case V.A. Winfrey V.P. Olson G.E. Dev. Biol. 1995; 167: 338-349Crossref PubMed Scopus (32) Google Scholar, 15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar), these sizes are similar to the differences in the properties of the C4BPs from other mammalian species compared with mouse (44Barnum S.R. Immunol. Res. 1991; 10: 28-42Crossref PubMed Scopus (16) Google Scholar). Furthermore, AM67 and sp56 are soluble at low pH, a commonly used condition for the extraction of acrosomal proteins (45Meizel S. Mukerji S.K. Biol. Reprod. 1976; 14: 444-450Crossref PubMed Scopus (46) Google Scholar). However, our finding that AM67 is intracellular contrasts with previous reports characterizing mouse sp56 as a cell-surface protein (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 46Bleil J.D. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5563-5567Crossref PubMed Scopus (183) Google Scholar). To examine the discrepancy in the localizations of AM67 and sp56, we used the post-embedding immunoelectron microscopic method, but found that both AM67 and sp56 were localized to the acrosomal contents in mature guinea pig and mouse sperm, respectively, while neither was observed on the plasma membrane. It seems unlikely that there is a pool of sp56 on the mouse sperm surface that was not detected by this method since both surface and intracellular antigens should be detected equally well. This conclusion was further substantiated by results from pre-embedding immunoelectron microscopy, a standard method used to localize cell-surface antigens, showing that sp56 was associated with acrosomal material in damaged sperm, but not on the plasma membrane (data not shown). It is possible that the colloidal gold labeling of sp56 interpreted as surface labeling by the immunogold surface replica method (15Cheng A. Le T. Palacios M. Bookbinder L.H. Wassarman P.M. Suzuki F. Bleil J.D. J. Cell Biol. 1994; 125: 867-878Crossref PubMed Scopus (143) Google Scholar, 36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar) could, in fact, be due to the exposure of acrosomal sp56 on the surface by permeabilization of the outer acrosomal and plasma membranes. The outer acrosomal membrane and the plasma membrane over the acrosome are known to be susceptible to mechanical and chemical disruption, and these membranes are also destabilized during capacitation (47Langlais J. Zollinger M. Plante L. Chapdelaine A. Bleau G. Roberts K.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7266-7270Crossref PubMed Scopus (121) Google Scholar). Furthermore, the mouse sperm utilized for the localization of sp56 by the immunogold surface replica technique were capacitated and lightly fixed with 1% paraformaldehyde, but not embedded prior to immunolabeling (36Suzuki-Toyota F. Maekawa M. Cheng A. Bleil J.D. J. Electron Microsc. 1995; 44: 135-139PubMed Google Scholar). Since formaldehyde fixation is reversible (48Barka T. Anderson P.J. Histochemistry: Theory, Practice, and Bibliography. Harper & Row Publishers, Inc., New York1963: 28-29Google Scholar), it appears that the “surface labeling” of sp56 may actually be attributable to the exposure of intra-acrosomal sp56 caused by the permeabilization, rupture, or partial fusion of the destabilized outer acrosomal and plasma membranes during the course of the sample manipulations. These results do not negate the results showing the ability of mouse sp56 to bind to the zona pellucida, but should cause us to reassess our concepts regarding sperm capacitation, sperm-egg recognition, and the acrosome reaction. Evidence from a variety of systems indicates that small dynamic fusion pores form in a common bilayer created by the apposing leaflets of the vesicular and plasmalemmal membranes (reviewed by Monck and Fernandez (49Monck J.R. Fernandez J.M. Curr. Opin. Cell Biol. 1996; 8: 524-533Crossref PubMed Scopus (119) Google Scholar)). These small pores are thought to occasionally close after the release of small non-quantal amounts of secretory products. In an application of this “dynamic fusion pore” hypothesis to sperm, capacitation may represent a state where the small fusion pores are dynamically opening and closing between the outer acrosomal and plasma membranes, thereby exposing acrosomal components. Thus, it might be possible that a capacitated sperm encountering an egg may actually bind to the zona via the transiently exposed acrosomal proteins, not a cell-surface molecule. Binding to the zona in this manner might “lock” the fusion pore into the static open state, driving exocytosis to completion. In consideration of the resistance of the acrosomal matrix to Triton X-100 solubilization, we propose that acrosomal matrix components such as AM67 and AM50 help form the dense core of the acrosome and establish the different compartments (M1, M2, and M3 domains) within this organelle. As proposed for the dynamic fusion pore hypothesis, the relative distribution of these proteins may position them for binding to other molecules such as the zona pellucida glycoproteins, and the affinity of AM67 and AM50 for other acrosomal components may regulate the differential release of certain secretory products following the acrosome reaction. Other acrosomal proteins such as dipeptidyl peptidase, autoantigen 1, and soluble hyaluronidase may not associate tightly with the matrix, and thus, they would be freely released following the acrosome reaction (5Hardy D.M. Oda M.N. Friend D.S. Huang Jr., T.T.F. Biochem. J. 1991; 275: 759-766Crossref PubMed Scopus (105) Google Scholar, 6de Vries J.W.A. Willemson R. Geuze H.J. Eur. J. Biochem. 1985; 37: 81-88Google Scholar, 8DiCarlantonio G. Talbot P. Gamete Res. 1988; 21: 425-438Crossref PubMed Scopus (35) Google Scholar, 34Talbot P. DiCarlantonio G. J. Histochem. Cytochem. 1985; 33: 1169-1172Crossref PubMed Scopus (35) Google Scholar). Proteins such as proacrosin would be more firmly associated with certain components of the matrix (e.g. AM67, AM50, and proacrosin-binding protein) (50Green D.P.L. Hockaday A.R. J. Cell Sci. 1978; 32: 177-184PubMed Google Scholar, 51Green D.P.L. J. Cell Sci. 1978; 32: 153-164PubMed Google Scholar, 52Green D.P.L. J. Cell Sci. 1978; 32: 137-157PubMed Google Scholar). The gradual dissolution of the matrix may result from the dissociation of matrix components resulting from partial proteolysis following the progressive conversion of the zymogen proacrosin to enzymatically active acrosin. With the activation of proteolysis, AM50 would be processed to AM50AR by proteolysis, leading to the destabilization of the AM50 oligomers and continued dissolution of the matrix (12Westbrook-Case V.A. Winfrey V.P. Olson G.E. Biol. Reprod. 1994; 51: 1-13Crossref PubMed Scopus (43) Google Scholar). This working hypothesis opens up new avenues for examining the release of materials following acrosomal exocytosis. In studies in progress, the binding properties of AM67 and AM50 are being examined to identify ligands for these large oligomers and to determine their roles in acrosomal biogenesis and fertilization. We thank Drs. Stuart B. Moss, Gregory S. Kopf, and Bayard T. Storey for comments and critical evaluation of the manuscript. We also greatly appreciate the preparation of capacitated mouse sperm by Dr. Cindi Ward and Sonya Martin." @default.
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W2034281339 title "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" @default.
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