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We previously identified an insoluble 50-kDa acrosomal matrix protein (AM50) localized to the ventral region of the guinea pig sperm apical segment. AM50 is converted to a 42-kDa polypeptide and released during matrix dispersion in acrosome-reacting sperm. This study examines the sorting pathways and assembly processes which generate the domain-specific distribution of AM50 in the acrosome. AM50 was expressed during early acrosome development and localized to the matrix of proacrosomal granules and the acrosomal vesicle. Initial sorting of AM50 occurred in Golgi phase spermatids, where it became concentrated in the matrix surrounding the acrosomal granule. AM50 remained restricted to the apical segment of acrosome phase spermatids and was finally sorted to the ventral matrix of the apical segment in maturation phase spermatids. By reducing SDS-PAGE testicular AM50 exhibited a slightly higher Mr of 52 kDa than the 50-kDa form of cauda epididymal spermatozoa. Nonreducing SDS-PAGE demonstrated that testicular and epididymal AM50 were assembled into homomeric complexes of 480 and 450 kDa respectively. Cauda epididymal sperm apical segments contained a second disulfide-cross-linked homomeric complex of 520 kDa composed of a 68-kDa subunit. These studies indicate that AM50 is first assembled into a disulfide-cross-linked complex and subsequently processed into mature AM50. These data also suggest that disulfide-linked complexes of different structural proteins may assemble into distinct acrosomal matrix compartments.

The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35-kD and 52-kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase-separation analysis with Triton X-114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore-induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. © 1994 Wiley-Liss, Inc.

The sperm acrosome contains a variety of hydrolytic enzymes that exhibit differential release during the acrosome reaction. The interaction between hydrolases and the acrosomal matrix, and their compartmentalization into chemically unique matrix domains may represent mechanisms by which this ordered release is achieved. This study characterizes an acrosomal matrix protein that is restricted to the ventral-most region of the apical segment of guinea pig cauda epididymal spermatozoa. Polyclonal antiserum, prepared against an SDS-PAGE-purified apical segment protein, recognized a 50-kDa band, termed AM50, on Western blots. Native AM50 is resistant to solubilization. However, during the acrosome reaction, AM50 is converted into a 42-43-kDa doublet protein (AM50AR) and released into the incubation medium. AM50 and AM50AR are not recognized by antiserum to guinea pig proacrosin and do not exhibit protease activity in a gelatinolytic SDS-PAGE assay. However, AM50AR does bind to p-aminobenzamidine affinity columns, suggesting that it may remain associated with proteases after the acrosome reaction. Light and electron microscopic immunocytochemistry established that AM50 was exclusively localized to the ventral aspect of the apical segment of the acrosome.

The anti-sperm monoclonal antibody (mAb) S19 was previously demonstrated to agglutinate human spermatozoa, inhibit sperm penetration of cervical mucus, and inhibit sperm-zona pellucida binding. These results implicated the cognate S19 antigen, designated sperm agglutination antigen-1 (SAGA-1), in gamete interactions and identified SAGA-1 as an attractive candidate for immunocontraceptive development. In the present study, evaluation of sperm agglutination with video microscopy showed that the S19 mAb rapidly and completely agglutinated human spermatozoa in a "tangled" pattern of agglutination. One- and two-dimensional immunoblot analyses identified SAGA-1 as a highly acidic, polymorphic sperm protein with an apparent molecular mass of 15-25 kDa and an isoelectric point of 2.5-3.0. Periodate treatment abolished this immunoreactivity, demonstrating that the S19 mAb reacted with a carbohydrate epitope and indicating that SAGA-1 is a glycoprotein. Absence of S19 immunoreactivity in postvasectomy seminal fluid implicated the testis, epididymis, and/or proximal vas deferens in the expression of SAGA-1. In solubility and phase partitioning assays, SAGA-1 was extracted from spermatozoa in Triton X-114 and exhibited the hydrophobic characteristics of integral and glycosylphosphatidyl inositol-anchored membrane proteins. These results identify SAGA-1 as a hydrophobic, highly acidic sperm glycoprotein that is localized on the entire sperm surface and has potential significance as a target for antibodies that inhibit sperm function and gamete interactions.

The sperm acrosome contains a variety of hydrolytic enzymes that exhibit differential release during the acrosome reaction. The interaction between hydrolases and the acrosomal matrix, and their compartmentalization into chemically unique matrix domains may represent mechanisms by which this ordered release is achieved. This study characterizes an acrosomal matrix protein that is restricted to the ventral-most region of the apical segment of guinea pig cauda epididymal spermatozoa. Polyclonal antiserum, prepared against an SDS-PAGE-purified apical segment protein, recognized a 50-kDa band, termed AM50, on Western blots. Native AM50 is resistant to solubilization. However, during the acrosome reaction, AM50 is converted into a 42-43-kDa doublet protein (AM50AR) and released into the incubation medium. AM50 and AM50AR are not recognized by antiserum to guinea pig proacrosin and do not exhibit protease activity in a gelatinolytic SDS-PAGE assay. However, AM50AR does bind to p-aminobenzamidine affinity columns, suggesting that it may remain associated with proteases after the acrosome reaction. Light and electron microscopic immunocytochemistry established that AM50 was exclusively localized to the ventral aspect of the apical segment of the acrosome.