Isolation and characterization of MPM-2-reactive sperm proteins: homology to components of the outer dense fibers and segmented columns.
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abstract
Sperm from most mammalian species catalyze the formation of an aster of microtubules in the oocyte after fertilization. One component that may be involved in the regulation of sperm centrosomal activity in the oocyte is a phosphorylated protein complex (MPM-2-reactive sperm protein; MSP) with a molecular mass of 77-85 kDa identified by the MPM-2 antibody. The objective of this study was to compare the MSPs to a previously identified 85-kDa complex (ODF/CP85) that is a component of the outer dense fibers of the sperm midpiece and the segmented columns of the connecting piece. MSPs were isolated from boar sperm using a differential extraction procedure and preparative gel electrophoresis. Three mouse monoclonal and rabbit polyclonal antibodies were made to the isolated complex, and these antibodies labeled similar proteins in rabbit, bull, boar, and mouse sperm. Extraction and solubilization procedures for MSPs and ODF/CP85 required harsh chaotropic and reducing conditions. In addition to migrating at the same molecular mass on gels, proteins from each preparation labeled with MPM-2, an anti-ODF/CP antibody, and the anti-MSP antibody prepared in this study. Amino acid composition was similar to that reported previously for rat and bull ODF/CP85. Furthermore, immunolocalization by both fluorescent and transmission electron microscopy indicated that the MSPs are components of the outer dense fibers and probably the segmented columns of the connecting piece. Taken together, these results indicate that the MSPs are the previously identified 85-kDa complex of the outer dense fibers and connecting piece. Therefore, it is likely that any involvement of these proteins in the regulation of sperm centrosomal activity is through the process of connecting piece disassembly.
