Co-localization of the inositol 1,4,5-trisphosphate receptor and calreticulin in the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa.

Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.

Molecular mapping of thrombin-receptor interactions.

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G-protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90-fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies.

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Sperm mitochondria-associated cysteine-rich protein (SMCP) is an autoantigen in Lewis rats.

A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22-25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.

Post-obstruction rat sperm autoantigens identified by two-dimensional gel electrophoresis and western blotting.

Although antisperm autoantibody responses to obstruction of the male reproductive system have been documented, information on the nature of the cognate sperm autoantigens has been limited. In the present study, the patterns of sperm autoantigens recognized by sera from rats after obstruction of the vas deferens or epididymis were studied by high resolution two-dimensional (2-D) gel electrophoresis and western blotting. Comparisons of patterns of autoantigens stained on 2-D western blots of sera from prepubertal vasectomy, prepubertal epididymal ligation and adult vasectomy groups revealed both similarities and differences. Sera from sham-operated animals showed no detectable reaction or much lighter staining of a small number of spots. Visualization of sperm autoantigens on 2-D western blots supported the hypothesis that there is a relatively small set of sperm proteins that can be regarded as dominant post-obstruction sperm autoantigens because they are recognized by multiple post-obstruction sera. The 2-D analysis revealed previously undetected distinctions in the autoantigens recognized after adult and prepubertal vasectomy, as well as variations with the site of obstruction. These differences in the response may be due in part to changes in antigens of spermatozoa in different parts of the tract and at different ages, as well as variations in exposure of sperm cell proteins to the immune system resulting from the sites of spermatic granulomas. Preparative 2-D gels and western blotting with post-obstruction sera are now being used to identify specific sperm autoantigens by microsequencing of selected proteins.

Distribution of leukocytes in the epithelium and interstitium of four regions of the Lewis rat epididymis.

BACKGROUND: Leukocytes expressing different surface markers were studied in four regions of the epididymis of Lewis rats. Cells resembling lymphocytes or monocytes had been described in the epididymis, but previous studies differed as to their nature and immunologic significance. METHODS: Frozen sections were immunocytochemically stained with monoclonal antibodies W3/25, OX-8, OX-42, and RLN-9D3, which are directed toward markers on CD4+ cells, CD8+ cells, macrophages, and B lymphocytes, respectively. The concentration of stained cells in the epithelium and interstitial tissue of the initial segment, caput, proximal cauda, and distal cauda regions was determined by a procedure based on the optical disector method. RESULTS: CD4+ leukocytes were present in greater concentration than CD8+ cells or macrophages in both the epithelium and interstitial tissue of all four regions. In the epithelium, the concentration of CD8+ leukocytes was greater than that of macrophages in the initial segment, caput, and distal cauda. In the interstitium, however, the concentration of macrophages exceeded that of CD8+ cells in both parts of the cauda. Macrophages and T lymphocytes were generally present in greater concentrations in the interstitium than in the epithelium, especially in the more proximal parts of the epididymis. In contrast to T cells, B lymphocytes were not detected in the interstitium or epithelium of any of part of the epididymis, despite prominent staining of B cells in other locations. CONCLUSIONS: The epididymal epithelium of the Lewis rat contains many T lymphocytes, which may correspond to 'halo' cells. CD4+ leukocytes predominate in all regions of the epididymis. The interstitium may function as a reservoir of leukocytes for the epithelial compartment. The epididymis is not normally a site for local immunoglobulin synthesis.

Antisperm autoantibody responses to vasectomy and vasovasostomy in Fischer and Lewis rats.

Antisperm autoantibodies were studied in Fischer and Lewis strains of rats after either vasectomy, vasectomy followed one month later by vasovasostomy, or sham operations. The time course of antibody response to sperm protein autoantigens was assayed by Western blot analysis of sera obtained at intervals up to 3 months. Rats of both strains responded to immunization with isologous spermatozoa with production of high titer hyperimmune sera. Sera from vasectomized Fischer rats showed antisperm antibodies on Western blots, but bands were stained with less intensity and frequency than for Lewis rats. In both Fischer and Lewis strains, major protein autoantigens were observed at 75-83, 68-71, 63, 57, 51, 41, and 21-23 kDa, lending support to the hypothesis that there is a set of dominant sperm autoantigens recognized by a consensus of postvasectomy rat sera. The lesser response of Fischer rats to vasectomy was not due to absence of dominant postvasectomy sperm autoantigens in Fischer sperm extracts, nor was it attributable to inability of Fischer rats to mount an immune response to these antigens, since immunization with isologous sperm was successful in raising antibodies to the dominant autoantigens. Vasovasostomy did not result in a general decrease in antisperm antibodies, and reactions to some antigens actually increased.

Temporal recognition of sperm autoantigens by IgM and IgG autoantibodies after vasectomy and vasovasostomy.

Temporal patterns of IgM and IgG autoantibodies to sperm proteins were studied by western blot analysis at intervals after bilateral vasectomy, vasectomy followed one month later by vasovasostomy, or sham operations. Responses were detected to eight major autoantigens at 21-23, 36, 41, 51, 57, 63, 68-71 and 75-83 kDa, by study of staining patterns of sequential serum samples from individual animals and by analysis of the incidence of reaction to each protein. The four lower molecular weight antigens (21-23, 36, 41 and 51 kDa) provoked mainly IgG responses. The strongly stained set of higher molecular weight antigens (57, 63, 68-71 and 75-83 kDa) tended to show more clearly defined temporal patterns of IgM followed by IgG response, including a high incidence of IgM antibody at the 2-week interval. Three of the larger peptides (57, 63 and 68-71 kDa) appeared highly immunogenic, since some reactions were detected even in sham-operated rats. The classical patterns of IgM and IgG antibody responses to the majority of the dominant sperm autoantigens are in accord with the hypothesis that vasectomy mimics immunization with spermatozoa. The high incidence of IgM antibodies in the earliest sample, taken 2 weeks after vasectomy, suggests that the initial immunizing event takes place within about a week after the operation. Vasovasostomy did not bring about a decrease in antisperm antibodies. Instead, some animals demonstrated an increased reaction to certain antigens after reversal of vasectomy, even though the vasovasostomies were anatomically successful.

PIP: The production of antisperm antibodies is common subsequent to vasectomy and antisperm antibodies frequently persist following the reversal of vasectomy. The number of such antibodies may even increase after vasovasostomy. Using adult male Lewis rats, the authors analyzed the dominant autoantigens which evoke IgM and/or IgG autoantibodies after vasectomy by western blotting (WB) methods, the temporal patterns of IgM and IgG autoantibodies to specific sperm proteins, and the influence of vasovasostomy upon IgM and IgG antisperm autoantibodies. The temporal patterns were studied by WB at intervals after bilateral vasectomy, vasectomy followed 1 month later by vasovasostomy, and fake operations. Responses were detected to 8 major autoantigens of 21-23, 36, 41, 51, 57, 63, 68-71, and 75-83 kDa through the study of staining patterns of sequential serum samples from individual animals and by analysis of the incidence of reaction to each protein. The 4 lower-molecular-weight antigens provoked mainly IgG responses, while the strongly stained higher-molecular-weight antigens showed more clearly defined temporal patterns of IgM followed by IgG response, including a high incidence of IgM antibody at the 2-week interval. The peptides of 57, 63, and 68-71 kDa seemed to be highly immunogenic, since some reactions were detected even in rats which received only a fake operation. Results support the hypothesis that vasectomy mimics immunization with spermatozoa, while the high incidence of IgM antibodies in the earliest sample, taken 2 weeks after vasectomy, suggests that the initial immunizing event occurs within approximately 1 week after the operation. Vasovasostomy caused no decrease in antisperm antibodies.