Human sperm coating antigen from seminal plasma origin.

Sperm surface glycoproteins may be involved in sperm-zona pellucida recognition. Some of these coating proteins are of seminal plasma origin and their expression may change in the process of capacitation and acrosome reaction. Sperm specific monoclonal antibodies (mAb) define the presence and role of sperm membrane associated proteins. We have isolated a monoclonal antibody (SEM-12) specific for human sperm that shows, by indirect immunofluorescence, a discontinuous distribution of the antigen on the head and tail surfaces of non capacitated sperm. This antigen is also present in human seminal plasma as detected by ELISA. The antigen is detectable in sperm of goat, ram, and mouse. Two proteins in the range of 80-84 kDa have been isolated by affinity chromatography with SEM-12 mAb. The same result is obtained by immunoprecipitation. This antibody inhibits sperm motility and acrosome reaction (spontaneous and A23187 ionophore induced.

Characterization of monoclonal antibodies specific for human sperm: effect of CRL-10 on acrosome reaction.

Monoclonal antibodies to human sperm were obtained from hyperimmunized BALB/c mouse spleen cells fused with myeloma NS-1 cells. Each antibody recognized definite regions in fresh unfixed sperm: equatorial region, acrosome, postacrosome, midpiece, tail. All the antibodies were specific for sperm. We selected CRL-10 monoclonal antibody, specific for acrosome, for a detailed study. The expression of the CRL-10 antibody-bound antigen was detected in other mammalian species. When CRL-10 antibody was added prior to sperm incubation in a capacitating medium, promotion of the acrosome reaction was observed.

Characterization and regional binding of human sperm monoclonal antibodies.

Five monoclonal antibodies (MAb) with specific binding for tail, midpiece, equatorial region, and postacrosome of human sperm were selected. The reactivity of the MAbs was tested by three techniques: an ELISA with human sperm, an indirect immunostaining analyzed by a cell sorter, and immunofluorescence on the cell where the antigens were localized. The localization or exposure of the antigens changes throughout the in vitro capacitation. These antigens are distributed among other mammalian species: mouse, ram, and goat. Their presence was also analyzed in some human tissues: peripheral blood mononuclear cells, spleen, pancreas, thyroid, heart, skin, intestine, and lung. No cross-reactivity was detected. Some of the MAbs showing tail staining severely inhibited sperm motility after 5 hr of incubation with sperm, although no agglutination was present. This immobilizing activity of MAbs would prevent spermatozoa from reaching the oocyte in vivo.

Inhibition by anti-sperm monoclonal antibodies of the penetration of zona-free hamster oocytes by human spermatozoa.

Fourteen mAb specific for human sperm membrane antigens were selected to investigate their inhibitory effect on fertilization. The antigens were not expressed in other human somatic tissues but were present in sperm from other species. The antibodies were purified from ascites fluid produced in mice. The zona-free hamster egg penetration assay was used for the evaluation of the blocking ability of these antibodies. The inhibition rate was generally related to a decrease in the number of adherent sperm. Three groups of antibodies were distinguished: (i) four mAb that have high inhibition at any concentration; (ii) four mAb with an intermediate inhibitory effect, that is more dependent on the antibody concentration tested; and (iii) six mAb with little or no effect at any concentration. The presence of antibodies leads to a lower penetration index, or number of penetrating sperm per oocyte. MAb specific for head antigens promote high inhibition of fertilization; these antibodies show a patchy staining on the sperm head. The antibodies localized in the midpiece have an intermediate inhibitory effect. No inhibition is detected with the equatorial region binding pattern. Sperm agglutination does not play any role in the inhibition caused by the antibodies described here.

Effect of anti-human sperm monoclonal antibodies on mouse in vitro fertilization.

We employed anti-human sperm monoclonal antibodies to investigate how sperm membrane antigens are involved in gamete interactions. We have produced seven monoclonal antibodies specific for human sperm antigens, that showed reaction with mouse sperm by ELISA and by immunofluorescence. These antibodies did not react with zona pellucida or any other somatic human tissue. Some degree of toxicity was detected for oocytes at high antibody concentration and this was correlated with their inhibitory effect on fertilization. Unrelated to the degree of antigen expression or localization on sperm membrane, the antibodies showed several degrees of inhibition. The participation in sperm-zona pellucida interaction for every antigen could be evidenced by the impaired penetration of sperm caused by the presence of several concentrations of antibody. Thus, DAN-2, MOU-8 and VAC-4 inhibit mouse in vitro fertilization.

Characterization of a sperm-specific monoclonal antibody and isolation of 95-kilodalton fertilization antigen-2 from human sperm.

Monoclonal antibodies (mAbs) were raised in mice against human sperm. Of the eight hybridomas secreting mAbs that react with human sperm, one, the Vic-1 antibody, was selected for detailed analysis because of its high degree of tissue specificity. The Vic-1 antibody was of the IgG1 subclass and demonstrated binding predominantly with the acrosomal regions of viable but not methanol-fixed noncapacitated and capacitated human sperm cells. It also reacted with the acrosomal and mid-piece regions of viable capacitated as well as noncapacitated murine sperm, but not with methanol-fixed murine sperm. The Vic-1 antibody was germ-cell specific as it did not react with any human somatic cell, tissue, or secretion examined including seminal plasma. The Vic-1 antibody significantly (p = 0.0006) inhibited human sperm penetration of zona-free hamster oocytes in a concentration-dependent manner; at 15 g% concentration it almost completely blocked sperm penetration. The antibody significantly reduced the acrosome reaction and the release of acrosin activity in human sperm cells. There was no effect of the Vic-1 antibody on percentage of motile sperm, although it significantly affected motility characteristics such as linearity, amplitude of lateral head displacement, and beat frequency; motility parameters involved in the hyperactivation phenomenon related to capacitation and the acrosome reaction. The Vic-1 antibody recognized a predominant antigen of 95 kDa, designated fertilization antigen-2 (FA-2), in Western blot and immunoprecipitation procedures using human sperm preparations. The FA-2 antigen was isolated from human sperm preparations by using an immunoaffinity column containing the Vic-1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of anti-sperm antibodies in serum, seminal plasma and cervical mucus by the immunobead test.

Anti-sperm antibodies in serum and seminal plasma were detected by means of an indirect immunobead test (IBT). Immunobeads with separate specificites for each immunoglobulin class (IBT-IgG, IBT-IgM, and IBT-IgA) were used. Semen parameters were controlled in all sperm donors and Biggers-Whitten-Whittingham (BWW) medium supplemented with human serum albumin (HSA) was used to increase sperm motility. This technique was tested with high titre anti-human sperm sera induced in rabbits. Sperm tails showed a good response by IBT. We included in this study 178 men and 35 women evaluated for infertility and the sera were also tested by the Tray Agglutination Test (TAT). Although the presence of semen markers such as agglutination or trembling of spermatozoa is meaningful even by itself, the percentage of anti-sperm antibodies was increased in the patients with markers, both using IBT (21.4%) and using TAT (35.7%). At high titres of specific immunoglobulins (rabbit antisera and vasectomized men), the correlation between IBT and TAT techniques was better than in sera with very low titres, in which more positive TAT's were detected.