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When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin-conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC-PNA-labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA-labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC-PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh-extended or frozen-thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37 degrees C, then a second antibody (rabbit anti-mouse IgG-FITC) was added for 30 min at 37 degrees C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate-buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma-acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonfluorescent "acrosomes", the percentage of sperm with intact plasma-acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra- and interassay coefficients of variability were less than 6%.

The rabbit sperm plasma membrane autoantigen, RSA, is a zona binding protein that binds the spermatozoon to the zona pellucida both before and after the acrosome reaction. In the present study rabbit spermatozoa undergoing the acrosome reaction in vitro are described and monospecific polyclonal mouse anti-RSA and protein A-gold label is used with the label-fracture technique (Pinto de Silva and Kan, J Cell Biol, 99:1156-1161, 1984) to map the location of RSA at the ultrastructural level before and after the acrosome reaction. RSA is most concentrated in the plasma membrane over the postacrosomal-equatorial region border. The label appears to cluster over the anterior aspects of the postacrosomal region's tooth-like projections. Following the acrosome reaction, RSA is still present in the postacrosomal region and often appears clustered in the medial aspects of the equatorial region.

Observations, employing freeze fracture replication and electron microscopic immunochemistry, have been carried out to determine structural correlations of the plasma membrane domain occupied by a 210 kDa protein involved in the acrosomal reaction of sea urchin sperm and recognized by the monoclonal antibody, J10/14 (Trimmer et al.: Cell 40:697-703, 1985; Proceedings of the National Academy of Sciences of the United States of America 83: 9055-9059, 1986). Immunogold-J10/14 staining of acrosome-intact sperm was intense along the flagellum and a narrow collar just posterior to the sperm apex that surrounded the acrosomal complex (acrosomal vesicle and subjacent anterior nuclear fossa containing g-actin). Counts of gold particles revealed a density (average number of particles/micron2 of surface area) eightfold greater along the plasma membrane associated with the acrosomal complex than membrane delimiting the remainder of the sperm head. The collar of J10/14 staining was isomorphic with a dense aggregation of intramembranous particles in the P-face of the plasma membrane and a thin cytoplasmic region that surrounded the acrosomal complex. In acrosome-reacted sperm, intense J10/14 staining was distributed along the flagellum and sperm head; prominent anterior staining was not apparent in all specimens. The density of gold particles associated with plasma membrane delimiting components of the former acrosomal complex, nucleus and mitochondrion, as well as the total average number of particles along the entire sperm surface, were increased in sperm acrosome-reacted with A-23187. Concomitant with this change in staining was the disappearance/reduction of the collars of intramembranous particles and cytoplasm. These observations indicate that plasma membrane components (210 kDa protein and intramembranous particles) and the collar of cytoplasm which are associated with the acrosomal complex are functionally, as well as structurally related. Analyses of particle density distributions along acrosome- and non-acrosome-reacted sperm suggest that the different staining patterns observed may be brought about by the recognition of cryptic sites at the time of the acrosomal reaction.

Due to the central role the acrosomal region plays in sperm-egg interactions, monoclonal antibodies (mAbs) were used to identify components of this domain in mouse sperm. Several sperm proteins that localize specifically to the anterior acrosomal region are described here in terms of electrophoretic mobility, susceptibility to proteolytic degradation, and post-translational modification during epididymal transit. Six different mAbs were used, each recognizing a distinctive antigen (Ag) or set of Ags in cauda epididymal mouse sperm: a doublet of 185/200 Kd (M42 mAb); 150-160 Kd (M5 mAb); 105 Kd (W71 mAb); 21, 35, and 60 Kd (M41 mAb); 27 and 33 Kd (W33 mAb); and 57 and 86 Kd (W108 mAb). Previously reported work implicates two of these, M42 Ag and M5 Ag, as participants in sperm-zona interaction (Saling and Lakoski: Biol Reprod 33:527-536, 1985; Saling: Dev Biol 117:511-519, 1986; and Lakoski et al.: Biol Reprod 38:221-233, 1988). Recognition of some (M42, M5, W108), but not all (W33), of the Ags by their corresponding mAbs was affected by sperm incubation with proteases (trypsin or collagenase). Evidence of post-translational modification during epididymal maturation was suggested by altered electrophoretic mobility of several of the Ags (M42, M5, W33, and W108) accompanying sperm transit from proximal to distal epididymis. Retention of sperm within the caput epididymis prevented structural alterations for the four proteins examined, indicating that spatial rather than temporal factors are critical for Ag modification in maturing mouse sperm.

A procedure is described for the production of large amounts of ascites fluid containing specific H-Y antibody. The distribution of H-Y antigen on mouse epididymal spermatozoa, thymocytes, and splenocytes was carried out using this specific antibody in the microcytotoxicity test and ELISA. Employing the indirect immunofluorescent technique, the H-Y antigen was localized on the acrosomal membrane of mouse epididymal and washed ejaculated human spermatozoa and on the entire membrane of mouse splenocytes and thymocytes. Immunohistochemical localization of the antigen in the testicular section indicated its presence in the cytoplasm of Leydig cells and on the membrane of Sertoli cells and sperm heads.

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluorescence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.