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To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

The acrosome plays an important role in fertilization. This study was designed to examine the role and behavior of a molecule, equatorin (the antigenic molecule of the monoclonal antibody mMN9), localized at the equatorial segment of the acrosome. In vitro fertilization (IVF) investigation was conducted to examine the role of this molecule, by assessing the effect of mMN9 in TYH medium (a modified Krebs Ringer bicarbonate solution) containing mMN9 at 0 (control), 25, 50, and 100 microg/ml. Under these conditions, the IVF investigation was divided into two experiments: 1) the zona pellucida (zona)-intact experiment, in which capacitated sperm inseminated cumulus- and zona-intact oocytes; and 2) the zona-free experiment, in which acrosome-reacted sperm inseminated zona-free oocytes. It was found that mMN9 did not affect sperm motility, zona binding, or zona penetration, but it significantly inhibited fertilization, reducing the rates of pronucleus and two-cell embryo formation in both the zona-intact and zona-free oocyte experiments. In addition, when judged at 5 h after insemination in the zona-intact experiment, nearly half of the unfertilized oocytes had accumulated sperm in the perivitelline space (perivitelline sperm), and concurrently we confirmed by electron microscopy the presence of many unreleased cortical granules preserved beneath the oolemma, indicating no occurrence of sperm-oocyte fusion. Confocal laser scanning light microscopy with indirect immunofluorescence demonstrated that equatorin was localized at the equatorial segment in both capacitated and perivitelline sperm (acrosome-reacted sperm). These results suggest that equatorin that is preserved at the equatorial segment is involved in the process of sperm-oocyte fusion in mice.

We have identified a 26-kDa (P26h) epididymal hamster sperm glycoprotein with a species-specific affinity for zona pellucida glycoprotein. Two immunological procedures have been used to document the biological function of this sperm component; active immunization of males against P26h and inhibition of sperm-zona pellucida binding in vitro by anti-P26h antibodies. The immunized male hamsters produced circulating antibodies specific to P26h. Indirect immunofluorescence studies showed that these antibodies bind to the surface of the sperm covering the acrosome. These males were mated with superovulated females, and although spermatozoa were recovered from the genital tract, none of the 194 oocytes recovered were fertilized. In contrast, control males immunized with hamster albumin fertilized 97.4% of the oocytes. Unlike control spermatozoa, those recovered from the cauda epididymidis of males immunized with P26h were characterized by the presence of antibodies at the surface of the acrosome. To establish whether the inhibition of in vivo fertilization by active immunization was occurring at the level of sperm-zona pellucida interaction, a polyclonal antiserum against P26h was raised, and the IgG fraction was added to an in vitro sperm-zona pellucida assay. Compared to the preimmune serum, the IgG inhibited the binding of spermatozoa in a dose-dependent manner. The Fab fragments generated from these IgGs were almost as efficient in inhibiting the binding. These results are discussed with regard to a possible function of P26h in hamster gamete interaction.

Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

During epididymal transit, mammalian spermatozoa acquire new surface antigens that may participate in gamete interaction. We have previously described a 26-kDa (P26h) epididymal hamster sperm protein that we propose to be involved in fertilization. In this study, we have searched for an antigenically related protein in the human, and have found that an anti-P26h antiserum recognizes a 34-kDa (P34H) protein on Western blot of human sperm proteins. Immunostaining showed that this protein is localized on the acrosomal cap of human epididymal spermatozoa but not on testicular gametes. The effect of the anti-P26h antiserum on the fertilizing ability of human spermatozoa was evaluated by use of a human zona pellucida binding assay. Compared to the preimmune serum, the antiserum caused a highly significant decrease in the number of sperm bound per zona pellucida. This inhibition was not due to the induction of a premature acrosomal reaction nor to an effect on the motility of the spermatozoa. The antiserum recognizing the P34H human sperm protein had no effect on gamete fusion as determined by the zona-free hamster test. Our results suggest that the human spermatozoon acquires an epididymal protein that shares a common epitope(s) with the P26h hamster sperm protein. The possible involvement of this human sperm antigen in the binding to the zona pellucida is discussed.

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)

In this study we used previously characterized monoclonal antibodies to acrosin (ACR.2) and to an acrosomal matrix antigen (ACR.4) to analyze the acrosin-binding activity of a 28-kDa putative acrosin-binding protein from the acrosomal matrix. The 28-kDa protein bound proacrosin and the 49-kDa form of acrosin (alpha-acrosin) but it did not bind the 36-kDa acrosin form (beta-acrosin). The acrosin-binding activity of the 28-kDa protein was stimulated by Ca2+, inhibited by Mg2+, and removed by disulphide bond reduction. Induction of the acrosome reaction by a calcium ionophore resulted in proteolytic cleavage of the 28-kDa protein, giving rise to a 12-kDa degradation product that was the only form of ACR.4 antigen released to incubation media; the release of the ACR.4 antigen was closely correlated with that of acrosin. The release of alpha-acrosin to incubation media was accelerated in the presence of ACR.4 antibody. In a cell-free system, a limited cleavage of the purified 28-kDa protein into immunoreactive degradation products was catalyzed by acrosin but not by trypsin or chymotrypsin. The data suggest that the 28-kDa acrosomal protein helps to maintain acrosomal matrix integrity and controls the acrosin release from acrosome-reacted cells.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.

The distribution of the sperm protein SP-10 was investigated in plastic-embedded samples of human testes by light and electron microscopy. An immunogold and silver enhancement technique, in conjunction with a monoclonal antibody (MHS-10) raised against SP-10, was used to localize the protein. SP-10 was detected in spermatids at each of the six stages of the cycle of the seminiferous epithelium. Light microscopy showed immunoreactive material at the circumference of developing acrosomes in the early steps of spermiogenesis. As differentiation proceeded and cell shape changed from round to elongated, immunoreactive material appeared in an arc, which gradually became a V shape bordering the spermatid nucleus. The area of the immunoreactive material and its shape corresponded to that of the developing acrosome. At the electron microscopic level, gold particles indicative of the presence of SP-10 were detected on electron-dense material found within the developing acrosomal vesicle in early steps of spermiogenesis. As the electron density of the acrosome increased, a high concentration of gold particles was seen in the vesicle matrix. The gold particles gradually became associated with the inner and outer acrosomal membranes of the most mature spermatids.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.

The intra-acrosomal human sperm protein SP-10 was previously designated a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. In the present study, a monoclonal antibody to SP-10 (MHS-10) was employed on Western blots to identify immunoreactive SP-10 in sperm extracts from baboon (Papio cyanocephalus anubis) and two macaques (Macaca mulatta and Macaca fascicularis). In each of these primates, the MHS-10 monoclonal antibody recognized a polymorphic pattern of immunoreactive peptides similar to that in humans. Immunoreactive SP-10 was also demonstrated in pig sperm. Using purified preparations of the previously described intra-acrosomal molecules acrosin and sperminogen in the pig, we observed that the MHS-10 monoclonal antibody did not react with these proteins, indicating SP-10 is distinct from these known acrosomal components. Sperm from several common species including the rabbit, bull, rat, guinea pig and cat did not immunoreact with the MHS-10 monoclonal antibody. By use of a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on Northern blots of poly A + RNA obtained from testes of Macaca fascicularis, Papio papio, and Papio cyanocephalus anubis, a 1.35-kb mRNA of identical size to the mRNA from human testes was identified. These results indicate that baboons, macaques, and pigs may be appropriate models for testing an SP-10-based contraceptive vaccine.

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.

Previous studies showed that sperm auto- and alloantigens participate in guinea pig (GP) fertilization. In an effort to determine how alloantibodies to GP sperm acrosomal contents (AC) inhibit fertilization, we identified acrosomal auto- and alloantigens using Western blots. The predominant autoantigens migrated with Mr = 25,000, Mr = 51,000, and Mr = 55,000 under nonreducing conditions. The primary (Mr = 25,000) acrosomal autoantigen, AA1, was purified to homogeneity from AC by gel filtration, cation-exchange chromatography, chromatofocusing, and a final gel filtration. We also purified AA1 from an acidic glycerol extract of spermatozoa by gel filtration, chromatofocusing, and high-performance liquid chromatography on hydroxylapatite. AA1 is a protein and shares at least one antigenic determinant with a 51,000 Mr acrosomal component. AA1 is acrosome-specific, as determined by immunoabsorption and by indirect immunofluorescence on testicular cells. By quantitative enzyme-linked immunosorbent assay, AA1 comprises 6.4% of acrosomal protein in GP spermatozoa. On the basis of its physiochemical properties and localization, we conclude that AA1 is a unique sperm autoantigen. Surprisingly, several antibody preparations, including allo- and heteroantibodies with high anti-AA1 titers, did not inhibit fertilization in vitro. Thus, the mechanism by which alloantibodies to AC inhibit GP fertilization in vitro is not by binding to AA1.

Unheated rabbit and human sera were found to induce acrosomal loss in rabbit spermatozoa, while similar concentrations of heated sera did not. In addition, human serum did not induce acrosomal loss when pretreated with antiserum to complement component C8, suggesting that acrosomal loss in unheated serum is caused by the membrane attack complex of complement. Human serum complement anaphylatoxins did not induce acrosomal loss, although they are known to induce exocytosis of secretory granules in other cell types. When incubated directly in human or rabbit sera, rabbit spermatozoa fixed complement; i.e., reduced the potential hemolytic activity of the sera. Fixation was suppressed by adding EGTA to reduce free calcium. This indicates that rabbit spermatozoa fix complement by initiating the classical pathway to complement activation. Initiation requires the presence of cell-bound immunoglobulins and the subsequent binding of complement component C1q. Immunoglobulins were detected in detergent extracts of washed ejaculated spermatozoa by a solid-phase radioimmunoassay, and the binding of 125 I-human C1q was detected on samples of living ejaculated spermatozoa. Seminal plasma was found to inhibit complement-induced hemolysis of erythrocytes. These results suggest that, in the absence of seminal plasma, spermatozoa may activate complement where it is present in the male or female tract.

Utilizing hybridoma technology and highly purified acrosome stabilizing factor (ASF), six monoclonal antibodies (mAbs) specific for ASF were produced and characterized. Specificity and binding properties of each clone were examined by immunoperoxidase labeling of electrophoretic blots of rabbit serum, seminal plasma, cauda epididymal fluid and vasectomized seminal plasma separated on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels. All mAbs recognize ASF in seminal plasma and cauda epididymal fluid but do not bind components in serum or vasectomized seminal plasma. Purification of ASF by affinity chromatography utilizing the mAbs, has shortened the 6-day isolation procedure for ASF used previously to less than 2 h and has increased the yield from 2 micrograms to 300 micrograms of ASF obtained per ml of seminal plasma. Three mAbs were used in conjunction with Cleveland digest with Staphylococcus aureus V8 protease, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoperoxidase labeling of Western Blots, to identify peptides containing specific determinants recognized by each mAb. At least five separate determinants were recognized by the six mAbs. The sensitivity of the Western blotting technique in conjunction with the specificity of the mAbs was exploited to detect polymeric forms of ASF in seminal plasma and cauda epididymal fluid. ASF is shown to be a 360-kd dimer consisting of two identical 180-kd monomers. Tools are now available to develop sensitive qualitative and quantitative assays for ASF, thus providing rapid, extremely sensitive methods for evaluating experiments designed to probe the molecular mechanism of capacitation.

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.

Previous work has indicated that the guinea pig sperm membrane protein, PH-20, functions in sperm-egg adhesion and that its surface expression is regulated by the acrosome reaction. The PH-20 protein was purified by monoclonal antibody affinity chromatography. Sixty-seven to one hundred percent of the PH-20 antigenic activity present in an octylglucoside (OG) extract of sperm was recovered in the purified protein. From 10(10) sperm, approximately 0.4 mg of PH-20 protein was obtained, which was about 0.24% of the total protein in the OG extract. The purified protein retained the ability to bind the three anti-PH-20 monoclonal antibodies we have isolated. Silver staining of purified PH-20 on overloaded sodium dodecyl sulfate (SDS) gels allowed the estimate that silver-stainable contaminants were present at a level of one part in 2000. The purified PH-20 protein exists in three forms separable on SDS-polyacrylamide gel electrophoresis: a major form with a molecular mass of 64 kDa, a minor form of 56 kDa, and an endoproteolytically cleaved form composed of two disulfide-linked fragments of 41-48 kDa and 27 kDa. Cleveland digests of the 64 kDa and 56 kDa polypeptides indicated that they were structurally related. A proportion of the 64 kDa polypeptide in each purified preparation had undergone endoproteolysis at a specific site, so that it was cleaved into the two disulfide-linked fragments, 41-48 kDa and 27 kDa. It is speculated that the site-specific endoproteolysis of PH-20 may occur during the acrosome reaction and have biological significance.

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.

Development of the sperm's capacity to interact with the zona pellucida was investigated at the stage when the acrosome reaction (AR) is induced. The response of epididymal sperm to agents that affect the occurrence of the AR was used to monitor maturational changes. Despite the finding that sperm from the three main epididymal regions were competent to undergo ARs induced by the divalent cation ionophore A23187 (56% AR, 74% AR, and 83% AR in caput, corpus, and cauda, respectively), the cells' responses to solubilized zonae pellucidae were different. When challenged with 5 zonae equivalents/microliter, both corpus and cauda sperm shed their acrosomes in high numbers (75% AR and 86% AR, respectively), whereas caput sperm did not (23% AR). Previous work has shown that the presence of M42 monoclonal antibody (mAb) during in vitro and in vivo fertilization inhibits sperm penetration through the zona pellucida by specific interference with zonae pellucidae-induced ARs. In this study, presence of the M42 mAb did not affect the incidence of A23187-induced ARs, whereas the zona-induced ARs that occurred in both corpus and cauda sperm were inhibited fully with M42 immunoglobulin (Ig) G. In addition, the antigen recognized by M42 mAb on sperm, termed M42 Ag, was examined during epididymal maturation. Although antigen localization appeared indistinguishable by immunofluorescence on sperm taken from the caput, corpus, and cauda regions of the epididymis, modification of this antigen during epididymal transit was detected. Equilibrium-binding studies using 125I-M42 IgG demonstrated a progressive increase during epididymal transit in the amount of M42 mAb that bound to fixed cells. Corpus and cauda sperm bound 185% and 240%, respectively, of the 125I-M42 IgG detected on caput sperm. These changes in expression of M42 Ag paralleled a structural change: the Mr of the antigen decreased from a 195,000/210,000 doublet in caput sperm to a 185,000/200,000 doublet in corpus and cauda sperm, as determined by immunoblot analysis of sodium dodecyl sulfate (SDS)-extracted sperm. Results presented here demonstrate that mouse sperm develop the capacity to undergo a zona-induced AR during epididymal maturation. The M42 antigen, which is involved in the zona-induced AR, is modified during epididymal transit coincident with development of the sperm's responsiveness to zonae. Our working hypothesis, based on these results, is that development of the sperm's capacity to undergo a physiological AR is related to modification of M42 Ag.

Autoantibodies raised in guinea pigs (GP) by hyperimmunization with epididymal sperm recognize antigens present on the plasma membrane of intact sperm and antigens associated with acrosome-reacted sperm. Plasma membrane autoantigens were characterized by SDS-PAGE analysis of immune precipitates from detergent extracts of radiolabeled epididymal sperm. Three major protein bands with approximate molecular weights (MW) of 69,000, 62,000 and 40,000 daltons were exhibited. Galactose oxidase and periodate oxidation followed by tritiation revealed two major plasma membrane glycoprotein autoantigens with MWs of 35,000 and 32,000 daltons and one sialoglycoprotein of 34,000 daltons. Antigenic molecules of similar MW were also detected by autoantisera to guinea pig testicular homogenates. Immune precipitates of radiolabeled detergent extracts of acrosome-reacted sperm analyzed by SDS-PAGE exhibited two major carbohydrate-containing peaks, one with a MW of approximately 42,000 daltons and one of 6,000 daltons. Since the 6,000 dalton peak was immunoprecipitated from the organic phase of both chloroform:methanol 2:1 and n-butanol extracts, the antigen may be a glycolipid. The possible existence of glycolipid autoantigen in GP sperm was further supported by the reaction in a solid phase radioimmunoassay of autoantibodies to GP sperm with glycolipid extracts from GP testis. This study demonstrates the presence of multiple autoantigenic specificities in the sperm-plasma membrane, acrosome-reacted sperm and testis of guinea pigs. It also demonstrates for the first time glycolipid autoantigens in testis and sperm.

Monoclonal antibody 1D4 reacts with a glycoconjugate antigen of the developing mouse spermatid acrosome until the terminal steps of spermiogenesis. Although 1D4 does not label the acrosome of mouse epididymal spermatozoa, it does bind to the acrosomal region of guinea pig epididymal sperm. Here we report that the antigens recognized in extracts of guinea pig spermatogenic cells and sperm are different from those detected in mouse spermatids. Three major bands of reactivity with apparent molecular weights (Mr) of 97,000-145,000, 180,000, and greater than 200,000 were detected in extracts of guinea pig sperm. Soluble antigens with the same molecular weights were released after the acrosome reaction was induced with ionophore A23187. On two-dimensional immunoblots, 1D4 recognized a microheterogeneous population of molecules in guinea pig sperm extracts. The molecules recognized by this antibody are not major Concanavalin A receptors and do not react strongly with periodic acid-Schiff's stain or periodic acid-dansylhydrazine. However, comparisons of immunoblots of sperm extracts indicate that 1D4 reacted with antigens having similar molecular weights to glycoconjugates recognized by antibodies J1 and C6. Immunofluorescent labeling of guinea pig germ cells showed that 1D4 reacted only with the acrosomes of developing spermatids and sperm, and occasionally with the juxtanuclear region of spermatocytes. In general, staining was associated with the periphery of the acrosome and not with the acrosomal granule. Immunoblots of extracts of guinea pig spermatocytes, round spermatids, condensing spermatids, and sperm demonstrated that the antigens change during germ cell differentiation. Thus, 1D4 can be used as a marker of the developing acrosome for studies of the synthesis, structure, and assembly of the sperm organelle.