Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg

Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Monoclonal antibody AG7 inhibits fertilization post sperm-zona binding.

Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define spermatozoal antigens involved in the fertilization process. We have produced a number of anti-human sperm mAbs by immunization of female mice with the 100,000 x g supernatant of octylglycoside-solubilized washed human sperm. From a panel of mAbs, 1 antibody, AG7, was selected and characterized due to its fertilization-inhibiting characteristics. MAb AG7 defines a sperm acrosome antigen-1 (SAA-1) located in the acrosomal region of human sperm as evaluated by indirect immunofluorescence. Staining of life sperm cells indicated that the antigen is present on the sperm surface. SAA-1 was also found on sperm of several other mammalian species, implying evolutionary conservation of the antigen. SAA-1 was first observed on testicular sperm and can be followed through epididymal transit, ejaculation, and capacitation. When applied in a mouse in vitro fertilization assay, mAb AG7 inhibits fertilization by greater than 95%, and inhibition is dose dependent, with half-maximal inhibition at 0.8 micrograms/ml. The block to fertilization could not be attributed to sperm agglutination, inhibition of motility, interference with adhesion to the zona pellucida, or inhibition of fusion with the oocyte membrane. MAb AG7 was demonstrated to inhibit calcium influx in spermatozoa in vitro (measured using the fluorescent indicator fura 2), a prerequisite for the acrosome reaction. Initial biochemical characterization of the antigen suggests it is proteinlike in nature, with a molecular weight of approximately 220 kD. The results suggest that SAA-1, identified by mAb AG7, is a sperm antigen crucially involved in the fertilization process, possibly an atypical steroid receptor or ion channel located within the sperm plasma membrane.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

C-kit receptor and its possible function in human spermatozoa

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility.     

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.     

Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization.

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.     

Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)

β-N-Acetylglucosaminidase (β-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that β-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of β-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for β-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of β-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of β-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that β-NAGase can play a significant role in the fertilization process in teleosteans.     

Sperm motility-initiating activity in the egg jelly of the externally-fertilizing urodele amphibian, Hynobius lichenatus

Low osmolality initiates sperm motility during the external fertilization of aquatic anuran amphibians. It is thought that this process occurs also in urodeles, but this has not been fully examined in these species. We report here that fertilization was achieved in the externally fertilizing hynobiid, Hynobius lichenatus, by direct insemination onto the egg jelly surface without initial exposure of the sperm to a hypoosmotic solution. To identify the factors in addition to low osmolality that initiate sperm motility in Hynobius, we suspended the sperm of this amphibian in egg jelly extract (JE), and about 90% began to move within 1 min. This indicated the presence of a substance in JE that promotes motility initiation, as is also the case in the newt, Cynops pyrrhogaster. To examine whether this JE factor is homologous to the sperm motility-initiating substance (SMIS) in the newt, we tested for possible inter-species cross-reactivity of the JE. The percentage of moving Cynops sperm was increased to 67% in Hynobius JE at 5 min, and 65% of the Hynobius sperm began to move in Cynops JE within 1 min, indicating that JE is indeed cross-reactive between these species of salamander and newt. Concomitantly, pretreatment of Hynobius JE with Fab fragments of a Cynops SMIS monoclonal antibody resulted in a decreased number of moving Hynobius sperm. Immunoblotting further suggested that the substance in Hynobius JE responsible for motility initiation has an 18 kDa molecular mass, with an isoelectric point at 7.5.     

Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization

Egg activation in cross-fertilization between Xenopus eggs and Cynops sperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified from Cynops sperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation in Xenopus eggs. We inseminated unfertilized Xenopus eggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained from Xenopus sperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease in Xenopus sperm is involved in fertilization, most likely by participating in egg activation.     

Antibodies to sperm surface antigens and the c-myc proto-oncogene product inhibit early embryonic development in mice.

The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab' antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab' identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab' did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development.     

A role for phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.     

Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate-attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.     

Monoclonal antibodies, immunofluorometric assay, and detection of human semenogelin in male reproductive tract: no association with in vitro fertilizing capacity of sperm

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.     

c-Abl proto-oncoprotein is expressed and tyrosine phosphorylated in human sperm cell

The presence and possible role of c-Abl proto-oncoprotein was investigated in human sperm cell. The c-Abl monoclonal antibody (mAb), against the protein tyrosine kinase domain of v-Abl protein, reacted specifically with the acrosomal region of methanol-fixed capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The c-Abl mAb predominantly recognized two protein bands of 145 kD and 95 kD in detergent-solubilized (Triton X-100 and NP-40) sperm and testes preparations in the Western blot procedure. The 95 kD protein band reacted stronger than the 145 kD band and was the only band detected in the lithium diiodosalicylate (LIS)–solubilized sperm preparation, and even in the Triton X-100/NP-40 extracts of sperm of some men. In the in vitro kinase assay using the Triton X-100–solubilized capacitated sperm preparation, the 95 kD protein was autophosphorylated at the tyrosine residues, which was inhibited in the presence of c-Abl mAb. The tyrosine phosphorylation of sperm proteins, especially of the 95 kD protein, has been shown to have a vital role in human sperm function, namely, the sperm capacitation/acrosomal exocytosis and binding to zona pellucida of oocyte. These findings suggest that the c-Abl or c-Abl-like proteins are present in mature sperm cells that are tyrosine autophosphorylated and may have a role in human sperm cell function. Mol. Reprod. Dev. 51:210–217, 1998. © 1998 Wiley-Liss, Inc.     

The use of anti-VDAC2 antibody for the combined assessment of human sperm acrosome integrity and ionophore A23187-induced acrosome reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca(2+) increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca(2+) transmembrane transport.     

Anti-idiotype antibodies prevent antibody binding to mouse sperm and antibody-mediated inhibition of fertilization

Antibodies to components of sperm can interfere with sperm function and prevent fertilization by blocking specific steps of gamete interaction. It can be proposed that anti-idiotype antibodies (anti-Ids) that recognize determinants located close to or within the antigen-binding site of an anti-sperm antibody could block antibody binding to sperm antigen and antibody-mediated inhibition of fertilization. To test this hypothesis, rabbit polyclonal antibodies to idiotypic determinants of the monoclonal anti-sperm antibody M42.15 were developed and characterized. Previous studies demonstrated that M42.15 monoclonal antibody (mAb) inhibits fertilization in vitro and in vivo by inhibiting sperm-zona pellucida interaction. Anti-idiotype antibodies to M42.15 mAb (anti-Id M42) were isolated by affinity chromatography on immobilized M42.15 mAb. Binding specificity of anti-Id M42.15 was demonstrated in a solid-phase radioimmune binding assay and by specific immunoprecipitation of soluble M42.15 mAb. Anti-Id M42 competitively inhibited M42.15 mAb, but not P220.2 mAb, binding to mouse sperm, confirming that the anti-Id preparation contained antibodies directed against idiotopes within or adjacent to the antigen-binding site of the mAb. At equimolar concentrations, anti-Id M42 inhibited binding of 125I-labeled M42.15 mAb to sperm by greater than 80%. These results showed that anti-Id M42 efficiently blocked antibody binding to sperm and suggested that anti-Id M42 could be used to neutralize the anti-fertility activity of the M42.15 mAb. When tested in in vitro fertilization assays, anti-Id M42, but not rabbit immunoglobulin, prevented M42.15 mAb-induced inhibition of fertilization. Together, these results show that the inhibitory activity of anti-sperm antibodies capable of interfering with gamete interaction can be neutralized by anti-Id that recognize determinants close to the antigen-combining site of the anti-sperm antibody.