Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.

[Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis].

OBJECTIVE: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS: An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm.

Evidence has been presented that the PH-20 protein functions in sperm adhesion to the egg zona pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, 1985, J. Cell Biol., 101:2239-2244). The PH-20 protein migrates from its original surface domain to a new surface domain after the acrosome reaction (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). The acrosome reaction is an exocytotic event that results in insertion of a region of the secretory granule membrane, the inner acrosomal membrane (IAM), into the plasma membrane. After the acrosome reaction, PH-20 protein migrates to the IAM from its initial domain on the posterior head surface. We have now found a new dynamic feature of the regulation of PH-20 protein on the sperm surface; exocytosis increases the surface expression of PH-20 protein. After the acrosome reaction there is an approximately threefold increase in the number of PH-20 antigenic sites on the sperm surface. These new antigenic sites are revealed on the surface by insertion of the IAM into the plasma membrane. Our evidence indicates that before the acrosome reaction an intracellular population of PH-20 antigen is localized to the IAM. When migration of the surface population of the PH-20 protein is prevented, PH-20 protein can still be detected on the IAM of acrosome-reacted sperm. Also, PH-20 protein can be detected on the IAM of permeabilized acrosome-intact sperm by indirect immunofluorescence. Thus, the sperm cell regulates the amount of PH-20 protein on its surface by sequestering about two-thirds of the protein on an intracellular membrane and subsequently exposing this population on the cell surface by an exocytotic event. This may be a general mechanism for regulating cell surface composition where a rapid increase in the amount of a cell surface protein is required.

Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm.

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.

A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida.

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.

Sperm autoantibodies in vasectomized rats of different inbred strains.

An immune response to antigens of spermatozoa occurs after vasectomy in rats of some inbred strains, but not in others. Antibodies to rat spermatozoa were detected by indirect immunofluorescence in some of the serums of vasectomized rats of the following strains: 80 percent of Lewis, 47 percent of Brown Norway, 13 percent of Buffalo, 12 percent of Wistar-Furth, and 11 percent of ACI rats. No such antibodies were detected in the serums of vasectomized Fischer, Dark Agouti, and Sprague-Dawley rats.

Naturally occurring antibodies reactive with sperm proteins: apparent deficiency in AIDS sera.

A set of naturally occurring immunoglobulin M (IgM) antibodies that are reactive with a defined subset of proteins in the acrosomal cap region of human sperm has been identified. These antibodies are present in a broad spectrum of human sera from males and females, 1 day to 40 years of age, and are absent or markedly deficient in a large proportion of sera from individuals with the acquired immune deficiency syndrome (AIDS) or at risk for AIDS. The subset of proteins with which the IgM antibodies are reactive includes a factor (or factors) capable of inhibiting lectin-induced T-lymphocyte proliferation. The prevalence of the sperm-reactive IgM antibodies indicates that they are not elicited by sperm. Further, immunoreactivity of the sperm proteins resulting in depletion of specific circulating IgM antibodies, or other interactions between the sperm proteins and elements of the immune system, may be a factor in the suppressed state of the immune system in AIDS.

Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine.

Melanoma cells are significantly resistance to the current treatments. Therefore, the best option for high-risk populations is prevention. Recently, many preventive cancer vaccines have been developed. In our previous study, several bioinformatic tools were employed for selection of the most immunodominant epitopes of acrosin binding protein (ACRBP) and synaptonemal complex protein 1 (SYCP1) antigens to design multiepitope DNA and peptide cancer vaccines. In the current study, the final construct of the multiepitope DNA vaccine was placed into a pcDNA3.1 vector and then, subcloned into a pET-28a (+) expression vector for transfecting BL21 E. coli strain. The recombinant multiepitope peptide vaccine, weighing 6.35 kDa, was purified by Fast protein liquid chromatography technique (FPLC) and detected by western blotting. Subsequently, C57BL/6 mice were immunized by a mixture of the peptide vaccine and incomplete Freund's adjuvant (IFA) (four vaccinations with one-week intervals). Two weeks after the last vaccination, the serum levels of the peptide-specific IgG total, IgG2a, and IgG1 were measured by enzyme-linked immunosorbent assays (ELISA). Also, the immunized mice splenocytes efficacy for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) after stimulation with the peptide vaccine was evaluated. At last, the prophylactic effect of the peptide vaccine immunization was evaluated in B16-F10 murine melanoma model. The peptide vaccine immunization caused a significant increase in the serum levels of IgG1, IgG2a, and IgG2a. Also, the immunized mice splenocytes exhibited significantly higher ability to produce IL-4 (10-fold) and IFN-γ (16-fold) after stimulation with the peptide vaccine, in comparison with the PBS and IFA groups. The peptide immunized mice exhibited 50.2% and 43% decrease in the mean tumors' volume in comparison with PBS and IFA groups. Also, the mean survival time for the peptide immunized mice was 33 ±â€¯1.3 days which was 5 and 6 days more than the PBS and IFA groups, respectively. The obtained results exhibit high efficacy of the designed multiepitope peptide vaccine for the immune system activation and anti-tumor prophylactic effects in the murine melanoma model.

Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.

A vaccine formula comprised of five recombinant human intra-acrosomal sperm proteins was inoculated into female monkeys to test whether specific antibodies to each component immunogen could be elicited in sera and whether antibodies elicited by the vaccine affected in vitro fertilization. Acrosomal proteins, ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, were expressed with his-tags, purified by nickel affinity chromatography and adsorbed to aluminum hydroxide. Five female cynomolgus monkeys were inoculated intramuscularly three times at monthly intervals. All five monkeys developed both IgG and IgA serum responses to each recombinant immunogen on Western blots. Each serum stained the acrosome of human sperm and bound to the cognate native protein on Western blots of human sperm extracts. By ELISA, all monkeys developed IgG to each immunogen, with the highest average absorbance values to ESP, SAMP 32 and SP-10, followed by lower values for SLLP-1 and SAMP 14. IgA was also generated to each component immunogen with the highest average absorbance values to SLLP-1 and SP-10. For antigens that induced an IgA response, the duration of the IgA response was longer than the IgG response to the same antigens. This study supports the concept that a multivalent contraceptive vaccine may be administered to female primates evoking both peripheral (IgG) and mucosal (IgA) responses to each component immunogen following an intramuscular route of inoculation with a mild adjuvant, aluminum hydroxide, approved for human use.

CD55 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.

CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa.

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

Immunisation of the male tammar wallaby (Macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilisation rate.

Immunocontraception has been proposed as an effective and humane means of controlling overabundant kangaroo populations in Australia. We have examined the feasibility of using a sperm-based vaccine for this purpose using a model macropod species, the tammar wallaby (Macropus eugenii). This study has demonstrated immunocontraception in a marsupial species following immunisation of males with homologous spermatozoa. Serum anti-sperm IgG titres were associated with a significant reduction in fertilisation rates following mating with superovulated female wallabies. Antigen-specific IgG penetrated the reproductive tract at the rete testis and bound spermatozoa in vivo. IgG was detected bound to the acrosome and midpiece regions of both epididymal and ejaculated spermatozoa. The absence of adverse testicular pathology and sperm movement effects suggests that contraception may have been achieved by antibody-mediated blocking of sperm surface antigens essential for fertilisation. This study demonstrates that a contraceptive vaccine targeting sperm antigens has potential for fertility control in male macropods.

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa.

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization.

Monoclonal antibody from vasectomized mouse identifies a conserved testis-specific antigen TSA70.

Vasectomy results in the occlusion of testicular outflow, leading to autoimmunity characterized by the production of antisperm antibodies (ASA). Reports on the rise in ASA following vasectomy in several species are available; however, not much is known about the specific sperm autoantigens to which postvasectomy antibodies are directed. In the present study, monoclonal antibodies were generated using a vasectomized mouse. One of the monoclonal antibodies, D5E5, identified an approximately 70-kd antigen localized on the principal piece of the tail and also on the tip of the acrosome of mouse sperm. The cognate antigen was expressed postmeiotically in a stage-specific manner during spermiogenesis, starting from step 8 of elongating spermatids during spermiogenesis up to mature spermatozoa. The protein was conserved across the species, as observed by its presence in rat, bull, marmoset, and human sperm. Following capacitation, the antigen on the head was seen to shift to the acrosomal region and was lost after the acrosome reaction. However, the localization on tip of the acrosome still persisted, which indicates that the antigen may play a role post-acrosome reaction in sperm egg interaction. Resistance to Triton X-100 solubilization indicates that TSA70 could be an acrosomal matrix protein. In addition, we observed a significant reduction in forward progressive motility of mouse sperm treated in vitro with D5E5. In view of its testis specificity, acrosome and tail localization, and conserved nature, TSA70 is likely to play an important role in sperm function.

Study on the role of a sperm membrane-associated antigen in canine fertilization.

The Mab 4B12 produced by us against capacitated boar spermatozoa was found to recognize a protein located in the acrosome portion of capacitated boar spermatozoa which is shared by different animal species, dogs included. It was shown that Mab 4B12 might affect fertilizing ability in vitro of boar spermatozoa. Using indirect immunofluorescence (IIF) test, we provide evidence here that Mab 4B12 stained the acrosome of the capacitated but not of the ejaculated and acrosome-reacted canine spermatozoa. The biological experiments using hemizona assay functional test in this study provide evidence supporting the involvement of Mab 4B12 corresponding antigen in the functional steps required for canine sperm-zona pellucida binding. These results together with the data on cell and tissue specificity of the 4B12 antigen suggest its contraceptive potential for canine fertilization.

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization.

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes. A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30.000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.

Genetic control of humoral immunity to sperm acrosomal and cell surface antigens.

We have defined the nature of genetic control of humoral immunity to sperm cell surface (TSDA) and acrosome (Ac) antigens. A sperm immunization dose that distinguishes between high-responder (BALB/c) and low-responder (B10) strains of mice was identified. B10 mice were unresponsive, whereas BALB/c and F1 hybrid mice responded to sperm of both parental strains. The ratio of nonresponder to responder mice in the B10 X F1 backcross and F1 X F1 inbred generations indicates anti-TSDA or anti-Ac antibody responsiveness is controlled through more than a single gene. The significant correlation found between the anti-TSDA and anti-Ac responses is consistent with the possibility that one or more genes controls antibody responsiveness against determinants common to both the Ac and sperm cell surface. Neither anti-TSDA nor anti-Ac antibody responses were linked to the I-A subregion of the responder H-2d MHC haplotype. In addition to a genetic difference in the control of humoral immunity to sperm, a difference in sperm immunogenicity among strains was observed. Antigens of the Ac of B10 sperm are more immunogenic than those of BALB/c sperm, and this trait is linked to the B10 Y chromosome.