Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

Effect of anti-pig zona pellucida serum on fertilization in several mammals

The effect of goat antiserum against isolated pig zonae pellucidae on fertilization in vivo was examined in the pig, cow, sheep, rabbit, rat, and mouse. As shown by indirect immunofluorescence, anti-pig zona serum reacted strongly with the zonae of pig, cow, sheep, and rabbit, but the reaction with the zonae of mouse and rat was weak. Passive immunization with anti-pig zona serum significantly, or completely, inhibited fertilization in all species. However, inhibition of fertilization was more pronounced in the pig, cow, sheep, rabbit, and mouse than in the rat. Inhibition of fertilization in the rabbit was also observed after passive immunization with antiserum absorbed with rabbit liver and kidney. All of the zonae recovered from the pig, cow, sheep, rat, and mouse after passive immunization with anti-pig zona serum exhibited strong fluorescence, regardless of the incidence of fertilization. It was concluded that the pig and other mammalian zonae pellucidae tested have tissue-specific antigens.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.     

Anticomplement immunofluorescence for the titration of antibody to varicella-zoster virus

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.     

Immunocontraceptive potential of the Ig‐like domain of Izumo

To investigate whether the Ig‐like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig‐like domain of mouse Izumo. The anti‐PB antibody significantly inhibited the fusion of sperm with zona‐free mouse eggs with no effect on sperm motility, while anti‐PA and anti‐PC antibodies virtually had no effect on sperm–egg fusion at the same concentration. Furthermore, in the presence of anti‐PB antibody, the anti‐sperm reactivity could be competitively inhibited by recombinant PB protein. The PB‐specific antibody staining was restricted to the acrosome region in acrosome‐reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig‐like domain of Izumo plays an important role in the fertilization process, as verified by the dose‐dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig‐like domain of Izumo might be a new candidate for the development of a contraceptive vaccine. Mol. Reprod. Dev. 76: 794–801, 2009. © 2009 Wiley‐Liss, Inc.     

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.     

小鼠组蛋白去乙酰化酶2多肽抗血清的制备

目的利用人工合成小鼠组蛋白去乙酰化酶2（histone deacetylase 2,HDAC2）多肽制备特异性抗HDAC2抗血清,用于相关疾病的体内外诊断。方法根据HDAC2基因编码的氨基酸序列合成多肽,与载体偶联后免疫动物,所制备的抗血清用ELISA、Western blot及免疫组织化学方法鉴定。结果 ELISA检测表明所制备的抗血清可同多肽抗原发生阳性反应,效价1∶4 000;Western blot结果显示抗血清可与多肽抗原及APP/PS1转基因小鼠的脑组织发生反应;免疫血清1∶100,1∶200,1∶400,1∶800四个稀释度均能与小鼠脑组织中的HDAC2反应。结论所制备的多肽抗血清可识别组织及血清中的HDAC2,可应用于相关领域的体内外研究。     

Inhibition of fertilization of the rabbit ova in vitro by the antibody to the inner acrosomal membrane of rabbit spermatozoa

The antibody to the rabbit sperm inner acrosomal membrane, raised in guinea pig, completely inhibited the fertilization of rabbit ova in vitro. The F(ab')2 of the antibody was equally effective in inhibiting fertilization. The antibody appeared to exert its inhibitory effect by binding to the inner acrosomal membrane of acrosome-reacted sperm. The antibody-treated sperm did not attach to or penetrate the zona pellucida. Thus, anti-IAM offers a great potential as a contraceptive agent.     

In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.     

Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay

The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Inhibition of fertilization in cattle by passive immunization with anti-zona pellucida serum

Passive immunization with antiserum prepared against isolated bovine zonae pellucidae inhibited fertilization in the cow. The minimum dosage of antiserum (titer 2⁷ by immunofluorescence) required for complete inhibition was 2 ml/kg of body weight.     

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.     

Fertilization of zona-drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.     

Monoclonal antibodies against unfertilized zona-free mouse oocytes: Characterization and effects on fertilization

A panel of anti-oocyte antibodies was raised against unfertilized zona-free mouse oocytes by intrasplenic immunization and checked for their effects on in vitro fertilization. Four antibodies decreased the fertilization rate from about 90% in controls to 8% (B5-2 F7), 12% (A2-2 A7), 13% (4-G1), and 25% (A2-2 F2), when the sperm cell concentration was 1 × 10⁵ to 1 × 10⁶. Antigen localization: All the antibodies labelled components in the cell membrane of zona-free oocytes as demonstrated by indirect immunofluorescence and/or by complement-mediated oocyte lysis. In various patterns, the ooplasm and zona pellucida were also labelled with different intensities. Western blotting: A2-2 A7 and A2-2 F2 recognized a protein with a molecular weight of approximately 65 kDa, while antibody B5-2 F7 bound a 97 kDa protein. Complement activation and complement-mediated oocyte lysis: Systemically injected antibodies, C3 and C4 were detected on zona-free oocytes recovered from the mouse oviduct indicating the activation of C3 and C4 by antigen-antibody complexes. The recovered oocytes were not damaged, suggesting a presence of complement-regulating factors. In vitro, however, a large number of zona-free oocytes preincubated with antibodies were lysed or protruded ooplasma vesicles in complement-active serum. Stage, tissue, and species specificity: None of the antibodies, except A2-2 A7, showed a positive immunolabelling to the pronuclear stage. Antibodies 4-G1 and A2-2 F2 cross-reacted with the ovarian oocytes. No antibodies bound to any of the tissues tested, indicating that the corresponding antigen epitopes are not commonly expressed. A2-2 A7, A2-2 F2, and B5-2 F7 cross-reacted with hamster and human unfertilized oocytes, suggesting the presence of developmentally conserved molecules and the possibility to apply these antibodies in hamster and human in vitro fertilization. It is concluded that the approach used could be a useful strategy in searching for anti-fertilization antibodies for human contraception. © 1996 Wiley-Liss, Inc.     

Mouse sperm antigens that participate in fertilization. II. Inhibition of sperm penetration through the zona pellucida using monoclonal antibodies

To dissect the process of mammalian sperm interaction with the egg at a molecular level, we have generated monoclonal antibodies (mAbs) to mature mouse sperm using syngeneic mouse testis as the immunogen. In this paper, we report upon three members of a mAb family, all of which displayed identical immunofluorescence patterns on cauda epididymal mouse sperm. Each of these mAbs, termed M42, M5, and M41, localized to a restricted region of plasma membrane overlying the acrosome. When tested for an effect on the fertilization process in vitro, two of the mAbs, M42 and M5, demonstrated significant inhibition. The inhibitory capacity was dependent upon the presence of the zona pellucida; neither M42 nor M5 was capable of blocking fertilization when zona pellucida-free mouse eggs were used. Identification of the antigens recognized by this group of mAbs was achieved by immunologic detection of sodium dodecyl sulfate-extracted sperm components separated via electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose. M42, which blocked fertilization, recognized a high molecular weight cluster of bands with Mr of approximately 220,000 to 240,000. M5, which also prevented fertilization, specifically recognized a sperm component with subunit molecular weight of approximately 54,000. M41, which did not interfere with fertilization, did not interact with any high molecular weight components, but recognized components with Mr of approximately 60,000, 35,000, and 21,000. Taken together with the work presented in a companion paper (Saling, Irons, and Waibel, this issue), we have demonstrated that it is possible to describe particular cellular regions of mammalian sperm with respect not only to location and function, but also to the molecules that are candidates for a role in that function.     

Monoclonal antibodies with specificity for three different serotypes of Marek's disease viruses in chickens

A total of 50 antibody-secreting hybridoma cells against Marek's disease virus (MDV) and turkey herpesvirus (HVT) have been produced. Eleven hybridomas were used for serotyping a panel of 15 pathogenic and nonpathogenic strains of MDV and HVT, representing three serotypes. The antibodies from the culture medium have fluorescence antibody (FA) titers of up to 100 and those from mouse ascitic fluid have titers ranging from 10(4) to 10(6). Monoclonal antibody T81 is type-common, i.e., it reacts at equal titer with all MDV and HVT tested. Of the remaining 10 antibodies, eight react only with pathogenic and attenuated strains of MDV (presumably serotype 1), one reacts only with nonpathogenic MDV (presumably) serotype 2), and one reacts only with strains of HVT (presumably serotype 3). Two hybridomas belong to IgG2a and IgG2b subclasses, respectively, and the remaining nine belong to IgG1 subclass. None of the antibodies specific for MDV strains reacted with homologous viruses in serum neutralization (SN), agar gel precipitin (AGP), or membrane immunofluorescence tests. Antibody L78, which is specific for HVT, was reactive with its homologous virus in the SN test; antibody from the culture medium showed an SN titer of 10 and that from mouse ascites a titer of 10,000. None of the antibodies specific for MDV or HVT reacted with other avian or mammalian herpesviruses, avian leukosis viruses (ALV), reticuloendotheliosis viruses (REV), or Marek's disease tumor-associated surface antigen (MATSA) expressed in a lymphoblastoid cell line, MDCC-MSB-1.     

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.