1140406603The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo -/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.     

Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--II. Epitope analysis of monoclonal antibodies

The method for comparison of epitope specificity of different monoclonal antibodies to one antigen, using a panel of monoclonal antibodies to mouse and human alpha-fetoprotein is described. The method used exploits the special properties of electroendosmotic flow in nitrocellulose membranes under the conditions of anionic isotachophoresis. Electroendosmosis allows successive transfer of several immunoreagents to the dots of monoclonals previously bound to the nitrocellulose membrane. The inhibition of antigen binding to monoclonal dot, if the antigen is mixed with excess of another monoclonal antibody, demonstrates that both monoclonals are directed to the same epitope, and vice versa. The method requires neither purified monoclonals nor antigens, or radio labelling, and is performed semi-automatically. It was shown that each monoclonal antibody to mouse and human alpha-fetoprotein had its own epitope specificity.     

Natural and induced immunological infertility

Given the observation that naturally occurring antibodies to eggs and sperm can cause infertility, it seems feasible to pursue development of an infertility vaccine based on the induction of a specific immune response to gamete or early embryo antigens. Antibodies directed to the zona pellucida have been researched, but at current levels of purification, result in reduced ovarian hormone production. Of the numerous sperm antigens, LDH-C4 appears most promising for use in a vaccine. In the past decade, antisperm antibody investigations have focused on surface antibodies and sperm mixed agglutination reactions. It appears that antibodies in accessory fluids bind to sperm during ejaculation and/or antisperm antibodies enter the male tract at the epididymal level or higher. Antibodies directed against egg or sperm may prevent or modify the normal process of capacitation in which sperm undergo a series of biochemical and morphological transformations. Antisperm antibodies can suppress fertility by preventing sperm transport through cervical mucus or impeding the sperm-egg interaction during fertilization. The definition of sperm antigens associated with infertility--essential for development of a contraceptive vaccine--is being facilitated by monoclonal antibody techniques and DNA technology. Since the sperm surface is organized into highly specialized and distinct regions, cell recognition is an important research area. Most salient to the recognition and regulation of cell interaction are the components of the sperm plasma membrane and the zona pellucida.     

Monoclonal antibodies to human eosinophil plasma membrane antigens enhance the secretion of eosinophil cationic protein.

This study was done to examine the nature of the membrane constituents involved in the secretion of eosinophil cationic protein (ECP) from human blood eosinophils. Three mouse monoclonal antibodies were used, which showed greater binding to membrane antigens on activated, and light density eosinophils from patients with an eosinophilia, than on nonactivated or normal density eosinophils. All three antibodies (EoN4, EoN5 & EoN6) stimulated normal density human eosinophils to secrete ECP, either alone or in association with sepharose-C3b. The antibodies bound to at least two separate sites on the membrane, which were distinct from the receptors for immunoglobulins, C3b, and eosinophil activating factor. One combination of antibodies increased the amount of ECP which was secreted. The membrane antigen recognized by antibody EoN4 was a glycoprotein, molecular weight 75 kD. These findings showed that ECP secretion may be induced by a wider range of stimuli than has been previously recognized, and that the antigens recognized by these monoclonal antibodies may play an important role in the induction of eosinophil degranulation.     

Sites of antisperm antibody action.

Antisperm antibodies can affect sperm function in the cervical mucus, as they undergo capacitation, or during sperm-egg interaction. During capacitation, the sperm membrane destabilizes and antigens previously not available to antibodies may become exposed. Antibody attachment can reduce fertilization as measured by hamster egg preparation or the hemizona assay. A panel of monoclonal and polyclonal patient sera were tested for their ability to inhibit sperm-zona pellucida tight binding. We tested 25 monoclonals from a panel from the World Health Organization, but only nine were positive by indirect bead binding, indicating that the majority of the antibodies were not sperm surface antigens. The sera of 13 patients were used; three were negative and served as controls, nine had antibodies of the IgG isotype bound to 100% of the sperm heads examined, and seven also exhibited sperm specific IgA antibodies. Of the monoclonals tested, six inhibited zona binding by 60%. Sera of seven patients caused inhibition of greater than or equal to 50% zona binding; those from male patients caused the greatest inhibition. When further specific testing was conducted, one monoclonal caused the greatest inhibition of zona binding; sera of three patients inhibited zona binding by 70%. The antibodies, whether monoclonal or patient sera, were tested for their effect on capacitation. The ability to undergo hyperactivated motility after antibody exposure was assessed and no changes appeared due to antibody exposure. Serum from one of the patients appeared to affect sperm calcium influx to some extent. Sera from two patients appeared to reduce the number of spermatozoa capable of undergoing the acrosome reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human monoclonal anti-nuclear antibodies produced by human-mouse heterohybridomas

To obtain human monoclonal anticentromere antibodies, mouse myelomas were fused with unfractionated mononuclear cells from the peripheral blood of a patient diagnosed as having the CREST variant of scleroderma: with only anticentromere antibodies. After a single fusion an heterohybridoma secreting a human antibody specific for nuclear antigens, as detected by indirect immunofluorescence staining, was isolated. The monoclonal antibody secreted by the clone was of the human IgM class. Indirect immunofluorescence staining of the antibody on HEp-2 cells showed multiple nuclear dots or a discrete speckled pattern resembling that of an anticentromere antibody. Immunoblot analysis showed antibody binding to a 33 kD antigen derived from the nuclear protein fraction. Enzyme-immunoassay results clearly showed that the antibody reacted with the chromosomal protein fraction and not calf thymus DNA.     

Production of monoclonal human antibody to HLA-DR5 (DRw11) by mouse/human heterohybridomas

We describe here the production of a human monoclonal antibody to the HLA-DR5 antigen. A human B-cell line secreting cytotoxic antibody that reacted preferentially with DR5-positive targets was fused to the mouse myeloma P3X63Ag8.653 and the resulting heterohybridomas cloned twice. The clones secreted human IgM (lambda light chain), which showed specificity for the DR5 antigen in cytotoxicity assays and reacted with DRw11-positive but not DRw12-positive targets. These results demonstrate the potential of this approach to the production of human monoclonal antibodies to transplantation antigens.     

Current status of anti-zona pellucida antibodies

It is clear that the mammalian zona pellucida contains tissue-specific antigens that cross-react among certain species. Certain of these antigens generate antibodies that inhibit sperm attachment. Polyclonal antibody production may be an important aspect of this inhibition. In certain species there are other effects of anti-zona antibodies, such as direct action on the ovary. It is uncertain whether immunization with zona antigens will ever be a practical method of contraception in humans. Such vaccination might require unacceptable adjuvants or large amounts of antigen. The persistence and effectiveness of the antibody is not yet proven, and pregnancy has occurred in some despite presence of anti-zona autoantibodies. A safe and effective vaccine may still be found, however, given the large variety of zona pellucida antigens available. The cause of naturally occurring anti-zona pellucida antibodies in humans is unknown. The incidence of these antibodies depends on the assay used. The significance of positivity in a given individual is also uncertain. A number of patients will conceive if other concurrent fertility problems are treated. Positive results should be confirmed by a second method, preferably by testing the sera against human ova. Specific treatment by steroids or other immunosuppressive regimens remains controversial.     

Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Identity between SAGA-1, the H6-3C4 antigen, and CD52

PROBLEM: The correlation of anti-sperm antibodies (ASA) with some instances of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as a more complete understanding of the mechanism behind this phenomenon, are dependent on the identification and characterization of relevant sperm antigens. METHOD OF STUDY: In this article, we review literature on methods employed to identify sperm antigens using anti-sperm polyclonal and monoclonal antibodies from infertile patients and vasectomized men. Particular focus is given to approaches using human and mouse monoclonal antibodies to define the SAGA-1 human sperm antigen. RESULTS: ASA present in sera and genital tract secretions from infertile patients and vasectomized men have been employed in a variety of methods to identify sperm antigens. In an alternate approach, a monoclonal antibody (mAb), H6-3C4, was immortalized from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titers. Subsequently, the sperm-agglutinating, murine S19 mAb was shown to react with the H6-3C4 cognate antigen. The H6-3C4 S19 cognate antigen, designated Sperm Agglutination Antigen-1 (SAGA-1), was characterized as a polymorphic, highly acidic, GPI-anchored glycoprotein on the surface of human spermatozoa. Purification with the S19 mAb followed by microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Immunoblot analysis demonstrated that these two glycoproteins differed in carbohydrate composition. Thus, sperm SAGA-1 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core peptide but with different carbohydrate structures. CONCLUSIONS: Autoimmunity to the SAGA-1 and/or CD52 glycoforms may lead to infertility. Structural and immunologic differences between these glycoproteins may be important factors in the etiology of immunologic infertility and other autoimmune disorders.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

人红细胞血型A抗原的亲和层析纯化

ＡＢＯ抗原是人红细胞上一类重要的血型抗原，抗Ａ、Ｂ型抗体在临床检验中是必不可少的试剂。为了提高抗Ａ单克隆抗体的生产效率，用ＩｇＭ抗体亲和层析法提纯了Ａ糖脂蛋白的可溶性部分，并在用一些免疫生化的方法加以鉴定之后，将它通过抗体分子的中介与胶颗粒连结，成为颗粒性抗原免疫Ｂａｌｂ／ｃ小鼠，以探讨这种纯化和免疫方法的可行性。     

Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance

The mammalian zona pellucida has an important function in the fertilization process. The zona pellucida protein 3 (ZP3 or ZPC) is the ligand for primary sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The objective of these studies was to localize ZP3 protein using antisera generated against defined synthetic peptides that are specific for mouse or for human ZP3. Immunohistochemistry and transmission electron microscopy were applied to murine and human ovary sections. Immunochemical studies were performed in hemizonae pellucidae from microbisected human oocytes. Using the competitive hemizona assay and various anti-ZP3 antibodies, we further intended to identify human ZP3 epitopes of functional significance. Our results showed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein in mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) detected human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots. ZP3 material was also detected in cumulus cells by immunohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay revealed that none of the anti-ZP3 synthetic peptide antisera affected sperm binding suggesting that those epitopes are not involved in primary sperm binding. Anti-porcine ZP3 beta protein antibodies (polyclonal) blocked human sperm-zona pellucida binding. In summary, these anti-ZP3 synthetic peptide antibodies specifically reacted with intact ZP3 protein (murine and human) but did not inhibit human sperm-zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.     

Monoclonal autoantibodies from insulin-dependent diabetic patients: autoantibodies against beta-cell surface or cytoplasmic antigens.

To investigate the target antigen(s) recognized during the autoimmune process in insulin-dependent diabetes mellitus (IDDM), we produced human monoclonal antibodies by Epstein-Barr virus transformation of peripheral blood lymphocytes from a large number (n = 50) of newly diagnosed IDDM patients. Screening by indirect immunofluorescence assay, using the RINm5F rat insulinoma cell line and eight other human or rat tumour cell lines, was performed to identify monoclonal antibodies that reacted with either membrane or cytoplasmic antigens. Eighteen IgM monoclonal antibodies reacting with cytoplasmic antigens of RIN cells were obtained; 14 of them also showed a staining of the cytoplasm of various non-beta-cell lines, while four displayed a binding restricted to beta-cells among the panel tested. However, among three monoclonal antibodies reacting with the membrane of RIN cells, one (HMD-1) produced an IgG antibody with a binding restricted to the membrane of beta-cells (RIN, HIT, and normal rat islet cells). The membrane antigens of HMD-1 were identified in Western blotting as proteins with molecular weights of 64 and 70 kD. This antibody had no apparent cytotoxic effect on RIN cells. These data suggest that, apart from 'natural autoantibodies,' it is feasible to obtain human monoclonal antibodies from IDDM patients that bind specifically to the beta-cell cytoplasm or to the beta-cell membrane.     

重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

Monoclonal Antibodies Reacting Against Capacitated but not With Freshly Ejaculated Boar Spermatozoa

PROBLEM: The involvement of individual sperm proteins in differentiation of antigenically specific and functionally defined regions on sperm membrane has not yet been completely elucidated. METHOD: BALB/c mice were immunized with live capacitated boar spermatozoa and used for production of monoclonal antibodies (MAbs). ELISA, IIF, SDS-PAGE, IVF, and cytologic methods were used for selection and biological characterization of MAbs as well as for identification of corresponding antigens. RESULTS: MAb1F10, MAb2E2, and MAb4B12 react with antigens in the acrosome portion of live capacitated spermatozoa. MAb 1F10 reacted with human sperm cells along with those from bull, ram, mouse, dog, whereas MAb2E2—with mouse's spermatozoa and MAB4B12 - with bull's, mouse's, and dog's spermatozoa. Some glycolytic enzymes seemed to reduce mildly the reactions of the MAbs with enzyme treated sperm cells; proteolytic enzymes eliminated the binding of MAbs to the sperm acrosome. These MAbs have no sperm agglutinating and/or sperm-immobilizing activities and reduced the number of spermatozoa binding to zona pellucida. CONCLUSIONS: MAb1F10, MAb2E2, and MAb4B12 seemed to recognize membrane associated antigens with potential role in the initial stages of fertilization, specific for capacitated but not for freshly ejaculated spermatozoa.     

Biochemical analysis of the rat MHC class I antigens RT1.Aa, RT1.Fa and Pa

In DA strain rats, there are two other MHC class I loci (Pa and RT1.Fa) in the vicinity of the classical class I locus RT1.Aa. The Pa antigen is the pregnancy-associated antigen, and it was detected by antibodies elicited in WF females pregnant by DA males without any other immunization. The Fa antigen was detected by a monoclonal antibody raised by alloimmunization. In the present work, the Aa, Fa and the Pa antigens have been compared by HPLC peptide mapping and by isoelectric focusing after their isolation by appropriate monoclonal antibodies. All the three antigens are identical in primary structure with respect to lysine, methionine, asparagine and the aromatic amino acid residues, but they differ from one another with respect to glutamic acid and/or aspartic acid residues. The pI values of the antigens differ slightly. All three antigens have two identical N-linked glycans, but the Fa antigen has an additional N-linked glycan. Based on the available amino acid sequence of the Pa antigen, it can be concluded that both Aa and Pa antigens are devoid of glycosylation in the second domain. This lack of glycosylation of the classical antigen Aa is unique for the rat, since classical class I antigens of the mouse show glycosylation in the first and second, and sometimes in the third domain, and those in the human, in the first domain only. The high degree of similarity among the Aa, Fa, and Pa molecules that this study indicates is also unique for the rat, since antigens encoded by different class I genes of the same haplotype are quite disparate in the mouse and human.     

Murine monoclonal antibodies reactive with human heart and group A streptococcal membrane antigens.

Ten selected murine hybridoma cell lines that produce monoclonal antibodies against M type 5 Streptococcus pyogenes and human heart antigen were isolated. All of the monoclonal antibodies studied were determined to be the immunoglobulin M isotype. The antibodies were characterized on the basis of their reactions with Triton X-100-extracted whole human heart antigens, sodium dodecyl sulfate-extracted sarcolemmal antigens, and whole streptococci or their membranes. Enzyme-linked immunosorbent assays and Western immunoblotting techniques were used to compare the reactivity of the monoclonal antibodies. All 10 of the antibodies were first selected for their reactivity with Triton X-100-extracted heart antigens and whole group A, M type 5 streptococci. These antibodies were then divided into two categories: strong reactors or weak reactors with human sarcolemmal and streptococcal membranes. Among the strong reactors, two different types of monoclonal antibodies were observed on the basis of their immunobanding patterns with sarcolemmal and streptococcal membranes on Western blots. Monoclonal antibodies that were strong reactors with sarcolemmal and group A streptococcal membrane antigen were directed against a determinant on a family of proteins. The major reactants of sarcolemmal extracts were high-molecular-weight proteins near 200,000. Some monoclonal antibodies demonstrated more specificity for the heart than did others when reacted with separated Triton X-100-extracted tissue antigens from the heart, kidney, and skeletal muscle. One of the monoclonal antibodies that reacted with group A streptococci reacted with a Triton X-100-extracted heart antigen ca. 40,000 daltons in size. None of these monoclonal antibodies opsonized type 5 Streptococcus pyogenes, and in enzyme-linked immunosorbent assays most of the antibodies were found to react to a lesser degree with other groups of streptococci. Monoclonal antibody was used to probe normal and rheumatic sarcolemma for differences in reactivity. Although the rheumatic heart reacted more intensely, no major differences between the immunobanding patterns of normal and rheumatic hearts were observed.     

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Peripheral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody/antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.     

Characterization of boar spermadhesins by monoclonal and polyclonal antibodies and their role in binding to oocytes.

PROBLEM: The role of Ala-Trp-Asn (AWN) and Ala-Gln-Asn (AQN) families of spermadhesive sperm proteins in fertilization. METHOD OF STUDY: The preparation and characterization of polyclonal antibodies against AWN and AQN spermadhesins and one monoclonal antibody (MAb), designated Bo.5, against AWN spermadhesin. The use of biochemical and immunocytochemical methods for characterization of spermadhesins on the sperm membrane of boar spermatozoa and in the cryostat sections of boar reproductive organs. RESULTS: Polyclonal anti-AWN and anti-AQN antibodies specifically reacted with AWN and AQN proteins, respectively. MAb Bo.5 detected the 17-, 16-, and 14-kDa protein members of AWN subfamily. The monoclonal, as well as the polyclonal, AWN antibodies remarkably decreased the sperm binding to the egg surface in an in vitro sperm zona pellucida binding assay. CONCLUSIONS: Presented results demonstrate that polyclonal antibodies and MAb Bo.5 against spermadhesins specifically recognize the membrane-associated antigens and inhibit the binding of sperm to oocytes. Reduced binding of sperm to oocytes, due to the antibodies, indicates the role of these spermadhesins in sperm-egg primary binding.