RESUMO
Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.
Assuntos
Isoantígenos/análise , Espermatozoides/imunologia , Animais , Epididimo/ultraestrutura , Cobaias , Técnicas Imunoenzimáticas , Fragmentos Fab das Imunoglobulinas , Masculino , Microscopia Eletrônica , Espermátides/imunologia , Espermátides/ultraestrutura , Espermatozoides/ultraestruturaRESUMO
In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs of the Rockefeller and Hartley strains, immunized with different preparations of the guinea pig male reproductive tract (RMT) emulsified in complete Freund's adjuvant-H37Ra. The RMT preparations were purified guinea pig spermatozoal autoantigens S, P, and T; whole guinea pig spermatozoa, and extract from epididymal tissue. The cellular sensitivity in vivo was demonstrated by injecting the proper antigen into the skin of the tested animals and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with whole guinea pig spermatozoa cells were inhibited in vitro by the specific antigen, epididymal extract, and autoantigen-T. Autoantigen-S was found to be a weak immunogen. However, the migration of peritoneal exudate cells from guinea pigs sensitized with large amounts of antigen-S was inhibited by whole spermatozoa in vitro. This cross-reactivity revealed the possibility that the immunogenicity of purified autoantigen-S might be connected to its molecular size. According to the immunizing dose of the antigens, testicular lesions of either the aspermatogenic or orchitis type were found in the testes of sensitized guinea pigs. Lesions in the testes of the guinea pigs were not detectable by cross-immunization with heterologous human or rat spermatozoa, although some degree of in vitro cross-reactivity was detected by skin test studies.
Assuntos
Antígenos/análise , Antígenos/isolamento & purificação , Autoantígenos/análise , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Espermatozoides/imunologia , Animais , Autoantígenos/isolamento & purificação , Doenças Autoimunes/imunologia , Inibição de Migração Celular , Cobaias , Humanos , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Macrófagos/imunologia , Masculino , Orquite/imunologia , Especificidade de Órgãos , Ratos , Testículo/imunologiaRESUMO
Autologous or allogeneic spermatozoa in amounts of 10(8), 10(7), 10(6) or 10(5) incorporated in complete Freund's adjuvant were injected into hemiorchidectomized Hartley guinea-pigs. The course of autoimmune aspermatogenic orchi-epididymitis, delayed hypersensitivity and humoral responses to autologous and allogeneic spermatozoa and to spermatozoa autoantigens S, P and T (isolated from a large pool of spermatozoa) were investigated after 1,2,3 or 4 weeks of evolution. Testicular and epididymal lesions and the cellular and humoral responses to autoantigens S and T were nearly identical in autoimmunized and alloimmunized guinea-pigs; but the delayed hypersensitivity responses to whole spermatozoa was more frequent in alloimmunized than in autoimmunized guinea-pigs. This latter result might not be due to the addition of two responses on to autoantigens and one to alloantigens (both present in allogeneic spermatozoa) since the positive responses of the alloimmunized guinea-pigs to autologous spermatozoa compared to allogeneic were found more frequent. From the technical conditions of the experiments, especially due to the use of complete Freund's adjuvant, a helper effect originating in allotypic determinants on allogeneic spermatozoa seems an unlikely explanation. Autoimmunization or alloimmunization in incomplete Freund's adjuvant instead of complete adjuvant provoked a weak response. However, the antibody response to antigen S was significantly more frequent in alloimmunized than in autoimmunized animals; in this case allotypic determinants in autoantigen S responsible for a helper effect might be the cause.
Assuntos
Epididimite/imunologia , Espermatozoides/imunologia , Animais , Formação de Anticorpos , Antígenos , Autoantígenos , Doenças Autoimunes , Cobaias , Hipersensibilidade Tardia/imunologia , Isoantígenos , MasculinoRESUMO
Three different autoantigens (S, P and T), extracted and separated from guinea-pig spermatozoa, give rise to an autoimmune aspermogenic orchitis (AIAO) when injected with Freund's complete adjuvant (FCA). They also induce specific antibodies, such as anaphylactic (with S and P), complement-fixing (with P and T), spermotoxic (only with T) and precipitating and Arthus-inducing antibodies (only with P). Passive transfer of AIAO was attempted by injections of high total doses (15-20 ml per animal) of immune sera directed against one of the three antigens. Successful passive transfers were evaluated by the intensity of the epididymal and testicular lesions which were comparable to the actively induced ones, and by the rapid appearance of these lesions in less than 1 week and their lasting for at least 2 weeks. The disease was passively transferred with anti-P immune sera in as many as 64% of these cases and up to 40% with anti-T immune sera. Anti-S sera did not transfer AIAO more than did control normal and anti-DNP-BGG guinea-pig sera. The incidence and intensity of lesions were greatly for anti-P or slightly for anti-T increased by pretreating the future recipients with FCA. Hyperimmune sera are considerably more effective than early sera even when the latter are used in a time sequence reproducing that of the active reaction. The orchitogenic acitvity of anti-T sera appears to be localized in IgG2 DEAE fractions while that of anti-P has been found only in Ig1-containing DEAE fractions.
Assuntos
Antígenos/análise , Autoantígenos/análise , Orquite/imunologia , Espermatozoides/imunologia , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Reação de Arthus , Doenças Autoimunes/imunologia , Epididimite/imunologia , Cobaias , Imunização Passiva , Imunoglobulina G , Masculino , EspermatogêneseRESUMO
Use was made of water-soluble adjuvant fractions with homologous sperm, sperm extract or sperm autoantigens to induce autoimmune aspermatogenic orchiepididymitis (AIAO) in guinea-pigs. Guinea-pigs were injected with soluble sperm extract or sperm purified autoantigen S emulsified in incomplete Freund adjvant (IFA) together with two synthetic watersoluble adjuvants, Mur-Nac-L-Ala-D-isoGlu et Mur-Nac-L-Ala-acide D-isoGlu. These animals developed lesions of AIAO, delayed hypersensitivity reactions and specific anti-spermatozoa antibodies. These lesions and reactions were comparable to the ones induced in a control group treated with the same spermatozoa extract or autoantigen S in complet Freund adjuvants. No lesion was observed in the absence of IFA, Other guinea-pigs received a mixture of spermatoza or purified sperm autoantigens, S, P, T. and of polyadenylicpolyuridylic complex either in saline or emulsified in IFA. The recipients of sperm or autoantigen T mixed with Poly A:U, with ou without IFA, displayed lsdions of AIAO; autoantigen S and Poly A:U in IFA, provoked AIAO but did not do it in the absenceof IFA.
Assuntos
Adjuvantes Imunológicos , Doenças Autoimunes/etiologia , Epididimite/etiologia , Espermatogênese , Animais , Cobaias , Masculino , SolubilidadeRESUMO
Three approaches are utilized to study and characterize spermatozoal antigens. An immunological approach has demonstrated the presence of spermatozoal auto-, iso- and allo-antigens. Spermatozoal auto-antigens studies by several authors are able to induce the whole spectrum of immune reactions (delayed hypersensitivity, complement-fixing antibodies and anaphylactic antibodies0 as well as of autoimmune aspermatogenic orchiepididymitis (AIAO). Different extraction procedures result in various preparations and even in different independent autoantigens (at least four), one protein, one membrane-linked antigen and at least two glyco-proteins. Spermatozoal iso-antigens stricto sensu are determined by the Y chromosome and present on at least 50% of the spermatozoa. Spermatozoal allo-antigens are also present at the surface of spermatozoa, especially blood group antigens (ABO and MNS systems), transplantation antigens (HL-A, H-2) and also some other unidentified ones. A biochemical approach has mainly been directed towards spermatozoal enzymes that have been directed towards spermatozoal enzymes that have been shown to be antigenic even in the species of origin. This is the case for lactic dehydrogenase LDH-X (a mid-piece enzyme) and for acrosomal enzymes, e.g., hyaluronidase, possibly sorbitol dehydrogenase and trypsin-like acrosomal proteinase (the auto- and allo-antigenicity of the latter having not been established). At least three of these enzymes are known or supposed to play a role in the process of fertilization. A clinical approach has described the presence of spermatozoal-coating antigen(s), such as transferrin or blood group substances from secretors obtained following the admixture of the secretions of the seminal vesicles. Indications were also obtained for the existence of antibodies directed against defined antigens. Several types of localization of antibodies on spermatozoa were described: acrosome (front part), equatorial segment, post-nuclear region, mid-piece and tail. Attempts at fractionation of human psermatozoal antigens are still at a preliminary stage. Whatever the approach, the main interest of these antigens is that they are able to induce, in the species of origin or in a related species antibodies capable of interfering with the normal process of reproduction, especially fertilization..
Assuntos
Antígenos , Autoantígenos , Isoantígenos , Espermatozoides/imunologia , Oxirredutases do Álcool/metabolismo , Animais , Autoantígenos/análise , Sítios de Ligação de Anticorpos , Antígenos de Grupos Sanguíneos , Membrana Celular/imunologia , Testes de Fixação de Complemento , Epitopos , Feminino , Fertilização , Cobaias , Antígenos de Histocompatibilidade/análise , Humanos , Hialuronoglucosaminidase/metabolismo , Imunidade Celular , Isoantígenos/análise , Isoenzimas , L-Lactato Desidrogenase/imunologia , Masculino , Peptídeo Hidrolases/metabolismo , Cromossomos Sexuais , Sorbitol , Espermatozoides/enzimologia , Testículo/enzimologia , Testículo/imunologiaRESUMO
A quantitative ultrastructural study has been carried out on the lesions that are induced in vitro in guinea-pig spermatozoa by the action of auto-antispermatozoa antibodies and complement. The responsibility of three-independent autoantigen-autoantibody systems (S, P and T) has been explored. The only anti-T antibody known to fix complement and to be spermotoxic (T is a membrane-linked autoantigen), caused significant and important lesions, the immunologically specific origin of which was demonstrated. These lesions began a few seconds after complement had been added. The cytoplasmic membrane is first involved, then the acrosomal membranes, and then the acrosomal contents are lysed. The remarkable rapidity of action of complement on the antibody-sensitized target is emphasized. A typical dose-effect curve is obtained with dilutions of anti-T immune sera. Non-C1-fixing anti-S as well as C1-fixing anti-P antibodies (P has been shown to be intra-acrosomal) do not provoke any significant lesions, even in the presence fo complement, as compared to normal and various controls. However, anti-P serum, when added to non-damaging dilutions of anti-T in the presence of complement, was able to provoke significant lesions in the acrosomes. The bearing of these findings on the mechanisms of in vivo lesions is discussed.
Assuntos
Autoanticorpos , Proteínas do Sistema Complemento , Espermatozoides/imunologia , Animais , Reações Antígeno-Anticorpo , Cobaias , Soros Imunes , Masculino , Espermatozoides/ultraestruturaRESUMO
Inhibition by serum of experimental autoimmune orchitis (EAO) was studied in guinea-pigs. It was found that the capacity of an autoantigen, antigen P, purified from guinea-pigs' spermatozoa, to produce lesions of EAO could be inhibited by mixing antigen P with a small amount of normal human serum before injection into guinea-pigs. In protected animals, cell-mediated immunity and humoral antibodies to antigen P were also significantly suppressed.
Assuntos
Doenças Autoimunes/imunologia , Sangue , Orquite/imunologia , Animais , Formação de Anticorpos , Autoanticorpos , Autoantígenos , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Epididimo/patologia , Cobaias , Humanos , Hipersensibilidade Tardia , Imunidade Celular , Masculino , Orquite/patologia , Orquite/prevenção & controle , Espermatozoides/imunologia , Testículo/patologiaRESUMO
This report extends to the guinea pig the discovery of a lymphocyte plasminogen activator (LPA) previously described in the mouse. In the guinea pig, we have identified enzymatic activity similar to that in the mouse, but which has two distinct components: first, as in the mouse, a membrane-bound molecule present even in the quiescent lymphocyte (mLPA), and second, a previously unreported soluble molecule appearing in the culture medium after appropriate cell stimulation (sLPA). This sLPA, like most known lymphokines, was released by in vitro recall of sensitized lymphocytes by the antigen and by direct contact with a mitogen. There was a parallel evolution in the culture for sLPA and for some other well-known lymphokines (LT, LIF), and their detection thresholds were of the same order. A possible activation of plasminogen (Pg) by macrophages contaminating the lymphocyte cultures was carefully rule out. sLPA was produced by lymph node lymphocytes as well as by blood lymphocytes, but not by spleen cells, thymocytes, or peritoneal lymphocytes. A study of the kinetics of the release of sLPA, together with that of metabolic modifiers, suggested that an intracellular synthesis precedes the secretion of the molecule. Data obtained from the use of B- and T-enriched subpopulations or B- or T-dependent antigens and mitogens point to the B lymphocytes as the major, if not exclusive, source of sLPA. The choice of synthetic chromogenic substrates S 2251, S 2444, and S 2288 in some experiments led us to confirm most of the above properties of sLPA with still greater precision and reliability.
Assuntos
Linfócitos B/imunologia , Linfocinas/metabolismo , Ativadores de Plasminogênio/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Cobaias , Cinética , Linfonodos/citologia , Macrófagos/imunologia , Solubilidade , Especificidade por SubstratoRESUMO
Four autoantigens (S, P, T, Z), are known to be present in guinea pig spermatozoa. The only anti-T antibody is able to fix complement and is spermotoxic. Using immunoenzymatic technics, autoantigen T has been localized on the plasma membrane of spermatozoa and spermatids. A quantitative ultrastructural study has shown the anti-T induced irreversible, specific lesions on the germinal cells in presence of complement. A few minutes after addition of complement, almost all the cells are injured. Control sere (normal serum, anti-ova or anti-S or anti-P sera are inefficient. These findings are related to the mechanisms of autoimmune aspermatogenetic orchitis.
Assuntos
Especificidade de Anticorpos , Antígenos/análise , Autoanticorpos , Autoantígenos/análise , Proteínas do Sistema Complemento/metabolismo , Espermatozoides/imunologia , Animais , Cobaias , Técnicas Imunoenzimáticas , Masculino , Espermatozoides/ultraestruturaRESUMO
In this study, AIAO was adoptively transferred with a high proportion of success to syngeneic recipient guinea pigs. Donors of strains 2 and 13 were sensitized with a series of spermatozoal autoantigens (whole spermatozoa and three autoantigens isolated therefrom: S, P and T). Syngeneic (experimental) and allogeneic (control) recipients were all transferred by strictly i.v. injections of lymphoid cells. The damage observed in testis and epididymis (mainly in the latter) was identical to, but milder than, that seen in active forms of AIAO. The incidence and severity of the disease were dependent on: the type of inducing antigen, S, T, P in order of decreasing efficiency; the length of immunization in donors, with increasingly serious lesions as periods ranged from 1 to 4 weeks; the presence or not of a complementary treatment of recipients with bacterial adjuvant enhancing the disease. Other parameters were less important, such as the strain 2 or 13 specificities, the amount of immunogen or the addition of peritoneal cells to lymph node cells. Skin hypersensitivity was concomitantly transferred to a large majority of isogenic recipients. But the incidence and severity of the disease showed only a partial correlation with Arthus type or delayed type responses to autoantigens. Thus guinea pig AIAO, already known to be transferable by immune sera (mainly anti-P and also anti-T) may also be transferred in physiological conditions by sensitized lymphoid cells (mainly anti-S and also anti-T).