TESTIS ANTIGENS OF MAN AND SOME OTHER PRIMATES*

Transcription

1 .. FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society Vol. 26, No. 5, May 1975 Printed in U.SA. TESTIS ANTIGENS OF MAN AND SOME OTHER PRIMATES* ALAN C. MENGE, PH.D., AND BRYAN FULLER, B.Sc. Center for Research in Reproductive Biology, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan Since the demonstration of spermatozoal antigenicity, there have been numerous reports suggesting that some degree of human infertility may be a result of antibodies to spermatozoa. 1-3 The reported incidence of infertile men and women exhibiting serum antibodies to spermatozoa varies, largely due to the method used for detecting antibodies. 2 8 That the incidence of positive antibody tests among infertile patients was usually higher than that for control patients gave credence to the immunologic infertility hypothesis. Additional evidence, especially concerning female infertility, has come from animal experiments. Studies on animals have indicated that infertility can be induced by immunization with homologous seminal and testicular materials.9 11 The responsible antigen or antigens seem to be sperm-specific and of testicular origin. Investigations on sera 12 and fertility data 13 on infertile couples suggest that sperm-specific antigens also are of concern in human immunologic infertility. Although there are many reports of analysis of human seminal antigens, informative studies on the antigenicity of human testis and testes of other primates are lacking. Therefore, the present study was undertaken to examine the immunologic specificity and crossreactivity of testis antigens of man and, in Received July 15, *Supported by Grant NIH-HD from the United States Public Health Service, Center for Population Research. 473 addition, of some of the subhuman primates: chimpanzee (pan satyras), rhesus monkey (Macaca mulatta), and baboon (papio cynocephalus). MATERIALS AND METHODS The testes, epididymides, and sections of liver, kidney, and spleen of the subhuman primates were obtained immediately after the deaths of the animals and were stored frozen ( -60 C) until used. The human specimens were obtained at postmortem autopsies of accident victims and were also stored frozen. Testes homogenates were made by homogenization with an equal volume of cold saline (0.15 M NaCl) in a Waring Blendor. The extracts were prepared by mixing the homogenates with an equal volume of 0.05 M acetic acid containing 2% Triton X-100; the mixtures were stirred overnight in a cold room (5 C). The material was then centrifuged at approximately 27,000 x g for 30 minutes and the supernatant was dialyzed extensively against distilled water for 2 to 3 days in the cold room. The testis extract was lyophilized and dissolved as needed in saline or Tris HCl buffer (ph 7.8) (approximately 25 mg of powder/ml). Antisera were produced in male New Zealand White rabbits by intradermal or intramuscular and subcutaneous injections of the antigenic material mixed with an equal volume of Freund's complete adjuvant. One millilter of antigen-adjuvant mixture was injected into several

2 474 MENGE AND FULLER May 1975 sites per animal. The animals usually received weekly or biweekly injections for a total of three to five injections. The quantities of the different antigens used per injection were: 100 x 10 6 epididymal sperm (washed five times), 250 mg of homogenized testis (wet weight), and 5 to 10 mg of testis extract. Two to four rabbits per type of antigen material were used. Serum samples were obtained before immunization and at weekly intervals for 2 to 3 weeks after the third and subsequent injections. The rabbit antisera were absorbed with lyophilized serum powder (30 mglml) and equal volumes of washed and packed liver, kidney, and spleen cells from the respective species that the antisera had been raised against. Three young, sexually mature, male rhesus monkeys were immunized intradermally, two with rhesus testis homogenate and one with human testis extract. The animals received three biweekly injections; blood samples were obtained after the second injection. Human immune sera were obtained from men who had undergone vasectomy. The different immunologic methods used to detect antibodies and antigens were as follows: (1) the sperm immobilization test, 6 in which 0.2 ml of serum was serially diluted in saline, and 0.05 ml of diluted semen (40 x 106 sperm/ml) and 0.05 ml of complement (rabbit serum) were added; the mixture was incubated at 37o C for 1 hour and the percentage of motile sperm was estimated; (2) sperm immobilization inhibition tests, in which 0.1 ml of antigen was preincubated for 20 minutes at 37 C with diluted antiserum before the addition of sperm and complement; (3) immunoelectrophoresis (IEP), using 0.8% agarose in 0.01 M barbital buffer (ph 8.4) and approximately 3 rna/slide, for 60 to 75 minutes; (4) indirect immunofluorescence, using fluorescein isothiocyanate conjugates of goat antisera against rabbit 7 S y-gl~bulin, human y-globulin, and rhesus monkey y-globulin (Hyland Laboratories, Los Angeles, Calif.); and (5) testis proteinase activity inhibition, using the methods of Zaneveld et al. 17 and a gelatin membrane. For the protease inhibition studies, the y-globulins of the antisera were obtained by fractionation with 1fs volume of saturated (NH 4) 2S0 4 Equal volumes of antibody and antigen preparations were preincubated together for 30 minutes before 1 drop of the mixture was placed on a gelatin membrane, to be observed for proteolytic activity. Exposed, developed, and fixed Kodak projector slide plates were used as the gelatin membrane substrate. Extract of human testis was subjected to gel filtration on a column (1.6 x 60 em) containing Sephadex G-200 in 0.1 M Tris-HCl buffer (ph 7.8). Fractions (approximately 2.5 ml) were then collected. Rhesus monkey sperm were obtained from two normal males by electroejaculation, using a rectal probe and an SPE Ejaculator (Standard Precision Electronics, Denver, Col.). Human sperm samples were obtained from volunteers by masturbation. RESULTS The results of the immunoelectrophoresis, sperm immobilization tests, and protease inhibition studies are presented in Table 1. Rabbit antisera against testis homogenates and extracts showed numerous precipitin reactions with the specific testis antigenic materials against which they had been produced, as well as crossreactions with testis antigens of the other species. The results are composites of those obtained with the different antisera produced in rabbits immunized with the same antigenic material. The maximal number of testis-specific antigens detected was 8 to 10 for the different species. Immunization with the extracts induced antisera capable of detecting more antigens in IEP. The IEP procedure used to determine the common identity of testis

3 Vol. 26, No.5 TESTIS ANTIGENS OF PRIMATES 475 "' TABLE 1. Maximum Number of Precipitin Lines in Immunoelectrophoresis, Sperm-Immobilizing Titers, and Anti-Proteinase Activity of Different Antisera Determination Rabbit antisera against Rhesus monkey antisera against HTH HTE HES CTE RhTH RhTE RhES BTH BTE HTH RhTH Testis extract Human Chimpanzee Rhesus monkey Baboon Immobilizin titer Proteinase NT + + NT inhibition HTH, human testis homogenate; HES, human epididymal sperm; CTE, chimpanzee testis extract; RhTH, rhesus monkey testis homogenate; RhTE, rhesus monkey testis extract; RhES, rhesus monkey epididymal sperm; BTH, baboon testis homogenate; BTE, baboon testis extract; NT, not tested;+, inhibition... antigens among the primate species is illustrated in Figure 1. The antigens detected were presumed to be testisspecific, since the antisera had been absorbed with serum and tissue antigens of the respective species. After the absorbed antisera had been allowed to react with normal serum and saline extracts of liver, kidney, and spleen in IEP, no precipitin lines were found. The rhesus monkey antisera against human testis reacted in IEP with two similarly located antigens in extracts of human, rhesus monkey, and baboon testes. Rhesus monkey anti-rhesus monkey testis antisera showed cross-reactions with one and two antigens, respectively, in human and baboon testis extracts. None of the rhesus monkey antisera reacted with the extract of chimpanzee testis. The immunogen capable of inducing sperm-immobilizing antibody appeared to have common antigenicity among the primate species. The antisera, both rabbit and rhesus monkey, reacted equally as well with rhesus monkey sperm as with human sperm, which were used to obtain the titers presented in Table 1. The testis extract from each species absorbed the sperm-immobilizing activity of rabbit and rhesus monkey antisera against the sperm testis preparations of the homologous and the heterologous systems. These extracts also absorbed the immobilizing antibody of serum samples from two men who had had vasectomies. FIG. 1. Immunoelectrophoresis slide. Rabbit anti-baboon testis extract serum is in the troughs, baboon testis extract is in the top well, and rhesus monkey testis extract is in the lower well.

4 476 MENGE AND FULLER May 1975 fraction: antigen: 4 HTE a 2!~ 5._. 6 0 _ / / IEP ~-_:~:::::::::::::=:~:!;:;:~;;7=:::::: ---:==:9=:! ~Ht~'.o2sM I 2,4,6 5,7, 9,10 ][ 5,10 Abs.RAHTE :I E 0 CD ('I HTE Sephadex G-200 Tris-HCI O.lM 1.6x60cm. 40 FIG. 2. Composite graph illustrating (1) the protein profile of HTE obtained through a Sephadex G- 200 column and location of the sperm-immobilizing antigen (SlAg) and proteinase, and (2) IEP ofhte and of Sephadex G-200 fractions of HTE against absorbed rabbit antisera against HTE (JlAHTE). A similar cross-reaction was observed among the y-globulins from the different antisera, as judged by their ability to inhibit the proteolytic action of human testis extracts on gelatin membranes. y-globulins from normal sera of rabbits and monkeys and from a rabbit antiserum against human serum did not inhibit the proteolytic effects of the extract. Fractionation of the crude human testis extract (HTE) by gel filtration on a Sephadex G-200 column yielded poor separation but repeatable results (Fig. 2). The column eluates were arbitrarily divided into six different fractions (fractions I to VI). The fractions were subjected to immunoelectrophoresis and procedures to detect the proteinase activity and spermimmobilizing antigen. The proteolytic and sperm-immobilizing antigen activities were separated; sperm-immobilizing antigen was largely confined to fraction II, and proteolytic activity was confined to fractions III and IV. With these crude preparations, however, neither of the activities could be assigned to specific precipitin lines in IEP. Indirect immunofluorescent methods indicated that the rabbit and monkey immune sera reacted strongly with the acrosomes and main tail pieces and weakly or not at all with the midpiece and postnuclear cap area of human and rhesus monkey sperm. Obvious differences in staining intensities and patterns were not apparent among the rabbit antisera against testis preparations of the different species, except that anti-baboon testis antisera gave weak reactions with human sperm. Absorption of rabbit and rhesus antisera against human and rhesus monkey testis preparations and epididymal sperm with the dify

5 Vol. 26, No.5 TESTIS ANTIGENS OF PRIMATES 477 ferent HTE fractions from gel filtration indicated that acrosomal fluorescence was inhibited by fractions III and IV and tail fluorescence was affected by fractions II through V. DISCUSSION The immunoelectrophoretic analysis of extracts of testes from four primate species indicated a complexity of testicular antigens. There were at least 10 antigens of human testis that were specific, inasmuch as they did not cross-react with serum, liver, spleen, or kidney. With immunodiffusion methods, the detection of sperm-specific antigens with antisera directed against semen and epididymal sperm has proved difficult. Whereas Rao et al.18 reported finding three antigens specific for human sperm, other workers15 19 have failed to detect specific antigens. However, several antigens were found to be specific to seminal plasma, and they adsorbed onto sperm. Of the 10 testis antigens that were detected, 3 were identifiable by absorbed antisera against washed epididymal sperm. Additional absorption of the antisera with pooled seminal plasma from vasectomy patients did not reduce the number of precipitin lines. Absorption of anti-hte serum with the seminal plasma, however, did remove one to two precipitin bands with the testis extract. The number of immunoprecipitin lines was reduced to four with HTE against rabbit anti-hte serum that had been further absorbed with packed, washed, ejaculated sperm cells. These results imply that there is a minimum of four to five specific antigens of human sperm. As was expected from reports on other antigenic systems, there was a high degree of cross-activity of testis antigens among the four primate species. Investigations have been concerned with similarities among different primate species for the antigens of serum, 20 red blood cells,21 and leukocytes.22 Our study, based on the number of cross-reacting antigens, suggests that chimpanzee, baboon, and rhesus monkey testis antigens, in that order, more closely resemble those of the human. This is similar to reports for the antigenic systems. The immobilizin-inducing immunogen appeared to be antigenically identical among the four species. Our results confirm the observation by other workers 6 12 that the immobilizing antibody is directed against antigen originating in the testis. This antigen is probably membranebound, as observed by the swelling and lysis of human sperm,23 lysis and perforation of rabbit sperm plasma and outer acrosomal membranes, 24 and immunofluorescent staining of acrosomal membranes of guinea pig sperm25 caused by the action of the immobilizing antibody. It seems that, once the membrane integrity is violated, immobilization of the sperm rapidly follows. The human testis immobilizing antigen is acid-soluble and relatively heat-stable (60 C for 1 hour) and may be equivalent to the T antigen reported in guinea pig sperm. 26 The proteolytic enzymes of the testis extracts detectable by the gelatin membrane method appeared largely to have trypsin- and chymotrypsin-like activities. 27 Similar to the immobilizing antigen, the proteinases were immunogenic and cross-reactive among the species of primates examined. Human sperm aerosomal proteinase is immunogenic and is antigenically distinguishable from pancreatic tryspinp We did not determine the hyaluronidase activity of the preparations, although hyaluronidase has been shown to be immunogenic. Human testis hyaluronidase, however, appears to be antigenically distinctive in comparison with testis proteinases, since it is both tissue- and species-specific. 28 Most of the proteolytic activity observed in the extracts appeared to be chymotypsin-like,

6 478 MENGE AND FULLER May 1975 inasmuch as the testis trypsin-like activity was very labile to the effects oflyophilization, glassware, and neutral ph. Gel filtration of HTE, using Spehadex G-200, yielded poor resolution of the different testis antigens, possibly because of the vast number of different proteins in the supernatant. Initial purification steps seem to be necessary before cleaner separation by gel filtration is possible. There was partial separation of the spermimmobilizing antigen and proteinase activity. Absorption of rabbit antisera against HTE with those fractions containing the protease activity caused a significant reduction in sperm acrosomal fluorescence. This is in accord with the hypothesis that the site of origin of the proteolytic enzymes is in the acrosome of the mature sperm cell. SUMMARY Rabbit antisera raised against testis preparations of human, chimpanzee, rhesus monkey, and baboon origin were used to study testis-specific antigens within and among the four primate species. Antisera were absorbed with serum, liver, kidney, and spleen preparations of the respective species against which they had been produced. Immunoelectrophoretic analysis of testis extracts, using the absorbed antisera, indicated the following minimum numbers of testisspecific antigens for each species: man, 10; chimpanzee, 8; rhesus monkey, 10; and baboon, 8. Most of the testis antigens were cross-reactive among species. The results suggest that human spermatozoa possess at least four to five specific antigens which originate in the testis. The antigen that induces sperm-immobilizing antibody cross-reacted among the four species of primates. Human and rhesus monkey sperm reacted equally in the immobilization system. Testis extracts from each primate species were capable of removing the sperm-immobilizing activity of human immune sera by absorption. Testis proteinase activity, as determined by using a gelatin membrane substrate, was inhibited by the y-globulin fractions of rabbit and rhesus monkey antisera and also appeared to be cross-reactive among the species. Acknowledgments. We express our graditude to Dr. R. G. Snyder, University of Michigan Highway Safety Research Institute, for the generous gift of the subhuman primate tissues, to Dr. W. L. Johnson for assistance with the immunofluorescent techniques, and to Miss W. L. Hsiao for her technical asbistance. REFERENCES 1. Katsh S: Immunology, fertility, and infertility: an historical survey. Am J Obstet Gynecol 77:946, Behrman SJ: The immune response and infertility: experimental evidence. In Progress in Infertility, Edited by SJ Behrman, RW Kistner. Boston, Little Brown and Co, 1968, p Fjallbrant B: Sperm antibodies and sterility in men. Acta Obstet Gynecol Scand 4 7 [Suppl4] :13, Franklin RR, Dukes CD: Antispermatozoal activity and unexplained infertility. Am J Obstet Gynecol 89:6, Schwimmer WB, Ustay KA, Behrman SJ: An evaluation of immunologic factors of infertility. Fertil Steril18:167, lsojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. Am J Obstet Gynecol101:677, Isojima S: Relationship between antibodies to spermatozoa and sterility in females. In Immunology and Reproduction, Edited by RG Edwards. London, International Planned Parenthood Federation, 1969, p Ansbacher R, Manarang-Pangan S, Srivannaboon S: Sperm antibodies in infertile couples. Fertil Steril 22:298, Katsh S: Infertility in female guinea pigs induced by injection of homologous sperm. Am J Obstet Gynecol 78:276, Edwards RG: Immunological control of fertility in female mice. Nature 203:50, Menge AC: Immune reactions and infertility. J Reprod Fertil [Suppl]10:171, Manarang-Pangan S, Behrman SJ: Spermatotoxicity of immune sera in human infertility. Fertil Steril 22:145, 1971

7 Vol. 26, No.5 TESTIS ANTIGENS OF PRIMATES Ansbacher R, Keung-Yeung K, Behrman SJ: Clinical significance of sperm antibodies in infertile couples. Fertil Steril 24:305, Hekman A, Riimke P: The antigens of human seminal plasma, with special reference to lactoferrin as a spermatozoa-coating-antigen. Fertil Steril 20:312, Li TS, Behrman SJ: The sperm and seminal plasma specific antigens ofhuman semen. Fertil Steril 20:312, Shulman S, Bronson P: Immunochemicalstudies on human seminal plasma. I. Cha:l;lges in composition during storage as demonstrated by electrophoresis. Fertil Steril 19:549, Zaneveld LID, Schumacher GFB, Travis J: Human sperm acrosomal proteinase: antibody inhibition and immunological dissimilarity to human pancreatic trypsin. Fertil Steril24:479, Rao SS, Sheth AR, Sadri KK: Antigens of human spermatozoa. J Reprod Fertil 2:204, Weil AJ, Rodenburg JM: Immunological differentiation of human testicular (spermatocele) and seminal spermatozoa. Proc Soc Exp Bioi Med 105:43, Goodman M: Serological analysis of the systematics of recent hominoids. Hum Bioi 35:377, Weiner AS, Moor-Jankowski J, Socha WW: Principles of blood grouping in apes and monkeys: human, simian and cross-immune types. Transplant Proc 4:101, van Rood JJ, van Leeuwen A, Bainer H: HL-A and ChL-A: similarities and differences. Transplant Proc 4:55, Jeffrey W, Parish WE: Allergic infertility: laboratory techniques to detect anti-spermatozoal antibodies. Clin Allergy 2:261, Russo J, Metz CB: The ultrastructural lesions induced by antibody and complement in rabbit spermatozoa. Bioi Reprod 10:293, Toullet F, Voisin GA, Nemirowsky M: Histoimmunochemical localization of three guinea pig spermatozoal autoantigens. Immunology 24:635, Voisin GA, Toullet F: Relation between hypersensitivity responses to auto-antigens and tissue damage in the male reproductive tract. In Immunology and Reproduction, Edited by RG Edwards. London, International Planned Parenthood Federation, 1969, p Johnson WL, Menge AC: Unpublished data 28. Metz CB: Role of specific sperm antigens in fertilization. Fed Proc 32:2057,