IN 1899, Landsteiner discovered a sperm-immobilizing antibody in guinea

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1 Mechanism of Experimental Sterility Induced in Guinea Pigs by Injection of Homologous Testis and Sperm I. Relationship Between Sterility and Anaphylactic Reaction of the Uterus YOSHIO ASHITAKA, M.D., SHINZO ISOJIMA, M.D.," and HISASHI UKITA, M.D. IN 1899, Landsteiner discovered a sperm-immobilizing antibody in guinea pigs which had been injected intraperitoneally with bovine sperm. Since that time, there have been a number of reports concerning the antisperm antibody and its relationship to sterility after injection of sperm. Specific precipitins, agglutinins, and spermatotoxins against testis and sperm were found by many other workers According to Isojima and Stepus, 12 guinea pig testis has three kinds of organ-specific antigens which are absent in female guinea pigs and, hence, capable of eliciting antibody production in them. Nevertheless, results have been contradictory concerning the sterility induced by immunization of female animals with testis and sperm McCartney discovered in 1923 that, after subcutaneous injection of sperm into female rats, a sperm-immobilizing antibody appeared in the blood and vagina, and that there was a decrease in conception. In 1940, however, Henle concluded that no tendency towards infertility existed in female guinea pigs after immunization with sperm, even though antisperm antibodies were found in the serum. In 1959, Freund's adjuvant was introduced by Isojima et aly in the immunization of female guinea pigs with testis homogenates, and the development of sterility in these animals From the Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka, Japan. Presented, in part, at the 17th meeting of the Japanese Association for Research on Sterility, Kansai Region, Kyoto, Japan, June 11, *Present address: Department of Biochemistry Research, Roswell Park Memorial Institute (New York State Department of Health), Buffalo, N. Y. 206

2 VoL. 15, No.2, 1964 STERILITY: UTERINE ANAPHYLAXIs 207 was conclusively demonstrated. Katsh 17 obtained similar results with guinea-pig sperm and Freund's adjuvant. In 1957 and 1958, Katsh used the Schultz-Dale technic and found that a guinea-pig uterus immunized with testis or sperm would contract upon contact with a suspension of sperm. On the basis of this finding, Katsh suggested that the contraction induced would impair the implantation of the fertilized egg in the endometrium. The following experiments were carried out to demonstrate the possible relationship of uterine anaphylaxis to sterility. Preparation of Sperm Suspension MATERIALS AND METHODS Testes, together with the epididymides, were taken from male guinea pigs ( gm.) under ether anesthesia, and the epididymides were separated. The sperm was washed out of the seminiferous ducts with 2 ml. of Tyrode's solution, and diluted with Tyrode's solution until the concentration of sperm was in the range of 2.0 X 10 7 jml. to 1.0 X 108 jml. Immunization One milliliter of sperm suspension mixed with 1 gm. of testis was homogenized in a Potter-Elvehjem homogenizer and then emulsified with an equal volume of complete Freund's adjuvant. 8 Female guinea pigs ( gm.) were injected intradermally with 0.1 ml. of the resulting mixture in each of six different sites on the back (total dose, 0.6 ml.). The immunization was repeated twice: 2 weeks later, and 3 weeks after that. Anaphylactic Reaction An adult guinea-pig uterus, immunized with homologous testis and sperm, was extirpated along with an attached portion of the vagina, and was connected with a glass tube (Fig. 1) so that the cervix and the tip of the glass tube were facing each other (Fig. 2). After one uterine horn was tied and the end of the other clamped, the uterus was placed in a perfusion medium. of Tyrode's solution saturated with oxygen and kept at 37 C. The reaction, whether anaphylactic or negative, was recorded by means of a kymograph. RESULTS When 0.2 ml. of guinea-pig sperm suspension ( 4.0 X 107 jml.) was added by means of a long injection needle to the bottom of the curved glass tube

3 208 AsHITAKA ET AL. FERTILITY & STERILITY containing 3.0 ml. of Tyrode's solution, the immunized uterus showed only slight, irregular contractions (Fig. 3). Seven minutes later, however, upon the addition of 1.0 ml. of sperm suspension to the 215 ml. of perfusion medium in the beaker, a typical anaphylactic reaction occurred. The ratio of the ' Fig. 1 (left). Glass tube for producing anaphylactic reaction of guinea-pig uterus. The tip (diameter, 2 mm.) of the glass tube is connected with attached portion of vagina or inserted into uterine cavity. Fig. 2 (right). Attached portion of vagina connected to tip of glass tube, and one horn of uterus to kymograph. A indicates addition of sperm suspension to bottom of glass tube; B, addition of sperm suspension to perfusion bath. sperm concentration in the glass tube to that in the perfusion bath was 14.3:1. Similar results were obtained (Fig. 4) with a uterus having no irregular contraction. In this case, 0.5 ml. of sperm suspension ( 4.0 X 10 7 jml.) was added to 1.5 ml. of Tyrode' s solution in the glass tube, and 10 min. later, 0.5 ml. of the same sperm suspension was added to the 197 ml. of perfusion bath. The ratio of the sperm concentrations was 131:1. In the next experiment, the tip of the glass tube was inserted into the uterine cavity, so that the sperm would be directly in contact with the endometrium by motility or diffusion. The uterus did not contract on the addition of sperm through the glass tube. Some of the uteri were examined after 5 min., and spermatozoa were found in the uterine cavity (Fig. 5). In another version of the same experiment, after sperm suspension was added to the outer perfusion bath 15 min. later, contractions resulted (Fig. 6). The ratio of the sperm concentrations was 126:1. As a control, a nonimmunized guinea-pig uterus was used in the same manner, but no uterine contraction occurred. Acetylcholine was then substituted for sperm suspension in a normal guinea-pig uterus preparation with the glass tube in contact with the cervix. The uterus did not show any contraction upon addition of 0.1 ml. of acetyl-

4 VoL. 15, No. 2, STERILITY: UTERINE ANAPHYLAXIS choline ( 1:1,000) to 1.2 ml. of Tyrode's solution in the glass tube, but it contracted remarkably when 0.1 ml. of acetylcholine ( 1:1,000) was added to the 180 ml. of the outer perfusion bath (Fig. 7). The ratio of acetylcholine concentrations was 150:1. When the sperm suspension was poured into the uterine cervix through the glass tube after the acetylcholine-induced contraction of the uterus, smear preparations 30 min. later showed sperm in the uterine cavity. Fig. 3. Kymogram of guineapig uterus sensitized with guinea-pig testis and sperm. A in-dicates addition of sperm suspension ( 0.2 mi.) to perfusion medium ( 3.0 mi.) in glass tube; B, addition of sperm suspension ( 1.0 ml.) to perfusion medium (215 mi.) in bath. Fig. 4. Kymogram of guineapig uterus sensitized with guinea-pig testis and sperm, and showing no irregular contraction. A indicates addition of sperm suspension ( 0.5 mi.) through glass tube; B, addition of sperm suspension ( 0.5 mi.) to perfusion medium ( 197 ml. ) in bath. Fig. 5. Presence of spermatozoa in smear preparation from uterine cavity 5 min. after addition of sperm suspension to perfusion medium in glass tube (tip of tube inserted into uterine cavity). {l ta,...- t 10.,.;... B --,

5 210 AsHITAKA ET AL. FERTILITY & STERILITY r Fig. 6. Kymogram of guineapig uterus sensitized with guinea-pig testis and sperm. A indicates addition of sperm suspension (0.3 ml.) to perfusion medium in glass tube (tip of tube inserted into uterine cavity); B, addition of sperm suspension (0.3 ml. ) to perfusion medium ( 126 ml.) in bath. Fig. 7. Kymogram of normal guinea-pig uterus. A indicates addition of 0.1 ml. of acetylcholine (1:1,000) to perfusion medium ( 1.2 ml.) in glass tube; B, addition of 0.1 ml. of acetylcholine (1:1,000) to perfusion medium ( 180 ml.) in bath. DISCUSSION From the results obtained in the present study, it appears that the immunized uterus of the female guinea pig will not contract upon contact with sperm under natural conditions in the vagina. From an immunological standpoint, the immune reaction causing uterine contraction cannot continue until all the sperm in the vagina are dead, because, in most cases, relaxation will follow within 20 min. (as we have observed), and spermatozoa can survive longer than 40 min. in the vagina. Also, anaphylactic contraction of a uterus will not occur in response to the same antigen after relaxation from the first contraction. As a consequence, sperm can then pass through the cervix into the uterine cavity. From our present study, it is also clear that sperm can pass through the cervical canal even during uterine contraction following acetylcholine administration. It was demonstrated that a guinea-pig uterus immunized with testis and sperm did not contract upon contact of the sperm with the surfaces of the cervix and endometrium. Any sperm which might pass into the abdominal cavity through the fallopian tubes would not be enough to induce an anaphylactic reaction and, thus, to cause the uterus to contract. In fact, we found no spermatozoa in the fallopian tubes and only a few at the base of

6 VoL. 15, No. 2, 1964 STERILITY: UTERINE ANAPHYLAXIS 211 the uterine horn 3 hr. after instillation of 0.2 mi. ( 2 X 10 7 jml.) of sperm suspension into the vagina. Six and 12 hr. after instillation of sperm into the vagina, we found no sperm in any part of the fallopian tubes. In the present study, we have tested the suggestion made by Katsh that uterine anaphylaxis might be a cause of sterility in guinea pigs immunized with testis and sperm, but we have found this suggestion unsatisfactory from the standpoint of immunological desensitization as well as on the basis of the results of our own study. SUMMARY 1. The uterus in guinea pigs immunized with homologous testis and sperm was shown to contract upon contact of sperm with the serosal surface of the uterus, but not upon contact of sperm with the surfaces of the cervix and endometrium. 2. Sperm migration ~rom the vagina into the uterine cavity could be demonstrated in a guinea-pig uterus following contraction caused by acetylcholine. 3. Our findings indicate that uterine anaphylaxis is not responsible for the sterility of female guinea pigs immunized with testis and sperm. S.I. Roswell Park Memorial Institute Buffalo, N. Y. REFERENCES 1. BASKIN, M. J. Temporary sterilization by the injection of human spermatozoa. A preliminary report. Am. ]. Obst. & Gynec. 24:892, BRUNNER, E. K. Experiment in temporary immunization against pregnancy. Human Fertil. 6:10, DITTLER, R. Die Sterilisierung des weiblichen Tierkorpers durch parenterale Spermatufuhr. Munchen med. Wchnschr. 67:1495, EASTMAN, N. J., GuTTMACHER, A. F., and STEWART, E. H. Experimental observations on "sperm immunity" in rat. ]. Contracept. 4:141, EscUDER, C. J. Temporary sterilization of women by injections of semen. ].A.M.A. 107:916, FARNUM, C. G. The biologic test for semen. ].A.M.A. 37:1721, FoGELSON, S. J. The non-specific antigenic effect of spermatozoa upon fertility. Surg. Gynec. & Obst. 42:314, FREUND, J., and McDERMOTT, K. Sensitization to horse serum by means of adjuvants. Proc. Soc. Exper. Biol. & Med. 49:548, GUYER, M. F. Studies on cytolysins. III. Experiments with spermatotoxins. ]. Exper. Zool. 35:201, HENLE, W., HENLE, G., and CHAMBERS, L. A. Studies on the antigenic structure of some mammalian spermatozoa. ]. Exper. Med. 68:335, lla. HENLE, W., and HENLE, G. Spermatozoal antibodies and fertility. I. Attempt to

7 212 AsHITAKA ET AL. FERTILITY & STERILITY induce temporary sterility in female white mice by passive immunization with spermatozoal antisera. ]. Immunol. 38:91, llb. HENLE, W., and HENLE, G. Spermatozoal antibodies and fertility. II. Attempt to induce temporary sterility in female guinea pigs by active immunization against spermatozoa. ]. Immunol. 38:105, IsoJIMA, S., and STEPUS, S. Antigenicity of guinea pig testis and ovary. Internat. Arch. Allergy 15:350, IsoJIMA, S., GRAHAM, R. M., and GRAHAM, J. B. Sterility in female guinea pigs induced by injection with testis. Science 129:44, }ARCHO, J. Artificial production of sterility; with special reference to experimental temporary sterility biologically induced in the female. Am. ]. Obst. & Gynec. 16: 813, KATSH, S. In vitro demonstration of uterine anaphylaxis in guinea pigs sensitized with homologous testis or sperm. Nature 180:1041, KATSH, S. Demonstration in vitro of anaphylactoid response of the uterus and ileum of guinea pigs injected with testis or sperm. ]. Exper. Med. 107:95, KATSH, S. Infertility in female guinea pigs induced by injection of homologous sperm. Am. ]. Obst. & Gynec. 78:216, KENNEDY, W. P. The production of spermatoxins. Quart.]. Exper. Physiol. 14:219, KovAs, F. K. The influence of the male sex gland on the female. An experimental study to determine the sex ratio of the offspring. Am. ]. Obst. & Gynec. 10:527, LAMOREUX, W. F. Spermatozoal antibodies and infertility in the fowl. ]. Exper. Zool. 85:419, LANDSTEINER, K. Zur Kenntnis der spezifisch auf Blutkorperchen wirkenden Sera. Zentralbl. Bakt. 25:546, MACHIDA, K. Studies on the sperm immunization. ]ap. ]. Obst. & Gynec. 8:1429, a. McCARTNEY, J. L. Studies on the mechanism of sterilization of the female by spermatoxin. Am.]. Physiol. 63:201, b. McCARTNEY, J. L. Further observations on the antigenic effects of semen. Mechanism of sterilization of female rat from injection of spermatozoa. Am. ]. Physiol. 66:404, METCHNIKOFF, E. Sur!'influence de l'organisme sur les toxines. Sur la spermotoxine et l'antispermotoxine. Ann. Inst. Pasteur 14:1, OsLUND, R. M. Physiological effects of spermatoxin. ].A.M.A. 86:1755, PARSON, E. I., and HYDE, R. R. An evaluation of spermatoxic sera in the prevention of pregnancy. Am.]. Hyg. (sect. B.) 31:89, PFEIFFER, H. Beitrage zur losung des biologisch forensischen Problems der Unterscheidung von Spermaeiweiss gegeniiber den anderen Eiweissarten derselben Spezies durch die Prazipitinmethode. Wien. klin. Wchnschr. 18:631, PoMMERENKE, W. T. Effects of sperm injections into female rabbits. Physiol. Zool. 1:91, SAVINI, E., and CASTANO, T. Immunite spermotoxique et fecondation. Compt. Rend. Soc. biol. 71:22, UcHIGAKI, S. Biological studies on sterility. Kinki Fufinkaishi (Japanese ed.) 10: 1061, VoGT, E. Sterilitat und Spermaimmunitat. Klin. Wchnschr. 1:1144, WANG, Y. Effect of spermatozoan injections on the fertility of female albino rats. Chinese]. Physiol. 10:53, 1936.