EXPERIMENTAL AUTOALLERGIC ASPERMATOGENESIS IN THE RAT Thesis for the Degree of Pn. D. MICHIGAR STATE UNSVERSITY Salah Eldin lmbabi 1967 W's-3‘: kill,” a .‘y c- “I" . t! I. 4"; '3 r. la“. AMA.“ ~‘Ak 1’ . filial} :gggn $3333 Ungversicy THU. “I“ This is to certify that the thesis entitled EXPUT INFN’IAL AUTOA LLFRGIC AF. PITRNAT 0C TTNS IS IN TIN? RAT presented by SALAH FLDIN INBABI has been accepted towards fulfillment of the requirements for PT lD degree in PA'FIIOTDGY W4 Major p;ofessor 13ml“ vbmary. 24. 1967 0-169 JUL 2 (I 2005 ABSTRACT EXPERIMENTAL AUTOALLERGIC ASPERMATOGENESIS IN THE RAT by Salah Eldin Imbabi Autoallergic aspermatogenesis in albino rats was studied in 4 experimental models. In this study 178 males and 63 females were used. Fertility of male and female rats was assessed by comparing numbers and sizes of litters produced and litter frequency before and after inoculation. Experimental animals were killed at the end of each eXperiment and their organs examined for gross and microscopic lesions. Lesions in affected testicles were graded as types 1, 2, 3 or u according to the severity and extent of testicular damage. Sera of inoculated rats were tested for passive cutaneous anaphylaxis (PCA) reactivity in normal young male and female albino rats. Study of the pathogenesis of autoallergic aspermato- genesis was attempted in models employing lymphoid cells, parabiosis or immune heterologous sera. Autologous or isologous testicular homogenates incor— porated in complete Freund's adjuvant induced aspermato— genesis of varying severity (types 1-u) in unilaterally Salah Eldin Imbabi orchiectomized and nonorchiectomized rats. The fertility of these rats was suppressed to varying extents with an average of 40% reduction in litters sired. Infertility was more closely associated with testic— ular damage than with PCA reactivity of sera. Autologous testicle was more effective in inducing testicular damage and infertility than isologous testicle. Temporary periods of infertility were induced in female rats inoculated with rat testicular homogenates in complete adjuvant. A lyophylizable material was prepared from rat testicles and epididymal spermatozoa by acid extraction, ammonium sulphate precipitation and trichloracetic acid purification (TCPM). This.TCPM fraction incorporated in complete adjuvant and inoculated intradermally into male rats was highly effective in inducing testicular damage. The protein residue precipitated by trichlorace- tic acid was ineffective. Histamine or serotonin incorporated with TCPM in complete adjuvant heightened the skin reaction but had no influence on the development of testicular lesions. Heparin and to a lesser degree, hyaluronic acid had suppressive effects on both the skin reaction and. the subsequent development of testicular lesions. Killed Leptospira canicola inoculated with TCPM in incomplete adJuvant, failed to induce aspermatogenesis. Salah Eldin Imbabi Aspermatogenesis was transferred by lymphoid cells and by parabiosis. Recipient rats in this model were neonatally grafted with parental lymphoid cells in order to render them tolerant to subsequent lymphoid cell in- oculation or parabiosis. Type 1 testicular damage was induced in l rat 8 days after inoculation with sensitized lymphoid cells. Similar lesions were noted in l parabiotic recipient 10 days after it was Joined to a sensitized donor. More pronounced tes— ticular lesions up to type 4 were seen in 6 of 10 lymphoid— cell—inoculated and 4 of 7 parabiotic recipient rats killed approximately 4 weeks or more after treatment. Splenomegaly, enlargement of thymus and lymph nodes, and weight and hair loss were noted in lymphoid—cell— inoculated and parabiotic rats. These were suggestive of graft~versus—host reactions. Sera of-male and female rabbits immunized with rat testicular homogenates in complete adjuvant failed to sup— press the fertility of either male or female rats. Testi~ cular damage was not demonstrated in male rats inoculated with such sera. Sera of actively immunized male rats inoculated into young females did not influence their conception rate in any measurable manner. ACKNOWLEDGMENTS The author wishes to express his sincere thanks and appreciation to Dr. D. A. Schmidt for his guidance, assistance, and continued encouragement throughout this investigation. The author also wishes to express his sincere gratitude to Dr. C. C. Morrill and Dr. R. F. Langham for their critical review of this manuscript. Many thanks are due to Dr. R. C. Belding, Depart- ment of Microbiology and Public Health for his encourage— ment and valuable council. The author is indebted to Mrs. M. Sunderlin, Mrs. F. M. Whipple, and Mrs. N. L. Miller for their technical assistance in preparing tissue sections. 11 EXPERIMENTAL AUTOALLERGIC ASPERMATOGENESIS IN THE RAT By Salah Eldin Imbabi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1967 TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . vi Chapter I. INTRODUCTION . . . . . . . . . . I II. LITERATURE REVIEW. . . . . . . . . 3 Historical . . . . . . . 3 Mechanism of Self Injury . . . . . 21 III. MATERIALS AND METHODS . . . . . . . 3O Experiment I . . . . . . . . . 33 Experiment II . . . . . . . . . 36 Protein Extraction . . . 36 Purification of the Aspermatogenic FaCtOI' o o o o 37 Inoculation of Rats with TCPM. . . 38 EXperiment III. . . . . . A0 Preparation of Heterologous Immune Sera . . . . . . . . A0 Inoculation of Female Rats with Immune Sera . . . . A0 Inoculation of Male Rats with Immune— Rabbit Serum. . . . . . Al Experiment IV . . . . . A2~ Preparation of Lymphoid Cell Suspension. . . . A2 Transfer of Sensitized Lymphoid Cells . . . . . A3 Parabiosis . . .. . . . . . . A3 IV. EXPERIMENTAL RESULTS. . . . . . . . A6 Testicular Lesions . . . . . . . 51 Experiment I . . . . . . 60 Female Rat Inoculations. . . . . 65 Experiment II . . 69 Results of Testicular TCPM Inocula— tion . . . . . . . . . . 69 iii Chapter Experiment III Experiment IV . Lymphoid Tissue Inoculations Parabiosis . . Histopathologic Examination. Testicular Lesions. . . . V. DISCUSSION Active Immunization with Testicular Homogenates . . Reduced Fertility in Female Rats Purified Aspermatogenic Factor. Histamine Inoculation. Mediation of Autoallergic Aspermato- genesis . Transfer of Autoallergic .Aspermato- genesis . Transfer by Lymphoid Cell Inoculation. Transfer by Parabiosis VI. SUMMARY AND CONCLUSIONS Summary . . . Conclusions . . . . BIBLIOGRAPHY . . . . . . . . . . . VITA O O O O O O 0 O O O O O O 0 iv Page 77 8O 80 85 88 9O 98‘ 98 102 10A 109 11A 119 119 121 125 125 127 129 Table 10. LIST OF TABLES Summary of Litters in Unilaterally Orchiectomized and Nonorchiectomized Male Rats Before and After Inoculation Testicular Damage Induced in Unilaterally Orchiectomized and Nonorchiectomized Male Rats Inoculated with Testicular Homogenate. . Summary of PCA Reactivity of Sera of Control and Inoculated Male and Female Rats . . . . . Summary of Litters by Female Control Rats and Those Inoculated with Testicular Hemogenate in Complete Adjuvant . . . . . . . . . Testicular Lesions in Inoculated and Control Rats Testicular Lesions in Male Rats Inoculated Intra— dermally with Killed L. Canicola, Histamine, Serotonin, Heparin or Hyaluronic Acid, in Addition to TCPM in AdJuvant . . . . . . Summary of Litters Sired by Male Rats Inoculated with Normal and Anti-Rat-Testicle Rabbits’ Sera Summary of Litters by Female Rats Inoculated with Immunized— and Control— Rat or Rabbit Sera. . Gross Changes of Thymus, Spleen, Lymph Nodes and Testicles and Microscopic Testicular Lesions in Rats Inoculated with Lymphoid Tissue Suspensions from TCPM-Sensitized and Control Rats . .~ . . . . . . . . . Summary of Gross Changes in Thymus, Spleen, Lymph Nodes, Testicle and Epididymis and Microscopic Testicular Lesions in Parabiotic Rats . . . Page 61 6A 66 67 70 72 78 79 81 97 LIST OF FIGURES Figure Page 1. Superficial skin ulcer, 1 day after inoculation of TCPM in complete adjuvant . . . . . . A8 2. Granulomatous lesion, 70 days after inoculation of TCPM in complete adjuvant . . . . . . A9 3. Granulomatous reaction at the site of inocula— tion of heparin— supplemented TCPM in complete adjuvant. . . . . . . . 50 A. Perivascular infiltration with mononuclear cells in a skin lesion A0 days after inocula- tion of TCPM in complete adjuvant . . . . 52 5. Type 1 testicular lesion 35 days after intra- dermal inoculation of TCPM in complete ‘ adj uvarlt 0‘ o o o o o o o I o o o o 5“ 6. Type 2 testicular lesion in rat inoculated intradermally with TCPM in complete adjuvant. 56 7. Type 3 testicular damage in rat inoculated intradermally with TCPM in complete adjuvant. 57 8. Type 3—A damage in testicle of a rat 120 days after intradermal inoculation of TCPM in complete adjuvant. . . . . . . . . . 57 9. Exfoliated cells in epididymis of same rat as in Figure 8. . . . . . . . . . . 58 10. Type A damage 180 days after inoculation of testicular homogenate in complete adjuvant . 58 11. Higher magnification of Figure 10 . . . . . 59 12. Histamine— inoculated testicle 1 day after injection . . . . . . . . . . . 7A 13. ' Histamine- inoculated testicle 21 days after injection . . . . .y . . . . . . 7A vi Figure Page 1A. Type 3-A damage in seminiferous tubules adjoin— ing the area of histamine necrosis . . . . 75 15. Type A-damage in seminiferous tubules at some distance from the area of histamine necrosis. 75 16. Thymus from a lymphoid—cell—inoculated rat . . 83 17 Spleen of lymphoid-cell—inoculated rat . . . 83 18. Peyers patch from lymphoid-cell—inoculated rat. 8A 19. Severely damaged germinal epithelium in testicle of lymphoid-cell—inoculated rat . . 85 20. Parabiotic pair, killed 60 days after surgery . 87 21. Enlarged spleens and lymph nodes of donor (A) and.recipient (B) of a parabiotic pair of rats . . . . . . . . . 87 22. Testicles of parabiotic recipient (A) and donor (.8) e o o o o o o e e o o o o o 89 23. Suture granuloma with giant cells at the skin junction line of a parabiotic pair of rats . 89 2A. Lymphoid and reticulo—endothelial hyperplasia in spleen of parabiotic recipient rat . . . 91 25. Spleen of parabiotic recipient rat. . . . . 91 26. Reticulo—endothelial hyperplasia in lymph node of a parabiotic rat . . . . . . . . . 92 27. Peyers patch of same rat-as in Figure 25. . . 92 28. Thymus of a parabiotic recipient rat . . . . 93 29. Peripherolobular necrosis in liver of a parabiotic rat. . . . . . . . . . . 93 30. Testicle of normal rat. . . . . . . . . 95 31. Type-A damage in testicle of a parabiotic recipient rat . . . . . . . . . . . 95 32. Epididymis of same rat as in Figure 31 . . . 96 vii I. INTRODUCTION The fascinating aspects of autoimmunity and the many unanswered questions in the field provided the main stimulus for undertaking the present experimental work. Autoallergic aspermatogenesis was experimentally pro- duced in guinea pigs with regularity, through the use of Freund's mycobacterial adjuvants. Reports of induction of aspermatogenesis in the rat were rather limited. On the other hand, a few statements were encountered in the litera- ture alluding to the relative unresponsiveness of this animal to delayed hypersensitive reactions and its insuscep— tibility to autoallergic aspermatogenesis. Such statements were previously challenged by the high susceptibility of the rat to autoallergic encephalomyelitis in which delayed hypersensitivity was believed to have an instrumental role. If induction of autoallergic aspermatogenesis is limited to guinea pigs, a breach between theoretical and applied aspects of the disease becomes evident. Observed auto-antigenicity of the testicle was explained on the basis of sequestration of the organ early in fetal life. If this were true, one fails to see why laboratory animals beside the guinea pig should not respond to subsequent unmasking of the antigen. A factor that induced aSpermatogenesis when inoculated into guinea pigs was isolated from guinea pig testicle. There were no reports in the literature of any attempts to purify or isolate such a factor from rat testicle. Many reports were encountered in the literature relat- ing to the effects of testicular antigen inoculations on the fertility of females. However, there was no reference to the effects of autoimmune testicular lesions on the actual fertility of the male animal as measured by breeding capacity. Theoretical aspects relatingtx)the pathogenesis of autoallergic aspermatogenesis were discussed by various workers. There seemed to be general agreement that the disease, like other autoallergic diseases might be produced by sensitized lymphoid cells rather than serum antibodies. Reports of experimental work to test such a hypothesis were lacking in the literature. In View of this, the present experimental work was conducted in an attempt to provide experimental data to correct the mentioned deficiencies. The rat was chosen because of the controversy which was raised about its susceptibility to induced aSpermatoF genesis. Furthermore it was suitable for fertility tests due to the prolificacy of the female. II. LITERATURE REVIEW Historical Early in the history of immunology, Bordet (1898) heteroimmunized guinea pigs by inoculating them with rabbits' red blood cells. He demonstrated the development in the guinea pig's sera of a new material which he called "sub— stance sensibilicatrice” or sensitizing substance. In the following year, Ehrlich and Morgenroth (1899) produced hemolytic isoantibodies in goats by injecting them with red blood cells from other goats. Ehrlich's repeated efforts to induce antibody formation by inoculating goats with their own red cells were, however, unsuccessful, which led him to believe that it was unthinkable for the body to destroy itself or in his words produce a state of "horror autotoxicus" (Ehrlich 1899). Donath and Landsteiner,(l9OA) discovered an autOF hemolysin in clinical cases of paroxysmal cold hemoglobin- urea and thus provided the first clinical link for the development of concepts of autoimmunization. Following that, red cell autohemolysins and autoagglutinins were demonstrated in certain clinical cases of acquired hemolytic anemia (Chauffard and Troiser, 1908; Widal g§_a1, 1908; and Widal and Weisenbach, 1913). Experimental induction of hemolytic anemia in the guinea pig by injection of anti-guinea—pig—red-cell rabbit serum was first accomplished by the Italian pathologist Banti (1913) who believed that the spleen was the main cause of the condition. Early work with spermatozoa demonstrated their anti- genicity in heterologous Species (Landsteiner, 1899). Metchnikoff (1900) induced serum spermotoxins in rabbits by inoculating them with isologous spermatozoa. Metalnikoff (1900) found that guinea pigs inoculated with spermatozoa from other guinea pigs developed spermotoxins in their sera. These were lethal 12,11239 to isologous as well as homologous spermatozoa. The same author noted that while the spermotoxin was active against autologous spermaotzoa in vitrg it did not seem to be active in 1132. He speculated that phagocytic cells in the blood contained the active substance which could only be released as a result of the death and disinte- gration of these cells in zitrg. Landsteiner (1902) utilized hanging drop preparations to study the toxic effects of anti-spermatozoa-sera on viable spermatozoa. He demonstrated that such sera caused immobiliz— ation and death, without lysis, of homologous spermatozoa. In contrast to whole spermatozoa, Taylor (1908) found that iso- lated nucleic acids, protamines and other extracts of Sperma— tozoa, were ineffective in inducing spermotoxin formation. Guyer (1922) found that spermotoxic serum produced by repeatedly injecting fowls with rabbit spermatozoa was active in 11232 against both fowls' and rabbits' spermatozoa. He also reported that when rabbit spermatozoa were injected intravenously into male rabbits, at four- to five-week intervals, partial or complete sterility resulted, with in— activation, reduction or disappearance of spermatozoa and disintegration of the seminiferous tubules. Sterility in female rats induced by active immunization with rat Spermatozoa was also reported by McCartney (1923). He demonstrated spermatozoa agglutinating antibodies in the genital tracts of these females and thought that in rodents spermatozoa might penetrate the epithelium of the genital tract and invade the underlying connective tissue. In that location, spermatozoa would be able to induce spermotoxin production. He speculated that the same could happen in women which might account for the sterility of common prosti— tutes. It is interesting to note that a similar idea was conceived before autoimmunization was recognized (Darwin, 1898). In his work "The Descent of Man and Selection in Relation to Sex" Darwin stated that "The profligacy of the women may in part account for their small fertility." The previous statement, immunologically interpreted (McCartney, 1923; Katsh, 1959) was taken to suggest that repeated exposure to antigenic material from spermatozoa could induce an immune sterility in the female. Female rabbits were rendered sterile for four months or longer following repeated intravenous injections of pro— gressively increasing doses of rabbits' ejaculation fluid collected after coitus from the female's vagina (Dittler, 1920). Although active immunization of female rabbits caused inhibition of their fertility, there was no hindrance to ovulation and no anaphylaxis observed (Dittler, 1920). Similarly, pregnant rats grafted with rat testicle had normal pregnancies and did not suffer from any physiologic or somatic deleterious effects as a result of the trans— plantation (Moor, 1921). Previously fertile female rats and hens were tempor- arily sterilized for periods of 2 to 22 weeks by repeated subcutaneous injections respectively of rats' and roosters' spermatozoa in Ringer's solution (McCartney, 1923). He reported that human semen, used in place of rat spermatozoa, was equally effective in producing sterility in female rats, and sterile ejaculates, gross enlargement and fatty degener- ation of the testicles in the male rat. In the actively immunized female rats that became pregnant, there was a reduction in the number of young per litter from an average of 6.8 to 5.8. Early investigators tended to consider spermatozoa as completely Species nonspecific. Thus McCartney (1923) found rat and human spermatozoa to be equally effective in steril— izing male and female rats. Fogelson (1926) also reported induction of sterility in female rats for variable periods, by using either rat, guinea pig or human spermatozoa and found that sera of such females reacted equally well with all three antigens. Subsequent work in which use was made of heterologous antisera and cataphoretic methods (Mudd and Mudd, 1929) or agglutination, precipitation and immobilization tests (Lewis, 193A) revealed a marked species and organ selectivity of the spermatozoa antigens. Cross reactions were observed among bull, ram and guinea pig testicles (Mudd and Mudd, 1929) and among those of bull, ram, rats, mice and rabbits (Henle, 1938). It was demonstrated that, while cross reactions between spermatozoa of the various species occurred, the reactivity was weak and decreased as the species became genetically unrelated (Mudd and Mudd, 1929; and Henle, 1938). Lewis (193A) inoculated rabbits with the alcohol- soluble fraction of rabbit testicle. The serum antibody produced reacted strongly with rabbit testicle and brain and to a lesser degree with these organs from other species. Similarly, anti-beef—brain serum reacted with the alcohol- soluble fractions of both brain and testicle from various species. He thus postulated the existence of the same or of identical antigens in testicle and brain. Using vibration— fragmented spermatozoa and heterologous antisera, Henle and Henle (1938) demonstrated, by slide agglutination and comple— ment-fixation, the presence of both head— and tail-specific antigens and one antigen common to both heads and tails. In the early literature, the only reference to any pathologic changes in the testicle was made by McCartney (1923) and even in his report the evidence was inconclusive and histopathologic studies were lacking. In later years attempts to correlate the antitesticular or antihyaluronidase serum antibodies with any specific lesions in the testicle were unsuccessful (Freund at al., 1953, 195A, 1955; Waksman, 1959; Bishop et a1., 1961). Similarly, use of heterologous or isologous antisera for passive induction of aspermato- genesis associated with testicular injury were not fruitful, (Freund, 1953). The induction of autoimmune aspermatogenesis was suc- cessful after the advent of mycobacterial adjuvants (Freund at al., 1953). In the guinea pig, inoculation of homologous or autologous spermatozoa, mitochondrial fractions or testic- ular homogenates, incorporated in Freund's complete adjuvant, regularly produced aspermatogenesis in the guinea pig (Freund et al., 1953, 1955). This has been reported more recently by Katsh and Bishop (1958), Waksman (1959), Bishop et a1. (1961), Boughton and Spector (1963), Bishop and Carlson (1965). Freund et al. (1953, 1955) were unable to induce testic— ular damage in guinea pigs inoculated with testicular antigens in incomplete adjuvant. Complete adjuvant and antigen, inocu— lated separately on opposite sides of the animal were also ineffective. The authors were, however, able to produce aSpermatogenesis when the antigen and adjuvant were inoculated separately but on the same side of the animal which suggested the involvement of lymph drainage. The intradermal route of inoculation and the complete adjuvant were reported to be indispensible for the successful induction of aspermatogenesis, other routes being ineffective, or much less effective (Freund gt a1., 1953, 1955; Waksman, 1959). Waksman (1959, 1960) speculated that the importance of the intradermal route might be due either to slow dis- sipation of the antigen or to some unknown factors that might combine with and alter the antigen in such a way as to make it more effective in inducing immune responses. Whatever the mechanism, the skin reaction at the site of inoculation was found to be reminiscent of the hypersensitivity type of tuberclin reaction (Freund at al., 1953; Waksman, 1959; Boughton and Spector, 1963). Because of this mode of induction and the lack of cor- relation between lesions and circulating antibodies, a delayed hypersensitivity similar to that elicited by tuber— culin was postulated for the mediation of the autoimmune process by Freund at al. (1955), Waksman (1959) and Bishop et a1. (1961). For the same reasons, Waksman (1959, 1960, 1962) suggested the name autoallergic instead of autoimmune aspermatogenesis. In addition to the use of whole testicle or spermatozoa, crude and purified extracts were prepared and used in the guinea pig. Alcoholic extractions were found to be ineffec- tive in producing the disease (Freund 33 a1. 1953). On the other hand, acid extracts of guinea pig testicle and sperma- tozoa, subsequently precipitated with ammonium sulphate to give an ammonium sulphate precipitated material (ASPM), had about 100 times the potency of testicular homogenate (Freund at al., 1955); Carlson and Bishop, 1962). Deproteinization 10 of the ASPM by treatment with trichloracetic acid gave a supernatant fluid containing a trichloracetic acid purified material (TCPM) which was a partially dialyzable and lyophi- lizable material of high aspermatogenic qualities (Freund et al., 1955; Bishop gt 31., 1961; Carlson and Bishop, 1962). Further purification of the TCPM with a chloroform-butanol mixture yielded the most potent aspermatogenic factor, which was called the chloroform purified material (CPM) (Freund 33 a1., 1955). The Freund's purified extract contained A% nitrogen, 10% reducing material and significant amounts of aromatic amino acids as demonstrated by their absorption spectra in ultraviolet light and hence it was suggested that the asper- matogenic factor behaved like a polysaccharide—polypeptide complex (Freund-gt a1., 1955). The same authors also found that the aspermatogenic prOperties of the factor were not affected by autoclaving at 15 pounds of pressure for one hour, formamide treatment or enzymatic digestion. Katsh gt a1. (1960) and.Katsh and Katsh (1961) employed diethylaminoethyl (DEAE)--cellulose column for separation of testicular extracts and homogenates and studied the eluates by electrophoresis, paper chromatography, infrared Spectro- photography and enzymatic digestion. They reported finding four different antigenic moieties, two of which were asso— ciated with hyaluronidase and nucleic acids, reSpectively; the third was a protein and the fourth was a polysaccharide. 11 The chloroform purified material (CPM) containedpoly- peptides and polysaccharides in the amounts of 70% and 20%, respectively (Carlson and Bishop, 1962; Bishop, 1963). Accord— ing to these authors the polysaccharide appeared to be solely galactose and, when separated by glucose gradient, was found to correspond closely with the aspermatogenic factor. Extracts, comparable in activity to Freund's TCPM, were obtained from guinea pig testicles by autoclaving, digestion with papain, phenol treatment and trichloracetic acid depro- teinization. Such treatment yielded a partially dialyzable and lyophylizable material containing 7.7%.nitrogen and 11.5% reducing material (Brown gt gt., 1963; Glynn and Holborow, 1965). Sera of aspermatogenic guinea pigs immunized by whole testicular homogenates, spermatozoa or ASPM contained anti- body that was demonstratable by conventional serologic tests, by immunodiffusion and by passive cutaneous anaphylaxis (Freund gt gl., 1955; Katsh and Bishop, 1958; Katsh and Katsh, 1961). While this was the case with crude homogenates and Proteinaceous extracts, highly purified aspermatogenic frac— tions of the order-of the TCPM or CPM were incapable of eliciting antibody that could be demonstrated by any of the Previous tests (Bishop, 1963; Bishop and Carlson, 1965). Immunoelectrophoretic separation of ASPM from guinea pig t€Sticle yielded one aspermatogenic, non—antibody—forming fraction and four non-aspermatogenic, antibody-inducing com— ponents (Katsh and Katsh, 1961; Bishop, 1963). 12 Until recently, questions were raised as to whether spermatozoa really have distinct antigens of their own and how much of this antigenicity is conferred by absorption of secretions of the male accessory glands. Seminal plasma antigens and thrice-washed rabbit seminal Spermatozoa were so closely related that they were not distinguishable by heterologous antisera prepared in the guinea pig (Neil and Finkler, 1958). A spermatozoa—coating antigen (SCA), derived from the seminal plasma, was demonstrated on rabbits' seminal sperm but not on testicular or epididymal spermatozoa (Well and Rodenberg, 1962). This was confirmed by fluorescent anti- seminal-plasma—antibody which stained seminal spermatozoa and epithelial cells lining the seminal chambers and the glandular epithelium within the muscular layer of the seminal vesicles (Well and Rodenberg, 1962). A.SCA was also demonstrated, by fluorescent antibody, on human seminal spermatozoa and was similarly found to originate from the seminal vesicles and to be highly species— and organ-specific (Weil, 1965). In human secretor individuals, seminal plasma may con- tain blood-group antigens (Raffel, 1961; Dumonde, 1966). In bulls, out of all the blood groups, the J factor was the only one demonstrated in the semen (Matousek, 196A). Similar SCA were recognized for the guinea pig (Pernot, 1965), buffalo, (Rao and Sadri, 1960) and ram (Hathaway and Hartree, 1963). Common antigenic factors between bovine serum, seminal fluid and seminal spermatozoa were revealed through l3 heteroantigenic analysis by Hunter and Hafs (196A) who sug— gested that spermatozoa might acquire serum antigens while in the vasa deferentia and before absorbing the vesicular antigens. This might account for earlier findings of cross reactivity between spermatozoa and red blood cells (Mudd and Mudd, 1929). The presence of tissue- and species—specific antibody- inducing antigens in various mammalian spermatozoa and tes— ticles was amply demonstrated by heteroantigenic analysis (Mudd and-Mudd, 1929; Lewis, 193A; Henle and Henle, 1938; Katsh and Bishop, 1958). Tested by immunoelectrophoresis, spermatozoa of the Chinese hamster gave at least 16 bands that were predominantly species—specific (Lajos and Tyler, 1963). Antigens specific for the acrosomal portions were demonstrated by fluorescent-antibody technique in the mouse, rat and guinea pig (Barth and Russel, 196A). Demonstration of the tissue specificity and localiza— tion of the apsermatogenic antigen was problematic because induction of circulating antibody was usually dissociated from aspermatogenesis and serologic methods were therefore futile (Freund gt-gt., 1955; Bishop gt gt., 1961; Bishop, 1963). Aspermatogenic guinea pig sera, tagged with fluorescein gave specific immunofluorescence with the acrosomes of homo— logous spermatozoa (Baum gt_gt., 1961). The central area of the seminiferous tubules, consisting of secondary spermato— cytes, spermatids and Spermatozoa also stained with fluorescein 1A while other surrounding elements and epididymal epithelium did not (Brown gt gt., 1963). Katsh (1959) inoculated mature and immature guinea pigs with testicular homogenates derived from guinea pigs of various ages and found that only the homogenates containing Spermatocytes and their derived cell types were able to induce aspermatogenesis. Homogenized fetal guinea pig testicles, injected into mature guinea pigs, did not induce aspermatogenesis which indicated that, with maturation, antigen is elaborated at the stage of secondary spermatocytes and eventually imparted to the sperm acrosome (Katsh, 1959). Testicular damage following immunization, involved secondary Spermatocytes, spermatids and spermatozoa. The testicles thus damaged failed to induce aspermatogenesis when injected into other guinea pigs. This provided further evidence for the localization of the antigen in these elements (Bishop gt gt., 1961). When newborn guinea pigs were inoculated with either testicular homogenates or purified antigen in complete Freund's adjuvant, it was found that induction of-aspermato— genesis awaited, not development of immunologic competence, but rather sexual maturation, differentiation and acquisition of the antigen (Katsh, 1959; Bishop gt gt., 1961). According to these authors, the disease was not an inhibition of-sperm— atogenesis as was previously suggested by Freund gt gt. (1955), but was due to specific destruction following matura— tion and differentiation of the germinal epithelium. 15 Conversely, aspermatogenesis was induced by extracts from lA—day-old guinea pig testicles which were in the spermatogonia and early primary spermatocytic stages (Freund gt gt., 1953). Primary spermatocytes of the guinea pig testicle were also found to be susceptible to the action of cytotoxic sera in tissue culture (Chutna and Rychlikova, 196Aa, b). In severe cases of aspermatogenesis, testicles were described in which all germinal epithelium was lost and seminiferous tubules were like empty rings lined only with Sertoli cells (Katsh and Bishop, 1958; BishOp gt gt., 1961). This might suggest that primary spermatocytes and spermatogonia contain precursers which share with the antigen some configurational characteristics (Dumonde, 1966). Most of the work on autoallergic aspermatogenesis as reported in the literature was done on guinea-pigs because of their reSponsiveness and the comparative ease and regular— ity with which the disease could be produced (Bishop and Carlson, 1965). In this animal, responsiveness was found to increase with the amount of inbreeding, but-even then, two equally inbred strains could vary widely in their suscepti— bility to the disease, the Wright strain being more responsive than Strain 2 (Bishop and Carlson, 1965). Individual guinea pigs within a strain varied greatly in their response to the antigen and while in some the damage was slight and patchy, others had severe lesions and sometimes damage of all the germinal elements in the testicle (Freund gt gt., 1953, 1955; 16 Katsh and Bishop, 1958; Bishop gt _t., 1961). Even differ— ent tubules within the same testicle varied in the extent of damage manifested, Katsh and-Bishop (1958). Severity of lesions increased with the age of the animal (Katsh, 1959) and with the length of time after inoculation (Bishop gt gt., 1961). Damage was consistently bilateral with both testicles affected to a comparable extent (Waksman, 1959; Boughton and Spector, 1963). Occasionally when the antigen—adjuvant mix— ture was given intramuscularly, the two testicles of the same animal failed to react to the same.extent (Bishop and Carlson, 1965). Guinea pigs.were usually immunized by a single dose of antigen incorporated in complete Freund’s adjuvant and admin— istered intradermally in several sites (Freund gt_gt., 1955; Bishop gt gt., 1961). Waksman (1959) stressed the importance of the intradermal inoculations, having found other routes far less effective. Bishop and Carlson (1965) however obtained good results, comparable to those resulting from intradermal inoculation, by the intramuscular route. Wentworth and; Mellen (196A) reported the effectiveness of the intraperitoneal and intramuscular inoculations in suppressing spermatogenesis in the male Japanese quail. Freund gt gt., (1953) produced limited injury in the contralateral testicles of guinea pigs by injecting antigen in adjuvant in the other testicle. Injection of-turpentine in one testicle and complete Freund's adjuvant intradermally on the same side of the animal caused 17 severe necrosis in the inoculated testicle but failed to pro- duce recognizable injury to the other testicle (Broughton and Spector, 1963). Severe injury was produced in the left testicles of rats, rabbits, guinea pigs and monkeys by intra- testicular injections of Freund‘s complete adjuvant, while the non-injected right testicles had scattered but recogniz— able damage (Eyquem and Nrieg, 1965). The authors believed the damage to the right testicles to be the result of an autoimmune process. In the guinea pig the earliest changes in the testicular parenchyma were observed A-6 days following the inoculation of testicular homogenate in complete Freund's adjuvant (Boughton and Spector, 1963). Freund gt gl;4_(l953, 1955) observed severe testicular lesions in guinea pigs 1A days postinocula- tion. Generally there was a long and variable latent period followed by explosive damage and the severest lesions were seen 2 to A months after inoculation (Bishop gt_gtt, 1961). The lesions were always confined to the germinal elements, usually secondary spermatocytes, spermatids and spermatozoa; but in severe cases, all germinal layers were damaged and exfoliated leaving empty tubules lined with a few Sertoli cells (Freund et 1., 1955; Katsh and Bishop, 1958; Bishop gt §_1_. , 1961) . Degeneration of the germinal cells was indicated by loss of cytoplasm (Katsh and Bishop, 1958). This was followed by necrosis in which nuclei became pyknotic, fragmented andtthe dead cells were exfoliated into the lumens 18 of the seminiferous tubules and epididymis. Reduced numbers or the complete absence of spermatozoa and the presence of exfoliated cells in the epididymal tubules were among lesions described (Freund gt gt., 1953, 1955; Katsh and Bishop, 1958; Waksman, 1959). Hyperplasia of Sertoli cells and their sub- sequent degeneration and destruction of the basement membrane (Freund gt gt., 1953) and detachment of the deeper layers from the basement membrane (Bishop gt gl., 1961) were reported. Giant, multinucleate cells appearing in the region of secondary spermatocytes were described by Freund gt gt. (195A, 1955), Katsh and Bishop (1958), Boughton and Spector (1963) and Bishop and Carlson (1965). Severe damage was followed by atrophy and subsequent fibrosis (Freund gt gt., 1953) or atrophy without fibrosis (Waksman, 1959). No antiandrogenic activity was observed judging by the intactness of interstitial cells and the lack of degenerative lesions in the accessory male glands (Freund gt gt., 1955; Katsh and Bishop, 1958; and Bishop gt gt., 1961). Lesions in the brain following inoculation of testicu— lar antigens in the guinea pig were rarely encountered (Wakeman, 1959). These comprised a slight infiltration of the meninges and cuffing of some of the vessels in the brain. with mononuclear cells. Conversely, slight degenerative lesions in the testicle following administration of brain tissue in adjuvants was-reported by Katsh and Bishop (1958). Bishop and Carlson (1965), using trichloracetic acid and- 19 chloroform—butanol—purified brain antigen in complete Freund's adjuvant, were able to induce recognizable damage to guinea pig testicles in addition to severe encephalomyelitis. Testicular lesions associated with induced autoimmune aspermatogenesis in guinea pigs and rats were not inflamma— tory in nature (Freund gt gt., 1953, 195A). Few mononuclear cells were seen in severely affected testicles and were con- sidered to be consequential to the damage (Freund gt gt., 1955). Bishop and Katsh (1958), Bishop gt gt., 1961), and Bishop and Carlson (1965) reported slight infiltration with mononuclear cells following administration of large doses of testicular antigens in complete adjuvants. On the other hand, Waksman (1959) reported severe inflammation and infil- tration of the interstitial tissue with many lymphocytes and histiocytes before any damage to the germinal epithelium was demonstrable. He therefore considered the infiltrating cells to be responsible for the germinal cell destruction and stated that for such a reaction to be seen, inoculated animals should be killed early in the course of the experi— ment. Boughton and Spector (1963), killing their animals at regular intervals starting on the day following inocula— tion, reported that the cellular infiltration when present followed destruction of the germinal epithelium and was probably a tissue reSponse to the necrosis. Temporary sterility in female rats and testicular lesions in male rats were induced by inoculation of saline 20 suSpensions of homologous and heterologous Spermatozoa (McCartney, 1923). Recently Freund gt gt. (195A) produced aspermatogenesis in rats by successive intracutaneous injec- tions of rat testicular homogenates in complete Freund's adjuvant. They described patchy testicular damage occurring in isolated tubules and severe damage involving large numbers of tubules or the whole testicle. The damage, as in the guinea pig, was confined to the germinal epithelium and characterized by degeneration, necrosis and exfoliation of dead cells into the lumens. Rarely and.only in advanced cases was there any intertubular inflammatory reaction but large mononucleate andnwfltinucleategfiant cells were seen in the germinal layers of the tubules (Freund gt gt., 195A). Although no other reports were encountered in the literature about attempts to reproduce Freund's findings in the rat, there were a few comments about the comparative insensitivity of this animal to autoallergic aspermatogenesis (Bishop gt gt.,1961; Bishop and Carlson, 1965). According to Bishop gt gt. (1961), the aspermatogenic response could not be elicited in animals with greater embryologic prestige than the guinea pig. However in rabbits, sensitization was reportedly possible although experimental data were not provided (Katsh and Bishop, 1958). Since early in this century, females of various rodents have been rendered temporarily sterile by injections of homolo— gous or heterologous spermatozoa (Dittler, 1920; Moor, 1921; McCartney, 1923; Pommerenke, 1928). 21 Recently, inhibition of fertility in the female guinea pig for up to 320 days following inoculation of homologous spermatozoa with or without adjuvant was reported (Katsh, 1959). tg ttttg anaphylactoid response of uterine strips from sensitized female guinea pigs was closely correlated in intensity and duration with the extent of infertility (Katsh, 1957, 1958, 1959). Katsh (1959) speculated that immobilizing and cytolytic antibodies in the female genital tract, in addition to tg tttg uterine contractions in response to ejaculated spermatozoa might cause the observed infertility of the sensitized female. In a comprehensive discussion of female infertility, Cushing and Campbell (1957) state with considerable emphasis that the active immunization of females, including women, against homologous spermatozoa would not bring about sterility even though antibodies against spermatozoa could be demon— strated in the serum. Mechanism of Self Injury Dameshek and Schwartz (1938a) induced various types of heterohemolytic anemia in the guinea pig with an anti—guinea— pig-red—cell—rabbit—serum and pointed out many similarities between the experimental heteroimmune and the clinical auto— immune disorders. The classical experiments of Landsteiner (1936) estab- lished that a simple chemical substance, nonantigenic by itself, could become antigenic by combining with proteins or 22 cellular material. Landsteiner's experimental findings were verified by clinical cases of hemolytic diseases such as aminopyrine agranulocytosis (Dameshek and Colms, 1936), the well established sedormid” thrombocytopenia (Ackroyd, 19A9), and the quinidine thrombocytopenic purpura (Bolton, 1956). In some diseases, autoantibodies which reacted with the tissues were demonstrated. Among these were antibodies against thyroglobulin and thyroid tissue found in association with Hashimoto's disease and other clinical forms of thyroid— itis (Witebskey gt _t., 1957; Halburg, 196A). Three per cent of sterile men examined by Rfimke and Hellinga (1959) had anti- sperm antibodies in their sera. Similarly, autoantibodies against the eye lens were revealed in cases of sympathetic ophthalmia (Colins, 1953; Hackett and Thompson, 19A6) and against nervous tissue in patients with nervous disorders (Shamri, 1963; Paterson, 1966). Anti-heart autoantibodies were found in association with rheumatic heart diseases (Anon., 1961; Van der geld, 196A) and antikidney antibodies in cases of nephritis (Yagi and Pressman, 1958). The significance of these antibodies and their role in the pathogenesis of the respective diseases was not always clear (Dumonde, 1966). In experimentally induced glomeru— lonephritis in rats, light and electron microscopic studies revealed that inoculation of nephrotoxic serum caused definite swelling of the glomerular basement membrane and endothelium, *Sedormid: allyl is0propy1 acetylurea : a tranquillizer. 23 resulting in contraction of the vessel (Bohle gt gt., 1958). Baum gt gt. (196A) demonstrated thrombus formation and localization of fluorescent rheumatoid factor—antibody com- plexes in the blood vessels of exposed rat mesentery. It was postulated that in these cases soluble antigen—antibody complexes formed and could localize at the site of the anti- gen in the tissue, bringing about the harmful effects (Bohle gt gt., 1958; Baum gt gt., 196A; Dumonde, 1966). In the case of autoimmune responses to the optic lens, testis, thyroid or brain, it was theorized that the antigens become sequestered from the immune apparatus early in fetal life so as to make them unrecognizable as "self" if unmasked at a later date (Burnet, 1961; Steiner and Volpe, 1961; Dameshek, 1963). In this connection it was shown that the testicular aspermatogenic antigens, at least in the guinea pig, were elaborated about three or four weeks after birth, which might explain the intolerance of the host to them if they were to be encountered by immunologically competent cells (Katsh, 1959; Bishop gt gt., 1961). Aspermatogenesis, thyroiditis, ophthalmia and enceph- alomyelitis were induced in experimental animals by inocula- tion of the respective isologous tissues incorporated in Freund's complete adjuvant (Freund gt gt., 1955; Witebsky and Rose, 1956; Collins, 1953; Lipton and Freund, 1953). Failure to transfer these diseases passively by hyperimmune sera, the need for Freund's complete adjuvant and presence of delayed 2A sensitive responses of the tuberculin type, have fostered a theory for pathogenesis based on delayed hypersensitivity. A pure delayed hypersensitivity was defined (Gell and Benacerraf, 1961) as an immunologically specific, erythema- tous and/or indurated reaction, demonstrable histologically, taking a few hours to reach a maximum of induration. Furthermore, the reaction occurs in the absence of demon— strable antibodies of the conventional type and is trans- ferable by cells and not by serum. It was stipulated (Waksman, 1959, 1960, 1962; Dixon, 1963) that for any disease to be considered the result of delayed hypersensitivity, there should be a correlation between the hypersensitivity reaction and some organ-specific lesions accompanied by histiocytic and lymphocytic infiltra- tions around small blood vessels. Such correlation, however, was not found to be always absolute. Delayed hypersensitiv— ity to homologous optic lens and to thyroid without the occurrence of demonstrable organ lesions was observed by Paterson (1966) and Dumonde (1966). On the other hand, aspermatogenesis could be dissociated from the occurrence of a delayed hypersensitivity to testicular extracts (Rhmke, 1959; BishOp gt gt., 1961). Similarities between the reactions noticed in experi- mental aspermatogenesis, thyroiditis, uveitis, encephalomye- litis and peripheral neuritis, and those classical allergic responses mediated by reaginic antibody, induced Waksman (1959, 1960 and 1962) to describe the former diseases as autoallergic rather than autoimmune. 25 Besides this distinction, there is a subtle difference in meaning between the two terms although they have been so often used interchangeably. Literally, autoimmune means safe from self or inability to respond against one's own tissues as implied by Ehrlich's remark "horror autotoxicus," while autoallergic is defined as altered reactivity against self (Paterson, 1966). However no serious objections were raised about using the two terms interchangeably provided the difference in their meanings is recognized (Dameshek, 1963; Paterson, 1966). Emphasis was repeatedly placed on the use of adjuvant of the Freund's complete type which was considered of prim- ary importance in the production of a hypersensitive state (Freund, 1957; Waksman, 1960, 1962). Pearson gt gt.(l96l) demonstrated that Freund's complete adjuvant by itself, when injected into rats, induced a well defined cellular prolif- eration of reticulo—endothelial cells and lymphocytes which by itself might be important in developing immunologic phenomena. While the role of adjuvant in induction of autoallergic diseases was generally recognized (Dumonde, 1966), saline suspensions of isologous brain tissue (Rivers gt gt., 1933) and of testicular tissue (Bishop, 1958), effectively induced encephalomyelitis in rats and aspermatogenesis in guinea pigs, respectively. Katsh (1960) induced aspermatogensis in guinea pigs by inoculating them with isologous testicle mixed with Corynebacterium rubrum. Testicular damage demonstrated in 26 the inoculated guinea pigs was as severe and uniform as when mycobacterial adjuvants were used. What factors in both bacteria are responsible for eliciting the response is not yet known. Tuberculin sensitivity was passively transferred by labeled lymphoid cells from guinea pigs, sensitized with purified protein derivative, to normal guinea pigs (Najarian and Feldman, 1961). In two autoallergic diseases, namely thyroiditis (Sclare and Tayler, 1961) and encephalomyelitis (Paterson, 1960), lymphoid cells were capable of passively transferring the diseases. Activity of sensitized lymphoid cells against thyroid cells was demonstrated tg ttttg (Pulvertaft, 196A) and against nervous tissue (Paterson, 1966). There is no record, as yet, of the successful transfer of aspermatogenesis or uveitis. Conversely, cytotoxic effects of antithyroid sera on the homologous thyroid cells were seen by Pulvertaft gt gt. (1959). Similarly, cytotoxic properties of antiteSticle sera on testicular tissue in culture were described (Chutna and Rychlikova, 196Aa, 196Ab). It was only in the hands of Waksman (1959) that mono— nuclear cells and lymphocytes were found to infiltrate the testicular tissue and bring about damage to the germinal epithelium. Other workers did not find this to be true and repeatedly made the observation that lymphocytes and other inflammatory cells were conspicuous by their absence; they nevertheless attributed the condition to delayed 27 hypersensitivity mediated by a cellular rather than an anti- body mechanism (Freund gt gt., 1955; Katsh and Bishop, 1958; Bishop gt gt., 1961). Other immunologic manifestations did not lend them- selves to explanation on bases of either altered or sequestered antigens or altered host reactivity. They were better understood, however, after the emergence of Burnet's "Clonal Selection" theory (Burnet, 1959). On several occasions, Burnet (1959, 1962, 196A) and Burnet gt gt. (196A) stressed the importance of groups of immunologically com— petent cells or clones and their role in immunologic phenomena. In the homograft rejection, large groups of lymphocytes weregseen surrounding the rejecting graft (Medaware, 1958). Andre gtggt. (1962) demonstrated the development of large primitive~appearing cells during immune and homograft rejec- tion phenomena. Such proliferation in response to antigen was shown to take place in the lymphoid apparatus, including the spleen, and particularly in the germinal follicles (Cogdon, 1961; Burnet, 1962, 1963). Similarities between autoallergic aspermatogenesis and the graft rejection phenomenon were pointed out by Bishop gt gt. (1961). They found these conditions to be immuno- logically produced but nontransferable by immune serum alone. They also believed that delayed hypersensitivity of the tuberculin type was involved in the induction mechanism of both conditions. .The aspermatogenic antigen was found to be a polypeptide—polysaccharide complex and the skin antigen was 28 a closely related amino acid—polysaccharide complex (Bishop gt gt., 1961). However, the authors observed the difference between aspermatogenesis where the reaction was against the host's own testicle and the host-against-graft reaction when foreign skin was rejected. Persons with chronic lymphocytic leukemias or general— ized lymphosarcomatosis were found to develop an autoimmune hemolytic anemia at some time during the course of the leukemic disease usually following a triggering mechanism (Rosenthal gt gt., 1955; Dameshek and Schwartz, 1959; Dameshek gt gt., 1961; Agreev, 1963). In these conditions, the leukemic cells were viewed as abnormal but immunologically competent "clones" proliferating in the host, becoming sensitized to its tissues and subsequently attacking them. In runt disease, which is a graft-versus-host reaction, the transplantation of competent cells from a genetically dissimilar animal but one whose tissues were tolerated, was followed by clinical manifestations including hemolytic anemia and wasting. This was demonstrated in immature mice (Belling— ham gt gt., 1953, 1962; Trentin, 1958; Trentin and Session, 1961, 1963; Oliner gt gt., 1961; Burnet, 1962) and in rats (Gowans gt gt., 1963). It was visualized that here, although the host was tolerant of the grafted cells, these were intol— erant of the host and eventually attacked it. As an outcome of these observations, Dameshek (1963) postulated that it was conceivable to think of autoimuniza- tion as a proliferation of abnormal yet immunologically 29 competent cells in a leukemic though not necessarily invasive manner, and that they might eventually attack the host. In this connection Dameshek (1963) pointed out that it might be more realistic to attempt production of autoimmune diseases using models like those used to produce runt disease rather than attempting to modify the antigens by denaturing them or by use of adjuvants. III. MATERIALS AND METHODS The experimental work was mostly done with albino rats.1 A total of 2A1 rats was used, of which 178 were males and 63 females. In one part of the study, 5 male and 3 female Dutch rabbits were used. The rats were kept in metal cages and water and food provided gg libitum. They were fed a pelleted mouse and rat diet.2 Pregnant females were provided with glass trays half filled with wood shavings where they littered and kept their young. The majority of the rats used, were bred during the early part of the experiment. Twenty—eight males and 28 females were obtained, ear-marked and then left for a period of 7 days to get accustomed to the food and environment. After 7 days each male was mated with a female to test their fertility prior to treatment. Near the end of the gestation period the males were removed to separate cages, and the females left alone to litter. Each litter was counted and weighed collectively as soon after birth as it was discovered. A total of 337 lSpartan Research Animal, Haslett, Michigan. 2Rockland lab animal diets. Complete mouse/rat diet, distributed by Tekland Inc., Monmouth, Illinois. 30 31 baby rats were born, out of which 175 males and 25 females were preserved. At weaning time, 20 days later, 70 males and the 25 females were ear—marked and preserved for use in future experiments. The remaining males were subsequently killed and their testicles and epididymises used for antigen prep— arations. At 65 days of age, 15 male rats were bred each with a littermate female. The first generation by this cross was designated as the F1 generation. Eighty F male and 1 10 female rats were ear—marked and subsequently used in the experiment. To avoid repetition, methods and procedures uniformly used and common to all four sections will be described first. Freund's complete and incomplete adjuvantsl were the only adjuvants used. The adjuvant selected comprised 50% of the total inoculum which was usually 1.00 ml. for the rat, the other half being the antigen suspended or dissolved in distilled water. Experimental animals were usually inoculated while they were anesthetized with ether. For intradermal inocula- tion, the back and sides of each animal were shaved, dis- infected with alcohol and the inoculum given in 12 to 15 sites on both sides of the animal along two lines extending from the shoulders to the flanks and at a distance of about 1 in. from the vertebral column. 1Difco Laboratories, Detroit, Michigan. 32 Experimental animals were killed by exsanguination while under ether anesthesia. Terminal blood samples were collected and the serum removed after clotting. Immediately following euthanasia, necropsy was done and portions of various tissues were collected for histopathologic examina- tion. Testicle, epididymis, prostate, Cowpers gland, adrenal, pancreas, thyroid and salivary gland were fixed in Bouin's solution. The testicle was weighed and-dropped intact into Bouin's solution and left for about 30 minutes to harden. It was then cut in two and returned to the fixative. Portions of skin at the site of inoculation, brain, thymus, lymph node, lung, spleen, liver, kidney and ileum were collected and fixed in Zenker's-solution. Sera obtained by terminal bleeding of experimental animals were tested for their capacity to induce passive cutaneous anaphylaxis (PCA) in other test rats (Ovary, 196A). Young, healthy, male and female rats weighing 150—225 grams were used as test animals. These were shaved by electric clippers on the back and sides, A to 5 hours prior to intra- dermal inoculation of serum. For anesthesia, phenobarbital sodiuml was injected intraperitoneally, at the rate of 0.15 to 0.25 ml. per rat. Each rat was used to test 8 different serum samples. Four injections were made on each side along a line from lJensen-Salisbury Laboratories, Kansas City, Missouri. Contains 1 gr. phenobarbital sodium per 1.00 ml. 33 the shoulder to the point of the sacrum and at a distance of 1.5 cm. from the vertebral column and 2.5 cm. from each other. The undiluted serum to be tested was inoculated intradermally in 0.25 ml. quantities. Twenty—five minutes later a mixture of antigen and dye was injected. The challenge injection was prepared by mixing 0.5 m1. of a 1% solution of Evan's blue with 1.00 ml. of_a 1% solution or suspension of the antigen depending on whether soluble extract or homogenate was used. Half of the challenge dose was given by slow intracardial injection and the other half intraperitoneally. The cutaneous reactions were examined every 5 minutes for up to 35 minutes and then the animal was killed and the underside of the skin observed and the reaction recorded. Experiment I This experiment was conducted to obtain information on the following: 1. Susceptibility of the rat to autoallergic aspermato- genesis following inoculation of testicular homogenates. 2. The effectiveness of autologous testicle in inducing aspermatogenesis. 3. The relative importance of complete and incomplete adjuvants on the induction of the disease. A. To measure the outcome of the disease, if any, by its effects on the fertility of the male. 5. To assess the influence of testicular antigens on the fertility of the female. 3A Twenty-eight pairs of rats that were previously mated and proved to be fertile, were used in this experiment. Twenty of the male rats were unilaterally orchiectomized. The orchiectomy operations were performed while the rats were in pentobarbital anesthesia. The right testicle was approached through a flank incision. The spermatic cord was ligated and then severed distal to the ligature. The testicle and epididymis were then removed and the abdominal wound closed by interrupted sutures. Epididymal spermatozoa were obtained by forcing dis— tilled water from a syringe through a 27—gauge needle into the vas deferens until the epididymis became turgid. The epididymis was then slit open and the contents collected. The testicle was decapsulated, weighed and added to the spermatozoal suspension; they were then homogenized together with addition of an equal volume of distilled water in a Ten Broeck grinder. Homogenates were stored at -70 C until the time of inoculation. Three weeks after unilateral orchiectomy, all males were mated for the second time with their previous mates. Close to term, the males were removed for inoculation. When litters were born they were weighed, counted and killed by ether. Sixteen unilaterally orchiectomized males were inocu— lated intradermally, each with his own testicular homogenate. The inoculum contained approximately 50 mg. of testicular 35 tissue on a wet basis. In 10 and 6 of these rats, respec— tively, the homogenates were incorporated in complete and incomplete Freund's adjuvants. Four nonorchiectomized rats were injected with a pooled testicular homogenate in complete Freund's adjuvant. Four unilaterally orchiectomized and A nonorchiectomized control rats were inoculated intradermally with 1.0 ml. sterile distilled water. Seven days after inoculation, all males were again mated with their respective mates and the pairs were left together until the end of the experiment, approximately 6 months later. For the duration of the experiment, the males were repeatedly inoculated at A-week intervals. The female rats were observed frequently for pregnancy, litters were counted and weighed as soon as they were noticed and the young subsequently killed. Ten females, 7 of which were inoculated intradermally with pooled testicular homogenate in Freund's complete adjuvant, and 3 controls were subsequently added to the experiment. They were mated to healthy males of known fertility and their rate of conception and number of young born per litter were recorded in a manner similar to that described for the inoculated males. Terminal blood sera of inoculated and control animals were tested for PCA reactivity. 36 Experiment 11 An acid extract of rat testicular tissue and spermatozoa was prepared and subsequently deproteinized. The purification was done by the general method used by Freund gt gt. (1955) for the purification of guinea-pig testicular antigens. The experiment was intended to investigate the following: 1. The possibility of isolating an aspermatogenic antigen from rat testicle. 2. The capacity of such purified material to induce auto- allergic aspermatogenesis in rats. 3. The effects of certain substances in initiating or modifying the immunologic response. Ninety—eight rats, 75-85 days old and 16 other rats weighing A00-A25 Gm. were killed by exsanguination after they were anesthetized. Pieces of spleen, lymph node and thymus were collected from certain parents of F1 litters intended for use in an- other experimental model to be described later. Protein Extraction Testicles and epididymises of all llA rats were collected aseptically. Pooled decapsulated testicles weigh- ing 302 Gm. were mixed with epididymal contents obtained as described earlier. To this, 325 m1. of 0.1N acetic acid were added and the mixture homogenized in a Waring blender and the homogenate left overnight in the refrigerator. On the following day, the homogenate was centrifuged at 2200 rpm 37 for 15 minutes. Solid ammonium sulphate was added to the supernatant fluid to a concentration of 30% and the solution left for 1 hour and centrifuged once more. Further amounts of solid ammonium sulphate were added to the supernatant fluid to reach a concentration of 70% of the total volume. This was left overnight in the refrigerator after which it was centrifuged at 6000 rpm for 8 minutes. The sediment was resuspended in distilled water and dialyzed against distilled water for 2A hours at approxi- mately A C. The dialysis bags were then suspended in front of an electric fan to evaporate the water and concentrate the solution to about 1/10 its volume. Purification of the Aspermatogenic Factor Lyophylization of the ammonium sulphate-precipitated material which was described by Freund gt gt. (1955) was eliminated in the present procedure. The concentrated protein solution was collected and to each A volumes, 5 volumes of 20% trichloracetic acid were added and the mixture left for 18 hours in the refrigerator. It was then centrifuged at 8000 rpm for 10 minutes after which the supernatant fluid and the sediment were collected separately and each dialyzed for 2A hours against distilled water. Finally each solution was concentrated, 1y0phylized in small portions and stored. The trichloracetic acid soluble material will be referred to as TCPM (trichloracetic 38 acid purified material). The trichloracetic acid precipi— tated material will be referred to as testicular protein. Inoculation of Rats with TCPM In this experiment, male rats were used. The rats weighed 200—250 Gm. at the time of the first inoculation. TCPM was administered at the rate of 3 mg. for the first injection and subsequently 1 mg. every A weeks until the animal was killed. The TCPM was dissolved in 0.5 ml. of_ distilled water. When an additive was used it was first dissolved in 0.5 ml. of distilled water and the resulting solution used to further dissolve the TCPM. The adjuvant was then added in 0.5 m1. quantities and thoroughly mixed to give 1.0 m1. of a uniform emulsion. TCPM incorporated in complete adjuvant was inoculated intradermally into 30 and intramuscularly into 10 rats. Five rats were inoculated intradermally with TCPM in incom- plete adjuvant. Complete adjuvant in distilled water was injected intradermally into 5 other rats. Testicular protein at the rate of 20 mg. per rat in complete adjuvant was similarly inoculated into 10 male rats. Ten milligrams of histamine base1 in addition to the standard dose of TCPM in complete adjuvant were inoculated intradermally into each of 12 rats. A group of 6 rats was given histamine-supplemented TCPM in incomplete adjuvant. lMatheson, Coleman and Bell, Highland Avenue, Norwood, Ohio. 39 Three other groups of 6 rats each, in turn, were given 50 units of heparin,1 0.5 mg. hyaluronic acid2 or 20 mg. serotonin creatinine sulphate3 in addition to TCPM and com- plete adjuvant. Five control rats were inoculated with a mixture of TCPM, hyaluronic acid» serotoninand.heparin in the same dosage incorporated in incomplete adjuvant. Six rats were inoculated intradermally with TCPM and 0.5 ml. of a killed 7-day culture of Leptospira canicola incorporated in incomplete adjuvant. The leptospiral culture was killed by autoclaving at 18 lbs. of pressure for 90 minutes. Six control rats were each given 1 ml. of dis— tilled water intradermally. One milligram of histamine was inoculated intratesticu— larly into the right testicle of each of A rats that were originally inoculated with histamine-supplemented-TCPM in complete adjuvant. Three rats that were inoculated with histamine—supplemented-TCPM in incomplete adjuvant and 3 control rats received the same treatment. The intratesticular inoculations were made A0 days following the intradermal injection. Rats from the various groups, along with appropriate controls, were killed at regular intervals until the termin— ation of the experiment 185 days after the first inoculation. lUpjohn, Kalamazoo, Michigan. 2Nutritional Biochemicals, Cleveland, Ohio. 3Nutritional Biochemicals, Cleveland, Ohio. A0 Experiment III This was conducted to obtain information on the effect of passively transferred anti—rat—testicle serum antibodies on the fertility of male and female rats. Young male and female rats of demonstrated fertility were inocu- lated. Preparation of Heterolggous Immune Sera Eight Dutch rabbits, 5 males and 3 females, were bled prior to their inoculation. Sera were obtained from the blood samples and used for inoculation of control male and female rats. The rabbits were then inoculated at 2—weeks intervals with rat spermatozoa—testicle homogenate in complete adju— vant. A 2.0 ml. inoculum was divided between intradermal, intramuscular, and intarperitoneal routes. It contained approximately 100 mg. of testicle on a wet weight basis, homogenized with distilled water and incorporated in complete adjuvant. The rabbits were bled 30 days after the first inoculation and again 3 weeks later. Serum separated from this blood was injected into groups of male and female rats as will be described later. Inoculation of Female Rats with Immune Sera Female rats intended for inoculation were mated to healthy male rats. Each female was observed closely for signs of pregnancy. Soon after parturition, each female Al was inoculated subcutaneously with 1.5 ml. of serum and 1 day later remated with her previous mate. The females were then observed for signs of pregnancy and, when parturition occurred, litters were counted, weighed and killed with ether. The experiment was terminated for each female when the first litter after inoculation was produced. Inoculation with Isologous Sera.-—Serum samples were obtained at the time of necropsy from actively immunized male rats. These rats from Experiment I had been repeatedly inoculated intradermally with isologous testicular homogenates in complete adjuvant. The sera obtained from these males were pooled and 1.5 m1. injected into each of 7 female rats. Normal-rat serum was injected into each of 3 other female rats. Inoculation with Immune-Rabbit Serum.——A group of 5 female rats were inoculated with immune male-rabbit serum. A second group of 5 female rats was inoculated with sera of immunized female rabbits. Normal-rabbit serum was inoculated into 2 other females. Inoculation of Male Rats with Immune-Rabbit Sergm Young males known to be fertile were mated to young normal females. Pregnancies were followed and close to the end of the gestation period, the male was removed to a separate cage. When a litter was born, it was weighed, counted A2 and killed with ether. The male rat was inoculated on the same day on which the litter it had sired was born. It was remated with its previous mate 3 days after serum inoculation. The females were then observed for signs of pregnancy and soon after parturition the litters were counted, weighed and killed. The sire rats were killed a short time after this and portions of various organs collected for histo— pathologic examination as described at the beginning of this chapter. Experiment IV Preparation of Lymphoid Cell SuSpension Portions of spleen, lymph node and thymus from parents of 6 Fl litters were collected and homogenized with an equal volume of 0.85% NaCl. The homogenate was centrifuged and the sediment washed twice by resuspension in 0.85% NaCl. and centrifugation. Finally serial ten-fold dilutions of the resuspended sediment were made and the cells counted by use of a hemocytometer. An amount of 0.1 ml. of a diluted suspen— sion containing approximately 5 x 107 cells per ml. was inoculated into each of 65 baby rats within the first 2 hours after birth. The baby rats were anesthetized with ether and half the cell suspension inoculated intracardially and the remainder intraperitoneally. About 20% of those inocu— lated died within a short time after inoculation, apparently from shock and handling. Of the survivors 28 males were preserved and the rest were killed. “3 Transfer of Sensitized Lympgoid Cells At necropsy of male rats that were repeatedly sensi— tized with TCPM in complete Freund's adjuvant, portions of their lymph nodes, Spleen and thymus were collected. These were homogenized with an equal volume of 0.85% NaCl., washed and packed in graduated centrifuge tubes. The sediment was then resuspended in 0.85% NaCl. containing 5 mg. TCPM per ml. and the suspension incubated at 37 C for 1 hour. Follow— ing this, the cell suspension was washed twice and resuspended in three times its volume of 0.85% NaCl. This lymphoid cell suspension was inoculated into 10 F1 male rats that had been neonatally injected with parental lymphoid cells. The rats were 60 days old at the time of the second injection. Each one was given the equivalent of 0.A ml. packed cells in 0.85% NaCl. In ether anesthesia, approximately one—half the dose was slowly injected intra- cardially and the remainder intraperitoneally. Four control rats were injected with a suspension of lymphoid cells from normal controls. ' Rats in this experiment were killed 8, 15, 25, 35 or A5 days after inoculation. Parabiosis Ten male Fl rats, 7 of which were repeatedly inoculated with TCPM in Freund's complete adjuvant and 3 nonsensitized controls were used as donors. These were joined in para- biosis each with an F1 litter mate male recipient which was AA not sensitized with TCPM but had been neonatally inoculated with parental lymphoid cells. The parabiosis operation was performed under pheno— barbital anesthesia. Each rat was weighed and 0.15 to 0.25 ml. pentobarbital solution was injected intraperitoneally. The left side of the donor and the right Side of the recipi— ent were shaved from the neck to the root of the tail. The animals were placed on their sides with the shaved sides up and the backs touching. In each rat, a long skin incision was made from the cheek to about 1 cm. from the root of the tail, parallel to the vertebral column. The skin edges were separated and the underlying facia exposed along the incision line.' In the area between the last rib and the sacrum a A cm. long incision through the ademinal muscles and ulti— mately through the peritoneum was made in each rat. Starting with the innermost peritoneal layers, the two opposing edges, one from each of the participating rats, were joined together by interrupted sutures. The lower edges for each layer were sutured first and then the upper side, thus forming a common peritoneal cavity. Similarly the abdominal muscles were sutured and finally the four skin edges were sutured all the way from the cheeks to the roots of the tails. Supporting sutures were inserted in the region of the two opposing scapulas and made to include the supra- spinatus muscle and overlying skin from each rat. A5 Following recovery, each pair was placed in a separate cage until it was killed. Parabiotic pairs having immunized donors were killed at 8, 10, 15, 20, 27, 37 and 60 days postoperation, respec— tively. Controls were killed at 10, 27 and 60 days, respectively. At necropsy, blood samples were collected in EDTA (Ethylene—diamine—tetrachloracetate) for red and white cell and differential counts. Testicles, spleens and thymuses were removed and weighed prior to their fixation. IV. EXPERIMENTAL RESULTS All rats in the various experiments were in good condi- tion and apparently not adversely affected by long confine— ment. They had good appetite except those in which adjuvant was used. Anorexia was then noted for l or 2 days after inoculation. No signs of concurrent diseases were noticed throughout the period of experimental work. Body weights of rats in Experiments I-III were similar to those of the controls of Similar age. Rats inoculated with lymphoid tissue suspensions and those jointed in para- biotic pairs did not weigh as much as untreated controls of similar age. They did not consume the same amounts of food as did rats in the 3 other experiments. Signs of concurrent disease were not noted except for occasional diarrhea but on the whole the rats were not lively and the hair was rough and lusterless and came out easily. Lesions unrelated to the experimental procedure in- cluded 2 cases of pneumonia, one of which was in a control and the other in a male rat inoculated with testicular homo- genate in Freund's complete adjuvant. In 1 rat there was an adenocarcinoma metastatic in the thymus, but no primary tumor was detected. Rats inoculated intradermally with either complete or incomplete adjuvant developed ulcers at the A6 A7 sites of inoculation. An eruption 0.5-1.0 cm. in diameter developed 6-12 hours after inoculation. The covering epithe- lium was eroded usually on the day following inoculation leaving a raw surface. The ulcers persisted for long periods especially in rats inoculated with complete adjuvant. In these, raw granulation tissue with a moist appearing core was usually observed whenever the covering eschar was removed. Subsequent inoculations, which were usually administered close to the peripheries of previous ulcers elicited a more pronounced reaction and resulted in the same type of nonheal- ing ulcers. Ulcer formation was also noted when Freund's complete adjuvant alone was inoculated. There was a noticeable enhancement of the initial inflamatory reaction at the site of inoculation of histamine- supplemented antigen when mixed with either adjuvant. Swelling was evident 1 hour after inoculation, but subsequent changes were the same as in rats that were not injected with histamine. Histopathologic examination of skin from inoculation sites of rats killed at various periods after inoculation revealed definite changes. The epidermis of rats killed as early as the day following inoculation with TCPM in either adjuvant contained superficial ulcers at the site of inocula- tion. The epidermis in the ulcerated area was necrotic and infiltrated with neutrophils. Below the ulcerated area, large vacuoles,possibly due to oil droplets, were seen extend- ing down to the muscular layer of the dermis. Collections of mononuclear cells were seen between the vacuoles (Figure 1). A8 Figure l.--Superficial skin ulcer, 1 day after inocu- lation of TCPM in complete adjuvant. Note necrosis (A) and infiltration with macrophages (B). H & E. x 750. In rats killed 7 days postinoculation, the reaction was granulomatous and layers of macrOphageS were surrounding drop— lets of the inoculum. Plasma cells, mast cells and lympho— cytes were scattered outside the granulomatous reaction area and in the areolar tissue network beneath the muscularis. There was a marked difference in the cellular reaction induced by either complete or incomplete adjuvant in rats killed later than postinoculation day 7. While in both instances the basic granulomatous reSponse was of the same nature, it was more intense when complete adjuvant was used. Moreover the lymphocytic infiltration was more prominent with complete adjuvant while it was either slight or not present with the incomplete adjuvant. A9 The numbers of lymphocytic and other mononuclear cells seemed to increase with time and in many cases persisted for over 6 months (Figure 2). Figure 2;—-Granulomatous 1eSion, 70 days after5 inoculation of TCPM in complete adjuvant. Note infiltration with lymphocytes. H & E. x 178. Similar infiltration with lymphocytes was seen in rats inoculated with either histamine, serotonin or hyaluronic acid mixed with TCPM and complete adjuvant, although it was not as pronounced with the hyaluronic acid. Marked lymphocytic infiltration was also observed with a mixture of TCPM, Leptospira canicola and incomplete adjuvant. A mixture of heparin, TCPM and complete adjuvant elicited a Similar granu- loma but lymphocytes were few (Figure 3). 50 11 1 _, Figure 3.——Granulomatous reaction at the site of inoculation of heparin-supplemented TCPM in com- plete adjuvant. Note vacuoles-which contained the adjuvant; and the paucity of lymphocytes. H & E. x A70. 51 Giant cells were not seen in the skin at the site of inoculation of antigens in either adjuvant. However, many giant cells were seen in the granulomatous reaction around the suture material in parabiotic rats. In the skins of rats killed A0 days or more after in— oculation, some perivascular cuffing with mononuclear cells was observed in the connective tissue close to the granu— lomatous reaction (Figure A). Testicular Lesions As a rule, lesions believed to be the direct result of an immunologic response to testicular antigens, were confined to the testicle and epididymis. All such leisons were basically the same and varied only in extent and severity. To avoid repetition these will be described first. Other lesions that were seen in association with this response or as a result of a specific treatment will be described in their appropriate places with regard to the particular experimental model. Encapsulated testicles obtained from a large number of rats approximately 3 months old weighed 1.5—1.95 Gm., with an average of 1.72 Gm./testicle. Epididymises of the same testicles weighed 0.5—0.75 Gm. with an average of 0.61 Gm./ epididymis. Testicles of control rats killed at various ages of 3-11 months, weighed 1.8-2.3(hng with an average of 1.88 Gm. There was approximately a 0.3 Gm. increase in the weight of the testicle for every month of age after the 52 Figure A::;Perivascular infiltratIon with mononuclear cells in a skin lesion A0 days after inoculation of TCPM in complete adjuvant. H & E. x 750. 53 third month. Epididymises of the same rats weighed 0.6—0.75 Gm. with an average of 0.7 Gm. ' Testicles inoculated with histamine were of normal size in rats killed shortly after inoculation but shrunken and hard in those killed 3 weeks or later. Histopathologic changes were confined to the seminifer- ous tubules and, in the majority of cases, only to the germinal epithelium within these tubules without involvement of sup- porting elements. Degenerative changes were noted in a portion of seminiferous tubules of some uninoculated rats. These changes were indistinguishable in character from those seen in inoculated rats and presumably occurred as a result of inoculation. The changes involved only spermatids and secondary spermatocytes without involvement of the deeper layers. Signs of degeneration included loss of stainability and vacuolation of the cytoplasm and pyknosis of some of these cells. Such degenerative changes determined by actual counts of healthy and affected tubules involved up to 1.5% of the total number of tubules in control rats. Any testicle having double this number or 3% affected tubules was considered abnormal and the degeneration classified as pathologic in origin. Testicles having 3 to 10% of tubules with degenerative changes in the superficial layers of the germinal epithelium were classified as having type I injury (Figure 5). Those 5A Figure 5.-—Type l testicular lesion 35 days after intradermal inoculation of TCPM in complete adjuvant. Note pyknosis of nuclei in the region of spermatids and secondary spermatocytes. H & E. x A70. 55 having 10 to 20% of the tubules with similar degeneration and a few exfoliated cells in the lumens of tubules or in the epididymises were considered as having type 2 damage. In some testicles less than 10% of the tubules had superficial degen- eration and in addition the primary spermatocytes in a portion of tubules were similarly degenerated. This was also classi- fied as type 2 damage (Figure 6). Type 3 damage was seen in testicles with 20—50% of the tubules having damage in the region of secondary spermatocytes with accompanying degeneration or necrosis in the region of the primary spermatocytes. In this type, numerous exfoliated cells were present in the lumens of the seminiferous and epi— didymal tubules. In addition, varying numbers of multinucleate giant cells were seen in the regions of primary and secondary spermatocytes and in the lumens of epididymal and seminiferous tubules (Figures 7, 8, 9). Also classified in this category were testicles having less than 20% type 3 damaged tubules but in which spermatogonia were partially or completely damaged and exfoliated. Damage of a more subtle nature where germinal epithelium in the majority or all of the seminiferous tubules had been damaged was classified as type A. In the most severely affected testicles all germinal epithelium including spermatogonia was lost leaving tubules looking like empty circles lined only with Sertoli cells and occasional Spermatogonia. The lumens of seminiferous tubules and those of the epididymis either con- tained-desquamated dead cells or were completely empty (Figures 10, ll). 56 Figure 6.——Type 2 testicular lesion. Rat was inoculated intradermally with TCPM in complete adjuvant. Note severe vacuolation of the cytoplasm of secondary and primary sperm- atocytes (A), fading of nuclei (B) and multincleate cells (C). H & E. x 750 Figure 7.—-Type 3 testicular damage in rat inoculated intra— dermally with TCPM in complete adjuvant. Note pyknosis of nuclei, exfoliation of cells in the lumen (A) and loss of germinal epithelium (B). H & E. x 187. Figure 8.——Type 3-A damage in testicle of a rat 120 days after intradermal inoculation of TCPM in complete adjuvant. (A) Com— plete loss of germinal epithelium and (B) binucleate giant cell. H & E. x 187. ‘ \ . g - ' A" 1.: .', N , . ~ M ' , ’g_ ~ > . _ n . O O ’ .. ‘ :1 Figure 9.——Same rat as in Figure 8. The epididymal tubules contain fragmented spermatozoa and exfoliated cells. H & E. x 187. .. "a; . ,~"'”‘EVLF Figure 10.-—Type A damage 180 days after inoculation of testic- ular homogenate in complete adjuvant. Note reduced size of seminiferous tubules and loss of germinal epithelium. H & E. x 187. 59 Sertoli cells were not affected except in a few instances in which they were fewer in number than in the controls. No damage to the basement membranes of tubules was noticed in any of the experimental rats. In certain instances in which severe lesions were observed, there was detachment of the basement membrane from the deeper layer of cells in the tubules (Figure 7). Similarly, Leydig cells were not affected. In some testicles which were significantly reduced in size, the number of interstitial cells seemed to be increased (Figure 11). Inflammatory reactions were not observed except in testicles inoculated with histamine. In a few other testicles mononuclear cells that looked like small lymphocytes were occasionally seen in the interstitial spaces in the vicinity of blood vessels or inside them. Figure ll.——Higher magnification of Figure 10. Note lack of nuclei in region of Sertoli cells (A) and large number of interstitial cells (B). H & E. x 750. 60 Experiment I In this experiment, 20 unilaterally orchiectomized and 8 nonorchiectomized male rats were mated. They produced litters 22-28 days later. Litters by unilaterally orchiecto— mized males were closely similar in size to those produced before orchiectomy. They also compared closely to litters by nonorchiectomized males (Table 1). No adverse effects attributable to unilateral orchiectomy were observed at any stage of the experiment. In the 6 months following inoculation of autologous testicular homogenate in Freund's complete adjuvant, 10 uni- laterally orchiectomized male rats sired 1-5 litters each with an average of 3.5. Each rat sired a total number of young in the whole period ranging from 1—50 with an average total of 31.7 and an average number per litter of 8.A6 young. A group of 6 unilaterally orchiectomized rats that were inoculated with autologous testicular homogenate in in— complete adjuvant, sired A-6 litters each. The group had a total of 30 litters with 320 young which amounted to 10.66 young/litter/rat. In a total of 2A litters, A control unilaterally orchiectomized rats sired 259 young. On the average, each rat sired 6 litters with a total of approximately 65 young or 10.8 young/litter. Four nonorchiectomized rats inoculated with pooled rat testicular homogenate in complete adjuvant sired 17 litters with a total of 1A7 young. The average was 5.6 young/litter. pcn>3wvw opmadsooefl ll <1: 0 C H o ucm>shcm muoHQEoo ll <1: 0 mpuemwoeon smHSOABmcu .EOm .pmmem 61 coma m.mH :.mH mm.m ma.:H nun amps: teapomfl UmHHHpmHQ Iconoeoz : umua mu.mm oo.m mm.z mm.mH It: .< .o tachomfi + .Eom.pmme taosocoz.z m.:a m.oa oo.w ootma mm.ma scum; vmuHEOB nmaafiumfin nomfinoso.: 5.5m mm.oa oo.m mm.:a mo.ma o.< .osH nmNHSOp + .Eom.pmoe tomfinoso.m o: m:.m m.m 0.:H m.ma w.¢.o ooNflEOp +m.Eom pmoe tomflnoao 0H mumppfiq swam beam nopuflq coapmHzSOCH ouflm soppaq smppfiq CH soapwHSoocH meom conpwazoocfi ouwm scppfiq soaposemm tumom mo nonssz Imam msouomfinosomsm soaumHSSOeH mpwm no peoosom mwdao>¢ owdno>< omwum>¢ omwno>< no.0QzB coapafiaomoo .usm nonesz asepomfinosopmom soapeHSoocH scpm< use muommm mama can: chHEOpocHnosocoz new coaanuamHnoso zHHesmpmHHea ea msmppaq no zswsssmtu.a mqm3wcm mpmHoEoocH u .¢ .och HmHhome omHQHnso vHom QHpmompOHconp u Smoen pcm>shvm ouoHQEoo u .< .o 0 zoom who: mGOHmoH on n o m oo oo oo o. m o. o. o. o. m .0 o. oo 00 N hHHoQEnHmUMCHPCH .4 .o + .ponm .pmme 0H 0 o o o N H H o o o o o o H H o o o o H o o m %H-H.QH:OWSE¢.H9CH .< .o + Smoe OH 0. o. oo 00 H .0 oo oo o. N o. o. on to N hHHMEthMoHpGH «pmpmg omHHHpmHQ + .d .o m o. on co co H 0. oo o. 0. H o. o. oo o. m hHHMEhmumanC-fi o.< .ocH + Smoe m m H H .. H H H H m .m .. H H m m zHHmEpmomppCH o..a.o + ozdoe mm a m m H o z m m H o z m .m H do COHpmHSoocH mo ooze mumm COHmmH mo mama :OHmoa no ooze COHmoq mo come no nonszz omHuHmH ammo omHnHo mama oouH mama COHuMHsoochmom QOHpMHSOOQHpmom COHpmHsoochmom r’ 1 mpwm Hoppcoo 65w oopstoocH CH mQOHmoH hmHBOHpmmBII.m mqm<9 71 months after inoculation had the same type of severe damage. None of the rats inoculated either with TCPM in incomplete adjuvant or with complete adjuvant alone had any testicular lesions. TCPM in complete adjuvant inoculated intramuscularly into 10 rats induced types 1 and 2 damage only. Type 2 was seen in 2 rats killed 150 and 170 days after inoculation (Table 5). Histamine—supplemented-TCPM in complete adjuvant induced testicular lesions of types 3 and 4 in 2 of 3 rats killed 4—6 months after inoculation. Type 2 damage was seen in 2 rats killed on postinoculation days 62 and 90. Rats killed between postinoculation days 1-30 were without testicular lesions (Table 6). Four rats previously inoculated with histamine-supple— mented—TCPM in complete adjuvant, developed severe lesions in a testicle when histamine was subsequently injected in that testicle. There was an extensive area of necrosis which probably correSponded to the area of histamine injection. Lymphocytes and a few macrophages and neutrophils were seen in the interstitial spaces. Leydig cells and other elements in the intertubular spaces appeared normal. Fourteen days or later after the intratesticular injection of histamine, all cellular elements within seminiferous tubules in the area of necrosis were not discernible. The tubular contents resembled a homogeneous 72 pcm>3wcm ouoHoEoocH m<.och mHooHcmo thomOmeq n .o.qH ommn ocHsmumHn u.pmHmU UHom OHCOMSHmzc u .<.D.mn pcm>3nom mpmHoEoo u .¢.oo chmomn u .pmommw HmHamme omHmeso oHom OHpmomLOHEOHLp n Smash :HCOponmm u .popmmn comm who: mCOHmmH o: n o . m .. .. .. .. H .. .. .. .. m .. .. .. .. m CHCOpoamm + .< .D .m + .pmomm + .< .ocH + Smoa m .. .. .. .. H .. .. .. .. m .. .. .. .. m .o.q + .< .ocH + SmoB m .. .. .. .. H .. .. H H .. .. .. .. .. m s.<.D.m + .¢ .0 + Emoe m .. .. H .. m .. .. .. .. H .. .. .. .. m m.pmomm + .¢ .0 + Smoe m H H .. .. .. .. .. m .. .. .. .. .. .. m m.popmm + .¢ .0 + SmoB m .. .. .. .. H .. .. .. .. H .. .. .. .. H .pmHm m.¢ .ocH + Smoe m H H .- H .. .. .. m .. .. .. .. .. .. m U.pmHm + o.¢.o + namoe m z m m H o a m m H o z m m H mo COHmoH no maze COHmoa mo mums :onoq no maze, COprHzoocH mo ooze mumm owHuHmH mama omHuHo mama oth when do hoossz coHumHzoOCHpmom COHumHSoocHumom COHpmHSOQQHpmom mpcm>3mc< afilzmoe on coHuHoo< CH .oHo¢ oHcoszmzm no chwomm .chouopmm .mcHEmpmHm .hHooHomo .q ooHHax Boa: sHHoehooohocH ooooHsooeH whom oHoz ca nooanoq hoHooHonoe-:.o mqmee 73 mass bound by a basement membrane which appeared to be unaffected (Figures l2, 13). In rats killed later than 14 days following intrates— ticular inoculation, the zone immediately adjoining the area of necrosis had typical type 3 or type 4 damage (Figures 14, 15). The left noninoculated testicles of 2 rats in this group had types 1 and 2 damage, respectively. These were killed 28 and 45 days, respectively, after intratesticular injection of histamine. ‘ Similar necrosis was also demonstrated in histamine— inoculated testicles of 3 rats initially inoculated with histamine-supplemented-TCPM in incomplete adjuvant. Testicular damage outside the area of necrosis was not noticed. The contralateral testicles of these rats were comparable to those of noninoculated controls. Testicular lesions were noted in 4 of 6 rats inocu— lated with serotonin—supplemented—TCPM in complete adjuvant. Two of these, killed 69 and 98 days, reSpectively, after inoculation, had type 2 lesions. Those killed on post- inoculation days 136 and 165 had types 3 and 4, respectively (Table 6). One rat of 6, inoculated with heparin-supplemented— TCPM in complete adjuvant and killed on postinoculation day 140 had type 2 damage. The other 5 rats were completely without lesions (Table 6). 7n Figure 12.—-Histamine—inoculated testicle 1 day after injec— tion. Note pyknosis of nuclei in a necrotic seminiferous tubule (A) and infiltration with lymphocytes (B). H & E. x 187. Figure l3.-—Histamine-inocu1ated testicle 21 days after injec— tion. Note homogeneous material in seminiferous tubules (A) and connective tissue and a few macrophages in the interstitial Srbaces (B). H & E. x 187. 75 Figure l4.——Histamine-inoculated testicle. Type 3-4 damage in seminiferous tubules adjoining the area of histamine necrosis. A necrotic tubule is seen at A. H & E. x 187. Figure l5.——Histamine-inoculated testicle. Type 4-damage in Seminiferous tubules at some distance from the area of histamine necrosis. H & E. x 187. 76 Types 1 and 2 damage were observed in 2 rats inocu— lated with hyaluronic acid-supplemented—TCPM in complete adjuvant. These were killed 61 and 90 days, reSpectively, following the initial inoculations. The remaining 4 rats killed on postinoculation days 3, 14, 45 and 156, respectively, did not have any testicular lesions (Table 6). Lesions were not seen in testicles of rats inoculated with a mixture of heparin, serotonin, hyaluronic acid and TCPM in incomplete adjuvant. Similarly, no lesions were demonstrated in any of the 6 rats injected with TCPM and L. canicola in incomplete adjuvant (Table 6). Testicular protein in complete adjuvant also failed to induce any lesions in 10 rats killed between postinocu— lation days 7-178 (Table 5). Sera of 26 TCPM—inoculated rats, killed after post inoculation day 40 and representing various groups in this experiment, failed to induce PCA in normal rats. Sera of 9 rats, inoculated with testicular protein in complete adjuvant, had moderate to strong PCA reactivity. 77 Experiment III Rabbits, injected with rat testicular homogenate in complete adjuvant and killed 50 days later, had no gross nor microscopic lesions. Their sera were effective in induc- ing moderate to strong PCA in normal rats. Male rats, inoculated with either male- or female- rabbits' sera and subsequently mated to female rats of known fertility, produced litters 23-41 days after inoculation. The average time from inoculation to the birth of a litter among rats injected with sensitized—male—rabbit serum was 28.5 days. In those injected with sensitized-female-rabbit serum, this period averaged 32 days. Control rats injected with normal~rabbit serum had litters in an average of 26.5 days. Data of litter sizes before and after inoculation are summarized (Table 7). At necropsy, no gross, nor microscopic lesions were seen in any organ including the testicles. Similar groups of female rats inoculated with sera from either sensitized male, or female rabbits and mated one day later had litters in 20-39 days. Controls, injected with normal~rabbit scrum, littered 24~40 days later. Data on lit- ters before and after inoculation are shown (Table 8). Table 8 also illustrates similar findings for 7 female rats injected with sensitized-male-rat serum and 3 controls injected with serum from normal male rats. Lesions were not observed in any of the serum-inoculated females, nor were abortions, still-births, or other defects noted. 78 om.wm 0:.mm m>.HH .MHIQH oo.mH MHIHH Esumm .mUHDQmm Hassoz m oz.om Hmnmm om.OH mHum om.HH mH-OH npaooom mHmEmm omNHcseeH m . u . I . I mpHnnmm 00 mm mm mm om HH :H OH 0: HH mH OH mHmz ooNHcsesH m mwmnm>< owcmm mwmnm>¢ mmcmm mwwpo>¢ wwcmm Enumm mo oomsom copmHzoocH mpmm nouqu paw COHumHsoocw mmfiw ampqu mNHm nopqu mo ponesz cmmzuom mama coaumHsoochmom COHpmHSoOCHmnm .ohom .mofiooom oHoHomoe. noomuHoca one Hosaoz so“: oohoHsoocH whom oHoz so ooaam whopqu no assessmuu.a mamas 79 oo.wm ozlmm mm.oa HHIm mN.HH MHIm ESMmm .mpHnnmm coHuwHSOOCHmnm m oo.mm mmumm om.m HHuo om.OH mHuoH mpHnnmm mHmEmm vmpmHSOOGH m om.mm mmIHm o:.m OHIm oo.OH NHIN mpHnnmm mHmz copmHsoocH m oo.nm mmlom oo.OH mHIm om.0H mHIm mpmm Hoppcoo m om.om nmlom mm.m mHlm m:.0H mHnm mpmm ocpmHzoocH N mwmnm>< omcmm mmmpm>¢ mmswm owmpm>¢ mmcmm Espmm mo condom ompmHSOOWm hoooaq och coHooHsoocH ohHm hoooaq oNHm hoooaq no sommmm coozomm mama COHpmHSOOQHpmom COHpmHSQQQHmLm whom pHonmm Lo pmm IHthcoo ccm IUmNHCSEEH ssz pmpmHSQOCH mpmm mHmEom mp whopqu mo NHLmEESmIId mqm59 OHCoHQm QOHumHsoocH oHocoqu mpmm mo mo mama mwmpm>< psmsomuchm mmmho>< ommpo>< upmom mo condom hmossz mpmm Hoppcoo cam UoNHpHmsmmuzmoa scum mCOHmchmsm mzmeB cHonoqu 00H: ompwHSOOQH mpmm cH mQOHmmq pmHSOHpmmB oHdoomOAOHz 0cm mmHoHpmmB new mmooz noEzq .cmoHom .m55239 no mowcmco mmoh0nn.m mqm<. owwac>< pCmsmemHCm owwpo>< mwmnm>< mHeHoHoHam eds oHoHpmoa spam oHpoHnoCum CH «Conoq CoHsoHpnoa.oHCoomonon.0Co .noooz Caszq .CmoHam .nsesna CH nomCuC0_mmohc no muoessmtt.0H mqmea V. DISCUSSION Various degrees of temporary sterility were induced in female experimental animals by inoculation of testicular antigens (McCartney, 1923; Katsh, 1959). Conversely, auto- allergic aspermatogenesis was induced in experimental animals but the effects were measured only by gross and histopathologic lesions in the testicles. Effects of such lesions on the fertility of the male, as measured by actual mating experiments, were not encountered in the literature. Rumke and Hellinga (1959), demonstrated spermatozoa- agglutinating antibody in the sera of 3% of 2000 sterile. men examined. They speculated that the serum antibody was a true autoantibody and could be held responsible for sterility of these men. Once more, experimental data was lacking to demonstrate how much sterility could be attributed to the action of the antispermatozoa antibody Egg s9. Two models utilizing rats were designed to correct, at least in part, deficiencies in the literature pertaining to immunologically induced sterility. Active Immunization With Testicular Homogenates Unilateral orchiectomy of 20 male rats did not seem to affect their fertility (Table 1). Temporary sterility for periods of 6-22 weeks was induced in unilaterally 98 99 orchiectomized rats by inoculating them with autologous testicular homogenates in complete adjuvant. The average number of postinoculation litters sired by these rats was 3.5 in approximately 6 months, and litter sizes averaged 60% of those sired by the same males before inoculation. Comparable reductions in numbers and sizes of litters by nonorchiectomized male rats inoculated with isologous testicular homogenates in complete adjuvant were also noted. The fertility of unilaterally orchiectomized rats in- oculated with autologous testicular homogenate in incomplete adjuvant was also impaired but to a much less extent than in those in which complete adjuvant was used. The maximum period of observed sterility was 6 weeks in 2 of 6 rats. There was, however, slight but progressive reduction in litter sizes in 4 other rats in this group. Testicular injury was demonstrated in a proportion of unilaterally orchiectomized and nonorchiectomized rats in- oculated with testicular homogenates in complete adjuvant (Table 2). Mild testicular lesions were also noted in l unilaterally orchiectomized rat inoculated with testicular homogenate in incomplete adjuvant. PCA reactivity was demon- strated in sera from the majority oerats inoculated with testicular homogenates in either complete or incomplete adjuvants. In individual rats, infertility was, to a con- siderable extent, associated with testicular damage rather than PCA reactivity. Some sera that induced strong and moderate PCA reactions were obtained from rats in which 100 testicular damage was not seen and whose fertilities were only slightly impaired. Conversely, other rats with demon— strable testicular lesions and reduced fertilities, had sera that induced weak to moderate PCA reactions. It may be speculated that in these rats, reduced fertility or temporary sterility was the outcome of testicular damage and the role of serum antibody was secondary. The dissociation of testicular damage from the presence of high serum antibody titers was reported by Freund gt—gi. (1955), Waksman (1959) and BishOp gt a1. (1961). Bishop and Carlson (1965) found that testicular damage was induced in guinea pigs by an antigenic factor different from that reSpon- sible for induction of PCA reactive antibody. In the present experiment, reduced fertility demonstrated in the absence of testicular damage may suggest a mediatory role for serum anti- body. However, injection of anti—rat—testicle sera into male rats did not seem to influence their fertility (Table 7). In view of this, one may speculate that the same factor or } factors that induced testicular damage were also responsible for reduction of fertility. Reduced fertility may have been a manifestation of impaired spermatogenesis which fell short of causing demonstrable necrosis. Such impairment may be visualized as the first stage in the path leading to degener- ation and-necrosis. Freund 33 gl. (1953, 1955) regarded autoallergic asper- matogenesis in guinea pigs to be the result of inhibition or 101 suppression of spermatogenesis. This was questioned by Katsh (1959) and Bishop gt gt. (1961) who considered it to be the result of specific destruction of the germinal epithelium containing the antigen. Multinucleate giant cells in the regions of secondary and primary spermatocytes of affected testicles were reported by Freund gt gt. (1955), Waksman “g (1959), andeoughton and Spector (1963). Similar changes ,P- I were seen in rats in the present experiment and were noted relatively earlier than destruction of the germinal epithelium. The presence of such multinucleate cells may indicate an im— ( 9. mm «EMA: IE; . pairment of cellular division perhaps due to inhibition to some vital enzyme systems. It may, therefore, be hypothesized that specific inhi— bition independent of PCA—reactive—antibody may cause derangement of Spermatogenesis leading to reduced fertility. Such inhibition may, at least in part, lead to necrosis. Autologous testicle seemed to be more effective in inducing testicular damage and suppressed fertility in rats than isologous testicle. This is in agreement with similar observations reported by Bishop and Carlson (1965) in con- nection with autoallergic aspermatogenesis in guinea pigs. It seems likely that the closer the configuration of the inoculated antigenic determinant to that present in the inoculated animal, the more effective the response will be. 102 Reduced Fertility in Female Rats Reduced fertility marked either by temporary sterility or reduced litter sizes, was induced in female rats inoculated with rat testicular homogenate in complete adjuvant. Both the total number of litters and the number of young/litter were reduced considerably (Table 4). Temporary sterility ' over periods of 6-22 weeks was induced by McCartney (1923) in a female rats inoculated with rat spermatozoa in Ringer's solution. In the present experiment, temporary sterility for a maximum of 80 days was noted in female rats. Reductions E; in the sizes of litters, on the other hand, extended over periods of up to 153 days. McCartney (1923) and Fogelson (1926) rendered female rats temporarily sterile for up to 22 and 26 weeks following inoculations of isologous and heterologous spermatozoa, respectively, in Ringer's solution. These workers attributed the reduced fertility to circulating antibodies invading the female genital tract and causing agglutination of spermatozoa. Edwards (1960) was unable to demonstrate local antibodies in the genital tracts of nonpregnant female rabbits inoculated with rabbit semen in-complete adjuvant. However he did demonstrate such local antibodies in pregnant females. If the same phenomenon also occurs in female rats, it is dif- ficult to understand how serum antibodies can act on sperma- tozoa during copulation of a nonpregnant-animal. Delay in-conception of female guinea pigs inoculated with spermatozoa in either saline or complete adjuvant was 103 reported by Katsh (1959). He found that spermatozoa sus— pended in saline were far less effective in inducing infertility than spermatozoa incorporated in complete adju— vant, although comparable levels of serum antibodies were demonstrated. It-was observed that although inoculated female rats in this experiment had strongly PCA—reactive sera, they 5%. {"1 IA varied widely with respect to induced infertility. Also PCA—reactive sera from sensitized male rats and male and female rabbits did not seem to have any recognizable effect ti on the fertility of female rats. _ Such a discrepancy between levels of serum antibody and observed female infertility is difficult to explain. Until a better explanation is offered, the hypothesis intro— duced by Katsh (1959) seems to be promising. He found that strips of uteri from sensitized female guinea pigs contracted for at least 4 hours when challenged tg_tittg with guinea» pig spermatozoa. Whether similar anaphylactoid reactions can occur in the living female following copulation can only be inferential at this moment. It may be possible to specu- late that such contractions taking place in the uterus may cause expulsion of spermatozoa introduced during copulation. Furthermore a contracting uterus may not be a favorable place for embedding a fertilized ovum. On the other hand, mediation of female infertility through uterine contractions alone may be subject to the magnitude and duration of such con— tractions, the mechanics of which are not yet known. 104 Even if such contractions play a part in impairing conception, the role of serum antibody should not be over— looked. A hypothesis that may reconcile the various obser- vations calls for a synergistic combination between local uterine anaphylaxis and serum antibody. The anaphylactoid reaction accompanied by histamine release (Katsh, 1959) may allow serum-antibody to pass out into the uterine lumen where it may act on spermatozoa. Purified Aspermatogenic Factor The lyophylizable trichloracetic acid purified material (TCPM) that was prepared from rats' testicles and spermatozoa had highly aspermatogenic prOperties. The exact chemical nature was not.determined but in a preliminary test it gave a positive reaction with the Schiffs'reagent. This may indicate that TCPM contained a carbohydrate, probably a polysaccharide fraction. Since TCPM was prepared by trich- 1oracetic acid deproteinization, it was assumed to be protein free. In the dosage used, TCPM was found to be at least twice as effective as testicular homogenates in inducing aspermato— genesis in rats. This may seem to be a comparatively modest increase in efficiency due to purification and concentration. The comparison was made on the basis of the percentage of rats which developed severe lesions 4-6 months after inocu- lation of-either testicular homogenates or TCPM. Freund gt gt. (1955) reported that TCPM prepared from guinea pig 105 testicle was 50 to 100 times more effective than testicular homogenates in inducing aspermatogenesis in guinea pigs. They apparently arrived at this conclusion by varying the dose of TCPM which was found to be effective in very small doses in contrast to the homogenates. Such dose variation was not attempted in the present experiment and, therefore, the actual potency of rat testicular TCPM has not been determined. Inoculation of testicular protein residue into male rats induced the formation of PCA-reactive antibody but Li. testicular lesions were not seen in any of these rats. 6 This was suggestive that, as in guinea pigs (Katsh and‘ Katsh, 1961), the aspermatogenic factor is an entity differ— ent from antibody-inducing antigens. Testicular lesions of varying severity were induced in male rats by intradermal inoculations of either testicu— lar homogenates or TCPM in complete adjuvant. The earliest lesions in rats inoculated with TCPM in complete adjuvant were observed 35 days after inoculation. In guinea pigs, mild testicular lesions were demonstrable as early as 7 days (Boughton and Spector, 1963). Freund gt gt. (1955) described guinea pig testicles with lesions 14 to 21 days following inoculation of TCPM in complete adjuvant. Severe lesions in which most or all of the germinal epithelium was affected developed after an incubation period of 4 to 6 months (Bishop gt al., 1961). 106 Severe testicular lesions in TCPM—inoculated rats took over 4 months to develop. Unlike guinea pigs which were injected only once, the rats in the present experiment were given repeated injections at 4—week intervals. In the majority of rats, 4—5 injections had to be given before severe damage was noted. Not all rats inoculated with _ ‘3 fink—J either TCPM or testicular homogenate developed testicular lesions. Some repeatedly inoculated rats had testicles com— parable to those of noninoculated controls and others, receiving fewer injections, had severe type-3 or type-4 gfl damage. Similar variations in responsiveness were recognized in association with autoallergic aspermatogenesis in guinea pigs (Bishop and Carlson, 1965). These authors found that sensitivity and responsiveness varied from one breed of .guinea pigs to another and that it increased with the degree of inbreeding. The same phenomenon was reported in other autoimmune diseases (Dumonde, 1966). Freund gt gt. (1954) induced varying degrees of testicu- lar damage in 19 of 24 rats inoculated with testicular homogenate in complete adjuvant. The authors concluded that, although severe lesions were induced, the rat was much less sensitive than the guinea pig. Bishop gt gt. (1961) stated that laboratory animals other than guinea pigs would not be susceptible to autoallergic aspermatogenesis. In comparing results encountered in this experiment with those reported for guinea pigs, it appeared that rats 107 were more difficult to sensitize. Long incubation periods and repeated inoculations were needed to induce aspermato- genesis. The proportion of rats developing severe testicu— lar lesions was not as high as that reported in guinea pigs. In the final analysis, however, severe lesions com- parable to those occurring in guinea pigs were induced by 31 either particulate homogenates or a purified extract. E Mild testicular damage was also induced in rats by TCPM in complete adjuvant inoculated intramuscularly. This does not conform with the hypothesis that skin sensitization i} has an indispensable role in the development of autoallergic W aspermatogenesis (Waksman, 1959). Testicular lesions were not as extensive nor as severe as in rats inoculated intra- dermally (Table 5). Testicular lesions comparable in severity and cytopathy to those produced by the intradermal route were induced in guinea pigs by intramuscular injection of testicular antigens in complete Freund‘s adjuvant (Bishop and Carlson, 1965). Complete adjuvant was found necessary for production of testicular lesions by inoculation of either testicular homogenates or TCPM. Testicular homogenate or TCPM incor- porated in incomplete adjuvant induced minimal lesions in the testicles of only 1 rat. Early in the history of autoallergic aspermatogenesis Freund gt gt. (1953) stressed the importance of complete adju— vant for the induction of immune responses. Similarly 108 complete adjuvant was considered necessary for induction of other autoallergic diseases (Dumonde, 1966). It was evident in the present experiment that the activity of the complete adjuvant was related to the presence of the mycobacterium. An antigen may be altered by the adjuvant in such a way as to increase its foreignness and thereby its capacity to induce responses against itself and in turn against normal ‘- self antigens (Dameshek, 1963). This may not be the case with testicular antigens because of their early sequestration which renders them foreign to the immune mechanism from the k: start. Moreover it was demonstrated (Bishop gt gt., 1961) that the closer the antigenic determinants in the inoculated antigen to those of the animals own tissue the more.effec— tive the result would be. One may speculate that the adjuvant functions by influencing certain immunologically competent cells. Such an influence-may render these cells specifically sensitized to the inoculated antigen and ultimately more capable of self agression. These cells may be adequately described as specifically conditioned competent cells. As may be seen, such a hypothesis leans to a great extent on the theory of altered clones first introduced by Burnet (1959) and since adopted by many workers to explain various auto— immune phenomena (Dameshek, 1963). Lesions resulting from sensitization with testicular antigens were confined to the testicle. In affected testicles, lesions were almost entirely confined to the various layers of germinal epithelium. The fact that other cellular elements 109 like Sertoli and Leydig cells were not affected may-indicate the specificity of the immunologic response. Brain lesions like those reported in guinea pigs by Freund gt gt. (1955), Waksman (1959), and Bishop and Carlson (1965) were not encountered in rats. Presence of multinucleate cells within the regions of r_ secondary and primary spermatocytes was of special interest. .1 As mentioned earlier, they were suggestive of derangement of spermatogenesis, probably defective division. Two to 9 g nuclei were present in some of these cells. This may indi— a; cate that the cell nuclei were dividing without the accompany— ing cytoplasmic division. This seems to suggest suppression of spermatogenesis at least in the initial phase of the immunologic process which was to culminate in necrosis. Jacobson (1964) described changes in the testicle following the administration of anti—leukemic drugs like amethopterin and methotrexate. Cell division was impaired resulting in severe clumping of the chromosomes and formation of multinucleate giant cells. The two drugs are folic acid antagonists which probably act by delaying a step between metaphase and anaphase. It-may not be groundless to speculate on the presence and participation of factors having similar effects in the mediation of autoallergic aspermatogenesis in the rat. Histamine Inoculation Histamine, added to the inoculum of TCPM in complete adjuvant, did not seem to modify the development of testicular 110 lesions in any manner. The results were comparable to those elicited by inoculation of TCPM alone in complete adjuvant. Histamine is a powerful vasodepressant produced by decarboxyl- ation of histidine, a process that occurs in many tissues, including mast cells (Dagliesh, 1955). Histamine release by injured tissues was accepted as the mediator of vascular permeability at least in the first hour after injury (Spector and Willoughby, 1964). Release of histamine along with a certain slowly reacting substance during anaphylactic re- actions has been demonstrated (Chakravarty and Uvnas, 1960; Li Broklehurst, 1960). Jancso (1947) found that histamine activated the retic- ulo—endothelial system. He demonstrated that histamine introduced in the skin or liberated therein produced changes in small veins such that intravenously injected particles became localized in the skin at the site of histamine injec— tion. However, histamine is not known to take part in reactions of delayed hypersensitivity although these may be accompanied by immediate hypersensitivity to the same antigen (Gell and Benacerraf, 1961). In rats, histamine was found to have very little or no role in mediation of vascular changes accompanying hypersen— sitive reactions. Serotonin was thought to be the pharma- cologically active substance in this species (Spector and Willoughby, 1964). Serotonin is abundant in brain tissue, blood platelets and mast cells. Certain drugs that produced lll marked alterations in the central nervous system were found. to be potent serotonin antagonists tg ytttg. Histamine and serotonin were used in this experiment in an endeavor to study any role that they may have in modi— fying responses to the inoculated antigen. It was antici- pated that inoculation of these substances might heighten the local reaction and, in turn, the autoallergic condition. . 7‘3 It was found, however, that neither histamine nor serotonin had a measurable effect on the development of autoallergic testicular lesions. In the site of skin inocu— E; lation lymphocytes and other mononuclear cells appeared to be more numerous when histamine was used. Histamine inoculated intratesticularly had a definite and pronounced effect. There was extensive necrosis in- volving large numbers of tubules at the site of histamine deposition- Whether necrosis was the result of direct toxicity of histamine to germinal epithelium or partially due to ischemia resulting from collapse of blood vessels could not be determined. It seems more likely that necrosis was attributable to a great extent to direct effect of histamine since damage was more or less confined to the area where histamine was injected. Testicular changes in tubules outside the area of necrosis were similar to types 3 and 4 damage seen in-other aspermatogenic testicles. These changes along with milder lesions seen in the contra- lateral noninoculated testicle were considered to be lesions of autoallergy. 112 The severe types of damage demonstrated in the inocu— lated testicle may have been due to superimposed histamine injury and the accompanying cellular infiltration. The rats were inoculated with TCPM in complete adjuvant approxi— mately 40 days prior to intratesticular injection. It is speculated that sensitized cells were among those present in the inflammatory reaction and, being in the vicinity of antigen containing cells, were able to exert their effect locally. The enhancing effect of similar triggering mech— anisms on percipitation of lesions in the target organ was stressed by Dumonde (1966). Heparin, on the other hand, seems to have exerted a suppressive effect on the inflammatory reaction at the site of inoculation. Infiltration with lymphocytes and other mononuclear cells was less pronounced than in rats inoculated with TCPM and complete adjuvant alone. This relative defi~ ciency in mononuclear cells was apparently associated with a general lack of severe testicular lesions. Production of heparin by mast cells and its general function as an anticoagulant has been established (Asboe— Hansen and Glick, 1955). Heparin was also found to exert an inhibitory action on various enzyme systems involved in pro- tein synthesis (Spensley and Rogers, 1954; Riley gt gt., 1955). Halpern gt gt. (1965) found that heparin exerted a protective effect in experimental immune nephritis. The retarding effect of heparin on the autoallergic response was noted in this experiment although the mechanism 113 of such retardation was not clear. The inoculated antigen was a protein-free extract and the mycobacterial enzymes were assumed to be inactive. Enzymes available for inhibition at the inoculation site were those in various cellular elements in the skin and inflammatory exudate. If such in— hibition can take place in enzyme systems of cells involved in the immune mechanism, sensitization of such cells or their reactivity may be interfered with. Like heparin, hyaluronic acid is an aminated mucopoly- saccharide but it has a simpler structure (Dorfman, 1955; Whistler, 1957).- The intercellular cement which binds paren- chymal cells together was found to be.a gel—like material containing highly polymerized hyaluronic acid. When hyalur— onic acid is depolymerized by hyaluronidase or when production of polymerized hyaluronic acid is impaired, the.intercellular cement may break allowing spreading of intracellular material (Duran—Renals, 1950). Proliferative lesions of rheumatic arthritis were associated with partial depolymerization or incomplete poly- merization of hyaluronic acid, which was corrected by the administration of corticosteroids (Dorfman and Schiller, 1958). Hyaluronic acid was also associated withlretardation of the skin inflammatory reaction and the subsequent testi- cular lesions. It may be speculated that hyaluronic acid may replenish the polymerized form thus strengthening the intercellular cement and thus retard spreading of the antigen or other sensitizing material. "(YET "_ T U pin-1 . '.3 It)”: r 114 An oversimplified speculation depends on an indirect effect by stimulation of corticosteroid production which ultimately suppresses proliferative and immunologic responses. In both instances where either heparin or hyaluronic acid was injected, a relatively mild infiltration of the inoculation site with lymphocytes was noted. In the same rats subsequent testicular lesions were limited to slight f: degeneration. It must not be inferred that development of . testicular lesions was absolutely dependent on the abundance of lymphocytes in the inoculation site. If this were true, i; it would be very difficult to explain the lack of testicular lesions, in spite of pronounced lymphocytic infiltration, at the site of inoculation of Leptospira canicola with TCPM and incomplete adjuvant. It is speculated that the mere attraction of lympho—\ cytes to the inoculation site where they can encounter the antigen.is not the important factor which determines the development of the immune response. It seems that for such responses to develop, a more subtle and specific sensitiza- tion of immunologically competent cells to the antigen must occur. Such a sensitizing influence seems to be an attribute of the complete adjuvant, or more specifically, the myco- bacterium contained in it. Mediation of Autoallergic Aspermatogenesis_ In view of the method of induction, type of lesions and failure of passive transfer of the disease by serum, 115 autoallergic aspermatogenesis was regarded to be the product of hypersensitivity of the delayed type (Freund gt gt., 1955; Waksman, 1950; Bishop gt gt., 1961). Lack of correla— tion between serum antibody and testicular damage has led investigators to believe that the condition was mediated by cells which were probably lymphocytes (Bishop and Carlson, 1965). ..1__” Similar mediation was suggested for various other autoimmune diseases in which delayed hypersensitivity re— actions to the antigens were believed to occur (Dumonde, EJ- 1966). In 2 of these systems, namely thyroiditis (Sclare~ and Taylor, 1961) and autoallergic encephalomyelitis (Pater— son, 1960), the diseases were successfully transferred to unimmunized-hosts by lymphoid cells. The role of lymphocytes in the mediation of pure de—p layed hypersensitivity to tuberculin was established by Najarian and_Feldman (1961). These authors transferred skin reactivity to tuberculin by labeled lymphocytes and subse— quently demonstrated their presence in the skin reaction site. Bishop gt gt. (1961) pointed out the many similarities between the homograft rejection phenomenon and autoallergic aspermatogenesis. The main difference between the 2 condi- tions is that in homograft rejection a skin graft essentially foreign to the host-is rejected while in aspermatogenesis the animal's own testicle is damaged. The role of lymphoid cells and particularly the small lymphocyte in the homograft 116 rejection phenomenon was illustrated by Gowans gt _t. (1963). Granger and Weiser (1964) demonstrated the ta ttttg destruc— tion of homograft target cells by immune macrophages. Many attempts were made to associate delayed hyperw sensitivity with organ-specific lesions (Waksman, 1959, 1962). Such correlation was not always absolute. Delayed hyper— sensitivity to thyroid or optic lens was observed to occur without recognizable organ lesions while aspermatogenesis could occur without demonstrable delayed hypersensitivity (Dumonde, 1966). The presence of lymphoid cell infiltrations in affected organs was considered to be indicative of a delayed hyper- sensitivity mechanism (Waksman, 1957, 1959, 1962). In the experience of other investigators (Freund gt gt., 1953, 1955; Bishop gt gt., 196; Bishop and Carlson, 1965) such lymphocytic infiltrations were not seen. In the various models of the present experiment the lesions of aspermatogenesis were remarkably acellular. The only lesions resembling those described for delayed hyper- sensitivity were noticed in the skin at the site of inocu- lation. Lymphocytic infiltrations of varying extent were demonstrated within and below the muscular layer' of the dermis. Paterson (1966) stated that even the ability to trans- fer an autoallergic disease with lymphoid cells should not exclude the role of free antibody molecules in the mechanism of tissue damage. High titers of serum antibody demonstrable 117 by conventional methods did not correlate with testicular damage in autoallergic aspermatogenesis (Freundgt gt., 1955; Bishop gt gt., 1961). On the other hand Chutna and Rychlikova (196M) demonstrated cytotoxic properties of sera from aspermatogenic guinea pigs against testicular epithelium in tissue culture. They found that testicular damage was correlated more closely with cytotoxicity of the sera than with delayed skin reactivity. A cytotoxic factor in Hashimoto serum was also demon— strated against human thyroid cells in tissue culture by Pulvertaft __t__ 3;. (1959) and Halberge (1964).. Pulvertaft (196“) demonstrated the activity of lymphocytes against thyroid cells in tissue culture. Kucharski and Favour (196U) found the factor for passive transfer of the delayed sensi— tivity to tuberculin in the supernatant fluid from spleen cell washings. Heightened immunity to skin homografts in rabbits was passively transferred to normal rabbits by lymphoid cell RNA (Mannick and Dahl, 196“). In a study of graft~versus—host reactions, Batchelor and Howard (1965) found that immune serum against host cells acted synergistically with parental spleen cells in poten— tiating graft-versus—host reactions in F1 hybrid mice. It may, therefore, be advisable to draw a line distinguishing between cytotoxic factors in sera and conventional circulat- ing antibody when speaking about activities of sera in connection with certain diseases. There seems to be enough evidence that cytotoxic factors may be produced in lymphoid 118 tissue. This is in contradistinction to conventional anti- body which is probably produced by plasmocytas. In various diseases in which lymphocytic infiltrations were demonstrated in association with organ—specific lesions, the connection between the 2 manifestations can easily be seen. Lesions of autoallergic aspermatogenesis in guinea pigs and rats were noted for their lack of cellular infil- trations. Is it possible that lymphocytes, at a distance from the testicle, elaborate such a factor which does its damage when the blood containing it ultimately reaches the testicles? If this is the case, such a factor must be highly potent to withstand the great dilution to which it must be subjected in the circulatory system. It must also be endowed with a high order of specificity to bypass various organs and exert its effect only on target cells. Further- more it must be of relatively small molecular size to facilitate its passage through cell membranes. Alternatively it must have the property of active transport across cell membranes, for which function a special configuration may be needed. What-single entity possesses all these attributes, and needs for its elaboration a highly specific antigenic determinant? In all autoallergic diseases it was found that the closer the configuration of the antigen determinant to that of the animal's own antigen, the more effective the sensitization will be (Dumonde, 1966). Enzymes are known v-r.l' . ‘ 3 I. 1.. _’?5~o \ 119 to possess all these properties (White gt gt., 1959). Enzymes possess high specificity, function in infinitesimal concentrations and are involved in active transport across cell‘membranes. It is, therefore, speculated that the factor involved in cytotoxicity of-the testicle has several of the attri— butes of an enzyme and may possibly be one. If this is true, then a ready explanation for the retarding effects of heparin will be found because of its demonstrated properties in enzyme inhibition. Transfer of Autoallergic Aspermatogenesis Transfer bytLymphoid Cell Inoculation _ Testicular lesions varying in severity from types 1 to 4 were demonstrated in 6 of 10 F1 rats inoculated with lymphoid.cells from TCPM—sensitized parents. As in rats actively sensitized with testicular antigens, testicular damage was confined to the germinal epithelium. In some testicles occasional lymphocytes.and other mononuclear cells were seen in the intertubular spaces, mainly in the vicinity of blood vessels. Lymbhocytic infiltration beyond these few cells was not seen in any of the testicles. Injected lymphoid cells were not marked and thus there was no way of tracing them in recipient rats. Their role in precipitating testicular damage may be assumed since they constituted the only-inoculum. Under the circumstances it may be difficult to determine whether the injected cells 120 were responsible directly for the lesions or indirectly by transferring their sensitivity to host cells. Certain features support the assumption that the inocu— lated parental cells were directly responsible for the damage: 1. The host Fl rats were grafted immediately after birth with parental strain lymphoid cells in an attempt to render them tolerant to similar injections on future dates. Effectiveness of such treatment in inducing tolerance was reported by Medaware (1961) and Brent and Cowland (1961, 1963). There is, therefore, no reason to assume that parental cells subsequently inocu- lated were unable to survive because of host intolerance. Type 1 testicular lesions were demonstrated in l rat, 8 days after inoculation of parental lymphoid cells. This short period was highly suggestive of passive trans— fer of the immunologic response (Paterson, 1960). Wasting, irregular hair loss, splenomegaly and enlarge— ment of thymuses were noticed in inoculated Fl rats. These lesions were described for runt disease or graft- versus—host reactions. Billingham gt gt. (1953), Trentin (1958) and Oliner gt _t. (1961) demonstrated that the newborn, immunologically immature mouse developed a wasting disease after transplantation with immunologically competent cells. The cells were obtained from a genet- ically different animal whose tissues were tolerated by the host. Similar findings in rats were reported by Billingham (1962). 121 Proliferation of reticulo-endothelial cells was seen in rats inoculated with parental lymphoid cells. This may suggest the development of‘a graft—versus—host reaction in these rats. Burnet (1963) has stressed the importance of reticula— endothelial and thymocytic proliferation in the thymus, in the development of autoimmune disease. The relation between such proliferation and the occurrence of runt disease in mice inoculated with parental cells was demonstrated by the same author (Burnet and Holmes, 1964). Congdon (1961) ' ki- stated that proliferation of-immunologically competent cells, Whether reticulum, lymphoid or plasmocytic seemed to take place in lymphoid apparatus, including the spleen. Lesions indicative of a graft—versus—host reaction. observed in this model suggested that the grafted cells did survive and, being intolerant of the host, reacted against it. In addition to being intolerant of the host, these cells were specifically sensitized with testicular antigen. It is speculated that testicular damage demon— strated in this model may be regarded as part of a graft— versus—host reaction or specific runt disease. Transfer by Parabiosis In this model, F donor and recipient rats were litter l mates. In addition, recipient rats were neonatally inocu— lated with parental lymphoid cell suspensions. 122 Enlargement of spleens, lymph nodes and thymuses, particularly during the first 2 weeks after surgery, was noted in inoculated and control donors as well as recipients. This was suggestive of the development of immunologic re- sponses probably against parabiotic partners. This mani- festation in addition to the observed weight and hair loss may be analogous to those seen in association with lymphoid cell inoculation in the previous model. Lymphoid organ enlargement seemed to decrease with time and in parabiotic rats killed 37 or 60 days after surgery, the spleens, lymph nodes and thymuses were almost normal in size. This may be due to gradual development of tolerant states in partners to each other. Induction of specific tolerance in adult rats by means of parabiosis was reported by Nakic gt gt. (1962). In parabiotic rats, participation of lymphoid cells in transferring the immunologic response from donor to recipient can only be inferential. Since all tissues, blood. and peritoneal fluids of one rat were practically available to the other, there was no way of determining whether . lymphoid cells, other cells or serum factors were the mediators of the transfer. On the other hand, parabiosis was employed to transfer immune status which could not be transferred by serum alone. Lipton and Freund (1953) and Paterson (1966) successfully transferred experimental autoallergic encephalomyelitis by , (“'43 w I! ,.' 123 means of parabiosis. Activity of sensitized lymphoid cells against nerve tissue tg ttttg was demonstrated (Paterson, 1966). Type 1 testicular lesions were seen in l recipient rat 10 days after surgery while the sensitized donor had type 2 degeneration. Active sensitization of the recipient rat from residual antigen in the donors skin cannot be al— together excluded. In this rat the short time required after surgery for testicular lesions to develop was sug— gestive of a passive transfer. Lipton and Freund (1953) stipulated that lesions develOping in recipients within 10 days of parabiosis may be considered to be the result of passive rather than active transfer. More pronounced testicular lesions were demonstrated in 3 other TCPM—sensitized donors and 2 of their respective recipient partners, killed 27 or more days after parabiosis. In l.pair the recipient had type 4 and the donor type 3 testicular damage. This was interesting since the cyto— toxic factor was assumed to come from the sensitized donor rat. This may reflect on the great variation in responsive- ness reported even among litter mates (Bishop and Carlson, 1965). Attempts to produce testicular lesions by inoculating rats with immunized male and female rabbits' sera were not successful. Male and female rabbits' sera were equally ineffective in precipitating testicular damage in spite of 12“ of their strong to moderate PCA reactivity. The ineffec— tiveness of conventional serum antibodies relative to aspermatogenesis and other autoallergic diseases has already been mentioned. The rabbits were inoculated with rat testicular homogenates in complete adjuvant. Testicular lesions were not seen in any of the males and hence lack of anti—testicle ‘I cytotoxic factors in the serum may be assumed. This sug— gests the high order of species specificity of the rat’s aspermatogenic factor. J VI. SUMMARY AND CONCLUSIONS Summary Autoallergic aspermatogenesis in albino rats was studied in 4 experimental models. In this study 178 males and 63 females were used. Fertility of male and female rats was assessed by a comparing numbers and sizes of litters produced and litter Bi frequency before and after inoculation. EXperimental animals were killed at the end of each experiment and their organs examined for gross and microscopic lesions. Lesions in affected testicles were graded as types 1, 2, 3, or u according to the severity and-extent of testicular damage. Sera of inoculated rats were tested for passive cutane— ous anaphylaxis (PCA) reactivity in normal young male' and female albino rats. Study of the pathogenesis of autoallergic aspermato— genesis was attempted in models employing lymphoid cells, parabiosis or immune heterologous sera. Autologous or isologous testicular homogenates incor— porated in complete Freund's adjuvant induced aspermatogenesis of varying ,severity (types 1—4) in unilaterally orchiectomized and nonorchiectomized rats. The fertility of these rats was suppressed to varying extents with an average of “0% reduction in litters sired. 125 126 Infertility was more closely associated with testicular damage than with PCA reactivity of sera. Autologous testicle was more effective in inducing testicular damage and infertil— ity than isologous testicle. Temporary periods of infertility were induced in female rats inoculated with rat testicular homogenates in ! complete adjuvant. #1 A lyophylizable material was prepared from rat testicles A and epididymal spermatozoa by acid extraction, ammonium sulphate precipitation and trichloracetic acid purification E; (TCPM). This TCPM fraction incorporated in complete adju- vant.and inoculated intradermally into male rats.was highly effective in inducing testicular damage. The protein residue precipitated-by trichloracetic acid was ineffective. Histamine or serotonin incorporated with TCPM in com- plete adjuvant heightened the skin reaction but had no influence on the development of testicular lesions. Heparin and to a lesSer degree, hyaluronic acid had suppressive effects on both the skin reaction and the sub— sequent development of testicular lesions. Killed Leptogpira canicola inoculated with TCPM in incomplete adjuvant, failed to induce aspermatogenesis. Aspermatogenesis was transferred by lymphoid cells and- by parabiosis. Recipient rats in this model were neonatally grafted with parental lymphoid cells in order to render them tolerant to subsequent lymphoid cell inoculation or parabiosis. 127 Type 1 testicular damage was induced in l rat 8 days after inoculation with sensitized lymphoid cells. Similar lesions were noted in 1 parabiotic recipient 10 days after it was joined to a sensitized donor. More pronounced tes— ticular lesions up to type A were seen in 6 of 10 lymphoid- cell-inoculated and 4 of 7 parabiotic recipient rats killed “ approximately u weeks or more after treatment. fa Splenomegaly, enlargement of thymus and lymph nodes, 3 and weight and hair loss were noted in lymphoid-cell— E inoculated and parabiotic rats. These were suggestive of Li. graft-versus-host reactions. Sera of male and female rabbits immunized with rat testicular homogenates in complete adjuvant failed to sup— press the fertility of either male or female rats. Testicular damage was not demonstrated in male rats inoculated with such sera. Sera of actively immunized-male rats-inoculated- into young females did not influence their conception rate in any measurable manner. Conclusions l. Rats are susceptible to autoallergic aspermatogenesi8~ but may be less sensitive than guinea pigs. 2. Infertility and testicular damage may be produced by inoculating male rats with autologous or isologous testicle. 3. Autologous testicle was more effective than isologous testicle in inducing infertility and testicular damage. 10. 11. 128 Infertility in the male rat may be a result of testicu— lar damage rather than serum antibody. Female infertility could not be induced by isologous or heterologous immune sera alone. It is speculated that local uterine anaphylaxis in combination with serum antibody may be associated with female infertility. The aspermatogenic factor in rat testicle may be purified and lyophylized. Complete adjuvant of the Freund's type was necessary for sensitization of_rats with TCPM or with testicular homogenates. Aspermatogenesis in the rat was transferred passively by lymphoid cells and parabiosis. Aspermatogenesis in rats may be associated with graft— versus—host reactions. 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Yagi, Y., and Pressman, D. (1958) Multiplicity of the com— ponent of rat kidney antigen responsible for the local— ization of rat kidney antibodies. J. Immunol., 81:7-13. VITA The author was born on August 15, 1926 in Omdurman, Sudan, where he received his elementary, intermediate and secondary education. He graduated.from the University College of Khartoum With a diploma in Veterinary medicine in December, 1950. Between 1951—1958, the author worked with the Depart— ment of Veterinary services which eventually became the Ministry of Animal Resources in the Sudan. He occupied the positions of field officer, district, and province Veterinary officer. During 1957, he attended the school of Veterinary Medicine, University of Edinburgh for graduate study and received a diploma in Tropical Veterinary Medicine. In 1958 the author_joined the faculty of Veterinary Science, University onghartoum as senior lecturer in internal medicine. Since September, 1962, he has been engaged in graduate training at Michigan State University. He received a Master's degree for work done in the Depart- ment of Surgery and Medicine in 196“. Salah Eldin Imbabi l"Alllllllllm