SUMMARYExperimental autoimmune orchitis (EAO) was produced in C3H/He mice with as high as 100% incidence by two or ihrce s,c. injections of I x 10' viable syngeneic lesticular germ ceils (TC) without resorting lo adjuvants. Bordetella pertu.\si.s vaccine, or other immunological manipukuions. On day 40 after the lirst injection ofTC. the lesions induced were characterized by interstitial infiltration ol' inflammatory cells and severe hypospermatogenesis in the testis with resulting whole organ atrophy and. in contrast, by a complete lack ofepididymitis. Immunological studies revealed thai ihis form of immunization caused both delayed-typc hypersensitivity and humoral antihody responses to syngeneic TC. We compared the susceptibilities lo the induction of this lype of EAO among six different strains ofinbred mice comprising A'J.AKR. BALB/c. C3H/Hc.C57BL/6 and DBA/2 mice. All strains except for DBA/2 mice developed lesions of EAO to a greater or lesser extent, and severe disease was induced with high frequency in two strains, C3H/'He and A'J. As this murine model of EAO can be induced withoui the use of Freund's complete adjuvant and B. pertus.u.t vaccine, it is simply "autoimmune* in nature and may provide new ways for further investigation into the immunological mechanisms which regulate deleterious autoimmune reactions to germ cell antigens leading to the male infertility.

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by two or three subcutaneous injections of viable syngeneic testicular germ cells, without the use of any adjuvants or immunopotentiators and that the disease induced was characterized by a complete lack of epididymitis despite a definite orchitis with hypospermatogenesis. In this report, immunohistochemical characterization of immune cells in the fully-developed orchitic lesion was carried out using monoclonal antibodies and immunoperoxidase staining. Thy-1.2+ cells, Mac-1+ cells, B220+ cells and cytoplasmic Ig-bearing cells in the lesion were estimated to be approximately 30, 15, 20 and 30% of all inflammatory cells, respectively. Major phenotype of T cells in the lesion was CD4+ (approximately 85%) with the remainder (approximately 15%) being CD8+. The percentages of cytoplasmic IgG-, IgA- and IgM-bearing cells were estimated as approximately 35, 60 and 5% of all cytoplasmic Ig-bearing cells, respectively. Deposits of immunoglobulins and third component of complement were identified on the basement membrane of the seminiferous tubules, interstitium between the tubules, vessel endothelium and degenerated germ cells in the lesion. Circulating antibodies directed against the acrosomal portion of germ cells were detected in IgG and IgM classes but not in IgA class. Inflammatory cells (including macrophages, B cells and, probably, activated T cells) in the lesion were Ia+, but Leydig cells, Sertoli cells and germ cells did not stain for Ia at all.

Sequential changes in the distribution of lymphocyte subpopulations of the thymus and spleen in BALB/c mice (male, 8-weeks old) during 2 weeks after a single i.p, injection of cyclophosphamide (CY, 200 mg kg body weight) were studied mainly through the use of CD4, CD8 and CD3-E markers together with single-or two-color flow cytofluorometry. In the thymus on Day 2 after CY treatment, a marked decrease in the number and proportion of PNAh', CD3-and CD4+CD8+ double positive (DP) subpopulation was observed in parallel with a marked reduction in the thymus weight, cortical area and total thymocyte number. This phenomenon might be associated with the decrease in the percentage of thymocytes at the S phase of the cell cycle on Day 1 and 2 after CY treatment, owing to the depletion of the rapidly dividing cells in DP subset. There was a significant reduction in the number and proportion in CD4+ single positive(SP) subset on Day 7. The cell number of CD8+SP subset continued to decrease during Day 7 to Day 14. This contrasted with the behaviors of DP, CD4+SP and CD4-CD8-double negative (DN) subsets in which a considerable recovery was attained by Day 14. The spleen from CY-treated mice showed a marked decrease in the DN subset and surface immunoglobulin-positive cells, perhaps B lymphocytes, in both the percentage and the absolute cell number on Days 2 and 7, which paralleled the marked reduction in its weight and total cell number. The absolute cell numbers of CD4+SP and CD8+SP subsets in the spleen were also reduced on Days 2 and 7. The reduction of the CD4+SP/CD8+SP ratio was found in the thymocytes on Days 2 and 7 but not in spleen cells. Our results suggest that the principal target cell population of CY is the PNAh', CD3-and DP immature cortical thymocytes as well as splenic B cells. The sustained decrease in the number of CD8+SP thymocytes after CY treatment might be in part relevant to the potentiating effect of CY pretreatment on immune responses.

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