Serum autoantibodies in mice immunized with syngeneic testicular germ cells alone.

PROBLEM: The aim of this study was to identify the characteristics of serum autoantibodies in experimental autoimmune orchitis (EAO) induced by immunization with syngeneic testicular germ cells (TGC) alone in mice. METHOD OF STUDY: The serum autoantibodies were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical staining. RESULTS: Serum autoantibody titers against TGC and epididymal spermatozoa (ES) gradually increased with the development of EAO. At the initial stage of EAO, serum autoantibodies reacted with only a few antigens of TGC and ES, while in the later stage, the reactions with various antigens of TGC and ES occurred. However, the autoantibodies were specific to the testis and epididymis but not to other organs. CONCLUSION: These results indicate that titers and kinds of autoantibodies in TGC-immunized mice increase with EAO development and they are specific to TGC and ES.

Lymphangiogenesis in chronic inflammation in the testis.

Lymphangiogenesis occurs in various organs under inflammatory conditions. Recently, it was demonstrated that activated macrophages play an important role in the process of lymphangiogenesis. However, lymphangiogenesis during testicular inflammation has not yet been studied. Here, we investigated lymphangiogenesis in experimental autoimmune orchitis, a immunologic male infertility model, in mice. Histological changes were observed using immunohistochemical staining with the monoclonal antibodies against F4/80 (mature macrophage marker), lymph vessel endothelium HA-receptor 1 (LYVE-1) (lymphatic endothelial cells marker) and CD31 (endothelial cells marker). The expression of angiogenesis and lymphangiogenesis factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D and TNF-α, which are secreted by activated macrophages, were examined using real-time RT-PCR. The results showed that lymphangiogenesis occurred along the undersurface of the tunica albuginea but not into the interstitium proper between the seminiferous tubules (STs) during the orchitis. It was noted that some F4/80-positive macrophages expressed LYVE-1 at the undersurface of the tunica albuginea and also in the testicular interstitium proper. RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-D and TNF-α were significantly increased but that of VEGF-C remained unchanged in the inflammatory testes. This study suggests that testicular macrophages are involved in the specific lymphangiogenesis in the chronic inflammation.

The effects of adjuvants on autoimmune responses against testicular antigens in mice.

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund's adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.

Involvement of Fas/Fas-L and Bax/Bcl-2 systems in germ cell death following immunization with syngeneic testicular germ cells in mice.

Experimental autoimmune orchitis (EAO) is characterized by T cell-dependent lymphocytic inflammation and seminiferous tubule damage, which can result in the death of germ cells. The aim of the present study is to investigate the roles of the Fas/Fas-L and Bax/Bcl-2 systems in the death of germ cells in mice with EAO that is induced by immunization with syngeneic testicular germ cells (TGC). The results using real-time reverse transcription-polymerase chain reaction and immunostaining show that many terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining germ cells were present in seminiferous tubules during the active inflammation stage, and these cells were persistently observed in the seminiferous epithelium until the postactive inflammation stage. Intratesticular mRNA expression levels of both Fas and Bax were increased during the active inflammation stage and were dramatically decreased during the post-active inflammation stage. In contrast, the intratesticular mRNA expression levels of both Fas-L and Bcl-2 did not show significant changes during the active inflammation stage but showed extreme increases during the post-active inflammation stage. Immunohistochemically, some Fas- and Bax-positive germ cells were detected during the active inflammation stage, but these were hardly found during the post-active inflammation stage. In contrast, some Fas-L- and Bcl-2-positive germ cells were found during the active inflammation stage, and many of these were also observed during the post-active inflammation stage. These results indicate that germ cell death during TGC-induced EAO is mediated by the Fas/Fas-L and Bax/Bcl-2 systems during the active inflammation stage but not during the post-active inflammation stage.

Postinflammation stage of autoimmune orchitis induced by immunization with syngeneic testicular germ cells alone in mice.

We previously established an immunological infertility model, experimental autoimmune orchitis (EAO), which can be induced by two subcutaneous injections of viable syngeneic testicular germ cells on days 0 and 14 in mice without using any adjuvant. In this EAO model, CD4+ T-cell-dependent lymphocytic infiltration and immune deposits were found with spermatogenic disturbance on day 120. However, the late stage of EAO (= postactive inflammation stage on day 365) has not yet been investigated. Therefore, we investigated the histopathological characteristics of the late stage. The results revealed that the lymphocytic infiltration finally resolved; however, the seminiferous epithelium persistently showed maturation arrest and the Sertoli cell-only feature. In the seminiferous tubules showing maturation arrest, both proliferation and apoptosis of germ cells had occurred simultaneously. It was also noted that there were deposits of immunoglobulin G and the third component of complement on the thickened basement membrane of seminiferous tubules in the late stage of EAO. These results indicate that histopathology after active inflammation in EAO comprises persistent damage to the seminiferous epithelium and may resemble the histopathology of "idiopathic disturbance of spermatogenesis" in man.

The origin of lymphatic capillaries in murine testes.

It is known that the seminiferous tubules are bathed in a sea of lymph in mice, which are commonly used in reproductive and immunological studies. Although testicular lymphatic vessels arising from the tunica albuginea can be macroscopically observed in mice, the exact distribution of the lymphatic capillaries remains unclear. In the present study, we investigated the distribution of lymphatic capillaries in normal testes by immunohistochemical staining with monoclonal antibodies against lymph vessel endothelium HA-receptor 1 (LYVE-1) and a platelet endothelial cell adhesion molecule (CD31). Moreover, normal lymphocytes were locally injected into the testes of recipient mice, and their migration was investigated with the use of LYVE-1 and CD31. The results showed that lymphatic capillaries were in and just beneath the tunica albuginea but not in the interstitium between the seminiferous tubules. It was also noted that these were abundant in the thickened tunica albuginea adjacent to the epididymis, but they were scarce in the thin tunica albuginea opposite the epididymis. When normal lymphocytes were locally injected into testes, the injected lymphocytes migrated between the seminiferous tubules and then drained into the lymphatic vessels in the tunica albuginea. These results suggest that tissue fluid might drain from lymphatic capillaries that arise just beneath the tunica albuginea.

Experimental model of autoimmune orchitis with abdominal placement of donor's testes, epididymides, and vasa deferentia in recipient mice.

Haploid germ cells (spermatids and spermatozoa) develop in the testis after immune tolerance has been established. Therefore, they contain various autoimmunogenic antigens, but the testis is known to be an immunologically privileged organ. In particular, the blood-testis barrier formed by Sertoli cells protects autoimmunogenic haploid germ cells from attack by the autoimmune system. Experimental autoimmune orchitis (EAO), a breakdown of the testicular immune privilege leading to immunological male infertility, has been ordinarily induced in mice by immunization twice with testicular antigens+complete Freund's adjuvant (CFA)+Bordetella pertussis (BP). We previously found that two subcutaneous injections of viable syngeneic testicular germ cells induced murine EAO without the use of CFA+BP. In both EAO models, the lesions are characterized by spermatogenic disturbance with lymphocytic inflammation, and a second immunization with testicular antigens is critical for the disease induction. In the present study, we found that only one placement of a syngeneic donor's testes, epididymides and vasa deferentia (TEV) into the abdominal cavity or subcutaneous space was sufficient to induce EAO on the recipient's testes in mice. It was also noted that the placement of TEV induced only orchitis without epididymo-vasitis, while the serum autoantibodies were reactive with haploid germ cells existing throughout the TEV. Furthermore, the TEV placed in the abdominal cavity rather than the subcutaneous space was effective in inducing severe EAO, and the A/J strain was most susceptible to the TEV-induced EAO among the three strains examined. The model of EAO induced by the placement of the donor's TEV into the abdominal cavity in A/J mice will be helpful for the further analyses of testicular autoimmunity.