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.

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.

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 transcriptionpolymerase 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.

SummaryLymphangiogenesis 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.

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