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We previously established an experimental model of autoimmune orchitis (EAO) by means of immunization with testicular germ cells (TGC) alone in mice and confirmed that the disease can be transferred to mice that had received CD4+ but not CD8+ or B lymphocytes obtained from TGC-immunized donor mice. The tubuli recti (TR) are special in that lymphocytes first accumulate around them before spreading to the peripheral seminiferous tubules in EAO. However, the minute changes in the TR remain unknown. Therefore, we investigated the histopathology of the TR before the induction of spermatogenic disturbance. The results revealed that the first infiltrating lymphocytes around the TR were not only of T-cell but also of B-cell lineage. Moreover, it was also shown that some of these infiltrating lymphocytes migrated into the TR, with resultant degeneration of the TR epithelium before damage to the seminiferous epithelium. These findings indicate that TR epithelial cells are the first targets of autoreactive T and B lymphocytes in EAO.

Spermatoza contain various autoimmunogenic materials, which are recognized as foreign by the self immune system. Therefore, the blood-tesits-barrier (BTB) formed by Sertoli cells, basal lamina and myoid cells protects autoimmunogeneic spermatozoa from attack by the self immune system. However, the BTB at the tubuli recti (TR) and the rete testis (RT) is known to be incomplete against humoral substances. We investigated here whether the BTB is physiologically penetrated by lymphocytes in mice. We performed light and electron microscopical observation of the seminiferous tubules (ST), the TR and the RT in normal C3H/IHe mice. Although no lymphocytes were observed inside the ST, a very few lymphocytes could be found beyond the basal lamina of the TR and the RT. These lymphocytes were close to testicular spermatozoa in the TR lumen. These findings provide a possibility that lymphocytes may gain access to autoantigens of spermatozoa inside the TR and RT under physiological conditions in mice.

The tubuli recti (TR) are immunologically special, because the lymphocytes preferably accumulate around them during the course of T-cell dependent testicular autoimmunity in mice. This finding implies that the testicular interstitium around the TR is where autoreactive lymphocytes can gain access to autoimmunogenic germ cell antigens. In the present study, the histoarchitecture of the TR was minutely examined in normal mice. Three-dimensional analysis showed that 14-16 TR appeared to be connected to the rete testis (RT). Electron microscopical analysis revealed that the epithelial cells in the TR formed protruding cytoplasmic strings, with active endocytosis of degenerated spermatozoa, and exhibited three different morphological characteristics. Furthermore, a few macrophages were found to have penetrated into the TR. Immunohistochemical image analysis revealed that more macrophages specifically accumulated around the TR than the RT or seminiferous tubules. These findings indicate that macrophages preferentially accumulate around the TR, where contact between germ cell antigens and macrophages may occur under normal and pathological conditions.

Background: Most testicular and epididymal lymphocytes express T-cell markers, but their cytotoxic potential and activation status have not been reported. In this study, distribution of the cytotoxic cells was compared between normal and cryptorchid testes stratified into two groups: the first with complete absence of germ cells [Sertoli cell-only (SCO)] and the second with arrested spermatogenesis (SCA)., Methods: Immunohistochemistry for the T-lymphocyte marker CD3 and cytotoxic markers CD8, TIA-1 and granzyme B was performed on paraffin-embedded sections., Results: The number of CD8+ and CD3+ intra-epithelial lymphocytes (IELs) increased distally throughout the normal epididymis. TIA-1 immunostaining revealed that a significant proportion of IELs exhibited cytotoxic potential, whereas granzyme B staining disclosed a subpopulation of activated cytotoxic lymphocytes (CTLs). TIA-1/CD8 and granzyme B/CD8 double immunostaining revealed that the vast majority of TIA-1+ and granzyme B+ cells were CD8+. The proportion of activated granzyme B+ lymphocytes increased distally throughout the normal epididymis. The number of TIA-1+ and granzyme B+ intra-epithelial and stromal lymphocytes was significantly increased in the normal as opposed to the SCO cryptorchid epididymis and proximal vas deferens., Conclusions: These results suggest that exposure of the testicular excurrent ducts to spermatozoa or immature germ cells triggers the activation and recruitment of CTLs. Cytotoxic granule effector mechanisms may contribute to the immunological barrier preventing the immune response to spermatozoa in testicular ducts.

Problem: Previous studies demonstrated that F4/80 antigen (murine macrophage-specific antigen)-positive cells in testes of normal adult mice accumulate particularly in the interstitium adjacent to the tubuli recti and rete testis (i.e., the central region). However, it remains unknown whether this accumulation is a congenital or acquired phenomenon., Method of Study: The distribution of F4/80-positive cells on frozen sections of testes obtained from various aged mice was immunohistochemically examined to determine when the positive cells specifically accumulate in the central region., Results: F4/80-positive cells were homogeneously distributed throughout the testicular interstitium with no specific accumulation until 2 weeks of age. However, at 3 weeks of age, the density of positive cells in the central region became slightly, but significantly, higher than that in the interstitium between the seminiferous tubules. Between 3 and 4 weeks of age, the cell density in the central region increased rapidly, the density at 4 weeks of age reaching the level of the mature testes of 8-week-old mice., Conclusion: These results demonstrate that the specific accumulation of F4/80-positive cells in the central region is an acquired phenomenon, which starts and ends before puberty.

Using a monoclonal immunoperoxidase technique on frozen tissue sections of normal testis and epididymis, a distinctive distribution of lymphocytes has been identified. Almost all of the intra-epithelial lymphocytes were T cells and the majority of these belonged to the T8+ ("suppressor/cytotoxic") subset. The distribution contrasted with the predominance of the T4+ ("helper/inducer") subset in the interstitial tissues and in normal peripheral blood. The predominance of the "suppressor/cytotoxic" subset within the epithelium of the epididymis may exist in order to prevent the development of the immune response to auto-antigens on spermatozoa.