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CD4+ CD25+ regulatory T cells (Tregs) strongly influence the early and late autoimmune responses to meiotic germ cell antigens (MGCA) and the gonadal immunopathology in vasectomized mice. This is supported by the published and recently acquired information presented here. Within 24h of unilateral vasectomy (uni-vx) the ipsilateral epididymis undergoes epithelial cell apoptosis followed by necrosis, severe inflammation, and granuloma formation. Unexpectedly, vasectomy alone induced MGCA-specific tolerance. In contrast, uni-vx plus simultaneous Treg depletion resulted in MGCA-specific autoimmune response and bilateral autoimmune orchitis. Both tolerance and autoimmunity were strictly linked to the early epididymal injury. We now discovered that testicular autoimmunity in uni-vx mice did not occur when Treg depletion was delayed by one week. Remarkably, this delayed Treg depletion also prevented tolerance induction. Therefore, tolerance depends on a rapid de novo Treg response to MGCA exposed after vasectomy. Moreover, tolerance was blunted in mice genetically deficient in PD-1 ligand, suggesting the involvement of induced Treg. We conclude that pre-existing natural Treg prevents post-vasectomy autoimmunity, whereas vasectomy-induced Treg maintains post-vasectomy tolerance. We further discovered that vasectomized mice were still resistant to autoimmune orchitis induction for at least 12-16 months; thus, tolerance is long-lasting. Although significant sperm autoantibodies of low titers became detectable in uni-vx mice at 7 months, the antibody titers fluctuated over time, suggesting a dynamic "balance" between the autoimmune and tolerance states. Finally, we observed severe epididymal fibrosis and hypo-spermatogenesis at 12 months after uni-vx: findings of highly critical clinical significance., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Experimental allergic orchitis is an organ-specific autoimmune disease characterized by inflammatory infiltrates associated with the seminiferous tubules of the testes. Orchitis is often, but not always, accompanied by aspermatogenesis in susceptible strains of mice. In this study, various strains of H-2 congenic mice were used to examine the relationship between orchitis and aspermatogenesis, and as a result, a genetic predisposition to spontaneous aspermatogenesis has been defined. A high correlation was seen between orchitis and aspermatogenesis in B10.D2/nSnJ mice, however, the two conditions were uncorrelated in C57BL/10J mice. Subsequent analysis of C57BL/10J congenic strains showed their aspermatogenesis to be spontaneous, rather than due to either testis-specific antigen or adjuvants. Further studies using other H-2 congenic strains revealed that the aspermatogenesis seen in C57BL/10J mice is linked to H-2 and influenced by C57BL/10J background genes. Finally, spontaneous aspermatogenesis was shown not to be a function of differences in the level of testicular testosterone.

In this study, immunochemical techniques were employed to examine the guinea pig (GP) testicular proacrosin-acrosin system. Monospecific polyclonal antibodies to the highly stable enzymatically active 34,000 molecular weight form of GP testicular acrosin were generated. Western blot analysis of acid extracts prepared from snap-frozen freshly excised GP testes revealed two major immunoreactive bands with mol. wts of approximately 62,000 and 48,000 and one minor band with an approximate mol. wt of 54-56,000. The 62,000 mol. wt molecule identified is in close agreement with the previously reported mol. wt for purified GP testicular proacrosin. Western blot analysis of different species of testicular acid extracts demonstrated the evolutionary relatedness of the proacrosin-acrosin systems since immunoreactivity was observed primarily with acid extracts from rodent species (rat, mouse and hamster) and not with extracts from evolutionarily less-related species (goat, ram and bovine). The majority of the cross-reactivity observed was characterized by immunoreactivity with the 62,000 and 48,000 mol wt molecules. The only species which exhibited cross-reactivity with the 54-56,000 mol. wt protein was rat. In addition, the iso-immunogenic and aspermatogenic properties of the 34,000 mol. wt form of GP testicular acrosin were examined. One out of five Hartley and one out of seven Strain 2 female GPs immunized and boosted with a total of 200 micrograms of purified protein exhibited increased levels of circulating anti-acrosin iso-antibodies. The antigenic specificity of the iso-antibodies observed in the two animals was verified by Western blot analysis. All other female animals, including two strain 13 GPs, exhibited very low or undetectable levels of such antibodies following immunization. One out of three male Hartley GPs immunized with 50 micrograms of the purified protein exhibited typical lesions of experimental allergic orchitis while none of a group of three animals developed lesions at a 5 micrograms dose. Taken together, these results suggest that the 34,000 mol. wt form of GP testicular acrosin is neither a highly potent iso-immunogen nor aspermatogenic autoantigen.

Active experimental allergic orchitis (EAO), characterized by inflammation of the testes (autoimmune orchitis), aspermatogenesis, epididymitis and vasitis was induced in mice using a panel of tissue antigens as immunogens. Immunization with allogeneic murine tissue homogenates emulsified in complete Freund's adjuvant (CFA) accompanied by the injection of pertussigen revealed that only adult murine testicular and epididymal homogenates are capable of eliciting murine EAO. All other tissue antigens studied including prepubertal mouse and epididymal homogenates failed to elicit significant disease. Immunization with xenogeneic testicular antigens also failed to elicit significant disease indicating that the major murine aspermatogenic autoantigen(s) is also highly species specific. Sensitization with allogeneic mouse testicular homogenates (MTH) from different disease resistant strains was for the most part no less potent in inducing significant disease than was immunization with mouse testicular homogenates from disease susceptible strains. However, testicular homogenates from NZB/B1NJUnm and MRL/MpJ-/+Unm mice were significantly less potent at inducing autoimmune epididymitis as compared to other strains, indicating possible interstrain differences in the immunogenicity of the aspermatogenic autoantigen(s) relevant to eliciting epididymitis. Attempts at solubilization and purification of the major murine aspermatogenic autoantigen(s) utilizing techniques employed for the purification of aspermatogenic autoantigens such as AP3 from guinea pig (GP) testes were unsuccessful. Additional extraction procedures resulted in solubilization of the relevant autoantigen(s) only after reduction in the presence of 6 M guanidine hydrochloride. These data suggest that: (1) there may be a much more limited number of aspermatogenic autoantigens in murine testes as compared to GP testes; (2) the disease inducing determinant(s) may be expressed as either a sequential antigenic determinant(s) or as an antigenic determinant(s) in the carbohydrate portion of a glycoprotein or glycopeptide; and (3) the disease inducing autoantigen(s) may be present in situ in a highly insoluble form requiring active processing within the target organ in order to generate soluble antigen capable of being seen by immune reactants.

Significant differences in susceptibility to the induction of experimental allergic orchitis (EAO) were found to exist among the various substrains of BALB/c mice. Substrains which were susceptible to disease induction include BALB/cKa, BALB/cByJ, BALB/cWtJ, BALB/cCmcCr, BALB/cCrgl, BALB/cHeA, BALB/cAnNCr, BALB/cPt, BALB/cWehiUnm, BALB/cOrnl and BALB/cArg whereas BALB/cJ and BALB/cSki BoyPt were resistant to EAO. Aspermatogenesis and autoimmune vasitis, two additional lesions characteristic of murine EAO, were found to correlate with susceptibility to autoimmune orchitis. Susceptibility and/or disease resistance did not segregate within any of the evolutionary branches of the BALB/c genealogic tree nor did it correlate with any of the previously described allelic differences distinguishing BALB/c substrains. Therefore, differential disease susceptibility may represent the manifestation of mutational and/or retroviral induced genotypic differences in the regulatory genes controlling testicular autoimmunity.