Testicular biopsies from infertile men have been found to contain immunopathological evidence of orchitis and testicular immune complexes, which are also found in EAO. At present, respective epidemiological data are very scarce. Clinically, testicular biopsy specimens from men with spermatogenic disturbance of immunologic origin are supposed to represent the final stage of the pathological process (post-active inflammation stage) in which the infiltration of immune reactants has already ceased and only the spermatogenic disturbance remains. In the majority of patients, diagnosis is hampered by an asymptomatic course of the disease and unspecific clinical signs. Therefore, immunological factors may be more often involved in male infertility than has been suspected if the patients can receive the medical examination more early. Furthermore, examination sites in the testis may affect the incidence of inflammation. Many histopathological studies revealed the frequent involvement of inflammatory cell infiltration at the mediastinum (the tubuli recti and the rete testis) (Hatakeyama 1984; Tung et al. 1987; Itoh et al. 1995b). Actually, differing from the common biopsy cases of convoluted seminiferous tubules at the peripheral testis, the sampling of tissue from the tubuli recti and the rete testis is very difficult because of the danger that biopsy-induced tissue injury may block TGC transport to the ductuli efferentes. However, if biopsy tissue can be taken from the tubuli recti and the rete testis regions safely, the incidence of lymphocytic infiltration in the testes of infertile men should become higher than reported. It should be also kept in mind that testicular biopsy artificially breaks BTB with the resultant exposure of TGC autoantigens to the immune system. Therefore, development of noninvasive test for diagnosis of orchitis is needed. Several different imaging methods such as color Doppler sonography and magnetic resonance imaging have been tried (Moraes et al. 2010).

As another noninvasive test, identification of specific markers of testicular autoimmunity has been awaited. Even though only anti-sperm antibodies, which represent humoral immunity, have been taken into account by many urologists or andrologists when assessing immunologic male infertility (Shibahara et al. 2005; Shiraishi et al. 2009), many reports on EAO imply that DTH response, cellular immunity, is more critical for the disease induction than humoral immunity in spite of no use of DTH-inducing CFA (Sakamoto et al. 1985; Sakamoto and Nomoto 1986; Itoh et al. 1991a, b, c, 1992b; Itoh 2009). Considering that the DTH intensity against TGC well correlated with EAO development (Qu et al. 2014), in vivo assay of DTH response on injection of some specific TGC autoantigens or in vitro assay of cytokines secretion or cell proliferation of isolated lymphocytes stimulated with TGC autoantigens may be effective for screening and predicting methods to detect whether testicular autoimmunity is involved or not in infertile male patients. In immunologically infertile women, a precise and objective method for the diagnosis was developed and examined, based on a one-step agarose leukocyte migration inhibition factor assay (Dimitrov et al. 1992). The migration areas are evaluated by a computer-assisted image analysis system. The radial migration indexes and area migration indexes are computed and expressed as a migration index percentage for each patient and control. Therefore, development of a reliable, standardized testing protocol for diagnosis of autoimmune orchitis will contribute to grasp precise epidemiology of testicular autoimmunity in men.

Most recently, a new noninvasive biomarker for the diagnosis of testicular inflammation was reported (Fijak et al. 2014). In sera from infertile azoospermic patients with histologically confirmed testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60), were found. Proteins in lysates of normal testicular tissue have 14 proteins that immunoreacted with a majority of sera of patients with testicular inflammation. Of these 14 proteins, ER-60, transferrin, and chaperonin containing T-complex protein 1, subunit 5, were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. Therefore, measurement of ER-60 autoantibody titers in serum could be a novel noninvasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances.

Although many patients with idiopathic male infertility are asymptomatic, chronic genitourinary inflammation often results in leukocytospermia, an elevated number of leukocytes in semen. Hagen et al. (2015) evaluated expression of TLR-2, TLR-4, cyclooxygenase-2, and nuclear factor-like 2 in semen samples of age-matched infertile patients with and without leukocytospermia by the usage of specific Western blot evaluation, cytokine array, and immunofluorescence microscopy followed by computer-based analysis. Differential cytokine profiling of seminal plasma by antibody array revealed upregulation of the four pro-inflammatory chemokines in leukocytospermia. Therefore, TLR-2, TLR-4, cyclooxygenase-2, and nuclear factor-like 2 in semen can serve as novel biomarkers for idiopathic and asymptomatic male infertility patients.

Investigation of initial stage of testicular autoimmunity should lead to understanding of first contact between immune cells and testicular autoantigens in the testis. There are many EAO models nowadays, but there is still limited information of mode of EAO onset. Investigation of start of inflammatory cell infiltration in various EAO models may be useful for understanding its pathophysiology. Spermatids and spermatozoa are apparently foreign cells for females, prepubertal males, and neonatally castrated males. Therefore, transfer of lymphocytes obtained from syngeneic females, prepubertal males, or neonatally castrated males into adult male animals may be also valid to study the onset of contact between lymphocytes and TGC autoantigens in the testis.

In the testis, testis-specific effector CD4⁺ T cells can recognize TGC autoantigens on antigen-presenting cells in the testis before EAO induction. Under normal condition, many TGC are degenerated in the seminiferous tubules, and their autoantigens should latently leaked, followed by endocytosis by various testicular somatic cells, such as Sertoli cells, peritubular myoid cells, testicular macrophages/dendritic cells, Leydig cells, and capillary endothelia. It is supposed that testicular macrophages/dendritic cells expressing MHC class II antigens surrounding the tubuli recti and rete testis are most important antigen-presenting cells for EAO induction; however, it remains unclear how TGC autoantigens leak outside the germ cell ducts and picked up by macrophages/dendritic cells in the testicular interstitium under physiological condition. It is also noted that mouse testicular cells express Fas ligand and have the potential to induce apoptotic death of activated T cells expressing Fas (CD95). To establish EAO, the orchitogenic T cells must escape the T cell killing mechanism by testicular cells. One possible mechanism is that the testis-specific effector T cells are resistant to the Fas-mediated cell death induced by the Fas ligand on testicular cells because Fas-mediated signaling does not always induce apoptosis. Alternatively, there is another possibility that EAO induction modulates expression of the Fas ligand on the testicular cells, which allow the testis-specific T cells to escape from apoptosis induction.

The role of humoral immunity in EAO induction still remains obscure. The histopathologic distribution of the initiation of inflammation differs between active and passive EAO (Tung et al. 1987; Itoh et al. 1991c, 1992b). Active EAO induced by testicular homogenate+CFA+BP immunization initially affects subcapsular seminiferous tubules far from the rete testis. In contrast, passive EAO by transfer of CD4⁺ T cells obtained from testicular homogenate+CFA+BP-immunized donor mice preferentially induce inflammation in the tubuli recti adjacent to the rete testis. In TGC-induced EAO, active EAO is characterized by orchitis that preferentially affects the tubuli recti and the rete testis and by the absence of epididymo-vasitis; however, passive EAO is consistently accompanied by epididymo-vasitis. It is supposed that both cellular and humoral immune responses are induced in active EAO, while cellular rather than humoral immunity is critical for passive EAO. This indicates that EAO is generally CD4⁺ T cell dependent, but B cells and plasma cells also affect the start of EAO.

Although Th1 cells were thought to be the main drivers of organ-specific autoimmunity, animals lacking the Th1 signature cytokines and molecules are not resistant, but more susceptible to multiple autoimmune diseases (Ferber et al. 1996; Jones et al. 1997; Matthys et al. 1999). In these animals, the generation of a unique CD4⁺ T cell subset, named Th17 cells, is involved. Th17 cells produce the effector cytokine IL-17, which promote tissue inflammation and neutrophil recruitment for host defense and also are primarily associated with autoimmune inflammation (Korn et al. 2009). Actually, in testicular antigens+CFA+BP-induced EAO lesion, CD4⁺IL-17⁺ and CD8⁺IL17⁺ were immunohistochemically identified (Jacobo et al. 2011a, b). The addition of IL-17A to normal rat Sertoli cell cultures induced a significant decline in transepithelial electrical resistance and a reduction of occluding expression and redistribution of occludin and claudin-11, altering the Sertoli cell tight junction barrier (Perez et al. 2014). Also in vivo, intra-testicular injection of recombinant rat IL-17A in rats induced increased the BTB permeability and delocalization of occludin and claudin-11. Therefore, involvement of Th17 cells for the start of testicular inflammation will be studied in other EAO models.

The target finding by orchitogenic lymphocytes was antigen-specific, but the following tissue damage leading to spermatogenic disturbance may be produced by an antigen nonspecific fashion in EAO. Previously, the pathogenesis for TGC depletion in EAO lesion was often attributed to an influx of specific autoantibodies into the germ cell ducts with a leaky BTB; however, recently, much attention is being paid to the effect of cytokines on the function of Sertoli cells, Leydig cells, and the developing TGC (Itoh et al. 1992b, 1993). IL-1, IL-6, IL-17, IFN-gamma, TNF-alpha, and transforming growth factor-beta have been reported to affect Sertoli cells and the inter-Sertoli cell junctions as the BTB (Banks and Kastin 1992; Plotkin et al. 2000; Lui et al. 2003; Li et al. 2006; Sarkar et al. 2008; Wang and Lui 2009; Lie et al. 2011; Lydka et al. 2012; Perez et al. 2012, 2014; Zhang et al. 2014b). If this hypothesis is correct, the depletion of TGC should be induced without a direct contact of TGC with specific autoantibodies or specific CD4⁺ T cells during EAO.

The vitro experiments on seminiferous tubule cultures showed that IFN-gamma and TNF-alpha induced apoptosis of TGC through the Fas/Fas ligand system (Riccioli et al. 2000). It is indicated that the Fas/Fas ligand system mediates apoptosis of TGC in the injured testis but not in the normal testis having spontaneous apoptosis (Koji 2001; Koji et al. 2001). It was also demonstrated that IL-6 induced apoptosis of TGC in isolated seminiferous tubules in rats (Rival et al. 2006). Clinically, the semen IL-6 level is increased in vasectomy reversal patients (Nandipati et al. 2005). Local injection of IFN-gamma into the testis in vivo induced a direct cytotoxic effect on TGC, indicating that IFN-gamma is also harmful to the seminiferous epithelium (Natwar et al. 1995). In addition, IFN-gamma was shown to increase the expression of Fas in Sertoli cells and makes these cells susceptible to Fas ligand-mediated cytotoxicity in the seminiferous epithelium (Riccioli et al. 2000). IFN-alpha is produced predominantly by leukocytes in man, and most cell types are able to produce IFN-alpha in the mouse. In transgenic mice using a plasmid containing IFN-alpha gene, aberrant expression of the introduced IFN-alpha gene occurs only in the testis, in which an ongoing degeneration of TGC leading to calcium deposits and atrophy of the seminiferous tubules were found (Hekman et al. 1988). Intra-testicular injection of recombinant rat IL-17A to rats induced focal inflammatory cell infiltration in the interstitium and germ cell sloughing in adjacent seminiferous tubules. ED1⁺ macrophages were the main population infiltrating the interstitium following IL-17A injection (Perez et al. 2014). Therefore, IL-17A may facilitate the recruitment of immune cells to the testicular interstitium, resulting in the spermatogenic disturbance.

Coxsackievirus and adenovirus receptor is a junction molecule that is expressed on Sertoli cells and TGC. It mediates Sertoli cell-TGC adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the BTB, suggesting that coxsackievirus and adenovirus receptor-based cell adhesion and migration are crucial for spermatogenesis (Gao and Lui 2014). Combined treatment with IFN-gamma+TNF-alpha exerts a synergistic effect by downregulating mRNA of coxsackievirus and adenovirus receptor and its protein levels. Therefore, downregulation of coxsackievirus and adenovirus receptor by IFN-gamma+TNF-alpha treatment may provide an explanation of how TGC are sloughing in the seminiferous epithelium during testicular inflammation.

It became evident that damaged TGC products induce expression of various inflammatory mediators, including TNF-alpha, IL-1-beta, IL-6, and monocyte chemotactic protein-1, in Sertoli cells. Notably, the damaged TGC products-induced inflammatory gene expression was significantly reduced by knockout of TLR-2 and TLR-4 (Zhang et al. 2013a). Monocyte chemotactic protein-1 secreted by Sertoli cells after stimulation with damaged TGC products promotes migration of macrophages around the seminiferous tubules. Accumulated macrophages might deteriorate the spermatogenic disturbance. Indeed, busulfan-induced spermatogenic disturbance in vivo upregulates TNF-alpha and monocyte chemotactic protein-1 expression in Sertoli cells and facilitates macrophage migration into the testis in wild-type mice. These phenomena were not observed in TRL2^−/−TLR4^−/− mice. It indicates that damaged TGC products induce inflammatory gene expression in Sertoli cells via the activation of TLR-2 and TLR-4, which may initiate prolonged inflammatory responses in the testis. However, clinical reports of the detection of various cytokines in testis biopsy specimens from infertile men have been still limited. To elucidate the mechanisms underlying the spermatogenic disturbance during human immune orchitis, functional analyses of cytokines and establishment of cytokine therapies in various EAO models will be expected.

Additionally, factors of damage to the spermatogenesis other than cytokines must also be kept in mind. Testicular inflammation may elevate the organ temperature and also disturb microcirculation in the testis, resulting in heat and ischemic damage to the seminiferous epithelium. Preferential accumulation of lymphocytes around the tubuli recti in EAO may cause dysfunction of valve structure of modified Sertoli cells inside the tubuli recti and then increase intra-seminiferous tubular pressure. A recent study showed that an impaired removal of apoptotic TGC induce noninfectious testicular inflammation, thus favoring testicular autoimmunity (Schuppe and Meinhardt 2005; Schuppe et al. 2008; Pelletier et al. 2009). The meaning of phagocytic removal of apoptotic TGC by Sertoli cells should be also an interesting topic to be investigated in field for maintaining testicular immune microenvironment.

During EAO, the spermatogenic disturbance may involve both failure of TGC differentiation and facilitation of TGC death (Kuerban et al. 2012). It remains unclear whether TGC death is by necrosis, apoptosis, or cell death involving autophagy. Recently, autophagy has been focused in various cells (Enomoto et al. 2007; Yokoyama et al. 2008; Kawakita et al. 2009; Ohtomo et al. 2010; Kawaguchi et al. 2011; Komatsu et al. 2012). It may be that TGC leaked out of BTB receive necrosis; however, TGC death inside the BTB results from not only apoptosis and but also autophagy. Besides initiating apoptotic pathways, EAO also may induce autophagic pathways in TGC and/or Sertoli cells, including autophagosome formation (Bustamanter-Marin et al. 2012; Eid et al. 2012, 2015; Han et al. 2015).

Testicular autoantigens expressed in haploid TGC appear after puberty when immunocompetence has been already established. A large number of testis-specific autoantigens are present not only on spermatids and spermatozoa but also on spermatogonia, spermatocytes, Sertoli cells, Leydig cells, and basement membrane of the tubular walls (Sato et al. 1981; Lustig et al. 1982, 1987; Ichinohasama et al. 1986; Yule et al. 1988). For a more complete understanding of the immune pathogenesis of EAO, molecular and biochemical approaches to TGC autoantigens from mice at different weeks after birth are needed. However, the molecular structure of autoantigens relative to EAO has been little characterized. Considering that testicular homogenate+CFA+BP-induced EAO involves immune responses against various antigens, it may be difficult to clear which proteins are target antigens that are critical for EAO induction. To identify the target autoantigens effectively, its investigation using EAO models induced by no use of any chemical or bacterial agents such as active EAO induced by immunization with testicular antigens alone and spontaneous EAO after manipulation of immune system may be easy and useful.

Although EAO-inducing TGC are highly species-specific, there are also common testicular antigens among different species for eliciting anti-TGC DTH with or without involving EAO (Yoshida et al. 1979, 1981; Adekunle et al. 1987; Qu et al. 2017). Moreover, sporadic orchitis and spermatogenic disturbance were observed with immunization with non-testicular antigens such as brain, spinal cord, thyroid gland, kidney, adrenal gland, and ovary emulsified in CFA and concomitant intravenous injection with BP (Adekunle et al. 1987). Therefore, EAO-inducing T cells can be also activated by stimulation with non-testicular peptides that cross-react with testicular autoantigens at the level of the TCR. This molecular mimicry depends in part on the sharing between unrelated peptides of the few critical amino acids that are required for activation of pathogenic orchitogenic T cells. If the candidate purified autoantigen-specific CD4⁺ T cell clone is established and shown to induce EAO by adoptive transfer to naïve recipients, we can identify the purified protein as the target autoantigen of EAO in the future.

Identification of susceptible and resistant strains of inbred mice and the phenotyping of segregating populations derived from them allow an estimation of the number of genes involved. Previous studies have demonstrated that genetic predisposition is a major contributor to EAO susceptibility and resistance. It should be also determined whether immune responses to relevant testicular autoantigens are primarily regulatory or active in nature for each strain. Research based on molecular linkage analysis of inbred mice has been embarked upon for mapping genes that influence the susceptibility and resistance to EAO. In general, the MHC is the primary genetic determination of autoimmune disease susceptibility with multiple additional interacting loci required.

In regard to EAO, Balb/c strain (H-2^d) is susceptible to testicular antigen+CFA+BP immunization but resistant to TGC immunization or neonatal thymectomy. C3H/He (H-2^d) strain is susceptible to TGC immunization but resistant to testicular antigen+CFA+BP immunization or neonatal thymectomy. C57BL/6 (H-2^b) strain is susceptible to testicular antigen+CFA+BP immunization or neonatal thymectomy but resistant to TGC immunization. In contrast, A/J strain (H-2^a) is susceptible to the all three EAO models. Therefore, genetic susceptibility to EAO differs among the three disease models (Itoh et al. 1991a; Tokunaga et al. 1993a, b; Kojima and Prehn 1981; Kojima and Spencer 1983; Teuscher et al. 1985a, b). Identification of susceptible and resistant strains of inbred mice and the phenotyping of segregating populations derived from them allow an establishment of the number of genes involved in each EAO model. Genetic analysis of inbred mouse strains until now indicates that susceptibility or resistance to EAO is polygenic and strongly influenced by both H-2-linked and non-H2-linked genes (Person et al. 1992). However, the identification and characterization of non-MHC genes have been problematic, since most autoimmune diseases are polygenic with the individual genes exhibiting only partial or minimal penetrance.

There are also some EAO models developed by gene manipulations such as rearranged TCR alpha-chain gene transfer, HLA-B27 gene transfer, TAM gene knockout, Aire gene knockout, and p35 gene knockout (Hammer et al. 1990; Sakaguchi et al. 1994; Lu et al. 1999; Hubert et al. 2009; Sun et al. 2010; Taurog et al. 2012; Zhang et al. 2013b; Terayama et al. 2014). Trials of these gene manipulations on various murine inbred strains may also contribute to the understanding of genetic control of EAO.

The effective treatment of organ-specific inflammatory disorders of putative autoimmune origin is an ongoing goal in clinical medicine. Because the lymphocytic infiltration of the testis, the disturbance of spermatogenesis and the occurrence of DTH responses against testicular antigens can be observed in cases of human immunologic infertility (Anderson and Hill 1998); EAO should be used as in vivo tool for studying immuno-inflammatory pathways and immunotherapeutic approaches for the treatment of the human orchitis.

Cyclosporine A and deoxyspergualin have been used for the EAO treatment (Hojo and Hiramine 1985; Ablake et al. 2002). For specific inhibition of disease, adoptive transfer of tissue antigen-specific Treg may offer one of the effective treatments. In the normal state, there may be pathogenic clones reactive to autoantigens of TGC and clones to regulate such autoreactive cells, but these clones may keep silent in the appropriate balance between the clones. Strong proliferation of TGC-specific effector clones may occur after two or three injections with TGC, but the predominance of TGC-specific Treg clones may be generated in the course of more repeated immunization (Itoh et al. 1992a, b). Antigen-specific regulation of EAO has been also demonstrated following repeated intravenous injection of deaggregated, soluble testis antigens (Mukasa et al. 1992). Mice treated in this manner developed long-lasting resistance to the induction of EAO from subsequent challenge with TGC. In EAO developed in mice that received various immune-manipulations by neonatal thymectomy, irradiation, or drugs, specific deletion or dysfunction of T cell population, both naturally occurring Treg and inducible Treg, should illustrate the complex nature and balance of immunoregulation. Therefore, multiple Treg populations may be present and participate for EAO regulation.

Although the mechanisms for the development of autoimmune diseases remains obscure, accumulating evidence suggests that these increasingly recognized disorders result from environmental or occupational exposures of noninfectious agents in genetically susceptible individuals (Miller 2014). Impaired health status such as malnutrition, obesity, alcohol, tobacco, or illicit drug use may affect spermatogenesis (Eid et al. 2012, 2015). Nowadays, we can take various medicines and are daily exposed to various environmental toxicants of low doses (Ishihara et al. 2000; Gu et al. 2003; Ablake et al. 2004; Miyaso et al. 2010, 2014a, b; Kitaoka et al. 2013; Ogawa et al. 2013; Hirai et al. 2015). Some of them have ability to affect testicular function or immune function. To investigate whether various chemicals such as cadmium, uranium, phthalic acid esters, tamoxifen, diethylstilbestrol, bisphenol A, decabromodiphenyl ether, flutamide, alcohol, and cigarette smoking affect the incidence and severity of testicular autoimmunity is also valid (Fig. 8.1). The increased output of these chemicals has drawn a large number of people into contact with these elements. Malenchenko et al. (1978) were the first to investigate the effect of an environment chemical on the EAO induction by the use of uranium. Recently, it has appeared that low dose exposure of cadmium or phthalic acid significantly increased susceptibility of EAO (Ogawa et al. 2013; Hirai et al. 2015). Neonatal treatment with estrogen developed severe epididymo-vasitis later (Naito et al. 2014). It was also found that neonatal exposure to diethylstilbestrol, artificial estrogenic compound, induced epididymitis in all treated mice after 5 weeks of age and also evoked orchitis in some mice after 12 weeks of age (Miyaso et al. 2014a, b). These inflammatory lesions may be of “autoimmune/autoinflammatory syndrome induced by adjuvants (ASIA),” in which estrogen acts as an adjuvant in male mice. A study of the effect of di-(2-ethylhexyl) phthalate on the testicular immune microenvironment revealed that lymphocytes and F4/80- and MHC class II antigen-positive cells were significantly increased with the elevation of IL-10 and IFN-gamma mRNA expressions in the testes (Kitaoka et al. 2013). Histochemical analyses involving horseradish peroxidase as a tracer showed that a little blood-borne horseradish peroxidase had infiltrated into the lumen of a few seminiferous tubules beyond the BTB. Age- and species-dependent infiltration of macrophages into the testes of rats and mice exposed to phthalate was also demonstrated (Murphy et al. 2014). In that study, a significant increase in monocyte chemoattractant protein-1 by peritubular myoid cells occurred 12 h after phthalate exposure.

In regard to the effect of other environmental factors on testicular autoimmunity, it may be also valuable whether exposure to an electromagnetic wave and field could affect EAO (Grigorev et al. 1981; Naito et al. 2012; Hanci et al. 2013). Exposure to a 900-MHz electromagnetic field in the prenatal term on the 21-old-day rat induced irregularities in seminiferous epithelium and tubular basal membrane accompanied by decreased diameter of the seminiferous tubules. Apoptotic index, lipid peroxidation, and DNA oxidation were higher than controls (Hanci et al. 2013). The increased TGC apoptosis may give specific lymphocytes a chance to react with TGC autoantigens more easily.

In the “autoimmune/autoinflammatory syndrome induced by adjuvants” (ASIA), different conditions induced by various adjuvants such as infectious fragments, hormones, aluminum, silicone, and metal are included, and the syndrome is characterized by common signs and symptoms, resulting in boosting the immune response and triggering the development of autoinflammatory phenomena (Loyo et al. 2012; Cruz-Tapias et al. 2013; Lujan et al. 2013; Colafrancesco et al. 2014).

CFA and BP have been used as adjuvants for EAO induction. Treatment with CFA and BP releases various pro-inflammatory cytokines involving IL-1-beta, IL-6, IL-12 TNF-alpha, and IFN-gamma in vivo (Mielcarek et al. 2001; Tonon et al. 2002; Raghavendra et al. 2004). In mice that were intravenously injected with BP alone, systemic inflammation involving hepatosplenomegaly and lymphadenopathy was induced, and the ductuli efferentes, the epididymis, the vas deferens, and the accessory sex glands in BP-injected mice received inflammatory cell infiltration (Itoh et al. 1995a). The mycobacterial components within CFA signal T cells assume a Th1 profile so that various lymphocytic clones should be activated, resulting in enhancement of the systemic inflammation (Billiau and Matthys 2001). Indeed, the employment of CFA and BP has proved to be instrumental in the breakdown of testicular immune privilege and microcircumstance for the spermatogenesis indirectly, as well as in augmenting of DTH (Pelletier et al. 1981; Sewell et al. 1986; Adekunle et al. 1987; Lustig et al. 1987; Mealy et al. 1990; Perez et al. 2011, 2012). Furthermore, the number of MHC class II antigen-bearing macrophages increased in the testis after the CFA+BP treatment (Tung et al. 1987), and both production of anti-TGC autoantibodies and development of cytolytic activity of T cells against TGC are inducible without the use of testicular antigens for sensitization (Ben et al. 1986; Musha et al. 2013). Therefore, CFA+BP treatment also may induce ASIA-like condition. Therefore, there is a possibility that EAO may be inducible with CFA+BP treatment alone if some optimal conditions such as more prolonged, chronic, and repeated sensitization with the adjuvants or other xenogeneic antigens are devised.

It is of considerable interest to know what happens in the host associated with the failure in the elaborate systemic immune system. Presumably such a critical situation could be created experimentally in animals by enforced overwork of the immune system, e.g., by repeated immunizations with an appreciable but not tolerogenic amount of xenoantigens over an extended period. Historically, Okabayashi and his colleagues (1980) had innovatively investigated prolonged sensitization with xenoantigens such as ovalbumin, hemolytic streptococci, Escherichia coli, bovine serum albumin, and horse serum over a year (Fig. 8.2). They had carried out a series of experiments in the experimental animals to clarify the overall sequence of events taking place in the immune system of the host by means of chronic and prolonged antigenic stimulation by using foreign proteins or bacterial antigens (Okabayashi 1964, 1967a, b, 1972, 1973, 1979). With such repeated immunization, the animals showed an enhanced immune cell proliferation in the lymphoid organs, together with accelerated hematopoiesis in the bone marrow. However, the reactivity of the immune system may not be permanent. Following continued antigen administration, the systemic immune reaction developed to the highest level during the middle stage but gradually became less reactive. A wasting in the systemic immune reaction thus ensued in the terminal stage as evidenced by progressive exhaustion of lymphoid responses. The bone marrow likewise showed prominent hypoplasia with or without fibrosis, and a variety of diseases of the blood, organs, and tissues such as mucoid, fibrinoid, hyaline, and amyloid degeneration occurred in sequence. Okabayashi had emphasized that before entering into the overt anergic or exhausted terminal phase, certain immunologic disorders develop in the later stage of prolonged antigenic stimulation which are significantly different from those observed during the early stages. Alterations of the organs and tissues gradually became degenerative in nature; however, under conditions of the imbalance in the wasting immune system, there appeared not only the degenerative conversion of the inflammatory process but also the development of “autoimmunization.” The resulting dysplasia of lymphoid tissues and imbalance of the immune cell population, which could be interpreted in terms of the disturbance in immunological regulation, may induce “autoimmune diseases in the wasting immunity.” Therefore, the chronically sensitized host is affected by systemic immune reaction for a long period, and prolonged antigenic sensitization induces all four immunopathies composed of immunoproliferative diseases, allergic and hypersensitivity diseases, immunodeficiency diseases, and autoimmune diseases (Fig. 8.3). Although the heart, kidneys, and various lymphoid organs have been well investigated (Okabayashi et al. 1980), it should be of interest to investigate whether and how the testis and other male reproductive organs are immunopathologically affected during prolonged sensitization with xenoantigens.

In animals that received allogeneic or xenogeneic cells, tissues, or organs, “graft-versus-host reaction” or “transplant rejection” occurs if some immunosuppressive treatments are not given. In testicular immunology, four transplantation models described below may help the investigation on physiology of testicular immune privilege.