If the BTB is severely damaged, the autoimmunogenic TGC leaks out beyond the BTB, causing a continuous supply of autoantigens with the resultant testicular inflammation. Fainboim et al. (1976) reported immunologic response in the testes of guinea pigs after unilateral traumatic orchitis (Fig. 6.1). They found that foci showing the classical picture of EAO appeared in the contralateral testis. Later, in mice, Naito et al. (2009a) also demonstrated that rupture of one testis by a scissor induced EAO in the contralateral testis (=sympathetic EAO). Traumatic rupture of unilateral testis induced significant DTH against TGC (Sakamoto et al. 1995; Naito et al. 2009a). The DTH response to TGC by testicular trauma was induced without any active immunization with TGC and was further enhanced by treatment with cyclophosphamide before the trauma and was significantly suppressed by cyclosporine A (Sakamoto et al. 1995, 1998). The traumatized testes undergo early degeneration of the seminiferous epithelium followed by neutrophilic inflammation and later fibrosis with little lymphocytic infiltration. In the contralateral testes, EAO characterized by both lymphocytic inflammation and spermatogenic disturbance was induced.

In this sympathetic EAO model, adeno-associated virus-mediated human IL-10 gene transfer suppressed the disease development (Watanabe et al. 2005). A single intramuscular injection of IL-10 gene into mice with the testicular trauma significantly suppressed both EAO and DTH against TGC. In the EAO-suppressed mice, serum IL-10 peaked at 3 weeks after the injection, and numbers of IFN-gamma and IL-2-expressing cells in the spleen and testes were significantly fewer. The results are in contrast to the aggravating effect of exogenously administered IL-10 on TGC-induced EAO (Kaneko et al. 2003).

Torsion of the spermatic cord results in vascular changes that can range from partial venous obstruction to complete occlusion of the testicular artery (Hirai et al. 2017). The damage of BTB undergoing experimental torsion of the spermatic cord also induced sympathetic EAO in the contralateral testis of rats and rabbits (Nagler and White 1982; Cerasaro et al. 1984; Rodriguez et al. 2006) (Fig. 6.1). In this model, animals were subjected to 720 degree unilateral spermatic cord torsion. In rats, the maximal degree of the spermatogenic disturbance was seen 30 days after torsion in the contralateral testis, in which T cells, mast cell, and macrophages increased in number. TNF-alpha concentration and the number of TNF receptor-positive TGC were also increased in the contralateral testis (Rodriguez et al. 2006). This contralateral EAO was prevented if splenectomy was performed on the rats with unilateral twisted testis (Nagler and White 1982). In rabbits, there were no differences of severity of contralateral EAO between animals that had received unilateral testis torsion alone and animals that had received detorsion at 36 h or 96 h. Salvaged unilateral testes after detorsion became atrophic and that detorsion did not protect the contralateral testis from EAO induction (Cerasaro et al. 1984). However, orchiectomy of the unilateral twisted testis appeared to protect the contralateral testis from EAO (Nagler and White 1982).

Vasectomy, a common male contraceptive approach, often results in immune responses to sperm antigens. In vasectomized animals and humans, the epididymis underwent epithelial cell apoptosis followed by necrosis, severe inflammation, and granuloma formation (spermatic granuloma) as a result of extravasated spermatozoa by vasectomy, creating a localized endogenous danger signal (Rival et al. 2013). In vasectomized rabbits, guinea pigs, monkeys, and mice, post-vasectomy EAO has been documented (Bigazzi et al. 1976; Tung 1978; Tonsy et al. 1979; Tung and Alexander 1980; Anderson and Alexander 1981) (Fig. 6.1). The post-vasectomy EAO is associated with deposition of IgG and complement on the basal lamina of the seminiferous tubules. These lesions are frequently accompanied by mononuclear cell infiltration and destruction of seminiferous epithelium. Some studies revealed the induction of both humoral and cellular autoimmune responses directed against sperm antigens after vasectomy (Ansbacher 1973; Alexander and Tung 1977; Herr et al. 1987; Nashan et al. 1990; McDonald and Halliday 1992; Flickinger et al. 1994, 1995, 1996). The post-vasectomy EAO is likely resulted from continuous stimulation by exposed sperm antigens coming from the inflamed epididymis. Indeed, testicular lymph and lymph nodes contained spermatozoa in men, rams, and boars after vasectomy (Ball et al. 1982; Ball and Setchell 1983). Such direct access of spermatozoa to lymph nodes is likely to provide a powerful stimulus to the development of anti-sperm autoimmunity.

In guinea pigs, EAO is developed 14 months after vasoligation. Furthermore, a similar histopathology was found in unoperated testes after unilateral vasoligation, and peritoneal exudate cells from vasoligated guinea pigs transferred identical lesions to syngeneic recipients (Tung 1978). Blood flow or lymphatic drainage in the testis and epididymis may be changed by vasectomy or vasoligation (Itoh et al. 1998a). It is known that the ducts of the caput epididymis are the sites of absorption of various materials leaving the testis. Therefore, vasectomy or vasoligation may allow the epididymal ducts to absorb testis-secreting materials, including autoantigens of mature spermatids, more strongly and to leak or excrete some of the absorbed autoantigens to the outside of the ducts. Actually, Johnson and Howards (1975, 1976, 1977) reported the leakage of epididymal spermatozoa from the caput epididymal ducts after vasectomy in guinea pigs and hamsters. It was also found that the levels of IFN-gamma, IL-6, and IL-10 in the epididymides strikingly increased with vasectomy alone (Qu et al. 2008).

Serum autoantibodies from vasectomized rats bound autoantigens of approximately 86 kDa, 63 kDa, 43 kDa, and 20 kDa of sperm extract and autoantigens of approximately 76 kDa, 60 kDa, and 42 kDa of testicular extracts (Handley et al. 1988). Immunohistochemically, autoantigens of 63 kDa and 43 kDa were synchronously expressed in the cytoplasm of spermatids (Handley et al. 1991). On immunization with syngeneic TGC alone, EAO is induced in mice with no involvement of autoimmune epididymitis (Itoh et al. 1991a, b). In contrast, autoimmune epididymitis with no EAO can be induced in vasectomized mice by immunization with TGC (Qu et al. 2008). The appearance of autoantigens relevant to EAO or autoimmune epididymitis was investigated by reaction of each immune serum with testicular and epididymal extracts from normal mice of various ages by immunoblotting. The results showed that the antisera obtained from mice with TGC-induced EAO lesions specifically defined testicular antigens with molecular weights of 15 kDa, 40 kDa, 75 kDa, and >200 kDa from 4 weeks of age, but the antisera obtained from mice with autoimmune epididymitis induced by vasectomy+TGC-immunization strongly defined testicular antigens of 25 kDa from 5 weeks of age and epididymal antigens of 25 kDa from 8 weeks of age (Qu et al. 2010). This suggests that vasectomy changes the target autoantigens in TGC-induced autoimmunity.

It has been demonstrated that autoreactive lymphocytes could preferentially gain access to the tubuli recti and the rete testis where BTB is incomplete. (Dym and Fawcett 1970; Aoki and Fawcett 1975; Itoh et al. 1995a, b, 1998b). Indeed, a focal lymphocytic infiltration was faintly and occasionally found in vasectomized mice (personal observation). However, vasectomy may increase the hydrostatic pressure in the epididymal ducts rather than the tubuli recti and the rete testis. This may result in possible leakage of more germ cell autoantigens into the epididymal interstitium, followed by autoimmune epididymitis (Qu et al. 2008). Therefore, ligation of ductuli efferentes rather than vasectomy may affect the integrity of BTB. The permeability of Sertoli cell tight junctions to lanthanum, a blood-borne tracer, has been compared in rats after ligation of the vas deferens and after ligation of the ductuli efferentes (Neaves 1978). In vasectomized rats, lanthanum penetrated only short distance into the Sertoli cell tight junctions; thereby, the BTB prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In contrast, lanthanum completely penetrated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment in rats that had received ligation of the ductuli efferentes. Therefore, ligation of the ductuli efferentes is very effective in eliciting change in the BTB. A later study demonstrated the breakdown of the BTB by ligation of the ductuli efferentes is reversible (Tao et al. 2000). Clinically, a spermatocele refers to the cystic accumulation of semen in the male reproductive tract because of the blockade of the germ cell flow. Considering that the spermatocele is thought to be caused by narrowing of the lumen of the excurrent duct with resultant cystic dilatation of the duct, the ductuli efferentes should be most vulnerable to the lumen occlusion. Actually, a senile change of the seminiferous epithelium, which releases agglutinated germ cells into the lumen of the seminiferous tubules, occupied the very narrow lumen of the ductuli efferentes with TGC, resulting in the blockade of TGC flow at the rete testis (Itoh et al. 1999). Therefore, occlusion of the ductuli efferentes may also break down the BTB, increasing susceptibility to EAO.

It is also noted that there are still conflicting reports on the immunological long-term effects of vasectomy. Vasectomized males represent a population whose immune potential may be compromised by leakage of sperm into the blood vascular system. Diminished T cell function after vasectomy has been reported in rhesus monkeys (Wilson et al. 1979), but none was revealed in mice (Anderson and Alexander 1981). The effect of vasectomy on EAO was investigated in the neonatal thymectomy model, in which neonatal thymectomy on day 3 induces multiple organ-localized autoimmune diseases involving EAO. The incidence of EAO was found to increase when day 3-thymectomized mice received vasectomy on day 60 after birth (Taguchi and Nishizuka 1981) (Fig. 6.2). Kojima and Spencer (1983) also reported that vasectomy increased the incidence of testicular atrophy in day 3-thymectomized mice.

Qu et al. (2008) demonstrated that vasectomized mice became resistant to active EAO induction; mice receiving sham-vasectomy and the following TGC immunization had EAO with no epididymitis. In sharp contrast, no EAO was found in the testes of any mice receiving vasectomy and the following TGC immunization. Instead, caput epididymitis involving infiltration of CD4⁺ T cells, CD8⁺ T cells, B cells, and macrophages was induced in them with striking elevation of the epididymal tissue levels of both IL-6 and IL-10 mRNA (Qu et al. 2008). Therefore, vasectomy suppressed EAO and alternatively induced caput epididymitis (Fig. 6.2). Later, in other studies, it was demonstrated that vasectomized mice did not develop any post-vasectomy EAO lesion. Instead, they became resistant to EAO induction by testicular antigens+CFA+BP-immunization but are sensitive to experimental autoimmune encephalomyelitis (Wheeler et al. 2011) (Fig. 6.2). This testis-specific tolerance is long lasting and has continued for at least 12–16 months; however, the specific tolerance in vasectomized mice switched over to pathologic autoimmunity following concomitant depletion of CD4⁺ CD25⁺ Foxp3⁺ Treg, unilaterally vasectomized mice of which the Treg had been simultaneously depleted developed both cellular and humoral autoimmune responses against meiotic TGC antigens, resulting in post-vasectomy EAO bilaterally in spite of no active immunization with testicular antigens+CFA+BP. However, this post-vasectomy EAO did not occur in unilaterally vasectomized mice when Treg depletion was delayed by 1 week. Therefore, post-vasectomy EAO depends on a rapid “natural” Treg depletion just after vasectomy. However, the delayed Treg depletion at week 1 allowed active EAO induction by testicular antigens+CFA+BP-immunization at week 3 in unilaterally vasectomized mice, although unilaterally vasectomy alone showed resistance to this active EAO. Therefore, a dynamic and fine balance between tolerance and autoimmunity was demonstrated in this disease model. Moreover, tolerance was blunted in unilaterally vasectomized mice that are genetically deficient in PD-ligand1. The PD-1 receptor and its cognate PD-ligand1 are one of the major inhibitory ligand-receptor pairs that are highly expressed on Treg. The interaction of PD-1 and PD-ligand1 on T cells and antigen-presenting cells is critical for peripheral tolerance and autoimmunity prevention (Riella et al. 2012). Similar to the wild-type vasectomized mice, PD-1 knockout mice that had been unilaterally vasectomized exhibited the resistance to active EAO by immunization with testicular antigens+CFA+BP. In contrast, PD-ligand1 knockout mice that had been unilaterally vasectomized were sensitive to this active EAO. Since previous studies have demonstrated that PD-ligand1 is required for the generation of “induced” Treg and maintenance of Foxp3 expression (Riella et al. 2012), the findings suggest the involvement of “induced” Treg on suppression of active EAO in vasectomized mice. Therefore, there may be two types of Treg in vasectomized mice, in which preexisting “natural” Treg may prevent post-vasectomy EAO, whereas “vasectomy-induced Treg” may maintain post-vasectomy tolerance (Rival et al. 2013).

Testis heating suppresses spermatogenesis which is marked by TGC loss via apoptotic pathways. Recent studies demonstrated that heat triggers autophagy and apoptosis in TGC (Zhang et al. 2012). EAO is induced in the untreated contralateral testis by unilateral heating injury in the guinea pig (Rapaport et al. 1969; Fernandez-Collazo et al. 1972) (Fig. 6.1). This sympathetic EAO is accompanied by humoral response and DTH against TGC. In thermally injured left testis, congested vessels, interstitial infiltration of both polymorphonuclear and mononuclear cells, and their infiltration into lumen of the seminiferous tubules were observed. Later, a fibrotic reaction appeared, followed by sclerosis of the testis. In the contralateral right testis, the lesions consisted of seminiferous tubules with sloughing of TGC, vacuolization of Sertoli cells, and mononuclear infiltration in the interstitial tissue, but no alteration in Leydig cells was observed (Fernandez-Collazo et al. 1972).

It is known that the environmental temperature in the abdominal cavity or inguinal canal is 2 degrees higher than in the scrotum and is the reason for the damage to an undescended testis. It is noted that experimental unilateral cryptorchidism induces pathologic changes leading to the spermatogenic disturbance in “both” testes. A high non-physiological environmental temperature makes the BTB permeable by immune cells. In the model of experimental unilateral testicular cryptorchidism using azathioprine as an immunosuppressant, the total number of spermatogonia in the ectopic as well as in the contralateral orthotopic testis increased significantly (Mengel and Zimmermann 1982).

The effect of 43 C warming on cell junctions in the seminiferous epithelium was examined. Expressions of adherence junction-associated molecules, such as N-cadherin and beta-catenin, and tight junction-associated molecule zonula occludens protein 1 were significantly reduced in 24–48 h after heat treatment, indicating to disruption of the BTB (Chen et al. 2008). Analyses using chromium Cr⁵¹-EDTA as a tracer also revealed that the BTB was less effective during the period of spermatogenic disruption following local 43 C heating of the testis (Setchell et al. 1996).

Unilateral multiple in situ freezing of the rabbit testis was shown to be an effective antigenic stimulus as evidenced by production of tissue-specific antibodies against testicular antigens (Ablin et al. 1971). In the unilateral testes receiving cryoinjury, coagulation necrosis is evidenced by homogenous eosinophilia of the tubular and vascular elements. Around the tubules, a moderate and diffuse fibroblastic-histiocytic infiltration, as well as a perivascular cuff of lymphocytes, was recognized. Histological studies of testicular biopsies of the contralateral testis of frozen animals revealed modest to extensive alterations consisting of degenerative changes in the seminiferous tubules accompanied by interstitial edema, interstitial hyperplasia, and the spermatogenic disturbance (Ablin 1972; Ablin and Soanes 1972). Mild to moderate interstitial mononuclear cell infiltration was also induced in the contralateral testis following the unilateral cryoinjury, indicating the induction of sympathetic EAO (Zappi et al. 1973, 1974) (Fig. 6.1).

Turpentine is a fluid produced by the distillation of resin obtained from live trees. It can cause a local and prolonged inflammation when injected into the target tissues. In the guinea pigs that had received local injection with turpentine into unilateral testis and intradermal injection with CFA, histological examination of the locally injected unilateral testis showed a large central core of coagulation necrosis, in which the outlines of the seminiferous tubules remained visible (Boughton and Spector 1963). Beyond the area of necrosis, the testis showed a zone of inflammatory cell infiltration, with fibrosis, extensive degeneration of the seminiferous tubules, and the interstitial edema. On the other hand, contralateral testis showed a patchy tubular degeneration among a great bulk of normal tubular zone on day 7 (Fig. 6.1). In the affected tubules, there were no spermatozoa or spermatids, and primary and secondary spermatocytes were disintegrating. The spermatogonia were relatively less affected, and the primary damage appeared to be in the spermatocytes. The Sertoli cells seemed unaffected, as did the interstitial tissues. In contrast, in animals receiving intra-testicular turpentine without intradermal CFA injection, contralateral testis showed no tubular degeneration. Furthermore, in animals that had received intradermal injection with CFA without intra-testicular turpentine, the testes were macroscopically and microscopically normal (Boughton and Spector 1963).

Eyquem and Krieg (1965) produced bilateral EAO by injecting a small amount of CFA alone into the left testis in rats, guinea pigs, and monkeys (Fig. 6.1). In CFA-injected unilateral testes, infiltration of mononuclear cells, resorption of the inoculated product in the granuloma, spermatogenic disturbance, and exudation of fibrinoid products were induced. In the contralateral testes, the spermatogenic disturbance was noted, but there was no apparent inflammatory cell response and granuloma. Boughton and Spector (1963) also tried similar experiments in guinea pigs; however, little damage in CFA-injected left testis and no detectable pathological changes in the contralateral testis were found.

CFA was injected into cauda epididymides bilaterally in rats, in which the cauda was heavily infiltrated with polymorphonuclear leukocytes, lymphocytes, plasma cells, and macrophages (Tonsy et al. 1979). The cellular inflammatory infiltrate was also present in the regions of the corpus and caput epididymides. Furthermore, there was marked testicular degeneration. The testicular interstitium was infiltrated with lymphocytes, plasma cells, and macrophages, which seemed to be crossing the wall of the seminiferous tubules. Multinucleated large macrophages were also noted in the seminiferous tubules. The basal lamina of the seminiferous tubules was externally thickened. There was almost complete absence of all TGC except for Sertoli cells. Occasionally, some tubules were distended with large numbers of spermatozoa and no other immature TGC. Sera of the injected rats were all positive for anti-sperm autoantibodies.

Infection is a candidate of a trigger of autoimmune diseases that occur in the natural course of the diseases. Guinea pigs were inoculated into the testis with viable Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus (Sanui et al. 1982, 1983). DTH skin reactions against TGC were carried out 1 week later and significantly detected only when guinea pigs were locally injected with Listeria monocytogenes. The DTH reactions involved many lymphocytes, macrophages, and basophils with some neutrophils and eosinophils at subdermal tissue and were specific for TGC since the significant DTH reaction could not be detected by the elicitation with sheep red blood cells as sensitizing antigens. Significant anti-TGC DTH reaction was not detected when guinea pigs were injected with Listeria monocytogenes intravenously or subcutaneously. However, the local injection of viable Listeria monocytogenes into unilateral testis induced EAO in the contralateral testis and epididymis (=sympathetic EAO) in guinea pigs (Fig. 6.1). The pathological changes in the contralateral testis were characterized by the severe spermatogenic disturbance and severe interstitial infiltration of mononuclear cells. In a small number of animals, polymorphonuclear leukocytes were detected in or around the seminiferous tubules. Mononuclear cells also invaded into some seminiferous tubules; however, basophils were not detected in the EAO lesion. In the rete testis, massive infiltration of mononuclear cells was observed. In the lumens of the rete testis, degenerated and desquamated TGC and macrophages phagocytosing many spermatozoa were found. Most seminiferous tubules were empty or sometimes contain a central mass of degenerated TGC. However, Sertoli cells still remained a normal appearance. Moreover, hypertrophic Leydig cells with eosinophilic cytoplasm appeared prominent when inflammatory cell infiltration was subsided (Sanui et al. 1982, 1983).

Listeria monocytogenes are facultative intracellular pathogens that grow in the cytoplasm of infected host cells. The virulent strain of Listeria monocytogenes is able to survive in the cytoplasm by escaping from phagosomes. In the sites of local infection with Listeria monocytogenes in the unilateral testis, apparent destruction of architecture of the seminiferous tubules was seen, and enhancement of both cellular and humoral immune responses was expected due to adjuvant effects of the bacterial components. Listeria monocytogenes was not detected in the EAO-affected contralateral testis, liver, or spleen during the period of experiments, showing that the bacterial growth was limited to the inoculated unilateral testis. These data show a possibility that the macrophages/dendritic cells accumulate in virulent Listeria monocytogenes-infected site and then uptake, process, and present TGC autoantigens released from the damaged testis with the infection (Sanui et al. 1982, 1983). On the other hand, avirulent Listeria monocytogenes, which lacks the expression of listeriolysin O, failed to induce any anti-TGC immune responses and sympathetic EAO even when inoculated at a high dose into the testis.

The unilateral testicular inoculation of virulent Listeria monocytogenes in mice also induced specific DTH response against TGC and caused sympathetic EAO (Fig. 6.1). A unilateral infection of Listeria monocytogenes into the testis of mice induced not only Listeria-specific T cells but also autoreactive T cells that can transfer EAO into naïve mice (Mukasa et al. 1995, 1997, 1998; Matsuzaki et al. 1997). In mice unilaterally infected with Listeria monocytogenes, the contralateral testis developed inflammatory lesion without detectable microorganisms, providing evidence for an autoimmune phenomenon, like in the guinea pig model. This model therefore gives the opportunity to investigate to two types of inflammation in one mouse: a bacterial-induced reaction and an autoimmunity-induced inflammatory reaction. The EAO can be adoptively transferred to naïve syngeneic mice by intravenous injection with TGC-specific T cell clones that were derived from spleens of the intra-testicular Listeria-infected mice. All EAO-inducing clones expressed both CD4 and TCR alpha-beta and showed proliferative response to TGC but did not cross-react to the Listeria antigen. They produced both IFN-gamma and TNF-alpha when stimulated with TGC, but IL-2, IL-4, and IL-10 were undetectable. Therefore, alpha-beta T cells play a central pathogenic role in causing EAO, as in other EAO models.

The TGC-specific T cells transferred EAO without histological changes in the liver and kidney. Therefore, the orchitogenic T cell clones recognize testis-specific antigens that are not expressed by other organs, although the antigenic molecules have not yet been identified. Furthermore, the increased number of gamma-delta T cells was found in both infected and sympathetic EAO-affected testes. The gamma-delta T cell ratio in infiltrated T cells in testes reached up to 20–30% in whole T cells. Surprisingly, when gamma-delta T cells were depleted, inflammation in both sides was significantly augmented. Thus, gamma-delta T cells in this model may function as Treg in the disease progress and may have some immunoregulatory control on alpha-beta T cells (Mukasa et al. 1997, 1998). It was also shown that the gamma-delta T cells reduced alpha-beta pathogenic T cell function through cytokines. The negative regulatory role of gamma-delta T cells in inflammation may be due to the production of IL-10 or transforming growth factor-beta, which is known to exert a suppressive function on alpha-beta T cells.

There is little information about whether sympathetic orchitis accompanied by cellular and humoral immune responses against TGC can be experimentally induced by other local infection in the testis. In mice, intra-seminiferous tubular injection of live Escherichia coli in unilateral testis resulted in that the bacteria actively propagated and reached a maximal level in a day but started to decrease after day 5 and completely disappeared within 2 months. The expression of macrophage inflammatory protein-2 and TNF-alpha became evident in testicular macrophages as early as 1–3 h. Neutrophils were first accumulated in the testicular interstitium at 9–12 h, and the spermatogenic disturbance was observed on day 1 and seemed unrecoverable and irreversible even after the bacteria were eliminated (Nagaosa et al. 2009). However, the spermatogenic disturbance was not evident in the contralateral testis. Additionally, in mice that received intra-testicular injection with lipopolysaccharide or dead Escherichia coli, the spermatogenic disturbance was not evident (Nagaosa et al. 2009).