Immunological microenvironment in the testis

Abstract Background The testis is specific in that it produces haploid germ cells of which autoantigens newly appear long after the neonatal immune tolerance. Under normal condition, these autoantigens are protected by the blood‐testis barrier formed by Sertoli cells. Thus, the testis is an immunologically privileged site where haploid cells are protected from autoimmune attack. Methods The immunological microenvironment in the testis was experimentally investigated using mice and rats. Main findings Not only the blood‐testis barrier but also various immuno‐suppressive factors are involved in the immune‐privileged testis. Indeed, germ cells transplanted into the xenogeneic seminiferous tubules could proliferate and differentiate with no aid of artificial immunosuppression. On the other hand, autoimmune orchitis could be experimentally produced by various methods of immunization with syngeneic or xenogeneic germ cell antigens. Conclusion Our results indicate that the testis is immunologically privileged but also immunologically fragile organ. Therefore, the dual nature is critical for immunoregulation of testicular function.


| APPE AR AN CE OF S PERMATIDS AND S PERMATOZOA AF TER THE E S TAB LIS HMENT OF IMMUNE TOLER AN CE
The reproductive system has evolved to allow self to interact with non-self, whereas immune system has evolved to make distinction from non-self to self, thereby allowing the emission of non-self. It in males, only a small number of spermatogonia develop within the testes from fetal to pre-pubertal period; however, once puberty is reached, active spermatogenesis begins, and approximately 100 million spermatozoa are then produced, transported, and excreted on daily basis until gerontic period 1 ( Figure 1A).
The developmental phase is different between lymphoid and gonadal tissues. It is well known that the lymphoid organs are the earliest to degenerate and develop in the embryo, but the reproductive organs are the slowest to mature ( Figure 1A). The seminiferous tubules of mice contain spermatogonia at the day of birth, preleptotene spermatocytes a 1 week of age, pachytene spermatocytes at 2 weeks of age, round and oval spermatids at 3 weeks of age, the elongating spermatids at 4 weeks of age, and most mature (elongated) spermatid at 5 weeks of age. 2 Spermatids and spermatozoa do not appear in the testis until puberty (starts at around 35 days of postnatal age) that far later than the period of neonatal immune tolerance from fetal to infant period. Therefore, autoantigens of these haploid cells may be targeted for immunological elimination ( Figure 1B). Different from spermatogonia and preleptotene spermatocytes having 46 chromosomes, spermatids and spermatozoa emerging from meiosis have only 23 chromosomes but express various new differentiation autoantigens. 3 Thus, the male reproductive organs have larger amounts of new autoantigens than those in the female ones.

| TE S TICUL AR IMMUNO -ENVIRONMENT FOR G ERM CELL D IFFERENTIATI ON
Spermatogenesis takes place within the convoluted seminiferous tubules, which then connect the tubuli recti (TR) and terminate at the rete testis (RT) (Figure 2A). Testicular germ cells (TGC) then leave the rete testis and are transported to the ductuli efferentes, epididymal ducts, and vas deferens ( Figure 2A). Some studies show the development of testis in mice during the postnatal period. The tight junction between Sertoli cells develops between 10 and 16 days of age in the mouse and the presence of Sertoli cell junctions and seminiferous tubule lumen at 18 days of age. 4,5 Lee et al demonstrate that the area of RT can be detected at 10 days of age and the RT areas are significantly increased from 18 days of age. 6 Autoimmunogenic spermatids and spermatozoa are believed to be protected from detrimental immune attacks by blood-testis barrier (BTB), which is located at the base of the seminiferous tubules ( Figure 2B). The BTB, established at 15 days of age in mice, is mainly composed of inter-Sertoli cell junctions including tight junction, basal ectoplasmic specializations, gap junctions, and desmosome-like junctions. 7,8 It is well known that the BTB protects post-meiotic germ cells and is essential for the spermatogenesis. Recently, transplantation of rat spermatogenesis was demonstrated inside the BTB of immune competent mice. 9 This indicates that the BTB protects not only autologous but also xenogeneic TGC from immunological elimination.
However, based on the findings of some researches, spermatids and spermatozoa are not completely isolated from immune system by the BTB in mammals; instead, these cells are not normally rejected by the individual's own immune system because of being maintained in a fine and subtle state of immune balance. [10][11][12][13][14][15][16] All testicular cells, involving TGC, Sertoli cells, Leydig cells, testicular macrophages, peritubular myoid cells, endothelia of blood, and lymph capillaries

| B RE AK DOWN OF TE S TI CUL AR IMMUNE PRIVILEG E RE SULTS IN TE S TICUL AR AUTOIMMUNIT Y
At the puberty, the male immune system first perceives intolerant haploid cell antigens in the testis as completely different from any other previously encountered and already tolerated diploid cell antigens. In particular, spermatids, which have more abundant cytoplasm than spermatozoa, should contain various autoimmunogenic antigens of large amounts. Therefore, the testes in which spermatids emerge from miosis may be more sensitive to autoimmune inflammation than the vas deferens and epididymis in which mature spermatozoa but not spermatids are compacted.
If the testicular immune privilege is upset under some condition, immune responses against the TGC autoantigens should be induced.
The characteristics of testicular autoimmunity include the detection of inflammatory cell infiltration into the testis, disturbed spermatogenesis, testicular antigens-specific T-cell response, the specific serum autoantibodies, and binding of the autoantibodies and complements in the testis, 28  The increase in testicular mast cells closely contacted to the seminiferous tubules indicates a relationship between mast cell proliferation and the BTB dysfunction. 42 Testicular germ cells autoantigens F I G U R E 3 Various immuno-suppressive factors in the testis leak beyond the BTB when the BTB is functionally damaged. This leads to a continuous supply of the autoantigens to the immune system, with the resultant chronic inflammation in the testis for a prolonged spermatogenic disturbance. 19,43 Particularly, the BTB is demonstrated to be incomplete at the TR and the RT. 44,45 This implies that the testicular tissue around the TR is a site where autoreactive lymphocytes can gain access to autoimmunogenic TGC antigens.
Furthermore, in rodent study, it was found that many macrophages accumulate around the TR and a few of them penetrate into the TR. 17 Under normal condition, they may take the materials leaked from the TR to inhibit the induction of inflammatory responses. 46,47 However, when the testicular immune privilege becomes unstable and upset, the TR should be immunological-specific region, where lymphocytes are attracted. Because diagnostic biopsy of the TR and the RT is clinically impossible, therefore, it is quite difficult to know the histological appearance in infertile patients. 19,48 That is why the clinical data on the relation between autoimmune orchitis and male infertility have been still unclear.
On the other hand, as a model of acquired idiopathic spermatogenetic failure, research on experimental autoimmune orchitis (EAO) has been widely conducted in mice, rats, guinea pigs, and other animals (Table 1) As EAO can be induced by only exposing TGC to the immune system outside the BTB without any artificial immune enhancement, 11 it is noted that this disease model is closer to the clinical cases, in which focal injuries in the testis such as ischemia and trauma damage the seminiferous tubules, followed by leakage of the TGC to the outside of the tubules. Furthermore, the area of predilection of lymphocytic infiltration is in and near the interstitial tissue adjacent to the TR and the RT in both the human cadavers and the mouse EAO model. 43,52 Therefore, even if no inflammatory lesion is found on biopsy at some testis regions far from the TR and RT of infertile men, a possibility of inflammation involving the mediastinum testis remains.
It is clinically general that the presence of anti-sperm antibodies is a key for diagnosis of male infertility of immunologic origin, 53 and measurement and analysis of anti-sperm antibodies have been extensive. 54 Moreover, autoantibodies against the other testicular cells and components such as Sertoli cells, Leydig cells, and basement membrane of the seminiferous tubules were also detected in male infertility. 55,56 However, it is insufficient to diagnose testicular autoimmunity only with detection of the autoantibodies on sera and/or semen. The role of autoantibodies in EAO induction still remains obscure. Active EAO is induced by immunization with testicular antigens, and passive EAO is inducible by transfer of testisspecific lymphocytes. Therefore, both cellular and humoral immune responses are induced by immunization for active EAO while cellular but not humoral immunity is critical for passive EAO. Indeed, the histopathologic patterns of the initiation of inflammation in active and passive EAO differ from each other. 13,57,58 This indicates that EAO is generally CD4 + T-cell dependent, but B cells, plasma cells, and their production of autoantibodies should affect the inflammatory pattern of EAO.

| HOW THE TE S TICUL AR IMMUNE PRIVILEG E AG AIN S T XENOG ENEI C G ERM CELL S IS EFFEC TIVE OR B ROK EN?
Previously, it has been reported that delayed type hypersensitivity (DTH) against allogeneic or xenogeneic TGC was elicited in syngeneic TGC-immunized mice 59 and the following studies revealed that DTH rather than humoral immunity against TGC antigens is critical for TGC-induced EAO induction. 12,14,[60][61][62] Recently, it was found that immunizations with rat TGC alone can induce murine EAO without using adjuvants 63 (Table 1; Figure 4A). The DTH against murine TGC was significantly elevated in mice immunized with syngeneic or xenogeneic TGC (rat TGC). Serum autoantibodies to murine TGC determined by enzyme-linked immunosorbent assay were significantly On the other hands, it has been known that rat spermatogenesis can occur in the seminiferous tubules of congenitally immunodeficient recipient mice after transplantation of rat spermatogonial stem cells (SSCs). 64 Experimentally immunosuppressed adult mice were found to be also useful as the recipients. When hamster SSCs were transplanted into the testes of infant rats with immature immune system, hamster spermatogenesis could be detected within the rat seminiferous tubules. 65 Later, transplantation of rat SSCs into immunocompetent mice was investigated. The results showed that transplanted rat SSCs could undergo complete spermatogenesis in recipient mouse testes, and many rat spermatozoa could be detected in the recipient epididymides 9 ( Figure 4B). This implies that transplanted rat spermatogonia could undergo complete spermatogenesis in normal immune system of the recipient mice. 66   without using adjuvants in both immunocompetent and immunodeficient animals. This ease of disease induction is far different from other experimental organ-specific autoimmune diseases. Therefore, although the testis is regarded as an immunologically privileged organ and resistant to inflammatory responses, it is also highly susceptible to autoimmune inflammation. Normal spermatogenesis appears to be dependent on the sheltered microenvironment for TGC and also on a fine balance between effective and suppressive immunity against physiologically leaked TGC antigens in "natural autoimmunity."

| CON CLUS ION
This article does not contain any studies with human subjects performed by the authors. All the experimental protocols in this study were carried out in accordance with the guidelines of the National Institutes of Health and were approved by the Tokyo Medical University Animal Committee.

ACK N OWLED G EM ENTS
This work was supported by a Grant-in-Aid for General Science Research (C: 15K08159 and C: 15K08937) from the Ministry of Education Science Sports and Culture in Japan.

CO N FLI C T O F I NTE R E S T
All authors have no conflicts of interest to declare.