Immunodeviation towards T cell-mediated immune response in the testes of LPS-induced mouse epididymo-orchitis.

The testicular consequences of acute epididymo-orchitis remain largely unelucidated in long-term damage, which might be a neglected factor for male infertility. In this study, the differential phenotype of testicular immune cell subpopulations in lipopolysaccharide (LPS)-induced mouse epididymo-orchitis were analyzed by flow cytometry on day 1, day 7, and day 28. The number of macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) steadily decreased in the testes with inoculation. Total F4/80-CD11c+ dendritic cells (DCs) maintained a relatively stable level, whereas conventional type 1 dendritic cells (cDC1) increased gradually from day 1 to day 28. There was a lower number of CD4+ and CD8+ T cells at day 1 and day 7, and they had similar results with a ceiling level at day 28. The testes displayed a higher level of CD3+ T cells but a lower frequency of macrophages, cDC2, and neutrophils at 28 days post-inoculation compared with the epididymis. In summary, our data indicates acute epididymo-orchitis could lead to long-term damage in the testes, which is characterized by CD3+ T cell (including CD4+ and CD8+ T cells)-mediated immune responses.

Varicocele-Mediated Male Infertility: From the Perspective of Testicular Immunity and Inflammation.

Background: Varicocele (VC) is present in 35 - 40% of men with infertility. However, current surgical and antioxidant treatments are not completely effective. In addition to oxidative stress, it is likely that other factors such as testicular immune microenvironment disorder contribute to irreversible testicular. Evidence suggests that VC is associated with anti-sperm antibodies (ASAs), spermatogenesis and testosterone secretion abnormalities, and testicular cytokine production. Moreover, inhibition of inflammation can alleviate VC-mediated pathogenesis. The normal function of the testis depends on its immune tolerance mechanism. Testicular immune regulation is complex, and many infectious or non-infectious diseases may damage this precision system. Results: The testicular immune microenvironment is composed of common immune cells and other cells involved in testicular immunity. The former includes testicular macrophages, T cells, dendritic cells (DCs), and mast cells, whereas the latter include Leydig cells and Sertoli cells (SCs). In animal models and in patients with VC, most studies have revealed an abnormal increase in the levels of ASAs and pro-inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha in the seminal plasma, testicular tissue, and even peripheral blood. It is also involved in the activation of potential inflammatory pathways, such as the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing (NLRP)-3 pathway. Finally, the development of VC-mediated infertility (VMI) may be facilitated by abnormal permeability of proteins, such as claudin-11, that constitute the blood-testis barrier (BTB). Conclusions: The testicular immune response, including the production of ASAs and inflammatory factors, activation of inflammatory pathways, and destruction of the BTB may be involved in the pathogenesis of VMI it is necessary to further explore how patient outcomes can be improved through immunotherapy.

Transient systemic inflammation in adult male mice results in underweight progeny.

PROBLEM: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes. METHOD OF STUDY: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality. RESULTS: Our findings demonstrated a significant induction of testicular TNF-α, IL-1ß and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier. CONCLUSION: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development.

Relevance of angiogenesis in autoimmune testis inflammation.

Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. This model reflects testicular pathological changes reported in immunological infertility in men. Progression of EAO in rodents is associated with a significantly increased percentage of testicular endothelial cells and interstitial testicular blood vessels, indicating an ongoing angiogenic process. Vascular endothelial growth factor A (VEGFA), the main regulator of physiological and pathological angiogenesis, can stimulate endothelial cell proliferation, chemotaxis and vascular permeability. The aim of this study was to explore the role of VEGFA in the pathogenesis of testicular inflammation. Our results found VEGFA expression in Leydig cells, endothelial cells and macrophages in testis of rats with autoimmune orchitis. VEGFA level was significantly higher in testicular fluid and serum of rats at the end of the immunization period, preceding testicular damage. VEGF receptor (VEGFR) 1 is expressed mainly in testicular endothelial cells, whereas VEGFR2 was detected in germ cells and vascular smooth muscle cells. Both receptors were expressed in testicular interstitial cells. VEGFR2 increased after the immunization period in the testicular interstitium and VEGFR1 was downregulated in EAO testis. In-vivo-specific VEGFA inhibition by Bevacizumab prevented the increase in blood vessel number and reduced EAO incidence and severity. Our results unveil relevance of VEGFA-VEGFR axis during orchitis development, suggesting that VEGFA might be an early marker of testicular inflammation and Bevacizumab a therapeutic tool for treatment of testicular inflammation associated with subfertility and infertility.

Corticosterone Enhances the AMPK-Mediated Immunosuppressive Phenotype of Testicular Macrophages During Uropathogenic Escherichia coli Induced Orchitis.

Testicular macrophages (TM) play a central role in maintaining testicular immune privilege and protecting spermatogenesis. Recent studies showed that their immunosuppressive properties are maintained by corticosterone in the testicular interstitial fluid, but the underlying molecular mechanisms are unknown. In this study, we treated mouse bone marrow-derived macrophages (BMDM) with corticosterone (50 ng/ml) and uncovered AMP-activated protein kinase (AMPK) activation as a critical event in M2 polarization at the phenotypic, metabolic, and cytokine production level. Primary TM exhibited remarkably similar metabolic and phenotypic features to corticosterone-treated BMDM, which were partially reversed by AMPK-inhibition. In a murine model of uropathogenic E. coli-elicited orchitis, intraperitoneal injection with corticosterone (0.1mg/day) increased the percentage of M2 TM in vivo, in a partially AMPK-dependent manner. This study integrates the influence of corticosterone on M2 macrophage metabolic pathways, phenotype, and function, and highlights a promising new avenue for the development of innovative therapeutics for orchitis patients.

Prokineticin 2 via Calcium-Sensing Receptor Activated NLRP3 Inflammasome Pathway in the Testicular Macrophages of Uropathogenic Escherichia coli-Induced Orchitis.

Reproductive tract infections contribute to the development of testicular inflammatory lesions, leading to male infertility. Previous research shows that the activation of the NLRP3 inflammasome in orchitis promotes the secretion and maturation of IL-1ß and, thus, decreases male fertility. The calcium-sensing receptor (CaSR) is closely related to the secretion of proinflammatory cytokines. An increase in the CaSR level promotes the assembly and activation of the NLRP3 inflammasome. However, the role of CaSRs in orchitis is unknown. We first constructed a uropathogenic Escherichia Coli (UPEC) rat orchitis model and then detected the expression of CaSR and NLRP3 inflammatory pathway proteins in testicular macrophages (TM) through RT-PCR and WB, calcium levels in TM through flow cytometry, and proinflammatory factor IL-1ß through ELISA. In addition, testosterone levels in the serum samples were detected using liquid chromatography-mass spectrometry (LC-MS). Here, we show that CaSR upregulation after infection in TM in a rat model of UPEC induces the activation of the NLRP3 inflammasome pathway and thereby enhances IL-1ß secretion and reduces the testosterone level in the blood. Moreover, CaSR inhibitors can alleviate inflammatory impairment. After UPEC challenge in vitro, CaSR promoted NLRP3 expression and released IL-1ß cleaved from TM into the supernatant. Overall, elevated CaSR levels in TM in testes with UPEC-induced orchitis may impair testosterone synthesis through the activation of the NLRP3 pathway and PK2 is an upstream regulatory protein of CaSR. Our research further shows the underlying mechanisms of inflammation-related male infertility and provides anti-inflammatory therapeutic targets for male infertility.

Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions.

Interleukin (IL)-18 is an inflammasome-mediated cytokine produced by germ cells, Leydig cells, and resident macrophages that is indispensable in the maintenance of homeostasis in the testis. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are very high. However, the impact of acute inflammation and IL-18 on Leydig cells remained unclear. TM3 cells, a mouse Leydig cell line, and RAW264.7 cells, a mouse macrophage cell line, were stimulated with lipopolysaccharide (LPS) or recombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage, and markers of apoptotic pathways. In Leydig cells, caspase 3 cleavage was increased and death-receptor-mediated apoptotic pathways were activated after LPS stimulation. However, LPS stimulation did not increase IL-18 expression in the Leydig cell line. When high-dose rIL-18 was administered to the Leydig cell line to mimic levels seem after inflammation, rIL-18 upregulated Tnf-α mRNA, Fadd mRNA, and Fas protein, promoted cleavage of caspase-8 and caspase-3, and induced apoptosis. Low-dose rIL-18 did not stimulate apoptosis. To determine if the high level of IL-18 seen in the testes after inflammation was derived from immune cells, we examined IL-18 protein expression in a macrophage cell line, RAW264.7. In contrast to the TM3 cells, IL-18 was significantly increased in RAW264.7 cells after LPS stimulation. These results suggest that high-dose IL-18 derived from macrophages is harmful to Leydig cells. Reducing the overexpression of IL-18 could be a new therapeutic approach to prevent Leydig cell apoptosis as a result of acute inflammation.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord.

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

Fluoride Induces Autoimmune Orchitis Involved with Enhanced IL-17A Secretion in Mice Testis.

Fluoride (F) widely exists in the water and food. Recent studies reported that F induced testicular toxicity via inflammation reaction. This study was aimed to explore the mechanism of F-induced inflammation in testis. 100 healthy male mice (BALB/cJ strain) were randomly divided into five groups including: control, experimental autoimmune orchitis (EAO), and three F groups (25, 50, and 100 mg/L sodium fluoride (NaF)). After 150 d, the results showed a significant increase in testicular cytokines levels including of IL-17A, IL-6, IFN-γ, and TNF-α in NaF and EAO groups compared with control group. Interestingly, the presence of specific antisperm autoantibodies in antitesticular autoantibodies and the notable recruitment of immunocyte (T cells and dendritic cells) were also observed in NaF and EAO groups. In addition, findings showed that in NaF and EAO groups macrophages and T cells both significantly secreted IL-17A, and the protein and mRNA levels of cytokines (IL-6 and TGF-ß) were significantly increased. From these results, it can be concluded that autoimmune orchitis and IL-17A are implicated in F-induced testicular inflammation.

Activation of the NLRP3 Inflammasome Pathway by Prokineticin 2 in Testicular Macrophages of Uropathogenic Escherichia coli- Induced Orchitis.

Infections of the reproductive tract are known to contribute to testicular inflammatory impairment, leading to an increase of pro-inflammatory cytokines such as IL-1ß, and a decline in sperm quality. Prokineticin 2 (PK2), a secretory protein, is closely associated with the secretion of pro-inflammatory cytokines in inflamed tissue. It was reported that increased PK2 is related to the upregulation of IL-1ß, but the underlying mechanism remains elusive. Here, we illustrated that PK2 was upregulated in testicular macrophages (TM) in a rat model of uropathogenic Escherichia coli (UPEC) infection, which induced the activation of the NLRP3 inflammasome pathway to boost IL-1ß secretion. Administration of PK2 inhibitor alleviated the inflammatory damage and suppressed IL-1ß secretion. Moreover, PK2 promoted NLRP3 expression and the release of cleaved IL-1ß from TM to the supernatants after the challenge with UPEC in vitro. IL-1ß in the supernatants affected Leydig cells by suppressing the expression of genes encoding for the enzymes P450scc and P450c17, which are involved in testosterone production. Overall, we revealed that increased PK2 levels in TM in UPEC-induced orchitis may impair testosterone synthesis via the activation of the NLRP3 pathway. Our study provides a new insight into the mechanisms underlying inflammation-associated male infertility and suggests an anti-inflammatory therapeutic target for male infertility.

Germ cell apoptosis and survival in testicular inflammation.

Male infertility is due to genetics, hormonal or environmental causes, or is idiopathic. Azoospermia is linked to local testicular microenvironment deregulation, with inflammatory cells present in the 15% of testicular biopsies of infertile patients. As widely reported, spermatogenesis and steroidogenesis are controlled by local immunoregulatory agents produced by immune and nonimmune cells. Moreover IL-6R, TNFR1, Fas and IL-1R are expressed on germ cells, indicating a direct action of pro-inflammatory agents on these cells. Beyond the known function of cytokines and nitric oxide on testicular function at the stable levels present in the normal testis, this review focalises on the effect of pro-inflammatory factors on germ cell survival and death when inflammatory conditions are established in the testis. As no cure for male infertility has been found up to the present, intracytoplasmic sperm injection is the therapeutic option for azoospermic patients who wish to achieve genetic parenthood. Therapies with antioxidant and anti-inflammatory agents in experimental models of testicular damage have been successful. However, clinical implementation is uncertain in cases with a prolonged inflammatory state of the testis. Therapies offering multiple approaches to treat infertility by restoring the spermatogonial stem cell niche and protecting germ cells from apoptosis should be considered.

Microenvironmental signals govern the cellular identity of testicular macrophages.

Testicular macrophages (TM) comprise the largest immune cell population in the mammalian testis. They are characterized by a subdued proinflammatory response upon adequate stimulation, and a polarization toward the immunoregulatory and immunotolerant M2 phenotype. This enables them to play a relevant role in supporting the archetypical functions of the testis, namely spermatogenesis and steroidogenesis. During infection, the characteristic blunted immune response of TM reflects the need for a delicate balance between a sufficiently strong reaction to counteract invading pathogens, and the prevention of excessive proinflammatory cytokine levels with the potential to disturb or destroy spermatogenesis. Local microenvironmental factors that determine the special phenotype of TM have just begun to be unraveled, and are discussed in this review.

Microcystin-leucine arginine exhibits immunomodulatory roles in testicular cells resulting in orchitis.

Microcystin-leucine arginine (MC-LR) causes testicular inflammation and hinders spermatogenesis. However, the molecular mechanisms underlying the immune responses to MC-LR in the testis have not been elucidated in detail. In this study, we show that MC-LR induced immune responses in Sertoli cells (SC), germ cells (GC), and Leydig cells (LC) via activating phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa B (NF-κB), resulting in the production of pro-inflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and chemokine (C-X-C motif) ligand 10 (CXCL10). The observed effects were attributed to reduced activity of protein phosphatases 2A (PP2A) as a result of binding of MC-LR to the catalytic subunit of PP2A in SC and GC. By contrast, innate immune responses were triggered by Toll-like receptor 2 (TLR2) in LC because MC-LR could not enter into the LC and subsequently inhibit the PP2A activity. PI3K/AKT/NF-κB were also activated in SC, GC, and LC in vivo, with the enrichment of TNF-α, IL-6, MCP-1, and CXCL10 in the testis. Following chronic exposure, MC-LR-treated mice exhibited decreased sperm counts and abnormal sperm morphology. Our data demonstrate that MC-LR can activate innate immune responses in testicular cells, which provides novel insights to explore the mechanism associated with MC-LR-induced orchitis.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin.

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells.

STUDY QUESTION: What is the underlying mechanism of Sertoli cell (SC) resistance to cell death? SUMMARY ANSWER: High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli. WHAT IS KNOWN ALREADY: In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways. STUDY DESIGN, SIZE, DURATION: In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC- 51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.

Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone.

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body's immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography-tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

Exposure to TiO2 Nanoparticles Induces Immunological Dysfunction in Mouse Testitis.

Although TiO2 nanoparticles (NPs) as endocrine disruptors have been demonstrated to be able to cross the blood-testis barriers and induce reproductive toxicity in male animals, whether the reproductive toxicity of male animals due to exposure to endocrine disruptor TiO2 NPs is related to immunological dysfunction in the testis remains not well understood. This study determined whether the reproductive toxicity and immunological dysfunction induced by exposure to TiO2 NPs is associated with activation or inhibition of TAM/TLR-mediated signal pathway in mouse testis. The results showed that male mice exhibited significant reduction of fertility, infiltration of inflammatory cells, rarefaction, apoptosis, and/or necrosis of spermatogenic cells and Sertoli cells due to TiO2 NPs. Furthermore, these were associated with decreased expression of Tyro3 (-18.16 to -66.6%), Axl (-14.7 to -57.99%), Mer (-7.98 to -72.62%), and IκB (-11.25 to -63.16%), suppression of cytokine signaling (SOCS) 1 (-21.99 to -73.8%) and SOCS3 (-8.11 to -34.86%), and increased expression of Toll-like receptor (TLR)-3 (21.4-156.03%), TLR-4 (37.0-109.87%), nuclear factor-κB (14.75-69.34%), interleukin (IL)-lß (46.15-123.08%), IL-6 (2.54-81.98%), tumor necrosis factor-α (6.95-88.39%), interferon (IFN)-α (2.54-37.25%), and IFN-ß (10.19-80.56%), which are involved in the immune environment in the testis. The findings showed that reproductive toxicity of male mice induced by exposure to endocrine disruptor TiO2 NPs may be associated with biomarkers of impairment of immune environment or dysfunction of TAM/TLR3-mediated signal pathway in mouse testitis. Therefore, the potential risks to reproductive health should be attended, especially in those who are occupationally exposed to TiO2 NPs.

Syphilitic orchitis mimicking a testicular tumor in a clinically occult HIV-infected young man: a case report with emphasis on a challenging pathological diagnosis.

BACKGROUND: Syphilitic orchitis is a rare manifestation of gumma in tertiary syphilis, microscopically typically characterized by multiple discrete granulomas with central necrosis and peripheral fibrosis. We report a case of syphilitic orchitis mimicking a testicular tumor with atypical histological features. CASE PRESENTATION: A 33-year-old clinically occult HIV-infected man had a testicular tumor. A radical orchiectomy was performed, and a histological examination showed an acute and chronic interstitial inflammatory lesion as well as spindle cell proliferation, without typical gumma formation, necessitating the differential diagnosis having to be made from a panel of etiological factors. Syphilitic orchitis was confirmed by both an immunohistochemical study and PCR testing for the Treponema pallidum DNA polymerase I gene using paraffin-embedded tissues. However, serology tests, including both the Venereal Disease Research Laboratory (VDRL) test and Treponema pallidum partical agglutination (TTPA), demonstrated false-negative results. CONCLUSION: Syphilitic orchitis may present atypical and unusual histological features, and should be included in the differential diagnoses of nonspecific interstitial inflammatory lesions of the testes by pathologists, especially in immunocompromised patients.

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion.

BACKGROUND: Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO. OBJECTIVE: The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion. METHOD AND RESULTS: EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect. CONCLUSIONS: We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.