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Busulfan is an alkylating agent commonly used in cancer chemotherapy. It is also an ideal agent for preparing transplant recipients of spermatogonial stem cells because of its high efficiency in destroying endogenous germ cells in the testis. However, its toxicity mechanism remains unclear, affecting its clinical use and applications. Based on reports of busulfan causing orchitis and a previous study by our team, this article summarizes the relationship between busulfan and orchitis, cytokines, the blood-testis barrier, and the cytoskeleton, unravels the regulatory pathways and mechanism behind busulfan-induced orchitis, and reveals the molecular mechanism underlying impaired spermatogenic function in orchitis, providing new ideas for the clinical application of busulfan while reducing its testicular toxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dong Wang reports financial support was provided by Jilin Agricultural University. Dong Wang reports financial support was provided by Foundation for Innovative Research Groups of the National Natural Science Foundation of China., (Copyright © 2022. Published by Elsevier Inc.)

A man in his late 50s, with uncontrolled type 2 diabetes mellitus (T2DM) and morbid obesity, presented to the hospital with complicated epididymo-orchitis. The onset of symptoms (scrotal pain, erythema and swelling) occurred after the use of empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, for 2 months. His baseline antidiabetic medications were insulin, glipizide and metformin. Initially, he had failed treatment of epididymo-orchitis with oral levofloxacin for 3 weeks, followed by 2 weeks of doxycycline therapy. At the presentation to the hospital, an ultrasound of the scrotum revealed scrotal and right testicular abscess. The patient underwent right inguinal orchiectomy. Postoperatively, pus culture was positive for Enterococcus faecalis and Candida glabrata , and hence, he was treated with oral antibiotics including high-dose antifungal medications. Adequate wound care and regular follow-up demonstrated resolution of infection. This case highlights the risk of severe urogenital infection associated with the use of SGLT2 inhibitors in the setting of uncontrolled T2DM., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

As an important source of air pollutant, airborne particulate matter (PM) has become a major threat to public health. Orchitis is characterized by acute or chronic testicular inflammation and is a primary cause of male infertility. Although accumulating evidence indicates that PM exposure is associated with increased male infertility rates, the mechanism by which PM is involved is not well understood. Here, we found that short-term PM exposure activated NF-κB signaling in mouse Leydig cells and testes and leading to asymptomatic orchitis. Analyzing the mitochondrial abundance and cGAMP levels in PM exposed mouse Leydig cells, we found that PM exposure induced mitochondrial injury and mtDNA release, leading to inflammation via the cGAS-STING axis. We also found that aspirin-induced acetylation of cGAS inhibited the inflammation in mice after PM exposure, especially in the testes. Moreover, aspirin pretreatment rescued offspring growth in PM-exposed mice. In summary, our study not only provides evidence that PM-induced asymptomatic orchitis in mice may be amenable to aspirin pre-treatment by acetylating cGAS, but also provides a potential explanation for male infertility caused by air pollutants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhu, Chen, Qiu, Liu, Mwangi, Zhao, Ding, Lai and Jin.)

Background: Phthalates such as di (2-ethylhexyl) phthalate (DEHP) are well known exogenous substances, disrupting reproductive system function and structure. The current research demonstrated the effect of ellagic acid (EA) on DEHP-induced testicular injury in mice., Methods: Thirty-five healthy adult male mice were randomly divided to five groups; normal saline receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) and EA receiving groups (25, 50 and 100 mg/kg/day, p.o.). Treatment duration of animals was 14 days. Body and testes weights and sperm characteristics and histological changes of testes were evaluated. Serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were analyzed. In the testicular tissue, oxidative/nitrosative stress markers and inflammatory cytokine levels were measured., Results: Ellagic acid significantly reduced DEHP-induced reduction of body and testes weights. The DEHP-induced reduction of spermatogonia, primary spermatocyte and sertoli cells numbers as well as reduction of sperm vitality and progressive motility were reversed by EA. Furthermore, EA inhibited DEHP-induced alterations in serum hormone levels. These effects were associated with the reduction of DEHP-induced increased level of oxidative stress and inflammatory responses., Conclusions: Ellagic acid considerably inhibits testicular toxicity of DEHP through reducing oxidative/nitrosative stress and inflammatory responses. Our data suggest that EA may be considered as a promising agent to inhibit male reproductive toxicity induced by endocrine disrupting chemicals such as DEHP., (© 2021. The Author(s).)

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis., (© 2021 Wiley Periodicals LLC.)

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) have anti-inflammatory effects. One specific PUFA, alpha-linolenic acid (ALA), shows both anti-inflammatory and antioxidant properties. In the testes, inflammatory mediators are known to increase when orchitis is induced by bacterial lipopolysaccharide (LPS). This study aimed to determine whether the anti-inflammatory properties of ALA could have a protective effect against LPS-induced orchitis in mice. The mice were divided into untreated control, orchitis and ALA-treated orchitis groups. Orchitis was induced by intraperitoneal injection of LPS. The ALA-treated group was administered ALA by gavage three days before intraperitoneal LPS injection. Cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), inducible nitric oxide synthase (iNOS) enzymes and nuclear factor kappa-B (NF-κB) in the testes, as well as serum interleukin 6 (IL-6) and tumour necrosis factor-alpha (TNF-α), were analysed using enzyme-linked immunosorbent assay (ELISA) tests. LPS administration increased the expression of several inflammatory mediators, including IL-6, TNF-α and NF-κB, as well as the COX-2, cPLA2 and iNOS enzymes. ALA administration significantly prevented the LPS-induced increases in these inflammatory mediators and enzymes (p < .05). The anti-inflammatory and antioxidant effects of ALA may make it a useful candidate for the treatment of orchitis caused by bacterial LPS., (© 2020 Blackwell Verlag GmbH.)

Objective: The present study is aimed at investigating the anti-inflammatory, antioxidative, and antiapoptotic effects of methane on lipopolysaccharide- (LPS-) induced acute orchitis and its potential mechanisms., Methods: Adult male rats were intraperitoneally (i.p.) injected with methane-rich saline (MS, 20 mL/kg) following LPS (5 mg/kg, i.p.). The survival rate was assessed every 12 h until 72 h after LPS induction, and surviving rats were sacrificed for further detection. The wet/dry ( W / D ) ratio was determined, and testicular damage was histologically assessed. Inflammatory cytokines in the testes and serum, including interleukin-1 β (IL-1 β ), IL-6, IL-10, and tumor necrosis factor- α (TNF- α ), were measured using ELISA and RT-qPCR. Oxidative stress was evaluated by the level of superoxide dismutase (SOD) and malondialdehyde (MDA). Testicular apoptosis was detected via TUNEL staining. The expression of prokineticin 2 (PK2)/prokineticin receptor 1 (PKR1) was also analyzed using RT-qPCR, western blotting, and immunohistochemistry., Results: It was found that methane significantly prolonged rat survival, decreased the W/D ratio, alleviated LPS-induced histological changes, and reduced apoptotic cells in the testes. Additionally, methane suppressed and promoted the production of pro- and anti-inflammatory cytokines, respectively. Furthermore, methane significantly increased SOD levels, decreased MDA levels, and decreased testicular expression of PK2 and PKR1. Therefore, methane exerts therapeutic effects on acute orchitis and might be a new and convenient strategy for the treatment of inflammation-related testicular diseases., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Chao Huang et al.)

This study aimed to compare acacetin adverse effect besides a cisplatin low dose on male reproductive and urinary systems on mice. In this study, 36 male Balb/c mice were received Dimethyl sulfoxide, cisplatin (1 mg/kg) or acacetin (10, 25, 50 mg/kg) for 3 days, while the sixth group, treated with acacetin (50 mg/kg) for 10 days. All treatments were done consequence daily and intraperitoneally. Histological and biochemical factors to male reproductive and urinary systems were assayed. Only in cisplatin exposed group, significant differences were seen with the others. So that, some reproductive criteria were significantly decreased; serum levels of follicle-stimulating hormone, luteinizing hormone, testosterone, sperm parameters, the diameters of seminiferous tubules, Johnsen's score and from the urinary system; renal corpuscles' space, Mg 2+ concentration (p < .01). Moreover, significant increases were seen in serum levels of tumour necrosis factor-alpha, Blood urea nitrogen, creatinine, testis myeloperoxidase activity and tumour necrosis factor-alpha expression, kidney histological damage and renal corpuscle diameter (p < .01). Cisplatin exposure disrupts histological and functional characteristics of either male reproductive or urinary systems, suggesting by inflammation-promoting. Acacetin does not induce any harmful impact on these two systems and could be considered as a safe flavonoid by high dose and prolonged usage., (© 2019 Blackwell Verlag GmbH.)

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-β) were significantly increased. From these results, it can be concluded that autoimmune orchitis and IL-17A are implicated in F-induced testicular inflammation.

It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon β (IFNβ) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.

Objective: To search for a method of establishing a reliable mouse model of orchitis and investigate the association of orchitis with the activation of the inflammasome., Methods: We equally randomized 40 adult male KM mice into groups A (sham operation), B (intraperitoneal injection of lipopolysaccharide ［LPS］), C (unilateral testicular injection of glacial acetic acid ［GAA］), and D (unilateral testicular injection of LPS). At 3 weeks after modeling, we measured the sperm concentration and percentage of progressively motile sperm (PMS) in the epididymis by computer-assisted semen analysis, observed the pathological changes in the testis tissue by HE staining, and determined the expressions of the Caspase-1 and interleukin (IL)-1β proteins by Western blot., Results: The sperm concentration in the epididymis was significantly decreased in groups B (［25.74 ± 3.19］ ×10⁶/ml), C (［17.16 ± 4.41］ ×10⁶/ml) and D (［16.92 ± 7.13］ ×10⁶/ml) as compared with that in group A (［28.20 ± 1.63］ ×10⁶/ml) (all P < 0.05), even more significantly in B than in C and D (P < 0.01), and so was PMS in groups B (［29.57 ± 2.16］%), C (［18.10 ± 2.38］%) and D (［7.34 ± 1.63］%) in comparison with group A (［59.34 ± 1.10］%) (P < 0.01), even more significantly in B and C than in D (P < 0.01). Light microscopy revealed different degrees of pathological changes in the testis tissue, most significant in group D, followed by C and B. Both the expressions of Caspase-1 and IL-1β were remarkably up-regulated in groups B, C and D compared with those in group A (P < 0.01), even more markedly in D than in B and C (P < 0.05)., Conclusions: Unilateral testicular injection of LPS is a more efficient method than either unilateral testicular injection of GAA or intraperitoneal injection of LPS for establishing the mouse model of orchitis. Orchitis may be pathologically associated with the activation of the NLRP3 inflammasome.

Background: Immune checkpoint inhibitors such as pembrolizumab and nivolumab have emerged as active treatment options for patients with many cancers, including metastatic melanoma, but can also cause symptomatic or life-threatening immune-related adverse events, including encephalitis. Epididymitis and orchitis are rare complications of these therapies., Case Presentation: We describe herein a patient with metastatic melanoma who developed epididymo-orchitis followed by encephalitis while receiving pembrolizumab. The patient developed testicular pain and fever after his third dose of pembrolizumab; ultrasound evaluation demonstrated bilateral epididymo-orchitis. He then developed headaches, fever, and altered mental status over the next week and was admitted to the hospital. Lumbar puncture revealed inflammatory changes consistent with meningoencephalitis; he did not improve with broad-spectrum antibiotics, and an extensive workup for infectious etiologies, including cerebrospinal fluid testing using a clinical metagenomic next-generation sequencing assay, was negative. He received high-dose steroids for suspected autoimmune encephalitis, and both his orchitis and meningoencephalitis improved rapidly after one dose. He fully recovered after a 5-week taper of oral steroids., Discussion: Here, we report a case of epididymo-orchitis complicating immune checkpoint inhibitor therapy. This patient subsequently developed severe encephalitis but rapidly improved with steroids. Clinicians should be aware of rare complications of these agents., Key Points: Epididymo-orchitis is a rare and potentially life-threatening complication of anti-programmed death protein 1 (anti-PD-1) therapy.For patients on anti-PD-1 therapy who develop either epididymo-orchitis or epididymitis without clear infectious cause, immune-related adverse events should be considered in the differential diagnosis.If severe, epididymo-orchitis related to anti-PD-1 therapy may be treated with high-dose corticosteroids., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2019.)

Increasing evidence shows that 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) enhances inflammation, and inflammation has a significant negative impact on fertility. Therefore, the aim of this study was to investigate the effects of TCDD on testis inflammation. Pregnant mice and primary Sertoli cells were treated with TCDD, and male offspring and Sertoli cells were treated with lipopolysaccharides(LPS). We then measured testis apoptotic cells, proinflammatory cytokines, and observed the Klotho/PDLIM2/p65 pathway. In vivo results revealed that TCDD further enhanced LPS-increased testis apoptotic cells and concentrations of testicular proinflammatory cytokines (IL1β, IL18, and IL12) (p < 0.05). An in vitro investigation showed the levels of proinflammatory cytokines were increased in TCDD + LPS-treated cells compared with LPS-treated cells (p < 0.05). Compared with the LPS-treated cells, expression of Klotho and PDLIM2 was significantly decreased in TCDD + LPS-treated cells (p < 0.05), while expression of p65 and NLRP3 were significantly increased in the cotreatment cells (p < 0.05). However, the addition of Klotho to the TCDD + LPS-cotreated cells significantly increased PDLIM2 and decreased p65 activation and NLRP3 (p < 0.05). Meanwhile, mRNA levels and the secretion of proinflammatory cytokines were both suppressed by exogenous Klotho (p < 0.05). Administration of Klotho decreased TCDD + LPS-induced cytokines and apoptosis in mice (p < 0.05). Taken together, TCDD may increase testicular inflammation by affecting the secretion of proinflammatory cytokines in Sertoli cells via the Klotho/PDLIM2/p65 pathway, which influences the testicular microenvironment and induces germ cell apoptosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although sodium nitrite has been widely used as food preservative, building bases of scientific evidence about nitrite continues to oppose the general safety in human health. Moreover, thymoquinone (TQ) has therapeutic potential as antioxidant, anti-inflammatory, antibacterial and anticancer. Therefore, we investigated the effects of both sodium nitrite and TQ on testicular tissues of rats. Forty adult male Sprague Dawley rats were used. They received either 80 mg kg(-1) sodium nitrite or 50 mg kg(-1) TQ daily for twelve weeks. Serum testosterone was measured. Testis were weighed and the testicular tissue homogenates were used for measurements of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-6, IL10, caspase-3, caspase-8 and caspase-9. Sodium nitrite resulted in significant reduction in serum testosterone concentration and elevation in testis weight and Gonado-Somatic Index. We found significant reduction in testicular tissues levels of IL-4 and IL-10 associated with elevated levels of TNF-α, IL-1β, IL-6, caspase-3, caspase-8 and caspase-9. In conclusion, chronic oral sodium nitrite induced changes in the weight of rat testis accompanied by elevation in the testicular tissue level of oxidative stress markers and inflammatory cytokines. TQ attenuated sodium nitrite-induced testicular tissue damage through blocking oxidative stress, restoration of normal inflammatory cytokines balance and blocking of apoptosis., (© 2015 Blackwell Verlag GmbH.)

Exposure to di-(2-ethylhexyl) phthalate (DEHP) induces spermatogenic disturbance (SD) through oxidative stress, and affects the immune system by acting as an adjuvant. Recently, we reported that in mice, a low dose of DEHP, which did not affect spermatogenesis, was able to alter the testicular immune microenvironment. Experimental autoimmune orchitis (EAO) can be induced by repeated immunization with testicular antigens, and its pathology is characterized by production of autoantibodies and SD. In the present study, we investigated the effect of a low-dose DEHP on the susceptibility of mice to EAO. The exposure to DEHP-containing feed (0.01%) caused a modest functional damage to the blood-testis barrier (BTB) with an increase in testicular number of interferon gamma (IFN-γ)-positive cells and resulted in the production of autoantibodies targeting haploid cells, but did not affect spermatogenesis. While only single immunization with testicular antigens caused very mild EAO, the concurrent DEHP exposure induced severe EAO with significant increases in number of interferon gamma-positive cells and macrophages, as well as lymphocytic infiltration and serum autoantibody titer accompanied by severe SD. To summarize, the exposure of mice to the low-dose DEHP does not induce significant SD, but it may cause an increase in IFN-γ positive cells and modest functional damage to the BTB in the testis. These changes lead to an autoimmune response against haploid cell autoantigens, resulting in increased susceptibility to EAO., (Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Orchitis (testicular swelling) often occurs during systemic inflammatory conditions, such as sepsis. Interleukin 18 (IL18) is a proinflammatory cytokine and is an apoptotic mediator during endotoxemia, but the role of IL18 in response to inflammation in the testes was unclear. WT and IL18 knockout (KO) mice were injected lipopolysaccharide (LPS) to induce endotoxemia and examined 12 and 48  h after LPS administration to model the acute and recovery phases of endotoxemia. Caspase activation was assessed using immunohistochemistry. Protein and mRNA expression were examined by western blot and quantitative real-time RT-PCR respectively. During the acute phase of endotoxemia, apoptosis (as indicated by caspase-3 cleavage) was increased in WT mice but not in IL18 KO mice. The death receptor-mediated and mitochondrial-mediated apoptotic pathways were both activated in the WT mice but not in the KO mice. During the recovery phase of endotoxemia, apoptosis was observed in the IL18 KO mice but not in the WT mice. Activation of the death-receptor mediated apoptotic pathway could be seen in the IL18 KO mice but not the WT mice. These results suggested that endogenous IL18 induces germ cell apoptosis via death receptor mediated- and mitochondrial-mediated pathways during the acute phase of endotoxemia and suppresses germ cell apoptosis via death-receptor mediated pathways during recovery from endotoxemia. Taken together, IL18 could be a new therapeutic target to prevent orchitis during endotoxemia., (© 2015 Society for Reproduction and Fertility.)

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.

Diethylstilbestrol (DES), an endocrine-disrupting chemical, is an infamous artificial estrogenic compound. Although neonatal exposure to DES has been shown to result in inflammation of the male reproductive system, it has not, to our knowledge, been reported to induce testicular inflammation. Here we report that neonatal exposure to DES caused granulomatous orchitis with spermatogenic disturbance in 4 of 17 ICR male mice at 12 weeks of age. In the animals with spermatogenic disturbance, we observed either seminiferous tubules containing only cells with Sertoli cell features (likely Sertoli cell syndrome), or tubule cells in maturation arrest that contained only spermatogonia and/or spermatocytes. Following neonatal DES exposure, 5-week-old mice exhibited inflammation in cauda epididymis; by 8 weeks, the inflammation had spread to all segments of epididymis but not the testis; by 12 weeks, inflammation of the epididymis was observed in all mice. These data indicated that cauda epididymis has increased sensitivity to neonatal DES exposure compared to other segments of epididymis and testis. The data also implied that neonatal DES exposure-induced inflammation in cauda epididymis extended gradually to the testis via corpus and caput during development.

Intratesticular injection of EsterilSol (zinc gluconate neutralized with arginine) is a chemical sterilant for male dogs sometimes used in population control campaigns. Adverse reactions have been reported in 1% to 4% of treated dogs, but detailed histomorphologic descriptions are lacking. During a behavioral study conducted in the Chilean Patagonia in 2012, severe necrosuppurative orchitis and ulcerative dermatitis were observed in 2 of 36 (6%) dogs sterilized with EsterilSol according to the manufacturer's instructions. Reactions were noted on days 8 and 7 postinjection and required scrotal ablation on days 8 and 13, respectively; neither reaction was associated with the injection site. Although self-trauma following administration may have contributed, the cause of the adverse reactions is uncertain. EsterilSol is a relatively uncomplicated method to sterilize male dogs, but the occurrence of severe adverse reactions several days after administration emphasizes the need for the provision of long-term monitoring and veterinary care during sterilization campaigns using this product., (© The Author(s) 2013.)

The inflammation of the reproductive system can affect reproduction causing partial or complete infertility. It is well known that lipopolysaccharide (LPS) triggers an inflammatory response in the whole organism, including immunologically privileged organs, e.g. the testicles. Adult male TCRalpha-/-, TCRdelta-/-, CD1d-/- and beta2m-/- on B10.PL (H-2(u)) and B10.PL control mice were intraperitonealy (i.p.) injected with lipopolysaccharide (LPS). The animals were killed 24h and 10 days post LPS treatment and their gonads were prepared for microscopic examination. Histological changes in the testes after LPS injection were found only in control B10PL and CD1d-/- mice. The experiments revealed disturbances in Leydig's glands structure, blood vessel dilatation in the interstitial tissue as well as degeneration of seminal tubule epithelium, disruption ofspermatogenesis and subsequent decrease of sperm cell number in the tubule lumen. These changes were noticed mainly 10 days after LPS treatment. Lack of either TCRalphabeta+ CD8+ or TCRgammadelta+ lymphocytes diminishes the response of testicular macrophages to LPS whereas the absence of CD1d-dependent NKT cells does not affect macrophage reactivity.

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood-testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ-specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2 ) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg(-1) body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra-testicular mRNA expression of interleukin (IL)-6, tumor necrosis factor-α and IL-1β was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity., (Copyright © 2012 John Wiley & Sons, Ltd.)

First reported as a vasoactive peptide in the cardiovascular system, intermedin (IMD), also known as adrenomedullin 2 (ADM2), is a hormone with multiple potent roles, including its antioxidant action on the pulmonary, central nervous, cardiovascular and renal systems. Though IMD may play certain roles in trophoblast cell invasion, early embryonic development and cumulus cell-oocyte interaction, the role of IMD in the male reproductive system has yet to be investigated. This paper reports our findings on the gene expression of IMD, its receptor components and its protein localization in the testes. In a rat model, bacterial lippolysaccharide (LPS) induced atypical orchitis, and LPS treatment upregulated the expression of IMD and one of its receptor component proteins, i.e. receptor activity modifying protein 2 (RAMP2). IMD decreased both plasma and testicular levels of reactive oxygen species (ROS) production, attenuated the increase in the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin 6 (IL6) and interleukin 1 beta (IL1β), rescued spermatogenesis, and prevented the decrease in plasma testosterone levels caused by LPS. The restorative effect of IMD on steroidogenesis was also observed in hydrogen peroxide-treated rat primary Leydig cells culture. Our results indicate IMD plays an important protective role in spermatogenesis and steroidogenesis, suggesting therapeutic potential for IMD in pathological conditions such as orchitis.

An outbreak of nine cases of mumps was reported from a total of 97 vaccinated nursing students at two medical colleges in Thailand in 2010, 16-26 days after administration of MMR vaccine containing the L-Zagreb mumps strain. Symptoms ranged in severity from fever and parotid swelling to orchitis. Clinical samples were obtained from seven patients and three were suitable for further study. Sequencing confirmed that the SH gene of the mumps virus in the unpassaged clinical specimens was identical to the L-Zagreb SH gene in the vaccine. Further analysis of the viral genome identified nucleotide position 5170 as a novel mutation which corresponds to an amino acid change in the fusion protein. This study provides another virologically confirmed example of mumps resulting from the L-Zagreb vaccine strain., (Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

We report the case of a 22-year-old Saudi male patient who was treated extensively in the past with various regimens for acute promyelocytic leukaemia that was refractory to all standard treatments. He was ultimately administered Gemtuzumab to induce remission and subjected to an allogeneic bone marrow transplant. However, he developed orchitis, which has not been previously reported with this agent.

Background: Male reproductive tract infection and inflammation are important aetiological factors of infertility. Experimental Autoimmune Orchitis (EAO) is a model of chronic inflammation useful to study mechanisms of inflammatory reactions leading to testicular impairment. EAO is characterised by interstitial cell infiltrate of lymphomonocytes, producers of pro-inflammatory cytokines involved in germ cell apoptosis. Nitric oxide (NO), a free radical promoting immune cell activation and apoptosis, is synthesised by conversion of l-arginine to l-citrulline catalysed by NO synthase (NOS). The NOS isoforms are: constitutively endothelial (e) and neuronal (n) NOS and inducible (i) NOS., Objectives: Although the NO-NOS system was found to be up-regulated by pro-inflammatory mediators in immune and non immune testicular cells, data on its regulation in chronic inflammatory states is lacking., Methods and Results: EAO was induced in rats by active immunisation with spermatic antigens and adjuvants; control (C) rats were injected with adjuvants. Untreated normal (N) rats were also studied. We demonstrated that iNOS, eNOS and nNOS was mainly expressed by interstitial cells in N and C rats and that in EAO NOS was up-regulated and also expressed by tubular cells. Constitutive and inducible NOS content (Western blot) as well as NO production and activity increased in the testis of rats with EAO. iNOS content and activity were selectively up-regulated in the testis of rats with orchitis. Flow cytometric analysis of NOS isoforms in testicular macrophages (M) showed that the percentage of ED1(+)ED2(-) and ED1(+)ED2(+) M subsets, expressing constitutive and iNOS isoforms was significantly higher in EAO, but no change in the percentage of ED1(-)ED2(+) resident M was observed compared to C rats. M from EAO rats also released more NO than C and N rats., Conclusions: In testis of rats with EAO, NO-NOS system was up-regulated and both testicular M and cells from seminiferous tubules contributed to NO increase. NO over production in orchitis was generated mainly by increased iNOS content and activity., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Bacterial Lipopolysaccharide (LPS) induced inflammation is implicated in the infection associated testicular tissue damage. Earlier, using a LPS induced acute endotoxemic rat model, we have shown the involvement of inflammation-induced oxidative stress in the impaired steroidogenesis and spermatogenesis. In the present study, we report a significant induction (more than 2-fold) of stress response proteins HSP-60, HMGB-1 and 2 in the testes, as early as 6 h after LPS injection with a later decrease. This induction of acute stress is closely followed by a significant reduction (74%) in Bcl2/Bax ratio along with leakage of cytochrome c (3 fold increase, p < 0.05) from mitochondria and increased caspase-3 activity levels (2.9 fold, p < 0.05) at 12 h and 24 h post LPS injection respectively. Further studies on PARP cleavage revealed a pattern similar to necrotic death during early periods (3 h to 24 h) and apoptosis at later periods (24 h to 72 h) after LPS treatment. In conclusion, the present study shows the involvement of stress response proteins and mitochondrial dysfunction in LPS-induced germ cell death in male rats.

We report 3 cases of orchitis following vaccination with mumps-measles-rubella (MMR) vaccine, two with an onset within 3 days following vaccination. Orchitis is a common complication of mumps infection, particularly in post-pubertal males, and is also recognized as a very rare complication of mumps vaccination. These cases, discussed together with a comprehensive review of the existing literature regarding post-vaccine orchitis, highlight uncertainty regarding the pathogenesis of post-vaccine orchitis., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Only a few cases of tuberculous epididymo-orchitis after bacillus Calmette-Guérin (BCG) therapy have been published. This report presents a case of granulomatous epididymo-orchitis after intravesical BCG therapy in a patient presenting with pain and unilateral swelling of the scrotal content. This complication should always be considered when a patient presents with these symptoms after BCG therapy. Isoniazid may be used as a first choice of treatment.

Xanthogranulomatous orchitis (XGO) is a rare histological finding that is generally diagnosed at orchiectomy. To date, fewer than 10 reports of XGO exist in the literature. The exact pathogenesis of XGO is unknown and patients may present with a rapid onset of symptoms or a longer, more indolent course. We report a patient who presented with symptoms of epididymoorchitis, leukocytosis, and fever after an instillation of bacillus Calmette-Guérin therapy for bladder cancer.

We report the case of an 18-year-old male patient with epididymo-orchitis following MMR vaccine. Salivary gland involvement, meningitis/encephalitis are well-known complications of the MMR vaccine, but involvement and infection of the testis is a rarity. A search for this complication on the medical databases showed no reported cases in the UK.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

Objective: To describe a case of epididymo-orchitis that developed four years after treatment with intravesical bacillus Calmette-Guérin (BCG) and to review the incidence of this adverse effect., Data Sources: Information about the patient was obtained from the medical chart. A MEDLINE search of English-language literature (from January 1976 to April 1999) was conducted., Study Selection: All case reports of BCG-related epididymo-orchitis were evaluated. Review articles describing complications of BCG therapy for bladder cancer and the prevention and treatment of these complications were reviewed., Data Extraction: Studies were evaluated for reports of BCG-related epididymo-orchitis and its treatment., Data Synthesis: Our case report is compared with others reported in the literature. The incidence of BCG-associated epididymoorchitis is rare., Conclusions: Epididymo-orchitis should be considered as a late complication of BCG therapy for bladder cancer. Proper patient selection may help decrease the risk of complications from BCG therapy.

Two cases of adverse reactions to intravesical treatment with bacillus Calmette-Guérin are reported. The first patient presented with a severe suppurating epididymo-orchitis three months after his last treatment. The second patient developed Reiter's disease with arthritis of one knee and conjunctivitis 1 week after the fourth instillation.

Between May 1993 and November 1995, 71 cryptorchid boys were treated with human chorionic gonadotropin (hCG); 42 were operated upon following unsuccessful hCG treatment. A routine orchiopexy was performed in each case. In 10 cases a testicular biopsy was made during orchipexy within 3 days following hCG treatment; in another 10 biopsies were taken 6 to 9 months after treatment. Testicular biopsies were taken at the time of orchiopexy in 5 cryptorchid boys who were not treated with hCG as a control group. A mild, inflammation-like reaction was found in the cryptorchid testes in the period immediately following the last hCG injections, but those studied 6 to 9 months after the last injection there were no apparent such reactions. In contrast to the inflammation-like reaction, the volume density of blood vessels, interstitial bleeding, and diameter of the seminiferous tubules had not regressed. The numbers of spermatogonia per tubular transverse section and the percentage of tubular transverse sections containing spermatogonia (the fertility index) were increased.

Removal of apoptotic cells from the tissues appears to be a major function of resident tissue macrophages. In order to investigate further the role of testicular macrophages after massive Leydig cell death, adult rats were injected intra-testicularly with liposome-entrapped dichloromethylene diphosphonate (Cl2MDP-lp, right testis) to deplete testicular macrophages, and with NaCl (left testis) as control. Ten days later, the animals were injected intraperitoneally with ethylene dimethane sulphonate (EDS) to induce Leydig cell apoptosis. In macrophage-containing testes there was a 2-fold increase in the number of macrophages on days 1-3 after EDS treatment and Leydig cells were completely eliminated from the interstitium by the second day after treatment. The main differences in the response to Leydig cell death in macrophage-depleted testes were: (1) an early rise in the concentration of small mononuclear, lymphocyte-like cells, (2) a greater influx of circulating monocytes, (3) the existence of variable inflammatory infiltrates on days 3-4, and (4) the disappearance of infiltrating monocytes by day 10. These results suggest that resident macrophages prevent the inflammatory reaction elicited by massive Leydig cell death.

Dynamic MRI tracking of rat T-cells in vivo is performed in rat testicles after labeling isolated rat T-cells in vitro with superparamagnetic dextran-coated iron-oxide particles, BMS180549. Tissue inflammation induced by the local injection of the calcium ionophore, A23187, is used to attract labeled T-cells. Gradient-echo MR images of rat testicles show a statistically significant decrease (4%) of the signal intensity in areas of injection of A23187 as early as 30 min after intravenous infusion of 2 x 10(8) labeled T-cells. The signal change reaches its maximum (6-7% decrease) at about 60-120 min after cell infusion. T2-mapping also shows a decrease of T2 in the areas with A23187. Image quantitation, which includes a chemical-shift effect, significantly enhances the sensitivity for detection of superparamagnetically labeled T-cells. Localization of labeled T-cells in rat testicles has been verified by fluorescence microscopy studies of T-cells co-labeled with a lipophilic fluorescent carbocyanine dye, 1,1-dioctadecyl-3,3,3',3'-tetramethyl-lindocarbocyanine perchlorate. These results represent the first successful demonstration of dynamic tracking of specific cells in vivo using MRI.

Occlusions of the rat efferent ductules were induced by an oral gavage of carbendazim and the capacity for regeneration or recanalization of the ductules was histologically evaluated between 2 and 70 days post-treatment. At 2 days post-treatment, the efferent ductules were occluded by sloughed materials and showed various degrees of inflammation. Severely damaged epithelium showed few regenerative features at later intervals. On the other hand, epithelia with medium inflammation often exhibited irregular epithelial growth along the luminal contents or the formation of multiple abnormal ductules. These abnormal ductules were formed by migrated and original epithelia at the periphery of the occluded lumen at 16 days post-treatment, indicating attempted recanalization. At later time periods, 32 and 70 days post-treatment, the occluded original lumen was filled in by fibrotic connective tissue and surrounded by a series of abnormal ductules. These abnormal ductules were characterized by cuboidal epithelia, a small luminal diameter, fewer cilitated cells than normal, less developed organelles in the epithelial cells, and basal laminae of irregular thickness. However, there was no evidence that occluded ductules formed patent re-connections via abnormal ductules. The results suggest that occluded efferent ductules have the ability to initiate epithelial regrowth and to form new ductules, but the newly formed ductules are abnormal and are not adequate to recover from azoospermia at least at 70 days post-treatment.

Experimental allergic orchitis (EAO) was induced in rats following the injection of homologous testicular homogenate (THr) emulsified in Freund's complete adjuvant. Immune RNA (iRNA) was extracted from the spleen and lymph nodes. Normal guinea-pig peritoneal exudate cells (GP-PEC), when incubated in vitro with iRNA, were able to specifically recognise and respond to the immunising antigens (THr and PPD) as assessed by the direct migration inhibition reaction (MIR). Positive MIR's were also observed when incubated peritoneal exudate cells were tested against testicular homogenates from mice and guinea-pigs. This would confirm the presence of a common testicular antigen(s) in the three species studied. The reaction would appear to be organ-specific, kidney homogenate being unable to cause migration inhibition. GP-PEC incubated with iRNA which had been pretreated with RNAse did not show antigen-specific migration inhibition. Similarly, GP-PEC treated with RNA extracted from non-immunised donor rats did not respond.

The influence of acute and chronic uranium poisoning on the induction and course of experimental autoimmune thyroiditis and orchitis was studied. It has been demonstrated that the testes and thyroid gland are involved in the general pathological process in uranium poisoning. Autoantibodies to the testes and to the thyroid gland were found to circulate in the blood of the poisoned animals. Uranium poisoning caused destructive changes in the testes and in the thyroid, similar to the picture of an autoimmune process. No summation of the effect was observed in the synthesis of autoantibodies during the induction of experimental autoimmune thyroiditis and orchitis on the background of chronic uranium poisoning although the most conspicuous histological changes were found in these groups and were characteristic of a pronounced autoimmune process.

A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions.