RESUMEN
Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.
RESUMEN
The main functions of the testis, steroidogenesis and spermatogenesis, depend on the endocrine axis and systemic and local tolerance mechanisms. Infectious or non-infectious diseases may disturb testicular immune regulation causing infertility. Literature has illustrated that bacterial and viral infections lead to autoimmune infertility: either sperm antibodies or autoimmune epidydimo-orchitis. However, little is known about the association between non-infectious testicular pathologic diseases and autoimmunity. Here we review the novel aspect of varicocele and testicular cord torsion pathology linked to inflammation and discuss how immune factors could contribute to or modulate autoimmunity in ipsi- and contralateral testis.
RESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes/patología , Neovascularización Patológica , Testículo/irrigación sanguínea , Testículo/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/prevención & control , Bevacizumab/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Orquitis/inmunología , Orquitis/metabolismo , Orquitis/prevención & control , Codorniz/embriología , Ratas Wistar , Transducción de Señal , Testículo/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Infection and inflammation of the male reproductive tract are relevant causes of infertility. Inflammatory damage occurs in the special immunosuppressive microenvironment of the testis, a hallmark termed testicular immune privilege, which allows tolerance to neo-antigens from developing germ cells appearing at puberty, long after the establishment of systemic immune tolerance. Experimental autoimmune orchitis (EAO) is a well-established rodent model of chronic testicular inflammation and organ specific autoimmunity that offers a valuable in vivo tool to investigate the pathological and molecular mechanisms leading to the breakdown of the testicular immune privilege. The disease is characterized by the infiltration of the interstitium by immune cells (mainly macrophages, dendritic cells, and T cells), formation of autoantibodies against testicular antigens, production of pro-inflammatory mediators such as NO, MCP1, TNFα, IL6, or activins and dysregulation of steroidogenesis with reduced levels of serum testosterone. EAO leads to sloughing of germ cells, atrophic seminiferous tubules and fibrotic remodeling, parameters all found similarly to changes in human biopsies from infertile patients with inflammatory infiltrates. Interestingly, testosterone supplementation during the course of EAO leads to expansion of the regulatory T cell population and inhibition of disease development. Knowledge of EAO pathogenesis aims to contribute to a better understanding of human testicular autoimmune disease as an essential prerequisite for improved diagnosis and treatment.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Orquitis/inmunología , Animales , Humanos , MasculinoRESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes/patología , Epididimitis/patología , Antagonistas de los Receptores Histamínicos H1/farmacología , Cetotifen/farmacología , Mastocitos/efectos de los fármacos , Orquitis/patología , Torsión del Cordón Espermático/patología , Testículo/efectos de los fármacos , Animales , Enfermedades Autoinmunes/inmunología , Recuento de Células , Epidídimo/efectos de los fármacos , Epidídimo/inmunología , Epidídimo/patología , Epididimitis/inmunología , Hipersensibilidad Tardía , Inmunidad Celular/efectos de los fármacos , Masculino , Mastocitos/inmunología , Mastocitos/patología , Orquitis/inmunología , Ratas , Índice de Severidad de la Enfermedad , Torsión del Cordón Espermático/inmunología , Testículo/inmunología , Testículo/patología , VacunaciónRESUMEN
Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.
Asunto(s)
Privilegio Inmunológico , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Testículo/enzimología , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Epidídimo/patología , Privilegio Inmunológico/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Quinurenina/análisis , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/metabolismo , Masculino , Orquitis/metabolismo , Orquitis/patología , Ratas , Ratas Wistar , Células de Sertoli/citología , Células de Sertoli/metabolismo , Índice de Severidad de la Enfermedad , Testículo/metabolismo , Testículo/patología , Triptófano/análogos & derivados , Triptófano/análisis , Triptófano/metabolismo , Triptófano/farmacología , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismoRESUMEN
Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology.
Asunto(s)
Diferenciación Celular , Epidídimo/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Glicoproteínas de Membrana/deficiencia , Proteínas de Plasma Seminal/genética , Animales , Epidídimo/patología , Epitelio/metabolismo , Epitelio/patología , Infertilidad Masculina/patología , Masculino , Ratones , Ratones NoqueadosAsunto(s)
Genitales Masculinos/inmunología , Infertilidad Masculina/inmunología , Inflamación/inmunología , Autoinmunidad , Genitales Masculinos/patología , Humanos , Infertilidad Masculina/patología , Infertilidad Masculina/prevención & control , Inflamación/complicaciones , Inflamación/patología , MasculinoRESUMEN
In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/deficiencia , Espermatogénesis , Espermatozoides/fisiología , Animales , Peso Corporal , Epidídimo/metabolismo , Femenino , Fertilidad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Espermatozoides/ultraestructura , Testículo/metabolismoRESUMEN
Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) have been described in several tissues, where they regulate cellular proliferation, differentiation, motility and apoptosis. Although FGF2/FGFRs expression in the male reproductive tract has been reported, there is scarce evidence on their presence in the female reproductive tract and their involvement in the modulation of sperm function. Therefore, the objective of this study was to determine the expression of FGF2 in the female reproductive tract and to assess the role of the FGF2/FGFRs system in the regulation of sperm physiology using the murine model. FGF2 was detected in uterus and oviduct protein extracts, and it was immunolocalized in epithelial cells of the uterus, isthmus and ampulla, as well as in the cumulus oophorus-oocyte complex. The receptors FGFR1, FGFR2, FGFR3 and FGFR4 were immunodetected in the flagellum and acrosomal region of sperm recovered from the cauda epididymis. Analysis of testis sections showed the expression of FGFRs in germ cells at different stages of the spermatogenesis, suggesting the testicular origin of the sperm FGFRs. Sperm incubation with recombinant FGF2 (rFGF2) led to increased sperm motility and velocity and to enhanced intracellular Ca2+ levels and acrosomal loss compared to the control. In conclusion, this study shows that FGF2 is expressed in tissues of the female reproductive tract. Also, the fact that functional FGFRs are present in mouse sperm and that rFGF2 affects sperm motility and acrosomal exocytosis, suggests the involvement of this system in the in vivo regulation of sperm function.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Genitales Femeninos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Espermatozoides/fisiología , Animales , Femenino , Masculino , Ratones , Testículo/metabolismoRESUMEN
Galectin-1 (Gal-1), a proto-type member of galectin family, is highly expressed in immune privileged sites, including the testis. However, in spite of considerable progress the relevance of endogenous and exogenous Gal-1 in testis pathophysiology have not yet been explored. Here we evaluated the in vivo roles of Gal-1 in experimental autoimmune orchitis (EAO), a well-established model of autoimmune testicular inflammation associated with subfertility and infertility. A significant reduction in the incidence and severity of EAO was observed in mice genetically deficient in Gal-1 (Lgals1(-/-)) versus wild-type (WT) mice. Testicular histopathology revealed the presence of multifocal testicular damage in WT mice characterized by an interstitial mononuclear cell infiltrate and different degrees of germ cell sloughing of seminiferous tubules. TUNEL assay and assessment of active caspase-3 expression, revealed the prevalence of apoptotic spermatocytes mainly localized in the adluminal compartment of seminiferous tubules in EAO mice. A significant increased number of TUNEL-positive germ cells was detected in EAO testis from WT compared with Lgals1(-/-) mice. In contrast, exogenous administration of recombinant Gal-1 to WT mice undergoing EAO attenuated the severity of the disease. Our results unveil a dual role of endogenous versus exogenous Gal-1 in the control of autoimmune testis inflammation.
Asunto(s)
Apoptosis/inmunología , Enfermedades Autoinmunes/inmunología , Galectina 1/inmunología , Orquitis/inmunología , Túbulos Seminíferos/inmunología , Espermatocitos/inmunología , Animales , Apoptosis/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Galectina 1/genética , Masculino , Ratones , Ratones Noqueados , Orquitis/genética , Orquitis/patología , Túbulos Seminíferos/patología , Espermatocitos/patologíaRESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Inhibidores Enzimáticos/farmacología , Células Intersticiales del Testículo/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Orquitis/prevención & control , Espermatozoides/metabolismo , Acetilcisteína/farmacología , Adyuvantes Inmunológicos , Animales , Apoptosis/efectos de los fármacos , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Técnicas de Cocultivo , Mezclas Complejas , Regulación de la Expresión Génica , Humanos , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/inmunología , Células Intersticiales del Testículo/patología , Hormona Luteinizante/sangre , Hormona Luteinizante/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Óxido Nítrico/biosíntesis , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Orquitis/inducido químicamente , Orquitis/inmunología , Orquitis/patología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Espermatozoides/efectos de los fármacos , Espermatozoides/inmunología , Espermatozoides/patología , Testosterona/biosíntesis , Testosterona/metabolismo , Triazenos/farmacologíaRESUMEN
PROBLEM: The phenotype and function of regulatory T (Treg) cells in rats with experimental autoimmune orchitis (EAO) was evaluated. METHOD OF STUDY: Distribution of Treg cells in draining lymph nodes from the testis (TLN) and from the site of immunization (ILN) was analysed by immunohistochemistry. The number, phenotype and proliferative response (5-bromo-2'-deoxyuridine incorporation) of Treg cells were evaluated by flow cytometry and Treg cell suppressive activity by in vitro experiments. TGF-ß expression was evaluated by immunofluorescence. RESULTS: Absolute numbers of Treg cells and BrdU+ Treg cells were increased in LN from experimental compared to normal and control rats. These cells displayed a CD45RC(-), CD62L(-), Helios(+) phenotype. CD4(+) CD25(bright) T cells from TLN of experimental rats were able to suppress T cell-proliferation more efficiently than those derived from normal and control rats. Cells isolated from TLN and ILN expressed TGF-ß. CONCLUSION: Our results suggest that Treg cells with a memory/activated phenotype proliferate extensively in the inflamed testis and LN of rats with EAO exhibiting an enhanced suppressive capacity. TGF-ß may be involved in their suppressive mechanism.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Ganglios Linfáticos/inmunología , Orquitis/inmunología , Linfocitos T Reguladores/inmunología , Testículo/inmunología , Animales , Factores de Transcripción Forkhead/inmunología , Humanos , Masculino , Fenotipo , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
The purpose of this review is to describe how the immune cells present in the testis interact with the germinal epithelium contributing to survival or apoptosis of germ cells (GCs). Physiologically, the immunosuppressor testicular microenvironment protects GCs from immune attack, whereas in inflammatory conditions, tolerance is disrupted and immune cells and their mediators respond to GC self antigens, inducing damage of the germinal epithelium. Considering that experimental models of autoimmune orchitis have clarified the local immune mechanisms by which protection of the testis is compromised, we described the following topics in the testis of normal and orchitic rats: (1) cell adhesion molecule expression of seminiferous tubule specialized junctions and modulation of blood-testis barrier permeability by cytokines (2) phenotypic and functional characteristics of testicular dendritic cells, macrophages, effector and regulatory T cells and mast cells and (3) effects of pro-inflammatory cytokines (TNF-α, IL-6 and FasL) and the nitric oxide-nitric oxide synthase system on GC apoptosis.
RESUMEN
Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.
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Barrera Hematotesticular/metabolismo , Interleucina-6/farmacología , Interleucina-6/fisiología , Ocludina/metabolismo , Orquitis/inmunología , Células de Sertoli/ultraestructura , Uniones Estrechas/fisiología , Animales , Enfermedades Autoinmunes/metabolismo , Permeabilidad de la Membrana Celular , Proliferación Celular , Células Cultivadas , Masculino , Orquitis/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/química , Testículo/efectos de los fármacos , Testículo/inmunología , Uniones Estrechas/efectos de los fármacosRESUMEN
Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas-Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.
Asunto(s)
Apoptosis , Enfermedades Autoinmunes/patología , Proteína Ligando Fas/metabolismo , Orquitis/patología , Espermatozoides/metabolismo , Espermatozoides/patología , Testículo/patología , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Barrera Hematotesticular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Masculino , Orquitis/metabolismo , Permeabilidad/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Solubilidad , Espermatozoides/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.
Asunto(s)
Acrosina/inmunología , Autoanticuerpos/inmunología , Anticoncepción Inmunológica , Precursores Enzimáticos/inmunología , Fertilidad/inmunología , Inmunización , Vacunas Anticonceptivas/inmunología , Vacunas de ADN/inmunología , Acrosina/genética , Animales , Precursores Enzimáticos/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Interacciones Espermatozoide-Óvulo/inmunología , Espermatozoides/inmunología , Vacunas Anticonceptivas/genética , Vacunas de ADN/genéticaRESUMEN
AIMS: We previously reported that recombinant human Secretory Leukocyte Protease Inhibitor (SLPI) inhibits mitogen-induced proliferation of human peripheral blood mononuclear cells. To determine the relevance of this effect in vivo, we investigated the immuno-regulatory role of SLPI in an experimental autoimmune orchitis (EAO) model. MAIN METHODS: In order to increase SLPI half life, poly-ε-caprolactone microspheres containing SLPI were prepared and used for in vitro and in vivo experiments. Multifocal orchitis was induced in Sprague-Dawley adult rats by active immunization with testis homogenate and adjuvants. Microspheres containing SLPI (SLPI group) or vehicle (control group) were administered s.c. to rats during or after the immunization period. KEY FINDINGS: In vitro SLPI-release microspheres inhibited rat lymphocyte proliferation and retained trypsin inhibitory activity. A significant decrease in EAO incidence was observed in the SLPI group (37.5%) versus the control group (93%). Also, SLPI treatment significantly reduced severity of the disease (mean EAO score: control, 6.33±0.81; SLPI, 2.72±1.05). In vivo delayed-type hypersensitivity and ex vivo proliferative response to testicular antigens were reduced by SLPI treatment compared to control group (p<0.05). SIGNIFICANCE: Our results highlight the in vivo immunosuppressive effect of released SLPI from microspheres which suggests its feasible therapeutic use.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inmunidad Celular/efectos de los fármacos , Factores Inmunológicos/farmacología , Orquitis/tratamiento farmacológico , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Animales , Enfermedades Autoinmunes/inmunología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Composición de Medicamentos , Hipersensibilidad Tardía/tratamiento farmacológico , Inmunidad Celular/inmunología , Terapia de Inmunosupresión , Linfocitos/efectos de los fármacos , Masculino , Microesferas , Orquitis/inmunología , Ratas , Ratas Sprague-Dawley , Proteínas RecombinantesRESUMEN
Experimental autoimmune orchitis (EAO) is a useful model to study chronic testicular inflammation and infertility. EAO is characterized by severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. We previously reported an increase in CD4+ and CD8+ effector T cells in the testes of rats with EAO. Since cytokine patterns determine T cell effector functions, in the present work we analyzed the cytokines expressed by these cells during disease development. By flow cytometry, we detected an increase in the number of tumor necrosis factor-α (TNF) and interferon -γ (IFNG)-producing CD4+ T cells in the testis at EAO onset. As the severity of the disease progressed, these cells declined while CD8+ T cells producing TNF and IFNG increased, with the predominance of IFNG expression. As a novel finding, we identified by immunofluorescence CD4+ interleukin 17 (IL17)+ and CD8+ IL17+ cells in the testes of EAO rats, with CD4+ and CD8+ T cells predominating at the onset and in the chronic phase of EAO respectively. Moreover, IL17 (western blot) and IL23 content (ELISA) increased in EAO, with maximum levels in the chronic phase. These results suggest the involvement of CD4+ T helper (Th) 1 and Th17 subsets as co-effector cells governing EAO onset, as well as the central contribution of CD8+ T cells producing Th1 and Th17 cytokines in the maintenance of chronic inflammation. The expression of T-bet and RORγt (western blot) in the testis over the course of disease also supports the presence of Th1 and Th17 cells in the testes of EAO rats.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-17/metabolismo , Interleucina-1/metabolismo , Orquitis/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Western Blotting , Enfermedad Crónica , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas para Inmunoenzimas , Masculino , Orquitis/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas de Dominio T Box/metabolismoRESUMEN
Although the testis is an immunoprivileged organ, infection and inflammation may overwhelm immunosuppressor mechanisms inducing autoimmune reactions against spermatic antigens which result in aspermatogenesis and infertility. Autoimmune orchitis is a model of chronic inflammation useful for elucidating pathogenic mechanisms involved in testicular damage. We developed experimental autoimmune orchitis (EAO) in rats by active immunization with spermatic antigens and adjuvants characterized by interstitial inflammatory cell infiltrate, apoptosis and sloughing of germ cells. Quantitative and phenotypic analysis of testis-infiltrating cells revealed an increased number of macrophages, dendritic cells and T cell subsets that include effector Th1 and Th17 cells as well as Foxp3+ regulatory T cells (T(regs)). Immune cells secrete pro-inflammatory cytokines, TNF-α, IFN-γ, IL-6, IL-12, IL-17 and IL-23, which disrupt the normal testicular immunosuppressor microenvironment. As a consequence, increased numbers of germ cells expressing TNFR1, IL-6R and Fas undergo apoptosis. Functional analysis shows that dendritic cells in EAO testis have a mature immunogenic status and are able to induce immune responses to testicular antigens. We also observed that T(regs) accumulated in the inflamed testis are functionally suppressive but are unable to downregulate inflammation, probably due to the function limiting effect of pro-inflammatory cytokines.