RESUMO
BACKGROUND: Peritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21â¯day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs. METHODS: We established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors. RESULTS: Exogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1ß) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin. CONCLUSION: Melatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis. GENERAL SIGNIFICANCE: If transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.
Assuntos
Melatonina , Testículo , Animais , Colágeno/metabolismo , Cricetinae , Ciclo-Oxigenase 2/metabolismo , Masculino , Melatonina/metabolismo , Melatonina/farmacologia , Mesocricetus , Espermatogênese , Testículo/metabolismoRESUMO
We have previously shown an inverse correlation between testicular melatonin concentration and inflammation/oxidative stress-related markers levels in infertile men showing unexplained azoospermia. Here, we evaluated the impact of melatonin oral supplementation (daily 3 mg dose used to treat sleep disorders) in the incidence of local inflammation, oxidative stress, and tubular wall fibrosis development in young and middle-aged infertile adult men. Compared with testes without histological alterations, gonads with morphological abnormalities showed lower melatonin concentration along with increased macrophage numbers, TBARS generation, and expression levels of inflammation-related markers and antioxidant enzymes, as well as tubular wall collagen fibers disorganization and thickening. Melatonin oral supplementation not only increased its own testicular levels but also decreased inflammation- and oxidative stress-related markers levels, and improved the tubular wall aspect. Overall, our work provides insights into the potential benefits of melatonin on the inflammatory and oxidative status in testes of patients suffering from unexplained infertility.
Assuntos
Inflamação/tratamento farmacológico , Melatonina/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Adulto , Antioxidantes/uso terapêutico , Suplementos Nutricionais , Humanos , MasculinoRESUMO
Continuously elevated levels of growth hormone (GH) during life in mice are associated with hepatomegaly due to hepatocytes hypertrophy and hyperplasia, chronic liver inflammation, elevated levels of arachidonic acid (AA) at young ages and liver tumors development at old ages. In this work, the hepatic expression of enzymes involved in AA metabolism, cPLA2α, COX1 and COX2 enzymes, was evaluated in young and old GH-transgenic mice. Mice overexpressing GH exhibited higher hepatic expression of cPLA2α, COX1 and COX2 in comparison to controls at young and old ages and in both sexes. In old mice, when tumoral and non-tumoral tissue were compared, elevated expression of COX2 was observed in tumors. In contrast, exposure to continuous lower levels of hormone for a short period affected COX1 expression only in males. Considering the role of inflammation during liver tumorigenesis, these findings support a role of alterations in AA metabolism in GH-driven liver tumorigenesis.
Assuntos
Fosfolipases A2 do Grupo IV/genética , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Regulação para Cima/genética , Alanina Transaminase/sangue , Animais , Peso Corporal , Bovinos , Proliferação de Células , Feminino , Fosfolipases A2 do Grupo IV/metabolismo , Hepatócitos/citologia , Fígado/anatomia & histologia , Masculino , Camundongos Transgênicos , Tamanho do Órgão , Fosforilação , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/metabolismoRESUMO
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.
Assuntos
Privilégio Imunológico , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Testículo/enzimologia , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Epididimo/patologia , Privilégio Imunológico/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/análise , Linfonodos/enzimologia , Linfonodos/metabolismo , Masculino , Orquite/metabolismo , Orquite/patologia , Ratos , Ratos Wistar , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Índice de Gravidade de Doença , Testículo/metabolismo , Testículo/patologia , Triptofano/análogos & derivados , Triptofano/análise , Triptofano/metabolismo , Triptofano/farmacologia , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismoRESUMO
Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes src/genética , Genes src/fisiologia , Hormônio do Crescimento/genética , Humanos , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Current GH administration protocols imply frequent s.c. injections, resulting in suboptimal compliance. Therefore, there is interest in developing delivery systems for sustained release of the hormone. However, GH has different actions depending on its continuous or pulsatile plasma concentration pattern. GH levels and circulating concentration patterns could be involved in the regulation of epidermal growth factor receptor (EGFR) expression in liver. Aberrant expression of this receptor and/or its hyperactivation has been associated with the pathogenesis of different types of carcinoma. Considering that one of the adverse effects associated with GH overexpression and chronic use of GH is the increased incidence of malignancies, the aim of this study was to analyze the effects of GH plasma concentration patterns on EGFR expression and signaling in livers of mice. For this purpose, GH was administered by s.c. daily injections to produce an intermittent plasma pattern or by osmotic pumps to provoke a continuously elevated GH concentration. Intermittent injections of GH induced upregulation of liver EGFR content, augmented the response to EGF, and the induction of proteins involved in promotion of cell proliferation in female mice. In contrast, continuous GH delivery in male mice was associated with diminished EGFR in liver and decreased EGF-induced signaling and expression of early genes. The results indicate that sustained delivery systems that allow continuous GH plasma patterns would be beneficial in terms of treatment safety with regard to the actions of GH on EGFR signaling and its promitogenic activity.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Hormônio do Crescimento/administração & dosagem , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Esquema de Medicação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/sangue , Bombas de Infusão , Injeções , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
We have previously described that melatonin inhibits androgen production in hamster testes via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. This study attempted to determine the initial events of the melatonin/CRH signaling pathway. In Leydig cells from reproductively active Syrian hamsters, Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a colorimetric assay demonstrated that melatonin and CRH activate tyrosine phosphatases and subsequently reduce the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk), down-regulate the expression of c-jun, c-fos and steroidogenic acute regulatory (StAR), and inhibit the production of testosterone. These effects were prevented by a highly selective CRH antagonist, thus indicating that melatonin does not exert a direct role. Specific mitogen-activated protein kinase kinase (MEK) and jnk blockers inhibited expression of c-jun, c-fos, StAR and the production of testosterone, confirming that these are events triggered downstream of erk and jnk. In Leydig cells from photoperiodically regressed adult hamsters, CRH inhibited the production of androstane-3α,17ß-diol (3α-diol), the main androgen produced, through the same signaling pathway. Testicular melatonin concentration was 3-4-fold higher in reproductively inactive hamsters than that detected in active animals. Since melatonin, CRH, and their receptors are present not only in hamster testes but also in testicular biopsies of infertile men, we can conjecture about the relevance of this previously uncharacterized pathway in human fertility disorders. In summary, our study identifies crucial intracellular events triggered by melatonin/CRH in the testis that lead to a down-regulation of the steroidogenic process.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Células Intersticiais do Testículo/metabolismo , Melatonina/metabolismo , Animais , Cricetinae , Masculino , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: In non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO) and hypospermatogenesis (H) are commonly found. In these pathologies, Leydig cell hyperplasia (LCH) is detected in some patients. Since TGF-ß1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-ß1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), and the co-receptor endoglin in human biopsies from patients with idiopathic infertility. METHODS: Specific immunostaining of TGF-ß1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-ß1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR. RESULTS: Immunohistochemical studies revealed that TGF-ß1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-ß1 signaling, are also detected in both their phosphorylated (activated) and dephosphorylated form in all samples TGF-ß1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed. CONCLUSIONS: In conclusion, the high levels of mRNA and protein expression of the TGF-ß1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.
Assuntos
Infertilidade Masculina/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Síndrome de Células de Sertoli/metabolismo , Doenças Testiculares/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Receptores de Activinas Tipo II/biossíntese , Adulto , Antígenos CD/biossíntese , Endoglina , Humanos , Hiperplasia/metabolismo , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Superfície Celular/biossíntese , Proteínas Smad/metabolismo , Testículo/metabolismoRESUMO
We have previously observed expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the key enzyme in the biosynthesis of prostaglandins (PGs), in reproductively active Syrian hamster Leydig cells, and reported an inhibitory role of PGF(2alpha) on hamster testicular steroidogenesis. In this study, we further investigated PTGS2 expression in hamster Leydig cells during sexual development and photoperiodic gonadal regression. Since PTGS2 is mostly expressed in pubertal and reproductively active adult hamsters with high circulating levels of LH and androgens, we studied the role of these hormones in the regulation/maintenance of testicular PTGS2/PGF(2alpha). In active hamster Leydig cells, LH/hCG and testosterone induced PTGS2 and PGF(2alpha) production, and their actions were abolished by the antiandrogen bicalutamide (Bi). These results indicate that LH does not exert a direct effect on PG synthesis. Testosterone also stimulated phosphorylation of the mitogen-activated protein kinase isoforms 3/1 (MAPK3/1) within minutes and hours, but the testosterone metabolite dihydrotestosterone had no effect on PTGS2 and MAPK3/1. Because Bi and U0126, an inhibitor of the MAP kinase kinases 1 and 2 (MAP2K1/2), abolished testosterone actions on MAPK3/1 and PTGS2, our studies suggest that testosterone directly induces PTGS2/PGF(2alpha) in hamster Leydig cells via androgen receptors and a non-classical mechanism that involves MAPK3/1 activation. Since PGF(2alpha) inhibits testosterone production, it might imply the existence of a regulatory loop that is setting a brake on steroidogenesis. Thus, the androgen environment might be crucial for the regulation of testicular PG production at least during sexual development and photoperiodic variations in hamsters.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Células Intersticiais do Testículo/enzimologia , Testosterona/metabolismo , Antagonistas de Androgênios/farmacologia , Animais , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Cricetinae , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Indução Enzimática , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Masculino , Mesocricetus , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fotoperíodo , Inibidores de Proteínas Quinases/farmacologia , Receptores Androgênicos/metabolismo , Desenvolvimento Sexual , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
We have previously found that cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is present in the testicular interstitial cells of infertile men, whereas it is absent in human testes with no evident morphological changes or abnormalities. To find an animal model for further investigating COX-2 and its role in testicular steroidogenesis, we screened testes from adult species ranging from mice to monkeys. By using immunohistochemical assays, we found COX-2 expression only in Leydig cells of the reproductively active (peripubertal, pubertal, and adult) seasonal breeder Syrian hamster. COX-2 expression in hamster Leydig cells was confirmed by RT-PCR. In contrast, COX-1 expression was not detected in hamster testes. Because COX-2 expression implies PG synthesis, we investigated the effect of various PGs on testosterone production and found that PGF2 alpha stood out because it significantly reduced human chorionic gonadotropin-stimulated testosterone release from isolated hamster Leydig cells in a dose-dependent manner. This mechanism involves a decreased expression of testicular steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase. Testicular concentration and content of PGF2 alpha in reproductively active hamsters as well as production of PGF2 alpha from isolated hamster Leydig cells were also determined. Moreover, PGF2 alpha receptors were localized in Leydig cells of hamsters and testicular biopsies from patients with Sertoli cell only and germ arrest syndromes. Thus, in this study, we described a COX-2-initiated pathway that via PGF2 alpha production, PGF2 alpha receptors, steroidogenic acute regulatory protein, and 17beta-hydroxysteroid dehydrogenase represents a physiological local inhibitory system of human chorionic gonadotropin-stimulated testosterone production in the Syrian hamster testes.