RESUMO
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
Leydig-cell tumours (LCTs) are rare endocrine tumours of the testicular interstitium, with recent increased incidence. Symptoms include precocious puberty in children; and erectile dysfunction, infertility and/or gynaecomastia, in adults. So far, scientific evidence points to aromatase (CYP19) overexpression and excessive oestrogen and insulin-like growth factor (IGF) -1 production as responsible for Leydig-cell tumourigenesis. LCTs are usually benign; however, malignant LCTs respond poorly to chemo/radiotherapy, highlighting the need to identify novel targets for treatment. Herein, we investigated the potential role of the histamine receptor H4 (HRH4) as a therapeutic target for LCTs using R2C rat Leydig tumour cells, a well-documented in vitro model for Leydigioma. Also, we studied for the first time the expression of CYP19, IGF-1R, oestrogen receptor (ER) α, ERß, androgen receptor (AR) and HRH4 in human prepubertal LCTs versus normal prepubertal testes (NPTs). HRH4 agonist treatment inhibited steroidogenesis and proliferation in R2C cells and also negatively affected their pro-angiogenic capacity in vitro and in vivo, as assessed by evaluating the proliferative activity of human umbilical vein endothelial cells and by means of the quail chorioallantoic membrane assay, respectively. Moreover, E2 and IGF-1 inhibited HRH4 mRNA and protein levels. In human prepubertal LCTs, CYP19, IGF-1R, ERα and ERß were overexpressed compared with NPTs. In contrast, HRH4 staining was weak in LCTs, but moderate/strong and confined to the interstitium in NPTs. Importantly, HRH4 was absent or barely detectable in seminiferous tubules or germ cells. Overall, our results point to HRH4 as a novel therapeutic target in LCTs.
Assuntos
Antineoplásicos/farmacologia , Guanidinas/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Imidazóis/farmacologia , Tumor de Células de Leydig/tratamento farmacológico , Receptores Histamínicos H4/agonistas , Neoplasias Testiculares/tratamento farmacológico , Tioureia/análogos & derivados , Fatores Etários , Inibidores da Angiogênese/farmacologia , Animais , Aromatase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Coturnix/embriologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lactente , Tumor de Células de Leydig/metabolismo , Tumor de Células de Leydig/patologia , Masculino , Terapia de Alvo Molecular , Neovascularização Patológica , Ratos , Receptor IGF Tipo 1 , Receptores Androgênicos/metabolismo , Receptores Histamínicos H4/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores da Síntese de Esteroides/farmacologia , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Tioureia/farmacologiaRESUMO
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
As we previously reported, testes of men suffering from hypospermatogenesis and germ cell arrest or Sertoli cell-only syndrome show a major increase in the number of macrophages expressing interleukin-1ß (IL-1ß) and abundant expression of cyclooxygenase-2 (COX-2), the inducible isoform of the key enzyme in the biosynthesis of prostaglandins (PGs), in Leydig cells. In the present study we report [1] a positive correlation between IL-1ß levels and COX-2 expression in testes of infertile patients, [2] the induction of COX-2 by IL-1ß in mouse Leydig cells (TM3) and human macrophages (THP-1), and therefore [3] evidence for an IL-1ß-dependent induction of testicular inflammatory states.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Infertilidade Masculina/metabolismo , Interleucina-1beta/metabolismo , Prostaglandinas/metabolismo , Testículo/metabolismo , Adulto , Animais , Biópsia , Células Cultivadas , Humanos , Infertilidade Masculina/patologia , Interleucina-1beta/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Síndrome de Células de Sertoli/metabolismo , Síndrome de Células de Sertoli/patologia , Testículo/patologiaRESUMO
The aim of this study was to analyze whether di(2-ethylhexyl) phthalate (DEHP), a Sertoli and Leydig cell toxicant, is able to induce alterations in the expression of testicular gap and tight junction proteins. DEHP was administered by gavage (1 g/5 mL corn oil/kg body weight/day) to 25-day-old male Sprague-Dawley rats for 2 days (DEHP-27d) and control rats were treated with corn-oil vehicle for 2 days (C-27d); animals were killed 24 h after the last treatment. Testes of DEHP-27d rats showed different degrees of germ cell sloughing of seminiferous tubules (ST). No alterations of the blood testis barrier (BTB) by lanthanum tracer study were observed. ST of DEHP-27d rats showed a milder immunofluorescence and more restricted expression of connexin-43 (Cx43) in the adluminal and basal compartment compared to C-27d. In DEHP-27d rats, we found a discontinuous immunofluorescent (IF) pattern for zonula occludens (ZO-1), contrasting with the continuous IF profile observed in C-27d, and a delocalization of claudin-11. A decrease in Cx43 and ZO-1 and no changes in occludin expression were detected by Western blot in the testes of DEHP-27d rats. Results from 57-day-old rats treated with DEHP for 2 days and held for 30 days without treatment showed that the alterations in protein expression induced by DEHP are reversible. However, a delay of spermatogenesis compared to C-57d rats, occurred. Data demonstrated that DEHP does not impair BTB permeability but induces germ cell sloughing that might respond to a down regulation of Cx43 and ZO-1 that alters cell junction proteins.
Assuntos
Conexinas/biossíntese , Dietilexilftalato/toxicidade , Junções Comunicantes/efeitos dos fármacos , Túbulos Seminíferos/efeitos dos fármacos , Testículo/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacosRESUMO
Immunoexpression of IGF-I, IGF-II, type 1 IGF receptor (IGFR), insulin receptor (IR), and GH receptor (GHR) was analyzed in human testis, in three age groups (Gr): Gr1 (neonates), Gr2 (postnatal testicular activation), and Gr3 (early prepuberty). In interstitial cells, low IGF-I and GHR, but moderate IR immunoexpression was observed in all Grs. However, high expression of IGF-II in Gr1, and moderate expression of IGFR in Gr1 and Gr2 were found. In Leydig cell (LC), high expression of IGF-II, moderate expression of IGFR and GHR, and undetectable IGF-I was found. Moreover, IR was highly expressed in Gr2. The effect of IGF-I on cell proliferation (PI) and apoptosis (AI), induction of cytochrome P450 side chain cleavage (cP450scc) immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA and testosterone (T) secretion was evaluated in human testis cell cultures. IGF-I increased P450scc immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA, T secretion, and PI, but decreased AI. We propose that IGF-II, mainly through IR, is involved in functional LC differentiation. In some interstitial cells, probably in LC precursors, IGF-II/IR could be involved, among other factors, in the stimulation of PI and/or inhibition of AI, and in LC differentiation.
Assuntos
Diferenciação Celular , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Células Intersticiais do Testículo/metabolismo , Transdução de Sinais , Testículo/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Fatores Etários , Apoptose , Autopsia , Proteínas de Transporte/metabolismo , Proliferação de Células , Células Cultivadas , Pré-Escolar , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Humanos , Lactente , Recém-Nascido , Células Intersticiais do Testículo/enzimologia , Masculino , RNA Mensageiro/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes/metabolismo , Testículo/citologia , Testículo/enzimologia , Testículo/crescimento & desenvolvimento , Testosterona/metabolismoRESUMO
The expression of aromatase, estrogen receptor alpha (ERalpha) and beta (ERbeta), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERalpha by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERbeta and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERbeta, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERbeta were immunoexpressed strongly whereas no expression of ERalpha, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.
Assuntos
Aromatase/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Receptores Androgênicos/metabolismo , Testículo/metabolismo , Aromatase/genética , Diferenciação Celular , Pré-Escolar , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Células Germinativas/metabolismo , Humanos , Lactente , Recém-Nascido , Células Intersticiais do Testículo/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Células de Sertoli/metabolismoRESUMO
This study investigated the effect of DEHP exposure on N-cadherin and alpha-, beta- and p120-catenin immunoreactivities in the rat testis. DEHP was administered by daily gavage to 25-day-old male Sprague-Dawley rats at a dose of 2 g DEHP/5 ml corn oil/kg body weight for 2 days or 7 days. Control rats were treated with corn oil vehicle under the same conditions. Animals were killed at 24h after the last treatment. Another group of rats treated with DEHP or corn oil vehicle (control group) for 2 days were held for 30 days without treatment to observe recovery. Testes were analyzed for histopathology, TUNEL staining, immunofluorescence (IF) and Western blot analyses. Animals exposed to DEHP for 2 days or 7 days showed severe alterations of seminiferous tubules characterized by germ cell sloughing. Animals from the longer term recovery group treated with DEHP showed foci of delayed spermatogenesis. A linear and continuous pattern of N-cadherin was observed in the basal compartment of the seminiferous tubules. A similar pattern but with higher IF intensity was observed for N-cadherin in rats treated with DEHP for 2 days or 7 days, compared to control animals. The alpha-, beta- and p120-catenins were detected in the basal compartment of seminiferous tubules in similar localization and IF pattern for DEHP and control groups. A significant increase in testicular N-cadherin and alpha-catenin levels was detected by Western blot analysis in DEHP-exposed versus control rats. No variations in N-cadherin or catenin expression were detected in the recovery groups. These findings demonstrate that DEHP induces an up-regulation of N-cadherin and alpha-catenin expression and may perturb cell-cell adhesion phenomena in the seminiferous tubule.
Assuntos
Caderinas/metabolismo , Cateninas/metabolismo , Dietilexilftalato/toxicidade , Células de Sertoli/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Peso Corporal/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Dietilexilftalato/administração & dosagem , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Intubação Gastrointestinal , Masculino , Microscopia Eletrônica de Transmissão , Tamanho do Órgão/efeitos dos fármacos , Fosfoproteínas/metabolismo , Plastificantes/administração & dosagem , Plastificantes/toxicidade , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/patologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/ultraestrutura , Espermatócitos/efeitos dos fármacos , Espermatócitos/patologia , Espermatogênese/efeitos dos fármacos , Espermatogônias/efeitos dos fármacos , Espermatogônias/patologia , Testículo/efeitos dos fármacos , Testículo/patologia , Testículo/ultraestrutura , delta CateninaRESUMO
La célula. Componentes químicos de la célula. Métodos de estudio en biología celular. Membrana celular. Matriz citoplasmática y citoesqueleto. Superficie celular. Señalización intercelular. Sistema de endomembranas. Mitocondrias. Peroxisomas. Núcleo interfásico. Estructura de los genes. Transcripción del ADN. Traducción del ARN. Replicación del ADN. Mitosis. Control del ciclo celular. Meiosis. Fecundación. Citogenética. Diferenciación celular. Muerte celular. La célula vegetal. Cloroplastos
Assuntos
Biologia Molecular , BiologiaRESUMO
Fueron estudiados los efectos de un antiandrógeno puro, la flutamida, y un inhibidor de la 5Ó-reductasa, la finestarida, sobre el desarrollo prenatal de los genitales externos y la próstata de la rata. En los grupos controles la distancia anogenital fue de 3.1 ñ 0.24mm en los recién nacidos machos, y de 1.2 ñ 10mm en las hembras. En los machos se observaron esbozos prostáticos en la porción cefálica de la uretra. Una dosis de 6mg/Kg/día de flutamida produjo una disminución signficativa de la distancia anogenital de los machos, pero no alteró el desarrollo de la próstata. A dosis más altas, la flutamida produjo la completa feminización de los genitales externos e inhibió la formación de la próstata. La administración de 2,8 y 16mg/Kg/día de finestarida condujo a una progressiva disminución de la distancia anogenital de los recién nacidos machos, pero no produjeron la abolición del desarrollo prostático. Sin embargo, la administración conjunta de 2mg/Kg/día de finestarida y 6mg/Kg/día de flutamida produjo una completa inhibición en el desarrollo de los esbozos prostáticos
Assuntos
Animais , Masculino , Feminino , Gravidez , Ratos , Diferenciação Sexual , Finasterida/administração & dosagem , Flutamida/administração & dosagem , Flutamida/farmacologia , Genitália Masculina/embriologia , Próstata/embriologia , Ratos WistarRESUMO
Fueron estudiados los efectos de un antiandrógeno puro, la flutamida, y un inhibidor de la 5O-reductasa, la finestarida, sobre el desarrollo prenatal de los genitales externos y la próstata de la rata. En los grupos controles la distancia anogenital fue de 3.1 ñ 0.24mm en los recién nacidos machos, y de 1.2 ñ 10mm en las hembras. En los machos se observaron esbozos prostáticos en la porción cefálica de la uretra. Una dosis de 6mg/Kg/día de flutamida produjo una disminución signficativa de la distancia anogenital de los machos, pero no alteró el desarrollo de la próstata. A dosis más altas, la flutamida produjo la completa feminización de los genitales externos e inhibió la formación de la próstata. La administración de 2,8 y 16mg/Kg/día de finestarida condujo a una progressiva disminución de la distancia anogenital de los recién nacidos machos, pero no produjeron la abolición del desarrollo prostático. Sin embargo, la administración conjunta de 2mg/Kg/día de finestarida y 6mg/Kg/día de flutamida produjo una completa inhibición en el desarrollo de los esbozos prostáticos (AU)