RESUMO
The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that results from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing Glutaminase isoform 2 and reducing Glutamine synthetase expression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of Glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signalling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.
RESUMO
The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH increases the rate of proliferation of Sertoli cells; however, little is known about the transcription factors that are activated by the hormone in order to regulate Sertoli cell proliferation. On the other hand, Hypoxia Inducible Factors (HIFs) are master regulators of cell growth. HIFs are dimers of HIF-ß and HIF-α subunits. Considering that HIF-ß is constitutively expressed, HIF transcriptional activity is regulated through the abundance of HIF-α subunits. To date, three HIF-α isoforms have been described. The association of the different HIF-α subunits with HIF-ß subunit constitutes three active transcription factors -HIF-1, HIF-2 and HIF-3- which interact with consensus hypoxia-response elements in the promoter region of target genes. Hypoxia has been classically considered the main stimulus that increases HIF transcriptional activity, however, regulation by hormones under normoxic conditions was also demonstrated. The aim of this work has been to investigate whether HIFs participate in the regulation of rat Sertoli cell proliferation by FSH. Sertoli cells obtained from 8-day old rats were cultured in the absence or presence of FSH. It has been observed that FSH increases HIF transcriptional activity and HIF-2α mRNA levels without modifying either HIF-1α or HIF-3α expression. Incubations with FSH have been also performed in the absence or presence of a pharmacological agent that promotes HIF-α subunit degradation, LW6. It has been observed that LW6 inhibits the FSH effect on proliferation, CCND1 expression and c-Myc transcriptional activity. Altogether, these results suggest that HIFs might be involved in the regulation of Sertoli cell proliferation by FSH.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hormônio Foliculoestimulante/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Hormônio Foliculoestimulante/farmacologia , Genes bcl-1/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Células de Sertoli/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Metformin (MET) is one of the most widely used anti-hyperglycemic agents for treating patients with type 2 diabetes and it has started to be used in pediatric population at ages when Sertoli cells are still proliferating. It is well known that follicle-stimulating hormone (FSH) is the major Sertoli cell mitogen. The aim of the study is to investigate a possible effect of MET, which has been shown to have anti-proliferative properties, on FSH regulation of postnatal Sertoli cell proliferation and on the molecular mechanisms involved in this regulation. The present study was performed in eight-day-old rat Sertoli cell cultures. The results obtained show that MET in the presence of FSH increases phosphorylated acetyl-CoA carboxylase and decreases phosphorylated p70S6K levels. Moreover, we show that MET decreases FSH-stimulated Sertoli cell proliferation, and this decrease is accompanied by a reduction in FSH-stimulated Ccnd1 and Ccnd2 expression and an increase in cell cycle inhibitor p21Cip expression. Altogether, these results suggest that MET can, at least in part, counteract the effect of FSH on postnatal Sertoli cell proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hormônio Foliculoestimulante , Hipoglicemiantes/efeitos adversos , Metformina/efeitos adversos , Células de Sertoli/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Animais , Masculino , Cultura Primária de Células , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Células de Sertoli/metabolismoRESUMO
The presence of lipid droplets (LD) and the utilization of fatty acids (FA) as a source of energy are Sertoli cell (SC) putative characteristics. It is well known that SCs can phagocyte and degrade apoptotic germ cells (AGC) resulting in increasing lipid content and ATP levels. A relationship between the regulation of lipid storage and of lipid oxidation in SC might be envisaged. The aim of this study was to analyze whether AGC and FA are able to simultaneously regulate molecular mechanisms involved in lipid storage and in FA oxidation in SC. The experimental model utilized in this study consisted in SC cultures obtained from 20-day-old rats that were co-cultured with AGC or treated with palmitic acid (PA, 500 µM) for 24 and 48 h. AGC and PA increase LD, triacylglycerol (TAG) content and mRNA levels of Plin1, Plin2, Plin3 (proteins involved in TAG storage). Simultaneously, AGC and PA rise the extent of FA oxidation and mRNA levels of Cpt1 and Lcad (proteins involved in FA degradation). Results also show that peroxisome proliferator-activated receptor (PPAR) transcriptional activity, transcription factor which participate in lipid metabolism regulation, increases by AGC and PA treatment in SC. Additionally, the presence of a PPARg antagonist decreases the upregulation of LD content and Plin1 expression. Similarly, the presence of a PPARb/d antagonist reduces the increase in FA oxidation and Cpt1 mRNA levels. Altogether these results suggest that AGC and FA, which probably generate PPAR ligands, regulate lipid storage and fatty acid utilization, contributing to the energy homeostasis in the seminiferous tubules.
Assuntos
Apoptose , Comunicação Celular , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/farmacologia , Células de Sertoli/efeitos dos fármacos , Espermatozoides/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Técnicas de Cocultura , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Oxirredução , Ácido Palmítico/metabolismo , Perilipina-1/genética , Perilipina-1/metabolismo , Perilipina-2/genética , Perilipina-2/metabolismo , Perilipina-3/genética , Perilipina-3/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos Sprague-Dawley , Células de Sertoli/metabolismo , Transdução de Sinais , Espermatozoides/patologia , Triglicerídeos/metabolismoRESUMO
Paracrine regulation of Sertoli cell function by germ cells is an outstanding characteristic of testicular physiology. It has been demonstrated that Sertoli cells produce ketone bodies and that germ cells may use them as energy source. The aim of the study was to analyze a possible regulation by germ cells of ketogenesis in Sertoli cells. Cultures of Sertoli cells (SC) obtained from 31-day-old rats were co-cultured with germ cells (GC). The results presented herein show that the presence of GC stimulated 3-hydroxybutyrate production and increased mRNA levels of two enzymes involved in ketogenesis-carnitine palmitoyltransferase 1a (CPT1a) and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (mHMGCoA) synthase- in SC. Additionally, GC increased monocarboxylate transporter 4 (Mct4) expression in SC, a transporter involved in ketone bodies exit. To evaluate if the observed effects might be mediated by soluble factors, SC cultures were incubated with germinal cell-conditioned medium (GCCM) or with two growth factors, bFGF and IGF1, which are known to be secreted by GC. We observed that GCCM and bFGF stimulated ketone bodies production but that IGF1 did not modify it. Also, we observed that GCCM and bFGF increased Cpt1a and Mct4 mRNA levels. In summary, results presented herein demonstrate that Sertoli cells are able to produce ketone bodies and that its production is regulated in a paracrine way by germ cells. This study adds new information about communication between Sertoli cells and developing germ cells.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Células Germinativas/metabolismo , Células de Sertoli/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Corpos Cetônicos/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacosRESUMO
The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the physiological relevance of this mechanism. As for the regulation of PDC, analysis of pyruvate dehydrogenase kinase (Pdk) expression showed that FSH increases Pdk3 and decreases Pdk4 mRNA levels while bFGF increases the expression of all Pdks. In addition, we showed that bFGF increases phosphorylated PDC levels and that bFGF-stimulated lactate production is partially inhibited in the presence of a PDK inhibitor. Altogether, these results add new information regarding novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells. Considering that lactate is essential for the production of energy in spermatocytes and spermatids, these mechanisms might be relevant in maintaining spermatogenesis and male fertility.
Assuntos
Hormônios/fisiologia , Ácido Láctico/metabolismo , Células de Sertoli/metabolismo , Animais , Células Cultivadas , Fertilidade , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hormônio Foliculoestimulante/farmacologia , Masculino , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Sprague-Dawley , EspermatogêneseRESUMO
The purpose of this study was to investigate if FSH and bFGF regulate fatty acid (FA) metabolism and mitochondrial biogenesis in Sertoli cells (SC). SC cultures obtained from 20-day-old rats were incubated with 100ng/ml FSH or 30ng/ml bFGF for 6, 12, 24 and 48h. The expression of genes involved in transport and metabolism of FA such as: fatty acid transporter CD36 (FAT/CD36), carnitine-palmitoyltransferase 1 (CPT1), long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases (LCAD, MCAD), and of genes involved in mitochondrial biogenesis such as: nuclear respiratory factors 1 and 2 (NRF1, NRF2) and transcription factor A (Tfam), was analyzed. FSH stimulated FAT/CD36, CPT1, MCAD, NRF1, NRF2 and Tfam mRNA levels while bFGF only stimulated CPT1 expression. A possible participation of PPARß/δ activation in the regulation of gene expression and lactate production was then evaluated. SC cultures were incubated with FSH or bFGF in the presence of the PPARß/δ antagonist GSK3787 (GSK; 20µM). bFGF stimulation of CPT1 expression and lactate production were inhibited by GSK. On the other hand, FSH effects were not inhibited by GSK indicating that FSH regulates the expression of genes involved in FA transport and metabolism and in mitochondrial biogenesis, independently of PPARß/δ activation. FA oxidation and mitochondrial biogenesis as well as lactate production are essential for the energetic metabolism of the seminiferous tubule. The fact that these processes are regulated by hormones in a different way reflects the multifarious regulation of molecular mechanisms involved in Sertoli cell function.
Assuntos
Ácidos Graxos/metabolismo , Hormônio Foliculoestimulante/metabolismo , Oligopeptídeos/metabolismo , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Animais , Expressão Gênica , Humanos , Masculino , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
Experimental autoimmune orchitis is a useful model for studying testicular inflammation and germ/immune cell interactions. Th17 cells and their hallmark cytokine IL17A were reported to be involved in the development of autoimmune orchitis. The aim of the present work is to investigate the pathogenic role of IL17A in rat testis. In vitro experiments were performed in order to analyze effects of IL17A on Sertoli cell tight junctions. The addition of IL17A to normal rat Sertoli cell cultures induced a significant decline in transepithelial electrical resistance and a reduction of occludin expression and redistribution of occludin and claudin 11, altering the Sertoli cell tight junction barrier. Intratesticular injection of 1 µg of recombinant rat IL17A to Sprague-Dawley rats induced increased blood-testis barrier permeability, as shown by the presence of biotin tracer in the seminiferous tubule adluminal compartment, and delocalization of occludin and claudin 11. Results showed that IL17A induced focal inflammatory cell infiltration in the interstitium and germ cell sloughing in adjacent seminiferous tubules. Moreover, an increase in TUNEL+ apoptotic germ cells was also observed. Inflammatory ED1+ macrophages were the main population infiltrating the interstitium following IL17A injection. This correlated with an increase in mRNA expression of the monocyte chemoattractant protein Ccl2, its receptor Ccr2 and the vascular cell adhesion molecule Vcam1. Overall results suggest a relevant role of IL17A in the development of testicular inflammation, facilitating the recruitment of immune cells to the testicular interstitium and inducing impairment of blood-testis barrier function.
Assuntos
Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/patologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-17/metabolismo , Animais , Apoptose/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Claudinas/metabolismo , Impedância Elétrica , Células Germinativas/efeitos dos fármacos , Células Germinativas/patologia , Linfócitos/metabolismo , Macrófagos/metabolismo , Masculino , Ocludina , Orquite/metabolismo , Orquite/patologia , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR2/metabolismo , Receptores de Interleucina-17/metabolismo , Células de Sertoli/metabolismo , Junções Íntimas/metabolismo , Regulação para Cima/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Sertoli cells (SCs) are essential to maintaining germ cell development. Metformin, the main pharmacologic treatment for pediatric type 2 diabetes, is administered to children during SC maturation. The present study aimed to analyze whether metformin affects SC energy metabolism and blood-testis barrier (BTB) integrity. Primary SC cultures were used for the in vitro studies. In vivo effects were studied in Sprague-Dawley rats treated with 200 mg/kg metformin from Pnd14 to Pnd30. Metformin decreased fatty acid oxidation and increased 3-hydroxybutyrate production in vitro. Moreover, it decreased the transepithelial electrical resistance across the monolayer and induced ZO-1 redistribution, suggesting an alteration of cell junctions. In vivo, a mild but significant increase in BTB permeability and ZO-1 expression was observed in the metformin group, without changes in testicular histology and meiosis progression. Additionally, adult rats that received metformin treatment during the juvenile period showed no alteration in BTB permeability or daily sperm production. In conclusion, metformin exposure may affect BTB permeability in juvenile rats, but this seems not to influence spermatogenesis progression. Considering the results obtained in adult animals, it is possible to speculate that metformin treatment during the juvenile period does not affect testicular function in adulthood.
RESUMO
The definitive number of Sertoli cells (SCs), achieved during the proliferative periods, defines the spermatogenic capacity in adulthood. It is recognized that FSH is the main mitogen targeting SC and that it exerts its action, at least partly, through the activation of the PI3K/Akt/mTORC1 pathway. mTORC1 controls a large number of cellular functions, including glycolysis and cell proliferation. Interestingly, recent evidence revealed that the glycolytic flux might modulate mTORC1 activity and, consequently, cell cycle progression. Although mature SC metabolism has been thoroughly studied, several aspects of metabolism regulation in proliferating SC are still to be elucidated. The objective of this study was to explore whether aerobic glycolysis is regulated by FSH through mTORC1 pathway in proliferating SC, and to assess the involvement of glycolysis in the regulation of SC proliferation. The present study was carried out utilizing 8-day-old rat SC cultures. The results obtained show that FSH enhances glycolytic flux through the induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and lactate dehydrogenase A (LDHA) in an mTORC1 dependent manner. In addition, PFKFB3 and LDH inhibitors prevent FSH from activating mTORC1 and stimulating SC proliferation and glycolysis, presumably through mTORC1 pathway inhibition. In summary, FSH simultaneously regulates SC proliferation and glycolysis in an mTORC1 dependent manner, and glycolysis seems to cooperate with FSH in the stimulation of both cellular functions through the modulation of the same signalling pathway. Therefore, a positive feedback between the mTORC1 pathway and glycolysis triggered by FSH is hypothesized.
Assuntos
Hormônio Foliculoestimulante , Fosfatidilinositol 3-Quinases , Masculino , Ratos , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proliferação de Células , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , GlicóliseRESUMO
It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.
Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Glicina/análogos & derivados , Herbicidas/administração & dosagem , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Barreira Hematotesticular/metabolismo , Claudinas/metabolismo , Conexinas/metabolismo , Glicina/administração & dosagem , Masculino , Ocludina/metabolismo , Ratos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/metabolismo , Testosterona/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , GlifosatoRESUMO
BACKGROUND: The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. OBJECTIVE: The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. MATERIALS AND METHODS: Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200 mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. RESULTS: MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. DISCUSSION AND CONCLUSION: These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hipoglicemiantes/efeitos adversos , Metformina/efeitos adversos , Células de Sertoli/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Gravidez , Ratos Sprague-DawleyRESUMO
Roundup (R), a formulation that contains glyphosate (G) as the active ingredient, is a commonly used nonselective herbicide that has been proposed to affect male fertility. It is well known that an adequate Sertoli cell function is essential to maintain germ cell development. The aim of the present study was to analyze whether G and R are able to affect Sertoli cell functions, such as energy metabolism and blood-testis barrier (BTB) integrity. Sertoli cell cultures from 20-day-old rats were exposed to 10 and 100â¯ppm of G or R, doses which do not decrease cell viability. Neither G nor R caused impairment in lactate production or fatty acid oxidation. G and R decreased Transepithelial Electrical Resistance, which indicates the establishment of a Sertoli cell junction barrier. However, neither G nor R modified the expression of claudin11, ZO1 and occludin, proteins that constitute the BTB. Analysis of cellular distribution of claudin11 by immunofluorescence showed that G and R induced a delocalization of the signal from membrane to the cytoplasm. The results suggest that G and R could alter an important function of Sertoli cell such as BTB integrity and thus they could compromise the normal development of spermatogenesis.
Assuntos
Glicina/análogos & derivados , Herbicidas/toxicidade , Células de Sertoli/efeitos dos fármacos , Animais , Barreira Hematotesticular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Claudinas/biossíntese , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glicina/toxicidade , Junções Intercelulares/efeitos dos fármacos , Ácido Láctico/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espermatogênese/efeitos dos fármacos , GlifosatoRESUMO
Sertoli cells are somatic cells present in seminiferous tubules which have essential roles in regulating spermatogenesis. Considering that each Sertoli cell is able to support a limited number of germ cells, the final number of Sertoli cells reached during the proliferative period determines sperm production capacity. Only immature Sertoli cells, which have not established the blood-testis barrier, proliferate. A number of hormonal cues regulate Sertoli cell proliferation. Among them, FSH, the insulin family of growth factors, activin, and cytokines action must be highlighted. It has been demonstrated that cAMP/PKA, ERK1/2, PI3K/Akt, and mTORC1/p70SK6 pathways are the main signal transduction pathways involved in Sertoli cell proliferation. Additionally, c-Myc and hypoxia inducible factor are transcription factors which participate in the induction by FSH of various genes of relevance in cell cycle progression. Cessation of proliferation is a pre-requisite to Sertoli cell maturation accompanied by the establishment of the blood-testis barrier. With respect to this barrier, the participation of androgens, estrogens, thyroid hormones, retinoic acid and opioids has been reported. Additionally, two central enzymes that are involved in sensing cell energy status have been associated with the suppression of Sertoli cell proliferation, namely AMPK and Sirtuin 1 (SIRT1). Among the molecular mechanisms involved in the cessation of proliferation and in the maturation of Sertoli cells, it is worth mentioning the up-regulation of the cell cycle inhibitors p21Cip1, p27Kip, and p19INK4, and of the gap junction protein connexin 43. A decrease in Sertoli cell proliferation due to administration of certain therapeutic drugs and exposure to xenobiotic agents before puberty has been experimentally demonstrated. This review focuses on the hormones, locally produced factors, signal transduction pathways, and molecular mechanisms controlling Sertoli cell proliferation and maturation. The comprehension of how the final number of Sertoli cells in adulthood is established constitutes a pre-requisite to understand the underlying causes responsible for the progressive decrease in sperm production that has been observed during the last 50 years in humans.
RESUMO
Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.
Assuntos
Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 3/biossíntese , Células de Sertoli/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/fisiologia , Transportador de Glucose Tipo 1/efeitos dos fármacos , Transportador de Glucose Tipo 3/efeitos dos fármacos , Interleucina-1beta/farmacologia , Interleucina-1beta/fisiologia , Ácido Láctico/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacosRESUMO
Hypoxia Inducible Factors (HIFs) are master regulators of glycolytic metabolism. HIFs consist of a constitutive HIFbeta (HIFß) subunit and a HIFalpha (HIFα) subunit, whose half-life depends on prolyl-hydroxylases activity. Inhibition of prolyl-hydroxylases by hypoxia or transition metals, or augmentation of HIFα subunit levels by hormonal stimuli lead to a higher HIF transcriptional activity. On the other hand, it is well known that lactate produced by Sertoli cells is delivered to and used by germ cells as an energy substrate. The aim of this work was to investigate whether HIFs participate in the regulation of lactate production in rat Sertoli cells and whether they are involved in the FSH mechanism of action. In order to reach a higher HIF transcriptional activity, Sertoli cells were treated with CoCl2. We observed that a higher HIF transcriptional activity leads to an augmentation of: lactate production, glucose uptake and LDH activity. Besides, an increase in Glut1, Pkm2 and Ldha mRNA levels was observed. These findings suggested that HIFs may participate in the modulation of Sertoli cell nutritional function. As FSH regulates lactate production, we evaluated whether HIFs were involved in FSH action. Sertoli cells were stimulated with FSH in the absence or presence of LW6, a drug which promotes HIFα subunit degradation. On the one hand, we observed that FSH increases HIF1α protein, Hif1α and Hif2α mRNA levels and, on the other hand, that LW6 inhibits FSH-stimulated lactate production, glucose uptake, Glut1, Pkm2 and Ldha expression. It is proposed that HIFs are key components of the intricate pathways utilized by FSH to regulate the provision of lactate for germ cells. Considering that FSH is the master endocrine regulator of Sertoli cells, it is not surprising that this hormone may employ several regulatory mechanisms to fulfill the nourishing functions of this cell type.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lactatos/metabolismo , Células de Sertoli/metabolismo , Acetanilidas/farmacologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Northern Blotting , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cobalto/farmacologia , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Fatores de Tempo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Besides giving structural support, Sertoli cells regulate the fate of germ cells by supplying a variety of factors. These factors include hormones, several pro- and anti-apoptotic agents and also energetic substrates. Lactate is one of the compounds produced by Sertoli cells, which is utilized as an energetic substrate by germ cells, particularly spermatocytes and spermatids. Beyond its function as an energy source, some studies have proposed a role of lactate in the regulation of gene expression not strictly related to the energetic state of the cells. The general hypothesis that motivated this investigation was that lactate affects male germ cell function, far beyond its well-known role as energetic substrate. To evaluate this hypothesis we investigated: 1) if lactate was able to regulate germ cell gene expression and if reactive oxygen species (ROS) participated in this regulation, 2) if different signal transduction pathways were modified by the production of ROS in response to lactate and 3) possible mechanisms that may be involved in lactate stimulation of ROS production. In order to achieve these goals, cultures of germ cells obtained from male 30-day old rats were exposed to 10 or 20 mM lactate. Increases in lactate dehydrogenase (LDH) C and monocarboxylate transporter (MCT)2 expression, in Akt and p38-MAPK phosphorylation levels and in ROS production were observed. These effects were impaired in the presence of a ROS scavenger. Lactate stimulated ROS production was also inhibited by a LDH inhibitor or a NAD(P)H oxidase (NOX) inhibitor. NOX4 expression was identified in male germ cells. The results obtained herein are consistent with a scenario where lactate, taken up by germ cells, becomes oxidized to pyruvate with the resultant increase in NADH, which is a substrate for NOX4. ROS, products of NOX4 activity, may act as second messengers regulating signal transduction pathways and gene expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Ácido Láctico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células de Sertoli/metabolismo , Transdução de Sinais/fisiologia , Espermatozoides/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Láctico/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Transdução de Sinais/efeitos dos fármacos , Espermatozoides/citologiaRESUMO
Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.
Assuntos
Interleucina-1beta/farmacologia , Lactatos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting/métodos , Butadienos/farmacologia , Células Cultivadas , Cromonas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imunossupressores/farmacologia , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Nitrilas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Estimulação Química , Transferrina/análise , Transferrina/metabolismo , WortmaninaRESUMO
The aim of the present study was to investigate whether the AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in Sertoli cells and whether its activation by 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside (AICAR) results in the regulation of cell metabolism to ensure lactate supply for germ cell development. Sertoli cell cultures from 20-day-old rats were used. Western blot analysis for the alpha-subunit of AMPK showed that high levels of AMPK are present in Sertoli cells. Treatment of the cultures with AICAR resulted in a dose- and time-dependent increase of P-AMPK levels indicating activation of the enzyme. A possible effect of AICAR on Sertoli cell lactate production was then analyzed. A dose- and time-dependent increment in lactate secretion was observed. The participation of AMPK activation in different biochemical processes that may be implicated in the regulation of lactate production was also analyzed. AICAR stimulated glucose uptake in a dose- and time-dependent manner. Additionally, AICAR increased the glucose transporter 1 (GLUT1) and decreased the glucose transporter 3 (GLUT3) mRNA levels. As for the role of AMPK in the regulation of the monocarboxylate transporters 1 and 4 (MCT1 and MCT4), it has been observed that AICAR treatment decreased MCT1 and increased MCT4 mRNA levels. In summary, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells.