RESUMEN
Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes.
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ATPasas Asociadas con Actividades Celulares Diversas , Enfermedad del Hígado Graso no Alcohólico , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Hepatocitos/enzimología , Humanos , Hígado/enzimología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias Hepáticas/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Enfermedad del Hígado Graso no Alcohólico/enzimología , Ratas , Triglicéridos/metabolismoRESUMEN
The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.
Asunto(s)
Células Intersticiales del Testículo , Dinámicas Mitocondriales , Ratones , Masculino , Ratas , Animales , Células Intersticiales del Testículo/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , Proteínas Mitocondriales/metabolismo , Colesterol/metabolismo , Testosterona/metabolismoRESUMEN
Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.
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Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Células Intersticiales del Testículo/enzimología , Testosterona/metabolismo , Expresión Génica , Humanos , Células Intersticiales del Testículo/ultraestructura , Masculino , TranscriptomaRESUMEN
Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.
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Células Intersticiales del Testículo/metabolismo , Fosfoproteínas/metabolismo , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Diglicéridos/metabolismo , Eliminación de Gen , Células Intersticiales del Testículo/ultraestructura , Ligandos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/ultraestructura , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Progesterona/metabolismo , Ratas Sprague-Dawley , Receptores de GABA/metabolismo , Transducción de Señal , Esteroides/biosíntesisRESUMEN
Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2 mg/kg/day M1 for 6 weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.
RESUMEN
BACKGROUND AND AIMS: The mitochondrial translocator protein (TSPO, 18 kDa) is pivotal in binding cholesterol and facilitating its transfer from the outer to the inner mitochondrial membrane. Atriol is a TSPO ligand disrupting cholesterol binding by targeting the cholesterol-recognition amino acid consensus domain. Prior research has shown that TSPO deficiency improved metabolic-associated steatohepatitis (MASH). We hypothesized that Atriol may have the potential to alleviate MASH. METHODS AND RESULTS: In vitro cell culture studies revealed that Atriol treatment effectively mitigated MASH by restoring mitochondrial function, inhibiting the NF-κB signaling pathway, and reducing hepatic stellate cell (HSC) activation. SD male rats were fed a GAN diet for 10 months to induce MASH. During the final two weeks of feeding, rats received intraperitoneal Atriol administration daily. Atriol treatment significantly ameliorated MASH by reducing lipid accumulation, diminishing hepatic lobular inflammation and fibrosis, decreasing cell death, and inhibiting excessive bile acid synthesis. Moreover, Atriol restored mitochondrial function in primary hepatocytes isolated from MASH rats. In search of the mechanism(s) governing these effects, we found that Atriol downregulated the proinflammatory chemokine CXCL1 through the NF-κB signaling pathway or via myeloperoxidase (MPO) in HSCs and Kupffer cells. Additionally, in vitro, studies further suggested that CXCL1 treatment induced dysfunctional mitochondria, inflammation, HSCs activation, and macrophage migration, whereas Atriol countered these effects. Finally, the mitigating effects of Atriol on MASH were reproduced by pharmacological inhibition of NF-κB or MPO and neutralization of CXCL1. CONCLUSION: Atriol ameliorates MASH both in vitro and in vivo, demonstrating its potential therapeutic benefits in managing MASH.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, continues to produce mounting infections and fatalities all over the world. Recent data point to SARS-CoV-2 viral infections in the human testis. As low testosterone levels are associated with SARS-CoV-2 viral infections in males and human Leydig cells are the main source of testosterone, we hypothesized that SARS-CoV-2 could infect human Leydig cells and impair their function. We successfully detected SARS-CoV-2 nucleocapsid in testicular Leydig cells of SARS-CoV-2-infected hamsters, providing evidence that Leydig cells can be infected with SARS-CoV-2. We then employed human Leydig-like cells (hLLCs) to show that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 is highly expressed in hLLCs. Using a cell binding assay and a SARS-CoV-2 spike-pseudotyped viral vector (SARS-CoV-2 spike pseudovector), we showed that SARS-CoV-2 could enter hLLCs and increase testosterone production by hLLCs. We further combined the SARS-CoV-2 spike pseudovector system with pseudovector-based inhibition assays to show that SARS-CoV-2 enters hLLCs through pathways distinct from those of monkey kidney Vero E6 cells, a typical model used to study SARS-CoV-2 entry mechanisms. We finally revealed that neuropilin-1 and cathepsin B/L are expressed in hLLCs and human testes, raising the possibility that SARS-CoV-2 may enter hLLCs through these receptors or proteases. In conclusion, our study shows that SARS-CoV-2 can enter hLLCs through a distinct pathway and alter testosterone production.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Masculino , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Testosterona/metabolismo , Células Intersticiales del Testículo/metabolismo , Testículo/metabolismo , Peptidil-Dipeptidasa A/metabolismoRESUMEN
Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long-Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2-20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6-30 cells increased in all treatment groups, with 29 +/- 2% in control vs. 37 +/- 3, 35 +/- 3, and 56 +/- 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.
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Dietilhexil Ftalato/toxicidad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Exposición Materna , Animales , Femenino , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor Inhibidor de Leucemia/biosíntesis , Masculino , Embarazo , Preñez , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Factor de Células Madre/metabolismoRESUMEN
The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).
Asunto(s)
Colesterol/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Receptores de GABA/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Colesterol/análogos & derivados , Colesterol/química , Química Clic/métodos , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Proteínas de Transporte de Membrana Mitocondrial/química , Elastasa Pancreática/metabolismo , Procesos Fotoquímicos , Receptores de GABA/químicaRESUMEN
Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.
RESUMEN
Both fibroblast growth factor 2 (FGF2) and luteinizing hormone (LH) have been reported to regulate androgen production in Leydig cells in progenitor Leydig cells. The objective of the present study is to examine the regulation of androgen production in rat immature Leydig cells (ILCs). ILCs were isolated from 35-day-old rat testes and cultured in DMEM/F12 medium with LH (1 ng/ml) or FGF2 (10 ng/ml). 5alpha-Androstane-3alpha, 17beta-diol (3alpha-DIOL), the primary androgen in ILCs, and testosterone (T) were measured by Radioimmuno assay. The results showed the LH stimulated androgen production in ILCs, and FGF2 did not. However, FGF2 decreased the LH-stimulated androgen production. Real-time PCR and enzyme assay showed that FGF2 decreased levels of several steroidogenic enzymes, inhibited the expressions of steroidogenic acute regulatory (StAR) protein and steroidogenic factor 1 (Nr5a1) in LH-stimulated ILCs. FGF2-mediated inhibition of Nr5a1gene expression may be the mechanism through which FGF2 inhibits LH-stimulated androgen production.
Asunto(s)
Andrógenos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/farmacología , Fosfoproteínas , Factor Esteroidogénico 1 , Andrógenos/biosíntesis , Animales , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células Intersticiales del Testículo/efectos de los fármacos , Hormona Luteinizante/metabolismo , Masculino , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Factor Esteroidogénico 1/efectos de los fármacos , Factor Esteroidogénico 1/metabolismo , Testículo/citología , Testosterona/biosíntesisRESUMEN
Although the role of translocator protein (TSPO) in cholesterol transport in steroid-synthesizing cells has been studied extensively, recent studies of TSPO genetic depletion have questioned its role. Amhr2-Cre mice have been used to generate Leydig cell-specific Tspo conditional knockout (cKO) mice. Using the same Cre line, we were unable to generate Tspo cKO mice possibly because of genetic linkage between Tspo and Amhr2 and coexpression of Amhr2-Cre and Tspo in early embryonic development. We found that Amhr2-Cre is expressed during preimplantation stages, resulting in global heterozygous mice (gHE; Amhr2-Cre+/-,Tspo -/+). Two gHE mice were crossed, generating Amhr2-Cre-mediated Tspo global knockout (gKO; Tspo -/-) mice. We found that 33.3% of blastocysts at E3.5 to E4.5 showed normal morphology, whereas 66.7% showed delayed development, which correlates with the expected Mendelian proportions of Tspo +/+ (25%), Tspo -/- (25%), and Tspo +/- (50%) genotypes from crossing 2 Tspo -/+ mice. Adult Tspo gKO mice exhibited disturbances in neutral lipid homeostasis and reduced intratesticular and circulating testosterone levels, but no change in circulating basal corticosterone levels. RNA-sequencing data from mouse adrenal glands and lungs revealed transcriptome changes in response to the loss of TSPO, including changes in several cholesterol-binding and transfer proteins. This study demonstrates that Amhr2-Cre can be used to produce Tspo gKO mice instead of cKO, and can serve as a new global "Cre deleter." Moreover, our results show that Tspo deletion causes delayed preimplantation embryonic development, alters neutral lipid storage and steroidogenesis, and leads to transcriptome changes that may reflect compensatory mechanisms in response to the loss of function of TSPO.
RESUMEN
In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.
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Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/fisiología , Células Madre Adultas/metabolismo , Células Madre Adultas/fisiología , Factores de Edad , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Embrión de Mamíferos , Perfilación de la Expresión Génica , Masculino , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Células Madre/metabolismoRESUMEN
Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP), which are commonly found in cosmetics and in flexible plastics distributed by the food, construction, and medical products industries, have been classified as anti-androgens. High-dose DEHP exposure in utero is associated with decreased androgen levels. However, when administered after birth, low doses of DEHP (eg, 10 mg/kg body weight) may stimulate androgen production. In the present study, the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 +/- 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 +/- 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 +/- 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 +/- 0.37 ng/mL) and seminal vesicle weights (0.33 +/- 0.02 g) compared with control serum T (1.98 +/- 0.20 ng/mL) and seminal vesicle weight (0.26 +/- 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 +/- 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 microM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels.
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Dietilhexil Ftalato/farmacología , Maduración Sexual/fisiología , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Exposición a Riesgos Ambientales , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/fisiología , Hormona Luteinizante/sangre , Masculino , Tamaño de los Órganos , Próstata/anatomía & histología , Próstata/crecimiento & desarrollo , ARN/genética , Ratas , Ratas Long-Evans , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Seminales/anatomía & histología , Vesículas Seminales/crecimiento & desarrollo , Maduración Sexual/efectos de los fármacos , Testículo/anatomía & histología , Testículo/crecimiento & desarrolloRESUMEN
Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Glucocorticoides/metabolismo , Células Intersticiales del Testículo/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/antagonistas & inhibidores , Animales , Corticosterona/metabolismo , Regulación Enzimológica de la Expresión Génica , Masculino , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato , Testosterona/biosíntesisRESUMEN
The testis is known to be a site of corticosterone action, and testosterone production in Leydig cells is directly inhibited by glucocorticoids. Glucocorticoids bind to both glucocorticoid receptors (GRs) and to mineralocorticoid receptors (MRs). In Leydig cells, selective mineralocorticoid binding could result from oxidative inactivation of glucocorticoid by type 1 and/or 2 11beta-hydroxysteroid dehydrogenase (11betaHSD), as both isoforms are expressed. However, it remains unclear whether Leydig cells express MRs and respond directly to mineralocorticoid action. Therefore, the aims of the present study were to ascertain: (1) whether MR mRNA, protein and receptor binding are present in Leydig cells; and (2) if the mineralocorticoid modulates testosterone production. The mRNA encoding MR, as well as protein, and binding activity were each observed in adult rat Leydig cells. MR-ligand binding specificity within isolated Leydig cells was evaluated further by measuring displacement of MR binding to aldosterone by corticosterone in the presence and absence of carbenoxolone, an inhibitor of 11betaHSD1 and 2 that decreases conversion to biologically inert 11-dehydrocorticosterone. Carbenoxolone inhibited 11betaHSD oxidative activity, and reduced corticosterone-binding by 50%. Mineralocorticoid effects on steroidogenesis were assessed in the presence of aldosterone (0.01-10 nM) with or without the MR antagonist, RU28318. Aldosterone induced dose-dependent increases in both basal and luteinizing hormone-stimulated testosterone production. RU28318 eliminated the increase, indicating that these effects of aldosterone were mediated by the MR. The effects of aldosterone and luteinizing hormone (0.1 ng/ml) on testosterone production were synergistic, suggesting that the two hormones increased steroidogenesis through separate pathways. We conclude that Leydig cells express MRs and that testosterone production is subject to regulation by aldosterone.
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Aldosterona/farmacología , Células Intersticiales del Testículo/fisiología , Receptores de Mineralocorticoides/fisiología , Testosterona/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Aldosterona/metabolismo , Animales , Sitios de Unión , Carbenoxolona/farmacología , Corticosterona/análogos & derivados , Corticosterona/farmacología , Sinergismo Farmacológico , Técnicas In Vitro , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacología , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Espironolactona/farmacologíaRESUMEN
The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[14C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[14C]citrulline accumulation. Increased intracellular Ca2+ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.
Asunto(s)
Andrógenos/biosíntesis , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Óxido Nítrico/farmacología , Comunicación Paracrina/fisiología , Testosterona/biosíntesis , Animales , Arginina/farmacología , Perfilación de la Expresión Génica , Macrófagos/metabolismo , Masculino , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/citologíaRESUMEN
Only occupying about 1%-5% of total testicular cells, the adult Leydig cell (ALC) is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS). In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA) profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.
Asunto(s)
Antiespermatogénicos/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Mesilatos/farmacología , Regeneración/efectos de los fármacos , Testículo/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Familia 2 del Citocromo P450 , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Análisis por Micromatrices , Modelos Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Regeneración/fisiología , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Testículo/citología , Testículo/efectos de los fármacos , Testosterona/genética , Testosterona/metabolismoRESUMEN
Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 microg/kg.d (a dose that approximates BPA levels in the environment) from postnatal d 21-35 suppressed serum LH (0.21 +/- 0.05 ng/ml; vs. control, 0.52 +/- 0.04; P < 0.01) and testosterone (T) levels (1.62 +/- 0.16 ng/ml; vs. control, 2.52 +/- 0.21; P < 0.05), in association with decreased LHbeta and increased estrogen receptor beta pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nm BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17alpha-hydroxylase/17-20 lyase. BPA decreased serum 17beta-estradiol levels from 0.31 +/- 0.02 ng/ml (control) to 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.23 +/- 0.03 ng/ml in rats exposed to 2.4 microg, 10 microg, or 100 mg/kg.d BPA, respectively, from 21-35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 +/- 34 (control) to 261 +/- 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility.
Asunto(s)
Estrógenos no Esteroides/farmacología , Células Intersticiales del Testículo/enzimología , Hormona Luteinizante/metabolismo , Fenoles/farmacología , Hipófisis/metabolismo , Esteroides/biosíntesis , Envejecimiento , Andrógenos/biosíntesis , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Aromatasa/genética , Aromatasa/metabolismo , Compuestos de Bencidrilo , Relación Dosis-Respuesta a Droga , Estradiol/sangre , Receptor beta de Estrógeno , Femenino , Expresión Génica/efectos de los fármacos , Células Intersticiales del Testículo/efectos de los fármacos , Hormona Luteinizante/sangre , Hormona Luteinizante de Subunidad beta/genética , Masculino , Tamaño de los Órganos/efectos de los fármacos , Hipófisis/efectos de los fármacos , Embarazo , ARN Mensajero/análisis , Ratas , Ratas Long-Evans , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Seminales/crecimiento & desarrollo , Testículo/embriología , Testículo/crecimiento & desarrollo , Testosterona/biosíntesisRESUMEN
Physical and psychosocial stress challenge homeostasis, increasing glucocorticoid secretion (in rodents, corticosterone [CORT]) while decreasing testosterone (T) levels. The dynamics of stress-induced changes in T, CORT, and luteinizing hormone (LH) concentrations in mice have not been investigated previously. In particular, it remains to be established whether there is a rapid effect of CORT that is directly mediated by glucocorticoid receptors (GRs) in the testis. Therefore, serum and intratesticular T, serum CORT, and LH levels were measured during acute immobilization (IMO) stress, using the C57BL/6 strain of mice. The effects of testicular GR blockade were investigated by administration of the GR antagonist, RU486, via intratesticular (IT) or intraperitoneal (IP) injection. CORT levels increased in stressed males starting at 15 minutes, reaching a fivefold higher plateau by 1 hour compared with controls (P < .01). Conversely, starting from 30 minutes on, both serum and intratesticular T levels decreased in stressed males to 30% and 8% of control values, respectively, by 6 hours (P < .01). In contrast, LH was unchanged by IMO stress for up to 6 hours. Intratesticular treatment with RU486 partially prevented the IMO-induced decline in T levels. CORT treatment reduced intracellular cyclic adenosine monophosphate (cAMP) content in Leydig cells by 15 minutes and T production by 30 minutes in vitro. We conclude that 1) the rapid changes in T suggest a suppression of T biosynthesis by glucocorticoid through a nongenomic mechanism, lowering the production of cytoplasmic cAMP; 2) changes in gonadotropic stimulation of Leydig cells are unlikely to explain the suppression of T levels during acute stress; and 3) the results are consistent with a direct inhibitory action of CORT on Leydig cells.