RESUMO
The transcription factor steroidogenic factor 1 (SF-1) is exclusively expressed in the brain in the ventral medial hypothalamic nucleus (VMH) and is required for the development of this nucleus. However, the physiological importance of transcriptional programs regulated by SF-1 in the VMH is not well defined. To delineate the functional significance of SF-1 itself in the brain, we generated pre- and postnatal VMH-specific SF-1 KO mice. Both models of VMH-specific SF-1 KO were susceptible to high fat diet-induced obesity and displayed impaired thermogenesis after acute exposure to high fat diet. Furthermore, VMH-specific SF-1 KO mice showed significantly decreased LepR expression specifically in the VMH, leading to leptin resistance. Collectively, these results indicate that SF-1 directs transcriptional programs in the hypothalamus relevant to coordinated control of energy homeostasis, especially after excess caloric intake.
Assuntos
Gorduras na Dieta/administração & dosagem , Ingestão de Energia , Leptina/fisiologia , Fator Esteroidogênico 1/fisiologia , Termogênese , Núcleo Hipotalâmico Ventromedial/fisiologia , Animais , Homeostase , Camundongos , Camundongos Knockout , Fator Esteroidogênico 1/genéticaRESUMO
Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage.
Assuntos
Células-Tronco Fetais/citologia , Células-Tronco Fetais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Dosagem de Genes , Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologiaRESUMO
Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation.
Assuntos
Transtornos do Desenvolvimento Sexual/metabolismo , Feto , Proteínas Hedgehog/metabolismo , Células Intersticiais do Testículo/metabolismo , Ovário/metabolismo , Animais , Diferenciação Celular/genética , Transtornos do Desenvolvimento Sexual/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Feto/citologia , Feto/metabolismo , Proteínas Hedgehog/genética , Imuno-Histoquímica , Hibridização In Situ , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Diferenciação Sexual , Transdução de Sinais/genéticaRESUMO
During mammalian sex determination, expression of the Y-linked gene Sry shifts the bipotential gonad toward a testicular fate by upregulating a feed-forward loop between FGF9 and SOX9 to establish SOX9 expression in somatic cells. We previously proposed that these signals are mutually antagonistic with counteracting signals in XX gonads and that a shift in the balance of these factors leads to either male or female development. Evidence in mice and humans suggests that the male pathway is opposed by the expression of two signals, WNT4 and R-SPONDIN-1 (RSPO1), that promote the ovarian fate and block testis development. Both of these ligands can activate the canonical Wnt signaling pathway. Duplication of the distal portion of chromosome 1p, which includes both WNT4 and RSPO1, overrides the male program and causes male-to-female sex reversal in XY patients. To determine whether activation of beta-catenin is sufficient to block the testis pathway, we have ectopically expressed a stabilized form of beta-catenin in the somatic cells of XY gonads. Our results show that activation of beta-catenin in otherwise normal XY mice effectively disrupts the male program and results in male-to-female sex-reversal. The identification of beta-catenin as a key pro-ovarian and anti-testis signaling molecule will further our understanding of the mechanisms controlling sex determination and the molecular mechanisms that lead to sex-reversal.
Assuntos
Transtornos do Desenvolvimento Sexual , Regulação da Expressão Gênica no Desenvolvimento , Ovário/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , beta Catenina/metabolismo , Animais , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovário/metabolismo , Fenótipo , Diferenciação Sexual , Transdução de Sinais , Testículo/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt4 , beta Catenina/genéticaRESUMO
Steroidogenic factor 1 (SF-1) is a nuclear receptor that plays important roles in the hypothalamus-pituitary-steroidogenic organ axis. Global knockout studies in mice revealed the essential in vivo roles of SF-1 in the ventromedial hypothalamic (VMH) nucleus, adrenal glands, and gonads. One limitation of global SF-1 knockout mice is their early postnatal death from adrenocortical insufficiency. To overcome limitations of the global knockout mice and to delineate the roles of SF-1 in the brain, we used Cre/loxP recombination technology to genetically ablate SF-1 specifically in the central nervous system (CNS). Mice with CNS-specific knockout of SF-1 mediated by nestin-Cre showed increased anxiety-like behavior, revealing a crucial role of SF-1 in a complex behavioral phenotype. Our studies with CNS-specific SF-1 KO mice also defined roles of SF-1 in regulating the VMH expression of target genes implicated in anxiety and energy homeostasis. Therefore, this review will focus on our recent studies defining the functional roles of SF-1 in the VMH linked to anxiety and energy homeostasis.
Assuntos
Sistema Nervoso Central , Fator Esteroidogênico 1/metabolismo , Animais , Ansiedade/metabolismo , Comportamento Animal/fisiologia , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/metabolismo , Metabolismo Energético , Homeostase , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Fator Esteroidogênico 1/genéticaRESUMO
Steroidogenic factor 1 (SF-1) plays key roles in adrenal and gonadal development, expression of pituitary gonadotropins, and development of the ventromedial hypothalamic nucleus (VMH). If kept alive by adrenal transplants, global knockout (KO) mice lacking SF-1 exhibit delayed-onset obesity and decreased locomotor activity. To define specific roles of SF-1 in the VMH, we used the Cre-loxP system to inactivate SF-1 in a central nervous system (CNS)-specific manner. These mice largely recapitulated the VMH structural defect seen in mice lacking SF-1 in all tissues. In multiple behavioral tests, mice with CNS-specific KO of SF-1 had significantly more anxiety-like behavior than wild-type littermates. The CNS-specific SF-1 KO mice had diminished expression or altered distribution in the mediobasal hypothalamus of several genes whose expression has been linked to stress and anxiety-like behavior, including brain-derived neurotrophic factor, the type 2 receptor for CRH (Crhr2), and Ucn 3. Moreover, transfection and EMSAs support a direct role of SF-1 in Crhr2 regulation. These findings reveal important roles of SF-1 in the hypothalamic expression of key regulators of anxiety-like behavior, providing a plausible molecular basis for the behavioral effect of CNS-specific KO of this nuclear receptor.
Assuntos
Ansiedade/genética , Sistema Nervoso Central/metabolismo , Fator Esteroidogênico 1/genética , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Sítios de Ligação , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Fator Esteroidogênico 1/metabolismo , Fator Esteroidogênico 1/fisiologiaRESUMO
The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Mitocôndrias/metabolismo , Fosfoproteínas/fisiologia , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/sangue , Animais , Southern Blotting , Corticosterona/sangue , Feminino , Técnicas de Transferência de Genes , Gônadas/metabolismo , Immunoblotting , Masculino , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Ovário/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/metabolismoRESUMO
The nuclear receptor steroidogenic factor 1 (SF-1) plays essential roles in the development and function of the ventromedial hypothalamic nucleus (VMH). Considerable evidence links the VMH and SF-1 with the regulation of energy homeostasis. Here, we demonstrate that SF-1 colocalizes in VMH neurons with the cannabinoid receptor 1 (CB1R) and that a specific CB1R agonist modulates electrical activity of SF-1 neurons in hypothalamic slice preparations. We further show that SF-1 directly regulates CB1R gene expression via a SF-1-responsive element at -101 in its 5'-flanking region. Finally, we show that knockout mice with selective inactivation of SF-1 in the brain have decreased expression of CB1R in the region of the VMH and exhibit a blunted response to systemically administered CB1R agonists. These studies suggest that SF-1 directly regulates the expression of CB1R, which has been implicated in the regulation of energy homeostasis and anxiety-like behavior.
Assuntos
Regulação da Expressão Gênica , Receptor CB1 de Canabinoide/genética , Fator Esteroidogênico 1/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Ácidos Araquidônicos/administração & dosagem , Ácidos Araquidônicos/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Escuridão , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fosforilação/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Elementos de Resposta , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Núcleo Hipotalâmico Ventromedial/enzimologiaRESUMO
Leptin, an adipocyte-derived hormone, has emerged as a critical regulator of energy homeostasis. The leptin receptor (Lepr) is expressed in discrete regions of the brain; among the sites of highest expression are several mediobasal hypothalamic nuclei known to play a role in energy homeostasis, including the arcuate nucleus, the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus. Although most studies have focused on leptin's actions in the arcuate nucleus, the role of Lepr in these other sites has received less attention. To explore the role of leptin signaling in the VMH, we used bacterial artificial chromosome transgenesis to target Cre recombinase to VMH neurons expressing steroidogenic factor 1, thereby inactivating a conditional Lepr allele specifically in steroidogenic factor 1 neurons of the VMH. These knockout (KO) mice, designated Lepr KO(VMH), exhibited obesity, particularly when challenged with a high-fat diet. On a low-fat diet, Lepr KO(VMH) mice exhibited significantly increased adipose mass even when their weights were comparable to wild-type littermates. Furthermore, these mice exhibited a metabolic syndrome including hepatic steatosis, dyslipidemia, and hyperleptinemia. Lepr KO(VMH) mice were hyperinsulinemic from the age of weaning and eventually developed overt glucose intolerance. These data define nonredundant roles of the Lepr in VMH neurons in energy homeostasis and provide a model system for studying other actions of leptin in the VMH.
Assuntos
Adiposidade/genética , Síndrome Metabólica/genética , Receptores para Leptina/genética , Núcleo Hipotalâmico Ventromedial/metabolismo , Animais , Composição Corporal/genética , Peso Corporal/genética , Ingestão de Alimentos/genética , Sistema Endócrino/fisiologia , Metabolismo Energético/genética , Feminino , Glucose/metabolismo , Homeostase/genética , Integrases/genética , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Especificidade de Órgãos , Receptores para Leptina/metabolismoRESUMO
OBJECTIVE: Steroidogenic factor 1 (SF1), officially designated NR5A1, is a nuclear receptor that plays key roles in endocrine development and function. Previous reports of human SF1 mutations revealed a spectrum of phenotypes affecting adrenal function and/or gonadal development and sex differentiation. We present the clinical phenotype and functional effects of a novel SF1 mutation. PATIENT: The patient is a 22-year-old 46, XY Japanese patient who presented with dysgenetic testes, atrophic vasa deferentia and epididymides, lack of Müllerian structures, and clitoromegaly. Endocrine studies revealed normal adrenal function. RESULTS: Analysis of the SF1 gene revealed compound heterozygosity for a previously described p.G146A polymorphism and a novel missense mutation (p.R84C) in the accessory DNA-binding domain. The father carried the p.G146A polymorphism and the mother had the p.R84C mutation; both were clinically and reproductively normal. Functional studies demonstrated that the p.R84C SF1 had normal nuclear localization but decreased DNA-binding affinity and transcriptional activity compared with wild-type SF1; it did not exhibit any dominant negative activity. CONCLUSIONS: These results describe the human phenotype that results from compound heterozygosity of the p.G146A polymorphism and a novel p.R84C mutation of SF1, thereby extending the spectrum of human SF1 mutations that impair testis development and sex differentiation in a sex-limited manner while preserving normal adrenal function.
Assuntos
Proteínas de Ligação a DNA/genética , Disgenesia Gonadal 46 XY/genética , Proteínas de Homeodomínio/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Insuficiência Adrenal/genética , Insuficiência Adrenal/fisiopatologia , Adulto , Células Cultivadas , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunofluorescência , Disgenesia Gonadal 46 XY/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Luciferases/genética , Mutação/fisiologia , Plasmídeos/genética , Polimorfismo Genético/genética , Polimorfismo Genético/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator Esteroidogênico 1 , Receptores alfa dos Hormônios Tireóideos/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The ventromedial hypothalamic nucleus (VMH) plays an important role in the control of feeding and energy homeostasis. In contrast to other hypothalamic nuclei that are also known to regulate energy balance, there is a paucity of nucleus-specific marker genes for the VMH, limiting the application of molecular approaches for analyzing VMH information processing, function, and circuitry. Here, we report the use of laser-capture microdissection to isolate a set of cDNAs that are enriched in the VMH relative to two adjacent hypothalamic nuclei, the arcuate and dorsomedial hypothalamus. The relative expression levels of nine of the 12 most robustly expressed VMH-enriched genes were confirmed by real-time PCR analysis using separate RNAs from these three nuclei. Three of these VMH-enriched genes were further characterized by in situ hybridization histochemistry, including pituitary adenylate cyclase activating polypeptide, cerebellin 1, and an expressed sequence tag named LBH2. Finally, to test whether some of these genes were coordinately regulated, we monitored their expression in steroidogenic factor 1 (SF-1) knock-out mice. SF-1 is a transcription factor that controls the development of the VMH. The RNA levels for four of these genes were reduced in these knock-out animals, further suggesting that they are direct or indirect targets of this orphan nuclear receptor. The VMH-enriched genes identified here provide a basis for a functional analysis of VMH neuronal subpopulations via the use of bacterial artificial chromosome transgenics and related technologies. These results also demonstrate the utility of laser-capture microdissection coupled with microarray technology to identify nucleus-specific transcriptional networks.
Assuntos
Biomarcadores/metabolismo , Regulação da Expressão Gênica/fisiologia , Lasers , Microdissecção/métodos , Núcleo Hipotalâmico Ventromedial/metabolismo , Animais , Northern Blotting/métodos , Feminino , Citometria de Fluxo/métodos , Proteínas de Homeodomínio , Hibridização In Situ/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator Esteroidogênico 1 , Fatores de Transcrição/deficiência , Núcleo Hipotalâmico Ventromedial/anatomia & histologiaRESUMO
Recent data implicate locally produced steroids, termed neurosteroids, as regulators of neuronal function. Adrenal and gonadal steroidogenesis is controlled by changes in the steroidogenic acute regulatory protein (StAR); however, little is known about the regulation of neurosteroid production. We now demonstrate unequivocally that StAR mRNA and protein are expressed within glia and neurons in discrete regions of the mouse brain, and that glial StAR expression is inducible. Consistent with a role in de novo neurosteroidogenesis, StAR colocalizes with the cholesterol side-chain cleavage enzyme P450(scc) in both mouse and human brains. These data support a role for StAR in the production of neurosteroids and identify potential sites of active de novo steroid synthesis in the brain.
Assuntos
Encéfalo/metabolismo , Fosfoproteínas/metabolismo , Animais , Encéfalo/citologia , Química Encefálica , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/análise , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Neuroglia/metabolismo , Neurônios/metabolismo , Especificidade de Órgãos , Fosfoproteínas/análise , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Esteroides/biossíntese , Células Tumorais CultivadasRESUMO
The adrenal glands are comprised of two distinct endocrine organs: the outer cortex, which is derived from mesoderm and synthesizes steroid hormones, and the inner medulla, which contains neuroectodermal cells derived from the neural crest and produces the catecholamine hormones norepinephrine and epinephrine. The developmental program that gives rise to the adrenal gland begins early during embryogenesis and continues throughout gestation and well after birth. In this article, we review the molecular mechanisms of adrenal differentiation and development, focusing on the contributions of genes responsible for the development of the adrenal cortex as identified from studies of experimental animal models and human subjects with clinical diseases. These studies identify a hierarchical network of transcription factors, including Wilms' tumor-1, steroidogenic factor-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, X-linked-1, PBX1, and CITED2, that both give rise to the adrenal cortex and subsequently determine its subsequent function in steroidogenesis.
Assuntos
Córtex Suprarrenal/embriologia , Córtex Suprarrenal/fisiologia , Glândulas Suprarrenais/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Glândulas Suprarrenais/patologia , Animais , Receptor Nuclear Órfão DAX-1 , Proteínas de Ligação a DNA/fisiologia , Epinefrina/metabolismo , Proteínas de Homeodomínio , Humanos , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Norepinefrina/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas/fisiologia , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/fisiologia , Proteínas Repressoras/fisiologia , Fator Esteroidogênico 1 , Esteroides/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Proteínas WT1/fisiologiaRESUMO
Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Luminescentes/genética , Fatores de Transcrição/genética , Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Animais , Biomarcadores , Linhagem da Célula , Cromossomos Artificiais Bacterianos , Proteínas de Ligação a DNA/metabolismo , Sistema Endócrino/fisiologia , Feminino , Citometria de Fluxo/métodos , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica no Desenvolvimento , Engenharia Genética/métodos , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ovário/embriologia , Ovário/metabolismo , Adeno-Hipófise/embriologia , Adeno-Hipófise/metabolismo , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Testículo/embriologia , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células Intersticiais do Testículo/patologia , Ovário/fisiologia , Testículo/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proliferação de Células , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Corpo Lúteo/anormalidades , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica , Células da Granulosa/fisiologia , Proteínas de Homeodomínio , Infertilidade/genética , Integrases/genética , Células Intersticiais do Testículo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Ovário/patologia , Fosfoproteínas/genética , Receptores Citoplasmáticos e Nucleares , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta , Diferenciação Sexual/genética , Fator Esteroidogênico 1 , Testículo/anormalidades , Testículo/citologia , Fatores de Transcrição/genéticaRESUMO
The steroidogenic acute regulatory protein (StAR) is essential for the regulated production of steroid hormones, mediating the translocation of intracellular cholesterol to the inner mitochondrial membrane where steroidogenesis begins. Steroidogenic cells lacking StAR have impaired steroidogenesis and progressively accumulate lipid, ultimately causing cytopathic changes and deterioration of steroidogenic capacity. Developmental studies of StAR knockout (KO) mice have correlated gonadal lipid deposits with puberty, suggesting that trophic hormones contribute to this lipid accumulation. To delineate the role of gonadotropins in this process, we examined double mutant mice deficient in both StAR and gonadotropins [StAR KO/hpg (hypogonadal)]. Lipid accumulation was ameliorated considerably in StAR KO/hpg mice but was restored by treatment with exogenous gonadotropins, directly linking trophic hormones with gonadal lipid accumulation. To define the relative roles of exogenous vs. endogenous cholesterol in the lipid accumulation, we also examined mice lacking both StAR and apolipoprotein A-I (StAR KO/Apo A-I KO). Steroidogenic tissues of StAR KO/Apo A-I KO mice had markedly decreased lipid deposits, supporting the predominant role of high-density lipoprotein-derived cholesterol in the lipid accumulation caused by StAR deficiency. Finally, we used electron microscopy to compare mitochondrial ultrastructure in StAR KO and cholesterol side-chain cleavage enzyme (Cyp11a1) KO mice; despite comparable lipid accumulation within adrenocortical cells, the effects of StAR deficiency and Cyp11a1 deficiency on mitochondrial ultrastructure were markedly different. These findings extend our understanding of steroidogenic cell dysfunction in StAR KO mice and highlight key roles of trophic hormones and high-density lipoprotein-derived cholesterol in lipid deposits within StAR-deficient steroidogenic cells.
Assuntos
Gonadotropinas/metabolismo , Lipoproteínas HDL/metabolismo , Fosfoproteínas/genética , Córtex Suprarrenal/metabolismo , Fatores Etários , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Gonadotropinas/genética , Metabolismo dos Lipídeos , Lipoproteínas HDL/genética , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ovário/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Esteroides/sangue , Testículo/metabolismoRESUMO
Although recent research has focused on the fundamental role(s) of steroids synthesized de novo in the brain on development, the mechanism by which production of these neurosteroids is regulated remains unclear. Steroid production in peripheral tissues is acutely regulated by the steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroid biosynthesis: the intramitochondrial delivery of cholesterol to cytochrome P450scc for conversion to steroid. We recently demonstrated that StAR is present in discrete cell types in the adult brain, suggesting that neurosteroid production is mediated by StAR. Nevertheless, little is known regarding the presence of StAR in the developing brain. In the present study, the presence of StAR and for the first time, its homolog, the putative cholesterol transport protein metastatic lymph node 64 (MLN64), were defined in the neonatal mouse brain using immunocytochemical techniques. Both StAR and MLN64 were found to be present in the brain with staining patterns characteristic to each protein, indicating the authenticity of StAR and MLN64 immunoreactivity. Furthermore, we found MLN64 to be expressed in the adult brain as well, apparently at higher levels than StAR. Importantly, StAR protein is present in cells that also express P450scc. These data suggest that, as with the adult, neurosteroid production during development occurs through a StAR-mediated pathway.
Assuntos
Animais Recém-Nascidos/metabolismo , Encéfalo/metabolismo , Fosfoproteínas/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Imuno-Histoquímica , Camundongos , Distribuição TecidualRESUMO
Knockout (KO) mice lacking steroidogenic factor 1 (SF-1) exhibit a phenotype that includes adrenal and gonadal agenesis, impaired gonadotropin expression, and abnormalities of the ventromedial hypothalamic nucleus (VMH). Studies in rodents with lesions of the ventromedial hypothalamus have implicated the VMH in body weight regulation, suggesting that SF-1 KO mice may provide a genetic model of obesity. To prevent death, SF-1 KO mice were rescued with corticosteroid injections, followed by syngeneic adrenal transplants from wild-type (WT) littermates. Corticosterone and ACTH levels in WT and SF-1 KO mice were indistinguishable, documenting restoration of hypothalamic-pituitary-adrenal function. Although weights at earlier ages did not differ significantly from WT littermates, SF-1 KO mice were significantly heavier by 8 wk of age and eventually weighed almost twice as much as WT controls. Obesity in SF-1 KO mice predominantly resulted from decreased activity rather than increased food intake. Leptin was increased markedly, insulin was modestly elevated, and glucose was indistinguishable from WT mice. Although sex steroids in rodents affect weight, ovariectomy did not abolish the weight difference between WT and SF-1 KO mice. These SF-1 KO mice are a genetic model of late-onset obesity that may help elucidate the role of the VMH in weight regulation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Doenças Hipotalâmicas/genética , Obesidade/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Corticosteroides/sangue , Glândulas Suprarrenais/transplante , Animais , Peso Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Fatores de Transcrição Fushi Tarazu , Proteínas de Homeodomínio , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovariectomia , Fenótipo , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that plays key roles in endocrine development and function. Knockout mice lacking SF-1 have adrenal and gonadal agenesis, impaired gonadotropin expression, and structural abnormalities of the ventromedial hypothalamic nucleus. Previous studies have identified three human subjects with mutations in SF-1 causing adrenocortical insufficiency with varying degrees of gonadal dysfunction. We now describe a novel 8-bp microdeletion of SF-1, isolated from a 46, XY patient who presented with gonadal agenesis but normal adrenal function, which causes premature termination upstream of sequences encoding the activation function 2 domain. In cell transfection experiments, the mutated protein possessed no intrinsic transcriptional activity but rather inhibited the function of the wild-type protein in most cell types. To our knowledge, this is the first example of an apparent dominant-negative effect of a SF-1 mutation in humans. These findings, which define a SF-1 mutation that apparently differentially affects its transcriptional activity in vivo in the adrenal cortex and the gonads, may be relevant to the cohort of patients who present with 46, XY sex reversal but normal adrenal function.
Assuntos
Glândulas Suprarrenais/fisiopatologia , Proteínas de Ligação a DNA/genética , Transtornos do Desenvolvimento Sexual , Deleção de Genes , Fatores de Transcrição/genética , Adulto , Sequência de Bases , Feminino , Fatores de Transcrição Fushi Tarazu , Genes Dominantes , Genitália Feminina/anormalidades , Proteínas de Homeodomínio , Humanos , Ligantes , Mutação , Receptores Citoplasmáticos e Nucleares , Fator Esteroidogênico 1 , Transcrição GênicaRESUMO
Targeted gene disruption has produced knockout (KO) mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 KO mice lacked adrenal glands and gonads, and also had impaired expression of gonadotropins in pituitary gonadotropes and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). To define the roles of SF-1 within discrete sites of the hypothalamic-pituitary-steroidogenic organ axis, we have sought to make tissue-specific SF-1 KO mice (as reviewed here). We first used adrenal transplants to restore adrenal function in global SF-1 KO mice, providing a physiological form of a "VMH-specific" KO to study the roles of SF-1 in weight regulation. These adrenal-transplanted SF-1 KO mice became obese due to decreased locomotor activity, providing a novel model of hypothalamic obesity. Mice with a pituitary-specific KO of SF-1 mediated by the Cre-loxP recombination strategy exhibited hypogonadotropic hypogonadism, revealing essential roles of SF-1 in pituitary function in vivo. Ongoing studies seek to inactivate SF-1 in the brain or specific gonadal cell types, thereby defining its roles in development and function at these sites. In addition, we review our use of bacterial artificial chromosome transgenesis to develop a fluorescent marker for cells that express SF-1.