RESUMO
Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.
Assuntos
Hormônios Esteroides Gonadais/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Células Tecais/citologia , Células Tecais/metabolismo , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Feminino , Hormônios Esteroides Gonadais/metabolismo , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Células Tecais/efeitos dos fármacosRESUMO
Significant advances have taken place in our knowledge of the enzymes involved in steroid hormone biosynthesis since the last comprehensive review in 1988. Major developments include the cloning, identification, and characterization of multiple isoforms of 3beta-hydroxysteroid dehydrogenase, which play a critical role in the biosynthesis of all steroid hormones and 17beta-hydroxysteroid dehydrogenase where specific isoforms are essential for the final step in active steroid hormone biosynthesis. Advances have taken place in our understanding of the unique manner that determines tissue-specific expression of P450aromatase through the utilization of alternative promoters. In recent years, evidence has been obtained for the expression of steroidogenic enzymes in the nervous system and in cardiac tissue, indicating that these tissues may be involved in the biosynthesis of steroid hormones acting in an autocrine or paracrine manner. This review presents a detailed description of the enzymes involved in the biosynthesis of active steroid hormones, with emphasis on the human and mouse enzymes and their expression in gonads, adrenal glands, and placenta.
Assuntos
Corticosteroides/biossíntese , Colesterol/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Hidroxiesteroide Desidrogenases/metabolismo , Corticosteroides/química , Animais , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Hormônios Esteroides Gonadais/química , Humanos , Hidroxiesteroide Desidrogenases/genética , Camundongos , Especificidade de ÓrgãosRESUMO
The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD) is required for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each a product of a distinct gene. Two isoforms, 3betaHSD I and II, are expressed in a tissue-specific manner in humans. 3betaHSD I is the only isoform expressed in the placenta, where it is required for the biosynthesis of progesterone and thus essential for the maintenance of pregnancy. We recently identified two transcription factors, activating protein-2gamma (AP-2gamma) and the homeodomain protein, distaless-3 (Dlx-3), that are expressed in both human and mouse trophoblast cells that were shown to be required for trophoblast-specific expression of the orthologous murine 3betaHSD, 3betaHSD VI. Although we identified specific binding sites for AP-2gamma and Dlx-3 in the distal promoter of the human 3betaHSD I gene, HSD3B1, it was found that these transcription factors were not involved in determining placental-specific expression of human 3betaHSD I. Instead, a 53-bp placental-specific enhancer element located between -2570 and -2518 of the HSD3B1 promoter was identified. Within this 53-bp element, two potential placental transcription factor binding sites were found. EMSAs with a 20-bp oligonucleotide containing these two potential placental-specific binding sites identified one of the binding sites specific for the transcription enhancer factor (TEF)-5, which is highly expressed in human placenta and in placental choriocarcinoma-derived JEG-3 cells and the other overlapping binding site, specific for a GATA-like protein. Site-specific mutations in either the TEF-5 binding site or in the GATA binding site, each resulted in complete loss of enhancer activity. The data indicate that TEF-5 and the GATA-like protein act in a coordinate manner to determine the placental-specific expression of the human 3betaHSD I enzyme and therefore are critical for placental progesterone production required for the maintenance of pregnancy.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Placenta/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , 17-Hidroxiesteroide Desidrogenases/biossíntese , Região 5'-Flanqueadora/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Camundongos , Mutação/genética , Especificidade de Órgãos , Placenta/enzimologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição de Domínio TEA , Transcrição Gênica/genéticaRESUMO
A matured megakaryocyte releases thousands of platelets through a drastic morphological change, proplatelet formation (PPF). The megakaryocyte/erythrocyte-specific transcription factor, p45 NF-E2, is essential for initiating PPF, but the factor regulating PPF has not been identified. Here we report that estradiol synthesized in megakaryocytes triggers PPF. We demonstrate that a key enzyme for steroid hormone biosynthesis, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), is a target of p45 NF-E2, and rescues PPF of p45 NF-E2-deficient megakaryocytes. We also show that estradiol is synthesized within megakaryocytes, and that extracellular estradiol stimulates PPF, inhibition of 3beta-HSD activity blocks PPF, and estrogen receptor antagonists inhibit platelet production in vivo. We conclude that autocrine estradiol action regulates platelet production by triggering PPF.
Assuntos
Plaquetas/citologia , Estradiol/fisiologia , Megacariócitos/citologia , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fatores de Ligação de DNA Eritroide Específicos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Fatores de Transcrição/metabolismoRESUMO
Müllerian inhibiting substance (MIS) is produced by fetal Sertoli cells and causes regression of the Müllerian duct in male fetuses shortly after commitment of the bipotential embryonic gonad to testes differentiation. MIS is also produced by the Sertoli cells and granulosa cells of the adult gonads where it plays an important role in regulating steroidogenesis. We have previously shown that MIS can dramatically reduce testosterone synthesis in Leydig cells by inhibiting the expression of cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (Cyp17) mRNA in vitro and in vivo. To characterize the signal transduction pathway used by MIS to control expression of endogenous Cyp17 in a mouse Leydig cell line, we demonstrate that MIS inhibits both LH- and cAMP-induced expression of Cyp17 at concentrations as low as 3.5 nM and for as long as 18 h. The induction of steroidogenic acute regulatory protein (StAR) mRNA by cAMP, however, was slightly increased by addition of MIS. Protein kinase A (PKA) inhibition with H-89 blocked Cyp17 mRNA induction, suggesting that MIS interferes with the PKA signal transduction pathway. Inhibition of Cyp17 induction was not seen with added U0126, and wortmannin inhibited the induction incompletely. In addition, phosphorylation of the cAMP responsive element binding protein (CREB) was not detected following 50 micro M cAMP exposure, a concentration sufficient for Cyp17 mRNA induction. Moreover, CREB phosphorylation, which was observed with addition of 500 micro M cAMP, was not inhibited by coincubation with MIS. Taken together, these results suggest that cAMP induces expression of Cyp17 by a PKA-mediated mechanism and that this induction, which is inhibited by MIS signal transduction, does not require CREB activity, and is distinct from that used to induce steroidogenic acute regulatory protein expression.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Glicoproteínas , Inibidores do Crescimento/farmacologia , Células Intersticiais do Testículo/enzimologia , Esteroide 17-alfa-Hidroxilase/genética , Hormônios Testiculares/farmacologia , Animais , Hormônio Antimülleriano , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores do Crescimento/administração & dosagem , Cinética , Tumor de Células de Leydig , Masculino , Camundongos , Fosforilação , RNA Mensageiro/análise , Transdução de Sinais , Hormônios Testiculares/administração & dosagem , Células Tumorais CultivadasRESUMO
The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is essential for the biosynthesis of all active steroid hormones. The 3beta-HSD enzyme consists in multiple isoforms, each the product of a distinct gene. In the mouse, six tissue-specific isoforms have been identified. These isoforms are expressed in a tissue- and temporal specific manner. Mouse 3beta-HSD VI is the only isoform expressed in decidua and giant trophoblast cells during the first half of mouse pregnancy. The tissue- and temporal-specific expression of 3beta-HSD VI during mouse pregnancy, as determined by in situ hybridization and immunohistochemistry, shows that 3beta-HSD is expressed exclusively in the antimesometrial decidua on E6.5 and E7.5. By E9.5, expression of 3beta-HSD is observed in giant trophoblast cells with a marked increase in expression by E10.5. No expression of 3beta-HSD is seen in decidua after E7.5 and no expression of 3beta-HSD is seen in the embryo at any of the times investigated. Giant trophoblast cells in culture from E9.5 and E10.5 synthesize progesterone with cells from E10.5 producing about 3.5-fold more progesterone during the first 24 h in culture. Western blot analysis of 3beta-HSD VI protein demonstrates that the amount of 3beta-HSD VI protein correlates with the amount of progesterone biosynthesis in giant trophoblast cells from E9.5 and E10.5. We propose that progesterone produced during the first half of mouse pregnancy in decidua and giant trophoblast cells acts as an immunosuppressant at the fetal maternal interface to prevent rejection of the fetus.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Camundongos/fisiologia , Prenhez/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Feminino , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos/genética , Família Multigênica , Gravidez , Prenhez/genética , Progesterona/biossíntese , Progesterona/fisiologia , Trofoblastos/metabolismoRESUMO
The ontogeny and functional role of steroidogenesis during mammalian gestation is poorly understood. This review provides a summary of our recent findings on the spatio-temporal expression of key steroidogenic genes controlling progesterone synthesis in the uterus during mouse pregnancy. We have shown that onset of cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3beta-hydroxysteroid dehydrogenase/isomerase type VI (3betaHSD VI) expression occurs upon decidualization of the uterine wall induced by implantation. This unexpected early expression of the enzymes in the maternal decidua is terminated at mid-pregnancy when the steroidogenic ability reappears in the extraembryonic giant cells at the time of placentation. The giant cells express another protein indispensable for steroid hormone synthesis in the adrenal and gonads, Steroidogenic Acute Regulatory (StAR) protein. Unlike the human placenta, the steroidogenic genes are not expressed in the cells of the mature mouse placenta during the second half of gestation. Finally, our studies suggest that transcriptional regulation of P450scc is mediated by a non-SF-1 protein that substitutes SF-1 functions in the extraembryonic cells. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the maternofetal interface preventing the rejection of the fetal allograft.
Assuntos
Placenta/metabolismo , Prenhez/metabolismo , Roedores/fisiologia , Esteroides/biossíntese , Útero/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Expressão Gênica , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Gravidez , RatosRESUMO
The enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each the product of a distinct gene. Human 3 beta-HSD I in placenta is essential for placental progesterone biosynthesis and thus is essential for the maintenance of pregnancy. The murine ortholog, 3 beta-HSD VI, is the only isoform expressed in giant trophoblast cells during the first half of mouse pregnancy. This study was designed to identify the cis-acting element(s) and the associated transcription factors required for trophoblast-specific expression of 3 beta-HSD VI. Transfection studies in placental and nonplacental cells identified a novel 66-bp trophoblast-specific enhancer element located between -2896 and -2831 of the 3 beta-HSD VI promoter. DNase protection analysis of the enhancer element identified three trophoblast-specific binding sites, FPI, FPII, and FPIII. Electrophoretic mobility shift assays with oligonucleotides representing the protected sequences, FPI and FPIII, and nuclear extracts isolated from human JEG-3 cells and from mouse trophoblast cells, demonstrated the same binding pattern that was distinct from the binding pattern with mouse Leydig cell nuclear proteins. Further electrophoretic mobility shift assays identified AP-2 gamma and the homeodomain protein, Dlx 3, as the transcription factors that specifically bind to FPI and FPIII, respectively. Site-specific mutations in each of the binding sites eliminated enhancer activity indicating that AP-2 gamma and Dlx 3, together with an additional transcription factor(s) that are conserved between humans and mice, are required for trophoblast-specific expression of 3 beta-HSD VI.