RESUMO
Early expression of estrogen receptors (esr) and their role in regulating early expression of cyp19a1b encoding brain aromatase were examined in the brain of zebrafish. Using in toto hybridization and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), a significant increase in the expression of esr1, esr2a, and esr2b was observed between 24 and 48 hours postfertilization (hpf). In toto hybridization demonstrated that esr2a and esr2b, but not esr1, are found in the hypothalamus. Using real-time RT-PCR, an increase in cyp19a1b mRNAs occurs between 24 and 48 hpf, indicating that expression of cyp19a1b is temporally correlated with that of esr. This increase is blocked by the pure anti-estrogen ICI182,780. Furthermore, E2 treatment of cyp19a1b-GFP (green fluorescent protein) transgenic embryos results in appearance of GFP expression in the brain as early as 25 hpf. These results indicate that basal expression of cyp19a1b expression in the brain of developing zebrafish most likely relies upon expression of esr that are fully functional before 25 hpf.
Assuntos
Aromatase/metabolismo , Encéfalo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Estrogênio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Aromatase/genética , Encéfalo/embriologia , Encéfalo/enzimologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/enzimologia , Estradiol/análogos & derivados , Estradiol/metabolismo , Antagonistas de Estrogênios/metabolismo , Fulvestranto , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Steroid hormones are important physiological regulators in the body. Steroid hormones are mainly synthesized in the adrenal and gonads. Their synthesis is stimulated by pituitary hormones through cAMP as an intracellular mediator. The first and rate-limiting step for steroid biosynthesis is catalyzed by CYP11A1. Important regulatory elements for the control of the CYP11A1 gene expression have been characterized both in vitro and in vivo. The SF-1-binding sites are cis-acting elements controlling the basal and cAMP-stimulated gene expression. Our transgenic mouse studies showed that the 2.3kb promoter contains information controlling developmentally regulated gene expression. Finally, we present our results on the cloning of steroidogenic genes in zebrafish, a new model organism for genetic studies.
Assuntos
Regulação da Expressão Gênica , Camundongos Transgênicos/genética , Esteroides/biossíntese , Peixe-Zebra/genética , Glândulas Suprarrenais/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , AMP Cíclico/farmacologia , Gônadas/metabolismo , Camundongos , Modelos Animais , Hormônios Hipofisários/farmacologia , Regiões Promotoras GenéticasRESUMO
The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.
Assuntos
Aromatase/genética , Aromatase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Encéfalo/enzimologia , Células COS , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Expressão Gênica , Hibridização In Situ , Masculino , Ovário/enzimologia , Filogenia , Reprodução/genética , Comportamento Sexual Animal , Distribuição Tecidual , TransfecçãoRESUMO
The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.