RESUMO
In vertebrates, sex is determined essentially by two means, genetic factors located on sex chromosomes and epigenetic factors such as temperature experienced by the individual during development. Steroids, especially estrogens, are clearly involved in gonadal differentiation in non-mammalian vertebrates. In this regard, the expression of the estrogen-producing enzyme, aromatase, has been shown to be temperature-sensitive in species where temperature can reverse sex differentiation, especially in our model, the amphibian Pleurodeles waltl. We investigated here the regulation of aromatase expression in the brain during sex differentiation in Pleurodeles. We first isolated a brain isoform of aromatase mRNA which differs in its 5' untranslated region from the isoform previously isolated from adult gonads. In adult Pleurodeles, the brain isoform is mainly expressed in brain tissue while the other isoform is gonad specific. Thus, regulation of aromatase expression in P. waltl could occur by alternative splicing of non-coding exon 1 as previously described in mammals. We then investigated aromatase expression in the brain of male and female larvae and found no differences with regard to sex. Measures of aromatase activity in the brain also showed no differences between sexes at larval stages whereas activity markedly increases in the ovary concomitant with the start of gonadal differentiation. These results support the hypothesis that aromatase could be a target of a temperature-sensitive sex-reversing effect in the gonads but not in the brain.
Assuntos
Aromatase/metabolismo , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Gônadas/enzimologia , Pleurodeles/crescimento & desenvolvimento , Pleurodeles/genética , Diferenciação Sexual , Envelhecimento/fisiologia , Sequência de Aminoácidos , Animais , Aromatase/química , Aromatase/genética , Sequência de Bases , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TemperaturaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Metais/farmacologia , Pleurodeles/metabolismo , Xenopus laevis/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Sítios de Ligação , Cádmio/metabolismo , Cádmio/farmacologia , Células Cultivadas , Cobre/metabolismo , Cobre/farmacologia , DNA Complementar/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Modelos Moleculares , Dados de Sequência Molecular , Pleurodeles/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Xenopus laevis/genética , Zinco/metabolismo , Zinco/farmacologiaRESUMO
The Ikaros gene encodes a family of transcription factors which plays a crucial role in hematopoiesis. To improve our knowledge about the immune system of Pleurodeles waltl, we sequenced the cDNA coding for the Ik-1 isoform of that salamander and analyzed its tissue expression by semi-quantitative RT-PCR. Ikaros transcripts are abundant in the thymus and the spleen, thereby confirming that these organs are, respectively, the primary and secondary lymphoid tissues of Pleurodeles. Analysis of the isoforms produced by this animal revealed two isoforms characteristic of amphibians in which an alternative internal splicing site deletes the 3' half of exon 3 which interestingly comprises the first Zn finger of Ikaros. Ikaros transcripts were found at the earliest stages of development of Pleurodeles indicating that Ikaros has a function at the very early lymphopoietic stages. Moreover, the presence of Ikaros transcripts in spermatozoa suggests that this protein could have another and yet unknown function.