RESUMEN
Growth hormone (GH) excess causes an increment in the metabolic rate and in reactive oxygen species generation, which accelerate the ageing process in mammals. Considering that there is no information on this subject in fish, the aim of the present study was to evaluate the excess GH effect on senescence in a zebrafish (Danio rerio) transgenic model. In order to reach this objective, we analyzed the phenotype of spinal curvature and expression of genes related to the anti-oxidant defense system and myogenesis in muscle of 8 and 30 months old GH-transgenic males. Gene expression analyses revealed that both superoxide dismutase isoforms were down-regulated only in 30 months old animals, while glutamate cysteine ligase was down-regulated in GH-transgenic zebrafish. Acceleration of the spinal curvature and a reduction in the expression of miogenin at both ages and MyoD in the old fish were also observed. Although neurolipofuscin accumulation was not significant in GH-transgenic zebrafish, the estimation of maximum longevity based on the von Bertalanffy growth function was significantly lower in this group. The results obtained here indicate that GH overexpression reduces the transcription of anti-oxidant defense system and myogenesis-related genes, which probably accelerates senescence in the zebrafish transgenic model used.
Asunto(s)
Glutamato-Cisteína Ligasa/genética , Hormona del Crecimiento/metabolismo , Desarrollo de Músculos/genética , Curvaturas de la Columna Vertebral/metabolismo , Superóxido Dismutasa/genética , Pez Cebra/metabolismo , Envejecimiento/fisiología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Antioxidantes/metabolismo , Carpas/genética , Amplificación de Genes , Regulación del Desarrollo de la Expresión Génica , Glutatión/genética , Glutatión/metabolismo , Gráficos de Crecimiento , Hormona del Crecimiento/genética , Hormona del Crecimiento/fisiología , Peroxidación de Lípido/genética , Lipofuscina , Masculino , Músculo Esquelético/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Radiografía , Curvaturas de la Columna Vertebral/diagnóstico por imagen , Pez Cebra/genéticaRESUMEN
Homozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NT and HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO (p<0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p<0.01) and 4.3 times (p<0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins.
Asunto(s)
Hormona del Crecimiento/fisiología , Hígado/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Homocigoto , Factor I del Crecimiento Similar a la Insulina/metabolismo , Janus Quinasa 2/metabolismo , Hígado/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio) were produced using two transgenes, both comprised of the carp beta-actin promoter driving the expression of either the green fluorescent protein (GFP) reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.