RESUMO
Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na(+), K(+)-ATPase, H(+)-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na(+), K(+)-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na(+). Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na(+) import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.
Assuntos
Animais Geneticamente Modificados , Hormônio do Crescimento/genética , Equilíbrio Hidroeletrolítico , Peixe-Zebra , Trifosfato de Adenosina/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Metabolismo Energético/genética , Água Doce , Brânquias/metabolismo , Brânquias/patologia , Hormônio do Crescimento/fisiologia , Salinidade , ATPase Trocadora de Sódio-Potássio/biossíntese , Equilíbrio Hidroeletrolítico/genética , Equilíbrio Hidroeletrolítico/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
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.
Assuntos
Hormônio do Crescimento/fisiologia , Fígado/metabolismo , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Homozigoto , Fator de Crescimento Insulin-Like I/metabolismo , Janus Quinase 2/metabolismo , Fígado/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Most biological actions of growth hormone (GH) are mediated by the insulin-like growth factor I (IGF-I) that is produced after the interaction of the hormone with a specific cell surface receptor, the GH receptor (GHR). Even though the GH excess on fish metabolism is poorly known, several species have been genetically engineered for this hormone in order to improve growth for aquaculture. In some GH-transgenic fish growth has been dramatically increased, while in others high levels of transgene expression have shown inhibition of the growth response. In this study, we used for the first time different genotypes (hemizygous and homozygous) of a GH-transgenic zebrafish (Danio rerio) lineage as a model for studying the GH resistance induced by different GH transgene expression levels. The results obtained here demonstrated that homozygous fish did not grow as expected and have a lower condition factor, which implies a catabolic state. These findings are explained by a decreased IGF-I and GHR gene expression as a consequence of GH resistance. Together, our results demonstrated that homozygous GH-transgenic fish showed similar characteristics to the starvation-induced fish and could be an interesting model for studying the regulation of the GH/GHR/IGF-I axis in fish.
RESUMO
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.