Marcio Azevedo Figueiredo

SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio).

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.

GH overexpression modifies muscle expression of anti-oxidant enzymes and increases spinal curvature of old zebrafish

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.

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Even though growth hormone (GH) transgenesis has demonstrated potential for improved growth of commercially important species, the hormone excess may result in undesired collateral effects. In this context, the aim of this work was to develop a new model of transgenic zebrafish (Danio rerio) characterized by a muscle-specific overexpression of the GH receptor (GHR) gene, evaluating the effect of transgenesis on growth, muscle structure and expression of growth-related genes. In on line of transgenic zebrafish overexpressing GHR in skeletal muscle, no significant difference in total weight in comparison to non-transgenics was observed. This can be explained by a significant reduction in expression of somatotrophic axis-related genes, in special insulin-like growth factor I (IGF-I). In the same sense, a significant increase in expression of the suppressors of cytokine signaling 1 and 3 (SOCS) was encountered in transgenics. Surprisingly, expression of genes coding for the main myogenic regulatory factors (MRFs) was higher in transgenic than non-transgenic zebrafish. Genes coding for muscle proteins did not follow the MRFs profile, showing a significant decrease in their expression. These results were corroborated by the histological analysis, where a hyperplasic muscle growth was observed in transgenics. In conclusion, our results demonstrated that GHR overexpression does not induce hypertrophic muscle growth in transgenic zebrafish probably because of SOCS impairment of the GHR/IGF-I pathway, culminating in IGF-I and muscle proteins decrease. Therefore, it seems that hypertrophy and hyperplasia follow two different routes for entire muscle growth, both of them triggered by GHR activation, but regulated by different mechanisms.

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

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.

GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males

Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis.

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

Impairment of the immune system in GH-overexpressing transgenic zebrafish (Danio rerio)

Growth hormone (GH) is an important regulator of immune functions in vertebrates, and it has been intensively reported a series of stimulatory actions of this hormone over on the immune system. Within aquaculture, overexpression of GH has been considered a promising alternative for promoting higher growth rates in organisms of commercial interest. Considering the various pleiotropic effects of GH, there are still few studies that aim to understand the consequences of the excess of GH on the physiological systems. In this context, our goal was to present the effects of the overexpression of GH on immune parameters using a model of zebrafish (Danio rerio) that overexpress this hormone. The results showed that GH transgenic zebrafish had 100% of mortality when immunosuppressed with dexamethasone, revealing a prior weakening of the immune system in this lineage. Morphometric analysis of thymus and head kidney revealed a reduction in the area of these structures in transgenic zebrafish. Moreover, the phenotypic expression of CD3 and CD4 thymocytes was also depreciated in transgenic zebrafish. Furthermore, a decrease was noted in the expression of genes RAG-1 (60%), IKAROS (50%), IL-1β (55%), CD4 (60%) and CD247 (40%), indicating that development parameters, of innate and acquired immunity, are being harmed. Based on these results, it can be concluded that the excess of GH impairs the immune functions in GH transgenic zebrafish, indicating that the maintenance of normal levels of this hormone is essential for the functioning of immunological activities.

Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model.

The aim of this study was to evaluate the effects of growth hormone (GH) overexpression on the gene expression profile of multiple components of the antioxidant defense system (ADS) of different genotypes of a GH-transgenic zebrafish (Danio rerio) model. Several ADS-related genes were analyzed by semiquantitative reverse transcription–PCR in the liver of hemizygous (HE) and homozygous (HO) transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotype-dependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse, which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

High level of GHR nuclear translocation in skeletal muscle of a hyperplasic transgenic zebrafish

It has been reported that nuclear translocation of growth hormone receptor (GHR) may directly activate cell proliferation in mammals and birds. However, this phenomenon has not yet been described in fish. Recently, we have developed a transgenic zebrafish that overexpresses GHR in a muscle-specific manner. Considering that this transgenic model exhibits hyperplasic muscle growth, the present work aims at verifying the relationship between GHR nuclear translocation and muscle cell proliferation. This relationship was evaluated by the phosphorylation state of the proliferative MEK/ERK pathway, expression of nuclear import-related genes, immunostaining of phospho-histone H3 (PH3) as a proliferation marker, and nuclear GHR localization. The results showed a significant decrease in the phosphorylation state of ERK1/2 proteins in transgenics. Moreover, there was an increase in expression of three out of four importin genes analyzed parallel to a large flow of GHR displacement toward and into the nucleus of transgenic muscle cells. Also, transgenics presented a marked increase in PH3 staining, which indicates cell proliferation. These findings, as far as we know, are the first report suggesting a proliferative action of GHR in fish as a consequence of its increased nuclear translocation. Thus, it appears that the nuclear migration of cytokine receptors is a common event among different taxonomic groups. In addition, the results presented here highlight the possibility that these membrane proteins may be involved more directly than previously thought in the control of genes related to cell growth and proliferation.

Food intake and appetite control in a GH-transgenic zebrafish

Abstract The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. The regulatory network for GH is complex and includes many central and peripheral endocrine factors as well as that from the environment. It is known that GH transgenesis results in increased growth, food intake, and consequent metabolic rates in fishes. However, the manner in which GH transgenesis alters the energetic metabolism in fishes has not been well explored. In order to elucidate these consequences, we examined the effect of GH overexpression on appetite control mechanisms in a transgenic zebrafish (Danio rerio) model. To this, we analyzed feeding behavior and the expression of the main appetite-related genes in two different feeding periods (fed and fasting) in non-transgenic (NT) and transgenic (T) zebrafish as well as glycaemic parameters of them. Our initial results have shown that NT males and females present the same feeding behavior and expression of main appetite-controlling genes; therefore, the data of both sexes were properly grouped. Following grouped data analyses, we compared the same parameters in NT and T animals. Feeding behavior results have shown that T animals eat significantly more and faster than NT siblings. Gene expression results pointed out that gastrointestinal (GT) cholecystokinin has a substantial contribution to the communication between peripheral and central control of food intake. Brain genes expression analyses revealed that T animals have a down-regulation of two strong and opposite peptides related to food intake: the anorexigenic proopiomelanocortin (pomc) and the orexigenic neuropeptide Y (npy). The down-regulation of pomc in T when compared with NT is an expected result, since the decrease in an anorexigenic factor might keep the transgenic fish hungry. The down-regulation of npy seemed to be contradictory at first, but if we consider the GH’s capacity to elevate blood glucose, and that NPY is able to respond to humoral factors like glucose, this down-regulation makes sense. In fact, our last experiment showed that transgenics presented elevated blood glucose levels, confirming that npy might responded to this humoral factor. In conclusion, we have shown that GT responds to feeding status without interference of transgenesis, whereas brain responds to GH transgenesis without any effect of treatment. It is clear that transgenic zebrafish eat more and faster, and it seems that it occurs due to pomc down-regulation, since npy might be under regulation of the humoral factor glucose.