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.

Silencing of Gonad-Inhibiting Hormone Transcripts in Litopenaeus vannamei Females by use of the RNA Interference Technology.

The method usually employed to stimulate gonadal maturation and spawning of captive shrimp involves unilateral eyestalk ablation, which results in the removal of the endocrine complex responsible for gonad-inhibiting hormone (GIH) synthesis and release. In the present study, RNAi technology was used to inhibit transcripts of GIH in Litopenaeus vannamei females. The effect of gene silencing on gonad development was assessed by analyzing the expression of GIH and vitellogenin, respectively, in the eyestalk and ovaries of L. vannamei females, following ablation or injection with dsRNA-GIH, dsRNA-IGSF4D (non-related dsRNA), or saline solution. Histological analyses were performed to determine the stage of gonadal development and to assess the diameter of oocytes throughout the experimental procedure. Only oocytes at pre-vitellogenesis and primary vitellogenesis stages were identified in females injected with dsRNA-GIH, dsRNA-IGSF4D, or saline solution. Oocytes at all developmental stages were observed in eyestalk-ablated females, with predominance of later stages, such as secondary vitellogenesis and mature oocytes. Despite achieving 64, 73, and 71% knockdown of eyestalk GIH mRNA levels by 15, 30, and 37 days post-injection (dpi), respectively, in dsRNA-GIH-injected females, the expected increase in ovary vitellogenin mRNA expression was only observed on the 37th dpi. This is the first report of the use of RNAi technology to develop an alternative method to eyestalk ablation in captive L. vannamei shrimps.

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-1ß (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.

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.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach.

The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

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.

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.

Increased growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor I (IGF-I) gene transcription after hyperosmotic stress in the Brazilian flounder Paralichthys orbignyanus.

Growth hormone (GH) action is the result of an intracellular cascade initiated just after its interaction with the growth hormone receptor (GHR) located on the surface of target cells. This cascade culminates with the transcription of target genes, such as the insulin-like growth factors (IGFs), which are responsible for most GH biological effects. In addition to its central role in growth, fish GH is also involved with osmoregulatory control. Within this context, the objective of the present work was to isolate GH, GHR, and IGF-I cDNAs from the Brazilian flounder Paralichthys orbignyanus and evaluate whether these genes are induced by hyperosmotic stress. The obtained results indicated that GH mRNA had a significant peak only 24 h after hyperosmotic stress. In gills, GHR mRNA was significantly increased after 7 days. In liver, GHR and IGF-I mRNAs were significantly increased in 72 h and both reached even higher levels after 7 days. These results indicate that hyperosmotic stress can increase GH sensitivity in the gills and liver of P. orbignyanus and, consequently, improve IGF-I production. The management of this parameter could be useful in achieving better growth performance for this and other commercially important species in which GH has a direct correlation with osmoregulatory mechanisms.