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.     

Reproductive parameters of double transgenic zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) males overexpressing both the growth hormone (GH) and its receptor (GHR)

Growth hormone (GH) transgenesis presents a high potential application in aquaculture. However, excess GH may have serious consequences due to pleiotropic actions. In order to study these effects in zebrafish (Danio rerio), two transgenic lines were developed. The first expresses GH ubiquitously and constitutively (F0104 line), while the second expresses the GH receptor in a muscle-specific manner (Myo-GHR line). Results from the F0104 line showed accelerated growth but increased reproductive difficulties, while Myo-GHR did not show the expected increase in muscle mass. Since the two lines appeared to display complementary characteristics, a double transgenic (GH/GHR) was created via crossing between them. This double transgenic displayed accelerated growth, however reproductive parameters remained uncertain. The objective of the present study was to determine the reproductive capacity of males of this new line, by evaluating sperm parameters, expression of spermatogenesis-related genes, and reproductive tests. Double transgenics showed a strong recovery in almost all sperm parameters analyzed when compared to the F0104 line. Gene expression analyses revealed that Anti-Müllerian Hormone gene (amh) appeared to be primarily responsible for this recovery. Reproductive tests showed that double transgenic males did not differ from non-transgenics. It is possible that GHR excess in the muscle tissues of double transgenics may have contributed to lower circulating GH levels and thus reduced the negative effects of this hormone with respect to reproduction. Therefore, it is clear that GH-transgenesis technology should take into account the need to obtain adequate levels of circulating hormone in order to achieve maximum growth with minimal negative side effects.     

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.     

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon,Oncorhynchus kisutch

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.     

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 P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85alpha, p85beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85alpha or p85beta, respectively (p85alpha (+/-) and p85beta(-/-)). Wild type and p85beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85alpha(+/-)mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85alpha expression. These findings demonstrated the importance of increased p85alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.     

Growth hormone,insulin-like growth factor I,and motoneuron size

In this study we asked whether growth hormone (GH) and one of its key mediators, insulin-like growth factor I (IGF-I), influence spinal motoneuron size in conjunction with whole body size. We present evidence that GH has such a role, possibly without the mediation of IGF-I. Both lumbar motoneuron and body size were found to be increased relative to littermate controls in transgenic mice overexpressing GH, while body size, but not motoneuron size, was increased in mice overexpressing IGF-I. GH overexpression coordinately increased nucleolar, nuclear, and cell body size in lumbar spinal motoneurons, so that their normal size relationships were preserved in the transgenic mice. In addition, spinal cord and brain weights were significantly increased in both types of transgenic animal. We conclude that GH can regulate motoneuron, central nervous system, and body size in the same animal, and that IGF-I can mimic the effects of GH on at least two of these three parameters. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 202–212, 1997.     

Ageing genetic signature of hypersomatotropism

Acromegaly is a pathological condition that is caused by over-secretion of growth hormone (GH) and develops primarily from a pituitary adenoma. Excess GH exposure over a prolonged period of time leads to a wide range of systemic manifestations and comorbidities. Studying the effect of excess GH on the cellular level could help to understand the underlying causes of acromegaly health complications and comorbidities. In our previous publications, we have shown that excess GH reduces body side population (SP) stem cells and induces signs of premature ageing in an acromegaly zebrafish model. Here, we study acromegaly ageing in greater depth at the level of gene expression. We investigated whether acromegaly induces an ageing genetic signature in different organs. Using the GenAge database, our acromegaly model showed a significant enrichment of ageing genetic datasets in the muscle but not in other organs. Likewise, the hierarchical clustering of wild type (WT), acromegaly and aged RNA data from various organs revealed the similarity of gene expression profiles between the acromegaly and the aged muscles. We therefore identified overlapping differentially expressed genes (DEGs) in different organs between acromegaly and aged zebrafish. Importantly, about half of the muscle, liver and brain acromegaly DEGs overlapped with aged zebrafish DEGs. Interestingly, overlapping was observed in the same way; acromegaly-up DEGs overlapped with aged zebrafish up DEGs, not down DEGs, and vice versa. We then identified the biological functions of overlapping DEGs. Enrichment database analysis and gene ontology showed that most overlapping muscle genes were involved in ageing metabolism, while overlapping liver DEGs were involved in metabolic pathways, response to hypoxia and endoplasmic reticulum stress. Thus, this study provides a full ageing genetic signature of acromegaly at the gene expression level.     

The role of insulin-like growth factor-I in neuroendocrine function and the consequent effects on sexual maturation: inferences from animal models

It is known that growth hormone (GH) plays an important role in growth and development.Additionally, emerging evidence suggest that it also influences hypothalamic-pituitary-gonadal function. We have found that GH from different species has different effects in mice. In rodents, human GH (hGH) binds to both GH and prolactin (PRL) receptors; it has both somatotrophic and lactotrophic effects. Since PRL has a profound effect on neuroendocrine function, the results obtained from hGH treatment or from transgenic animals expressing the hGH gene reflect PRL-like effects of this hormone. However, bovine GH (bGH) is purely somatogenic and therefore the effects of bGH represent the function of the natural GH produced in rodents. Furthermore, our studies in mice and rats have shown that not all effects of GH are stimulatory and the duration of exposure of the hypothalamo-hypophyseal-gonadal system to GH might influence the secretions of gonadotropins and gonadal steroids. In humans, excess productions of GH in acromegaly and GH resistance in Laron syndrome adversely affect reproduction. Similarly, it has been demonstrated that in transgenic mice expressing various GH genes, in insulin-like growth factor-I (IGF-I) gene-knockout mice, in GH receptor gene-disrupted (GHR-KO) mice, and in Ames dwarf mice the onset of puberty and/or fertility is altered. Therefore, excess or subnormal secretion of GH can affect reproduction. We have shown that the hypothalamic-pituitary functions are affected in transgenic mice expressing the GH genes, Ames dwarf mice and in GH receptor gene knockout mice. The majority of the GH effects are mediated via IGF-I and the aforementioned effects may be due to the GH-induced IGF-I secretion or due to the absence of this peptide production. It is important to realize that the syntheses and actions of IGF binding proteins are controlled by IGF-I. Furthermore, some IGF binding proteins can inhibit IGF-I action. Therefore, the concentrations of IGF binding proteins and the ratio of these binding proteins and IGF-I within the body might play a pivotal role in modulating IGF-I effects on the neuroendocrine-gonadal system.     

Regulation of feeding behavior and food intake by appetite-regulating peptides in wild-type and growth hormone-transgenic coho salmon

Survival, competition, growth and reproductive success in fishes are highly dependent on food intake, food availability and feeding behavior and are all influenced by a complex set of metabolic and neuroendocrine mechanisms. Overexpression of growth hormone (GH) in transgenic fish can result in greatly enhanced growth rates, feed conversion, feeding motivation and food intake. The objectives of this study were to compare seasonal feeding behavior of non-transgenic wild-type (NT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch), and to examine the effects of intraperitoneal injections of the appetite-regulating peptides cholecystokinin (CCK-8), bombesin (BBS), glucagon-like peptide-1 (GLP-1), and alpha-melanocyte-stimulating hormone (α-MSH) on feeding behavior. T salmon fed consistently across all seasons, whereas NT dramatically reduced their food intake in winter, indicating the seasonal regulation of appetite can be altered by overexpression of GH in T fish. Intraperitoneal injections of CCK-8 and BBS caused a significant and rapid decrease in food intake for both genotypes. Treatment with either GLP-1 or α-MSH resulted in a significant suppression of food intake for NT but had no effect in T coho salmon. The differential response of T and NT fish to α-MSH is consistent with the melanocortin-4 receptor system being a significant pathway by which GH acts to stimulate appetite. Taken together, these results suggest that chronically increased levels of GH alter feeding regulatory pathways to different extents for individual peptides, and that altered feeding behavior in transgenic coho salmon may arise, in part, from changes in sensitivity to peripheral appetite-regulating signals.     

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96?h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24?h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.     

Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.     

Fertility of transgenic female mice expressing bovine growth hormone or human growth hormone variant genes

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of overexpression of growth hormone on T cell activity in transgenic mice

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overexpressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.     

Spatial learning and memory in male mice with altered growth hormone action

Growth hormone (GH) has a significant influence on cognitive performance in humans and other mammals. To understand the influence of altered GH action on cognition, we assessed spatial learning and memory using a Barnes maze (BM) comparing twelve-month old, male, bovine GH (bGH) and GH receptor antagonist (GHA) transgenic mice and their corresponding wild type (WT) littermates. During the acquisition training period in the BM, bGH mice showed increased latency, traveled longer path lengths and made more errors to reach the target than WT mice, indicating significantly poorer learning. Short-term memory (STM) and long-term memory (LTM) trials showed significantly suppressed memory retention in bGH mice when compared to the WT group. Conversely, GHA mice showed significantly better learning parameters (latency, path length and errors) and increased use of an efficient search strategy than WT mice. Our study indicates a negative impact of GH excess and a beneficial effect of the inhibition of GH action on spatial learning and memory and, therefore, cognitive performance in male mice. Further research to elucidate GH's role in brain function will facilitate identifying therapeutic applications of GH or GHA for neuropathological and neurodegenerative conditions.