Endocrine effects of growth hormone overexpression in transgenic coho salmon

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.     

Central administration of growth hormone-releasing hormone and corticotropin-releasing hormone stimulate downstream movement and thyroxine secretion in fall-smolting coho salmon (Oncorhynchus kisutch)

The ultimate signal triggering downstream migration in anadromous salmonids is unknown. A plasma surge of T₄ (T₄ surge) occurs during downstream migration in salmonids; however, the causal relationship between migratory behavior and the T₄ surge is not well known. We first examined the progression of smolt indicators (skin silvering, condition factor (CF), gill Na⁺, K⁺-ATPase (NKA) activity and plasma T₄ levels) in underyearling, fall-smolting coho salmon (Oncorhynchus kisutch) from August to December. In November, the fish showed the characteristics of fully developed smolts, i.e. the skin completely covered with silvery scales, CF at a nadir, and peak NKA activity and plasma T₄ levels. Based on these results, we examined the effects of four neuropeptides, thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), and gonadotropin-releasing hormone (GnRH), on the downstream movement (negative rheotaxis) and T₄ surge in fully smoltified underyearling coho salmon. The experiment was run in circular-shaped channel tanks and the neuropeptide treatment was performed as intracerebroventricular (ICV) injections. ICV injection of GHRH and CRH stimulated both downstream movement and plasma T₄ level. TRH injection stimulated plasma T₄ level but suppressed downstream movement. GnRH injection had no effect. It is hypothesized that GHRH and CRH play key roles in triggering downstream migration of anadromous salmonids, and that the accompanying T₄ surge is a consequence of the neuroendocrine processes that trigger migration.     

Cortisol effects on plasma electrolytes and thyroid hormones during smoltification in coho salmon Oncorhynchus kisutch

Plasma concentrations of Na+, K+, triiodothyronine (T3), and thyroxine (T4) and muscle water content were measured in yearling coho salmon. Oncorhynchus kisutch, after injection of cortisol in April, May, and June in fresh water (FW) and during acclimation to seawater (SW). Cortisol (17-21 micrograms/g), injected intraperitoneally in a melted cocoa butter suspension, suppressed the rise of plasma Na+ during SW acclimation in April but not in May or June. Muscle water content increased during SW acclimation in cortisol-treated fish in April and June. These observations suggest a hypoosmoregulatory function for cortisol during SW acclimation. Cortisol treatment also induced elevations of plasma K+ in FW (April only) and SW (April and May only). Cortisol treatment increased plasma T3 during SW acclimation in June and T4 in FW in May. The results suggest that cortisol may modify osmoregulatory processes and thyroid hormone activity during smoltification and acclimation to SW in yearling coho salmon.     

Changes in plasma corticosteroids during smoltification of coho salmon,Oncorhynchus kisutch

Plasma corticosteroid concentrations were determined by competitive protein binding assay in coho salmon, Oncorhynchus kisutch, during smoltification. Between January and April plasma corticosteroid levels decreased from 8.6 to 3.5 ng/ml, but increased to 24.0 ng/ml by the end of May in smolts retained in fresh water. This nearly eightfold increase in corticosteroids occurred as plasma thyroxin levels declined. Thus plasma corticosteroids appear to be increasing during the period when coho salmon normally migrate to sea.     

Androgen effects on plasma GH, IGF-I, and 41-kDa IGFBP in coho salmon (Oncorhynchus kisutch)

Among many species of salmonids, fast growing fish mature earlier than slow growing fish, and maturing males grow faster than non-maturing ones. To study the potential endocrine basis for this reciprocal relationship we examined the in vivo effects of the androgens, testosterone (T) and 11-ketotestosterone (11-KT), on plasma growth hormone (GH), insulin-like growth factor-I (IGF-I) and 41-kDa IGF binding protein (41-kDa IGFBP) (putative IGFBP-3) in coho salmon, Oncorhynchus kisutch. Immature male and female, two-year old fish (avg. wt. 31.7 +/- 0.63 g) were injected with coconut oil containing T or 11-KT at a dose of 0.1, 0.25, or 1 microg/g body weight. Blood samples were taken 1 and 2 weeks postinjection, and analyzed by immunoassay for T, 11-KT, GH, IGF-I, and 41-kDa IGFBP. Steroid treatments elevated the plasma T and 11-KT levels to physiological ranges typical of maturing fish. Plasma IGF-I and 41-kDa IGFBP levels increased in response to both T and 11-KT in a significant and dose-dependent manner after 1 and 2 weeks, but GH levels were not altered. These data suggest that during reproductive maturation, in addition to the previously demonstrated effects of the IGFs on steroidogenesis, the gonadal steroids may in turn play a significant role in regulating IGF-I and its binding proteins in fish. The interaction between the reproductive and growth axes may provide a regulatory mechanism for bringing about the dimorphic growth patterns observed between maturing and non-maturing salmonids and other species of fish.     

Effects of growth hormone and cortisol on the downstream migratory behavior in masu salmon, Oncorhynchus masou

The effects of ovine growth hormone (oGH) and cortisol on downstream migratory behavior in yearling (1⁺) smolts and underyearling (0⁺) parr of masu salmon, Oncorhynchus masou, were examined during the downstream migratory period in spring using artificial raceways. In May, each of 22 1⁺ smolts and 0⁺ parr were implanted with cholesterol pellets containing 250 μg of oGH and/or 2 mg of cortisol. Their downstream migratory behavior was subsequently observed in artificial raceways, along with control groups 4-23 days after implantation. In 1⁺ smolts, the frequency of downstream migratory behavior was 23%, 18%, 72%, and 82% in the control, oGH, cortisol, and oGH + cortisol-treated groups, respectively. The frequency was significantly higher in the cortisol and oGH + cortisol-treated groups than in the control and oGH-treated groups. In 0⁺ parr, the frequency of downstream migratory behavior in the cortisol (82%) and cortisol + oGH-treated (90%) groups was significantly higher than in the control (18%) and oGH-treated (0%) groups. These results indicate that cortisol is an important endocrine factor inducing downstream migratory behavior in both 1⁺ smolt and 0⁺ parr of masu salmon.     

cDNA cloning of thyroid hormone receptor betas from the conger eel,Conger myriaster

Two distinct TRbetas cDNA clones were generated by RACE from a conger eel (Conger myriaster). The deduced amino acid sequences of the conger eel TRbetas (cTRbetas) showed higher homologies to the known TRbetas of other vertebrate animals than to TRalphas. Two cTRbetas possessed an insertion sequence in the hinge region similar to other teleost fish TRbetas. Variation in cTRbeta due to differential splicing in the hinge region of the cTRbetas genes was found using PCR analysis. The determination of TRbeta mRNA levels in eel tissue was performed using competitive RT-PCR. The cTRbeta1 mRNA was widely expressed, whereas cTRbeta 2 mRNA was most highly expressed in the brain and pituitary. The expression pattern of cTRbeta1 and cTRbeta2 in tissues were similar to that of TRbeta1 and TRbeta2 in mammals.     

Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog

The current review focuses on the molecular mechanisms and developmental roles of thyroid hormone receptors (TRs) in gene regulation and metamorphosis in Xenopus laevis and discusses implications for TR function in vertebrate development and diversity. Questions addressed are: (1) what are the molecular mechanisms of gene regulation by TR, (2) what are the developmental roles of TR in mediating the thyroid hormone (TH) signal, (3) what are the roles of the different TR isoforms, and (4) how do changes in these molecular and developmental mechanisms affect evolution? Even though detailed knowledge of molecular mechanisms of TR-mediated gene regulation is available from in vitro studies, relatively little is known about how TR functions in development in vivo. Studies on TR function during frog metamorphosis are leading the way toward bridging the gap between in vitro and in vivo studies. In particular, a dual function model for the role of TR in metamorphosis has been proposed and investigated. In this model, TRs repress genes allowing tadpole growth in the absence of TH during premetamorphosis and activate genes important for metamorphosis when TH is present. Despite the lack of metamorphosis in most other vertebrates, TR has important functions in development across vertebrates. The underlying molecular mechanisms of TR in gene regulation are conserved through evolution, so other mechanisms involving TH-target genes and TH tissue-sensitivity and dependence underlie differences in role of TR across vertebrates. Continued analysis of molecular and developmental roles of TR in X. laevis will provide the basis for understanding how TR functions in gene regulation in vivo across vertebrates and how TR is involved in the generation of evolutionary diversity.     

Sexual dimorphism of plasma sex steroid levels in juvenile coho salmon, Oncorhynchus kisutch, during smoltification

Concentrations of plasma sex steroids, cortisol, and thyroxine were measured by radioimmunoassay in hatchery coho salmon (Oncorhynchus kisutch during winter and early spring. Mean plasma 11-ketotestosterone (11-KT) and estradiol levels fell into two distinct categories: 11-KT was 181-373% higher in males than in females, and estradiol was 109-143% higher in females than in males. No changes in plasma levels of estradiol in fish of both sexes, or in levels of 11-KT in males, were evident during spring when plasma thyroxine and cortisol were markedly elevated, indicating that the fish were undergoing smoltification. Although plasma 11-KT in females appeared to be lower in late April than in February, it showed no correlation with plasma thyroxine or cortisol in these individuals. Our finding of sexual dimorphism in 17 alpha-20 beta-dihydroxy-4-pregnen-3-one was inconsistent between stocks of fish and among sampling dates, thus making interpretation of the results difficult. However, no relationship between this steroid and plasma thyroxine or cortisol was observed. Therefore, plasma levels of sex steroids do not seem to be related to the changes in plasma thyroxine or cortisol observed during smoltification of coho salmon.     

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 microM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.     

Molecular and genetic studies suggest that thyroid hormone receptor is both necessary and sufficient to mediate the developmental effects of thyroid hormone

Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopuslaevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general.     

甲状腺功能减退对新生早期大鼠各脑区甲状腺激素受体mRNA表达的影响

目的探讨甲状腺功能减退（甲减）对新生早期大鼠各脑区甲状腺激素受体（TR）mRNA表达的影响。方法建立甲减Wistar大鼠动物模型，分别于仔鼠0、14、21、45d，用实时荧光定量PCR方法检测大脑、小脑、脑干和海马TR mRNA的表达。结果与对照组相比，各时间点甲减仔鼠各脑区TRα1 mRNA的表达量呈总体下调趋势，而甲减0d仔鼠大脑、小脑、脑干TRα2 mRNA表达量明显增高（t=8．18、6．23、3．68，P〈0．01），且45d仔鼠各脑区TRα2 mRNA表达量仍高于对照组（t大脑=5．50、t小脑=5．46、t脑干=4．10、t海马=11．83，P〈0．01），TRα1、TRα2 mRNA表达峰（21d）均延迟于对照组（14d）出现。甲减仔鼠TRβ1 mRNA表达变化趋势与对照组相一致，但45d仔鼠各脑区TRβ1 mRNA表达量均低于对照组（t大脑=4．64、t小脑=2．73、t脑干=3．90、t海马=5．07，P〈0．01或〈0．05）。结论甲减时甲状腺激素受体mRNA表达峰值的延迟出现以及异常的表达变化与克汀病脑损伤机制密切相关。     

Changes in plasma estradiol and effects of triiodothyronine on plasma estradiol during smoltification of coho salmon, Oncorhynchus kisutch

Plasma estradiol-17 beta levels were measured by radioimmunoassay in untreated coho salmon of both sexes or in fish fed triiodo-L-thyronine during the smoltification period. Mean plasma estradiol increased between February 25 and April 1 from 94 to 142 pg/ml, and then by May 13, it had decreased to 80 pg/ml. This hormonal cycle was followed by a second significant increase to 219 pg/ml on June 22. Plasma thyroxine level covaried with that of estradiol. Treatment with triiodothyronine had no effect on plasma estradiol or thyroxine levels. Plasma estradiol surges during the smoltification period must be considered along with other significant endocrine changes for a possible role in the developmental phenomena that characterize this period.     

Effects of thyroxin, cortisol, growth hormone, and prolactin on lipid metabolism of coho salmon, Oncorhynchus kisutch, during smoltification

Juvenile coho salmon, Oncorhynchus kisutch, were either immersed in thyroxin-containing water (T4; 20 micrograms/ml) or implanted with cortisol (5 mg), bovine growth hormone (GH; 1.5 microgram/g body wt), or ovine prolactin (PRL; 1.5 microgram/g body wt), both early and late in smoltification. T4 and cortisol treatment stimulated lipid mobilization in parr. T4-stimulated lipid mobilization was indicated by decreased total lipids, primarily as triacylglycerols, and increased lipolytic enzyme (triacylglycerol lipase) activity in the liver and dark muscle. T4-stimulated lipid mobilization from mesenteric fat was indicated by decreased total tissue mass and by increased lipase activity. Cortisol caused significant reductions in total lipid concentration and triacylglycerol content of the liver and dark muscle; these effects were accompanied by increased lipase activity. Cortisol treatment did not affect mesenteric fat total lipid concentration, total tissue mass, or triacylglycerol content. However, cortisol implantation did enhance mesenteric fat lipase activity. Thyroxin and cortisol treatment failed to elicit alterations in the pattern of tissue lipid mobilization of smolts. GH stimulated lipid mobilization from coho salmon parr. Depletion of liver total lipids was accompanied by increased lipolytic enzyme (triacylglycerol lipase) activity. GH had limited effects on dark muscle and mesenteric fat. In smolts, GH had virtually no effect on lipid mobilization. PRL strongly stimulated lipid mobilization in parr; this effect was evident in all depots studied (liver, dark muscle, mesenteric fat). Decreases in total lipid concentration, or in total tissue mass (mesenteric fat), were accompanied by increased lipase activity and generally resulted in reduced tissue triacylglycerol content. Smolts appeared refractory to PRL treatment. Smolts (characteristically possessing elevated liver lipase activity) that were hypophysectomized exhibited low levels of liver lipase activity. Cortisol replacement restored enzyme activity to approximately the same levels as those observed in sham-operated controls. GH replacement restored lipase activity, but not to the levels observed in sham-operated controls. These results indicate that T4, cortisol, GH, and PRL all stimulate lipid mobilization in developing salmon by enhancement of lipolysis and suggest that T4, cortisol, GH, and PRL are among the factors which contribute to smoltification-associated lipid depletion.     

Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis

The Chinese giant salamander (Andrias davidianus) is one of the largest and ‘living fossil’ species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.     

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5'-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The beta-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.