Growth Hormone Receptor Transcripts Research Articles

THU545 Growth Hormone Receptor Antagonist As A Novel Combination Therapy Against Triple Negative Breast Cancer

Abstract Disclosure: P. Mora-Criollo: None. R. Basu: None. J.J. Kopchick: None. Triple Negative breast (TNBC) cancer is classified based on the absence of ER-, PR-, and HER2- receptors and is associated with high metastatic potential. TNBC accounts for approximately 15-20% of new breast cancer diagnoses and is responsible for most breast cancer-related deaths. First-line treatment of anticancer drugs can be effective in 30-70% of patients with TNBC, but during treatment, more than half of the patients develop resistance and progress into metastatic disease. Previous studies have demonstrated the link between growth hormone receptor (GHR) expression and mammary carcinogenesis and established that GHR signaling promotes breast cancer development and progression. Conversely, inhibition of GHR expression has been shown to arrest tumor initiation and reduce metastasis of mammary carcinoma cells. Here we present the feasibility studies for employing GHR as a target for novel combination therapeutic approaches in TNBC. We performed retrospective analysis of GHR transcript expression and survival in TNBC patients, followed by experimental data in cellular models using combinations of Pegvisomant (an FDA-approved GHR antagonist) with chemotherapeutic agents, as a potential therapeutic option in TNBC. Survival probability plots from the TCGA dataset of 405 TNBC samples showed that patients expressing high levels of the GHR showed a significantly poor survival (27 months, p=0.038) compared to patients expressing low GHRs (40.37 months). Additionally, human breast cancer MDA-MB-453 cells, when treated with human (hGH)(2.5 nM) increased activation of JAK2, STAT5, SRC, ERK1/2, and AKT, associated with cancer progression. Importantly, treatment of the cells with Paclitaxel (1 nM) + (hGH)(2.5 nM) increased ABCG2 expression and the epithelial-to-mesenchymal transition (EMT) makers (SLUG, SNAIL, N-cad) associated with drug resistance and tumor progression. On the contrary, when cells were treated with Pegvisomant (500 nM) in combination with Paclitaxel + GH, a significant decrease in protein and RNA expression of ABCG2 as well as EMT markers was observed. Next, serum collected from GH antagonist (GHA) mice and WT as control, were used to treat PY8119 mouse cells in combination with Paclitaxel (1 nM) + bovine (bGH)(2.5 nM). Downregulation of protein and RNA expression levels of ABCG2 as well EMT makers in GHA serum treatment compared to WT was also found. This preliminary data shows that the GHR antagonist, Pegvisomant, in combination with anticancer drugs, can be effective in attacking drug resistance in TNBC. Key words: Pegvisomant, growth hormone receptor Presentation: Thursday, June 15, 2023

Effect of quercetin on muscle growth and antioxidant status of the dark sleeper Odontobutis potamophila.

Quercetin is a flavanol beneficial in reducing fat, promoting muscle growth, and Anti-oxidation. To study its effects in freshwater fish, the full-length cDNA of the follistatin (FST) and myostatin (MSTN) genes of the dark sleeper Odontobutis potamophila were cloned for the first time. Juvenile individual O. potamophila was exposed to quercetin at one of four concentrations (0, 2.5, 5, and 10 mg/L) for 21 days. The expression level of MSTN which inhibits muscle growth in the quercetin solution was lower than in the unexposed control group. The genes that promote muscle growth are in TGF-β superfamily like FST, TGF-β1 (transforming growth factor-beta 1), and Myogenic regulatory factors (MRFs) like Myf5 (myogenic factor 5), MyoD (myogenic differentiation), MyoG (myogenin), were higher than in the control group. Apolipoprotein and growth hormone receptor transcription levels in the quercetin-treated fish were significantly lower than in the control group. The concentrations of triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in the muscle tissue decreased, and the lipid-lowering function of quercetin was also demonstrated at the biochemical level. In this study, we analyzed the mRNA levels of AKT, Keap1 (kelch-like ECH-associated protein 1), Nrf2 (NF-E2-related factor 2) oxidation-related genes in the Nrf2/ARE antioxidant pathway, and Malondialdehyde (MDA), catalase (CAT) activity and glutathione (GSH) content in the hepatopancreas of O. potamophila after quercetin treatment, the mRNA expression of AKT, Nrf2 and CAT activity and GSH content are higher than in the control group. Quercetin enhances antioxidant properties and positively affects muscle growth. The results showed that quercetin has no significant effects on the growth performance of O. potamophila, but is effective in increasing muscle growth rate and lowering muscle fat content.

Long-term effect of early nutrition on endocrine parameters and liver and endometrial gene expression of the members of the somatotrophic axis in Hereford heifers.

This study compared the effect of different management systems on endocrine parameters, and gene expression of members of the somatotrophic axis in the liver and endometrium of beef heifers. Twenty-two 709-days-old heifers submitted to Early Weaning (EW, n=8), Traditional Weaning (TW, n=7) and TW plus creep feeding (TW+CF, n=7) were used. Animals were synchronized with two prostaglandin (PG) injections at 11-day interval (Oestrus=Day 0). Blood samples were collected daily for progesterone (P4) determination, and endometrial and liver biopsies on Days 7 and 16 for transcript determination of members of the somatotrophic axis. Progesterone concentrations were greater on Days 15 and 16 (p<.02) of the cycle in TW+CF than TW and EW heifers. On Day 7, TW+CF heifers expressed greater liver total growth hormone receptor transcripts than TW heifers (p=.05) and greater insulin-like growth factor (IGF)-binding protein 3 mRNA than both EW and TW groups (p<.05). On Days 7 and 16, TW+CF expressed more endometrial IGF1 mRNA than the other groups (p<.01). We conclude that increasing the plane of nutrition of nursing calves may have a long-term effect on the functioning of the somatotrophic axis both in the liver and in the endometrium.

Generation of a miniature pig disease model for human Laron syndrome.

Laron syndrome is a rare disease caused by mutations of the growth hormone receptor (GHR), inheriting in an autosomal manner. To better understand the pathogenesis and to develop therapeutics, we generated a miniature pig model for this disease by employing ZFNs to knock out GHR gene. Three types of F0 heterozygous pigs (GHR+/4bp, GHR+/2bp, GHR+/3bp) were obtained and in which no significant phenotypes of Laron syndrome were observed. Prior to breed heterozygous pigs to homozygosity (GHR4bp/4bp), pig GHR transcript with the 4 bp insert was evaluated in vitro and was found to localize to the cytoplasm rather than the membrane. Moreover, this mutated transcript lost most of its signal transduction capability, although it could bind bGH. GHR4bp/4bp pigs showed a small body size and reduced body weight. Biochemically, these pigs exhibited significantly elevated levels of GH and decreased levels of IGF-I. These results resemble the phenotype observed in Laron patients, suggesting that these pigs could serve as an ideal model for Laron syndrome to bridge the gaps between mouse model and human.

Acute heat stress and dietary methionine effects on IGF-I, GHR, and UCP mRNA expression in liver and muscle of quails

This study evaluated the expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), and uncoupling protein (UCP) mRNA in muscle and liver of quails that were in thermal comfort or exposed to heat stress and that were fed diets with or without methionine supplementation. Meat quails were fed a diet that either met the nutritional demands for methionine (MS) or did not meet this demand (methionine-deficient diet, MD). The animals were either kept at a thermal comfort temperature (25°C) or exposed to heat stress (38°C for 24 h starting on the 6th day). RNA was extracted from liver and breast muscle, and cDNA was synthesized and amplified using quantitative reverse transcription-polymerase chain reaction. Animals that were fed the MS diet and remained at the thermal comfort temperature exhibited increased IGF-I mRNA expression in the liver (0.56 AU). The GHR mRNA expression in the liver and muscle was influenced by both the study variables. Animals receiving the MS diet showed higher GHR expression, while increased expression was observed in animals at the thermal comfort temperature. The UCP mRNA expression in the muscle was influenced by both methionine supplementation and heat stress. Higher expression was observed in animals that received the MD diet (2.29 vs 3.77 AU) and in animals kept in thermal comfort. Our results suggest that heat stress negatively affects the expression of growth-related genes and that methionine supplementation is necessary to appropriately maintain the levels of IGF-I, GHR, and UCP transcripts for animal metabolism.

Expression profiles of growth hormone receptor and insulin-like growth factor I in cattle and yak tissues revealed by quantitative real-time PCR

Abstract. The goals of this study were to compare the mRNA expression profiles of growth hormone recep tor (GHR) and insulin-like growth factor I (IGF-I) in various tissues of cattle and the semi-wild yak (Datong yak) and to find out whether the mRNA levels of the two genes are correlated. The mRNA levels of GHR and IGF-I in heart, lung, liver, spleen, pancreas, kidney, muscle, mammary gland and ovary of cattle and yak were investigated by using quantitative real-time po ly mer ase chain reaction (PCR). The experiments showed that the transcript levels of the two genes were signif icantly higher in liver (P&lt;0.05) than in the other tissues for both species and that IGF-I levels varied more among tissues (P&lt;0.01) than did GHR levels. The GHR tran script level in pancreas was higher in yak (P&lt;0.05) than in cattle. There was no statistically sig nif i cant difference in IGF-I tran script levels among all the tissues of both bovine groups. Growth hormone receptor and IGF-I transcript levels were positively correlated in mammary gland (P&lt;0.01), lung (P&lt;0.05) and muscle (P&lt;0.05) in yak, negatively correlated in cattle heart (P&lt;0.05) and not correlated in the other tissues. The results indicate that the two genes are reg u lated differently in various tissues under normal physiological conditions in these two bovine species.

Endometrial expression of leptin receptor and members of the growth hormone—Insulin-like growth factor system throughout the estrous cycle in heifers

The growth hormone (GH)—insulin-like growth factor (IGF) system is expressed in bovine uterus during the estrous cycle and early pregnancy and is acknowledged to play an important role in regulating the development of the embryo and uterus. The leptin receptor (LEPR) is also expressed in the bovine uterus although it is not known whether its expression varies during the estrous cycle. In this study, the expression of the IGF-I and -II, the type 1 IGF receptor (IGF-1R), GH receptor (GHR) and LEPR transcripts was determined on endometrial transcervical biopsies collected on days 0 (estrus), 5, 12 and 19 of the cow estrous cycle ( n = 8). The expression of mRNA was determined by RT real time PCR using ribosomal protein L19 as a housekeeping gene. It has been demonstrated for the first time that LEPR mRNA is expressed in the bovine uterus throughout the estrous cycle and that it presents a cycle-dependent variation, with higher levels observed during the luteal phase. The expression of IGF-I mRNA was greatest at estrus and day 5 (100%), and decreased on days 12 and 19 to 47% and 35% of the initial values. IGF-II mRNA increased on day 12 and decreased sharply thereafter (to one-third of day 12 values). Interestingly, IGF-1R showed the same pattern as IGF-II: increased 50% on day 12 compared to values at estrus and presented a sharp decrease on day 19. The expression of GHR transcript was greatest at estrus and on day 5 and progressively decreased thereafter. These results show that the GH–IGF system components are distinctively regulated during the estrous cycle suggesting that modulation of the IGF system may influence uterine activity during this period. The increase in the uterine sensitivity to IGFs during the late luteal phase – as demonstrated by the increased IGF-1R expression – concomitant with the increased IGF-II mRNA expression may reinforce the role of IGF-II during early pregnancy. Moreover, leptin is also likely to play roles during early embryo development.

Growth hormone receptors in zebrafish ( Danio rerio): Adult and embryonic expression patterns

Growth hormone receptor (GHR) is a critical regulator of growth and metabolism. Although two GHRs have been characterized in many fish species, their functional characteristics, mechanisms of regulation and roles in embryonic development remain unclear. The zebrafish ( Danio rerio) is an excellent model organism to study both developmental and physiological processes. In the present work, we characterized the complete cDNA sequences of zebrafish GHRs, ghra and ghrb, and their gene structures. We studied the expression of both receptors in adult tissues, and during embryonic development and larval stages by means of RT-PCR and whole-mount in situ hybridization. We determined that both transcripts are maternal ones, with specific expression patterns during development. Both GHR transcripts are mainly expressed in the notochord, myotomes, anterior structures and in the yolk cell. Interestingly, their expression became undetectable at 96 h post-fertilization. Unlike other reports in fish, ghrs expression could not be detected in brain when adult tissues were used, and we detected ghrb but not ghra transcripts in muscle. In addition, we determined alternative transcript sequences for ghra with specific domain deletions, and alternative transcripts for ghrb that generate a premature stop codon and codify for truncated isoforms. These isoforms lack intracellular regions necessary for the activation of signal transducers and activators of transcription (STAT) family transcription factors 5.

Seasonal regulation of vitellogenin by growth hormone in the goldfish liver

Goldfish, like many other fish species, undergo well-documented annual cycles, which include well-defined somatic and reproductive states. It is known that part of the year is spent in a somatotropic state (usually from late spring to late fall), followed by transition into the reproductive state (usually from late Fall to late Spring). The onset of reproductive cycle is characterized by synthesis of phosphoglycoprotein vitellogenin (Vg), a precursor to the egg yolk proteins in the liver and its uptake in the oocyte. Hormones regulate the process of vitellogenesis and circulating 17-β-estradiol is known to be a key factor during the reproductive period. During vitellogenesis, pituitary produces growth hormone (GH), but the fish divert most of the energy towards reproduction rather than growth. In this study we investigated the role of GH in the regulation of Vg in goldfish. The goldfish Vg cDNA was partially cloned (1348 amino acids), which included a highly conserved lipoprotein N-terminal domain, and used as a probe for Northern blot analysis. Treatment of female liver in vitro with recombinant goldfish GH increased Vg mRNA in goldfish at early stages of recrudescence (September), but was without effect in mid-late stages of vitellogenesis and post-spawning period (February–June). There was a correlation between GH activity and basal expression levels of the growth hormone receptor (GHR). Like Vg, GHR transcript was increased by treatment with GH in September but not February or June. Overall, the present study provides novel information on the role of GH in the season-dependant regulation of Vg and GHR gene expression.

Growing in Antarctica, a challenge for white adipose tissue development in Adélie penguin chicks (Pygoscelis adeliae)

Rapid growth is of crucial importance for Adélie penguin chicks reared during the short Antarctic summer. It partly depends on the rapid ontogenesis of fat stores that are virtually null at hatching but then develop considerably (x40) within a month to constitute both an isolative layer against cold and an energy store to fuel thermogenic and growth processes. The present study was aimed at identifying by RT-PCR the major transcriptional events that chronologically underlie the morphological transformation of adipocyte precursors into mature adipocytes from hatching to 30 days of age. The peak expression of GATA binding protein 3, a marker of preadipocytes, at day 7 posthatch indicates a key proliferation step, possibly in relation to the expression of C/EBPalpha (C/EBPalpha). High plasma total 3,5,3'-triiodo-l-thyronine (T(3)) levels and high levels of growth hormone receptor transcripts at hatching suggested that growth hormone and T(3) play early activating roles to favor proliferation of preadipocyte precursors. Differentiation and growth of preadipocytes may occur around day 15 in connection with increased abundance of transcripts encoding IGF-1, proliferator-activated receptor-gamma, and C/EBPbeta, gradually leading to functional maturation of metabolic features of adipocytes including lipid uptake and storage (lipoprotein lipase, fatty-acid synthase) and late endocrine functions (adiponectin) by day 30. Present results show a close correlation between adipose tissue development and chick biology and a difference in the scheduled expression of regulatory factors controlling adipogenesis compared with in vitro studies using cell lines emphasizing the importance of in vivo approaches.

The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill)

BackgroundQuantitative reaction norm theory proposes that genotype-by-environment interaction (GxE) results from inter-individual differences of expression in adaptive suites of genes in distinct environments. However, environmental norms for actual gene suites are poorly documented. In this study, we investigated the effects of GxE interactions on levels of gene transcription and growth by documenting the impact of rearing environment (freshwater vs. saltwater), sex and genotypic (low vs. high estimated breeding value EBV) effects on the transcription level of insulin-like growth factor (IGF-1) and growth hormone receptor (GHR) in brook charr (Salvelinus fontinalis).ResultsMales grew faster than females (μ♀ = 1.20 ± 0.07 g·d-1, μ♂ = 1.46 ± 0.06 g·d-1) and high-EBV fish faster than low-EBV fish (μLOW = 0.97 ± 0.05 g·d-1, μHIGH = 1.58 ± 0.07 g·d-1; p < 0.05). However, growth was markedly lower in saltwater-reared fish than freshwater sibs (μFW = 1.52 ± 0.07 g·d-1, μSW = 1.15 ± 0.06 g·d-1), yet GHR mRNA transcription level was significantly higher in saltwater than in freshwater (μSW = 0.85 ± 0.05, μFW = 0.61 ± 0.05). The ratio of actual growth to units in assayed mRNA ('individual transcript efficiency', iTE; g·d-1·u-1) also differed among EBV groups (μLOW = 2.0 ± 0.24 g·d-1·u-1; μHIGH = 3.7 ± 0.24 g·d-1·u-1) and environments (μSW = 2.0 ± 0.25 g·d-1·u-1; μFW = 3.7 ± 0.25 g·d-1·u-1) for GHR. Males had a lower iTE for GHR than females (μ♂ = 2.4 ± 0.29 g·d-1·u-1; μ♀ = 3.1 ± 0.23 g·d-1·u-1). There was no difference in IGF-1 transcription level between environments (p > 0.7) or EBV groups (p > 0.15) but the level of IGF-1 was four times higher in males than females (μ♂ = 2.4 ± 0.11, μ♀ = 0.58 ± 0.09; p < 0.0001). We detected significant sexual differences in iTE (μ♂ = 1.3 ± 0.59 g·d-1·u-1; μ♀ = 3.9 ± 0.47 g·d-1·u-1), salinities (μSW = 2.3 ± 0.52 g·d-1·u-1; μFW = 3.7 ± 0.53 g·d-1·u-1) and EBV-groups (μLOW = 2.4 ± 0.49 g·d-1·u-1; μHIGH = 3.8 ± 0.49 g·d-1·u-1). Interaction between EBV-group and environment was detected for both GHR (p = 0.027) and IGF-1 (p = 0.019), and for iTE in the two genes (p < 0.0001; p < 0.05, respectively), where increased divergence in levels of GHR and IGF-1 transcription occurred among EBV-groups in the saltwater environment.ConclusionOur results show that both environment and sex have major impacts on the expression of mRNA for two key genes involved in the physiological pathway for growth. We also demonstrate for the first time, at least in fish, genotype-by-environment interaction at the level of individual gene transcription. This work contributes significantly to ongoing efforts towards documenting environmentally and sexually induced variance of gene activity and understanding the resulting phenotypes.

Insulin regulation of growth hormone receptor gene expression: involvement of both the PI-3 kinase and MEK/ERK signaling pathways

The mechanism(s) of insulin's effects on growth hormone receptor (GHR) gene expression are poorly understood. Using rat hepatoma cells, we have previously shown that insulin treatment reduces GHR mRNA and protein in a time- and concentration-dependent manner, at least in part via down-regulation of GHR transcription. The present study determines whether the phosphatidylinositol-3 kinase (PI-3 kinase) and mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways are involved in mediating these effects of insulin. Inhibition of the PI-3 kinase pathway partially blocked insulin's reduction of GHR mRNA, as did inhibition of the MEK/ERK pathway, resulting in higher GHR mRNA levels. Inhibition of both pathways was necessary to completely block insulin effects. Similar results were obtained for GHR protein. Collectively, these data suggest that insulin signaling via either the PI-3 kinase or MEK/ERK pathway may result in partial reduction of GHR gene expression, whereas signaling via both pathways may be required to achieve the full insulin effect.

Effect of nutrition on the GH responsiveness of liver and adipose tissue in dairy cows

Dairy cows enter a period of energy insufficiency after parturition. In liver, this energy deficit leads to reduced expression of the liver-specific GH receptor transcript (GHR1A) and decreased GHR abundance. As a consequence, hepatic processes stimulated by GH, such as IGF-I production, are reduced. In contrast, adipose tissue has been assumed to remain fully GH responsive in early lactation. To determine whether energy insufficiency causes contrasting changes in the GH responsiveness of liver and adipose tissue, six lactating dairy cows were treated for 4 days with saline or bovine GH when adequately fed (AF, 120% of total energy requirement) or underfed (UF, 30% of maintenance energy requirement). AF cows mounted robust GH responses in liver (plasma IGF-I and IGF-I mRNA) and adipose tissue (epinephrine-stimulated release of non-esterified fatty acids in plasma, IGF-I mRNA, and p85 regulatory subunit of phosphatidylinositol 3-kinase mRNA). Reductions of these responses were seen in the liver and adipose tissue of UF cows and were associated with decreased GHR abundance. Reduced GHR abundance occurred without corresponding reductions of GHR1A transcripts in liver or total GHR transcripts in adipose tissue. In contrast, undernutrition did not alter the abundance of proteins involved in the early post-receptor signaling steps. Thus, a feed restriction reproducing the energy deficit of early lactation depresses GH actions not only in liver but also in adipose tissue. It remains unknown whether a similar reduction of GH action occurs in the adipose tissue of early lactating dairy cows.

Insulin regulation of growth hormone receptor gene expression: Evidence for a transcriptional mechanism of down-regulation in rat hepatoma cells

The role of insulin in regulating responsiveness to growth hormone (GH) remains unclear. Continuous insulin treatment reduces GH binding, which suggests that insulin may effect growth hormone receptor (GHR) levels. The present study used rat hepatoma cells to examine the effects of insulin and GH on GHR gene expression. Prolonged insulin treatment (greater than 3 h) significantly reduced GHR mRNA, and removal of insulin led to a gradual recovery. This effect of insulin occurred at physiologic concentrations, occurred many hours before the insulin-regulated decrease in GHR protein, and was mediated by reduction of GHR transcription. GH treatment dramatically reduced GHR protein, but caused only a modest reduction in GHR mRNA. These findings indicate that the heterologous reduction of GHR by insulin occurs via transcriptional downregulation, and the homologous reduction of GHR by GH occurs via a different mechanism. Furthermore, with insulin, extended time of exposure may be necessary for appreciable reduction of GHR.

Differential regulation of membrane associated-growth hormone binding protein (MA-GHBP) and growth hormone receptor (GHR) expression by growth hormone (GH) in mouse liver

Growth hormone (GH) binding to GH receptor (GHR) is the initial step that leads to the physiological functions of the hormone. Proteolytical cleavage of the GHR in humans and rabbits and alternative processing of the GHR transcript in rodents generates circulating growth hormone binding protein (GHBP). Moreover, other GHR truncated forms that result from alternative processing of the GHR mRNA transcript have been described. These GHR short forms are inserted in the plasma membrane but they are unable to transduce the signal. In rodents, membrane associated-GHBP (MA-GHBP), which accounts for a significant proportion of liver GH binding capacity, represents the main GHR short form found in membranes, and may therefore function as a negative form of the receptor. In the present study, GHR and MA-GHBP content in liver were analyzed using mutant and transgenic mice expressing different concentrations of growth hormone to evaluate the correlation between GH levels, body weight (BW), GHR and MA-GHBP expression. It was found that GH deficiency was associated with diminished BW, GHR and MA-GHBP expression, while increased GH concentration led to increased BW, GHR and MA-GHBP expression, but MA-GHBP upregulation was more pronounced than the observed increase in GHR expression. Since GHR and MA-GHBP both contribute to liver GH binding capacity, GH-induced enrichment of the dominant negative form would represent a compensatory mechanism triggered by high levels of the hormone. This attempt to attenuate the effects of supraphysiological concentrations of GH may be critical to reduce or prevent their plausible damaging effects on the organism.

Hormone receptors in gills of smolting Atlantic salmon, Salmo salar: Expression of growth hormone, prolactin, mineralocorticoid and glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase type 2

This is the first study to report concurrent dynamics in mRNA expression of growth hormone receptor (GHR), prolactin receptor (PRLR), gluco- and mineralocorticoid receptor (GR and MR) and the 11β-hydroxysteroid dehydrogenase type-2 enzyme (11β-HSD2) in Atlantic salmon ( Salmo salar) gill during smoltification. Transcript levels were analysed by quantitative PCR in fresh water (FW) fish and after a 24-h salt water (SW) challenge. GHR transcript levels increased concurrent with gill Na +, K +-ATPase activity in FW fish consistent with the SW-adaptive role of GH. SW-transfer induced an increased GHR expression levels in the early stages of smoltification but a decrease in expression at the peak of smoltification. PRLR transcript levels decreased steadily during smoltification in agreement with the recognized hyper-osmoregulatory role of PRL. Surprisingly, PRLR levels increased after SW transfer during the course of smoltification. GR mRNA levels were low early on during smoltification but increased at the peak of smoltification and remained high during de-smoltification, indicative of increased cortisol signalling at this point. Coherently, SW transfer increased GR levels to smolt levels prior to the smoltification peak. 11β-HSD2 levels increased at the smoltification peak and MR levels increased during de-smoltification, suggesting a need for protection of MR from cortisol signalling during smoltification. This is supported by the fact that SW-transfer results in a profound up-regulation of 11β-HSD2, whereas SW transfer down-regulates MR levels. The study concludes that GR and MR may have distinctive roles in developing hypo- and hyper-osmoregulatory mechanisms during smoltification and de-smoltification, respectively.

Regulation of the somatotropic axis by dietary factors in rainbow trout (Oncorhynchus mykiss)

The activity of the somatotropic axis was analysed in juvenile rainbow trout (Oncorhynchus mykiss) fed either a fishmeal-based diet (FM) or graded levels of plant proteins to replace 50% (PP50 diet), 75% (PP75 diet) or 100% (PP100 diet) of the fishmeal protein. For this purpose, partial cloning and sequencing of the gene encoding rainbow trout growth hormone receptor (GHR) was first accomplished by RT-PCR, using degenerate primers based on the sequences of non-salmonid fish GHR. Growth rates and energy retention were lowered by the PP75 and PP100 diets and a concurrent and progressive increase in plasma levels of growth hormone (GH) was found. However, no changes in hepatic GH binding and total plasma insulin-like growth factor (IGF)-I levels were observed among the four experimental groups. This fact agrees with the lack of changes in hepatic measurements of GHR and IGF-I transcripts. No consistent changes in IGF transcripts were found in peri-visceral adipose tissue and skeletal muscle, but GHR mRNA was up-regulated in the peri-visceral adipose tissue of fish fed the PP75 and PP100 diets, which would favour the lipolytic action of GH. Two specific bands (47 and 33 kDa) of IGF-binding proteins were found in the plasma of all analysed fish, but the sum of the two integrated areas increased progressively with plant protein supply, which might reflect a reduced free IGF availability. Therefore, in our experimental model, the growth impairment could be due, at least in part, to a lowered availability of biologically active IGF (free IGF fraction) rather than to liver GH desensitization or defect in IGF synthesis and release at the systemic and/or paracrine-autocrine level.

Plasma Hormones and Expression of Growth Hormone Receptor and Insulin-Like Growth Factor-I mRNA in Hepatic Tissue of Periparturient Dairy Cows

Growth hormone plays a central role in the change in nutrient metabolism that occurs during the initiation of lactation. The actions of growth hormone are mediated by the growth hormone receptor (GHR) whose mRNA is present in three alternatively spliced forms (GHR 1A, 1B, and 1C). Liver-specific GHR 1A mRNA is transiently decreased around parturition, but the exact timing of the decline is not known. Our objective was to generate a daily profile for total GHR (GHRtot; all GHR transcripts), GHR 1A, and IGF-I mRNA expression in liver of periparturient Holstein cows and evaluate these daily mRNA profiles relative to daily profiles for periparturient hormones and metabolites. Liver biopsies and blood samples (n=139) were collected from 65 Holstein cows at the University of Missouri Dairy Farm. At least two cows were sampled on each day from 14 d before to 14 d after parturition. Total cellular RNA was isolated and reverse transcribed to cDNA. Target cDNA were measured by quantitative real-time polymerase chain reaction. Plasma was assayed for progesterone, estradiol, insulin, growth hormone, IGF-I, glucose, and nonesterified fatty acids. The GHR 1A mRNA declined 2 d before parturition, was lowest 3 to 4 d after parturition, and then increased. The IGF-I mRNA declined 1 d after parturition, was lowest 2 to 5 d after parturition and then increased. Total GHR mRNA was not affected by day. The decrease in GHR 1A mRNA was associated with a decrease in progesterone and an increase in estradiol shortly before parturition. A detailed profile of GHR 1A, IGF-I, and GHRtot mRNA expression during the periparturient period was provided. The decreases in GHR 1A and IGF-I during the transition period occurred immediately before (GHR 1A) or shortly after (IGF-I) parturition. Rapid changes in placental and ovarian steroids before parturition were coincident with changes in GHR 1A mRNA.

Growth hormone (GH) binding and expression of GH receptor 1A mRNA in hepatic tissue of periparturient dairy cows.

Growth hormone (GH) plays a role in metabolic adaptations that occur during lactogenesis. Liver GH receptor transcript (GHR 1A) is transiently decreased near parturition and may reduce GH-dependent signaling leading to low blood insulin-like growth factor I (IGF-I) concentrations in periparturient dairy cattle. We hypothesized that the decrease in GHR 1A mRNA at parturition was associated with decreased GH binding (i.e., GHR protein concentration) in liver. Blood and liver biopsy samples were collected from 12 Holstein cows on d -12 +/- 1, 3, and 17 relative to parturition. Total cellular RNA was isolated from a sub-sample of liver. Quantitative real-time polymerase chain reactions were used to measure GHR 1A, total GHR, IGF-I, and cyclophilin mRNA. Microsomal membranes were isolated from the remaining liver tissue and assayed for 125I-bGH binding. Plasma was assayed for GH and IGF-I concentrations. Liver GHR 1A mRNA, specific 125I-bGH binding to liver membranes, liver IGF-I mRNA, and plasma IGF-I concentrations were lower on d 3 relative to d -12. The GHR 1A mRNA, 125I-bGH binding, and plasma GH concentrations increased on d 17 but liver IGF-I mRNA and plasma IGF-I concentrations did not change between d 3 and 17. Total GHR mRNA and cyclophilin mRNA amounts were similar on d -12, 3, and 17. Across all days, 125I-bGH specific binding in liver was highly correlated with liver GHR 1A mRNA (R2 = 0.68) but not with total GHR mRNA. Saturation binding analysis showed that GHR concentration (Bmax) in liver on d 3 had decreased to only 5% of the amount on d -12. We conclude that decreased GHR 1A mRNA leads to decreased GHR protein concentration in liver. Reduced GHR in liver likely contributes to a decrease in liver IGF-I production and reduced concentrations of IGF-I in blood of periparturient cows.

Human oocytes and preimplantation embryos express mRNA for growth hormone receptor

Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species.