Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs

There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations.     

Regulation of the genes for insulin-like growth factor (IGF) I and II and their receptors by steroids and gonadotropins in the ovary

Insulin-like growth factors (IGFs) I and II are two single-chain polypeptide hormones that are structurally related to each other and to proinsulin. Among the large number of growth factors involved in ovarian physiology, IGF-I and IGF-II are considered to be important progression factors for ovarian follicular development. To explore the ovarian expression of IGF-I, IGF-II and their receptor genes, a solution hybridization/RNase protection assay, was used. IGF-I mRNA was seen in the granulosa cells, and IGF-II mRNA in the theca-interstitial compartment. To study the hormonal regulation of the IGF-I and IGF-II gene, immature (21-day-old) hypohysectomized rats were treated with FSH (10 μg/day),GH (150 μg/day) and diethylstilbestrol (DES subcutaneous implant/5 days). Estrogen differentially regulated ovarian IGF-I and IGF-II gene expression. In concert with GH, estrogen up-regulated ovarian IGF-I mRNA, but significantly decreased hepatic IGF-I gene expression. Both IGF receptors (type I and type II) as well as the insulin receptor gene, were expressed in both ovarian cells. The expression of the type IIGF receptor gene (but not the type II IGF gene) was up-regulated by FSH and estrogen in vivo. In conclusion, these studies may serve to better understand the auto paracrine role of IGF, and their receptors in the pathophysiology of follicle recruitment, oocyte maturation and potentially embryo development.     

NO plays a role in LPS-induced decreases in circulating IGF-I and IGFBP-3 and their gene expression in the liver

In this study, we administered aminoguanidine, a relatively selective inducible nitric oxide synthase (iNOS) inhibitor, to study the role of nitric oxide (NO) in LPS-induced decrease in IGF-I and IGFBP-3. Adult male Wistar rats were injected intraperitoneally with LPS (100 microg/kg), aminoguanidine (100 mg/kg), LPS plus aminoguanidine, or saline. Rats were injected at 1730 and 0830 the next day and killed 4 h after the last injection. LPS administration induced an increase in serum concentrations of nitrite/nitrate (P < 0.01) and a decrease in serum concentrations of growth hormone (GH; P < 0.05) and IGF-I (P < 0.01) as well as in liver IGF-I mRNA levels (P < 0.05). The LPS-induced decrease in serum concentrations of IGF-I and liver IGF-I gene expression seems to be secondary to iNOS activation, since aminoguanidine administration prevented the effect of LPS on circulating IGF-I and its gene expression in the liver. In contrast, LPS-induced decrease in serum GH was not prevented by aminoguanidine administration. LPS injection decreased IGFBP-3 circulating levels (P < 0.05) and its hepatic gene expression (P < 0.01), but endotoxin did not modify the serum IGFBP-3 proteolysis rate. Aminoguanidine administration blocked the inhibitory effect of LPS on both IGFBP-3 serum levels and its hepatic mRNA levels. When aminoguanidine was administered alone, IGFBP-3 serum levels were increased (P < 0.05), whereas its hepatic mRNA levels were decreased. This contrast can be explained by the decrease (P < 0.05) in serum proteolysis of this binding protein caused by aminoguanidine. These data suggest that iNOS plays an important role in LPS-induced decrease in circulating IGF-I and IGFBP-3 by reducing IGF-I and IGFBP-3 gene expression in the liver.     

Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats

Estradiol sulfamate (J995), estradiol-17beta with a substituted sulfamate group in position 3, has much higher systemic estrogenic activity after oral administration than 17beta-estradiol (E2) due to reduced hepatic metabolism of the drug. The lower dose necessary for achievement of adequate systemic estrogenic effects results in a substantial reduction of otherwise commonly observed hepatic side-effects. This makes J995 a strong candidate as an estrogen suitable for oral administration. The present study was performed to examine and compare the effects of J995 and E2 on the uterus after oral or subcutaneous administration to ovariectomized or ovariectomized+hypophysectomized female rats, in particular on the levels of the estrogen receptor (ER) (alpha+beta), ERalpha mRNA and insulin-like growth factor-I (IGF-I) mRNA. The ER levels were determined using a ligand binding assay and the mRNA levels using solution hybridization. The doses of J995 or E2 were chosen to achieve comparable uterotrophic activity. The rats were treated with hormones for 7 days and the treatment was initiated 14 days after surgery. We conclude that there are no major differences in the uterine response to treatment with J995 or E2 with respect to the effects on ER and ERalpha mRNA levels. The IGF-I mRNA level though, is more affected by J995 than by E2 after 7 days of treatment, indicating a prolonged effect of J995.     

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor genes in the brain of masu salmon from immaturity to spawning

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, -R2, -R3, -R4, and -R5, was examined in the brain of masu salmon (Oncorhynchus masou). In addition, responses of these genes to GnRH were examined in a GnRH analog (GnRHa) implantation experiment. Brain samples were collected one week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single forebrains was determined by real-time PCR assays. Among the five genes, R4 and R5 were dominantly expressed in both sexes. R1, R4, and R5 mRNAs showed similar changes throughout the experimental period in both sexes. Levels tended to be high in winter and low in the pre-spawning season, followed by elevations in the spawning period. The mRNA levels had weak to moderate negative correlations with the plasma level of estradiol-17beta (E2) in females. The effects of GnRHa on msGnRH-R mRNAs were not apparent for all the subtypes. These results indicate that the msGnRH-R1, -R4, and -R5 genes are synchronously expressed during sexual maturation. There was a trend toward decreased levels of their expression prior to the spawning period and then increased levels at spawning, possibly causing GnRH target neurons to sensitize to a GnRH stimulus. Furthermore, E2 may be involved in msGnRH-R gene expression in the brain of female masu salmon during sexual maturation.     

Effect of pulsatile growth hormone administration to the growth-restricted fetal sheep on somatotrophic axis gene expression in fetal and placental tissues

We have previously reported (Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. J Endocrinol 177: 83-92, 2003) that a chronic pulsatile infusion of growth hormone (GH) to intrauterine growth-restricted (IUGR) ovine fetuses increased fetal circulating IGF-I levels without increasing fetal growth. We hypothesized a cortisol-induced upregulation of fetal hepatic GH receptor (GH-R) mRNA levels, secondary increases in IGF-I mRNA levels, and circulating IGF-I levels, but a downregulation of the type I IGF receptor (IGF-IR) as an explanation. We, therefore, measured mRNA levels of genes of the somatotrophic axis by real-time RT-PCR in fetal and placental tissues of fetuses with IUGR (induced by uteroplacental embolization from 110- to 116-days gestation) that received either a pulsatile infusion of GH (total dose 3.5 mg/day) or vehicle from 117-126 days and in control fetuses (n = 5 per group). Tissues were collected at 127 days (term, 145 days). Fetal cortisol concentrations were significantly increased in IUGR fetuses. However, in liver, GH-R, but not IGF-I or IGF-IR, mRNA levels were decreased in both IUGR groups. In contrast, in placenta, GH-R, IGF-I, and IGF-IR expression were increased in IUGR vehicle-infused fetuses. GH infusion further increased placental GH-R and IGF-IR, but abolished the increase in IGF-I mRNA levels. GH infusion reduced IGF-I expression in muscle and increased GH-R but decreased IGF-IR expression in kidney. IUGR increased hepatic IGF-binding protein (IGFBP)-1 and placental IGFBP-2 and -3 mRNA levels with no further effect of GH infusion. In conclusion, the modest increases in circulating cortisol concentrations in IUGR fetuses did not increase hepatic GH-R mRNA expression and, therefore, do not explain the increased circulating IGF-I levels that we found with GH infusion, which are likely due to reduced clearance rather than increased production. We demonstrate tissue-specific regulation of the somatotrophic axis in IUGR fetuses and a discontinuity between GH-R and IGF-I gene expression in GH-infused fetuses that is not explained by alterations in phosphorylated STAT5b.     

Estrogen induces insulin-like growth factor-I expression in the rat uterus

The inability to convincingly demonstrate a mitogenic effect of estrogen on isolated uterine cells in culture suggests that autocrine or paracrine growth factors may be important in the estrogen-induced uterine proliferative response. Here we report that uterine expression of insulin-like growth factor-I (IGF-I), an important mediator of GH action, is increased after 17 beta-estradiol (5 micrograms/100 g bw, ip) administration to ovariectomized prepubertal rats. An increase in uterine IGF-I mRNA abundance, approximately 14-fold above untreated controls, was apparent 6 h after estrogen administration and the level achieved exceeded that seen in the uterus from intact mature rats during diestrus. In contrast to the increase in IGF-I expression in the uterus, no significant change in serum IGF-I concentration or hepatic or renal IGF-I mRNA abundance was demonstrable after 17 beta-estradiol injection of ovariectomized prepubertal rats. The increase in uterine IGF-I expression, was similar in both pituitary-intact and hypophysectomized, ovariectomized rats. We believe this is the first report of induction of IGF-I expression by estrogen in vivo. As such, the finding expands the role and significance of IGF-I as a mediator of growth beyond that related to GH.     

Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10(-8) M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17beta (E(2)) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E(2) in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.     

Dietary daidzein influences laying performance of ducks (Anas platyrhynchos) and early post-hatch growth of their hatchlings by modulating gene expression

Our previous studies demonstrated that dietary supplementation of daidzein improves egg production in duck breeders during late periods of the laying cycle. The present study was aimed to clarify whether the growth of ducklings hatched from eggs laid by daidzein-treated hens would be affected, and to elucidate the mechanisms underlying potential trans-generational effects, by determining changes of hormone levels and mRNA expression of relevant genes. Daidzein was added to the basal diet of 415-day-old duck breeders at the level of 5 mg/kg. During 9 weeks of daidzein treatment, laying rate increased by 7.70%, average egg mass tended to increase, whereas yolk/albumen ratio decreased significantly. These changes were accompanied by significantly elevated plasma T4 and E2 levels, enhanced gonadotropin releasing hormone (GnRH) mRNA, but diminished estrogen receptor (ER)-beta mRNA expression in hypothalamus of daidzein-treated hens. Ducklings hatched from daidzein-treated eggs were significantly smaller at hatching, but they caught up with their control counterparts by 4 weeks of age. Serum levels of T4, pituitary GH, hepatic GH receptor (GHR) and type-1 IGF receptor (IGF-1R) mRNA expression were all suppressed markedly in the daidzein-treated group at hatching, but this suppression proved to be temporary, as at 4 weeks of age, expression levels of all investigated genes were restored. However, it is noteworthy that at 4 weeks of age an obvious down-regulation of hypothalamic GnRH mRNA expression was detected in ducklings maternally exposed to daidzein. Our results provide evidence that maternal exposure to daidzein affects post-hatch growth in the duck with accompanying changes in the secretion of metabolic hormones and expression of growth-related genes. Although the negative effect of maternal daidzein on embryonic growth could be eliminated 4 weeks after hatching, the long-term effect of maternal daidzein on reproductive function is not to be ignored and awaits further investigation.     

Effects of a high plant protein diet on the somatotropic system and cholecystokinin in Atlantic salmon (Salmo salar L.)

To investigate the endocrine signalling from dietary plant protein on somatotropic system and gastrointestinal hormone cholecystokinin (CCK), two iso-amino acid diets based on either high plant or high fish meal protein were fed to Atlantic salmon. Salmon with an average starting weight of 641 ± 23 g (N = 180), were fed a fish meal (FM) based diet (containing 40% FM) or diets mainly consisting of blended plant proteins (PP) containing only 13% marine protein, of which only 5% was FM for 3 months. mRNA levels of target genes GH, GH-R, IGF-I, IGF-II, IGFBP-1, IGF-IR in addition to CCK-L, were studied in brain, hepatic tissue and fast muscle, and circulating levels of IGF-I in plasma of Atlantic salmon were measured. We detected reduced feed intake resulting in lower growth, weight gain and muscle protein accretion in salmon fed plant protein compared to a diet based on fish meal. There were no significant effects on the regulation of the target genes in brain or in hepatic tissues, but a trend of down-regulation of IGF-I was detected in fast muscle. Lower feed intake, and therefore lower intake of the indispensable amino acids, may have resulted in lower pituitary GH and lower IGF-I mRNA levels in muscle tissues. This, together with higher protein catabolism, may be the main cause of the reduced growth of salmon fed plant protein diet. There were no signalling effects detected either by the minor differences of the diets on mRNA levels of GH, GH-R, IGF-IR, IGF-II, IGFBP-1, CCK or plasma protein IGF-I.     

Caffeic acid phenethyl ester ameliorates changes in IGFs secretion and gene expression in streptozotocin-induced diabetic rats

The protective effect of caffeic acid phenethyl ester (CAPE) against diabetes-induced alteration of IGFs protein and gene expression was investigated in serum, liver, heart, and kidney. In the present study, diabetic rats exhibited the decrease of IGF-I content in serum, liver and heart but the increase of that in kidney and CAPE blocked them. Diabetic rats also manifested the increase of IGF-II content in serum, liver, heart, and kidney and CAPE prevented them. CAPE prevented the diabetes-induced decrease of liver IGF-I mRNA and IGF-II mRNA, which is similar to pattern of IGFs mRNA in kidney. Moreover, diabetic rats exhibited the decrease of heart IGF-I mRNA but the increase of IGF-II mRNA and CAPE blocked them. In conclusion, CAPE, in part, prevented diabetes-induced alteration of IGF-I and IGF-II protein and gene expression in liver, heart, and kidney in rats.     

Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.     

Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mRNA in various organs of a GH-overexpressing transgenic Nile tilapia

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4–4-fold; IGF-II: 1.7–4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.     

Distribution and regulation of rat insulin-like growth factor I messenger ribonucleic acids encoding alternative carboxyterminal E-peptides: evidence for differential processing and regulation in liver

Alternative splicing of insulin-like growth factor I (IGF-I)/somatomedin C mRNAs generates two IGF-I mRNAs coding for IGF-I peptides with different sequences in the E domain of the IGF-I prohormone. These two mRNAs encode alternative E peptides due to the presence (IGF-Ib) or absence (IGF-Ia) of a 52-base insert in the region coding for the E domain. We have used a solution hybridization/RNase protection assay to determine the tissue distribution and regulation by GH of the expression of these alternative IGF-I mRNAs. IGF-Ib mRNAs are present in low abundance (representing approximately 2.5% of the total IGF-I mRNA) in heart, lung, muscle, testes, stomach, kidney, and brain, but represent approximately 13% of the IGF-I mRNA in liver. GH treatment of hypophysectomized rats increased steady-state IGF-I mRNA levels in liver, kidney, lung, and heart. In kidney, lung, and heart, IGF-Ia and IGF-Ib mRNA levels were coordinately regulated by GH, but, in liver, the fold increase in IGF-Ib mRNA levels was approximately three times greater than the fold increase in IGF-Ia mRNA levels. These data suggest that the processing of IGF-I mRNA in liver is different than in nonhepatic tissues. These results also further elucidate the organization of the rat IGF-I gene as well as the generation of multiple IGF-I mRNAs by alternative splicing.     

Identification of two insulin-like growth factor IIs in the Japanese eel, Anguilla japonica: cloning, tissue distribution, and expression after growth hormone treatment and seawater acclimation

To better understand the role of IGFs in Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-II (IGF-II) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-II cDNAs, eIGF-II-1 and eIGF-II-2, were cloned from the liver. A signal peptide and a mature peptide of both preproIGF-IIs were composed of 47 amino acids (aa) and 69 aa, but they differed at 17 aa and 13 aa, respectively. The E domain of eIGF-II-1 was 49 aa longer than that of eIGF-II-2, and differed at 22 aa within 52 aa. The highest eIGF-II-1 and II-2 mRNA levels were observed in the liver, with detectable levels also found in all tissues examined. The eIGF-II-1 mRNA levels in the liver, heart, and muscle were higher in females than in males, whereas those in the stomach and intestine were lower in the females. The eIGF-II-2 mRNA levels in the liver and swim-bladder were also higher in females than in males whereas those in the stomach, spleen, and intestine were lower in the females. The eIGF-II-1 mRNA levels in the liver were higher in large compared to small glass eels, while the eIGF-II-2 mRNA levels did not correlate with body weight. Both eIGF-II mRNA levels in the liver increased after eel GH treatments in vivo and in vitro. No differences in both eIGF-II mRNA levels were observed in the gills, liver, stomach and whole kidney between seawater- and freshwater-reared eels.     

Pituitary hormones dependent expression of insulin-like growth factors I and II in the immature hypophysectomized rat testis

Since insulin-like growth factors I (IGF-I) and II (IGF-II) appeared involved in paracrine or autocrine regulation of both cell multiplication and differentiation of the rat testis, we have investigated the pituitary hormonal dependence of IGF-I and IGF-II mRNA production in the testis of immature hypophysectomized rats (22 days old) supplemented with highly purified FSH, LH, GH or PRL. Our data show that testicular expression of IGF-I mRNA as measured by dot-blot hybridization, is increased by LH, FSH or GH treatments of 7-, 6-, and 4-fold, respectively, above controls. Intensity of the signal was 3-fold lower after PRL treatment than in hypophysectomized control rats. On the contrary, IGF-II mRNA expression, was found low in the immature hypophysectomized rat testis and unmodified by any hormonal treatment. In contrast to the increase of IGF-I expression in the testis no significant change in the IGF-I plasma concentration was observed after LH or FSH supplementation. GH treatment, as expected, increased 4-fold the IGF-I plasma concentration of the experimental animals. Since we have previously shown that LH, FSH, and GH exhibit selective cell multiplication and differentiation in the testis of our animal model, it is proposed that testicular IGF-I expression could be the tissue response to pituitary hormone in these phenomena.