Estrogen receptor (ER)alpha, but not ERbeta, gene is expressed in growth hormone-releasing hormone neurons of the male rat hypothalamus

GH synthesis and release from pituitary somatotropes is controlled by the opposing actions of the hypothalamic neuropeptides, GH-releasing hormone (GHRH), and somatostatin (SS). There is a striking sex difference in the pattern of GH secretion in rats. Early reports indicate that gonadal steroids have important imprinting effects during the neonatal period. Recently, our laboratory and others have reported that the GH secretory pattern is altered by short-term gonadal steroid treatment in adult rat, suggesting that gonadal steroids are also important determinants of the pattern of GH secretion during adult life. However, the site of action of gonadal steroids in the adult rat hypothalamus is still unknown. In this study, we used in situ hybridization in the adult male rat brain to determine whether GHRH neurons and/or SS neurons coexpress estrogen receptor alpha (ERalpha) and ERss genes. In the medial basal hypothalamus of adult male rat, the ERalpha messenger RNA (mRNA) was located in medial preoptic area (MPA) and arcuate nucleus (ARC), whereas ERss mRNA was detected in MPA, supraoptic nucleus, and paraventricular nucleus. From studies using adjacent sections, the distribution of ERalpha mRNA-containing cells appeared to overlap in part with those of GHRH and SS expressing cells only in the ARC. On the other hand, the distribution of ERss mRNA-containing cells does not appear to overlap with GHRH cells or SS cells. The double label in situ hybridization studies showed that in the ARC, 70% of GHRH neurons contain ERalpha mRNA, whereas less than 5% of SS neurons expressed the ERalpha gene. These results indicated that GHRH neurons are direct target cells for estrogens, and estrogens may act directly on GHRH neurons through ERalpha during adult life to modify GH secretory patterns.     

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.     

Differential expression of IGF-I mRNA and peptide in the male and female gonad during early development of a bony fish, the tilapia Oreochromis niloticus

Insulin-like growth factor I (IGF-I) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. The major source of circulating IGF-I is liver, but IGF-I-producing cells also occur in other organs, including the gonads. Because no data are available on the potential production sites of IGF-I in gonad development, developmental stages of monosex breedings of male and female tilapia from 0 day postfertilization (DPF) to 90 DPF were investigated for the production sites of IGF-I at the peptide (immunohistochemistry) and mRNA (in situ hybridization) level. IGF-I mRNA first appeared in somatic cells of the male and female gonad anlage at 7 DPF followed by IGF-I peptide around 9-10 DPF. Gonad anlagen were detected from 7 DPF. Starting at 7 DPF, IGF-I peptide but no IGF-I mRNA was observed in male and female primordial germ cells (PGCs) provided that IGF-I mRNA was not under the detection level, this observation may suggest that IGF-I originates from the somatic cells and is transferred to the PGCs or is of maternal origin. While in female germ cells IGF-I mRNA and peptide appeared at 29 DPF, in male germ cells both were detected as late as at 51-53 DPF. It is assumed that the production of IGF-I in the germ cells is linked to the onset of meiosis that in tilapia ovary starts at around 28 DPF and in testes at around 52-53 DPF. In adult testis, IGF-I mRNA and peptide occurred in the majority of spermatogonia and spermatocytes as well as in Leydig cells, the latter indicating a role of IGF-I in the synthesis of male sex steroids. In adult ovary, IGF-I mRNA and IGF-I peptide were always present in small and previtellogenic oocytes but only IGF-I peptide infrequently occurred in oocytes at the later stages. IGF-I expression appeared in numerous granulosa and some theca cells of follicles at the lipid stage and persisted in follicles with mature oocytes. The results suggest a crucial role of local IGF-I in the formation, differentiation and function of tilapia gonads.     

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14 weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.     

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.     

The relationship between growth hormone (GH) messenger ribonucleic acid levels and hormone release from individual cells derived from human GH-secreting pituitary adenomas

GH mRNA expression and GH release by individual cells derived from four GH-secreting pituitary adenomas were studied by in-situ hybridization and the reverse haemolytic plaque assay, respectively. In addition the percentage of PRL mRNA-containing cells was determined in these cell suspensions. The percentages of GH mRNA-containing cells varied between 52 and 89 while the percentages of GH plaque forming cells varied between 25 and 77. Frequency distributions of GH mRNA levels in individual cells and of individual GH plaque areas showed a majority of the cells having low GH mRNA levels and secreting low amounts of GH respectively, while there is a low proportion of cells expressing high GH mRNA levels and forming large GH plaques. There was a significant correlation between the GH mRNA levels and the GH plaque areas of individual cells from the four adenomas (P less than 0.001). The percentages of PRL mRNA-containing cells in the four different adenomas amounted to less than 1, 5, 2 and 18. Cultured cells from the adenomas consisting of 5 and 18% PRL mRNA-containing cells also contained and released measurable amounts of PRL. Our data show that individual cells from GH-secreting pituitary adenomas are heterogeneous with respect to GH mRNA expression, a small proportion of the cells expressing a high amount of GH mRNA. The heterogeneity in GH mRNA expression is correlated with the heterogeneity in GH release. These observations suggest that a considerable part of GH secreted from a GH-secreting pituitary adenoma is produced by a minority of the GH-secreting tumour cell population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin-releasing factor receptor subtype 1 and somatostatin modulating hypoxia-caused downregulated mRNA of pituitary growth hormone and upregulated mRNA of hepatic insulin-like growth factor-I of rats

The study aims to examine the effects of restraint, cold, and in combination of hypoxia on pituitary GH mRNA and hepatic IGF-I mRNA and its protein in rats, and the potential involvement of corticotropin-releasing factor receptor subtype 1 (CRFR1) and SS in mediating the effects of continual hypoxia. Continual or intermittent hypoxia of 5 km (10.8% O2) was simulated in a hypobaric chamber. The mRNAs and peptides were determined using RT-PCR and Elisa or histochemistry. Continual hypoxia of 5 km markedly enhanced immunostaining pituitary GH and hepatic IGF-I for 1 and 2 days restoring afterward. The hypoxia for 5 days significantly reduced the pituitary GH mRNA and increased the hepatic IGF-I mRNA. Intermittent hypoxia of 5 km 4 h/day for 2 days, cold (4 degrees C) 4h/day for 2 days, and restraint 4 h/day for 2 days alone or in combination significantly enhanced immunostaining pituitary GH and hepatic IGF-I (except cold). The combined stresses had greater effects than single stresses alone. CRFR1 antagonist (CP154526) or SS antagonist (cysteamine) markedly blocked hypoxia-reduced pituitary GH mRNA and hypoxia-activated hepatic IGF-I mRNA, and further reduced hypoxia-reduced plasma IGF. In conclusion, hypoxia (continually or intermittently), restraint, cold alone or in combination modulate pituitary GH and hepatic IGF-I. The pituitary GH/GH mRNA and hepatic IGF-I/IGF-I mRNA, and plasma IGF-I are modified by hypoxia through SS and CRFR1 mediation.     

IGF-I is distinctly located in the bony fish pituitary as revealed for Oreochromis niloticus, the Nile tilapia, using real-time RT-PCR, in situ hybridisation and immunohistochemistry

In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4+/-3.3 x 10(-3)pg/microg total RNA) was 1000-times lower than in liver (7.5+/-3.1 pg/microg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In alpha-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and alpha-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine cells.     

Effects of cycloheximide on hepatic and uterine insulin-like growth factor-I mRNA

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.     

Neonatal imprinting and regulation of estrogen receptor alpha and beta mRNA expression by estrogen in the pituitary and hypothalamus of the male rat

In male rodents, complete reproductive function is critically dependent on adequate estrogen action at different levels of the hypothalamic-pituitary-testicular axis. However, administration of high doses of estrogen during the critical period of neonatal differentiation results in multiple defects in the reproductive axis that disrupt male fertility, the molecular mechanism(s) behind such a phenotype remaining poorly characterized. The aim of this study was twofold: (1) to characterize the effects of neonatal estrogenization upon the pattern of estrogen receptors ERalpha and ERbeta mRNA expression in the pituitary and hypothalamus of the male rat, and (2) to evaluate the ability of estrogen to acutely regulate pituitary and hypothalamic ERalpha and ERbeta mRNA expression in the male rat. To achieve the first goal, groups of male rats were treated on the first day of life with estradiol benzoate (EB; 500 microg/rat), and pituitary and hypothalamic expression of ERalpha and ERbeta mRNA levels were assayed by semiquantitative RT-PCR at different ages from the neonatal period until adulthood (days 1-75 of life). In addition, the mechanism(s) of altered pituitary expression of ERalpha and ERbeta messages in neonatally estrogenized rats was explored by comparison of the effects of neonatal treatment with estrogen or a potent gonadotropin-releasing hormone (GnRH) antagonist. For the second goal, the acute effects of a single dose of EB (12.5-25 microg/rat) on hypothalamic and pituitary ERalpha and ERbeta mRNA expression were assessed in prepubertal and adult male rats. Neonatal estrogenization permanently decreased pituitary ERalpha and ERbeta mRNA expression levels respective to control values at all ages studied. This pattern of response was similar to the short-term effects of neonatal blockade of endogenous GnRH actions. In contrast, neonatal exposure to estrogen transiently increased the hypothalamic expression levels of ERalpha and ERbeta messages. This effect was detected in neonatal (5-day-old), infantile (15-day-old) and prepubertal (30-day-old) rats, and it disappeared at puberty. In addition, estrogen was able to acutely regulate mRNA expression levels of its cognate receptor subtypes in the pituitary and hypothalamus of intact male rats. In adult (75-day-old) males, EB administration (25 microg/rat) induced a significant time-dependent decrease in pituitary ERalpha and ERbeta mRNA levels. In contrast hypothalamic expression of ERalpha and ERbeta messages was increased after acute exposure to EB (12.5 microg/rat) in prepubertal males (30 days old); yet differences in the time course of the response to estrogen were noticed between targets. In conclusion, our data indicate that estrogen is able to neonatally imprint and acutely regulate mRNA expression levels of ERalpha and ERbeta in the pituitary and hypothalamus of the male rat. Regulation of pituitary and hypothalamic ERalpha and ERbeta gene expression by the cognate ligand represents a novel mechanism whereby estrogen modulates its own biological actions upon different levels of the male reproductive axis throughout the life span.     

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia,Oreochromis mossambicus

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.     

Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells

GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH-stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression.