Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Peptide insertion in the DNA-binding domain of fish glucocorticoid receptor is encoded by an additional exon and confers particular functional properties.

The trout glucocorticoid receptor (rtGR) contains an additional sequence of nine amino acids located between the two zinc fingers of the DNA-binding domain (DBD) (Endocrinology 136 (1995) 3774). Polymerase chain reaction on trout genomic DNA and sequencing were performed in the DBD region, demonstrating that this peptide is encoded by an additional exon of 27 nucleotides between the two exons encoding the two zinc fingers of other nuclear receptors. This additional sequence in the rtGR confers a better binding affinity of the receptor to a single GRE, as shown by gel shift experiments with GST-DBDrtGR fusion proteins, deleted or not of the nine amino acids (Delta9). This higher affinity is correlated with a higher constitutive transcriptional activity of the receptor on a reporter gene driven by a single GRE, but not with the ligand-induced transcriptional activity. Nevertheless, on a double GRE, the wild type and rtGR-Delta9 are equally active on both constitutive or dexamethasone-induced transcriptional activity. This original DBD structure could have emerged during evolution such as to allow better regulation of glucocorticoid dependent genes in relation to the large spectrum of cortisol physiological functions in fish.

Glucocorticoid-induced glucose release is abolished in trout hepatocytes with elevated hsp70 content.

The metabolic potential of cells with elevated heat shock protein 70 (hsp70) content was examined by measuring unstimulated and glucocorticoid-stimulated glucose release in trout hepatocytes maintained in primary culture. Exposure of hepatocytes to either heat shock (HS;+15 degrees C) or sodium arsenite (50 microM) did not affect cell viability, but resulted in significantly higher hsp70 levels over a 24 h recovery period. Hsp70 accumulation had no significant impact on unstimulated glucose release, but completely abolished cortisol-induced glucose release in trout hepatocytes. This lack of glucocorticoid responsiveness corresponded with lower glucocorticoid receptor protein levels. Together, our results suggest that stressor-induced hsp70 accumulation, while important for maintaining cellular homeostasis, may impair metabolic adjustments to subsequent stressors in animals, especially those that are glucocorticoid-dependent.

Glucocorticoid receptors and serotonin N-acetyltransferase activity in the fish pineal organ.

This study aimed to determine whether glucocorticoid receptors are expressed in the photosensitive trout pineal organ, and whether glucocorticoids modulate melatonin secretion. On Western blots from pineal extracts, an antibody directed against trout glucocorticoid receptor labeled a single band at the expected size (approximately 100 kDa). Dexamethasone inhibited pineal arylalkylamine N-acetyltransferase activity (AANAT2; serotonin --> N-acetylserotonin) in a dose-dependent manner after 6 h of culture in the dark (IC50 2.10(-8) M). RU486 (10(-7) M) alone had a partial agonistic activity, whereas it antagonized the effects of 10(-8) M dexamethasone. Hydroxyindole-O-methyltransferase activity (N-acetylserotonin --> melatonin) remained unaffected. This is the first demonstration that glucocorticoid receptors are present in the pineal organ and that glucocorticoids modulate melatonin production.

Effects of copper on cortisol receptor and metallothionein expression in gills of Oncorhynchus mykiss.

Effects of waterborne Cu (2.4 microM) on the expression of glucocorticoid receptor (GR) and metallothionein (MT) in the branchial epithelium of freshwater rainbow trout (Oncorhynchus mykiss) was studied by immunocytochemistry. After 5 days of Cu exposure, the number of GR-immunoreactive (GR-ir) cells in the gill epithelium had decreased, whereas the number of MT-ir cells had increased. Localization of GR in chloride cells was achieved by double staining for Na(+)/K(+)-ATPase; other cell types were identified on the basis of their topology. GRs were present in the chloride cells in both the filaments and lamellae, in respiratory cells in the lamellae, in pavement cells, basal layer cells and undifferentiated cells in the filaments. Co-localization of Na(+)/K(+)-ATPase and MT revealed chat MT was expressed in chloride cells, both in filaments and lamellae. Occasionally, MT immunoreactivity was found in pavement cells and in undifferentiated cells. By double staining for Na(+)/K(+)-ATPase and GR, for Na(+)/K(+)-ATPase and MT and for GR and MT, we can conclude that after 5 days of Cu stress there are chloride cells that express GR and MT, GR or MT alone or neither of the two proteins. This apparent functional heterogeneity of branchial chloride cells may reflect a limited window when chloride cell subpopulations show an adaptive response to Cu.

Transcriptional interference between glucocorticoid receptor and estradiol receptor mediates the inhibitory effect of cortisol on fish vitellogenesis.

In oviparous species, the synthesis of vitellogenin (Vg) takes place in the liver according to a strictly estrogen-dependent mechanism that first involves an up-regulation of the estrogen receptor (ER) by its own ligand. However, reports from the literature indicate that in trout stress or cortisol may cause a reduction of cytosolic E2-binding sites in the liver and a decrease in plasma Vg levels. To investigate the mechanisms underlying these effects, in vivo and in vitro experiments were designed in rainbow trout (Oncorhynchus mykiss). The results demonstrate that cortisol implanted into maturing females caused a marked decrease of rainbow trout ER (rtER) and rainbow trout Vg (rtVg) mRNA levels in the liver. In vitro experiments on hepatocyte aggregates also showed that dexamethasone (Dex) caused a strong decrease in the basal and E2-stimulated rtER mRNA and to a lesser extent rtVg mRNA. These effects were specific as no other hormones were able to mimic the inhibitory action of Dex. A study of rtER mRNA stability indicated that the effects of glucocorticoids are likely to take place at the transcriptional level. This was further indicated by transfection experiments in CHO-K(1) cells, which showed that rainbow trout glucocorticoid receptor (rtGR) strongly inhibited the E2-stimulated transcriptional activity of the rtER promoter. Taken together, these results indicate that the rtGR exerts a transcriptional interference on the expression of the rtER that may explain some of the negative effects of stress or cortisol on vitellogenesis.

Glucocorticoid receptor immunoreactivity in neurons and pituitary cells implicated in reproductive functions in rainbow trout: a double immunohistochemical study.

In order to identify the nature of the glucocorticoid receptor (GR)-expressing neurons and pituitary cells that potentially mediate the negative effects of stress on reproductive performance, double immunohistochemical stainings were performed in the brain and pituitary of the rainbow trout (Oncorhynchus mykiss). To avoid possible cross-reactions during the double staining studies, combinations of primary antibodies raised in different species were used, and we report here the generation of an antibody raised in guinea pig against the rainbow trout glucocorticoid receptor (rtGR). The results obtained in vitellogenic females showed that GnRH-positive neurons in the caudal telencephalon/anterior preoptic region consistently exhibited rtGR immunoreactivity. Similarly, in the anterior ventral preoptic region, a group of tyrosine hydroxylase-positive neurons, known for inhibiting gonadotropin (GTH)-2 secretion during vitellogenesis, was consistently shown to strongly express GR. Finally, we show that a large majority of the GTH-1 (FSH-like) and GTH-2 (LH-like) cells of the pituitary exhibit rtGR immunoreactivity. These results indicate that cortisol may affect the neuroendocrine control of the reproductive process of the rainbow trout at multiple sites.

Identification of potential sites of cortisol actions on the reproductive axis in rainbow trout.

The full length cDNA encoding a rainbow trout glucocorticoid receptor (rtGR) has been obtained from rainbow trout liver and intestine libraries. Northern blot analysis showed that the corresponding messengers are detected in the brain of trout with a size 7.5 kb similar to the size of rtGR mRNA in other target tissues. The distribution of the rtGR mRNA and protein was studied in the forebrain of the trout by means of both in situ hybridization and immunohistochemistry and compared with that of the oestrogen receptor (rtER). The GR and ER mRNAs and proteins were detected with a strong overlapping mainly in the: (a) preoptic region; (b) mediobasal hypothalamus; and (c) anterior pituitary, confirming their implication in the neuroendocrine control of pituitary functions. In both diencephalon and pituitary, the peptidergic phenotype of some neuron or cell categories expressing either type of receptors could be determined by double staining. Furthermore, double staining studies have demonstrated colocalization of the two receptors in the same neurons or pituitary cells. The rtER and rtGR were found to be co-expressed in the dopaminergic neurons inhibiting GTH2 secretion and in pituitary cells of the anterior lobe--notably the gonadotrophs. Given that the promoter of the ER gene contains several potential glucocorticoid-responsive elements (GRE) and that cortisol inhibits the oestradiol-stimulated ER expression in the liver, the possibility exists for modulation of ER gene expression by GR in the hypothalamo-pituitary complex. This could explain some of the well documented effects of stress on the reproductive performance in salmonids.

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss).

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH2-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions.

Transcriptional regulation of expression of the rainbow trout albumin gene by estrogen.

Estrogens modulate the expression of many liver-specific genes in oviparous species. For instance, expression of the estrogen receptor and vitellogenin genes is strongly up-regulated by estradiol in rainbow trout liver. Using hepatocyte primary cultures, we demonstrate that trout albumin (Alb) gene is also regulated by this hormone. Indeed, treatment of hepatocytes with 1 microM estradiol led, after 24 h, to a dramatic decrease in Alb mRNA level. To investigate the mechanism of this down-regulation, run-off experiments were performed and mRNA half-lives were determined in the presence and absence of estradiol. The results show that the down-regulation of Alb mRNA expression by estrogens occurs only at the transcriptional level.

Rainbow trout glucocorticoid receptor overexpression in Escherichia coli: production of antibodies for western blotting and immunohistochemistry.

Fragments of cDNA that encode the N-terminal and DNA-binding domains (DBD) of the rainbow trout glucocorticoid receptor (rtGR) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The fusion proteins induced by IPTG could readily be detected as 45- and 40-kDa bands, respectively, in crude extracts, as well as in proteins purified on glutathione-agarose. These purified hybrid proteins were used to immunize rabbits. The antisera produced were tested for specificity by Western blot analysis using extracts from COS-1 cells transfected with an rtGR expression vector and from trout liver cells. The antisera raised against the DBD domain did not detect any bands on Western blots, even at low antiserum dilution. However, the purified DBD fusion protein specifically bound GRE-containing DNA fragments in gel-shift assays, and the retarded complexes were supershifted by these antibodies. The antisera raised against the N-terminal domain consistently detected two protein bands at 104 and 100 kDa in the two cell extracts and allowed specific immunohistochemical staining in fish brain and pituitary. For the first time in fish, these antibodies will allow analysis of GR expression in different cortisol target tissues.

Differential regulation of two genes implicated in fish reproduction: vitellogenin and estrogen receptor genes.

In rainbow trout as well as in other species, variability of estrogen receptor (ER) gene expression according to the cell type and the physiological state reflects a differential cell and gene sensitivity to estrogen. We previously demonstrated that expression of the rainbow trout estrogen receptor (rtER) and vitellogenin (Vg) genes were induced differently by estrogens in rainbow trout liver. Therefore, these two genes offered a suitable model to study the influence of ER concentration on gene transcriptional activities. In the present study we show that the transcription rate of rtER and Vg genes during an estrogenic treatment are affected differently by variation of cellular ER concentration. We demonstrate that rtER gene exhibits a low threshold response to loaded estrogen receptor, and increasing ER amounts do not affect the transcriptional response of this gene during an estrogenic stimulation. On the contrary, Vg gene expression requires the presence of a higher loaded estrogen receptor level to be induced, and its transcriptional response is directly proportional to the amount of synthesised ER.

Distribution and expression of glucocorticoid receptor mRNA in the forebrain of the rainbow trout.

The expression and distribution of glucocorticoid receptor mRNA was studied in the forebrain of mature female and immature undifferentiated rainbow trout (Oncorhynchus mykiss) by means of Northern blotting and in situ hybridization. A single mRNA species of 7.5 kb was detected in mRNA polyA+ prepared from the anterior brain. In situ hybridization was carried out using a 35S-labelled riboprobe corresponding to the A/B-domain (between nucleotides 1224 and 1763) of the recently cloned rainbow trout glucocorticoid receptor cDNA. Comparison of adjacent sections hybridized with the sense and antisense probes allowed detection of a specific signal with a similar distribution pattern in all animals studied. In the telencephalon, a specific hybridization was detected in scattered cells of the dorsal telencephalic hemisphere, but the stronger signal was consistently observed in the dorsal nucleus, and to a lesser degree in the ventral nucleus of the ventral telencephalon. Heavy hybridization staining was consistently observed in all subdivisions of the preoptic nucleus and the nucleus lateralis tuberis, which are the main hypophysiotrophic regions in fish. A weaker signal was detected in the nucleus anterioris periventricularis, nucleus suprachiasmaticus and thalamic region. The presence of a strong signal in virtually all magnocellular neurons of the nucleus preopticus, known for producing vasotocin, isotocin and corticotropin-releasing factor favors a direct function of cortisol in regulating its own secretion.

Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression.

Rainbow trout hepatocyte primary culture was used to test the influence of some xenobiotics on the expression of two genes implicated in reproduction, those for the estrogen implicated in reproduction, those for the estrogen receptor (ER) and vitellogenin (Vg). We showed that chlordecone, nonylphenol, a polychlorobiphenol (PCB) mixture (Aroclor 1245) and lindane were able to induce ER and Vg mRNA accumulation. Antiestrogens, 4-hydroxytamoxifen and ICI 164,384, prevented the effects of the xenobiotics, indicating that the induction of gene expression is mediated by the ER. Among these four xenobiotics, only chlordecone and nonylphenol were able to displace the binding of [3H]estradiol to ER-enriched COS-1 extracts, and to activate an estrogen-dependent reporter gene (ERE-TK-CAT) cotransfected with an expression vector containing ER cDNA. The results suggest that chlordecone and nonylphenol are direct inducers of rainbow trout ER and Vg gene expression, whereas PCBs and lindane act through their hepatic metabolites. Moreover, pentachlorophenol acts as an antagonist of the induction by estradiol of rainbow trout ER and Vg gene expression.

Cloning of a teleost fish glucocorticoid receptor shows that it contains a deoxyribonucleic acid-binding domain different from that of mammals.

In the teleost fish, physiological and biochemical studies suggest that glucocorticoids regulate both salt balance and metabolic activities. In mammals, however, these functions are divided between glucocorticoids and mineralocorticoids. In mammals, separate receptors for these two classes of steroid hormone have been cloned and sequenced. To begin to understand the regulation in fish of the vital processes ascribed to glucocorticoids, we have cloned, sequenced, expressed, and studied the steroid-binding and transcriptional activation capabilities of the rainbow trout (Onchorhynchus mykiss) glucocorticoid receptor. Northern blot analysis shows a single rainbow trout GR messenger RNA species of 7.5 kilobases expressed in gill, intestine, skeletal muscle, kidney, and liver. The trout GR 2274-nucleotide coding sequence provides for a protein of 758 amino acids, with appropriate similarities to mammalian GR, with one striking exception. As in other members of the steroid/thyroid/retinoid receptor family, the DNA-binding domain contains two putative zinc fingers. These have high homology with those of other GRs. However, between the zinc fingers in the trout GR are found 9 more amino acids than are seen in mammalian GRs, raising questions as to the functional form of the fish, as opposed to the mammalian, GR. It has been proposed that as fish appear to use glucocorticoids for both metabolic and salt control, presumably through a single GR, GR would prove to be the evolutionary precursor to mammalian GR and mineralocorticoid receptor (MR). Computer analysis of the known sequences of GRs and MRs, however, suggests that the fish GR did not give rise to the MR of higher animals, but that both subfamilies of receptor arose from some earlier gene.

The activity of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and 5 alpha-reductase, together with androgen levels in male rat pituitary during sexual maturation.

In all subcellular pituitary fractions, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-ol dehydrogenase) activity is high (1 to 3 pmol/mg/h) with NADH or NADPH as cofactor, and 3 beta-hydroxysteroid dehydrogenase (3 beta-ol dehydrogenase) activity much lower. The highest activity of the latter (0.15 pmol/mg/h) is detected in cytosol with NADH as cofactor. During sexual maturation, cytosolic (NADH-dependent) 3 alpha- and 3 beta-ol dehydrogenase activities remain constant, whereas the 5 alpha-reductase activity is maximum at 37 days. The levels of different pituitary androgens were evaluated by radioimmunoassay. At 28 days, testosterone level is 4 ng/g of tissue, then after 42 days the level remains between 4.5 and 6 ng/g at a level higher than the DHT level. In all cases during the maturation of the rat, the different 5 alpha-reduced androgens are in the same ratio: DHT greater than 3 alpha-diol greater than 3 beta-diol, and the sum of these three 5 alpha-reduced androgens decreases between the 28th and the 90th day.

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.

[Evidence of 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol and of 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol in the anterior pituitary of the prepuberal male rate]. / Mise en évidence du 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol et du 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol dans l'hypophyse antérieure du rat mâle prépubère.

Incubation of 3H-5 alpha-androstane-3 beta, 17 beta-diol with anterior pituitaries from immature male Rats led to the major production of polar radio-metabolites. Of these, 53 and 27% were identified with 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol, respectively. The formation of these trihydroxysteroids may be involved in the intracellular regulation of 5 alpha-androstane-3 beta, 17 beta-diol levels.

[Hormonal regulation of 5 alpha-reductase activity in anterior pituitary gland microsomes of the male rat]. / Régulation hormonale de l'activité de la 5 alpha-réductase des microsomes de l'hypophyse antérieure du rat mâle.

It was previously shown that the microsomal 5 alpha-reductase activity in the male rat pituitary was increased by castration. Subcutaneous administration of androgens to castrated rats prevented the rise in 5 alpha-reductase activity. Their relative efficiency was as follows: 5 alpha-dihydrotestosterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than testosterone. Under our experimental conditions 5 alpha-androstane-3 beta, 17 beta-diol and estrogens were inefficient. The rise in 5 alpha-reductase activity following castration is exclusively located in hypophysis and it is probably due to an increased of the enzyme biosynthesis.