Effects of nonylphenol on estrogen receptor conformation, transcriptional activity and sexual reversion in rainbow trout (Oncorhynchus mykiss).

Estrogenic potency of 4-n-nonylphenol diethoxylate, 4-n-nonylphenol (NP) and metabolites were tested using two bioassays: rainbow trout hepatocyte culture and recombinant yeast stably expressing rainbow trout estrogen receptor (rtER) and containing estrogen-dependent reporter genes. Since NP was the only compound active in both systems, its interaction with rtER was studied in more detail. Qualitative and quantitative differences were observed in the presence of 17beta-estradiol (E2) or NP when estrogen-dependent promoters containing one to three estrogen-responsive elements were used in yeast. Moreover, limited proteolysis of rtER after E2 or NP binding presented different patterns after SDS-PAGE analysis suggesting that NP induces a differential conformation of rtER compare to E2. This finding may have important implications with respect to the biological activity of NP. Thus, the effects of NP on the activation of an E2-dependent gene and on sexual differentiation were assessed on all-male trout embryos exposed to NP for 1 h per day for 10 days. Although in situ hybridization demonstrated that E2, and to a lesser extend NP, were able to increase rtER mRNA level in the liver of embryos, no indication of total or partial sexual reversion was observed (even in E2 treated fishes) when the gonads were examined 8 months after hatching.

Function of N-terminal transactivation domain of the estrogen receptor requires a potential alpha-helical structure and is negatively regulated by the A domain.

Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region. These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved. Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function. Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential alpha-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential alpha-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and, interestingly, ERs with an inverted alpha-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures. Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtER(L) and rtER(S), respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor. In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.

The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function.

The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E domains with human estrogen receptor alpha (hER) [NR3A1]. A previous study has shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae. To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D or E/F regions of rtER were replaced by corresponding regions of hER and expressed in yeast cells. Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D domains play an important role in the magnitude of transactivation of ER. Two other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affect hybrid receptor activities. Moreover, a chimeric hER carrying the C domain of rtER showed maximal transcriptional activity similar to that observed with rtER. Gel shift assays showed that, whereas rtER and hER present a similar binding affinity to an estrogen response element (ERE) element, the rtER C domain is responsible for a weaker DNA binding stability compared to those of hER. In addition, the human C domain allows approximately 2 times faster association of ER to an ERE. Utilization of reporter genes containing one or three EREs confirms that rtER requires protein-protein interactions for its stabilization on DNA and that the C domain is involved in this stabilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs. Interestingly, although E domains of these two receptors are much less conserved, replacement of this domain in rtER by its human counterpart resulted in higher estradiol sensitivity but no increase in the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcription in the presence of estradiol similar to both AF-1 and AF-2 hER. This implies that these domains, which show poor sequence homology, may interact with similar basal transcription factors.

Inhibition of rainbow trout (Oncorhynchus mykiss) estrogen receptor activity by cadmium.

This study was conducted to determine if the cadmium-mediated inhibition of vitellogenesis observed in fish collected from contaminated areas or undergoing experimental exposure to cadmium correlated with modification in the transcriptional activity of the estrogen receptor. A recombinant yeast system expressing rainbow trout (Oncorhynchus mykiss) estradiol receptor or human estradiol receptor was used to evaluate the direct effect of cadmium exposure on estradiol receptor transcriptional activity. In recombinant yeast, cadmium reduced the estradiol-stimulated transcription of an estrogen-responsive reporter gene. In vitro-binding assays indicated that cadmium did not affect ligand binding to the receptor. Yeast one- and two-hybrid assays showed that estradiol-induced conformational changes and receptor dimerization were not affected by cadmium; conversely, DNA binding of the estradiol receptor to its cognate element was dramatically reduced in gel retardation assay. This study provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter through a direct effect on estradiol receptor transcriptional activity and may affect a number of estrogen signaling pathways.

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.

Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene.

A characteristic of all estrogen receptors (ER) cloned from fish to date is the lack of the first 37-42 N-terminal amino acids specific to the A domain. Here we report the isolation and characterization from trout ovary of a full-length complementary DNA (cDNA) clone encoding an N-terminal variant form of the rainbow trout ER (rtER). Sequence analysis of open reading frame of this cDNA predicts a 622-amino acid protein. The C-terminal region of this protein, from amino acid position 45 to the end, was very similar to the previously reported rtER (referred to as the short form, or rtER(S)). In contrast, this novel rtER cDNA (referred to as the long form, or rtER(L)) contains an additional in-frame ATG initiator codon that adds 45 residues to the N-terminal region of the protein. This new N-terminal region may represent the A domain of ER found in tetrapod species. The first 227 bp of this new cDNA were similar to the 3'-end intronic sequence of the rtER gene intron 1. These data together with S1 nuclease, primer extension, and RT-PCR experiments demonstrate that the rtER(L) represents a second isoform of rtER that arises from an alternative promoter within the first intron of the gene. Transcripts encoding both rtER forms were expressed in the liver. In vitro translation of the rtER(L) cDNA produced 2 proteins with molecular masses of 71 and 65 kDa, whereas rtER(S) cDNA produced 1 65-kDa protein. Interestingly, Western blot analysis with a specific antibody against the C-terminal region of rtER revealed 2 receptor forms of 65 and 71 kDa in trout liver nuclear extracts, in agreement with the presence of the 2 distinct classes of rtER messenger RNA in this tissue. Functional analysis of both rtER isoforms revealed that although rtER(S) consistently exhibited a basal (estrogen-independent) trans-activation activity that could be further increased in the presence of estrogens, the novel isoform rtER(L) is characterized by a strict estrogen-dependent transcriptional activity. These data suggest that the additional 45 residues at the N-terminal region of rtER(L) clearly modify the hormone-independent trans-activation function of the receptor.

Cloning and sequencing of the gilthead sea bream estrogen receptor cDNA.

We report here the complete nucleotide sequence of a cDNA clone containing the full-coding sequence of the Sparus aurata estrogen receptor (ER) isolated from an expression library prepared from gilthead sea bream liver poly A+ RNA. The library was screened using a single strand rainbow trout ER cDNA probe, corresponding to the C-D domain. The cDNA sequence containing an insert of 2369 nucleotides was found to encode a protein of 579 amino acids. The 5'- and 3'-untranslated regions of the message are 186 and 392 nucleotides long, respectively. The gilthead sea bream ER shows the higher homology with the ER of another perciform, Chrysophrys major (93%), moderate to high homology with Oreocromis aureus (78%) medaka (77%) and rainbow trout (70.7%) ERs and lower homology with japanese eel (45%), amphibian (47%), avian (48.5%) and mammalian (47-47.5%) ERs. The sequence homologies and phylogenetic analysis of the various ERs suggest that gilthead sea bream ER should be considered as a ER alpha-like.

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

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.

The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation.

The rainbow trout estrogen receptor (rtER) is a positively autoregulated gene in liver cells. In a previous report, we showed that upregulation is mediated by an estrogen response element (ERE) located in the proximal promoter of the gene and that a half binding site for nuclear receptors (5'-TGACCT-3') located 15 bp upstream of the ERE is involved in the magnitude of the estrogen response. We now report that the human orphan receptor COUP-TF and a COUP-TF-like protein from trout liver are able to bind to the consensus half-site. When cotransfected with the rtER gene proximal promoter, COUP-TF had no regulatory functions on its own. Interestingly, COUP-TF enhanced rtER transactivation properties in the presence of estradiol in a dose-dependent manner when cotransfected with the rtER gene promoter. Unliganded retinoid receptor heterodimers had the same helper function as COUP-TF in the presence of estradiol but were switched to repressors when the ligand all-trans-retinoic acid was added. Mutation of the consensus half-site only slightly reduced COUP-TF helper function, suggesting that it actually results from a complex mechanism that probably involves both DNA binding of COUP-TF to the promoter and protein-protein interaction with another transcription factor bound to the promoter. Nevertheless, a DNA-binding-defective mutant of COUP-TF was also defective in ER helper function. Competition footprinting analysis suggested that COUP-TF actually establishes contacts with the consensus upstream half-site and the downstream ERE half-site that would form a DR-24-like response element. Interaction of COUP-TF with the DR-24 element was confirmed in footprinting assays by using nuclear extracts from Saccharomyces cerevisiae expressing COUP-TF. Finally, interaction of COUP-TF with mutants of the rtER gene promoter showed that COUP-TF recognizes the ERE when the upstream half-site is mutated. These data show that COUP-TF may activate transcription through interaction with other nuclear receptors. This cross-talk between liganded nuclear receptors and orphan receptors is likely to modulate the spectrum of action of a particular ligand-receptor complex and may participate in the cell-type specificity of the ligand effect.

Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen.

RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.

Two complementary bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout estrogen receptor and trout hepatocyte cultures.

A relation between the chemical structure of a xenobiotic and its steroidal action has not yet been clearly established. Thus, it is not possible to define the estrogenic potency of different xenobiotics. An assessment may be accomplished by the use of different bioassays. We have previously developed a yeast system highly and stably expressing rainbow trout estrogen receptor (rtER) in order to analyze the biological activity of the receptor. The recombinant yeast system appears to be a reliable, rapid and sensitive bioassay for the screening and determination of the direct interaction between ER and estrogenic compounds. This system was used in parallel with a more elaborate biological system, trout hepatocyte aggregate cultures, to examine the estrogenic potency of a wide spectrum of chemicals commonly found in the environment. In hepatocyte cultures, the vitellogenin gene whose expression is principally dependent upon estradiol was used as a biomarker. Moreover, competitive binding assays were performed to determine direct interaction between rtER and xenobiotics. In our study, 50% of the 49 chemical compounds tested exhibited estrogenic activity in the two bioassays: the herbicide diclofop-methyl; the fungicides biphenyl, dodemorph, and triadimefon; the insecticides lindane, methyl parathion, chlordecone, dieldrin, and endosulfan; polychlorinated biphenyl mixtures; the plasticizers or detergents alkylphenols and phthalates; and phytoestrogens. To investigate further biphenyl estrogenic activity, its principal metabolites were also tested in both bioassays. Among these estrogenic compounds, 70% were able to activate rtER in yeast and hepatocytes with variable induction levels according to the system. Nevertheless, 30% of these estrogenic compounds exhibited estrogenic activity in only one of the bioassays, suggesting the implication of metabolites or different pathways in the activation of gene transcription. This paper shows that it is important to combine in vivo bioassays with in vitro approaches to elucidate the mechanism of xenoestrogen actions.

Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression.

Estrogen receptor (ER) and vitellogenin (Vg) gene expression are strongly up-regulated by estrogens in rainbow trout liver. In this paper, we have used primary cultured hepatocytes to examine the mechanisms implicated in estrogen regulation of ER and Vg gene expression. Treatment of hepatocytes with 1 microM estradiol (E2) led to a rapid increase in ER and mRNA level (15 fold) followed by Vg and mRNA induction. Transcription rate and mRNA half-life determination carried out in the presence or absence of E2, demonstrated that E2 increases both the ER and Vg gene transcriptional activity and mRNA stability (ca. 3 fold). The effect of E2 was inhibited by an excess of antiestrogen, showing that E2-stimulation of ER and mRNA level is mediated by the estrogen receptor. Our data show that ER and Vg genes have different hormonal sensitivity. In fact, the Vg gene required a higher concentration of E2 to be stimulated compared to the ER gene. Examination of the mechanisms involved in post-transcriptional regulation of ER mRNA showed that the setting up and maintenance of this regulation process implies that estrogen receptor and the general translational activity within the cells, suggesting that ER mRNA depends on the synthesis of an estrogen-dependent protein. However, the cis and trans elements involved in E2-stabilization process remain to be identified.

Cooperation between the human estrogen receptor (ER) and MCF-7 cell-specific transcription factors elicits high activity of an estrogen-inducible enhancer from the trout ER gene promoter.

The human estrogen receptor (hER) is expressed in breast cancer MCF-7 cells and plays a major role in tumorigenic processes. In this report, we demonstrate that MCF-7-specific factors can cooperate with the hER to increase its transactivation activity. We previously demonstrated that the rainbow trout ER (rtER) gene is up-regulated by the rtER protein itself, through an enhancer that contains an imperfect estrogen-responsive element (FP1 area). By performing footprinting experiments, we have delineated two other regulatory regions (FP2 and FP3 areas) in the 0.2-kb enhancer. We show, by transient transfections, that hER poorly transactivates this enhancer in CHO-K1 and Ishikawa cells whereas, in MCF-7 cells, transcriptional activation occurs at a level about 20-fold higher than when the enhancer estrogen-responsive element (FP1) is the only regulatory region included in the reporter gene. These results indicate that areas other than FP1 are important regulatory sites of this enhancer. Site-directed mutagenesis demonstrated that the FP1 area is absolutely necessary for induction by estradiol as well as for basal activity of this enhancer in MCF-7 cells. Gel shift experiments showed that MCF-7 cells contain a factor that binds to the FP3 area and is poorly expressed in all other tested cell lines. As suggested by site-directed mutagenesis and deletion experiments, this FP3-binding protein may enhance the hER transactivation ability in MCF-7 cells. These data reinforce the idea that cell-specific transcription factors cooperate with steroid receptors to achieve maximal induction of hormone-responsive genes.

Characterization of the transcription start point of the trout estrogen receptor-encoding gene: evidence for alternative splicing in the 5' untranslated region.

The estrogen receptor (ER)-encoding gene (ER) regulates many genes implicated in the reproductive functions. Moreover, rainbow trout ER (rtER) is itself up-regulated by its own product. We have used Northern blot, RNase protection, primer extension and reverse transcription-polymerase chain reaction (RT-PCR) to study the position of the rtER mRNA transcription start point (tsp) in liver. This analysis has revealed the presence of a tsp positioned at the beginning of the cloned rtER cDNA. Functionality of this tsp was tested in transient transfections in CHO-K1 cells. The characterization of the rtER 5' untranslated region (UTR) showed that two transcripts exist in liver which differ in their 5'-UTR. The first one is 100% homologous to the cloned rtER cDNA sequence. The other one contains a 41-bp insertion. The isolation and sequencing of the first intron showed that this insertion arises from alternative splicing, due to the use of a splicing site internal to the first intron.

Preliminary evidence suggesting variations of GtH 1 and GtH2 mRNA levels at different stages of gonadal development in rainbow trout, Oncorhynchus mykiss.

To study the variations of alpha and beta GtH 1 and 2 gene expression during gonadal development in male and female rainbow trout, chum salmon alpha and beta GtH 1 and 2 probes were used. The alpha subunit cDNA probe used is identical to the cDNA encoding the alpha subunit common to both GtH 1 and 2. Total RNAs were prepared from pooled pituitaries and the validation of the use of these probes for studying the variation of GtH mRNAs was made by Northern blot analysis. The quantitative determination of GtH mRNAs employed slot blot hybridization. In males and females, beta GtH 1 predominates in early stages of gonadal development (spermatogonia A and previtellogenesis), beta GtH 2 being weakly expressed. Both beta GtH 1 and beta GtH 2 are expressed during prespermiation, spermiation, and the periovulatory period with a predominance of beta GtH 2. In both sexes alpha GtH variations follow beta GtH 2 variations.

Differential functional activities of rainbow trout and human estrogen receptors expressed in the yeast Saccharomyces cerevisiae.

The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Saccharomyces cerevisiae, was used to analyse the biological activity of the receptor. The rtER mRNA encoded a 65-kDa protein which was immunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (Kd = 1.35 nM) was very similar to the value measured from trout liver extracts but 3-5-fold higher than the Kd found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor. While the hER Kd remained unchanged at both 4 degrees C or 22 degrees C, it was slightly modified at 30 degrees C. The Kd measured for rtER at 22 degrees C and 30 degrees C were about 2-fold, and 12-fold higher, respectively, than the Kd obtained at 4 degrees C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrogen-receptor-mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used. The reporter genes consist of one, two, or three copies of estrogen-responsive elements (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for beta-galactosidase. The induction of beta-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activation of transcription mediated by rtER responded in an estradiol-dose-dependent manner as in animal cells. However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10-fold higher. This is probably a consequence of the lower estradiol-affinity for rtER compared to hER. The levels of induction of the reporter genes containing two or three ERE were strongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect previously described for multiple ERE. The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used. In this case transcriptional activation indicated by rtER was 10-20 fold lower. This suggests that rtER requires protein/protein interaction for its stabilization on DNA. Antiestrogens were able to bind rtER and promote gene transcription. However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required. This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery.