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.

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.

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.

One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides.

POMC is the precursor for a number of biologically active peptides such as ACTH, alpha-MSH, beta-MSH, and beta-endorphin. It is well known that some of these peptides, especially beta-endorphin, are involved in the regulation of reproductive functions in mammals. In order to investigate the possible role of POMC-derived peptides in the control of fish reproduction, we have cloned and sequenced two different trout POMC cDNAs called POMC A and POMC B. These cDNAs exhibited limited sequence homology (44%). The deduced amino acid sequences also showed weak similarity (43%), despite the high conservation of some peptide sequences (alpha-MSH, beta-MSH, and beta-endorphin). The POMC A coding sequence exhibited an unusual length, generating the longest endorphin ever sequenced. The long carboxy-terminal part of the beta-endorphin A contained three potential dibasic cleavage sites, allowing the occurrence of three new peptides: EQWGREEGEE, ALGE, and YHFQG. Using in situ hybridization, we found that the two POMC genes were expressed in the same pituitary cells. POMC A mRNA was the only one detectable in the hypothalamus of sexually inactive fish, whereas the two POMC genes were expressed in the hypothalamus of sexually active fish. These results indicate that two functional POMC genes are present in the rainbow trout. In POMC neurons, the expression of the POMC B gene is likely to be under the control of sexual steroids.

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.

Identification and estrogen induction of two estrogen receptors (ER) messenger ribonucleic acids in the rainbow trout liver: sequence homology with other ERs.

The estrogen-binding region of the cDNA for chicken ER reveals a mRNA of 3.5 kilobases (kb) in rainbow trout liver. The level of this messenger, which is very low in the liver of naive male animals, can be increased by estrogen stimulation. With this chicken probe, we have isolated a clone from a lambda gt10 trout liver cDNA library. The partial cDNA sequence, which encompasses most of the coding region, shows two domains of striking amino acid homology with human, avian, and Xenopus estrogen receptors (ERs) (DNA binding region: 90%, Hormone binding region: 60%). With this specific probe rainbow trout ER, we detected another messenger (4.5 kb) that is less expressed than the 3.5 kb messenger. The kinetics of stimulation of the two messengers is compared with the kinetics of accumulation of vitellogenin mRNA after E2 administration. This report constitutes the first identification of ER mRNA from a fish.

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 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.

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.

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.

[Characterization of specific receptors for estradiol, induction of vitellogenin and its mRNA in the liver of rainbow trout (Salmo gairdnerii)]. / Caractérisation de récepteurs spécifiques à l'oestradiol, induction de la vitellogénine et de son mRNA dans le foie de truite arc-en-ci el (Salmo gairdnerii).

In all egg laying vertebrates, synthesis and use of vitellogenin (Vg) are intimately bound to the active phase of reproduction. In the liver of the rainbow trout (Salmo gairdnerii), Vg synthesis is influenced by estradiol (E2) which, we believe, acts through the classical mechanism of steroid hormone action. After binding of the hormone to a soluble specific receptor protein, the estradiol-receptor complex can interact with chromatin and modulate the expression of Vg genes, leading to increased synthesis of specific mRNA and Vg. We show here: (i) the presence of specific oestrogen receptors (dissociation constant KD congruent to 1.5 X 10(-9) M for E2) in the cytosol of the male trout liver. (ii) The male liver, offering, an ideal experimental control of "zero" background, we followed-in the liver of male trout--the kinetics of induction of Vg mRNA by hybridization with Vg cDNA, after E2 stimulation, and (iii) the apparition of Vg in the serum by using an original rocket immuno-electrophoretic technique. The male trout liver vitellogenin model and the original techniques we developed will be very useful to study the influence of endogenous and exogenous factors on the different steps (receptors, transcription, translation) of vitellogenesis regulation.

Early increases in RNA polymerase I activity and 18S and 28S rRNA synthesis in the male rat pituitary after oestradiol treatment.

This article reports the effect of a single injection of 17 beta-oestradiol on RNA synthesis, in the male rat pituitary. An increase in RNA polymerase I activity, with a maximum effect between 10 and 15 hours, is described. No modification in RNA polymerase II activity was detected. These results were extended and confirmed, using in vitro double labelling of RNA, following in vivo oestrogen treatment. Polyacrylamide gel electrophoresis of nuclear and cytoplasmic RNA showed an increased incorporation of adenine into 28S and 18S rRNA, in the pituitaries of oestrogen-treated animals. The 5S rRNA was not modified by the hormonal treatment. These effects on RNA polymerase I activity and on 28S and 18S rRNA synthesis were closely correlated with the long-term nuclear retention of receptor-oestradiol complexes, in vivo. Taken together, these observations argue in favor of the nucleolus as a preferential target for receptor-bound oestradiol, in the cell nucleus of the male rat pituitary.

Effect of in vivo oestradiol treatment on cell-free transcription in trout liver nuclear extracts.

In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potential of trout liver nuclear extracts in a cell-free transcription system. As reporter genes, we used DNA sequences without G (G-free cassettes) under the control of three promoters derived from the 5' flanking sequence of the Xenopus vitellogenin B1 gene; two of them were responsive to the oestrogen receptor (ER) through oestrogen responsive elements (ERE). Maximal transcriptional activity was obtained within a range of 40-130 micrograms protein per extract depending on the extract preparation. Transcription was maximal in reactions carried out at 25 degrees C. Similar transcriptional activities for the three promoters were observed when transcription was performed in extracts from untreated male trout. In contrast, we observed a 4.5- to 6-fold increase in the transcription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradiol-treated male trout were used. This effect was correlated with the increase in the nuclear ER concentration induced by in vivo hormonal treatment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor. These data demonstrated, therefore, that transcription was ERE-dependent in this system and suggest strongly that it was mediated by the trout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxifen or ICI 164384 had no effect on the transcriptional activity of the two ERE-containing promoters, indicating that transcription was hormone-independent in trout liver nuclear extracts.

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.

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.

Effect of somatostatin on prolactin in rainbow trout (Oncorhynchus mykiss) pituitary cells in primary culture.

To study the control of prolactin secretion in fish, an in-vitro technique using a monolayer cell culture system of rainbow trout pituitary glands was developed. Such secretion was characterized by measurement of both prolactin release and prolactin mRNA content using a trout prolactin cDNA as a probe. This cell culture technique, already used to study the regulation of gonadotrophin secretion in rainbow trout, was further validated by measuring total DNA and protein content. Both parameters appeared to be stable after 2 days of culture. Studying the effect of somatostatin (SRIF) on prolactin cells indicated that a maximal inhibitory effect (62%) was observed after 24 h of treatment. Significant inhibition of prolactin release was obtained for SRIF doses ranging from 50 nM to 1 microM. However, in the same experiment, SRIF was much more potent as an inhibitor of growth hormone release. Short-term (< 12 h) incubation with SRIF did not induce a significant change in prolactin release, whereas growth hormone release was reduced at as early as 1 h after SRIF exposure. SRIF did not have a significant effect on total prolactin content or prolactin mRNA levels, suggesting the absence of an effect on prolactin synthesis. No increase in the magnitude of the inhibitory effect of SRIF was observed when using pituitary cells from immature, mature male or mature female trout. When comparing effects on primary cultures containing cells from the whole pituitary with a prolactin cell-enriched population, SRIF appeared to have the same inhibitory effect on prolactin release, supporting a direct action of SRIF on prolactin cells. These results provide further support for SRIF being a prolactin-inhibiting factor in rainbow trout and acting as a modulator of a dominant stimulatory control of prolactin release.

Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene.

We previously reported that the expression of the rainbow trout estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5' flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized an estrogen-responsive element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This element confers estrogen responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0.2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting element involved in the regulation of the gene by estrogen.

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.

Demonstration of a cytoplasmic receptor protein for oestrogen in the canine prostate gland.

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid--receptor complex was found to have a sedimentation coefficient of 4--5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3.8 +/- 1.53 (S.D.) X 10(-10) mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

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.