Farzad Pakdel

Aromatase in the brain of teleost fish: expression, regulation and putative functions.

Unlike that of mammals, the brain of teleost fish exhibits an intense aromatase activity due to the strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. In situ hybridization, immunohistochemistry and expression of GFP (green fluorescent protein) in transgenic tg(cyp19a1b-GFP) fish demonstrate that aromatase B is only expressed in radial glial cells (RGC) of adult fish. These cells persist throughout life and act as progenitors in the brain of both developing and adult fish. Although aromatase B-positive radial glial cells are most abundant in the preoptic area and the hypothalamus, they are observed throughout the entire central nervous system and spinal cord. In agreement with the fact that brain aromatase activity is correlated to sex steroid levels, the high expression of cyp19a1b is due to an auto-regulatory loop through which estrogens and aromatizable androgens up-regulate aromatase expression. This mechanism involves estrogen receptor binding on an estrogen response element located on the cyp19a1b promoter. Cell specificity is achieved by a mandatory cooperation between estrogen receptors and unidentified glial factors. Given the emerging roles of estrogens in neurogenesis, the unique feature of the adult fish brain suggests that, in addition to classical functions on brain sexual differentiation and sexual behaviour, aromatase expression in radial glial cells could be part of the mechanisms authorizing the maintenance of a high proliferative activity in the brain of fish.

Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.

Compared with other vertebrates, the brain of adult teleost fish exhibits two unique features: it exhibits unusually high neurogenic activity and strongly expresses aromatase, a key enzyme that converts aromatizable androgens into estrogens. Until now, these two features, high neurogenic and aromatase activities, have never been related to each other. Recently, it was shown that aromatase is expressed in radial glial cells of the forebrain and not in neurons. Here, we further document that Aromatase B is never detected in cells expressing the markers of postmitotic neurons, Hu and acetylated tubulin. By using a combination of bromodeoxyuridine (BrdU) treatment and immunohistochemical techniques, we demonstrate for the first time to our knowledge that aromatase‐positive radial cells actively divide to generate newborn cells in many forebrain regions. Such newborn cells can further divide, as shown by BrdU‐proliferating cell nuclear antigen double staining. We also demonstrate that, over time, newborn cells move away from the ventricles, most likely by migrating along the radial processes. Finally, by using antisera to Hu and acetylated tubulin, we further document that some of the newborn cells derived from radial glia differentiate into neurons. These data provide new evidence for the mechanism of neurogenesis in the brain of adult fish. In addition, given that estrogens are well‐known neurotrophic and neuroprotective factors affecting proliferation, apoptosis, migration, and differentiation, the expression of aromatase in the neural stem cells of the adult strongly demonstrates that the fish brain is an outstanding model for studying the effects of estrogens on adult neurogenesis and brain repair. J. Comp. Neurol. 501:150–167, 2007.

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.

In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout

We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.

A Dynamic Structural Model for Estrogen Receptor-α Activation by Ligands, Emphasizing the Role of Interactions between Distant A and E Domains

The functional interplay between different domains of estrogen receptor-alpha (ERalpha, NR3A1) is responsible for the overall properties of the full-length protein. We previously identified an interaction between the N-terminal A and C-terminal domains, which we demonstrate here to repress ligand-independent transactivation and transrepression abilities of ERalpha. Using targeted mutations based on ERalpha structural models, we determine the basis for this interaction that defines a regulatory interplay between ERalpha A domain, corepressors, and ERalpha Helix 12 for binding to the same C-terminal surface. We propose a dynamic model where binding of different ligands influences the A/D-F domain interaction and results in specific functional outcomes. This model gives insights into the dynamic properties of full-length ERalpha and into the structure of unliganded ERalpha.

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. Mol. Reprod. Dev. 48:317–323, 1997.

Inhibition of Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor Activity by Cadmium

Abstract 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.

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin.

SummaryThe complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.

Rainbow trout estrogen receptor presents an equal specificity but a differential sensitivity for estrogens than human estrogen receptor.

The estrogen receptor is a transcription factor that mediates the actions of estrogens which plays a major role in salmonid vitellogenesis. Previously, we cloned and described the estradiol receptor in rainbow trout. To study the transactivation characteristics, the full length cDNA was inserted in an expression vector and tested by transfection on an estrogen-dependent promoter (pERE-TK-CAT). For the first time, direct comparison between the trout receptor (rtER) and human receptor (hER) in an analogous system has been possible. We demonstrate that rtER can, like hER, transactivate transcription in a strictly hormone-dependent manner. The specificity and sensitivity of the rtER response to different steroids have been studied. With rtER, the E2 concentration needed for half maximal activation is 10 times higher than with hER. In addition, we show that rtER has a weak affinity for androgens and transactivation could be induced using high testosterone concentration. Experiments show that both rtER and hER have an equal specificity for estrogens but that the rtER binds its ligand with a lower sensitivity than hER. Several hypotheses concerning the role of main amino acids within the receptor protein are proposed based on the different properties of the receptors and comparison of sequences.

Androgens Upregulate cyp19a1b (Aromatase B) Gene Expression in the Brain of Zebrafish (Danio rerio) Through Estrogen Receptors

Abstract The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects.