Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17ß-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17ß-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish.

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERß1 or zfERß2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain.

Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA). The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4- or 7-day post-fertilization (dpf) zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B), expressed in the brain, using three different in situ/in vivo strategies: (1) Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols; (2) Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus); and (3) Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay) at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF, or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα). Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP) did not show estrogenic activity in our model.

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

Steroid modulation of neurogenesis: Focus on radial glial cells in zebrafish.

Estrogens are known as steroid hormones affecting the brain in many different ways and a wealth of data now document effects on neurogenesis. Estrogens are provided by the periphery but can also be locally produced within the brain itself due to local aromatization of circulating androgens. Adult neurogenesis is described in all vertebrate species examined so far, but comparative investigations have brought to light differences between vertebrate groups. In teleost fishes, the neurogenic activity is spectacular and adult stem cells maintain their mitogenic activity in many proliferative areas within the brain. Fish are also quite unique because brain aromatase expression is limited to radial glia cells, the progenitor cells of adult fish brain. The zebrafish has emerged as an interesting vertebrate model to elucidate the cellular and molecular mechanisms of adult neurogenesis, and notably its modulation by steroids. The main objective of this review is to summarize data related to the functional link between estrogens production in the brain and neurogenesis in fish. First, we will demonstrate that the brain of zebrafish is an endogenous source of steroids and is directly targeted by local and/or peripheral steroids. Then, we will present data demonstrating the progenitor nature of radial glial cells in the brain of adult fish. Next, we will emphasize the role of estrogens in constitutive neurogenesis and its potential contribution to the regenerative neurogenesis. Finally, the negative impacts on neurogenesis of synthetic hormones used in contraceptive pills production and released in the aquatic environment will be discussed.

Aromatase, estrogen receptors and brain development in fish and amphibians.

Estrogens affect brain development of vertebrates, not only by impacting activity and morphology of existing circuits, but also by modulating embryonic and adult neurogenesis. The issue is complex as estrogens can not only originate from peripheral tissues, but also be locally produced within the brain itself due to local aromatization of androgens. In this respect, teleost fishes are quite unique because aromatase is expressed exclusively in radial glial cells, which represent pluripotent cells in the brain of all vertebrates. Expression of aromatase in the brain of fish is also strongly stimulated by estrogens and some androgens. This creates a very intriguing positive auto-regulatory loop leading to dramatic aromatase expression in sexually mature fish with elevated levels of circulating steroids. Looking at the effects of estrogens or anti-estrogens in the brain of adult zebrafish showed that estrogens inhibit rather than stimulate cell proliferation and newborn cell migration. The functional meaning of these observations is still unclear, but these data suggest that the brain of fish is experiencing constant remodeling under the influence of circulating steroids and brain-derived neurosteroids, possibly permitting a diversification of sexual strategies, notably hermaphroditism. Recent data in frogs indicate that aromatase expression is limited to neurons and do not concern radial glial cells. Thus, until now, there is no other example of vertebrates in which radial progenitors express aromatase. This raises the question of when and why these new features were gained and what are their adaptive benefits. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Effects of estradiol in adult neurogenesis and brain repair in zebrafish.

The brain of the adult teleost fish exhibits intense neurogenic activity and an outstanding capability for brain repair. Remarkably, the brain estrogen-synthesizing enzyme, aromatase B, is strongly expressed, particularly in adult fishes, in radial glial cells, which act as progenitors. Using zebrafish, we tested the hypothesis that estrogens affect adult neurogenesis and brain regeneration by modulating the neurogenic activity of radial glial cells. To investigate this, the estrogenic environment was modified through inhibition of aromatase activity, blockade of nuclear estrogen receptors, or estrogenic treatments. Estrogens significantly decreased cell proliferation and migration at the olfactory bulbs/telencephalon junction and in the mediobasal hypothalamus. It also appears that cell survival is reduced at the olfactory bulbs/telencephalon junction. We also developed a model of telencephalic lesion to assess the role of aromatase and estrogens in brain repair. Proliferation increased rapidly immediately after the lesion in the parenchyma of the injured telencephalon, while proliferation at the ventricular surface appeared after 48 h and peaked at 7 days. At this time, most proliferative cells express Sox2, however, none of these Sox2 positive cells correspond to aromatase B-positive radial glial cells. Interestingly, aromatase B expression was significantly reduced 48 h and 7 days after the injury, but surprisingly, at 72 h after lesion, aromatase B expression appeared de novo expressed in parenchyma cells, suggesting a role for this ectopic expression of aromatase in brain repair mechanisms. Altogether these data suggest that estrogens modulate adult, but not reparative neurogenesis, in zebrafish.

Nuclear progesterone receptors are up-regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish.

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation.

The brain of teleost fish, a source, and a target of sexual steroids.

Neurosteroids are defined as steroids de novo synthesized in the central nervous system. While the production of neurosteroids is well documented in mammals and amphibians, there is less information about teleosts, the largest group of fish. Teleosts have long been known for their high brain aromatase and 5α-reductase activities, but recent data now document the capacity of the fish brain to produce a large variety of sex steroids. This article aims at reviewing the available information regarding expression and/or activity of the main steroidogenic enzymes in the brain of fish. In addition, the distribution of estrogen, androgen, and progesterone nuclear receptors is documented in relation with the potential sites of production of neurosteroids. Interestingly, radial glial cells acting as neuronal progenitors, appear to be a potential source of neurosteroids, but also a target for centrally and/or peripherally produced steroids.

Sexual dimorphism in the brain aromatase expression and activity, and in the central expression of other steroidogenic enzymes during the period of sex differentiation in monosex rainbow trout populations.

Using genetic monosex male and female rainbow trout populations, the potential sex differences in the central expression of estrogen receptors (esr1, esr2a, esr2b), brain aromatase (cyp19a1b) and some other steroidogenic enzymes was studied over the period of sex differentiation (from 35 to 63 dpf: days post-fertilization) using quantitative polymerase chain reaction (q-PCR). In addition, aromatase activity was evaluated during this period. The results indicated that brain aromatase (cyp19a1b) expression and activity showed a clear and significant sexually dimorphic pattern with higher levels in male brain between 35 and 53 dpf before the time of gonad morphological differentiation. At that time the expression of a key enzyme involved in the conversion of cholesterol into steroids, the cyp11a1 (p450scc), as well as the estrogen receptors were also sexually dimorphic. The dimorphism was lost from 56 dpf onwards. Transcription factors such as nr5a1b (sf1) and nr0b1 (dax1), but not foxl2a were also higher in males than in females. These results demonstrate that, before or during the early period of morphological gonad differentiation, the brain exhibits a clear sexual dimorphism with respect to the expression and activity of aromatase as well as of certain enzymes and factors involved in steroid synthesis as p450scc and sf1. The results suggest a higher potentiality to produce estrogens by male brains during sex differentiation time.

Aromatase, brain sexualization and plasticity: the fish paradigm.

In contrast to mammals, teleost fish have a very labile genetic sex determination. Sex differentiation is influenced by a combination of hormonal, social and environmental factors and teleost fishes exhibit many examples of hermaphroditism. This means that the brain of fish is not irreversibly sexualized early in life. This review aims at highlighting some unique features of fish that may explain their brain sexual plasticity. Unlike mammals, in which brain aromatase activity decreases after birth, adult teleosts exhibit an intense aromatase activity due to strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. Interestingly, 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. In agreement with the fact that brain aromatase activity is correlated with sex steroid levels, the high expression of cyp19a1b is due to an autoregulatory loop through which estrogens and aromatizable androgens upregulate aromatase expression. Given the well-established roles of estrogens and aromatase on brain sexualization, these features suggest that the brain of fish conserves properties of embryonic mammalian brain throughout life - high neurogenic activity and high aromatase expression in progenitor cells correlated with sex steroid levels. The permanent dialogue between the brain and the gonad would permit sex changes and thus the emergence of a variety of reproductive strategies. Other hypotheses are also discussed.

Cxcr4 and Cxcl12 expression in radial glial cells of the brain of adult zebrafish.

Unlike that of mammals, the brain of adult teleost fish exhibits an intense and widespread neurogenic activity as a result of the persistence of radial glial cells acting as neural progenitors throughout life. Because chemokines, notably CXCL12, and their receptors, such as CXCR4, play key roles in mammalian embryonic neurogenesis, we investigated Cxcr4 and Cxcl12 expressions in the brain of adult zebrafish and their potential relationships with cell proliferation. Cxcr4 expression was found to be restricted to radial glial cells in the adult zebrafish, where it is co-expressed with established radial glial cell markers, such as brain lipid-binding protein (Blbp) or the estrogen-synthesizing enzyme aromatase B (Cyp19a1b). Double stainings combining proliferating cell nuclear antigen (PCNA) and Cxcr4 immunolabelling indicated that there is no obvious association between Cxcr4 expression and radial glial cell proliferation. Interestingly, cxcl12a messengers were detected in ventricular regions, in cells corresponding to aromatase B-immunoreactive radial glial cells. Altogether, our data demonstrate Cxcl12 and Cxcr4 expression in radial glial cells of the brain of adult zebrafish, supporting important roles for the Cxcl12/Cxcr4 pair in brain development and functioning.

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.

[Oestrogens and neurogenesis: new functions for an old hormone. Lessons from the zebrafish]. / Oestrogènes et neurogenèse : de nouvelles fonctions pour une vieille hormone. Leçons tirées du poisson zèbre.

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fish exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

A cyp19a1b-gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells.

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens.

Suicidal ideation among Viennese high school students.

OBJECTIVE: Suicidality spans a spectrum ranging from suicidal thoughts to suicidal acts, and suicidal ideation is more prevalent in youth populations, suggesting important developmental issues. We assessed the prevalence of lifetime suicidal ideation and associated psychosocial factors among Viennese high school students. METHOD: An anonymous self-report survey assessing demographic characteristics and the major psychiatric risk factors of teenage suicide was completed by students at three Viennese high schools (n = 214; mean age 15.4 years). RESULTS: Eighty-one (37.9%) high school students reported having had suicidal thoughts at some point in their lives, girls significantly more often than boys (48.5% vs. 29.1%, P = 0.004). Furthermore, lifetime suicidal ideation in Viennese high school students was associated with living in broken-home families, cigarette smoking, substance problems (alcohol/drugs), self-reported depression, and high school type (the highest prevalence was in grammar school). After adjusting for confounders, we found that female gender, substance problems, school type and cigarette smoking were significantly associated with lifetime suicidal ideation in Viennese high school students. CONCLUSIONS: The psychosocial factors associated with adolescent suicidal ideation require attention in the contexts of suicide prevention and mental health promotion.

The SrtA Sortase of Streptococcus agalactiae is required for cell wall anchoring of proteins containing the LPXTG motif, for adhesion to epithelial cells, and for colonization of the mouse intestine.

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.

Relationships between aromatase and estrogen receptors in the brain of teleost fish.

Teleost fish are known for exhibiting a high aromatase activity mainly due to the expression of the cyp19b gene, encoding aromatase B (AroB). Recent studies based on both in situ hybridization and immunohistochemistry have demonstrated in three different species that this activity is restricted to radial glial cells. In agreement with measurements of aromatase activity, such aromatase-expressing cells are more abundant in the telencephalon, preoptic area, and mediobasal hypothalamus, although positive cells are also found in the midbrain and hindbrain. Comparative distribution of AroB and estrogen receptor (ERalpha, ERbeta1, and ERbeta2) expression indicates that the preoptic region and hypothalamus are major target for locally produced estradiol (E2) which is likely involved in controlling expression of genes implicated in neuroendocrine regulations. However, AroB and ER have never been reported to be co-expressed in the same cells which is intriguing given that, at least in some species, AroB is strongly up-regulated by E2 itself in agreement with the presence of an estrogen-responsive element (ERE) in the proximal promoter of the cyp19b gene. In vivo data in zebrafish have shown that E2 up-regulates AroB only in radial glial cells. This is in agreement with in vitro transfection experiments indicating that this ERE is functional, but not sufficient, as the E2 regulation of AroB only occurs in glial cell contexts, suggesting a cooperation between ER and so far unidentified glial-specific factors. These data also suggest that radial glial cells may express low amounts of ER that escaped detection until now. The expression of AroB in radial cells, well known for their roles in neurogenesis and now considered as progenitor cells, suggests that local E2 production within these cells could influence the well-documented capacity of the brain of teleosts to grow during adulthood.

Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene.

Compared with adult mammals, the brain of teleost fish is characterized by an extremely high capacity to aromatize androgens into estrogens, and this metabolic activity results from the expression of a specific brain aromatase (AroB) generated by the cyp19b gene. In this study, we first generated antibodies to zebrafish AroB and used them to map AroB-positive structures in the brain of adult zebrafish. We show that AroB is exclusively expressed in radial glial cells, mainly in the olfactory bulbs, telencephalon, preoptic area, and hypothalamus. Second, we investigated in vivo and in vitro the mechanisms involved in the estradiol (E2) regulation of the cyp19b gene. By means of whole-mount hybridization and immunohistochemistry on zebrafish embryos and larvae, we confirmed the E2-dependent upregulation of the cyp19b gene, and we show that E2 triggers AroB expression in radial glial cells mainly in the preoptic area and mediobasal hypothalamus of 48 hpf (hours post fertilization) and 108 hpf larvae. In addition, an in vitro analysis of 0.5 kb of the promoter region of the cyp19b gene demonstrated that this E2-dependent regulation involves a direct transcriptional action of estrogen receptors requiring estrogen-responsive elements. However, the data obtained on different cell lines demonstrate that a glial cell context is necessary for full E2 induction. The correlation between our in vivo and in vitro data suggests that the E2-dependent upregulation of AroB is favored by a glial cell context.