Colette Vaillant

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.

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon.

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.

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.

Vitellogenin gene expression in primary culture of male rainbow trout hepatocytes

Using a primary culture of trout hepatocytes we compare the kinetics of accumulation of vitellogenin and its messenger RNA after estrogen administration. We found that the cells were more sensitive to estradiol than to other estrogens. The lowest effective concentration of estradiol was 10(-9) M. At 10(-6) M the androgens have no effect. Comparison of the primary and secondary stimulation with E2 shows that the initial rate of accumulation of vitellogenin is very much higher in the secondary stimulation. Over a time course of primary stimulation we show that after estradiol withdrawal the rate of accumulation of vitellogenin mRNA in the secondary is a function of time of the first stimulation.

Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development.

Early expression of estrogen receptors (esr) and their role in regulating early expression of cyp19a1b encoding brain aromatase were examined in the brain of zebrafish. Using in toto hybridization and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), a significant increase in the expression of esr1, esr2a, and esr2b was observed between 24 and 48 hours postfertilization (hpf). In toto hybridization demonstrated that esr2a and esr2b, but not esr1, are found in the hypothalamus. Using real‐time RT‐PCR, an increase in cyp19a1b mRNAs occurs between 24 and 48 hpf, indicating that expression of cyp19a1b is temporally correlated with that of esr. This increase is blocked by the pure anti‐estrogen ICI182,780. Furthermore, E2 treatment of cyp19a1b‐GFP (green fluorescent protein) transgenic embryos results in appearance of GFP expression in the brain as early as 25 hpf. These results indicate that basal expression of cyp19a1b expression in the brain of developing zebrafish most likely relies upon expression of esr that are fully functional before 25 hpf. Developmental Dynamics 238:2641–2651, 2009.

Activity and expression of steroidogenic enzymes in the brain of adult zebrafish

The brain of adult teleost fish exhibits several unique and interesting features, notably an intense neurogenic activity linked to persistence of radial glial cells acting as neural progenitors, and a high aromatase activity supported by strong expression of the cyp19a1b gene. Strikingly, cyp19a1b expression is restricted to radial glial cells, suggesting that estrogens are able to modulate their activity. This raises the question of the origin, central or peripheral, of C19 androgens available for aromatization. This study aimed to investigate the activity and expression of other main steroidogenic enzymes in the brain of adult zebrafish. We demonstrate by high‐performance liquid chromatography that the zebrafish brain has the ability to convert [3H]‐pregnenolone into a variety of radiolabeled steroids such as 17OH‐pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydro‐testosterone, estrone, estradiol, progesterone, and dihydro‐ and tetrahydro‐progesterone. Next, we show by in situ hybridization that messengers for key steroidogenic enzymes, such as Cyp11a1 (P450SCC), 3β‐Hsd, Cyp17 and Cyp19a1b, are widely expressed in the forebrain where they exhibit an overall similar pattern. By combining aromatase B immunohistochemistry with in situ hybridization, we show that cyp11a1, 3β‐hsd and cyp17 messengers are found in part in aromatase B‐positive radial processes, suggesting mRNA export. This set of results provides the first demonstration that the brain of fish can produce true neurosteroids, possibly in radial glial cells. Given that radial glial cells are brain stem cells during the entire lifespan of fish, it is suggested that at least some of these neurosteroids are implicated in the persisting neurogenic process.

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.

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. J. Comp. Neurol. 518:4855–4876, 2010.

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.

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.