Isabelle Anglade

Molecular Characterization of Three Estrogen Receptor Forms in Zebrafish: Binding Characteristics, Transactivation Properties, and Tissue Distributions

Abstract There are two estrogen receptor (ER) subtypes in fish, ERα and ERβ, and increasing evidence that the ERβ subtype has more than one form. However, there is little information on the characteristics and functional significance of these ERs in adults and during development. Here, we report the cloning and characterization of three functional ER forms, zfERα, zfERβ1, and zfERβ2, in the zebrafish. The percentages of identity between these receptors suggest the existence of three distinct genes. Each cDNA encoded a protein that specifically bound estradiol with a dissociation constant ranging from 0.4 nM (zfERβ2) to 0.75 nM (zfERα and zfERβ1). In transiently transfected cells, all three forms were able to induce, in a dose-dependent manner, the expression of a reporter gene driven by a consensus estrogen responsive element; zfERβ2 was slightly more sensitive than zfERα and zfERβ1. Tissue distribution pattern, analyzed by reverse transcription polymerase chain reaction, showed that the three zfER mRNAs largely overlap and are predominantly expressed in brain, pituitary, liver, and gonads. In situ hybridization was performed to study in more detail the distribution of the three zfER mRNAs in the brain of adult females. The zfER mRNAs exhibit distinct but partially overlapping patterns of expression in two neuroendocrine regions, the preoptic area and the mediobasal hypothalamus. The characterization of these zfERs provides a new perspective for understanding the mechanisms underlying estradiol actions in a vertebrate species commonly used for developmental studies.

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.

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. J. Comp. Neurol. 485:304–320, 2005.

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.

Distribution of aromatase mRNA and protein in the brain and pituitary of female rainbow trout: Comparison with estrogen receptor α

Recent data indicate that estrogens locally produced in the brain by aromatization of androgens could be important for neurogenesis and brain repair. In this respect, fish are interesting because of the extremely high aromatase activity of their brain. In this study, the rainbow trout brain aromatase was cloned and riboprobes were used to map the distribution of cells expressing the corresponding mRNAs. A very strong hybridization signal was detected in the pituitary and in cells bordering the ventricles in the telencephalon and ventral diencephalon, with the highest expression in the preoptic area and hypothalamus. A weaker signal was detected in the ependymal layer bordering the torus semicircularis and optic tectum. This localization was fully confirmed by immunohistochemistry using antibodies against a teleost aromatase. In addition, this antibody showed that aromatase expression in fact corresponds to radial glial cells because immunoreactive cells had long cytoplasmic processes extending toward the pial surface. Because brain aromatase was shown to be upregulated by estradiol in fish, the distribution of aromatase mRNAs was compared with that of rainbow trout estrogen receptor α (rtERα) on adjacent sections. Although the highest aromatase expression was found in regions expressing rtERα, no obvious coexpression was found, as rtERα was never observed in radial cells. However, reverse transcriptase‐polymerase chain reaction experiments performed on brain cell cultures enriched in glial cells suggest that a weak expression of rtERα in glial cells cannot be excluded. The possible role of the high brain aromatase content in fish could be related to the continuous growth of their central nervous system during adulthood. J. Comp. Neurol. 462:180–193, 2003.

Origin of the pituitary innervation in the goldfish

Despite the large number of studies devoted to the pituitary of teleosts, the origin of the direct pituitary innervation is still largely unknown. Although such a model is ideal for applying retrograde transport techniques, these methods involve the difficult in vivo injection of tracers into the pituitary and have never been applied. Recently, a lipophilic fluorescent dye (1-1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanin; DiI) has been introduced and shown to have the capacity of being transported by the membranes of paraformaldehydefixed tissues. Microcrystals of DiI were implanted via a ventral approach into the pituitary of goldfish previously fixed by intracardiac perfusion of paraformaldehyde. The goldfish heads were kept immersed in paraformaldehyde for various periods of time (2–6 weeks). The brains were then dissected and cut transversally using a Vibratome. The results demonstrate that hypophysiotrophic areas are essentially restricted to the preoptic region, the mediobasal hypothalamus and the nucleus dorsolateralis thalami. In addition, cell bodies probably containing gonadotrophin releasing-hormone were also retrogradely stained along a pathway that can be traced up to the olfactory bulbs. The results also confirm that cell bodies, located around the ventral aspect of the preoptic recess and probably corresponding to dopaminergic neurons, project to the pituitary. Large neurons have also been observed in the rostral dorsal midbrain tegmentum just caudal to the posterior commissure. Most neurons of the so-called paraventricular organ remain unstained. Finally, a fiber tract originating from an undetermined territory of the posterior brain has been observed. The results are discussed in relation to the possible chemical nature of the hypophysiotropic neurons.

Differential expression of three different prepro‐GnRH (gonadotrophin‐releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax)

The expression sites of three prepro‐gonadotrophin‐releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser8‐mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp7Leu8‐mGnRH), and chicken GnRH‐II (cGnRH‐II: His5Trp7Tyr8‐mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH‐associated peptide (GAP)‐coding regions of the prepro‐GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP‐II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti‐sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA‐expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP‐positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations. J. Comp. Neurol. 429:144–155, 2001.

Estrogen Receptors Are Expressed in a Subset of Tyrosine Hydroxylase-Positive Neurons of the Anterior Preoptic Region in the Rainbow Trout

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

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.

Characterization and Expression of the Nuclear Progestin Receptor in Zebrafish Gonads and Brain

Abstract The zebrafish nuclear progestin receptor (nPR; official symbol PGR) was identified and characterized to better understand its role in regulating reproduction in this well-established teleost model. A full-length cDNA was identified that encoded a 617-amino acid residue protein with high homology to PGRs in other vertebrates, and contained five domains characteristic of nuclear steroid receptors. In contrast to the multiplicity of steroid receptors often found in euteleosts and attributed to probable genome duplication, only a single locus encoding the full-length zebrafish pgr was identified. Cytosolic proteins from pgr-transfected cells showed a high affinity (Kd = 2 nM), saturable, single-binding site specific for a native progestin in euteleosts, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). Both 17,20beta-DHP and progesterone were potent inducers of transcriptional activity in cells transiently transfected with pgr in a dual luciferase reporter assay, whereas androgens and estrogens had little potency. The pgr transcript and protein were abundant in the ovaries, testis, and brain and were scarce or undetectable in the intestine, muscle, and gills. Further analyses indicate that Pgr was expressed robustly in the preoptic region of the hypothalamus in the brain; proliferating spermatogonia and early spermatocytes in the testis; and in follicular cells and early-stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV) in the ovary. The localization of Pgr suggests that it mediates progestin regulation of reproductive signaling in the brain, early germ cell proliferation in testis, and ovarian follicular functions, but not final oocyte or sperm maturation.