Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.

Actions of sex steroids on kisspeptin expression and other reproduction-related genes in the brain of the teleost fish European sea bass.

Kisspeptins are well known as mediators of the coordinated communication between the brain-pituitary axis and the gonads in many vertebrates. To test the hypothesis that gonadal steroids regulate kiss1 and kiss2 mRNA expression in European sea bass (a teleost fish), we examined the brains of gonad-intact (control) and castrated animals, as well as castrated males (GDX) and ovariectomized females (OVX) that received testosterone (T) and estradiol (E2) replacement, respectively, during recrudescence. In GDX males, low expression of kiss1 mRNA is observed by in situ hybridization in the caudal hypothalamus (CH) and the mediobasal hypothalamus (MBH), although hypothalamic changes in kiss1 mRNA levels were not statistically different among the groups, as revealed by real-time PCR. However, T strongly decreased kiss2 expression levels in the hypothalamus, which was documented in the MBH and the nucleus of the lateral recess (NRLd) in GDX T-treated sea bass males. Conversely, it appears that E2 evokes low kiss1 mRNA in the CH, while there were cells expressing kiss2 in the MBH and NRLd in these OVX females. These results demonstrate that kisspeptin neurons are presumably sensitive to the feedback actions of sex steroids in the sea bass, suggesting that the MBH represents a major site for sex steroid actions on kisspeptins in this species. Also, recent data provide evidence that both positive and negative actions occur in key factors involved in sea bass reproductive function, including changes in the expression of gnrh-1/gonadotropin, cyp19b, er and ar genes and sex steroid and gonadotropin plasma levels in this teleost fish.

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.

Synthesis of estrogens in progenitor cells of adult fish brain: evolutive novelty or exaggeration of a more general mechanism implicating estrogens in neurogenesis?

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fishes 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.

Spontaneous and gonadotropin-releasing hormone-stimulated cytosolic calcium rises in individual goldfish gonadotrophs.

The cytosolic free calcium concentration, [Ca2+]i, was monitored in single isolated goldfish gonadotrophs with the fluorescent probe Indo-1. It was found that goldfish gonadotrophs exhibit both spontaneous and secretagogue-induced [Ca2+]i rises. Spontaneous [Ca2+]i transients showed striking kinetic features and a sensitivity to external Ca2+ suggesting that they were the consequence of transient Ca2+ entries. Two kinetically distinct patterns of [Ca2+]i rises were generated in response to the two native forms of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a part of the gonadotrophs, GnRHs triggered a plateau [Ca2+]i rise whereas in other responsive cells they induced a series of [Ca2+]i bursts, each consisting of grouped [Ca2+]i transients. Both plateau and burst [Ca2+]i response patterns were due to Ca2+ entry through plasma membrane Ca2+ channels, inasmuch as they were suppressed with external Ca2+ removal. No contribution of Ca2+ release from thapsigargin-sensitive stores was observed in either response pattern. While in mammalian gonadotrophs GnRH rises [Ca2+] by mostly acting on internal Ca2+ sequestering stores, our results show that GnRH-stimulated goldfish gonadotrophs rapidly increase Ca2+ entry to enhance their [Ca2+]i levels.

Peptide YY (PYY) and fish pancreatic peptide Y (PY) expression in the brain of the sea bass (Dicentrarchus labrax) as revealed by in situ hybridization.

Tetrapod vertebrates express three neuropeptide Y (NPY)-related peptides: NPY, peptide YY (PYY), and pancreatic polypeptide (PP). Both NPY and PYY mRNA have been localized in the brain of tetrapods whereas PP expression is restricted to the pancreas. Some teleost fish commonly produce NPY and PYY but pancreatic peptide Y (PY) instead of PP. Both NPY and PYY mRNAs are widely distributed in the brain of non-tetrapod species, but no information about PY central expression is available. In the present study, molecular riboprobes were used to study PYY and PY mRNA central distribution in the sea bass (Dicentrarchus labrax). PYY and PY gene expression was predominantly detected within the sea bass forebrain. Telencephalic PYY gene expression was restricted to the ventral part of the ventral telencephalon, and no PY expression was detected in the cerebral hemispheres. Both PYY and PY mRNAs were found within the preoptic area and lateral hypothalamus. Distinct PY or PYY mRNA cell groups were localized in the pretectal area and synencephalon or posterior tubercle, respectively. Caudally, PY gene expression was found in the medial reticular formation, whereas PYY transcripts were localized within the vagal lobe. The results demonstrate that vertebrate brain expresses three NPY-related genes and further support the hypothesis that PP and PY arose by independent gene duplications from PYY. The receptor system of the NPY family as well as gene expression within the main hypophysiotropic and feeding behavior areas suggest an involvement of both peptides in the control of food intake and pituitary secretion.

Characterization of trout galanin and its distribution in trout brain and pituitary.

Galanin was purified from an extract of the stomach of the rainbow trout, Oncorhynchus mykiss, and its primary structure was established as Gly-Trp-Thr-Leu-Asn-Ser- Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Gly-Ile-Asp-Gly-His-Arg20- Thr-Leu-Ser-Asp- Lys-His-Gly-Leu-Ala. Trout galanin shows six amino acid substitutions compared with pig galanin, but the N-terminal region (residues 1-14) has been fully conserved. The distribution of galanin-immunoreactive (GAL-IR) structures in the trout brain and pituitary was studied via immunohistochemistry. GAL-IR cell bodies were observed only in the caudal telencephalon, the preoptic region, and the mediobasal hypothalamus. GAL-IR fibers, however, are widely distributed throughout the brain, with a much lower density in the midbrain and posterior brain than in the tel- and diencephalon. Particularly dense innervation of the mediobasal hypothalamus, the ventral and supracommissuralis parts of the caudal telencephalon, and the region above and below the anterior commissure was observed. A heavy innervation of the pituitary was consistently detected. GAL-IR fibers were present in neurohypophyseal digitations of both the anterior and intermediate lobes with highest density in the region of the proximal pars distalis, where growth hormone and gonadotropic cells are located. Fibers were also seen in digitations of the rostral pars distalis, in particular between the prolactin follicles. The distribution of GAL-IR neurons in the central nervous system and pituitary of the trout suggests that the peptide may exercise an important role in the regulation of neuroendocrine functions, particularly those related to reproduction.

Comparative distribution of mammalian GnRH (gonadotrophin-releasing hormone) and chicken GnRH-II in the brain of the immature Siberian sturgeon (Acipenser baeri).

The brain of the sturgeon has recently been shown to contain at least two forms of GnRH (gonadotropin-releasing hormone), mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). In this study, we compared the distribution of immunoreactive (ir) mGnRH and cGnRH-II in the brain of immature Siberian sturgeons (Acipenser baeri). The overall distribution of mGnRH was very similar to the distribution of sGnRH in teleosts such as salmonids or cyprinids. mGnRH-ir perikarya were observed in the olfactory nerves and bulbs the telencephalon, the preoptic region, and the mediobasal hypothalamus. All these cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the hypothalamopituitary interface. No ir-fibers were observed in the anterior lobe of the pituitary, but a few were seen to enter the neurointermediate lobe. mGnRH-ir fibers were detected in many parts of the brain, particularly in the forebrain. mGnRH-ir cerebrospinal fluid-contacting cells were observed in the telencephalon, the preoptic region, and the mediobasal hypothalamus. In contrast, cGnRH-II was present mainly in the posterior brain, although a few ir axons were seen in the above-mentioned territories. In particular, cGnRH-II-ir cells bodies, negative for mGnRH, were consistently observed in the nucleus of the medial longitudinal fasciculus of the midbrain tegmentum. The cGnRH-II innervation in the optic tectum, cerebellum, vagal lobe, and medulla oblongata was more abundant than the mGnRH innervation in the same areas. This study provides evidence that the organization of the GnRH systems in a primitive bony fish is highly similar to that reported in teleosts and further documents the differential distribution of two forms of GnRH in the brain of vertebrates.

Immunohistochemical localization of gonadotropin-releasing-hormone-associated peptide in the brain of the frog.

Gonadotropin-releasing-hormone (GnRH)-associated peptide (GnRH-AP) is a 56 amino acid neuropeptide derived from the GnRH prohormone. GnRH-AP corresponds to the C-terminal fragment flanking the GnRH peptide. Using an antiserum raised against human GnRH-AP [1-56], or against human GnRH, we have investigated the neuronal systems containing either peptide in the central nervous system of the frog Rana ridibunda by immunohistological techniques. A main group of GnRH-AP-containing perikarya was found in a dorsoventral orientation of the supra anterior preoptic area (SAPA) just in front of the preoptic recess. Fibers originating from these perikarya projected rostrally toward the medial septal nucleus and the diagonal band of Broca. A network of GnRH-AP-immunoreactive (ir) fibers runs caudally from the SAPA toward the ventral hypothalamus. A high density of GnRH-AP-ir terminals was found in the median eminence. A few positive fibers were detected in the neural lobe of the pituitary, particularly in the region bordering the pars intermedia. Labelling of consecutive sections by either GnRH-AP or GnRH antibodies showed that GnRH and GnRH-AP-like irs were contained in the same cells of the SAPA. The double-staining technique with electrophoretic elution confirmed the colocalization of GnRH and GnRH-AP within the same neurons. Such a coexistence indicates that frog GnRH originates from a high molecular weight precursor which is closely related to rat pro-GnRH. The relative preservation of the C-terminal sequence of the pro-GnRH during evolution suggests that GnRH-AP may possess intrinsic biological activity. The high density of GnRH-AP-containing neurons projecting through the external zone of the median eminence would support the concept that GnRH-AP is involved in the modulation of pituitary hormone secretion.

Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distributions of estrogen receptor-immunoreactive cells and gonadotrophin-releasing hormone-immunoreactive neurons on the same sections of the brains of adult male and female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the ventral and lateral telencephalon, the preoptic region, the mediobasal hypothalamus, and the ventromedial thalamic nucleus. Gonadotrophin-releasing hormone-immunoreactive perikarya were detected in the olfactory bulbs, the ventral telencephalon, the preoptic area, and the mediobasal hypothalamus. Double-staining studies showed that, although some estrogen receptor-positive cells were in close proximity to gonadotrophin-releasing hormone-immunoreactive perikarya, careful examination of 550 gonadotrophin-releasing hormone-positive cells from five adult females and two adult males failed to demonstrate any evidence that gonadotrophin-releasing hormone neurons coexpress estrogen receptor in the brain of the rainbow trout. The present study provides, for the first time in teleosts, morphological evidence that gonadotrophin-releasing hormone neurons do not represent major direct targets for estradiol, suggesting that the positive feedback effects of estradiol onto the gonadotrophin-releasing hormone system are likely to be conveyed via other cell populations.

Expression of clock gene in the brain of rainbow trout: comparison with the distribution of melatonin receptors.

To identify brain structures potentially acting as biological clocks in rainbow trout (Oncorhynchus mykiss), the expression sites of a trout homolog of the mouse clock gene were studied and compared with that of melatonin receptors (Mel-R). For this purpose, a partial sequence of the trout clock gene, including a PAS domain, was obtained by reverse transcription-polymerase chain reaction and used to perform in situ hybridization. The highest density of clock transcripts was observed in the periventricular layer (SPV) of the optic tectum, but a weaker expression was detected in some pretectal nuclei, such as the posterior pretectal nucleus (PO) and the periventricular regions of the diencephalon. Comparison of the hybridization signal in fish sacrificed at 08:00 and 17:00 did not indicate major changes in clock expression levels. Comparison of adjacent sections alternatively treated with clock and Mel-R probes suggests that both messengers are probably expressed in the same cells in the SPV and PO. In addition, in situ hybridization with a glutamate decarboxylase 65 probe, demonstrates that cells expressing clock and Mel-R in the optic tectum are gamma-aminobutyric acid neurons. The tight overlapping between the expression of Mel-R and clock transcripts in cells of the PO and SPV suggests a functional link between these two factors. These results indicate that the optic tectum and the pretectal area of the rainbow trout are major sites of integration of the melatonin signal, express the clock gene, and may act as biological clocks to influence behavioral and endocrine responses in trout.

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: Ser(8)-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp(7)Leu(8)-mGnRH), and chicken GnRH-II (cGnRH-II: His(5)Trp(7)Tyr(8)-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.

Comparative distribution of neurotensin-like immunoreactivity in the brain of a teleost (Carassius auratus), an amphibian (Hyla meridionalis), and a reptile (Gallotia galloti).

The distribution of neurotensin (NT) was studied in the brain of three species belonging to the three major classes of cold-blooded vertebrates: teleost fishes (Carassius auratus), anuran amphibians (Hyla meridionalis), and reptiles (Gallotia galloti; Lacertidae). By using antibodies directed against synthetic bovine NT in the three species, immunoreactive cell bodies were discovered mostly in the telencephalon and diencephalon, in particular at the level of the preoptic region the mediobasal hypothalamus, and the thalamus. In the frog and the lizard, additional immunoreactive (ir) structures were observed in the optic tectum and the tegmentum of the mesencephalon. In the goldfish pituitary, an extensive innervation was consistently observed at the level of the rostral pars distalis, whereas in both frog and lizard, positive fibers were only detected in the external layer of the median eminence. In the three species there is a striking overlap between the distribution of the NT-ir cell bodies and that of the target cells for sexual steroids. The results are discussed in relation with those reported in birds and mammals, and with the possible interactions among NT, sexual steroids, and the neuroendocrine control of pituitary hormone release, in particular prolactin and gonadotrophin.

Distribution of glutamic acid decarboxylase mRNA in the forebrain of the rainbow trout as studied by in situ hybridization.

By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.

Central melatonin receptors in the rainbow trout: comparative distribution of ligand binding and gene expression.

To better define the role of melatonin in fish, we have compared in detail the distribution of 2-[125I]iodomelatonin binding sites with gene expression for melatonin receptor subtypes in a widely studied seasonal species, the rainbow trout. Three distinct partial sequences of the melatonin receptor gene were cloned from trout genomic DNA. Two of the sequences corresponded to the Mella receptor subtype, and one corresponded to the Mellb receptor subtype. Analysis of numerous clones failed to find a sequence equivalent to the Mel1c receptor subtype. Comparison of receptor gene expression with 2-[125I]iodomelatonin binding distribution indicated dendritic transport of the receptor. Melatonin receptors were associated predominantly with visually related areas of the trout brain, such as the thalamic region, the pretectal area, and the optic tectum. The pituitary was devoid of 2-[125I]iodomelatonin binding, and melatonin receptor gene expression was not detectable. It would appear from the results of the present study that melatonin in this species is involved primarily in the processing of visual signals. How melatonin interacts with circannual rhythms of growth and reproduction is unclear, although a direct interaction between melatonin and the hypothalamo-pituitary axis is not clearly indicated.

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss).

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH2-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions.

Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2.

We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.

The GnRH system in the European sea bass (Dicentrarchus labrax).

The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.

Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry.

Complementary DNA fragments that encode central and C-terminal domains of rainbow trout estrogen receptor (rtER) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). Both fusion proteins were induced by IPTG and could readily be detected as a 53-55 kDa band in crude extracts or in insoluble fraction after polyacrylamide gel electrophoresis and Coomassie blue staining. These recombinant proteins were solubilized and partially purified (ca. 60-75%) using centrifugation and different concentrations of urea. Gel mobility shift assays revealed that the hybrid protein containing ER central domain forms a specific complex with a synthetic estrogen-response-element. Similarly, we showed by steroid-binding assays that the hybrid protein containing the ER C-terminal domain binds specifically estrogen and not other steroids. These hybrid receptors were further isolated by electroelution after electrophoresis and used to immunize rabbits. Polyclonal antibodies from each antiserum were purified using GST-rtER fusion proteins. The specificity of these purified antibodies was confirmed by Western blot analysis using extracts from yeast and COS-1 cells transfected with rtER cDNA expression vectors. In these cells, rtER level was about 300-500 fmol/mg of protein, and the receptor was found as a single band migrating as a 65 kDa polypeptide. Interestingly, Western blot analysis with both purified antibodies directed against central or C-terminal regions of rtER revealed two receptor forms in trout liver nuclear extracts: a major form migrating as 65 kDa protein also observed in transfected cells, and a minor band at 71 kDa specific to the liver. Both receptor form levels were strongly induced by estradiol whereas they were virtually undetectable in untreated male trout livers. Immunocytochemistry performed on brain and pituitary of female trout revealed the presence of rtER in neurons located in the ventral telencephalon, preoptic area and mediobasal hypothalamus, as well as cells in the proximal pars distalis of the pituitary.

Sexual dimorphism in the distribution of alpha-neoendorphin-like immunoreactivity in the anterior pituitary of the rat.

The localization of the opioid peptide alpha-neoendorphin (alpha-Neo-E) was studied in the anterior pituitary of normal and castrated male and normal female rats. Immunoreactive (ir) cells were noted in both sexes. These alpha-Neo-E-ir cells were further characterized using double immunostaining with an elution-restaining procedure. It was seen that in males, alpha-Neo-E-ir cells corresponded mainly to luteinizing hormone/follicle-stimulating hormone cells and a few thyroid-stimulating hormone (TSH) cells, whereas in females, virtually all alpha-Neo-E-ir cells corresponded to TSH cells. Castration of male rats caused, within 3 to 5 days a dramatic decrease in the number of alpha-Neo-E-ir gonadotrophs, whereas the number of alpha-Neo-E-ir TSH cells tended to increase. Two weeks after castration, however, most alpha-Neo-E-ir cells were also follicle-stimulating hormone-ir. This study demonstrates that in the anterior lobe of the rat, alpha-Neo-E-ir is located within gonadotrophs and/or thyrotrophs, depending on the sex. In addition, results obtained following castration suggest that the expression of this peptide in the anterior pituitary depends upon the steroid environment, possibly indicating that alpha-Neo-E is implicated in the regulation of the pituitary-gonadal axis.