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.

The LIM/homeodomain protein islet-1 modulates estrogen receptor functions.

LIM/Homeodomain (HD) proteins are classically considered as major transcriptional regulators which, in cooperation with other transcription factors, play critical roles in the developing nervous system. Among LIM/HD proteins, Islet-1 (ISL1) is the earliest known marker of motoneuron differentiation and has been extensively studied in this context. However, ISL1 expression is not restricted to developing motoneurons. In both embryonic and adult central nervous system of rodent and fish, ISL1 is found in discrete brain areas known to express the estrogen receptor (ER). These observations led us to postulate the possible involvement of ISL1 in the control of brain functions by steroid hormones. Dual immunohistochemistry for ISL1 and ER provided evidence for ISL1-ER coexpression by the same neuronal subpopulation within the rat hypothalamic arcuate nucleus. The relationship between ER and ISL1 was further analyzed at the molecular level and we could show that 1) ISL1 directly interacts in vivo and in vitro with the rat ER, as well as with various other nuclear receptors; 2) ISL1-ER interaction is mediated, at least in part, by the ligand binding domain of ER and is significantly strengthened by estradiol; 3) as a consequence, ISL1 prevents ER dimerization in solution, thus leading to a strong and specific inhibition of ER DNA binding activity; 4) ISL1, via its N-terminal LIM domains, specifically inhibits the ER-driven transcriptional activation in some promoter contexts, while ER can serve as a coactivator for ISL1 in other promoter contexts. Taken together, these data suggest that ISL1-ER cross-talk could differentially regulate the expression of ER and ISL1 target genes.

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.

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.

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.

Distribution of estrogen receptor-immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss).

Using antibodies against the hormone binding domain of the trout estrogen receptor (ER), the distribution of ER-immunoreactive (ER-IR) cells was studied in the brain of maturing diploid and triploid female rainbow trout using a streptavidin-biotin-peroxidase method followed by a nickel-intensified diaminobenzidine reaction. This technique resulted in an excellent signal/background ratio allowing unambiguous identification of positive cells. In all animals, ER-IR cells were consistently located in three brain regions, the ventral telencephalon, the anterior ventral preoptic region, and the mediobasal hypothalamus. About 250 ER-IR cells were observed in the ventral and dorsal parts of the ventral telencephalon. In the anterior nucleus preopticus periventricularis, about 2400 ER-IR cells were observed surrounding the preoptic recess. In the posterior hypothalamus, approximately 2700 ER-IR cells were located in the anterior, posterior and inferior divisions of the nucleus lateralis tuberis and in the nucleus saccus vasculosus. In these regions cell nuclei exhibiting different densities of staining were observed and absolutely no labeling of cytoplasmic processes was detected. These results are in partial agreement with those obtained either after injection of tritiated-estradiol in other teleots species or in situ hybridization of ER mRNAs in trout. In particular, no immunoreactivity was observed in the thalamic region nor in the nucleus posterioris periventricularis. These data indicate that target cells for estradiol are essentially located in brain regions involved in the neuroendocrine control of pituitary functions and having direct connections with the hypophysis.

Characterization of neuropeptide Y expression in the brain of a perciform fish, the sea bass (Dicentrarchus labrax).

The distribution of neuropeptide Y (NPY) gene expression was mapped in the brain of the sea bass (Dicentrarchus labrax) by in situ hybridization with 35S-UTP labeled cRNA probes. Gene expression was mainly detected within the forebrain, although NPY mRNA transcripts were also localized in the tectum and tegmentum mesencephali and posterior brain. New NPY-expressing nuclei were found in the dorsal and ventral telencephalon, preoptic area, tuberal hypothalamus, synencephalon, tegmentum mesencephali and posterior brain. The profuse NPY gene expression within the main neuroendocrine areas of the teleost fish further supports a physiological role in the control of the pituitary secretion. In addition, NPY gene was expressed within the primary visual, olfactory and gustatory circuits of teleost which, subsequently, project to hypothalamic feeding center in teleost fish. Our results extend the NPY-expressing areas known in teleost species.

Identification of potential sites of cortisol actions on the reproductive axis in rainbow trout.

The full length cDNA encoding a rainbow trout glucocorticoid receptor (rtGR) has been obtained from rainbow trout liver and intestine libraries. Northern blot analysis showed that the corresponding messengers are detected in the brain of trout with a size 7.5 kb similar to the size of rtGR mRNA in other target tissues. The distribution of the rtGR mRNA and protein was studied in the forebrain of the trout by means of both in situ hybridization and immunohistochemistry and compared with that of the oestrogen receptor (rtER). The GR and ER mRNAs and proteins were detected with a strong overlapping mainly in the: (a) preoptic region; (b) mediobasal hypothalamus; and (c) anterior pituitary, confirming their implication in the neuroendocrine control of pituitary functions. In both diencephalon and pituitary, the peptidergic phenotype of some neuron or cell categories expressing either type of receptors could be determined by double staining. Furthermore, double staining studies have demonstrated colocalization of the two receptors in the same neurons or pituitary cells. The rtER and rtGR were found to be co-expressed in the dopaminergic neurons inhibiting GTH2 secretion and in pituitary cells of the anterior lobe--notably the gonadotrophs. Given that the promoter of the ER gene contains several potential glucocorticoid-responsive elements (GRE) and that cortisol inhibits the oestradiol-stimulated ER expression in the liver, the possibility exists for modulation of ER gene expression by GR in the hypothalamo-pituitary complex. This could explain some of the well documented effects of stress on the reproductive performance in salmonids.

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 paraformaldehyde-fixed 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.

Cloning, tissue distribution, and central expression of the gonadotropin-releasing hormone receptor in the rainbow trout (Oncorhynchus mykiss).

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the brain of rainbow trout. This cDNA encodes a protein of 386 amino acids (aa) exhibiting the typical arrangement of the G-protein-coupled receptors in seven transmembrane domains. However, a second ATG could give rise to a receptor with a 30-aa longer extracellular domain. As already shown in other fish and Xenopus, this protein possesses an intracellular domain, in contrast with its mammalian counterparts. In the case of rainbow trout, this intracellular carboxy-terminal tail consists of 58 residues. Northern blotting experiments carried out in the brain, the pituitary, and the liver only resulted in a single band of 1.9-2 kilobases in the pituitary, although reverse transcription-polymerase chain reaction amplification products were found in the brain, the pituitary, the retina, and the ovary. In situ hybridization using a probe corresponding to the full-length coding region of the receptor was performed on vitellogenic or ovulating females and allowed to detect a weak but specific signal in the proximal pars distalis of the pituitary, the preoptic region, the mediobasal hypothalamus, and the optic tectum. However, the strongest signal was consistently detected in a mesencephalic structure, the nucleus lateralis valvulae, the significance of which is presently open to speculation.

The reproductive brain in fish.

In fish as in other vertebrates, the brain is actively involved in the control of reproduction, first by participating, under the influence of external factors, in the establishment of an appropriate endocrine status, but also by allowing synchronization of the partners by the time of spawning. It is now well established that the pituitary gonadotropic function is controlled by multiple stimulatory and inhibitory factors, originating mainly from the preoptic region and the mediobasal hypothalamus, both target regions for sexual steroids. Little is known about the mechanisms involved in the mediation of external and internal factors, however there is indication that internal factors, such as androgens and melatonin, known to trigger particular behavioural and endocrine responses, act both at the level of neuroendocrine territories, but also on sensorial systems, which are the actual sites of action for external factors. This paper represents an attempt to summarize and integrate the recent literature devoted to the different aspects of the brain as a major participant in the complex endocrine and behavioural mechanisms of reproduction in fish.