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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 ...

In oviparous species, in addition to a full-length estrogen receptor alpha (ERα), another ERα isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5′ untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ERα gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers...

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.

In order to identify the nature of the glucocorticoid receptor (GR)-expressing neurons and pituitary cells that potentially mediate the negative effects of stress on reproductive performance, double immunohistochemical stainings were performed in the brain and pituitary of the rainbow trout (Oncorhynchus mykiss). To avoid possible cross-reactions during the double staining studies, combinations of primary antibodies raised in different species were used, and we report here the generation of an antibody raised in guinea pig against the rainbow trout glucocorticoid receptor (rtGR). The results obtained in vitellogenic females showed that GnRH-positive neurons in the caudal telencephalon/anterior preoptic region consistently exhibited rtGR immunoreactivity. Similarly, in the anterior ventral preoptic region, a group of tyrosine hydroxylase-positive neurons, known for inhibiting gonadotropin (GTH)-2 secretion during vitellogenesis, was consistently shown to strongly express GR. Finally, we show that a large majority of the GTH-1 (FSH-like) and GTH-2 (LH-like) cells of the pituitary exhibit rtGR immunoreactivity. These results indicate that cortisol may affect the neuroendocrine control of the reproductive process of the rainbow trout at multiple sites.

There are two estrogen receptor (ER) subtypes in fish, ERalpha and ERbeta, and increasing evidence that the ERbeta 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, zfERalpha, zfERbeta1, and zfERbeta2, 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 (zfERbeta2) to 0.75 nM (zfERalpha and zfERbeta1). 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; zfERbeta2 was slightly more sensitive than zfERalpha and zfERbeta1. 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.

In oviparous species, in addition to a full-length estrogen receptor alpha (ER alpha), another ER alpha isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5' untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ER alpha gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the isoforms also was performed. The data indicated that the full-length ER alpha is expressed in liver, brain, pituitary, and ovary, whereas expression of the isoform with the truncated A domain is restricted to the liver, demonstrating a tissue-specific expression of these two ER alpha isoforms. The presence of a short liver-specific isoform in oviparous species suggests its role in the development and/or maintenance of the unique function of the liver in the vitellogenesis process.

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.