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Biology of Reproduction 60, 642-650 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Glucocorticoid Receptor Immunoreactivity in Neurons and Pituitary Cells Implicated in Reproductive Functions in Rainbow Trout: A Double Immunohistochemical Study1

Christine A. Teitsmaa, Isabelle Angladea, Christèle Lethimoniera, Gwenola Le Dréana, Dany Saligauta, Bernadette Ducoureta, and Olivier Kah2,a

a Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Institut rennais d'Ecologie et Biologie des Poissons, Campus de Beaulieu, 35042 Rennes cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In all vertebrates, stress is known to affect many important integrative functions such as appetite/growth, homeostasis, immunity, and reproduction. With respect to reproduction, it is well recognized that stress exerts negative effects on a number of reproductive parameters in humans [1] and laboratory or farm animals [2]. In mammals, all three levels of the brain-pituitary-gonadal axis appear to be implicated in the antireproductive effects of stress [3]. At the central level, studies in rodents have shown that decreased gonadotropin secretion under stressful conditions results from both altered GnRH synthesis and/or release of GnRH and decreased sensitivity of the gonadotrophs to GnRH [4]. The deleterious effects of stress on the reproductive performance in fish are also well documented [5], in particular with respect to salmonids, not only because of their important commercial interest but also due to their particular susceptibility to stressors and water quality [6]. For instance, stress or cortisol implants reduce gonadal size, plasma levels of gonadal steroids, and pituitary gonadotropin (GTH) content in sexually maturing male or female brown trout and suppress plasma GTH levels in maturing male rainbow trout [7, 8]. Many of these effects are likely to be mediated by cortisol at the brain and/or pituitary level, since it has been observed that cortisol in vitro has little or no effect on the steroidogenesis of ovarian follicles of several teleost fish [9]. However, the potential targets of cortisol at the brain/pituitary level still remain to be identified.

Many of the genomic effects of corticosteroids are mediated by specific intracellular receptors acting as ligand-dependent transcription factors on the expression of certain target genes. The recent cloning and characterization of a full-length cDNA encoding a rainbow trout glucocorticoid receptor (rtGR) [10], and the development of antibodies directed against the N-terminal portion of the rtGR protein [11], have opened new opportunities to investigate the distribution of rtGR mRNA and protein in the brain-pituitary complex of the rainbow trout. Using in situ hybridization, we have recently shown that rtGR transcripts are abundant within the neuroendocrine regions of the brain, i.e., the preoptic area and the mediobasal hypothalamus, and to a lesser extent in the ventral telencephalon [12]. Immunohistochemistry has enabled us to confirm these results with a high degree of tissue preservation using an antibody raised in rabbit against the NH2-terminal fragment of the rtGR [11, 13]. These studies have confirmed the high expression of rtGR protein in the ventral telencephalon, the preoptic parvicellular and magnocellular areas, the mediobasal hypothalamus, and the optic tectum. However, until now, the nature of the neurotransmitters or neuropeptides synthesized in these rtGR-expressing neurons has not been determined. Knowledge of the chemical content of the rtGR-expressing neurons is essential for understanding the mechanisms by which cortisol affects neuronal activities in these neuroendocrine brain regions. In this study, we have used double immunohistochemistry to elucidate the chemical nature of rtGR-immunoreactive neurons and pituitary cells, with special emphasis on the neuroendocrine systems controlling reproduction.

In teleosts, the pituitary gonadotrophic activity is regulated by a complex interplay of brain factors, neuropeptides and neurotransmitters [14, 15], and peripheral factors (sex steroids, gonadal peptides). In salmonids in general, and notably in rainbow trout, it is well established that the pituitary secretes two gonadotropins, GTH-1 and GTH-2, which are synthesized by two different cell types [16, 17]. GTH-1, the FSH-like gonadotropin, is expressed early during sexual development and is liberated throughout the whole cycle [18, 19]. However, the neuroendocrine mechanisms implicated in the control of GTH-1 synthesis and release are still poorly understood and are likely to involve both gonadal steroids and peptides [20]. In contrast, much more information is available with respect to the control of expression and release of the maturational LH-like gonadotropin GTH-2. As in any vertebrate, GnRH is the main neuropeptide triggering GTH-2 secretion, whereas dopamine exerts a tonic inhibition of GTH-2 secretion in a number of teleosts, including the rainbow trout [21, 22]. In this species, it is known that a group of hypophysiotropic dopaminergic neurons in the anterior ventral preoptic region maintains GTH-2 secretion under an estrogen-dependent inhibitory tone throughout vitellogenesis [22]. Although more precise information is still needed, it is believed that the drop in estradiol levels at the end of vitellogenesis reduces the dopaminergic turnover in this cell population, which will cause removal of the dopaminergic inhibition [21] and the triggering of a large preovulatory surge of GTH-2 under GnRH stimulation.

In the present study, the potential expression of rtGR in GnRH neurons, preoptic dopaminergic neurons, and gonadotrophs was examined by double immunohistochemistry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Rainbow trout (Oncorhynchus mykiss) were supplied by a local fish farm (INRA, Le Drennec, Finistère, France) and kept in the laboratory in a recirculating water system at 12–15ÅC under an artificial light regime mimicking the natural photoperiod (46ÅN). The animals were fed a trout diet ad libitum. These animals were females at late stages of vitellogenesis (1 kg; n = 10).

Tissue Preparation

Animals were treated in agreement with the European Union regulation concerning the protection of experimental animals. Investigations were conducted in accordance with the Guiding Principles for the Care and use of Research Animals promulgated by the Society for the Study of Reproduction. The fish were rapidly anesthetized with phenoxyethanol (Sigma Chemical Co., St. Louis, MO; 4 ml/10 L fresh water) and perfused through the aortic bulb with a saline (0.65% NaCl) solution to eliminate the blood cells, followed by a fixative solution made of 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (0.1 M, pH 7.4). After dissection, the brain and pituitary were postfixed for 3 h in the same fixative at 4ÅC and immersed overnight at the same temperature in phosphate buffer containing 15% sucrose.

Antisera for Immunohistochemistry

To avoid potential cross-reactions between primary antibodies raised in the same species during the double staining protocols, an antiserum directed against the N-terminus portion of the rtGR fused with glutathione S-transferase was raised in guinea pig according to a procedure similar to that used to raise rtGR antibodies in rabbits [11]. Briefly, the 5' terminal fragment (496 base pairs [bp]) of the coding part of the rtGR cDNA was subcloned into a pGEX-3X expression vector (Pharmacia, Uppsala, Sweden). The vector was expressed in Escherichia coli to obtain a fusion protein consisting of glutathione S-transferase and the NH2-terminal fragment of the rtGR. Protein synthesis was induced by isopropylthiogalactoside (ICN, Costa Mesa, CA), and the resulting rtGR protein was purified by means of agarose-glutathione beads (Sigma). Guinea pigs were immunized by a first injection with the fusion protein (25 µg protein in 50 µl saline phosphate buffer) in complete Freund's adjuvant (50 µl; Gibco, Grand Island, NY), followed by a series of injections (every 3 wk for 3 mo) with the fusion protein (25 µg protein in 50 µl saline phosphate buffer) in incomplete Freund's adjuvant (50 µl; Gibco).

The characteristics of the rabbit antisera to salmon (s)GnRH [23, 24], salmon GTH-1 [17], GTH-2 [25], and those of the mouse anti-tyrosine hydroxylase (TH; Institut Jacques Boy, Reims, France; [22]) have been reported previously. When used for immunohistochemistry, the sGnRH antibodies recognize both sGnRH and chicken GnRH-II [24].

Single Staining Immunohistochemistry

Immunohistochemistry was performed on both coronal and longitudinal frozen sections (14 µm) obtained from trout brains embedded in Tissue-Tek (Bayer, Elkart, IN) and collected on gelatin-coated slides. Prior to overnight incubation with the anti-rtGR antibody, the sections were pretreated with 0.5% milk powder in Veronal buffer (0.1 M diethylmalonylurea containing 0.85% NaCl, pH 7.6) containing 0.2% Triton X-100 (TX). After exposure to the anti-rtGR antibody (1:1000), the sections were rinsed (3 times, 10 min each) in Veronal buffer containing TX and incubated with a peroxidase-coupled anti-guinea pig secondary antibody (1:200; Biosys, Compiègne, France) for 90 min. Before peroxidase visualization, the sections were rinsed twice (two times, 10 min each) in Veronal buffer with TX and once (2 min) in acetate buffer (0.1 M, pH 6).

The peroxidase-conjugated secondary antibody was visualized using the glucose oxidase method according to Shu et al. [26]. This method consists of placing the slides into 0.1 M acetate buffer (pH 6) containing ammonium nickel (II) sulfate (25 mg/ml; Sigma), glucose (2 mg/ml), ammonium chloride (0.4 mg/ml), 3,3'-diaminobenzidine (0.25 mg/ml; Sigma), and glucose oxidase (15 µg/ml; Sigma). After appearance of immunoreactivity as a dark blue precipitate, the reaction was stopped using distilled water. The slides were then mounted with phosphate buffer/glycerol (1:1), examined using an Olympus (Tokyo, Japan) Provis photomicroscope, and photographed using either Agfa (Mortsel, Belgium) Pan 25 ASA or Fujichrome (Fuji, Tokyo, Japan) Sensia 100 ASA films.

Double Staining Immunohistochemistry

In the case of sGnRH-rtGR and GTH-rtGR stainings, the slides were exposed overnight at room temperature to a combination of the two primary antibodies mixed at a final dilution of 1:1000. After washing, a mixture of Texas Red- or fluorescein-labeled anti-rabbit (Biosys; final dilution 1:200) and peroxidase-coupled guinea pig antibodies to rabbit IgG (Biosys; final dilution 1:200) was applied for 2 h at room temperature. After washing, the peroxidase activity was reacted as described above. The same field of the slides was then photographed twice under fluorescence and brightfield illumination using Ilford (Mobberley, UK) HP5 400 ASA and Agfapan 25 ASA films, respectively. In the case of the GTH-rtGR double staining, an Olympus Fluoview Confocal Microscope allowed the acquisition of the two signals on two different channels that could then be combined using the Fluoview program.

In the case of TH-rtGR, the two primary antibodies, rabbit anti-rtGR and mouse anti-TH, were mixed at a final dilution of 1:1000 and applied to the slides overnight at room temperature. After washing, the slides were exposed to peroxidase-coupled anti-rabbit IgG, which was reacted as described above, resulting in a dark blue precipitate. Peroxidase-coupled anti-mouse antibodies were then applied and reacted using the classical 3-3'-diaminobenzidine method, resulting in a brownish precipitate. Pictures were taken using Kodak (Eastman Kodak, Rochester, NY) Gold 100 ASA film. Alternatively, a combination of fluorescence and enzyme immunohistochemistry was used as described above (data not shown).

Control Reactions

The specificity of the rtGR antibodies was checked by Western blotting analysis on liver extracts or monkey COS-1 cells, either untransfected or transfected with a cytomegalovirus expression vector containing the full-length glucocorticoid receptor (GR) cDNA. Proteins were fractionated by 10% polyacrylamide-SDS gel electrophoresis and then electrotransferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). For immunodetection, the chemiluminescent substrate protein detection kit (CSPD; Tropix, Bedford, MA) was used under the conditions recommended by the manufacturer.

In addition, adjacent sections were alternatively treated with the primary antibody preincubated with the fusion protein (2 x 10-5 M), with the preimmune serum, or in the absence of primary or secondary antibody. The specificity of the GTH-1, GTH-2, TH, and GnRH antisera has been described previously [17, 23, 25].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of rtGR Antibodies in Guinea Pig

The production of glutathione S-transferase-rtGR-NH2 recombinant proteins in E. coli and their purification on glutathione-agarose beads have been reported previously [11]. The fusion protein of the expected size of 45 kDa was used for immunization of 2 guinea pigs. The resulting antisera were checked for specificity by Western blotting on rainbow trout liver cytosol extracts and on extracts from COS-1 cells transfected or not with the full-length rtGR cDNA. As shown in Figure 1, a 1:5000 dilution of antiserum 2, which was used subsequently in immunohistochemistry, was able to detect a single protein band in the liver extracts, two protein bands in the rtGR-transfected COS-1 cell extracts, and none in the untransfected control COS-1 cells. The sizes of these two protein bands were found to be 100 and 104 kDa (Fig. 1). Antiserum 1 produced similar results. The preimmune sera did not show any band on identical immunoblots (data not shown).



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  		FIG. 1. Specificity of guinea pig rtGR antibodies tested by immunoblot: the rtGR immunoserum recognized a single protein species in liver extracts (lane L) and two bands in extracts from COS-1 cells transfected with the full-length rtGR cDNA (lane Tr), whereas control COS-1 cell (lane C) did not show any positive signal. Molecular weights (on right) are x 10-3.

The results obtained with the rtGR guinea pig antiserum fully confirmed those obtained with antisera generated in rabbits [13]. The rtGR protein was widely expressed in both the telencephalon and diencephalon of vitellogenic rainbow trout (Fig. 2) and was always detected in the nucleus of the cells. In particular, rtGR-positive cells were detected in the ventral telencephalon and all subdivisions of the preoptic region, notably in the magnocellular preoptic nucleus. In the mediobasal hypothalamus, rtGR-immunoreactive cells were present in all components of the nucleus lateralis tuberis, in the nucleus recessus lateralis, in the nucleus recessus posterioris, and in the nucleus of the saccus vasculosus. In the pituitary, numerous positive cells were detected in the rostral and proximal pars distalis, while the neurointermediate lobe was devoid of immunoreactivity.



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  		FIG. 2. Diagram summarizing the distribution of rtGR-immunoreactive cells (dots) on a longitudinal section of the forebrain of the rainbow trout. Lines a, b, c indicate the transverse sections at the levels of a) the telencephalon, b) the preoptic region, and c) the mediobasal hypothalamus. Arrows with numbers point to the areas shown on Figures 3, 4, and 5, respectively. Dc, Area dorsalis telencephali pars centralis; Dd, area dorsalis telencephali pars dorsalis; DI, area dorsalis telencephali pars lateralis; MT, tegmentum of the midbrain; NDLI, nucleus diffusus lobi inferioris; NDTL, nucleus diffusus tori lateralis; NH, nucleus habenularis; NLTp, nucleus lateralis tuberis pars posterior; NPOav, nucleus preopticus pars anteroventralis; NPOmc, nucleus preopticus pars magnocellularis; NPOpc, nucleus preopticus pars parvocellularis; NVM, nucleus ventromedialis thalami; OB, olfactory bulb; OC, optic chiasma; OT, optic tectum; pit, pituitary gland; rl, lateral recess; rp, posterior recess; VC, valvula cerebelli; Vd, area ventralis telencephali pars dorsalis; Vv, area ventralis telencephali pars ventralis.

A Subset of GnRH Neurons Expressed rtGR

In the forebrain of rainbow trout, sGnRH-positive neurons are observed in the ventral olfactory bulbs, the ventral telencephalon, the anteroventral preoptic area, and the anterior hypothalamus [24, 27]. Based on the respective localization of the two antigens, potential colocalization of sGnRH and rtGR could be expected in the ventral telencephalon and the anterior preoptic region. In the caudal preoptic region, GnRH neurons are located lateral to the rtGR-expressing cells.

Accordingly, double staining carried out in vitellogenic females, using guinea pig antibodies to rtGR and rabbit antibodies to sGnRH, showed that a majority of GnRH neurons of the caudal ventral telencephalon and anterior preoptic region were simultaneously stained by the two antibodies. As the sGnRH staining was restricted to the perikarya, it could easily be distinguished from the nuclear localization of the rtGR (Fig. 3).



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  		FIG. 3. Double staining of longitudinal sections treated with the guinea pig anti-rtGR antibodies (a and c: immunoperoxidase) and the rabbit anti-sGnRH antibodies (b and d: immunofluorescence). Pairs a–b and c–d correspond to two different areas photographed twice and show that nuclei of sGnRH-immunoreactive neurons (large white arrows) are rtGR-immunopositive (large black arrows). Arrowheads point to rtGR-immunopositive nuclei corresponding to other unidentified cells. Bar = 25 µm.

None of the sGnRH neurons of the olfactory bulbs, anterior telencephalon, caudal preoptic area, and midbrain tegmentum were found to express rtGR.

TH Neurons of the Preoptic Region Expressed rtGR

The ventral wall of the preoptic recess, above the optic chiasma, contains a discrete population of TH-expressing cell bodies corresponding to the nucleus preopticus pars anteroventralis [22]. More caudally, TH cell bodies are located in a subependymal position in the nucleus anterioris periventricularis. Double staining studies indicated that both populations were immunoreactive for rtGR, and the nuclear staining of the rtGR could be unambiguously distinguished from the cytoplasmic staining of TH. However, there was a striking difference between the weak rtGR staining of the TH cells in the nucleus anterioris periventricularis and the strong rtGR labeling in the TH-positive cells of the nucleus preopticus pars anteroventralis. In this latter location, virtually 100% of the TH neurons expressed the rtGR (Fig. 4).



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  		FIG. 4. a) Double staining of a transverse section at the level of the anterior part of the preoptic recess (rpo), showing (in purple) the distribution of the rtGR-immunoreactive cell nuclei and (in brown) that of TH-positive cell bodies and fibers. Note that the rtGR staining is stronger in the nucleus preopticus pars anteroventralis (NPOav) and overlaps perfectly with the distribution of TH-immunoreactive cells, whereas the rtGR labeling is weaker in the nucleus preopticus pars parvicellularis (NPOpc), which lacks TH immunoreactivity. Bar = 100 µm. b) High-power view of the area framed in a showing that virtually all TH-immunoreactive neurons (brown staining) exhibit rtGR-immunostaining in the nucleus (purple staining). Arrows point to clearly double-stained neurons. Bar = 10 µm.

Gonadotrophs Expressed rtGR

In rainbow trout, the gonadotrophs, either GTH-1 and GTH-2 cells, are located in the proximal pars distalis, which exhibited consistently a large number of rtGR-positive cells. Double stainings indicated that a large majority, if not the totality, of both GTH-1 and GTH-2 cells are also rtGR-positive in these vitellogenic females (Fig. 5). Occasionally, rtGR-positive cells were also detected in the rostral pars distalis, but double staining studies indicated that these cells were either GTH-2 cells or corticotrophs (data not shown) and did not correspond to prolactin cells.



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  		FIG. 5. a) Double staining at the level of the pituitary showing that most GTH-1 cells (labeled in green with a rabbit anti-GTH-1 immunoserum) have a rtGR-positive nucleus (labeled in black). Simultaneous dual-channel confocal microscopy (fluorescein isothiocyanate [FITC] + interferential contrast). NH: neurohypophysis. Bar = 25 µm. b) Double staining at the level of the pituitary showing that most GTH-2 cells (labeled in green with a rabbit anti-GTH-2 immunoserum) have a rtGR-positive nucleus (labeled in black). Simultaneous dual-channel confocal microscopy (FITC + interferential contrast). Bar = 25 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides further information on the central expression of GR in a teleost species and demonstrates that the main neuronal populations involved in the regulation of GTH-2 secretion are likely to be regulated by cortisol in rainbow trout. In addition, we show that both GTH-1 and GTH-2 cells strongly express rtGR. Although the precise molecular targets of cortisol in these different cell populations are presently unknown, these results open new avenues for future investigations.

Similar to what was already reported for the antibodies raised in rabbit, those obtained in guinea pigs allowed the identification, by Western blotting, of two protein species of 100 and 104 kDa in COS-1 cells transfected with the rtGR cDNA. The sizes of these denaturated proteins are larger than the expected value of 83 kDa deduced from the amino acid sequence of the cloned rtGR cDNA [10]. The difference can be explained by the important posttranslational maturation of the proteins such as phosphorylation and/or glycosylation as already described for other nuclear receptors [28]. The guinea pig antibodies resulted in an excellent background/staining ratio and revealed an overall pattern of distribution similar to that previously reported with the rabbit antibodies [13].

With respect to GnRH neurons, our results clearly show that in these vitellogenic females, a large majority (at least 70%) of the sGnRH-producing neurons in the ventro-caudal telencephalon and anterior preoptic region express rtGR. Results in rats have already shown that, in the homologous regions, medial septum-lateral band of Broca and preoptic area, a subset of GnRH neurons colocalized nuclear GR [29], providing evidence that GR could directly regulate GnRH gene expression and thus modulate the hypothalamic-pituitary-gonadal axis. More recently, it has been shown in immortalized GnRH-secreting cell lines, which express a functional GR, that dexamethasone represses both the endogenous GnRH gene, decreasing the levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors [30]. Further studies on the mouse GnRH promoter have identified negative glucocorticoid-responsive elements (GRE) that bind a multiproteic complex involving the GR and are required for the repressive effects of glucocorticoids on GnRH expression [31]. In addition to altering GnRH expression, it has also been shown that dexamethasone affects GnRH secretion from GT1–7 cells [32]. Our morphological results, together with the fact that the promoter of GnRH has been shown to contain putative GRE in the Atlantic salmon [33], Masu salmon [34], and the stripped bass [35], indicate that the conditions exist for a potential regulation of GnRH expression by glucocorticoids in teleosts. A recent study in the African cichlid (Haplochromis burtoni) has shown a relationship between social status, cortisol levels, and size of GnRH perikarya in the preoptic area [36], indicating that GnRH expression might be one of the factors involved in the suppression of reproductive maturation in subordinate versus dominant fish in territorial species. Effects of dominance status on sex hormone levels have also been reported in laboratory and wild-spawning trout [37], but GnRH expression was not monitored in this latter study. Although further studies will be required to dissect the molecular mechanisms potentially implicating GR in sGnRH expression in fish, it is likely that such interactions have been conserved throughout the vertebrate lineage, indicating that they are functionally important. This alone could explain some of the negative effects of stress and/or cortisol on the reproductive performance of salmonids [5, 8]. Our results also show that the large GnRH-immunoreactive neurons of the midbrain that express chicken GnRH-II [24, 27] do not express rtGR, further indicating that these cells represent another set of GnRH neurons whose functions remain to be established.

A second potential cortisol target evidenced in this study concerns the TH neurons of the nucleus preopticus pars anteroventralis. There is strong evidence that these neurons are the source of the dopamine that inhibits GTH-2 secretion in both goldfish and rainbow trout [14, 22, 38]. First, electrolytic lesions of this particular area in goldfish cause a large increase in GTH-2 plasma level and ovulation in female goldfish [39] associated with the disappearance of the TH-positive fibers in the anterior lobe of the pituitary [38]. Second, retrograde tracing studies in goldfish [40], rainbow trout [22], and Atlantic salmon [41] have clearly indicated that neurons in this area are hypophysiotropic. Furthermore, in Atlantic salmon, retrograde studies combined with TH immunohistochemistry have unambiguously demonstrated that these TH neurons are hypophysiotropic [41]. In goldfish [38], rainbow trout [22], and Atlantic salmon [41], it is also known that these TH-positive neurons are dopaminergic. Extensive studies in goldfish and rainbow trout have demonstrated the inhibitory role of dopamine on GTH-2 secretion, and the role of estradiol in modulating this inhibitory tone is also well documented [15, 22]. In rainbow trout, there is evidence that the intensity of the dopaminergic inhibition is in direct relationship with the levels of circulating estradiol and that the drop in estradiol at the end of vitellogenesis is one of the signals triggering the preovulatory GTH-2 surge [21, 42]. As it has been shown that, in vitellogenic females, estrogen receptors are expressed in about 70% of the TH cells of this nucleus [22], it is believed that these cells represent the morphological substrate of the estrogen-dependent dopaminergic inhibition of GTH-2 secretion. After the cloning of a full-length cDNA of TH from rainbow trout [43], we have recently shown, by in situ hybridization, the presence of TH mRNA in this cell population (unpublished results and personal communication with T. Bailhache). In mammals, extensive studies have documented the presence of GR immunoreactivity [44] in catecholaminergic neurons [45], and it is well known that glucocorticoids and/or dexamethasone increase the steady-state levels of TH mRNA under various stress conditions in vivo and in vitro [46–48]. However, the precise sites for GR binding on the TH promoter remain unidentified, raising the possibility that multiple elements on the TH gene act in a cooperative fashion to promote transcriptional regulation by glucocorticoids [46–48]. Therefore, as TH is the rate-limiting enzyme for catecholamine synthesis, it is possible that cortisol increases the TH transcription and ultimately the dopamine stores in the ventral preoptic area. This could eventually lead in rainbow trout to increased dopaminergic inhibition of GTH-2 secretion, although this remains to be demonstrated.

The present study also shows that virtually all GTH-1 and GTH-2 cells express rtGR in vitellogenic female rainbow trout, indicating that those cells are potentially subject to direct cortisol regulation. Similar observations have been reported in the rat pituitary gland, in which a large majority of both FSH and LH cells were shown to exhibit GR immunoreactivity [49]. Such results may account for some of the known physiological effects of glucocorticoids on GTH synthesis and release. Although it has been shown in vivo that glucocorticoids inhibit both spontaneous and GnRH-stimulated LH secretion [50, 51], the opposite results were obtained in vitro [52]. Glucocorticoids were also shown to inhibit GnRH-induced LH{alpha} and LHß mRNA transcription, but not that of FSHß [50]. At the present stage, the effects of glucocorticoid on GTH synthesis and release in fish are poorly documented, although there is indication of lower GTH content under stressful conditions in salmonids [8]. On the other hand, late in maturation, stress is also known to advance spawning in trout [53]. It has been suggested that accelerated ovulation under stressful conditions late in maturation can occur as a switch in tactic for reproductive strategy when environmental conditions are unstable [53, 54].

The distribution of the rtGR clearly overlaps that of the estrogen receptor in the brain and pituitary of the trout [55, 56]. This work also indirectly demonstrates that estrogen and GRs coexist in some hypothalamic cells, such as the TH neurons of the anterior preoptic region, and in the GTH-2 cells, indicating that potential interactions between these two receptors may occur in these cell populations. In the liver of rainbow trout, the estrogen receptor gene is positively regulated by estradiol [57], and it is known that both basal and estradiol-stimulated expression of the estrogen receptor in the liver are inhibited by dexamethasone—an effect that is likely to take place at the transcriptional level (unpublished results). Therefore, it is possible that glucocorticoids also repress estrogen receptor expression at both central and pituitary levels, which could eventually interfere with the expression of estradiol-regulated genes. Such transcriptional interferences between endogenous steroid receptors on the expression of estrogen-dependent genes have already been documented in MCF-7 cells in the case of either progesterone or glucocorticoid receptors, an effect probably due to the fact that endogenous steroid receptors compete for factors that mediate their enhancer function [58]. As estrogen receptor is required for the expression of the ß subunit of GTH-2 in salmonids [59], such interactions could result in decreased GTH-2 synthesis under stressful conditions. Cortisol-induced inhibition of estrogen receptor and vitellogenin expression in the liver could explain the reduced quality of gametes observed in stressed rainbow trout [60].

In conclusion, this study confirms the widespread expression of rtGR in the brain-pituitary complex of the rainbow trout and shows that neurons and pituitary cells involved in the control of the reproductive axis are likely to be targets for cortisol. It is well known that the effects of steroid hormone receptors are directly linked to the intracellular receptor concentration [61]. Therefore, the fact that rtGR immunoreactivity in the various target cells studied in this work is quite robust tends to indicate that these receptors are likely to induce significant physiological effects in those cells. Although it was initially believed that DNA binding through GRE was a prerequisite for GR-mediated genomic actions, there is growing evidence that many of the GR effects are mediated by protein-protein interactions [62]. This is notably the case for the transrepression of GR on AP-1 driven genes [63]. Therefore, the present study opens new research avenues for future work aimed at investigating at the molecular level the role of glucocorticoids on the expression of genes involved in the regulation of reproductive functions.


   ACKNOWLEDGMENTS
 
We thank Dr. Penny Swanson for the gift of salmon GTH-1 antibodies. The assistance of L. Communier and J. Chauvin in preparing the illustrations is greatly appreciated.


   FOOTNOTES
 
1 Supported by the European Union (FAIR GT 95–2549), the CNRS, the INRA, and the Fondation Langlois. Back

2 Correspondence. FAX: 33 2 99 28 67 94; olivier.kah{at}univ-rennes1.fr Back

Accepted: October 13, 1998.

Received: July 22, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Negro-Vilar A. Stress and other environmental factors affecting fertility in men and women: overview. Environ Health Perspect 1993; 101:59–64.
  2. Clarke IJ, Hemsworth PH, Barnett JL, Tilbrook AJ. Stress and reproduction in farm animals. In: Sheppard KE, Boublik JH, Funder JW (eds.), Stress and Reproduction, Seporo Symposia NÅ 86. New York: Raven Press; 1993: 239–251.
  3. Rivier C, Rivest S. Effect of stress on the activity of hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms. Biol Reprod 1991; 45:523–532.[Abstract]
  4. Rosen H, Jameel ML, Barkan AL. Dexamethasone suppresses gonadotrophin-releasing hormone (GnRH) secretion and has direct pituitary effects in male rats: differential regulation of GnRH receptor and gonadotropin response to GnRH. Endocrinology 1988; 122:2873–2880.[Abstract/Free Full Text]
  5. Wendelaar-Bonga SE. The stress response in fish. Physiol Rev 1997; 77:591–625.[Abstract/Free Full Text]
  6. Schreck CB. Stress and rearing of salmonids. Aquaculture 1982; 28:241–249.
  7. Pickering AD, Pottinger TG, Sumpter JP. The effects of acute and chronic stress on the levels of reproductive hormones in the plasma of mature male brown trout, Salmo trutta. Gen Comp Endocrinol 1987; 48:249–259.
  8. Carragher JF, Sumpter JP, Pottinger TG, Pickering AD. The deleterious effects of cortisol implantation in two species of trout, Salmo trutta L. and Salmo gairdneri Richardson. Gen Comp Endocrinol 1989; 76:310–321.[CrossRef][Medline]
  9. Pankhurst NW, van der Kraak G, Peter RE. Evidence that the inhibitory effects of stress on reproduction in teleost fish are not mediated by the action of cortisol on ovarian steroidogenesis. Gen Comp Endocrinol 1995; 99:249–257.[CrossRef][Medline]
  10. Ducouret B, Tujague M, Ashraf J, Mouchel N, Servel N, Valotaire Y, Thomsson EB. Cloning of a teleost fish glucocorticoid receptor shows that it contains a DNA binding domain different from that of mammals. Endocrinology 1995; 136:3774–3783.[Abstract]
  11. Tujague M, Saligaut D, Teitsma C, Kah O, Valotaire Y, Ducouret B. Rainbow trout glucocorticoid receptor overexpression in Escherichia coli: production of antibodies for western blotting and immunohistochemistry. Gen Comp Endocrinol 1998; 110:201–211.[CrossRef][Medline]
  12. Teitsma C, Bailhache B, Anglade I, Balment B, Ducouret B, Kah O. Distribution of glucocorticoid receptor mRNA in the diencephalon of the rainbow trout. Neuroendocrinology 1997; 66:294–304.[Medline]
  13. Teitsma CA, Anglade I, Toutirais G, Muñoz-Cueto JA, Saligaut D, Ducouret B, Kah O. Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss). J Comp Neurol 1998; (in press).
  14. Kah O, Anglade I, Leprêtre E, Dubourg P, de Monbrison D. The reproductive brain in fish. Fish Physiol Biochem 1993; 11:85–98.[CrossRef]
  15. Trudeau VL. Neuroendocrine regulation of gonadotrophin II release and gonadal growth in the goldfish, Carassius auratus. Rev Reprod 1997; 2:55–68.[Abstract]
  16. Suzuki K, Kawauchi H, Nagahama Y. Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands. Gen Comp Endocrinol 1988; 71:292–301.[CrossRef][Medline]
  17. Naito N, Hyodo S, Okumoto N, Urano A, Nakai Y. Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout, Oncorhynchus mykiss, during ovarian development. Cell Tissue Res 1991; 266:457–467.[CrossRef]
  18. Weil C, Bougoussa-Houadec M, Gallais C, Itoh S, Sekine S, Valotaire Y. Preliminary evidence suggesting variations of GtH 1 and GtH 2 mRNA levels at different stages of gonadal development in rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1995; 100:327–333.[CrossRef][Medline]
  19. Prat F, Sumpter JP, Tyler CR. Validation of radioimmunoassays for two salmon gonadotropin (GtH I and GtH II) and their plasma concentrations throughout the reproductive cycle in male and female rainbow trout (Oncorhynchus mykiss). Biol Reprod 1996; 54:1375–1382.[Abstract]
  20. Ge W, Peter RE. Evidence for non-steroidal gonadal regulator(s) of gonadotropin release in the goldfish, Carassius auratus. Zool Sci 1994; 11:717–724.
  21. Linard B, Bennani S, Saligaut C. Involvement of estradiol in a catecholamine inhibitory tone of gonadotropin release in the rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 1995; 99:192–196.[CrossRef][Medline]
  22. Linard B, Anglade I, Navas JM, Corio M, Jego P, Saligaut C, Kah O. Coexistence of tyrosine hydroxylase- and estrogen receptor-immunoreactivity in a subset of neurons in the preoptic region of the rainbow trout. Neuroendocrinology 1996; 63:156–165.[Medline]
  23. Kah O, Zanuy S, Pradelles P, Cerdá J, Carrillo M. An immunoenzymoassay for salmon GnRH and its application to the study of the effects of diet on brain and pituitary GnRH in the sea bass (Dicentrarchus labrax). Gen Comp Endocrinol 1994; 95:464–474.[CrossRef][Medline]
  24. Navas JM, Anglade I, Bailhache T, Pakdel F, Breton B, Jégo P, Kah O. Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study. J Comp Neurol 1995; 363:461–474.[CrossRef][Medline]
  25. Govoroun MS, Huet JC, Pernollet JC, Breton B. Use of immobilized metal ion affinity chromatography and dye ligand chromatography for the separation and purification of rainbow trout pituitary gonadotropins, GTH I and GTH II. J Chromatogr 1997; 698:35–46.
  26. Shu S, Ju G, Fan L. The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 1988; 85:169–171.[CrossRef][Medline]
  27. Bailhache T, Arazam A, Klungland H, Aleström P, Breton B, Jego P. Localization of salmon GnRH mRNA and peptide in the brain of Atlantic salmon and rainbow trout. J Comp Neurol 1994; 347:444–454.[CrossRef][Medline]
  28. Orti E, Bodwell JE, Munk A. Phosphorylation of steroid hormone receptors. Endocr Rev 1992; 13:105–128.[Abstract/Free Full Text]
  29. Ahima RS, Harlan RE. Glucocorticoid receptors in LHRH neurons. Neuroendocrinology 1992; 56:845–850.[Medline]
  30. Chandran UR, Attardi B, Friedman R, Dong KW, Roberts JL, DeFranco DB. Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1 hypothalamic cell line. Endocrinology 1994; 134:1467–1474.[Abstract/Free Full Text]
  31. Chandran UR, Attardi B, Friedman R, Zheng ZW, Roberts JL, DeFranco DB. Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor. J Biol Chem 1996; 271:20412–20420.[Abstract/Free Full Text]
  32. Attardi B, Tsuji T, Friedman R, Zeng Z, Roberts JL, Dellovade T, Pfaff DW, Chandran UR, Sullivan MW, DeFranco DB. Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations. Mol Cell Endocrinol 1997; 131:241–255.[CrossRef][Medline]
  33. Klungland H, Lorens JB, Andersen O, Kisen GO, Alestrom P. The atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA. Mol Cel Endocrinol 1992; 84:167–174.[CrossRef][Medline]
  34. Higa M, Kitahashi T, Sasaki Y, Okada H, Ando H. Distinct promoter sequences of two precursors genes for salmon gonadotropin-releasing hormone in masu salmon. J Mol Endocrinol 1997; 19:149–161.[Abstract/Free Full Text]
  35. Chow MM, Kight KE, Gothilf Y, Alok D, Stubblefield J, Zohar Y. Multiple GnRHs present in a teleost species are encoded by separate genes: analysis of the sbGnRH and cGnRH-II genes from the striped bass, Morone saxatilis. J Mol Endocrinol 1998; (in press).
  36. Fox HE, White SA, Kao HF, Fernald RD. Stress and dominance in social fish. J Neurosci 1997; 17:6463–6469.[Abstract/Free Full Text]
  37. Cardwell JR, Sorensen PW, Van der Kraak GJ, Liley NR. Effect of dominance status on sex hormone levels in laboratory and wild-spawning male trout. Gen Comp Endocrinol 1996; 101:333–341.[CrossRef][Medline]
  38. Kah O, Dulka J, Dubourg P, Thibault J, Peter RE. Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldfish: existence of a dopaminergic preoptico-hypophyseal pathway. Neuroendocrinology 1987; 45:451–458.[Medline]
  39. Peter RE, Paulencu C. Involvement of the preoptic region in gonadotropin release inhibition in the goldfish, Carassius auratus. Neuroendocrinology 1980; 31:337–346.
  40. Anglade I, Zandbergen AM, Kah O. Origin of the pituitary innervation in the goldfish. Cell Tissue Res 1993; 273:345–355.[CrossRef]
  41. Holmqvist BI, Ekström P. Hypophysiotrophic systems in the brain of the Atlantic salmon: neuronal innervation of the pituitary and the origin of pituitary dopamine and nonapeptides identified by means of combined carbocyanine tract tracing and immunocytochemistry. J Chem Neuroanat 1995; 8:122–145.
  42. Kah O, Anglade I, Linard B, Pakdel F, Salbert G, Bailhache T, Ducouret B, Saligaut C, Le Goff P, Valotaire Y, Jégo P. Estrogen receptors in the brain-pituitary complex and the neuroendocrine control of gonadotrophin release in rainbow trout. Fish Physiol Biochem 1997; 17:53–62.[CrossRef]
  43. Linard B, Pakdel F, Marmignon MH, Saligaut C. Cloning of a cDNA coding for tyrosine hydroxylase in the rainbow trout (Oncorhynchus mykiss): comparison with other hydroxylases and enzymatic expression. J Neurochem 1998; (in press).
  44. Fuxe K, Wikström AC, Okret S, Agnati L, Härfstrand A, Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson JA. Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Endocrinology 1985; 117:1803–1812.[Abstract/Free Full Text]
  45. Härfstrand A, Fuxe K, Cintra A, Agnati L, Zini I, Wikström AC, Okret S, Yu ZY, Goldstein M, Steinbusch H, Verhofstadt A, Gustafsson JA. Glucocorticoid receptor immunoreactivity in monoaminergic neurons of rat brain. Proc Natl Acad Sci USA 1986; 83:9779–9783.[Abstract/Free Full Text]
  46. Lewis EJ, Harrington CA, Chikaraishi DM. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP. Proc Natl Acad Sci USA 1987; 84:3550–3554.[Abstract/Free Full Text]
  47. Fossom LH, Sterling CR, Tank AW. Regulation of tyrosine hydroxylase gene transcription rate and tyrosine hydroxylase mRNA stability by cyclic AMP and glucocorticoid. Mol Pharmacol 1992; 42:898–908.[Abstract]
  48. Kim KT, Park DH, Joh TH. Parallel up-regulation of catecholamine biosynthetic enzymes by dexamethasone in PC12 cells. J Neurochem 1993; 60:946–951.[CrossRef][Medline]
  49. Kononen J, Honkaniemi J, Gustafson JA, Pelto-Huikko M. Glucocorticoid receptor colocalization with pituitary hormones in the rat pituitary gland. Mol Cel Endocrinol 1993; 93:97–103.[CrossRef][Medline]
  50. Rosen H, Dalkin A, Haisenleder D, Friberg RD, Ortolano G, Barkan A. Dexamethasone alters responses of pituitary gonadotropin-releasing hormone (GnRH) receptors, gonadotropin subunit messenger ribonucleic acids, and gonadotropins to pulsatile GnRH in male rats. Endocrinology 1991; 128:654–660.[Abstract/Free Full Text]
  51. Briski KP, Sylvester PW. Antagonism of type II, but not type I glucocorticoid receptors results in elevated basal luteinizing hormone release in male rats. Neuroendocrinology 1994; 60:601–608.[Medline]
  52. Baldwin DM, Srivastava PS, Krummen LA. Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol. Biol Reprod 1991; 44:1040–1050.[Abstract]
  53. Contreras-Sanchez WN, Schreck CB, Fitzpatrick MS, Pereira CB. Effects of stress on the reproductive performance of rainbow trout (Oncorhynchus mykiss). Biol Reprod 1998; 58:439–447.[Abstract/Free Full Text]
  54. Wooton RJ. Introduction: tactics and strategies in fish reproduction. In: Potts GW, Wootton RJ (eds.), Fish Reproduction: Strategies and Tactics. London: Academic Press; 1984: 1–12.
  55. Salbert G, Bonnec G, Le Goff P, Boujard D, Valotaire V, Jego P. Localization of the estradiol mRNA in the forebrain of the rainbow trout. Mol Cell Endocrinol 1991; 78:173–180.
  56. Anglade I, Pakdel F, Bailhache T, Petit F, Valotaire Y, Jego P, Kah O. Distribution of estrogen receptor-immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss). J Neuroendocrinol 1994; 6:573–583.[CrossRef][Medline]
  57. Flouriot G, Pakdel F, Ducouret B, Le Dréan Y, Valotaire Y. Differential regulation of two genes implicated in fish reproduction: vitellogenin and estrogen receptor genes. Mol Reprod Dev 1997; 48:317–323.[CrossRef][Medline]
  58. Meyer ME, Gronemeyer H, Turcotte B, Bocquel MT, Tasset D, Chambon P. Steroid hormone receptors compete for factors that mediate their enhancer function. Cell 1989; 57:433–442.[CrossRef][Medline]
  59. Le Dréan Y, D Liu D, Wong AOL, Xiong F, Hew CL. Steroidogenic factor 1 and estradiol receptor act in synergism to regulate the expression of the salmon gonadotropin IIß subunit gene. Mol Endocrinol 1996; 10:217–229.[Abstract/Free Full Text]
  60. Campbell PM, Pottinger TG, Sumpter JP. Stress reduces the quality of gametes produced by rainbow trout. Biol Reprod 1992; 47:1140–1150.[Abstract]
  61. Vanderbilt JN, Miesfield R, Maler BA, Yamamoto KR. Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity. Mol Endocrinol 1987; 1:68–74.[Abstract/Free Full Text]
  62. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Shütz G. DNA binding of the glucocorticoid receptor is not essential for survival. Cell 1998; 93:531–541.[CrossRef][Medline]
  63. Karin M. New twist in gene regulation by glucocorticoid receptor: is DNA binding indispensable? Cell 1998; 93:487–490.



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