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Endocrinology Vol. 140, No. 3 1228-1235
Copyright © 1999 by The Endocrine Society

ARTICLES

Cortisol Selectively Stimulates Pituitary Gonadotropin ß-Subunit in a Primitive Teleost, Anguilla anguilla1

Yung-Sen Huang, Karine Rousseau, Miskal Sbaihi, Nadine Le Belle, Monika Schmitz and Sylvie Dufour

Laboratoire de Physiologie Générale et Comparée (Y.-S.H., K.R., M.S., N.L.B., S.D.), Muséum National d’Histoire Naturelle, URA 90, Centre National de la Recherche Scientifique, 75231 Paris Cedex 05, France; and the Department of Aquaculture, Swedish University of Agriculture (M.S.), 90183 Umea, Sweden

Address all correspondence and requests for reprints to: Dr. Sylvie Dufour, Laboratoire de Physiologie Générale et Comparée, Muséum National d’Histoire Naturelle (MNHN), URA 90, Centre National de la Recherche Scientifique, 7 rue Cuvier, 75231 Paris Cedex 05, France. E-mail: dufour{at}mnhn.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that in mammals, glucocorticoids, beside their stress-related inhibitory effects on reproductive function, may also play a stimulatory role at the onset of puberty. Using the juvenile female eel as a model, we investigated the potential stimulatory role of cortisol (F) on pituitary gonadotropin (GtH-II). GtH-II levels were measured by RIA, and messenger RNA (mRNA) levels for {alpha}- and GtH-II ß-subunits were determined by dot blot using homologous probes. F treatment increased eel pituitary GtH-II content in vivo and in vitro. Using a long term, serum-free primary culture of pituitary cells, we studied the direct effect of F on GtH-II production. F increased the GtH-II cellular content in vitro in a dose- and time-dependent manner. The relative potencies of various corticosteroids on GtH-II were: triamcinolone acetonide > dexamethasone > F >> cortisone and aldosterone, indicating a glucocorticoid-specific receptor (GR). F stimulated GtH-II production through a selective increase in mRNA levels for GtH-II ß-subunit; no significant effect was observed on {alpha}-subunit mRNA levels. This stimulatory effect of F on GtH-II ß, played out directly at the pituitary cell level, recalls that of F on FSHß in the rat. The present study, performed in a primitive teleost at the juvenile stage, suggests that the role of F in the positive regulation of gonadotropins at puberty may have arisen early in vertebrate evolution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISRUPTION of reproductive function is a well known consequence of chronic stress in mammals (1). Glucocorticoids [cortisol (F) or corticosterone (B)] are regarded as the main mediators for the response characteristic of adaptations to chronic stress. Both stress and excess glucocorticoids have been demonstrated to suppress LH secretion and inhibit ovulation in rats and monkeys and to delay sexual maturation in rats (1). F has been shown to block basal or GnRH-induced LH release in the rat in vivo (2, 3) and in vitro (4). Furthermore, glucocorticoids have been shown to inhibit gonadal steroidogenesis (5) and suppress GnRH release in the rat (6).

In fish, F is the main corticosteroid produced by the interrenals and plays the roles of both gluco- and mineralocorticosteroids seen in higher vertebrates (7, 8). As in mammals, an elevation of plasma F is the main indicator of stress in fish, and high levels of F have been shown to reduce growth rate, reproduction, and immune function in teleosts (9). In the case of reproductive function, implantation of F had deleterious effects on gonad weight and vitellogenin and steroid levels in two species of trout (Salmo trutta andOncorhynchus mykiss) (10). Acute and chronic stress have been shown to concomitantly increase plasma F and depress sex steroid levels and reproductive performance in the trout, Salmo trutta (11), and in the sparid fish, Pagrus auratus (12). In vitro data also showed a dose-dependent inhibitory effect of F on ovarian steroidogenesis in the trout, Oncorhynchus mykiss (13).

However, glucocorticosteroids are not always associated with negative effects on reproduction. It has been proposed that adrenal steroids may play an important positive role in determining the onset of puberty in mammals, including humans (1, 14). Indeed, during the process of sexual maturation in mammals, the adrenals are the first glands to be activated (15), and in normal puberty, adrenarche and gonadal maturation are closely related (14). On the one hand, administration of glucocorticoids has been shown to advance the onset of puberty in the female rat (16). On the other hand, adrenalectomy induced a delay in puberty, which was corrected by B replacement in the female rat (17). The delay of puberty may result from the reduction of FSH levels induced by adrenalectomy (1, 18). The synthesis of FSH, but not LH, is selectively stimulated by F and B in vivo or in vitro by a direct effect at the pituitary level in rats of both sexes (2, 3, 4, 19, 20, 21, 22). Furthermore, F stimulated basal and GnRH-induced FSH release, whereas basal or GnRH-induced LH release was inhibited by F in vitro (4).

In teleosts, as in mammals, besides their stress-related inhibitory effects, glucocorticoids have also been shown to stimulate reproductive function. In sexually mature fish, glucocorticoids can induce or enhance oocyte maturation (23). In Pacific salmon (Oncorhynchus spp.), sexual maturation is accompanied by interrenal hyperplasia and increased F secretion (24). In juvenile fish, F implantations or injections increased the pituitary content of gonadotropin-II (GtH-II; teleost gonadotropin homologous to tetrapod LH) (25). This has been shown in the rainbow trout (26) and in the European eel (Anguilla anguilla) (27). These data suggest a possible positive role of glucocorticoids on gonadotropic function during the first sexual maturation ("puberty") in fish as in mammals. However, until now no data are available in teleosts concerning the possible direct effect of F on the production of gonadotropins.

In the present study, we looked for a possible direct pituitary effect of F on the production of GtH-II in the European eel. This fish has a striking life-cycle, with a long period of juvenile growth and a prepubertal blockade, related to a low production of GtH-II, before reproductive migration (28). This animal model has proven particularly appropriate for experimental studies of stimulation of GtH-II production (27, 29, 30). Furthermore, the phylogenetic position of the eel, a member of the group Elopomorphs, which is considered to be an early emerging group among teleosts, may provide information on ancestral endocrine regulatory processes in the vertebrates (31).

To study the direct pituitary effect of F on eel GtH-II synthesis, we used a long term, serum-free system of primary cultures of eel pituitary cells (29). The effects of F on GtH-II were followed both at the protein GtH-II level by RIA of GtH-II cell content and release and at the messenger RNA (mRNA) level by dot blot using homologous complementary DNA (cDNA) probes for eel {alpha}- and GtH-II ß-subunits. The effects of various corticosteroids were compared to test the specificity of F action. In addition, the ability of F to stimulate GtH-II was validated by in vivo treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Juvenile female European eels, Anguilla anguilla L. (100–200 g BW), were netted in ponds in the north and west of France. The animals were transferred to the laboratory and kept in running aerated freshwater for a short time (1 to a few weeks) until experimentation. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Hormones
F, cortisone (E), aldosterone, dexamethasone (Dex), triamcinolone acetonide (9{alpha}-fluoro-11ß,16{alpha},17{alpha},21-tetrahydroxy-1,4-pregnadiene-3,20- dione 16,17-acetonide; TA), testosterone (T), and estradiol (E2) were obtained from Sigma Chemical Co. (Saint-Quentin Fallavier, France).

In vivo F treatment
Fish received three perivisceral injections per week of F (2 µg/g BW) suspended in saline (0.15 M NaCl) or of saline alone (controls) for 1 month. The day after the last injection, eels were killed by decapitation. Sera were obtained from blood samples allowed to clot overnight. Pituitaries were quickly removed and frozen until extraction.

Primary cultures of eel pituitary cells and in vitro steroid treatments
Dispersion of pituitary cells were performed using an enzymatic and mechanical procedure as previously described (32). About 100 eels were used for each experiment. The number of viable cells, as ascertained by trypan blue (Sigma Chemical Co.) exclusion test, represented more than 90% of the total dispersed cells. Cells were cultured on poly-L-lysine (Sigma Chemical Co.)-precoated plates in serum-free culture medium (CM; medium 199 with Earle’s salt, sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone; Life Technologies, Cergy-Pontoise, France) at 18 C under 3% CO2 and saturated humidity, according to the method of Huang et al. (29). Cells were plated on 96-well plates (Costar, Cambridge, MA) at a density of 62,500 cells/well for measuring GtH-II or GH by RIA or on 12-well plates at a density of 500,000 cells/well for measuring mRNAs by dot blot. After 1 day of culture to allow cell attachment, medium was changed, and steroid treatments were started (day 0).

The effects of various natural steroids and synthetic glucocorticoids were tested. Stock solutions of steroids (10-3 M) were prepared in 100% ethanol and kept at 4 C; they were further diluted with CM just before culture medium renewal. The final concentration of ethanol in culture wells never exceeded 0.2%; control wells were treated with the same concentration of ethanol in CM. Media were collected, and treatments were renewed every third day for up to 12 days. Media were kept frozen (-20 C) until RIA of GtH-II or GH. Cultures were stopped before the addition of hormones (day 0) and at various times of culture up to 12 days, according to the experiment, for measuring GtH-II or GH cellular content or mRNA levels for GtH-II subunits.

RIAs of GtH-II and GH
GtH-II pituitary contents from in vivo experiments were extracted by sonication (Bioblock Scientific, Illkirch, France) in 0.15 M NaCl. GtH-II or GH cellular contents from in vitro experiments were obtained by submitting cells to osmotic shock (distilled water) and two repeated cycles of freezing and thawing (29, 33).

GtH-II levels from pituitary extracts, sera, cellular contents, and culture media were assayed using a RIA for carp GtH-II ß-subunit validated for eel GtH-II (34). GH levels from cellular contents and culture media were assayed using a homologous RIA for eel GH (35).

Dot blot assay of {alpha} and GTH-II ß mRNAs
Cells were scraped in ice-cold PBS (Life Technologies) using a cell scraper (Costar) and collected by pipetting. After centrifugation (12,000 x g) for 5 sec at 4 C, supernatant was removed, 45 µl TE (10 mM Tris and 1 mM EDTA, pH 7.2) buffer was added, and cells were kept at -80 C until RNA extraction. Total RNA was extracted, and dot blots were performed according to the method of Huang et al. (29). Cells were vortexed after the addition of 5 µl 5% Nonidet P-40 (Sigma Chemical Co.) to disrupt cell walls after centrifugation (12,000 x g) for 15 min at 4 C, and supernatant containing total RNA was collected. Total RNAs were denatured at 60 C for 15 min after adding 30 µl 20 x SSC (standard saline citrate) and 20 µl 37% formaldehyde, then loaded on Hybond-N, Nylon membrane (Amersham, Les Ulis, France) through a Hybri-blot manifold (Schleicher & Schuell, Inc., Keen, NH). The membrane was air-dried, and RNAs were immobilized by baking at 80 C for 2 h.

The membrane was prehybridized for 5 h at 42 C in hybridization solution (Amersham) supplemented with 100 µg/ml yeast total RNA (Boehringer Mannheim, Meylan, France) and then hybridized in fresh hybridization solution with 32P- labeled eel GtH-II ß-subunit cDNA probe (36) overnight at 42 C. A random priming kit (High Prime, Boehringer Mannheim) was employed for labeling cDNA probes with [{alpha}-32P]deoxy-CTP (3000 Ci/mM) to a specific activity of 109 dpm/µg. After high stringency washing (0.1 x SSC and 0.1% SDS for 30 min at 60 C), the membranes were autoradiographed. Scanning densitometry and data processing were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The membranes were stripped by boiling in 0.5% SDS and subsequently hybridized under the same conditions with 32P-labeled eel {alpha}-subunit cDNA probe (37) and eel cytochrome b probe (38) as an internal standard.

Statistical analysis
For in vitro experiments, replicates of 6 wells (62,500 cells/well) for RIA studies or of 4 wells (500,000 cells/well) for mRNA studies were used for each treatment, and the mean ± SEM are given. For in vivo experiments, groups of eight eels were used for each treatment, and the mean ± SEM are given. Homogeneity of variance was assessed by Bart-lett’s test, and data were compared by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test, using InStat (GraphPad Software, Inc., San Diego, CA). The median effective dose (ED50) for corticosteroid stimulation of GtH-II cellular content was calculated by the analysis of dose-response curves (GraphPad Software, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo effect of F treatment on pituitary GtH-II content (Fig. 1Go)
Pituitary GtH-II content was low (2.2 ± 0.8 ng/pituitary) in control eels in accordance with the juvenile (sexually immature) stage of the eels. F significantly (P < 0.01) increased eel pituitary GtH-II content, which reached a mean value 10 times higher than that in controls after 1 month of treatment (Fig. 1Go). Serum GtH-II levels remained undetectable (<0.5 ng/ml) in cortisol-treated fish as in controls (data not shown).



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  		Figure 1. Effect of in vivo treatment with F on pituitary GtH-II content. Eels received three weekly injections of 2 µg F in saline/g BW for 1 month. Controls were injected with saline alone (0.15 M NaCl). Pituitary GtH-II content was measured by RIA. The mean ± SEM are given (n = 8 eels/group). **, P < 0.01 vs. controls (by Student’s t test).

 
Kinetics of in vitro effects of F on GtH-II cellular content and release (Fig. 2Go)
We investigated whether the stimulatory effect of F on GtH-II pituitary content could result from a direct pituitary action. Cells were cultured in serum-free medium 199 to avoid any interaction with hormones and growth factors contained in serum (29). In control wells, a time-dependent increase in GtH-II cellular content was observed (Fig. 2Go). Compared with the value on day 0 (1.18 ± 0.14 ng/62,500 cells), GtH-II cellular content was significantly increased on day 12 (P < 0.01). This increase in GtH-II cellular content together with a low, but detectable, amount of GtH-II released into the medium (0.13 ng/62,500 cells·12 days) reflect an active production of GtH-II during the culture. This trend was also shown in our previous studies (29).



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  		Figure 2. Kinetics of the effects of F on GtH-II cellular content and release in primary cultures of eel pituitary cells. Cells were cultured in the presence or absence (controls) of 10-6 M F. Culture media and hormone treatment were renewed every 3 days. Cultures were stopped before the addition of F (day 0) or after 6 or 12 days of treatment, and GtH-II cellular contents in control cells (open circles, dotted line) or in cells treated by F (closed circles) were measured by RIA. Total GtH-II release was calculated by cumulating GtH-II released every 3 days up to 12 days by control (open triangles, dotted line) or treated cells (closed triangles). The mean ± SEM are given (n = 6 wells/group; 62,500 cells/well). *, P < 0.05; ***, P < 0.001 (vs. controls at the same culture time). a, P < 0.01 (vs. day 0 controls, by variance analysis).

 
The addition of F (10-6 M) significantly (P < 0.001) increased GtH-II cellular content on days 6 and 12 (240% and 400% compared with controls at the same culture time, respectively; Fig. 2Go). Assay of GtH-II in medium also indicated a stimulatory effect of F on GtH-II release; cumulative GtH-II release over 12 days of culture was slightly, but significantly (P < 0.05), increased compared with control values (Fig. 2Go). These results indicate a stimulatory effect of F on GtH-II production, increasing both cellular content and release.

Dose-dependent effects of T, E2, and F on GtH-II cellular content in vitro (Fig. 3Go)
The effects of various doses (10-9–10-6 M) of T, E2, and F were tested over 12 days of cell treatment. T increased GtH-II cellular content in a dose-dependent manner, whereas E2 had no significant effect. This is in agreement with our previous demonstration of an androgen-specific stimulatory effect on GtH-II cellular content (29). In addition, F increased GtH-II cellular content in a dose-dependent manner (160%, P > 0.05; 270%, P < 0.05; 340%, P < 0.001; 580%, P < 0.001; from 10-9–10-6 M, compared with controls).



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  		Figure 3. Dose-dependent effects of T, E2, and F on GtH-II cellular content in primary cultures of eel pituitary cells. Cells were treated for 12 days with various doses of hormones (10-9–10-6 M). Culture media and hormone treatments were renewed every 3 days. Cultures were stopped on day 12, and GtH-II cellular contents were measured by RIA. The mean ± SEM are given (n = 6 wells/group; 62,500 cells/well). *, P < 00.5; ***, P < 0.001 (vs. controls, by variance analysis).

 
Lack of effect of F on GH cellular content and release in vitro (Fig. 4Go)
To test whether the positive effect of F on eel GtH-II production was caused by a nonspecific trophic effect, we also studied the effect of F on GH production (cellular content and release) by pituitary cells in the same cultures. As previously shown (33), GH cellular content (6.03 ± 0.25 µg/62,500 cells on day 0) and release (15.7 ± 0.4 µg/12 days·62,500 cells) in control wells were much higher than those for GtH-II, in accordance with the physiological stage (body growth phase) of the juvenile eels used. The present data showed that F did not have any significant effect on GH cellular content and release over 12 days of cell culture at any dose tested (10-9–10-6 M; Fig. 4Go), whereas F increased eel GtH-II cellular content in a dose-dependent manner (Fig. 3Go). This indicates a specific stimulatory effect of F on eel GtH-II production.



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  		Figure 4. Lack of effect of F on GH cellular content and release in primary cultures of eel pituitary cells. To evaluate whether the stimulatory effect of F on GtH-II production was specific, we measured GH levels by RIA in the same culture samples as those shown in Fig. 3Go. GH cellular content was measured at the end of the treatment (day 12). Total GH release was calculated by cumulating GH released every 3 days over 12 days. The mean ± SEM are given (n = 6 wells/group; 62,500 cells/well). No significant differences were found between groups (P > 0.05, by variance analysis).

 
Comparative effects of various corticosteroids on GtH-II cellular content in vitro (Fig. 5Go)
To study the specificity of F action, we compared the dose-dependent effects of various corticoids on eel GtH-II cellular content: F, E (the main metabolite of cortisol in the eel) (39), aldosterone (a mineralocorticoid), as well as two synthetic glucocorticoids (Dex and TA). F and the two synthetic glucocorticoids increased GtH-II cellular content in a dose-dependent manner, over 12 days of cell treatment and produced a similar maximal effect (500–600% of stimulation compared with controls) at the highest doses tested (Fig. 5Go). F increased GtH-II cellular content with an ED50 of 8 x 10-9 M and a maximal effect at 10-6 M. Dex and TA were significantly more effective than F, with ED50 of 2 x 10-9 and 1 x 10-9 M, respectively, and maximal effects at 10-8 M. In contrast, neither E nor aldosterone had any significant effect on GtH-II cellular content at any dose tested (ED50 >10-6 M; Fig. 5Go).



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  		Figure 5. Comparative effects of various corticosteroids on GtH-II cellular content in primary cultures of eel pituitary cells. The dose-dependent effects of F, E, aldosterone (Aldo), Dex, and TA on GtH-II cellular content were compared over 12 days of cell treatment. GtH-II cellular contents were measured by RIA on day 12. Results are expressed as a percentage of the control value. The mean ± SEM are given (n = 6 wells/group; 62,500 cells/well).

 
Effects of F on mRNA levels for {alpha}- and GtH-II ß-subunits in vitro (Figs. 6Go and 7Go)
We investigated whether the stimulatory action of F on GtH-II production implicated effects on mRNA levels for GtH-II subunits. The effects of F (10-6 M) on GtH-II cellular content and on mRNA levels for {alpha}- and GtH-II ß-subunits were compared with the effects of T (10-6 M), which was previously shown to stimulate GtH-II ß mRNA levels (29). Cultures were stopped after 12 days of treatment and used for both GtH-II RIA (cells plated on 96-well plates) and mRNA quantification (cells plated on 12-well plates). The dot-blot for GtH-IIß mRNA is shown in Fig. 6Go. Quantitative analysis (Fig. 7Go) showed significant stimulatory effects of F and of T on GtH-II cellular content (Fig. 7aGo) (600% and 450% compared with controls, respectively; P < 0.001) as well as on GtH-II ß mRNA levels (Fig. 7bGo; 520% and 420% compared with controls, respectively; P < 0.001). In contrast, no significant effects were observed on {alpha}-subunit mRNA levels (Fig. 7cGo).



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  		Figure 6. Effects of F and T on mRNA levels for GtH-II ß-subunit in primary cultures of eel pituitary cells: dot blot. Cells were cultured for 12 days in the absence (controls; CTL) or presence of 10-6 M F or T (4 wells/group; 500,000 cells/well). Cultures were stopped on day 12, and GtH-II ß-subunit mRNA was measured by dot blot, using a homologous cDNA probe for eel GtH-IIß. For quantitative and statistical analysis, see Fig. 7bGo.

 


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  		Figure 7. Comparative effects of F and T on GtH-II cellular content and mRNA levels for {alpha}- and GtH-II ß-subunits in primary cultures of eel pituitary cells. Cells were cultured for 12 days in the absence (controls) or presence of 10-6 M F or T. Cultures were stopped on day 12. GtH-II cellular content was measured by RIA (a). The mean GtH-II content ± SEM are given (n = 6 wells/group; 62,500 cells/well). The mRNAs for {alpha}- and GtH-II ß-subunits were measured by dot blot, using homologous cDNA probes for eel GtH-II{alpha} (c) and GtH-IIß (b). The mean mRNA levels (arbitrary units) are given ± SEM (n = 4 wells/group; 500,000 cells/well). ***, P < 0.001 vs. controls, by variance analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Juvenile eels have low pituitary GtH-II contents and undetectable GtH-II circulating levels (27, 40). In vivo treatment with F increased pituitary GtH-II content, in agreement with previous cytological observations indicating an activation of gonadotropic cells in F-treated eels (41). A stimulatory effect of F was also observed on GtH pituitary content in the juvenile trout, Oncorhynchus mykiss, after brain, but not perivisceral, implantation, suggesting that the positive action of F on GtH was exerted centrally and was not mediated by its peripheral metabolic effects (26). Our present study on primary cultures of eel pituitary cells demonstrates that F is able to stimulate GtH-II production by a direct action at the pituitary cell level.

Indeed, in vitro studies showed F to increase GtH-II cellular content in a time- and dose-dependent manner. At 12 days of culture, the ED50 was 8 x 10-9 M, which is largely compatible with the plasma levels of F in the European eel at the silver stage (prepubertal stage; mean level, 20 ng/ml) (42) and in the prepubertal rainbow trout (peak level up to 40 ng/ml) (43). The maximal stimulatory effect of F (400–900% of stimulation compared with controls, according to the experiments) was produced between 10-6 and 10-7 M. The low release of GtH-II into the medium was also significantly increased in F-treated wells.

To test whether the stimulatory effect of F on GtH-II production resulted from a specific effect on gonadotroph activity, we studied the effect of F on GH production by somatotrophs in the same culture conditions. In fact, this culture system has already been applied for studying GH regulation in vitro (33). Our present data show that F had no significant effect on GH production (cellular content and release) over at least 12 days of culture and at any dose tested (10-9–10-6 M). This indicates that the positive effect of F on eel GtH-II does not result from a general stimulatory action on pituitary cell activity, but from a specific effect on GtH-II production by gonadotrophs.

We investigated the specificity of F action by comparing the activities of various corticoids. Two synthetic glucocorticoids (Dex and TA) were able, like F, to increase GtH-II cellular content in a dose-dependent manner, with similar maximal stimulatory effects. Their relative potencies, as indicated by their ED50, were: TA > Dex > F. In contrast, cortisone, the main metabolite of F in the eel (39), had no significant effect up to 10-6 M. A mineralocorticoid, aldosterone, which is not produced in the eel (7), was also without effect on GtH-II production. These comparative abilities of various corticoids to stimulate GtH-II production indicate the implication of a receptor specific for glucocorticoids. In mammals, two types of corticosteroid receptors have been characterized and cloned [mineralocorticoid receptor and glucocorticoid receptor (GR)]. Although F is able to bind with high affinity to both types of receptors, aldosterone has a low affinity for GR (44). In salmonids, binding studies in various tissues (liver, muscle, gill, and brain) revealed the presence of a single type of receptor with a high affinity for natural or synthetic glucocorticoids and a low affinity for aldosterone (45, 46). Recently, a single type of glucocorticoid receptor has been cloned from the liver and detected in various tissues in the rainbow trout (O. mykiss). Its sequence and binding properties are more related to those of mammalian GR than mineralocorticoid receptor (8). These data support the hypothesis that cortisol in fish acts on various target tissues through a single receptor type, homologous to mammalian GR. In the eel, the relative potencies of various corticoids to stimulate GtH-II production from pituitary cells, as shown in the present study, are in good agreement with their respective binding affinities on eel gill cell cytosol (47). Localization of GR in the rainbow trout pituitary, using antibodies to recombinant trout GR, revealed staining in the proximal pars distalis where gonadotrophs are clustered (48). Therefore, the stimulatory effect of F on GtH-II production, shown in our study, could result from a direct action on gonadotrophs through binding to GR.

This is the first demonstration in a nonmammalian vertebrate of a stimulatory effect of F on gonadotropin production exerted directly at the pituitary cell level. In the sheep, B stimulated FSH cellular content in primary cultures of pituitary cells (49). Moreover, as in our model, a positive effect on FSH cellular content was produced by androgens, but not by E2 (49). In the rat, glucocorticoids (F or B) have been shown to increase FSH production from primary cultures of pituitary cells. This stimulatory effect was exerted specifically on FSH, with no increase in LH production observed (19, 20, 50, 51).

Measurement of mRNA levels for eel GtH-II {alpha}- and ß-subunits demonstrated that the stimulatory effect of F on GtH-II production was exerted through a specific increase in mRNA levels for GtH-II ß-subunit, with no significant effect observed on mRNA levels for {alpha}-subunit. The stimulatory effect of F on FSH production in the rat is also mediated by a selective increase in mRNA levels for FSH ß-subunit, with no effect on mRNA levels for {alpha} or LHß (22, 51). In the rat, studies on the mechanism of in vitro stimulation of FSH synthesis indicated that T increased FSH ß-subunit mRNA levels through an effect on mRNA half-life (52). However, B had no effect on mRNA stabilization, and it was suggested that B was probably acting at the transcriptional level (51).

Two gonadotropins (GtH-I and GtH-II) have been characterized in several teleosts (53). Sequence analysis of their ß-subunits suggests that fish GtH-I and GtH-II would be, respectively, homologous to tetrapod FSH and LH (25, 53). Our data indicate that the effects of glucocorticoids on eel GtH-IIß are similar to those they exert on mammalian FSHß. We also previously demonstrated an androgen-specific stimulation of eel GtH-IIß, which appeared closer to androgen regulation of mammalian FSHß than to those of LHß (29). These data suggest that despite a higher sequence identity between teleost GtH-IIß and mammalian LHß than between GtH-IIß and FSHß, direct pituitary regulation of eel GtH-IIß is more related to that of mammalian FSHß than LHß.

In fish, gonadal growth represents a major energetic challenge. For instance, in the eel, ovarian development accounts for up to half of the body weight at the end of sexual maturation (54). Hormones regulating intermediary metabolism, in particular F, therefore play an important indirect role in reproductive function. Furthermore, in relation to the particular life cycles of some species, gonadal development can occur concomitantly with migratory activity and a long period of fasting. This is the case for the lamprey (Petromyzon marinus), salmon (Oncorhynchus spp.), and eel (Anguilla spp.) (55). In these species, the metabolic energy necessary for migration and gonadal development comes exclusively from body stores. F, which has been shown to induce mobilization of lipid stores and to stimulate hepatic neoglucogenesis in the eel (56) as well as in other vertebrates (57), is likely to have a crucial role. We may speculate that during the transatlantic migration of the eel, fasting, swimming, as well as environmental factors such as deep ocean pressure (58) could activate the hypothalamic-pituitary-adrenal axis and then contribute to the onset of sexual maturation.

In conclusion, our study demonstrates that F in the eel, in addition to its indirect positive role in reproduction by its peripheral effects on intermediary metabolism, exerts a direct positive role on GtH-II production. This stimulatory effect on GtH-II, played out directly at the pituitary cell level, recalls that of F on FSH in mammals. The present study, performed in a primitive teleost at the juvenile stage, suggests that the role of F in the positive regulation of gonadotropins at puberty may have arisen early in vertebrate evolution.


   Acknowledgments
 
We are grateful to Drs. E. Burzawa-Gérard and J. Marchelidon (Centre National de la Recherche Scientifique, Paris, France) for hormones and antisera for eel GtH-II and GH RIAs, and to Drs. B. Quérat (Centre National de la Recherche Scientifique, Paris, France) and P. Vernier (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France) for cDNA probes to eel {alpha}, GtH-IIß, and cytochrome b. We thank Prof. B. Demeneix (MNHN, Paris, France) for English corrections.


   Footnotes
 
1 This work was supported by research grants from Centre National de la Recherche Scientifique (PICS Taiwan 487; CNRS/NSF 3900) and Conseil Supérieur de la Pêche (no. 97/442) (to S.D.), a visiting fellowship from MNHN (to M.S.), a Ph.D. fellowship (to Y.S.H.) from the Ministry of Foreign Affairs (France) and the Ministry of Education (Taiwan), a Ph.D. fellowship from the Ministry of Research and Education (France; to K.R.), and a Ph.D. fellowship (to M.S.) from the Ministry of Education (Syria). Back

Received August 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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