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Endocrinology Vol. 140, No. 5 2054-2062
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Effect of Insulin-Like Growth Factor I on in Vitro Gonadotropin (I and II) and Growth Hormone Secretions in Rainbow Trout (Oncorhynchus mykiss) at Different Stages of the Reproductive Cycle1

C. Weil, F. Carré, O. Blaise, B. Breton and P.-Y. Le Bail

Institut National de la Recherche Agronomique/Station Commune de Recherches en Ichtyophysiologie, Biodiversité et Environnement, Equipe Croissance et Qualité de la Chair des Poissons, Campus de Beaulieu, 35042 Rennes cedex, France

Address all correspondence and requests for reprints to: Dr. Claudine Weil, INRA/SCRIBE, Equipe Croissance et Qualité de la Chair des Poissons, Campus de Beaulieu, 35042 Rennes cedex, France. E-mail: weil{at}beaulieu.rennes.inra.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The short-term effect of insulin-like growth factor I (IGF-I) on GTH I (FSH-like), GTH II (LH-like), and GH production by cultured rainbow trout pituitary cells was studied in immature fish of both sexes, at early gametogenesis and in spermiating and periovulatory animals. IGF-I had no effect on basal GTH I and GTH II release, whereas it always inhibited basal GH, showing decreasing intensity with the gonad maturation. In absence of IGF-I, GTH I and GTH II cells were always responsive to GnRH, whereas no response was observed for GH cells whatever the sexual stage. The action of IGF-I on the sensitivity to GnRH differs between GTH and GH cells. The former requires a coincubation with IGF-I (10-6 M)/GnRH to show an increase in sensitivity, independent of the sexual stage. To be responsive to GnRH, the GH cells require longer exposure to IGF-I, the efficiency of which decreases with gonad maturation. The action of IGF-I (10-6 M) on GTH cell sensitivity to GnRH does not seem to be related to a mitogenic effect or to an improvement in cell survival. It seems to be IGF-I specific, not passing via the insulin receptor. Certain hypotheses on the putative role of IGF-I and GnRH as a link between growth and puberty are suggested.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PUBERTY is known to be closely related to growth rate in mammals (1) as well as in fish (2), indicating interactions between the somatotropic and gonadotropic axes. One of the possible candidates for such interactions could be IGF-I, the circulating levels of which increase strikingly during puberty in different mammalian species (3). IGF-I could act on the central nervous system (CNS) and/or at the gonadic level (4), as indicated by the presence of binding sites at both the CNS level (5, 6, 7) and the gonadal levels (4). With regard to the CNS level, IGF-I has been demonstrated both in vitro (8) and in vivo (3), to activate the release of GnRH in rats at the time of puberty which in turn increases plasma LH levels. Furthermore, in postpubescent rats, two short-term in vitro studies (9, 10) have shown that IGF-I acts directly at the pituitary level by positively modifying LH and FSH basal levels, as well as GnRH induced LH level. The former study was conducted with pituitaries from females in diestrous at high doses (approximately 10-6 M) of IGF-I (9); in the latter study, pituitary donors were normal males in spermatogenesis and doses of IGF-I tested were lower (5 x 10-9 M and 10-8 M) and corresponded to circulating levels (10).

These two studies give rise to several questions concerning IGF-I action at the pituitary level. Is this action species specific, influenced by the sex and the sexual status of pituitary donors, specific to gonadotropin production, or is it the consequence of a more general effect of IGF-I on cell metabolism? Another question concerns the mode of action of IGF-I: is IGF-I active in a similar way for gonadotropin and GH production, through the same endocrine or paracrine/autocrine mechanisms? Paracrine/autocrine action is possible because the presence of IGF-I messenger RNAs (mRNAs) has been reported at the pituitary level in rats (11, 12) and, at the brain level, in coho salmon (13).

The aim of the present work was to answer these different questions. The work was conducted on rainbow trout cultured pituitary cells collected from fish at different sexual stages. The effect of IGF-I on GTH production was analyzed by studying its effect on basal and GnRH-induced release of GTH I (FSH-like) and GTH II (LH-like); in parallel, its action on basal and GnRH-induced GH secretion was studied in the same cultures. Indeed, GnRH action on GH secretion has been reported in normal fish of different species, in trout in the presence of IGF-I, and in human pathologies often characterized by high levels of GH and IGF-I (14). Furthermore, the effect of IGF-I was compared with that of another growth factor, insulin. To partly answer the question of possible endocrine or autocrine/paracrine actions of IGF-I, the doses tested in the present study were 10-8 M and 10-6 M, corresponding, respectively, to circulating (15, 16) and possible paracrine/autocrine levels. Cells were submitted to the lower dose during a 48-h preincubation period, thus mimicking in situ conditions, whereas a 24-h coincubation with GnRH was carried out in the presence of IGF-I at 10-6 and 10-8 M.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were carried out on 1+-yr-old rainbow trout (Oncorhynchus mykiss) at different stages of the sexual cycle. All fish originated from the same strain (Mirwart), were reared at our experimental fish farm (SEDII, Sizun, France) under natural photoperiod (48 degree N) and water temperature, and, were fed with commercially prepared pellets (Aqualim, Nersac, France) at a rate recommended by the manufacturer.

The sexual stage, the means (± SEM) of body weight (BW in grams), gonado-somatic factor (GSI% = 100 x gonad weight/BW) and condition factor (K = 100 x BW/(standard length)3) of the fish for the different experiments were as follows:

1) Effect of IGF on GTH and GH production and influence of the sexual stage. Immature pooled males and females (BW = 330 ± 13; GSI % = 0.09 ± 0.05); females at the beginning of gametogenesis (BW = 487 ± 26; GSI % = 0.27 ± 0.09); males at the beginning of spermatogenesis (BW = 534 ± 36; GSI % = 0.29 ± 0.23); spermiating males (1 month after the beginning of spermiation; BW 1517 ± 58); females in the periovulatory period (8 to 15 days before ovulation; BW = 1266 ± 91). Fish from all five experiments had comparable condition factors with a mean value of 1.72 ± 0.11. Experiments were conducted from March to December 1996.

2) Specificity of IGF-I action. Pooled prepubertal males and females (BW = 460 ± 26; female GSI% = 0.21 ± 0.02, male GSI = 0.19 ± 0.04; K = 1.49 ± 0.02). This experiment was conducted in May 1997.

Cell cultures
Pituitary cell cultures were prepared according to a method validated for GTH II (17) and GH secretion studies (18). Briefly, pituitary glands were removed from anaesthetised fish (phenoxy-ethanol, 0.3 ml/liter, Aquaveto, La Loupe, France) and submitted to 0.1% collagenase (Boehringer Mannheim, Meylan, France) dispersion for 20 h at 12 C. At the end of dispersion, cells were washed twice with RPMI medium (Sigma Chemical Co., L’Isle D’Abeau Chesnes, France) and suspended in RPMI supplemented with 2% ultroser (IBF-Sepracor, Villeneuve-la-Garenne, France) and 1% of antibiotic-antimicotic mixture (Sigma Chemical Co., L’Isle D’Abeau Chesnes, France). A 250-µl aliquot of suspension containing 50,000 cells was plated into each well of 96-multiwell plates precoated with poly-L-lysine (5 µg/cm2 Sigma Chemical Co., L’Isle D’Abeau Chesnes, France). After 24 h of culture, cells were washed twice with ultroser and antibiotic-free RPMI.

Cells were then submitted to preincubation and incubation protocols. Preincubation was performed for 48 h in 250 µl medium with gentamycin (50 µg/ml; Sigma Chemical Co., L’Isle D’Abeau Chesnes, France), in either the absence or presence of 10-8 M recombinant human IGF-I (gift from Ciba-Geigy, Summit, NJ). At the end of this preincubation period, cells were washed twice with 200 µl of RPMI and incubated for 24 h in 250 µl of medium without antibiotics and either, supplemented or not with hormones, according to the required experimental regime. The medium contained either increasing doses (10-10 to 10-6 M) of salmon GnRH (sGnRH, Sigma Chemical Co., L’Isle D’Abeau Chesnes, France) in either the absence or presence of IGF-I (10-8 M and 10-6 M) or sGnRH 10-9 M in the presence of increasing doses ranging from 10-10 M to 10-6 M of IGF-I or bovine insulin (Sigma Chemical Co., L’Isle D’Abeau Chesnes, France).

For every experiment, at the end of the incubation period, plates were centrifuged (200 x g, 10 min). Media were collected, then frozen, either pure or diluted in hormone assay buffer containing 1% (final concentration) BSA (RIA grade, Sigma Chemical Co., L’Isle D’Abeau Chesnes, France) for further respective GTH or GH determination, respectively. Cells were lysed by incubation with 200 µl medium containing 0.1% Triton X-100 (Sigma Chemical Co., L’Isle D’Abeau Chesnes, France) and 1% BSA for 1 h, after which the lysate was frozen for future GTH and GH determination.

GTH and GH RIAs
The GTH I and II that were used as standards and for iodination were purified as previously described (19). GTH I and II levels were determined by specific RIAs, using antibodies raised against rainbow trout GTH Iß and chinook salmon GTH IIß (20). The antibody used for the GTH I assay presented 3.7% of cross-reaction with GTH II but no cross-reaction with GH and PRL. The antibody used for GTH II did not present any cross-reactions with GTH I, GH, or PRL. The intraassay and interassay variations were 4.6% and 9.8%, respectively, for GTH I and 5.9% and 8.3%, respectively for GTH II.

The GH RIA used has been previously described (21). A specific antibody for salmonid GH, which presents no cross-reaction with GTH I and GTH II, was used. Intraassay and interassay coefficients of variation are 4% and 8.3%, respectively.

Parameters studied and statistical analyses
Basal (dose 0 of GnRH) of GTH I and GTH II releases were measured after the 48-h preincubation period (in either the absence or presence of IGF-I 10-8 M), and, also, after the 24-h incubation period with the different doses of IGF-I (0, 10-8 M, and 10-6 M). In each case, the means were compared by the Wilcoxon-Mann-Whitney U test.

The effect of IGF-I on the GTH I, GTH II, and GH sensitivities to GnRH was analyzed by determining the minimal observed effective dose (moed) of GnRH able to induce a release that was statistically different from the basal level (by an ANOVA followed by Scheffe’s multiple range test) and by comparing the amplitude of the response (by the Wilcoxon-Mann-Whitney U test). In the case of a positive effect of IGF-I on the sensitivity to GnRH (assessed by a decrease of the moed), we have indicated the ratio (R50) between the ED50 of GnRH estimated from the control curve and the ED50 of GnRH estimated from the curve obtained in the presence of IGF-I.

Basal GTH I and GH releases and the responses to GnRH 10-9 M as a function of increasing doses of IGF-I and insulin were expressed as the percentage of basal releases, or of the response to GnRH observed in control medium, and was analyzed by ANOVA, followed by a Scheffe’s multirange test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1) Effect of IGF-I on GTH I and GTH II basal releases in immature fish
Basal GTH I and GTH II releases were studied at the end of the 48-h preincubation period in either control medium or in medium supplemented with IGF-I 10-8 M, as well as at the end of the further 24-h incubation period in either the control medium or in the medium supplemented with IGF-I 10-8 M and 10-6 M. GTH I is always detectable at both incubation periods. No significant effect of IGF-I was observed on GTH I release whatever the dose of IGF-I (10-8 M or 10-6 M) and the duration of the treatment (24 h, 48 h, and 72 h i.e. 48 + 24 h). The mean values observed after the preincubation and the incubation periods were 7.9 ± 0.37 and 5.57 ± 0.45 ng/well, respectively. GTH II levels were not detectable (<0.06 ng/well) at 24 h. A 48-h pretreatment with IGF-I 10-8 M did not modify GTH II release, and the mean value observed was 0.08 ± 0.005 ng/well.

2) Effect of IGF-I on GTH I and GTH II releases and production in response to GnRH in immature fish
As data were comparable whether in the presence or not of IGF-I 10-8 M during the 48-h incubation period, we have only reported results of the first case. GnRH induced a dose-dependent release of both GTH I and II (Fig. 1Go). In the absence of IGF-I during the 24-h incubation period, the minimal effective dose able to induce a significant release of GTH I and GTH II when compared with the basal level was 10-8 M. The elevation observed for GTH I and GTH II was 4-fold or about 10-fold, respectively. The presence of IGF-I 10-8 M during the incubation did not modify the sensitivity of the pituitary cells to GnRH, whereas IGF-I 10-6 M increased sensitivity because the minimal effective dose able to induce GTH I and GTH II releases was 10-9 M instead of 10-8 M. R50 were 7.5 and 10 for GTH I and GTH II, respectively. On the other hand, the amplitude of the response is unchanged.



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  		Figure 1. GTH I and GTH II responses to a 24-h coincubation with different doses of IGF-I (0 M, 10-8 M and 10-6 M) and increasing doses of GnRH by pituitary cultures preincubated for 48 h with IGF-I 10-8 M. Fish of both sexes were immature (see the characteristics of the fish in material and methods). Values are means ± SEM of five replicate wells of the same cell preparation. Limit of GTH II detection (0.06 ng/well). * and **, P < 0.05 and P < 0.01 vs. dose 0 of GnRH, respectively.

 
GTH I and GTH II productions (cell plus media contents) were not significantly modified by the different doses of GnRH, independently of the presence or not of IGF-I.

3) Influence of the sexual stage on the effect of IGF-I on basal and induced-GnRH GTH I and GTH II releases and production
Basal GTH I and GTH II releases were studied at the end of the 48-h period of preincubation either in control medium or in medium supplemented with IGF-I 10-8 M, as well as at the end of the following 24-h incubation period in either the control medium or in the medium supplemented with IGF-I 10-8 M or 10-6 M. Levels of both hormones increased with the maturation of the gonads for both sexes (see GTH I and GTH II basal releases observed during 24 h in Figs. 2Go and 3Go, respectively). Whatever the sexual stage, no significant (P > 0.05) effect of IGF-I was observed independently of the duration of its exposure (24 h, 48 h and 72 h i.e. 48 + 24 h), or the dose used (10-8 M or 10-6 M).



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  		Figure 2. Influence of the stage of gametogenesis on the GTH I response to a 24-h coincubation with different doses of IGF-I (0 M and 10-6 M) and increasing doses of GnRH by pituitary cultures preincubated for 48 h with IGF-I 10-8 M. Fish were males and females in early gametogenesis and in the periovulatory and spermiation period (see the characteristics of the fish in material and methods). Values are means ± SEM of five replicate wells of the same cell preparation. * and **, P < 0.05 and P < 0.01 vs. dose 0 of GnRH, respectively.

 


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  		Figure 3. Influence of the stage of gametogenesis on the GTH II response to a 24-h coincubation with different doses of IGF-I (0 M and 10-6 M) and increasing doses of GnRH by pituitary cultures preincubated for 48 h with IGF-I 10-8 M. Fish were males and females in early gametogenesis and in the periovulatory and spermiation period (see the characteristics of the fish in material and methods). Values are means ± SEM of five replicate wells of the same cell preparation. * and **, P < 0.05 and P < 0.01 vs. dose 0 of GnRH, respectively.

 
As the releases of GTH I and GTH II in response to GnRH were independent of the presence or absence of IGF-I 10-8 M during the preincubation period, as observed in immature fish, we have only represented the results corresponding to the first case in Figs. 2Go and 3Go, respectively. In the absence of coincubation with GnRH/IGF, the minimal effective dose of GnRH able to induce GTH I release was 10-8 M in early gametogenesis for both males and females as well as in spermiating and periovulatory animals (Fig. 2Go). For GTH II, the minimal effective dose was 10-9 M for animals of both sexes at early gametogenesis and 10-8 M for mature animals (Fig. 3Go). Coincubation with GnRH/IGF 10-8 M (results not shown in Figs. 2Go and 3Go) did not modify the sensitivity of the GTH I and GTH II cells to GnRH in any sexual stage studied because the minimal effective dose of GnRH was unchanged. However, it must be pointed out that, in early stages of gametogenesis, this dose of GnRH induced a higher response (P < 0.01). On the other hand, the coincubation of GnRH with IGF 10-6 M increased the sensitivity of the GTH I cells to GnRH whatever the sexual stage. Indeed, the minimal effective dose of GnRH able to induce GTH I release decreased in every case (10-9 M instead of 10-8 M) and R50 varies between 10 and 12.5. The sensitivity to GnRH of GTH II cells was also increased by the copresence of GnRH with IGF 10-6 M at every sexual stage, except in males at early gametogenesis. Indeed, compared with control, the moed for GTH II decreased in early gametogenesis (moed = 10-10 M, R50 =7) and periovulatory females (moed = 10-10 M, R50 = 20), as well as in spermiating males (moed = 10-9 M and R50 = 10). In males at early gametogenesis, the moed is statistically unchanged, but the intensity of the response was increased (P < 0.01) and the value of R50 was high (=20). A maximal sensitivity was observed in early gametogenesis in females because the amplitude of the GTH I and GTH II responses significantly increased (P < 0.01).

GTH II production (cell content plus release) increased with the sexual stage of the fish, whereas for GTH I such a progression was not found. GTH II values, expressed in ng/well, are 5.3 ± 0.4 and 10.9 ± 1.0 for males and females in early gametogenesis, respectively, and 188.9 ± 11.1 and 187.1 ± 15.2 for mature males and females, respectively. GTH I production, expressed in the same way, was 203.1 ± 18.8 and 495.6 ± 68.1 in early gametogenesis males and females and 244.7 ± 20.4 and 259.8 ± 20.2 in mature males and females, respectively. GTH I and GTH II productions were not modified by IGF-I with respect to the different doses of GnRH (results not shown).

4) Specificity of IGF-I action on gonadotropin secretion
The specificity of IGF-I action on gonadotropin secretion was studied by comparing the effect of increasing doses of IGF-I and insulin on gonadotropin and GH basal levels (Fig. 4Go) and GnRH induced (Fig. 5Go) secretion. The experiment was performed with pituitary cultures of early gametogenesis males and females that have been submitted to a 48-h preincubation period without IGF-I 10-8 M to prevent any saturation of the receptors. This particular stage was chosen because this stage is the most sensitive to IGF-I and GnRH. Only GTH I levels are reported as GTH II values were below the detection limit.



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  		Figure 4. Effect of a 24-h incubation with increasing doses (10-10–10-6 M) of IGF-I and insulin on basal GTH I and GH releases by pituitary cells after a preincubation without IGF-I. Fish were in early gametogenesis (see the characteristics of the fish in Materials and Methods). Responses were normalized as percentage of control. Values are means ± SEM of five replicate wells of the same cell preparation. Letters indicate the difference between values.

 


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  		Figure 5. Effect of a 24-h incubation with increasing doses (10-10–10-6 M) of IGF-I and insulin on GnRH 10-9 M induced GTH I and GH releases by pituitary cells after a preincubation without IGF-I. Fish were in early gametogenesis (see the characteristics of the fish in Materials and Methods). Responses were normalized as percentage of control response to GnRH. Values are mean ± SEM of five replicate wells of the same cell preparation. Letters indicate the difference between values.

 
Increasing doses of IGF-I (10-10–10-6 M) did not significantly modify GTH I basal release (Fig. 4Go). In the same wells, a gradual inhibition of GH secretion was observed with a maximal inhibition of 40% observed between 10-8 M and 10-6 M. Insulin did not modify basal GTH I release for doses ranging from 10 -10 M to 10 -7 M, whereas a significant (P < 0.01) augmentation (250%) was observed at 10 -6 M. In the same wells, only a weak (20%) and not significant (P > 0.05) inhibition of GH release was observed.

The GTH I response to GnRH 10-9 M (Fig. 5Go) increased gradually for the doses of IGF-I ranging from 10-10–10-6 M, extreme values being significantly different from each other (P < 0.01). Insulin did not modify the GTH I response to GnRH for doses ranging from 10-10 M to 10-7 M, whereas a significant (P < 0.01) increase was observed at the dose of 10-6 M. However, when comparing this response with the basal level observed in the presence of insulin 10-6 M (Fig. 4Go), it represents only a 1.35-fold augmentation, which is not significantly (P > 0.05) different from this basal level. In the same wells, no GH response to GnRH was observed, whatever the doses of IGF-I or insulin.

5) Effect of IGF-I on basal and induced-GnRH GH releases in relation to the sexual stage
IGF-I 10-8 M inhibited basal GH release with an intensity that decreased from immaturity to maturity (Table 1Go), whereas in the same cultures basal GTH I and GTH II releases were not significantly affected whatever the sexual stage.


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  		Table 1. Variation of GH cell sensitivity to IGF-I in function of the sexual cycle. Cell sensitivity is expressed by the effect of 48-h pretreatment with IGF-I 10-8 M on GH basal level and on the subsequent response to GnRH 10-7 M during a 24-h incubation period in the presence or not of IGF-I 10-8 M and 10-6 M.

 
Contrary to gonadotropins, in the absence of preincubation with IGF-I 10-8 M, no GH response to GnRH was observed in the subsequent incubation period whatever the sexual stage of the fish or the dose of IGF-I (0, 10-8 M or 10-6 M, results not shown). On the other hand, a 48-h preincubation period with IGF-I (10-8 M) exerted a permissive effect on the subsequent GH response to GnRH, with an efficiency that decreased with the advancing sexual stage of the fish as indicated in Table 1Go. A 48-h preincubation with IGF-I was sufficient at immaturity and early gametogenesis to cause, in the subsequent incubation, an increase in GH for doses of GnRH ranging from 10-8–10-6 M, but the effect was more pronounced when IGF-I was present in the incubation. In mature male and female fish, no significant effect of IGF-I was observed on the GH response to GnRH, even if it was present during both preincubation and incubation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the aims of the present work was to study in a vertebrate different from the rat, the influence of IGF-I on pituitary GTH production with respect to the sexual cycle. In fish, IGF-I production is clearly modified by nutrition, smoltification, and hormonal conditions and for this latter point the role of GH is well established (22). This work was conducted in vitro to eliminate the in vivo GH-IGF regulation, thus establishing the role per se of IGF on GTH production. In mammals, congenital or induced GH alterations have shown that physiological levels of GH seem to be necessary for the maintenance of normal basal levels or GnRH-induced gonadotropin levels (23), whereas no data are available on the direct effect of GH on the gonadotropin response to GnRH in normal mammals or fish.

The study was performed using pituitary cultured cells from rainbow trout of both sexes. Immature stages are characterized by low levels of estrogens, androgens, and by the absence of progestins, whereas in spermiating and periovulatory females progestins are present, as well as androgens in males and estrogens in females (24). The IGF-I used was recombinant human IGF-I, salmonid IGF-I not being available in sufficient quantities. However, the complementary cDNAs of salmon and human peptides are highly homologous in their ligand binding domains (25, 26) and recombinant peptides are equally potent in an in vitro assay using salmon gill cartilage (27). The production of the two GTHs has been studied thanks to specific RIAs (20) and was estimated by measuring basal and GnRH-induced releases in the medium, as well as the cellular content.

In the absence of IGF-I, we observed that in vitro basal GTH I (FSH-like) and GTH II (LH-like) releases vary from immaturity to ovulation and spermiation. This variation is more pronounced for GTH II because levels are undetectable or low in immature cultures and higher at the time of spermiation and ovulation and is in close relationship with GTH II cell content. On the other hand, basal GTH I was detectable at every stage from immaturity to maturation. These observations are in accordance with circulating (28, 29, 30) and pituitary (30, 31, 32) GTH I and II contents observed throughout the reproductive cycle in salmonids. Pituitary cells were always responsive to increasing doses of GnRH whatever the stage studied because doses of GnRH ranging from 10-8–10-6 M were able to induce GTH I as well as GTH II releases. This constant response to GnRH for both hormones from immaturity to maturity differs from data obtained in vivo (33) in which no GTH II response could be detected in immature fish, whereas no significant GTH I response was observed at any stage. This discrepancy between results might be due to different reasons such as an undetectable in vivo GTH II response, the existence of episodic secretion, particularly for GTH I or to the presence of in vivo inhibitory factors.

The short-term addition of IGF-I into the medium did not significantly modify GTH I and GTH II total productions as observed in rat (9), indicating that no effect exists on gonadotropin pituitary accumulation. Furthermore, in trout this IGF-I treatment did not significantly modify basal GTH I and GTH II releases, whatever the dose used or the sex or sexual stages of the fish. This differs from results obtained in rats, in which an increase in basal level of gonadotropins is observed between 4 h and 72 h (9, 10). This difference between species might be due to a more rapid effect of IGF-I in relation to incubation temperature (37 C in rat vs. 18 C in rainbow trout). Longer treatment may cause an increase in the basal level in trout, and such an experiment should be conducted in further work.

In the present study, whatever the sexual stage of the trout, preincubation for 48 h with IGF 10-8 M was not sufficient to modify subsequent GTH I and II responses to GnRH. This observation is in accordance with previous work conducted in immature fish, using a GTH II RIA directed against the total GTH II heterodimeric molecule (14). On the other hand, this same treatment induced GH sensitivity to GnRH in immature fish, and to a lesser extent in early gametogenesis fish, as previously reported (14). This last observation indicates that the lack of gonadotropin response is not linked to a general failure of the culture sensitivity. To be modified by IGF-I, the gonadotropin responses to GnRH necessitate the coincubation of IGF-I with GnRH, whatever the type of preincubation (either in the presence or absence of IGF-I). In this case, the lower dose (10-8 M), corresponding to the circulating level, induced an increase of the intensity of the gonadotropin responses to the minimal effective dose of GnRH, in early gametogenesis fish only. On the other hand, whatever the sexual stage, the highest dose of IGF-I (10-6 M) is always efficient in increasing GTH sensitivity to GnRH, by decreasing the minimal observed effective dose of GnRH. Furthermore, a tendency to an increase in the amplitude of the plateau is observed in early gametogenesis females. This does not seem to be related to an increase in GTH productions by IGF-I because radioimmunoassayable content was not modified. However, the present work does not allow the determination of the effect of IGF-I on the accessibility to GnRH of various pools of GTH in relation to the particular sexual status or steroid milieu.

The significance of the efficiency of this high dose (10-6 M) of IGF-I on gonadotropin sensitivity to GnRH is questionable and gives rise to different hypotheses. We can reject right away a possible contamination by another hormonal factor, because recombinant IGF-I was used.

The first hypothesis concerns an action of IGF-I linked to a mitogenic effect on pituitary cells or to an improvement of cell survival, leading to an increase in the amount of releasable GTH. However, despite the mitogenic action of growth factors, and particularly IGF-I, has been described on different tissues, its role in pituitary normal cell growth has been elucidated only recently in a work conducted in mice (34). In this work it stimulates only the proliferation of corticotrophs and mammotrophs among the other cell types (34), whereas it modestly enhanced the growth of rat GH3 cells (35). In the present work, high doses of IGF-I have no positive effect on immunoassayable GTH total production (cell content plus release) at any sexual stage, and by microscopic observation no particular effects on cell number and condition could be noticed. In consequence, although we do not study the effect of IGF-I on DNA synthesis, or on GTH I and GTH II mRNAs, we think that no positive effect of IGF-I on mitogenesis and/or synthesis, or on cell survival occurred.

The second explanation could concern an action of this high dose of IGF-I through the insulin receptor because the gonadotropic in vivo action of insulin as well as its direct gonadal (36) and pituitary (10, 37) actions, have previously been reported. In rats, insulin provokes, in vitro, basal and GnRH-induced FSH and LH releases, in a dose-related manner. If such an action exists in trout, the effect obtained with IGF-I at the dose of 10-6 M is supposed to be equivalent to that obtained with a 100 times lower dose of insulin, because IGF-I is 100 times less potent in different tissues of fish (38, 39, 40). In the present work, it does not seem that the potentiation of the response to GnRH by IGF-I 10-6 M passes through the insulin receptor because no increase of the response to GnRH 10-9 M was observed for doses of insulin ranging from 10-10 to 10-7 M in early gametogenesis fish. Such an increase was observed at 10-6 M, when compared with the response to GnRH in the absence of insulin, but it represents only a 1.35-fold augmentation when compared with basal level which shows a dramatic increase for this dose of insulin (basal level not affected by such a high dose of IGF-I). This efficient dose of insulin is high compared with circulating levels (41) and a paracrine/autocrine action does not seem reliable because it is known that the salmonid brain does not produce insulin (42). These two remarks, as well as the absence of a gradual GTH I basal release in relation to increasing doses of insulin, as reported in mammals, led us to think more likely of a nonspecific action (contamination) of purified insulin on the release rather than of an effect linked to mitogenesis and/or synthesis of gonadotropins. Furthermore, this growth factor has been shown, in pituitary cultured cells, to have no effect on DNA synthesis in rat (37), or on the expression of FSH ß, LH ß and the common {alpha}-subunit in sheep (35).

Another hypothesis would be a possible paracrine/autocrine action of IGF-I because the presence of IGF-I mRNAs has been reported in the brain of different species (see introduction). Furthermore, the addition of IGF-I antibody to rat pituitary cultures reduces the LH response to GnRH (43), indicating the presence in the medium of this component. The use of IGF-I antibodies or binding proteins in trout pituitary cultured cells should be useful in establishing a paracrine effect of IGF-I. If such an effect is demonstrated, it should allow to reject the possibility of a pharmacological effect suggested by the use of high doses of IGF-I. In a second step, its cellular origin and its regulation should be elucidated.

The present work do not allow to determine the mechanisms involved in this positive effect of IGF-I on GTH production, but they do suggest that they are different from those acting on GH production. The efficient dose of IGF-I, the timing of its application with that of GnRH, as well as the relationships with sexual status of fish are different. Indeed, the basal secretion of GH is affected by low doses (10-9 - 10-8 M) of IGF-I (44), with decreasing intensity with the maturation of the gonad (14), as we have previously reported. On the other hand, basal GTH I and GTH II releases were not significantly influenced by IGF-I, independently of the sexual status. With regard to the effect of IGF-I on the sensitivity to GnRH, that of GH necessitates to be induced a long application of IGF-I. In the case of maximal sensitivity (immature fish), the lower dose (10-8 M) is sufficient and the copresence of GnRH with IGF is not required. Furthermore, GH sensitivity decreases with the maturation of the gonads, because even the prolonged presence of IGF-I (10-8 M plus 10-6 M) is not sufficient in mature fish to induce a response to GnRH. On the other hand, for GTH sensitivity to GnRH cells have to be incubated with a simultaneous exposure to IGF-I and GnRH. This sensitivity is constantly augmented by IGF-I 10-6 M, whatever the sexual stage of the fish (the mean of R50 for GTH I and GTH II among five experiments is 12. 5 ± 1.4). We have to point out that the sensitivity to lower doses (such as 10-8 M) seems to be variable with the sexual cycle of the animals because it was detected only in early gametogenesis fish. These observations on GH and GTH sensitivity to GnRH allow us to hypothesize on a direct effect of IGF-I on GTH cells (paracrine/autocrine and endocrine) and also on an endocrine and/or indirect effect on GH cells, which need to be tested.

In conclusion, the in vitro potentiating effect of IGF-I (10-6 M) on GTH responses to GnRH exists at every stage of gametogenesis and seems to be higher in early gametogenesis fish in which the lower dose (10-8 M), corresponding to circulating levels, is also efficient in increasing GTH sensitivity. Furthermore, at this time IGF-I is still efficient in inducing a GH response to GnRH. These observations indicate the putative in vivo roles of IGF-I and GnRH at the time of puberty and could explain, in part, the relationship between body weight and acquisition of the first gametogenesis. In prepubescent animals, IGF-I could induce GH sensitivity to GnRH, thus leading to an increase in GH levels, which in turn could elevate the production of IGF-I, which would then induce more growth and the induction of puberty by acting in part on the central nervous system. Before testing this hypothesis in vivo, in vitro work must be done at the pituitary level by studying the possible paracrine/autocrine action of IGF-I and the effect of IGF-I on pituitary GnRH receptor characteristics, which could explain the observed increase in GnRH sensitivity. IGF-I action should also be investigated at the level of the brain by looking in vitro at the effect of IGF-I on GnRH production because IGF-I is known in rat to increase GnRH release (see introduction).


   Acknowledgments
 
The authors are grateful to Drs Märki and Kaufmann from Ciba-Geigy for kindly supplying rhIGF-I and to Dr. N. Bury for improving the English quality of this article.


   Footnotes
 
1 This work was partly financed by INRA in the frame work of a GDR program "Régulation de la croissance chez les poissons d’intérêt aquacole". Back

Received June 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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