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Regular Article |
a University of Washington School of Fisheries, Seattle, Washington 98192
b Northwest Fisheries Science Center, Seattle, Washington 98112
ABSTRACT
The effects of insulin-like growth factor I (IGF-I) and insulin on the function of coho salmon gonadotropes in vitro were investigated. Dispersed pituitary cells from immature coho salmon (Oncorhynchus kisutch) were incubated with IGF-I for 1, 3, 7, or 10 days, then incubated with salmon GnRH for an additional 24 h. Medium FSH content before and after GnRH treatment and intracellular FSH content after GnRH treatment were measured. Incubation of pituitary cells with IGF-I for 7 or 10 days increased GnRH-stimulated FSH release and remaining cell content, but did not affect basal release. To examine the specificity of the effects of IGF-I, we compared FSH release and cell content of FSH and LH after 10-day incubation with a range of concentrations of IGF-I or insulin. Incubation with physiological concentrations of IGF-I resulted in significantly higher GnRH-stimulated FSH release and remaining cell content of FSH and LH. Conversely, supraphysiological concentrations of insulin were required to produce more moderate effects on gonadotropin levels. These results suggest that elevation of gonadotropin levels by IGF-I may be one mechanism by which somatic growth and nutrition promote pubertal development in salmon.
anterior pituitary, FSH, growth factors, insulin, LH, puberty
INTRODUCTION
In most species of salmonids, the age of reproductive maturation varies both between and within populations. Although age of maturation is in large part a heritable trait, it is clear that, as in many other vertebrates, it is also influenced by somatic growth, energy balance, or both at critical times of year [1, 2].
The mechanisms by which growth rate and energy balance influence reproductive maturation in salmonids are not known. However, there is increasing evidence that metabolic hormones and growth factors may act directly at multiple levels of the hypothalamic-pituitary-gonadal axis to promote onset of puberty in a variety of vertebrates. One such factor is insulin-like growth factor I (IGF-I).
In mammals, IGF-I may promote puberty via both central and peripheral pathways. For example, intraventricular injections of IGF-I in female rats significantly advances puberty [3] and, in mammals, exogenous IGF-I increases LH pulsatility, circulating levels, or both, likely via effects on GnRH neurons [3, 4]. In the periphery, IGF-I may directly stimulate proliferation and function of testicular and ovarian germ and somatic cells, as shown in vitro for several species (reviewed in [5–8]). IGF-I may also act directly at the pituitary; addition of IGF-I to rat pituitary cell cultures increases basal FSH and LH secretion and GnRH-stimulated LH secretion [9–11].
There is also evidence for roles of IGF-I in stimulating reproductive development at both the gonadal and pituitary levels in teleosts. IGF-I induces final maturation of red seabream (Pagrus major) oocytes in vitro [12], and stimulates steroid production in coho salmon (Oncorhynchus kisutch) granulosa cell culture, but inhibits steroid production by theca-interstitial cell culture [13]. IGF-I also stimulates spermatogonial proliferation in spermatogenetic rainbow trout (O. mykiss) testes in vitro [14, 15]. At the pituitary level, IGF-I increases intracellular LH (formerly GTH II) levels and stimulates LH release in European eel (Anguilla anguilla) pituitary cell culture [16, 17]. Coincubation of rainbow trout pituitary cells with IGF-I and salmon GnRH (sGnRH) increases sensitivity to sGnRH as measured by gonadotropin release [18].
Two types of gonadotropins have been characterized in salmonids: FSH (formerly GTH I) and LH. During the year of maturation in coho salmon, FSH levels rise gradually over many months, coincident with gonad growth, vitellogenesis, and spermatogenesis, then decrease at the time of final maturation of the gametes. LH levels rise more sharply, immediately preceding final maturation of the gametes and spawning [19]. Similar patterns have been observed in chinook salmon (O. tshawytscha) [20] and Atlantic salmon (Salmo salar) [21], whereas in female rainbow trout, FSH peaks early in vitellogenesis and again at the time of spawning [22–25]. Thus, it is widely believed that in salmonids, FSH regulates gonadal growth, and that LH controls final maturation and release of gametes [19, 22]. Little is known about the mechanisms controlling the observed rise in FSH levels in salmonids.
To determine proximate mechanisms mediating the observed relationship between growth and reproductive maturation in salmonids, we are investigating the effects of metabolic hormones and growth factors on the reproductive endocrine axis. In this paper we present the results of experiments testing the effects of IGF-I on FSH content and release from coho salmon pituitary cell cultures in vitro. We examined the effect of IGF-I on FSH levels for several reasons. As noted earlier, IGF-I stimulates gonadotrope activity in some mammals and teleosts. Furthermore, circulating IGF-I levels correlate with growth [26] and with long-term nutritional status [27] in salmonids, suggesting that IGF-I could signal growth and energy status to the reproductive axis. Finally, we focused on FSH rather than LH because it most likely mediates the early events of pubertal development in salmonids.
MATERIALS AND METHODS
Experimental Design
Two experiments were conducted to examine the effect of IGF-I on basal and subsequent GnRH-stimulated release and remaining cell content of FSH. In experiment 1, the kinetics of the effects of IGF-I were examined in male and female fish. Cells were preincubated for 1, 3, 7, or 10 days with blank medium (control), 100 nM sGnRH (as a positive control), or 1–100 nM IGF-I. These concentrations of IGF-I were selected on the basis of peripheral levels of IGF-I in salmon [28, 29] and are similar to those used in other in vitro studies in teleosts [12–18]. After this preincubation period, medium was removed and all cells were incubated with 100 nM sGnRH for 24 h. In cultures prepared from male fish, three concentrations of IGF-I (1, 10, and 100 nM) were tested. Medium FSH concentrations after preincubation and after 24 h of sGnRH treatment, and FSH and DNA content of cell lysates were measured. In cultures prepared from female fish, only one concentration of IGF-I (40 nM) was tested because of a limited numbers of cells, and medium concentrations of FSH after preincubation and after 24 h sGnRH treatment were measured.
In experiment 2, the concentration responses to both IGF-I and insulin were examined in males and females. Cells were preincubated for 10 days with 0.1–100 nM IGF-I or insulin, 100 nM sGnRH, or blank (control) medium, prior to 24 h exposure to sGnRH. Medium FSH concentrations after preincubation and after 24 h sGnRH treatment were measured. Cell contents of FSH, LH, and DNA were measured in cultures from male fish only.
Fish
Experiment 1 was conducted in January and February 1998 using 2-yr-old coho salmon (University of Washington stock) reared from fertilized eggs at the hatchery facilities at the Northwest Fisheries Science Center (NWFSC), Seattle, Washington. Fish were reared on a standard ration of Biodiet Grower pellets (Bioproducts, Warrenton, OR) in recirculated fresh water under a seasonally adjusted photoperiod and relatively constant temperature (11–13°C). Experiment 2 was conducted in March and April 1998 using 2-yr-old coho salmon (Domsea Broodstock, Rochester, WA); these fish had been transported to the NWFSC and maintained as described earlier for 2 mo prior to experiments. Mean body weights and gonadosomatic indices (GSI = 100 x gonad weight/body weight) ± SEM of females were 165 ± 5 g and 0.43 ± 0.02%, respectively, in experiment 1 (n = 35), and 221 ± 14 g and 0.45 ± 0.03% in experiment 2 (n = 12). Mean body weights ± SEM of males were 186 ± 10 g in experiment 1 (n = 47), and 239 ± 11 g in experiment 2 (n = 24); GSI of males was <0.10% in both experiments. These fish were expected to mature at age 3, in late autumn 1998.
Hormones
Human recombinant IGF-I (Peninsula Laboratories, Belmont, CA) and bovine insulin (Sigma, St. Louis, MO) were dissolved in 0.1 M acetic acid and diluted to 1 µM in Waymouths medium (Gibco BRL, Gaithersburg, MD), and stored at -70°C until day of use. Salmon GnRH (Peninsula Laboratories) was dissolved in 0.1 M acetic acid, diluted to 10 µM in Waymouths medium, and stored at -70°C until day of use. Hormones were thawed on ice and diluted to final concentrations in Waymouths medium immediately prior to addition to cell cultures.
Pituitary Cell Culture
Fish were killed by decapitation and pituitary glands were collected and placed in ice-cold modified Hanks balanced salt solution (HBSS) with 10 µg/ml gentamycin sulfate and 10 units/ml penicillin G (Gibco BRL). The pituitary glands of 12 to 25 fish were pooled according to sex for each experiment. Pituitary cell dispersions and viable cell counts were performed as previously described [30]. Cells were suspended in modified Waymouths medium [30] and plated at a density of 105/well in 24-well plates (Falcon Primaria, Becton-Dickinson, Lincoln Park, NJ).
After plating, cells were incubated at 15°C under atmospheric air for 72 h to allow cells to settle and attach to the wells. Medium was removed and replaced with 500 µl modified Waymouths medium + 0.1% BSA (RIA grade, Sigma) containing the designated treatments. Cells were incubated at 15°C under atmospheric air for the designated number of days, the medium was removed and stored at -70°C until assayed for basal (preincubation) FSH content, and 500 µl modified Waymouths + 0.1% BSA containing 100 nM sGnRH was added to each well. Cells were incubated for an additional 24 h at 15°C. Medium was collected and stored at -70°C until assayed for sGnRH-stimulated FSH content. To harvest cells, plates were stored at -70°C, thawed, and 500 µl Ca++-free HBSS was added to each well. After two additional cycles of freezing and thawing, medium was vigorously pipetted and cell suspension was collected from wells and stored at -70°C until assayed for intracellular content of FSH, LH, and DNA.
Four replicate wells were used for each treatment in each experiment. Each treatment and incubation time was tested twice in each sex using different pituitary pools, with two exceptions. The time course studies only tested the 10-day incubation once per sex, and the comparison of IGF-I and insulin was conducted on cells from females only once.
Measurement of FSH and LH
For measurement of intracellular FSH and LH, cell suspensions were thawed, briefly sonicated, and centrifuged for 10 min at 10 000 x g. The supernatant was transferred to new tubes and stored at -70°C.
Culture media and cell lysates were analyzed for FSH and LH content using radioimmunoassays developed for coho salmon FSH and LH [31]. When necessary, samples were diluted in assay buffer prior to measurement. All samples from an individual trial were run in the same assay. Samples were assayed in duplicate. LH was assayed only in cell lysates due to insufficient amounts of medium for both FSH and LH measurements.
Measurement of DNA Content
To evaluate the effects of the treatments on cell number, DNA was measured using CyQuant Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) with the following modifications: Cell suspensions were thawed and diluted 1:1 with 2x CyQuant lysis buffer containing RNAse A (Ambion, Austin, TX) and incubated in 96-well cytofluor microtiter plates for 1 h at room temperature. CyQuant-GR was added to 1x final concentration, and plates were incubated 5 min at room temperature. Fluorescence was measured using a CytoFluor II microplate reader (PerSeptive Biosystems, Framingham, MA) at 485/20 nm excitation, 530/25 nm emission, and gain of 90. Fluorescence values were converted to DNA concentrations using a standard curve.
Statistical Analysis
ANOVA, followed by Tukey's honestly significant difference test, was used to determine differences in FSH, LH, and DNA concentrations among hormone treatments and incubation times. Calculations were performed with SPSS 8.0 (SPSS, Inc., Chicago, IL). Differences were considered significant at P < 0.05.
RESULTS
Experiment 1: Kinetics of the Effects of IGF-I
Effect of IGF-I on basal FSH release In cultures of dispersed pituitary cells prepared from either males or females, medium FSH levels increased over time in all treatments during the preincubation period (Figs. 1a and 2a). For all incubation times, medium FSH levels were higher in the sGnRH-treatment groups than in the control or IGF-I-treatment groups. IGF-I did not consistently affect basal FSH release during preincubation relative to controls, although in one trial in males, 7- or 10-day treatment with 1 nM IGF-I resulted in significantly lower basal FSH release than controls (Fig.1a).
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Effect of IGF-I on sGnRH-induced FSH release In pituitary cell cultures prepared from males, there were significant concentration-dependent and time-dependent effects of preincubation with IGF-I on subsequent sGnRH-induced FSH levels (Fig. 1b). Preincubation with IGF-I for 1 or 3 days did not consistently alter subsequent sGnRH-induced FSH release. However, preincubation with 10 or 100 nM IGF-I for 7 days, and with 1–100 nM IGF-I for 10 days significantly increased sGnRH-induced FSH release compared with preincubation in control medium.
The effect of preincubation time on subsequent sGnRH-induced FSH release differed among treatments. Within controls, sGnRH-stimulated FSH release significantly decreased with preincubation time, whereas within IGF-I treatments, sGnRH-stimulated release significantly increased with preincubation time. Time of preincubation with sGnRH did not significantly alter the amount of FSH released during the subsequent 24-h sGnRH treatment (Fig. 1b).
Similarly, in females, FSH release in response to 24-h sGnRH exposure was significantly higher after 7- and 10-day preincubation with IGF-I than control medium (Fig. 2b). Within treatments, sGnRH-induced release decreased with preincubation time in controls, but sGnRH-induced release tended to increase with preincubation time with IGF-I.
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Effect of IGF-I on intracellular FSH levels In males, 3-, 7-, and 10-day preincubation with IGF-I resulted in significantly higher levels of intracellular FSH remaining after exposure to sGnRH than preincubation with control medium (Fig. 1c). After 3 days of preincubation, FSH content of cells treated with 100 nM IGF-I was significantly higher than in controls. After 7 and 10 days, all concentrations of IGF-I tested resulted in significantly higher levels than controls, and levels were concentration-dependent.
Intracellular FSH content decreased significantly with time of preincubation with control medium and sGnRH, but did not change over time with 1 nM IGF-I treatment. Within 10 nM and 100 nM IGF-I treatments, FSH content increased significantly with time of preincubation (Fig. 1c).
Effect of IGF-I on DNA content Neither time nor treatment caused significant, consistent effects on DNA concentrations in cultures prepared from males (Fig. 1d).
Experiment 2: Concentration-Response and Comparison of IGF-I and Insulin
FSH release In pituitary cell cultures prepared from males, neither IGF-I nor insulin affected FSH release during the 10-day preincubation period (Fig. 3a). However, preincubation of cells for 10 days with 1, 10, or 100 nM IGF-I increased subsequent sGnRH-induced FSH release in a concentration-dependent manner (Fig. 3b). Preincubation with insulin increased sGnRH-induced FSH release, but differences were only statistically significant after preincubation with 100 nM insulin (Fig. 3b).
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In females, there was no significant effect of any concentration of IGF-I or insulin on basal FSH levels in the medium (Fig. 4a). However, preincubation of cells with 10 nM or 100 nM IGF-I increased subsequent sGnRH-induced FSH release, while preincubation with insulin was ineffective (Fig. 4b).
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Intracellular FSH In pituitary cell cultures prepared from males, intracellular FSH content was significantly higher in cells preincubated with 1, 10, or 100 nM IGF-I than in controls (Fig. 3c). Treatment with 10 or 100 nM insulin also resulted in higher FSH content, but differences were not consistently significant. Preincubation with 10 or 100 nM IGF-I resulted in intracellular FSH levels that were significantly higher than any other treatment.
Intracellular LH In males, cellular content of LH was significantly higher after incubation with 1–100 nM IGF-I or with 10 or 100 nM insulin than with control medium, and the response was concentration-related (Fig. 3d).
DNA In pituitary cell cultures prepared from males, incubation with 10 or 100 nM IGF-I tended to increase DNA content, although such differences were not consistently significant (Fig. 3e).
DISCUSSION
Our results show that IGF-I may act directly at the pituitary to influence FSH levels in coho salmon; IGF-I increased not only sGnRH-stimulated release of FSH, but also the intracellular content of FSH. The effects were both concentration- and time-dependent. Whereas stimulatory effects of IGF-I on FSH and LH release in rat [9, 10] and on LH content and release in eel [16] have previously been described, to our knowledge, this is the first evidence of an effect of IGF-I on pituitary FSH content in any vertebrate.
We observed no increase in the basal (non-GnRH-stimulated) release of FSH during preincubation with IGF-I; in some instances, a slight decrease was observed. Lack of IGF-I effects on basal gonadotropin release in vitro agrees with a study of rainbow trout [18], but differs from studies of eel and rat. In long-term dispersed pituitary cell cultures from eel, increases in basal GTH II (LH) release were measurable by Day 9 [16]. In cultures of rat dispersed pituitary cells, incubation with IGF-I increased basal release of both FSH [9] and LH [9, 10].
It has recently been reported that incubation with IGF-I for 2–3 days increases sensitivity of rainbow trout pituitary cells to GnRH [18]. Our results extend the findings of that study, in that our longer period of culture with IGF-I (7–10 days) increased both the magnitude of GnRH-induced FSH release and the cell content of FSH and LH. Furthermore, in the trout, IGF-I was only effective in increasing sensitivity to GnRH when the two hormones were coincubated, while we observed an effect of preincubation of IGF-I on amplitude of subsequent GnRH response in the absence of IGF-I.
In salmonids, gonadotropes are specialized; individual cells produce either FSH or LH, but not both [32]. To test whether the effect of IGF-I was specific for FSH-producing gonadotropes, the intracellular content of LH remaining after sGnRH exposure was also measured. LH content was higher after preincubation with IGF-I, suggesting that IGF-I effects were not limited to FSH cells. However, because medium content of LH was too low to measure, it is unclear whether IGF-I enhanced GnRH-induced LH release.
In the current experiments, IGF-I was effective within a physiological range. In coho salmon, receptor binding and efficacy of mammalian IGF-I and insulin are approximately equivalent to that of the salmonid homologues [14, 33–35]; therefore, our results would likely be similar if salmonid hormones were used. In immature coho salmon, total IGF-I circulates at levels of approximately 4 nM, although free IGF-I levels are much lower [28]. IGF-I is also produced in the pituitary in mammals [36, 37] and in at least one teleost [38], suggesting that pituitary levels in salmonids may be higher than plasma levels. Whether IGF-I acts via paracrine or endocrine pathways in the pituitary in vivo will require further study.
We compared the responses to insulin and IGF-I to test for specificity and to evaluate whether the effects of IGF-I are mediated by IGF-type receptors. In salmonids, IGF-I may bind to both IGF-type receptors and, at high concentrations, insulin receptors [39, 40]. While long-term incubation with insulin also resulted in increased GnRH-induced FSH release and cell content of FSH and LH, these effects required concentrations 10- to 100-fold higher than that required for IGF-I. These results suggest that the effect of IGF-I was likely mediated via IGF receptors, rather than insulin receptors. The lack of response in salmon to physiologic concentrations of insulin is very different from that observed in rat, where lower concentrations of insulin than IGF-I were effective in inducing basal and GnRH-stimulated LH release, although both hormones were effective within their respective physiologic ranges [10].
The effects of IGF-I on gonadotropin levels may be a result of increased cell number. IGF-I may affect cell number by promoting proliferation, as demonstrated in many different mammalian cell types (reviewed in [5, 41]) as well as in some salmonid cell types [14, 15, 42]. However, whether IGF-I has a proliferative effect on pituitary cells in general, or on gonadotropes in particular, has not been established in teleosts. In mouse pituitary cell culture, IGF-I stimulates proliferation of corticotrophs and mammotrophs, but not LH-positive or FSH-positive gonadotropes [43]. IGF-I may also affect cell number by inhibiting apoptosis [41]. Evidence of an antiapoptotic effect of IGF-I on gonadotropes is lacking, although IGF-I does inhibit apoptosis of pituitary somatotrophs in the teleost tilapia hybrid, Oreochromis aureus x O. niloticus [38].
In the present study, total DNA content was measured as an index of cell proliferation or survival. In experiment 1, there was no trend in DNA levels after IGF-I treatment relative to controls. In experiment 2, DNA content tended to increase after incubation with the highest concentrations of IGF-I; however, significant differences in FSH release were observed after incubation with 1 nM IGF-I, despite a lack of increased DNA. This suggests that the effects of IGF-I are not solely a result of an effect on total cell number, although it remains possible that IGF-I affected proliferation or survival of gonadotropes, but the assay could not detect such a change in a mixed-cell culture.
In rats, IGF-I stimulates LH release without apparent effect on total pituitary cell number [10, 11]. In eels, IGF-I stimulates LH content and release without an apparent increase in LH-positive cells relative to controls, as measured by immunohistochemistry [16]. Based on previously reported functions of IGF-I, cell number-independent mechanisms of action could include effects of glucose and amino acid uptake, gene transcription, protein synthesis, and differentiation (reviewed in [5, 6]).
The results presented here suggest that IGF-I, acting at the pituitary, may directly link growth and nutritional state with the reproductive endocrine axis in salmonids, and therefore be a contributing factor in the inverse correlation between growth and age of maturity in salmon. High levels of IGF-I, such as those found in fast-growing or nutritionally replete fish, may increase the ability of pituitary to respond to seasonal cues with sufficient FSH release, thereby permitting initiation or continuation of pubertal development. While further study is required to determine the mechanism by which IGF-I stimulates FSH and LH content and FSH release, it should be considered that any of these mechanisms could be biologically relevant. That is, whether IGF-I increases numbers or differentiation of gonadotropes, or specifically or nonspecifically promotes gonadotropin synthesis, the result would be similar: increased levels of gonadotropins available to stimulate maturation of the gonads.
ACKNOWLEDGMENTS
We thank Bradley A. Gadberry and Timothy W. Newcomb for rearing the fish used in these studies.
FOOTNOTES
First decision: 9 January 2000.
1 This work was supported by Bonneville Power Administration (Projects 92-022 and 93-056) and by the Joint Institute for the Study of the Atmosphere and Ocean under National Oceanic and Atmospheric Administration cooperative agreement NA67RJO155, contribution 758. D.M.B. was supported in part by the H. Mason Keeler Fellowship of the University of Washington School of Fisheries. 
2 Correspondence: Dianne M. Baker, University of Washington School of Fisheries, Box 355020, Seattle, WA 98195. FAX: 206 860 3267; dibaker{at}u.washington.edu
Accepted: April 25, 2000.
Received: December 29, 1999.
REFERENCES
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