INTRODUCTION

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IN TELEOST FISHES, there is increasing evidence to suggest that the control of growth hormone (GH) release is influenced by gonadal steroid hormones (9). In a number of species, the administration of exogenous gonadal steroid hormones to immature animals has been shown to increase plasma GH levels (8, 17, 25), suggesting an action of steroid hormones on GH release. Moreover, elevated plasma GH levels have been reported in several species during sexual maturation (1, 7, 11, 12, 22, 23, 25). It is not clear how steroid hormones influence GH release in teleosts. Gonadal steroid hormones may directly affect GH release at the level of the somatotroph or have an indirect mechanism of action mediated via altered synthesis and release of hypothalamic GH regulatory factors and/or changes in the responsiveness of the pituitary gland to hypothalamic stimuli.

Recent work in teleost fishes suggests that one potential means of gonadal steroid hormone regulation of GH release is via altered somatostatin (SS) inhibitory tone (21). In the goldfish (Carassius auratus), it has been shown that there was a decrease in immunoreactive (ir) SS in the hypothalamus when serum GH levels were the highest (15). This period corresponded to the phase of sexual recrudescence and maturation (21). Similarly, in the rainbow trout (Oncorhynchus mykiss), plasma SS-14 levels declined progressively with advancing gonadal development, and plasma SS-14 levels were negatively correlated with plasma GH concentrations (11). These data suggest that during sexual maturation, the gonadal steroid hormones may alter both hypothalamic SS inhibitory tone and circulating SS levels. Moreover, we previously showed that in vivo administration of 17-estradiol (E₂) significantly increased plasma GH levels and decreased plasma SS-14 levels in rainbow trout (10), further indicating that gonadal steroid hormones may increase GH levels indirectly via altered SS inhibitory tone.

It is currently not known if the observed decline in plasma SS-14 after E₂ treatment reflects a decreased SS-14 release alone or if E₂ also alters peripheral and/or hypothalamic SS-14 synthesis. Therefore, in this study we examine the effects of E₂ on peripheral and hypothalamic SS mRNA levels, as well as plasma SS concentrations. Because it was recently reported that there are three distinct mRNAs in rainbow trout that encode for different forms of SS (13, 19), we have examined the possibility that these three mRNAs are differentially regulated by E₂.

	MATERIALS AND METHODS

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Experimental animals. Juvenile rainbow trout, Oncorhynchus mykiss, were obtained from the Alma Research Station (Alma, ON). The fish were held in 80-liter fiberglass tanks supplied with constantly running aerated well water at 9 ± 2°C, held under a 13:11-h light-dark photoperiod, and were fed commercial pelleted trout food (Martin's Feed Mills, Elmira, ON, Canada) daily to satiety. The fish were acclimated to these conditions for at least 2 wk before the experiments.

Steroid pellet implantation. Juvenile rainbow trout (n = 20 for both treatment groups) were given either a slow-release implant of E₂ (Sigma Chemical, St. Louis, MO) dissolved in coconut oil or coconut oil alone (control) to deliver a dose of 10 µg/g body wt in a volume of 0.5 ml, after Flett and Leatherland (6). The initial mean body weight of the fish was 404.1 ± 15.14 g. Two weeks after steroid implantation, each fish was anesthetized in MS-222 (125 mg/l), weighed, and bled into heparinized tubes after caudal severance. Plasma was stored at 70°C until analyzed for GH and SS concentrations. The endocrine pancreas (Brockmann body) and hypothalamus were removed from each animal, and ~100 mg of each tissue were placed in a 2-ml microcentrifuge tube, immediately frozen on dry ice, and stored at 90°C until analysis.

Assays. Plasma GH was analyzed using a noncompetitive two-site ELISA for oncorhynchid GH developed by Farbridge and Leatherland (5), and plasma SS-14 and SS-25 concentrations were measured by RIA validated for use with rainbow trout plasma (3).

SS mRNA quantitation. Total RNA was extracted from the Brockmann bodies and hypothalamus by a modification of the RNAzol method (16). Preprosomatostatin (PPSS) I, PPSS II', and PPSS II" mRNA levels were measured by a quantitative slot-blot technique (19) in which in vitro-synthesized cRNA standards were blotted onto nylon membranes along with sample RNA. The membranes were first hybridized with gene-specific ³²P-labeled oligonucleotide probes and quantified using the Packard Cyclone Phosphor System (Meriden, CT). Sample blots were then stripped and rehybridized with a ³²P-labeled full-length gamma-actin probe and requantitated. After correction for differences in probe specific activity and normalization to gamma-actin, mRNA levels were expressed as molecules of somatostatin mRNA × 10⁸/µg total RNA.

Statistical analyses. Plasma GH, SS-14, and SS-25 levels and the tissue levels of PPSS I, PPSS II', and PPSS II" mRNA were compared between the E₂-primed and oil-primed treatment groups using SigmaStat (Jandel Scientific, San Rafael, CA). Data were examined for normality and equal variance. When the data passed these tests, they were analyzed using Student's t-test (P = 0.05); if data failed either test, they were compared using the Mann-Whitney rank sum test.

	RESULTS

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The HSI in the E₂-primed fish was significantly (P < 0.005) elevated relative to the control (E₂: 1.51 ± 0.09; control: 1.16 ± 0.06). Plasma GH levels were significantly (P < 0.05) elevated in the E₂-treated fish (4.33 ± 0.71 and 2.90 ± 0.20 ng/ml for E₂-treated fish and controls, respectively). Plasma SS-14 levels were significantly (P < 0.001) depressed in E₂-primed fish (0.375 ± 0.009 and 0.556 ± 0.017 ng/ml for E₂-treated and controls, respectively). Similarly, E₂ treatment significantly reduced plasma SS-25 levels (P < 0.001) (0.359 ± 0.014 and 0.440 ± 0.014 ng/ml for E₂-treated and controls, respectively) (Fig. 1).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effects of 17-estradiol on the concentrations of growth hormone (GH; A), somatostatin (SS)-14 (B), and SS-25 (C) in the plasma of rainbow trout. Data are presented as means ± SE (n = 20). * Significant difference compared with the oil-treated control group (P < 0.05).

In the hypothalamus, E₂ treatment significantly (P < 0.001) decreased the levels of PPSS I and PPSS II" mRNA (PPSS I: 1.417 ± 0.111 and 0.587 ± 0.036; PPSS II": 1.768 ± 0.121 and 0.965 ± 0.0811 molecules mRNA × 10⁸/µg total RNA for control and E₂-treated animals, respectively). However, there was no effect of E₂ treatment on PPSS II' mRNA levels (1.620 ± 0.141 and 1.397 ± 0.153 molecules mRNA × 10⁸/µg total RNA for the control and E₂-treated animals, respectively) (Fig. 2). In the pancreas, E₂ treatment had no significant effect on the levels of either PPSS II' mRNA (control: 8.116 ± 0.604; E₂-primed 8.465 ± 0.729 molecules mRNA × 10⁸/µg total RNA) or PPSS II" (control: 4.226 ± 0.242; E₂-primed 4.052 ± 0.335 molecules mRNA × 10⁸/µg total RNA). However, E₂ treatment significantly (P < 0.005) decreased levels of PPSS I mRNA (control: 4.098 ± 0.083; E₂-primed 2.629 ± 0.322 molecules mRNA × 10⁸/µg total RNA) (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of 17-estradiol on the abundance of mRNAs encoding preprosomatostatin (PPSS) I (A), PPSS II" (B), and PPSS II' (C) in the hypothalamus of rainbow trout. The results are presented as a representative slot blot (insets) or as means ± SE (n = 20). * Significant difference compared with the oil-treated control group (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of 17-estradiol on the abundance of mRNAs encoding PPSS I (A), PPSS II" (B), and PPSS II' (C) in the Brockmann bodies (endocrine pancreas) of rainbow trout. The results are presented as a representative slot blot (insets) or as means ± SE (n = 20). * Significant difference compared with the oil-treated control group (P < 0.05).

	DISCUSSION

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This study provides further evidence for the regulation of somatostatinergic inhibitory tone by E₂ in vivo in rainbow trout, both at the level of circulating SS and tissue-specific SS synthesis. This study confirms a previous report of an inhibitory action of E₂ administration on plasma SS-14 levels in rainbow trout (10) and provides the first evidence for an inhibitory effect of E₂ on plasma SS-25 concentrations.

It has been suggested that, as in mammals, circulating SS-14 may contribute to the overall GH-inhibitory tone in teleost fish (10). However, the role of circulating plasma SS-25 is less clear. In goldfish, salmonid SS-25 did not inhibit GH release from pituitary fragments in vitro (18); however, there is no information available regarding the effect of SS-25 on GH release in salmonid species. Therefore, it is possible that the decreased levels of circulating SS-25 could also contribute to an overall decrease in somatostatinergic inhibition of pituitary GH release; however, further studies are required to determine if SS-25 has GH-inhibitory actions in rainbow trout.

Although in vivo studies suggest that circulating SS may inhibit pituitary GH release (2, 24), the relative importance of the plasma SS pool, of primarily peripheral origin, compared with hypothalamically derived SS to GH secretion is not known. Moreover, measurement of plasma SS levels does not resolve whether the inhibition of circulating SS levels after E₂ treatment is a result of decreased peripheral and/or hypothalamic SS synthesis or merely reflective of decreased SS release. Therefore, the main objective of this study was to examine the effect of E₂ on the synthesis of SS in both the hypothalamus and the endocrine pancreas (Brockmann body).

In mammals, gonadal steroid hormones have been shown to affect both hypothalamic SS content as well as SS synthesis (4). Moreover, the mean SS release and hypothalamic SS content vary during the estrous cycle in rats concomitant with fluctuations in plasma levels of gonadal steroid hormones (4). Similarly, in goldfish, there are seasonal alterations in levels of PPSS mRNA in the brain (14). The study reported here provides the first evidence that E₂ administration can affect SS synthesis in both the brain and the Brockmann bodies of rainbow trout. Moreover, E₂ administration results in differential regulation of the expression of the three previously identified rainbow trout SS mRNAs, PPSS I, PPSS II', and PPSS II" (13, 19), in the hypothalamus and pancreas. In the present study, we have identified PPSS II' mRNA in the hypothalamus of the rainbow trout, but its expression was not affected by E₂ treatment. These data contrast with a previous study where PPSS II' was not identified in whole brain preparations (19). This discrepancy may stem from the nature of the preparation or from variations in the physiological state of the animals used in these studies, but this remains to be determined. The present study has shown that in the hypothalamus, E₂ administration significantly decreases the levels of PPSS I mRNA, which encodes for a PPSS that gives rise to SS-14 with a sequence identical to mammalian SS-14 (13). These data are consistent with a recent study in goldfish that reported that seasonal alterations in PPSS I mRNA levels (encoding for SS-14 identical to the mammalian form) in the hypothalamus show a close association with the seasonal variations in irSS-14 (14). Taken together, these data suggest that E₂-stimulated increases in plasma GH (8, 25, 26) and pituitary GH content (26) might be due, in part, to a downregulation of the hypothalamic SS-14 inhibitory tone. In addition to a reduction in PPSS I mRNA, E₂ treatment also significantly reduced levels of PPSS II" mRNA, which encodes for a PPSS that could be processed to SS-25 or [Tyr⁷,Gly¹⁰]-SS-14 (19). However, because it is currently not known whether SS-25 or [Tyr⁷,Gly¹⁰]-SS-14 can inhibit GH secretion in rainbow trout, the significance of this reduction in PPSS II" to the regulation of GH is unknown. In goldfish, [Pro²]-SS-14 is able to inhibit basal GH secretion from perifused pituitary fragments in vitro (14), suggesting that some SS-14 variants are biologically active; however, further studies are required to determine if SS-25 or [Tyr⁷,Gly¹⁰]-SS-14 have GH-regulatory actions. E₂ administration did not alter PPSS II' mRNA in the hypothalamus.

It has been suggested that in mammals, SS-14 produced in peripheral tissues, as well as the SS-14 of hypothalamic origin, may influence pituitary GH release (20). Therefore, steroid hormone-induced changes in SS synthesis in peripheral tissues may also contribute to an overall alteration in the endogenous somatostatinergic inhibitory tone. Three distinct SS mRNAs have been identified in the endocrine pancreas of rainbow trout: PPSS I (encoding for SS-14), PPSS II' (encoding for SS-28 and [Tyr⁷,Gly¹⁰]-SS-14), and PPSS II" (encoding for SS-25 and [Tyr⁷,Gly¹⁰]-SS-14) (19). E₂ treatment significantly reduced PPSS I mRNA levels in the endocrine pancreas, similar to its effect in the hypothalamus. This result is consistent with the inhibition of plasma SS-14 levels after E₂ treatment. These results suggest that E₂ may increase GH release by decreasing synthesis of hypothalamic and pancreatic SS-14 and reducing plasma levels of SS-14. By contrast, E₂ treatment did not affect levels of PPSS II' or PPSS II" mRNA levels in the pancreas. These data suggest that the decreased levels of circulating SS-25 are not a result of decreased peripheral synthesis and therefore may reflect changes in secretion and/or clearance of SS-25.

In conclusion, this study has shown that E₂ administration to juvenile rainbow trout results in a significant reduction in plasma SS-14 and SS-25 levels. In addition, E₂ causes a decrease in both hypothalamic and pancreatic PPSS I mRNA and hypothalamic, but not pancreatic, PPSS II" mRNA. E₂ had no effect on either hypothalamic or pancreatic levels of PPSS II' mRNA. This is the first report of differential regulation of SS genes in rainbow trout. These data suggest that E₂ acts, in part, to increase plasma GH levels in rainbow trout by decreasing the endogenous inhibitory somatostatinergic tone by inhibiting both plasma levels of SS-14 and SS-25 and hypothalamic levels of mRNA encoding these proteins.