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In vitro experiments were conducted to test the hypothesis that 17alpha,20beta-dihydroxy-4-prengnen-3-one (17,20-P) regulates cortisol metabolism in Pacific salmon. In both rainbow trout and coho salmon, cortisol metabolism was significantly higher in the kidney compared to the liver. The rainbow trout kidney converted cortisol primarily into an unidentified water-soluble metabolite with a molecular mass of 354. The coho salmon kidney converted cortisol primarily into cortisol-21-sulfate. High physiological concentrations of 17,20-P had no effect on cortisol metabolism by the rainbow trout kidney, but almost completely inhibited the production of cortisol-21-sulfate by the coho salmon kidney. This was accompanied by a coincident increase in the production several neutral cortisol metabolites, including cortisone. Cortisone was also found to inhibit renal sulfotransferase (SULT) activity suggesting that there could be a local positive feedback mechanism initiated by the rise in 17,20-P that quickly reduces SULT activity as follows: the pre-spawning rise in 17,20-P inhibits SULT, cortisol is metabolized to cortisone instead of cortisol-21-sulfate, cortisone further inhibits SULT, more cortisone is produced, and so on. If SULT normally acts as a gatekeeper enzyme to protect the cell from cortisol excess, this mechanism would rapidly remove enzymatic protection and expose tissues to high local concentrations of cortisol. In addition, the inhibition of peripheral cortisol metabolism by 17,20-P could increase circulating concentrations of the corticosteroid. These events could be a part of the mechanism that leads to the symptoms of cortisol excess associated with the post-spawning mortality of semelparous Pacific salmon.

Physiological levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20-P) stimulated time- and dose-dependent increases in cortisol production by rainbow trout (Oncorhynchus mykiss) interrenal tissue cultured in vitro. Significant stimulation occurred in response to 100, 300, and 1000 ng/ml of 17,20-P. Lower doses were ineffective. Elevated cortisol levels were observed 1 hr after addition of 300 ng/ml 17,20-P. No additive or synergistic interaction was evident between human adrenocorticotropin fragment 1-24 (ACTH1-24) and 17, 20-P in stimulating cortisol secretion, although 300 ng/ml 17,20-P could further enhance cortisol production above levels already stimulated by 300 ng/ml ACTH. 17alpha, 20alpha-dihydroxy-4-pregnen-3-one also stimulated cortisol secretion, but was only half as effective as 17,20-P. Estradiol-17beta, testosterone, and 11-ketotestosterone had no effect on cortisol secretion. Inhibitors of mRNA and protein synthesis had no effect on 17,20-P-stimulated cortisol production. Radiotracer studies demonstrated that the bioconversion of 17,20-P to cortisol could fully account for the cortisol produced by the interrenal in response to 17,20-P and demonstrated that rainbow trout interrenal cells contain an active 20beta-hydroxysteroid dehydrogenase. These data suggest that 17,20-P may be a regulator of cortisol production during the periovulatory period in salmonid fishes.

Interrenal tissue from embryonic and larval rainbow trout (Oncorhynchus mykiss) was cultured in vitro and exposed to various doses of adrenocorticotropin (ACTH) to document the ontogeny of interrenal responsiveness to tropic stimulation. Resting and acute stress-induced changes in whole-body cortisol levels in vivo were also measured to determine if the corticosteroid stress response first develops with the onset of interrenal responsiveness to ACTH. No evidence was found that the hypothalamic-pituitary-interrenal (HPI) axis of rainbow trout is transiently activated prior to hatching. In vivo, a corticosteroid stress response was first observed 2 weeks after hatching, and stress-induced cortisol levels (at 1 hr poststress) were significantly higher 3 weeks after hatching than they were at 2 or 4 weeks after hatching. In contrast, cultured interrenal tissue produced significant levels of cortisol in response to ACTH at the time of hatching, and in vitro cortisol production by the interrenal increased significantly between 3 and 4 weeks after hatching. Interrenal sensitivity to ACTH did not change appreciably with development. We conclude that (1) the final maturation of the corticosteroid stress response in rainbow trout occurs at the level of the brain and/or sensory inputs and not at the level of the interrenal cell; (2) negative feedback mechanisms within the HPI axis develop 3 to 4 weeks after hatching; and (3) the period between 3 and 4 weeks after hatching may be homologous to the stress hyporesponsive period after birth in mammals and thus could be a stage when environmental influences can permanently alter the development of the corticosteroid stress response in rainbow trout.

The ontogeny of the interrenal stress response in rainbow trout was characterized by measuring resting and acute-stress-induced changes in whole-body cortisol levels in embryos and larvae at different early developmental stages. In Experiment 1, resting cortisol levels averaged 6.0 ng/g in newly fertilized eggs, fell to less than 0.3 ng/g by the time of hatching at Week 4 (incubation at 10 degrees), and increased to 1.4 ng/g by Week 5. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. In Experiment 2, resting cortisol averaged 1.4 ng/g in newly fertilized eggs, fell to less than 0.03 ng/g by Week 2, and then steadily increased between Weeks 3 and 6 to a peak of 4.8 ng/g before falling to 1.2 ng/g by Week 7. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. Six-week-old fish showed a 2.3-fold increase in cortisol levels at 1 hr poststress, indicating that the hypothalamic-pituitary-interrenal axis first develops responsiveness to stress 2 weeks after hatching and 1 week before the onset of exogenous feeding. The stress hyporesponsive period after hatching in rainbow trout may be homologous to the 2-week stress hyporesponsive period after birth in rodents, the function of which may be to maintain low, constant corticosteroid levels during a critical developmental period when these steroids can have permanent effects on neural organization. As suggested for mammals, this period may be a time when rainbow trout are particularly vulnerable to environmental effects on their subsequent development.