Effects of 17alpha,20beta-dihydroxy-4-pregnen-3-one on cortisol production by rainbow trout interrenal tissue in vitro.

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.

In vitro effects of ACTH on interrenal corticosteroidogenesis during early larval development in rainbow trout.

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.

Ontogeny of the cortisol stress response in larval 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.

Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost,Stizostedion vitreum.

The annual reproductive cycle of walleye (Stizostedion vitreum) was characterized by documenting changes in gonadal development and serum levels of estradiol-17ß (E2), testosterone (T), 17α,20ß-dihydroxy-4-pregnen-3-one (17,20-P), and 11-ketotestosterone (11-KT) in wild fish captured from upper midwestern lakes and rivers throughout the year. Fish from the populations used in this study spawn annually in early- to mid-April. Walleye showed group synchronous ovarian development with exogenous vitellogenesis beginning in autumn. Oocyte diameters increased rapidly from ∼ 200 µm in October to ∼ 1,000 µm in November, and reached a maximum of 1,500 µm just prior to spawning. Changes in gonadosomatic indices (GSIs) paralleled changes in oocyte diameters. Serum E2 levels in females increased rapidly from low values in October (< 0.1 ng ml(-1)) to peak levels of 3.7 ng ml(-1) in November, coinciding with the period of the most rapid ovarian growth. Subsequently, E2 levels decreased from December through spawning. Serum T levels exhibited a bimodal pattern, increasing to 1.6 ng ml(-1) in November, and peaking again at 3.3 ng ml(-1) just prior to spawning. We detected 11-KT in the serum of some females at concentrations up to 5.6 ng ml(-1), but no seasonal pattern was apparent. In this study (unlike our results in a related study) 17,20-P was not detected. In males, differentiation of spermatogonia began in late August, and by January the testes were filled (> 95% of germ cells) with spermatozoa. Mature spermatozoa could be expressed from males from January through April. GSIs ranged from 0.2% (post-spawn) to 3.2% (pre-spawn). Serum T levels rose from undetectable levels in post-spawn males to 1.6 ng ml(-1) by November, remained elevated throughout the winter, and peaked at 2.8 ng ml(-1) I prior to spawning. Levels of 11-KT in males remained low (< 10 ng ml(-1), from post-spawning through January, then increased significantly by March and peaked just prior to spawning at 39.7 ng ml(-1). Our results indicate that vitellogenesis and spermatogenesis are complete or nearly so, in walleye by early winter, and suggest that it may be possible to induce spawning in this species several months prior to the normal spawning season by subjecting fish to relatively simple environmental and hormonal treatments.

Effects of the uterus and mid-pregnancy conceptus on luteal lifespan of the rat.

Hysterectomy on or after Day 10 resulted in a quick return of pseudopregnant rats to oestrus but the interval to oestrus in pregnant rats was significantly longer (P less than 0.01), indicating that the gravid uterine horn interfered with uterine-induced luteolysis by Day 10. When pseudopregnant rats were injected with extracts of conceptuses or pregnant rat serum (PRS), samples collected on Days 10 or 12 of pregnancy delayed the return to oestrus (P less than 0.01 for conceptus extracts, P less than 0.05 for PRS) and serum progesterone concentrations on Day 13 of pseudopregnancy were higher (P less than 0.01) than in controls. Bioassays utilizing mammary development or inhibition of oestrus in virgin cyclic rats as endpoints failed to detect lactogenic activity in Day 10 conceptus extracts or PRS, and radioreceptor assays using bovine prolactin (NIH-B5) as a standard showed that Day 10 extracts had only about 2--3% of the lactogenic activity found in Day 12 extracts. Injections of dilutions of Day 12 extracts with 2 or 4 times the lactogenic activity as Day 10 extracts did not extend pseudopregnancy. These results suggest that by Day 10 the conceptus produces a factor capable of interfering with luteolysis and that this factor is not placental lactogen.