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Notes: This study examines the effect of the steroid analogue, RU486, on the physiological responses of fed and chronically fasted rainbow trout to an acute handling stressor. This potent ligand of the glucocorticoid receptor was administered as a slow-release implant either alone, or in combination with cortisol. There were temporal changes in plasma cortisol concentrations following administration of cortisol implants in both fed and fasted trout. By day 14, plasma cortisol levels in fed fish were similar in all treatment groups, but in fasted fish, the effect of cortisol administration on plasma cortisol concentrations was still evident; RU486 administered with cortisol, did not affect this response. Cortisol administration also elicited a small, but significant increase in plasma GH concentrations in fed rainbow trout and in plasma glucose concentrations in fasted animals. RU486-treatment prevented these responses. Conversely, whereas RU486 alone had no effect on hepatic 5′-monodeiodinase activity, when administered with cortisol it enhanced the marked suppressive effect of cortisol evident in both fed and fasted groups, suggesting that it may exert an interactive effect with cortisol on this process. Stressor-related changes in plasma cortisol, glucose, GH and thyroid hormone concentrations were evident in both fed and fasted groups; however, there was no evidence of a suppressive effect of RU486 treatment on any of the measured plasma parameters. Although RU486 did not prevent the stressor-related changes, the post-stressor cortisol profiles in RU-treated trout were extremely erratic compared with the oil-treated controls. This implies a disturbance of the normal interactions of the components of the hypothalamus-pituitary-interrenal tissue axis.

Notes: The present study investigates the effect of cannulation and chronic‘black-box’ confinement, as well as epinephrine administration (4–0 μg kg−1), on the degree and time-course of alterations in trout (Oncorhynchus mykiss) catecholamine and cortisol concentrations. Plasma cortisol concentrations in seawater trout acclimated to 3–6° C reached 104 ng ml−1 1 day after cannulation/confinement and remained elevated above resting levels (8 ng ml−1) until 6 days post-confinement. Although plasma epinephrine and norepinephrine generally declined over the period of confinement (day 1 approx. 12 nM; day 7 approx. 6 nM), norepinephrine titres were usually higher and more variable. Epinephrine injection caused elevations in plasma epinephrine levels but not in norepinephrine levels; epinephrine titres reaching 107 ± 26 nM (range 65–238 nM) at 2 min post-injection and returning to pre-injection levels by 30 min post-injection. Plasma cortisol increased by 20 ng ml−1 following epinephrine administration. Based on the time-course for post-confinement alterations in plasma cortisol, it appears that up to a week may be required before cannulated fish are completely acclimated to ‘black-box’ confinement. The findings suggest that meaningful results from experiments utilizing epinephrine injection and ‘black-box confinement are contingent upon: (1) knowledge of circulating epinephrine levels shortly after injection (i.e. within 2 min post-injection); and (2) an experimental design that takes into account the elevated cortisol titres that are inherent with cannulation/confinement and epinephrine injection.

Notes: The ability of developing rainbow trout Oncorhynchus mykiss embryos to compensate for elevated oocyte triiodothyronine (T3) content and whether elevation of oocyte T3 content within a physiologically meaningful range affects growth rates of the embryo or the expression of genes encoding for thyroid hormone receptors α(TRα) and β(TRβ) were examined. Oocytes were immersed in ovarian fluid alone (control) or T3-enriched ovarian fluid prior to fertilization and water hardening, to induce a dose-dependant increase in oocyte T3 content of c. 3 (control), c. 30 (LT3) or c. 110 ng egg−1(HT3). To examine the interaction of embryo somatic growth with altered thyroid state more effectively, the embryos were reared at two ambient temperatures (8·5 and 5·5°C ) to induce different growth rates. A significant decline in whole embryo T3 content was measured in the T3-treatment groups reared at both water temperatures by 3 weeks post-fertilization (dpf), and may have reflected the action of outer ring monodeiodinase, which was present in microsomes prepared from embryos 23 dpf. Whole embryo T3 levels in the HT3 group, however, remained higher than controls until phase 2 of development [the onset of endogenous thyroid hormone (TH) release]. This suggested that the embryos exerted some control over their response to exogenous TH, but that there was a limit to the level of control exerted by the embryonic tissues. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for the two TR isoforms as early as 26 dpf, and quantitative real-time RT-PCR (qPCR) was used to examine the effect of elevated oocyte T3 content on the expression of these TR genes in embryos raised at 8·5 and 5·5° C, and sampled at similar developmental stages prior to the onset of embryonic TH synthesis, to ensure that the oocyte T3 was the only source of TH exposure to the embryo. There was a suppression of the TRα gene expression in the control 5·5° C group relative to the control 8·5° C group. In addition, both TRα and TRβ mRNA accumulation was lower, relative to the controls, in the LT3 treatment group reared at 8·5° C suggesting a suppressive effect of the lower level of T3 treatment on the TR gene expression. Conversely, there were no differences from controls in the HT3 treatment group, possibly indicating that this level of exposure overrides the down-regulating capacity of the embryo. Similar patterns were seen for TRα and TRβ mRNA accumulation in embryos reared at 5·5° C, but because of the temperature suppressed level of TRα mRNA in the controls, significant affects of the LT3 treatment were only found for TRβ. There were no measurable effects of T3 treatment on oocyte fertility or embryo somatic growth for either temperature treatment group, nor was somatic growth hormone content (measured only in the 8·5° C treatment group) apparently related to in ovo T3 levels. The results suggest that altered in ovo T3 levels, within the ranges used here, do not induce marked affects on embryo development, probably because of the ability of the embryo to maintain the integrity of its TH milieu.

Notes: A novel fibroblast-like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation of the stress activated member of the mitogen-activated protein kinase family, p38MAPK and induction of heat shock protein (Hsp70) were evident. The time-course of the p38MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38MAPK-specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress.

Notes: Hepatocytes in primary culture from fed and 2 month fasted Arctic charr Salvelinus alpinus were exposed to physiological doses of either cortisol, salmon growth hormone (GH), salmon insulin-like growth factor-I (IGF-I) or a combination of salmon GH and salmon IGF-I. Fasting significantly lowered medium glucose levels compared to the fed fish, but had no significant effects on hepatocyte glycogen content or on the activities of enzymes involved in the intermediary metabolism. Cortisol treatment had no effect on hepatocyte glycogen content or on the enzyme activities investigated, but resulted in a significant increase in medium glucose concentration in hepatocytes isolated from fasted, but not fed fish. GH and IGF-I treatments, both singly and in combination, significantly increased the glycogen content of hepatocytes isolated from fed fish, with less pronounced effects on hepatocytes isolated from fasted fish. The combination of GH and IGF-I significantly increased lactate dehydrogenase activity regardless of the feeding state and significantly reduced the phosphenolpyruvate carboxykinase activity and medium glucose concentration in hepatocytes isolated from fed fish. Further, GH and IGF-I significantly increased the activities of alanine aminotransferase and aspartate aminotransferase in hepatocytes isolated from fasted fish, but not fed fish. There were no effects of GH, IGF-I, or their combination, on glucose 6-phosphate dehydrogenase or 3-hydroxyacyl-CoA dehydrogenase activities. The results demonstrated that nutritional status of the animal modulates hepatocyte responsiveness to metabolic hormones, and suggested a role for GH and IGF-I in hepatic glycogen conservation.