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.
This study explored several physiological criteria that could be used to assess the steroidogenic condition of the ovarian follicles of individual fish of an asynchronously ovulating captive rainbow trout Oncorhynchus mykiss stock. In these fish, the date of sampling, morphological variables such as gonado‐somatic index or ovarian follicle mass and visual assessment of the ovary provided accurate indications of the maturational condition of an individual. The physiological variables measured included the in vitro basal and cyclic adenosine monophosphate (cAMP)‐stimulated synthesis by ovarian follicles of 17 β ‐oestradiol (E 2 ) and testosterone (T); in addition, quantitative reverse‐transcription (RT)‐PCR was used to measure the relative expression of star and p450scc genes by ovarian follicles. The ratios of cAMP‐stimulated E 2 and T synthesis to basal E 2 and T synthesis provided a reliable indication of differences in the steroidogenic status of the follicles of individual animals. On the basis of these criteria, together with the use of gene expression profiles, it was possible to classify individual fish as being at an early, mid or late‐vitellogenic stage.
The objective of this study was to investigate the potential of hepatocytes for cortisol uptake and metabolism in 3,3{prime},4,4{prime}-tetrachlorobiphenyl (TCBP) treated trout. Two groups of rainbow trout (Oncorhynchus mykiss) were either given an intraperitoneal implant of peanut oil alone or peanut oil containing TCBP (10 mg.kg{sup {minus}1} body weight) and sampled six weeks later. The toxicant exposed fish had significantly lower condition factor and plasma glucose concentration, whereas plasma cortisol, protein and hepatocyte protein concentration and liver ethoxyresorufin-O-deethylase (EROD) activity were significantly higher in the TCBP compared to the sham group. There was no significant difference in plasma lactate and amino acid concentration, hepatocyte glycogen content or liver cytosolic cortisol binding affinity or capacity between the two groups. The uptake of [{sup 3}H] cortisol was significantly higher in the hepatocytes of TCBP treated fish compared to the sham fish. Also, there was enhanced catabolism of [{sup 3}H] cortisol by hepatocytes of TCBP treated fish; the major metabolite appeared to be tetrahydrocortisone. The results indicate that the potential for cortisol clearance is enhanced in hepatocytes of TCBP treated trout. The data also tend to suggest in vivo regulatory mechanisms that might possibly prevent the increased clearance of the hormone from circulationmore » in toxicant exposed fish.« less
Cortisol rapidly activates cell signalling in trout hepatocytes; however, the mechanisms are far from clear. We tested the hypothesis that rapid action of cortisol involves modulation of intracellular Ca 2+ levels in trout hepatocytes. Stress level of cortisol reported in fish plasma elicits a rapid increase in intracellular Ca 2+ level as determined by Fura‐2AM ratiometric imaging. The increase in intracellular Ca 2+ level in trout hepatocytes was also seen with the membrane impermeable form of the steroid (cortisol‐BSA), and this response was not affected by mifepristone (glucocorticoid receptor antagonist). We determined the role of extracellular and intracellular stores of calcium in affecting cortisol‐induced elevation in Ca 2+ levels by using EGTA (a chelator) and BAPTA‐AM (inhibitor), respectively. Furthermore, we determined if IP3R pathway was involved in the activation of intracellular stores by using various inhibitors, including PLC inhibitor (U73122), thapsigargin (SERCA blocker) and Rynodine (RyR blocker). Also, L‐type calcium channel blocker Nifedipine and Ca 2+ release‐activated Ca 2+ (CRAC) channel blocker Cpd5J‐4 were utilized to determine the mode of entry of extracellular calcium in response to cortisol stimulation. Overall, our results suggest that cortisol rapidly increases intracellular calcium levels and this may be mediated by the activation of CRAC channel in trout hepatocytes. Support or Funding Information This study was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV.
