Prostaglandins in the kidney, urinary bladder and gills of the rainbow trout and European eel adapted to fresh water and seawater.

Prostaglandins in kidney, gills, and urinary bladder of freshwater-adapted and seawater-adapted rainbow trout, Oncorhynchus mykiss (= Salmo gairdneri), and European eel, Anguilla anguilla, were determined by solid-phase extraction of tissue homogenates and high-pressure liquid chromatography. Prostaglandins E2, E1, F1 alpha, F2 alpha, and D2 and the more stable metabolite of prostacyclin, 6-keto F1 alpha, occurred in these osmoregulatory tissues. In gill filaments and kidneys of both eel and trout, prostaglandins D2 and 6-keto F1 alpha were major prostaglandins. Concentrations of these prostaglandins were significantly lower in the eel after seawater adaptation, but not in the trout. The urinary bladder of the trout contained the highest levels of prostaglandins; bladders of seawater-adapted trout contained prostaglandin D2 at 6.7 ng/mg wet tissue, the highest level of any prostaglandin determined in the present studies. Prostaglandin D2 was not detected in bladders of freshwater-adapted trout.     

Metabolism of 25-hydroxycholecalciferol in a teleost fish, the rainbow trout (Salmo gairdneri)

Plasma concentrations of vitamin D3 (cholecalciferol) metabolites have been studied in rainbow trout (Salmo gairdneri) adapted to varying environmental calcium concentrations in both fresh water and artificial seawater, and in natural seawater. In vivo, intraarterial injection of tritiated 25-hydroxycholecalciferol was followed by its transformation to a number of metabolites including compounds that cochromatographed on high performance liquid chromatography (HPLC) with 1,25-dihydroxycholecalciferol and 25,26-dihydroxycholecalciferol. Hypercalcaemia and increased environmental calcium were associated with a greater transformation to the compound cochromatographing with 25,26-dihydroxycholecalciferol, while hypocalcaemia and reduced environmental calcium concentrations induced more conversion to the 1,25-dihydroxycholecalciferol-like compound. In vitro, both metabolites were produced by liver but not by kidney preparations, and the difference in conversion ratios observed in vivo associated with changes in plasma calcium were also seen in vitro. It is concluded that the metabolism of 25-hydroxycholecalciferol in the trout can be influenced by calcium status, but at present the physiological importance of this metabolism and the mechanisms and site(s) of action of the metabolites are unknown.     

Daily variations in plasma cortisol levels of individual female rainbow trout Salmo gairdneri: Evidence for a post-feeding peak in well-adapted fish

Blood sampling through a catheter caused a significant and variable increase in the plasma cortisol concentration of most tested female rainbow trout. In well-adapted catheterized individuals sampled at 24- or 28-hr intervals, baseline cortisol levels were less than 8 ng/ml. A peak (15–50 ng/ml) repeatedly appeared shortly after feeding and was still present when the fish could not see the experimenter at feeding time. Other cortisol increases (12–24 ng/ml) occasionally occurred but were not reproducible. In non-feeding chronically disturbed trout, cortisol concentrations fluctuated widely (2–100 ng/ml), with no indication of periodicity.     

Structure of a fish (rainbow trout) growth hormone gene and its evolutionary implications.

We have isolated and sequenced a clone from a rainbow trout (Salmo gairdneri) genomic library that carries a gene encoding a fish growth hormone (GH). This gene spans a region of approximately equal to 4 kilobases, nearly twice that of mammalian GH genes. The trout GH gene is comprised of six exons, in contrast with five exons in mammals. The additional intron in the fish gene interrupts translated regions that are analogous to the last exon of its mammalian counterpart. In addition, the alleged internally repeating sequence in mammalian GH, prolactin (Prl), or placental lactogen (PL) is not observed in the predicted polypeptide sequence of fish GH. Direct repeats that flank exons I, III, and V of the mammalian GH, Prl, and PL genes are absent in the fish GH gene. These findings indicate that the rainbow trout GH gene structure does not support the current hypothesis that internally repeated regions in GH, Prl, and PL arose from a small primordial gene.     

Effect of pulsatile flow on gas exchange in the fish gill: theory and experimental data.

A model for gas exchange in the fish gill allowing for time-varying water and blood flow is presented. An analysis based on this mathematical model shows that pulsatile water and blood flow potentially may reduce the efficiency of gas exchange significantly. The degree of inefficiency imposed on gas exchange is, however, determined by the physical dimensions of the gill and the gas capacitance coefficients of water and blood. Using anatomical and physiological data it is shown to be likely that for a large group of fishes, including the salmonids, pulsatility of water and blood flow affects gas exchange efficiency only marginally. A close coupling between cardiac and respiratory rhythms is therefore only of marginal advantage to gas exchange efficiency. Due to their exceptional gill dimensions tunas, and to a lesser extent mackerels, are susceptible to the negative effect of pulsatility on gas exchange, which may be one of the factors favouring ram ventilation in these species.     

Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout

Stanniocalcin (STC) is a hormone that is synthesized and secreted by the corpuscles of stannius (CS), endocrine glands that are unique to the bony fishes. The hormone inhibits Ca2+ transport from the aquatic environment into the bloodstream by way of the gills. Previous in vitro studies by our laboratory have shown that STC secretion is positively regulated by Ca2+ in a time- and dose-dependent fashion. In this report, we have examined circulating levels of STC in free-swimming, cannulated rainbow trout and how hormone levels are affected by surgical stress and intra-arterial infusions of mono and divalent cations. In addition, the plasma hormone has been concentrated by immunoaffinity chromatography and characterized by Western blot analysis. The results suggest that the in vivo response of the CS is extremely rapid and Ca(2+)-specific and that STC circulates in multiple molecular weight forms.     

Steroid secretion of rainbow trout testis in vitro: variation during the reproductive cycle

Testicular tissue collected at different stages of gonadal development was incubated with a pituitary extract (PE) from mature salmon. Three androgens (11-ketotestosterone, OT; 11 beta-hydroxytestosterone, OHT; and testosterone, T) and 17 alpha,20 beta-dihydroxyprogesterone (17-20 beta P) were quantified by radioimmunoassay in incubation media. OHT and OT were secreted in larger quantities than T and 17-20 beta P. The PE dose that evoked a half-maximal response (ED50), the ratios of maximum stimulated vs baseline secretion, and total testicular steroid output all changed during the reproductive cycle. Androgen secretion in response to PE was low in immature and spent fish, both in terms of ED50 and the ratio of maximum stimulated vs baseline secretion. This ratio increased in testes showing the first signs of maturation and remained elevated during rapid testicular growth, before reaching maximum values at full maturity. The lowest ED50 values were found at the end of spermatogenesis and during the peak spawning period. 17-20 beta P secretion could not be stimulated noticeably until the fish had entered the spawning period and, as opposed to androgens, remained stimulable in spent fish. ED50 values for 17-20 beta P ranged, without showing clear-cut variations, above those calculated for androgens. The changes in PE reactivity and steroid secretion capacity during the reproductive cycle are likely to contribute to the changes in circulating steroid concentrations and may allow modulations of testicular steroid production without large changes in circulating GTH levels.     

Metabolic consequences of hypercapnia in the rainbow trout, Salmo gairdneri: beta-adrenergic effects

The metabolic consequences of external hypercapnia (1% CO2) were assessed in rainbow trout (Salmo gairdneri) in the presence or absence of circulating levels of the beta adrenoceptor antagonist, propranolol. External hypercapnia caused a severe extracellular respiratory acidosis and a less pronounced reduction of hepatic intracellular pH (pHi). pHi was restored to prehypercapnic values after 48 hr of continuous hypercapnia due to elevation of bicarbonate levels. In the presence of propranolol, hypercapnia elicited a pronounced activation of pyruvate kinase (PyK) (measured at both low and high phosphoenolpyruvate (PEP) concentrations) and inactivation of both total glycogen phosphorylase (GPase) and glycogen phosphorylase a (GPase a). In the absence of propranolol, the changes in enzyme activities were significantly reduced (low PEP PyK activity) or totally absent (GPase inactivation). These results suggest that beta adrenoceptor-mediated phenomena offset disruptive effects of hypercapnia on PyK and GPase activities and may be important in the control of gluconeogenesis and glycogenolysis during this acid-base disturbance. The adrenergic effects were not related to modification of hepatic intracellular acid-base status. Hypercapnia induced a rapid depletion of liver glycogen and concomitant hyperglycemia. These effects were not prevented by pretreating fish with propranolol and appeared to be unrelated to changes in GPase a activity. These results suggest that factors other than adrenergic activation of GPase a are involved in the enhancement of liver glycogenolysis.     

Estrone and estradiol participation during exogenous vitellogenesis in the female rainbow trout, Salmo gairdneri

Ovarian steroids were treated for their ability to induce vitellogenin synthesis in the liver of the rainbow trout, Salmo gairdneri. These steroids were chosen because they had been reported to induce vitellogenin in the plasma of teleosts, i.e., estrone, estradiol, and testosterone, or because they were synthesized during vitellogenesis, as was the case with the above steroids and androstenedione and 17α-hydroxyprogesterone. Immature trout were ovariectomized and injected daily for 7 days. The administered doses of steroid were 100, 250, and 500 ng/g body wt. Only estradiol and estrone induced vitellogenin in the plasma; estrone had about 5% of the potency of estradiol. A dose-effect curve was determined for estradiol in the range from 25 to 1000 ng/g body wt. A maximal amount of 7 mg vitellogenin/ml plasma was found following the administration of a dose of 250 ng/g estradiol. Vitellogenin was not present in the plasma of animals treated with saline, nor could it be detected in the liver, not even after the administration of estradiol. Estradiol administration increased the total plasma protein concentration from 35 to maximally 51 mg/ml and decreased the total piver protein concentration from 163 to minimally 118 mg/g. The relative weight of the liver increased from 12.9% to maximally 22.4%. Vitellogenin was not detected in the liver of any of the experimental animals, indicating a low storage and rapid secretion of vitellogenin. The other steroids influenced some of the variables, but never was the total pattern of effect comparable to that of estradiol. Estradiol is found to be the ovarian steroid that physiologically regulates the synthesis of vitellogenin in the liver of the rainbow trout; estrone is less active. Experiments undertaken to determine the effects of the combined presence of estrone and estradiol revealed that estrone was capable of boosting the vitellogenic effect of estradiol when compared to the induced vitellogenin levels following treatment with a combination of testosterone and estradiol. The vitellogenin concentrations induced by a certain dose of one of the combinations of estrone and estradiol approximated the concentrations induced by the same dose of estradiol alone. This effect was independent of the ratio in which estrone and estradiol were administered. These findings could not be explained by conversions of estrone into estradiol or by the vitellogenic activity of estrone alone. The estrone, estradiol, and vitellogenin concentrations in the plasma of the experimental animals were in the same range as determined previously in untreated mature vitellogenic females. Vitellogenin and estradiol levels were found to correlate in experimental animals treated with estradiol (r = 0.627; N = 20). This was not the case in animals treated with combinations of estrone and estradiol. In these animals, however, the sum of estrone and estradiol levels in the plasma correlated with vitellogenin levels (r = 0.724; N = 42). Vitellogenin was not detected in the liver of any of the experimental animals, which indicates a low storage and rapid secretion of vitellogenin. The importance of viewing the sum of estrone and estradiol plasma levels in connection to physiological studies of the regulation of exogenous vitellogenesis in the rainbow trout is discussed.     

The involvement of calcitonin in the reproductive physiology of the rainbow trout

Plasma from rainbow trout (Salmo gairdneri) of both sexes was sampled every month throughout an annual reproductive cycle in order to elucidate possible relationships between plasma calcitonin and free and protein-bound calcium and magnesium. This was then studied in greater detail around the time of ovulation in the female fish. The plasma levels of the parameters studied were stable in males during the whole annual cycle and were similar to the levels found in females during at least 6 months of the cycle. Plasma levels of protein-bound calcium and magnesium as well as calcitonin were raised in the females for 6, 4 and 3 months respectively. These increases occurred concomitantly during the months before and after ovulation, but no correlations between the protein-bound ion and calcitonin levels were found during this period. With the exception of a decrease observed in December, the free plasma levels of calcium and magnesium were stable in both males and females throughout the cycle. This decrease was attributed to the high production rate of the yolk-protein precursor vitellogenin, which binds both calcium and magnesium. In the detailed study on the ovulating females, plasma calcitonin levels were high 4 weeks before, and continued to increase until the time of ovulation, when a sharp decrease towards normal was noted. The free plasma calcium and magnesium levels were not affected, while the protein-bound levels of calcium and magnesium were found to decline towards normal for the duration of the experiment, without any obvious correlation with the time of ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonrelationship between plasma melatonin and background adaptation in the rainbow trout (Salmo gairdneri)

Adult female rainbow trout (Salmo gairdneri) were maintained outdoors on black, white, and neutral colored backgrounds. Reflectometry readings were used to assess the color adaptation of the fish. Plasma samples were collected from the same individuals both before and after exposure to the backgrounds, as well as at both 1200 and 2400 hr. Using a sensitive radioimmunoassay, no significant differences in plasma melatonin levels could be detected for the fish kept on the three backgrounds. Since the fish did show significant color modification in response to 18–21.5 days on the backgrounds, it is concluded that melatonin may not participate in background adaptation in trout. A consistent nocturnal elevation in melatonin levels was noted on all backgrounds. The stress associated with taking multiple blood samples from the same fish may have caused a significant increase in post-treatment compared to pretreatment nocturnal melatonin titers.     

Differences between rainbow trout and brown trout in the regulation of the pituitary-interrenal axis and physiological performance during confinement.

The responses of rainbow trout and brown trout to the same stressor were compared by measuring primary and secondary stress responses during and after a 5.5-h net confinement. Basal levels of adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), and glucose were higher in brown trout than in rainbow trout. While confinement induced transient increases in plasma ACTH and cortisol levels in both species, the magnitude of these responses, but not the time course, was greater in brown trout. Brown trout, but not rainbow trout, showed a reduction in plasma alpha-MSH levels after 5.5 h confinement before returning to control values, and the glucose levels in the brown trout were elevated throughout the confinement and recovery periods. Confinement also resulted in increased hematocrit values and reduced plasma sodium and chloride levels in both species. Rainbow trout appeared to recover faster from the confinement, as glucose and hematocrit values in the brown trout remained elevated and ionoregulatory disturbances persisted even after 46 h. During recovery effects on the immune system were more pronounced in brown trout than in rainbow trout. Circulating white blood cell numbers were reduced in both species following 23 h recovery, but remained low in the brown trout. Elevated alternative complement activity and oxygen radical production were found after 23 h recovery, and reduced lysozyme activity was found after 46 h, in brown trout only. Results indicate that differences in the stress response of these closely related species, as illustrated by the intensity of the cortisol response, originate at the level of the pituitary and are also manifested through secondary stress responses.     

Diurnal rhythms in hypothalamic/pituitary AVT synthesis and secretion in rainbow trout: evidence for a circadian regulation

Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptide hormones for which a role in adaptation to environmental changes has been suggested in fish. In teleosts, there are only a few available studies about circadian changes of AVT and IT levels, and a role of those peptides in the circadian system has been mainly suggested on the basis of the role of the homologous hormone AVP in mammals. Herein, we evaluated the diurnal rhythms in plasma AVT, pituitary AVT and IT content and the hypothalamic pro-vasotocin (pro-VT) expression in rainbow trout kept under a natural photoperiod, as well as their persistence in constant darkness as a tool for defining circadian dependence. Trout kept under a natural light cycle showed clear diurnal rhythms in both circulating and pituitary AVT levels with peak values around the last hours of the light phase. Hypothalamic pro-VT mRNA was also rhythmically expressed with similar peak characteristics. These rhythms persisted in fish kept under constant darkness for nearly two consecutive days, although peaks were progressively attenuated and phase-advanced. An IT rhythm was also found in pituitary of the trout maintained under a natural photoperiod, but not in those kept under continuous darkness. These results suggest that rhythms of hypothalamic AVT synthesis might be regulated by endogenous circadian mechanisms, and these rhythms contribute to maintain a similar fluctuation in pituitary AVT secretion into the blood. A potential role for AVT in the circadian and seasonal time-keeping system of teleost fish, either as a component of the neural machinery that participates in the adaptation to cyclic environmental changes, or as a circadian/seasonal output signal, is also discussed.     

Effects of natural androgens and corticosteroids on gonad differentiation in the rainbow trout, Salmo gairdneri

Rainbow trout fry were treated with equimolar quantities of cortisol, cortisone, androstenedione, and 11β-hydroxyandrostenedione, added to the aquarium water during a 4-week period. All four steroids inhibited ovarian growth. Androstenedione did not influence gonadal sex differentiation; the other steroids pushed the sex ratio in the male direction. However, a near to normal sex ratio was observed 300 days after the 11β-hydroxyandrostenedione treatment. When added to the food in two different doses during 8 weeks, 11β-hydroxyandrostenedione had a pronounced masculinizing effect, and androstenedione did not modify the gonads at all. It is suggested that the two exogenous corticosteroids were probably converted into 11-oxygenated androstenedione derivatives, and that these 11-oxygenated androstenedione derivatives are particularly important in sustaining the differentiation and early development of the testes in rainbow trout. Testosterone does not seem to be indispensable for these processes, because two different doses of testosterone-blocking cyproterone acetate, added to the food for 8 weeks, failed to affect early gonad development.     

Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss)

In this study, we investigated the effects of one melanocortin receptor (MCR) agonist and two antagonists on food intake in juvenile rainbow trout. Baseline food intake was established prior to 1 μl intracerebroventricular injection (ICV) of the non-specific agonist MTII, the MC4R antagonist HS024 and the MC3/4R antagonist SHU9119 at concentrations of 0.3, 1 or 3 nM. Saline-injected fish and untreated fish served as controls. Changes in food intake were observed 1 h after the ICV injections. Our results showed that treatment with MTII significantly decreased food intake at 3 nM compared to control, HS024 significantly increased food intake at 3 nM compared to control and saline-treated fish, and SHU9119 significantly increased food intake at 3 nM compared to saline-treated fish. In conclusion, our study provides further evidence, and hence strengthens the hypothesis, that MC4R participates in the control of energy balance in fish in the same manner as in mammals. Our findings that HS024 is more potent than SHU9119 in increasing food intake suggest that the effects of melanocortin on energy balance in rainbow trout are mainly regulated by activation of MC4R. Hence, HS024 seems an excellent tool as a MC4R antagonist in rainbow trout.     

The corticosteroid receptor and the action of various steroids in rainbow trout fibroblasts

Corticosteroid binding sites with the characteristics of steroid receptors were detected with the synthetic corticosteroid, [3H]triamcinolone acetonide (TA), in monolayers of the rainbow trout fibroblast cell line, RTG-2. The sites had low capacity as saturation was achieved at approximately 5 nM. Scatchard plots of the data suggested a single population of high-affinity binding sites. The number of receptors per cell was approximately 20,000; the dissociation constant, 1 nM. Changes in [3H]thymidine incorporation and cellular morphology were monitored as potential corticosteroid-sensitive metabolic responses. Only cortisol and 11-deoxycortisol among 14 naturally occurring steroids and TA, fluocinolone acetonide, dexamethasone, and prednisolone among 6 synthetic corticosteroids inhibited [3H]thymidine incorporation and altered the morphology in RTG-2 cells. Two observations suggested that the corticosteroid receptor mediated these responses. The synthetic steroid, RU 38486, which is an antiglucocorticoid in mammals, did not elicit these responses, had a high affinity for the receptor, and blocked the ability of cortisol and TA to change [3H]thymidine incorporation and cellular morphology. Second, the affinity of various natural steroids for the receptor correlated with their ability to elicit a cellular response. Cortisol, and to lesser extent 11-deoxycortisol, showed strong affinity for the receptor. Cortisone, aldosterone, and the sex steroids had no affinity and did not elicit cellular responses.     

Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400-450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 10(-10) M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10(-2) M.