Effects of cortisol and triiodo-L-thyronine on the steroidogenic capacity of rainbow trout ovarian follicles at two stages of oocyte maturation

The in vitro basal and salmon gonadotropin (sGTH)-stimulated steroidogenic capacity of rainbow trout follicles was examined at four stages [early (EV)-, mid (MV)- and peak-vitellogenic (PV), and pre-ovulatory, post-vitellogenic (PO)] of gonadal recrudescence using radioimmunoassays (RIAs) to measure 17-estradiol (E₂) and testosterone (T) production. In addition, follicles were incubated in the presence of [³H]pregnenolone ([³H]P⁵) and the radiolabelled steroid metabolites produced were separated using high performance liquid chromatography (HPLC). Peak basal and sGTH-stimulated E₂ and T production was found in PV stage follicles and lowest in PO stage follicles, and there were marked differences in the HPLC profiles of steroid metabolites. For EV stage follicles the major metabolite eluted as a peak that co-eluted with the androstenedione (A₄) and 17-hydroxyprogesterone (17-OHP) standards. A smaller peak that co-eluted with 11-hydroxyandrostenedione (11-OHA₄) and very small peaks co-eluting with 20-dihydroprogesterone (20-DHP) and E₂ were also seen. MV and PV stage follicles produced predominantly E₂, together with a small combined A₄ + 17-DHP peak, traces of 11-OHA₄ and two peaks that did not co-elute with any of the reference standards. The PO stage follicles produced only 17, 20-dihydroxy-4-pregnene-3-one (17,20-P).In addition, the effects of cortisol and triiodothyronine (T₃) on steroidogeneis were investigated in PV and PO stage ovarian follicles. For PV stage follicles, cortisol at 100 ng ml^–1 in the incubation medium significantly suppressed both basal and sGtH- stimulated T and E₂ production relative to control treatments. T₃ at 10 ng ml^–1 in the medium had no significant effect on either basal or sGtH-stimulated T or E₂ production compared to the controls, nor did it have any beneficial effect over the suppressive effect of cortisol. PO phase follicles taken 1 to 2 weeks prior to anticipated spawning had very low E₂ and T production, and there was no effect of cortisol or T₃, alone or in combination, on E₂ or T production. For PV stage follicles incubated in the presence of [³H]P⁵, cortisol suppressed T and E₂ production, but did not block the steroid pathway at any specific level; T₃ had no apparent affect on the metabolism of [³H]P⁵. The PO stage follicles produced little or no E₂; the major metabolite was 17,20-P. Cortisol and T₃ had no apparent effect on either basal or sGtH-stimulated 17,20-P production by the follicles at this stage of maturation.     

Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

Cytochrome P-450 mediated O-dealkylation of 7-alkoxycoumarins in liver microsomes from rainbow trout (Salmo gairdneri)

Evidence has recently been presented for variation in the inducibility of various 7-alkoxycoumarin-O-dealkylase activities in liver microsomes from a number of mammalian species by -naphthoflavone (NF). In the present study we have investigated the inducibility of hepatic microsomal 7-methoxycoumarin-O-demethylase, 7-ethoxycoumarin-O-deethylase, 7-propoxycoumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities in rainbow trout by NF. O-demethylase activity was increased approximately 17-fold, O-deethylase and O-depropylase activities approximately 9-fold and O-debutylase activity approximately 25-fold. The kinetics of the various hepatic microsomal 7-alkoxycoumarin-O-dealkylase activities were investigated in control and NF-treated rainbow trout. The O-demethylase-, O-depropylase- and O-debutylase activities exhibited monophasic Michaelis-Menten kinetics in liver microsomes from both control and NF-treated rainbow trout, whereas the O-deethylase activity exhibited biphasic Michaelis-Menten kinetics in control liver microsomes and monophasic Michaelis-Menten kinetics in liver microsomes from NF-treated rainbow trout.     

Melatonin, but not somatostatin-14 influences in vitro steroidogenesis by ovarian follicles of rainbow trout

Ovarian follicles taken from sexually maturing rainbow trout at the mid-vitellogenic stage of ovarian development were incubated in vitro in the presence or absence of melatonin or somatostatin-14 (SRIF-14) to determine whether there is evidence of a direct action of these factors on gonadal steroidogenesis in fishes. The steroidogenic capacity of the ovarian follicles was assessed by measuring testosterone (T) and 17-estradiol (E₂) release into the incubation medium, and by examining the steroid metabolites produced following incubation of follicles with radiolabelled steroid precursors.Melatonin appears to elicit a biphasic effect on steroidogenesis by in vitro rainbow trout ovarian follicles; at a concentration of 1 × 10^–3 M, melatonin stimulated basal T and E₂ production, but at a concentration of 1 × 10^–2 M there was an inhibition of basal and sGtH-stimulated T and E₂ Melatonin may act to reduce the activity of specific steroidogenic enzymes, since there was evidence of melatonin at 1 × 10^–2 M enhancing the accumulation of [³H]17-hydroxyprogesterone in the medium following incubation with [³H]pregnenolone, possibly suggesting the inhibition of C_17,20-lyase activity. In contrast, SRIF-14, used at concentrations of 1 × 10^–8 M and 1 × 10^–6 M, had no effect on basal or sGtH-stimulated E₂ or T production by ovarian follicles, incubated in vitro.     

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17-estradiol, 17-20-dihydroxy-4-pregnen-3-one (17-20-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17-estradiol levels. Inversely, plasma 17-20-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17-20-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.     

Reproduction related immunoglobulin changes in rainbow trout

Annual changes in plasma immunoglobulin (IgM) levels were investigated in three strains of rainbow trout Oncorhynchus mykiss which have different spawning periods, i.e., September–October, November–December, and January, reared under constant water temperature and natural day length. Plasma IgM levels decreased during the spawning season in all strains tested. The IgM changes became reversed in response to significant increases in plasma testosterone (T) and estradiol-17 in females and T and 11-ketotestosterone in males. Though the IgM decline showed a connection with suppressed immunocompetence, since many mature fish caught fungal diseases, no clear differences were observed in the plasma IgM levels between infected and noninfected fish during the spawning season. Incidentally, plasma IgM levels in infection prone fish were higher than in noninfection prone fish prior to the spawning season, whereas coincident differences in the plasma steroid levels were observed. Immature fish reared under lower water temperatures showed lower IgM levels. The effect of water temperature may have to be considered when analyzing the defense mechanism during the spawning season in rainbow trout.     

Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss

The immunosuppressive effects of steroid hormones were evaluated as the response against implanted steroid hormones, cortisol (F), testosterone (T), estradiol-17 (E2), and 11- ketotestosterone (11-KT), in juvenile rainbow trout. In long term experiments (5 weeks), fish were given a single intraperitoneal implant of F or T. A clear suppressive effect of plasma IgM levels with F and T was not necessarily obtained, although mucus IgM levels were reduced corresponding to the elevated plasma steroid hormone levels. In short term experiments (1 week), intraperitoneal implantation of T, 11-KT and E2 suppressed plasma and mucus IgM levels, although the effects were not dose-dependent. When administered through diet, F and T caused a suppression of plasma IgM levels; F administration at both high and low dosages caused a significant decrease in plasma IgM levels, while only a high dose of T caused the suppression. These results suggest that sex steroid hormones, as well as F, have immunosuppressive functions in rainbow trout.     

In vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, Bester

In this study, developmental changes in the steroidogenic capacity of testicular fragments and isolated ovarian follicles of a hybrid sturgeon, Bester, at a variety stage of developments were examined. Testicular fragments or isolated ovarian follicles were incubated in L-15 medium in the presence or absence of different concentrations of five preparations; forskolin, human chorionic gonadotropin (HCG), pregnenolone (P₅), 17-hydroxyprogesterone (17OHP) and testosterone (T) for 18 h at 15 °C. After incubation, concentrations of 11-ketotestosterone (11 KT) (testis) and, 17-estradiol (E₂) (ovarian follicles) and 17,20-dihydroxy-4-pregnen-3-one (DHP) (testis and ovarian follicles) were measured. 11KT was detected in the media following incubation with P₅, 17OHP and T. Its concentration was higher during late spermatogenesis and prespermiation and lower at the degeneration stage. Both P₅ and 17OHP were converted to DHP during the prespermiation stage. Forskolin had little stimulatory effect on the synthesis of 11KT and DHP and HCG did not induce the production of these steroids.E₂ was detected in the medium following incubation of follicles with P₅, 17OHP and T at all stages of oocyte development. The concentration of E₂ in the medium increased during vitellogenesis with the peak production occurring at the tertiary yolk stage. In contrast, the potencies of follicles to produce steroids shifted to the production of DHP during migratory nucleus stage. Forskolin and HCG had little effect on the synthesis of E₂ and DHP. These results demonstrated that the failure of spontaneous spermiation or ovulation is not due to the insufficient synthesis of DHP, but may due to the lack of availability of precursors.     

Inhibition of HPI axis response to stress in gilthead sea bream (Sparus aurata) with physiological plasma levels of cortisol

This study investigates the effect of corticosteroid (cortisol) administration on the stress response of the gilthead sea bream Sparus aurata subjected to a 48 h confinement. The effect of (in-vitro and in-vivo ) cortisol administration on the in-vitro ACTH sensitivity of the interrenal tissue; the plasma levels and tissue concentration of cortisol; and the plasma levels of ACTH, -MSH, -endorphin and glucose were determined. Confinement caused a transient and concomitant increase in plasma cortisol and ACTH levels. However, in cortisol-fed fish the plasma ACTH levels were lower, indicating a suppresion of the ACTH release from the corticotropes by cortisol. In contrast to the activation of the corticotropes, the levels of plasma melanotrope derived peptides were not affected. In spite of the fact that interrenal cells of cortisol-fed gilthead sea bream released less cortisol than controls, the interrenal sensitivity to ACTH was not affected by in-vivo and in-vitro cortisol administration. This suggests that the interrenal sensitivity to ACTH in stressed (confinement) sea bream is probably not regulated by -MSH, N-ac--END, or by cortisol. Thus, in gilthead sea bream the interrenal sensitivity to ACTH could be regulated at the hypothalamus and/or pituitary and communicated via circulating ACTH levels.     

Effects of modulatory agents on neurally-mediated responses of trout intestinal smooth musclein vitro

Mediators and mechanisms responsible for the inhibitory modulation of trout intestinal smooth muscle were examined using a series of putative mediators and substances known to modulate neurotransmission in mammalian systems. Frequency response relationships to transmural stimulation and concentration response relationships to 5-hydroxytryptamine, carbachol, and substance P were established on paired segments of rainbow trout intestinein vitro in the presence and absence of putative modulatory agents. Modulation of neurally-mediated contractions of trout intestine was achieved with dibutyryl cyclic AMP and forskolin, agents that increase intracellular levels of cyclic AMP. The effect appears to be at the level of the smooth muscle, since the adenylate cyclase activator, forskolin, inhibited muscarinic and serotoninergic contractions as well as transmurally stimulated contractions. Substance P-induced contractions were unaffected by forskolin. The endogenous agonists/neurotransmitters which would increase cyclic AMP levels in rainbow trout intestinal smooth muscle are as yet unknown. The effects do not appear to be modulated by vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), or agents that activate -adrenoceptors. Prostaglandin E₂ (PGE₂) and ₂-adrenergenic agonists are possible agents which will decrease contractility of the smooth muscle. They were only active in the proximal intestine and on transmurally stimulated contractions. The effects of both PGE₂ and ₂-agonists appear to be prejunctional, decreasing release of contractile neurotransmitters in the enteric nervous system.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.     

Effects of aromatase inhibitors on sexual maturation in Atlantic salmon,Salmo salar,male parr

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17, 20-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.     

Incorporation of yolk fatty acids into body lipids of goldfish (Carassius auratus L.) larvae raised at two different temperatures

In five separate experiments, eggs from a single female goldfish were fertilized at 20°C. They were incubated at 22°C for 6 hours, after which some of the eggs were transferred to 13°C. When a defined post-hatch developmental stage was reached, lipid extracts were prepared from larvae, both with yolk sacs intact and after removal of the yolk sac by dissection. Other larvae were sampled at yolk exhaustion. Gas chromatographic analysis of fatty acid profiles revealed that larvae incorporated 16:0, 18:0, 20:4 (n–6) and 22:6 (n–3) into their tissues in proportions higher than those present in the eggs from which they were derived. At 22°C, these trends were particularly apparent at yolk exhaustion. At 13°C, proportions of polyunsaturated fatty acids in the bodies of newly hatched larvae were higher than those in the 22°C larval bodies. Monounsaturated fatty acids were preferentially depleted during development, especially in larvae from high quality eggs. No dependence of egg quality, as assessed by larval viability at 22°C, on total egg lipid mass or fatty acid composition was found. Larvae from the lowest quality eggs showed a reduced preference for incorporation of (n–3) polyunsaturated fatty acids into their tissues.     

The presence of 17α,20β-dihydroxy-4-pregnen-3-one receptor activity in the ovary of the brook trout,Salvelinus fontinalis,during terminal stages of oocyte maturation

The presence of 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) oocyte receptor activity has been demonstrated in brook troutSalvelinus fontinalis. Scatchard analyses of the cytosol fraction during various terminal stages of oocyte maturation gave a high equilibrium association constant (K_a) value of 1.394±0.669 10⁸M^–1 (n=7) and low maximum binding capacities (N_max). The association kinetics of the receptor was second order k₊₁=2.292×10⁶M^–1 sec^–1. The dissociation rate constant k_a was 1.502×10^–2 sec^–1 for the first order dissociation reaction. The K_a=1.526×10⁸M^–1, when it was determined from k₊₁/k_–1 a value close to that found from the Scatchard analysis. Competition studies showed the following binding affinities testosterone > 17-HP > 17,20-DHP > Promegestone > progesterone > estradiol > pregnenolone; cortisol showed no competitive inhibition. Cytosolic extracts when pre-equilibrated with various labelled steroids and eluted from a Sephacryl S-300 column gave multiple specific binding peaks. On sucrose density gradient centrifugation specific binding was observed at 3.05 S in cytosol containing 0.15M sodium chloride buffer. The receptor lost binding activity when incubated with various proteases, but DNase and RNase had no effect. Blood plasma without heparin at (110) dilution also bound [³H]17,20-DHP, Ka was 8.04×10⁷ M^–1.The nuclear pellet extract (750×g) gave very little specific binding activity even at high radiolabelled steroid concentrations and a linear Scatchard plot was not obtained. Nevertheless the nuclear extract, after dextran-charcoal treatment, pre-equilibrated with [³H]17,20-DHP, bound specifically to DNA cellulose, and cytosol from the same oocytes also bound to DNA cellulose under similar conditions. Although specific binding to DNA cellulose was obtained the salt concentrations at which the steroid-receptor complex elution took place was not reproducible in both nuclear extracts and cytosol samples. Also binding activity was extremely small compared to the total cytosolic binding. The nuclear extract when pre-equilibrated with high concentrations (20 nM) of the labelled steroid and then chromatographed on Sephacryl S-300 column gave a specific binding peak which was similar to that of the cytosolic preparation.The receptor levels in cytosol decreased progressively during final maturation (Stages 1–7). There is preliminary evidence for the presence of 17,20-DHP receptor activity in cytosol of landlocked Atlantic salmonSalmo salar ouananiche, and rainbow troutSalmo gairdneri. The zona radiata fraction from late stages oocyes 5, 6, and 7 in brook and rainbow trout oocytes were isolated by ultracentrifugation; from this fraction a protein was characterized which covalently bound [³H]R5020 after photoaffinity labelling. The same protein also bound [³H]17,20-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [³H]17,20-DHP, although the molecular weights were different. The blood sample [³H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17,20-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.A. Maneckjee is presently NSERC postgraduate scholar at MSRL and Ph.D. candidate at Department of Biochemistry, Memorial University of Newfoundland.     

The cytochrome P450 system of atlantic salmon (Salmo salar): I. Basal properties and induction of P450 1A1 in liver of immature and mature fish

The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The K_m for 7-ethoxyresorufin was 0.4 µM, and V_max 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20–30 pmol/min/mg protein). Treatment with the P450 1A1 inducer -naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30–40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (M_r=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at –80°C in 20% glycerol without losing more than 20–40% of its catalytic activity.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goksøyr 1989).     

Variation in content of Ω3 fatty acids in farmed Atlantic salmon,with special emphasis on effects of non-dietary factors

The main aim of the present study was to examine the impact of some biological and environmental factors on the lipid and fatty acid compositions of farmed Atlantic salmon (Salmo salar), with special emphasis on 3 fatty acids. Two year groups of salmon at nine fish farms distributed along the Norwegian coast were fed the same diet and were sampled every second month. The data are believed to give a representative characterization of lipid and fatty acid content of salmon farmed in Norway.Multiple regression analysis revealed that variation in lipid content and body weight explained 80% of the variation found in 3 fatty acids in farmed salmon, and 22:6 3 showed greater variation than other 3 fatty acids. Further analysis of lipid-corrected values revealed only minor effects of latitude on the per cent content of highly unsaturated 3 fatty acids, and hardly any effect of seawater temperature, with the exception of 22:6 3, which decreased slightly with increasing temperature.The per cent 22:6 3 in the fillet became gradually reduced with increasing fish age and body weight, whereas the content of 20:5 3 and other 3 fatty acids remained relatively constant. The per cent content of 22:6 3 of young salmon was higher than in the feed, but approached the feed value gradually as body weight increased. The lipid content of the salmon increased with fish age, and the absolute quantitative contents of both 22:6 3 and 20:5 3 increased meanwhile, even though the per cent content of 22:6 3 decreased quite pronouncedly.The per cent 22:6 3 and other 3 fatty acids was higher in wild than in farmed salmon, but the absolute quantitative content was higher throughout in farmed salmon, which had higher lipid contents. The 3/6 ratio, which is important in human health evaluation, was lower in farmed than in wild salmon. The large flexibility of 3 fatty acids and lipid content of farmed salmon leave us with the option of producing a wide variety of salmon qualities requested by the market. Both per cent and absolute quantitative 3 contents, as well as the 3/6 ratio, may readily be manipulated.     

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.     

Synthesis of 17,20α- and 17,20β-dihydroxy-4-pregnen-3-ones, 11-ketotestosterone and their conjugates by gills of teleost fish

Goldfish, carp and trout gills were incubated with ³H-17-hydroxyprogesterone (17P). With goldfish gills, the metabolites were 17,20-dihydroxy-4-pregnen-3-one (17,20P; 82%), 17,20-dihydroxy-4-pregnen-3-one (17,20P; 8%), 11-ketotestosterone (KT) glucuronide (5.4%) and 17,20P glucuronide (0.2%). Sulfates were not detected. Carp gills converted 17P into 17,20P (11.2%), 17,20P (9.6%), KT (8.4%), glucuronides of 17,20P (1.3%) and 17,20P (1.6%) and sulfates of 17,20P (5.1%) and 17,20P (7.2%). 17,20P (38% free, 1.8% glucuronide and 21.1% sulfate) was the sole metabolite of ³H-17P in trout gill incubations. In the presence of high (10; µg ml^-1) substrate concentration, cyprinid gills gave predominantly free 17,20P, while trout gills yielded only free 17,20P. Production of 17,20P, predominantly as its sulfate, from endogenous precursors was demonstrated in trout gills but was not stimulated by trout primary extract. Our results demonstrate for the first time the steroidogenic potential of teleost gills and suggest that they may play a role in secretion of pheromones in some species.