A comparative study of picomolar affinity 2-[125I]iodomelatonin binding sites in the hearts of three salmonid species

The hearts of three cultured salmonid species, collected at either mid-light or mid-dark were studied for their binding to 2-[¹²⁵I]iodomelatonin, a specific melatonin agonist. The binding was saturable, reversible, and highly specific. The equilibrium dissociation constant (K_d) ranged from 30.1 ± 3.0 pmole 1⁻¹ in Arctic charr (Salvelinus alpinus) to 40.5 ± 2.3 pmole 1⁻¹ in rainbow trout (Oncorhynchus mykiss) indicating a high binding affinity. The maximum density of binding (B_max) was at the low femtomolar level of 0.57 to 0.87 fmole mg⁻¹ protein. Higher B_max appeared to be demonstrated in the mid-light samples when compared to the mid-dark samples but the difference was not significant (p > 0.05). Competition study with various indoles showed the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin ≫ N-acetylserotonin ⋙ serotonin. Guanosine 5′-O-(3-thiotriphosphate) (GTP_γS) strongly inhibited the binding (IC₅₀ = 0.66 μmole 1⁻¹) in the rainbow trout heart, suggesting that these binding sites belong to the superfamily of G-protein linked receptors. Our results suggest the presence of melatonin receptors in the fish heart. In addition, there was no marked intraspecies differences in K_d, B_max and specificity that could be correlated with the phylogeny or life history of the salmonid species.     

Specific binding of [3H]17α,20β-dihydroxy-4-pregnen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss)

Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to plasma membranes prepared from defolliculated oocytes of rainbow trout (Onchorhynchus mykiss) was identified and characterized. Binding was rapid and reached equilibrium in 30 min. 17α,20β-DP strongly inhibited [³H] 17α,20β-DP binding in a competitive manner. Scatchard analysis revealed two different binding sites: a high affinity binding site with a K_d of 18 nM and a B_max of 0.2 pmoles/mg protein; and a low affinity binding site with a K_d of 0.5 μM and a B_max of 1 pmoles/mg protein. This binding activity was successfully solubilized with n-heptyl-β-D-thioglucoside. [³H]17α,20β-DP binding to solubilized preparations reached equilibrium in 1h, and was competitively inhibited with 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one. However, Scatchard analysis showed a single binding site with a K_d of 0.3 μM. The reason for the disappearance of the high affinity binding site in solubilized preparations remains unclear. These results demonstrate that a specific binding site for 17α,20β-DP exists in the plasma membrane of rainbow trout oocytes.

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Résumé Une liaison spécifique de le [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), avec des membranes plasmiques d'ovocytes défollicularisés de truite arc-en-ciel (Onchorhynchus mykiss), a été identifiée et caractérisée. Sa cinétique est rapide et atteint son équilibre en 30 minutes. Le 17α,20β-DP inhibe fortement, et de manière compétitive, la liaison de la [³H] 17α,20β-DP. Une étude de Scatchard a mis en évidence deux sites diffŕents de liaison: un site de forte affinité, de K_d 18 nM et de B_max 0,2 pmoles/mg de protéine; et un site de faible affinité, de K_d 0,5 μM et de B_max 1 pmoles/mg de protéine. L'activité de liaison a été solubilisée, avec succés, par le n-heptyl-β-D-thioglucoside. Dans la fraction soluble, la liaison de le [³H]17α,20β-DP atteint un équilibre en 1h.; et elle est complétement inhibiée par la 17α,20β-DP et le 17α,20β,21-trihydroxy-4-pregnen-3-one. Cependant, une étude de Scatchard ne permet de déceler qu'un seul site de liaison, de K_d 0,3 μM. La disparition du site de liaison de forte affinité dans la fraction soluble reste inexpliquée. Ces résultats démontrent l'existence d'un site spécifique de liaison du 17α,20β-DP dans les membranes plasmiques des ovocytes de truite arc-en-ciel.

     

Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (<Emphasis Type="Italic">Monopterus albus</Emphasis> Zuiew)

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish rice field eel (Monopterus albus Zuiew) by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephacryl S-200 HR. The molecular masses of the three purified PGs were all estimated as 36 kDa using SDS–PAGE. Two-dimensional gel electrophoresis (2D-PAGE) showed that pI values of the three PGs were 5.1, 4.8, and 4.6, respectively. All the PGs converted into corresponding pepsins quickly at pH 2.0, and their activities could be specifically inhibited by aspartic proteinase inhibitor pepstatin A. Optimum pH and temperature of the enzymes for hydrolyzing hemoglobin were 3.0–3.5 and 40–45°C. The K _m values of them were 1.2 × 10⁻⁴ M, 8.7 × 10⁻⁵ M, and 6.9 × 10⁻⁵ M, respectively. The turnover numbers (k _cat) of them were 23.2, 24.0, and 42.6 s⁻¹. Purified pepsins were effective in the degradation of fish muscular proteins, suggesting their digestive functions physiologically.     

Influence of ionic composition on acid-base regulation in rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia

The influence of ambient calcium, bicarbonate and chloride levels on acid-base regulation was investigated in rainbow trout acclimated and exposed to hypercapnia in five different water types. In soft water (low [Ca⁺⁺] and [HCO₃ ⁻]), compensation of the respiratory acidosis was slow and incomplete within 72h. High ambient [HCO₃ ⁻] clearly improved extracellular HCO₃ ⁻ accumulation, and pH recovery was accomplished within 24h. This may result from stimulation of branchial HCO₃ ⁻ (influx)/Cl⁻ (outflux) exchange. Elevation of ambient [Cl⁻] had a small, positive effect on pH compensation. High ambient [Ca⁺⁺] improved the degree of pH compensation. Plasma [HCO₃ ⁻] and [Cl⁻] showed an inverse 1:1 relationship in all acclimation groups, revealing an ubiquitous chloride-mediated acid-base regulation. Ventilation activity was increased by hypercapnia and only returned to control values in hard water (high [HCO₃ ⁻]and [Ca⁺⁺]). During progressive hypercapnia (up to 3% CO₂), hard water acclimated fish obtained significantly higher plasma [HC0₃ ⁻] (51.2 mM) than fish acclimated to low [Ca⁺⁺]/high [HCO₃ ⁻] (44.7 mM). This suggests an additive effect of ambient Ca⁺⁺ on plasma HCO₃ ⁻ accumulation. At levels of CO₂ above 1%, some mortality was induced in low [Ca⁺⁺]/high [HCO₃ ⁻] water. Dying fish could be distinguished from surviving fish by an excessive Cl⁻loss and increasing extracellular anion gap.     

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes

The effects of porcine, scombroid, and salmon insulins, and bovine and anglerfish glucagons on glycogen depletion and glycogen phosphorylase (GPase) activities were examined in freshly isolated American eel (Anguilla rostrata) hepatocytes. Eel liver GPase in crude homogenates was activated (increase in % GPase a) by phosphorylating conditions and was rapidly inactivated (less than 1 h) when a phosphatase inhibitor (fluoride) was absent. Caffeine inhibits, and AMP activates, the b form of GPase consistent with their effects on rat liver GPase. Both mammalian and fish glucagons increased glucose production in eel hepatocytes, but had more ambiguous effects on glycogen levels and GPase activities. The magnitude of bovine glucagon effects were dependent on the initial glycogen content of the cells; only at glycogen concentrations less than approximately 70 μmoles.g⁻¹ did glucagon significantly increase % GPase a. Anglerfish glucagon significantly increased cyclic AMP (cAMP) concentrations by 90% at 10⁻⁷ M, but had no effects at 10⁻⁹ M and 10⁻⁸ M. Scombroid and salmon insulins maintained hepatocyte glycogen concentrations and decreased glucose production, with these effects more pronounced at low (10⁻⁹ to 10⁻⁸ M) rather than high (10⁻⁷ M) hormone concentrations. Porcine and salmon insulins decreased total GPase and % GPase a activities, and salmon insulin decreased CAMP levels, but only at 10⁻⁸ M (by 44%). Glycogen is, therefore, depleted by glucagon and maintained by insulin in freshly isolated American eel hepatocytes, and these changes are accomplished, at least in part, by changes in the activities of GPase. Changes in cAMP do not explain all of the observed hormone effects.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

Quantification of presumptive Na+/H+ antiporters of the erythrocytes of trout and eel

The presumptive Na⁺/H⁺ exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[³H]isobutyl)-amiloride (³H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte -adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC₅₀ = 20.1 ± 1.1 mol l^–1, N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific ³H-MIA binding (IC₅₀ = 257 ± 8.2 nmol l^–1, N = 3) than amiloride, which possessed a proton extrusion IC₅₀ of 26.1 ± 1.6 mol l^–1 (N = 6) and a binding IC₅₀ of 891 ± 113 nmol l^–1 (N = 3). The specific Na⁺ channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific ³H-MIA binding. Trout erythrocytes suspended in Na⁺-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable ³H-MIA binding sites per cell (B_max, presumptive Na⁺/H⁺ antiporters) with an apparent dissociation constant (K_D) of 244 ± 29 nmol l^–1 (N = 6). Acute hypoxia (PO₂ = 1.2 kPa; 30 min) did not affect the K_D, yet resulted in a 65% increase in the number of presumptive Na⁺/H⁺ antiporters. Normoxic eel erythrocytes, similarly suspended in Na⁺-free saline, possessed only 17,133 ± 3,716 presumptive Na⁺/H⁺ antiporters (N = 6), 45% of that of trout erythrocytes, with a similar K_D (246 ± 41 nmol l^–1, N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte -adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na⁺/H⁺ exchange sites.     

Thyroid hormone concentrations in the gonads of wild chum salmon during maturation

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T₄) were less than 5 ng ml⁻¹, and those of 3,5,3′-triiodo-L-thyroxine (T₃) were less than 2 ng ml⁻¹ I in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI). There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T₄ concentrations were undetectable (less than 0.2 ng g⁻¹) or less than 2 ng g⁻¹ when GSI was lower than 1%, but increased thereafter and reached a plateau of 8–10 ng g⁻¹ when GSI became 2%. The ovarian T₃ concentrations were about 5 ng g⁻¹ when GSI was 1%, increased to a maximum level (20 ng g⁻¹) when GSI was about 2%, and decreased to a constant level of 10 ng g⁻¹ thereafter. The T₄ and T₃ content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte. The T₄ concentrations in the testis were 1 ng g⁻¹ or less regardless of the GS1. On the other hand, the T₃ concentrations were highest (15 ng g⁻¹) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g⁻¹ I in testes containing spermatozoa, raising the possibility of a role for T₃ during early gamete and/or gonad maturation of testes.     

Effect of acid environments on cortisol and cortisol receptor activity in Atlantic salmon,Salmo salar

The cytosol and nuclear extract of gill tissue obtained from laboratory held Atlantic salmon,Salmo salar manifested saturable cortisol binding of high affinity and low capacity (cytosol: K_a = 0.198 ± 0.024 × 10⁹/M, N_max = 116.8 ± 20.8 fmol/mg protein; nuclear extract: K_a = 0.823 ± 0.057 × 10⁷/M, N_max = 1563 ± 330 fmol/mg protein; n = 4). The cytosol receptor activity displayed high steroid and tissue specificity and a single binding peak at 191,000 Da following gel permeation chromatography. Atlantic salmon exposed for 3 or 8 months to waters from the Medway River (pH about 5.1), the Westfield River (pH about 4.8) and calcium carbonate treated Westfield River (pH about 5.6) showed no gill cytosol receptor activity. Cortisol receptor activity in the gill nuclear extracts from fish in limed Westfield River water in December (3 months) was less than half the activity in the fish treated with Medway River water (p < 0.05) although the plasma cortisol values were not different. In May (8 months), the plasma cortisol of fish in limed water was almost twice that of the fish held in acid Westfield River water (p = 0.058).     

Cod CGRP and tachykinins in coeliac artery innervation of the Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>: presence and vasoactivity

The presence and vasoactive effects of native calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) were studied on isolated small branches of the coeliac artery from Atlantic cod, Gadus morhua, using immunohistochemistry and myograph recordings, respectively. Immunohistochemistry revealed nerve fibers containing CGRP- and SP/NKA-like material running along the wall of the arteries. CGRP induced vasorelaxation of precontracted arteries with a pD₂ value of 8.54 ± 0.17. Relaxation to CGRP (10⁻⁸ M) was unaffected by l-NAME (3 × 10⁻⁴ M) and indomethacin (10⁻⁶ M) suggesting no involvement of nitric oxide or prostaglandins in the CGRP-induced relaxation. SP and NKA (from 10⁻¹⁰ to 3 × 10⁻⁷ M) contracted the unstimulated arteries at concentrations from 10⁻⁸ M and above in 42% and 33%, respectively, of the vessels. It is concluded that the innervation of the cod celiac artery includes nerves expressing CGRP-like and tachykinin-like material, and that a vasodilatory response to CGRP is highly conserved amongst vertebrates while the response to tachykinins is more variable.     

Age and growth of Gerres sp. (Perciformes: Gerreidae) in Okinawa Island of Southern Japan

A total of 518 Gerres sp. were collected around Okinawa Island, Japan, from November 2002 to July 2005, with monthly sampling where the standard length of females (n=218) were 56.2–147.1 mm, and males (n=149) were 62.2–139.4 mm. The maximum ages observed for females were 5⁺ years and males were 4⁺ years, estimated by transverse sectioned sagittal otoliths. Mean marginal increment indicated that opaque rings were formed once a year during April to July. The standard length (SL; mm) — body wet weight (BW; g) relationships were described as BW=(3.26×10⁻⁵) SL^2.97 and BW=(3.13×10⁻⁵) SL ^2.98 for females and males, respectively, and the standard length at age described by von Bertalanffy growth function for females, L _t=137.1(1−e^{−0.80[t+0.80]}) and males, L _t=127.3(1−e^{−0.82[t+0.93]}).     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

Tolerance of striped trumpeter Latris lineata embryos to ozonated seawater

The tolerance of striped trumpeter, Latris lineata (Bloch and Schneider 1801) embryos to ozonated seawater was examined as a possible means of disinfection. The effect of a range of ozone doses and exposures (CT = concentration × exposure time) was tested at different stages of embryonic development. Three-day-old embryos two-thirds developed around the yolk were exposed for 0.5, 1 or 5 min to ozone concentrations of 0.5, 1, 2 and 5 mg O₃ l⁻¹ in a fully orthogonal factorial design. For each treatment there were four replicate 250 ml containers that each received 100 ± 15 embryos. There was no significant difference in hatching success between control-treated embryos or embryos ozonated at 0.5 or 1 mg O₃ l⁻¹ for 0.5, 1 or 5 min (P < 0.05). However, hatching success was significantly reduced when embryos were treated with 2 or 5 mg O₃ l⁻¹ for 5 min or 5 mg l⁻¹ O₃ for 1 min (P < 0.05). The tolerance of embryos to 0, 0.5 or 2 mg O₃ l⁻¹ for 1 min at different stages of development (Day 0, 1, 2, 3 or 4), was then examined. An ozone concentration of 0.5 mg l⁻¹ had no effect on hatching success at any stage of development, but a concentration of 2 mg l⁻¹ significantly reduced hatching success on all days except Day 3. A safe and tested hatchery practise is to disinfect striped trumpeter embryos with 1 mg O₃ l⁻¹ for 1 min on Day 3 post-fertilisation when the embryo is two-thirds developed around the yolk.     

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH₂, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 μmol l⁻¹in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 μmol l⁻¹: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH₂ in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 μmol l⁻¹was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Lack of arterial PO<Subscript>2</Subscript> downregulation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) during long-term normoxia and hyperoxia

Regulation of arterial partial pressure of O₂ (P_aO₂) in Atlantic salmon (Salmo salar) was investigated during resting conditions in normoxic and hyperoxic water. Dorsal aorta cannulated adult Atlantic salmon (1.2–1.6 kg, n = 8) were exposed to 2 week sequential periods of normoxia [16.7 ± 1.1 kPa (mean ± SD)] and hyperoxia (34.1 ± 4.9 kPa) in individual tanks containing seawater (33.7 ± 0.2 ppt) at stable temperature conditions (8.7 ± 0.7°C) and a light regime of L:D = 12:12. Tank design and sampling procedures were optimized to provide suitable shelter and current for the fish, and to allow repeated, undisturbed sampling of blood from free-swimming fish. Fish were sampled regularly through the experimental period. P_wO₂, P_aO₂, blood ion composition (Na⁺, K⁺, Cl⁻), acid–base status (pH, PCO₂, HCO₃ ⁻), haematocrit and glucose were measured. The most frequently observed P_aO₂ values were in the range of 60–80% of P_wO₂, both during normoxia and hyperoxia, and P_aO₂ values were significantly lower during normoxia than during hyperoxia. Blood pH, PCO₂ and HCO₃ ⁻ were significantly elevated during hyperoxia, while, Na⁺, Cl⁻ and Hct were significantly lower. K⁺ and glucose showed no significant differences. This study demonstrates a lack P_aO₂ regulation in Atlantic salmon to low partial pressures, in contrast to previous reports for many aquatic gill breathing animals. Both during normoxia and hyperoxia, P_aO₂ reflects P_wO₂, and alterations in external PO₂ consequently result in proportional arterial PO₂ changes. Physiological adaptation to hyperoxia, as illustrated by changes in several blood parameters, does not include down-regulation of P_aO₂ in Atlantic salmon. The lack of P_aO₂ regulation may make Atlantic salmon vulnerable to the oxidative stress caused by increased free radical formation in hyperoxic conditions.     

Characterization of putative steroid receptors in the membrane, cytosol and nuclear fractions from the olfactory tissue of brown and rainbow trout

Specific binding sites for testosterone have been detected in three compartments of olfactory tissue from brown and rainbow trout. Binding of³H-testosterone to the membrane fraction of olfactory tissue is of high affinity (K_d = 0.5–1.9 nM) and limited capacity (N_max = 30–60 fmol mg⁺¹ protein). Binding is reversible, and is eliminated by protease treatment. The membrane binding site exhibits a high degree of ligand specificity; 11β-hydroxytestosterone, 11-ketotestosterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, cortisol, and estradiol-17β all fail to displace testosterone at 20-fold excess while testosterone itself competes successfully. These attributes are consistent with the presence of specific steroid receptor proteins. Binding of testosterone within the cytosol is of moderate affinity (K_d = 9.0–23.0 nM) and high capacity (N_max = 0.5–2.9 pmol mg⁺¹ protein) and is more readily displaced by a number of steroid competitors than is the case for the membrane site. The rate of association and dissociation of testosterone from the cytosolic binding site is markedly more rapid than the equivalent processes in the membrane fraction. Binding of testosterone to the nuclear extract is of high affinity (K_d ∼3.0 nM) and limited capacity (N_max ∼50 fmol mg⁺¹ protein). There are no substantial differences between species or between sexes in the affinity or capacity of testosterone-binding sites in nuclear extract or membrane fraction. However, cytosolic testosterone-binding sites are three- to four-fold more abundant in rainbow trout than in brown trout, and female rainbow trout have more cytosolic binding sites than male rainbow trout, but a lower affinity for testosterone than male sites. Preliminary evidence supports the involvement of the membrane-associated testosterone-binding site in olfactory processes. Rainbow trout display an EOG response to testosterone at a concentration (≥ 10⁺⁹ M) which is consistent with the equilibrium dissociation constant (K_d) of the membrane-associated testosterone-binding site. Binding of³H-testosterone to the membrane-associated site shows a pH dependency which is comparable to the effects of pH on the EOG response to testosterone in intact fish. The attributes of the intracellular testosterone-binding sites are common to testosterone receptors in other fish tissues which are known androgen target tissues. This suggests that the development and/or function of salmonid olfactory tissue may be susceptible to influence by endogenous testosterone.     

Comparative growth of Pacific oyster (Crassostrea gigas) postlarvae with microfeed and microalgal diets

Two trials were carried out in the laboratory in order to assess the effect of microparticulated feed (F) and live (Thalassiosira pseudonana, M) diets on the growth of recently set (396 ± 13 μm shell height) and 2 mm Crassostrea gigas postlarvae. Different proportions of M and F (100:0, 75:25, 50:50; 25:75, 0:100) were delivered in a single dose of 3 h d⁻¹ in trial 1. Dietary M:F proportions of 100:0, 50:50, and 0:100 were delivered as a single pulse of 8 h d⁻¹ (P1) or two pulses of 4 h⁻¹ (P2) in trial 2. Maximal daily M ration was 296 cells μl⁻¹ d⁻¹ (trial 1), 150 M cells μl⁻¹ d⁻¹ (trial 2), or their equivalent F dry weight. Shell height (SH), dry (DW), and organic weight (AFDW) were evaluated weekly. Oysters from trial 1 significantly increased their size after 28 days, and exhibited no significant dietary differences in terms of DW (1.21 ± 0.15 to 2.01 ± 0.28 mg) or AFDW (0.091 ± 0.022 to 0.166 ± 0.029 mg). Newly set postlarvae (trial 2) also exhibited significant growth after 25 days. No dietary differences were observed in trial 2, yet P2 oysters attained significantly higher shell heights (825–912 μm) than P1 oysters (730–766 μm) after 25 d. Pulse effects were marginally not significant in terms of AFDW and growth rate. Together, these findings showed that balanced microfeeds have a practical potential for the culture of early C. gigas postlarvae, when they are delivered in pulse-feeding schemes     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.