Interaction of low density lipoproteins with liver cells in rainbow trout

Liver is the main catabolic tissue for low density lipoprotein in rainbow trout (Gjøen and Berg 1992). We have investigated the interaction of LDL with isolated trout liver cells and liver membranes. ¹²⁵I-TC labelled trout LDL bound to isolated trout liver cells in a time dependent and saturable manner with an apparant K_d of 20.1 g/ml, suggesting the existence of a specific binding site on the surface of these cells. The binding was Ca²⁺ dependent assessed by the 50% reduction obtained by 5 mM EDTA. Saturable binding to isolated trout liver membranes could also be demonstrated, but with lower affinity as compared to intact cells. Degradation of ¹²⁵I-TC-LDL in hepatocytes was also saturable as degradation could be inhibited about 60% by a 100 fold surplus of unlabelled LDL. The rate of degradation increased with temperature up to 20°C. Both cell association (binding + uptake) and degradation were reduced down to 20% of control in the presence of microtubular and lysosomal inhibitors. Hepatic catabolism of trout LDL therefore seems to depend on receptormediated endocytosis, followed by lysosomal degradation.Abbreviations TC tyramine cellobiose - LDL low density lipoproteins - MeLDL methylated low density lipoproteins - VLDL very low density lipoproteins - HDL high density lipoproteins - VTG vitellogenin - EDTA ethylenediamine tetraacetic acid - PBS phospate buffered saline - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - DMPC L--phosphatidylcholine-dimyristoyl     

Arginine effects on biochemical composition of sperm in rainbow trout,Oncorhynchus mykiss

The present study investigated the effect of arginine on seminal plasma composition in rainbow trout. Male rainbow trout broodstocks (2500 ± 200 g) were fed five practical diets (each consisting of three triplicates) supplemented with Arginine at 0.50%, 1.50% and 2.00%. The control group were fed without arginine. Broodstock feeding lasted for 90 days, and then fish semen was sampled. Results indicated no significant differences in LDH, ALP, Fe²⁺ and phosphorous content among the different treatments. The lowest levels of AST and ALT and the highest levels of Ca²⁺ and Mg²⁺ ions were observed in the treatment fed with 1.50% arginine, which showed significant differences from other treatments (P < 0.05). Moreover, the amount of Cl^?, Na⁺ and K⁺ ions was significantly increased in the seminal plasma in fish fed diets containing arginine in comparison with the control. As the amount of arginine was increased, the levels of uric acid became significantly greater in contrast to urea and glucose levels. The highest amounts of cholesterol, fructose and total protein were observed in treatments fed on 2.00%, 0.50% and 1.00% arginine, respectively, showing significant differences from other treatments (P < 0.05). The highest pH value was assayed in the 1.50% arginine treatment. Results indicated that arginine had a potential efficacy on semen quality in rainbow trout broodstocks.     

Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

Response to T3 treatment and changing environmental salinity of liver lipid composition, mitochondrial respiration and (Na++ ++ K++)-ATPase activity in rainbow trout Oncorhynchus mykiss Walbaum

Rainbow trout Oncorhynchus mykiss Walbaum were fed a pelleted diet (14.3% wet weight lipid) containing 9 p.p.m. 3, 5, 3′‐triiodo‐l ‐thyronine (T₃) for 1 month and then transferred from fresh water to brackish water (average 22 p.p.t. salinity), where they were maintained untreated for 22 days. Trout fed a control diet were subjected to the same protocol. For both treated and control trout, liver lipid and fatty acid composition, mitochondrial respiratory activity and oxidative phosphorylation and (Na⁺ + K⁺)‐ATPase activity were monitored in fish sampled periodically throughout the trial. No differences between treated and control trout occurred in liver total lipid, phospholipid and cholesterol content or fatty acid composition. Conversely, irrespective of T₃ administration, the trout from the two habitats showed adaptive changes to salinity, differing in phospholipids and in the fatty acid composition of total and neutral lipids and selected phospholipids. Liver membrane permeability and mitochondrial respiratory activity were affected by both T₃ treatment and salinity transfer. The latter was apparently greater than the former in affecting mitochondrial respiratory activity. The higher (Na⁺ + K⁺)‐ATPase activity in T₃‐treated trout after 22 days in brackish water may reflect long‐term effects of the hormone linked to salinity adaptation.     

Sodium dependent ion transporters in trout gills

Biochemical procedures developed to isolate plasma membranes from the branchial epithelium of rainbow trout (Oncorhynchus mykiss) yield membrane fractions that are specifically enriched in the plasma membrane marker enzyme Na⁺/K⁺-ATPase. As the bulk of the branchial Na⁺/K⁺-ATPase is assumed to be confined to the mitochondria-rich chloride cells, such membrane preparations must contain the essence of the enzymatic machinery of the chloride cells. Basal Na⁺ activity in branchial (chloride) cells is around 10 millimolar and, accordingly, we find a K_m for Na⁺ of the Na⁺/K⁺-ATPase of 13 millimolar, indicating that the enzyme may be regulated by changes in cytosolic sodium. The Na⁺-gradient across the serosal plasma membrane created by this pump provides energy for 3Na⁺/Ca²⁺-exchange and bumetanide-sensitive Na⁺/K⁺/2Cl^--cotransport. Here we further postulate the presence of a Na⁺/Cl^--cotransporter, indicated by thiazide-sensitive, bumetanide-insensitive transport of Na⁺ and Cl^-; this cotransporter activity awaits the characterization of its kinetics. The Na⁺/Ca²⁺-exchanger has kinetic characteristics compatible with a regulatory role of cytosolic Na⁺ in the activity of this carrier. Both Na⁺/Ca²⁺-exchange and Ca²⁺-ATPase activity may contribute to transport of Ca²⁺, the former having lower affinity for calcium but a higher capacity than the latter carrier. The Na⁺/K⁺/2Cl^--cotransporter has kinetics that favor a regulatory role for plasma K⁺ in the activity of this carrier. Seawater adaptation leads to increased activity of cotransporter molecules in the plasma membrane fractions (the activity increases relative to that of the Na⁺/K⁺-ATPase) and this may reflect a function in Cl^--extrusion performed by the chloride cells in a seawater environment. A function for the cotransporter in the gills of freshwater fish may be the regulation of cell volume.     

An analysis of the ion-exchange characteristics of fish-egg chorions

The chorions of rainbow trout were investigated to measure their ion-exchange characteristics. These characteristics have been incorporated into a model, based on ion-exchange theory, which attempts to describe interactions between chorions and a range of water qualities, in terms of the distribution of K⁺. The model was tested by comparing predicted K⁺ accumulation by chorions with that actually measured in experimental conditions. The model successfully predicted the influence of combinations of K⁺ and Ca²⁺ on K⁺ accumulation, and was able to predict the direction of changes in K⁺ accumulation in the presence of aluminium and low pH.     

The effect of postprandial changes in pH along the gastrointestinal tract on the distribution of ions between the solid and fluid phases of chyme in rainbow trout

An area of emerging importance is the role that the diet can play in alleviating the demands for ion uptake in fish living in a freshwater environment, by providing a highly concentrated supply of electrolytes. The availability of ions for uptake from the diet likely requires dissolution in the fluid phase of the chyme. However, the distribution of ions between the fluid and solid phases of chyme has not been well‐characterized in fish, and little is known about the effects of location along the gastrointestinal (GI) tract, or about the pH gradients found therein, on this distribution. Hence, the pH and ionic concentrations (Na⁺, K⁺, Cl^?, Ca²⁺ and Mg²⁺, in both fluid and solid phases) of the chyme in each GI tract section were measured at various time points during the digestion of a single meal of commercial pellets in freshwater rainbow trout (Oncorhynchus mykiss). Additionally, the presence of an inert reference marker (lead‐glass beads) in the diet was used to quantify the distribution of these ions between the solid and fluid phases of the chyme. The buffering capacity of the food was evident in the acidic stomach (ST), whereas the intestine provided an alkaline environment for further digestion. It appeared that pH had little influence on the distribution of the monovalent ions between the phases in all GI tract sections. However, the ST showed significant changes in the partitioning of both Ca²⁺ and Mg²⁺, with each mineral becoming highly dissolved as the gastric chyme pH decreased. This was followed by subsequent precipitation of both minerals in the alkaline environment of the intestine. The high degree of dissolution of Ca²⁺ and Mg²⁺ in the fluid phase of gastric chyme corresponded with large absorptive rates from the ST seen previously, however, this was not true of the monovalent ions.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Effect of different ions on larval metamorphosis of the mussel Mytilus galloprovincialis

Metamorphic responses of pediveliger larvae of Mytilus galloprovincialis to different ions were investigated through a series of bioassays. Effects of tetraethylammonium chloride (TEA) on inductive effects of these above ions were also investigated. Excess ions including Li⁺, Cs⁺, Rb⁺ and Ca²⁺ induced larval metamorphosis at 10^?3 M to 5 × 10^?2 M in 24‐h exposure assays. In continuous exposure assays, only excess Ca²⁺ showed inductive activity and induced 25% metamorphosis at 5 × 10^?2 M. Larval responses to Li⁺ and Rb⁺were inhibited by TEA, while induction of metamorphosis by Cs⁺and Ca²⁺ was independent of the presence of TEA. Thus, these ions used can be useful inducers of larval metamorphosis for application in the aquaculture industry. The finding provides new insights on the biochemical mechanism controlling larval metamorphosis in this species.     

Ionoregulatory responses to temperature change in two species of freshwater fish

We examined the ionoregulatory responses to temperature changes in two species of freshwater fish that differ in thermal preferences; the stenothermal, cold-water rainbow trout (Oncorhynchus mykiss) and the more eurythermal, warm-water common shiner (Notropis cornutus). We found that rainbow trout maintained constant plasma Na⁺ levels over the entire temperature regime (5–20 °C). Upon transfer from 15 °C (holding temperature) to 5 and 10 °C, rainbow trout experienced a significant drop in Na⁺ uptake (J_in ^Na), but after two weeks J_in ^Na had upregulated to warm temperature levels. Further, Na⁺ efflux (J_out ^Na) fell significantly at the colder temperatures. As a result, trout at the lowest temperatures were still in ion balance. When trout were exercised to exhaustion both O₂ consumption (MO₂) and J_out ^Na rose significantly at all temperatures, but while MO₂ continued to be dependent upon temperature, J_out ^Na was high and constant. In contrast to the trout, common shiners experienced a 20% drop in plasma Na⁺ at 5 °C. Upon exposure to cold temperatures they experienced a reduced J_in ^Na, and showed no signs of acclimation during the subsequent two weeks. Likewise J_out ^Na was constant at all temperatures. These findings raise questions regarding the degree to which fish employ homeostatic mechanisms designed to defend a set- point (i.e., steady-state) osmolarity and ionic composition.     

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.     

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.     

Glucose-stimulated lipolysis in rainbow trout,Oncorhynchus mykiss,liver

Rainbow trout, Oncorhynchus mykiss, were used to characterize further the influence of glucose on hepatic lipolysis. Liver was removed from fed fish, cut into 1 mm³ pieces and incubated for up to 5 h in Hanks medium containing either 2 mM, 5.5 mM, 10 mM, or 25 mM glucose. Glucose-stimulated lipolysis was indicated by tissue triacylglycerol (TG) lipase activity and by medium concentrations of glycerol and fatty acids (FA). Triacylglycerol lipase activity in liver pieces incubated in the presence of higher concentrations (25 mM) of glucose was significantly higher than that in liver pieces incubated in lower concentrations (2 mM) of glucose, rising from 0.075 ± 0.002 (mean ± SEM) nmol FA released/h/mg protein to 0.092 ± 0.004 units. Similarly, higher concentrations of glucose stimulated significantly more FA release and glycerol release from liver pieces than that stimulated by lower concentrations of glucose. Glycerol release from liver pieces incubated in the presence of 10 mM glucose and 25 mM glucose was ca. 2-fold to 2.8-fold, respectively, higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Fatty acid release from liver pieces incubated in the presence of 10 mM or 25 mM glucose was ca. 1.8-fold higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Notably, increased glycerol release was not accompanied by a parallel increase in FA. Fatty acid reesterification was more pronounced in liver pieces exposed to higher glucose (10 mM and 25 mM) than in liver pieces exposed to lower glucose (2 mM and 5.5 mM). ¹⁴C-incorporation studies indicated that glucose serves as a carbon source for reesterified FA in trout liver. The route of reesterification appears to be from glucose to glycerophosphate to phosphatidic acid to diacylglycerol to TG. Increasing concentrations of glucose did not affect glycerol kinase activity, indicating that glucose-stimulated lipolysis was not accompanied by increased glycerol recycling within the liver. These results suggest that glucose stimulates fatty acid reesterification and directly enhances net lipolysis in trout liver incubated in vitro.A part of this study was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1991, Atlanta, GA.     

Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K⁺ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na⁺, K⁺-ATPase was attained at 10 mol l^–1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K⁺ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K⁺ influx increased, but was inhibited to about 15% by 30 mol l^–1 sulfide. In RBCs of crucian carp neither Na⁺, K⁺-ATPase nor ouabain-resistant K⁺ influx were affected by sulfide concentrations up to 850 mol l^–1. Differences in sulfide-sensitivity of K⁺ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na⁺, K⁺-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K⁺ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species.     

Motility of spermatozoa ofAlburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism

The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na⁺, K⁺ and protein levels (negative correlations: % immotile spermatozoa-Na⁺, K⁺; positive correlations: % motile spermatozoa-osmolality, pH, Na⁺, K⁺, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na⁺, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase     

Polyunsaturated fatty acids do not activate protein kinase C in the testis of the goldfish (Carassius auratus)

Earlier studies have established that polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid and docosahexaenoic acid inhibit steroid production in the goldfish testis. As PUFA inhibit testicular steroidogenesis in the rat through activation of protein kinase C (PKC), the present studies were undertaken to characterize the properties of PKC in the goldfish testis and to test the effects of selected PUFA on PKC activity. PKC activity was quantified in goldfish testis homogenate following partial purification by DEAE-cellulose chromatography by determining the transfer of radiolabelled phosphate from [γ - ³²P]ATP to histone III-S. Testicular PKC activity was defined by the amount of protein phosphorylation in the presence of phosphatidylserine, phasphatidylcholine, Ca²⁺ ions and diolein (a 1,2-diacylglycerol analog) above that obtained in response to Ca²⁺ ions alone. Western blot analysis of a crude testis homogenate using an antibody specific to the α and β isoforms of mammalian PKC led to the identification a single band of protein (80 kD) that co-migrated with PKC from rabbit brain cytosol. Addition of arachidonic, eicosapentaenoic or docosahexaenoic acids failed to activate PKC. However, PKC activity stimulated by phospholipid, Ca²⁺ ions and diolein was inhibited in a dose related fashion by all of these fatty acids. These studies suggest that the inhibitory effects of EPA and DHA on testicular steroidogenesis are not mediated by activation of PKC. The lack of effect of PUFA on PKC activity in the goldfish testis suggests that either the distribution of PKC isoforms differs between the testis of mammals and fish or that PKC is not activated by PUFA in the goldfish.     

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of¹²⁵I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10¹⁰ M^–1 and a maximum binding capacity (B_max) of 9 fmol mg^–1 protein. Liver tissue displayed the highest¹²⁵I-rcGH binding of all the tissues examined. Displacement of¹²⁵I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 g rcGH g^–1 body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 g rPRL g^–1 body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.     

Response of rainbow trout gill (Na++K+)-ATPase and chloride cells to T3 and NaCl administration

With the aim of comparing the effects of oral T₃ and NaCl administration on trout hypoosmoregulatory mechanisms, three groups of rainbow trout (Oncorhynchus mykiss Walbaum) held in freshwater (FW) were fed a basal diet (C), the same diet containing 8.83 ppm of 3,5,3-triiodo-L-thyronine (T₃) (T) or 10% (w/w) NaCl (N) respectively for 30 d. They were then transferred to brackish water (BW) for 22 d and fed on diet C. Gill (Na⁺+K⁺)-ATPase activity and its dependence on ATP, Na⁺ and pH, number of gill chloride cells (CC), serum T₃ level as well as fish growth, condition factor (K) and mortality were evaluated. During the FW phase, as compared to C trout, T trout showed a two fold higher serum T₃ level, had unchanged gill (Na⁺+K⁺)-ATPase activity and increased CC number, whereas N trout showed higher gill (Na⁺+K⁺)-ATPase activity and CC number. At the end of the experiment the enzyme activity was in the order T>N>C groups and all groups showed similar CC number. Both treatments changed the enzyme activation kinetics by ATP and Na⁺. A transient increase in K value occurred in N group during the period of salt administration. In BW, T and N groups had higher and lower survival than C group respectively. Other parameters were unaffected by the treatments. This trial suggests that T₃ administration promotes the development of hypoosmoregulatory mechanisms of trout but it leaves the (Na⁺+K⁺)-ATPase activity unaltered till the transfer to a hyperosmotic environment.     

Changes in Sperm Production,Sperm Motility,and Composition of Seminal Fluid in Caspian Brown Trout,Salmo trutta caspius,Over the Course of a Spawning Season

This study was carried out to evaluate milt quality in male Caspian brown trout (Salmo trutta caspius) over the course of the winter spawning season. Milt samples were collected biweekly during December and January. Chemical composition of seminal fluid, sperm production (milt volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were measured. Milt volume, sperm density, osmolality, seminal minerals (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl^?), and total protein gradually decreased over the spawning season. Glucose and triglyceride content of milt did not show significant changes over the spawning season. Milt pH and the percentage and duration of motility were comparatively stable, declining only at the end of the season. Significant positive correlations were found between sperm density and seminal minerals, total protein and spermatocrit; percentage of motile spermatozoa and seminal minerals, total protein; and duration of motility and K⁺, Cl^?, total protein, and pH. Results show that season has a significant influence on milt quality in male Caspian brown trout, with the best milt being available at the beginning of spawning season.     

Ionoregulatory and respiratory responses of brown trout,Salmo trutta,exposed to lethal and sublethal aluminium in acidic soft waters

Soft water acclimated (Ca²⁺ 0.02 mM; Na⁺ 0.03 mM; K⁺ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l^–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l^–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l^–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (C_aO₂) but no changes in plasma osmolarity or the concentrations of plasma Na⁺, K⁺ and Cl^–. Physiological disturbance was more marked at the 50 g l^–1 Al concentration. The surviving fish at 25 g l^–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l^–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na⁺ and Cl^–1. Although C_aO₂ decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.