The teleost pseudobranch: a role for preconditioning of ocular blood supply?

The physiological relevance of the teleost pseudobranch as a remnant of a reduced gill arch is still unclear. Numerous hypotheses have been proposed regarding its physiological role, but direct confirmatory evidence is lacking. The close relationship by serial blood flow arrangement with the fish eye’s choroid rete has sparked the idea that pseudobranchial preconditioning of blood pH may facilitate initiation of the Root effect and thus support the establishment of high oxygen tensions for retinal diffusive supply. This idea was critically tested by studies on isolated pseudobranchs in situ (Oncorhynchus mykiss), perfused with RBC/Ringer or RBC/plasma suspensions of widely varied composition (pH 7.4–8.2). Detailed analysis of inflowing as compared to effluent perfusates indicated normal aerobic metabolism expressed by a rise in Pco₂ (+0.39 ± 0.13 mmHg ), an oxygen utilization of 25% and a high oxygen consumption of ∼400 nmol g⁻¹ min⁻¹. Upon passage of the pseudobranch, pH (corrected for Haldane effect) was only slightly acidified (−0.03 to −0.10), [HCO₃ ⁻] and [lactate] were slightly enhanced (+0.51 mmol l⁻¹ or 0.13 mmol l⁻¹, respectively). In order to test for yet unknown plasma components involved in pseudobranch function, a second series of experiments was conducted using RBC-suspensions in fresh plasma instead of Ringer, with results closely resembling those of the RBC/Ringer series. Lacking any physiologically significant correlation with the level of perfusate pH, the obtained data indicate pseudobranchial basic metabolic activity rather than pH regulatory characteristics. Also the observed absolute changes in pH are negligible in terms of pH regulation towards the Root-threshold. Accordingly, the present experiments as well as plausibility evaluation of mechanisms do not support the idea of blood pH pre-adjustment prior to entry into the choroid rete structure of the teleost eye to facilitate the Root-mediated oxygen release.     

Stimulation of ureogenesis in the perfused liver of an Indian air-breathing catfish, Clarias batrachus, infused with different concentrations of ammonium chloride

The pattern of changes of activity of the urea cycle enzymes and the rate of urea-N excretion were studied in the perfused liver of an Indian air-breathing ureogenic walking catfish, Clarias batrachus. The liver was perfused with different concentrations of NH₄Cl for a period of 60 min to determine the role of ammonia for stimulation of hepatic ureogenesis and the threshold level of ammonia loading needed to cause such stimulation. Both the urea-N excretion and the ammonia uptake by the perfused liver were found to be a saturable process. Ammonia accumulated significantly in the liver infused with 1.25 moles g liver ^–1 min^–1 of NH₄Cl, followed by a maximum accumulation of about 28.5 moles g wet wt^–1 with the infusion of 5.08 moles g liver^–1 min^–1. The Vmax of the urea-N excretion (0.47 mol g liver^–1 min^–1) was obtained with the addition of 5.08 moles g liver^–1 min^–1 of NH₄Cl. Both the tissue and the specific activity of the urea cycle enzymes, except ornithine transcarbamylase and arginase, were stimulated significantly with the infusion of either 1.25 or 5.08 moles g liver^–1 min^–1 of NH₄Cl. Maximum stimulation of tissue activity of carbamoyl phosphate synthetase (about 120%) was seen with the infusion of 5.08 mol g liver^–1 min^–1, and for argininosuccinate synthetase (about 135%), and argininosuccinate lyase (about 50%) with the infusion of 10.81 mol g liver^–1 min^–1 of NH₄Cl. Higher accumulation of ammonia of about 10–15 mol g wet wt^–1 from the physiological level in the perfused liver while infusing with NH₄Cl was suggested to be one of the major causes of stimulation of ureogenesis. The presence of such physiological adaptive strategy is probably necessary in this unique group of air-breathing walking catfish to survive under hyper-ammonia stress in their normal habitat or while living outside water or while burrowing inside mud.     

The effects of experimental anaemia on CO2 excretionin vitro in rainbow trout,Oncorhynchus mykiss

The effects of severe experimental anaemia on red blood cell HCO₃ ^– dehydrationin vitro were examined in rainbow trout,Oncorhynchus mykiss. After 5 days of anaemia (haematocrit=4.9±1.1%) induced by intraperitoneal injection of phenylhydrazine hydrochloride, fish displayed elevated arterial CO₂ tensions (anaemic PaCO₂=3.19±0.42 torrvs. control PaCO₂=1.35±0.17 torr) and a significant acidosis (anaemic pHa=7.73±0.04vs. control pHa=7.99±0.04). However, after 15–20 days of anaemia (hct=6.6±0.8%) induced by blood withdrawal, the arterial CO₂ tension was significantly lower than the control value, suggesting that physiological adjustments occurred within this time period to compensate for the lowered haematocrit. Compensation probably did not involve alterations in ventilation, which was unaffected by 5 days of anaemia (anaemic ;w=786±187 ml min^–1 kg^–1 vs. control ;w=945±175 min^–1 kg^–1), based on indirect Fick principle measurements.Potential adaptations to longer term anaemia at the level of the red blood cells were investigated using a radioisotopic HCO₃ ^– dehydration assay. Owing to the difference in haematocrits, the HCO₃ ^– dehydration rate for blood from anaemic fish was significantly lower than that for control fish following equilibration at the same CO₂ tension. This difference was eliminated when HCO₃ ^– dehydration rates were measured on blood samples adjusted to the same haematocrit, a result which implies that the intrinsic rate of CO₂ excretion at the level of the red blood cell was not up-regulated during anaemia. The difference was also eliminated by equilibrating the blood samples with CO₂ tensions appropriate for the group from which the sample was obtained,i.e., PCO₂=1.4 torr for control samples and PCO₂=3.2 torr for anaemic samples; each at the appropriate haematocrit. It is concluded that the elevated PaCO₂ helps to reset CO₂ excretion to the control level, but that some additional physiological adjustment occurs to lower the PaCO₂ after 15–20 days of anaemia.     

Oxygen consumption and responses to hypoxia of ammocoetes of the southern hemisphere lampreyGeotria australis

The standard rate of oxygen consumption of ammocoetes (larvae) ofGeotria australis with a mean weight of c. 0.5 g was 9.6, 31.4 and 59.4l g^–1 h^–1 at 4.5, 15.5 and 25.0°C respectively, which gives an overall Q₁₀ of 2.4. The regression coefficient for the logarithmic relationship between oxygen consumption and body weight at 15.5°C was 0.704. The ammocoetes ofG. australis have a much lower rate of oxygen consumption at 15.5 and 25.0°C than those of holarctic lampreys. This presumably reflects the lower oxygen delivery pressure to their tissues and helps account for their slow growth rate. At 15.5°C, ammocoetes ofG. australis emerged from the substrate at 21–25 mm Hg and, unlike those of the Northern HemisphereIchthyomyzon greeleyi, died at 14–17 mm Hg. Thus, despite having a thinner water/blood barrier in the gills and blood with a higher oxygen affinity and capacity than holarctic ammocoetes, the larvae ofG. australis cannot survive very low dissolved oxygen tensions. This is apparently related to an inability of larvalG. australis to meet the high oxygen requirements of the respiratory pump at these oxygen tensions. During metamorphosis, oxygen consumption at 15.5°C rose from approximately 27l g^–1 h^–1 at the beginning of transformation to 33.2l g^–1 h^–1 by Stage 3 and then rapidly to 66l g^–1 h^–1 at Stage 6. It remained near this level in Stage 7 and the downstream migrant.     

Relationship between metabolic rate in vitro and body mass in a marine teleost,porgy Pagrus major

The rate of oxygen consumption of minced whole body was determined volumetrically, as an indication of metabolic rate in vitro (M _{in vitro} ), at 20°C in porgy Pagrus major ranging from 0.0002 g (just after hatch) to 2.9 g (67 days old) in body mass. A triphasic relationship was found between M _{in vitro} of individual fish (l.min^–1) and wet body mass W (g). During the prolarval stage accompanied with the transitional period to the postlarval stage (0.00020–0.00023 g, 0–6 days old), the mass-specific metabolic rate in vitro (M _{in vitro} /W in l.g^–1.min^–1) increased with age (D in days) as expressed by an equation M _{in vitro} /W = 3.88 + 0.74/D. During the postlarval stage (0.00031–0.003 g, 8–22 days old), M _{in vitro} /W remained almost constant, independent of body mass following an equation M _{in vitro} /W = 5.24 W^–0.085. During the juvenile and adolescent stages (0.0047–2.9 g, 30–67 days old), M _{in vitro} /W decreased with increasing body mass following an equation M _{in vitro} /W = 1.66 W^–0.235. These results correspond with the triphasic relationship between metabolism in vivo and body mass observed in intact porgy of 0.0002–270 g (Oikawa et al. 1991). It is concluded, therefore, that the dependence of mass-specific metabolic rate on body size exists in vitro as well as in vivo, during the early stages in the porgy. Based on these results, factors controlling the metabolism-size relationship are discussed.     

Basal H2-receptor stimulated and pH-dependent acid secretion from an isolated stomach mucosa preparation of the cod,Gadus morhua,studied using a modified pH-static titration method

Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH⁺min^–1cm^–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC₅₀=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH⁺min^–1cm^–1. Ranitidine, a H₂-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H₁-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H₂-receptor and that histamine is secreted under basal conditions.     

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.     

Stereological Analysis of Blood Space and Tissue Types in the Pseudobranch of the Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

The teleost pseudobranch, a reduced mandibular gill arch, morphologically closely resembles a gill hemibranch. It consists of several filaments bearing numerous lamellae. The vascular system of the pseudobranch also matches that of a gill, with afferent and efferent filamental arteries and the lamellar blood space. Despite the pseudobranch’s gill-like appearance, however, a gas exchange function is excluded in many teleosts by overlying opercular epithelium, effectively eliminating diffusional exchange with the external medium. The physiological function of the pseudobranch is still unknown, though its conspicuously close association with the ocular choroid rete mirabile, a structure supporting elevated oxygen partial pressures in the eye, suggests a role related to the process of oxygen secretion into the eyes of teleosts. In this paper we present new stereological data on the pseudobranch of rainbow trout (average weight: 478 ± 114 g, x ± SD, n = 12). The total Cavalieri volume of the pseudobranch was 46.4 μl (97 μl kg⁻¹), consisting of lamellae 49%, connective tissue 43.8%, large blood vessels 4.5%, filamental cartilage 2.3%, and central venous sinus 1.3%. Lamellae were comprised of pillar and pseudobranchial cells 81%, blood space 12.3%, and lacunar tissue 6.4%. The inner surface of the blood space in the lamellae was 12.2 cm² (25.5 cm² kg⁻¹), and its volume was 2.81 μl (5.88 μl kg⁻¹). The results demonstrate the pseudobranch’s potential for physiological functions requiring a high surface area or surface-to-volume ratio, i.e. ion transfer, gas exchange and other regulatory processes. More detailed analysis of the function of the pseudobranch, however, has to remain subject of future morphological and physiological studies.     

Physiological and biochemical parameters for egg quality determination in lake trout,Salmo trutta lacustris

The present study investigated the relationships between egg viability and ovarian fluid composition, egg physiology and egg metabolism in lake trout, Salmo trutta lacustris, to obtain biomarkers for egg quality determination. The ovarian fluid pH, protein levels and activities of aspartate aminotransferase and -d-glucuronidase were significantly correlated with egg viability expressed as the number of eyed stage embryos. Regression models demonstrated that an ovarian fluid pH between 8.44 and 8.57, protein levels below 235.56 mg 100 ml^–1ovarian fluid, aspartate aminotransferase activity below 31.65 m min^–1 l^–1ovarian fluid and -d-glucuronidase activity below 8.62 m min^–1 l^–1 ovarian fluid characterized egg batches with high viability (80%).The increase in the egg wet weight during water hardening was also significantly correlated with the number of eyed stage embryos, and egg batches with high egg viability (80%) increased in wet weight by 13% during water hardening.From the investigated metabolic parameters the number of eyed stage embryos was significantly correlated with activities of NADP-dependent isocitrate dehydrogenase (egg viability 80% at 2.07 nM min^–1 mg^–1 protein) and NAD-dependent malate dehydrogenase (egg viability 80% at 47.25 nM min^–1 mg^–1 protein), with the respiration rate (egg viability 80% at 8.71 nM min^–1 mg^–1 protein), with the ratio of NADH to NAD levels (egg viability 80% 0.872), with the levels of free, non-esterified fatty acids (egg viability 80% 72.34 g mg^–1 protein), and the ratio of non esterified to esterified fatty acids (egg viability 80% at 0.749). Also, subjective and visual control methods were described to distinguish between batches with viable and non viable eggs.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Effects of sublethal, acidic aluminum exposure on blood ions and metabolites, cardiac output, heart rate, and stroke volume of rainbow trout, Oncorhynchus mykiss

Doppler flow probes were fitted around the ventral aorta of rainbow trout, which were exposed to combinations of pH and aluminum (pH 5.1–6.2, Al 0–80 g l^–1) for 60 h. Fish accumulated Al at the gill and exhibited decreased blood Na⁺, Cl^–, and Ca²⁺ concentrations, and increased K⁺, hematocrit, hemoglobin, glucose, and lactate, indicating increasing ionoregulatory disturbance with increasing Al concentration. Fish exposed to ambient water (6.2) or low-pH (5.3) water without Al exhibited slight reductions in heart rates, as well as increased stroke volume, resulting in little variation in cardiac output. In the presence of Al (20 to 40 g l^–1) at low pH (5.1–5.3), fish increased their heart rate slightly and generally maintained their stroke volume, resulting in increased cardiac output in the first two days of exposure. At the highest Al concentration (80 g l^–1, pH 5.1), tachycardia was observed, concomitant with a decrease in stroke volume. The ionoregulatory imbalance and resulting increased blood viscosity explain these increases in heart rate rather than stroke volume in fish exposed to high concentrations of Al.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

Gill Na+-K+ ATPase, carbonic anhydrase activities and plasma osmotic and ionic variations during smoltification of Atlantic salmon in the north of Portugal

Gill Na⁺-K⁺ ATPase and carbonic anhydrase activities were measured, on a fortnightly basis, from February to July, in 0₊ age Atlantic salmon (Salmo solar), hatched and reared in a freshwater experimental station in Covas, northern Portugal. Plasma osmolarity and ionic composition were also measured. Gill Na⁺-K⁺ ATPase activity increased slowly until April (15–19 moles Pi mg prot^–1 h^–1). From April to late May there was a great increase in activity (19–32 moles Pi mg prot^–1 h^–1) followed by a sharp decline in June (15 moles Pi mg prot^–1 h^–1). In contrast, carbonic anhydrase activity decreased significantly from early April to early June (170-70 moles p-nitrophenol mg prot^–1 h^–1) and increased in late June, suggesting the existence of a compensatory mechanism for the changes in Na⁺-K⁺ ATPase activity. Plasma osmolarity and sodium concentration showed lower levels during the period of high ATPase activity. On the other hand, plasma calcium concentrations showed an increase during the same period (3.47–5.98 mm1^–1 of plasma). A transitory decrease in osmolarity and plasma sodium and chlorine concentrations occurred in March, prior to the surge in Na⁺-K⁺ ATPase activity, suggesting that the physiological changes, characteristic of parr-smolt transformation can be a consequence of this loss of freshwater osmoregulatory capacity.     

Brackishwater carp culture in potentially waterlogged areas using animal wastes as pond fertilizers

In order to assess the possibilities of utilizing drainage effluents (salinity range 5.0–12.5), fish culture experiments were carried out. Experiments on polyculture using cow dung (24 000 kg ha^–1 y^–1) as pond fertilizer were conducted at five different salinity levels (0.3–8.5). Studies have revealed that carp perform well in salinities up to 7.5 and reasonably high fish production has been obtained. Even though the ponds had a high trophic status, higher salinities ( > 7.5) appear to repress fish growth probably due to low dissolved oxygen (DO), high BOD and high NH₄-N. Experiments on monoculture of common carp (Cyprinus carpio) conducted at two different salinity levels (0.3–0.9 and 6.0–7.0) using four different organic fertilizers (cow dung at 24 000 kg and 20 000 kg, poultry at 1500 kg, duck at 6000 kg and sheep/goat at 1500 kg ha^–1 y^–1) have revealed the highest fish growth to be in poultry-treated ponds, followed in decreasing order by duck and sheep/goat wastes. Similar trends in fish production were observed both in fresh- and salt-water ponds. However, fish production was lower in ponds having higher salinities ( > 7.5). Nevertheless, these studies indicated that inland saline waters can be utilized for fish culture. With minor modifications in the existing technology of fish culture in stagnant freshwater fish ponds, animal wastes could be used to fertilize brackishwater fish ponds.     

The effect of seawater flow and temperature on metamorphosis and postlarval development in great scallop

Variations in growth and survival of hatchery-reared post-metamorphicjuveniles of great scallop Pecten maximus prompted anexamination of settlement and postlarval development. The effects ofseawater flow and temperature on great scallop metamorphosis andpostlarvae were studied over a 4–5 week period. In allexperiments, and regardless of environmental conditions, great scallopmetamorphosed after a 2–3 week period with values of 35 to70%. Subsequently, spat numbers increased slightly. Spatmortality generally occurred from the third week onward and reachedlevels as high as 30% by the fifth week under standardconditions. At 20 °C, however, 60% mortality levels wererecorded. Differences in spat growth rate, ranging from 37 to 45 mday^–1, were noticed at different seawater flow ratesbut no clear tendency could be discerned. Temperature affected spatgrowth with an increase in size from 24 m day^–1 at15 °C to 35 m day^–1 at 18 °C. Conversely,growth was suppressed at 20 °C (14 m day^–1).For optimal metamorphosis and postlarval development in great scallop, aseawater flow of 4.3 L h^–1 per sieve and a temperatureof 15 °C are recommended.     

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.     

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.     

Seasonal changes in reproductive condition and plasma levels of sex steroids in the blue cod,Parapercis colias (Bloch and Schneider) (Mugiloididae)

Gonad and plasma samples were taken from blue cod captured throughout the reproductive cycle, gonad condition was assessed, and plasma levels of 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20P), testosterone (T), 17-estradiol (E₂) and estrone (E₁) were measured by radioimmunoassay. It was confirmed that spawning occurred over an extended period in late winter and spring, with individual fish being involved in multiple spawning events. Plasma levels of T were bimodal in both sexes with peaks (maximum of 6.0 ng.ml^–1) occurring 2 months prior to, and also during the early part of the spawning period. 17,20P was elevated in males (2.1 ng.ml^–1) in mid-spermatogenesis coinciding with the first T peak (4.9 ng.m.^–1). 17,20P was detectable but not significantly elevated (0.6–1.2 ng.ml^–1) at any sample time in females. E₂ was elevated in mature females (1.0 ng.ml^–1) early in the spawning period but remained at assay detection limits (0.3 ng.ml^–1) at all other sample times. Neither 17OHP nor E₁ were detectable in the plasma of either sex. It is suggested that bimodal increases in sex steroids prior to spawning may be a feature of species with rapid recrudescence.     

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 Zn2+ on Ca2+ uptake by mitochondria and endoplasmic reticulum in permeabilized tilapia gill cells

An intracellular ATP-dependent Ca²⁺ pumping mechanism, distinct from mitochondrial Ca²⁺ accumulation, was identified within tilapia gill cells. Cell suspensions treated with 0.003% saponin, which selectively permeabilizes the plasma membrane, were used to characterize the Ca²⁺ sequentering mechanisms as endoplasmic reticulum and mitochondria and to determine the effect of Zn²⁺ on their Ca²⁺ storing activity. Of the Ca²⁺ taken up by the endoplasmic reticulum, 80% was released by IP₃ (10 mol l^–1). The Ca²⁺ pump of the endoplasmic reticulum was 2.5 times less sensitive to Zn²⁺ (IC₅₀=0.05 nmol l^–1) than was the mitochondrial uptake mechanism (IC₅₀=0.20 nmol l^–1). The results indicate that Ca²⁺ is stored predominantly within the endoplasmic reticulum at 0.1 mol l^–1 and that this storing capacity is seriously attenuated by namomolar concentrations Zn²⁺.