Showing papers in "Fish Physiology and Biochemistry in 1997"

The biology of salmon growth hormone: from daylight to dominance

Abstract: The elucidation of the molecular structure of salmon growth hormone (GH) in the mid-1980's paved the way for a new era of endocrinological research. Establishment of homologous immuno- and receptor-assays have made studies of the secretion, tissue and plasma GH levels, GH turn-over and GH receptor concentrations possible. This overview attempts to summarize the present understanding of the biological roles of GH in salmon. Although the involvement of GH in the regulation of physiological processes throughout the salmon life history has yet to be comprehensively explored, the hormone has already been demonstrated to have several important functions. GH is a principal regulator of somatic growth in salmonids. The growth-stimulating effect of GH is probably integrated with that of insulin-like growth factor I (IGF-I), as in later vertebrates. GH stimulates protein synthesis and improves feed conversion during growth. The hormone also promotes lipid and glycogen breakdown as well as gluconeogenesis, functions which are probably of great importance during starvation when GH levels are seen to increase. During parr-smolt transformation of anadromous salmonids, circulating GH levels appear to be governed by environmental cues. Increasing springtime daylength elevates GH levels, and temperature modulates the photoperiod regulation of GH. The seawater-adapting role of GH during the parr-smolt transformation is complex. In freshwater, GH improves hypoosmoregulatory ability by stimulating branchial Na+,K+-ATPase activity and probably also acts in kidney and intestine. Following seawater entry, GH levels and turn-over increase transiently, probably to further increase seawater tolerance. Accumulating in vitro and in vivo data support the conclusion that GH is involved in the regulation of sexual maturation in salmonids although further studies are needed to establish the exact role of GH in this process. GH increases appetite but it is unclear whether the hormone effects the central nervous system directly, or acts indirectly through metabolic changes. GH increases swimming activity as well as dominant feeding behaviour and diminishes anti-predator behaviour of juvenile salmonids. The GH-induced changes of behavioural patterns imply that there exists an ecological trade-off between high growth rate and long-term survival which may explain why natural fish populations normally grow at sub-maximal rates. Current knowledge indicates that GH is an important and multi-functional hormone in salmon and a central mediator of seasonal changes in physiology and behaviour. The regulatory effects of GH are also of great applied interest as they are likely to affect both product quality in aquaculture and long-term survival of released fish.

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Abstract: Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20β-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g-1 BW week-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.

The role of growth in endocrine regulation of salmon smoltification

Abstract: Keywords: growth, salmon, smoltification, insulin, insulin-like growth factor I (IGF-I), growthhormone, thyroxine, photoperiod, temperature, nutritioncreases hepatic IGF-I mRNA (Cao et al. 1989;Sakamoto and Hirano 1993; Duan et al. 1994) andelevates blood IGF-I levels (Moriyama et al.1994). The majority of IGF-I in salmonid blood isprobably bound to specific binding proteins(IGFBPs), some of which are influenced by GH,insulin and nutrition (Kelley et al. 1992; Siharathand Bern 1993). The IGFBPs in blood and in tis-sues surrounding target cells undoubtedly play asignificant role in modifying the action of IGF-Iin fish, as they do in mammals, although this islargely unexplored in the context of salmon smol-tification. IGF-I inhibits GH release by negativefeedback, as shown

Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss)

Abstract: This study examined how muscle metabolic organization varied during an annual cycle in which rainbow trout (Oncorhynchus mykiss) were held in outdoor holding ponds in which they were exposed to natural changes in temperature (range 0.2 to 15.6°C) and photoperiod. We examined the activities of glycolytic and mitochondrial enzymes in red and white muscle to evaluate whether trout enhance their capacity for lipid and carbohydrate oxidation during cold-acclimization. When assayed at habitat temperature, the enzyme activities generally increased in spring to reach a maximum in summer followed by a decrease in the fall. This led to significantly higher activities at warm than cold periods for all enzymes measured in red muscle and all but one in white muscle. The activities at 10°C provided little evidence for compensatory adjustments of aerobic capacity. Particularly in red muscle, enzyme levels at 10°C were generally lower during cold than warm periods. The variation of enzyme activities throughout the cycle was not due to changes in protein concentration, as the same responses were observed when activities were expressed per g wet mass or per mg protein. Although the aerobic capacity did not increase with cold-acclimatization, the relative capacity for lipid oxidation was higher in winter than in summer trout. In contrast, the relative capacity for aerobic glycolysis was higher in summer than in winter trout. Thus, the metabolic capacities of trout muscle undergo seasonal reorganization.

Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

Abstract: The impact of 17β-estradiol (E2) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17β-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E2 and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E2-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E2- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na+,K+-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E2 and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na+,K+-ATPase activity, relative α-subunit Na+,K+-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E2- and 4-NP-treated fish was strongly correlated with a decreased gill Na+,K+-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17β-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E2 activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.

Photoperiodic manipulations of the reproductive cycle of sea bass (Dicentrarchus labrax) and their effects on gonadal development, and plasma 17ß-estradiol and vitellogenin levels

Abstract: The annual profile of plasma vitellogenin (VTG) and 17s-estradiol (E2) levels, as well as gonadal development and spawning characteristics were investigated in captive female sea bass (Dicentrarchus labrax). Endocrine and gonadal changes were studied in fish reared under natural conditions or exposed to manipulated photothermal cycles. In natural conditions of photoperiod and temperature, sea bass spawned from February through March (East coast of Spain, 40°N 0°E). One or two months of constant long-days (15L/9D) in a constant short-day photoperiod regime (9L/15D) all-year-round, given early in the year (March and March–April), advanced spawning by 3 months. The same treatment applied later in the year (September–October) delayed spawning by 1 month, compared to controls. In all groups, changes in plasma VTG levels were correlated with E2 levels, oocyte growth and spawning time. In control females, VTG was low (<100 ng ml-1) during the summer, until its first surge in plasma 4 months before the beginning of spawning. The VTG (3.1 ± 0.3 mg ml-1) and E2 (4.1 ± 0.5 ng ml-1) levels showed a single annual peak during late vitellogenesis, the time of the highest proportion of vitellogenic oocytes in the ovary. Constant high levels of VTG (1–1.4 mg ml-1) and E2 (1.6–1.9ng ml-1) were maintained during the entire spawning time, together with the presence of vitellogenic oocytes, suggesting the existence of several waves of oocyte growth in the ovary and thus, several spawns per female. Endocrine profiles and oocyte development in fish exposed to constant photoperiods were similar to controls, but were shifted in time in relation to the displacement of the spawning time. In the fish showing advanced spawns, the duration of the gametogenic proces was compressed when compared to controls. The differences observed in the evolution of the reproductive-related factors in the advanced groups, which were exposed to a reduction in temperature to 15°C, suggest an influence of the temperature in the early stages of the reproductive cycle in sea bass.

The role of cortisol in amino acid mobilization and metabolism following exhaustive exercise in rainbow trout (Oncorhynchus mykiss Walbaum)

Abstract: The role of cortisol in the mobilization and metabolism of amino acids following exhaustive exercise in rainbow trout (Onorhynchus mykiss) was investigated. Plasma cortisol levels were elevated for 4h following exercise and 6, of a total of 22 amino acids, showed significant responses. In white muscle, alanine and glutamine were elevated immediately after exercise through to 4h, whereas glutamate, and the branched-chain amino acids (BCAA), isoleucine, leucine and valine, all decreased over this time. In plasma all of these amino acids increased from 2-4h post-exercise, while in liver, alanine and glutamine increased, glutamate did not change and the BCAAs declined over this time. Blockade of the post-exercise elevation in plasma cortisol with either metyrapone (cortisol synthesis inhibitor) or dexamethasone (cortisol release blocker) prevented the changes in tissue amino acid levels. This study demonstrates that cortisol can act rapidly (within minutes to hours) to alter amino acid metabolism in fish. A model is presented to explain the action of cortisol on amino acid metabolism.

Plasticity of the properties of mitochondria from rainbow trout red muscle with seasonal acclimatization

Abstract: Cold-acclimation of rainbow trout brings only limited changes in muscle metabolic capacities, but marked modifications in membrane composition. Thus, we examined whether the functional properties of mitochondria from trout red muscle were modified by seasonal temperature acclimatization. Mitochondria from fall-acclimatized trout had higher maximal capacities (state 3 rates) for the oxidation of pyruvate and acyl carnitines at 12 and 20 °C than mitochondria isolated from summer-acclimatized trout. For these substrates, the increased oxidative capacity completely compensated for the seasonal drop in temperature. Pyruvate and palmitoyl carnitine were consistently the preferred substrates, while decanoyl and octanoyl carnitine were oxidized at higher rates than glutamine, particularly in fall trout. State 4 rates of oxygen uptake (obtained when all ADP has been converted to ATP) differed less among substrates, but varied seasonally. State 4 rates at 12 and 20 °C were higher in mitochondria isolated from fall than summer trout. At low temperatures, the Q10 of both maximal and state 4 rates of substrate oxidation tended to be higher for mitochondria from fall trout. The apparent Arrhenius activation energy (Ea) for mitochondrial pyruvate oxidation was higher in fall than summer trout whereas the Ea’s for palmitoyl carnitine and decanoyl carnitine oxidation did not change. The fatty acids of mitochondrial phospholipids from fall trout were more polyunsaturated than those from summer trout, with 12% more double bonds occurring than in summer trout, suggesting that membrane restructuring may be involved in the observed compensatory responses.

Sublethal effects of a carbamate pesticide on pheromonal mediated endocrine function in mature male Atlantic salmon (Salmo salar L.) parr

Abstract: The sublethal effect of the carbamate pesticide carbofuran on the priming pheromonal system of Atlantic salmon (Salmo salar L.) was investigated. Previous studies have demonstrated that ovulated female salmon release a priming pheromone in their urine (considered to be an F-type prostaglandin) which is subsequently detected by mature male salmon and results in increased levels of plasma sex steroids and expressible milt. In the present study, electrophysiological recordings from the olfactory epithelium of mature male salmon parr indicated that the responses to prostaglandin F2α (PGF2α) were significantly reduced at nominal concentrations of carbofuran as low as 1.0 µg l-1, and the threshold of detection was reduced 10-fold. A 5 day exposure to carbofuran significantly reduced the ability of male parr to respond to PGF2α stimulation. The priming effect of PGF2α on milt and plasma 17,20β-dihydroxy-4-pregnen-3-one levels were abolished at water concentrations at and above 2.7 µg l-1. In addition, the priming effect of PGF2α on plasma testosterone and 11-ketotestosterone concentrations was abolished at water carbofuran concentrations above 6.5 µg l-1. Exposure to similar concentrations of carbofuran also resulted in a reduction in the levels of free and glucuronidated steroids in the bile of PGF2α primed male parr. The effect of carbofuran on the priming response did not appear to be due to a direct effect on the testes, since the ability of testes to respond to pituitary extract stimulation in vitro was not impaired in carbofuran-exposed males. Carbofuran appeared to reduce significantly or abolish the priming pheromonal system in mature male parr by directly affecting the ability of the olfactory system to detect PGF2α, although the toxicological mechanism involved is as yet unknown. The results are therefore discussed in relation to the possible sublethal effects of carbofuran on reproduction in the Atlantic salmon.

Recovery from nitrite-induced methaemoglobinaemia and potassium balance disturbances in carp

Abstract: The ability of carp to recover from nitrite-induced methaemoglobinaemia and disturbances in potassium balance and cell volume was studiedin vivo andin vitro. Nitrite accumulated to a plasma concentration of 3 mM during 2 days of nitrite exposure was eliminated from the plasma within 2–3 days in clean water. The nitrite-induced methaemoglobinaemia disappeared after 3 days of recovery. During nitrite exposure, K+ was lost from the red blood cells (RBCs) and from skeletal muscle tissue, which led to reduced cell volume and an extracellular hyperkalaemia. Extracellular [K+] rose less than predicted if lost K+ had remained in the extracellular space, suggesting further transport of K+ to the environment. The intracellular K+ and water content were restored after few days of recovery in clean water, but this was paralleled by development of an extracellular hypokalaemia. This shows that intracellular K+ balance was reestablished at the expense of the extracellular compartment, and supports that an overall K+ deficit resulted from K+ loss to the environment during nitrite exposure. Ventricle tissue differed from skeletal muscle and RBCs by not loosing K+ and by having increased sodium and water contents during nitrite exposure. These changes were corrected by recovery in nitrite-free water. In vitro addition of nitrite to blood with low O2 saturation induced metHb formation and RBC K+ efflux. Subsequent reduction of metHb to functional Hb was similar in blood with low and high O2 tension. A net re-uptake of K+ was observed only in RBCs with low O2 saturation and when metHb reached low values.

Epidermal proteases of the Japanese eel

Abstract: A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch2Cl, chymostatin, CdCl2, CuCl2, HgCl2 and ZnCl2 inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.

Distinct expression of GnRH genes in the red seabream brain

Abstract: This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.

Binding characteristics and regulation of the 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor on testicular and sperm plasma membranes of spotted seatrout (Cynoscion nebulosus)

Abstract: The binding characteristics of 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) to plasma membranes prepared from the testes and sperm of spotted seatrout (Cynoscion nebulosus) were investigated using a filtration method to retain the bound 20β-S. A single class of high affinity (Kd = 17.9 nM), low capacity (Bmax = 0.072 nM g-1 testes) binding sites was identified by saturation and Scatchard analyses on testicular membranes of spermiating spotted seatrout. A corresponding receptor (Kd = 22.17 nM, Bmax = 0.00261 nM ml-1 milt) was also detected in spermatozoan membrane preparations. The rates of 20β-S association and dissociation were rapid, both had Thalfs of less than 1 min. Competition studies indicated that the receptor was highly specific for 20β-S. 17,20β-dihydroxy-4-pregnen-3-one, which had the highest affinity of the other steroids tested, had a relative binding affinity (RBA) of 14.3%. Progesterone, 11-deoxycortisol and testosterone competed with an order of magnitude less affinity (RBA's of 7.4, 1.8 and 1.1%, respectively). Estradiol displayed low affinity for the receptor (RBA = 0.4%) and cortisol did not cause any displacement at 1000-fold excess concentration. Specific 20β-S receptor binding was detected in plasma membranes from testes of both spermiating and non-spermiating seatrout and on spermatozoa. Prolonged incubation of testicular fragments from a spermiating fish with gonadotropin (15 IU ml-1 human chorionic gonadotropin) or forskolin (10 µM) caused a 2–3 fold increase in membrane receptor binding. Previous studies have shown that gonadotropin-induced upregulation of the 20β-S plasma membrane receptor in seatrout ovaries is required for the oocytes to become responsive to 20β-S and undergo final maturation. The existence of a 20β-S membrane receptor on sperm and its upregulation in the testes by gonadotropin raises the possibility that final maturation of spermatozoa in male seatrout may be regulated by a similar mechanism.

Reproduction related immunoglobulin changes in rainbow trout

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

The ovarian regulation of ovulation in teleost fish

Abstract: Ovulation is the release of a mature oocyte from its follicle wall enclosure in the ovary. This process requires the separation of the oocyte from the granulosa layer, the rupture of the follicle wall and the active expulsion of the oocyte through the rupture site. Results of experiments on various vertebrates, including fish, have shown that the control of these processes may involve the cooperative action of a number of ovarian regulators including proteases, protease inhibitors, progestational steroids, eicosanoids, catecholamines and vasoactive peptides. We have used two teleost models, the brook trout (Salvelinus fontinalis) and the yellow perch (Perca flavescens) to study the mechanism and control of ovulation in fish. Using subtractive cDNA cloning, a family of ovarian and ovulation specific mRNAs (“TOPs” = trout ovulatory proteins) was isolated from the brook trout ovary. These mRNAs have not previously been observed in the ovary of any vertebrate species; however, the proteins they code for have significant sequence homology to a group of mammalian protease inhibitors called antileukoproteinases. These inhibitors have been isolated from several mammalian mucosal tissues and their function may be to protect the mucosal lining from nonspecific degradation by proteases released from infiltrating leukocytes. The ovarian proteins encoded by the TOP mRNAs have now been characterized by Western blotting using antibodies derived against recombinant TOPs. Given the similarity of TOPs to antileukoproteinases, one function of TOPs may be to regulate proteolysis at the time of ovulation. In yellow perch, the maturational steroid, 17α,20β-dihydroxy-4-pregnen-3-one (17,20-PG), stimulates both germinal vesicle breakdown and ovulation in vitro. The stimulation of ovulation can be blocked by indomethacin, a prostaglandin endoperoxide synthase inhibitor. Thus, it appears that 17,20-PG acts through the production of an eicosanoid that is most likely a primary prostaglandin. This hypothesis is further supported by the observations that (1) a direct correlation exists between indomethacin levels that block ovulation and those that block primary prostaglandin synthesis in the ovary; (2) ovulation can be restored in indomethacin-blocked incubates with primary prostaglandins; (3) PGF levels increase at the time of ovulation in incubations of yellow perch follicles stimulated with 17,20-PG; and (4) the stimulation of PGF by steroids in the ovary is specific for 17,20-PG. Finally, 17,20-PG-stimulated ovulation and follicular prostaglandin synthesis requires the close interaction of extrafollicular tissue and other follicle wall layers.

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

Abstract: 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.

Sodium dependent ion transporters in trout gills

Abstract: 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 Km 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.

Effect of a dietary phospholipid supplementation on growth and fatty acid composition of European sea bass (Dicentrarchus labrax L) and turbot (Scophthalmus maximus L) juveniles from weaning onwards.

Abstract: Two 40-day feeding trials using extruded diets were conducted to assess the effect of a dietary phospholipid (PL) supplementation on growth, survival and fatty acid composition of European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) from weaning onwards. Two dietary treatments (FO and PL) were tested; both had an identical extruded basis (92.5% total diet weight) coated with a different lipid fraction (7.5% total diet weight). Diet PL contained 2% egg yolk PL (69% pure). In diet FO the PL was replaced by hydrogenated coconut oil. The isolipidic diets contained an equal amount of fish oil ethyl esters providing 1.6% (% diet dry weight) of n-3 highly unsaturated fatty acids (HUFA). A diet water stability test showed no effect of the PL supplementation on the leaching of the dietary fatty acids. In both fish species weight, but not survival, significantly increased as a result of PL supplementation. Weaning onto the experimental diets resulted in similar changes in the relative percent levels of fatty acids in both species. In general, the percentage of saturated fatty acids levelled off after a rapid increase, while monoenes increased after an initial decrease. Total n-3 polyunsaturated fatty acids (PUFA) decreased and total n-6 PUFA remained almost constant. The major effect of the dietary PL on fish fatty acid composition was a 50% increase in n-6 and n-3 HUFAs compared to the PL-free FO diet. The rise in n-6 HUFA may have reflected the higher moiety in the dietary PL. On the other hand this was not the case for the n-3 HUFA since they represented only low levels in the PL fraction (0.1%) compared to that provided by the ethyl esters (1.6%) suggesting a more efficient incorporation of the PL n-3 HUFA than of the ethyl ester n-3 HUFA. A second hypothesis is that the dietary PL may have favored the incorporation of the dietary ethyl ester n-3 HUFA.

Proton pumps in the fish gill and kidney

Abstract: The proton pump or vacuolar type H+-ATPase is an oligomeric protein responsible for electrogenic H+ secretion in a variety of acid-secreting epithelia Recently, the proton pump was identified in both the gill and kidney of freshwater-adapted rainbow trout (Oncorhynchus mykiss) Using immunocytochemistry, H+-ATPase has been localized in the pavement cells and chloride cells of the lamellar epithelium During periods of internal acidosis, there is a marked increase in the expression of the branchial proton pump as identified by Western analysis, immunocytochemistry and in situ hybridization This augmented expression of proton pumps occurs concomitantly with a marked increase in branchial acid excretion and Na+ uptake Immunocytochemical studies suggest that the pavement cell, rather than the chloride cell, is the predominant site of acid excretion during periods of acidosis These findings are consistent with the notion that in freshwater teleosts, Na+ uptake and H+ excretion are linked via the coupling of the electrogenic proton pump to apical membrane Na+ channels This mechanism may be controlled by hormones including cortisol and/or growth hormone The fish kidney plays an important role in regulating acidosis via the re-absorption of filtered HCO3 - Recently, we have demonstrated using Western analysis and immunocytochemistry, the presence of proton pump in rainbow trout kidney and observed increased H+-ATPase expression during respiratory acidosis These new findings suggest a role for the renal proton pump in acid-base regulation

Fast growth, protein turnover and costs of protein metabolism in yolk-sac larvae of the African catfish (Clarias gariepinus).

Abstract: In fish larvae the costs of rapid growth may be accommodated by a decrease in the rate of protein turnover or by a reduction in the costs of protein synthesis. Protein growth, synthesis and degradation were measured in yolk-sac larvae of Clarias gariepinus and the costs of protein synthesis and protein growth were estimated. Growth rates were over 100% protein weight day-1. Protein synthesis retention efficiency (retained protein per unit of synthesis) was estimated to be 69.6%, a value comparable to that of larger fish. The larvae used 43% of their oxygen consumption for protein synthesis. Nevertheless, protein synthesis costs were close to theoretical minima. Therefore, the high growth rates of catfish yolk-sac larvae seem to be possible through minimisation of the costs of protein synthesis. These low costs are associated with high rates of protein synthesis (138%protein weight day-1), and elevated RNA concentrations (107 µg RNA mg-1protein), which together suggest very high RNA efficiencies (12.9 g protein synthesized g-1RNA day-1).

Dietary spermine supplementation induces intestinal maturation in sea bass (Dicentrarchus labrax) larvae

Abstract: Sea bass (Dicentrarchus labrax) larvae were fed microparticulated compound diet containing 0 (FP0), 0.10 (FP10) and 0.33% (FP33) of a polyamine, spermine, from day 20 to day 38. LP group was fed live prey. This group exhibited the highest growth and survival. The addition of spermine did not lead to growth enhancement. A 33% survival improvement was obtained in FP33 group compared to FP0 group. The spermine addition affected the activity of pancreatic enzymes, trypsin, chymotrypsin and amylase, during larvae development. This non specific effect suggested that the action of spermine would be mediated by hormones. In the intestine, the FP33 group exhibited from day 31 higher activities of brush border membrane enzymes (leucine aminopeptidase and alkaline phosphatase) and lower level in a cytosolic enzyme (leucine-alanine peptidase) compared to FP10 and FP0 group. The diet containing the highest spermine level induced an enzymatic profile similar to that obtained in LP group and characteristic of a mature enterocyte. The initiation of enterocyte maturation at a proper development stage was associated to the survival improvement observed in FP33 group.

Environmental control of fish reproduction: a different perspective

Abstract: Nearly all studies on the environmental control of fish reproduction have, to date, focused on the roles played by various parameters of the natural environment, such as changes in water temperature and photoperiod. While these factors undoubtedly still play significant roles, other factors, which have arisen as a consequence of man's activities, also play major roles in at least some aquatic environments. One such factor is pollution; whereas attention is usually focused primarily on severe pollution and the fish mortality that can result, recently some emphasis has been given to the more subtle, but potentially serious, consequences of less severe pollution. Many freshwater habitats, particularly rivers in urban areas, now receive substantial volumes of effluent from various sources (both industrial and domestic). This effluent can contain chemicals capable of mimicking endogenous hormones, and hence has the potential to disrupt endocrine-mediated processes such as reproduction. This paper is concerned with the identity of these endocrine-disrupting chemicals, their mechanisms of action, and the effects they have on reproduction, both in wild fish and in fish maintained in the laboratory.

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Abstract: Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-14C]18:2n-6 and [1-14C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while Δ6 desaturase activity was increased significantly with cell age, Δ5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.

Mismatch between patterns of circulating and testicular androgens in African catfish, Clarias gariepinus

Abstract: 11-Ketotestosterone (11-KT) is an important plasma androgen in male African catfish. The quantitatively predominating androgen produced by the testis, however, is 11β-hydroxyandrostenedione (OHA). Our working hypothesis to explain this mismatch assumed that OHA is converted to 11-KT at extratesticular sites. First, we examined the in vivo metabolism of [3H]-OHA in mature males after sham-operation or removal of either the testes (TC), the seminal vesicles (SVC), or both (TSVC) by analysing the pattern of circulating [3H]-androgens two hours after intravenous injection of [3H]-OHA. [3H]-OHA was converted to [3H]-11-KT to the same extent in all groups, indicating that neither ablation of testes nor of seminal vesicles, or both attenuates this conversion. We then examined the in vitro metabolism of [3H]-OHA by several types of tissues. Liver and seminal vesicle tissue were found to produce significant amounts of [3H]-11-KT. The conversion capacity in vivo was assessed by injecting TSVC-castrated males with increasing doses of radioinert OHA, followed by the quantification of OHA and 11-KT plasma levels. Saturation of the conversion capacity was not reached but the 11-KT production capacity is at least 80 ng per ml of plasma per hour. Moreover, liver fragments were tested for their OHA to 11-KT conversion capacity in vitro using increasing concentrations of radioinert OHA. The 11-KT producing increased with time and OHA concentration. The production rate was 90 pg 11-KT mg-1 liver h-1. Considering the results of the surgical experiments and the fact that the total hepatic mass by far exceeds that of the seminal vesicles, we conclude that the hepatic conversion is of primary relevance in vivo.

Long-term starvation in the European eel: general effects and responses of pituitary growth hormone-(GH) and somatolactin-(SL) secreting cells

Abstract: European eels reaching the silver stage stop feeding in freshwater and during their spawning migration to the Sargasso sea (6000 km at least in the Ocean). The total duration of this exceptional fast is not well known. Few data are available on the general condition and the endocrine responses to starvation of migrating eels. In this study male (silver) and female (yellow and silver) eels were kept in freshwater without food for various lengths of time. Animals were killed after 7 months to 3 or 4 years. After a gradual decrease, the final body weight was reduced by 84% in males (52 months) and 67–69% in females (up to 4 years). The condition factor (K) followed a parallel curve. In the pituitary gland, GH cells were hypertrophied and highly stimulated. Their cross-sectional area was negatively correlated to K. Large GH cells remained well immunostained with an anti-eel GH serum after 7 to 12 months of starvation. In the leanest eels, the immunostaining was often reduced and many GH cells appeared degranulated, suggesting a low hormonal storage. In contrast, SL cells were reduced in size and number in the anterior half of the neurointermediate lobe (NIL), but showed a more heterogeneous picture in the caudal portion. GH and SL cell activities seemed to be negatively correlated in starved fish and controlled by different mechanisms. GH appears to play a major role in the long-term survival of fasted eels; SL does not seem to be involved.

Oxygen consumption and ventilatory reflex responses are influenced by dietary lipids in sturgeon

Abstract: The effects of one year's feeding with diets enriched either in highly unsaturated fatty acids of the w3 series (w3 HUFA) (fish oil-supplemented diet, FOD) or in saturated fatty acids (SFA) (coconut oil-supplemented diet, COD) on fatty acid composition of tissue lipids, on patterns of resting oxygen consumption and on responses to hypoxia and hypercapnia were investigated in the Adriatic sturgeon (Acipenser naccarii) FOD sturgeon had higher levels of w3 HUFA in liver and muscle lipids than COD fish, which had higher levels of SFA A frequency distribution of instantaneous oxygen uptake rates (M]O2), as measured every 10 min for 8 h, revealed a different pattern of resting oxygen consumption between the two groups The FOD sturgeon consumed oxygen in a narrow range of low rates, with a lower mean M]O2 than COD sturgeon, which showed a wide range of more elevated rates FOD sturgeon had a lower opercular pressure amplitude than COD fish in normoxia Exposure to three levels of hypoxia PO2s = 108±02; 66±02 and 46±02 kPa) or mild hypercapnia (PCO2 = 10±02 kPa) did not affect ventilation in FOD fish but elicited hyperventilation in COD animals Mild hypoxia (PO2 = 108±02 kPa) and hypercapnia caused less reduction in blood oxygen content in FOD as compared with COD sturgeon The effects of adding vitamin E supplements to the diets was investigated; groups fed vitamin E supplements had elevated M]O2 and hyperventilated in hypoxia The data indicate that dietary fatty acid composition influences resting M]O2 in sturgeon and that this influences the regulation of ventilation and blood O2 levels in hypoxia and hypercapnia The low resting M]O2 of fish fed w3 HUFA supplements (the FOD group) obviated the need for hyperventilation in hypoxia or hypercapnia, thereby making them less sensitive to these stresses than sturgeon fed SFA (COD group) or sturgeon fed either diet supplemented with vitamin E

Estrogen receptors in the brain-pituitary complex and the neuroendocrine regulation of gonadotropin release in rainbow trout

Abstract: In all vertebrates, steroid feedback is one of the mechanisms by which the brain is constantly kept informed on the physiological status of the periphery. It is, therefore, a very efficient system of synchronization between the different actors of the brain-pituitary-effector axis, particularly in the context of reproduction. The central effects of steroids are mediated by specific receptors belonging to the superfamily of intracellular receptors and acting as ligand dependant transcription factors to modulate the expression of certain target genes. In order to investigate the molecular events underlying steroid actions in the brain/pituitary complex, it is necessary, as a first step, to locate precisely steroid expressing cells in the brain/pituitary complex and to identify the phenotype of these cells on the basis of the neurohormone(s) or hormones they produce. This paper will review recent data obtained in our laboratory regarding the distribution and regulation of expression of estrogen receptors in the brain and pituitary gland of the rainbow trout (Oncorhynchus mykiss), using both in situ hybridization or immunohistochemistry. In addition, information concerning the phenotypic identification of cells expressing these receptors is presented, especially with respect to the main neuroendocrine systems controlling reproduction. The functional meaning of these data are discussed in the context of the reproductive physiology of trout.

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Abstract: Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I β and II β mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.

Pubertal development of male African catfish,Clarias gariepinus. In vitro steroidogenesis by testis and interrenal tissue and plasma levels of sexual steroids

Abstract: In fish, sex steroids initiate and/or accelerate the maturation of the brain-pituitary-gonad axis. In order to obtain information on the steroid milieu during the pubertal development of male African catfish, we have monitored the conversion of [3H]-pregnenolone and [3H]-androstenedione by testis and [3H]-pregnenolone by interrenal tissue fragmentsin vitro. Pubertal development occurs between two and six months of age. Testicular development proceeds through four main stages that are characterised histologically by the presence of spermatogonia (stage I), spermatogonia and spermatocytes (stage II), spermatogonia, spermatocytes and spermatids (stage III), and all germ cells including spermatozoa (stage IV). 11β-Hydroxyandrostenedione and cortisol were the main products of testes and interrenal tissue, respectively, in all stages of the pubertal development; a change in the steroidogenic pattern was not observed during this period. The interrenal tissue displayed no significant conversion of [3H]-pregnenolone to 11-oxygenated androgens. Blood plasma was analyzed for the presence of five androgens; testosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, androstenetrione, and 11-ketotestosterone. 11-Ketotestosterone was the quantitatively dominating androgen in the circulation at all stages of development, which was more pronounced in stages III and IV. The obvious differences between thein vitro andin vivo results, namely 11β-hydroxyandrostenedione being the main testicular productvs. 11-ketotestosterone dominating in the blood, may indicate that 11β-hydroxyandrostenedione is converted to 11-ketotestosterone at extratesticular sites.

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Abstract: Changes in the levels of plasma vitellogenin (Vg), estradiol (E2) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 µg kg−1 body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E2 and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml−1) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E2 (0.62 ± 0.13 ng ml−1 and T (0.33 ± 0.30 ng ml−1), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 µg ml−1 during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml−1 for E2 and 0.1 ng ml−1 for T); cGTH treatment did not significantly increase plasma E2 level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml−1 and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml−1 after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E2 to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10−6 to 10−5 M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E2 receptor.In vivo, androgens given alone at 50 µg kg−1 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E2-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E2.