Effects of long-term temperature acclimation on thyroid hormone deiodinase function, plasma thyroid hormone levels, growth, and reproductive status of male Atlantic cod, Gadus morhua.

The recent collapse of the Northwestern Atlantic cod fisheries has coincided with a cooling of water temperatures. During this time the condition factor of cod has been poor. The objective of the present study was to determine the effects of long-term temperature acclimation on growth reproduction and thyroid function in laboratory held Atlantic cod (Gadus morhua). One of the key parameters used to assess thyroid function is the peripheral metabolism of L-thyroxine (T4) by microsomal deiodinase enzymes. Deiodinase function has not been described for gadid fish. T4 outer-ring deiodinating activity (apparent K(m) 1-2 nM) was confined primarily to liver. Its properties resembled those for hepatic T4ORD activity of other teleosts and the mammalian type II deiodinase. The T4ORD activity of cod liver exceeded that of salmonids and could explain the high plasma T3 levels (10-18 ng/ml), which were 2-5 times greater than T4 levels. T4 and T3 inner-ring deiodination was confined mainly to brain. In order to determine the effects of long-term temperature acclimation on cod, somatic growth, reproduction, and thyroidal status were assessed monthly in 400-900-g satiation-fed male Atlantic cod captured in June from the St. Lawrence Estuary and then acclimated from August to the following June under a natural photoperiod at 2-4 degrees C (LT) or 6-10 degrees C (HT). Reproductive status was determined from the gonadosomatic index (GSI), plasma testosterone (T) and 11-ketotestosterone (11-KT) levels, and the appearance of milt; thyroidal status was determined from plasma T4 and 3,5,3'-triiodo-L-thyronine (T3) levels and hepatic T4ORD activity to produce biologically active T3. Testis maturation (high levels of 1 and 11-KT, and milt release) occurred in April and May and was uninfluenced by acclimation temperature. LT cod grew more slowly than HT cod. Differences in body weight were particularly evident from December to February. In conclusion, (i) cod possess outer- and inner-ring deiodinase activities, predominating respectively in liver and brain, and with properties resembling those of other teleosts, (ii) T4ORD activity of liver is unusually high and may account for the high plasma T3 levels in this species, (iii) T4ORD activity tends to increase during periods of increased somatic growth, and (iv) chronic acclimation of male cod to 2-4 degrees C, as opposed to 6-10 degrees C, decreases somatic growth but does alter circulating levels of thyroid hormones and androgens and it does not change the time of sexual maturation.

Gonadotropin action on brook trout sperm duct epithelium: ion transport stimulation mediated by cAMP and Ca2+.

The isolated sperm duct epithelium from the brook trout Salvelinus fontinalis serves as a model for gonadotropin (GtH) action and is the only example of direct GtH stimulation of epithelial ion transport. In response to purified salmonid carbohydrate-rich GtH added to either the luminal or blood side of the epithelium, the duct actively secretes K+ (measured as 86Rb+ fluxes) and actively reabsorbs Na+ (measured by 22Na+ fluxes or as short circuit current, Isc). As a consequence of the ion transport, the seminal plasma has low Na+ concentration and high K+ content that in turn keeps developing sperm quiescent prior to spawning. All of the increase in Na+ transport in response to GtH addition can also be evoked by 1.0 mM db-cAMP + 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), indicating that GtH action on Na+ transport is mediated by cAMP. In contrast, 86Rb+ efflux is only partially stimulated by db-cAMP + IBMX. K+ secretion can be stimulated fully by GtH or with the addition of the Ca2+ ionophore ionomycin (1 microM) in combination with db-cAMP + IBMX. Further, the cAMP-stimulated portion of K+ secretion is resistant to the K+ channel blocker Ba2+ (2.0 mM, added to the luminal side) while the ionomycin-stimulated K+ secretion is Ba(2+)- and quinidine (0.1 mM, luminal side)-sensitive. We conclude that GtH acts by two intracellular messengers in this system. The stimulation of Na+ active reabsorption and a Cl(-)-dependent K+ secretion are both mediated by cAMP. A second, Ba(2+)-sensitive K+ secretion is evoked by intracellular Ca2+ and likely represents a group of Ca(2+)-activated K+ channels on the apical membrane of the epithelium.

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.

Growth enhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone gene construct.

We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

Growth hormone heterogeneity in American plaice pituitaries: isolation, characterization, and partial amino acid sequence.

Growth hormones (GHs) have been isolated from pituitary glands of American plaice (Hippoglossoides platessoides), a marine flatfish, using affinity and gel filtration chromatography, followed by preparative polyacrylamide gel electrophoresis (PAGE). A bioassay based on serum triiodothyronine elevation in immature rainbow trout was used to monitor biological activity. These GHs originate from two molecular mass regions, 42K and less than 33K relative molecular mass (Mr), in their native state. The 42K Mr region yielded two forms of GH, which differ in terms of quantity and net charge as evidenced by native PAGE, a major variant with a relative mobility of (Rf) 0.22 and a lesser variant with Rf 0.28. The less than 33 Mr region has a single GH species with Rf 0.22. Upon sodium dodecyl sulfate-PAGE, without reduction, both GH variants from the 42K Mr region gave Mrs of 21K, while the GH from the less than 33K Mr region was 20K Mr, typical of monomeric vertebrate GHs. The proteins composing the 42K Mr region are proposed as GH dimers since they yield 21K Mr peptides. The less than 33K Mr region contains a GH monomer (20K Mr) in its native state. An amino-terminal amino acid sequence, identical for both the 42K and the 20K Mr Rf 0.22 forms, has good homology with other complete fish GH sequences near their carboxyl-terminal regions (between amino acids 130 and 196). The GH dimers (42K Mr) predominate in the plaice pituitary, contributing 93% of the total, of which 86% gives rise to the Rf 0.22 variant.

Isolation of sockeye salmon growth hormone utilizing serum triiodothyronine enhancement in rainbow trout to monitor biological activity.

Growth hormone (GH) was isolated from sockeye salmon (Oncorhynchus nerka) pituitary glands using established techniques of affinity and gel filtration chromatography, and preparative polyacrylamide gel electrophoresis. The GH activity was followed throughout the fractionation procedure with a bioassay based on the increase of serum triiodothyronine (T3) in rainbow trout (Oncorhynchus mykiss), measured by radioimmunoassay (RIA). Amino-terminal amino acid sequence analysis and subsequent comparison with established GH sequences from other Oncorhynchus sp. were used to confirm the isolation of sockeye salmon GH (ssGH). The bioassay was sensitive to a dose of 55 ng of purified ssGH/g fish. Monomeric GHs, located in the carbohydrate-poor protein fraction, were the only pituitary components that elevated serum T3. Twenty-four hours after GH injection was an appropriate and practical time to blood sample, allowing completion of the bioassay, including RIA, in 3 days. The generic homology, between the source of pituitaries and the bioassay animals used in this study, should permit the bioassay to be useful during GH isolation from pituitaries of all Oncorhynchus sp.

Stimulation of in vitro ovarian estradiol-17 beta synthesis in the rainbow trout by the carbohydrate-poor protein fraction from sockeye salmon pituitary glands.

The role of carbohydrate-poor (Con A I) and carbohydrate-rich (Con A II) pituitary protein fractions, isolated from sockeye salmon (Oncorhynchus nerka), were investigated pertaining to in vitro estradiol-17 beta (E2) production by rainbow trout (Oncorhynchus mykiss) ovarian follicles. During the early vitellogenic phase of the reproductive cycle, using defolliculated ovarian follicle preparations (outer epithelium-thecal layer absent), it was demonstrated that the Con A I fraction was capable of increasing E2 production, in the presence of exogenous testosterone (T) as the substrate. Under similar conditions the Con A II fraction (containing the maturational gonadotropin) was inactive. However the Con A II fraction or T, separately, increased E2 production by intact ovarian follicles, whereas the Con A I fraction did not. A mechanism proposed to explain the regulation of ovarian E2 synthesis involves the Con A I fraction enhancing aromatase activity in granulosa cells permitting an increased conversion of T to E2.

Demonstration of putative membrane and cytosol steroid receptors for 17α, 20ß-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020).

Cytosol from brook trout ovarian follicles (stages 1-3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([(3)H]R5020). The covalently bound cytosol protein had a relative mass of 501,000 Mr following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6-7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 Mr. The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [(3)H]17α,20ß-DHP and PA labelling to [(3)H]R5020. The association constant (Ka) was 2.0×10(7)M(-1) and maximum binding capacity (Nmax) 427 fmoles/mg protein with MIS [(3)H]17α,20ß-DHP.No evidence for nuclear binding of MIS [(3)H]17α,20ß-DHP or PA labelling of [(3)H]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17α,20ß-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.

Transport of 17α,20ß-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis ovarian follicles.

Extremely low levels of the maturation inducing steroid (MIS) 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17α,20ß-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17α,20ß-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.

Gonadotropin stimulation of K+ secretion and Na+ absorption by brook trout (Salvelinus fontinalis) sperm duct epithelium.

The sperm duct epithelium from mature spermiating brook trout (Salvelinus fontinalis) was mounted in vitro to examine control of Na+ absorptive and K+ secretory transport. Na+ absorption (measured as the short-circuit current) and K+ secretion (measured using 86Rb+ as tracer) were stimulated by 3-isobutyl-1-methylxanthine and cyclic AMP while unstimulated tissues had no net ion transport. Purified chum salmon (Oncorhynchus nerka) Con AII carbohydrate-rich gonadotropin produced a rapid, sustained rise in Rb+ secretion and Na+ uptake in a log linear dose-dependent manner. Addition of gonadotropin to either apical (mucosal) or basolateral (serosal) sides evoked the response, but addition to the apical side produced the more rapid effect, indicating that receptors for the hormone are present on both sides of the transporting cells and suggesting that subepithelial tissue may slow the response to serosally added hormone. This is the first indication that gonadotropin may directly regulate ion transport functions of the blood-testis barrier of vertebrates and in this way regulate seminal plasma ionic composition.

Regulation of antifreeze protein production in winter flounder: a unique function for growth hormone.

Salmon pituitary extract and the protein fraction unabsorbed on concanavalin A-Sepharose, the carbohydrate-poor fraction, depressed plasma levels of antifreeze proteins (AFP) when the pituitary fractions were administered to flounder in late fall or winter. The active pituitary protein occurred in the fraction with a mean molecular weight of 25,000. The two major isohormones of growth hormone (GH) were the only biologically active proteins identified from the pituitary. Hypophysectomized flounder synthesize AFP in the spring and the two isohormones of GH suppress the synthesis. The fraction of flounder pituitaries containing putative GH depressed flounder plasma levels of AFP in late fall.

Control of ion transport by the sperm duct epithelium of brook trout (Salvelinus fontinalis).

The sperm duct epithelium of brook trout (Salvelinus fontinalis), mountedin vitro in Ussing-style epithelial chambers actively absorbs Na(+) (measured as the short-circuit current, Isc) and secretes K(+) (measured using(86)Rb(+) as tracer). Dibutyryl-cyclic-adenosine monophosphate (db-cAMP) and 3-isobutyl-1-methylxanthine (IMX) produce a rapid, sustained stimulation of both ion transport processes, but the hormone connected to the response is unknown. Purified sockeye salmon CON A2 gonadotropin (GtH) produces a dose-dependent, rapid and sustained rise in Na(+) uptake and K(+) secretion. The time course, electrophysiological and transport characteristics are similar to those evoked by IMX. Carbohydrate-poor (chum salmon CON A1) GtH is ineffective. Pretreatment of fish with 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-P) significantly increases milt volume but is without effect on resting or stimulated (IMX + db-cAMP) levels of sperm duct ion transport. This is the first indication of a direct, rapid action of GtH on ion transport by the vertebrate blood-testis barrier. The results suggest direct involvement of GtH in control of later stages of sperm maturation.

Hormonal regulation of antifreeze protein gene expression in winter flounder.

The essential features of experiments carried out over the past fifteen years are brought together with new data to formulate a model describing the hormonal regulation of the annual plasma antifreeze polypeptide (AFP) cycle in winter flounder (Pseudopleuronectes americanus). The precise time of onset of antifreeze synthesis in the fall appears to be regulated by photoperiod acting through the central nervous system (CNS) on the pituitary gland. During the summer, growth hormone (GH) blocks transcription of the AFP genes. With the loss of long daylengths in the fall, the CNS inhibits the release of GH allowing AFP gene transcription in the liver to proceed. In the spring GH is again released from the pituitary and AFP gene transcription is blocked.

The presence of 17α,20ß-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 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-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 (Ka) value of 1.394±0.669 10(8)M(-1) (n=7) and low maximum binding capacities (Nmax). The association kinetics of the receptor was second order k+1=2.292×10(6)M(-1) sec(-1). The dissociation rate constant ka was 1.502×10(-2) sec(-1) for the first order dissociation reaction. The Ka=1.526×10(8)M(-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 > 17α-HP > 17α,20ß-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 (1∶10) dilution also bound [(3)H]17α,20ß-DHP, Ka was 8.04×10(7) 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 [(3)H]17α,20ß-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 17α,20ß-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 [(3)H]R5020 after photoaffinity labelling. The same protein also bound [(3)H]17α,20ß-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [(3)H]17α,20ß-DHP, although the molecular weights were different. The blood sample [(3)H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17α,20ß-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.

Molecular cloning and expression of salmon prolactin cDNA.

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.

Adrenocorticotropin- and opiate-like hormones from pituitaries of the sockeye salmon Oncorhynchus nerka.

The pituitaries of vitellogenic sockeye salmon (Oncorhynchus nerka) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate which formed upon the addition of a copious volume of acetone to the extract, designated acid acetone powder, was subjected to salt fractionation and desalting, followed by ion-exchange chromatography on CM-cellulose. An unadsorbed fraction (S-1) and four adsorbed fractions (S-2, S-3, S-4 and S-5) were obtained. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal decapsular cells to produce corticosterone and by their immunoreactivities in an adrenocorticotropin-specific radioimmunoassay. The steroidogenic activities of all fractions, except S-4, were blocked by corticotropin inhibiting peptide. Opiate activity was detected in the fractions by their ability to inhibit the binding of either [3H]naloxone or (D-ala2, D-leu5)-[3H]enkephalin to rat brain membranes. There was a discrepancy in the potencies of the five fractions in the two opiate radioreceptor assays, indicating the presence of opiate peptides with different affinities of binding to the micron- and delta-opiate receptors of the rat brain. There was a separation between adrenocorticotropic and opiate receptor binding activities, suggesting that the activities were due to separate molecular entities.

The cellular location of 1 alpha-hydroxycorticosterone binding protein in skate.

Antiserum to 1 alpha-hydroxycorticosterone binding protein was used to investigate its location in selected tissues of the skate Raja ocellata. Immunofluorescence, using an indirect technique, showed 1 alpha-hydroxycorticosterone binding sites in potential target tissue: gill. chloride cells, rectal gland parenchyma, and a portion of the kidney tubule. The binding protein was not detectable in the ventricle or the intestinal spiral valve but was associated with liver parenchyma and interrenal cells. The intracellular location of the binding protein is apparently tissue specific.