Salmon Prolactin Research Articles

Primary structure of chum salmon prolactins: Occurrence of highly conserved regions

The complete amino acid sequence of prolactin from the pituitaries of salmon ( Oncorhynchus keta) has been determined. Salmon prolactin comprised two variants, I and II, which were separated by reverse-phase high-performance liquid chromatography. Each variant was reduced, S-carboxymethylated, and then cleaved with cyanogen bromide and enzymes. The resulting fragments were separated by reverse-phase high-performance liquid chromatography, as well as gel filtration, and subjected to sequence analysis by the dansyl-Edman method. Both variants contain 187 amino acid residues with two disulfide linkages at residues 46–160 and 177–187, lack a linkage in the N-terminal portion of mammalian prolactins, and differ from each other by the replacement of only four amino acid residues. Salmon prolactin (sPRL) shows 31% sequence identity with ovine prolactin. Moreover, four restricted regions, i.e., sPRL (3–21), (46–60), (68–83), and (160–178), encompass this significant conservatism between the teleost and the mammalian hormone, with identities of 47, 87, 62, and 68%, respectively. Such considerable identity between these distant phylogenic species strongly suggests that these regions may be responsible for the biological activity of prolactin.

Development and validation of a salmon prolactin radioimmunoassay

A highly specific radioimmunoassay (RIA) for the measurement of prolactin (PRL) in the plasma and pituitary of salmonid fishes was developed using a rabbit antiserum to chinook salmon ( Oncorhynchus tschawytscha) PRL. The PRLs purified from chinook salmon and chum salmon ( O. keta) pituitaries showed exactly the same competitive inhibition curves in the RIA, regardless of iodination of either hormone. The displacement curves for pituitary extracts and plasma from several salmonids, including chum, coho, and amago salmon, rainbow trout, and Japanese charr, were parallel to the salmon PRL standard, whereas those from the eel, goldfish, carp, and tilapia showed negligible cross-reactivity. Negligible cross-reactivity was also seen with plasma from hypophysectomized rainbow trout or coho salmon. None of the mammalian PRL or growth hormone (GH) preparations, bullfrog PRL, or presumptive chum salmon “gonadotropin” and eel “PRL” cross-reacted in the PRL RIA. Presumptive chum salmon GH showed less than 0.05% cross-reactivity. The RIA sensitivity was less than 0.1 ng of the salmon PRL standard per milliliter. The immunoreactive reactive plasma PRL levels in mature chum salmon were below 1 ng/ml in seawater. The plasma PRL in females increased to about 8 ng/ml 1 day after transfer to fresh water, and high levels (2–4 ng/ml) were maintained during 3–7 days after the transfer. In contrast, when males were transferred to fresh water, an increase in plasma PRL was seen only 1 day after the transfer. A significant decrease in plasma osmolality was observed in both males and females after transfer to fresh water. No change was observed either in plasma PRL or osmolality, when fish were transferred from seawater to seawater.

Plasma prolactin level during transfer of rainbow trout ( Salmo gairdneri) and Atlantic salmon ( Salmo salar) from fresh water to sea water

Hypoosmoregulatory activity of two salmonid species (one smoltifying and one non-smoltifying) was studied by direct transfer of these fish into sea water. Two plasma parameters were followed during the experiments: osmotic pressure and sodium level. Using an homologous radioimmunoassay for salmon prolactin, the plasma level of this hormone was also measured. Seawater adaptation of rainbow trout appeared to be difficult, as shown by the high increase of osmotic pressure and sodium level. However, after 2 weeks the fish were able to regulate their hydromineral balance. Prolactin level dropped within 24 h after transfer and remained significantly lower than in the freshwater fish during the experiment. In contrast, Atlantic salmon smolts transferred to sea water were able to control their hydromineral balance within a few days after transfer, as shown by plasma osmotic pressure and sodium level. Moreover, prolactin level was not significantly different between freshwater and seawater fish from day 4 to the end of the experiment. Thus, by measuring plasma prolactin level, it was possible to confirm the two patterns of seawater adaptation presented by Atlantic salmon smolt and rainbow trout. These results are discussed in terms of a possible role of prolactin in salmonid osmoregulation.

Mode of Action of some Stimulants of the Hatching Enzyme Secretion in Fish Embryos*: (hatching/secretion/fish embryo/Medaka).

Mode of action of two stimulants of the hatching enzyme secretion, electric current (AC) and potassium cyanide, was analyzed by applying them to Medaka embryos in the presence or absence of suppressants of nervous system-mediated secretion, tetrodotoxin or MS-222. Electric current (AC) stimulated the secretion of the hatching gland of the embryos that had been treated with these suppressants, while potassium cyanide did not. These results strongly suggest that electric current acts as a stimulant of hatching enzyme secretion directly on the gland cell itself, while potassium cyanide stimulates the secretion indirectly, probably through nervous system of the embryo. In the present experiments, it was also shown that Ca2+ and ionophore, X-537A, when applied directly to the hatching gland extracellularly, induced a marked secretion-associated morphological change of the gland cells instantaneously. However, it was found that chum salmon prolactin did not induce the secretion-associated morphological changes in the hatching gland cells when it was applied directly to the gland cells in situ or indirectly through embryonic circulation.

Calcium fluxes and in vitro [ 3H]prolactin synthesis and release from the rostral pars distalis of coho salmon ( Oncorhynchus kisutch)

1. 1. Calcium fluxes, as measured by 45Ca and [ 3H]prolactin synthesis and release from the rostral pars distalis of coho salmon, were examined in vitro. 2. 2. db-cAMP tended to show stimulated synthesis but not release of [ 3H]prolactin. 3. 3. Medium of high osmotic pressure was shown to inhibit release of [ 3H]prolactin. 4. 4. In both of these treatments there was an association between [ 3H]prolactin synthesis with net 45Ca accumulation in the tissue. 5. 5. Chlorpromazine at two concentrations seemed to stimulate both synthesis and release of [ 3H]prolactin with less 45Ca accumulation than control tissues. 6. 6. An efflux study showed an increased efflux from the tissue under the db-cAMP treatment and that this effect is reversible when db-cAMP treatment ends. 7. 7. These results suggest that coho salmon prolactin release may not utilize calcium as suggested by stimulation-secretion coupling theory since: (i) both media of high osmotic pressure and db-cAMP show a net 45Ca accumulation without [ 3H]prolactin release, (ii) chlorpromazine can show [ 3H]prolactin release without net 45Ca accumulation; and (iii) efflux studies using db-cAMP indicate high intracellular calcium under a treatment shown here to have inhibited release.

Fish growth hormone enhances peripheral conversion of thyroxine to triiodothyronine in the eel ( Anguilla anguilla L.)

In the eel, a low dose of tilapia growth hormone (tGH) (45 ng/g body wt), like ovine GH (oGH), induces a decrease in plasma thyroxine and a concomitant increase in plasma triiodothyronine, which result from a stimulation of peripheral conversion of thyroxine to triiodothyronine. Salmon prolactin (sPrl), unlike ovine Prl (oPrl), has no such action. Recognition of this specific action of growth hormone (GH) on production of active thyroid hormone (T 3) opens up a new approach to the problem of the action of both hormones (GH, T 3) in growth and in seawater adaptation of fish.

Purification and biological characterization of chinook salmon prolactin

Prolactin from chinook salmon pituitaries was purified by acid acetone extraction, saline precipitation, chromatofocusing, and gel filtration. This procedure allowed us to recover highly purified prolactin as demonstrated by the presence of a single NH 2-terminal amino acid and a single band in sodium dodecyl sulfate gel electrophoresis. Chinook salmon prolactin appeared to be a basic protein of 22,500 molecular weight. Throughout the purification, prolactin bioactivity was followed by radioreceptor assay for lactogenic hormones. The prolactin character of the purified protein was established by its lactogenic activity in the rabbit mammary gland in vitro and its sodium-retaining activity in hypophysectomized Fundulus heteroclitus.

Bioassay for salmon prolactin using hypophysectomized Fundulus heteroclitus

A bioassay for salmon prolactin (PRL) is described. This assay which is based on the sodium-retaining action of PRL in the hypophysectomized killifish, Fundulus heteroclitus, has proved to be rapid, sensitive (250 pg PRL per gram of fish), and specific. The procedure has been used to characterize the biological activity of a highly purified PRL from the pituitaries of the chinook salmon, Oncorhynchus tschawytscha, and a similar PRL isolated (by acid buffer polyacrylamide disc gel electrophoresis) from pituitaries of coho salmon ( O. kisutch) (MW ca. 22,000; isoelectric point >9).

Immunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin

An antiserum raised to highly purified chum salmon ( Ocorhynchus keta) prolactin (sPRL) was used to identify prolactin-producing cells in the adenohypophysis of 15 species of teleosts by the immunocytochemical peroxidase-antiperoxidase method. In the chum salmon, the only pituitary cells that reacted with sPRL antibody were the PRL cells organized as follicular structures in the rostral pars distalis. When the antiserum was absorbed with sPRL, on the other hand, no immunoreactive cell was observed in the pituitary, indicating the specificity of the antiserum. Furthermore, the antibody to sPRL reacted only with PRL cells in the pituitaries of three species of salmonids, a plecoglossid, eel, carp, goldfish, killifish, tilapia, and five species of marine fishes, thus showing no species specificity of the antibody among the teleosts tested. The PRL cells of the eel decreased in number and also in immunoreactivity after adaptation to seawater for 1 month. On the other hand, highly immunoreactive PRL cells were observed in the pituitaries of marine fishes, although the cells were much fewer in number than in eels and in other fishes in fresh water.

Isolation and properties of chum salmon prolactin

A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mg/g of wet tissue. It was 10–15 times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with V e V o = 2.0 . Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an α-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH 2-terminal residue, isoleucine, and a single COOH-terminal residue, half-cystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH 2-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.

A comparison of prolactins from a marine, an estuarine, and a freshwater teleost

Prolactin was isolated from pituitaries of winter flounder Pseudopleuronectes americanus, American plaice Hippoglossoides platessoides, and carp Cyprinus carpio by chromatography using concanavalin A—Sepharose, gel filtration, and ion-exchange chromatography. All prolactins were unadsorbed on DEAE Bio-Gel A equilibrated with 5 m M NH 4HCO 3 at pH9, and were active in retaining plasma sodium level in hypophysectomized Poecilia latipinna in fresh water. Flounder and carp prolactins were similar in amino acid composition. An antiserum against salmon prolactin localized in the rostal pars distalis of the flounder pituitary.

Effect of L-DOPA, Bromocripton and Lergotrile on prolactin cell activity and hydromineral regulation in yearling coho salmon,Oncorhynchus kisutch

The prolactin inhibiting substances L-DOPA, Lergotrile and Bromocripton appeared to reduce the secretory activity of the prolactin cells in coho salmon (Oncorhynchus kisutch) yearlings acclimated to distilled water and effected significant reductions in plasma osmotic pressure and plasma Na+ and/or Cl− concentrations. This suggests that in coho salmon prolactin is involved in osmotic or ionic homeostasis in hyposmotic environments in a manner similar to that in other teleosts.

Isolation of prolactin from salmon pituitary

Prolactin(s) was isolated from salmon pituitary by chromatography on concanavalin A, gel filtration, and ion-exchange chromatography. The prolactin not adsorbed on DEAE Bio-Gel A (DEAE 1) had the same molecular weight by get filtration before and after ion-exchange chromatography. This salmon prolactin had a molecular weight of 24,300 by gel filtration and 20,500 by SDS gel electrophoresis. The isoelectric point was 6.0 when determined by isoelectric focusing. This prolactin was homogeneous by electrophoresis under acidic and basic conditions and essentially homogeneous by isoelectric focusing. The protein consistently maintained plasma sodium levels in hypophysectomized Poecilia latipinna at very low dosage (50 ng/fish). Antibodies against this prolactin localized specifically in the prolactin cells of rainbow trout pituitary. A protein fraction more strongly adsorbed on DEAE Bio-Gel A (DEAE 4) also had some prolactin activity in the Poecilia assay. Prior to DEAE chromatography this protein occurred in the same molecular weight fraction as the nonadsorbed protein but after DEAE chromatography the fraction contained three proteins with molecular weights of 23,000 (64%), 46,000 (21%), and 66,000 (15%). The results suggest that the higher molecular weight proteins are aggregates formed during isolation but the 23,000 molecular weight protein may be a modified form of the unadsorbed prolactin. The growth hormone fraction (DEAE 2) had no prolactin activity when tested at 10 times the minimum effective dose of salmon prolactin. The remaining DEAE fractions, 3 and 5, were also inactive in the Poecilia assay.