Expression of hormone genes and osmoregulation in homing chum salmon: a minireview

Pacific salmon migrate from ocean through the natal river for spawning. Information on expression of genes encoding osmoregulatory hormones and migratory behavior is important for understanding of molecular events that underlie osmoregulation of homing salmon. In the present article, regulation of gene expression for osmoregulatory hormones in pre-spawning salmon was briefly reviewed with special reference to neurohypophysial hormone, vasotocin (VT), and pituitary hormones, growth hormone (GH) and prolactin (PRL). Thereafter, we introduced recent data on migratory behavior from SW to FW environment. In pre-spawning chum salmon, the hypothalamic VT mRNA levels increased in the males, while decreased in the females with loss of salinity tolerance when they were kept in SW. The amounts of GH mRNA in the pituitary decreased during ocean migration prior to entrance into FW. Hypo-osmotic stimulation by SW-to-FW transfer did not significantly affect the amount of PRL mRNA, but it was elevated in both SW and FW environments along with progress in final maturation. Behaviorally, homing chum salmon continued vertical movement between SW and FW layers in the mouth of the natal river for about 12h prior to upstream migration. Pre-spawning chum salmon in an aquarium, which allowed fish free access to SW and FW, showed that individuals with the lower plasma testosterone (T) and higher estradiol-17beta (E2) levels spent longer time in FW when compared with the SW fish. Taken together, neuroendocrine mechanisms that underlie salt and water homeostasis and migratory behavior from SW to FW may be under the control of the hypothalamus-pituitary-gonadal axis in pre-spawning salmon.     

Attenuation of diurnal rhythms in plasma levels of melatonin and cortisol, and hypothalamic contents of vasotocin and isotocin mRNAs in pre-spawning chum salmon

In the present study, diurnal changes in plasma levels of melatonin and cortisol, and hypothalamic contents of neurohypophysial hormone mRNAs were examined in pre-spawning chum salmon, Oncorhynchus keta. From late November to early December, homing fish were captured at two sites along their migratory pathway on the Sanriku coast, Japan. Fish captured in the seawater (SW) environment were transferred to SW aquaria, and fish captured in the freshwater (FW) environment were to FW aquaria. They were maintained under natural photoperiod of approximately 10L:14D and sacrificed at 4-h interval through 24-h period. Plasma levels of melatonin were determined by radioimmunoassay, while cortisol levels were determined by enzyme immunoassay. Hypothalamic contents of vasotocin and isotocin mRNAs were determined by quantitative dot-blot hybridization assay. The melatonin levels showed weak nocturnal elevations in the SW and FW males, and FW females. The levels were maximal at 22:00 and minimal at 10:00 or 14:00, however the amplitudes were smaller than those reported in the previous studies using immature salmonids. The levels of vasotocin and isotocin mRNAs were higher in the males at all time points. The mRNA levels, however, did not show any diurnal variations in either of group. The same applied to plasma cortisol levels. These results indicate that the diurnal endocrine rhythms were attenuated in pre-spawning chum salmon, in contrast to the prominent diurnal rhythms in immature salmonids.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.     

Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldfish, Carassius auratus

Seasonal changes in the mRNA levels of glycoprotein alpha, gonadotropin (GTH) Ibeta and IIbeta, and thyrotropin (thyroid-stimulating hormone (TSH)) beta subunits in the pituitary of goldfish were quantified by Northern blot analysis and laser densitometry. Reproductive development and thyroid activity were monitored by measuring gonadosomatic index, plasma GTH II, testosterone (T), estradiol, 11-ketotestosterone, and thyroid hormones (T4 and T3). Plasma GTH II and steroids showed characteristic increases, while plasma thyroid hormones levels, in general, decreased in association with the reproductive period. In females, the mRNA levels of the alpha, GTH Ibeta, and GTH IIbeta subunits increased synchronously during early spawning period (April) and then decreased during ovarian regression (August). In males, the levels of the alpha and GTH IIbeta subunits showed changes similar to those in females, but the GTH Ibeta mRNA levels showed only a small increase during the late spawning period (May). In both sexes, TSHbeta mRNA levels were high during winter to early spring (February and April) and low during late spring to summer (May and August). These results suggest that in goldfish the gonadotropins may be synthesized synchronously in order for asynchronous gametogenesis to take place. Additionally, the data suggest a negative feedback relationship between synthesis of the TSHbeta subunit and the thyroid hormones.     

Kinetic studies of growth hormone and prolactin during adaptation of coho salmon, Oncorhynchus kisutch, to different salinities.

The kinetics of growth hormone (GH) and prolactin (PRL) in coho salmon (Oncorhynchus kisutch) transferred from fresh water (FW) to seawater (SW) and vice versa were examined to help clarify the osmoregulatory roles of the two hormones during periods of migration to different salinities. Chum salmon GH or PRL was administered by a single injection intraarterially, and metabolic clearance rate (MCR) and secretion rate (SR) of injected hormones were calculated from the disappearance of the hormones from the plasma. When coho salmon smolts were acclimated to SW, MCR, SR, and plasma level of GH in SW-adapted (2-3 weeks) fish were twice as great as those in fish in FW. On the other hand, there was no difference in the kinetics of GH between the adult coho salmon in SW and those adapted to FW (2-3 weeks). The transfer of the adult coho salmon from SW to FW was followed after 2 days by a rise in plasma level and SR of PRL, which tended to stay at high levels after 2-3 weeks. The MCR of PRL increased significantly after 2-3 weeks in FW. These results support the likelihood of an important role of GH in SW adaptation and of PRL in FW adaptation in coho salmon.     

The development of monoclonal antibodies against salmon (Oncorhynchus kisutch and O. keta) pituitary hormones and their immunohistochemical identification

Monoclonal antibodies (MCAs) directed against several salmon pituitary hormones were generated by the fusion of myeloma cells with spleen cells from mice that had been immunized with either chum salmon (Oncorhynchus keta) growth hormone (GH) or prolactin (PRL), or one of two purified protein preparations from coho salmon (O. kisutch) pituitaries. Hybridoma were cloned by limiting dilution and screened for MCA production using immunohistochemical procedures. MCAs were generated that bound specifically to GH, PRL, or gonadotropic cells. MCAs were generated that bound to either fine granular material or large globular inclusions in the cytoplasm of the "classical" strongly PAS-positive globular gonadotropic cell type found in mature fish. This suggests that these MCAs are directed against gonadotropin II (GTH II). A MCA was also generated that bound both granular and globular material in the globular gonadotrops and granular material in the weakly PAS-positive vesicular gonadotrops in pituitaries from mature fish and to a cell type in immature rainbow trout pituitaries which is tentatively identified as the gonadotropin I (GTH I) cell type. This MCA did not bind to thyrotrops in immature rainbow trout pituitaries.     

Development of salmon GTH I and GTH II radioimmunoassays

Radioimmunoassays (RIAs) for the measurement of two gonadotropins, GTH I and GTH II, in the plasma and pituitary of salmonid fish were developed using a rabbit antiserum to beta-subunits of chum salmon GTH I and GTH II. Intact GTH I and GTH II were used as standards and radioactive competitors. The displacement curves for plasma of salmonids including chum salmon, amago salmon, and rainbow trout were parallel to chum salmon GTH I and GTH II standards. Parallel displacement curves were obtained for pituitary extracts of chum salmon and amago salmon. The cross-reactivities of growth hormone, prolactin, and proopiomelanocortin (POMC)-related hormones were less than 1% in both RIAs. However, cross-reactivities of GTH I in the GTH II RIA and GTH II in the GTH I RIA were 10 and 12%, respectively. Plasma concentrations of both GTHs from salmonids at various stages of reproductive development were compared. In immature rainbow trout of both sexes (males: average (AV) gonadosomatic index (GSI) = 0.05; females: AV GSI = 0.24), plasma levels of GTH I and GTH II were low (less than 2 ng/ml). During prematurational stages of spermatogenesis and vitellogenesis in rainbow trout (males: AV GSI = 0.43; females: AV GSI = 2.8), the predominant GTH in the pituitary and plasma was GTH I. In contrast, plasma concentrations of GTH II were significantly higher than those of GTH I in postovulatory amago and chum salmon. Similarly, pituitary concentrations of GTH II were significantly higher than those of GTH I in postovulatory and spermiating amago salmon and postovulatory chum salmon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential response of plasma prolactin to freshwater transfer of smolts and postsmolts of seawater-adapted coho salmon (Oncorhynchus kisutch)

Prolactin (PRL) was purified from chum salmon, Oncorhynchus keta, pituitary glands and was used to develop a homologous radioimmunoassay for the measurement of PRL from salmon. The plasma PRL response to freshwater (FW) transfer differed in seawater (SW)-adapted postsmolt (250 g) and smolts (15 g) of coho salmon. Postsmolts had a pronounced and prolonged elevation of plasma titers of PRL with hypercalcemia and stable plasma sodium levels. The FW-transferred postsmolts had significantly lower pituitary gland PRL only at 0.5 and 2 hr post-transfer as compared to SW-SW. Smaller smolts showed stable plasma PRL levels after FW transfer, hypocalcemia 48 post-transfer, depressed plasma sodium concentrations, and lowered plasma osmotic pressure. This different response may be due to an increased osmoionic regulatory challenge encountered by the smaller smolts or possibly due to some other developmental change between the two different age classes.     

Seasonal changes of responses to gonadotropin-releasing hormone analog in expression of growth hormone/prolactin/somatolactin genes in the pituitary of masu salmon

Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/SL genes, in the pituitaries of growing and maturing masu salmon (Oncorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones.     

Disparate release of prolactin and growth hormone from the tilapia pituitary in response to osmotic stimulation

In most teleost fishes, prolactin (PRL) plays a key role in freshwater (FW) adaptation, whereas growth hormone (GH) is involved in seawater (SW) adaptation in salmonids and certain euryhaline species including the tilapia, Oreochromis mossambicus. Consistent with its osmoregulatory activity, PRL release increases in response to physiologically relevant reductions in extracellular osmolality. When dispersed PRL and GH cells from FW-acclimatized fish were incubated in media of varying osmolalities, PRL release increased significantly in response to a 12% reduction in medium osmolality during 1 and 4h of exposure. By contrast, cells from SW-acclimatized fish responded only to a 24% reduction in osmolality. Growth hormone release on the other hand increased whether medium osmolality was reduced or raised. Cell volume increased together with PRL release during the perifusion of dispersed PRL cells in direct proportion to the reduction in medium osmolality. Growth hormone release increased whether GH cell volume increased or decreased. In in vivo studies, circulating PRL levels increased as early as 1h after the transfer of fish from SW to FW, whereas GH levels remained unchanged during 24h of acclimatization. These results indicate that while PRL and GH cells are osmosensitive, the PRL cells respond to reductions in extracellular osmolality in a manner that is consistent with PRL's physiological role in the tilapia. While the rise in GH release following the reduction in osmolality is of uncertain physiological significance, the rise in GH release with the elevation of medium osmolality may be connected to its role in SW adaptation.     

Development and validation of a salmon growth hormone radioimmunoassay

A highly specific and sensitive radioimmunoassay (RIA) for the measurement of plasma and pituitary growth hormone (GH) levels in salmonid fishes was developed using an anti-serum raised in rabbit against chum salmon (Oncorhynchus keta) GH (sGH). Pituitary extracts and plasma from chum, coho, masu, and amago salmon, and from rainbow trout and Japanese charr, all exhibited displacement curves parallel to the sGH standard. Samples from the eel, carp, goldfish, and tilapia, as well as plasma from hypophysectomized chum salmon and rainbow trout, all showed negligible cross-reactivity. None of the mammalian or teleostean GH or prolactin preparations tested cross-reacted with the antibody in the assay system. RIA sensitivity was 0.6 ng sGH/ml of plasma when 100 microliter of plasma was employed. Intra- and interassay coefficients of variation were 3.9 and 4.1%, respectively. Plasma GH levels of the mature chum salmon caught in Otsuchi Bay were highly variable, especially in females (20.2 +/- 8.2 ng/ml) as compared with males (16.0 +/- 1.1 ng/ml), and there was no significant change after transfer to fresh water. Whereas there was no change in plasma GH levels in males kept in seawater, the levels in females increased with time in close correlation with the increase in plasma chloride.     

A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish

Two PRL-releasing peptides (PrRP20 and PrRP31) were recently identified from mammalian hypothalamus by an orphan receptor strategy, and a C-terminal RF (arginyl-phenylalamyl-) amide peptide (RFa), structurally related to mammalian PrRP, was also identified from the brain of the Japanese crucian carp (C-RFa) by an intestine-contracting assay. However, to date there have been no reported studies that have examined the PRL-releasing effects of RFa in fish. In the present study we determined the cDNA, primary structure, and function of a homolog of the mammalian PrRP20 in the chum salmon, Oncorhynchus keta. An RFa cDNA encoding a preprohormone of 155 amino acids was cloned from the hypothalamus of chum salmon by 3'- and 5'-rapid amplification of cDNA ends. A native RFa was purified from an acid extract of salmon hypothalami by a Sep-Pak C(18) cartridge, affinity chromatography using anti-synthetic C-RFa, and reverse phase HPLC on an ODS-120T column. The salmon RFa proved to be identical with C-RFa on the basis of elution position on reverse phase HPLC. Immunocytochemical staining in rainbow trout, Oncorhynchus mykiss, revealed that C-RFa-immunoreactive cell bodies were located in the posterior part of hypothalamus and C-RFa-immunoreactive fibers were abundant from the hypothalamus to the ventral telencephalon. A small number of immunoreactive fibers were projected to the pituitary and terminated close to the PRL cells in the rostral pars distalis and to the somatolactin (SL) cells in the pars intermedia. The hypophysiotropic effects of the fish homolog were determined on the release of PRL, SL, and GH from the pituitary of the rainbow trout. Plasma PRL and SL levels were increased at 3 and 9 h, respectively, after ip injection of the synthetic C-RFa into the rainbow trout at doses of 50 and 500 ng/g body weight. In contrast, plasma GH levels were decreased after 1 h at 500 ng/g body weight. Perifusion of the trout pituitaries with synthetic C-RFa at concentrations of 10 pM to 100 nM demonstrated maximum PRL release at 100 pM and maximum SL release at 10 and 100 nM. However, GH release was not affected. These data are the first to demonstrate that a homolog of mammalian PrRP (fish RFa) is a major hypothalamic peptide of PRL release in teleost fish.     

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 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.     

Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca(2+) through a stretch-activated Ca(2+) channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca(2+) ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.     

Immunomodulatory effects of prolactin and growth hormone in the tilapia,Oreochromis mossambicus

To clarify the roles of prolactin (PRL) and GH in the control of the immune system, the effects of environmental salinity, hypophysectomy, and PRL and GH administration on several immune functions were examined in tilapia (Oreochromis mossambicus). Transfer from fresh water (FW) to seawater (SW) did not alter plasma levels of immunoglobulin M (IgM) and lysozyme. The superoxide anion (O(2)(-)) production in head kidney leucocytes accompanied by phagocytosis was elevated in SW-acclimated fish over the levels observed in FW fish. Hypophysectomy of the fish in FW resulted in a reduction in O(2)(-) production in leucocytes isolated from the head kidney, whereas there was no significant change in plasma levels of IgM or lysozyme. Treatment with tilapia GH and PRLs (PRL(177) and PRL(188)) enhanced O(2)(-) production in vitro in head kidney leucocytes in a dose-related manner. Extrapituitary expression of two PRLs, GH and IGF-I mRNA was detected in lymphoid tissues and cells such as head kidney, spleen, intestine and leucocytes from peripheral blood and head kidney. PRL-receptor mRNA was detected in head kidney leucocytes, and the level of expression was higher in SW-acclimated fish than that in FW fish. Treatment with PRL(177) caused higher production of O(2)(-) in the head kidney leucocytes isolated from SW tilapia than that from FW fish. In view of the fact that PRL acts antagonistically to osmoregulation in SW, its immunomodulatory actions in this euryhaline fish would appear to be independent of its osmoregulatory action.     

Sodium-retaining activity of chum salmon prolactin in some euryhaline teleosts

Sodium-retaining activity of chum salmon prolactin (PRL) was examined in several euryhaline teleosts. Chum PRL was 100 times more potent than ovine PRL in maintaining plasma sodium levels in the hypophysectomized killifish, Fundulus heteroclitus, transferred from 50% seawater to fresh water. The effects of PRLs were parabolic, high doses of the hormones being less effective than low doses. When injected into seawater-adapted fry of the ayu, Plecoglossus altivelis, or into juvenile rainbow trout, Salmo gairdneri, adapted to 50% seawater, a dose-dependent increase in plasma sodium was observed. Chum PRL was 2-10 times more active than ovine PRL, and the effects in the ayu were also parabolic. An increase in plasma sodium also occurred when the PRLs were injected into the seawater-adapted eel, Anguilla japonica; the chum and ovine PRLs were equipotent, and hypercalcemia was also observed. In contrast, both chum and ovine PRLs were without effect on plasma sodium levels of chum salmon fry, either when injected into seawater-adapted fish kept in seawater or into fish subsequently transferred to fresh water. The absence of an effect of PRLs in chum salmon fry seems to be due, at least in part, to their good osmoregulatory ability during the period of seaward migration; effects of the exogenously administered PRLs may be compensated for by other hormones responsible for their hydromineral balance.