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To clarify the relationships between growth, endocrine status and habitat characteristics in Japanese eel (Anguilla japonica), plasma and stomach mRNA levels of ghrelin were examined in wild eels captured in the river and the bay, and in cultured eels during and after experimental fasting. Wild juvenile eels captured in freshwater habitats within the river showed significantly higher plasma and stomach mRNA levels of ghrelin than did fish obtained from brackish-water habitats within the bay. In cultured eels experimentally fasted for 4 weeks, plasma and stomach mRNA levels of ghrelin increased. After refeeding, the both parameters returned to the levels observed in continuously feeding control fish. In pigmented elvers, 2 months of feed restriction resulted in a significant increase in whole-body ghrelin mRNA. It is suggested that interaction between ghrelin and feeding is related to their habitats through differential food acquisition in fresh and brackish water environments.

Prolactin (PRL) is a vertebrate hormone with diverse actions in osmoregulation, metabolism, reproduction, and in growth and development. Osmoregulation is fundamental to maintaining the functional structure of the macromolecules that conduct the business of life. In teleost fish, PRL plays a critical role in osmoregulation in fresh water. Appropriately, PRL cells of the tilapia are directly osmosensitive, with PRL secretion increasing as extracellular osmolality falls. Using a model system that employs dispersed PRL cells from the euryhaline teleost fish, Oreochromis mossambicus, we investigated the autocrine regulation of PRL cell function. Unknown was whether these PRL cells might also be sensitive to autocrine feedback and whether possible autocrine regulation might interact with the well-established regulation by physiologically relevant changes in extracellular osmolality. In the cell-perfusion system, ovine PRL and two isoforms of tilapia PRL (tPRL), tPRL177 and tPRL188, stimulated the release of tPRLs from the dispersed PRL cells. These effects were significant within 5–10 minutes and lasted the entire course of exposure, ceasing within 5–10 minutes of removal of tested PRLs from the perifusion medium. The magnitude of response varied between tPRL177 and tPRL188 and was modulated by extracellular osmolality. On the other hand, the gene expression of tPRLs was mainly unchanged or suppressed by static incubations of PRL cells with added PRLs. By demonstrating the regulatory complexity driven by positive autocrine feedback and its interaction with osmotic stimuli, these findings expand upon the knowledge that pituitary PRL cells are regulated complexly through multiple factors and interactions.

This study investigated endocrine control of branchial ionoregulatory function in Nile tilapia (Oreochromis niloticus) by prolactin (Prl188 and Prl177), growth hormone (Gh) and cortisol. Branchial expression of Na(+)/Cl(-) cotransporter (ncc) and Na(+)/K(+)/2Cl(-) cotransporter (nkcc) genes were employed as specific markers for freshwater- and seawater-type ionocytes, respectively. We further investigated whether Prl, Gh and cortisol direct expression of two Na(+), K(+)-ATPase (nka)-α1 subunit genes, denoted nka-α1a and nka-α1b. Tilapia transferred to fresh water following hypophysectomy failed to adequately activate gill ncc expression; ncc expression was subsequently restored by Prl replacement. Prl188 and Prl177 stimulated ncc expression in cultured gill filaments in a concentration-related manner, suggesting that ncc is regulated by Prl in a gill-autonomous fashion. Tilapia transferred to brackish water (23 ‰) following hypophysectomy exhibited a reduced capacity to up-regulate nka-α1b expression. However, Gh and cortisol failed to affect nka-α1b expression in vivo. Similarly, we found no clear effects of Gh or cortisol on nkcc expression both in vivo and in vitro. When considered with patterns previously described in euryhaline Mozambique tilapia (O. mossambicus), the current study suggests that ncc is a conserved target of Prl in tilapiine cichlids. In addition, we revealed contrasting dependencies upon the pituitary to direct nka-α1b expression in hyperosmotic environments between Nile and Mozambique tilapia.

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproductive function in various vertebrates and invertebrates. In the present study, we have identified the GnRH-like peptide cDNA and peptide from the cerebral ganglion (CG) of the Pacific abalone, Haliotis discus hannai. Pacific abalone GnRH-like peptide (hdhGnRH-like peptide) cDNA encodes precursor, which possesses the typical organization of the known mollusk GnRH-like peptide precursors, including a hydrophobic signal peptide, GnRH-like peptide, and a cleavage site followed by a GAP-like peptide region. Three hdhGnRH-like peptides, pQNYHFSNGWHAamide (hdhGnRH-11amide), pQNYHFSNGWHA (hdhGnRH-11OH), and pQNYHFSNGWHAG (hdhGnRH-12OH), were determined from the acid/acetone extract of the CG by mass spectrometry (LC-MS/MS) analysis. The hdhGnRH-like peptide mRNA expression was detected not only in the CG but also in gonads, and hdhGnRH-11amide was also detected in the extract of gonads. The mRNA expression of hdhGnRH-like peptide in the CG was lower in spawned males than in non-spawned animals, while no change in hdhGnRH-like peptide mRNA expression was shown in both ovulated and non-ovulated abalone. The hdhGnRH-11amide induces spawning and ovulation of both mature males and females in a concentration-dependent fashion following intramuscular injection. These results indicate that three hdhGnRH-like peptides are yielded from a single hdhGnRH-like peptide precursor, and that at least hdhGnRH-11amide is involved in the control of reproduction of the Pacific abalone.

Growth hormone 1 (GH1), a pituitary hormone, plays a key role in the regulation of growth. Both excess GH1 treatment and overexpression of a GH1 transgene promote growth of salmon, but these animals exhibit physiological abnormalities in viability, fertility and metabolism, which might be related to pituitary function. However, the molecular dynamics induced in the pituitary by excess GH1 remain unknown. In this study, we performed iTRAQ proteome analysis of the amago salmon pituitary, with and without excess GH1 treatment, and found that the expression levels of proteins related to endocrine systems, metabolism, cell growth and proliferation were altered in the GH1-treated pituitary. Specifically, pituitary hormone prolactin (2.29 fold), and somatolactin α (0.14 fold) changed significantly. This result was confirmed by proteome and transcriptome analyses of pituitary from the GH1-transgenic (GH1-Tg) amago salmon. The dynamics of protein and gene expression in the pituitary of GH1-Tg amago salmon were similar to those of pituitary treated with excess GH1. Our findings suggest that not only excess GH1 hormone, but also the quantitative changes in other pituitary hormones, might be essential for the abnormal growth of amago salmon. These data will be useful in future attempts to increase the productivity of fish farming.

Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation ofigf1andigf2mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of bothigf1andigf2in a concentration-related and biphasic manner over the physiological range, with a greater effect onigf2mRNA level. Insulin increased basaligf2mRNA level, and strongly increased GH-stimulatedigf2mRNA level, but slightly reduced basaligf1mRNA level and did not affect GH-stimulatedigf1mRNA level. Cortisol inhibited GH stimulation ofigf1, but increased GH stimulation ofigf2mRNA level. The synergistic effect of insulin and GH onigf2mRNA level was confirmedin vivo. These results indicate that insulin and cortisol differentially modulate the response ofigf1andigf2mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami . The hagfish GPH consists of two subunits, α and β, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHα and -β were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein α-subunit 2 (GPA2) and β-subunit 5 (GPB5) gave rise to GPHα and GPHβ of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHα and -β subunits appear to be the typical gnathostome GPHα and -β subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

We examined whether gonadotropin-releasing hormone (GnRH)-like peptides are present in the neural ganglia of the gastropod Pacific abalone (Haliotis discus hannai) by reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) analysis and by immunohistochemistry. Cerebral ganglion extracts showed a similar retention time to lamprey GnRH-II (lGnRH-II) in rpHPLC combined with TR-FIA analysis. GnRH-like-immunoreactive (ir) cell bodies (which reacted with a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the peripheral region of the cerebral ganglion, and they were observed to send fibers into the neuropil. GnRH-like-ir fibers were also detected in the neuropil of the pedal ganglion, the visceral nerve, and the nerve originating from the pedal ganglion. Chicken GnRH-II (cGnRH-II)-like-ir fibers (which reacted with a rabbit polyclonal antibody raised against cGnRH-II) were also observed in the neuropil of the cerebral ganglion. GnRH-like-ir fibers and cGnRH-II-like-ir fibers were distinguishable in the neuropil of the cerebral ganglion by double-staining immunohistochemistry. These results suggest that multiple GnRH-like peptides exist in the neural ganglia of the Pacific abalone.

Growth hormone is a member of the GH/PRL/SL family produced by the anterior pituitary in all vertebrates. GH is a single-chain polypeptide containing two highly conserved intramolecular disulfide bonds that are important for biological activity. The actions of GH are initiated by its binding to a membrane-bound GHR that is located in various tissues, including the liver, muscle, adipose tissue, mammary gland, bone, kidney, and embryonic stem cells. GH in tetrapods as well as in fish is involved in the regulation of somatic growth primarily through the induction of IGF. It is also implicated in metabolism, reproduction, immunity, and seawater adaptation. The synthesis and release of GH is regulated by GHRH, ghrelin, somatostatin, GnRH, thyroid hormone, cortisol, and activin. Recombinant human GH is used for treatment of GH deficiency and dwarfism.

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

We have identified the first and perhaps only gonadotropin beta-like protein by cDNA cloning in sea lamprey, a member of the oldest lineage of vertebrates, the agnathans. Two pituitary gonadotropins (GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) have been identified in representative species of all classes of vertebrates except the agnathans. The present study was undertaken to identify GTH in sea lamprey, Petromyzon marinus, to gain a further understanding of the origin and evolution of reproductive pituitary hormones and their respective genes in vertebrates. Sea lamprey preGTHbeta-like cDNA was cloned from a plasmid cDNA library using an expressed sequence tag analysis. The preGTHbeta-like cDNA encoded 150 amino acids, in which the GTHbeta-like protein consisted of 134 amino acid residues. Sea lamprey GTHbeta-like protein contained 12 Cys residues and two N-glycosylation sites at homologous positions to those of FSHbeta and LHbeta. The region of the molecule that has been proposed to control receptor binding specificity (i.e., the region between the 10th and 12th Cys residues) suggests that the proposed heterodimer would be more like a FSH than a LH. Sea lamprey GTHbeta-like protein-producing cells were identified immunocytochemically in the ventral part of the proximal pars distalis of pituitary using antiserum prepared against a synthetic peptide of preGTHbeta-like protein (52-68). Intraperitoneal administration of sea lamprey GnRH-I and -III at 100 microg/g body weight (twice at a 24h interval) increased expression of GTHbeta-like protein in the pituitary of adult female sea lamprey during the final maturational period. Thus, these results are the first to demonstrate the presence of a single GTH-like system in lampreys. Because the sea lamprey GTHbeta-like protein is a clear out-group compared to those of the LH and FSH family based on phylogenic analysis, we propose that an ancestral glycoprotein hormone gave rise to only one GTH in lampreys and to the glycoprotein hormone family that gave rise to LH, FSH, and TSH during the early evolution of gnathostomes.

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 µg g−1 body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL−1 for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression is unclear even in mammals. To study the possible presence of PrRP–PRL axes not only in the brain-pituitary but also in peripheral organs, the expression patterns of PrRP, PRL and growth hormone (GH) were characterized in amphibious euryhaline mudskippers (Periophthalmus modestus). PrRP mRNA is abundantly expressed not only in the brain but also in the liver, gut and ovary, while less abundant expression was also detected in the skin and kidney. Corresponding to the distribution of PrRP mRNA, PRL mRNA was also detectable in these organs. During adaptation to different environments, the changes in mRNA levels of PrRP paralleled those in PRL in the brain-pituitary, liver and gut in an organ-specific manner. Brain PrRP mRNA and the pituitary PRL mRNA increased under freshwater and terrestrial conditions (PP

We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V13R14Q15) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.

In order to clarify the mechanisms of neoteny in the ice goby (Leucopsarion petersii), we examined effects of thyroid hormone and thiourea (TU) treatments on their neotenic characteristics and the pituitary-thyroid axis. Adult ice goby were exposed to 3, 5, 3'-triiodothyronine (T3, 0.1 ppm), TU (inhibitor of thyroid hormone synthesis, 30 ppm), or the combination of the two for 2 weeks. Observations of whole body T3 levels, thyroid follicles and TSH immunoreactive cells in the pituitary suggests the presence of a functioning thyroidal system. However, all of the neotenic features did not disappear in T3-treated fish, suggesting the absence of T3 responsiveness in peripheral tissues. These results indicate the similarity between neoteny of the ice goby and obligatory-type neoteny of urodeles.

The "cobalt" variant of rainbow trout (Oncorhynchus mykiss) lacks most of the pars intermedia of the pituitary, and shows significant obesity with an enlarged liver and a fat accumulation in the abdominal cavity. Plasma levels of growth hormone, prolactin, and somatolactin were significantly lower in the cobalt variant than those in the normal trout. In contrast, plasma insulin level was four times higher than that in the normal. Plasma levels of total protein, free cholesterol, and triacylglycerol were higher in the cobalt, while those of glucose and fatty acids were not different from the normal levels. In the white muscle, red muscle, liver, and mesenteric fat, the cobalt showed higher contents of triacylglycerol than the normal fish. There was no significant difference in tissue contents of phosphatidylcholine between the two groups of the trout, except for that in the mesenteric fat, exhibiting significantly lower content than in the normal fish. Activity of triacylglycerol lipase in the liver in vivo was lower in the cobalt than that in the normal trout, while there was no significant difference between the two in the cultured liver slices. Desacetyl-alpha-MSH stimulated lipolysis of triacylglycerol similarly in the cultured liver slices from the normal trout and from the cobalt variant. Results from this study suggest that the lack of pars intermedia and the increased plasma level of insulin are involved in a depression of lipid mobilization and obesity in this variant of rainbow trout.

Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the α-glycoprotein subunit (αGSU) and gonadotropin β subunits (Iβ and IIβ) of red seabream using the 5′- and 3′-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of αGSU, Iβ, and IIβ are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Iβ mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexua...

In this study, receptors for insulin and insulin-like growth factor I (IGF-I) in isolated theca-interstitial layers and granulosa cells of the coho salmon preovulatory ovary were characterized, and the effects of IGF-I on ovarian steroidogenesis were examined. Specific receptors for insulin and IGF-I were found in granulosa and theca-interstitial layers. In both follicular layers, IGF-I receptors were greater in number and higher in affinity than insulin receptors. The effects of IGF-I on in vitro production of testosterone (T) and 17 alpha-hydroxyprogesterone (17OH-P) by theca-interstitial layers and of 17 beta-estradiol (E2) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) by granulosa cell layers were evaluated during the preovulatory period. Both human and salmon recombinant IGF-I inhibited the basal and GTH II-stimulated T and 17OH-P production by theca-interstitial layers throughout the preovulatory period. In contrast, IGF-I stimulated the production of both E2 and 17,20 beta-P by granulosa cell layers prior to germinal vesicle breakdown (GVBD) but only stimulated the production of 17,20 beta-P by granulosa cell layers after GVBD. The inhibitory effects of IGF-I on steroid production by the theca-interstitial layer and the opposite stimulatory effects on steroid production by the granulosa cell layer, coupled by the presence of specific IGF-I receptors in both follicular layers, suggest that IGF-I may play a role in the regulation of steroidogenesis in the preovulatory coho salmon ovary.

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

There are many biochemical and molecular biological data indicating that IGF-I is present in teleost fish. The present study examines red seabream for the presence and cellular distribution of IGF-I in the ovarian follicles during oocyte growth and final maturation of oocytes by immunocytochemistry. Immunoreactivity appeared in the granulosa cells at the lipid stage. Immunoreactivity became strong at the primary yolk globule stage and diminished in intensity during development of maturational competence (responsiveness to maturation-inducing steroid) and final maturation of oocytes. Intense immunoreactivity was also localized in the outer layer of the zona radiata and the peripheral region of the ooplasm at the primary yolk globule stage. Postovulatory follicles had low immunoreactivity in the early phase of degeneration. These immunocytochemical observations show that the granulosa cell layer is the main site of IGF-I production. IGF-I may be involved in granulosa cell proliferation and differentiation.

A specific homologous radioimmunoassay (RIA) for measurement of insulin-like growth factor-I (IGF-I) in plasma of salmonid and a few non-salmonid fish species was developed using recombinant coho salmon IGF-I (rsIGF-I) as tracer and standard, and antiserum against this peptide raised in rabbits. The minimum detection level of IGF-I was 1.5 ng/ml and linearity was obtained in a range from 1.5 to 23 ng/ml. No cross-reaction was detected in the salmon IGF-I RIA with mammalian growth factors, salmon pituitary hormones, salmon or mammalian insulin, or any peptide in rat plasma. Although salmon IGF-I has high sequence similarity to mammalian IGF-I, it did not cross-react with anti-human IGF-I serum in human RIA and serial dilutions of plasma from salmon were not parallel to the human IGF-I standards in this assay system. In contrast, dilution curves for plasma of salmonids, such as coho (Oncorhynchus kisutch), Atlantic (Salmo salar), and sockeye (O. nerka) salmon, rainbow trout (O. mykiss), some other teleost fish, such as tilapia (Oreochromis mossabmica), carp (Cyprus carpio), eel (Anguilla rostrata), Atlantic halibut (Hippoglossus hippoglossus), and agnathan, the sea lamprey (Petromyzon marinus), assessed in salmon IGF-I RIA were parallel to the rsIGF-I standards. Acid-ethanol extraction of plasma samples altered the molecular weight, but not the quantity, of immunoreactive IGF-I, implying that IGF-I binding proteins in salmon plasma do not affect the performance of the salmon IGF-I RIA. Gel filtration of nonacidified plasma on a Sephadex G-75 superfine column produced two immunoreactive IGF-I peaks of molecular weights of approximately >70 k and 7 kDa, whereas acidification of plasma increased the relative amount of the 7-kDa peak (IGF-I) and the >70-kDa peak disappeared. The recoveries of rsIGF-I added to extracted or nonextracted plasma were 97.4 and 94.9%, respectively. Inter- and intraassay coefficients of variation were 3.6 and 3.3%, respectively. Plasma IGF-I levels in coho salmon smolts were 117.4 ± 19.1 ng/ml as compared to IGF-I levels in parr (45.3 ± 2.5 ng/ml) or in adult fish (45.2 ± 5.4 ng/ml) measured in the same assay. Injection of salmon growth hormone, but not prolactin or somatolactin, caused a significant and dose-dependent elevation of plasma IGF-I levels, while either fasting or injection of streptozotocin led to a significant decline in systemic IGF-I. We conclude that the salmon IGF-I RIA reported in this study is valid for measurement of IGF-I levels in salmonids and, likely, in a variety of other fish species.

The pituitary gland is present in all vertebrates, from agnathans (jawless vertebrates) to mammals, but not in invertebrates. Reproduction in gnathostomes (jawed vertebrates) is controlled by two pituitary gonadotropins (GTHs), luteinizing hormone and follicle-stimulating hormone, which are part of the pituitary glycoprotein hormone (GPH) family. Hagfishes, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Accordingly, they are of particular importance in understanding the evolution of the pituitary GPHs and their functions related to vertebrate reproduction. Nevertheless, key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Our current report has revealed the first identification of a functional GPH composed of two subunits that possess gonadotropic action at the pituitary of brown hagfish. It seems most likely that an ancestral GPH gave rise to only one GTH in hagfish pituitary and that multiplicity of GPHs arose later during the early evolution of gnathostomes. This paper briefly summarizes the latest findings on the hagfish GPH from an evolutionary point of view.

Maturing adult sockeye salmon Oncorhynchus nerka were intercepted while migrating in the ocean and upstream in freshwater over a combined distance of more than 1,300 km to determine physiological and endocrine changes associated with ionoregulation. Sockeye migrating through seawater and freshwater showed consistent declines in gill Na+/K+ -ATPase (NKA) activity, plasma osmolality and plasma chloride concentration. In contrast, plasma sodium concentration became elevated in seawater as fish approached the river mouth and was then restored after sockeye entered the river. Accompanying the movement from seawater to freshwater was a significant increase in mRNA for the NKA α1a subunit in the gill, with little change in the α1b subunit. Potential endocrine signals stimulating the physiological changes during migration were assessed by measuring plasma cortisol and prolactin (Prl) concentrations and quantifying mRNA extracted from the gill for glucocorticoid receptors 1 and 2 (GR1 and GR2), mineralocorticoid receptor (MR), growth hormone 1 receptor (GH1R), and prolactin receptor (PrlR). Plasma cortisol and prolactin concentrations were high in seawater suggesting a preparatory endocrine signal before freshwater entry. Generally, the mRNA expression for GR1, GR2 and MR declined during migration, most notably after fish entered freshwater. In contrast, PrlR mRNA increased throughout migration, particularly as sockeye approached the spawning grounds. A highly significant association existed between gill PrlR mRNA and gill NKA α1a mRNA. GH1R mRNA also increased significantly, but only after sockeye had migrated beyond tidal influence in the river and then again just before the fish reached the spawning grounds. These findings suggest that cortisol and prolactin stimulate ionoregulation in the gill as sockeye salmon adapt to freshwater.

1. 1. Recombinant salmon growth hormone at doses of 0.8 and 2.1 μg/g significantly enhanced linear growth in hypophysectomized male killifish, Fundulus heteroclitus , over that of controls and a significant regression was found between growth and the logarithm of dose. 2. 2. Bovine growth hormone elicited significant growth enhancement at all three dosages tested (1,4 and 10 μg/g) and a significant log/dose relationship was also observed. 3. 3. Observations on the relative weight of the gonads indicate that whole salmon pituitary extract (25 μg/g) possesses strong gonadotropic activity and that both bGH and rsGH had smaller but significant effects on the gonads. 4. 4. It is suggested that growth hormone may play a subsidiary synergistic role to other pituitary hormones in gonadal development.

Effects of salmon and eel growth hormones (GHs) on bone metabolism in rainbow trout (Oncorhynchus mykiss) were studied using histomorphometry of their pharyngeal bones. When salmon GH (sGH) was injected intraperitoneally at a dose of 0.01 or 0.1 microgram/g/week for 10 times into fed trout, body growth in both length and weight was accelerated. Formation and resorption of bone were enhanced only by the higher dose of sGH. When a cholesterol pellet containing recombinant eel GH (reGH-pellet) was implanted subcutaneously into starved trout for 1 week (37 micrograms reGH/18 g fish), formation and resorption of bone increased, although activity of bone formation was low. The results indicate that the GHs increased both formation and resorption of bone, regardless of the nutritional status of the fish.

Publisher Summary This chapter describes the neuroendocrine control of synthesis and secretion of two homologous pituitary hormones—prolactin (PRL) and somatolactin (SL), in teleost fish. PRL and SL are produced in the rostral pars distalis and the pars intermedia of Osteichthyes, respectively. The synthesis and secretion of PRL and SL are regulated by two antagonistic hypothalamic neurohormones. Many hypothalamic neurohormones participate in the regulation of PRL and SL. Moreover, these neurohormones and pituitary hormones exhibit versatile functions in teleosts. An individual neurohormone appears to exert a different function or sometimes just opposite functions or no function, depending not only on species or ontogeny but many other factors including the time of treatment, reproductive status, environmental conditions, and the route of administration. Specific action of each hypothalamic neurohormone is determined by the presence of specific G‐protein coupled receptors. Both PRL and SL have been implicated in many physiological processes through specific Janus kinase (JAK) associated receptors. The chapter also discusses the molecular characteristics and biological functions of these hormones and their receptors together with the mode of actions of hypothalamic stimulating and inhibiting factors, including some of our recent findings on PRL‐releasing peptide (PrRP).

Proopiomelanocortin (POMC) is cleaved into small peptides, such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormones (MSHs), and beta-endorphin (beta-END), by tissue-specific posttranslational processing in the corticotrophs of the pars distalis (PD) and melanotrophs of the neurointermediate lobe (NIL) of the pituitary. We examined the posttranslational processing of POMC in the pituitary of the banded houndshark Triakis scyllium by molecular cloning and subsequent mass spectrometric identification of the POMC-derived peptides in the pituitary extracts. One-fifth of the randomly selected clones from a Triakis pituitary cDNA library contained a cDNA encoding for POMC. Triakis prePOMC contained 4 MSHs and a single beta-END, as has been observed in case of other cartilaginous fish POMCs. These predicted hormonal segments were flanked by basic amino acid residues, which are the cleavage sites for the processing enzymes, i.e., protein convertases. Mass spectrometry was performed using PD (including most parts of the rostral and proximal PD) and NIL extracts to detect mass values corresponding to the POMC-derived peptides. Consequently, ACTH, beta-END, and the joining peptide (JP) were detected in the PD extract, while MSHs, processed beta-END, and some other POMC-derived peptides were identified in the NIL extract; however, neither acetylated alpha-MSH nor acetylated beta-END was detected in the latter. These tissue-specific POMC processing patterns are similar to those of the other vertebrate pituitaries; however, the absence of acetylated peptides suggests the lack of an acetylation system in the melanotrophs in the NIL of the Triakis pituitary.

Transport of recombinant salmon growth hormone (rsGH) into the circulation of rainbow trout following intragastric or rectal administration was investigated. Changes in plasma GH levels were analyzed by radioimmunoassay specific to chum salmon GH. Intragastric administration of rsGH by oral intubation resulted in elevation of GH immunoreactivity in plasma after 11h. The plasma GH increased maximally after 15h, and then declined rapidly to reach a normal level after 19h. On the other hand, oral intubation of rsGH to carp, which has no stomach, caused elevation of plasma GH levels after 1 h which lasted for 21 h. In sharp contrast, rectal administration of rsGH to rainbow trout significantly elevated plasma GH levels after 15 min, which reached a maximum after 30 min. The rsGH was labeled with fluorescein isothiocyanate (FITC) to distinguish exogenous from endogenous GH, and administered into rainbow trout rectally. Subsequent analysis of plasma samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two fluorescent bands at the same molecular weights as those of the monomeric and dimeric rsGHs. Intragastric and rectal administration of rsGH into juvenile trout resulted in significant increases in length and body weight compared to the control fish. This study strongly suggests that the rsGH is transported into the circulation of salmonids via the lower part of the intestine and takes part in growth stimulation.

The ability of recombinant salmon growth hormone (rsGH) to accelerate the gorwth of juvenile caho salmon Oncorhynchus kisutch and chum salmon Oncorhynchus keta by immersion was in-vestigated. Amdinistratio of rsGH by immersion at doses of 3 or 30mg/L for 60 min every 7 days resulted in a significant increase in the body length and body weight compared to control groups. The growth promoting effects of rsGH were also demonstrated in juvenile chum salmon, which were immersed in rsGH solution of 30mg/L for 30 or 60 min every 4 days. These results strongly suggest that the observed growth stimulation following immersion is due to a specific effect of GH.

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH2 from the goldfish brain. In this study, to clarify the physiological functions of these three LPXRFamide peptides (gfLPXRFa-1, -2, and -3), we analysed the localisation and hypophysiotrophic activity of these peptides using sockeye salmon, Oncorhynchus nerka, in which immunoassay systems for several anterior pituitary hormones have been developed. gfLPXRFa-immunoreactive cell bodies were detected in the nucleus posterioris periventricularis of the hypothalamus and immunoreactive fibres were distributed in various brain regions and the pituitary. We also detected gfLPXRFa-immunoreactivity in the pituitary by competitive enzyme-linked immunosorbent assay combined with reversed-phase HPLC. These three gfLPXRFamide peptides stimulated the release of FSH, LH and GH, but did not affect the release of prolactin (PRL) and somatolactin (SL) from cultured pituitary cells. These results suggest that novel fish hypothalamic LPXR-Famide peptides exist in the brain and pituitary of sockeye salmon and stimulate the release of gonadotrophins and GH from the pituitary.

Prolactin-releasing peptide (PrRP), recently isolated from the brain of mammals and teleosts, is a strong candidate for being a stimulatory hormone of pituitary prolactin secretion. The present study examined whether or not PrRP is capable of inducing prolactin gene expression and elevating plasma prolactin levels in vivo in cannulated rainbow trout. Following a single intra-arterial injection of chum salmon PrRP (40 nmol kg(-1)) through a dorsal aorta catheter, plasma prolactin levels increased (P

SUMMARY Atlantic salmon (Salmo salar) juveniles were reared under simulated conditions of normal photoperiod (LDN) or short days (LD 9:15) and ambient temperature (AMB: normal temperature increases in April) or an advanced temperature cycle (ADV: temperature increases in February). Under both photoperiod conditions, the timing of increased and peak levels of gill Na+,K+-ATPase activity were not altered by temperature,although the rate of increase was initially greater under ADV. ADV/LD 9:15 resulted in peak gill Na+,K+-ATPase activity that was half of that seen under normal photoperiod and temperature conditions. Plasma growth hormone (GH) levels increased threefold in late March under ADV/LDN,but not under ADV/LD 9:15, indicating that there is a photoperiod-dependent effect of temperature on levels of this hormone. Plasma insulin-like growth factor I (IGF-I) increased in spring in all groups, with increases occurring significantly earlier in the ADV/LDN group. In each photoperiod condition, the advanced temperature cycle resulted in large decreases in plasma thyroxine(T4) levels in March, which subsequently recovered, whereas plasma 3,5,3′-triiodo-L-thyronine (T3) levels were not substantially affected by either photoperiod or temperature. There was no consistent pattern of change in plasma cortisol levels. The results do not provide support for the role of temperature as a zeitgeber, but do indicate that temperature has a role in the timing of smolting by affecting the rate of development and interacting with the photoperiod.

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.

In the tilapia (Oreochromis mossambicus), as in many teleosts, prolactin (PRL) plays a major role in osmoregulation in freshwater. Recently, PRL-releasing peptides (PrRPs) have been characterized in mammals. Independently, a novel C-terminal RF (arginine-phenylalanine) amide peptide (Carrasius RF amide; C-RFa), which is structurally related to mammalian PrRPs, has been isolated from the brain of the Japanese crucian carp. The putative PrRP was purified from an acid extract of tilapia brain by affinity chromatography with antibody against synthetic C-RFa and HPLC on a reverse-phase ODS-120 column. The tilapia PrRP cDNA was subsequently cloned by polymerase chain reaction. The cDNA consists of 619 bp encoding a preprohormone of 117 amino acids. Sequence comparison of the isolated peptide and the preprohormone revealed that tilapia PrRP contains 20 amino acids and is identical to C-RFa. Incubation of the tilapia pituitary with synthetic C-RFa (100 nM) significantly stimulated the release of two forms of tilapia PRL (PRL188 and PRL177). However, the effect of C-RFa was less pronounced than the marked increase in PRL release in response to hyposmotic medium. The ability of C-RFa to stimulate PRL release appears to be specific, since C-RFa failed to stimulate growth hormone release from the pituitary in organ culture. In contrast, rat and human PrRPs had no effect on PRL release. C-RFa was equipotent with chicken GnRH in stimulating PRL release in the pituitary preincubated with estradiol 17beta. Circulating levels of PRL were significantly increased 1 h after intraperitoneal injection of 0.1 microg/g of C-RFa in female tilapia in freshwater but not in males. These results suggest that C-RFa is physiologically involved in the control of PRL secretion in tilapia.

We have examined the interaction of photoperiod and temperature in regulating the parr-smolt transformation and its endocrine control. Atlantic salmon juveniles were reared at a constant temperature of 10°C or ambient temperature (2°C from January to April followed by seasonal increase) under simulated natural day length. At 10°C, an increase in day length [16 h of light and 8 h of darkness (LD 16:8)] in February accelerated increases in gill Na+-K+-ATPase activity, whereas fish at ambient temperature did not respond to increased day length. Increases in gill Na+-K+-ATPase activity under both photoperiods occurred later at ambient temperature than at 10°C. Plasma growth hormone (GH), insulin-like growth factor, and thyroxine increased within 7 days of increased day length at 10°C and remained elevated for 5–9 wk; the same photoperiod treatment at 2°C resulted in much smaller increases of shorter duration. Plasma cortisol increased transiently 3 and 5 wk after LD 16:8 at 10°C and ambient temperature, respectively. Plasma thyroxine was consistently higher at ambient temperature than at 10°C. Plasma triiodothyronine was initially higher at 10°C than at ambient temperature, and there was no response to LD 16:8 under either temperature regimen. There was a strong correlation between gill Na+-K+-ATPase activity and plasma GH; correlations were weaker with other hormones. The results provide evidence that low temperature limits the physiological response to increased day length and that GH, insulin-like growth factor I, cortisol, and thyroid hormones mediate the environmental control of the parr-smolt transformation.

Immunoglobulin M (IgM) is a major component of the humoral immune system of teleosts. This study examines the effects of hypophysectomy and subsequent replacement with prolactin (PRL) or growth hormone (GH) upon the plasma IgM levels of the rainbow trout (Oncorhynchus mykiss). Plasma IgM levels of the hypophysectomized fish were decreased to 30% of those in sham-operated fish 1 or 4 weeks after operation. Implantation of a cholesterol pellet containing salmon PRL or GH restored plasma IgM levels of the hypophysectomized fish, suggesting important roles for PRL and GH in the regulation of circulating IgM level in trout.

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologous in vivo bioassay and by immunoblots using anti-human TSH beta-subunit antisera. In vivo treatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH beta subunit has 56% sequence identity to that of human TSH beta subunit and is identical to the deduced amino acid sequence of trout TSH beta subunit. The N-terminal sequence (25 residues) of the salmon TSH alpha subunit is identical to gonadotropin alpha-II subunit. Molecular sizes of the alpha and beta subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS-PAGE. Antiserum generated against salmon TSH, which was preadsorbed with alpha subunit using an alpha-subunit affinity column, detected only salmon TSH beta subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH beta subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I beta and GTH II beta subunits, alpha subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally, in vivo injection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.

Insulin-like growth factor-I (IGF-I) has been purified from plasma of adult rainbow trout, Oncorhynchus mykiss. Plasma samples were collected 48 hr following injection of recombinant tuna growth hormone at a dose of 0.5 microgram/g body weight. Acid-ethanol extract of plasma was fractionated by gel filtration on a Sephadex G-50 superfine column. Two-step ion-exchange chromatography on DEAE-52 and then Mono-S columns followed. Rainbow trout IGF-I was further purified by immunoaffinity chromatography with anti-recombinant coho salmon IGF-I (rsIGF-I) serum and HPLC on a reverse-phase C18 column. During purification, trout IGF-I was monitored by SDS-PAGE, immunoblotting with anti-IGF-I-serum, homologous radioimmunoassay (RIA) for salmon IGF-I, and sulfation bioassay. The trout IGF-I appeared on SDS-PAGE as a single band with a molecular weight of 7 kDa, the same size as rsIGF-I. The partial N-terminal amino acid sequence (residues 1-20) was identical to the predicted mature trout IGF-I cDNA sequence. Trout IGF-I cross-reacted with anti-rsIGF-I serum in immunoblotting and its dilution curve was parallel to the rsIGF-I standard curve in salmon RIA. In concentrations of 50 and 500 ng/ml, trout IGF-I significantly stimulated sulfation uptake by the cultured branchial cartilage of rainbow trout. This stimulatory effect of trout IGF-I was dose-dependent and similar in its biological potency to rsIGF-I.

Our previous studies have shown that mammalian and salmon insulins stimulate sulphate uptake by cultured eel cartilage, suggesting the possible involvement of insulin in the regulation of cartilage matrix synthesis. In the present study, homologous eel insulin was isolated and characterized, and its effects on cartilage matrix synthesis and DNA synthesis were examined in vitro. Insulin was extracted from eel pancreas with acid–ethanol, and subsequently purified by isoelectric precipitation at pH 5·3, gel filtration on Sephadex G-50, and reversed-phase high-performance liquid chromatography. The amino acid composition and complete sequence (50 residues) of eel insulin revealed high homology to teleostean and mammalian insulins. The isolated eel insulin produced a more pronounced and longer lasting hypoglycaemic effect than bovine insulin in the eel. Homologous eel insulin, like bovine insulin-like growth factor (IGF-I) and insulin, stimulated sulphate uptake by cultured eel cartilage in a dose-dependent manner (16–1000 ng/ml). Combination experiments using maximal concentrations of bovine IGF-I (250 ng/ml) and increasing amounts of eel insulin (10–250 ng/ml) showed no additive effects of insulin on sulphate uptake, suggesting that insulin and IGF-I may share a common mechanism(s) of action. Eel insulin and bovine IGF-I also enhanced thymidine incorporation by eel cartilage in a dose-dependent manner (4–1000 ng/ml); eel insulin was equipotent with bovine IGF-I. These results suggest that insulin, like IGF-I, may exert direct growth-promoting actions in branchial cartilage of the eel. Journal of Endocrinology (1992) 133, 221–230