Pituitary Of Fish Research Articles

Deciphering Posttranslational Processing Events in the Pituitary of a Neopterygian Fish: Cloning of a Gar Proopiomelanocortin cDNA

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The β-endorphin sequence had 91% sequence identity with sockeye A β-endorphin and 71% sequence identity withXenopus laevisβ-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar α-MSH sequence was identical to the α-MSH sequence in rat POMC. The gar β-MSH sequence had 77% sequence identity with salmonid forms of β-MSH and 53% sequence identity with tetrapod forms of β-MSH. The γ-MSH region of gar POMC only had 26% primary sequence identity with tetrapod γ-MSH sequences. Gar γ-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar γ-MSH is discussed in light of the absence of this sequence in salmonid fish.

Mode of action of (−)nicotine on GtH2 secretion from pituitary cells of common carp (Cyprinus carpio L.)

Abstract Enzymatically dispersed carp pituitary cells in a continuous perifusion system were used to investigate the mode of the action of nicotine on GtH2 secretion. Nicotine given in a 12-min pulse stimulated GtH2 release in a dose-dependent manner. Doses of 10−7 M and 10−6 M regularly evoked significant increase in GtH2 secretion up to 140% and to 250% of the baseline, respectively. Prolonged (60 min) administration of nicotine showed that the secretory response of the cells consists of two distinct phases: first-acute phase (4 to 14 min) and second-sustained release phase (20 to 40 min). The magnitude of the response during the first phase was dose-dependent and significantly different between doses. In contrast, the magnitude of the response during second phase showed no significant differences between doses and was stabilized at 140% of the baseline. Repeated administration of several 12-min pulses of nicotine showed distinct reaction of the cells according to the dose. Using higher doses (10−6 M) a progressive desensitization of the cells for consecutive stimuli was observed, including absence of response for the last (fourth) pulse. In the case of 10−7 M nicotine, the stimulatory action was observed only during the first pulse. All subsequent next pulses had no effect on the GtH2 secretion rate and this reaction cannot be explained by a desensitization phenomenon. Even though specific nicotinic receptors in fish pituitary secretory cells have been elusive to date, our results bring indirect proof that stimulatory action of nicotine on GtH2 secretion from carp pituitary cells is exhibited by a receptor mediated mechanism.

A V1-type receptor for mediating the neurohypophysial hormone-induced ACTH release in trout pituitary.

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss. Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D50) showed that these peptides affected ACTH release in the following order of potency: CRF (8 x 10(-13) M) > AVT (2 x 10(-10) M) > IT (10(-7) M). Maximal responses (Dmax) were obtained for hormonal concentrations of 10(-10) M, 10(-8) M and 10(-6) M respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides. Specific V1 or V2 agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V1 agonist, [Phe2, Orn8]-oxytocin, stimulated ACTH release while the V2 agonist, [deamino1, Val4, D-Arg8]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V1 agonist with 10(-7) M and 7 x 10(-9) M respectively, and were in the range of values obtained with natural peptides. The V1 antagonist, [d(CH2)5(1), O-Me-Tyr2, Arg8]-vasopressin, and the V2 antagonist, [d(CH2)5(1), D-Ile2, Ile4, Arg8, Ala9]-vasopressin, maximally reversed the 10(-9) M AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 x 10(-10) M concentration of the analogues and a D50 approximately 2 x 10(-11) M. These results demonstrated the presence of only one V1-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V1b-type.

Possible sites of dopaminergic inhibition of gonadotropin release from the pituitary of a teleost fish, tilapia

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED 50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 μM) totally abolished this stimulation. Dopamine (DA; 1 μM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 μM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D 2 type mode of action. The inhibition of GtH release in culture, caused by 1 μM APO, the specific DA D 2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 μM) in perifusion was not reduced by DA (1 μM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca 2+ ionophore A23187, however, their inhibitory effect was reversed by 10 μM ionomycin. The stimulatory effect of ionophore A23187 differed from that of ionomycin in that A23187 was able to stimulate GtH release only in the presence of Ca 2+ in the medium, whereas ionomycin stimulated the release also in the absence of the ion, and even in the presence of EGTA. It is assumed that ionomycin at 10 μM promotes the mobilization of Ca 2+ from intracellular stores. As neither ionomycin nor AA caused any leakage of lactic dehydrogenase from cultured pituitary cells, the GtH released in response to this agonist is specific and cannot be attributed to damage of the cells' membrane. It is proposed that, in addition to sites distal to cAMP formation and proximal to AA formation, DA may inhibit GtH release from the pituitary of tilapia at sites distal to Ca 2+ influx and at a step proximal to Ca 2+ mobilization from intracellular sources.

Cloning of a Growth Hormone from a Primitive Bony Fish and Its Phylogenetic Relationships

Growth hormone (GH), prolactin, and their relatives constitute a multigene family which is considered to have evolved from a common ancestor. The structural and functional domains of GH appear to be highly conserved among vertebrates. In order to investigate the phylogenetic relationships among GHs in the Actinopterygii and Sarcopterygii, we have cloned and sequenced GH from the pituitary of the primitive bony fish, Amia calva. Bony fishes (teleosts) and Amia (Halecomorphi) are purported sister-groups within the Neoptergii, hence studies on this perspective group of fish can provide insights into the evolution of GH. The deduced amino acid (aa) sequence from A. calva GH (amGH) cDNA revealed that the mature GH consists of 190 residues. Phylogenetic comparisons with GH aa sequences from blue shark, sturgeon, four teleosts (eel, carp, porgy, flounder), and two sarcopterygians (African lungfish and bullfrog) indicated, in the most parsimonious cladogram, that amGH clusters as the sister-group to the teleosts, that sturgeon is the sister-group to the Neopterygii, and that the African lungfish and bullfrog are in the same clade.

Distribution of Variant Forms of Immunoreactive Gonadotropin-Releasing Hormone and β-Gonadotropins I and II in the Platyfish, Xiphophorus maculatus, from Birth to Sexual Maturity

Immunoreactive (ir) lamprey (l), mammalian (m), chicken II (chII), and salmon (s) gonadotropin-releasing hormone (GnRH) and ir-coho salmon β-gonadotropins I and II (GTH I and GTH II) have been localized in the brain and pituitary gland of Xiphophorus maculatus, the platyfish, at various stages of development from birth to sexual maturity. Ir-GTH I was found in the pituitary gland at all stages examined; ir-GTH II was found only in animals in the pubertal and mature stages. Ir-sGnRH was seen only in the pituitary gland of neonatal and immature animals. In pubertal fish, it was found in the nucleus olfactoretinalis (NOR) of the brain as well as in the pituitary gland. In older animals it was found in the nucleus preopticus periventricularis (NPP) and the nucleus lateralis tuberis (NLT), as well as in the NOR and the pituitary gland. Ir-mGnRH was seen in the pituitary of neonatal and immature fish. It was restricted to the NOR and pituitary gland of pubertal animals. In mature animals, it had a similar, but not identical, distribution as that of ir-sGnRH. Antisera to mGnRH and sGnRH appeared to stain the same cells in the brain and pituitary gland. Ir-lGnRH was found only in the pituitary gland of animals of all ages where it stained the same cells as ir-mGnRH and ir-sGnRH. Ir-chIIGnRH was absent in all immature stages. In animals that were sexually mature it was seen in tracts in the NPP, nucleus preopticus (NPO), NLT, and pituitary gland. Ir-chIIGnRH seemed to colocalize with ir-sGnRH in the pituitary but there was no apparent colocalization with any other form of ir-GnRH in the brain. These results demonstrate that variant forms of GnRH and GTH are present at defined stages of development in specific regions of the brain and pituitary gland and suggest that different forms of GnRH and GTH regulate different aspects of reproductive system development and physiology.

Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

Detection of [formula omitted] forms of α-MSH and β-endorphin in the intermediate pituitary of the holostean fishes, Lepisosteus spatula, Lepisosteus osseus, and Amia calva

Acid extracts of the intermediate pituitaries of the gars, L. spatula and L. osseus, were fractionated by Sephadex G-50 column chromatography and analyzed by radioimmunoassay. This procedure revealed that immunoreactive forms of N- acetylated β-endorphin- and α-MSH-sized material were present in equimolar amounts and represented the major end products of the POMC biosynthetic pathway in these species. Cation-exchange chromatography indicated that multiple N- acetylated forms of β-endorphin were present in the intermediate pituitaries of the two species of gar, and that these forms differed in their net positive charge and in their apparent molecular weight. Reversed-phase HPLC analysis of the α-MSH-related material indicated that up to 90% of the total MSH in the pituitary of the gar was N- acetylated . Furthermore, the predominant form of α-MSH in both species of gar was N,O- diacetyl-ACTH(1–13)- NH 2 . Nearly identical results were obtained following the analysis of α-MSH-related peptides in the intermediate pituitary of the bowfin, A. calva. The pattern of posttranslational processing of POMC observed in the intermediate pituitaries of holostean fishes is very similar to the processing events observed in lungfishes, turtles, and mammals; hence, the processing of POMC has been remarkably conserved during vertebrate evolution.

Intracellular mediation of GnRH action on GTH release in tilapia

The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala(6), Pro(9)-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca(2+) ionophore (A23187; 1-100 μM) or ionomycin (0.02-10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1-10 μM) indicating that Ca(2+) influx via voltage-sensitive Ca(2+) channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 μM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25-125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33-330 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro(2,6), Trp(3)]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca(2+) influx through voltage-sensitive Ca(2+) channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03–3 mM) or forskolin (0.1–10 μM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [d-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01–1.0 μM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01–1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release. These results are consistent with the hypothesis that cAMP is involved as a mediator in the transduction of GnRH stimulation of gonadotropin release in tilapia, and operates in parallel or is interconnected with the system of Ca2+ influx, protein kinase C (PKC) activation and arachidonic acid, as previously described in this and other fish.

Somatolactin, a novel pituitary protein: purification and plasma levels during reproductive maturation of coho salmon.

Somatolactin (SL), a newly discovered fish pituitary protein belonging to the GH/prolactin family, was isolated from coho salmon (Oncorhynchus kisutch). Antibodies were raised to purified coho SL, and a homologous radioimmunoassay was developed and validated. The assay was specific for SL as indicated by the absence of cross-reactivity with coho salmon GH, gonadotrophins I and II and less than 0.2% cross-reaction to prolactin. Serial dilutions of plasma and pituitary extracts from Oncorhynchus species including coho salmon, chinook salmon and rainbow trout were parallel to the coho salmon SL standard curve. Displacement curves for dilutions of Atlantic salmon (Salmo salar) plasma, but not pituitary extract were parallel to the standards. Plasma levels of SL were measured in coho salmon throughout the final year of reproductive maturation. During the period of gonadal growth, plasma SL levels increased and were highly correlated to oestradiol levels in females and 11-ketotestosterone levels in males. Peak levels of SL were observed at the time of final maturation and spawning in both sexes. It is hypothesized that SL may regulate some physiological aspect of reproduction.

Immunocytochemical localization of a galanin-like peptidergic system in the brain and pituitary of some teleost fish

Immunostaining of brain and pituitary sections of teleost fishes (eels, salmonidae, cyprinidae, gourami, sculpin, mullet) with anti porcine galanin (GAL) revealed the presence of immunoreactive (ir) perikarya and a rich network of fibers. Ir-perikarya were located rostrodorsally to the recessus preopticus, and in the posterior tuberal hypothalamus. Ir-fibers were abundant in basal telencephalon and around diencephalic ventricular recesses but never contacted their lumen. Furthermore, they were observed in basal hypothalamus, brainstem and ventral medulla. Ir-fibers passed along corticotropic (ACTH), gonadotropic cells and somatotropes (GH cells) in eel and trout pars distalis, but rarely ended in caudal neurohypophysis. In goldfish pituitary ir-fibers occurred in neural digitations and among different cell types which however did not contain a GAL-like peptide. The relation GAL fibers/GH cells appeared more evident in species with a high growth rate. The other species showed a similar distribution of brain GAL. In eels and trout, ir-perikarya were not observed in areas containing somatostatin, GH- and ACTH-releasing factor, and ACTH-like perikarya, suggesting that GAL did not coexist with these peptides. The widespread distribution of a GAL-like peptide in teleost brain suggests that it could play a role of neurotransmitter and/or neuromodulator and regulate the secretion of adenohypophysial hormone(s).

Regulation of differential release of alpha-melanocyte-stimulating hormone forms from the pituitary of a teleost fish, Oreochromis mossambicus.

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of alpha-MSH (des-acetyl, mono-acetyl and di-acetyl alpha-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl alpha-MSH was, especially in comparison with monoacetyl alpha-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of alpha-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of alpha-MSH release, but did not modulate the relative abundance of the different forms of alpha-MSH released from the MSH cells. TRH was a potent stimulator of alpha-MSH release. It enhanced in vitro the release of di-acetyl alpha-MSH more than the release of mono-acetyl alpha-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro.

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [3H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45Ca2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. Our studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca2+ metabolism.

Hypo- and hypercalcemic peptides in fish pituitary glands

Immunoreactive parathyroid hormone (irPTH), PTH-related peptide (PTHrp), and stanniocalcin-like peptides were detected in Coho salmon pituitary glands. The PTH, PTHrp, and stanniocalcin immunoreactivity of the salmon pituitary was separated by high-performance liquid chromatography purification into distinct peptide fractions that coeluted with pure preparations of these hormones. Stanniocalcin was localized by immunocytochemistry in the neurohypophysis and preoptic area of the platyfish brain, in areas in which irPTH has previously been detected in other teleosts. Although it is well established that the fish pituitary exerts hypercalcemic control over calcium metabolism, these results demonstrate the presence of hyper- and hypocalcemic peptides in teleost pituitary glands. The detection of PTHrp in the fish pituitary is the first report of its presence outside of mammalian species and suggests an early evolutionary divergence of PTH and PTHrp from an ancestral gene.

Autoradiographic distribution of galanin binding sites in the brain and pituitary of the sea bass ( Dicentrarchus labrax)

Specific binding sites for galanin (GAL) were detected in brain and pituitary of a marine teleost fish, the sea bass, after in vitro incubation of tissue sections with [ 125I]GAL and light microscopic autoradiography. Binding conditions were optimized and as a result the binding was saturable and specific. In the brain, [ 125I]GAL binding was found to occur in all parts of the dorsal and ventral telencephalon, in the anterior, tuberal and posterior hypothalmus, in the thalamus and in the tectum opticum, in the inferior lobe and in the ventral medulla oblongata. In the pituitary dense [ 125I]GAL binding was confined to the area occupied by the prolactin cells in the rostral part of the adenohypophysis. These findings provide the first anatomical evidence for the presence of GAL specific binding sites in the teleost brain and pituitary.

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.

A competitive enzyme immunoassay for chum salmon growth hormone.

An enzyme immunoassay (EIA), based on a competitive assay system, for the measurement of growth hormone (GH) in the pituitary of salmonid fishes and for the hormone released into pituitary incubation medium was developed using a rabbit antiserum against natural chum salmon growth hormone (nsGH I) isolated from pituitary glands. Recombinant salmon GH (rsGH I), was coupled to horseradish peroxidase (HRP). Antibody-bound HRP-rsGH I was separated from free HRP-rsGH I by a double antibody precipitation method. The enzyme activity in the precipitate was detected by a colorimetric method, in which o-phenylenediamine and hydrogen peroxide were used as substrates. nsGH I and rsGH I showed different affinity to the antibody. The displacement curves for extracts from chum salmon and rainbow trout pituitaries, and incubation medium of the pars distalis of the trout were parallel to the GH standard, indicating that the present GH-EIA could estimate GH concentrations in those solutions. An alkaline solution was more effective than saline to extract GH from the pituitary gland. Among the chum salmon adenohy-phophyseal hormones examined, only gonadotropin I showed slight cross-reactivity (less than 0.1%). Neurohypophyseal hormones such as arginine vasotocin and isotocin showed slight (1%) and parallel cross-reactivities to the standard hormone.

Development and validation of a competitive enzyme immunoassay for chum salmon prolactin: A comparison to radioimmunoassay

An enzyme immunoassay (EIA), based on a competitive assay system, for the measurement of prolactin (PRL) in the pituitary of salmonid fishes and of hormone released in medium from incubated pituitary was developed using a rabbit antiserum to chum salmon PRL (PRL, a combination of PRL I and PRL II). Chum salmon PRL was coupled to horseradish peroxidase (HRP). The incubation procedure for the antigen-antibody reaction was analogous to that in the radioimmunoassay (RIA) for PRL. The antibody-bound HRP-PRL was separated by a double antibody method. The enzyme activity in the precipitate was followed by a colorimetric method, in which 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine were used as substrates. PRL, PRL I, and PRL II showed exactly the same competitive curves in the EIA system. PRL (127–158) showed the highest cross-reactivity among the fragments of PRL examined. Low cross-reactivity was seen with other hormones isolated from chum salmon pituitary. The displacement curves for pituitary extracts from several salmonids, including chum salmon, coho salmon, and rainbow trout, were parallel to that of the PRL standard, whereas those from the carp and tilapia showed negligible cross-reactivity. A parallel displacement curve to the PRL standard was also seen with incubation medium of the pars distalis of the chum salmon pituitary. Plasma from chum salmon, coho salmon, and rainbow trout gave nonspecific HRP activity in the EIA. The values of PRL-EIA were significantly correlated ( y = 0.99 x + 1.06, r = 0.942, P < 0.05, n = 24) with those obtained in PRL-RIA. The ED 50 of PRL-EIA was 11.5 ± 0.86 ng ( n = 10). The interassay coefficient was 23.7% at ED 50 ( n = 10). Intraassay coefficients of variation were 12.8% at 5 ng and 10.6% at 20 ng ( n = 10).

Correlations between pituitary and testis in sexually maturing male rainbow trout, Oncorhynchus mykiss, during spermatogenesis

Histological changes in the pituitary of sexually maturing male rainbow trout, Oncorhynchus mykiss, were correlated with spermatogenesis in the testes. Granular basophils in the pituitary appeared simultaneously with the development of spermatogonial cysts in the testis. There was no histological evidence that basophils released their granules during spermatogonial cyst formation. A coefficient of correlation was calculated to test the interaction between the numbers of granular basophils and cysts of spermatogonia. The correlation was not significant. These findings suggest that granules from pituitary basophils do not play a significant role in the development of spermatogonial cysts. Histological sections revealed that basophils degranulated rapidly with the appearance of cysts of spermatocytes. A significant correlation (r = −96) between basophils and cysts of spermatocytes occurred when the interaction between these two variables was tested. The negative correlation indicated that pituitary basophils decreased in number as cysts of spermatocytes in the testes increased. These findings provide support for the hypothesis that basophils may play a significant role in the development of spermatocyte cysts in rainbow trout testes. A second nongranular basophil observed in the pituitaries of fish in this study had no apparent correlation with any of the stages of spermatogenesis. While previous investigators have reported the appearance of granular basophils in the pituitary concurrent with the process of spermatogenesis, this marks the first study that compares histological changes in the testis directly with those in the pituitary in sexually maturing rainbow trout. In addition, it reports for the first time a significant correlation between basophils and a specific stage in the cycle of spermatogenesis.