Somatolactin Research Articles

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.

Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH(2) 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.

Involvement of somatolactin in background adaptation of the cichlid fish Cichlasoma dimerus

Somatolactin (SL) is a pituitary hormone present exclusively in fish that is involved in different physiological processes. The role of SL was evaluated in Cichlasoma dimerus (Teleostei, Perciformes) exposed for 10 days to a black and white background (BB and WB). Changes in alpha-melanophore stimulating hormone (alphaMSH) and melanin concentrating hormone (MCH) cells were also analyzed for comparison with SL. A melanin dispersing effect was observed in fish exposed to a BB, while a concentrating one was observed in those exposed to a WB. By Western blot, three SL-immunoreactive (ir) bands (32, 28 and 23.5 kD) were evidenced. Pituitary SL-ir levels were 2.66- and 2.67-fold greater in the 32 Kd and 28 kD bands, respectively, in BB fish compared with those of WB fish. The SL-ir 23.5 Kd band was not included in the analysis because of its unknown identity. In addition, SL-ir cell number and area were significantly higher in the BB condition (BB 22.73+/-1.46, WB 7.37+/-0.54 and BB 27.39+/-1.00 microm2; WB: 16.61+/-0.65 microm2). No significant differences were observed in the number of the hypothalamic MCH-ir neurons. However, a significant difference was observed in their nuclear area (BB 11.61+/-0.42 microm2, WB 17.80+/-0.84 microm2). alphaMSH-ir cells showed a marked increased in number (BB 35.96+/-1.22, WB 24.36+/-1.04), but no significant differences were observed in the cell area. In conclusion, this study presented clear evidence towards a possible involvement of SL in the adaptation to background colors in teleost together with alphaMSH and MCH.

Medaka double mutants for color interfere and leucophore free: characterization of the xanthophore–somatolactin relationship using the leucophore free gene

Somatolactin (SL) plays an essential role in body-color regulation in medaka and is encoded by the color interfere (ci) locus. The ci mutant fish possess constitutively increased numbers of leucophores and a concomitant decrease in visible xanthophores. However, the mechanism of action of SL on these cell types, and the role of SL in body-color regulation in other species, is unknown. In this study, we verified an SL-xanthophore relationship in ci mutant fish using the leucophore free (lf) gene. Histological observation of lf larvae indicated that these mutants do not possess differentiated leucophores. The ci-lf double mutant, whose genotype was confirmed using DNA markers, lacked leucophores; however, the number of xanthophores remained low, demonstrating that leucophores are not necessary for mediating SL signaling to xanthophores. This finding suggests a conserved function for SL in xanthophore regulation across species, rather than the evolution of a medaka-specific and leucophore-dependent role of SL in body-color regulation. Our results also demonstrate that the lf gene has an indispensable role in leucophore development epistatic to SL signaling. The lf gene has not been cloned. The high-resolution recombination map surrounding the lf locus constructed in this study, together with medaka whole genome sequences that will be released soon, will allow the rapid cloning of the lf gene by forward genetic approaches.

Distribution of Immunoreactivities for Adenohypophysial Hormones in the Pituitary Gland of the Polypteriform Fish, Polypterus endlicheri

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunoreactive adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian alpha-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-beta-endorphin II as well as anti-mammalian alpha-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHbeta and salmon LHbeta, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.

Effects of salmon GnRH and sex steroid hormones on expression of genes encoding growth hormone/prolactin/somatolactin family hormones and a pituitary-specific transcription factor in masu salmon pituitary cells in vitro

Expression of genes encoding growth hormone (GH), prolactin (PRL), and somatolactin (SL) in growing and maturing salmon was stimulated by gonadotropin-releasing hormone (GnRH) analog during particular periods of the life cycle. GnRH therefore appears to directly and/or indirectly regulate gene expression for GH, PRL, and SL in combination with the pituitary–gonadal axis, such as sex steroid hormones. Direct effects of salmon GnRH (sGnRH), estradiol-17β (E2), testosterone, and 11-ketotestosterone (11KT) on the amounts of GH, PRL, and SL mRNAs were thus examined using primary pituitary cell cultures of masu salmon at the four reproductive stages. We also determined the amounts of mRNA encoding pituitary specific POU homeodomain transcription factor (Pit-1) by real-time polymerase chain reactions. The amounts of GH, PRL, and SL mRNAs in the control cells elevated with gonadal maturation, coincidently with those of Pit-1 mRNA. sGnRH at 1.0 nM elevated the amounts of all mRNAs examined in the pre-spawning females, whereas significant effects were not observed with 100 nM sGnRH at any reproductive stages. Sex steroid hormones had no significant effects before initiation of gonadal maturation and at the maturing stage. In the males, E2 tended to decrease the amounts of SL mRNA in the pre-spawning stage. In the females, E2 and 11KT increased the amounts of PRL and SL mRNAs in the pre-spawning stage, but halved those of PRL mRNA in the spawning stage. The amounts of Pit-1 mRNA changed coincidently with those of PRL and SL mRNAs at all examined stages. The effects of E2 alone were abolished by 100 nM sGnRH. The present results indicated that both sGnRH and steroid hormones directly modulate synthesis of Pit-1, and further expression of PRL and SL genes. sGnRH may indirectly regulate GH/PRL/SL family hormone genes through the pituitary–gonadal axis, particularly in the late stage of gametogenesis.

Developmental ontogeny of prolactin and its receptor in fish

Prolactin (PRL) is a member of a family of structurally similar proteins which includes growth hormone (GH) and somatolactin (SL) in teleost fish. The genes encoding these proteins are expressed principally in the pituitary gland and sequence analysis reveals they share considerable similarity. GH, PRL, and SL bring about their physiological action by binding to specific receptors localised in the membrane of cells in target tissue. The PRL receptor (PRLR) and GH receptor (GHR) have been identified in a number of teleosts but the SL receptor remains to be characterised. On hormone binding, receptors dimerise, and signal transduction occurs via the JAK/STAT signalling pathway. The principal action of PRL in fish is freshwater osmoregulation, although it has also been implicated in reproduction, behaviour, growth, and immunoregulation. The role of PRL in early development and metamorphosis is well established, respectively, in mammals and amphibians, although its role in fish is not so well known. Studies have shown that PRL mRNA and protein are restricted to the developing pituitary gland in fish embryos and larvae. PRLR mRNA and protein is also present in fish embryos and has a widespread tissue distribution in larvae. The levels of PRLR and PRL mRNA vary throughout embryonic and early larval development. The potential role of PRL in fish embryos and larvae is considered in relation to their physiological status.

Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass ( Dicentrarchus labrax)

A specific radioimmunoassay (RIA) for European sea bass ( Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH–IGF–I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream ( Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass.

Genomic Structure and Functional Analysis of Promoter Region of Somatolactin Gene of Sea Bream (Sparus aurata)

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone-prolactin family and is produced in the intermediate lobe of teleosts. The SL gene was isolated from a sea bream genomic library and found to be composed of 5 exons distributed within a 9-kb length of DNA. Sequence analysis of the proximal promoter region showed the presence of a classical TATA box located 59 bp upstream from the initial start ATG codon, 5 consensus sequences corresponding to the Pit-1 binding element, and a putative CREB site. In CHO cells cotransfected with the DNA from 2 plasmids, one encoding sea bream Pit-1 under Rous sarcoma virus long terminal repeat regulation and one encoding the SL promoter driving the expression of luciferase, Pit-1 was found to enhance the expression of luciferase. Only one Pit-1 binding site was necessary for enhancement. Analysis by immunoblots of in vitro culture of pituitaries of Sparus aurata showed that several agents, including estradiol, verapamil, and phorbol myristate acetate, had different inhibitory effects on SL and growth hormone released to the culture medium.

Cloning of somatolactin alpha and beta cDNAs in zebrafish and phylogenetic analysis of two distinct somatolactin subtypes in fish.

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin superfamily, with recognizable homologues in all fish taxa examined to date. Although sequences from most fish share reasonably high sequence identity, several more highly divergent SLs have been reported. Goldfish SL and a second SL protein found in rainbow trout (rtSLP) are remarkably different from each other and also dissimilar to other SLs. It has been unclear whether rtSLP is a recent paralogue restricted to rainbow trout, or reflects a more ancient duplication of the SL gene, and whether it is related to the goldfish sequence. Here we report the cloning of two different zebrafish SL cDNAs, which share only 57.5% nucleotide and 47.7% deduced amino acid identities. One copy, designated zebrafish SLalpha (zfSLalpha), displays a typical range of sequence similarity to most other SLs. The other copy, zebrafish SLbeta (zfSLbeta), shows low identity to most other SLs; surprisingly, it is most similar to the divergent SL sequence from goldfish. The mRNAs of zfSLalpha and zfSLbeta were expressed specifically in two distinct regions of the pars intermedia in zebrafish. Cells expressing zfSLalpha are located at the posterior pars intermedia, bordering the neurohypophysis, whereas zfSLbeta is expressed in the anterior part of the pars intermedia, bordering the pars distalis. Phylogenetic analyses indicate that zfSLbeta, goldfish SL and rtSLP all belong to the SL hormone family; however, along with the genes from eel and catfish, these divergent sequences form a group that is clearly distinct from all other SLs. These results suggest the presence of two distinct SL families, SLalpha and SLbeta, which may trace back to a teleost genome duplication prior to divergence of the cyprinids and salmonids.

Nutritional assessment of somatolactin function in gilthead sea bream ( Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream ( Sparus aurata) has been analysed. First, a down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH) administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 μg/g fish) evoked a short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 μmol/g fish) on plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a daily and seasonal signal of abundant energy at a precise calendar time.

돌돔(Oplegnathus fasciatus) somatolactin cDNA의 분석

돌돔 (Oplegnathus fasciatus) SL을 암호화하는 cDNA clone을 뇌하수체로부터 RT-PCR 방법에 의해 획득하였다. 돌돔 SL cDNA의 길이는 1636 bp로서 24개의 아미노산인 signal peptide와 207개 의 aa으로 구성된 mature protein을 암호화하는 696 bp의 open reading frame을 갖고 있다. 또한, 돌돔 SL 아미노산에는 이황화 결합에 관여하는 7개의 시스테인 잔기 <TEX>$(Cys^{5},\; Cys^{15},\; Cys^{42},\; Cys^{65},\; Cys^{181},\; Cys^{198}\$</TEX> 과 <TEX>$Cys^{206})$</TEX>와 두 개의 potential N-glycosylation site인 <TEX>$Asn^{121}$</TEX> 과 <TEX>$Asn^{153}$</TEX>을 확인하였다. 돌돔 SL은 goldfish와 channel catfish를 제외한 다른 경골어류 SL에 아미노산 서열은 61.1∼92.6%, 뉴클레오타이드 서열은 63∼92.6%의 일치를 나타낸다. 아미노산 서열 alingment에서 돌돔 SL은 다른 어류 SL에 공통적인 4개의 conserved domain <TEX>$(A_{SL},\; B_{SL},\; C_{SL}$</TEX>과 <TEX>$D_{SL})$</TEX>을 갖고 있음을 확인하였다. 이들중 <TEX>$A_{SL},\; B_{SL}$</TEX>,과 <TEX>$D_{SL}$</TEX>,은 경골어류 growth hormone과 prolactin에도 잘 보존되어 있었다 재조합 돌돔 SL 단백질을 E. coli에서 생산하기 위해 돌돔 SL cDNA를 발현벡터에 클로닝하여 단백질의 발현을 유도하였다 발현된 단백질은 SDS-PAGE에 의해 분자량 약 27 kDa의 재조합 단백질의 발현을 확인하였다. cDNA encoding somatolactin (SL) was obtained by RT-PCR from pituitary glands of rock bream (Oplegnathus fasciatus). The full length cDNA of rock bream somatolactin (rbSL) is 1636 bp long. It contains a 696 bp open reading frame encoding a signal peptide of 24 amino acids (an) and a mature protein of 207 aa. rbSL has seven cysteine residues<TEX>$(Cys^{5},\; Cys^{15},\; Cys^{42},\; Cys^{65},\; Cys^{181},\; Cys^{198}\; $</TEX>and <TEX>$Cys^{206})$</TEX> and two potential N-glycosylation sites at positions <TEX>$Asn^{121}$</TEX>and <TEX>$Asn^{153}$</TEX>. The rbSL shares 61.1∼92.6% amino acid sequence similarities and 63∼92.6% nucleotide sequence identities with other teleost SLs, except for goldfish and channel catfish SL. Amino acid sequence alignment revealed that rbSL has four conserved domains <TEX>$(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$</TEX> common to all SLs. Out of these domains, <TEX>$(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$</TEX>, are also conserved in all teleost growth hormones and prolactins. The cDNA of rbSL has been cloned into pET expression vector in order to produce recombinant rbSL in E. coli BL2l(DE3) cells. The recombinant protein showed a molecular weight of 27 kDa in SDS-PAGE.

Overwinter fasting and re-feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilisation

This study investigated the roles of cortisol and growth hormone (GH) during a period of fasting in overwintering salmonid fish. Indices of carbohydrate (plasma glucose, liver glycogen), lipid (plasma free fatty acids (FFAs)) and protein metabolism (plasma protein, total plasma amino acids) were determined, together with plasma GH, cortisol and somatolactin (SL) levels at intervals in three groups of rainbow trout (continuously fed; fasted for 9 weeks then fed; fasted for 17 weeks). In fasted fish, a decline in body weight and condition factor was accompanied by reduced plasma glucose and hepatic glycogen and increased plasma FFA. No consistent elevation of plasma GH occurred until after 8 weeks of fasting when plasma GH levels increased ninefold. No changes were observed in plasma total protein and AA until between weeks 13 and 17 when both were reduced significantly. When previously fasted fish resumed feeding, plasma glucose and FFA, and hepatic glycogen levels rapidly returned to control values and weight gain resumed. No significant changes in plasma cortisol levels, related to feeding regime, were evident at any point during the study and there was no evidence that SL played an active role in the response to fasting. The results suggest that overwinter fasting may not represent a significant nutritional stressor to rainbow trout and that energy mobilisation during fasting may be achieved without the involvement of GH, cortisol or SL.

In vitro effect of leptin on somatolactin release in the European sea bass ( Dicentrarchus labrax): dependence on the reproductive status and interaction with NPY and GnRH

The aim of the present work was to investigate the neuroendocrine control of pituitary somatolactin (SL) release using dispersed pituitary cell culture obtained from male European sea bass ( Dicentrarchus labrax) at different stages of sexual development. The effect of mouse recombinant leptin, sea bream gonadotropin releasing-hormone (sbGnRH) and porcine neuropeptide Y (pNPY) and their potential interaction on the SL release were investigated. High doses of leptin (10 −8–10 −6 M) were differentially effective in inducing SL release depending on the sexual developmental stage. Porcine NPY alone was not effective on basal SL release, but it dose-dependently (0.1 and 1 nM) enhanced SL release induced by leptin (10 −6 and 10 −8 M) in late pre-pubertal but not in post-pubertal stages. No effect of sbGnRH in association or not with leptin was observed on SL release. These findings are the first evidences that leptin and pNPY can play an important role in the neuroendocrine control of pars intermedia function and SL release in fish. In addition, the sensitivity of SL producing cells to leptin and NPY only in prepubertal and pubertal stages, provides the potential role of SL in the nutritional control of the onset of puberty.

Ontogeny of adenohypophyseal cells in the pituitary of the American shad ( Alosa sapidissima)

The distribution and ontogeny of adenohypophyseal cells have been studied in the pituitary gland of embryos, larvae, and juveniles of the clupeid American shad ( Alosa sapidissima) using immunocytochemical techniques. In juvenile specimens, adenohypophysis was composed of rostral pars distalis (RPD), formed by cavities lined by prolactin (PRL), adrenocorticotropic hormone (ACTH), and gonadotropic hormone (GTH) cells; proximal pars distalis (PPD), containing growth hormone (GH), GTH, and putative thyroid stimulating hormone (TSH) cells; and pars intermedia (PI) with somatolactin (SL) and melanophore stimulating hormone (MSH) cells. At 3 days post-fertilization (3 days pre-hatching) the pituitary of embryos consisted of an oval mass of cells, close to the ventral margin of the diencephalon, divided in rostral and caudal regions. At this time PRL and ACTH cells appeared in the rostral region of the adenohypophysis, while SL cells were observed in the caudal region where MSH cells showed reactivity 1 day before hatching. At variance, GH cells showed a weak immunoreactivity in the rostral portion at hatching that increased 2 days latter. GTH cells also showed weak immunoreactivity in the rostral region of the adenohypophysis at hatching time. Two days later GTH cells were located in the rostral and central regions of the adenohypophysis. At hatching, the neurohypophysis was very small and no nerve processes were seen to penetrate the adenohypophysis tissue. After hatching, the pituitary gland elongated and in 7 days old larvae, the RPD showed a small lumen surrounded by a palisade of PRL, ACTH, and GHT cells; the PPD showed GH and GTH cells while the PI contained SL and MSH cells. The adenohypophysis and neural lobe increased in size with development and, in 42 days old larvae, they were similar to those of juvenile specimens.

Pituitary gene expression of somatolactin, prolactin, and growth hormone during Atlantic salmon parr–smolt transformation

Abstract Somatolactin (SL), prolactin (PRL) and growth hormone (GH) belong to the same family of adenohypophyseal hormones. In fish, these hormones have all been linked to several important physiological functions. During salmon smoltification, GH has an important role in stimulating growth and improving hypoosmoregulatory ability. The role of PRL in fish osmoregulation has mostly been linked to survival in fresh water (FW), whereas the physiological functions of SL is less clear, having been linked to reproduction, stress, Ca-regulation and acid–base balance. To obtain more information on the roles and interactions of these hormones during the parr–smolt transformation, juvenile Atlantic salmon in FW were sampled from early-February until late-June. Approximately every 2 weeks, fish were sacrificed and pituitaries sampled for assessment of SL, PRL, and GH mRNA. In addition, fish were transferred to seawater (SW) in late-May, and sampled after 1 and 4 weeks in SW. Somatolactin mRNA expression did not change during the parr–smolt transformation of the Atlantic salmon, but increased transiently during the first week in SW, a response which may be correlated to stress and/or Ca-balance. PRL and GH mRNA expression increased transiently during the latter part of the parr–smolt transformation of Atlantic salmon. Following SW entry, the PRL mRNA expression decreased, while GH mRNA expression increased further. This indicates that PRL is of importance during the parr–smolt transformation of the Atlantic salmon, probably by counteracting the hypoosmoregulatory effects of GH and cortisol, allowing the fish to maintain osmotic balance during the smoltification process.

Immunohistochemical localization of gonadotropin releasing hormones in the brain and pituitary gland of the Nile perch, Lates niloticus (Teleostei, Centropomidae)

In the present study we investigated the distribution of gonadotropin-releasing hormones (GnRH) in the brain of Lates niloticus and their association with different pituitary cell types using immunohistochemical techniques. We found immunoreactive (ir) chicken GnRH-II (cGnRH-II) and mammalian GnRH (mGnRH) as the main components of the GnRH-ir system within the brain of the Nile perch. The results indicate that mGnRH and cGnRH are localized in different neurons: mGnRH-ir perikaria were observed in the preoptic region particularly in the organum vasculosum laminae terminalis (OVLT) and in the nucleus lateralis tuberis pars posterior (NLTP) of the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. mGnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, and mesencephalon) and in the pituitary. cGnRH-ir cell bodies are restricted to the optic tract, but few scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II ir fibers, contrasting with an extensive mGnRH innervation. Moreover, mGnRH-ir fibers were targeting the three areas of the pituitary gland: rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). Double immunolabeling studies showed GnRH-ir fibers in close proximity with prolactin (PRL)- and adrenocorticotropic hormone (ACTH)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropins (GTHs)-producing cells in the PPD in the external border of the PI, and with somatolactin (SL)- and α-melanocyte stimulating hormone (α-MSH)-producing cells in the PI. Our results showed direct morphological evidence for a close association of GnRH-ir fibers with the different adenohypophysial cell types. These results suggest a multiple role of GnRH in the regulation of various pituitary hormones’ release.

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.

Changes in expression of genes encoding gonadotropin subunits and growth hormone/prolactin/somatolactin family hormones during final maturation and freshwater adaptation in prespawning chum salmon.

The pituitary levels of mRNAs encoding gonadotropin (GTH) subunits (GTH alpha2 and IIbeta), prolactin (PRL), and somatolactin (SL) increased in chum salmon during the last stages of spawning migration. In the present study, changes in pituitary levels of mRNAs encoding GTH alpha2, Ibeta, and IIbeta; growth hormone (GH); PRL; and SL were examined in homing chum salmon of Sanriku stock to clarify whether the changes are associated with final maturation or freshwater (FW) adaptation. In 1993, fish were caught at four areas: off the coast of Sanriku (off-coast), the mouth of Otsuchi Bay (ocean), inside of Otsuchi Bay (bay), and the Otsuchi River (river). In addition, effects of hypoosmotic stimulation by transition from seawater (SW) to FW were examined in 1994 and 1995. The amounts of mRNAs were determined by dot-blot analyses or real-time polymerase chain reactions. The levels of GTH alpha2 and IIbeta mRNAs in the ocean, bay, and river fish were two to five times those in the off-coast fish, and the levels of SL mRNAs in the bay fish were two to four times those in the off-coast fish. The levels of GH and PRL mRNAs in the ocean and bay fish were significantly lower than those in the off-coast fish, and those in the river fish were three to five times those in the ocean and bay fish. In the SW-to-FW transition experiment in 1994, the levels of GTH alpha2, Ibeta, and IIbeta mRNAs transiently increased, whereas changes were insignificant in 1995. The levels of GH, PRL, and SL mRNAs increased in both SW and FW environments, and no apparent effects of SW-to-FW transition were observed. The present study suggests that in prespawning chum salmon, expression of genes encoding GTH alpha2, IIbeta, and SL elevates with final maturation regardless of osmotic environment. Hypoosmotic stimulation by transition from the SW-to-FW environment is not critical to modulate expression of genes for PRL. PRL gene expression can be elevated in SW fish that were sexually almost matured.

Endocrine mediators of seasonal growth in gilthead sea bream ( Sparus aurata): the growth hormone and somatolactin paradigm

Regulation of somatolactin (SL) and the somatotropic axis was examined year-around at three different stocking times (spring, summer, and autumn) in a Mediterranean fish, the gilthead sea bream ( Sparus aurata). The overall timing of plasma growth hormone (GH) increase was similar among trials (late spring–early summer), but the range of variation year-around was different and followed changes in food intake. Total plasma insulin-like growth factor-I primarily followed changes on growth rates, and a close positive correlation between IGF-I and thermal-unit growth coefficient (TGC) was found irrespective of fish stocking time. Thus, the activation of the somatotropic axis preceded always warm growth spurts, whereas the rise of SL in concurrence with low plasma cortisol levels was found at late autumn. This up-regulation of circulating SL titres preceded the winter inhibition of feeding, and it was more severe in big fish (spring and summer stocking times) than in small fish (autumn stocking time), growing with a relative high efficiency during the cold season despite of a severe hypertriglyceridemia and a high hepatosomatic index. These new insights provide good evidence for a different timing of GH and SL increases, and it is likely that the dominant role of SL in energy homeostasis is to be a mediator of the adaptation to fasting after replenishment of body fat stores, whereas GH and IGF-I are perceived as growth-promoting signals in times of food intake and increasing temperature and day-length.