Tilapia Pituitary Research Articles

Transcriptomic responses of saline-adapted Nile tilapia (Oreochromis niloticus) to rearing in both saline and freshwater.

The Nile tilapia, Oreochromis niloticus, is an important species for global aquaculture. Recently, a single genetic line of Nile tilapia was developed using estimated breeding values (EBVs) for body weight under moderately saline water that showed significant improvement in growth performance. To explore the molecular mechanisms underlying this enhanced growth capacity, RNA-Seq was used to profile differences in gene expression in the liver and pituitary gland of high- and low-growth performance families of male Nile tilapia progeny, reared in either saline or freshwater environments. Comparisons of tissues from high- and low-EBV families, and also between fish reared in either saline or freshwater, revealed 142 and 2208 differentially expressed genes (DEGs), respectively. DEGs identified between the EBV groups comprised a number of genes involved in the regulation of growth and reproduction. We found an overexpression of hormone genes involved in growth-inhibition in the pituitary of Low-EBV tilapia including 2 somatostatin genes (GHIH), corticoliberin (CRH) and tachykinin-3-like protein. Furthermore, several genes associated with the cAMP pathway were underexpressed in low-EBV tilapia pituitary together with several early response genes. This study provides insight into the transcriptomic factors associated with growth performance in saline-adapted Nile tilapia reared in environments with high and low salinity levels and provides valuable knowledge for the future development of selection strategies to improve growth performance in this species.

Effects of Kiss2 on the Expression of Gonadotropin Genes in the Pituitary of Nile Tilapia (Oreochromis niloticus).

Kisspeptin, expressed mainly in the hypothalamus, stimulates gonadotropin-releasing hormone neurons to facilitate reproduction. In some model animals, the kisspeptin is also expressed in the pituitary. Recently, a pathway has been suggested in which kisspeptin acts directly on the pituitary to secretion of gonadotropin in mammals. In the present study, pituitaries of the Nile tilapia (Oreochromis niloticus) were cultured at different concentrations of kisspeptin-10 (Kp-10, FNYNPLSLRF) for 3 hours to observe the effect of kisspeptin on the expression of follicle-stimulating hormone β subunit (fshβ) gene and luteinizing hormone β subunit (lhβ) gene. Pituitary tissues were cultured with 0.1 μM of Kp-10, luteinizing hormone releasing hormone (LHRH), or LHRH+Kp-10 for 3, 6, 12, and 24 hours to investigate changes in the expression of fshβ and lhβ mRNA. Pituitaries cultured with high concentration of Kp-10 more than 0.1 μM for 3 hours exhibited a significant increase of fshβ mRNA expression, but not lhβ mRNA. The expression of both fshβ and lhβ mRNA increased after 6 hours in 0.1 μM of Kp-10 medium in comparison with that in the control medium. Tissues cultured in the LHRH medium however exhibited increased expression of both genes not only at 6 but also 12 hours. There were no significant differences of fshβ and lhβ gene expression in tissues cultured with LHRH+KP-10 medium compared with the control. These results suggested that although kisspeptin plays an important role in fshβ and lhβ expression in the pituitary of Nile tilapia, its action is far more complicated than expected.

Dunaliella salina extract alleviates the toxic impact of Dioxin induced endocrine disruption in Nile tilapia

Pollution of lakes and seas in Egypt become a severe problem as it produces a lot of wastes and toxic chemicals. The water pollution induces a lot of endocrine disruptions in aquatic organisms. Several endocrine organs are influencing by pollution such as testes, ovaries, pituitary, hypothalamus, thyroid, and adrenal organs. Propositions of upsetting endocrine chemicals (EDCs) can meddle with fish larval advancement affecting the delicate balance of the biological ecosystem. Endocrine disruption effects on reproductive cycles may affect reproductive behaviour and the development of secondary sexual characteristics. So the main objective of this research is to use marine algae, Dunaliella salina, extract in fish immunity enhancement against pollutants. Nile tilapia fish were exposed to Dioxin (as the main pollutant in the Egyptian environment) after feeding on algae extracts for one month. The results revealed that Dunaliella salina was able to protect the fish against pollution as it enhances the immune capability of Nile tilapia fish due to increase the levels of the antioxidant enzymes and decrease the protein oxidation. Additionally, Dunaliella salina extract was able to decrease the alteration in the expression of genes encoding endocrine hormones such as LH and FSH in the pituitary of Nile tilapia exposed to Dioxin in comparison to control fish. Moreover, the Dunaliella salina extract decreased significantly the DNA damage in brain tissues of Nile tilapia exposed to Dioxin compared with control fish. The results conclude that Dunaliella salina could be used as ameliorative compounds against toxicity of Dioxin.

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the expression of PACAP in cultured tilapia astrocytes.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone-releasing hormone (GHRH)-PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP(38) and tGHRH and a short form (166 bp) encoding only tPACAP(38). The short form was found to be more abundant in astrocytes. Addition of ovine PACAP(38) (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP(38) at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC(1)-R) mRNA in the astrocytes was not responsive to PACAP(38) treatment. The tPACAP(38) expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP(38) expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.

Irisin stimulates gonadotropins gene expression in tilapia (Oreochromis niloticus) pituitary cells

The link between energy metabolism and reproduction is well known in vertebrates. Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, plays an important role in energy homeostasis. However, biological actions of irisin on reproduction remain elusive. To address this gap, we examined the direct effects of irisin on luteinizing hormone β (LHβ) and follicle-stimulating hormone β (FSHβ) gene expression in tilapia pituitary cells. As a first step, the transcripts of FNDC5 were detected in the proximal pars distalis (PPD), but not in the rostral pars distalis (RPD) and neurointermediate lobe (NIL) of the tilapia pituitary by RT-PCR. In the tilapia pituitary, irisin immunoreactive signals were also detected in PPD region. In primary cultures of tilapia pituitary cells, irisin was effective in stimulating both LHβ and FSHβ mRNA levels in vivo and in vitro. In cultured pituitary cells of tilapia, removal of endogenous irisin by immunoneutralization using irisin antiserum inhibited LHβ and FSHβ gene expression. Salmon gonadotrophin releasing hormone (sGnRH) increased LHβ and FSHβ mRNA levels in tilapia pituitary but these stimulatory actions were not either enhanced by treatment with irisin or blocked by irisin antiserum. Furthermore, the stimulation on LHβ and FSHβ mRNA expression was coincident with the enhancement of LHβ and FSHβ mRNA stability after irisin treatment. These results provide evidence that irisin may serve as a novel intrapituitary factor maintaining gonadotropins gene expression in tilapia pituitary.

Kisspeptin2 stimulates the HPG axis in immature Nile tilapia (Oreochromis niloticus).

It has been suggested that kisspeptin influences reproduction and onset of puberty in fishes. Unlike mammals, which produce only one kisspeptin (Kiss1), some teleosts have two, Kiss1 and Kiss2, both thought to be involved in the stimulation of gonadotropin (GTH) secretion. In Nile tilapia (Oreochromis niloticus), however, only Kiss2 has been identified so far. The effect of Kiss2 on GTH release varies significantly depending on species and reproductive stage. Furthermore, its physiological function in this species is not clearly defined. In this study, kiss2 gene expression profiles were examined using quantitative real-time PCR (qRT-PCR) in the brain, pituitary, and gonads of Nile tilapia at different reproductive stages (male: immature, pre-spermiation, post-spermiation; female: immature, pre-spawning, post-spawning). The kiss2 mRNA expression profiles of the brain, pituitary, and gonads of both sexes shared a similar pattern their expression was significantly higher at the immature stage than at the mature or post-spawning stages, implying it is involved in early gonadal maturation in this species. To investigate the effect of kisspeptin on the hypothalamus-pituitary-gonad (HPG) axis in vivo, synthetic kisspeptin2 (FNYNPLSLRF) was injected into immature male and female tilapia intraperitoneally (i.p.) at a dose of 200pmol/g body weight. The results showed that synthetic Kiss2 administration increased the expression of GnRH I, fshβ and lhβ mRNA in the brain and increased 17β-estradiol (E2) and 11-ketotestosterone (11-KT) levels in the plasma. These results suggest that Kiss2 stimulates the expression of GnRH and GTH genes in immature Nile tilapia.

Stellate Cell Networks in the Teleost Pituitary.

The folliculostellate cells of the mammalian pituitary are non-endocrine cells that are implicated in long-distance communication and paracrine signaling, but to date, these cells have yet to be characterized in teleosts. We found that the stellate cells of the teleost pituitary share many common attributes with mammalian folliculostellate cells. By labeling of stellate cells in live preparations of tilapia pituitaries we investigated their distribution, association with other endocrine cells and their anatomical and functional coupling. In the pars intermedia, stellate cells were arranged around neuronal bundles and their processes extended into the pars distalis. Within the pars distalis, stellate cells formed close associations with FSH cells and, to a lesser degree, with GH and LH cells, suggesting differential paracrine regulation of the two gonadotrope populations. The production of follistatin by stellate cells further corroborates the notion of a paracrine role on FSH release. We also found stellate cells to form gap junctions that enabled dye transfer to neighboring stellate cells, implicating that these cells form a large-scale network that connects distant parts of the pituitary. Our findings represent the first wide-scale study of stellate cells in teleosts and provide valuable information regarding their functional roles in pituitary function.

Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors

Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (−)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism.

Adrenergic Regulation of Somatolactin Gene Expression in Tilapia Pituitary Cells

Epinephrine is an important neuroendocrine regulator to control growth hormone (GH) secretion in vertebrates. Somatolactin (SL), the latest member of the GH family, is a novel pituitary hormone with diverse functions in fish. In a previous report it was shown that epinephrine had a potent inhibitory effect on SL release in fish. However, very little is known about the mechanisms responsible for epinephrine inhibition of SL gene expression. In primary cultures of tilapia neurointermediate lobe (NIL) cells, epinephrine not only reduced SL mRNA levels, but could also abolish pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated SL gene expression. The inhibitory effects of epinephrine on SL gene expression were mimicked by additions of α2-adrenergic agonists clonidine and UK14304, whereas similar treatments with the α1-agonist cirazoline or the β-agonist isoproterenol had no effects in this regard. In parallel experiments, the SL response to epinephrine was significantly abolished by co-incubations with the α2- antagonist yohimbine, but the α1- or β-antagonist was not effective in this regard. In tilapia NIL cells, the α2-adrenergic agonist clonidine suppressed cAMP production and blocked forskolin and PACAP induction of total cAMP production. By using a pharmacological approach, the adenylate cyclase (AC) activator- and cAMP analog-stimulated SL responses were blocked treatment with clonidine. Furthermore, adrenergic inhibition of SL gene expression was also mimicked by inhibiting AC and blocking protein kinase A (PKA). These results, as a whole, suggest that α2-adrenergic stimulation can downregulate SL gene transcription by inhibiting the AC/cAMP-dependent mechanism at the tilapia pituitary level.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic–pituitary–interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes.

LPXRFa, the piscine ortholog of GnIH, and LPXRF receptor positively regulate gonadotropin secretion in Tilapia (Oreochromis niloticus).

LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive hypothalamo-pituitary-gonadal axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides that varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, whereas the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by ip injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, βLH cells were found to co-express both tilapia lpxrf and tilapia lpxrf-r mRNA, whereas some of the βFSH cells coexpressed only lpxrf-r mRNA. No coexpression of tilapia lpxrf-r was identified in GH-positive cells. These findings suggest that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.

Prolactin is a major inhibitor of hepatic Leptin A synthesis and secretion: Studies utilizing a homologous Leptin A ELISA in the tilapia

The present study identifies regulatory interactions between leptin A (LepA) and the pituitary hormone prolactin (PRL). In order to measure tilapia (Oreochromis mossambicus) LepA, an enzyme-linked immunosorbent assay (ELISA) utilizing a rabbit polyclonal antibody specific to tilapia LepA was first developed. The antibody shows strong cross reactivity to recombinant tilapia LepA (rtLepA), and a corresponding 16kDa protein in both tilapia and striped bass plasma, but not to recombinant human leptin (rhLep). The assay has a linear detection range of 0.25–1000nM, with intra- and interassay variability of 9% and 16%, respectively. Plasma LepA levels measured in tilapia ranged from 0.8 to 3.9nM, similar to that found for other vertebrates. Hypophysectomy (Hx) increased circulating LepA and lepa mRNA levels in the liver, the dominant source of hormone production. Adminstration of ovine PRL (oPRL, 5μg/g BW) to Hx fish restored circulating LepA and hepatic lepa mRNA levels to those of control fish. Additionally, oPRL reduced lepa mRNA levels in a dose-dependent fashion in cultured hepatocytes following an 18h incubation. Previous work in our lab indicates that rhLep stimulates PRL release in vitro from tilapia pituitaries. Here, both rtLepA and rhLep (0.5μg/g BW) increased mRNA expression of tilapia prolactin mRNAs (prl1, prl2) in the pituitary in vivo. These results demonstrate that LepA enhances pituitary prolactin synthesis and release, while PRL in turn inhibits hepatic leptin secretion and synthesis in teleosts. We postulate this regulatory interaction may be necessary for mobilizing energy reserves during acute hyperosmotic adaptation.

Stretch-activated cation channel TRPV4 mediates hyposmotically induced prolactin release from prolactin cells of mozambique tilapia Oreochromis mossambicus

In teleost fish, prolactin (PRL) is an important hormone for hyperosmoregulation. The release of PRL from the pituitary of Mozambique tilapia is stimulated by a decrease in extracellular osmolality. Previous studies have shown that hyposmotically induced PRL release is linked with cell volume changes, and that stretch-activated Ca(2+) channels are likely responsible for the initiation of the signal transduction for PRL release. In this study, we identified the stretch-activated Ca(2+) channel transient receptor potential vanilloid 4 (TRPV4) from the rostral pars distalis (RPD) of tilapia acclimated to freshwater (FW). TRPV4 transcripts were ubiquitously expressed in tilapia; the level of expression in RPDs of FW-acclimated fish was lower than that found in RPDs of seawater (SW)-acclimated fish. Immunohistochemical analysis of the pituitary revealed that TRPV4 is localized in the cell membrane of PRL cells of both FW and SW tilapia. A functional assay with CHO-K1 cells showed that tilapia TRPV4 responded to a decrease in extracellular osmolality, and that its function was suppressed by ruthenium red (RR) and activated by 4α-phorbol 12,13-didecanoate (4aPDD). Exposure of dissociated PRL cells from FW-acclimated tilapia to RR blocked hyposmolality induced PRL release. PRL release, on the other hand, was stimulated by 4aPDD. These results indicate that PRL release in response to physiologically relevant changes in extracellular osmolality is mediated by the osmotically sensitive TRPV4 cation channel.

Prolactin177, prolactin188 and prolactin receptor 2 in the pituitary of the euryhaline tilapia, Oreochromis mossambicus, are differentially osmosensitive

Two forms of prolactin (Prl), prolactin 177 (Prl(177)) and prolactin 188 (Prl(188)), are produced in the rostral pars distalis (RPD) of the pituitary gland of euryhaline Mozambique tilapia, Oreochromis mossambicus. Consistent with their roles in fresh water (FW) osmoregulation, release of both Prls is rapidly stimulated by hyposmotic stimuli, both in vivo and in vitro. We examined the concurrent dynamics of Prl(177) and Prl(188) hormone release and mRNA expression from Prl cells in response to changes in environmental salinity in vivo and to changes in extracellular osmolality in vitro. In addition, mRNA levels of Prl receptors 1 and 2 (prlr1 and prlr2) and osmotic stress transcription factor 1 (ostf1) were measured. Following transfer from seawater (SW) to FW, plasma osmolality decreased, while plasma levels of Prl(177) and Prl(188) and RPD mRNA levels of prl(177) and prl(188) increased. The opposite pattern was observed when fish were transferred from FW to SW. Moreover, hyposmotically induced release of Prl(188) was greater in Prl cells isolated from FW-acclimated fish after 6 h of incubation, while the hyposmotically induced increase in prl(188) mRNA levels was only observed in SW-acclimated fish. In addition, prlr2 and ostf1 mRNA levels in Prl cells from both FW- and SW-acclimated fish increased in direct proportion to increases in extracellular osmolality both in vivo and in vitro. Taken together, these results indicate that the osmosensitivity of the tilapia RPD is modulated by environmental salinity with respect to hormone release and gene expression.

Involvement of the cAMP messenger system and extracellular Ca 2+ during hyposmotically-induced prolactin release in the Mozambique tilapia

In accord with its role in freshwater osmoregulation, prolactin (PRL) release from the tilapia pituitary is stimulated by small, physiologically relevant reductions in plasma osmolality, a response that is mediated by an acute influx of intracellular Ca 2+ through stretch-activated Ca 2+channels. In the present study, the role of the calcium and cyclic AMP (cAMP) messenger system in the transduction of a response to a hyposmotic stimulus was examined using dispersed PRL cells and PRL cell membrane preparations from freshwater-acclimated tilapia. When PRL cells were treated with the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (100 μM), significant increases in cAMP levels and PRL release were observed at 1 h. Exposure to reduced medium osmolality (300 mOsmolal) in the presence of IBMX further augmented PRL release. Depletion of Ca 2+ from the incubation medium blocked PRL release even in the presence of IBMX. By contrast, exposure of PRL cells to cholera toxin (CTX), an activator of adenylyl cyclase (AC), stimulated PRL release and cAMP accumulation in both the presence and absence of extracellular Ca 2+. On the other hand, treatment with the Ca 2+ ionophore A23187, which elicits a large rise in intracellular free Ca 2+, reduced cAMP accumulation. Likewise, the AC activity of a PRL cell membrane preparation was reduced as extracellular Ca 2+ concentration increased from 0.1 to 1 μM. These results indicate that: (1) the stimulation of PRL release and cAMP formation by a fall in extracellular osmolality are Ca 2+-dependent; (2) large increases in intracellular Ca 2+ attenuate cAMP formation; (3) direct agonists of cAMP messenger system, such as cholera toxin, however, stimulate PRL release independently of the extracellular Ca 2+. These findings add to the evidence that the osmosensitive response of the tilapia PRL cell is mediated through a Ca 2+-dependent mechanism. Nevertheless, the present findings also suggest that tilapia PRL cells have the ability to rapidly augment release PRL both via a Ca 2+-dependent manner and via a cAMP-dependent pathway in the absence of extracellular Ca 2+.

Prolactin-releasing peptide receptor expressed in the pituitary in Mozambique tilapia Oreochromis mossambicus: An aspect of prolactin regulatory mechanisms

Prolactin (PRL)-releasing peptide (PrRP) has been reported as a strong candidate for a stimulating factor of both PRL secretion and expression in teleost species; however, there is no information available on its receptor. Here we report cDNA cloning and characterization of PrRP receptor expressed in the pituitary of Mozambique tilapia Oreochromis mossambicus. The deduced amino acid sequence of cDNA for tilapia PrRP receptor shared 50–83% homology with other vertebrate homologs. Intracellular calcium mobilization assay revealed that PrRP receptor responded to as low as 1 nM order of tilapia PrRP, indicating its high affinity to PrRP. The expression of PrRP receptor was detected in the brain, pituitary, heart, spleen, kidney and rectum of freshwater (FW)- and seawater (SW)-adapted fish. There was no significant difference between FW and SW fish in transcription levels of PrRP receptor in the rostral pars distalis (RPD) of the pituitary. Similarly, the PrRP expression level in the whole brain was not changed by environmental salinity. Immunohistochemistry with a specific antibody showed that PrRP receptor was mainly localized in the cells of the RPD and neurohypophysis in the pituitary of both FW and SW tilapia. We also examined the effects of PrRP on PRL expression in primary-incubated PRL cells of FW tilapia; PrRP failed to stimulate PRL expression in PRL cells in vitro. These results suggest that in vivo stimulatory effects of PrRP on PRL gene expression reported in teleosts are presumably mediated by an unknown regulator secreted from the neurohypophysis expressing PrRP receptor.

Further Insights into the Insulin‐like Growth Factor‐I System of Bony Fish Pituitary with Special Emphasis on Reproductive Phases and Social Status

Insulin-like growth factor (IGF)-I, which is crucially involved in fish growth, differentiation, and reproduction, occurs in tilapia pituitary. IGF-I peptide, which is probably produced in hypothalamic perikarya, is present in axons of the neurohypophysis, and IGF-I mRNA and peptide are present in the adenohypophysis in adrenocorticotrophic hormone cells, melanocyte-stimulating hormone cells, with interindividual differences in growth hormone cells, and, starting with puberty, in gonadotrophic hormone (GTH) cells. Subordinate males showed a high IGF-I but a lower beta-luteinizing hormone expression, while in dominant males the opposite was found. IGF-I from the GTH cells may act as auto/paracrine regulators of GTH cell proliferation and enhance GTH synthesis and release during puberty and reproduction, depending on the social status.

CDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca 2+ and acid–base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573 bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLα of advanced teleosts such as medaka and flounder, and around 50% identity with SLβ of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Orexin-B-like immunoreactivity localized in both luteinizing hormone- and thyroid-stimulating hormone-containing cells in the Nile tilapia ( Oreochromis niloticus) pituitary

Immunohistochemical techniques were employed to examine orexin-like immunoreactivities in the pituitary of the Nile tilapia ( Oreochromis niloticus). Rabbit anti-orexin-A serum and mouse anti-orexin-B monoclonal antibodies were used as primary antibodies. Orexin-B immunoreactive cells corresponded to luteinizing hormone (LH)- or thyroid-stimulating hormone (TSH)-containing cells, and all LH- and TSH-containing cells were immunoreactive for orexin-B. However, we found no orexin-A immunoreactive cells in the pituitary. In the Nile tilapia, an orexin-B-like substance may be secreted from LH- or TSH-containing cells and may regulate pituitary function, rather than the orexin-A-like substance in the pituitaries of Japanese seaperch and medaka.