Acute salinity tolerance and the control of two prolactins and their receptors in the Nile tilapia (Oreochromis niloticus) and Mozambique tilapia (O. mossambicus): A comparative study.

Osmoregulation in vertebrates is largely controlled by the neuroendocrine system. Prolactin (PRL) is critical for the survival of euryhaline teleosts in fresh water by promoting ion retention. In the euryhaline Mozambique tilapia (Oreochromis mossambicus), pituitary PRL cells release two PRL isoforms, PRL188 and PRL177, in response to a fall in extracellular osmolality. Both PRLs function via two PRL receptors (PRLRs) denoted PRLR1 and PRLR2. We conducted a comparative study using the Nile tilapia (O. niloticus), a close relative of Mozambique tilapia that is less tolerant to increases in environmental salinity, to investigate the regulation of PRLs and PRLRs upon acute hyperosmotic challenges in vivo and in vitro. We hypothesized that differences in the regulation of PRLs and PRLRs underlie the variation in salinity tolerance of tilapias within the genus Oreochromis. When transferred from fresh water to brackish water (20‰), Nile tilapia increased plasma osmolality and decreased circulating PRLs, especially PRL177, to a greater extent than Mozambique tilapia. In dispersed PRL cell incubations, the release of both PRLs was less sensitive to variations in medium osmolality in Nile tilapia than in Mozambique tilapia. By contrast, increases in pituitary and branchial prlr2 gene expression in response to a rise in extracellular osmolality were more pronounced in Nile tilapia relative to its congener, both in vitro and in vivo. Together, these results support the conclusion that inter-specific differences in salinity tolerance between the two tilapia congeners are tied, at least in part, to the distinct responses of both PRLs and their receptors to osmotic stimuli.

Control of leptin by metabolic state and its regulatory interactions with pituitary growth hormone and hepatic growth hormone receptors and insulin like growth factors in the tilapia (Oreochromis mossambicus).

Leptin is an important cytokine for regulating energy homeostasis, however, relatively little is known about its function and control in teleost fishes or other ectotherms, particularly with regard to interactions with the growth hormone (GH)/insulin-like growth factors (IGFs) growth regulatory axis. Here we assessed the regulation of LepA, the dominant paralog in tilapia (Oreochromis mossambicus) and other teleosts under altered nutritional state, and evaluated how LepA might alter pituitary growth hormone (GH) and hepatic insulin-like growth factors (IGFs) that are known to be disparately regulated by metabolic state. Circulating LepA, and lepa and lepr gene expression increased after 3-weeks fasting and declined to control levels 10days following refeeding. This pattern of leptin regulation by metabolic state is similar to that previously observed for pituitary GH and opposite that of hepatic GHR and/or IGF dynamics in tilapia and other fishes. We therefore evaluated if LepA might differentially regulate pituitary GH, and hepatic GH receptors (GHRs) and IGFs. Recombinant tilapia LepA (rtLepA) increased hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 from isolated hepatocytes following 24h incubation. Intraperitoneal rtLepA injection, on the other hand, stimulated hepatic igf-1, but had little effect on hepatic igf-2, ghr1, or ghr2 mRNA abundance. LepA suppressed GH accumulation and gh mRNA in pituitaries in vitro, but had no effect on GH release. We next sought to test if abolition of pituitary GH via hypophysectomy (Hx) affects the expression of hepatic lepa and lepr. Hypophysectomy significantly increases hepatic lepa mRNA abundance, while GH replacement in Hx fish restores lepa mRNA levels to that of sham controls. Leptin receptor (lepr) mRNA was unchanged by Hx. In in vitro hepatocyte incubations, GH inhibits lepa and lepr mRNA expression at low concentrations, while higher concentration stimulates lepa expression. Taken together, these findings indicate LepA gene expression and secretion increases with fasting, consistent with the hormones function in promoting energy expenditure during catabolic stress. It would also appear that LepA might play an important role in stimulating GHR and IGFs to potentially spare declines in these factors during catabolism. Evidence also suggests for the first time in teleosts that GH may exert important regulatory effects on hepatic LepA production, insofar as physiological levels (0.05-1 nM) suppresse lepa mRNA accumulation. Leptin A, may in turn exert negative feedback effects on basal GH mRNA abundance, but not secretion.

Rearing Mozambique tilapia in tidally-changing salinities: Effects on growth and the growth hormone/insulin-like growth factor I axis.

The growth hormone (GH)/insulin-like growth factor (IGF) axis plays a central role in the regulation of growth in teleosts and has been shown to be affected by acclimation salinity. This study was aimed at characterizing the effects of rearing tilapia, Oreochromis mossambicus, in a tidally-changing salinity on the GH/IGF axis and growth. Tilapia were raised in fresh water (FW), seawater (SW), or in a tidally-changing environment, in which salinity is switched between FW (TF) and SW (TS) every 6h, for 4months. Growth was measured over all time points recorded and fish reared in a tidally-changing environment grew significantly faster than other groups. The levels of circulating growth hormone (GH), insulin-like growth factor I (IGF-I), pituitary GH mRNA, gene expression of IGF-I, IGF-II, and growth hormone receptor 2 (GHR) in the muscle and liver were also determined. Plasma IGF-I was higher in FW and TS than in SW and TF tilapia. Pituitary GH mRNA was higher in TF and TS than in FW and SW tilapia. Gene expression of IGF-I in the liver and of GHR in both the muscle and liver changed between TF and TS fish. Fish growth was positively correlated with GH mRNA expression in the pituitary, and GHR mRNA expression in muscle and liver tissues. Our study indicates that rearing fish under tidally-changing salinities elicits a distinct pattern of endocrine regulation from that observed in fish reared in steady-state conditions, and may provide a new approach to increase tilapia growth rate and study the regulation of growth in euryhaline fish.

Regulation of gill claudin paralogs by salinity, cortisol and prolactin in Mozambique tilapia (Oreochromis mossambicus).

In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin.

Prolactin 177, prolactin 188, and extracellular osmolality independently regulate the gene expression of ion transport effectors in gill of Mozambique tilapia.

This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380, and 450 mosmol/kg H2O. The mRNA expression of Na(+)/K(+)-ATPase α1a (NKA α1a) and Na(+)/Cl(-) cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na(+)/K(+)/2Cl(-) cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL177 and tPRL188). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKAα1a, and PRLR1 expression; Na(+)/H(+) exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL177 and tPRL188 differentially induce expression of these ion transporters, according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors, as well as directly respond to PRL to modulate branchial ionoregulatory functions.

The effects of acute salinity challenges on osmoregulation in Mozambique tilapia reared in a tidally changing salinity.

This study characterizes the differences in osmoregulatory capacity among Mozambique tilapia, Oreochromis mossambicus, reared in freshwater (FW), in seawater (SW) or under tidally driven changes in salinity. This was addressed through the use of an abrupt exposure to a change in salinity. We measured changes in: (1) plasma osmolality and prolactin (PRL) levels; (2) pituitary expression of prolactin (PRL) and its receptors, PRLR1 and PRLR2; (3) branchial expression of PRLR1, PRLR2, Na(+)/Cl(-) co-transporter (NCC), Na(+)/K(+)/2Cl(-) co-transporter (NKCC), α1a and α1b isoforms of Na(+)/K(+)-ATPase (NKA), cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 3 (AQP3) and Na(+)/H(+) exchanger 3 (NHE3). Mozambique tilapia reared in a tidal environment successfully adapted to SW while fish reared in FW did not survive a transfer to SW beyond the 6 h sampling. With the exception of CFTR, the change in the expression of ion pumps, transporters and channels was more gradual in fish transferred from tidally changing salinities to SW than in fish transferred from FW to SW. Upon transfer to SW, the increase in CFTR expression was more robust in tidal fish than in FW fish. Tidal and SW fish successfully adapted when transferred to FW. These results suggest that Mozambique tilapia reared in a tidally changing salinity, a condition that more closely represents their natural history, gain an adaptive advantage compared with fish reared in FW when facing a hyperosmotic challenge.

Discovery of conventional prolactin from the holocephalan elephant fish, Callorhinchus milii.

The conventional prolactin (PRL), also known as PRL1, is an adenohypophysial hormone that critically regulates various physiological events in reproduction, metabolism, growth, osmoregulation, among others. PRL1 shares its evolutionary origin with PRL2, growth hormone (GH), somatolactin and placental lactogen, which together form the GH/PRL hormone family. Previously, several bioassays implied the existence of PRL1 in elasmobranch pituitaries. However, to date, all attempts to isolate PRL1 from chondrichthyans have been unsuccessful. Here, we cloned PRL1 from the pituitary of the holocephalan elephant fish, Callorhinchus milii, as the first report of chondrichthyan PRL1. The putative mature protein of elephant fish PRL1 (cmPRL1) consists of 198 amino acids, containing two conserved disulfide bonds. The orthologous relationship of cmPRL1 to known vertebrate PRL1s was confirmed by the analyses of molecular phylogeny and gene synteny. The cmPRL1 gene was similar to teleost PRL1 genes in gene synteny, but was distinct from amniote PRL1 genes, which most likely arose in an early amphibian by duplication of the ancestral PRL1 gene. The mRNA of cmPRL1 was predominantly expressed in the pituitary, but was considerably less abundant than has been previously reported for bony fish and tetrapod PRL1s; the copy number of cmPRL1 mRNA in the pituitary was less than 1% and 0.1% of that of GH and pro-opiomelanocortin mRNAs, respectively. The cells expressing cmPRL1 mRNA were sparsely distributed in the rostral pars distalis. Our findings provide a new insight into the studies on molecular and functional evolution of PRL1 in vertebrates.

In vivo and in vitro effects of high-K(+) stress on branchial expression of ROMKa in seawater-acclimated Mozambique tilapia.

Recently, a teleost ortholog of renal outer medullary K(+) channel (ROMK) expressed in gill ionocytes (ROMKa) has emerged as a primary K(+)-excreting pathway in fish. However, the mechanisms by which ROMKa expression is regulated in response to perturbations of plasma K(+) levels are unknown. In this study, we aimed to identify potential links between the endocrine system and K(+) regulation in a euryhaline fish. We assessed time-course changes in multiple endocrine parameters, including plasma cortisol and gene expression of branchial glucocorticoid and mineralocorticoid receptors (GR1, GR2, and MR) and pituitary hormones, in seawater (SW)-acclimated Mozambique tilapia (Oreochromis mossambicus) exposed to high-K(+) (H-K) SW. Exposure to H-K SW elicited little effects on plasma cortisol or mRNA levels of GRs and pituitary hormones. Since plasma K(+) and branchial ROMKa expression was increased within 6h after H-K treatment in vivo, the effect of high K(+) was subsequently tested in a gill filament incubation experiment using media with differing K(+) concentrations. ROMKa mRNA levels were induced following incubation of filaments in H-K medium for 6h. The present study is the first to demonstrate that the expression of ROMKa in teleost ionocytes can respond to high K(+) conditions independent from systemic signaling.

The osmoregulatory effects of rearing Mozambique tilapia in a tidally changing salinity.

The native distribution of Mozambique tilapia, Oreochromis mossambicus, is characterized by estuarine areas subject to salinity variations between fresh water (FW) and seawater (SW) with tidal frequency. Osmoregulation in the face of changing environmental salinity is largely mediated through the neuroendocrine system and involves the activation of ion uptake and extrusion mechanisms in osmoregulatory tissues. We compared plasma osmolality, plasma prolactin (PRL), pituitary PRL mRNA, and mRNA of branchial ion pumps, transporters, channels, and PRL receptors in tilapia reared in FW, SW, brackish water (BW) and in tidally-changing salinity, which varied between FW (TF) and SW (TS) every 6h. Plasma PRL was higher in FW tilapia than in SW, BW, TF, and TS tilapia. Unlike tilapia reared in FW or SW, fish in salinities that varied tidally showed no correlation between plasma osmolality and PRL. In FW fish, gene expression of PRL receptor 1 (PRLR1), Na(+)/Cl(-) cotransporter (NCC), aquaporin 3 (AQP3) and two isoforms of Na(+)/K(+)-ATPase (NKA α1a and NKA α1b) was higher than that of SW, BW or tidally-changing salinity fish. Gene expression of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC1a), and the cystic fibrosis transmembrane conductance regulator (CFTR) were higher in fish in SW, BW or a tidally-changing salinity than in FW fish. Immunocytochemistry revealed that ionocytes of fish in tidally-changing salinities resemble ionocytes of SW fish. This study indicated that tilapia reared in a tidally-changing salinity can compensate for large changes in external osmolality while maintaining osmoregulatory parameters within a narrow range closer to that observed in SW-acclimated fish.

Nutritional status and growth hormone regulate insulin-like growth factor binding protein (igfbp) transcripts in Mozambique tilapia.

Growth in teleosts is controlled in large part by the activities of the growth hormone (Gh)/insulin-like growth factor (Igf) system. In this study, we initially identified igf-binding protein (bp)1b, -2b, -4, -5a and -6b transcripts in a tilapia EST library. In Mozambique tilapia (Oreochromis mossambicus), tissue expression profiling of igfbps revealed that igfbp1b and -2b had the highest levels of expression in liver while igfbp4, -5a and -6b were expressed at comparable levels in most other tissues. We compared changes in hepatic igfbp1b, -2b and -5a expression during catabolic conditions (28days of fasting) along with key components of the Gh/Igf system, including plasma Gh and Igf1 and hepatic gh receptor (ghr2), igf1 and igf2 expression. In parallel with elevated plasma Gh and decreased Igf1 levels, we found that hepatic igfbp1b increased substantially in fasted animals. We then tested whether systemic Gh could direct the expression of igfbps in liver. A single intraperitoneal injection of ovine Gh into hypophysectomized tilapia specifically stimulated liver igfbp2b expression along with plasma Igf1 and hepatic ghr2 levels. Our collective data suggest that hepatic endocrine signaling during fasting may involve post-translational regulation of plasma Igf1 via a shift towards the expression of igfbp1b. Thus, Igfbp1b may operate as a molecular switch to restrict Igf1 signaling in tilapia; furthermore, we provide new details regarding isoform-specific regulation of igfbp expression by Gh.

Effects of salinity and prolactin on gene transcript levels of ion transporters, ion pumps and prolactin receptors in Mozambique tilapia intestine.

Euryhaline teleosts are faced with significant challenges during changes in salinity. Osmoregulatory responses to salinity changes are mediated through the neuroendocrine system which directs osmoregulatory tissues to modulate ion transport. Prolactin (PRL) plays a major role in freshwater (FW) osmoregulation by promoting ion uptake in osmoregulatory tissues, including intestine. We measured mRNA expression of ion pumps, Na(+)/K(+)-ATPase α3-subunit (NKAα3) and vacuolar type H(+)-ATPase A-subunit (V-ATPase A-subunit); ion transporters/channels, Na(+)/K(+)/2Cl(-) co-transporter (NKCC2) and cystic fibrosis transmembrane conductance regulator (CFTR); and the two PRL receptors, PRLR1 and PRLR2 in eleven intestinal segments of Mozambique tilapia (Oreochromis mossambicus) acclimated to FW or seawater (SW). Gene expression levels of NKAα3, V-ATPase A-subunit, and NKCC2 were generally lower in middle segments of the intestine, whereas CFTR mRNA was most highly expressed in anterior intestine of FW-fish. In both FW- and SW-acclimated fish, PRLR1 was most highly expressed in the terminal segment of the intestine, whereas PRLR2 was generally most highly expressed in anterior intestinal segments. While NKCC2, NKAα3 and PRLR2 mRNA expression was higher in the intestinal segments of SW-acclimated fish, CFTR mRNA expression was higher in FW-fish; PRLR1 and V-ATPase A-subunit mRNA expression was similar between FW- and SW-acclimated fish. Next, we characterized the effects of hypophysectomy (Hx) and PRL replacement on the expression of intestinal transcripts. Hypophysectomy reduced both NKCC2 and CFTR expression in particular intestinal segments; however, only NKCC2 expression was restored by PRL replacement. Together, these findings describe how both acclimation salinity and PRL impact transcript levels of effectors of ion transport in tilapia intestine.

Effects of salinity on metabolic rate and branchial expression of genes involved in ion transport and metabolism in Mozambique tilapia (Oreochromis mossambicus).

This study investigated the effects of two rearing salinities, and acute salinity transfer, on the energetic costs of osmoregulation and the expression of metabolic and osmoregulatory genes in the gill of Mozambique tilapia. Using automated, intermittent-flow respirometry, measured standard metabolic rates (SMRs) of tilapia reared in seawater (SW, 130 mg O2 kg⁻¹ h⁻¹) were greater than those reared in fresh water (FW, 103 mg O2 kg⁻¹ h⁻¹), when normalized to a common mass of 0.05 kg and at 25±1°C. Transfer from FW to 75% SW increased SMR within 18h, to levels similar to SW-reared fish, while transfer from SW to FW decreased SMR to levels similar to FW-reared fish. Branchial gene expression of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), an indicator of SW-type mitochondria-rich (MR) cells, was positively correlated with SMR, while Na⁺-Cl⁻ cotransporter (NCC), an indicator of FW-type MR cells, was negatively correlated. Principal Components Analysis also revealed that branchial expression of cytochrome c oxidase subunit IV (COX-IV), glycogen phosphorylase (GP), and a putative mitochondrial biogenesis regulator in fish, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), were correlated with a higher SMR, plasma osmolality, and environmental salinity, while expression of glycogen synthase (GS), PGC-1ß, and nuclear respiratory factor 1 (NRF-1) had negative correlations. These results suggest that the energetic costs of osmoregulation are higher in SW than in FW, which may be related to the salinity-dependent differences in osmoregulatory mechanisms found in the gills of Mozambique tilapia.

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.

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

Prolactin restores branchial mitochondrion-rich cells expressing Na+/Cl- cotransporter in hypophysectomized Mozambique tilapia.

Hypophysectomy and hormone replacement therapy were conducted to investigate the regulation of branchial mitochondrion-rich cell (MRC) recruitment and hormone receptor expression in euryhaline tilapia (Oreochromis mossambicus). Gene expression and immunolocalization of Na(+)/Cl(-) cotransporter (NCC) and Na(+)/K(+)/2Cl(-) cotransporter (NKCC) were used as markers for freshwater (FW)- and seawater (SW)-type MRCs, respectively. In FW fish, hypophysectomy resulted in a significant drop in plasma osmolality, an effect associated with a marked reduction of NCC gene expression and the disappearance of MRCs with apical-NCC immunoreactivity. In contrast, hypophysectomy in SW fish did not impact plasma osmolality, NKCC, or Na(+), K(+)-ATPase(alpha1) gene expression, or the recruitment of MRCs with basolateral-NKCC. Hypophysectomized fish in SW exhibited reduced mRNA levels of prolactin (PRL) receptor 1 and growth hormone (GH) receptor in the gill; GH receptor expression was also reduced following hypophysectomy in FW. PRL replacement therapy restored NCC gene expression and the appearance of MRCs with apical NCC in both FW and SW; there was no interaction of PRL with cortisol. In FW, cortisol modestly stimulated NKCC mRNA levels, while no effect of GH was evident. In SW, no clear effects of hormone replacement on gene expression of NKCC, Na(+), K(+)-ATPase(alpha1), or hormone receptors were detected. Taken together, the essential nature of PRL to survival of Mozambique tilapia in FW is derived, at least in part, from its ability to stimulate the recruitment of MRCs that express NCC, while recruitment of SW-type MRCs does not require pituitary mediation in this euryhaline tilapia.

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia.

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.

Ionoregulatory and endocrine responses to disturbed salt and water balance in Mozambique tilapia exposed to confinement and handling stress.

This study assessed the endocrine and ionoregulatory responses by tilapia (Oreochromis mossambicus) to disturbances of hydromineral balance during confinement and handling. In fresh water (FW), confinement and handling for 0.5, 1, 2 and 6h produced elevations in plasma cortisol and glucose; a reduction in plasma osmolality was observed at 6h. Elevations in plasma prolactins (PRL(177) and PRL(188)) accompanied this fall in osmolality while no effect upon growth hormone (GH) was evident; an increase in insulin-like growth-factor I (IGF-I) occurred at 0.5h. In seawater (SW), confinement and handling increased plasma osmolality and glucose between 0.5 and 6h; no effect on plasma cortisol was seen due to variable control levels. Concurrently, both PRLs were reduced in stressed fish with only transient changes in the GH/IGF-I axis. Next, the branchial expression of Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and Na(+)/Cl(-) cotransporter (NCC) was characterized following confinement and handling for 6h. In SW, NKCC mRNA levels increased in stressed fish concurrently with elevated plasma osmolality and diminished gill Na(+), K(+)-ATPase activity; NCC was unchanged in stressed fish irrespective of salinity. Taken together, PRL and NKCC participate in restoring osmotic balance during acute stress while the GH/IGF-I axis displays only modest responses.

Identification and genomic sequence of a ghrelin receptor (GHS-R)-like receptor in the Mozambique tilapia, Oreochromis mossambicus.

The growth hormone secretagogue-receptor (GHS-R) Is an endogenous receptor for the gut hormone ghrelin (GRLN). Two lsoforms of GHS-R have been Identified In several animals: functional GHS-R1a and a splice variant of unknown function, GHS-R1b. Here we report Identification of a GHS-R-like receptor (GHSR-LR) In the Mozambique tilapia, Oreochromis mossambicus. The cDNA is 1584 bp In length and encodes a 384-amino acid GHS-R1a ortholog. The amino acid sequence of tilapia GHS-R1a is 54, 60, 80 and 89% Identical to that of rat, chicken, pufferfish, and seabream GHS-R1a, respectively. Genomic PCR revealed that the tilapia GHS-R gene Is composed of two exons separated by a single intron. In addition, a GHS-R1b ortholog, which Is generated by alternative splicing of the GHS-R gene and contains part of the intron, was Identified and predicted to be a 298-amino acid protein. Functional analyses of tilapia GHS-R1a were conducted using mammalian HEK 293 and CHO cells, but the expected increase in intracellular calcium Ions by tilapia or rat GRLN was not observed. We found that the GHS-R1a ortholog Is expressed In greater quantities than the GHS-R1b ortholog in all tissues assayed. Further studies are required to conclude that our Identified protein Is the GHS-R for tilapia, although the gene structure and amino acid sequence showed high similarities to other GHS-R genes; thus, we designated this protein GHSR-LR.

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 (1573bp 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 SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta 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.