Darren T. Lerner

Switching of Na+, K+-ATPase isoforms by salinity and prolactin in the gill of a cichlid fish

We identified and investigated the changes in expression of two gill Na(+), K(+)-ATPase α-subunit isoforms (α-1a and α-1b) in relationship with salinity acclimation in a cichlid fish, Mozambique tilapia. Transfer of freshwater (FW)-acclimated fish to seawater (SW) resulted in a marked reduction in α-1a expression within 24 h and a significant increase in α-1b expression with maximum levels attained 7 days after the transfer. In contrast, transfer of SW-acclimated fish to FW induced a marked increase in α-1a expression within 2 days, while α-1b expression decreased significantly after 14 days. Hypophysectomy resulted in a virtual shutdown of α-1a mRNA expression in both FW- and SW-acclimated fish, whereas no significant effect was observed in α-1b expression. Replacement therapy by ovine prolactin (oPrl) fully restored α-1a expression in FW-acclimated fish, while cortisol had a modest, but significant, stimulatory effect on α-1a expression. In hypophysectomized fish in SW, replacement therapy with oPrl alone or in combination with cortisol resulted in a marked increase in α-1a mRNA to levels far exceeding those observed in sham-operated fish. Expression of α-1b mRNA was unaffected by hormone treatment either in FW-acclimated fish or in SW-acclimated fish. The mRNA expression of fxyd-11, a regulatory Na(+), K(+)-ATPase subunit, was transiently enhanced during both FW and SW acclimation. In hypophysectomized fish in FW, oPrl and cortisol stimulated fxyd-11 expression in a synergistic manner. The clear Prl dependence of gill α-1a expression may partially explain the importance of this hormone to hyperosmoregulation in this species.

Acute salinity challenges in Mozambique and Nile tilapia: Differential responses of plasma prolactin, growth hormone and branchial expression of ion transporters

The responses of Mozambique and Nile tilapia acclimated to fresh water (FW) and brackish water (BW; 17 per thousand) were compared following acute salinity challenges. In both species, plasma osmolality increased to above 450 mOsm by 2h after transfer from FW to seawater (SW); these increases in osmolality were accompanied by unexpected increases in plasma prolactin (PRL). Likewise, PRL receptor gene expression in the gill also increased in both species. In Nile tilapia, hyperosmotic transfers (FW to BW and SW) resulted in increased plasma growth hormone (GH) and in branchial GH receptor gene expression, responses that were absent in Mozambique tilapia. Branchial gene expression of osmotic stress transcription factor 1 (OSTF1) increased in both species following transfer from FW to SW, whereas transfer from BW to SW induced OSTF1 expression only in the Nile tilapia. Branchial expression of Na(+)/Cl(-) cotransporter was higher in FW in both species than in BW. Branchial gene expression of Na(+)/K(+)/2Cl(-) cotransporter (NKCC) increased after transfer from BW to SW in Mozambique tilapia, whereas expression was reduced in the Nile tilapia following the same transfer. The difference in the SW adaptability of these species may be related to a limited capacity of Nile tilapia to up-regulate NKCC gene expression, which is likely to be an essential component in the recruitment of SW-type chloride cells. The differential responses of GH and OSTF1 may also be associated with the disparate SW adaptability of these two tilapiine species.

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.

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.

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.

Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in Mozambique tilapia

Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ERα, ERβ1, and ERβ2. A primary function of 17β-estradiol (E(2)) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg). To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ERα and ERβ, and an antagonist for mammalian ERα, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-piperidino-pyrazole (MPP), respectively. ERα was more sensitive and responsive to PPT than ERβ1 or ERβ2 in transactivation assays. All ER isoforms indicated equivalent responsiveness to DPN compared with E(2), although sensitivity to DPN was lower. MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E(2) effect on Vg and ERα 48h post-injection. DPN increased ERα and Vg expression and plasma Vg post-injection, whereas PPT was without effect; DPN seems to stimulate Vg production through activation of ERα. The ligand binding domain of all tilapia ER forms shares only 60-65% amino acid identity with human ERα and ERβ. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs.

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-1β, 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.

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.

Endocrine regulation of prolactin cell function and modulation of osmoreception in the Mozambique tilapia

Prolactin (PRL) cells of the Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors by virtue of their intrinsic osmosensitivity coupled with their ability to directly regulate hydromineral homeostasis through the actions of PRL. Layered upon this fundamental osmotic reflex is an array of endocrine control of PRL synthesis and secretion. Consistent with its role in fresh water (FW) osmoregulation, PRL release in tilapia increases as extracellular osmolality decreases. The hyposmotically-induced release of PRL can be enhanced or attenuated by a variety of hormones. Prolactin release has been shown to be stimulated by gonadotropin-releasing hormone (GnRH), 17-β-estradiol (E2), testosterone (T), thyrotropin-releasing hormone (TRH), atrial natriuretic peptide (ANP), brain-natriuretic peptide (BNP), C-type natriuretic peptide (CNP), ventricular natriuretic peptide (VNP), PRL-releasing peptide (PrRP), angiotensin II (ANG II), leptin, insulin-like growth factors (IGFs), ghrelin, and inhibited by somatostatin (SS), urotensin-II (U-II), dopamine, cortisol, ouabain and vasoactive intestinal peptide (VIP). This review is aimed at providing an overview of the hypothalamic and extra-hypothalamic hormones that regulate PRL release in euryhaline Mozambique tilapia, particularly in the context on how they may modulate osmoreception, and mediate the multifunctional actions of PRL. Also considered are the signal transduction pathways through which these secretagogues regulate PRL cell function.