Temperature modulates the osmosensitivity of tilapia prolactin cells.

In euryhaline fish, prolactin (Prl) plays an essential role in freshwater (FW) acclimation. In the euryhaline and eurythermal Mozambique tilapia, Oreochromis mossambicus, Prl cells are model osmoreceptors, recently described to be thermosensitive. To investigate the effects of temperature on osmoreception, we incubated Prl cells of tilapia acclimated to either FW or seawater (SW) in different combinations of temperatures (20, 26 and 32 °C) and osmolalities (280, 330 and 420 mOsm/kg) for 6 h. Release of both Prl isoforms, Prl188 and Prl177, increased in hyposmotic media and were further augmented with a rise in temperature. Hyposmotically-induced release of Prl188, but not Prl177, was suppressed at 20 °C. In SW fish, mRNA expression of prl188 increased with rising temperatures at lower osmolalities, while and prl177 decreased at 32 °C and higher osmolalities. In Prl cells of SW-acclimated tilapia incubated in hyperosmotic media, the expressions of Prl receptors, prlr1 and prlr2, and the stretch-activated Ca2+ channel, trpv4,decreased at 32 °C, suggesting the presence of a cellular mechanism to compensate for elevated Prl release. Transcription factors, pou1f1, pou2f1b, creb3l1, cebpb, stat3, stat1a and nfat1c, known to regulate prl188 and prl177, were also downregulated at 32 °C. Our findings provide evidence that osmoreception is modulated by temperature, and that both thermal and osmotic responses vary with acclimation salinity.

Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia.

In estuarine environments, euryhaline fish maintain a narrow range of internal osmolality despite daily changes in environmental salinity that can range from fresh water (FW) to seawater (SW). The capacity of euryhaline fish to maintain homeostasis in a range of environmental salinities is primarily facilitated by the neuroendocrine system. One such system, the hypothalamic-pituitary-interrenal (HPI) axis, culminates in the release of corticosteroids such as cortisol into circulation. Cortisol functions as both a mineralocorticoid and glucocorticoid in fish because of its roles in osmoregulation and metabolism, respectively. The gill, a key site for osmoregulation, and the liver, the primary storage site for glucose, are known targets of cortisol's actions during salinity stress. While cortisol facilitates acclimation to SW environments, less is known on its role during FW adaptation. In this study, we characterized the responses of plasma cortisol, mRNA expression of pituitary pro-opiomelanocortin (pomc), and mRNA expression of liver and gill corticosteroid receptors (gr1, gr2, and mr) in the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity challenges. Specifically, tilapia were subjected to salinity transfer regimes from steady-state FW to SW, SW to FW (experiment 1) or steady state FW or SW to tidal regimen (TR, experiment 2). In experiment 1, fish were sampled at 0 h, 6 h, 1, 2, and 7 d post transfer; while in experiment 2, fish were sampled at day 0 and day 15. We found a rise in pituitary pomc expression and plasma cortisol following transfer to SW while branchial corticosteroid receptors were immediately downregulated after transfer to FW. Moreover, branchial expression of corticosteroid receptors changed with each salinity phase of the TR, suggesting rapid environmental modulation of corticosteorid action. Together, these results support the role of the HPI-axis in promoting salinity acclimation, including in dynamically-changing environments.

Osmosensitive transcription factors in the prolactin cell of a euryhaline teleost.

In euryhaline fish, prolactin (Prl) plays a key role in freshwater acclimation. Prl release in the rostral pars distalis (RPD) of the pituitary is directly stimulated by a fall in extracellular osmolality. Recently, we identified several putative transcription factor modules (TFM) predicted to bind to the promoter regions of the two prl isoforms in Mozambique tilapia, Oreochromis mossambicus. We characterized the effects of extracellular osmolality on the activation of these TFMs from RPDs, in vivo and in vitro. OCT1_PIT1 01, CEBP_CEBP 01 and BRNF_RXRF 01 were significantly activated in freshwater (FW)- acclimated tilapia RPDs while SORY_PAX3 02 and SP1F_SP1F 06, SP1F_SP1F 09 were significantly activated in seawater (SW)- counterparts. Short-term incubation of SW- acclimated tilapia RPDs in hyposmotic media (280 mOsm/kg) resulted in activation of CAAT_AP1F 01, OCT1_CEBP 01, AP1F_SMAD 01, GATA_SP1F 01, SORY_PAX6 01 and CREB_EBOX 02, EBOX_AP2F 01, EBOX_MITF 01 while hyperosmotic media (420 mOsm/kg) activated SORY_PAX3 02 and AP1F_SMAD 01 in FW- tilapia. Short-term incubation of dispersed Prl cells from FW- acclimated fish exposed to hyperosmotic conditions decreased pou1f1, pou2f1b, stat3, stat1a and ap1b1 expression, while pou1f1, pou2f1b, and stat3 were inversely related to osmolality in their SW- counterparts. Further, in Prl cells of SW- tilapia, creb3l1 was suppressed in hyposmotic media. Collectively, our results indicate that multiple TFMs are involved in regulating prl transcription at different acclimation salinities and, together, they modulate responses of Prl cells to changes in extracellular osmolality. These responses reflect the complexity of osmosensitive molecular regulation of the osmoreceptive Prl cell of a euryhaline teleost.

Sex-specific responses to growth hormone and luteinizing hormone in a model teleost, the Mozambique tilapia.

Across the vertebrate lineage, sexual dimorphism in body size is a common phenomenon that results from trade-offs between growth and reproduction. To address how key hormones that regulate growth and reproduction interact in teleost fishes, we studied Mozambique tilapia (Oreochromis mossambicus) to determine whether the activities of luteinizing hormone (Lh) are modulated by growth hormone (Gh), and conversely, whether targets of Gh are affected by the presence of Lh. In particular, we examined how gonadal morphology and specific gene transcripts responded to ovine GH (oGH) and/or LH (oLH) in hypophysectomized male and female tilapia. Hypophysectomized females exhibited a diminished gonadosomatic index (GSI) concomitant with ovarian follicular atresia. The combination of oGH and oLH restored GSI and ovarian morphology to conditions observed in sham-operated controls. A similar pattern was observed for GSI in males. In control fish, gonadal gh receptor (ghr2) and estrogen receptor ß (erß) expression was higher in females versus males. A combination of oGH and oLH restored erß and arß in females. In males, testicular insulin-like growth factor 3 (igf3) expression was reduced following hypophysectomy and subsequently restored to control levels by either oGH or oLH. By contrast, the combination of both hormones was required to recover ovarian igf3 expression in females. In muscle, ghr2 expression was more responsive to oGH in males versus females. In the liver of hypophysectomized males, igf2 expression was diminished by both oGH and oLH; there was no effect of hypophysectomy, oGH, or oLH on igf2 expression in females. Collectively, our results indicate that gene transcripts associated with growth and reproduction exhibit sex-specific responses to oGH and oLH. These responses reflect, at least in part, how hormones mediate trade-offs between growth and reproduction, and thus sexual dimorphism, in teleost fishes.

Tilapia prolactin cells are thermosensitive osmoreceptors.

Prolactin (PRL) cells within the rostral pars distalis (RPD) of euryhaline and eurythermal Mozambique tilapia, Oreochromis mossambicus, rapidly respond to a hyposmotic stimulus by releasing two distinct PRL isoforms, PRL188 and PRL177. Here, we describe how environmentally relevant temperature changes affected mRNA levels of prl188 and prl177 and the release of immunoreactive prolactins from RPDs and dispersed PRL cells. When applied under isosmotic conditions (330 mosmol/kgH2O), a 6°C rise in temperature stimulated the release of PRL188 and PRL177 from both RPDs and dispersed PRL cells under perifusion. When exposed to this same change in temperature, ∼50% of dispersed PRL cells gradually increased in volume by ∼8%, a response partially inhibited by the water channel blocker, mercuric chloride. Following their response to increased temperature, PRL cells remained responsive to a hyposmotic stimulus (280 mosmol/kgH2O). The mRNA expression of transient potential vanilloid 4, a Ca2+-channel involved in hyposmotically induced PRL release, was elevated in response to a rise in temperature in dispersed PRL cells and RPDs at 6 and 24 h, respectively; prl188 and prl177 mRNAs were unaffected. Our findings indicate that thermosensitive PRL release is mediated, at least partially, through a cell-volume-dependent pathway similar to how osmoreceptive PRL release is achieved.

Enhanced expression of ncc1 and clc2c in the kidney and urinary bladder accompanies freshwater acclimation in Mozambique tilapia.

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities via the activities of multiple osmoregulatory organs, namely the gill, gastrointestinal tract, skin, kidney, and urinary bladder. Teleosts residing in freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water, and, therefore, the kidney and urinary bladder reabsorb Na+ and Cl- to support the production of dilute urine. Nonetheless, the regulated pathways for Na+ and Cl- transport by euryhaline fishes, especially in the urinary bladder, have not been fully resolved. Here, we first investigated the ultrastructure of epithelial cells within the urinary bladder of FW-acclimated Mozambique tilapia (Oreochromis mossambicus) by electron microscopy. We then investigated whether tilapia employ Na+/Cl- cotransporter 1 (Ncc1) and Clc family Cl- channel 2c (Clc2c) for the reabsorption of Na+ and Cl- by the kidney and urinary bladder. We hypothesized that levels of their associated gene transcripts vary inversely with environmental salinity. In whole kidney and urinary bladder homogenates, ncc1 and clc2c mRNA levels were markedly higher in steady-state FW- versus SW (seawater)-acclimated tilapia. Following transfer from SW to FW, ncc1 and clc2c in both the kidney and urinary bladder were elevated within 48 h. A concomitant increase in branchial ncc2, and decreases in Na+/K+/2Cl-cotransporter 1a (nkcc1a) and cystic fibrosis transmembrane regulator 1 (cftr1) levels indicated a transition from Na+ and Cl- secretion to absorption by the gills in parallel with the identified renal and urinary bladder responses to FW transfer. Our findings suggest that Ncc1 and Clc2c contribute to the functional plasticity of the kidney and urinary bladder in tilapia.

Transcriptional regulation of prolactin in a euryhaline teleost: Characterisation of gene promoters through in silico and transcriptome analyses.

The sensitivity of prolactin (Prl) cells of the Mozambique tilapia (Oreochromis mossambicus) pituitary to variations in extracellular osmolality enables investigations into how osmoreception underlies patterns of hormone secretion. Through the actions of their main secretory products, Prl cells play a key role in supporting hydromineral balance of fishes by controlling the major osmoregulatory organs (ie, gill, intestine and kidney). The release of Prl from isolated cells of the rostral pars distalis (RPD) occurs in direct response to physiologically relevant reductions in extracellular osmolality. Although the particular signal transduction pathways that link osmotic conditions to Prl secretion have been identified, the processes that underlie hyposmotic induction of prl gene expression remain unknown. In this short review, we describe two distinct tilapia gene loci that encode Prl177 and Prl188 . From our in silico analyses of prl177 and prl188 promoter regions (approximately 1000 bp) and a transcriptome analysis of RPDs from fresh water (FW)- and seawater (SW)-acclimated tilapia, we propose a working model for how multiple transcription factors link osmoreceptive processes with adaptive patterns of prl177 and prl188 gene expression. We confirmed via RNA-sequencing and a quantitative polymerase chain reaction that multiple transcription factors emerging as predicted regulators of prl gene expression are expressed in the RPD of tilapia. In particular, gene transcripts encoding pou1f1, stat3, creb3l1, pbxip1a and stat1a were highly expressed; creb3l1, pbxip1a and stat1a were elevated in fish acclimated to SW vs FW. Combined, our in silico and transcriptome analyses set a path for resolving how adaptive patterns of Prl secretion are achieved via the integration of osmoreceptive processes with the control of prl gene transcription.

Sex, salinity and sampling period dependent patterns of growth hormone mRNA expression in Mozambique tilapia.

Tilapias comprise the second most aquacultured finfish group in the world. Such popularity stems in part from their tolerance to a wide range of environmental conditions and their sexually dimorphic nature, where males grow larger than females. As in other vertebrates, growth in tilapia is regulated by the growth hormone/ insulin like growth factor (GH/IGF) system. Moreover, environmental salinity has previously been shown to directly modulate growth in tilapia. Less is known, however, regarding how salinity may modulate sexually dimorphic growth. Utilizing a species of tilapia of high salinity tolerance, the Mozambique tilapia, Oreochromis mossambicus, we compared gh expression from the pituitary of male and female adults reared in fresh water (FW), seawater (SW), and a tidal regime (TR) characterized by dynamically changing salinities between FW and SW every six hours, over a 24 h period. We found significant effects of sex, salinity regime and whether fish were sampled during daylight or dark hours. In both sexes, gh expression was greater in fish reared in SW and TR compared with those in FW, and greater in fish sampled during dark hours, compared with those sampled in daylight hours. Pituitary gh expression was greater in males than in females reared in SW and TR, but not in FW. These results provide insight on the sex-specific modulation of gh expression by environmental factors in Mozambique tilapia.

Growth hormone regulates intestinal gene expression of nutrient transporters in tilapia (Oreochromis mossambicus).

Among the various ways that growth hormone (GH) underlies the growth physiology of teleost fishes, GH stimulates transport pathways that facilitate the absorption of nutrients across intestinal epithelia. The current study investigated the effects of GH on the gene expression of nutrient transporters in an omnivorous teleost, the Mozambique tilapia (Oreochromis mossambicus). We employed pituitary gland removal (hypophysectomy) and hormone replacement to assess whether GH directs the gene expression of the GH receptor (ghr2), the peptide transporters, pept1a, pept1b and pept2, the amino acid transporter, slc7a9, the Na+/glucose cotransporter, sglt1, the glucose transporter, glut2, and the myo-inositol transporter, smit2, in anterior, middle, and posterior intestine. ghr2 was predominantly expressed in posterior intestine, while pept1a, pept1b, slc7a9, sglt1, glut2, and smit2 exhibited the highest mRNA levels in anterior and/or middle intestine. While hypophysectomized tilapia exhibited diminished expression of ghr2, pept1a, pept1b, slc7a9, and glut2 compared with intact and sham-operated controls, only ghr2, pept1a, pept1b and glut2 levels were restored by GH replacement. Our findings indicate that GH supports growth, at least in part, by stimulating the gene expression of its cognate receptor and key nutrient transporters in the intestine.

Experimental Approaches for Characterizing the Endocrine-Disrupting Effects of Environmental Chemicals in Fish.

Increasing industrial and agricultural activities have led to a disturbing increase of pollutant discharges into the environment. Most of these pollutants can induce short-term, sustained or delayed impacts on developmental, physiological, and behavioral processes that are often regulated by the endocrine system in vertebrates, including fish, thus they are termed endocrine-disrupting chemicals (EDCs). Physiological impacts resulting from the exposure of these vertebrates to EDCs include abnormalities in growth and reproductive development, as many of the prevalent chemicals are capable of binding the receptors to sex steroid hormones. The approaches employed to investigate the action and impact of EDCs is largely dependent on the specific life history and habitat of each species, and the type of chemical that organisms are exposed to. Aquatic vertebrates, such as fish, are among the first organisms to be affected by waterborne EDCs, an attribute that has justified their wide-spread use as sentinel species. Many fish species are exposed to these chemicals in the wild, for either short or prolonged periods as larvae, adults, or both, thus, studies are typically designed to focus on either acute or chronic exposure at distinct developmental stages. The aim of this review is to provide an overview of the approaches and experimental methods commonly used to characterize the effects of some of the environmentally prevalent and emerging EDCs, including 17 α-ethinylestradiol, nonylphenol, BPA, phthalates, and arsenic; and the pervasive and potential carriers of EDCs, microplastics, on reproduction and growth. In vivo and in vitro studies are designed and employed to elucidate the direct effects of EDCs at the organismal and cellular levels, respectively. In silico approaches, on the other hand, comprise computational methods that have been more recently applied with the potential to replace extensive in vitro screening of EDCs. These approaches are discussed in light of model species, age and duration of EDC exposure.

Early-life exposure to 17ß-estradiol and 4-nonylphenol impacts the growth hormone/insulin-like growth-factor system and estrogen receptors in Mozambique tilapia, Oreochromis mossambicus.

It is widely recognized that endocrine disrupting chemicals (EDCs) released into the environment through anthropogenic activities can have short-term impacts on physiological and behavioral processes and/or sustained or delayed long-term developmental effects on aquatic organisms. While numerous studies have characterized the effects of EDCs on temperate fishes, less is known on the effects of EDCs on the growth and reproductive physiology of tropical species. To determine the long-term effects of early-life exposure to common estrogenic chemicals, we exposed Mozambique tilapia (Oreochromis mossambicus) yolk-sac fry to 17ß-estradiol (E2) and nonylphenol (NP) and subsequently characterized the expression of genes involved in growth and reproduction in adults. Fry were exposed to waterborne E2 (0.1 and 1 µg/L) and NP (10 and 100 µg/L) for 21 days. After the exposure period, juveniles were reared for an additional 112 days until males were sampled. Gonadosomatic index was elevated in fish exposed to E2 (0.1 µg/L) while hepatosomatic index was decreased by exposure to NP (100 µg/L). Exposure to E2 (0.1 µg/L) induced hepatic growth hormone receptor (ghr) mRNA expression. The high concentration of E2 (1 µg/L), and both concentrations of NP, increased hepatic insulin-like growth-factor 1 (igf1) expression; E2 and NP did not affect hepatic igf2 and pituitary growth hormone (gh) levels. Both E2 (1 µg/L) and NP (10 µg/L) induced hepatic igf binding protein 1b (igfbp1b) levels while only NP (100 µg/L) induced hepatic igfbp2b levels. By contrast, hepatic igfbp6b was reduced in fish exposed to E2 (1 µg/L). There were no effects of E2 or NP on hepatic igfbp4 and igfbp5a expression. Although the expression of three vitellogenin transcripts was not affected, E2 and NP stimulated hepatic estrogen receptor (erα and erß) mRNA expression. We conclude that tilapia exposed to E2 and NP as yolk-sac fry exhibit subsequent changes in the endocrine systems that control growth and reproduction during later life stages.

Systemic versus tissue-level prolactin signaling in a teleost during a tidal cycle.

Euryhaline Mozambique tilapia (Oreochromis mossambicus) are native to estuaries where they encounter tidal fluctuations in environmental salinity. These fluctuations can be dramatic, subjecting individuals to salinities characteristic of fresh water (FW < 0.5‰) and seawater (SW 35‰) within a single tidal cycle. In the current study, we reared tilapia under a tidal regimen that simulated the dynamic conditions of their native habitat. Tilapia were sampled every 3 h over a 24 h period to temporally resolve how prolactin (PRL) signaling is modulated in parallel with genes encoding branchial effectors of osmoregulation. The following parameters were measured: plasma osmolality, plasma PRL177 and PRL188 concentrations, pituitary prl177 and prl188 gene expression, and branchial prl receptor (prlr1 and prlr2), Na+/Cl--cotransporter (ncc2), Na+/K+/2Cl--cotransporter (nkcc1a), Na+/K+-ATPase (nkaα1a and nkaα1b), cystic fibrosis transmembrane regulator (cftr), and aquaporin 3 (aqp3) gene expression. Throughout the 24 h sampling period, plasma osmolality reflected whether tilapia were sampled during the FW or SW phases of the tidal cycle, whereas pituitary prl gene expression and plasma PRL levels remained stable. Branchial patterns of ncc2, nkcc1a, nkaα1a, nkaα1b, cftr, and aqp3 gene expression indicated that fish exposed to tidally changing salinities regulate the expression of these gene transcripts in a similar fashion as fish held under static SW conditions. By contrast, branchial prlr1 and prlr2 levels were highly labile throughout the tidal cycle. We conclude that local (branchial) regulation of endocrine signaling underlies the capacity of euryhaline fishes, such as Mozambique tilapia, to thrive under dynamic salinity conditions.