We identified and investigated the changes in expression of two gill Na C , K C -ATPase a-subunit isoforms (a-1a and a-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 a-1a expression within 24 h and a significant increase in a-1b expression with maximum levels attained 7 days after the transfer. In contrast, transfer of SWacclimated fish to FW induced a marked increase in a-1a expression within 2 days, while a-1b expression decreased significantly after 14 days. Hypophysectomy resulted in a virtual shutdown of a-1a mRNA expression in both FW-and SW-acclimated fish, whereas no significant effect was observed in a-1b expression. Replacement therapy by ovine prolactin (oPrl) fully restored a-1a expression in FW-acclimated fish, while cortisol had a modest, but significant, stimulatory effect on a-1a expression. In hypophysectomized fish in SW, replacement therapy with oPrl alone or in combination with cortisol resulted in a marked increase in a-1a mRNA to levels far exceeding those observed in sham-operated fish. Expression of a-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 C , K C -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 a-1a expression may partially explain the importance of this hormone to hyperosmoregulation in this species.

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. Journal of Endocrinology (2012) 213, 89-98Introduction A necessary component of the systems that direct osmoregulatory processes are osmoreceptors that detect deviations from salt and water balance and initiate adaptive responses to restore equilibrium. The prolactin (Prl)-secreting cells of the euryhaline Mozambique tilapia (Oreochromis mossambicus) comprise a nearly homogeneous portion of the rostral pars distalis (RPD) of the pituitary (conservatively O95%) and are directly stimulated by physiologically relevant decreases in extracellular osmolality in vitro , Seale et al. 2006b. Consistent with its role as an essential hormone for fresh water (FW) acclimation (reviewed by Hirano (1986), Manzon (2002) and Sakamoto & McCormick (2006)) plasma Prl levels increase in vivo when tilapia are transferred from seawater (SW) to FW (Yada et al. 1994, Seale et al. 2002. The operation of tilapia Prl cells as true osmoreceptors and the anatomical organization of these cells in tilapia RPD provide an accessible model to integrate osmosensing mechanisms with gene transcription, hormone synthesis and hormone secretion (Seale et al. 2005).Hyposmotically induced Prl release from tilapia lactotrophs is dependent on the entry of extracellular Ca 2C through stretch-activated ion channels upon cell swelling (Seale et al. 2003). There is evidence that the influx of extracellular Ca 2C may have distinct intracellular effects depending on the acclimation salinity of the animal fro...

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Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation of igf1 and igf2 mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of both igf1 and igf2 in a concentration-related and biphasic manner over the physiological range, with a greater effect on igf2 mRNA level. Insulin increased basal igf2 mRNA level, and strongly increased GH-stimulated igf2 mRNA level, but slightly reduced basal igf1 mRNA level and did not affect GH-stimulated igf1 mRNA level. Cortisol inhibited GH stimulation of igf1, but increased GH stimulation of igf2 mRNA level. The synergistic effect of insulin and GH on igf2 mRNA level was confirmed in vivo. These results indicate that insulin and cortisol differentially modulate the response of igf1 and igf2 mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.

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.

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The peptide hormone prolactin is a functionally versatile hormone produced by the vertebrate pituitary. Comparative studies over the last six decades have revealed that a conserved function for prolactin across vertebrates is the regulation of ion and water transport in a variety of tissues including those responsible for whole-organism ion homeostasis. In teleost fishes, prolactin was identified as the “freshwater-adapting hormone”, promoting ion-conserving and water-secreting processes by acting on the gill, kidney, gut and urinary bladder. In mammals, prolactin is known to regulate renal, intestinal, mammary and amniotic epithelia, with dysfunction linked to hypogonadism, infertility, and metabolic disorders. Until recently, our understanding of the cellular mechanisms of prolactin action in fishes has been hampered by a paucity of molecular tools to define and study ionocytes, specialized cells that control active ion transport across branchial and epidermal epithelia. Here we review work in teleost models indicating that prolactin regulates ion balance through action on ion transporters, tight-junction proteins, and water channels in ionocytes, and discuss recent advances in our understanding of ionocyte function in the genetically and embryonically accessible zebrafish (Danio rerio). Given the high degree of evolutionary conservation in endocrine and osmoregulatory systems, these studies in teleost models are contributing novel mechanistic insight into how prolactin participates in the development, function, and dysfunction of osmoregulatory systems across the vertebrate lineage.

Prolactin (PRL) is a well-known regulator of ion and water transport within osmoregulatory tissues across vertebrate species, yet how PRL acts on some of its target tissues remains poorly understood. Using zebrafish as a model, we show that ionocytes in the gill directly respond to systemic PRL to regulate mechanisms of ion uptake. Ion-poor conditions led to increases in the expression of PRL receptor (prlra), Na+/Cl− cotransporter (ncc; slc12a10.2), Na+/H+ exchanger (nhe3b; slc9a3.2), and epithelial Ca2+ channel (ecac; trpv6) transcripts within the gill. Intraperitoneal injection of ovine PRL (oPRL) increased ncc and prlra transcripts, but did not affect nhe3b or ecac. Consistent with direct PRL action in the gill, addition of oPRL to cultured gill filaments stimulated ncc in a concentration-dependent manner, an effect blocked by a pure human PRL receptor antagonist (Δ1-9-G129R-hPRL). These results suggest that PRL signaling through PRL receptors in the gill regulates the expression of ncc, thereby linking this pituitary hormone with an effector of Cl− uptake in zebrafish for the first time.