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-ß-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.

Regulation of growth hormone (GH) receptor (GHR1 and GHR2) mRNA level by GH and metabolic hormones in primary cultured tilapia hepatocytes.

Growth hormone (GH) regulates essential physiological functions in teleost fishes, including growth, metabolism, and osmoregulation. Recent studies have identified two clades of putative receptors for GH (GHR1 clade and GHR2 clade) in fishes, both of which are highly expressed in the liver. Moreover, the liver is an important target for the anabolic effects of GH via endocrine IGFs, and liver sensitivity to GH is modulated by metabolic hormones. We investigated the effects of GH, insulin, glucagon, cortisol and triiodothyronine on GHR1 and GHR2 mRNA levels in primary cultured tilapia hepatocytes. Physiological concentrations of GH strongly stimulated GHR2 mRNA level (0.5-50×10(-9) M), but did not affect GHR1 mRNA level. Insulin suppressed stimulation of GHR2 mRNA level by GH (10(-8)-10(-6) M). Insulin increased basal GHR1 mRNA level (10(-8)-10(-6) M). Cortisol increased basal GHR2 mRNA level (10(-7)-10(-6) M), but did not consistently affect GH-stimulated GHR2 mRNA level. Cortisol increased basal GHR1 mRNA level (10(-9)-10(-6) M). Glucagon suppressed GH-stimulated GHR2 mRNA level and increased basal GHR1 mRNA level at a supraphysiological concentration (10(-6) M). A single injection of GH (5 µg/g) increased liver GHR2 mRNA level, and insulin injection (5 µg/g) decreased both basal and GH-stimulated GHR2 mRNA levels after 6 h. In contrast, insulin and GH injection had little effect on liver GHR1 mRNA level. This study shows that GHR1 and GHR2 gene expression are differentially regulated by physiological levels of GH and insulin in tilapia primary hepatocytes.

Osmoreception: perspectives on signal transduction and environmental modulation.

Osmoregulation is essential to life in vertebrates and osmoreception is a fundamental element in osmoregulation. Progress in characterizing the mechanisms that mediate osmoreception has been made possible by using a uniquely accessible cell model, the prolactin (PRL) cell of the euryhaline tilapia, Oreochromis mossambicus. In addition to a brief historical overview, we offer a summary of our recent progress on signal transduction and osmosensitivity in the tilapia PRL cell model. Prolactin is a central regulator of hydromineral balance in teleosts in freshwater (FW). Consistent with its essential role in FW osmoregulation, PRL release in tilapia is inversely related to extracellular osmolality, both in vivo and in vitro. Osmotically-driven changes in PRL cell volume control PRL release. A decrease in extracellular osmolality increases cell volume, leading to a rapid influx of Ca(2+) through stretch-activated channels followed by a sharp rise in PRL release. Our recent studies also suggest that cAMP is involved in the osmotic signal transduction, and that acclimation salinity can modulate PRL cell osmosensitivity. Prolactin cells from FW tilapia show a larger rise in PRL release after a reduction in medium osmolality than those from SW fish. Paradoxically, hyposmotically-induced increase in PRL mRNA was observed only in cells from SW fish. Our studies have revealed differences in the abundance of the water channel, aquaporin 3 (AQP3), and the stretch activated Ca(2+) channel, transient receptor potential vanilloid 4 (TRPV4) in PRL cells of FW and SW fish that may explain their differing osmosensitivity and osmoreceptive output in differing acclimation salinities.

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.

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, ERß1, and ERß2. A primary function of 17ß-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 ERß, 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 ERß1 or ERß2 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 ERß. 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.

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 receptor, growth hormone receptor, and putative somatolactin receptor in Mozambique tilapia: tissue specific expression and differential regulation by salinity and fasting.

In fish, pituitary growth hormone family peptide hormones (growth hormone, GH; prolactin, PRL; somatolactin, SL) regulate essential physiological functions including osmoregulation, growth, and metabolism. Teleost GH family hormones have both differential and overlapping effects, which are mediated by plasma membrane receptors. A PRL receptor (PRLR) and two putative GH receptors (GHR1 and GHR2) have been identified in several teleost species. Recent phylogenetic analyses and binding studies suggest that GHR1 is a receptor for SL. However, no studies have compared the tissue distribution and physiological regulation of all three receptors. We sequenced GHR2 from the liver of the Mozambique tilapia (Oreochromis mossambicus), developed quantitative real-time PCR assays for the three receptors, and assessed their tissue distribution and regulation by salinity and fasting. PRLR was highly expressed in the gill, kidney, and intestine, consistent with the osmoregulatory functions of PRL. PRLR expression was very low in the liver. GHR2 was most highly expressed in the muscle, followed by heart, testis, and liver, consistent with this being a GH receptor with functions in growth and metabolism. GHR1 was most highly expressed in fat, liver, and muscle, suggesting a metabolic function. GHR1 expression was also high in skin, consistent with a function of SL in chromatophore regulation. These findings support the hypothesis that GHR1 is a receptor for SL. In a comparison of freshwater (FW)- and seawater (SW)-adapted tilapia, plasma PRL was strongly elevated in FW, whereas plasma GH was slightly elevated in SW. PRLR expression was reduced in the gill in SW, consistent with PRL's function in freshwater adaptation. GHR2 was elevated in the kidney in FW, and correlated negatively with plasma GH, whereas GHR1 was elevated in the gill in SW. Plasma IGF-I, but not GH, was reduced by 4 weeks of fasting. Transcript levels of GHR1 and GHR2 were elevated by fasting in the muscle. However, liver levels of GHR1 and GHR2 transcripts, and liver and muscle levels of IGF-I transcripts were unaffected by fasting. These results clearly indicate tissue specific expression and differential physiological regulation of GH family receptors in the tilapia.

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus.

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.

Disparate release of prolactin and growth hormone from the tilapia pituitary in response to osmotic stimulation.

In most teleost fishes, prolactin (PRL) plays a key role in freshwater (FW) adaptation, whereas growth hormone (GH) is involved in seawater (SW) adaptation in salmonids and certain euryhaline species including the tilapia, Oreochromis mossambicus. Consistent with its osmoregulatory activity, PRL release increases in response to physiologically relevant reductions in extracellular osmolality. When dispersed PRL and GH cells from FW-acclimatized fish were incubated in media of varying osmolalities, PRL release increased significantly in response to a 12% reduction in medium osmolality during 1 and 4h of exposure. By contrast, cells from SW-acclimatized fish responded only to a 24% reduction in osmolality. Growth hormone release on the other hand increased whether medium osmolality was reduced or raised. Cell volume increased together with PRL release during the perifusion of dispersed PRL cells in direct proportion to the reduction in medium osmolality. Growth hormone release increased whether GH cell volume increased or decreased. In in vivo studies, circulating PRL levels increased as early as 1h after the transfer of fish from SW to FW, whereas GH levels remained unchanged during 24h of acclimatization. These results indicate that while PRL and GH cells are osmosensitive, the PRL cells respond to reductions in extracellular osmolality in a manner that is consistent with PRL's physiological role in the tilapia. While the rise in GH release following the reduction in osmolality is of uncertain physiological significance, the rise in GH release with the elevation of medium osmolality may be connected to its role in SW adaptation.

Involvement of phospholipase C and intracellular calcium signaling in the gonadotropin-releasing hormone regulation of prolactin release from lactotrophs of tilapia (Oreochromis mossambicus).

Gonadotropin-releasing hormone (GnRH) is a potent stimulator of prolactin (PRL) secretion in various vertebrates including the tilapia, Oreochromis mossambicus. The mechanism by which GnRH regulates lactotroph cell function is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated, we examined whether GnRH might stimulate PRL release through an increase in phospholipase C (PLC), inositol triphosphate (IP3), and intracellular calcium (Ca(i)2+) signaling. Using Ca(i)2+ imaging and the calcium-sensitive dye fura-2, we found that chicken GnRH-II (cGnRH-II) induced a rapid dose-dependent increase in Ca(i)2+ in dispersed tilapia lactotrophs. The Ca(i)2+ signal was abolished by U-73122, an inhibitor of PLC-dependent phosphoinositide hydrolysis. Correspondingly, cGnRH-II-induced tPRL188 secretion was inhibited by U-73122, suggesting that activation of PLC mediates cGnRH-II's stimulatory effect on PRL secretion. Pretreatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of Ca2+ release from intracellular stores, impeded the effect of cGnRH-II on Ca(i)2+. To further address the possible involvement of intracellular Ca2+ stores, IP3 concentrations in the tilapia rostral pars distalis (RPD containing 95-99% PRL cells) was determined by a radioreceptor assay. We found that GnRH-II induces a rapid (<5min) and sustained increase in IP3 concentration in the RPD. Secretion of tPRL(188) in response to cGnRH-II was suppressed by Ca2+ antagonists (TMB-8 and nifedipine). These data, along with our previous findings that show PRL release increases with a rise in Ca(i)2+, suggest that GnRH may elicit its PRL releasing effect by increasing Ca(i)2+. Furthermore, the rise in Ca(i)2+ may be derived from PLC/IP3-induced mobilization of Ca2+ from intracellular stores along with influx through L-type voltage-gated Ca2+ channels.

Identification of the growth hormone receptor in an advanced teleost, the tilapia (Oreochromis mossambicus) with special reference to its distinct expression pattern in the ovary.

There is considerable evidence that the GH/IGF-I axis plays an important role in female reproduction. We report the isolation and characterization of the GH receptor (GH-R) and its gene expression profile during oogenesis in the tilapia, Oreochromis mossambicus. cDNA encoding GH-R was cloned and sequenced from the tilapia liver. The predicted GH-R preprotein consisted of 635 amino acids and contained a putative signal peptide, an extracellular region with a characteristic motif, a single transmembrane region, and a cytoplasmic region with conserved box 1 and 2 domains. The tilapia GH-R shared 34-74% identities with known GH-Rs in vertebrates. A binding assay using COS-7 cells showed that the cloned GH-R bound specifically to tilapia GH. Northern blot analysis showed a single mRNA transcript in the liver and ovary. In situ hybridization revealed intense signals of GH-R in the cytoplasm and nucleus of immature oocytes. The granulosa and theca cells surrounding vitellogenic oocytes also contained the GH-R mRNA signals. About a tenfold greater level of GH-R mRNA was found in the immature oocytes versus the mature oocytes, along with high levels of IGF-I mRNA. There were no significant changes in mRNA levels of GH-R and IGF-I in the liver or in plasma IGF-I levels during oocyte development. No correlation was found between hepatic GH-R mRNA and ovarian GH-R mRNA. These results suggest that the GH/IGF-I axis in the ovary may be involved in the early phases of oogenesis, under a different regulatory mechanism of GH-R gene expression from that of the liver.

Changes in plasma concentrations of immunoreactive ouabain in the tilapia in response to changing salinity: is ouabain a hormone in fish?

Ouabain, a cardiac glycoside and inhibitor of Na(+), K(+)-ATPase, is now believed to be a steroid hormone in mammals, involved in blood pressure and volume regulation and possibly acting as a natriuretic hormone. We have identified ouabain-like immunoreactivity in the plasma and tissues of a euryhaline teleost, the tilapia (Oreochromis mossambicus), by means of solid-phase extraction followed by a specific radioimmunoassay. Plasma concentrations of immunoreactive ouabain were 5-20pg/ml. Ouabain immunoreactivity was detected in all the tissues examined, with highest concentrations in the head kidney followed by intestine and body kidney. When the fish in fresh water were transferred to seawater, plasma osmolality increased significantly after 2, 4, 8, and 24h. Significant increases were observed in plasma ouabain immunoreactivity after 4 and 24h, and a significant correlation was seen between ouabain immunoreactivity and plasma osmolality. There was also a significant correlation between the plasma osmolality and cortisol concentrations. Upon transfer from seawater to fresh water, significant increases were seen in plasma cortisol after 4 and 8h and in immunoreactive ouabain after 4h. When the correlation was analyzed using all the data obtained during the two transfer experiments, plasma ouabain immunoreactivity and cortisol were significantly correlated with plasma osmolality, whereas there was a significant negative correlation between plasma prolactin and osmolality. A significant positive correlation was also seen between plasma cortisol and ouabain immunoreactivity. These results suggest that immunoreactive ouabain may be involved, together with cortisol, in the maintenance of hydromineral balance in the tilapia.

Effects of transfer from seawater to fresh water on the growth hormone/insulin-like growth factor-I axis and prolactin in the Tilapia, Oreochromis mossambicus.

The effect of freshwater (FW) transfer on growth and on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis was examined in the tilapia, Oreochromis mossambicus. Tilapia were raised in seawater (SW) for 5 months and then transferred to FW for an additional 40 days. The growth rate of the fish transferred to FW was significantly reduced compared with the growth rate of fish that remained in SW. Plasma levels of GH were significantly elevated in FW-transferred fish, as were plasma IGF-I levels. Pituitary GH and liver IGF-I mRNA levels, on the other hand, were significantly reduced in the fish transferred to FW. There was a significant correlation between body mass and mRNA levels of GH and IGF-I, but not with plasma levels of GH and IGF-I. Fish transferred to FW had significantly higher prolactin (PRL)(177) levels than the SW control fish, although there was no difference in plasma PRL(188) levels. Consistent with the hyperosmoregulatory effects of PRL, mRNA levels of both PRL(177) and PRL(188) were significantly higher in FW-transferred fish than in the fish in SW. These results suggest that transferring tilapia from SW to FW activates the GH/IGF-I axis, but growth is still inhibited, possibly due to the greater metabolic cost of osmoregulation in FW than in SW.

Dual mode of cortisol action on GH/IGF-I/IGF binding proteins in the tilapia, Oreochromis mossambicus.

Glucocorticoids are known to impede somatic growth in a wide range of vertebrates. In order to clarify the mechanisms through which they may act in an advanced teleost fish, we examined the effects of cortisol administration on the growth hormone (GH)/insulin-like growth factor-I (IGF-I)/IGF-binding protein (IGFBP) system in the tilapia (Oreochromis mossambicus). In a short-term experiment, fish were injected intraperitoneally with cortisol (2 or 10 microg/g), and killed at 2, 4, 8 and 24 h after the injection. In a longer-term experiment, fish were killed 24 and 48 h after cortisol injection (2, 10 and 50 microg/g). Cortisol at doses of 2 and 10 microg/g significantly increased IGFBPs of four different sizes (24, 28, 30, and 32 kDa) in the plasma within 2 h without altering plasma levels of IGF-I or GH. On the other hand, cortisol at doses of 10 and 50 microg/g significantly reduced plasma IGF-I levels after 24 and 48 h. IGF-I mRNA levels in the liver were also significantly reduced by cortisol at doses of 10 and 50 microg/g after 48 h, suggesting that a decrease in plasma IGF-I levels is mediated through the attenuation of IGF-I gene expression in the liver. In contrast, no significant change was observed in plasma or pituitary contents of GH at any time point examined, which would appear to indicate that cortisol reduces IGF sensitivity to GH (GH-resistance). These results clearly indicate that cortisol induces a rapid increase in plasma IGFBPs and a more delayed decrease in IGF-I production. The dual mode of cortisol action may contribute to the inhibitory influence of cortisol on somatic growth in teleosts.

Amidated fish ghrelin: purification, cDNA cloning in the Japanese eel and its biological activity.

We purified ghrelin from stomach extracts of a teleost fish, the Japanese eel (Anguilla japonica) and found that it contained an amide structure at the C-terminal end. Two molecular forms of ghrelin with 21 amino acids were identified by cDNA and mass spectrometric analyses: eel ghrelin-21, GSS(O-n-octanoyl)FLSPSQRPQGKDKKPP RV-amide and eel ghrelin-21-C10, GSS(O-n-decanoyl) FLSPSQRPQGKDKKPPRV-amide. Northern blot and RT-PCR analyses revealed high gene expression in the stomach. Low levels of expression were found only in the brain, intestines, kidney and head kidney by RT-PCR analysis. Eel ghrelin-21 increased plasma growth hormone (GH) concentrations in rats after intravenous injection; the potency was similar to that of rat ghrelin. We also examined the effect of eel ghrelin on the secretion of GH and prolactin (PRL) from organ-cultured tilapia pituitary. Eel ghrelin-21 at a dose of 0.1 nM stimulated the release of GH and PRL, indicating that ghrelin acts directly on the pituitary. The present study revealed that ghrelin is present in fish stomach and has the ability to stimulate the secretion of GH from fish pituitary. A novel regulatory pathway of GH secretion by gastric ghrelin seems to be conserved from fish to human.

Effects of fasting on growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus.

Effects of fasting on the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis were examined in the tilapia (Oreochromis mossambicus) acclimated to fresh water. Fasting for 2 weeks resulted in significant reductions in body weight, specific growth rate and hepatosomatic index in both males and females. Significant reductions in specific growth rates were observed after 1 and 2 weeks in both sexes, although the decrease in body weight was not significant in the female. A significant reduction was also seen in the condition factor of females after 2 weeks. No change was seen in the gonadosomatic index in either sex. Two weeks of fasting also produced a significant reduction in plasma IGF-I but not in plasma GH, prolactin (PRL(188)) or cortisol. Significant reductions in the hepatic IGF-I mRNA were seen in both sexes. On the other hand, a significant increase was observed in cortisol receptor mRNA in the female liver. Plasma IGF-I levels were correlated significantly with specific growth rate, condition factor and hepatosomatic index, indicating that plasma IGF-I is a good indicator of growth in the tilapia. No change was seen in plasma glucose or osmolality after 2 weeks of fasting. During fasting, tilapia appears to convert metabolic energy from growth to basal metabolism including maintenance of ion and water balance.

Cell volume increase and extracellular Ca2+ are needed for hyposmotically induced prolactin release in tilapia.

In the tilapia (Oreochromis mossambicus), as in many euryhaline teleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and water permeability and increase solute retention. Consistent with these actions, PRL release is stimulated as extracellular osmolality is reduced both in vivo and in vitro. In the current experiments, a perfusion system utilizing dispersed PRL cells was developed for permitting the simultaneous measurement of cell volume and PRL release. Intracellular Ca(2+) was monitored using fura 2-loaded cells under the same conditions. When PRL cells were exposed to hyposmotic medium, an increase in PRL cell volume preceded the increase in PRL release. Cell volume increased in proportion to decreases of 15 and 30% in osmolality. However, regulatory volume decrease was clearly seen only after a 30% reduction. The hyposmotically induced PRL release was sharply reduced in Ca(2+)-deleted hyposmotic medium, although cell volume changes were identical to those observed in normal hyposmotic medium. In most cells, a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) during hyposmotic stimulation was dependent on the availability of extracellular Ca(2+), although small transient increases in [Ca(2+)](i) were sometimes observed upon introduction of Ca(2+)-deleted media of the same or reduced osmolality. These results indicate that an increase in cell size is a critical step in the transduction of an osmotic signal into PRL release and that the hyposmotically induced increase in PRL release is greatly dependent on extracellular Ca(2+).

Evidence that signal transduction for osmoreception is mediated by stretch-activated ion channels in tilapia.

Prolactin (PRL) plays a central role in the freshwater osmoregulation of teleost fish, including the tilapia (Oreochromis mossambicus). Consistent with this action, PRL release from the tilapia pituitary increases as extracellular osmolality is reduced both in vitro and in vivo. Dispersed tilapia PRL cells were incubated in a perfusion chamber that allowed simultaneous measurements of cell volume and PRL release. Intracellular Ca(2+) concentrations were measured from fura 2-loaded PRL cells treated in a similar way. Gadolinium (Gd(3+)), known to block stretch-activated cation channels, inhibited hyposmotically induced PRL release in a dose-related manner without preventing cell swelling. Nifedipine, an L-type Ca(2+) channel blocker, did not prevent the increase in PRL release during hyposmotic stimulation. A high, depolarizing concentration of KCl induced a transient and marked increase of intracellular Ca(2+) and release of PRL but did not prevent the rise in intracellular Ca(2+) and PRL release evoked by exposure to hyposmotic medium. These findings suggest that a decrease in extracellular osmolality stimulates PRL release through the opening of stretch-activated ion channels, which allow extracellular Ca(2+) to enter the cell when it swells.

Immunomodulatory effects of prolactin and growth hormone in the tilapia, Oreochromis mossambicus.

To clarify the roles of prolactin (PRL) and GH in the control of the immune system, the effects of environmental salinity, hypophysectomy, and PRL and GH administration on several immune functions were examined in tilapia (Oreochromis mossambicus). Transfer from fresh water (FW) to seawater (SW) did not alter plasma levels of immunoglobulin M (IgM) and lysozyme. The superoxide anion (O(2)(-)) production in head kidney leucocytes accompanied by phagocytosis was elevated in SW-acclimated fish over the levels observed in FW fish. Hypophysectomy of the fish in FW resulted in a reduction in O(2)(-) production in leucocytes isolated from the head kidney, whereas there was no significant change in plasma levels of IgM or lysozyme. Treatment with tilapia GH and PRLs (PRL(177) and PRL(188)) enhanced O(2)(-) production in vitro in head kidney leucocytes in a dose-related manner. Extrapituitary expression of two PRLs, GH and IGF-I mRNA was detected in lymphoid tissues and cells such as head kidney, spleen, intestine and leucocytes from peripheral blood and head kidney. PRL-receptor mRNA was detected in head kidney leucocytes, and the level of expression was higher in SW-acclimated fish than that in FW fish. Treatment with PRL(177) caused higher production of O(2)(-) in the head kidney leucocytes isolated from SW tilapia than that from FW fish. In view of the fact that PRL acts antagonistically to osmoregulation in SW, its immunomodulatory actions in this euryhaline fish would appear to be independent of its osmoregulatory action.

Isolation and characterization of a homologue of mammalian prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary.

In the tilapia (Oreochromis mossambicus), as in many teleosts, prolactin (PRL) plays a major role in osmoregulation in freshwater. Recently, PRL-releasing peptides (PrRPs) have been characterized in mammals. Independently, a novel C-terminal RF (arginine-phenylalanine) amide peptide (Carrasius RF amide; C-RFa), which is structurally related to mammalian PrRPs, has been isolated from the brain of the Japanese crucian carp. The putative PrRP was purified from an acid extract of tilapia brain by affinity chromatography with antibody against synthetic C-RFa and HPLC on a reverse-phase ODS-120 column. The tilapia PrRP cDNA was subsequently cloned by polymerase chain reaction. The cDNA consists of 619 bp encoding a preprohormone of 117 amino acids. Sequence comparison of the isolated peptide and the preprohormone revealed that tilapia PrRP contains 20 amino acids and is identical to C-RFa. Incubation of the tilapia pituitary with synthetic C-RFa (100 nM) significantly stimulated the release of two forms of tilapia PRL (PRL188 and PRL177). However, the effect of C-RFa was less pronounced than the marked increase in PRL release in response to hyposmotic medium. The ability of C-RFa to stimulate PRL release appears to be specific, since C-RFa failed to stimulate growth hormone release from the pituitary in organ culture. In contrast, rat and human PrRPs had no effect on PRL release. C-RFa was equipotent with chicken GnRH in stimulating PRL release in the pituitary preincubated with estradiol 17beta. Circulating levels of PRL were significantly increased 1 h after intraperitoneal injection of 0.1 microg/g of C-RFa in female tilapia in freshwater but not in males. These results suggest that C-RFa is physiologically involved in the control of PRL secretion in tilapia.