PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p < 0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p < 0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p < 0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na⁺/K⁺-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.     

Effects of environmental osmolality on release of prolactin,growth hormone and ACTH from the tilapia pituitary

Prolactin (PRL) plays a central role in freshwater (FW) adaptation in teleost fish. Evidence now suggests that growth hormone (GH) acts in the seawater (SW) adaptation in at least some euryhaline fish. Reflecting its important role in FW adaptation, plasma levels of PRL(188) and PRL(177) are higher in tilapia (Oreochromis mossambicus) adapted to FW than in those adapted to SW. A transient but significant increase in plasma GH was observed 6h after transfer from FW to SW. Elevated plasma PRL levels were seen in association with reductions in plasma osmolality after blood withdrawal in FW fish whereas no significant change was seen in plasma GH levels. When pituitaries from FW tilapia were incubated for 7 days, secretion of both PRLs was significantly greater in hyposmotic medium than in hyperosmotic medium for the first 24h. Secretion of GH from the same pituitary was relatively low during this period compared with PRL secretion. No consistent effect of medium osmolality on GH release was seen for the first day, but its cumulative release was increased significantly in hyperosmotic medium after 2 days and thereafter. On the other hand, ACTH release was extremely low compared with the secretion of PRLs and GH and there was no consistent effect of medium osmolality. These results indicate that PRL release from the tilapia pituitary is stimulated both in vivo and in vitro as extracellular osmolality is reduced, whereas the secretion of GH increases temporarily when osmolality is increased. ACTH seems to be relatively insensitive to the changes in environmental osmolality.     

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.     

Effects of short-term acid and aluminum exposure on the parr-smolt transformation in Atlantic salmon (Salmo salar): disruption of seawater tolerance and endocrine status

Episodic acidification resulting in increased acidity and inorganic aluminum (Al_i) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7–6.9) or acid/Al (pH 5.4–6.3, 28–64 μg l⁻¹ Al_i) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al_i. There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24 h SW challenge exhibited greater plasma Cl⁻ levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na⁺,K⁺-ATPase (NKA) activity and Na⁺,K⁺,2Cl⁻ (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3′,5′-triiodo-l-thyronine (T₃) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T₄) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24 h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T₃.     

Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca(2+) through a stretch-activated Ca(2+) channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca(2+) ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.     

Estrogenic compounds decrease growth hormone receptor abundance and alter osmoregulation in Atlantic salmon

Exposure of Atlantic salmon smolts to estrogenic compounds is shown to compromise several aspects of smolt development. We sought to determine the underlying endocrine mechanisms of estrogen impacts on the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Smolts in freshwater (FW) were either injected 3 times over 10 days with 2 μg g⁻¹ 17β-estradiol (E2) or 150 μg g⁻¹ 4-nonylphenol (NP). Seawater (SW)-acclimated fish received intraperitoneal implants of 30 μg g⁻¹ E2 over two weeks. Treatment with these estrogenic compounds increased hepatosomatic index and total plasma calcium. E2 and NP reduced maximum growth hormone binding by 30-60% in hepatic and branchial membranes in FW and SW, but did not alter the dissociation constant. E2 and NP treatment decreased plasma levels of IGF-I levels in both FW and SW. In FW E2 and NP decreased plasma GH whereas in SW plasma GH increased after E2 treatment. Compared to controls, plasma chloride concentrations of E2-treated fish were decreased 5.5 mM in FW and increased 10.5 mM in SW. There was no effect of NP or E2 on gill sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) activity in FW smolts, whereas E2 treatment in SW reduced gill Na⁺/K⁺-ATPase activity and altered the number and size of ionocytes. Our data indicate that E2 downregulates the GH/IGF-I-axis and SW tolerance which may be part of its normal function for reproduction and movement into FW. We conclude that the mechanism of endocrine disruption of smolt development by NP is in part through alteration of the GH/IGF-I axis via reduced GH receptor abundance.     

Changes in gene expression levels of somatolactin in the pituitary and morphology of gill mitochondria-rich cells in Mozambique tilapia after transfer to acidic freshwater (pH 3.5)

Mozambique tilapia, Oreochromis mossambicus, is easily acclimated to highly acidic water, and thus presents a useful model to unravel endocrine regulation of adaptation to acidic water in fish. We analyzed gene expression of somatolactin (sl), growth hormone (gh) and prolactin (prl), in the pituitary gland and size distribution of mitochondria-rich (MR) cells in the gills after transfer from normal freshwater (FW, pH 7.2) to acidified freshwater (AW, pH 3.5). Plasma osmolality drastically decreased until 2 days after transfer to AW, but had restored to normal after 1 week of acclimation, and this confirmed the excellent acid tolerance of tilapia. Expression levels of sl, gh and prl were all up-regulated during short-term exposure to AW. The expression of sl remained elevated up to 7 days after transfer; the expression of gh and prl was back to initial levels at that time. These findings point to an important and specific role of SL in adaptation to acid water in this tilapia, although temporal contribution of GH and PRL cannot be ruled out. The size distribution of branchial MR cells changed drastically during acclimation to AW. The mean MR cell size was 1.5-fold larger in the fish exposed to AW for 7 days compared to controls in FW. The gills and their MR cells are a likely site of important acid-base regulation, and SL may change ion-transport functions of MR cells to correct plasma osmotic balance disturbed by acid exposure.     

Changes in Plasma Atrial and Ventricular Natriuretic Peptide Concentrations after Transfer of Eels from Freshwater to Seawater or Vice Versa

Changes in plasma atrial and ventricular natriuretic peptide (ANP and VNP) concentrations were examined in conscious eels,Anguilla japonica,after transfer from freshwater (FW) to seawater (SW) or vice versa. These changes were compared in ventral and dorsal aortic blood since clearance receptors are present in the gills. Plasma Na concentration and osmolality in the ventral aortic blood rose linearly after transfer of FW eels to SW. Six hours after transfer, plasma ANP levels increased from 28.1 ± 2.9 to 44.8 ± 5.4 fmol/ml (n = 6) (P < 0.05) and plasma VNP levels also increased from 63.6 ± 3.8 to 91.2 ± 6.2 fmol/ml (n = 6) (P < 0.05). However, the levels gradually returned to the FW levels despite plasma Na concentration and osmolality remaining elevated. Dorsal aotic plasma ANP and VNP levels were unchanged after transfer of FW eels to SW, possibly due to extraction of secreted peptides by the branchial clearance receptors. After transfer of SW-adapted eels to FW, plasma ANP and VNP levels were unchanged, while both plasma Na concentration and osmolality decreased. These results indicate that plasma ANP and VNP levels are increased by an increase in plasma Na concentration or osmolality after transfer of FW eels to SW. However, the sustained increase in plasma Na concentration or osmolality no longer stimulated ANP and VNP secretion, and plasma ANP and VNP levels gradually returned to the FW levels gradually on adaptation to SW.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.     

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia,Oreochromis mossambicus

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.     

Glucocorticoid receptor upregulation during seawater adaptation in a euryhaline teleost,the tilapia (Oreochromis mossambicus)

Cortisol is an important seawater (SW) osmoregulatory hormone in the Mozambique tilapia (Oreochromis mossambicus), a highly euryhaline cichlid able to live in environments ranging from fresh water (FW) to salinities well in excess of full-strength seawater. Previous studies indicate that cortisol may promote SW adaptation by increasing gill chloride cell differentiation, Na(+)/K(+)-ATPase activity and subsequent excretion of excess salt following seawater acclimation. Despite cortisol's widely accepted role as a SW-adapting hormone, cortisol receptor regulation during SW acclimation is not well understood. The purpose of these studies was to determine whether the intracellular glucocorticoid receptor (GR) might be regulated in a manner consistent with cortisol's actions in SW adaptation. Saturation radioligand binding assays were conducted on gill cytoplasm preparations from fish sampled 4 and 24h and 4 and 14 days after transfer from FW to 2/3 SW or FW (control). Affinity (K(d)) of the gill GR remained constant over the timecourse, while numbers of receptors (B(max)) in SW fish were significantly elevated compared with controls at 24h and 4 days after transfer. Plasma osmolality was higher in fish transferred to SW for 24h, 4 days, and 14 days compared with those animals moved to FW. Plasma cortisol levels and hepatic cortisol binding remained constant between SW and FW fish throughout the timecourse of the salinity challenge. These studies indicate that seawater acclimation is accompanied by a specific upregulation of intracellular GR numbers in gill tissue. The lack of increase in circulating cortisol following SW adaptation may reflect enhancement of clearance of the steroid. It appears that an increase in cortisol receptors, which is closely associated with the rise in blood osmotic pressure that accompanies SW exposure, is an important component of cortisol's ability to promote SW adaptation in the tilapia.     

Osmoregulatory action of PRL,GH, and cortisol in the gilthead seabream (Sparus aurata L)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.     

Is the primitive regulation of pituitary prolactin (tPRL177 and tPRL188) secretion and gene expression in the euryhaline tilapia (Oreochromis mossambicus) hypothalamic or environmental?

We examined the effects of environmental salinity on circulating levels of the two prolactins (tPRL177 and tPRL188) and levels of pituitary tPRL177 and tPRL188 mRNA in the euryhaline tilapia, Oreochromis mossambicus. Fish were sham-operated or hypophysectomized and the rostral pars distalis (RPD) autotransplanted onto the optic nerve. Following post-operative recovery in (1/4) seawater, tilapia were transferred to fresh water (FW), (1/4) seawater (SW) or SW. Serum tPRL177 and tPRL188 levels in sham-operated and RPD-autotransplanted fish were highest in FW and decreased as salinity was increased. tPRL177 and tPRL188 mRNA levels in RPD implants as well as in pituitaries from the sham-operated fish were also highest in FW and decreased with increasing salinity. Serum osmolality increased with salinity, with the highest levels occurring in the seawater groups. We conclude that some plasma factor (probably plasma osmolality), in the absence of hypothalamic innervation, exerts a direct regulatory action on prolactin release and gene expression in the pituitary of O. mossambicus. This regulation is in accord with the actions of the two prolactins in the freshwater osmoregulation of the tilapia.