Abstract
Two forms of prolactin (Prl), prolactin 177 (Prl177) and prolactin 188 (Prl188), 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 Prl177 and Prl188 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 Prl177 and Prl188 and RPD mRNA levels of prl177 and prl188 increased. The opposite pattern was observed when fish were transferred from FW to SW. Moreover, hyposmotically induced release of Prl188 was greater in Prl cells isolated from FW-acclimated fish after 6 h of incubation, while the hyposmotically induced increase in prl188 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.
Introduction
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 >95%) and are directly stimulated by physiologically relevant decreases in extracellular osmolality in vitro (Grau et al. 1994, 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 Ca2+ through stretch-activated ion channels upon cell swelling (Seale et al. 2003). There is evidence that the influx of extracellular Ca2+ may have distinct intracellular effects depending on the acclimation salinity of the animal from which pituitary cells are collected. For example, hyposmotically induced Prl synthesis (Yoshikawa-Ebesu et al. 1995) and release (Seale et al. 2006a) is greater in RPDs collected from fish acclimated to FW than in the RPDs collected from SW fish. One likely explanation for this pattern is the fact that Prl cells from FW tilapia contain more stored Prl than those from SW fish (Nagahama et al. 1975, Specker et al. 1993) and, hence, exhibit a greater capacity to respond rapidly with hormone secretion when faced with a hyposmotic stimulus. In addition, recent evidence suggests that this difference in responsiveness may result from the diminished capability of Prl cells from SW-acclimated tilapia to initiate osmotic signal transduction. The relative rate of change in cell volume and water influx into Prl cells following an osmotic challenge was found to be significantly lower in cells from SW tilapia compared to those in FW (Watanabe et al. 2009). The question remains, however, as to whether the responsiveness of prl gene expression differs between FW- and SW-acclimated fish.
The tilapia pituitary releases two Prl molecules that differ in length and amino acid composition, and are encoded by separate genes (Specker et al. 1985, Yamaguchi et al. 1988, Rentier-Delrue et al. 1989). While the existence of two distinct Prl molecules, designated Prl177 and Prl188, suggests an evolution of differing actions (Chen et al. 1994), a bioassay failed to identify any clear differences in their osmoregulatory activities (Specker et al. 1985). On the other hand, it has been reported that Prl177 exhibits somatotropic activity while Prl188 does not (Shepherd et al. 1997). The tilapia Prls have also been found to respond differently to acclimation salinity. Borski et al. (1992) reported significant differences in the relative amounts of Prl188 and Prl177 in the RPDs of tilapia acclimated to FW, SW, or during transition from one salinity to another. Specifically, the ratio of Prl188 to Prl177 is >1 in FW and <1 in SW (Borski et al. 1992). This disparate control suggests that the two Prls are potentially regulated through discrete mechanisms. To date, however, it is not known whether acclimation salinity affects only the synthesis and release of these two hormones, or whether it also exercises differential effects on their gene expression. In a broader context, understanding the regulatory mechanisms governing the release and expression of both Prls in vivo and in vitro is essential for understanding the intricacies of the tilapia Prl cell as a model to better understand vertebrate osmoreceptors.
The main objectives of this study were to determine whether there are differential effects of acclimation salinity in vivo and extracellular osmolality in vitro on the release and expression of Prl177, and Prl188. We also explored the effects of acclimation salinity and extracellular osmolality on the mRNA expression of two Prl receptors (prlr) in the RPD to explore possible feedback mechanisms on Prl expression/release. Originally cloned by Sandra et al. (1995) in Nile tilapia (Oreochromis niloticus), the tilapia Prl receptor (PrlR1) is expressed in an array of tissues, including gill, intestine and kidney (Pierce et al. 2007). prlr1 mRNA expression is sensitive to salinity, with enhanced expression occurring in the gill when Mozambique tilapia are transferred from SW to FW (Breves et al. 2011) and down-regulated expression after transfer from FW to SW (Breves et al. 2010a). Recently, a second Prl receptor, PrlR2, was identified in tilapia gill on the basis of its induced expression following a hyperosmotic challenge (Fiol et al. 2009). It is unknown whether either of these PrlRs exhibit changes in expression in the pituitary following salinity challenges. Lastly, we explored the effects of acclimation salinity and extracellular osmolality on the gene expression of osmotic stress transcription factor 1 (ostf1) in the RPD. Fiol et al. (2006) characterised ostf1 as an immediate response gene in tilapia gill following exposure to hyperosmotic conditions and suggested that it may participate in conferring osmotic tolerance (Fiol & Kultz 2005, Fiol et al. 2006). To our knowledge, environmental factors that modulate ostf1 expression have not been reported in extra-branchial tissues. In the current study, we examined the effects of salinity transfers on plasma osmolality, plasma Prl177 and Prl188 levels, and mRNA levels of prl177, prl188, prlr1, prlr2 and ostf1 from RPDs, in vivo. We also investigated in vitro effects of various incubation osmolalities on the release of Prl177, Prl188 and mRNA levels of prl177, prl188, prlr1, prlr2 and ostf1 of dispersed Prl cells isolated from fish acclimated to either FW or SW.
Materials and Methods
Fish
Mature Mozambique tilapia (O. mossambicus) of both sexes (50–300 g), were obtained from a population maintained at the Hawaii Institute of Marine Biology, University of Hawaii. Fish were reared in outdoor tanks (700 l) with a continuous flow of FW (14 mOsmolal) or SW (1042 mOsmolal) under natural photoperiod. SW-acclimated tilapia employed in this experiment were spawned and reared in SW, having never been previously exposed to FW. FW-acclimated tilapia, on the other hand, were spawned and reared in FW, having never been previously exposed to SW. Water temperature was maintained at 24–26 °C. Animals were fed ∼5% of their body weight per day with Silver Cup Trout Chow (Nelson and Sons Inc., Murray, UT, USA). All experiments were conducted in accordance with the principles and procedures approved by the Institutional Animal Care and Use Committee, University of Hawaii.
In vivo experiments
Four tanks were employed for salinity transfer experiments. Each tank contained 50 fish of both sexes ranging from 50 to 100 g. At the time of transfer, one tank was transitioned from SW to FW with minimal disturbance by opening an incoming FW valve and closing a SW valve, while the other tank was transitioned from FW to brackish water (BW; 27%) by reducing the FW valve flow while opening the SW valve. FW and BW conditions were reached after 60 min. After 48 h, the tank that was transitioned to BW was exposed to full strength SW. Two control tanks, which contained fish from the original pool raised in either FW or SW, were held at constant salinity throughout the duration of the experiment. Fish were fed for the duration of the experiment and water temperature was maintained between 24 and 26 °C. Fish were sampled (n=6–10) at 0 h (immediately before opening the FW or SW valves), 6 h, 48 h and 10 days after transfer. At the time of sampling, fish were netted and anaesthetised with a lethal dose of 2-phenoxyethanol (2 ml/l). Blood was collected from the caudal vasculature by a needle and syringe coated with ammonium heparin (200 U/ml, Sigma–Aldrich). Plasma was separated by centrifugation and stored at −80 °C until later analyses. Fish were rapidly decapitated and the pituitary extracted. The RPD was dissected under a microscope and placed in separate tubes containing 750 μl TRI Reagent (MRC, Cincinnati, OH, USA). Samples were stored at −80 °C before RNA extraction and analyses of gene expression.
Cell cultures
Incubations of dispersed Prl cells were carried out as previously described (Seale et al. 2006a) with minor modifications. After removal of pituitaries from tilapia (60–300 g) of both sexes, RPDs were placed in isosmotic (330 mOsmolal) modified Ringer's culture medium (Seale et al. 2011), diced into fragments and treated with 0.125% trypsin in PBS. After termination of trypsin treatment by adding trypsin inhibitor (0.125% w/v), cells were harvested, and viability determined using the trypan blue exclusion test. Cell yield was estimated using a haemocytometer. Cells were re-suspended in isosmotic medium, plated (4–6×104 cells/well on 96 well plates) and incubated at 26 °C under saturated humidity. Cells were pre-incubated for 1 h in isosmotic medium before exposure to a series of osmolalities (280, 300, 330, 355 and 420 mOsmolal). Medium osmolality was adjusted by varying the concentration of NaCl and verified using a vapor pressure osmometer (Wescor 5100C; Wescor, Inc., Logan, UT, USA). Culture medium was collected after 6 h of incubation and stored at −80 °C before RIA. At the end of the incubation period, 200 μl TRI Reagent was added to each well. The mixture of TRI Reagent and cells was added to a 1.5 ml tube containing an additional 500 μl TRI Reagent and stored at −80 °C before RNA extraction and gene expression analyses.
RIAs
Prl177 and Prl188 levels in plasma and culture medium were measured by homologous RIA (Ayson et al. 1993, Yada et al. 1994). Assay parameters, including inter-assay coefficients of variations, cross reactivity between Prl177 and Prl188, and competitive binding curves, are reported in a previous study (Ayson et al. 1993). For in vivo experiments, plasma values are reported as nanograms per milliliter. For dispersed cell cultures, values obtained as nanograms per milliliter were first converted to micrograms per 104 cells and then calculated as relative percent change from isosmotic (330 mOsmolal) SW treatments.
Quantitative real-time PCR
Total RNA was extracted from intact RPDs and dissociated Prl cells using TRI Reagent according to the manufacturer's protocols and then reverse transcribed using a cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). All primer pairs are previously described: prlr1 (Pierce et al. 2007), prlr2 (long isoform; Breves et al. 2010b), ostf1 (Breves et al. 2010a), prl177 and prl188 (Magdeldin et al. 2007) and ef1-α (Breves et al. 2010b). The quantitative real-time PCRs were set up as previously described (Pierce et al. 2007). Briefly, 200 nM of each primer, 3 μl cDNA and 12 μl SYBR Green PCR Master Mix (Applied Biosystems) were added to 15 μl final reaction volume. The following cycling parameters were employed: 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min using the StepOnePlus real-time PCR system (Applied Biosystems). After verification that mRNA levels did not vary across treatments and time, ef1-α levels were used to normalise target genes. Reference and target genes were calculated by the absolute quantification method, in which relative expression levels are obtained based on a standard curve produced by the amplification of target tissue at known concentrations. For in vivo experiments, expression of target genes is reported as fold-change from 0 h SW controls. For in vitro experiments, gene expression is reported as fold-change from the 330 mOsmolal SW group.
Statistical analysis
Comparisons among different treatments were analysed by two-way ANOVA, with treatment and time as main effects. Significant (P<0.05) main effects of treatment were followed up by a Student's t-test at all time points. Comparisons among slopes were carried out by linear regression and analysis of covariance (ANCOVA). Statistical calculations were performed using a statistical software program, Prism 5.0 (GraphPad, La Jolla, CA, USA).
Results
Effects of salinity transfers on plasma Prl177 and Prl188 in vivo
Plasma osmolality decreased when fish were transferred from SW to FW, with the greatest reduction (to ∼305 mOsmolal) occurring 48 h after transfer. Conversely, plasma osmolality markedly increased when fish were transferred from FW to SW, reaching ∼390 mOsmolal by 6 h after transfer (Fig. 1A). Plasma Prl188 increased by 6 h after transfer from SW to FW, and reached the highest level at 48 h (Fig. 1B). After 10 days, plasma Prl188 levels of fish transferred from SW to FW remained significantly elevated from plasma levels in SW controls, but at levels lower than fish maintained in FW. This difference disappeared by 72 days after transfer; Prl188 levels of SW–FW and FW–FW groups were statistically similar at 6–7 ng/ml (data not shown). This pattern changed when fish were transferred from FW to SW. Six hours after transfer from FW to BW, plasma Prl188 dropped to levels observed in fish maintained in SW and remained low throughout the duration of the experiment (Fig. 1B). Although less drastic, a similar trend was observed with respect to plasma Prl177. Prl177 was increased at all time points after transfer from SW to FW when compared with levels in SW–SW controls; Prl177 decreased at all time points upon transfer from FW to SW when compared with levels in FW–FW controls (Fig. 1C). The ratio of Prl188/Prl177 in plasma following salinity challenges confirmed differential responsiveness to changes in environmental salinity. While Prl188/Prl177 was increased 48 h after transfer from SW to FW, Prl188/Prl177 decreased 10 days after transfer from FW to SW (Fig. 1D).
Changes in (A) plasma osmolality, (B) Prl188, (C) Prl177 and (D) Prl188/Prl177 after transfer of Mozambique tilapia from FW to SW (circles) and SW to FW (triangles). Symbols represent ±s.e.m. (n=6–10). Control fish were maintained in FW or SW and sampled on the same time course as fish subjected to salinity change. Differences among groups were evaluated by two-way ANOVA. *, **, ***Significantly different from the corresponding FW–FW controls (open circles) at P<0.05, 0.01 and 0.001 respectively by Student's t-test. †, ††, †††Significantly different from corresponding SW–SW controls (open triangles) at P<0.05, 0.01 and 0.001 respectively by Student's t-test.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0384
Effects of salinity transfers on prl177 and prl188 gene expression in vivo
Expression of prl188 in RPD increased significantly by 6 h after transfer from SW to FW, and reached the highest value, tenfold higher than SW controls, after 48 h. These elevated levels were maintained at 10 days (Fig. 2A). An opposite pattern was observed when fish were transferred from FW to SW, where prl188 mRNA levels dropped significantly by 48 h and remained low throughout the duration of the experiment.
Changes in mRNA levels of (A) prl188 (B) prl177 and (C) prl188/prl177 after transfer of Mozambique tilapia from FW to SW (circles) and SW to FW (triangles). Symbols represent mRNA levels from individual RPDs ±s.e.m. (n=8–10). Control fish were maintained in FW (FW–FW) or SW (SW–SW) and sampled on the same time course as fish subjected to salinity change. Expression levels are presented relative to the time 0 SW–SW group. Differences among groups were evaluated by two-way ANOVA. *, ***Significantly different from the corresponding FW–FW controls (open circles) at P<0.05, and 0.001 respectively by Student's t-test. †, ††, †††Significantly different from the corresponding SW–SW controls (open triangles) at P<0.05 and 0.001 respectively by Student's t-test.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0384
Expression of prl177 in response to salinity transfers followed overall patterns that were similar to, though less drastic than, those observed with prl188 (Fig. 2B). Transfer from SW to FW elicited a ∼2.5-fold increase by 48 h, and a threefold increase by 10 days. Transfer from FW to SW, on the other hand, induced significant decreases in prl177 mRNA by 48 h, but levels in these fish did not reach the levels of SW–SW controls until 10 days. The ratio of mRNA for prl188 to prl177 increased significantly after 6 h, 48 h and 10 days of transfer from SW to FW. Transfer from FW to SW, by contrast, resulted in reduced prl188/prl177 mRNA ratios that were significantly different from FW–FW controls at all time points (Fig. 2C). This trend was similar to that observed with plasma levels of both Prls (Fig. 1D).
Effects of extracellular osmolality on Prl177 and Prl188 release and gene expression in vitro
Initially, prl188 and prl177 mRNA expression in dispersed Prl cells of FW and SW fish incubated in isosmotic conditions (330 mOsmolal) was compared between time 0 and 6 h. While there was no effect of incubation time on prl188 and prl177 mRNA expression in dispersed cells of SW fish, there was a twofold increase in mRNA expression of prl188 in Prl cells of FW fish incubated for 6 h when compared with cells of FW fish at time 0 (data not shown). There was no effect of incubation time on mRNA expression of prl177 in dispersed cells of FW fish.
Dispersed Prl cells from fish acclimated to either FW or SW were then incubated under a range of medium osmolalities. The effect of reducing medium osmolality on Prl188 release was more robust, i.e. a significantly steeper slope, in incubations of Prl cells from FW fish (slope=−0.809±0.161; R2=0.407) than that observed with Prl cells from SW fish (slope=−0.404±0.103; R2=0.288; Fig. 3A). The effect of medium osmolality on prl188 gene expression differed between the two acclimation salinities. In Prl cells from SW fish, prl188 mRNA levels were inversely related to medium osmolality (slope=−0.468±7.43×10−2; R2=0.511). With Prl cells from FW fish, on the other hand, already high prl188 mRNA levels were unresponsive to changes in medium osmolality (slope=−4.09×10−2±6.19×10−2; R2=0.006; Fig. 3B).
Linear regression of (A) Prl188 release and (B) mRNA levels, (C) Prl177 release and (D) mRNA levels and (E) Prl188/Prl177 release and (F) mRNA levels from dispersed Prl cells exposed to different medium osmolalities during a 6-h incubation. Open squares and a solid line represent Prl release or mRNA levels and linear regression respectively from fish acclimated to FW, whereas shaded squares and a dashed line represent these same parameters from fish acclimated to SW. Values represent percent Prl release (A and C) or percent mRNA levels (B and D) relative to the 330 mOsmolal incubation group of each respective salinity ±s.e.m. (n=8, SW; n=12–16, FW). Values presented in panels E and F represent ratios of Prl release and expression respectively. Insets represent the values relative to the SW 330 mOsmolal incubation groups ±s.e.m. †, †††Regression slopes significantly different from each other at P<0.05 and 0.001 respectively.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0384
Prl177 showed a different pattern of response to medium osmolality. Prl177 release from dispersed Prl cells was significantly and equally responsive to extracellular osmolality regardless of the salinity in which the tilapia were reared (SW slope=−0.291±7.78×10−2; SW R2=0.28; FW slope=−0.432±0.158; FW R2=0.172; Fig. 3C). The mRNA levels of prl177 in Prl cells from SW-acclimated fish increased as medium osmolality was reduced, while in Prl cells from FW fish, prl177 mRNA levels were unresponsive to the changes in medium osmolality (SW slope=−0.182±0.083; SW R2=0.111; FW slope=−7.81×10−2±0.121; FW R2=0.012; Fig. 3D). The ratio of Prl188/Prl177 release was inversely related to changes in medium osmolality in FW fish with more Prl188 being released relative to Prl177 as osmolality was reduced (SW slope=−2.72×10−4±1.66×10−4; SW R2=0.065; FW slope=−1.61×10−3±7.03×10−4; FW R2=0.124; Fig. 3E). This pattern was not observed when Prl cells from SW fish were exposed to various osmolalities. The mRNA ratio of prl188 to prl177 showed a response that was opposite to that for release, rising significantly when Prl cells from SW fish were exposed to lower medium osmolalities (SW slope=−3.07×10−3±7.79×10−4; SW R2=0.289; FW slope=−2.09×10−4±1.16×10−3; FW R2=0.001; Fig. 3F). This pattern was not observed with Prl cells from FW fish in which medium osmolality had no effect on the prl188/prl177 mRNA ratio.
Effects of salinity transfers and extracellular osmolality on ostf1, prlr1 and prlr2 gene expression in RPD
The expression of prlr1, prlr2 and ostf1 in RPD following salinity challenges was measured in vivo, and to changes in medium osmolality in vitro. Comparisons of the expression levels of the three genes at time 0 and 6 h of incubation were carried out. The levels of prlr1 mRNA increased by twofold and decreased by twofold, in Prl cells of FW and SW fish respectively by 6 h of incubation when compared with time 0 (data not shown). The levels of prlr2 mRNA decreased by 1.5-fold and by twofold in Prl cells of FW and SW fish respectively by 6 h of incubation when compared with time 0 (data not shown). Finally, the levels of ostf1 mRNA decreased by twofold and by threefold in Prl cells of FW and SW fish respectively by 6 h of incubation when compared with time 0 (data not shown). The levels of prlr1 mRNA in RPDs did not change in response to either FW or SW transfer (Fig. 4A), nor in response to changes in medium osmolality in vitro (SW slope=4.48×10−3±0.11; SW R2<0.001; FW slope=5.29×10−2±0.104; FW R2=0.007; Fig. 4B). The levels of prlr2 mRNA, on the other hand, increased in RPDs by 6 h after transfer from FW to SW compared to levels in FW–FW controls (Fig. 4C). The levels of prlr2 mRNA from Prl cells isolated from both FW- and SW-acclimated fish increased in direct proportion to extracellular osmolality (SW slope=1.24±0.178; SW R2=0.571; FW slope=0.955±0.094; FW R2=0.733; Fig. 4D). Similarly, ostf1 mRNA increased by 6 h after transfer from FW to SW compared to FW–FW controls (Fig. 4E). While the Prl cells isolated from both FW- and SW-acclimated fish showed increased ostf1 gene expression in response to increased medium osmolality, this response was more robust in Prl cells isolated from FW-acclimated fish (SW slope=0.399±0.133; SW R2=0.204; FW slope=2.148±0.377; FW R2=0.488; Fig. 4F).
Changes in mRNA levels of (A) prlr1 (C) prlr2 and (E) ostf1 after transfer of Mozambique tilapia from FW to SW (circles) and SW to FW (triangles). Symbols represent mRNA levels from individual RPDs (±s.e.m.; n=8–10). Control fish were maintained in FW (FW–FW) or SW (SW–SW) and sampled on the same time course as fish subjected to salinity change. Expression levels are presented relative to the time 0 SW–SW group. Differences among groups were evaluated by two-way ANOVA. **, ***Significantly different from the corresponding FW–FW controls (open circles) at P<0.01, and 0.001 respectively by Student's t-test. Linear regression of (B) prlr1 mRNA, (D) prlr2 mRNA and (F) ostf1 mRNA from dispersed Prl cells exposed to different medium osmolalities during a 6-h incubation. Open squares and a solid line represent mRNA levels and linear regression respectively from fish acclimated to FW, whereas shaded squares and a dashed line represent these same parameters from fish acclimated to SW. Expression levels are presented relative to the 330 mOsmolal group of each respective salinity (±s.e.m.; n=8, SW-acclimated groups; n=12–16, FW-acclimated groups). Insets represent the values relative to the SW 330 mOsmolal incubation groups ±s.e.m.†††Regression slopes significantly different from each other at P<0.001.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0384
Discussion
The present study examined the modulation of osmotic responsiveness in the tilapia Prl cell by environmental salinity. Our findings indicate that: 1) Prl cells from fish acclimated to FW exhibited a greater capacity for hormone release in response to hyposmotic stimulation than those from SW fish; 2) prl gene expression in Prl cells from fish acclimated to SW was increased as medium osmolality was reduced in vitro, this did not occur in Prl cells from FW-acclimated fish; 3) release and gene expression of Prl188 is more responsive to changes in medium osmolality than release and gene expression of Prl177; and 4) gene expression of prlr2 and ostf1 in Prl cells is induced in direct proportion to increases in plasma osmolality in vivo and culture medium osmolality in vitro respectively. The differences in osmosensitivity between Prl188 and Prl177 we document suggest that different osmotic signal transduction mechanisms regulate their release and gene transcription. Moreover, the osmosensitivity of prlr2 and ostf1 expression provides evidence that these two factors contribute to osmotic control of tilapia Prl cell function.
Prl-secreting cells of the RPD are true osmoreceptors in the Mozambique tilapia (Seale et al. 2006b). In tilapia, Prl177 and Prl188 are encoded by separate genes and differ in both amino acid number and composition, and consequently exhibit only 69% homology (Specker et al. 1985, Yamaguchi et al. 1988, Rentier-Delrue et al. 1989). While the discovery of two distinct Prl molecules in tilapia suggested the evolution of distinct actions, no clear differences in osmoregulatory action have been demonstrated (Specker et al. 1985). This issue has been recently revisited by Fiol et al. (2009), who reported that Prl177 and Prl188 activate distinct downstream signalling pathways in transfected HEK293 cells expressing tilapia PrlRs.
Early studies have shown greater RPD and Prl cell size (Dharmamba & Nishioka 1968), greater Prl cell activity (Nagahama et al. 1975), and greater hypophyseal Prl content (Nicoll et al. 1981) in tilapia maintained in FW compared to tilapia maintained in SW. Later, Yoshikawa-Ebesu et al. (1995) reported that hyposmotically induced Prl177 and Prl188 synthesis is more robust in RPDs of fish acclimated to FW than in RPDs of fish acclimated to SW. Likewise, mRNA levels for both prl genes were higher in pituitaries of tilapia acclimated to FW than in pituitaries of fish acclimated to ¼ SW or SW (Shepherd et al. 1999). More recently, we showed with dispersed Prl cells that Prl188 release in response to in vitro reductions in osmolality is more sensitive in Prl cells of FW fish than in Prl cells of fish acclimated to SW (Seale et al. 2006a). To specifically address the differential osmosensitivity of Prl177 and Prl188 in the current study, we examined the effects of salinity transfers and a range of physiologically relevant extracellular osmolalities on the release and gene expression of these hormones. Consistent with previous reports (Seale et al. 2002, Breves et al. 2011), plasma osmolality decreased, and plasma Prl177 and Prl188 levels increased, following transfer from SW to FW. Gene expression of both prl genes followed a similar pattern. On the other hand, when fish were transferred from FW to SW, increased plasma osmolality was associated with decreases in plasma Prl177 and Prl188, as well as in prl177 and prl188 mRNA levels in RPDs; this finding is in agreement with a previous SW acclimation study (Breves et al. 2010a). These patterns are consistent with the hyperosmoregulatory actions of Prls. Nevertheless, there are changes in the ratio of Prl188/Prl177 that indicate that Prl188 is more sensitive to changes in environmental salinity than Prl177. Previously, Borski et al. (1992) reported relative RPD Prl188/Prl177 ratios close to 1.5:1 in FW fish and 0.5:1 in SW and in fish that had been transferred from SW to FW or from FW to SW after 49 days. In the present study, plasma Prl188/Prl177 ratio increased to 3:1 by 48 h after transfer from SW to FW, and declined to 0.5:1 by 10 days after transfer from FW to SW. Changes in the relative expression of both prl genes followed a pattern that is similar to that seen with plasma Prls. When faced with a decrease in environmental salinity, plasma Prl188 and prl188 mRNA rose to a greater extent than plasma Prl177 and prl177 mRNA, whereas after transfer of tilapia from FW to SW, both plasma levels and mRNA for Prl188 declined to a greater extent than for Prl177. These results indicate that, in vivo, the mechanisms responsible for both transcription and release of Prl188 are more responsive to changes in environmental salinity than those of Prl177. The greater responsiveness of Prl188 to changes in environmental salinity may therefore be a reflection of greater osmosensitivity of the signal transduction mechanisms controlling release/transcription of this hormone.
We compared the relative osmosensitivity of the two Prls by exposing dispersed Prl cells from FW- and SW-acclimated fish to alterations in extracellular osmolality. Despite the fact that Prl cells from FW-acclimated fish released tenfold more Prl188 than did those of SW fish after 6 h, changes in extracellular osmolality elicited responses in cells of fish from both acclimation salinities. The sensitivity of Prl188 release in Prl cells from FW fish, however, was far stronger than that in Prl cells from SW fish, as indicated by the significant difference in the slopes of osmolality vs Prl188 (Fig. 3A). The greater responsiveness of Prl cells from FW fish from that of SW fish to hyposmotic stimulation in vitro has been previously observed in dispersed Prl cells (Seale et al. 2006a), but was not observed under the conditions provided by Borski et al. (1992), who employed intact RPDs in 18–20 h incubations. In that study, Prl release in vitro was clearly discernible between RPDs of FW and SW fish but not between RPDs incubated with hypo- and hyperosmotic media. Differences in experimental design and in the method of Prl assay—electrophoresis in the previous case and RIA in this study—may have ultimately prevented the detection of differences in FW and SW Prl cell osmosensitivity in the earlier study. In the current study, different osmosensitivity regarding the release of the two Prls was observed in FW fish. As medium osmolality decreased, the Prl188/Prl177 release ratio increased. Overall, hyposmotically induced release of both Prls from SW-acclimated tilapia showed a lower sensitivity that may be derived from the lower quantity of Prl stored in the pituitaries of these fish (Nagahama et al. 1975). Recent evidence also suggests that this difference may result from the diminished ability of Prl cells from SW-acclimated tilapia to initiate osmotic signal transduction. The relative rates of cell volume increase and water influx into Prl cells following an osmotic challenge—essential steps of the osmotic signal transduction mechanism (Seale et al. 2005)—were found to be significantly lower in cells from SW tilapia compared to those in FW (Watanabe et al. 2009). These disparities were associated with lower gene expression, protein content and immunoreactivity for the water channel aquaporin 3 (Aqp3) in the Prl cells of SW fish as compared to those of FW fish (Watanabe et al. 2009).
Contrary to release patterns, the hyposmotically induced rise in prl188 mRNA only reached statistical significance in Prl cells of SW fish. Absolute levels of prl188 mRNA, however, were 4.5-fold higher in Prl cells of FW compared to those of SW fish after 6 h incubation in isosmotic conditions (330 mOsmolal). The fact that mRNA levels of the two Prls are much higher in the RPD of FW fish, and perhaps maximal, may render them unable to respond further to osmotic stimulation in vitro. The difference observed between prl188 and prl177 mRNA in response to salinity transfer in vivo was also observed in vitro. The ratio of the gene expression of prl188 to that of prl177, in Prl cells of SW fish, but not of FW fish, rose as medium osmolality was reduced. In earlier studies, employing 3H-leucine to label newly synthesised Prls, it was shown that the ratio of the appearance of newly synthesised 3H-Prl188 to that of 3H-Prl177 was 1.3 in FW tilapia RPD and 0.5 in SW RPD after exposure to a hyposmotic stimulus (Yoshikawa-Ebesu et al. 1995). This nearly threefold difference between FW and SW ratios is similar to the difference in ratios of plasma Prls between FW and SW fish in vivo, as well as the ratios of release and mRNA levels between Prl cells of FW and SW fish observed in vitro.
The current study provides the most in-depth analysis of differential Prl188 and Prl177 regulation by environmental salinity and extracellular osmolality, and is the first to identify the differences in Prl188 and Prl177 osmosensitivity in vitro. Both Prls have been shown to co-localise in the same secretory granules (Specker et al. 1993), indicating that differences in the release of both isoforms are unlikely to result from different Prl cell types or stored hormone pools within the same cell type. It is possible, therefore, that both hormones may be regulated by separate transduction pathways, or are differentially sensitive to intracellular spikes in Ca2+ and cAMP—second messengers that are known to be activated during osmotic signal transduction (Seale et al. 2005, 2011).
In the present study, we also measured the expression of two prlrs in the RPDs of fish transferred from SW to FW and vice versa, and compared these responses with those exhibited by Prl cells in vitro. Branchial expression of prlr2 has been shown to be higher in the gill of tilapia abruptly transferred to SW than in those maintained in FW (Fiol et al. 2009). HEK293 cells transfected with prlr2 exhibited greater tolerance to hyperosmotic stress when compared with empty vector controls or those transfected with prlr1 (Fiol et al. 2009). Expression of branchial prlr1 decreases with transfer from FW to SW (Breves et al. 2010a) and increases with transfer from SW to FW (Breves et al. 2011). Furthermore, branchial prlr1 levels decrease after hypophysectomy in SW but not in FW, suggesting that prlr1 expression is regulated, at least in part, by the pituitary (Breves et al. 2010b). We found prlr1 in the RPD to be unresponsive either to salinity transfers in vivo or to changes in extracellular osmolality in vitro. Nagahama et al. (1975) suggested that Prl secretion may be subject to negative feedback from Prl itself; this finds support in the basic observation that prlr genes are expressed in Prl cells. In mammalian lactotrophs, autocrine or paracrine inhibitory responses to local Prl are reported under physiological (lactation) and pathological (prolactinoma) conditions (Freeman et al. 2000). Autoregulation of Prl cell activity by Prl, through PrlRs, may play a similar role in tilapia to fine-tune hormone synthesis and secretion. This mode of autocrine or paracrine regulation is supported by a recent study in zebrafish embryos reporting that PrlR knockdown by morpholino induced drastic increases in Prl expression in lactotrophs (Liu et al. 2006). We therefore propose that increased prlr2 expression in tilapia RPDs in response to hyperosmotic conditions (both in vivo and in vitro) represents a mechanism to maintain low plasma Prl levels in hyperosmotic environments.
Levels of ostf1 mRNA in RPDs increased by 6 h after transfer from FW to SW and in response to an increase in medium osmolality in vitro. After 48 h and 10 days of transfer from FW to SW, however, ostf1 and prlr2 levels subsided to pre-transfer levels, possibly indicating that changes in ostf1 and prlr2 may be part of a transient response targeted to mitigate the marked increase in plasma osmolality (∼390 mOsmolal) observed at 6 h. The time course of ostf1 induction in the RPD following transfer from FW to SW is similar to that reported in the gills of tilapia exposed to the same hyperosmotic challenge (Fiol & Kultz 2005). Rapid induction of ostf1 in response to hyperosmotic stress has also been shown to occur in branchial cell cultures (Fiol et al. 2006). This response was shown to occur independently from systemic factors, which instead appear to prevent sustained transcriptional induction of ostf1 (Fiol et al. 2006). While induction of ostf1 in the RPDs of fish transferred from FW to SW in vivo is restricted to the first 6 h, further studies are required to determine whether hyperosmotic induction of ostf1 in Prl cells in vitro is also transient in nature. The greater hyperosmotically induced increase of ostf1 observed in Prl cells from FW fish suggests that these cells are more sensitive to hyperosmotic stimuli than those from SW fish.
Studies executing in vitro knockdowns of ostf1 and prlrs will be useful to reveal whether these genes support the adaptive osmotic responses of Prl cells. In order to explore the potential adaptive roles that prlr2 plays in tilapia Prl cells, further studies must identify the effects of the two Prls on both prlr2 expression and the osmosensitivtiy of hormone release and gene expression in Prl cells isolated from fish acclimated to various salinities.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded in part by the National Research Initiative Competitive Grants (grant numbers 2008-35206-18785 and 2008-35206-18787) from the USDA National Institute of Food and Agriculture and by the National Science Foundation (grant numbers IOS-0517769, OISE-0852518 and IOS-1119693).
Acknowledgement
We are grateful to Prof. Tetsuya Hirano for discussions and encouragement during the course of this study.
References
Ayson FG, Kaneko T, Tagawa M, Hasegawa S, Grau EG, Nishioka RS, King DS, Bern HA & Hirano T 1993 Effects of acclimation to hypertonic environment on plasma and pituitary levels of two prolactins and growth hormone in two species of tilapia, Oreochromis mossambicus and Oreochromis niloticus. General and Comparative Endocrinology 89 138–148. doi:10.1006/gcen.1993.1017.
Borski RJ, Hansen MU, Nishioka RS & Grau EG 1992 Differential processing of the two prolactins of the tilapia (Oreochromis mossambicus), in relation to environmental salinity. Journal of Experimental Zoology 264 46–54. doi:10.1002/jez.1402640108.
Breves JP, Fox BK, Pierce AL, Hirano T & Grau EG 2010a Gene expression of growth hormone family and glucocorticoid receptors, osmosensors, and ion transporters in the gill during seawater acclimation of Mozambique tilapia, Oreochromis mossambicus. Journal of Experimental Zoology 313 432–441. doi:10.1152/ajpregu.00213.2010.
Breves JP, Watanabe S, Kaneko T, Hirano T & Grau EG 2010b Prolactin restores branchial mitochondrion-rich cells expressing Na+/Cl− cotransporter in hypophysectomized Mozambique tilapia. American Journal of Physiology 299 R702–R710. doi:10.1152/ajprenal.00432.2010.
Breves JP, Seale AP, Helms RE, Tipsmark CK, Hirano T & Grau EG 2011 Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia. Comparative Biochemistry and Physiology 158 194–200. doi:10.1016/j.cbpa.2010.10.030.
Chen TT, Marsh A, Shamblott M, Chan K-M, Tang Y-L, Cheng CM & Yang B-Y 1994 Structure and evolution of fish growth hormone and insulin-like growth factor genes. In Fish Physiology, pp 179–209. Eds Sherwood Nancy M, Choy I, Farrell Hew Anthony P, J Randall David. London: Academic Press.
Dharmamba M & Nishioka RS 1968 Response of "prolactin-secreting" cells of Tilapia mossambica to environmental salinity. General and Comparative Endocrinology 1 409–420. doi:10.1016/0016-6480(68)90051-8.
Fiol DF & Kultz D 2005 Rapid hyperosmotic coinduction of two tilapia (Oreochromis mossambicus) transcription factors in gill cells. PNAS 102 927–932. doi:10.1073/pnas.0408956102.
Fiol DF, Chan SY & Kultz D 2006 Regulation of osmotic stress transcription factor 1 (Ostf1) in tilapia (Oreochromis mossambicus) gill epithelium during salinity stress. Journal of Experimental Biology 209 3257–3265. doi:10.1242/jeb.02352.
Fiol DF, Sanmarti E, Sacchi R & Kultz D 2009 A novel tilapia prolactin receptor is functionally distinct from its paralog. Journal of Experimental Biology 212 2007–2015. doi:10.1242/jeb.025601.
Freeman ME, Kanyicska B, Lerant A & Nagy G 2000 Prolactin: structure, function, and regulation of secretion. Physiological Reviews 80 1523–1631.
Grau EG, Richman NH & Borski RJ 1994 Osmoreception and a simple endocrine reflex of the prolactin cell of the tilapia, Oreochromis mossambicus. In Perspective in Comparative Endocrinology, pp 251–256. Eds Davey KG, Peter RE, Tobe SS. Ottawa: National Research Council of Canada.
Hirano T 1986 The spectrum of prolactin action in teleosts. In Comparative Endocrinology: Developments and Directions, pp 53–74. Ed. Ralph CL. New York, NY: A. R. Liss.
Liu NA, Liu Q, Wawrowsky K, Yang Z, Lin S & Melmed S 2006 Prolactin receptor signaling mediates the osmotic response of embryonic zebrafish lactotrophs. Molecular Endocrinology 20 871–880. doi:10.1210/me.2005-0403.
Magdeldin S, Uchida K, Hirano T, Grau EG, Abdelfattah A & Nozaki M 2007 Effects of environmental salinity on somatic growth and growth hormone/insulin-like growth factor-I axis in juvenile tilapia, Oreochromis mossambicus. Fisheries Science 73 1023–1032. doi:10.1111/j.1444-2906.2007.01432.x.
Manzon LA 2002 The role of prolactin in fish osmoregulation: a review. General and Comparative Endocrinology 125 291–310. doi:10.1006/gcen.2001.7746.
Nagahama Y, Nishioka RS, Bern HA & Gunther RL 1975 Control of prolactin secretion in teleosts, with special reference to Gillichthys mirabilis and Tilapia mossambica. General and Comparative Endocrinology 25 166–188. doi:10.1016/0016-6480(75)90187-2.
Nicoll CS, Wilson SW, Nishioka R & Bern HA 1981 Blood and pituitary prolactin levels in tilapia (Sarotherodon mossambicus; Teleostei) from different salinities as measured by a homologous radioimmunoassay. General and Comparative Endocrinology 44 365–373. doi:10.1016/0016-6480(81)90014-9.
Pierce AL, Fox BK, Davis LK, Visitacion N, Kitahashi T, Hirano T & Grau EG 2007 Prolactin receptor, growth hormone receptor, and putative somatolactin receptor in Mozambique tilapia: tissue specific expression and differential regulation by salinity and fasting. General and Comparative Endocrinology 154 31–40. doi:10.1016/j.ygcen.2007.06.023.
Rentier-Delrue F, Swennen D, Prunet P, Lion M & Martial JA 1989 Tilapia prolactin: molecular cloning of two cDNAs and expression in Escherichia coli. DNA 3 261–270. doi:10.1089/dna.1.1989.8.261.
Sakamoto T & McCormick SD 2006 Prolactin and growth hormone in fish osmoregulation. General and Comparative Endocrinology 147 24–30. doi:10.1016/j.ygcen.2005.10.008.
Sandra O, Sohm F, de Luze A, Prunet P, Edery M & Kelly PA 1995 Expression cloning of a cDNA encoding a fish prolactin receptor. PNAS 92 6037–6041. doi:10.1073/pnas.92.13.6037.
Seale AP, Riley LG, Leedom TA, Kajimura S, Dores RM, Hirano T & Grau EG 2002 Effects of environmental osmolality on release of prolactin, growth hormone and ACTH from the tilapia pituitary. General and Comparative Endocrinology 128 91–101. doi:10.1016/S0016-6480(02)00027-8.
Seale AP, Richman NH, Hirano T, Cooke I & Grau EG 2003 Evidence that signal transduction for osmoreception is mediated by stretch-activated ion channels in tilapia. American Journal of Physiology 284 C1290–C1296. doi:10.1152/ajpcell.00532.2002.
Seale A, Hirano T & Grau EG 2005 Stimulus-secretion coupling in the osmoreceptive prolactin cell of the tilapia. In Mechanosensitivity of the Cells from Various Tissues 1st edn, pp 371–389. Eds Kamkin A, Kiseleva I. Moscow: Academia.
Seale AP, Fiess JC, Hirano T, Cooke IM & Grau EG 2006a Disparate release of prolactin and growth hormone from the tilapia pituitary in response to osmotic stimulation. General and Comparative Endocrinology 145 222–231. doi:10.1016/j.ygcen.2005.09.006.
Seale AP, Hirano T & Grau EG 2006b Osmoreception: a fish model for a fundamental sensory modality. In Fish Endocrinology, pp 419–440. Eds Zaccone G, Reinecke M, Kapoor BK. Oxford: IBH Publishing Company.
Seale AP, Mita M, Hirano T & Gordon Grau E 2011 Involvement of the cAMP messenger system and extracellular Ca2+ during hyposmotically-induced prolactin release in the Mozambique tilapia. General and Comparative Endocrinology 170 401–407. doi:10.1016/j.ygcen.2010.10.022.
Shepherd BS, Sakamoto T, Nishioka RS, Richman NH, Mori I, Madsen SS, Chen TT, Hirano T, Bern HA & Grau EG 1997 Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia, Oreochromis mossambicus. PNAS 94 2068–2072. doi:10.1073/pnas.94.5.2068.
Shepherd BS, Sakamoto T, Hyodo S, Nishioka RS, Ball C, Bern HA & Grau EG 1999 Is the primitive regulation of pituitary prolactin (tPRL177 and tPRL188) secretion and gene expression in the euryhaline tilapia (Oreochromis mossambicus) hypothalamic or environmental? Journal of Endocrinology 161 121–129. doi:10.1677/joe.0.1610121.
Specker JL, King DS, Nishioka RS, Shirahata K, Yamaguchi K & Bern HA 1985 Isolation and partial characterization of a pair of prolactins released in vitro by the pituitary of cichlid fish, Oreochromis mossambicus. PNAS 82 7490–7494. doi:10.1073/pnas.82.22.7490.
Specker JL, Kishida M, Huang L, King DS, Nagahama Y, Ueda H & Anderson TR 1993 Immunocytochemical and immunogold localization of two prolactin isoforms in the same pituitary cells and in the same granules in the tilapia (Oreochromis mossambicus). General and Comparative Endocrinology 89 28–38. doi:10.1006/gcen.1993.1006.
Watanabe S, Hirano T, Grau EG & Kaneko T 2009 Osmosensitivity of prolactin cells is enhanced by the water channel aquaporin-3 in a euryhaline Mozambique tilapia (Oreochromis mossambicus). American Journal of Physiology 296 R446–R453. doi:10.1152/ajpregu.90435.2008.
Yada T, Hirano T & Grau EG 1994 Changes in plasma levels of the two prolactins and growth hormone during adaptation to different salinities in the euryhaline tilapia (Oreochromis mossambicus). General and Comparative Endocrinology 93 214–223. doi:10.1006/gcen.1994.1025.
Yamaguchi K, Specker JL, King DS, Yokoo Y, Nishioka RS, Hirano T & Bern HA 1988 Complete amino acid sequences of a pair of fish (tilapia) prolactins, tPRL177 and tPRL188. Journal of Biological Chemistry 263 9113–9121.
Yoshikawa-Ebesu JSM, Borski RJ, Richman NH & Grau EG 1995 Effects of acclimation salinity and in vitro medium osmotic pressure on the incorporatin of 3H-leucine into the two prolactins of the tilapia, Oreochromis mossambicus. Journal of Experimental Zoology 271 331–339. doi:10.1002/jez.1402710502.
