Effects of Osmotic Stress on the mRNA Expression of prl, prlr, gr, gh, and ghr in the Pituitary and Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii

In euryhaline teleost black porgy, Acanthopagrus schlegelii, the glucocorticoid receptor (gr), growth hormone receptor (ghr), prolactin (prl)-receptor (prlr), and sodium–potassium ATPase alpha subunit (α-nka) play essential physiological roles in the osmoregulatory organs, including the gill, kidney, and intestine, during osmotic stress. The present study aimed to investigate the impact of pituitary hormones and hormone receptors in the osmoregulatory organs during the transfer from freshwater (FW) to 4 ppt and seawater (SW) and vice versa in black porgy. Quantitative real-time PCR (Q-PCR) was carried out to analyze the transcript levels during salinity and osmoregulatory stress. Increased salinity resulted in decreased transcripts of prl in the pituitary, α-nka and prlr in the gill, and α-nka and prlr in the kidney. Increased salinity caused the increased transcripts of gr in the gill and α-nka in the intestine. Decreased salinity resulted in increased pituitary prl, and increases in α-nka and prlr in the gill, and α-nka, prlr, and ghr in the kidney. Taken together, the present results highlight the involvement of prl, prlr, gh, and ghr in the osmoregulation and osmotic stress in the osmoregulatory organs (gill, intestine, and kidney). Pituitary prl, and gill and intestine prlr are consistently downregulated during the increased salinity stress and vice versa. It is suggested that prl plays a more significant role in osmoregulation than gh in the euryhaline black porgy. Furthermore, the present results highlighted that the gill gr transcript’s role was solely to balance the homeostasis in the black porgy during salinity stress.


Introduction
The end product of the hypothalamic-pituitary-interrenal (HPI) axis, "cortisol", is a key endogenous glucocorticoid that has versatile function such as in respiration, osmoregulation, reproduction, immune responses, growth, and metabolism in teleosts [1]. It is well-known that during stress, cortisol forms a complex with the ligand-activated transcriptional factor, glucocorticoid receptor (gr), to bind specifically to the glucocorticoid response elements (GREs), which increases the transcription of target genes and takes a role in energy metabolism, inflammation, and neuromodulation [2][3][4]. In teleosts, there are two forms of gr genes (gr1 and gr2), and one mineralocorticoid receptor (mr) gene has been identified [5,6]. The trout gr1 had an additional nine amino acid residues (WRARQNTDG) in the DNA-binding domain [7]. An additional nine residues in gr1 confer a stronger

Serum Osmolality and Chloride Concentrations
Both serum osmolality and chloride concentrations were significantly (p < 0.05) increased when the fish were transferred from FW to SW on day 8 (Figure 1a, b). On the other hand, during the transfer of fish from SW to 4 ppt and SW to FW, both serum osmolality and chloride concentrations were significantly (p < 0.05) reduced on day 4 and day 8 as compared to the SW group (Figure 1c,d). There was no significant difference in serum osmolality and chloride concentrations during the transfer from FW to 4 ppt on day 4 (Figure 1a,b). The results are expressed as the mean ± SEM (standard error of the mean). Asterisks (*) indicate significant differences (p < 0.05) between control and treated groups.

The Expression of prl and gh Transcripts in the Pituitary
The quantitative real-time PCR (Q-PCR) analysis showed that the prl mRNA expressions were significantly reduced when the fish were transferred from FW to 4 ppt and SW on day 4 (0.5-fold, p < 0.05) and day 8 (2.8-fold, p < 0.05), respectively (Figure 2a). On the other hand, the prl transcripts were notably increased when the transfer was from SW to 4 ppt on day 4 (1.8-fold, p < 0.05) and from SW to FW on day 8 (3.1-fold, p < 0.05) ( Figure  2c). Exposure to SW increased gh mRNA in the pituitary on day 8 at 34 ppt (1.3-fold, p < 0.05) in black porgy (Figure 2b). There was no significant difference in gh mRNA Figure 1. Analysis of the serum osmolality (a,c) and chloride concentrations (b,d) in black porgy on day 4 and 8 (n = 9, in each group). The results are expressed as the mean ± SEM (standard error of the mean). Asterisks (*) indicate significant differences (p < 0.05) between control and treated groups.

The Expression of prl and gh Transcripts in the Pituitary
The quantitative real-time PCR (Q-PCR) analysis showed that the prl mRNA expressions were significantly reduced when the fish were transferred from FW to 4 ppt and SW on day 4 (0.5-fold, p < 0.05) and day 8 (2.8-fold, p < 0.05), respectively (Figure 2a). On the other hand, the prl transcripts were notably increased when the transfer was from SW to 4 ppt on day 4 (1.8-fold, p < 0.05) and from SW to FW on day 8 (3.1-fold, p < 0.05) (Figure 2c). Exposure to SW increased gh mRNA in the pituitary on day 8 at 34 ppt (1.3-fold, p < 0.05) in black porgy (Figure 2b). There was no significant difference in gh mRNA expression in the pituitary during the transfer from FW to 4 ppt on day 4 ( Figure 2b) and from SW to 4 ppt on day 4 and FW on day 8 (Figure 2d). expression in the pituitary during the transfer from FW to 4 ppt on day 4 ( Figure 2b) and from SW to 4 ppt on day 4 and FW on day 8 (Figure 2d). are expressed as the mean ± SEM. Asterisks (*) indicate significant differences (p < 0.05) between the control and treated group.

The Expression of α-nka, prlr, gr, and ghr Transcripts in the Gill
The α-nka (0.32-fold in 4 ppt, p < 0.05; 0.41-fold in SW, p < 0.05) and prlr (0.75-fold in 4 ppt, p < 0.05 and 0.52-fold in SW, p < 0.05) transcripts were similarly decreased during the transfer from FW to 4 ppt on day 4 and SW on day 8 (Figure 3a,b). Interestingly, the SW acclimation significantly raised the GR transcripts (1.44-fold in SW, p < 0.05) on day 8 ( Figure 3c), but no difference was found during the transfer from FW to 4 ppt on day 4 ( Figure 3c). Similarly, the ghr transcripts were unaffected by increasing the salinity on day 4 and day 8 (Figure 3d). On the other hand, gill prlr transcripts were increased during the transfer from SW to 4 ppt on day 4 (1.44-fold in 4 ppt, p < 0.05) and from SW to FW on day 8 (1.70-fold in FW, p < 0.05) (Figure 3f). α-nkamRNA was increased in fish gills from SW to FW on day 8 (2.11-fold in FW, p < 0.05) but did not increase in fish from SW to 4 ppt on day 4 ( Figure 3e). mRNA expression of gr and ghr in the gill was not changed by the osmoregulatory stress from SW to 4 ppt on day 4 (Figure 3g,h) and FW on day 8 ( Figure  3g,h).  The α-nka (0.32-fold in 4 ppt, p < 0.05; 0.41-fold in SW, p < 0.05) and prlr (0.75-fold in 4 ppt, p < 0.05 and 0.52-fold in SW, p < 0.05) transcripts were similarly decreased during the transfer from FW to 4 ppt on day 4 and SW on day 8 (Figure 3a,b). Interestingly, the SW acclimation significantly raised the GR transcripts (1.44-fold in SW, p < 0.05) on day 8 ( Figure 3c), but no difference was found during the transfer from FW to 4 ppt on day 4 ( Figure 3c). Similarly, the ghr transcripts were unaffected by increasing the salinity on day 4 and day 8 (Figure 3d). On the other hand, gill prlr transcripts were increased during the transfer from SW to 4 ppt on day 4 (1.44-fold in 4 ppt, p < 0.05) and from SW to FW on day 8 (1.70-fold in FW, p < 0.05) (Figure 3f). α-nkamRNA was increased in fish gills from SW to FW on day 8 (2.11-fold in FW, p < 0.05) but did not increase in fish from SW to 4 ppt on day 4 ( Figure 3e). mRNA expression of gr and ghr in the gill was not changed by the osmoregulatory stress from SW to 4 ppt on day 4 (Figure 3g,h) and FW on day 8 (Figure 3g     . Q-PCR was performed to analyze the transcripts of α-nka, prlr, gr, and ghr in the intestine during the transfer of black porgy (a-d) from FW to 4 ppt and SW, and (e-h) from SW to 4 ppt and FW on day 4 and day 8. The relative gene expression was normalized to the control (FW or SW group) value on day 4. The results (n = 9) are expressed as the mean ± SEM. Asterisks (*) indicate significant differences (p < 0.05) between the control and treated groups.

2.5The Expression of α-nka, prlr, gr, and ghr in the Kidney
During the transfer from FW to SW, the α-nka (0.81-fold in SW, p < 0.05) and prlr (0.62fold in SW, p < 0.05) transcripts were significantly decreased in the kidney on day 8 ( Figure  5a,b). On the other hand, the α-nka (1.3-fold in FW, p < 0.05), prlr (2.2-fold in FW, p < 0.05), and ghr (1.66-fold in FW, p < 0.05) transcripts were elevated during the transfer of fish from SW to FW on day 8 (Figure 5e,f,h). When the black porgy were transferred from FW to 4 ppt and SW, the gr and ghr mRNA showed no significant difference in the kidney (Figure 5c,d), and during the transfer from SW to 4 ppt and FW, fish did not show a significant difference in gr mRNA levels in the kidney on day 4 and day 8 (Figure 5g). . Q-PCR was performed to analyze the transcripts of α-nka, prlr, gr, and ghr in the intestine during the transfer of black porgy (a-d) from FW to 4 ppt and SW, and (e-h) from SW to 4 ppt and FW on day 4 and day 8. The relative gene expression was normalized to the control (FW or SW group) value on day 4. The results (n = 9) are expressed as the mean ± SEM. Asterisks (*) indicate significant differences (p < 0.05) between the control and treated groups.

The Expression of α-nka, prlr, gr, and ghr in the Kidney
During the transfer from FW to SW, the α-nka (0.81-fold in SW, p < 0.05) and prlr (0.62-fold in SW, p < 0.05) transcripts were significantly decreased in the kidney on day 8 (Figure 5a,b). On the other hand, the α-nka (1.3-fold in FW, p < 0.05), prlr (2.2-fold in FW, p < 0.05), and ghr (1.66-fold in FW, p < 0.05) transcripts were elevated during the transfer of fish from SW to FW on day 8 (Figure 5e,f,h). When the black porgy were transferred from FW to 4 ppt and SW, the gr and ghr mRNA showed no significant difference in the kidney (Figure 5c,d), and during the transfer from SW to 4 ppt and FW, fish did not show a significant difference in gr mRNA levels in the kidney on day 4 and day 8 (Figure 5g). Figure 5. Q-PCR was performed to analyze the transcripts of α-nka, prlr, gr, and ghr in the kidney during the transfer of black porgy from FW to 4 ppt and SW (a-d), and from SW to 4 ppt and FW (e-h) on day 4 and day 8. The relative gene expression was normalized to the control value on day 4. The results (n = 9) are expressed as the mean ± SEM. Asterisks (*) indicate significant differences (p < 0.05) between the control and treated group.

Discussion
The current study was carried out to investigate the expression pattern of hormones such as prl, gh, prlr, ghr, gr, and α-nka that are responsible for salinity and osmoregulatory stress in a euryhaline teleost, black porgy, during the transition from FW to 4 ppt and SW, and from SW to 4 ppt and FW acclimation at different time intervals. On day 4 and day 8, when the fish were transferred from SW to 4ppt and FW, their serum osmolality and chloride concentrations were significantly lower. On the other hand, these osmolality and chloride concentrations increased significantly on day 8, after transferring fish from 4 ppt to SW. When compared to FW-maintained fish, SW acclimation significantly increased serum osmolality and chloride levels in black porgy. As in other teleosts, serum chloride concentrations and osmolality decreased following transfer to FW [50][51][52]. During salinity changes, euryhaline teleosts face significant osmotic challenges. The endocrine system mediates osmoregulatory responses to salinity changes in the environment, which further initiates osmoregulatory tissues to modulate ion transport. Nevertheless, the gill is regarded as the primary site of total current sodium and chloride transport, which is controlled and mediated by chloride cell activity [53]. The results (n = 9) are expressed as the mean ± SEM. Asterisks (*) indicate significant differences (p < 0.05) between the control and treated group.

Discussion
The current study was carried out to investigate the expression pattern of hormones such as prl, gh, prlr, ghr, gr, and α-nka that are responsible for salinity and osmoregulatory stress in a euryhaline teleost, black porgy, during the transition from FW to 4 ppt and SW, and from SW to 4 ppt and FW acclimation at different time intervals. On day 4 and day 8, when the fish were transferred from SW to 4ppt and FW, their serum osmolality and chloride concentrations were significantly lower. On the other hand, these osmolality and chloride concentrations increased significantly on day 8, after transferring fish from 4 ppt to SW. When compared to FW-maintained fish, SW acclimation significantly increased serum osmolality and chloride levels in black porgy. As in other teleosts, serum chloride concentrations and osmolality decreased following transfer to FW [50][51][52]. During salinity changes, euryhaline teleosts face significant osmotic challenges. The endocrine system mediates osmoregulatory responses to salinity changes in the environment, which further initiates osmoregulatory tissues to modulate ion transport. Nevertheless, the gill is regarded as the primary site of total current sodium and chloride transport, which is controlled and mediated by chloride cell activity [53].
The expression of gh transcripts in osmoregulation varies among species [23]. The unaffected pituitary gh mRNA levels in SW and FW were reported in the euryhaline Mozambique tilapia [53][54][55], sea bass, Dicentrarchus labrax [56], and olive flounder, Paralichthys olivaceus [17]. Similarly, there was no significant difference in the mRNA expression of gh in the pituitary and ghr in the gill during FW to 4 ppt and SW to 4 ppt and FW transfer of black porgy at any time point in this study. However, the long-time exposure to the high salinity significantly increased the gh mRNA in the pituitary on day 8 during the transfer of black porgy from 0 ppt to 34 ppt. The lack of change in the gh levels in black porgy does not rule out a possible role in osmoregulation in this species. On day 4, kidney ghr mRNA expression was also not changed in the response to salinity and osmoregulatory stress. Long-term exposure of SW to FW resulted in the changes in ghr transcripts in the kidney of the black porgy on day 8. The FW transfer induced the increases in ghr gene expression in the black porgy kidney on day 8. Continuous ghr mRNA expression in the kidney from 6 h after transfer to the end of the experiment indicated the possibility of a centralized gh/Igf axis action, as previously described in salmon [57].
Previous research in teleosts indicates that the pituitary hormone prl regulates salt and water homeostasis by influencing ion retention and water uptake via peripheral osmoregulatory organs [39]. Consistently, the present study revealed that prl mRNA transcripts increased from SW to 4 ppt and FW transfer on day 4 and day 8, and decreased significantly from FW to 0 ppt and SW transfer on day 4 and day 8 in the pituitary. The pituitary prl contributes to the fish systemic response to salinity changes [58]. prl regulates ion-conserving and water-secreting processes in osmoregulatory tissues such as the gills, kidney, intestine, and urinary bladder to mediate FW acclimation [48]. prl gene expression rises in response to the decreases in environmental salinity [59,60]; in some cases, these rises are triggered by direct extracellular osmolality [61]. A decrease in prl mRNA levels was shown in black chin tilapia beginning on the first post-experimental day after SW exposure and lasting throughout the SW period, which was restored in FW to baseline control levels [62]. This is consistent with the observation made in isolated prl cells that fish salinity acclimation history influences osmotic responsiveness [63][64][65][66][67]. Based on the wide range of tissues known to respond to prl across teleosts, it is widely assumed that prl is a conserved regulator of physiological responses to low-salinity environments [23].
As with the prl transcripts in the pituitary, a similar pattern of prlr mRNA expressions was observed in the gill when the black porgy transferred from SW to 4 ppt and FW, and from FW to 4 ppt and SW. The increased mRNA expression of prl in the pituitary and prlr in the gill of FW black porgy suggests that prl and prlr may have a potential function in FW adaptation. At 12 and 24 h, gill prlr mRNA levels were lower in SW-transferred fish compared to FW controls. Despite previous research showing that prlr gene expression is reduced in long-term SW-acclimated tilapia [46], this study concludes that rapid downregulation of prlr occurs with SW acclimation [53]. It should also be stated that other factors, such as extracellular osmolality, may play a role in the regulation of prlr expression in teleost gills [53]. The concurrent changes in pituitary prl and gill prlr mRNA levels observed during the transfer of fish from SW to 4 ppt and FW on day 4 and 8 in this study support a role for prl in promoting FW acclimation in black porgy.
In the present study, increased prlr and α-nka transcripts in the gill were associated with the transfer of fish from high salinity to low salinity. The expression of α-nka mRNA in the intestine increased when the fish were transferred from FW to SW on day 8. When the black porgy were transferred from SW to FW, the transcripts of prlr, α-nka, and ghr were significantly increased in the kidney. Branchial α-nka mRNA has been reported to be higher in FW in black porgy [68], which is consistent with the current study's findings that NKA levels increased on day 8 in FW compared to SW. The expression of the αnka gene in the intestine of the euryhaline black porgy increases during the transition from FW to SW on day 8, demonstrating the importance of this enzyme in successful marine osmoregulation [69]. Euryhaline fishes can maintain a narrow range of internal osmolality in response to a wide range of environmental salinities. Fish acclimated to FW experience hypoosmotic conditions with osmotic water gain and diffusive ion loss, whereas fish accustomed to SW experience dehydration. Teleost fishes have evolved complex physiological functions at the level of osmoregulatory organs such as the gill, kidney, and intestine, which are primarily governed by the endocrine system, to deal with such challenges [33,70,71].
The SW adaptation increased gill gr expression within 24 h and maintained it for up to 4 days in SW, indicating the importance of osmoregulation in a hyperosmotic environment [9]. In the gill of rainbow trout, the gr mRNA expression was significantly increased during acute netting stress [72] and SW acclimation [72,73]. On the other hand, the gr mRNA expressions of black porgy were unaffected by salinity and osmoregulatory stress on day 4 at 4 ppt. When the fish were transferred from FW to SW, the gr transcripts in the gills were significantly increased on day 8, implying that the gr transcript played a significant role in black porgy to cope with the homeostasis during SW acclimation. The intestine of fish, as with the gills, is an important osmoregulatory organ. Cortisol is a key regulator of fish osmoregulation, promoting intestinal ion and water absorption [74][75][76], and the SW adaptation of euryhaline teleosts, with a significant increase in gr mRNA in the intestine [77,78]. gr transcripts were significantly increased in the intestine and kidney of the SW fish on day 1 in tilapia [9], whereas neither kidney nor intestinal gene mRNA expression of gr changed during the course of salinity and osmoregulatory stress on day 4 and day 8. The data revealed the differences in the regulation and expression of the gr among organs such as the kidney, gill, and intestine that have different functions [6,79,80].
Comparative approaches of the present study concluded that the transfer of black porgy from SW to 4 ppt and FW, and from FW to 4 ppt and SW acclimation showed the opposite expression in the prl in the pituitary and prlr in the gill of black porgy. αnka transcripts were increased in the fish gill acclimated to FW as compared to SW. The current study findings showed that the gill gr transcript was solely employed to reconcile homeostasis in the black porgy under salinity stress. Overall, during the transfer of black porgy from FW to 4 ppt and SW to 4 ppt, neither the kidney nor intestinal gene mRNA expression of prlr, α-nka, gr, and ghr changed during the course of the transfer from FW to 4 ppt and from SW to 4 ppt on day 4. Long-term salinity and osmoregulatory stress exposure, i.e., day 8, only affects the above transcripts in the black porgy intestine and kidney. The delayed increase in prlr, gr, α-nka, and ghr might be caused by the initiation of the endocrine process depending on the environmental salinity as well as the exposure time intervals among the various osmoregulatory organs of the black porgy.

Experimental Fish
Black porgy (2-year-old, n = 90; body weight = 375.70 ± 13.65 g, body length = 27.90 ± 0.30 cm) were cultured in SW and natural light system at a university aquarium (water temperature ranged from 19 to 24 • C). Fish were given pelleted dry food ad libitum at 1% (w/v) of their estimated body weight per day. The fish were anesthetized with 1% (v/v) glycophenol monophenyl ether and decapitated for each experiment. Pituitary, gill, kidney, and intestine samples were collected and stored in liquid nitrogen at −80 • C. The current experiments were carried out in accordance with the principles and procedures established by the Institutional Animal Care and Use Committee at National Taiwan Ocean University in Taiwan.

Experiment of Osmotic Transfer
In this study, two salinity experiments were carried out. Fish (n = 9 per group) were kept at 33 ppt for the experiment. The fish were randomly divided into two groups and kept in either SW or FW. After a 30-day acclimation period, the water salinity of fish (n = 9 per group) kept in FW was gradually changed to 4 ppt within 4 days (treated group; 4 day group, n = 9) and then from 4 ppt to 34 ppt SW) within another 4 days (treated group; 8 day group, n = 9). Similarly, fish kept in SW (n = 9 per group) were gradually changed to 4 ppt within 4 days (treated group; 4 day group, n = 9) and then from 4 ppt to FW within another 4 days (treated group; 8 day group, n = 9). Respective control groups (n = 9 per group) received the transfer but no salinity changes. The fish were sampled four and eight days after salinity transfer. Every day, the salinity of the water was checked and corrected as needed. The fish were anesthetized in 2-phenoxyethanol (0.4 mL/L) at the end of the experiment, and blood was collected from caudal vessels with a syringe in less than 5 min. The fish were then decapitated, and samples of the gills, intestine, kidney, and pituitary gland were taken, snap-frozen in liquid nitrogen, and stored at −80 • C until RNA extraction. Serum was obtained by centrifugation at 1000× g for 5 min at 4 • C and stored at −20 • C until further analysis.

RNA Extraction, the First-Strand cDNA Synthesis, and Cloning
TRIzol ® reagent (Gibco BRL; Grand Island, NY, USA) was used to isolate RNA from the pituitary, gill, intestine, and kidney, which was then reverse transcribed according to the manufacturer's protocol. The resulting cDNA was used as a template for subsequent PCR amplification of the genes used in this study.
The prl (accession number EU 165342), prlr (EF 467927), α-nka (EF 621407), ghr (AF 502071), and gr (AY 921612) genes were cloned from the black porgy gill cDNA. Multiple alignments of previously published sequences of the respective genes were constructed using the CLUSTAL X program (Modified version 1.81, 10-06-2010) to identify the conserved region; primers were then designed based on this information (Table 1) The PCR products were validated using 1.5% (w/v) agarose gel electrophoresis and ethidium bromide staining, and DNA fragments were excised using a Gel-M TM Gel Extraction system Kit (Bio 101) (VIOGENE; La Jolla, CA, USA) and cloned into pGEM ® -T Easy vector (Promega; Madison, WI, USA). The insert was sequenced on a plasmid using a dye terminator cycle sequencing kit (Perkin Elmer; Foster City, CA, USA) and submitted to BLAST for comparison to known sequences in the NCBI database. Table 1. Specific primers used for Q-PCR analysis. F: forward primer, R: reverse primer.

Gene
Orientation Nucleotide Sequences Usage 4.4. Quantification of α-nka, prl, prlr, gr, and ghr by Q-PCR Analysis Using the iQ TM Multicolor Real-Time PCR Detection system (Bio-Rad Co., Hercules, CA, USA), Q-PCR was used to examine the gene expression of α-nka, prl, prlr, ghr, and gr in the pituitary, gill, kidney, and intestine during FW and SW acclimation. Primer expression software (Version 3.0, 30-06-2008, Applied Biosystem; Foster City, CA, USA) was used to design the primers (Table 1). According to our previous study, gene quantification of standards, samples, and controls was performed simultaneously in a Q-PCR machine (iQTM Multicolor Real-Time PCR Detection System; Bio-Rad Co.) using iQTM SYBR green (Bio-Rad Co.) as a dsDNA minor-groove binding agent, forward and reverse primers, and water [81]. The slope of the relationship between log input cDNA (transcript concentrations) vs. Ct was used to calculate PCR efficiency. The relative expression levels of α-nka, prl, prlr, ghr, and gr in the pituitary, gill, intestine, and kidney were calculated. The control data of the SW or FW group on day 4 were calculated as 1 and then calibrated with other data in the same experiment. The standard curve correlation was −0.999.

Statistical Analysis
Data are presented as means standard deviation of the mean (mean ± SEM). The Student t-test was also used to determine whether there were significant differences (p < 0.05) between the control and treated group, which are denoted by asterisks (*).

Conflicts of Interest:
The authors declare no conflict of interest.