Evidence for an Osmoregulatory Role of Thyroid Hormones in the Freshwater Mozambique Tilapia Oreochromis mossambicus

General and Comparative Endocrinology 120, 157–167 (2000) doi:10.1006/gcen.2000.7542, available online at http://www.idealibrary.com on

Evidence for an Osmoregulatory Role of Thyroid Hormones in the Freshwater Mozambique Tilapia Oreochromis mossambicus M. C. Subash Peter, 1 Robert A. C. Lock, and Sjoerd E. Wendelaar Bonga Department of Animal Physiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Accepted July 24, 2000

0.26 nmol 䡠 L ⴚ1, respectively. This study shows that physiological concentrations of T 3 (<10.57 nmol 䡠 L ⴚ1) and T 4 (<6.64 nmol 䡠 L ⴚ1) enhance branchial Na ⴙ pump activity and chloride cell morphometric dynamics, favoring hyperosmoregulatory capacity in freshwater tilapia. These data are consistent with the hypothesis that thyroid hormones perform a role in hydromineral regulation in freshwater teleosts. © 2000 Academic Press Key Words: thyroid hormones; osmoregulation; teleost fish; sodium pump; chloride cell; Na ⴙ,K ⴙ-ATPase; ion transport.

The existing equivocal reports on the osmoregulatory role of triiodothyronine (T 3) and thyroxine (T 4) in teleosts prompted a reinvestigation of their osmoregulatory function in the euryhaline teleost Oreochromis mossambicus. Evidence is presented for thyroidal involvement in hydromineral balance in freshwater tilapia. Dose- and tissue-related responses to various T 3 and T 4 concentrations were observed in the branchial and renal tissues. The branchial Na ⴙ,K ⴙ-ATPase activity, known to reflect sodium pump dynamics, increased significantly after the administration of low doses of T 3 (20 and 40 ng 䡠 g ⴚ1) or T 4 (40 and 80 ng 䡠 g ⴚ1). Higher doses of T 3 and T 4 (>160 ng 䡠 g ⴚ1) did not change the enzyme activity, compared to sham-injected fish. Conversely, the specific activity of renal Na ⴙ,K ⴙ-ATPase decreased significantly at all doses of T 3 or T 4. Further, immunoreactive Na ⴙ,K ⴙATPase in T 4-treated fish increased in branchial chloride cells and this was coupled with a significant increase in the size of chloride cells. T 4 treatment, however, did not change branchial chloride cell density. Plasma osmolality, [Na ⴙ], and [Cl ⴚ] increased, whereas [K ⴙ] decreased following low doses of T 3 or T 4. As expected, plasma levels of T 3 and T 4 increased significantly in a dose-dependent manner after a single injection of either T 3 or T 4. The basal levels of T 3 and T 4 were 4.45 ⴞ 0.49 and 1.25 ⴞ

INTRODUCTION In teleosts, thyroid hormones (THs) influence many aspects of development and growth. Further, triiodothyronine (T 3) and thyroxine (T 4), the principal thyroid hormones in teleosts, are believed to have some involvement in osmoregulation. However, despite the relatively early postulated hypothesis on the thyroidal involvement in osmotic and ionic balance in fish (Gorbman et al., 1969) and the wide interest in these hormones in the following decades, its osmoregulatory role in fish remains to be defined (Eales, 1979; Folmar and Dickhoff, 1980; Milne and Leatherland, 1980; Omeljaniuk and Eales, 1986; Grau, 1987, 1988; Madsen, 1990; Leatherland, 1994; Mancera and McCormick, 1999; Schreiber and Specker, 1999a).

1

To whom correspondence should be made at the above current address. Permanent address: Department of Zoology, Fatima Mata National College, University of Kerala, Kollam 691 001, Kerala, India. E-mail: [email protected].

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Na ⫹,K ⫹-ATPase is involved in ion transport and is widely used as an index of osmoregulation in fishes. It also provides the driving force for vectorial Na ⫹ transport in a variety of osmoregulatory epithelia including kidney, where the enzyme is essential in reducing the urinary loss of monovalent ions in freshwater-acclimated fish (Madsen et al., 1995). The effect of THs on Na ⫹,K ⫹-ATPase actvity in seawater-adapted fish has been the subject of numerous studies. For example, a number of investigations demonstrated the influence of THs on Na ⫹,K ⫹-ATPase during salinity challenges in both Oncorhynchus kisutch (Folmar and Dickhoff, 1979) and Salmo salar (Prunet et al., 1989; Persson et al., 1998). A conclusive effect of THs has not yet been demonstrated, however. On the one hand, THs were reported to maintain Na ⫹ and osmotic balance during an osmotic challenge in Fundulus heteroclitus (Knoeppel et al., 1982; Grau, 1987), but in a number of other studies no effect was found on Na ⫹,K ⫹-ATPase activity in tilapia (Dange, 1986), as well as in other fish (Saunders et al., 1985; Madsen, 1990; Shrimpton and McCormick, 1998; Mancera and McCormick, 1999). Recent studies on summer flounder larvae revealed that T 4 probably plays a more important role in the development of hypoosmoregulatory ability than in hyperosmoregulation (Schreiber and Specker, 1999a, 2000). Very little is known about the effect of THs on renal Na ⫹,K ⫹-ATPase activity in fishes, though renal tissue is reported to be an important target of THs action in mammals (Ismail-Beigi, 1988). It has been observed that T 3 or T 4 administration in the air-breathing perch, Anabas testudineus, decreases the activity of renal Na ⫹,K ⫹-ATPase (M. C. S. Peter, unpublished). It is well known in mammals that T 3, in addition to its stimulation on hepatic Na ⫹,K ⫹-ATPase activity, favors entry of Na ⫹ into the liver cells, suggesting changes in membrane permeability (Ismail-Beigi et al., 1986; Ismail-Beigi, 1988). However, this effect of THs has not yet been explored in fishes. Interestingly, some reports on fish have revealed a synergistic interaction of THs with other hormones with well-established osmoregulatory actions. One explanation for the inconsistencies seen in the effects of THs in osmoregulation is that their primary action may be synergistic rather than direct. For example, THs potentiate the osmoregulatory activities of corti-

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Peter, Lock, and Wendelaar Bonga

sol (Dange, 1986; Shrimpton and McCormick, 1998) and growth hormone (Leloup and Lebel, 1993), although other studies did not demonstrate such an effect (Madsen, 1990; Mancera and McCormick, 1999). In addition, a thyrotropic action of growth hormone has been postulated in F. heteroclitus (Grau and Stetson, 1979), and in Salmo trutta and Oncorhynchus mykiss (Leloup and Label, 1993). In a recent study on mummichog, however, no interaction between T 3 and growth hormone for increasing gill Na ⫹,K ⫹-ATPase activity could be demonstrated (Mancera and McCormick, 1999). The baso-lateral tubular membranes of the chloride cells are the primary locations of Na ⫹,K ⫹-ATPase, which is involved in monovalent ion secretion in sea water (Foskett et al., 1983; De Renzis and Bornancin, 1984; Li et al., 1995; Madsen et al., 1995) and Na ⫹ uptake in freshwater (Perry, 1997; Fenwick et al., 1999). The activity of Na ⫹,K ⫹-ATPase normally correlates positively with chloride cell density (McCormick, 1990; Wendelaar Bonga et al., 1990; Van der Heijden et al., 1997) and there is convincing evidence that the number and Na ⫹,K ⫹-ATPase activity of chloride cells determine the branchial capacity of freshwater fish to absorb external Na ⫹ (Perry, 1997; Li et al., 1998). Data on potential thyroidal influences on chloride cell dynamics are scarce (Madsen, 1990). The evaluation of thyroid function in fish is complicated because the hypothalamo–pituitary–thyroid axis is dependent on a number of biotic (sex, age, and feeding status) and abiotic (photoperiod and temperature) variables (Eales, 1985; Grau, 1988; Leatherland, 1988, 1994). Furthermore, the dose of hormone, the duration of treatments, and the hormone carrier used can all influence the effects of THs, making it difficult to define specific thyroid hormonal effects (Eales, 1979; Leatherland, 1994). At present there is no convincing evidence available for a role of THs in fish hydromineral performance. This has prompted a study to determine whether T 3 and T 4 exert any control on the plasma electrolytic levels or Na ⫹,K ⫹ATPase activity in branchial and renal tissues of freshwater tilapia. Branchial Na ⫹,K ⫹-ATPase activity was assessed biochemically and immunocytochemically. The number of chloride cell was quantified microscopically.

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MATERIAL AND METHODS Fish Mature tilapia of both sexes from our own laboratory stock and weighing 51 ⫾ 5 g were used in this study. Fish were kept in separate 100-L glass aquaria containing Nijmegen tapwater at 26°. The water was continuously aerated, filtered (Eheim 2213), and recirculated at a rate of 600 L/h. The concentration of ions in the water (in mmol 䡠 L ⫺1) were Na ⫹ (0.4), Ca 2⫹ (0.8), Mg 2⫹ (0.2), K ⫹ (0.06), and Cl ⫺ (0.6), and the water pH was 7.6. Fish were fed daily with Trouvit pellets at 2% of total body weight. The photoperiod was set at 12 h light.

Administration of Hormones Three series of experiments were performed. In Experiment I the dose-related effects of triiodo-l-thyronine (T 3, free acid; Sigma, St. Louis, MO) were tested. Thirty-six fish were divided into six groups of 6 each and were placed in separate aquaria. Group 1 fish received 100 ␮l of hormone solvent (saline) and served as the control. The fish of groups 2 to 6 were injected with 20, 40, 80, 160, and 320 ng 䡠 g ⫺1 of T 3, respectively. In Experiment II the effects of l-thyroxine were tested using a similar protocol. In Experiment III the effects of 40 ng 䡠 g ⫺1 of T 3 were examined over time. Five groups of six fish each were treated with the hormone and an equal number received the solvent only. Sampling was done at 0, 6, 12, 24, and 48 h after injection. Injections were given intraperitoneally in 100 ␮l of solvent (0.9% NaCl) with or without T 3 or T 4, and all fish, with the exception of those in Experiment III, were sampled after 24 h. Feeding was stopped 24 h prior to the experiments.

Sampling Procedure Fish were anesthetised in 0.1% phenoxyethanol solution and blood was collected by caudal puncture with a heparinized syringe fitted with a 23-gauge needle. Plasma was separated immediately by centrifugation (3 min, 1200g). Fish were then killed by spinal transsection, and the gill arches and the kidney were

excised, placed in 2 ml ice-cold SEI buffer (0.3 M sucrose, 20 mM Na 2EDTA, 0.1 M imidazole, pH 7.4), and stored at ⫺20°. For light microscopy, pieces of second branchial arches from five fish were immediately fixed in Bouin fixative for 24 h.

Tissue and Plasma Analyses The specific activity of ouabain-sensitive Na ⫹,K ⫹ATPase was measured in homogenates (H o) prepared from branchial and renal tissues as described previously (Flik et al., 1983; Verbost et al., 1994). Branchial epithelium was scraped off from the gill filaments and homogenized in SEI buffer in a Potter device. Homogenates were centrifuged at 2000g for 10 min and the supernatant (H o) obtained was vortexed with saponin (0.2 mg/mg protein; Sigma) to render the membrane leaky. The protein concentration in homogenates was measured with a commercial Biuret protein assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin as standard. Phosphate release was quantified spectrophotometerically and the specific activity expressed in ␮mol P i 䡠 h ⫺1 䡠 mg protein ⫺1. Plasma Na ⫹ and K ⫹ concentrations were measured with a flame-photometric Auto Analyzer (Technicon Model IV). The Cl ⫺ concentration was determined spectrophotometrically via formation of ferrothiocyanate complex. Plasma osmolality (mOsm 䡠 kg ⫺1) was measured with a microosmometer (Roebling, Munich, Germany).

Immunocytochemistry Bouin-fixed branchial arches from saline- and T 4treated fish were dehydrated in alcohol and embedded in paraffin. Sections were cut at 5 ␮m, mounted on poly-l-lysine-coated slides (Sigma), and processed according to the peroxidase–antiperoxidase complex (PAP) technique (Witters et al., 1996) to visualize Na ⫹,K ⫹-ATPase-immunoreactive (ir) cells or chloride cells. Deparaffinated sections were rinsed in two changes of Tris-buffered saline (TBS; pH 7.6) and incubated with 20% normal goat serum for 30 min at 20°. A mouse monoclonal antibody raised against avian Na ⫹,K ⫹-ATPase (IgG; Johns Hopkins University, dilution 1:300) was applied and incubated overnight in a humid chamber. After two rinses of TBS, sections

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were incubated in goat anti-mouse IgG (Nordic, Denmark; dilution 1:100) for 1 h, and mouse PAP (Nordic, dilution 1:750) was applied for 1 h at 20°. The reaction product was visualized with 0.02% 3,3⬘-diaminobenzidine tetrachloride (Sigma) and 0.015% H 2O 2 in saline-free TBS (TB) for 15 min. The reaction was terminated by several rinses in TB. Sections were dehydrated, cleared in xylene, and mounted with Entellan (Merck, Darmstadt, Germany). Analysis was carried out with a Leica DM-RB/E microscope. To determine the relative differences in density and size of the branchial chloride cells in each fish, five randomly chosen areas were viewed. Cell areas were measured with an X–Y tablet (MOB-System; Kontron, Munich, Germany).

Radioimmunoassay (RIA) Plasma total T 4. RIA of tilapia plasma total T 4 was performed using rabbit anti-T 4 antiserum purchased from Henning GmbH (Berlin, Germany) to a final dilution of 1:32,000 and T 4 tracer from Amersham (Little Chalfont, England). Hormone-free tilapia plasma was prepared by repeatedly mixing plasma with charcoal and centrifuging. This procedure was tested for its efficacy in removing T 4 by means of tracer addition and for the absence of charcoal fines by microscopic examination. The assay was performed on 100-␮l samples. Standards, to which 100 ␮l hormone-free plasma was added, ranged from 0.125 to 100 nmol 䡠 L ⫺1. After 2 h of incubation at room temperature, the antibody-bound radioactivity was separated by means of sheep anti-rabbit antiserum in polyethylene glycol. The average limit of detection was 0.55 nmol 䡠 L ⫺1. Intra- and interassay coefficients of variation of duplicate means were 3.0 and 2.4%, respectively, at an average level of 2.5 nmol 䡠 L ⫺1. Plasma total T 3. RIA of tilapia plasma total T 3 was carried out using an Amerlex RIA kit from Amersham with modified standard curves. These contained 50 ␮l hormone-free tilapia plasma and 0.125–100 nmol 䡠 L ⫺1 T 3. The average limit of detection was 0.063 nmol 䡠 L ⫺1. Intra- and interassay coefficients of variation of duplicate means were 6.8 and 7.2%, respectively, at an average level of 0.65 nmol 䡠 L ⫺1.

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Peter, Lock, and Wendelaar Bonga

Statistics Significant differences among groups were tested by ANOVA followed by a multiple mean comparison test (Kramer, 1956). Significance between groups was accepted at P ⬍ 0.05.

RESULTS Na ⴙ ,K ⴙ -ATPase The effects after 24 h of increasing doses of T 3 or T 4 on the specific activity of Na ⫹,K ⫹-ATPase in branchial and renal tissues are shown in Fig. 1. T 3 at 20 and 40 ng 䡠 g ⫺1 and T 4 at 80 ng 䡠 g ⫺1 significantly increased branchial Na ⫹,K ⫹-ATPase activity. T 3 and T 4 above 160 ng 䡠 g ⫺1 had no effect. A significant decrease in the renal Na ⫹,K ⫹-ATPase activity was observed for all doses of T 3 and T 4 employed. Tilapia receiving 40 ng 䡠 g ⫺1 of T 3 produced a time-related increase in the activity of branchial Na ⫹,K ⫹-ATPase (Fig. 2A) and a decrease in renal Na ⫹,K ⫹-ATPase (Fig. 2B).

Plasma Parameters Experiments I and II increased the plasma concentration of Na ⫹ and Cl ⫺ in fish treated with low doses of either T 3 or T 4 compared to the respective salinetreated fish (Tables 1 and 2). Higher doses (⬎160 ng 䡠 g ⫺1) of T 3 or T 4 did not produce any significant effects. The plasma concentration of K ⫹ significantly decreased after 40 ng of either T 3 or T 4. Higher doses were ineffective. Plasma osmolality increased significantly at low doses of T 3 (40 ng 䡠 g ⫺1) and T 4 (80 ng 䡠 g ⫺1) compared to saline-treated fish but higher doses of T 3 and T 4 had no effect. The concentration of Na ⫹ and Cl ⫺ in Experiment III significantly increased 24 h after T 3 injection (Tables 3 and 4). A significant decrease in the concentration of K ⫹ was observed at 24 h in T 3-treated tilapia compared to saline-treated fish (Table 4). Plasma osmolality significantly increased 24 h after T 3 injection (Table 3). Except at 24 h, the other time points gave un-

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Plasma T 3 and T 4 Plasma concentration of T 3 was significantly elevated following various doses of T 3 injections, but there were significantly reduced plasma T 4 levels (Fig. 4A). Various doses of T 4 injections, although significantly increasing plasma T 4 levels, did not change the T 3 concentration in the plasma (Fig. 4B).

DISCUSSION This study for the first time provides evidence for osmoregulatory effects of both T 3 and T 4 in freshwater

FIG. 1. Na ⫹,K ⫹-ATPase specific activities 24 h after injection of increasing concentrations (20, 40, 80, 160, 320 ng 䡠 g ⫺1) of T 3 (A) and T 4 (B) in branchial and renal tissues of freshwater tilapia (Experiments I and II). Symbols represent mean ⫾ SE (n ⫽ 6). Statistics were based on Kramer’s multiple means comparison test. *P ⬍ 0.05; **P ⬍ 0.01.

changed values for all the plasma parameters tested after T 3 injection.

Branchial Chloride Cell Branchial chloride cell density in T 4-treated (40 ng 䡠 g for 24 h) tilapia did not differ from that of controls. A significant increase in chloride cell area measurement was, however, obtained in T 4-treated fish compared to control (Table 5). Moreover, a substantial immunoreactivity was observed in T 4-treated fish compared to saline-treated fish (Fig. 3). ⫺1

FIG. 2. Changes in specific activities of branchial (A) and renal (B) Na ⫹,K ⫹-ATPase after various time intervals (0, 6, 12, 24, 48 h) following a single injection of saline (control) and T 3 (40 ng 䡠 g ⫺1) in freshwater tilapia (Experiment III). Symbols represent mean ⫾ SE (n ⫽ 6). Statistics were based on Kramer’s multiple means comparison test. *P ⬍ 0.05; **P ⬍ 0.01.

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TABLE 1 Changes in the Levels of Plasma Osmolality and Plasma Ions in Freshwater Tilapia Following a Single Injection of T 3 at Various Doses after 24 h (Experiment I)

Control 20 ng 䡠 g ⫺1 40 ng 䡠 g ⫺1 80 ng 䡠 g ⫺1 160 ng 䡠 g ⫺1 320 ng 䡠 g ⫺1

Osmolality (mOsm 䡠 kg ⫺1)

Na ⫹ (mmol 䡠 L ⫺1)

Cl ⫺1 (mmol 䡠 L ⫺1)

K ⫹ (mmol 䡠 L ⫺1)

313 ⫾ 4.3 325 ⫾ 2.3 326 ⫾ 2.5* 320 ⫾ 2.1 310 ⫾ 3.4 308 ⫾ 3.1

159.8 ⫾ 2.3 170.4 ⫾ 1.6* 167.7 ⫾ 1.5 162.3 ⫾ 1.4 160.5 ⫾ 1.4 163.2 ⫾ 1.5

135.5 ⫾ 3.1 140.3 ⫾ 2.1 144.1 ⫾ 1.2* 138.6 ⫾ 2.1 133.5 ⫾ 2.5 134.6 ⫾ 2.1

2.72 ⫾ 0.18 2.46 ⫾ 0.05 2.18 ⫾ 0.09* 2.59 ⫾ 0.11 2.81 ⫾ 0.18 2.79 ⫾ 0.09

Note. Values are mean ⫾ SE for six fish. Significance of differences between control and different doses was statistically compared according to the multiple range test (Kramer, 1956). * P ⬍ 0.05.

tilapia. Significant dose- and organ-related effects on the Na ⫹,K ⫹-ATPase activity and plasma ion concentration were observed following single injections of low doses of T 3 and T 4. The increased specific activity of branchial Na ⫹,K ⫹-ATPase in tilapia in response to low doses of both T 3 and T 4 likely reflected an elevated Na ⫹ pump activity in these fish. Interestingly, the higher doses of THs used had no effect on branchial Na ⫹,K ⫹-ATPase activity. It is therefore reasonable to assume that the earlier reported lack of effects of either T 3 or T 4 on branchial Na ⫹,K ⫹-ATPase activity in tilapia (Dange, 1986) and in other teleosts (Madsen, 1990; Mancera and McCormick, 1999) can be ascribed to the high doses of these hormones (⬎1000 ng 䡠 g ⫺1) employed in those studies. An earlier report on juvenile S. salar, however, showed that T 4 (⬎1000 ng 䡠 g ⫺1) treatment over 11 days increases branchial Na ⫹,K ⫹ATPase (Madsen and Korsgaard, 1989). In this context,

it is equally plausible that the TH response to Na ⫹,K ⫹ATPase activity might be species specific. It is known that THs trigger osmoregulatory adaptation to seawater in salmonids and possibly in other migratory fishes (Young et al., 1989; Prunet et al., 1989; Wendelaar Bonga, 1993). A surge of T 3 and T 4 was reported during the parr–smolt transformation in coho salmon, O. kisutch, in freshwater, days or weeks before a peak in branchial Na ⫹,K ⫹-ATPase activity (Folmar and Dickhoff, 1979). The endocrine regulation of renal Na ⫹,K ⫹-ATPase has received little attention in fish, although some attention has been paid to the effects of cortisol (McCormick, 1995). In the present study, THs exerted a dose-related decrease in renal Na ⫹,K ⫹-ATPase activity in a freshwater fish. Even the higher doses of both T 3 and T 4, which failed to elicit a response to branchial Na ⫹,K ⫹-ATPase activity, brought down the renal en-

TABLE 2 Changes in the Levels of Plasma Osmolality and Plasma Ions in Freshwater Tilapia Following a Single Injection of T 4 at Various Doses after 24 h (Experiment II)

Control 20 ng 䡠 g ⫺1 40 ng 䡠 g ⫺1 80 ng 䡠 g ⫺1 160 ng 䡠 g ⫺1 320 ng 䡠 g ⫺1

Osmolality (mOsm 䡠 kg ⫺1)

Na ⫹ (mmol 䡠 L ⫺1)

Cl ⫺1 mmol 䡠 L ⫺1)

K ⫹ (mmol 䡠 L ⫺1)

312 ⫾ 3.8 319 ⫾ 2.6 323 ⫾ 3.5 326 ⫾ 3.6* 311 ⫾ 3.5 309 ⫾ 4.1

161.7 ⫾ 2.1 169.5 ⫾ 1.9 171.3 ⫾ 1.5* 169.8 ⫾ 1.4 166.5 ⫾ 1.4 163.8 ⫾ 1.9

133.3 ⫾ 2.2 143.8 ⫾ 2.4 143.2 ⫾ 1.6* 137.4 ⫾ 1.5 133.5 ⫾ 1.6 133.0 ⫾ 1.8

2.89 ⫾ 0.04 2.59 ⫾ 0.18 2.08 ⫾ 0.05* 2.15 ⫾ 0.07 2.68 ⫾ 0.09 2.74 ⫾ 0.18

Note. Values are mean ⫾ SE for six fish. Significance of differences between control and different doses was statistically compared according to the multiple range test (Kramer, 1956). * P ⬍ 0.05.

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TABLE 3 Changes in the Levels of Plasma Osmolality and Plasma Na ⫹ Level in Freshwater Tilapia Following a Single Injection of Saline (Control) and T 3 (40 ng 䡠 g ⫺1) and Sampled after Different Time Points (Experiment III)

TABLE 5 Comparison of the Density and the Size of Branchial Chloride Cells (CC) of Freshwater Tilapia Following Saline and T 4 (40 ng 䡠 g ⫺1) Injections after 24 h Saline

Osmolality (mOsm 䡠 kg ⫺1)

Na ⫹ (mmol 䡠 L ⫺1)

Sampling times (h)

Control

T3

Control

T3

0 6 12 24 48

304 ⫾ 2.5 308 ⫾ 2.5 308 ⫾ 1.5 312 ⫾ 3.0 308 ⫾ 2.1

— 310 ⫾ 3.2 313 ⫾ 1.5 321 ⫾ 1.5* 316 ⫾ 2.4

158 ⫾ 1.8 164 ⫾ 1.1 163 ⫾ 1.5 160 ⫾ 0.9 159 ⫾ 1.0

— 162 ⫾ 0.8 167 ⫾ 2.5 169 ⫾ 1.1* 161 ⫾ 1.5

Note. Values are mean ⫾ SE for six fish. Significance of differences between control and T 3 was statistically compared according to the multiple range test (Kramer, 1956). * P ⬍ 0.05.

zyme activity, which indicates differential actions of THs in osmoregulatory tissues. These observations support an osmoregulatory effect of THs in fish. The observed decrease in renal Na ⫹,K ⫹-ATPase activity indicates a reduction of the capacity of renal tubules to reabsorb monovalent ions. It is possible that the reduction of renal ion reabsorption is a response that counteracts the increase in branchial ion uptake. An inhibition of the renal Na ⫹,K ⫹-ATPase activity has been reported by Nolan et al. (1999) in tilapia and in the mummichog (Epstein et al., 1967) after seawater challenge. Further, tissue-specific changes in the

TABLE 4 Changes in the Levels of Plasma Cl ⫺ and K ⫹ in Freshwater Tilapia Following a Single Injection of Saline (Control) and T 3 (40 ng 䡠 g ⫺1) and Sampled after Different Time Points (Experiment III) Cl ⫺ (mmol 䡠 L ⫺1)

K ⫹ (mmol 䡠 L ⫺1)

Sampling times (h)

Control

T3

Control

T3

0 6 12 24 48

136.5 ⫹ 3.1 138.2 ⫹ 2.6 137.6 ⫹ 1.6 134.4 ⫹ 1.9 135.6 ⫹ 2.1

— 137.8 ⫹ 1.6 138.4 ⫹ 1.9 144.5 ⫹ 2.1* 137.1 ⫹ 2.7

2.85 ⫾ 0.13 2.69 ⫹ 0.09 2.65 ⫹ 0.08 2.71 ⫹ 0.13 2.81 ⫹ 0.09

— 2.37 ⫹ 0.12 2.34 ⫹ 0.12 2.07 ⫹ 0.04* 2.37 ⫹ 0.15

Note. Values are mean ⫾ SE for six fish. Significance of differences between control and T 3 was statistically compared according to the multiple range test (Kramer, 1956). * P ⬍ 0.05.

Branchial CC density (cells 䡠 mm ⫺2) Chloride cell area (␮m 䡠 cell ⫺2)

115

⫾ 12

54.5 ⫾ 5.0

T4 123 ⫾ 19 82.2 ⫾ 11.2*

Note. Cell size was determined as the maximum cross-sectioned surface area of the cell. Significance of differences between groups was analyzed using t test. * P ⬍ 0.05.

Na ⫹,K ⫹-ATPase activity have been reported in seawater-adapted tilapia stressed by confinement, where kidney Na ⫹,K ⫹-ATPase activity increased and Na ⫹,K ⫹-ATPase in the gill decreased (Nolan et al., 1999). THs have also been implicated in seawater tolerance, although evidence for a direct role of TH in this process remains unclear (McCormick, 1995). Recent studies on summer flounder, Paralichthys dentatus (Schreiber and Specker, 1999a), clearly indicate an involvement of thyroid in the development of osmoregulatory ability which corroborates the present results in tilapia. The significant rise in plasma Na ⫹ and Cl ⫺ and osmolality in response to T 3 and T 4 treatments, which is associated with an elevated branchial Na ⫹,K ⫹ATPase activity, is consistent with an involvement of THs in Na ⫹ pump activity and the uptake of ions in tilapia. It appears that administration of exogenous THs, which results in elevated plasma levels of THs within their normal physiological range decreases plasma K ⫹. Furthermore, these observations implied that THs might, in addition to stimulating Na ⫹,K ⫹ATPase activity, increase the permeability of target cell membranes to Na ⫹ and K ⫹, as has been demonstrated in mammalian experimental systems (IsmailBeigi et al., 1986; Ismail-Beigi, 1993). However, such an effect of THs in fish target tissues has not yet been investigated. It is reasonable, however, to suggest that in freshwater tilapia, both T 3 and T 4 may favor influx of Na ⫹ from the external medium as a result of either increased permeability or increased Na ⫹/H ⫹ exchanger activity. A decrease of plasma K ⫹ and an increase of plasma Cl ⫺ have been found in the eury-

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Peter, Lock, and Wendelaar Bonga

FIG. 3. Light microscopical branchial sections of freshwater tilapia immunostained with a monoclonal antibody against Na ⫹,K ⫹-ATPase following saline (A and B; magnification ⫻320 for A and ⫻2240 for B) and T 4 (C; magnification ⫻2240) injection (40 ng 䡠 g ⫺1) after 24 h.

haline elasmobranch Dasyatis sabina following TH treatment (de Vlaming et al., 1975). The elevated plasma Na ⫹ could be due to the increased uptake of Na ⫹ from the external medium via the gills. This view gains support from the inference that branchial chloride cells of freshwater fish absorb Na ⫹ and Cl ⫺ from the water (Perry, 1997, 1998). The osmoregulatory effect of T 3 and T 4 in freshwater tilapia is also evident from the changes in Na ⫹,K ⫹ immunoreactivity and the morphometric dynamics of branchial chloride cells. The substantial increase in chloride cell size after T 4 treatment correlates well with the observed increase in branchial Na ⫹,K ⫹ATPase activity, even though branchial chloride cell density remained unchanged. The highly elevated immunoreactivity observed in branchial chloride cells of T 4-treated fish clearly supports the biochemical evidence for the increased ionoregulatory capacity induced by THs. This observation is in agreement with the reported changes in Na ⫹,K ⫹ immunoreactivity of mitochondria-rich cells of summer flounder during

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metamorphosis (Schreiber and Specker, 1999b). A lack of change in branchial chloride cell numbers after T 4 injection was reported in freshwater-adapted rainbow trout (Madsen, 1990). The size and density of chloride cells usually account for their ionoregulatory ability, as an increase in chloride cell size and density in combination with stimulated ion transport and increased Na ⫹,K ⫹-ATPase density has been reported following cortisol administration (Foskett et al., 1981; McCormick, 1990; Dang et al., 2000). In the present study, T 4 treatment, while increasing the size of chloride cells, did not change the density of these cells, suggesting that T 4 may increase the sodium pump activity per chloride cell rather than the differentiation of new chloride cells. Further, it is known in summer flounder that THs mediate morphometric changes in gill mitochondria-rich cells (Schreiber and Specker, 2000). The higher branchial chloride cell area, concomitant with enhanced branchial Na ⫹,K ⫹-ATPase activity, indicates increased ion transporting capacity induced by

165

Thyroid and Osmoregulation in Fish

ACKNOWLEDGMENTS The authors thank Mr. Wim Atsma and Mr. Tony Coenen for technical assistance and Mr. Tom Spanings for animal care and sampling. We are grateful to Professor Jim Fenwick, University of Ottawa for critical reading of the manuscript and to Dr. Alec Ross for measuring the T 3 and T 4 levels. The financial support of the Graduate School for Environmental Chemistry and Toxicology (M&T) is gratefully acknowledged.

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FIG. 4. Plasma T 3 and T 4 levels after single injection (20, 40, 160, 320 ng 䡠 g ⫺1) of T 3 (A) or T 4 (B) in freshwater tilapia. The basal levels of T 3 and T 4 are 4.45 ⫾ 0.49 and 1.25 ⫾ 0.26 nmol 䡠 L ⫺1, respectively. Symbols represents mean ⫾ SE (n ⫽ 6). *P ⬍ 0.05; **P ⬍ 0.01.

THs in freshwater tilapia. The increase in branchial Na ⫹,K ⫹-ATPase activity associated with the rise in net plasma ion levels in THs-treated tilapia suggests that in freshwater fish THs might favor hyperosmoregulatory function. Furthermore, there is evidence that in seawater fish, the availability of THs is crucial during the development of hypoosmoregulatory ability (Schreiber and Specker, 2000). In conclusion, it is evident from these biochemical and immunocytochemical observations that both T 3 and T 4 at plasma concentrations within the physiological range stimulate branchial Na ⫹ pump activity and affect plasma ion levels in freshwater tilapia.

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