Fish Physiology and Biochemistry 17: 397403, 1997. 1997 Kluwer Academic Publishers. Printed in the Netherlands.

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Immunohistochemical study on changes in gill Na+/K+-ATPase -subunit during smoltification in the wild masu salmon, Oncorhynchus masou
K. Ura, S. Mizuno, T. Okubo, Y. Chida, N. Misaka1, S. Adachi and K. Yamauchi Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate 041, Japan; 1Kumaishi Branch, Hokkaido Fish Hatchery, Ayukawa 189-43, Japan Keywords: masu salmon, Na+/K+-ATPase -subunit, smoltification, gill, immunohistochemistry, chloride cell

Abstract Changes in immunoreactivity of Na+/K+-ATPase -subunit in gill sections of wild masu salmon (Oncorhynchus masou) during the parr-smolt transformation (smoltification) were compared with changes in gill Na+/K+-ATPase specific activity. Gill Na+/K+-ATPase specific activity increased from April and peaked in May. Immunohistochemical analysis, using an antiserum against a synthetic oligopeptide based on the conserved region of the Na+/K+-ATPase -subunit, revealed that immunoreactivity was confined to chloride cells in the surface layer of primary lamellae and the proximal end of secondary lamellae. The size and number of these cells increased gradually from February to May; however, the number of chloride cells of the secondary lamellae decreased in May. These data suggest that the synthesis of Na+/K+-ATPase and the proliferation of chloride cells occur prior to the elevation of enzyme activity. Moreover, it is likely the proliferation and hypertrophy of chloride cells on primary lamellae prepare smolts for entry into seawater and migration in the ocean.

Introduction The anadromous salmonids undergo a parr-smolt transformation (smoltification) before migrating to the sea. The transition from fresh to sea water necessitates an alteration in osmoregulatory mechanisms. These mechanisms develop coincidentally with smoltification (Hoar 1988). The activity of gill sodium-potassium activated adenosinetriphosphatase (Na+/K+-ATPase) increases in smolts prior to entry into sea water (Virtanen and Soivio 1985; Langhorne and Simpson 1986), while a further increase can be observed after the transfer (Folmar and Dickhoff 1979; McCormick et al. 1989). Na+/K+-ATPase is present in all animal cell membranes and is composed of two subunits, and (Lingrel 1992). In the gill of teleosts, this enzyme is localized on the membrane of tubular
Correspondence to: K. Ura, at the above address.

systems in chloride cells (CCs) (Hootman and Philpott 1979). During smoltification, the development of the CC is correlated with an increase in Na+/K+-ATPase activity and seawater tolerance (Threadgold and Houston 1964; Langdon and Thorpe 1984; Richman et al. 1987; Lubin et al. 1989). However, it is unclear whether the elevation in enzyme activity is caused by an increase in enzymes concomitant with the proliferation of CCs, or by activation of the enzyme itself. Recently, Witters et al. (1996) demonstrated cellular sites of active Na+ transport in the gill of rainbow trout (Oncorhynchus mykiss), using monoclonal antibody raised against avian Na+/K+-ATPase subunit. In addition, the Na+/K+-ATPase -subunit was immunolocalized in gills and kidneys of salmonid and non-salmonid fishes using a specific polyclonal antibody against a synthetic oligopeptide corresponding to part of the -subunit

398 (Ura et al. 1996). The main objective of the present study is to examine quantitative changes in Na+/K+-ATPase activity and immunoreactivity during smoltification in wild masu salmon (Oncorhynchus masou).

Materials and methods Fish Wild masu salmon were collected monthly from the Kenichi River, Hokkaido, Japan, by either a cast net or an electric shocker (Benkei Turbo, Frontier Electric, Japan) from January to May 1995. Samples of resident (non-smolting) fish were not available in sufficient numbers for the study. Wild fish and released fish from the single hatchery in this system were distinguished by the absence of the adipose fin in the latter. The age of the fish was judged from growth rings in the scales. Only yearling fish were used in this study. Average of fork length (FL) and body weight (BW) gradually increased from January to April (FL, 10.212.1 cm; BW, 10.016.5 g), and reached maximum values in May (FL, 13.4 cm; BW, 24.4 g; Fig. 1). Parr marks were evident without any body silvering in the parr (JanuaryMarch) and were obscured in pre-smolts (April). In fullsmolts, parr marks were completely absent, while the fins were clear with intense black pigment at the outer extremities of dorsal and caudal fin lobes in May.

Fig. 1. Changes (JanuaryMay) in fork length and body weight of wild masu salmon during smoltification. Values represent means SEM (n = 1020).

Na+/K+-ATPase analysis Na+/K+-ATPase activity was measured according to the modified method of Soyano et al. (1988). The release of inorganic phosphate (Pi) was measured according to the method of Goldenberg and Fermandoz (1966). Protein concentrations were determined by the method of Lowry et al. (1951) and enzyme activity was expressed as mol Pi mg1 protein h1. Immunohistochemical analysis Gills were removed and fixed for 48h in 4% PFA in 0.1 M phosphate buffer (pH 7.2) on ice. Immunostaining was performed according to the method of Ura et al. (1996). Tissue was cut in 5 m sections parallel to the long axes of the primary lamellae and perpendicular to the secondary lamellae. Immunoreactive-chloride cell (ir-CC) numbers were counted in the interlamellae region on primary lamellae and at the free surface on secondary lamellae for 300 cells from 3 individuals at random. Cell size was determined by measuring the long axis of 150 randomly chosen cells from 3 individuals. Statistical analysis The data presented are expressed as means SEM. One-way analysis of variance followed by

Sampling procedure Fish were anaesthetized in ethyl 4-aminobenzoate, and the first gill arch on the right side was removed and rinsed in cold Ringer buffer (0.3 M sucrose, 0.02 M Na2 EDTA, 0.05 M imidazole, pH 7.3). Tissue was frozen in liquid N2 and stored at 80 C until assay for Na+/K+-ATPase activity. For histological examination, the first gill arch on the left side was excised and fixed in cold 4% paraformaldehyde fluid.

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Fig. 2. Changes (JanuaryMay) in gill Na+, K+-ATPase activity during smoltification in wild masu salmon. Values represent means SEM (n = 5). * p < 0.05, significantly different from the value in January.

the Student-Newman-Keuls multiple range test were conducted on the data.

Results Na+/K+-ATPase activity The profile of gill Na+/K+-ATPase activity during smoltification in wild masu salmon is shown in Figure 2. Gill Na+/K+-ATPase activity was 0.48 0.06 mol Pi mg1 protein h1 in January, and remained low until March (0.51 0.09 mol Pi mg1 protein h1. Thereafter, the activity increased markedly and reached a maximum level in May (2.26 0.38 mol Pi mg1 protein h1, p < 0.05 compared with the value in January). Immunohistochemical analysis Immunohistochemical staining of gill sections of the masu salmon in January, March and May is shown in Figure 3. The antibody stained CCs situated on the surface of primary lamellae and at the proximal ends of secondary lamellae. Enlarged irCCs observed on primary and secondary lamellae in March and May, were associated with strong immunoreactivity compared to gill sections in January. Furthermore, a large oval nucleus was observed in the basal part of ir-CCs in gill sections sampled in May.

Fig. 3. Immunostaining of gill sections using the antibody to Na+, K+-ATPase -subunit. Panel A, B and C show gill sections from January, March and May, respectively. Bar = 25 m.

The changes in ir-CC numbers on primary and secondary lamellae during smoltification is shown in Figure 4A. The number of ir-CCs on the primary lamellae was 1.36 0.05 per interlamellae region in January, and gradually increased, to 2.89

400 in January, and significantly increased (p < 0.05) to a maximum in May (14.8 0.1 m). Similar tendencies and values for CC size were seen on secondary lamellae (12.1 0.6 m in January, 14.9 0.1 m in May, p < 0.05).

Discussion In the present study, gill Na+/K+-ATPase activity, which is one of the indicators of hypoosmoregulatory ability, gradually increased as smoltification progressed, reaching a maximum value in May. In other anadromous salmonids, the activity of this enzyme peaked at the smolt stage (Virtanen and Soivio 1985; Langhorne and Simpson 1986), and was elevated further after transfer of smolts to sea water (Folmar and Dickhoff 1979; McCormick et al. 1989). In masu salmon in Hokkaido, maximum seawater adaptability and downstream migratory behavior occurs from late April to May (Kubo 1980; Yamauchi et al. 1984, 1985). Taken together, there is good evidence to suggest that the increase in gill Na+/K+-ATPase activity occurring during smoltification is a preparatory step for seawater entry. In our immunohistochemical study with an antibody against the Na+/K+-ATPase -subunit, the immunoreactivity is almost exclusively confined to the CCs on the surface of primary lamellae and at the proximal ends of secondary lamellae. Recent ultrastructural analyses have clearly distinguished two types of CCs, on the basis of their location, shape, and ultrastructural features (Pisam and Rambourg 1991). In wild masu salmon, two types of CCs were also observed (Mizuno et al. unpublished data), but it is not clear whether our antibody stained all CCs. This issues is under investigation at the ultrastructural level. To data, the vital mitochondrial stain DASPEI (2-p-dimethylaminostyrylethyl-pyridiniumiodide), as well as Champy-Maillets fixative, a general stain for plasma membranes and extensive tubular systems, have been used commonly to identify and quantify mitochondria-rich cells or CCs at the light-microscope level (Bereiter-Hahn 1976; Avella et al. 1987). However, unlike the present study, their methods do not necessarily indicate the amount of Na+/K+-ATPase protein present in the cell. Both the number and size of immunoreactive-

Fig. 4. Changes (JanuaryMay) in immunoreactive-chloride cell number (A) and size (B) on the primary and secondary lamellae of wild masu salmon during smoltification. A total of 300 chloride cells were counted in the interlamellae region of primary lamellae, and the free surface of secondary lamellae, respectively, using gills from 3 individuals on month. Values represent means SEM. Cell number is expressed as number of chloride cells on interlamellae region (primary lamellae) and on free surface (secondary lamellae). * p < 0.05, significantly different from the value in January.

0.08 in May (p < 0.05 compared with the value in January). In contrast, the number of ir-CCs on the secondary lamellae gradually increased and peaked in April (1.83 0.07 per free surface; p < 0.05 compared with the value in January), followed by a decrease in May (1.40 0.07). Changes in ir-CC size during smoltification are shown in Figure 4B. The size of ir-CC on both primary and secondary lamellae gradually increased as smoltification proceeded. On primary lamellae, the average CC size was 11.1 0.1 m

401 chloride cells (ir-CCs) on primary lamellae increased as smoltification progressed and they peaked in May. Similarly, on the secondary lamellae, the size of ir-CCs increased as smoltification developed, while the number gradually increased until April, followed by a decrease in May. Moreover, in gills of resident, non-smolting fish hatchery stock during this season, the size of irCCs on both lamellae were small and did not vary in number in spring (Ura et al. unpublished data). According to previous studies, CCs located in lamellae may be involved in ion regulation in freshwater trout (Perry and Wood 1985; Avella et al. 1987), whereas the increased number of CCs in primary lamellae in seawater-adapted fish reflects adaptation to elevated external salinity (Thomson and Sargent 1977; Laurent and Dunel 1980; Avella et al. 1993). Indeed, enlarged ir-CCs were mainly observed on primary lamellae in gill sections in seawater-adapted masu salmon, Japanese eel (Anguilla japonica) and rockfish (Sebastes schlegeli) (Ura et al. 1996), suggesting that proliferation and hypertrophy of CCs on primary lamellae are preparatory for entry into sea water and migration to the ocean. Moreover, Laurent et al. (1994) suggested that chloride cells may migrate during cell development. Thus, a decrease in chloride cell number on secondary lamellae in May may have occurred as a result of migration to primary lamellae, although much more research is needed to determine the kinetics of chloride cells. It is notable that the increase in ir-CC number and size occurring from February onwards is accompanied by increased immunoreactivity. It is likely that the synthesis of Na+/K+-ATPase occurred from February, prior to the activation of this enzyme. Moreover, an elaboration of rough endoplasmic reticulum in CCs (Mizuno et al. unpublished data), and high expression of Na+/K+ATPase -subunit mRNA were observed in March and April in masu salmon gills (Ura et al. unpublished data). Likewise, high expression of Na+/K+ATPase -subunit mRNA was found in the gills of smolting Atlantic salmon (Salmo salar) (DCotta et al. 1996). Na+/K+-ATPase is a membrane-bound enzyme, composed of and subunits, which becomes activated as an -complex (mature enzyme) (Noguchi et al. 1987; Horisberger et al. 1991). In gill, Hootman and Philpott (1979) indicated clearly that the Na+/K+-ATPase is located on the surface of tubular systems. In the CCs of wild masu salmon, tubular systems developed from March, and extensive development was observed in May at the time of a peak in Na+/K+-ATPase activity (Mizuno et al. unpublished data). Smoltification-associated changes in gill morphology have been reported for other salmonids (Threadgold and Houston 1964; Langdon and Thorpe 1984; Richman et al. 1987; Lubin et al. 1989). Langdon and Thorpe (1984) and Richman et al. (1987) reported that changes in gill enzyme activity and chloride cell density are not necessarily concurrent events. Moreover, in the study of Doyle and Epstein (1972), elevated Na+/K+ATPase activity correlated with the maturation of new chloride cells rather than an increase in cell number. Our results indicate that the synthesis of Na+/K+-ATPase and proliferation occurs early in the process of smoltification, and suggest that the elevation of activity is caused by enzyme maturation (formation of the complex) concomitant with the development of tubular systems in chloride cells. Currently, there is great interest in cloning of the Na+/K+-ATPase gene and the status of this enzyme mRNA in gill or other tissues of fish (Kisen et al. 1994; Cutler et al. 1995a, b). In many animals, the presence of isoforms of each subunit have been reported (Lingrel 1992). We also observed at least three isoforms of -subunit mRNA of different size in gill of smolting masu salmon by Northern blot analysis (Ura et al. unpublished data). Since the -subunit has an important role for enzyme maturation, information on changes in this subunit are required for a more complete understanding of the dynamics of Na+/K+-ATPase during smoltification. During smoltification in anadromous salmonids, endogenous hormone levels change in distinct patterns (Young et al. 1989; Prunet et al. 1989; Bern and Nishioka 1993). Recently, Moriyama et al. (1994) reported higher plasma IGF-I levels in coho salmon (Oncorhunchus kisutch) smolts than those in parr, and Sakamoto et al. (1995) demonstrated that levels of IGF-I mRNA in the liver and the gill increased during smoltification in coho salmon. The hormonal regulation of osmoregulatory mechanism in salmonids and non-salmonids has been studied extensively. However, studies have focused on changes in Na+/K+-ATPase activity, other enzyme activity, chloride cell develop-

402 ment or ion flux (McCormick 1995), and there is only one report on hormonal effects on quantitative changes in Na+/K+-ATPase protein. Madsen et al. (1995) showed that cortisol, growth hormone and IGF-I enhanced expression of Na+/K+-ATPase -subunit mRNA. In order to clarify the osmoregulatory mechanisms in salmonids, further work is needed at the protein and mRNA levels of both and -subunits. Acknowledgements We thank Dr Mark Lokman, University of Otago, Dunedin, New Zealand, for critical reading of the manuscript. Thanks are also due to Mr. Yoshio Itoh, Kumaishi Branch, Hokkaido Fish Hatchery. This study was supported in part by a grant from the Fisheries Agency of Japan, and by a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References cited
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