Fish Physiology and Biochemistry 22: 45–50, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Free plasma thyroxine levels in coho salmon, Oncorhynchus kisutch, during parr-smolt transformation: comparison with total thyroxine, total triiodothyronine, and growth hormone levels Lars O.E. Ebbesson1,2 , Björn Th. Björnsson3, Sigurd O. Stefansson4 and Peter Ekström1 1 Department
of Zoology, Lund University, Helgonavägen 3, SE-22362 Lund, Sweden (Phone: +46 46 2229340; Fax: +46 46 2224425; E-mail:
[email protected]); 2 Seward Marine Center, Institute of Marine Science, University of Alaska Fairbanks, P.O. Box 730, Seward, AK 99664, USA; 3 Fish Endocrinology Laboratory, Department of Zoology, Göteborg University, Medicinaregatan 18, Box 463, SE-405 30 Göteborg, Sweden; 4 Department of Fisheries and Marine Biology, University of Bergen, High Technology Centre, N-5020 Bergen, Norway Accepted: October 9, 1999
Key words: development, endocrine regulation, environment, fish, plasma proteins, radioimmunoassay, smoltification, teleost
Abstract Free plasma thyroxine (FT4 ) levels were measured in coho salmon, Oncorhynchus kisutch, during parr-smolt transformation (smoltification) using an equilibrium dialysis system followed by a radioimmunoassay. The FT4 data were correlated to total plasma thyroxine (TT4), triiodothyronine (TT3), and growth hormone (GH). Plasma samples were taken weekly from early April to late May, when the salmon where released from the hatchery. Free thyroxine and GH levels increased gradually through smoltification. TT4 levels increased significantly in mid-April and in mid-May. TT3 levels increased in April and remained elevated until late-May after which they declined to the lowest levels. During the first increase in TT4 levels, FT4 levels remained low and TT3 levels did not increase until FT4 levels increased in late-April. In addition, after TT4 levels decreased in late May, FT4 levels remained elevated. These data show that there are differences between the plasma FT4 and TT4 profiles during smoltification. Nevertheless, regression analysis indicates that FT4 levels are highly correlated to the increases in the levels of GH (r=0.73) and TT4 (r=0.70). In addition, GH is less correlated to TT4 and TT3 (r = 0.24 and r = −0.46, respectively) compared with FT4 (r = 0.73), suggesting a close relationship between the increases of FT4 and GH. In addition, these data suggest that this method of measuring free plasma thyroxine may provide a new tool for studying the timing of thyroid hormone action and regulation during parr-smolt transformation in salmonids.
Introduction The first histological observations of changes in the thyroidal system during parr-smolt transformation (smoltification) were described by Hoar (1939), and since, the physiological roles of thyroid hormones and the regulatory mechanisms which control the thyroidal state in anadromous salmonids have been extensively investigated. Thyroid hormones have been implicated in a broad range of physiological functions during this period, although indisputable evidence for these functions is scarce. The discrepancies often occur due to
a lack of correlation between the ‘classical’ smoltification plasma TT4 peak and specific physiological changes which can be induced by thyroid hormone treatment (for review, see Dickhoff and Sullivan 1987; Hoar 1988; Leatherland 1993). These differences are at least partially due to the use of pharmacological doses, duration of exposure and/or the developmental stage of the experimental animals (Donaldson et al. 1979; Dickhoff and Sullivan 1987; Ebbesson et al. 1998). Nevertheless, the search continues for the mechanisms controlling the timing of thyroid hor-
46 mone actions during smoltification and many refer to the characteristic total plasma thyroxine (TT4 ) peak(s) that appear during smoltification. In salmonid fishes, as in other vertebrates, the thyroidal functional state involves the complex interrelationship between thyroxine (T4 ) production, T4 availability to tissues, conversion of T4 to triiodothyronine (T3 ), and the presence of T4 and/or T3 receptors. Consequently, studies have dealt with: the relationship between total and free-thyroid hormone levels; the regulation of enzymatic mechanisms which control the metabolism and degradation of thyroid hormones in hepatic and other tissues; the localization and changes of thyroid hormone binding sites in tissues and plasma; and the tissue uptake of thyroid hormones (see Eales 1995; Ebbesson et al. 1998). Free plasma thyroxine (FT4 ) levels have previously been recognized as an important factor in determining changes in thyroxine availability in teleosts (Eales and Shostak 1985; Eales and Shostak 1986). In addition, Boeuf et al. (1989) showed that free and total plasma thyroxine fluctuations mimic each other during smoltification in Atlantic salmon, Salmo salar. However, differences in the timing of TT4 increases in plasma compared with liver and brain have been observed during smoltification in coho salmon,Oncorhynchus kisutch (Specker et al. 1992). This suggests that, at least in coho salmon, T4 levels in tissues may be dependent on T4 availability rather than the TT4 levels and/or smoltification related differences in degradation and clearance rate between blood and various tissues. These observations rekindle the hypothesis that the availability of T4 to tissues may arise from changes in free plasma thyroxine levels and not from the total levels. In search of new insights into the timing of thyroid hormones influence, the present study sought to determine the relationship of plasma free thyroxine levels to total plasma thyroxine, triiodothyronine, and growth hormone (GH) levels in coho salmon during parr-smolt transformation and using a new method of measuring FT4 levels in fish (Ebbesson et al. 1998).
Materials and methods Coho salmon, Oncorhynchus kisutch, were sampled at Trail Lake Hatchery, Alaska, from an outdoor raceway maintained at 5 ± 1 ◦ C well water and exposed to natural photoperiod (60◦N). Plasma samples were taken weekly from mid-April until late-May, when
Figure 1. Changes in body weight, fork length, and condition factor during parr-smolt transformation in coho salmon. The statistical significances were defined using a one-way ANOVA followed by Fisher’s least significant difference test for multiple pair-wise comparisons and accepted at p = 0.05. Each data point represents the mean ± S.E.M. (n = 10) and points are significantly different if they are labelled with different letters.
47 creasing correlation: fork length (r = 0.82), GH (r = 0.73), body weight (r = 0.72), TT4 (r = 0.70).
the salmon where released from the hatchery. The fish were sampled between 10:00 and 12:00 at each sampling date. They were anaesthetized with MS222 (Finquell, USA) and blood collected from the caudal vessels using heparinized capillary tubes (VWR, USA). In order to obtain enough volume for the subsequent plasma analyses, blood from 8–10 fish was pooled for each sample, and three pooled samples were collected for each data point. Blood was subsequently centrifuged and plasma collected and stored at −80 ◦ C until assayed. The hormone assays were performed twice and in duplicate, except the GH assay, which was run once in duplicate. Commercially available radioimmunoassay (RIA) kits were used to measure TT4 and TT3 (RIA-gnost T4 - and T3 -coated tube kits, CisBio, France). The RIA kits for TT4 , TT3 were used according to manufacturer’s instruction manual with intra-assay coefficient of variation < 8% and
