Nuclear receptors for l -triiodothyronine in trout erythrocytes

GENERAL

AND

COMPARATIVE

Nuclear

ENDOCRINOLOGY

Receptors

65, 149- 160 (1987)

for L-Triiodothyronine

in Trout

Erythrocytes

CRAIG V. SULLIVAN,* DOUGLAS S. DARLING,?.~ AND WALTON W. DICKHOFF*+ *School of Fisheries rind tDepartment of ZoologyLv. Unil,ersity of Washington, Seattle, Washington #National Marine Fisheries Service, Coastal Zone and Estuurine Studies Di\aision. 2725 Montlake East, Northwaest and Alaska Fisheries Center. Seattle, Wushington 98112

98195. and Borrlelwrd

Accepted September 8. 1986 We have developed an in vitro assay to evaluate saturable specific binding of triiodothyronine (T,) by erythrocyte (RBC) nuclei isolated from rainbow trout. Our results indicate that the nuclei contain a TX-saturable protein which binds T, with temperature, and pH dependency, high T, affmity (K, = 1.6 x IO9 M-l), and relative thyroid hormone (TH) analog affinities (TRIAC > T, > T, > rT, > Tz) which are characteristic of TH receptors in other vertebrates. Our estimate of the maximal T, binding capacity (MBC) of nuclei isolated from heterogeneous populations of RBCs at different maturational stages (MBC = 3.6 fM/mg DNA; 13 sites/nucleus) was IO- 100 times lower than would be expected of a TH-responsive tissue. Differential cell counts revealed that I% of the RBCs in our trout were immature (pro-RBCs). Pro-RBCs, in contrast to mature RBCs. contain abundant heterochromatin, mitochondria, and polyribosomes, and synthesize hemoglobin. Evaluation of binding data for RBC nuclei taken from trout in which erythropoiesis was stimulated by prior bleeding indicated that MBC was directly proportional to the absolute number of pro-RBC nuclei in the incubation. Our maximum estimate of MBC for pro-RBC nuclei (458 fmol/mg DNA: 1781 sites/nucleus) falls within the range of MBC values reported for other vertebrate THresponsive tissues. These data indicate that RBCs of rainbow trout contain a nuclear protein (putative receptor) which binds T, with characteristics similar to the TH receptor of higher vertebrates, and that nuclear T, binding may be diminished during RBC maturation. ‘C 19x7 Academic

Press. Inc.

Thyroid hormones (THs) have been shown to stimulate erythropoiesis in representative species from each vertebrate class including mammals (Donati et al., 1964; Hollander et al., 1967; Golde et al., 1977; Popovic et al., 1977; Daniak et al., 1978), birds (Domm and Taber, 1946; Sturkie, 1951; Gilbert, 1963; Thapliyal et al., 1977, 1982), reptiles (Charipper and Davis, 1932; Eggert, 1933; Saint Girons, 1961; Thapliyal and Kaur, 1976), and amphibians (Meints and Carver, 1973; Thomas, 1974). In fishes radiothyroidectomy decreases erythrocyte (RBC) count and hematocrit and blood hemoglobin levels, and in vim treatment with thyroxine t Present address: Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, III. 60631.

or thyroid-stimulating hormone increases RBC count (reviewed by Eales, 1979). Increases in blood hematocrit occur during the spring in young salmon (Vernidub and Kolobova, 1971; Hardig and Hoglund, 1984; Zaugg and McLain, 1986), at which time their thyroid is also activated (reviewed by Folmar and Dickhoff, 1980). Thyroid hormone treatment can accelerate the switch from juvenile to adult forms of hemoglobin which normally occurs during salmonid development (Koch et al., 1964; Sullivan et al., 1985). The endogenous concentration of thyroid hormones in fishes apparently stimulates erythropoiesis. However, it is not known whether the erythroid tissues of fish are capable of responding directly to thyroid hormones or whether the effects of thyroid hormone treatment are mediated by other hormonal factors. 149 00 16-6480/87 $1.50 Copyright All right\

D 1987 by Academic Press. Inc. of reproductmn in any form resrved.

150

SULLIVAN,

DARLING,

Most of the biological effects of thyroid hormones are believed to be mediated by an interaction of the hormones with a nuclear receptor protein located within the target tissues (reviewed by Samuels et al., 1982). In thyroid-hormone-responsive tissues this nuclear protein binds 3,5,3’triiodo-L-thyronine (T,) with high affinity and limited capacity and displays affinities for TH and various TH analogs which mimic the relative biological potencies of the hormones and analogs in viva (reviewed by DeGroot et al., 1978; Oppenheimer, 1979). The TH-binding properties of the nuclear receptors have been highly conserved during vertebrate evolution (Darling et al., 1982: Weirich and McNabb, 1984; Eales, 1985). Since association of thyroid hormones with a nuclear receptor appears to be the first step in initiation of hormone action, we have chosen to investigate whether trout RBC nuclei possess saturable binding sites for T,, as do immature mammalian erythrocytes (Boussios et LII., 1982) and amphibian erythrocytes (Galton. 1984, 1985: Galton and St. Germain, 1985; Moriya et al., 1984). We developed an in vitro assay to evaluate specific binding of T, by nuclei isolated from RBCs of rainbow trout (Saho gnirdneri). Furthermore, to make more detailed evolutionary comparisons of TH receptor characteristics, we investigated the relative binding activities of several TH analogs in our assay. Finally, to examine the relationship between nuclear TH binding and erythropoiesis, we investigated the effects of stimulating erythropoiesis by bleeding on binding of T, by the trout RBC nuclei. MATERIALS

AND METHODS

Animals. Male and female rainbow trout (S. gclirdneri) were obtained from the School of Fisheries experimental fish hatchery at the University of Washington and were held in filtered, aerated Lake Washington water at ambient temperature (range 8-18”) under natural photoperiod. The fish were fed twice daily to satiation with a high-protein moist-pellet

AND

DICKHOFF

salmon diet (Satia and Brannon, 1975). The trout used in this study were I-2 years old (sexually immature) and ranged from 250 to 750 g in weight. Isolation ofnuclei. Trout were killed by concussion and bled from the caudal blood vessels. After centrifugation the plasma was stored at -70” for later determination of L-thyroxine (T,) and 3,5.3’-triiodo-L-thyronine concentrations by radioimmunoassay (Dickhoff et al.. 1978. 1982). The blood cells were weighed and washed twice in 0.9% NaCl 17 ml/g cells) and then once in the same volume of SMCT buffer (0.32 M sucrose. 3 mM MgCl,, 2 .mM CaC12, and 10 mM Tris buffer. pH 7.6) at O-4” by repeated resuspension and centrifugation. Following each centrifugation the supernatant and the buffy layer, containing the white blood cells. were aspirated and discarded. During isolation of RBC nuclei all centrifugations were done at 1000~ for 10 min at O-4”. The RBCs were weighed and resuspended in 0.2% Triton X-100 (Triton) in SMCT (14 ml/g RBC), incubated for 15 min at O-4”, and then centrifuged. The supernatant was discarded and the resulting crude nuclear pellet was washed once in 0.2% Triton X-100 in SMCT (14 ml/g RBC) and then once in the same volume of sterile ice-cold SMCT buffer containing 5 mM dithiothreitol and 0.002%. bovine serum albumin (assay buffer: ABI. The purified RBC nuclei were resuspended in AB (1.0 x 109-2.1 x IO9 nuclei/ml) and 0.3 ml of the nuclear suspension. containing 2-4 mg DNA, was dispensed into I2 x 75-mm glass tissue culture tubes for the TH binding assay. Nuclear DNA content was measured (Richards, 1974) using salmon sperm DNA (Sigma Chemical Co.. St. Louis, MO.) as a standard and nuclei were counted in a hemocytometer. The nuclei contained 6.4 t 0.3 pg DNA per nucleus (mean ? SE: N = IO). Nuclei purified in the above fashion appeared intact and free of debris or contamination with white blood cell nuclei when observed with the light microscope after staining in cell-counting diluent (see below). The purity of the nuclear preparation was further verified by phase contrast microscopy. The conditions for isolation of RBC nuclei described above were found to be optimal for obtaining maximal specific binding of T, to the nuclei. These conditions included the number of nuclei per incubation. the Triton X-100 concentration. the number and sequence of washes in Triton X-100, SMCT or AB. and the centrifugation forcr and duration Hor,none-binding c~sscl.v. The following methods were used for estimation of the association constant (K,) and maximal binding capacity (MBC) of T, by the nuclei using Scatchard analysis (Scatchard, 1949). In initial analyses (method A) 8 x IO8 nuclei were resuspended in 0.45 ml AB containing 2.5-5 nM labeled T, ([iz51]T,. sp act 550 mCi/mg. Industrial Nuclear. St. Louis, MO.) and concentrations of nonradioactive hormone (T,) to give a 0.1 to lOOO-fold molar ratio of T, to [iz51]T,. Incubations were done in triplicate and

NUCLEAR

RECEPTORS

were carried out for 90 min with constant shaking at 22”. The incubations were terminated by placing the tubes on ice for 15 min. Nuclear-bound hormone was separated from free hormone by adding 3.0 ml of icecold 0.5% Triton X-100 in SMCT, or 3.0 ml of 20% polyethylene glycol (PEG) in SMCT, to the incubation, mixing thoroughly, and centrifuging at 25OOg for 20 min. The radioactivity of an aliquot (0.7 ml) of the supernatant (free hormone) and the entire nuclear pellet (bound hormone) were counted separately to 99% confidence in a Micromedic gamma counter (Micromedic Systems, Horsham, Pa.). Under these conditions radioactive iodide, which may be present as a contaminant of the [iz51]T,, would cause a slight overestimate of the concentration of free hormone. Data were corrected for nonspecific binding and the K, and MBC of T, binding were estimated by use of Scatchard analysis as described by Chamness and McGuire (1975). MBC was expressed relative to the DNA content measured in the incubations. In later experiments (method B) incubations were done as before but RBC nuclei were incubated in concentrations of [iZ51]T, from 0.1 to 5 nM with (nonspecific binding) or without (total binding) a 200-fold molar excess of T,. Scatchard analysis was done on specific binding data calculated by subtracting nonspecific binding from total binding. Free hormone was calculated by subtraction of bound hormone from total hormone in the incubations. This procedure rests on the assumption that extranuclear hormone is not bound to protein or glass. This assumption appeared valid since calculated free hormone concentrations did not differ from free hormone concentrations measured in aliquots of supernatant. and because supernatant hormone was not precipitable by prior incubation with 20% PEG and centrifugation. The two methods for Scatchard analysis gave similar results (see Res&s). The latter method was used to investigate chemical analog affinities. Optitnization of binding assay. The effect of varying the concentration of Triton X-100 or PEG between 0 and 10% Triton or between 0 and 40% PEG was tested. These solutions were used as a wash after the incubation. Specific binding of [‘251]T, to the nuclei increased with increasing Triton or PEG concentration to plateaus between 0.1 and 5% Triton or between 20 and 40% PEG with apparent optima at 0.5% Triton or 20% PEG. The percentage of [iZSI]T, bound nonspecifically to the nuclei was decreased from 33 to 8% by use of 20% PEG or to 3% by use of 0.5% Triton (in place of SMCT), but attempts to further reduce nonspecific binding without diminishing specific binding using additional Triton, PEG, or SMCT washes were unsuccessful. Since specific binding was generally 20% greater using PEG rather than Triton, PEG was used routinely for Scatchard analysis. Specific binding of T, to the nuclei was pH-dependent; maxima occurred at pH 7.5 in one experiment

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151

and pH 7.8 in another (range, pH 6.5-8.5). The binding assay was routinely run at pH 7.6. A plateau phase of maximal specific binding (indicative of equilibrium) was evident at 1 to 4 hr of incubation at 22”. Incubations longer than 90 min did not consistently yield levels of specific binding equal to those obtained with 60- or 90-min incubations. possibly due to loss or degradation of binding sites (Bernal and DeGroot, 1977). Therefore, 90-min incubations at 22” were used in the binding assay. Specific binding at 0” did not reach maximal levels even after 4 hr of incubation. Specific binding of T, by the nuclei was linearly related to nuclear concentration between 1 and 4 mg DNA when increasing concentrations of purified nuclei were incubated with 1.O or 0.1 nM [iZSI]T, with or without a 200-fold excess of nonradioactive T,. Binding assays were routinely done using 2-4 mg DNA (3.1-6.2 x IO* nuclei) per incubation. Specific binding of T, to the nuclei was saturable. Additions of increasing amounts of [izSI]T, resulted in a linear increase in nonspecific binding but specific binding attained a plateau at 2.5 n&i [iZsI]T,. Chemical analog affinities. The binding aftinities of several TH analogs relative to T, were determined from their ability to compete with [1251]T, for the nuclear binding sites. The analogs used were T,, T,. 3,3’,5-triiodothyroacetic acid (TRIAC), 3.5diiodo-Lthyronine (T?; Sigma Chemical), and 3,3’,5’-triiodo+ thyronine (“reverse” T,, or rT,; CalbiochemBehring, La Jolla, Calif.). On the day of use analogs were dissolved (1 mgiml) in 10 ml of stock solution (9.9 ml methanol/O.1 ml 1.5 M NaOH). Further dilutions were made in AB. Analog competition studies were done using 2.5 nM [lz51]T, with or without O-2.5 (*M T,, T,, or hormone analog. PEG was used to separate bound hormone from free hormone. The data were corrected for nonspecific binding. The presence of nonlabeled analog caused a decline in specific binding of [12SI]T, to the nuclei at equilibrium. The rank-order relative binding affinities of the different analogs were estimated graphically as the molarity of the hormone analog necessary to displace 50% of the [iZ51]T, specifically bound to the nuclei, relative to the molarity of T, needed to displace 50% of the specifically bound labeled hormone. Extraction and enzytnaric characterization clear TH binding sites. Trout RBC nuclei

of nu-

were purified and incubated with [1251]T) and nonlabeled T, as described above except that incubations were of larger volume (10.6 ml), done in duplicate, containing 5 mg DNA/ml (8 x IO8 nuclei/ml) and 3 nM [iZ51]T, with or without a 200-fold molar excess of unlabeled T,. Incubations were done in 30-ml glass centrifuge tubes. Following incubation each tube was mixed and triplicate 0.45-ml samples of the incubate in each tube were transferred to 12 x 75-mm glass tubes, and the radioactivity of the samples (total hormone) was deter-

1.52

SULLIVAN,

DARLING,

mined. The samples were then incubated on ice for 15 min and processed as described above for the hormone-binding assay to determine specific binding. PEG was used to separate bound and free hormone. An equal volume of 0.4% Triton X-100 in AB was added to the nuclear suspension remaining in the 30-ml tubes, after which the contents of the tubes were mixed thoroughly. The tubes were placed on ice for 15 min, and they were then centrifuged at 1OOOgfor 10 min at O-4”. The supernatant was discarded and the nuclear pellet was washed once in 20 ml 0.2% Triton X-100 in AB and once in 20 ml AB by repeated resuspension and centrifugation (IOOOg, 10 min, O-4”). The nuclear pellet was then resuspended in 11 ml of extraction buffer (EB; 0.4 KCI, 3 mM MgCI,, 5 mM D’IT. and 10 mM Tris, pH 7.6) and incubated on ice with constant shaking for 1 hr. The radioactivity of triplicate 0.5 ml aliquots of the nuclear suspension in each tube was determined and the aliquots were returned to the incubation. The tubes were then centrifuged at 15,OOOgfor 30 min at 0”, and the supernatant was decanted, its volume was noted, and the radioactivity in triplicate 0.5ml aliquots of the supernatant from each tube was determined for calculation of specific binding as described above for nuclei. The efficiency of the extraction of specific binding, calculated by comparing specific binding in the original nuclear suspension (in EB) with specific binding in the extraction supernatant, was 27.6%. Since this efficiency was sufticient to provide solubilized binding sites for enzymatic characterization studies, no attempt was made to further refine the extraction procedure. The chemical nature of the binding sites was investigated by examining the effects of various enzymes on specific binding in the extract. The enzymes used were Pronase (B-grade protease from Streptomyes griseus. 45,000 PUKlg; Calbiochem. San Diego. Calif.), trypsin (type I from bovine pancreas, 9700 BAEE unitsimg) with or without trypsin inhibitor (type II-L from lima bean, 1 mg inhibits approx. 1.4 mg trypsin), a-chymotrypsin (type II from bovine pancreas, 40-50 units/mg: Sigma Chemical), ribonuclease A (RNAase, phosphate free, 4285 unitsimg), and deoxyribonuclease I (DNAase, RNAase free. 2414 units/ mg; Worthington Biochemical, Freehold. N.J.). Triplicate samples (0.2 ml) of the extract were mixed thoroughly with 0.1 ml of enzyme solution (1 mg enzyme/ml of AB) in 12 x 75mm glass tubes, the radioactivity in the tubes was determined, and they were then incubated at 5” for 1 hr and then at 0” for 10 min. Three volumes (0.6 ml) of dextran-coated charcoal (15 g/liter in 3 mM Tris buffer, pH 7.5) was then added to the tubes to bind free hormone. The tubes were then incubated on ice for 10 min and centrifuged at IOOOgfor 10 min at O-4”. The radioactivity in a 670~1 sample of the supernatant was determined, and specific binding, corrected for dilution, was calculated as before. Recovery of specific binding in the superna-

AND DICKHOFF tant ranged from 14 to 100% depending on the enzyme preparation used. Stimulation of etythropoiesis. The relationship between the erythropoietic status of the trout and the TH-binding characteristics of their RBC nuclei was examined. We stimulated erythropoiesis by bleeding (Lane. 1980; Lane and Tharp. 1980) and evaluated the K, and MBC of RBC nuclei isolated from control and bled trout. Total blood volume was calculated from the body weight of each trout (Huggel et al.. 1969) and 12% of the blood volume was removed by bleeding the trout from the caudal vein with a syringe containing 20% disodium citrate (20 pi/ml blood) as an anticoagulant. Hematocrits were determined and the blood concentration of mature RBCs and immature RBCs was evaluated as follows. Whole blood (25 ~1) was added to 5 ml of cell-counting diluent (125 ml Burr’s geimsastain stock solution (Biomedical Specialties. Santa Monica, Calif.), I.25 ml gluteraldehyde. 2.19 g NaCI, H,O to 250 ml) and the cells were mixed thoroughly by inversion, incubated overnight at 5”. resuspended, and counted in a hemocytometer. Two types of RBCs were identified based on their morphology and staining characteristics. Mature erythrocytes appeared as elongated elliptical cells. I?- 16 pm long x 6- 10 pm wide, containing a darkly staining elliptical nucleus and a homogeneous pink cytoplasm. A second RBC type was less elliptical with a less compacted and lighter staining nucleus, and a basophilic. blueish-gray cytoplasm. This latter RBC type has been identified as the immature RBC (pro-RBC) of salmonid fishes (Hardig, 1977, 1978; Lane et al.. 1982: Yasutake and Wales, 1983). The percentage of ProRBCs in the blood samples was further verified by differential cell counts of blood smears.

RESULTS Characteristics of Nuclear TH Binding

Two experiments in which Scatchard analysis was done using method A indicated an average binding affinity of 1.5 x lo9 M- * and an average maximal binding capacity of 3.4 fmol T,/mg DNA. Five other experiments in which Scatchard analysis was done using method B indicated a K, of 1.7 k 0.3 x lo9 M-l (mean L SE) and an MBC of 3.7 2 0.3 fmol T,/mg DNA (mean k SE). The results of a representative experiment are shown in Fig. 1. The K, and MBC derived from analyses in which method A was used fell well within the range of values obtained for K, (range 1.0-2.2 x IO9 M-t) and MBC (2.7-4.8

NUCLEAR

RECEPTORS

153

IN TROUT

nuclei. T, and rT, did not compete effectively with labeled T, for the nuclear binding site even at a lOOO-fold molar excess relative to [1251]TJ. Saturable binding in 0.4 M KC1 extracts of the nuclei was significantly reduced by incubation with Pronase, trypsin, or a-chymotrypsin, but not by incubation with RNAase, DNAase, or trypsin with trypsin inhibitor (Fig. 3). Effects of Bleeding ;

1’5

2'5

pMBOUND

FIG. 1. Scatchard plot of the binding of T, to trout erythrocyte nuclei. The data are from a representative assay (method B) in which nuclei were incubated in various concentrations of [i*~l]T, from 0.1 to 5.0 nM with or without a 200-fold excess of T, (see Materials and Methods). Nonspecific binding has been subtracted from all points. Symbols (0) represent the mean of triplicate incubations. Incubations contained 3.3 mg DNA (5.3 x 10s RBC nuclei) and were done for 90 min at 22”. Bound hormone was separated from free hormone using polyethylene glycol (see Materials and Methods). The regression line was fitted using least-squares analysis (r2 = 0.90. P c 0.05). See Results for average values for the binding afftnity and maximal binding capacity of the nuclei.

Pmol T,/mg DNA) using method B. Therefore the results of the seven experiments were pooled to give an estimated K, of 1.6 + 0.3 x lo9 M-l and an estimated MBC of 3.6 t 0.3 fmol T,/mg DNA (mean 5 SEM) or 13 + 1 sites/nucleus. The average correlation coefficients (r2) of the individual Scatchard plot linear regressions was 0.9 (N = 7). Hormones and analogs other than TRIAC had significantly different binding activity than did T, (Fig. 2). The rank-order relative binding potency of the hormones and analogs was TRIAC > T, > T, > rT, or T,. TRIAC was consistently (but not significantly) more effective than T, in inhibiting specific binding of [1251]T3 to the nuclei. Higher concentrations of T, than T, were needed for the same degree of inhibition of specific binding of labeled T, to the

The effects of bleeding on hematocrit and the concentration of mature RBCs (mRBCs) and immature RBCs (pro-RBCs) in trout are shown in Fig. 4. In control trout in our experiment hematocrit was 39.7 + 1.3% and the blood contained 1.6 ? 0.1 x lo6 mRBC/ml and 1.3 ir 3.4 x lo4 proRBCs/ml (mean ? SE; N = 20). In bled trout both hematocrit and the concentration of mRBCs were decreased greatly on Day 14 after bleeding. The concentration of pro-RBCs in blood samples taken from trout 14 days after bleeding was more than

I MOLAR

10 RATIO ANALOG:

1000

100 LABELED

T3

FIG. 2. Inhibition of specific binding of [i251]T, to trout erythrocyte nuclei by TRIAC, T,, T,, rT,, and T, (see Materials and Methods). Symbols (0) represent the mean of triplicate incubations. Vertical lines indicate SEM. Incubations were done for 90 min at 22 and contained 3.2 mg DNA (5.2 x lo* RBC nuclei) and 3.4 nM [iz51]T, and a 0- to IOOO-fold molar excess of nonisotopic hormone or analog. Horizontal dashed line indicates point of 50% reduction in specific binding of [izsI]T, to the nuclei used to estimate relative affinities of the hormones and analogs (see Results).

SULLIVAN,

1.54

DARLING,

100 z 2 m I

60

2z

40

1 b?

20

60

C

P

1

aC T/,

0

R

FIG. 3. The effect of various enzymes (C, control: P. Pronase: T, trypsin; aC. cr-chymotrypsin: T/l, trypsin with trypsin inhibitor; D. DNAase; R, RNAase) on specific binding (percentage of maximum) in a 0.4 A4 KCI extract (see Materials and Methods) of the trout erythrocyte nuclei. Triplicate 0.2-ml samples of extract were incubated with 0. I ml of the enzyme solution (I mgiml) at 5” for 1 hr and then at 0” for IO min. Free hormone was precipitated from the supernatant using dextran-coated charcoal. Recovery of specific binding in the supernatant ranged from 14 to 100% depending on the enzyme preparation used. Height of the bars represents the average specific binding in triplicate incubations. Vertical lines indicate SEM.

four times greater than control values. Twenty-four days after bleeding the hematocrit and concentration of mRBCs had returned to control values, but the concentration of pro-RBCs in bled trout was still more than twofold greater than control values on Day 24. We were unable to purify nuclei from 500

400

= 300 =: p: z 200

100

lma

0

18 DAY

AND DICKHOFF

RBCs obtained from bled trout 14 days after bleeding when the concentration of pro-RBCs was very high. Problems with nuclear purification were due to nuclear fragility. Figure 5 shows the results of Scatchard analysis of binding of T, to nuclei isolated from control and bled trout 24 days after the fish were bled. Bleeding had no effect on the K, of T, binding to trout RBC nuclei. However, the MBC of nuclei isolated from bled fish (7.8 fmolimg DNA) was 229% of the control value (3.4 pmol/g DNA), and was well outside the range of MBC values (2.7-4.8 fmolimg DNA) observed in seven previous Scatchard analyses. In bled fish the percentage of RBCs that were immature (1.89%) was 228% of that in controls (0.83%; see inset, Fig. 5). Hence bleeding caused both a 2.3-fold increase of pro-RBCs and a 2.3-fold increase of MBC. The plasma T, and T, concentrations in pooled control plasma samples (T3, 4.1 rig/ml; T,, 4.8 rig/ml) were similar to those in pooled plasma samples obtained from bled fish (T,, 5.9 rig/ml; T,, 2.5 ngiml) when the Scatchard analysis was performed (Day 24). These values fall within the range of plasma TH concentrations observed in our trout during development of the binding assay (T,, 3.3-6.3 ngiml; T, 2.4-13.9 rig/ml).

IL 24

FIG. 4. Effect of removal of 12% of the blood volume of trout on their hematocrit (open bars) and concentration of mature erythrocytes (cross-hatched bars) and immature erythrocytes (stippled bars) at 0 (N = 20), 14 (N = lo), and 24 (N = 5) days after bleeding. The data are expressed as a percentage of values in unbled trout. Vertical lines indicate SEM. See Results for average absolute values for hematocrit and mRBC and pro-RBC concentrations.

DISCUSSION

The erythroid tissues of vertebrates are useful for study of TH receptor function. For example, both thyroxine and 3,5,3’triiodo-L-thyronine stimulate erythropoiesis in human, murine, and canine bone marrow cells in vitro (Golde er al., 1977; Popovic er al., 1977; Daniak et al., 1978) and Boussios et al. (1982) have described in ~itvo high affinity, low capacity, saturable specific binding of T, and T, by erythrocytic nuclei isolated from hypoxic hamsters. In Boussios et al.‘s (1982) study the tissue response to TH (erythroid colony growth) was correlated with the extent of

NUCLEAR

2 tmoles

4 Bound/mg

6

RECEPTORS

8

DNA

FIG. 5. Scatchard plots of the binding of T, to erythrocyte nuclei isolated from blood of control trout (closed circles) or from trout which had been bled (12% of blood volume removed) 24 days prior to the assay (open circles). The nuclei were incubated in various concentrations of [1Z51]T, from 0.1 to 5.0 nM (method B) with or without a 200-fold excess of T, (see Materials and Methods). Nonspecific binding has been subtracted from all points. Symbols (0, 0) represent the mean of triplicate incubations. Incubations contained 3.2 mg DNA (5.1 x 108 RBC nuclei) and were done for 90 min at 22”. Bound hormone was separated from free hormone using polyethylene glycol (see Materials and Methods). The regression lines were fitted using least-squares analysis (control. r* = 0.80. P < 0.05: bled, r* = 0.95, P < 0.05). See Results for values for the binding affinity and maximal binding capacity of the nuclei of the two groups of fish. Inset indicates that the percentage of RBCs which were immature (pro-RBC) was proportional to MBC in control (C) and bled (B) trout. The height of the bars represents the mean value for five fish. The vertical bars indicate SEM.

hormone occupancy of the nuclear TH binding sites. Occupancy of nuclear receptors which bind TH with these characteristics is believed to result in the initiation of hormone action (reviewed by Oppenheimer, 1979). Nuclei of erythrocytes isolated from the peripheral blood of bullfrog tadpoles and adults specifically bind T, with high affinity and limited capacity in vitro (Moriya et al., 1984; Galton, 1984, 1985; Galton and St. Germain, 1985). Thyroid hormones stimulate erythropoiesis and also stimulate the change in erythropoietic cell line which underlies the transition from larval RBCs (containing larval hemoglobins) to adult (frog) RBCs (containing

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frog hemoglobins) which occurs during metamorphosis of anuran amphibians (Moss and Ingram, 1965; Forman and Just, 1980). We are unaware of any previous studies of nuclear TH receptors in erythroid tissues of birds, reptiles, or fishes. In our study an in vitro assay for thyroid hormone binding to trout RBC nuclei was developed allowing a direct analysis of the TH-binding characteristics of RBC nuclei. The assay was based on an in vitro nuclear T,-binding assay developed by DeGroot and Torresani (1975) for rat tissues as modified by Darling et al. (1982) for use with salmon tissues. However, the conditions for isolation of the RBC nuclei differed considerably from those used by Darling et al. for salmon liver and were developed independently. Specifically bound T, was extractable from the nuclei by 0.4 M KC1 and was shown to be bound to saturable sites. Nuclear T,-saturable macromolecules have previously been isolated from mammalian, avian, amphibian, and teleostean tissue nuclei by extraction with 0.4 h4 KCL (Surks et al., 1973; DeGroot et al., 1974; Samuels et al., 1974; Schwartz and Oppenheimer, 1978; Toth and Tabachnick, 1979; Van Der Kraak and Eales, 1980; Bellabarba and Lehoux, 1981). Specific binding in our nuclear extract was significantly reduced by incubation with Pronase, trypsin, and a-chymotrypsin but not by incubation with DNAase or RNAase. This indicates that the specific binding entity in the trout RBC nuclei is a protein. Similar results have been obtained with enzymatic digests of nuclear extracts from mammalian, avian, and trout tissues (Surks et a/., 1973; DeGroot et al., 1974; Samuels et al., 1974; Schwartz and Oppenheimer, 1978; Toth and Tabachnick, 1979; Van Der Kraak and Eales, 1980; Bellabarba and Lehoux, 1981). In mammalian tissues the TH receptor has been recently characterized as a nonhistone nuclear protein of molecular weight 50,000 which is an integral component of a receptor-chromatin complex and has been

156

SULLIVAN,

DARLING,

partially purified (reviewed by Oppenheimer, 1985). The binding of T, to the trout RBC nuclei displayed an appropriately high binding affinity expected of a hormone receptor. The linear Scatchard plots indicated that T, was bound to a single class of noncooperative high affinity sites. The affinity of the RBC nuclei for T, (K, = 1.6 -+ 0.3 x IO9 M-i) is similar to that previously reported by Van Der Kraak and Eales (1980) for T, binding in viva to rainbow trout hepatic nuclei (0.22 x lo9 M-l), and to that reported by Bres and Eales (1985) for T, binding to rainbow trout hepatic nuclei in l&o (7.2 x lo9 M-l). These findings suggest that the nuclear binding sites in different trout tissues are similar, and perhaps the same, protein(s). Furthermore, the affinity of trout RBC nuclei for T, is well within the range of affinities reported by other investigators (range OS-25 x lo9 M-l) using similar methods (e.g., in vitro incubations of isolated nuclei or nuclear extracts) for evaluation of nuclear TH binding in a variety of tissues obtained from fish, amphibians, birds, and mammals (DeGroot and Torresani, 1975; Spindler et al., 1975; Lindenberg et al., 1978; Schwartz and Oppenheimer, 1978; Morishige and Guernsey, 1978; Gonzales and Ballard, 1981; Darling ef al., 1982; Bellabarba and Lehoux, 1981; Cheng, 1983; Galton and Schaafsma, 1983; Lintlop and Youson, 1983; Anselmet et al., 1984; Weirich and McNabb, 1984; Bres and Eales, 1985; Chakraborti et al., 1986). The relative binding affinities (TRIAC > T, > T, > rT, > T,) of the thyroid hormones and hormone analogs that we tested are similar to those reported for several mammalian, avian, and teleostean tissues (see references cited above). In mammals and amphibians, the relative biological potencies of the various hormones and hormone analogs have been reported to correspond well with their relative nuclear binding affinities (Greenberg et al., 1963; Frieden, 1967; Koerner et al., 1974: Jor-

AND

DICKHOFF

gensen, 1976). However, the relative biological potencies of the various thyroid hormones and hormone analogs have not been adequately defined in fish. In coho salmon liver (Darling et al., 1982) and in rainbow trout liver (Bres and Eales, 1985) T, and T, have been shown to bind to the same nuclear site, since T, in sufficient concentration could displace all specifically bound T,, and T, could displace all specifically bound T, from the nuclei. In their comprehensive study of the chemical-structural requirements of the nuclear TH binding site in trout liver, Bres and Eales (1985) evaluated the relative binding affinities of 15 TH analogs and found that the requirements of the T, and T, binding site(s) were very similar. T, was several times more potent in competing with [12SI]T, for the nuclear site than was T,. Similar results were obtained with coho salmon liver nuclei by Darling et al. (1982) and with trout RBC nuclei (this study). These results suggest that in salmonids there may be a single nuclear TH receptor site and that the T, may be the active TH at the nuclear level. In teleosts T, is considered to be a less biologically potent “prohormone” for T,, and is apparently the only TH secreted by the thyroid (reviewed by Eales, 1979). T, can be converted by a peripheral 5’-deiodinase enzyme to T, in trout (reviewed by Eales, 1985). The maximal nuclear capacity for saturable T, binding of a tissue may be an index of the potential responsiveness of the tissue to TH (reviewed by Oppenheimer, 1979). For example, in adult bullfrogs (Rana catesbeiana) or neotenous urodeles (Nectunts maculosa), which display only limited responsiveness to TH in viva, the MBC of liver and RBC nuclei are greatly reduced relative to the hepatic and erythrocyte MBC of TH-sensitive bullfrog larva (Galton, 1985). In the studies cited above, estimates of in vitro MBC for nuclei isolated from several mammalian, avian, amphibian, and teleostean tissues range from

NUCLEAR

RECEPTORS

100 to > 10,000 fmol/mg DNA. Our estimated MBC of trout RBC nuclei was 3.6 fmol/mg DNA (13 sites/nucleus). This is a minimal estimate of MBC since our data were not corrected for occupancy of specific TH binding sites by endogenous hormone or loss or inactivation of receptors during extraction and incubation of the nuclei. Even assuming a 50% reduction of MBC due to these factors the MBC of trout RBC nuclei appears to be 10 to 100 times lower than would be expected of a TH-responsive tissue. Since our incubations contained a heterogeneous population of nuclei isolated from RBCs at different maturational stages, we hypothesized that specific TH binding might be restricted to the pro-RBCs. In contrast to mature RBCs, pro-RBCs contain organelles required for aerobic respiration and protein synthesis (mitochondria, polyribosomes) characteristically found in TH-responsive tissues (Lane el al., 1982). We were unable to confirm this hypothesis by evaluating TH binding by separate populations of proRBCs and mature RBCs, since we were unable to separate the two RBC types on Renograffin density gradients (data not shown) as described by Forman and Just (1981). We therefore evaluated the MBC of RBCs isolated from trout in which we had stimulated erythropoiesis by prior bleeding. Bleeding stimulated erythropoiesis in trout as made evident by the increase in concentration of pro-RBCs and decrease in concentration of mature RBCs in their peripheral blood. These results corroborate the previous reports by Lane (1979) and Lane and Tharp (1980) that bleeding stimulates erythropoiesis in trout. The MBC of RBC nuclei isolated from bled trout increased in direct proportion to the increase in pro-RBC nuclei in the incubations. The MBC of RBC nuclei isolated from bled trout was more than twice the MBC of control nuclei and was well above the range of MBC values calculated from seven previous experiments with control

IN TROUT

157

nuclei. These results suggest that in trout specific binding may be largely restricted to the early maturational RBC stages, and that TH may play a role in RBC maturation. A maximum estimate of MBC in trout RBCs, assuming that saturable TH binding is limited to the pro-RBCs, would be 458 fmollmg DNA (1781 sites/pro-RBC nucleus). These values fall within the range of MBC reported for nuclei isolated from other TH-responsive vertebrate tissues. In summary, our investigation of TH binding by trout RBC nuclei indicates that the nuclei display characteristics of TH binding which satisfy several criteria for a hormone receptor, including high affinity, limited capacity and chemical analog specificity. The binding site is extractable with 0.4 M KC1 and appears to be a nuclear protein. The relative affinities of the nuclei for TH and various TH analogs fit the general vertebrate pattern for TH receptors. The MBC of heterogeneous populations of mature RBC and pro-RBC nuclei is low, but specific binding may be limited to proRBC nuclei and fall within the range of MBC values previously reported for nuclei isolated from other vertebrate TH-responsive tissues. Our data imply that there is a decrease in the number of nuclear TH receptors during RBC maturation and that TH may play a role in RBC maturation in trout. ACKNOWLEDGMENTS The authors thank Professor Aubrey Gorbman, Department of Zoology. and Dr. William K. Hershberger, School of Fisheries. for use of their laboratory facilities. and Ms. Susan Hobbs, Department of Zoology, University of Washington, for her excellent technical assistance. This work was supported in part by a grant (S-P-30-ES-02190-04) to C.V.S. from the Marine/ Freshwater Biomedical Center of the National Institutes of Environmental Health Sciences. W.W.D. was supported by a Washington Sea Grant (Project R/A 42) and by the National Science Foundation (DCB-8416224) during the course of this work. This is contribution No. 706 from the School of Fisheries, College of Ocean and Fisheries Sciences, University of Washington, Seattle, Wash.

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REFERENCES Anselmet, A.. Gharbi-Chihi, inghelli, O., and Santelli, dothyronine receptor in cytes cloned from obese nology

J., Torresani. J.. GhirS. (1984). Nuclear triiodifferentiating preadipoand lean mice. Endocri-

114, 450-456.

Bellabarba, D.. and Lehoux, J. G. (1981). Triiodothyronine nuclear receptors in chick embryo: Nature and properties of hepatic receptor. Endocrinology 109; 1017-1025. Bernal, .I., and DeGroot, L. J. (1977). Thyroid hormone receptors: Release of receptor to the medium during in vitro incubations of isolated rat liver nuclei. Endocrinology 100, 648-655. Boussios, T.. McIntyre. W. R., Gordon, A. S.. and Bertles, J. F. (1982). Receptors specific for thyroid hormones in nuclei of mammalian erythroid cells: Involvement in erythroid cell proliferation. Brit.

J. Huematol.

51, 99- 106.

Bres, 0.. and Eales, J. G. (1985). Thyroid hormone binding to isolated trout (Salmo gairdneri) liver nuclei in vitro: Binding affinity, capacity, and chemical specificity. Gen. Camp. Endocrinol. 61, 29-39. Chakraborti, P.. Maitra. G., and Bhattacharya, S. (1986). Binding of thyroid hormone to isolated ovarian nuclei from a freshwater perch, An~bns testudineus.

Gen.

Comp.

Endocrinol.

62, 239-

246. Chamness. G. C., and McGuire, W. L. (1975). Scatchard plots: Common errors in correction and interpretation. Sreroids 26, 538-541. Charipper, H. A., and Davis, D. (1932). Studies on the Arneth count. XX. A study of the blood cells of Pseudenys elegans with special reference to the polymorphonuclear leucocytes. Q. FI. Esp. Physiol.

21, 371-382.

Cheng. S. (1983). Characterization of binding and uptake of 3.5,3’-triiodo-L-thyronine in cultured mouse fibroblasts. Endocrinology 112, 17541762. Daniak, N.. Hoffman, R., Maffei. L. A., and Forget. B. G. (1978). Potentiation of human erythropoiesis in vitro by thyroid hormone. Nurure (London)

212, 260-262.

Darling, D. S., Dickhoff, W. W.. and Gorbman, A. (1982). Comparison of thyroid hormone binding to hepatic nuclei of the rat and a teleost (Ontorhynchus

kisutch).

Endocrinology

111.

1936- 1943. DeGroot, L. J., Refetoff, S.. Bernal. J.. Rue, P. A., and Coleoni, A. H. (1978). Nuclear receptors for thyroid hormone. J. Endocrinol. Invest. 1, 79-88. DeGroot, L. J., Refetoff. S., Strausser. J., and Barasano, C. (1974). Nuclear triiodothyronine-binding protein: Partial characterization and binding to

AND

DICKHOFF

chromatin. Proc. Narl. Acad. Sci. USA 71, 4042-4046. DeGroot, L. J.. and Toressani, J. (1975). Triiodothyronine binding to isolated liver cell nuclei. Endocrinology

96, 357-369.

Dickhoff. W. W.. Folmar, L. C., and Gorbman. A. (1978). Changes in plasma thyroxine during smoltification of coho salmon (Oncorhyrwhus kistrtch). Gen.

Camp.

Endocrinol.

36, 229-232.

Dickhoff, W. W.. Folmar. L. C.. Mighell, J. L., and Mahnken. C. V. W. (1982). Plasma thyroid hormones during smoltification of yearling and underyearling coho salmon and yearling chinook salmon and steelhead trout. Aquaculture 28, 39-48. Domm. L. V., and Taber, E. (1946). Endocrine factor controlling erythrocyte concentration in the blood of the domestic fowl. Physiol. Zool. 19, 258-28 I. Donati. R. M., Warnecke, M. A., and Gallaher. N. L. (1964). Effect of triiodothyronine administration on erythrocyte radioiron incorporation in rats. Proc.

Sot.

Exp.

Biol.

Med.

115, 405-407.

Eales, J. G. (1977). In vii.0 determination of thyroxine deiodination rate in rainbow trout, &(//no guirdneri Richardson. Geh. Camp. Endocrinol. 33, 541-546. Eales. J. G. (1979). Thyroid hormones in cyclostomes and Evolution’* and fishes. In “Hormones (E. J. W. Barrington. ed.). Vol. I. pp. 341-436. Academic Press, New York. Eales, J. G. (1985). The peripheral metabolism of thyroid hormones and regulation of thyroidal status in poikilotherms. Cancld. J. Zool. 63, 1217- 123 I. Eggert, B. (1933). Uber die histologischen und physiologischen Beziehungen Zwischen Schilddruse und Hautung bei den Eineimischen Eidechsen. Zoo/.

An:.

105, 1-Y.

Folmar. L. C.. and Dickhoff, W. W. (1980). The parrsmelt transformation (smoltification) and seawater adaptation in salmonids: A review of selected literature. Aqunculfwe 21, l-37. Forman, L. J., and Just. J. J. (1981). Cellular quantitation of hemoglobin transition during natural and thyroid hormone induced metamorphosis of the bullfrog, Rnntr ccrte.sheiunu. Gen. Camp. Endocrino/.

43. I-12.

Frieden, E. (1967). Thyroid hormones and the biochemistry of amphibian metamorphosis. Rec. Prog. Hormone Res. 23, 139-194. Galton. V. A. (1984). Putative nuclear triiodothyronine receptors in tadpole erythrocytes: Regulation of receptor number by thyorid hormones. Endocrinology

114, 735-742.

Galton. V. A. (1985). 3,5,3’-triiodothyronine receptors and thyroxine 5’.monodeiodinating activity in thyroid hormone-insensitive amphibia. Gen. Comp. Endocrinol. 51, 465-471. Galton. V. A.. and St. Germain. D. L. (1985). Putative

NUCLEAR

RECEPTORS

nuclear thyroid hormone receptors in tadpole erythrocytes during metamorphic climax. Endocrinology

116, 99- 104.

Galton. V. A.. and Schaafsma. J. (1983). Binding of thyroid hormones to isolated hepatic nuclei from Rana catesbeiana tadpoles. Endocrinology 112, 1999-2006. Gilbert, A. B. (1963). The effect of estrogen and thyroxine on the blood volume of the domestic cock. J. Endocrinol. 26, 41-49. Golde, D. W., Bersch. N., Chopra. I. J.. and Chne. M. J. (1977). Thyroid hormones stimulate erythropoiesis in ~,ifro. Brit. .I. Haemrrtol. 37. 173-177. Gonzales, L. W., and Ballard. P. L. (1981). Identification and characterization of nuclear 35.3’.triiodothyronine-binding sites in fetal human lung. J. C/in. Endocrinol. Metab. 53, 21-28. Greenberg, C. M., Blank, B.. Pfeiffer. F. R.. and Pauls, J. F. (1963). Relative activities of several 3’- and 3’:5’ alkyl and aryl thyromimetic agents. Amer.

.I. Physiol.

201, 732-736.

Hardig, J. (1977). Maturation of circulating red blood cells in young Baltic salmon (Salmo salar. L.). Acta

Physiol.

Stand.

102, 290-300.

Hardig, J. (1978). Autoradiography on erythrokinesis and multihemoglobins in juvenile Salmo salar L. at various respiratory gas regimes. Acta Physiol. Stand.

103, 240-251.

Hardig, J.. and Hoglund. L. B. (1984). Seasonal variation in blood components of reared Baltic salmon. Salmo salar L. J. Fish Biol. 24, 565-579. Hollander, C. S., Thompson, R. H.. Berret, P. V. D., and Berlin, N. I. (1967). Repair of anemia and hyperepidemia of the hypothyroid dog. Endocrinology

81, 1007-

1017.

Huggel, H. J., Lane, H. C., and Ducret, C. G. (1969). Determination de la courbe d’homogeneisation et du volume sanguin circulant de la truite Salmo gairdneri rich. Par la method de la dilution isotopique de I’ isi1 et du “CR. J. Physiol. 61, 145- 1.54. Jorgensen, E. C. (1976). Structure activity relationships of thyroxine analogs. Pharmacol. Ther. B 2, 661-682. Koch, H. J., Bergstrom, E., and Evans, J. C. (1964). The microelectrophoretic separation on starch gel of the hemoglobins of Salrno salar. Mededel. Koninkl.

Vlaamse

Acad.

Wetenschap.

26, 1-33.

Koerner, D., Schwartz, H. L., Surks, M. I., and Oppenheimer, J. H. (1974). Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei; High correlation between structural requirements for nuclear binding and biological activity. J. Biol. Chem. 250, 64176423. Lane, H. C. (1979). Some haematological responses of normal and splenectomized rainbow trout,

159

IN TROUT

Salmo gairdneri, Biol. 14, 159-164.

to a 12% blood

IOSS.

J. Fish.

Lane, H. C. (1980). The response of the hemoglobin system of fed and starved rainbow trout, Sahno gairdneri Richardson, to bleeding. J. Fish Bioi. 16, 405-411.

Lane, H. C.. and Tharp. T. P. (1980). Changes in the population of polyribosomal-containing red cells of peripheral blood of rainbow trout, Salmo gairdneri Richardson, following prolonged starvation and bleeding. J. Fish Biol. 18, 661-668. Lane, H. C., Weaver, J. W., Benson, J. A., and Nichols, H. A. (1982). Some age related changes in adult rainbow trout. SuDno gairdneri Rich., peripheral erythrocytes separated by velocity sedimentation at unit gravity. J. Fish Biol. 21, l-13. Lindenberg. J. A., Brehier, A., and Ballard, P. L. (1978). Triiodothyronine binding in fetal and adult rabbit lung and cultured lung cells. Endocrinology 103, 1725-1731.

Lintlop, S. P., and Youson, J. H. (1983). Binding of triiodothyronine to hepatocyte nuclei from sea lampreys. Petromyzon marintrs L.. at various stages of the life cycle. Gen. Comp. Endocrinol. 49. 428-436. Meints, R. H.. and Carver, E J. (1973). Triiodothyronine and hydrocortisone effects on Rana pipiens erythropoiesis. Gen. Cornp. Endocrinol. 21, 9-15. Morishige, W. K., and Guernsey, D. L. (1978). Triiodothyronine receptors in rat lung. Endocrinology 102, 1628-

1632.

Moriya, T., Thomas. C. R., and Frieden. E. (1984). Increase in 3,5,3’-triiodothyronine (T&binding sites in tadpole erythrocyte nuclei during spontaneous and TX-induced metamorphosis. Endocrinology B..

114, 170-175.

and Ingram. V. M. (1965). The repression and induction by thyroxine of hemoglobin synthesis during amphibian metamorphosis. Proc. Nutl. Acad. Sri. USA 54, 967-974. Oppenheimer, J. H. (1979). Thyroid hormone action at the cellular level. Science 203, 971-979. Oppenheimer, J. H. (1985). Thyroid hormone action at the nuclear level. Ann. Int. Med. 102, 374-384. Popovic, W. J., Brown, W. E.. and Adamson, J. W. (1977). The influence of thyroid hormones on in Vitro erythropoiesis. J. C/in. Invest. 60, 907-913. Richards, G. M., (1974). Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Ano/. Bioc-hem. 57, 369-372. Saint Girons, H. (1961). Particularites anatomiques et histologiques de e’hypophyse chez le squamatas. Moss,

Arc,h. Biol.

Paris

72, 2 I l-299.

Samuels. H. H.. Perlman, A. J.. Raaka. B. M.. and Stanley. F. (1982). Organization of the thyroid

SULLIVAN,

160 hormone Horm.

receptor

in chromatin.

Rec.

DARLING, Prog.

Res. 38, 557-599.

Samuels, H. H., and Tsai, J. S. (1974). Thyroid hormone action: Demonstration of similar receptors in isolated nuclei of rat liver and cultured GH, cells. J. Ch. InrBest. 53, 656-659. Samuels, H. H., Tsai, J. S., Cassanova, J., and Stanley, F. (1974). In \lifro characterization of solubilized nuclear receptors from rat liver and cultured GH, cells. J. C/in. Inquest. 54, 853-865. Satia, B. P., and Brannon. E. L. (1975). The value of certain fish-processing wastes and dogfish (Syu&s sucklepi) as food for coho salmon (@Icorhynch1ts kisutch) fry. Prog. Fisi7 Cult. 37, 76-80. Schatchard, G. (1949). The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Schwartz, H. L.. and Oppenheimer, J. H. (1978). Ontogenesis of 3.5.3’-triiodothyronine receptors in neonatal rat brain: Dissociation between receptor concentration and stimulation of oxygen consumption by 3.5,3’-triiodothyronine. Endocrinology 103, 943-948. Spindler, B. J., MacLeod, K. M., Ring, J.. and Baxter, J. D. (1975). Thyroid hormone receptors -Binding characteristics and lack of hormonal dependency for nuclear localization. J. Biol. Chem. 250,4113-4119. Sturkie, P. D. (1951). Effects of estrogen and thyroxine upon plasma proteins and blood volume in the fowl. Endocrinology 49, 565-572. Sullivan, C. V.. Dickhoff, W. W.. Mahnken. C. V. W.. and Hershberger. W. K. (1985). Changes in the hemoglobin system of the coho salmon Oncorhynchus kisutch during smoltification and triiodothyronine and propylthiouracil treatment. Camp. Biochem. Physiol. A 81, 807-813. Surks, M. I., Koerner, D. H., Dillman, W., and Oppenheimer, J. H. (1973). Limited capacity binding sites for L-triiodothyronine in rat liver nuclei. J. Biol.

Chem.

248, 7066-7072.

AND DICKHOFF Thapliyal, J. P.. and Kaur, R. J. (1976). Effect of thyroidectomy, L-thyroxine. and temperature on hemopoiesis in the chequered water snake, Natrix piscutor. Gen. Comp. Endocrino[. 30. 182-188. Thapliyal. J. P.. Oommen, 0. V., Kaur. R. J.. and Garg. R. K. (1977). Effects of surgical thyroidectomy and L-thyroxine on the oxidative metabolism and hemopoiesis of spotted munia. Lonchuru punctulota. Gen. Comp. Endocrinol. 31, 486491. Thapliyal, J. P.. Pati. A. K.. Singh. V. K., and Lal. P. (1982). Thyroid, gonad, and photoperiod in the hemopoiesis of the migratory red-headed bunting, Emherizcr

hrlrniceps.

Gen.

Camp.

Endocrinol.

46,

327-332. Thomas. D. (1974). The influence of L-thyroxine in red blood cell type in the axolotl. Dev. Biol. 38, 187-194. Toth. E.. and Tabachnick, M. (1979). In \si\jo demonstration of the presence of specific saturable binding sites for triiodothyronine in nuclei of tadpole liver. Gen. Comp. Endocrinol. 38, 345-355. Van Der Kraak, G. J.. and Eales, J. G. (1980). Saturable 3.5,3’-triiodo-L-thyronine binding sites in liver nuclei of rainbow trout (So/m> guirdneri). Gen.

Camp.

Endocrinol.

42, 437-448.

Vernidub. M. E. and Kolobova, N. N. (1971). Seasonal variations in thyroid gland and erythropoiesis intensity of young salmons (Sulrno sultrr) during first year of their life. Dokl. Akud. Nmrk USSR, 201, 500-502. Weirich, R. T., and McNabb, E M. A. (1984). Nuclear receptors for L-triiodothyronine in quail liver. Gen. Comp. Endocrinol. 53, 90-99. Yasutake, W. T., and Wales, J. H. (1983). Microscopic anatomy of salmonids: An atlas. U.S. Dept. of the Interior. Fish and Wildlife Service Resource Publ. No. 150. Zaugg. W. S.. and McLain, L. R. (1986). Changes in blood levels of nucleoside triphosphates, hemoglobin, and hematocrits during Parr-smolt transformation of coho salmon (Oncorhynchus kisutcI7).

487-493.

Con7p.

Biochem.

Physiol.,

A.

84,