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cDNA encoding the beta type of thyroid hormone receptor (THR) was cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder (Paralichthys olivaceus). The deduced amino acid sequence of the flounder THR beta (fTHR beta 1) showed higher homologies to the known THR beta s of other vertebrate animals than to THR alpha s, including flounder THR alpha s. Unlike any other THR, fTHR beta 1 possessed an insertion sequence composed of nine amino acids in the region prior to the hormone-binding domain. PCR analyses suggested the presence of transcripts for another THR beta (fTHR beta 2) that had a longer insertion sequence than fTHR beta 1. The analyses further suggested that the cDNA sequences of the two flounder THR beta s most likely shared a constant sequence except for a 60-base additional sequence found only in fTHR beta 2. The flounder genome possessed a single gene for both fTHR beta 1 and beta 2, suggesting that the two THR beta variants are produced from the same gene through an alternative splicing system.

Thyroidal control of the transition of myosin isoforms during flounder metamorphosis was examined by the administration of either only thiourea (TU), a potent inhibitor of thyroid hormone synthesis, or thyroxine (T4) together with TU into premetamorphic larvae. Immersion of premetamorphic larvae in 400 microM of TU inhibited the appearance of the adult-type DTNB (5,5'-dithio-bis-nitrobenzoic acid) light chain, LC2, whereas administration of T4 with TU induced a precocious appearance of LC2 and decreased the relative amount of the larval-type DTNB light chain, LC2*. TU was administered into juveniles just after completion of metamorphosis. The treatment did not affect the composition of the myosin light chains. These results suggest that thyroid hormone irreversibly turns on the switch for transition of the DTNB light chains from larval to adult type during metamorphosis of the flounder.

Two distinct cDNAs encoding thyroid hormone receptors (THRs) were cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder larvae (Paralichthys olivaceus). Deduced amino acid sequences of the two isolated cDNAs shared 96% and 92% homologies in their DNA- and hormone-binding domains, respectively. These were highly conserved when compared to THRs for other vertebrates: 88-96% in the DNA-binding domain and 84-94% in the hormone-binding domain. Other receptors in the nuclear receptor family showed lower homologies than those of THRs. Both THRs for the flounder had higher homologies with the alpha-type THRs of other vertebrates than with the beta-type. Thus, the two THRs for flounder were designated as fTHR alpha A and fTHR alpha B.

Prolactin has an antimetamorphic action in amphibian tadpoles. In the Japanese flounder, Paralichthys olivaceus, ovine prolactin (oPRL, 100 ng/ml) antagonized the stimulatory effect of triiodothyronine (T3, 1 ng/ml) on the resorption of the dorsal fin rays of prometamorphic larvae in vitro. Ovine growth hormone (oGH) at the same dose was without effect. Injections with oPRL (50 ng/fish, six times) into prometamorphic larvae also delayed the resorption of the dorsal fin rays without affecting the rates of eye migration and settling, while oGH was again without effect. The changes in the expression of both PRL and GH mRNAs in the pituitary during metamorphosis were monitored by in situ hybridization using cDNA probes. Both PRL and GH genes were increasingly expressed during successive metamorphic stages. These results are discussed in light of the possible interactions of PRL and GH with thyroid hormones in the control of development in the flounder.

Both cortisol and thyroid hormones were detected in newly fertilized eggs of the Japanese flounder, Paralichthys olivaceus. Mean cortisol levels ranged around 2.5 ng/g wet weight. Cortisol concentrations declined to about one-tenth of their initial levels by 2 days before hatching. The concentrations of triiodothyronine (T3; 7 ng/g) were greater than those of thyroxine (T4; 0.4 ng/g). The T3 levels decreased gradually in the eggs until the time of hatching and then decreased rapidly to undetectable levels within 2 days, whereas T4 remained at more or less constant levels during early development. The significance of the "selective" clearance of the three hormones from eggs remains unclear. Tissue concentrations of cortisol during premetamorphosis were about 4 ng/g and increased to a peak level of 11 ng/g at climax. After climax, cortisol declined by 50%. The changes in thyroid hormone levels were in parallel with the changes in cortisol. T4 remained below 1 ng/g during prematamorphosis but increased gradually during prometamorphosis, reaching peak levels (12 ng/g) during metamorphic climax, and then declined by approximately 50%. T3 remained at low levels through most of the metamorphosis. Histologically, the interrenal tissue was activated during the metamorphosis. The coincident increases in cortisol and thyroid hormones support previous results of a synergistic action of both hormones during metamorphosis in the flounder.

Morphological and biochemical changes in the muscular tissue of metamorphosing flounder were studied in relation to the regulatory role of thyroid hormone. Premetamorphic larvae were reared in seawater alone or seawater containing either thyroxine (T4) or an antithyroid drug (thiourea, TU). Histological changes in the muscle were examined and biochemical changes in the muscle proteins were evaluated by SDS-PAGE and immunoblotting for troponin T (TNT). The muscle tissue of premetamorphic larvae was characterized by abundant vacuoles and basophilic sarcoplasm. In control fish, the larval muscle transformed into the adult type during metamorphic climax; the fibers were filled with abundant myofibrils and the vacuoles disappeared. Analysis by SDS-PAGE showed that the bands at 41.5, 35.5, 34.0, 33.5, 25.5, 23.0, 20.0, and 19.0 kDa clearly increased in density from the climax stage. Premetamorphic larvae possessed two immunoreactive TNT isoforms of 41.5 and 34.0 kDa, the former being predominant. At the climax stage an additional isoform appeared at 33.5 kDa, and the 34.0- and 33.5-kDa TNT became predominant. The administration of T4 precociously induced these histological and biochemical changes in the muscle tissue of flounder larvae. In contrast, TU treatment inhibited these developmental changes in the larval muscle. Our results suggest that the developmental changes in the muscular tissue of metamorphosing flounder are regulated by thyroid hormone.

Effects of thyroid hormones and cortisol on fin-ray resorption of the larval flounder were studied in vitro. Dorsal fin rays were dissected from the prometamorphic larvae of the Japanese flounder (Paralichthys olivaceus) and were cultured for 7-8 days in a medium supplemented with different concentrations of thyroxine (T4) or triiodothyronine (T3) or combinations of either thyroid hormone or cortisol (0.1 micrograms/ml). Both T4 and T3 induced shortening of the second fin ray. T3 was more potent than T4; doses of 0.1 and 1.0 micrograms/ml T4 significantly accelerated shortening of the fin rays, whereas T3 was effective at concentrations ranging from 0.001 to 1.0 micrograms/ml. Cortisol further enhanced the effects of both T4 and T3 but was ineffective in the absence of thyroid hormones. When the prometamorphic larvae were reared in water containing T4 (0.01 micrograms/ml), T4 + cortisol (0.10 micrograms/ml), or cortisol alone for 15 days, both the T4- and T4 + cortisol-treated groups showed an advancement of the morphological changes associated with metamorphosis relative to the control group without hormone treatment. However, no clear synergistic effect of cortisol was observed in the in vivo experiment; a sufficient quantity of cortisol may be produced by the larval interrenal.

Bovine thyroid-stimulating hormone (TSH) was microinjected (0.2 microliter/fish) into prometamorphic flounder larvae and the effects on metamorphosis as well as the tissue thyroxine (T4) and triiodothyronine (T3) levels were studied. After a single injection of TSH (5 mIU/g), the tissue T4 concentration increased markedly after 5 hr, reached a peak after 10 hr, and decreased subsequently. T4 concentration after 24 hr was still higher than in saline-injected fish but returned to the control level 48 hr after the injection. On the other hand, tissue T3 concentration was kept lower than the detectable level (0.2 ng/g) throughout the experimental period of 72 hr after a single injection of TSH (5 mIU/g). TSH treatment also accelerated the process of metamorphic climax, such as shortening of the second fin ray and eye migration. These results suggest that an increased secretion of TSH from the pituitary stimulates the thyroid, resulting in a surge of the tissue T4 concentration which induces the climax of the flounder larvae.

The tissue concentration of thyroxine (T4) in larval flounder (Paralichthys olivaceus) was studied at various stages of their metamorphosis using a specific radioimmunoassay developed for samples obtained by quantitative extraction of the hormone from fish eggs and larvae. T4 concentrations were below the limit of detection in extracts of fertilized eggs and larvae until the end of prometamorphosis. Several dorsal fin rays continued growing from premetamorphosis until the end of prometamorphosis. The hormone became detectable at the onset of metamorphic climax when the elongated dorsal fin rays stopped growing. A sharp increase in tissue T4 concentration was observed in midclimax of metamorphosis, and the high level of T4 (11-13 ng/g body weight) was maintained until the end of the climax. The period of elevated T4 levels coincided with resorption of the elongated dorsal fin rays. The right eye completed translocation from the right to the left side of the body during metamorphic climax. The tissue T4 level declined to about a half of the peak value during postclimax, and then increased moderately in later adult-type juveniles. The results of an additional experiment, in which fertilized eggs and newly hatched larvae were treated with T4 and/or thiourea, suggested the presence of negative feedback regulation of pituitary-thyroid axis, implying an involvement of thyroid hormones even in early premetamorphic larvae. These results suggest that metamorphic climax is induced by a surge of thyroid hormone, and that thyroid hormone may also regulate development before and after the metamorphosis.

Two experiments were carried out to clarify the effects of thyroxine (T4) and triiodothyronine (T3) on flounder metamorphosis. In the first experiment, three doses of T4 were administered to premetamorphic flounder larvae together with thiourea (TU). Thiourea treatment (30 ppm in ambient seawater) alone caused metamorphic stasis in the fish, although the beginning of the right eye migration was not completely inhibited. T4 at 100 and 10 ppb in ambient seawater induced metamorphosis in the thiourea-treated larvae. The fish of these groups completed translocation of the right eye to the left side and resorption of elongated dorsal fin rays. However, 1 ppb T4 failed to induce metamorphic climax. The elongated dorsal fin rays continued to grow until the end of the experiment in the group treated with TU alone and in combination with 1 ppb T4. In the second experiment, premetamorphic flounder larvae were treated with two doses of T4 and three doses of T3. The T3 treatment was several times more potent than T4 treatment in the induction of metamorphosis. The results of the present study suggest that the metamorphic events of flounder larvae are dependent on the thyroid hormone concentration, and the relative biological potency of T4 and T3 on flounder metamorphosis is similar to that observed in mammals and amphibia.