Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone.

We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.

Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus).

We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

Thyroid hormones promote early metamorphosis in grouper (Epinephelus coioides) larvae.

The response of grouper larvae to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), was examined. Two-, 3-, and 4-week-old grouper larvae were reared in seawater containing either T4 or T3 at 0.01, 0.1, and 1 ppm. T4 or T3 induced metamorphosis in all age groups in a dose-dependent manner. Regardless of the size of the larvae, metamorphosis was completed in 2 days in larvae treated with 1 ppm of either T4 or T3; 3-4 days in larvae exposed to 0.1 ppm; and 5-6 days in larvae immersed in 0.01 ppm. None of the fish in the control group completed metamorphosis during this period. Compared with the control fish, survival rates were higher in groups exposed to 0.01 ppm and lower in those exposed to 1 ppm of T3. In 4-week-old larvae, T4 treatment (0.01 to 1.0 ppm) resulted in higher survival compared to the control. These results suggest that a dose of 0.01 ppm is appropriate for acceleration of metamorphosis and improvement of survival in 3- and 4-week-old grouper larvae. A lower dose may be apropriate for earlier stages.

The antimetamorphic effect of prolactin in the Japanese flounder.

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.

Localization of mRNAs for a pair of prolactins and growth hormone in the Tilapia pituitary using in situ hybridization with oligonucleotide probes.

Oligonucleotide probes were synthesized for the mRNAs of a pair of tilapia prolactins (tPRL177 and tPRL188) and growth hormone (tGH) based on cDNAs for the hormones of Oreochromis niloticus and amino acid sequences for the hormones of O. mossambicus. The three 45mer probes were labeled with 35S for hybridization studies on pituitary sections of O. mossambicus adapted to fresh water (FW) or seawater (SW). Expression of tPRL mRNA in the rostral pars distalis was clearly evident with either PRL probe in adjacent sections in PRL cells of the rostral pars distalis; mRNAs of both PRLs were colocalized in the same cells. In addition, the tGH probe demonstrated expression of tGH mRNA specifically in GH cells in the proximal pars distalis. The hybridization signals for both PRLs were significantly greater in the rostral pars distalis of FW fish than in that of SW fish, as judged by computer-aided analysis. In addition, grain concentration for both PRLs was significantly greater over centrally located PRL cells of FW fish. In addition, although overall grain concentrations were lower in SW fish, there were significantly more grains over the centrally located PRL cells with the tPRL177 probe, whereas there was no difference with the tPRL188 probe. There was no detectable difference in the occurrence of tGH mRNA between FW and SW fish.

Flounder metamorphosis: its regulation by various hormones.

Metamorphosis in the flounder has often been compared with the transition of tadpoles into frogs. The dorsal fin rays of the Japanese flounder (Paralichthys olivaceus) elongate during prometamorphosis when thyroid hormone levels are low, and are resorbed during metamorphic climax when thyroid hormone levels are high. Using an in vitro system for the culture of the flounder fin rays, we have examined how various hormones affect the resorption process. Both thyroxine (T4) and triiodothyronine (T3) directly stimulated fin ray shortening, T3 being more potent than T4. Other hormones, such as prolactin, cortisol and sex steroids, did not directly affect the resorption process but modified the tissue's response to thyroid hormones. Similar observations were obtained from in vivo studies. We also monitored the changes in the whole body concentrations of various hormones during early development and metamorphosis, and related these with the thyroid hormone profiles in order to get a better picture of their interactions. The gaps in the present status of research on the role of thyroid hormones during metamorphosis in the Japanese flounder are also discussed.

Changes in whole body concentrations of cortisol, thyroid hormones, and sex steroids during early development of the chum salmon, Oncorhynchus keta.

Significant amounts of cortisol (20 ng/g) and thyroid hormones thyroxine (T4, 20 ng/g) and triiodothyronine (T3, 10 ng/g), as well as estradiol (8 ng/g) and testosterone (4 ng/g), are present in fertilized eggs of chum salmon. Changes in the concentrations of these hormones in the developing embryo and larvae were monitored until after the emergence of the fry from the gravel bed. Cortisol concentrations in the developing embryo fell steadily from 20 ng/g at fertilization to 2.5 ng/g after 3 weeks, increased slightly to 10 ng/g by 1 week before hatching, and maintained this level during the early stages of yolk sac absorption. During the later stages of yolk sac absorption, cortisol concentrations increased markedly and remained at about 30 ng/g until emergence, when they declined to 10-15 ng/g. In contrast, both T4 and T3 levels were stable during early development, decreased gradually during yolk sac absorption, and increased slightly during emergence. Estradiol and testosterone profiles were similar to that of cortisol during early development. Testosterone levels remained low throughout emergence. Likewise, estradiol levels were low during yolk sac absorption but showed a transient increase at the time of emergence. These hormonal changes are discussed in the context of egg development and subsequent downstream migration.

Changes in cortisol and thyroid hormone concentrations during early development and metamorphosis in the Japanese flounder, Paralichthys olivaceus.

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.

Cortisol enhances the stimulating action of thyroid hormones on dorsal fin-ray resorption of flounder larvae in vitro.

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.