Cloning of Bullfrog Thyroid-Stimulating Hormone (TSH) β Subunit cDNA: Expression of TSHβ mRNA during Metamorphosis

General and Comparative Endocrinology 119, 224 –231 (2000) doi:10.1006/gcen.2000.7515, available online at http://www.idealibrary.com on

Cloning of Bullfrog Thyroid-Stimulating Hormone (TSH) ␤ Subunit cDNA: Expression of TSH␤ mRNA during Metamorphosis Reiko Okada, Takeo Iwata, Takafumi Kato, Motoshi Kikuchi, Kazutoshi Yamamoto, and Sakae Kikuyama Department of Biology, School of Education, Waseda University, Nishi-waseda 1-6-1, Shinjuku-ku, Tokyo 169-8050, Japan Accepted May 10, 2000

A thyroid-stimulating hormone ␤ subunit (TSH␤) cDNA encoding both signal peptide and mature TSH␤ molecule was cloned from a cDNA library constructed from total RNA of the bullfrog (Rana catesbeiana) adenohypophysis. The bullfrog TSH␤ mRNA was estimated by Northern blot analysis to be approximately 1 kb. The deduced amino acid sequence showed 40 – 61% homologies with the sequences of TSH ␤ subunits of other vertebrates. Using the cDNA as a probe, we measured changes in mRNA expression in metamorphosing tadpoles of R. catesbeiana. The TSH ␤ subunit mRNA level increased progressively throughout prometamorphic stages, reaching its maximum at the end of prometamorphosis. The maximum level was maintained throughout early and mid climax, declining at late climax. These results, together with previously obtained data on plasma prolactin and pituitary prolactin mRNA levels, as well as thyroid hormone levels, are discussed in relation to metamorphic changes occurring in the bullfrog larvae. © 2000 Academic Press Key Words: TSH␤ cDNA; bullfrog; metamorphosis; TSH␤ mRNA expression.

The thyroid–pituitary function during metamorphosis has been studied mainly in the bullfrog (Rana catesbeiana) and the African clawed toad (Xenopus laevis) as model animals, mainly because the former is big, which facilitates collection of ample blood and tissue samples, and the latter can be raised in the laboratory and be available at any time of the year. In fact, changes in plasma thyroid hormone levels during metamorphosis have been measured mostly in the bullfrog. During metamorphosis, thyroid hormone levels in the bullfrog larvae begin to elevate during prometamorphosis and reach maximum at metamorphic climax, declining at the end of the climax stage (Miyauchi et al., 1977; Mondou and Kaltenbach, 1979; Regard et al., 1978; Suzuki and Suzuki, 1981). Yoshizato and Frieden (1975) first described the nuclear receptor for thyroid hormone in amphibian tissue. They observed that the nuclear receptor for triiodothyronine (T 3) in the tail fin cells of the bullfrog larvae showed twofold increase in maximum binding capacity, with a slight increase in K d value, during metamorphosis from premetamorphosis to metamorphic climax. More recently, changes in the thyroid hormone receptor mRNA level have been studied in Xenopus larvae. According to Yaoita and Brown (1990), the levels of thyroid hormone ␣-receptor mRNA in the whole body of X. laevis larvae increased throughout the premetamorphic stage and reached maximum by

It has been well documented that metamorphosis of amphibians is mainly under the control of three hormones, namely, thyroid hormone, adrenal corticoid, and prolactin (PRL) (Kikuyama et al., 1993). Among them, thyroid hormone has long been known as the key hormone to induce metamorphic changes. 224

0016-6480/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Bullfrog TSH␤ cDNA

prometamorphosis. On the other hand, the thyroid hormone ␤-receptor mRNA level reached its peak at metamorphic climax and declined at the end of climax (Yaoita and Brown, 1990) in parallel with thyroid hormone levels (Leloup and Buscaglia, 1977). Thyroid function in amphibian larvae has been considered to be under the control of thyroid-stimulating hormone (TSH), for metamorphic arrest by hypophysectomy is prevented by treatment with mammalian TSH (Dodd and Dodd, 1976) and immunoneutralization of endogenous TSH with antiserum against ovine TSH blocks spontaneous metamorphosis (Eddy and Lipner, 1976). Several years ago we obtained a thyrotropic glycoprotein fraction, which was virtually free of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from bullfrog pituitaries and demonstrated that it stimulated the release of thyroxine from the larval thyroid gland several times as potently as bovine TSH (Sakai et al., 1991). Up to the present, however, no data on the circulating levels and pituitary contents of TSH in metamorphosing tadpoles are available, since a homologous RIA for amphibian TSH has never been developed due to the lack of an ample amount of highly purified amphibian TSH. Under these circumstances, information about the mRNA levels of TSH, especially of the TSH ␤ subunit, in the pituitaries of metamorphosing tadpoles would be suitable to fill this gap. In this experiment, molecular cloning of bullfrog TSH␤ cDNA was conducted, and expression of TSH␤ mRNA during metamorphosis of bullfrog larvae was assessed using the TSH␤ cDNA as a probe.

MATERIALS AND METHODS Amplification of Partial cDNA Encoding Bullfrog TSH␤ by the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA (about 3 ␮g) was extracted from the bullfrog anterior pituitaries. Reverse transcription reactions were performed using Superscript II reverse transcriptase (Life Technologies, Grand Island, NY). Degenerate oligonucleotide primers were designed, based on conserved regions of TSH␤ from other species. PCR was performed in Ex-Taq buffer, containing

225

0.2 mM concentration each dNTP, 50 pmol each synthetic degenerated primer (primer 1, 5⬘TGCCTI(A/ G)CCAT(C/T)AA(C/T)ACIAC(C/T)(A/G)T(C/T)TG3⬘; primer 2, 5⬘-G(C/T)C(A/T)GT(A/G)TT(A/G)CAI(G/T)T(G/T)(C/T)CACACTI(A/G)CAGCT-3⬘) with 0.5 units Ex-Taq polymerase (Takara Shuzo, Kyoto, Japan) per 20 ␮l of reaction solution. The conditions of the PCR amplification were denaturation at 94° for 2 min followed by denaturation (94°, 20 s), annealing (42°, 20 s), and extension (60°, 1 min) reactions for 30 cycles. The amplified cDNA was subcloned into the pT7-blue T-vector (Novagen, Madison, WI). The plasmid containing the cDNA encoding TSH␤ was used to transform JM109 competent cells (Takara Shuzo) and was subjected to sequence analysis.

Construction of a cDNA Library and Screening of Bullfrog TSH␤ cDNA Total RNA was extracted from anterior pituitaries of bullfrogs by use of ISOGEN RNA extraction reagent (Nippon Gene, Tokyo, Japan), and then poly(A) ⫹ RNA was selected from the total RNA by use of OligotexdT30 super (Takara Shuzo). cDNA was synthesized from the poly(A) ⫹ RNA by reverse transcription. After ligation with an EcoRI/NotI adaptor, the cDNA was ligated with EcoRI-digested ␭ZAP II (Stratagene, La Jolla, CA) and packaged by using Gigapack III extracts (Stratagene). The PCR-amplified bullfrog TSH␤ cDNA fragment (210 bp long) was labeled with [␣- 32P]dCTP by the random-priming method (Feinberg and Vogelstein, 1983) using a BcaBEST Labeling Kit (Takara Shuzo) and was used to screen the cDNA library of the anterior lobes of bullfrog pituitaries. The phages of the cDNA library were plated on four large (diameter, 140 mm) LB agar plates with LB containing 0.7% agarose at a concentration of 1 ⫻ 10 4 pfu per plate. After having been transferred to membrane filters, the cDNAs were denatured and then fixed to the membranes. The filters were prehybridized in a hybridization solution consisting of 6-fold standard saline citrate (SSC), 0.2% (w/v) bovine serum albumin, 0.4% (w/v) Ficoll 400, 0.4% (w/v) polyvinylpyrrolidone, and 1% (w/v) sodium dodecyl sulfate (SDS) at 60° for 2 h. Hybridizations were performed with the labeled cDNA encoding partial bullfrog TSH␤ in the same solution at 60° for 16 h. The filters were washed once

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

226

in 2-fold SSC/0.1% SDS at room temperature, twice in 0.1-fold SSC/0.1% SDS at 60° for 30 min, and then placed in contact with an imaging plate using BAS2000II (Fujifilm, Tokyo, Japan) for 20 min. The clones giving positive signals were obtained by in vivo excision.

Okada et al.

FUJIX Bio-Imaging Analyzer BAStation (Fujifilm). The data were normalized with those for the bullfrog ␤-actin mRNA and expressed as an analysis of variance and arbitrary unit. Statistical analysis was performed by Duncan’s multiple range test.

DNA Sequence Analysis The cDNA sequences were analyzed by a cyclesequence method, using fluorescence-labeled primers with a Thermosequenase cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The cDNAs were analyzed on a dNA sequencer Model 4000L (LI-COR, Lincoln, NE).

Northern Blot Analysis Total RNA was isolated from bullfrog organs (testis, kidney, liver, intestine, hypothalamus, brain, and neurointermediate and anterior lobes of the pituitary). Ten micrograms of total RNA from each organ was separated by electrophoresis on a denaturing gel containing 10% agarose and 2.2 M formaldehyde and then transferred to a nylon membrane. The RNAs were fixed on the membrane by UV crosslinking. Quantification of TSH␤ mRNA was performed for RNAs from the anterior pituitaries of prometamorphic and climax tadpoles and of adult frogs. Total RNA was extracted from each sample, consisting of 20 pituitaries of tadpoles at stages XII, XVII, XIX, XX, XXII, and XXIV (Taylor and Korllos, 1946), as well as from pituitaries of adult frogs, as described above. Each sample was prepared from 20 larval pituitaries or from a single adult pituitary. Total RNA was quantified, and equal amounts of RNA (3 ␮g) were electrophoresed and blotted onto membranes, followed by UV fixation. After having been boiled in 1-fold SSC for 3 min, the membranes were prehybridized for 2 h at 60° in hybridization solution. Hybridization with the radiolabeled cDNAs was performed for 16 h at 60°, with the probe added to the prehybridization solution. The cDNA was labeled by use of a BcaBEST labeling kit (Takara Shuzo). The filters were washed once in 2-fold SSC/0.1% SDS at room temperature and twice in 0.1fold SSC/0.1% SDS at 60° for 30 min each time and placed in contact with an imaging plate using BAS2000II for 20 min. The results were analyzed with a

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

RESULTS Isolation of Bullfrog TSH␤ cDNA A full-length TSH␤ cDNA was obtained from a cDNA library of anterior lobes of bullfrog pituitary using the partial bullfrog TSH␤ cDNA as a probe. Sequencing analysis revealed that a clone, consisting of 930 bases, encoded the entire sequence of the open reading frame of bullfrog TSH␤ cDNA. The nucleotide sequence obtained and the amino acid sequence deduced are shown in Fig. 1. The deduced amino acid sequence indicates that this clone contained a signal peptide sequence and the entire sequence of the mature TSH␤ molecule. Comparison of amino acid sequences of ␤ subunits of bullfrog, mammal, chicken, Xenopus, and teleost TSHs are shown in Fig. 2.

Northern Blot Analysis To determine whether the bullfrog TSH␤ cDNA was specific to the anterior pituitary, we performed Northern blot analysis using various organs from the bullfrog. TSH␤ mRNA was revealed to be 1.0 kb long and to be expressed exclusively in the anterior pituitary (Fig. 3). No hybridization signals were detected for mRNAs from other organs such as testis, kidney, liver, intestine, hypothalamus, brain, and neurointermediate lobe of the pituitary. The TSH␤ mRNA concentrations in anterior pituitaries from tadpoles at various developmental stages (stages XII–XXIV) and adult frogs were assessed by Northern blot analysis. There was a linear relationship between the amount of total RNA and the density measured. The TSH␤ mRNA levels of the anterior lobe increased progressively during prometamorphosis (stages XII–XIX). At the end of prometamorphosis (stage XIX), the TSH␤ mRNA level reached its maximum, the values being two times higher than those for stage XII. The plateau level of TSH␤ mRNA was main-

Bullfrog TSH␤ cDNA

227

FIG. 1. The nucleotide sequence of the bullfrog TSH ␤ subunit cDNA. The predicted amino acids are shown below the nucleotide sequence. An asterisk indicates the termination codon. The signal peptide region is underlined.

tained during early and mid climax (stages XX–XXII) and declined at late climax (stage XXIV). The adult pituitaries showed extremely low values for TSH␤ mRNA expression (Fig. 4).

DISCUSSION Previously an attempt was made to isolate TSH from the bullfrog pituitaries by our group (Sakai et al., 1992). As a result, a highly purified TSH ␤ subunit

preparation was obtained, and its partial amino acid sequence was determined (Sakai, 1992). The first 10 amino acid residues of its N-terminal sequence, FLCMLTEYTM, coincide with the amino acid sequence 20 –29 deduced from the nucleotide sequence of TSH␤ cDNA obtained in this experiment. Accordingly, the amino acid sequence 1–19 is considered to be the signal peptide region of the bullfrog TSH␤. The sequence homology between the bullfrog TSH␤ and the bovine (Maurer et al., 1984), rat (Chin et al.,

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

228

Okada et al.

FIG. 2. Comparison of the amino acid sequences of vertebrate TSH ␤ subunit. The amino acid residues which match those of bullfrog TSH␤ are shown in white letters. Gaps as indicated by dashed lines have been introduced to obtain maximum homology. Sequences for bovine (Maurer et al., 1984), rat (Chin et al., 1985), mouse (Gurr et al., 1983), chicken (Gregory and Porter, 1997), Xenopus (Buckbinder and Brown, 1993), European eel (Salmon et al., 1993), and rainbow trout (Ito et al., 1993) are shown.

1985), mouse (Gurr et al., 1983), chicken (Gregory and Porter, 1997), Xenopus (Buckbinder and Brown, 1993), European eel (Salmon et al., 1993), and rainbow trout (Ito et al., 1993) TSH␤ subunits was 51.9, 50.4, 51.2,

FIG. 3. Northern blot analysis of bullfrog TSH␤ mRNA. Total RNAs (10 ␮g) prepared from various organs including the pituitary gland were electrophoresed in 1% agarose gel containing 2.2 M formaldehyde. The positions of ribosomal RNAs (28S and 18S) and 1 kb are indicated. Lane 1, anterior lobe of the pituitary; lane 2, neurointermediate lobe of the pituitary; lane 3, brain; lane 4, hypothalamus; lane 5, intestine; lane 6, liver; lane 7, testis; lane 8, kidney.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

50.0, 61.4, 39.5, and 43.5%, respectively. As in the case of other pituitary hormones of the bullfrog (Kobayashi et al., 1991; Takahashi et al., 1990, 1992; Yasuda et al., 1991), the primary structure of bullfrog TSH␤ was closer to that of other tetrapod animals than to that of teleosts. Buckbinder and Brown (1993) cloned a Xenopus TSH␤ cDNA. Using this cDNA as a probe, they measured TSH␤ mRNA levels in the pituitaries of metamorphosing Xenopus larvae. In Xenopus, TSH␤ mRNA levels increased throughout prometamorphosis, reaching maximum around the end of prometamorphosis (stage 58/59) and at early climax (stage 61); thereafter, the levels declined considerably. Thyroid hormone levels in the plasma of Xenopus larvae had earlier been shown to rise during early climax stages (60 – 62) and to decline thereafter (Leloup and Buscaglia, 1977). The TSH␤ mRNA expression profiles in the bullfrog larvae as revealed in this experiment are somewhat different from those in Xenopus larvae. In the bullfrog larvae, TSH␤ mRNA levels increased dur-

Bullfrog TSH␤ cDNA

FIG. 4. (A) Developmental changes in TSH␤ mRNA in the pituitary gland of metamorphosing tadpoles. Total RNA from pituitaries of stages XII–XXIV larvae and of adult frogs was subjected to Northern blot analysis for TSH␤ mRNA. Each sample was prepared from 20 pituitaries except for the case of adult frogs, for which each sample was prepared from a single gland. Total RNA was quantified, and equal amounts of RNA (3 ␮g) were electrophoresed. Each densitometry datum for TSH␤ mRNA was normalized by that for bullfrog ␤-actin mRNA and expressed relative to that of the stage XII group. Each column and vertical bar represents the mean value for 7 samples ⫾ SE. Values with different superscripts are significantly different at the 5% level (Duncan’s multiple range test). (B) Representative profiles of Northern blots of TSH␤ mRNA and ␤-actin mRNA. Lane 1, stage XII; lane 2, stage XVII; lane 3, stage XIX; lane 4, stage XX; lane 5, stage XXII; lane 6, stage XXIV; lane 7, adult.

ing prometamorphosis, reaching maximum at the end of prometamorphosis (stage XIX); and the maximum value was maintained throughout early and mid climax (stages XX–XXII). A considerable decline in TSH␤ mRNA levels occurred when the larvae reached the late climax stage (XXIV). In parallel with the TSH␤ mRNA levels, thyroid hormone levels also remained relatively high until the end of mid climax (Miyauchi et al., 1977; Mondou and Kaltenbach, 1979; Regard et al., 1978; Suzuki and Suzuki, 1981). The discrepancy in the pattern of changes in TSH␤ mRNA levels in Xenopus and R.

229

catesbeiana larvae seems to be attributable to the difference in the time required for the completion of metamorphosis. In the bullfrog, tail resorption occurs more gradually than in Xenopus. PRL is known to antagonize thyroid hormone to suppress the tail resorption both in vivo (Bern et al., 1967; Etkin and Gona, 1967) and in vitro (Derby and Etkin, 1968). More recently, overexpression of PRL in transgenic Xenopus larvae was shown to produce tailed frogs (Huang and Brown, 2000). By immunoneutralization experiments using antisera against mammalian (Eddy and Lipner, 1975) and bullfrog (Clemons and Nicoll, 1977; Yamamoto and Kikuyama, 1982) PRLs, endogenous PRL was demonstrated to cause a retardation of tail resorption in bullfrog tadpoles. In this species plasma PRL levels start to rise throughout early and mid climax, reaching maximum at the beginning of late climax (stage XXIV) and showing a moderate decline at the end of metamorphosis. In parallel with the change in plasma PRL levels, PRL mRNA levels in the pituitary exhibited a similar pattern of changes that started one stage in advance and ended one stage earlier (Takahashi et al., 1990). In Xenopus, PRL mRNA levels abruptly rise around the end of climax (Buckbinder and Brown, 1993). Unfortunately, there are no available data on plasma PRL concentrations in metamorphosing Xenopus larvae. Judging from the expression profiles of PRL mRNA, plasma PRL levels would probably be high during the late climax period when metamorphic changes are almost complete. In Xenopus, therefore, metamorphosis may proceed rapidly with little interaction between thyroid hormone and PRL. In the bullfrog larvae, on the other hand, PRL may antagonize thyroid hormone to some extent, resulting in relatively slow metamorphic changes. This may account for the existence of thyroid hormone in relatively high concentrations and the elevated expression of TSH mRNA levels throughout early and mid climax periods to ensure the completion of metamorphosis. In the present experiment, we did not assess the changes in TSH ␣ subunit mRNA levels in the bullfrog larvae. TSH is composed of two different subunits, designated ␣ and ␤. The TSH ␣ subunit is considered to be identical in amino acid sequence to the ␣ subunit of luteinizing hormone and follicle-stimulating hormone within a given species (Pierce and Parsons, 1981). Moreover, the ␣ subunit was revealed to be

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

230

contained in the PRL secretory granules in the bullfrog (Tanaka et al., 1992). Therefore, the ␣ subunit mRNA levels may scarcely reflect the synthesis of TSH molecules in the pituitary.

ACKNOWLEDGMENTS We thank Professor S. Ishii for his advice during the experiment. This study was supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan and by research grants from Waseda University, Asahi Glass Research Foundation, and Uehara Memorial Lifescience Foundation to S.K.

REFERENCES Bern, H. A., Nicoll, C. S., and Strohman, R. C. (1967). Prolactin and tadpole growth. Proc. Soc. Exp. Biol. Med. 126, 518 –520. Buckbinder, L., and Brown, D. D. (1993). Expression of the Xenopus laevis prolactin and thyrotropin genes during metamorphosis. Proc. Natl. Acad. Sci. USA 90, 3820 –3824. Chin, W. W., Muccini, J. A. J., and Shin, L. (1985). Evidence for a single rat thyrotropin-␤-subunit gene: Thyroidectomy increases its mRNA. Biochem. Biophys. Res. Commun. 128, 1152–1158. Clemons, G. K., and Nicoll, C. S. (1977). Effects of antisera to bullfrog prolactin and growth hormone on metamorphosis of Rana catesbeiana tadpoles. Gen. Comp. Endocrinol. 31, 495– 497. Derby, A., and Etkin, W. (1968). Thyroxine-induced tail resorption in vitro as affected by anterior pituitary hormones. J. Exp. Zool. 169, 1– 8. Dodd, M. H. I., and Dodd, J. M. (1976). The biology of metamorphosis. In “Physiology of the Amphibia” (J. A. Moore, Ed.), Vol. 3, pp. 467–599. Academic Press, New York. Eddy, L., and Lipner, H. (1975). Accelation of thyroxine-induced metamorphosis by prolactin antiserum. Gen. Comp. Endocrinol. 25, 462– 466. Eddy, L., and Lipner, H. (1976). Amphibian metamorphosis: The role of thyrotropin-like hormone. Gen. Comp. Endocrinol. 29, 333– 336. Etkin, W., and Gona, A. G. (1967). Antagonism between prolactin and thyroid hormone in amphibian development. J. Exp. Zool. 165, 149 –258. Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 –13. Gregory, C. C., and Porter, T. E. (1997). Cloning and sequence analysis of a cDNA for the ␤ subunit of chicken thyroid-stimulating hormone. Gen. Comp. Endocrinol. 107, 182–190. Gurr, J. A., Catterall, J. F., and Kourides, I. A. (1983). Cloning of cDNA encoding the pre-␤ subunit of mouse thyrotropin. Proc. Natl. Acad. Sci. USA 80, 2122–2126.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Okada et al.

Huang, H., and Brown, D. D. (2000). Prolactin is not a juvenile hormone in Xenopus laevis metamorphosis. Proc. Natl. Acad. Sci. USA 97, 195–199. Ito, M., Koide, Y., Takamatsu, N., Kawauchi, H., and Shiba, T. (1993). cDNA cloning of the ␤ subunit of teleost thyrotropin. Proc. Natl. Acad. Sci. USA 90, 6052– 6055. Kikuyama, S., Kawamura, K., Tanaka, S., and Yamamoto, K. (1993). Aspects of amphibian metamorphosis: Hormonal control. Int. Rev. Cytol. 145, 105–148. Kobayashi, T., Yasuda, A., Yamaguchi, K., Kawauchi, H., and Kikuyama, S. (1991). The complete amino acid sequence of growth hormone of the bullfrog (Rana catesbeiana). Biochem. Biophys. Acta 1078, 383–387. Leloup, J., and Buscaglia, M. (1977). La triiodothyronine, hormone de la me´tamorphose des amphibiens. C. R. Acad. Sci. 284D, 2261– 2263. Maurer, R. A., Croyle, M. L., and Donelson, J. E. (1984). The sequence of a cloned cDNA for the ␤ subunit of bovine thyrotropin predicts a protein containing both NH 2- and COOH-terminal extensions. J. Biol. Chem. 259, 5024 –5027. Miyauchi, H., LaRochell, J. F. T., Suzuki, M., Freeman, M., and Frieden, E. (1977). Studies on thyroid hormones and their binding in bullfrog tadpole plasma during metamorphosis. Gen. Comp. Endocrinol. 33, 254 –266. Mondou, P. M., and Kaltenbach, J. C. (1979). Thyroixine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen. Comp. Endocrinol. 39, 343–349. Pierce, J. G., and Parsons, T. F. (1981). Glycoprotein hormones: Structure and function. Annu. Rev. Biochem. 50, 465– 495. Regard, E., Taurog, A., and Nakashima, T. L. (1978). Plasma thyroxine and triiodothyronine levels in spontaneously metamorphosing Rana catesbeiana tadpoles and in adult Anuran Amphibia. Endocrinology 102, 674 – 684. Sakai, M. (1992). “Purification, Characterization and Biological Activity of Amphibian Thyroid Stimulating Hormone.” Thesis, Waseda University. Sakai, M., Hanaoka, Y., Tanaka, S., Hayashi, H., and Kikuyama, S. (1991). Thyrotropic activity of various adenohypophyseal hormones of the bullfrog. Zool. Sci. 8, 929 –934. Sakai, M., Hayashi, H., Hanaoka, Y., Tanaka, S., and Kikuyama, S. (1992). Isolation and characterization of bullfrog thyrotropin. Zool. Sci. 9, 1263. [abstract] Salmon, C., Marchelidon, J., Fontaine, Y.-A., Huet, J.-G., and Querat, B. (1993). Cloning and sequence of tyrotropin ␤-subunit of a teleost fish, the eel (Anguilla anguilla L.). C. R. Acad. Sci. 316, 749 –753. Suzuki, S., and Suzuki, M. (1981). Changes in thyroidal and plasma iodine compounds during and after metamorphosis of the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 45, 74 – 81. Takahashi, N., Kikuyama, S., Gen, K., Maruyama, O., and Kato, Y. (1992). Cloning of a bullfrog growth hormone cDNA: Expression of growth hormone mRNA in larval and adult bullfrog pituitaries. J. Mol. Endocrinol. 9, 283–289.

Bullfrog TSH␤ cDNA

Takahashi, N., Yoshihama, K., Kikuyama, S., Yamamoto, K., Wakabayashi, K., and Kato, Y. (1990). Molecular cloning and nucleotide sequence analysis of complimentary DNA from bullfrog prolactin. J. Mol. Endocrinol. 5, 281–287. Tanaka, S., Mizutani, F., Yamamoto, K., Kikuyama, S., and Kurosumi, K. (1992). The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullfrog (Rana catesbeiana) pituitary gland. Cell Tissue Res. 267, 223–231. Taylor, A. C., and Korllos, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7–23.

231 Yamamoto, K., and Kikuyama, S. (1982). Effect of prolactin antiserum on growth and resorption of tadpole tail. Endocrinol. Jpn. 29, 81– 85. Yaoita, Y., and Brown, D. D. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes. Dev. 4, 1917–1924. Yasuda, A., Yamaguchi, K., Kobayashi, T., Yamamoto, K., Kikuyama, S., and Kawauchi, H. (1991). The complete amino acid sequence of prolactin from the bullfrog. Rana catesbeiana. Gen. Comp. Endocrinol. 83, 218 –226.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.