10 citations found

Mol Cell Endocrinol 113 (2): 235-243 (1995)

Contrasting patterns of expression of thyroid hormone and retinoid X receptor genes during hormonal manipulation of Xenopus tadpole tail regression in culture.

Iwamuro S, Tata JR

Division of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, UK.

The precocious induction of amphibian metamorphosis is an ideal system for analyzing the developmental action of TH, while the hormonal activation of tadpole tail regression offers the further advantage of studying programmed cell death. One of the striking features of thyroid hormone (TH)-induced tail regression (as with morphogenetic responses of all tadpole tissues) in Xenopus is the rapid autoinduction of TRbeta gene, but it is not known how TH would affect the expression of the genes encoding TR's heterodimeric partner, retinoid X receptor (RXR). Here we first show that the synthetic glucocorticoid dexamethasone (Dex) potentiates and prolactin (PRL) suppresses, 3,3',5-triiodothyronine (T3)-induced regression of pre-metamorphic Xenopus tadpole tails in organ culture. T3 strongly upregulated (11-35-fold) the concentration of Xenopus TRbeta (xTRbeta) mRNA in these cultures while downregulating by 50% that of Xenopus RXRgamma (xRXRgamma) mRNA in the same samples of tail RNA. DEX and PRL enhanced or diminished the T3-regulated expression of these two transcripts, respectively, which parallels their other effects in whole tadpoles or cultured tails. The contrasting effects of the three hormones on the steady-state levels of xTRbeta and XRXRgamma mRNAs were time- and dose-dependent. T3 and DEX also strongly upregulated the transcription of xTRbeta gene transfected into Xenopus XTC-2 cells but PRL failed to prevent this autoinduction. The actions of these three hormones involved in amphibian metamorphosis, as judged by the expression of xTRbeta and xRXRgamma genes, reveal a new facet of hormonal interplay underlying their developmental actions. 


Gen Comp Endocrinol 99 (1): 28-34 (1995)

Development and application of a homologous radioimmunoassay for Xenopus prolactin.

Yamamoto K, Yamashita K, Hayakawa Y, Hanaoka Y, Kikuyama S

Department of Biology, School of Education, Waseda University, Tokyo, Japan.

A specific and sensitive homologous radioimmunoassay (RIA) for Xenopus prolactin (xPRL) was developed. PRL isolated from X. laevis pituitary glands was used for generating antiserum in a rabbit, for radioligand and for the standard. Pituitary homogenates and plasma from adult Xenopus produced displacement curves parallel to the xPRL standard. Plasma from hypophysectomized Xenopus showed negligible cross-reactivity. Purified PRLs from other amphibian species such as the bullfrog (Rana catesbeiana) and toad (Bufo japonicus) gave inhibition curves which did not parallel the standard. Ovine PRL, mouse PRL, newt (Cynops pyrrhogaster) PRL, bullfrog GH, and bullfrog LH showed no inhibition of binding even at relatively high doses in this RIA. The sensitivity of the RIA was 0.122 +/- 0.005 ng xPRL/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 2.46 and 6.65%, respectively. Histological studies of Xenopus adenohypophyses revealed that the cells which reacted immunologically with the antiserum against xPRL corresponded to those positively stained with antiserum against bullfrog PRL. A preliminary application of this homologous RIA for xPRL was performed by evaluating plasma and pituitary PRL levels in subadult and adult Xenopus of both sexes. 


Biochem Cell Biol 72 (11-12): 581-588 (1994)

Hormonal regulation of programmed cell death during amphibian metamorphosis.

Tata JR

Division of Developmental Biochemistry, National Institute for Medical Research, London, U.K.

Extensive programmed cell death (PCD) is initiated at the onset of amphibian metamorphosis, resulting in 100% of cells dying in some larval tissues, as during total regression of tail and gills. All cell death during metamorphosis is under the control of thyroid hormone (TH), which can initiate the process precociously in whole tadpoles or in individual tissues in culture. The hormone prolactin (PRL), given exogenously, prevents natural and TH-induced metamorphosis. We have exploited this dual hormonal regulation in premetamorphic Xenopus tails in organ culture to identify and characterize early genes that are TH-induced and considered important for initiating cell death. Among the earliest genes activated by TH are those encoding the two thyroid hormone receptors TR alpha and TR beta. This autoinduction of TR genes is considered important since, in blocking this process, PRL also inhibited the expression of other TH-inducible genes and prevented cell death. The expression of early genes other than TR genes, which are known to promote cell death or survival, is also considered to be important for the initiation of PCD during amphibian metamorphosis. We are, therefore, working on the identification, characterization, and expression of members of the Xenopus bcl-2-like gene family, as well as other genes, such as nur-77 and ICE, which may act as early genes during tadpole tail regression. 


Biochimie 76 (3-4): 320-328 (1994)

Modulation of the biological activity of thyrotropin-releasing hormone by alternate processing of pro-TRH.

Ladram A, Bulant M, Delfour A, Montagne JJ, Vaudry H, Nicolas P

Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Paris, France.

Thyrotropin-releasing hormone prohormone contains multiple copies of TRH linked together by connecting sequences. Like other plurifunctional prohormone proteins, pro-TRH undergoes differential proteolytic processing in various tissues to generate, beside authentic TRH, several other novel peptides corresponding to C-terminally extended forms of TRH and connecting fragments. The pro-TRH connecting peptides are, together with TRH, predominant storage forms of TRH-precursor related peptides in the rat hypothalamus. Connecting peptides are co-localized with TRH in the median eminence nerve endings and co-released through a mechanism involving voltage-operated Ca2+ channels. The connecting peptide Ps4 is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo through interactions with a specific pituitary cell receptor coupled to dihydropyridine and omega-connotoxin sensitive Ca2+ channels of the L-type. It also causes dose-dependent increases in the steady state levels of mRNAs of TSH and prolactin through stimulation of the respective gene promoter activities. These findings indicate that Ps4 and TRH, two peptides which originate from a single multifunctional biosynthetic precursor, can function on the same target tissues in a coordinate manner to promote hormonal secretion. This suggests that differential processing of the TRH prohormone may have the potential to modulate the biological activities of TRH. 


Gen Comp Endocrinol 91 (3): 307-317 (1993)

Isolation and characterization of two forms of Xenopus prolactin.

Yamashita K, Matsuda K, Hayashi H, Hanaoka Y, Tanaka S, Yamamoto K, Kikuyama S

Department of Biology, School of Education, Waseda University, Tokyo, Japan.

Two forms of highly purified prolactin (PRL) were obtained from pituitary glands of Xenopus laevis by extraction of acetone-dried powder with acid acetone and high-performance liquid chromatography on anion exchange, gel filtration, and reverse-phase columns. Purification was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis employing antiserum against bullfrog PRL. The Xenopus prolactins (xPRL-I and xPRL-II) thus obtained were shown to have similar molecular weights of 23,000 as determined by SDS-PAGE. The isoelectric points of xPRL-I and xPRL-II determined by isoelectric focusing were 5.6 and 5.3, respectively. Both hormones blocked T4-induced shrinkage of Xenopus tadpole tail fin in vitro. The amino acid compositions of the xPRLs resembled that of bullfrog PRL. The partial amino acid sequences of xPRL-I and of xPRL-II showed 78 and 68% homology with the comparable portion of the sequence of bullfrog PRL, respectively. Homology between xPRL-I and xPRL-II was 90%. 


Proc Natl Acad Sci U S A 90 (9): 3820-3824 (1993)

Expression of the Xenopus laevis prolactin and thyrotropin genes during metamorphosis.

Buckbinder L, Brown DD

Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210.

The cDNAs encoding Xenopus laevis prolactin (PRL) and the alpha and beta subunits of thyroid-stimulating hormone (TSH alpha and TSH beta, respectively) have been cloned from a pituitary library. Results of developmental RNA blot analysis contradict the long-held biological role for PRL as a juvenilizing hormone in amphibia. The pituitary gland of a premetamorphic tadpole expresses PRL mRNA at very low levels. The abundance of PRL mRNA increases late in metamorphosis as a response to thyroid hormone (TH), suggesting that PRL is more likely to have a function in the frog than in the tadpole. TSH alpha and -beta mRNA levels increase through prometamorphosis; this rise does not appear to be regulated directly by TH. At climax, both TH and TSH mRNA levels drop. The sequential morphological changes that characterize prometamorphosis depend upon the gradual increase of endogenous TH, which peaks at climax. This increase in TH in turn depends upon the lack of a traditional thyroid-pituitary negative-feedback loop throughout prometamorphosis. 


Dev Biol 149 (2): 463-467 (1992)

Prolactin prevents the autoinduction of thyroid hormone receptor mRNAs during amphibian metamorphosis.

Baker BS, Tata JR

National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.

We have recently reported that prolactin (PRL) inhibits both morphogenesis and cell death in thyroid hormone (T3)-induced amphibian metamorphosis (Tata et al., 1991), and that the autoinduction of T3 receptor (TR alpha and beta) mRNA is among the most rapid responses of premetamorphic Xenopus tadpoles to T3 (Kawahara et al., 1991). We now demonstrate that PRL prevents the rapid T3-induced upregulation of TR alpha and beta mRNAs in stages 50-54 Xenopus tadpoles and in organ cultures of tadpole tails. This effect is followed by the inhibition of the de novo activation of 63-kDa keratin gene by T3. We present an experimentally testable model whereby PRL exerts its juvenilizing action by preventing the amplification of TR by its autoinduction by T3. 


Dev Biol 146 (1): 72-80 (1991)

Prolactin inhibits both thyroid hormone-induced morphogenesis and cell death in cultured amphibian larval tissues.

Tata JR, Kawahara A, Baker BS

Laboratory of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, United Kingdom.

We describe for the first time the successful organ culture, in a serum-free chemically defined medium, of hind limb buds from stage 54/55 Xenopus laevis tadpoles in which 2 x 10(-9) M triiodothyronine (T3) precociously induces morphogenesis to give rise to morphologically normal limbs within 7 days. It was important to retain the mesenchymal tissue joining the two limb buds in order to obtain limb development in culture. T3 added to tail explants from the same larvae, cultured in parallel with limb buds, induced regression and cell loss at rates comparable to those seen during T3-induced metamorphosis in intact tadpoles. We also demonstrate for the first time that 0.2 units of prolactin (PRL) added at the same time as 2 x 10(-9) M T3 totally blocked both limb development and tail regression over 8 days in culture. When added after T3 had initiated its metamorphic action. PRL arrested further morphogenesis and regression of these two tissues, respectively. Retinoic acid at 10(-7) M had only a marginal effect. Histological examination showed that T3 added to limb buds produced normal chondrogenesis and osteogenesis in vitro as well as skin, muscle, and digit formation, while it produced a rapid and marked histolysis of fin and connective tissue of the tail. The ease of hormonally manipulating both morphogenesis and cell death in culture in opposite directions offers a simple, effective model system for molecular analysis of mechanisms underlying hormone-regulated postembryonic developmental processes. 


Horm Behav 25 (2): 128-136 (1991)

Thyrotropin-releasing hormone facilitates display of reproductive behavior and locomotor behavior in an amphibian.

Taylor JA, Boyd SK

Department of Biological Sciences, University of Notre Dame, Indiana 46556.

In the amphibian brain, thyrotropin-releasing hormone (TRH) is present in many regions outside the hypothalamus. The functions of this extrahypothalamic TRH however are unknown. We sought to determine whether TRH or its metabolites altered reproductive behaviors (amplectic clasping behavior) or locomotor behaviors of the male South African clawed frog, Xenopus laevis. TRH-injected (100 micrograms; dorsal lymph sac injection) male Xenopus displayed significantly fewer amplectic clasp attempts and longer clasp durations than saline-injected controls. The TRH metabolites, TRH acid and histidylproline diketopiperazine, similarly altered clasping behavior. Several hormones released by TRH, including thyroid-stimulating hormone, melanocyte-stimulating hormone, prolactin, and dopamine, had no significant effect on clasp frequency or duration. Locomotor activity in Xenopus males was increased significantly after 15 min following TRH injection (150 micrograms); this effect persisted for at least 1 hr. The metabolites did not alter locomotion. These studies indicate that TRH can facilitate the display of two behaviors in the South African clawed frog. Effects of TRH on locomotor and reproductive behaviors thus appear in several vertebrate classes. These behavioral actions of TRH likely occur through different mechanisms or at different sites. 


Gen Comp Endocrinol 77 (2): 202-211 (1990)

Gonadal hormones inhibit the induction of metamorphosis by thyroid hormones in Xenopus laevis tadpoles in vivo, but not in vitro.

Gray KM, Janssens PA

Department of Zoology, Australian National University, Canberra, ACT.

Although the major hormones controlling amphibian metamorphosis are those of the thyroid, other hormones, notably prolactin and the adrenal steroids, modulate the effects of thyroid hormones (TH). Some authors report that the gonadal steroids stimulate the metamorphic actions of TH whereas others report inhibition. The aims of the present study were to determine the effects of gonadal steroids on TH-induced metamorphosis in Xenopus laevis and to determine the site of action of these steroids. In all cases, hormones were added to the water in which the tadpoles were swimming. The gonadal steroids, testosterone and 17 beta-estradiol, inhibited triiodothyronine (T3)-induced metamorphosis in living, premetamorphic tadpoles of X. laevis. Both steroids, at 3.4 microM, prevented the reduction in body weight and the shrinkage of head and alimentary canal brought about by 1 nM T3. In contrast, 3.4 microM corticosterone stimulated T3-induced metamorphosis. Addition of 100 nM T3 to the medium induced a large reduction in size of X. laevis tails cultured in vitro. The antagonistic effects of testosterone were not reproduced in such cultures, whereas the synergistic action of corticosterone was maintained. Testosterone had no effect upon the specific binding of T3 to X. laevis tail tissue, whereas corticosterone increased such binding. These findings indicate that, while corticosterone stimulates the metamorphic actions of T3 by acting directly in the peripheral tissues, the gonadal steroids, particularly testosterone, inhibit T3 by acting at a more central site. Prolactin is known to antagonize the metamorphic actions of T3 and one such central action could be the stimulation of prolactin synthesis. However, testosterone inhibited the prometamorphic actions of bromocriptine, which stimulates metamorphosis by inhibiting production of prolactin. Thus the central action of testosterone is unlikely to be a stimulation of prolactin production.