Molecular and genetic studies suggest that thyroid hormone receptor is both necessary and sufficient to mediate the developmental effects of thyroid hormone

Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopuslaevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general.     

Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larval Xenopus laevis

Effects of bisphenol A (BPA) on embryonic and larval development were investigated. In Xenopus laevis blastulae treated with 2.5-3.0 x 10(-5) M BPA or with 10(-5) M 17beta estradiol (E2), malformation of the head region, scoliosis (curved vertebrate), and suppression of organogenesis were observed. In addition, 10(-5)-10(-4) M BPA blocked tri-iodothyronine (T3)-inducible resorption of the tail segments from premetamorphic (stage 52-54) larvae in vitro. When stage 52 tadpoles were immersed in 1.0-2.5 x 10(-5) M BPA, deceleration of both spontaneous and thyroxin (T4)-induced metamorphic changes occurred. Furthermore, BPA suppressed thyroid hormone receptor (TR) beta gene expression both in vivo and in vitro. Thus, we concluded that BPA at the concentrations examined affects both embryonic development and larval metamorphosis.     

Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog

The current review focuses on the molecular mechanisms and developmental roles of thyroid hormone receptors (TRs) in gene regulation and metamorphosis in Xenopus laevis and discusses implications for TR function in vertebrate development and diversity. Questions addressed are: (1) what are the molecular mechanisms of gene regulation by TR, (2) what are the developmental roles of TR in mediating the thyroid hormone (TH) signal, (3) what are the roles of the different TR isoforms, and (4) how do changes in these molecular and developmental mechanisms affect evolution? Even though detailed knowledge of molecular mechanisms of TR-mediated gene regulation is available from in vitro studies, relatively little is known about how TR functions in development in vivo. Studies on TR function during frog metamorphosis are leading the way toward bridging the gap between in vitro and in vivo studies. In particular, a dual function model for the role of TR in metamorphosis has been proposed and investigated. In this model, TRs repress genes allowing tadpole growth in the absence of TH during premetamorphosis and activate genes important for metamorphosis when TH is present. Despite the lack of metamorphosis in most other vertebrates, TR has important functions in development across vertebrates. The underlying molecular mechanisms of TR in gene regulation are conserved through evolution, so other mechanisms involving TH-target genes and TH tissue-sensitivity and dependence underlie differences in role of TR across vertebrates. Continued analysis of molecular and developmental roles of TR in X. laevis will provide the basis for understanding how TR functions in gene regulation in vivo across vertebrates and how TR is involved in the generation of evolutionary diversity.     

The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor

We investigated the effect of endocrine disrupting chemicals (EDCs), including medical, industrial, and agricultural chemicals, on 3,3',5-L-[125I]triiodothyronine ([125I]T3) binding to purified Japanese quail transthyretin (qTTR), a major thyroid hormone-binding protein in plasma, and to the ligand-binding domain of thyroid hormone receptor beta (qTR LBD). Scatchard plots of T3 binding to qTTR and qTR LBD revealed two classes of binding sites, with Kd values of 6.9 and 185 nM, and a single class of binding sites, with Kd value of 0.31 nM, respectively. Among the test chemicals, diethylstilbestrol was the most powerful inhibitor of [125I]T3 binding to qTTR (IC50 < 0.4 nM). Diethylstilbestrol, ioxynil (IC50 =1.1+/-0.5 nM) and pentachlorophenol (IC50 = 6.3+/-3.8 nM) displaced [125I]T3 from qTTR more effectively than unlabeled T3 (IC50 = 9.7+/-0.9 nM) did. Although malathion, 4-nonylphenol, bisphenol A and n-butylbenzyl phthalate were effective inhibitors of [125I]T3 binding to qTTR, their potency was two orders of magnitude less than that of T3. All test chemicals except for diethylstilbestrol had either a weak or no effect on [125I]T3 binding to qTR LBD. These results show that several EDCs tested in this study target qTTR rather than qTR LBD.     

Mate calling behavior of male South African clawed frogs (Xenopus laevis) is suppressed by the antiandrogenic endocrine disrupting compound flutamide

Several environmental pollutants have been identified as antiandrogenic endocrine disrupting chemicals (EDC), with flutamide (FLU) being a model compound for this type of action. Despite impacts of EDC interfering with sexual differentiation and reproduction in amphibians, established information about suggested effects on sexual behavior is still lacking. In this study adult male Xenopus laevis were injected with human chorionic gonadotropin (hCG) to initiate mate calling behavior. After one day hCG-stimulated frogs were treated via aqueous exposure over three days without and with FLU at concentrations of 10⁻⁸ and 10⁻⁶ M in comparison to untreated frogs. Androgen controlled mate calling behavior was recorded during the 12 h dark period. At the end of exposure circulating levels of testosterone (T) and 17β-estradiol (E2) were determined and furthermore gene expression was measured concerning reproductive biomarkers such as hypophysial luteinizing hormone (LH), follicle-stimulating hormone (FSH), testicular aromatase (ARO), 5α reductase type 1 (SRD5α1) and 5α reductase type 2 (SRD5α2). Both concentrations of FLU caused a significant decrease in calling activity starting at the second day of exposure. HCG injected positive controls had elevated levels of T compared to negative control frogs while in parallel treatment with FLU did not affect significantly the hCG elevated sex steroid levels. Furthermore, hCG treatment led to significantly decreased levels of gene expression for ARO and SRD5α2 but no impacts were detected on LH, FSH or SRD5α1 mRNA levels compared to negative controls. In summary, the behavioral parameter mate calling is the most sensitive biomarker detecting antiandrogenic modes of action in this challenge-experiment indicating that this non-invasive method could markedly contribute for sensitive assessment of antiandrogenic EDC.     

The thyroid gland and thyroid hormones in Senegalese sole (Solea senegalensis) during early development and metamorphosis

We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea senegalensis), and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Under our rearing conditions at 19.5 degrees C, most larvae entered metamorphosis in stage 1 at 15 days post-hatching (dph), and completed metamorphosis in stage 4 at 25dph. The onset of metamorphosis coincided with surges in whole body T4 and T3 concentrations. Crossmon's trichrome stain colored the lumen of follicular structures brightly red, and this co-localized with a T4-immunoreactivity. Thyroid follicles were first observed in stage 0 pre-metamorphic larvae at 5dph of age, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Increases in whole body thyroid hormone levels coincided with a 2(1/2)-fold increase in the total thyroidal colloid area in stage 1 larvae (aged 15dph) compared to stage 0 larvae (12dph). This was preceded by an approximately 40%-increase in the follicles' epithelial cell height in stage 0 larvae at 12dph compared to larvae at 5dph, and by an increase in the whole body T3/T4 ratio, indicative of an increase in outer ring deiodination. We conclude that in S. senegalensis there is a clear chronology in the activation of the thyroid gland that starts in early pre-metamorphic larvae.     

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 μg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

A salient feature of the thyroid hormones. L-thyroxine (T4) and triiodo-L-thyronine (T3), is the multiplicity of their physiological and biochemical actions in a wide variety of vertebrates. Important among these is their profound effects on postembryonic development. Analysis of their developmental action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process (morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. It is therefore an ideal model for studying the mechanisms underlying the hormonal regulation of postembryonic development. This article will first summarize the key features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the important role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in mammals and other vertebrate species.     

Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector

We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.     

Aqueous leaf extracts display endocrine activities in vitro and disrupt sexual differentiation of male Xenopus laevis tadpoles in vivo

The occurrence of natural substances acting as endocrine disrupting compounds (EDC) in the environment is to date poorly understood. Therefore, (anti)androgenic and (anti)estrogenic activities of three different aqueous leaf extracts (beech, reed and oak) were analyzed in vitro using yeast androgen and estrogen screen. The most potent extract was selected for in vivo exposure of Xenopus laevis tadpoles to analyze the potential effects on development and reproductive biology of amphibians. Tadpoles were exposed from stage 48 to stage 66 (end of metamorphosis) to aqueous oak leaf extracts covering natural occurring environmental concentrations of dissolved organic carbon. Gene expression analyses of selected genes of the hypothalamus-pituitary-gonad and of the hypothalamus-pituitary-thyroid axis as well as histological investigation of gonads and thyroid glands were used to evaluate endocrine disrupting effects on the reproductive biology and development. Female tadpoles remained unaffected by the exposure whereas males showed severe significant histological alterations of testes at the two highest oak leaf extract concentrations demonstrated by the occurrence of lacunae and oogonia. In addition, a significant elevation of luteinizing hormone β mRNA expression with increasing extract concentration in male tadpoles indicates an involvement of hypothalamus-pituitary-gonad axis mainly via antiandrogenic activity. These results suggest that antiandrogenic EDC of oak leaf extract are responsible for inducing the observed effects in male tadpoles. The present study demonstrates for the first time that in surface waters, natural occurring oak leaf compounds at environmentally relevant concentrations display antiandrogenic activities and have considerable effects on the endocrine system of anurans affecting sexual differentiation of male tadpoles.     

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-β antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.     

Sex reversal effects on Caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol A

Exposure to environmental contaminants known as endocrine disruptors (EDs) alters the development and function of reproductive organs in several species. Bisphenol A (BPA) is an estrogenic chemical that leaches from dental materials and plastic food and beverage containers. BPA has been found in sewage, surface and drinking water, and therefore poses a potentially significant risk for human and wildlife. Prenatal exposure of rodents to environmentally relevant doses of BPA alters the development of the reproductive organs of male and female offspring. Species with temperature dependent sex determination (TSD) could act as sentinels of ecosystem health by providing sensitive biomarkers of endocrine disruptor's effects. We selected Caiman latirostris as an animal model to study endocrine disruption caused by BPA. The aim of this study was to determine whether exposure in ovum to BPA could cause estrogen-like effects on the reproductive system of C. latirostris. Sex determination and gonadal histoarchitecture were the endpoints evaluated after in ovum exposure to different doses of BPA and 17beta-estradiol (E(2)). We confirmed that C. latirostris is a species with TSD and additionally demonstrated that BPA causes estrogen-like developmental effects by reversing gonadal sex and altering gonadal histoarchitecture. Differences in responses to BPA and E(2) in our in vivo system were on the order of 100-fold. In contrast published in vitro studies have reported differences on the order of 10,000x or more. These results support the utility of C. latirostris, a species in which sex determination is temperature dependent, as a tool in assessing estrogenic activity in vivo and as a sentinel to monitor EDs in aquatic environment.     

Xenopus laevis as a model for studying thyroid hormone signalling: from development to metamorphosis

Amphibian metamorphosis is a well-established model for dissecting the mechanisms underlying thyroid hormone (TH) action. How the pro-hormone, T(4), the active form, T(3), the deiodinases and the nuclear receptors (TRs) contribute to metamorphosis in Xenopus has been extensively investigated. Our recent work has concentrated on two key ideas in TH signalling in Xenopus: first, that there could be active roles for both liganded and unliganded receptors, and second, that ligand availability is a determining factor orchestrating these actions and is tightly controlled in target tissues. Recently, we addressed these questions at stages preceding metamorphosis, i.e. during embryogenesis, before differentiation of a functional thyroid gland. We show that repression by unliganded TR is essential to craniofacial and eye development during early development and that at these stages all three deiodinases are active. These results open new perspectives on the potential roles of TH signalling during embryogenesis.     

Bisphenol A acts differently from and independently of thyroid hormone in suppressing thyrotropin release from the bullfrog pituitary

The objective of this investigation was to ascertain whether bisphenol A (BPA), which has a structural resemblance to thyroid hormone (TH), acts as a TH agonist or antagonist in terms of affecting the release of thyrotropin (TSH). To this end, we exposed adult bullfrog (Rana catesbeiana) pituitary cells to BPA and/or TH in the presence or absence of corticotropin-releasing factor (CRF), which is known to have a potent TSH-releasing activity in amphibians. BPA (10(-9)-10(-4)M) did not affect the basal release of TSH. However, it suppressed CRF-inducible TSH release at 10(-4)M, but not at 10(-5)M. Triiodothyronine (T(3)) at 10(-7)M and l-thyroxine (T(4)) at 10(-6)M also suppressed the CRF-inducible release of TSH. The combination of T(3) (10(-7)M) or T(4) (10(-6)M) with BPA (10(-4)M) had an additive effect in suppressing TSH release. A comparison of the suppressive effects of BPA and T(3) on the release of TSH following the addition of actinomycin D or cycloheximide to the culture medium revealed that both of the latter compounds blocked T(3)-inducible but not BPA-inducible suppression of TSH release. The results indicate that the mechanism of action of BPA is different from that of T(3) in that T(3) action involves RNA and protein synthesis, whereas BPA action does not involve either of these processes. Furthermore, BPA was found to suppress the thyrotropin-releasing hormone-inducible release of both prolactin (PRL) and TSH. Our results suggest that BPA acts not only as a blocker of TSH secretagogues but also as a blocker of a PRL secretagogue at the pituitary level. Estradiol affected neither the release of TSH nor the release of PRL in the presence or absence of their secretagogues, suggesting that the suppression of the release of TSH and PRL caused by BPA may not be derived from its estrogenic activity.     

Characterization of thyroid hormones and thyroid hormone receptors during the early development of Pacific bluefin tuna (Thunnus orientalis)

We studied the profiles of 3,5,3'-l-triiodothyronine (T3), thyroxine (T4), and thyroid hormone receptors (TRs) in Pacific bluefin tuna (Thunnus orientalis) during embryonic and post-embryonic development. Both T3 and T4 were detected in embryos just before hatching, and it was found that the levels of both were increased in postflexion fish. The thyroid follicles were increased in both size and number in postflexion fish compared with preflexion fish. A TRbeta cDNA clone was generated by RACE. Two TRalpha cDNA clones were also partially identified and analyzed by real-time RT-PCR in this study. The TR mRNA levels in embryos were determined, and these were found to be lower than those in preflexion fish. Therefore, we considered that thyroid hormones function during early post-embryonic development as well as during embryonic development. Moreover, there was a peak in the TR mRNA level during postflexion stages, as seen during metamorphosis in Japanese flounder and Japanese conger eel. It is possible that thyroid hormones control the early development of scombrid fish through TRs, as they do for Pluronectiformes and Anguilliformes.