Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Abstract

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone (TH) signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor (TR) agonist (T3) and antagonist (amiodarone) by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2 days induced more dramatic increases of TRβ expression in stage 27 than in stage 34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax.Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

“…Overall, TH/bZIP, SHH and MMP2 as well as TRs in the intestine displayed roughly similar expression profiles during P. nigromaculatus metamorphosis, with a similar trend of BTEB expression (Lou et al, 2014a). The expression profiles of these genes parallel changes in endogenous TH levels, which remain low at premetamorphic and most prometamorphic stages, increase beginning in late prometamorphic stages, peak at the metamorphic climax, and decline towards the end of metamorphosis (Ge et al, 2014;Lou et al, 2014a).…”

“…The expression profiles of these genes parallel changes in endogenous TH levels, which remain low at premetamorphic and most prometamorphic stages, increase beginning in late prometamorphic stages, peak at the metamorphic climax, and decline towards the end of metamorphosis (Ge et al, 2014;Lou et al, 2014a). It is suggested that changes in TH-response gene expression levels reflect to some degree the responsiveness of TH-response genes to endogenous TH during P. nigromaculatus metamorphosis.…”

“…nigromaculatus frogs (from our amphibian house) were maintained in glass tanks containing charcoal-filtered tap water and fed with Terzebrio molitor daily. Housing and breeding conditions were reported in our previous study (Lou et al, 2014a). The water quality was as follows: without chlorine, iodine concentration 2-4 μg/L, pH 6.5-7.0, dissolved oxygen concentration > 5 mg/L, and water hardness (CaCO 3 ) approximately 150 mg/L.…”

“…Here, we chose these genes as candidate TH-response genes in P. nigromaculatus intestines, and investigated the developmental expression profiles during metamorphosis, in order to understand the relationship between their expression and endogenous TH levels. Four genes (TH/bZIP, MMP2, SHH and BTEB) were analyzed except for TRα and TRβ, which were reported in our previous study (Lou et al, 2014a).…”

“…According to our previous reports (Lou et al, 2014a), TR expression was regarded as one of the molecular biomarkers for assaying TH signaling disruption in P. nigromaculatus, with the intestines of stage 27 tadpoles as test tissues. Here, we chose an appropriate time for T3 induction in the screening assay by comparing the response of TR expression for 24 hr-, 48 hr-, and 72 hr-T3 induction in the intestines of stage 27 tadpoles.…”