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 stage34 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.
Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals(EDCs). Although ribosomal protein L8(rpl8) is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rpl8 c DNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 c DNA consisted of919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of m RNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rpl8 gene was found in all tissues examined(brain, liver, intestine, tail, testis and ovary) during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 m RNA expression was found under exposure to thyroid hormone(T4) and estrogen(estradiol), whereas expression of the corresponding biomarker genes was induced.Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus.
