Supplementary Material for: Developmental Profiles and Thyroid Hormone Regulation of Brain Transcripts in Frogs: A Species Comparison with Emphasis on <i>Physalaemus</i> <i>pustulosus</i>

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of <i>Silurana tropicalis</i> and <i>Rana pipiens</i>; however, the gene expression changes differed between species. We chose to study a third anuran species, <i>Physalaemus</i><i>pustulosus,</i> to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of <i>P. pustulosus.</i> Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic<i> P.</i><i>pustulosus</i> and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta <i>(trbeta)</i> and deiodinase type 3 <i>(dio3),</i> mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha <i>(tralpha)</i> and deiodinase type 2 <i>(dio2)</i> mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In <i>P. pustulosus</i>, T3 reduced the expression of aromatase <i>(cyp19)</i> while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in <i>R. pipiens</i> but differ from data for <i>S. tropicalis,</i> for which we found an increase in androgen synthesis enzymes but no effect on <i>cyp19.</i> Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes <i>(S. tropicalis)</i> or by decreasing estrogen synthesis (due to a decrease in <i>cyp19</i> in <i>P. pustulosus</i> and <i>R. pipiens</i>). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.