Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation

• Gonadal differentiation of Xenopus laevis at stages 45/46 or earlier is very sensitive to lower concentrations of EDCs.
• Stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis.
• The stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis.

Xenopus laevis is an important model for detecting feminizing effects of endocrine disrupting chemicals (EDCs) on amphibians because its genetic males can be induced to phenotypic females by estrogenic chemicals. It is crucial that chemical exposures begin at sensitive developmental stages for gonadal sex-reversal in X. laevis. To determine the optimal stages for initiating exposures, we investigated gonadal sex-reversal induced by low concentrations of 17α-ethinylestradiol (EE2) when exposures were initiated at different stages (3/4, 45/46, 48 and 50) until stage 58. We found that 0.1 nM EE2 resulted in 85%, 86%, 43%, and 19% intersex, whereas 1 nM EE2 caused 77%, 81%, 17%, and 8% phenotypic females, when genetic male tadpoles were exposed from stages 3/4, 45/46, 48 and 50, respectively. The data show the sensitivity of X. laevis gonads to EE2 at stages 45/46 is similar with that at stages 3/4, but the sensitivity decreases at stage 48 and stage 50, displaying a developmental stage-dependent manner. In another experiment using the offspring of another pair of frogs, we confirmed high sensitivity of X. laevis gonads at stages 45/46 to low concentrations of EE2. Considering that stages 45/46 tadpoles are easier to manipulate and have higher survival rates than earlier embryos, we propose that stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis. The developmental stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis. Also, our study suggests that gonadal differentiation in X. laevis possibly begins at stages 45/46, but not at later stages.