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Our recent study revealed some early molecular and cellular events in which 17β-estradiol (E2) disrupted testis differentiation and resulted in feminization in Xenopus laevis (the African clawed frog), an ideal species for studying reproductive endocrine disruption by estrogenic endocrine disrupting chemicals (EDCs). On this basis, we aimed to develop multiple biomarkers for early detection of testis differentiation disruption by estrogenic EDCs in X. laevis. Tadpoles at stage 45/46 were exposed to four known estrogenic EDCs with different