Ontogenetic Characterization of Müllerian Duct Development and Expression of anti-Müllerian hormone and Other Genes : New Endpoints in the Frog Xenopus tropicalis Test System

Abstract: The amphibian model-species Xenopus tropicalis is used in reproductive toxicology studies. Lifecycle studies are requested but they are time consuming and susceptible to several of the risks attributed to long-term studies. Herein, the ontogenetic expression of genes associated to gonads and Müllerian ducts and the early Müllerian duct development were characterized as well as explored as suitable early juvenile endpoints after developmental levonorgestrel (LNG) exposure. The urogenital mRNA expression of the anti-Müllerian hormone (amh) was male-biased while the cytochrome P450 19a1 (cyp19a1) mRNA expression was female-biased from the Nieuwkoop and Faber developmental stage (NF) 50, to 4 weeks post-metamorphosis. amh expression in combination with cyp19a1 expression is proposed to constitute a robust marker of phenotypic sex during gonadal differentiation in amphibians. The expression of the amh receptor 2 (amhr2) was characterized for the first time in amphibians. amhr2 was expressed higher in putative females than in males from NF 56, suggesting a role in ovarian development. The mRNA levels of intracellular and membrane progesterone receptors (ipgr and mpgr beta) respectively increased and decreased as the tadpole developed into a juvenile frog, indicating different roles for these genes during development. Exposure to LNG modulated the mRNA expression of the amh and pgrs in a sex and developmental stage dependent manner, suggesting the involvement of these genes in the LNG mode of action. The Müllerian ducts were first observed at NF 64 in both sexes and grew into distinct tubules, larger in females than in males at 4 weeks post-metamorphosis. Therefore, 4 weeks post-metamorphosis is suggested as a suitable early juvenile stage for investigation into disruptions of female reproductive organ development. In conclusion, the protein Amh is suggested to be involved in testicular differentiation and to be a reliable testicular marker during gonadal differentiation, even after developmental LNG exposure, in amphibians.