Ex vivo development of the entire mouse fetal reproductive tract by using microdissection and membrane-based organ culture techniques

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Ex vivo explant culture is an appealing alternative to in vivo studies on fetal reproductive organ development. There is extensive literature on ex vivo methods of growing the fetal gonad. However, a method for culturing the whole fetal reproductive tract that has a different shape and size has not been documented. Here, with careful dissection and proper tissue orientation, we successfully cultured the entire bicornuate reproductive tracts from mouse embryos of both sexes on the Transwell insert membrane. The cultured reproductive tract system undergoes sexually dimorphic establishment and region-specific morphogenesis comparable to in vivo development of their counterparts. To test this culture method's applications, we used chemical treatment (dihydrotestosterone and BMS 564929) and genetic cellular ablation mouse model (Gli1-CreER; Rosa-DTA) to investigate the roles of androgen signaling and Gli1+ mesenchyme in Wolffian duct development. Dihydrotestosterone and BMS 564929 promoted the ectopic maintenance of Wolffian ducts in cultured XX tissues. The efficient and specific elimination of Gli1+ mesenchyme was successfully achieved in the cultured tissues, resulting in defective coiling of Wolffian ducts. These results demonstrate the amenability of this organ culture method for chemical and genetic manipulations that are otherwise difficult to study in vivo. Taken together, the establishment of this organ culture method provides a valuable tool complementary to in vivo studies for understanding fetal reproductive tract development in mice.