Mechanisms of androgen-dependent Wolffian duct differentiation

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Project Summary A major cause of male infertility is defective and atypical development of the Wolffian duct, the embryonic structure that give rise to male internal reproductive tract organs. It is known that Wolffian duct differentiation is predominantly driven by the action of the testis-derived androgens. However, how the androgen signaling coordinates the process of the Wolffian duct differentiation remains unclear in the field of reproduction. The androgen action in Wolffian duct differentiation is mediated by the androgen receptor (AR), which is expressed in both Wolffian duct epithelium and mesenchyme. Using a new mesenchyme-specific Ar knockout mouse model, we provided the first genetic evidence that the AR in the mesenchyme is essential for Wolffian duct differentiation. By comparing chromatin accessibilities and transcriptomes of Wolffian duct mesenchymes from female and male embryos, we discovered a set of androgen- induced chromatin accessible regions and a new androgen-induced mesenchymal factor R- Spondin 3 (Rspo3). RSPO3 is a WNT signaling activator secreted from the mesenchyme to activate epithelial Wnt signaling that is essential for Wolffian duct morphogenesis. While the mesenchyme governs epithelial differentiation, the epithelium has the reciprocal inductive effects on the mesenchyme by synthesizing a paracrine growth factor WNT9B. We found that the loss of Wnt9b caused Wolffian duct degeneration at the sexual differentiation window when the androgen signaling was supposed to promote Wolffian duct survival. These observations lead to and support our central hypothesis: the androgen-dependent Wolffian duct differentiation requires the stimulation of the epithelium-derived WNT9B, and the androgen signaling in Wolffian duct differentiation is mediated by the mesenchymal AR and executed by the androgen-induced mesenchymal factor RSPO3 via epithelial-mesenchymal interactions. We will determine the mechanisms of WNT9B, AR, RSPO3 actions in promoting Wolffian duct differentiation by utilizing a combination of tissue-specific gene knockouts, gene expression assays, fluorescence-activated cell sorting, RNA-seq, ATAC-seq, and single cell mRNA-seq. AR and WNT9B variants in humans have been associated with defective androgen-dependent male reproductive tract differentiation. Therefore, the completion of our proposal will not only yield fundamental knowledge of basic mechanisms underlying androgen-dependent Wolffian duct differentiation but also provide knowledge directly relevant to our understanding of disorders of male sexual development in humans.