# Mechanisms of androgen-dependent Wolffian duct differentiation

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $421,711

## Abstract

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.

## Key facts

- **NIH application ID:** 10841678
- **Project number:** 5R01HD111425-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Fei Zhao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $421,711
- **Award type:** 5
- **Project period:** 2023-06-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10841678

## Citation

> US National Institutes of Health, RePORTER application 10841678, Mechanisms of androgen-dependent Wolffian duct differentiation (5R01HD111425-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10841678. Licensed CC0.

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