1Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA
2Comparative Biomedical Sciences Graduate Program, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, US
Abstract Text: Androgen actions have been known as the predominant driver of male reproductive tract development. During fetal development, testicular androgens promote the elongation and coiling of the Wolffian duct, the precursor of the epididymis. Proper Wolffian duct morphogenesis is important for the epididymal function in supporting sperm transport, maturation, and storage. The Wolffian duct is surrounded by undifferentiated connective tissues also known as the mesenchyme, which governs the morphogenesis and differentiation of ductal epithelium in developing organs. We recently discovered that GATA2 was specifically expressed in Wolffian duct mesenchyme during the fetal masculinization window. GATA2 belongs to the GATA family of transcription factors that bind to the characteristic “GATA” DNA sequence. Among the six members of the GATA family, GATA2 is the only member that is highly expressed in the Wolffian duct mesenchyme. GATA2 has been known to play crucial roles in cell differentiation and tubular morphogenesis of the mammalian ureter and inner ear. We therefore hypothesize that mesenchymal GATA2 is essential for Wolffian duct development. To test this hypothesis, we used the tissue-specific Cre-LoxP mouse model (Osr2-Cre; Gata2-flox) to delete Gata2 in the Wolffian duct mesenchyme. When Gata2 was absent in the mesenchyme, the Wolffian duct became shortened and failed to coil in the corpus and cauda regions. Wolffian duct elongation and coiling have been predominantly linked with androgen actions; however, we didn’t observe any deficiency in androgen production or androgen receptor expression. Then, we turned our attention to androgen-regulated Inhba, a mesenchyme-derived morphogen that is essential for epididymal elongation and coiling. By performing RNAscope and RT-qPCR, we discovered that Inhba expression was downregulated, and its expression localization was shifted from inner mesenchyme to the outer mesenchymal layer surrounding the Wolffian duct. Consistent with the reduction of Inhbaexpression, we observed reduced percentage of Ki67+ proliferating cells in the epithelium of Gata2cKOWolffian ducts. These results indicate that the defective coiling observed in Gata2cKO is at least in part due to decreased Inhbaexpression and/or inappropriate localization of Inhba. To gain more insights into the mechanisms of GATA2 action, we compared transcriptomes between and control and Gata2cKO Wolffian duct by bulk RNA-seq. Among 698 differentially expressed genes, we focused on the epididymal tissue marker, Itga4. Itga4 encodes the integrin subunit alpha 4, a subunit of the receptor for fibronectin. In the control tissue, ITGA4 is strongly expressed in the mesenchyme of the epididymal region. However, its expression was significantly reduced in the Gata2cKO, suggesting that GATA2 might control the identity of the epididymal mesenchyme. Therefore, our discovery not only demonstrates that GATA2 regulates paracrine signaling and identity of epididymal mesenchyme in promoting epididymal morphogenesis, but also prompts the investigation of the potential effects of human GATA2 mutations on epididymal morphogenesis and the predisposition to male infertility.
