SIGNALING PATHWAYS IN TESTICULAR DESCENT AND FUNCTION - Project Summary: Cryptorchidism (undescended testicles, UDT) affects 1-3% of full-term newborns, with 80% presenting unilaterally. After early surgical correction (orchiopexy) before age 2, the risks for testicular cancer and infertility remain elevated, with nearly 50% of patients with unilateral UDT having defects in sperm production, and 13% of them lacking sperm regardless of the presence of a contralateral descended testis (CDT). Despite its significant health and economic burden, over $1 billion annually in U.S. healthcare costs, the underlying mechanism of UDT remains largely unknown. The pathogenesis of UDT thus appears to transcend mere anatomical location. Indeed, a variety of environmental and genetic factors are linked to UDT. Members of the WNT family of genes often play critical roles in the organogenesis of left-right asymmetries and gonadal development. Until recently, it was considered that the Wnt4 signaling in the gonads suppresses testis development, allowing female organogenesis. This paradigm is shifting. WNT4 mutations are associated with testicular dysgenesis. We recently demonstrated that 100% of male mice lacking Wnt4 expression in the gubernaculum exhibit left UDT and profound sperm and fertility defects, despite normal levels of testosterone and a CDT. Thus, in the mouse, Wnt4 is required for symmetrical testis descent, proper testis and epididymal development, and fertility. This mouse model does not explain all of human cryptorchidism. However, we do believe that the Wnt pathway is likely to be an important player, especially in asymmetric UDT, which includes most patients with UDT. Our central hypothesis is that Wnt4 signaling is required for testicular position and function through three interconnected mechanisms: gubernacular and/or epididymal structural integrity, hormonal regulation, and genetic pathway coordination. To test these hypotheses, we propose two aims: (1) We will define the cause of infertility in Wnt4 gubernaculum-deficient mice and distinguish between the roles of testicular position, genetic defect, and hormone imbalance in both testes. Since Wnt4 can play different roles in the gubernaculum and the testes, we will determine its potential effects on Leydig cells after generating a conditional knockout mouse. (2) We will test the mechanism by which Wnt4 suppresses asymmetric gene expression by generating a spatial transcriptomics and proteomics profile of the gubernaculum and the epididymis in Wnt4-deficient mice and test the functionality of the signaling pathway in tissue-derived cell models. Finally, because Wnt4-deficient mice show severely impaired sperm motility in both testes, we will also test whether Wnt4 is required for epididymal development. This proposal is innovative in that it recognizes Wnt4 for promoting both proper testis development and symmetry in organogenesis. The proposed studies address a major gap in testicular development, which will enrich the understanding of factors predisposing patients to UDT and could improve current treatments.
