Evolutionary dampening of Wnt signaling may contribute to naturally ascrotal testes in mammals: Dampened Wnt pathway under ascrotal testes.

The testicular descent leading to the exteriorization of male gonads is a complex sexual dimorphic process observed in numerous mammals, whereas some species, such as elephants and cetaceans, retain the lifelong ascrotal testes. Despite the implication of Wnt signaling in testicular development and descent, the evolutionary mechanisms underlying ascrotal testes have not been adequately addressed. Here, we examined selection signatures and unique amino-acid substitutions in genes of the Wnt signaling pathway. We identified an ascrotal mammal-specific substitution (S406G) in ZNRF3 located within the DVL-interaction region. Functional assays showed that this substitution enhances the affinity of ascrotal ZNRF3 for DVLs and suppresses Wnt3a-induced Wnt signaling. Comparative transcriptomics of the gubernaculum between male fetal rats from wild-type and spontaneous cryptorchid (orl) strains revealed upregulation of Ctnnb1 and Gsk3b in orl rats, along with ascrotal-specific evolutionary changes in regulatory elements. Collectively, these findings suggest that Wnt signaling may be dampened in ascrotal mammals. This study provides insights into the pathogenesis of cryptorchidism in humans and domestic animals.