Evolutionary relaxation and functional change of INSL3 and RXFP2 may underlie natural cryptorchidism in mammals.

Cryptorchidism is a common congenital abnormality that increases infertility and testicular cancer risk in adulthood. However, a few mammals exhibit naturally undescended testes while maintaining normal reproduction. The mechanisms underlying this natural cryptorchidism remain unclear. Here, we found evolutionary relaxation in INSL3 and RXFP2 of cryptorchid mammals, with the highest dN/dS ratio observed in cetaceans. Cellular experiments demonstrated that cetacean INSL3 downregulated the cAMP-PKA-CREB pathway, thereby reducing gubernacular cell proliferation and contraction. Cetacean INSL3 knock-in mice exhibited groin-located testes, nearly perfectly mimicking cryptorchid phenotypes in cetaceans and other mammals with incompletely descended testes. Collagen and muscle fibers in the gubernaculum of transgenic mice were reduced, with differentially expressed genes enriched in muscle development and contraction pathways. Additionally, the knock-in mice displayed male sterility, impaired testicular development, and upregulated inflammatory pathways in the testes. Our findings reveal how evolutionary changes in the INSL3/RXFP2 pathway contribute to natural cryptorchidism in mammals and provide insights for investigating reproductive health and cancer resistance in cryptorchid species.