nphp4 deficiency disrupts testicular homeostasis and impairs male fertility in medaka ( Oryzias latipes )

Testicular homeostasis relies on intricate germ cell-somatic cell interactions, yet underlying regulatory mechanisms remain poorly understood. While cilia-related nphp4 mutations cause male infertility, phenotypes show notable interspecies differences and the driving mechanism remains unknown. In this study, we used the Japanese medaka (Oryzias latipes), a model with unique cystic spermatogenesis, to investigate the role of nphp4 in male reproduction. In situ hybridization localized nphp4 transcripts within the spermatogonial niche, particularly in gsdf-positive Sertoli cells. CRISPR/Cas9-mediated deficiency of nphp4 led to age-dependent infertility, characterized by a significant decline in fertilization rates. Although sperm retained normal flagellar structure, they exhibited severe motility defects, head abnormalities, and mitochondrial damage by 6 months of age. Notably, somatic cell dysfunction, including Sertoli cell apoptosis and altered expression of functional markers, occurred early and contributed to premature germ cell depletion. Transcriptomic profiling revealed a coordinated disruption of signaling pathways critical for spermatogonial stem cell maintenance, alongside genes involved in cell communication, ion homeostasis, cytoskeletal organization, and adhesion. Our findings suggest nphp4 is a key regulator of the testicular niche and highlight the utility of the medaka model in uncovering conserved mechanisms of infertility associated with cilia-related genes.