Single-cell analyses reveal impaired type B spermatogonia differentiation and meiotic entry in C-Nap1-null testes

Sperm development is critical for male reproductive capability; any disruption during the process of spermatogenesis will result in male infertility. In this research, we used the C-Nap1 encoded by the gene of Cep250 knockout mouse line as the model to evaluate the impact of absent C-Nap1 on spermatogenesis. To investigate the interaction between C-Nap1 and spermatogenesis, we utilized single-cell RNA sequencing to analyze 10,332 C-Nap1 ^+/+ and 13,308 C-Nap1 ^-/- testicular cells. We identified five main cell types within seminiferous tubules, including spermatogonia, Sertoli cells, spermatogonia stem cells, Leydig cells, and spermatocytes. We found a critical reduction in testicular spermatogonia and spermatocytes in C-Nap1-null testes, compared to its C-Nap1 ^+/+ controls. By combining uniform manifold approximation and projection clustering and psedotime ordering, we distinguished five spermatogonial stages/subtypes, demonstrating that type B spermatogonia differentiation and meiotic initiation are impaired during C-Nap1-null spermatogenesis. Following gene ontology enrichment analysis, meiosis-specific genes downregulated in the C-Nap1 ^-/- testicular cells were further verified by reverse transcription polymerase chain reaction (RT-PCR). Based on the differential gene expression, certain downregulated genes such as Ctnnb1 and Aurka encoding C-Nap1-binding potential β-Catenin and Aurka are encountered, which may account for defective type B spermatogonia differentiation and meiotic entry in C-Nap1-null testes.