CG31200 depletion disrupts germ cell differentiation during Drosophila spermatogenesis

Serine proteases are essential for diverse physiological functions, including digestion, blood clotting, immune response, fertilization, and cancer metastasis. Here, we report that CG31200, a predicted serine protease-related protein in Drosophila, plays an essential role in spermatogenesis. The depletion of CG31200 severely impaired male fertility, characterized by the complete absence of mature sperm and disrupted differentiation of spermatogonia. Single-cell RNA sequencing (scRNA-seq) analysis revealed extensive transcriptional dysregulation in both the late spermatogonia cluster and the spermatogonia-to-spermatocyte transitional cluster of CG31200-depleted testes. These changes were characterized by reduced expression of genes involved in flagellar assembly and axoneme formation, together with increased expression of mitochondrial and oxidative phosphorylation-related programs. Functional assays further showed reduced ATP content together with increased ROS levels and LPO in CG31200-knockdown testes, supporting mitochondrial dysfunction and oxidative stress. Additionally, pseudotime analysis indicated that CG31200-knockdown germ cells accumulated in early inferred developmental states. Knockdown Not1, a poly(A)-specific ribonuclease, partially phenocopied the CG31200 RNAi phenotype, and CG31200 depletion was associated with altered Not1 transcript features. Together, these findings indicate that CG31200 depletion disrupts germ-cell differentiation and sperm morphogenesis and is associated with mitochondrial dysfunction, oxidative stress, altered transcriptional states, and defective axoneme formation. This study provides a phenotypic and scRNA-seq framework for understanding the role of SPH-related proteins in Drosophila spermatogenesis.