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Here, we report that CG31200, a predicted serine protease\u2010related protein in\n                    <jats:italic>Drosophila<\/jats:italic>\n                    , plays an essential role in spermatogenesis. The depletion of\n                    <jats:italic>CG31200<\/jats:italic>\n                    severely impaired male fertility, characterized by the complete absence of mature sperm and disrupted differentiation of spermatogonia. Single\u2010cell RNA sequencing (scRNA\u2010seq) analysis revealed extensive transcriptional dysregulation in both the late spermatogonia cluster and the spermatogonia\u2010to\u2010spermatocyte transitional cluster of\n                    <jats:italic>CG31200<\/jats:italic>\n                    \u2010depleted 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\u2010related programs. Functional assays further showed reduced ATP content together with increased ROS levels and LPO in\n                    <jats:italic>CG31200<\/jats:italic>\n                    \u2010knockdown testes, supporting mitochondrial dysfunction and oxidative stress. Additionally, pseudotime analysis indicated that\n                    <jats:italic>CG31200<\/jats:italic>\n                    \u2010knockdown germ cells accumulated in early inferred developmental states. Knockdown\n                    <jats:italic>Not1, a poly(A)\u2010specific ribonuclease<\/jats:italic>\n                    , partially phenocopied the\n                    <jats:italic>CG31200<\/jats:italic>\n                    <jats:italic>RNAi<\/jats:italic>\n                    phenotype, and\n                    <jats:italic>CG31200<\/jats:italic>\n                    depletion was associated with altered\n                    <jats:italic>Not1<\/jats:italic>\n                    transcript features. Together, these findings indicate that\n                    <jats:italic>CG31200<\/jats:italic>\n                    depletion disrupts germ\u2010cell 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\u2010seq framework for understanding the role of SPH\u2010related proteins in\n                    <jats:italic>Drosophila<\/jats:italic>\n                    spermatogenesis.\n                  <\/jats:p>","DOI":"10.1111\/1744-7917.70326","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T11:32:11Z","timestamp":1785324731000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<i>CG31200<\/i>\n                    depletion disrupts germ cell differentiation during\n                    <i>Drosophila<\/i>\n                    spermatogenesis"],"prefix":"10.1111","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7536-7546","authenticated-orcid":false,"given":"Yang","family":"Fang","sequence":"first","affiliation":[{"name":"Hunan Key Laboratory of Biomedical Nanomaterials and Devices, School of Biological Science and Medical Engineering Hunan 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