Abstract
BACKGROUND: The heterodimeric FACT complex (SSRP1/Ssrp and SUPT16H/Dre4) is primarily recognized as a chromatin remodeler. While mutations in this complex are linked to human intellectual disability and it maintains neural stem cell fate in flies, single-cell RNA sequencing reveals robust FACT complex expression in human testicular germ cells. Nevertheless, its specific functions during spermatogenesis remain unexplored. METHODS: This study utilized Drosophila melanogaster as a model to investigate the roles of FACT complex during spermatogenesis. Germline-specific and somatic-specific knockdowns of Ssrp and dre4 combined with immunostaining were performed to assess their functions. Bulk and single-cell RNA sequencing analyses were conducted on Ssrp-deficient testes to investigate transcriptomic changes. RESULTS: Our study uncovers context-dependent functions for Ssrp. In testis somatic cells, Ssrp collaborates with Dre4 to sustain cyst stem cell populations via estrogen-related receptor-mediated glycolytic activation. Remarkably, germline Ssrp operates independently of Dre4, governing transit-amplifying divisions and meiotic progression. Germline-specific Ssrp depletion, but not dre4 knockdown, induces male sterility, characterized by spermatogonial accumulation, mitotic asynchrony, and meiotic arrest within primary spermatocytes. Bulk and single-cell RNA sequencing analyses of Ssrp-deficient testes reveal systemic transcriptomic dysregulation, including suppression of metabolic programs (glycolysis and oxidative phosphorylation) and activation of MAPK/EGFR signaling. Ssrp loss disrupts sister centromere cohesion during meiosis I, as well as diminished ATP levels and aberrant CENP-A accumulation, suggesting a dual regulatory nexus linking energy metabolism to chromosomal stability. CONCLUSIONS: This work reveals previously unknown, context-dependent functions of Ssrp during Drosophila spermatogenesis. Ssrp emerges as a multifunctional orchestrator essential for both somatic and germline compartments of germline development. These findings provide crucial foundational insights into reproductive disorders associated with Ssrp dysfunction and underscore the importance of the FACT complex in male germ cell development.
