FAM65B Regulates Proliferation, Cell Junctions, and Cytoskeletal Organization in Chicken Sertoli Cells

Semen quality in roosters is a critical reproductive trait in poultry, yet its genetic regulatory mechanisms remain poorly understood. Sertoli cells (SCs) are essential for establishing the blood–testis barrier and supporting spermatogenesis. Our previous single-cell transcriptomic analysis identified FAM65B (Family with sequence similarity 65, member B) as a SC-specifically enriched gene that is significantly upregulated in testes of roosters with high semen quality. Given its known roles in cell proliferation, adhesion, and cytoskeletal remodeling, we systematically investigated the spatiotemporal expression pattern of FAM65B in chicken testes and its functional roles in SC physiology using a combination of in vitro and tissue-based approaches. Our results demonstrate that FAM65B is predominantly localized in the SC cytoplasm. Transcriptomic analysis revealed that FAM65B knockdown downregulated genes involved in cell adhesion, cytokine signaling, and the PI3K-Akt and NF-κB pathways. Knockdown of FAM65B significantly inhibited SC proliferation, reduced the expression of junction-associated molecules, and impaired cytoskeletal rearrangement, while its overexpression promoted proliferation, enhanced F-actin filamentous structures, and strengthened cell–cell contacts. Collectively, our findings establish FAM65B as a novel positive regulator of SC function in chickens. These cellular findings, together with the association between FAM65B expression and semen quality traits, provide a foundation for further investigation of SC biology in avian male reproduction.