TNXB Regulates Spermatogenesis by Maintaining Cytoskeleton and Cell Junction Stability

Tenascin-X (TNXB) is an extracellular matrix glycoprotein known for its structural and regulatory roles in connective tissues, yet its pathophysiological role in testicular development and spermatogenesis remains uncharacterized. Here, we identified TNXB as clinically implicated in human spermatogenic disorder, with a significant downregulation in the testes of patients with non-obstructive azoospermia (NOA) revealed by isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics. Developmental profiling in mice revealed a progressive increase in TNXB expression with testicular maturation, with strong localization in germ cells. To elucidate its function in spermatogenesis, we generated conditional knockout (cKO) mice with germ cell-specific deletion of the Tnxb gene by using the Cre-LoxP system (Ddx4-Cre⁺, Tnxb^LoxP/Null). Tnxb deficiency led to reduced testis size, severe tubular degeneration, excessive germ cell loss, impaired spermatogenesis, and decreased sperm count and motility. Transcriptomic profiling of cKO testes revealed that dysregulated genes were mainly associated with meiosis and germ cell development, cytoskeletal and tight junction organization, and cell junction assembly, which were validated by quantitative real-time PCR, western blotting, and immunofluorescence. Further investigation demonstrated mislocalization and altered expression of key proteins regulating the cytoskeleton, cell junction, and extracellular matrix organization in the cKO testes. Notably, TNXB demonstrated an interaction with Collagen IV, a crucial extracellular matrix component. Deficiency of Tnxb led to an abnormal association between the Sertoli cell cytoskeleton and the junctional structure. These findings confirm that TNXB is indispensable for preserving the integrity and interactions among the extracellular matrix, cytoskeleton, and cell junction within the testis, and its deficiency impairs testicular architecture and spermatogenesis.