FBXO39 knockdown promotes spermatogenesis impairment by inducing mitochondrial dysfunction and ferroptosis via inhibiting KDM5A ubiquitination and regulating H3K4me3 demethylation.

The global decline in male fertility highlights the need to understand the mechanisms of spermatogenesis. Mitochondrial dysfunction and ferroptosis have emerged as key contributors to spermatogenic impairment, although the molecular basis of this process is still poorly defined. F-Box Protein 39 (FBXO39), a testis-enriched F-box protein, has been preliminarily associated with cell survival. However, whether FBXO39 participates in mitochondrial functional regulation or ferroptosis signaling during spermatogenesis remains largely unexplored. In our study, FBXO39 knockdown resulted in abnormal testicular development, impaired spermatogenesis, abnormal sperm morphology, and reduced testicular cell viability. Further analysis revealed that FBXO39 deficiency caused mitochondrial dysfunction and ferroptosis, as reflected by decreased ATP production, reduced mitochondrial DNA content, elevated eactive oxygen species (ROS) levels, diminished expression of key mitochondrial proteins, and elevated lipid peroxidation. Mechanistically, FBXO39 maintains mitochondrial homeostasis by targeting lysine-specific demethylase 5A (KDM5A) for ubiquitination-dependent degradation. Conversely, the accumulation of KDM5A upon FBXO39 loss suppressed single-stranded DNA-binding protein 1 (SSBP1) levels through demethylation of Histone H3 lysine 4 trimethylation (H3K4me3) at the SSBP1 promoter. Importantly, restoration of SSBP1 expression functionally ameliorated mitochondrial dysfunction induced by FBXO39 knockdown. Overall, FBXO39 regulates mitochondrial function and ferroptosis in testicular cells through ubiquitinating KDM5A, which affects SSBP1 expression by modulating H3K4me3 demethylation at the SSBP1 promoter. This study elucidates the role of FBXO39 in spermatogenesis and suggested that targeting this regulatory axis may offer novel therapeutic strategies for male infertility.