The S-depalmitoylase ABHD10 is essential for sperm mitochondrial sheath formation and male fertility.

Palmitoylation is the only fully reversible post-translational lipid modification that impacts 10-20% of the human proteome, but its role during spermatogenesis remains enigmatic. In this study, through generating HA-tagged Abhd10 knock-in mice, Abhd10-null mice, and combining super-resolution fluorescence imaging and electron microscopy, we identify that the S-depalmitoylase ABHD10 (abhydrolase domain containing 10) is a mitochondrial matrix protein, specifically expressed in testis and is essential for male fertility. Abhd10 knockout mice manifest severe sperm motility defects accompanied by malformed mitochondrial sheaths of sperm. Mitochondrial proteomic analysis reveals that ABHD10 deficiency downregulates respiratory chain complex proteins and mitochondrial sheath formation factors SPATA19 and GK2. Using mass spectrometry-based mitochondrial acyl-biotin exchange assays, we systematically identify that loss of ABHD10 leads to the hyper-palmitoylation of multiple functionally critical proteins, including mitochondrial sheath formation factors (SPATA19 and GK2) and aerobic respiration regulators (PDHX, NDUFV1 and SDHB). Co-immunoprecipitation and proximity labeling assays reveal the physical interactions between ABHD10 and its substrates (SPATA19, GK2, PDHX). Collectively, ABHD10 may bind to and mediate the S-depalmitoylation of SPATA19, GK2, and PDHX, thereby regulating the formation of the sperm mitochondrial sheath and mitochondrial function. This work not only identifies S-depalmitoylase ABHD10 as a key determinant of male fertility but also advances our understanding of post-translational regulation during spermatogenesis.