TBC1D21 is an essential factor for sperm mitochondrial sheath assembly and male fertility

Abstract During spermiogenesis, the formation of the mitochondrial sheath is critical for male fertility. The molecular processes that govern the development of the mitochondrial sheath remain unknown. Whether TBC1D21 serves as a GTPase-activating protein (GAP) for GTP hydrolysis in the testis is unclear, despite recent findings indicating that it collaborates with numerous proteins to regulate the formation of the mitochondrial sheath. To thoroughly examine the property of TBC1D21 in spermiogenesis, we applied the CRISPR/Cas9 technology to generate the Tbc1d21−/− mice, Tbc1d21D125A R128K mice with mutation in the GAP catalytic residues (IxxDxxR), and Tbc1d21-3xFlag mice. Male Tbc1d21−/− mice were infertile due to the curved spermatozoa flagella. In vitro fertilization is ineffective for Tbc1d21−/− sperm, although healthy offspring were obtained by intracytoplasmic sperm injection. Electron microscopy revealed aberrant ultrastructural changes in the mitochondrial sheath. Thirty-four Rab vectors were constructed followed by co-immunoprecipitation, which identified RAB13 as a novel TBC1D21 binding protein. Interestingly, infertility was not observed in Tbc1d21D125A R128K mice harboring the catalytic residue, suggesting that TBC1D21 is not a typical GAP for Rab-GTP hydrolysis. Moreover, TBC1D21 was expressed in the sperm mitochondrial sheath in Tbc1d21-3xFlag mice. Immunoprecipitation-mass spectrometry demonstrated the interactions of TBC1D21 with ACTB, TPM3, SPATA19, and VDAC3 to regulate the architecture of the sperm midpiece. The collective findings suggest that TBC1D21 is a scaffold protein required for the organization and stabilization of the mitochondrial sheath morphology.