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, Tbc1d21^{D125A 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 Tbc1d21^{D125A 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.

Summary sentence

The formation of the mitochondrial sheath is critical for male fertility, but the molecular processes that govern the development of the mitochondrial sheath remain unknown. By using multiple transgenic mouse models, we demonstrated that TBC1D21 is an essential protein for sperm mitochondrial sheath assembly and male fertility. The Tbc1d21-/- mice were infertile due to the malformation of the mitochondrial sheath assembly. Importantly, Tbc1d21^{D125A R128K} mice were fertile, suggesting that TBC1D21 may not be a classic Rab-GTP hydrolysis protein. Tbc1d21-3XFlag mice elucidated that TBC1D21, ACTB, and TPM3 complex in the form of a double helix around the mitochondrial sheath to maintain its assembly structure. This research sheds light on the pivotal role of TBC1D21 in the organization and stabilization of the mitochondrial sheath morphology.

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