Proteomic profiling of TBC1 domain family member 21-null sperms reveals the critical roles of TEKT 1 in their tail defects.

BACKGROUND: Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown. RESULTS: Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21-/- sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures. CONCLUSIONS: These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation.

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

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.

TBC1D21 Potentially Interacts with and Regulates Rap1 during Murine Spermatogenesis.

Few papers have focused on small guanosine triphosphate (GTP)-binding proteins and their regulation during spermatogenesis. TBC1D21 genes (also known as male germ cell RAB GTPase-activating protein MGCRABGAP) are related to sterility, as determined through cDNA microarray testing of human testicular tissues exhibiting spermatogenic defects. TBC1D21 is a protein specifically expressed in the testes that exhibits specific localizations of elongating and elongated spermatids during mammalian spermiogenesis. Furthermore, through co-immunoprecipitation (co-IP) and nano liquid chromatography⁻tandem mass spectrometry (nano LC⁻MS/MS), Rap1 has been recognized as a potential TBC1D21 interactor. This study determined the possible roles of Rap1 and TBC1D21 during mammalian spermiogenesis. First, the binding ability between Rap1 and TBC1D21 was verified using co-IP. Second, the stronger signals of Rap1 expressed in elongating and elongated murine spermatids extracted from testicular sections, namely spermatogonia, spermatocytes, and round spermatids, were compared. Third, Rap1 and TBC1D21 exhibited similar localizations at postacrosomal regions of spermatids and at the midpieces of mature sperms, through isolated male germ cells. Fourth, the results of an activating Rap1 pull-down assay indicated that TBC1D21 overexpression inactivates Rap1 activity in cell models. In conclusion, TBC1D21 may interact with and potentially regulate Rap1 during murine spermatogenesis.

The Possibility of TBC1D21 as a Candidate Gene for Teat Numbers in Pigs.

Based on a quantitative traits locus (QTL) study using a F2 intercross between Landrace and Korean native pigs, a significant QTL affecting teat numbers in SSC7 was identified. The strong positional candidate gene, TBC1D21, was selected due to its biological function for epithelial mesenchymal cell development. Sequence analysis revealed six single nucleotide polymorphisms (SNPs) in the TBC1D21 gene. Among these, two SNP markers, one silent mutation (SNP01) for g.13,050A>G and one missense mutation (SNP04) for c.829A>T (S277C), were genotyped and they showed significant associations with teat number traits (p value = 6.38E-05 for SNP01 and p value = 1.06E-07 for SNP04 with total teat numbers). Further functional validation of these SNPs could give valuable information for understanding the teat number variation in pigs.