Doublet microtubule-associated tektins and enzymes differentially regulate sperm flagellar integrity and motility

Abstract

Abstract Doublet microtubule (DMT)-associated proteins assemble and drive sperm flagella, which are essential for successful fertilization. However, the exact roles of different DMT-associated proteins in sperm function and the underlying molecular mechanisms remain elusive. Here, we generate four gene-knockout mice based on high-resolution structures targeting distinct DMT components: two intermediate filament-like tektins (TEKT1, TEKT5) and two enzymes (TSSK6, DUSP21). The depletion of TEKT1, shared by sperm flagella and motile cilia, causes male infertility characterized by impaired sperm motility and loss of the tektin bundle, whereas sperm-specific Tekt5 knockout (KO) mice remain fertile with largely normal flagellar function, indicating functional divergence within the tektin family. Tssk6 KO spermatozoa exhibit severely disturbed morphology and motility, resulting in homozygote infertility and heterozygote subfertility. Phosphoproteomics reveals dysregulated phosphorylation of axonemal proteins, highlighting the critical role of kinase-mediated signaling in regulating sperm motility. Conversely, Dusp21 KO mice display no fertility or sperm motility defects, suggesting compensatory phosphatase activity. Phenotypic comparisons between Tekt1 and Tssk6 KO mice suggest their involvement in distinct subtypes of asthenozoospermia. Overall, this study elucidates how filamentous and enzymatic DMT proteins govern sperm function through divergent mechanisms, which have implications for molecular diagnosis of male infertility.