<i>Tmem67</i> Is Required for Spermiogenesis and Male Fertility in Mice

ABSTRACT Spermiogenesis dysfunction is a major cause of male infertility; however, the underlying molecular mechanisms involved remain incompletely elucidated. Although transmembrane protein 67 (TMEM67), a ciliary transition zone protein implicated in ciliopathies, is highly enriched in mouse testes, its cell type–specific functional relevance in spermatogenesis is unclear. Here, we generated germ cell‐specific ( Stra8 ‐ Tmem67 f/f ) and Sertoli cell‐specific ( Amh ‐ Tmem67 f/f ) Tmem67 knockout mice to investigate the function of TMEM67 in spermatogenesis and male fertility. Amh ‐ Tmem67 f/f mice maintained normal fertility and exhibited normal spermatogenesis, with no significant differences in testicular histology or sperm count, morphology, or motility compared with wild‐type (WT) controls. However, Stra8 ‐ Tmem67 f/f males were completely infertile, manifesting severe oligoasthenoteratozoospermia (OAT) characterized by a drastic reduction in sperm count, total loss of sperm motility, and global sperm malformation. Further investigations revealed that TMEM67 deletion did not impair spermatogonial proliferation or meiosis, but instead disrupted key spermiogenic events, including manchette dynamics, acrosome biogenesis, and flagellum development. Proteomic analysis indicated that TMEM67 knockout altered the expression of numerous spermiogenesis‐related proteins. Furthermore, our experiments confirmed that TMEM67 deficiency led to profound perturbations in both the expression levels and subcellular localization of key spermiogenic regulators in the testis. Collectively, our findings demonstrate that TMEM67 is indispensable for spermiogenesis and male fertility, revealing its critical role in coordinating manchette function, axonemal integrity, and spermiogenesis‐related protein regulation, providing novel insights into OAT pathogenesis.