Li, F.-Q. | bioRxiv Science

bioRxiv2025

Loss of cilia drives centriole clustering and elimination during mammalian spermatogenesis

Cilia are microtubule-based organelles essential for signaling and motility, and their dysfunction causes ciliopathies often associated with infertility. In male germ cells, two types of cilia are present: zygotene primary cilia and sperm flagella. To define the role of cilia in spermatogenesis, we conditionally ablated the distal appendage protein CEP164, required for basal body docking and ciliogenesis, in male germ cells. CEP164 localized to the mother centriole/basal body throughout spermatogenesis, and its loss led to male infertility accompanied by absence of both zygotene cilia and sperm flagella. Despite defective ciliogenesis, meiotic chromosome pairing and DNA double-strand break repair proceeded normally. However, round spermatids exhibited basal body docking and flagellogenesis defects, and frequently formed supernumerary centriole clusters that were subsequently eliminated via residual bodies. Live-cell imaging revealed that centrioles were highly mobile, and centriole pairs from neighboring cells often associated through intercellular bridges, forming aggregates. These results establish that basal body docking is crucial for retaining centrioles within spermatids, and its disruption leads to centriole clustering and loss. In contrast, zygotene cilia are dispensable for meiotic chromosome pairing and DNA repair during mammalian spermatogenesis.

developmental biology↗

bioRxiv2020

The Distal Appendage Protein CEP164 Is Essential for Efferent Duct Multiciliogenesis and Male Fertility

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. Multiciliated cells (MCCs) are a specialized cell type with hundreds of motile multicilia, lining the brain ventricles, airways, and reproductive tracts to propel fluids/substances across the epithelial surface. In the male reproductive tract, MCCs in efferent ducts (EDs) move in a whip-like motion to stir the luminal contents and prevent sperm agglutination. Previously, we demonstrated that the essential distal appendage protein CEP164 recruits Chibby1 (Cby1), a small coiled-coil-containing protein, to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in MCCs (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility, however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. Consistent with these findings, multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that deletion of CEP164 in the MCCs of EDs causes basal body docking defects and loss of multicilia, leading to sperm agglutination, obstruction of EDs, and male infertility. Our study therefore unravels an essential role of the distal appendage protein CEP164 in male fertility. Author SummaryMulticilia are tinny hair-like microtubule-based structures that beat in a whip-like pattern to generate a fluid flow on the apical cell surface. Multiciliated cells are essential for the proper function of major organs such as brain, airway, and reproductive tracts. In the male reproductive system, multiciliated cells are present in the efferent ducts, which are small tubules that connect the testis to the epididymis. However, the importance of multiciliated cells in male fertility remains poorly understood. Here, we investigated the role of the critical ciliary protein CEP164 in male fertility using a mouse model lacking CEP164 in multiciliated cells. Male mice are infertile with reduced sperm counts. We demonstrate that, in the absence of CEP164, multiciliated cells are present in the efferent ducts but fail to extend multicilia due to basal body docking defects. Consistent with this, the recruitment of key ciliary proteins is perturbed. As a result, these mice show sperm agglutination, obstruction of sperm transport, and degeneration of germ cells in the testis, leading to infertility. Our study therefore reveals essential roles of CEP164 in the formation of multicilia in the efferent ducts and male fertility.

genetics↗