ANKEF1 is a key axonemal component essential for murine sperm motility and male fertility
Academy for Advanced Interdisciplinary Studies, Peking University, China
National Institute of Biological Sciences (NIBS), China
State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, China
The School of Public Health, Xinxiang Medical University, China
Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, China
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ANKEF1 is a key axonemal component essential for murine sperm motility and male fertility
study reports a critical role of the axonemal protein ANKRD5 in sperm motility and male fertility.
data were presented to support the main conclusion. This work will be of interest to biomedical researchers who study ciliogenesis, sperm biology, and male fertility.
https://doi.org/10.7554/eLife.105321.4.sa0
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Sperm motility is essential for male fertility and depends on the structural integrity of the sperm axoneme, which features a canonical ‘9 + 2’ microtubule arrangement. This structure comprises nine outer doublet microtubules (DMTs) that are associated with various macromolecular complexes. Among them, the nexin–dynein regulatory complex (N-DRC) forms crossbridges between adjacent DMTs, contributing to their stabilization and enabling flagellar bending. In this study, we investigated Ankyrin repeat and EF-hand domain containing 1 (ANKEF1, also known as ANKRD5), a protein highly expressed in the sperm axoneme. We found that ANKEF1 interacts with DRC5/TCTE1 and DRC4/GAS8, two key components of the N-DRC, and these interactions occur independently of calcium regulation. Male
mice exhibited impaired sperm motility and infertility. Cryo-electron tomography revealed a typical ‘9 + 2’ axoneme structure with intact DMTs in
null sperm; however, the DMTs showed pronounced morphological variability and increased structural heterogeneity. Notably, ANKEF1 deficiency did not alter ATP levels, reactive oxygen species levels, or mitochondrial membrane potential. These findings suggest that ANKEF1 may attenuate the N-DRC’s mechanical buffering—akin to a ‘car bumper’—between adjacent DMTs, thereby compromising axonemal stability under high mechanical stress during vigorous flagellar beating.
The interaction between sperm and egg, culminating in embryo formation, is fundamental to sexual reproduction and the continuation of species (
). Male infertility affects approximately 8–12% of the global male population, with defects in sperm motility accounting for over 80% of these cases (
). Fertilization requires successful spermatogenesis and normal sperm motility (
). In mammals, sperm acquire motility and fertilizing capacity during transit through the epididymis (
). This maturation process is essential for generating functionally competent sperm. Asthenozoospermia, characterized by reduced sperm motility, is a leading cause of clinical infertility; however, its underlying mechanisms remain poorly understood (
). Men with poorly motile or immobile sperm are typically infertile unless assisted reproductive techniques (ART), such as gamete intrafallopian transfer, in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI), are employed (
). Nevertheless, these ART methods may transmit underlying genetic defects to offspring. Deeper insights into the molecular mechanisms of sperm motility could yield targeted therapies for asthenozoospermia. Rather than bypassing the defect with ICSI, such strategies could directly correct it via modulation of key signaling pathways or gene therapy, potentially offering a cure (
Sperm motility is powered by the rhythmic beating of the flagellar, which is subdivided into the midpiece, principal piece, and endpiece (
). These segments share a conserved core structure—the central axoneme—comprising ~250 proteins that form the main components of the flagellum (
). The axoneme exhibits a characteristic ‘9 + 2’ ultrastructure, featuring nine outer doublet microtubules (DMTs) encircling a central pair of singlet microtubules. Adjacent DMTs are interconnected by the nexin–dynein regulatory complex (N-DRC) (
). The structure and molecular composition of the N-DRC are evolutionarily conserved and central to the regulation of sperm motility (
The N-DRC is a ~1.5 MDa macromolecular complex composed of two primary subdomains: the linker and the base plate (
). It also interacts with the outer dynein arms (ODA) via outer–inner dynein linkers, thereby contributing to the regulation of both ODAs and inner dynein arms (IDAs) (
). Although the N-DRC was initially believed to consist of 11 protein subunits (
), a twelfth component, CCDC153 (DRC12), was later identified through its interaction with DRC1 (
). In situ cryoelectron tomography (cryo-ET) studies in
have elucidated the three-dimensional architecture of the N-DRC, revealing that DRC1, DRC2/CCDC65, and DRC4/GAS8 form the core scaffold (
). Proteins DRC3/5/6/7/8/11 associate with this core and mediate interactions with other axonemal complexes (
). Biochemical analyses corroborate these findings and validate the proposed structural model (
). Functionally positioned between DMTs, the N-DRC converts microtubule sliding into coordinated axonemal bending by restricting the relative displacement of outer DMTs (
). Genetic mutations in N-DRC subunits demonstrate that its structural integrity is crucial for sperm motility. Specifically, mutations in DRC1, DRC2/CCDC65, and DRC4/GAS8 are associated with ciliary motility disorders, leading to primary ciliary dyskinesia (PCD) (
). Biallelic truncating mutations in DRC1 induce MMAF in humans, including disassembly of outer DMTs, disorganization of the mitochondrial sheath, and incomplete axonemal assembly (
). Similarly, loss of CCDC65 destabilizes the N-DRC, resulting in disorganized axonemes, global microtubule dissociation, and complete asthenozoospermia (
). Recent mammalian knockout studies further confirmed that loss of DRC2 or DRC4 results in severe sperm flagellar assembly defects, multiple morphological abnormalities of the sperm flagella (MMAF), and complete male infertility, highlighting their indispensable roles in spermatogenesis and reproduction (
). Homozygous frameshift mutations in DRC3 impair N-DRC assembly and intraflagellar transport, causing severe motility defects despite normal sperm morphology (
). In contrast, TCTE1 knockout mice exhibit normal sperm axoneme structure but impaired glycolysis, leading to reduced ATP levels, diminished sperm motility, and male infertility (
knockout mice display disrupted ‘9 + 2’ axonemal architecture, complete sperm immotility, and male infertility (
). Although the N-DRC is critical for sperm motility, whether additional regulatory components coordinate its function remains unclear. Here, we demonstrate that ANKEF1 is a novel N-DRC component essential for maintaining sperm motility. Absence of ANKEF1 results in diminished sperm motility and consequent male infertility.
Based on NCBI and single-cell RNA sequencing data,
exhibits testis-specific expression, with particularly high enrichment in the male reproductive system (
). In mice, ANKEF1 is a protein of 775 amino acids with a molecular weight of 86.9 kDa. Cross-species sequence comparison revealed that ANKEF1 is evolutionarily conserved (
), and alignment via Clustal Omega demonstrated 86% similarity between mous