Su-Ren Chen

The assembly and physiological function of cilia and flagella depend on the stable association of A- and B-tubules, which form axonemal doublet microtubules (DMTs). However, the mechanisms underlying the connection of B-tubules to A-tubules to form DMTs in mammalian cilia/flagella are unclear. CFAP77 encodes an outer junction (OJ) protein within DMTs that is conserved across many species and cell types. In this study, Cfap77 -KO mice were generated to reveal that CFAP77 is essential for sperm progressive motility and male fertility. Loss of CFAP77 led to opened B-tubules specifically at the OJ regions of axonemal DMTs as revealed by conventional transmission electron microscopy. Cryo-electron tomography was used to further resolve the in situ structure of sperm axonemal DMTs directly from Cfap77 -KO mice, which exhibited a loss of large filamentous density corresponding to the CFAP77-CCDC105-TEX43 ternary subcomplex at the OJ regions. Additionally, sperm proteomic analysis confirmed that CFAP77 knockout led to the complete loss of this ternary complex. Our work not only explores the physiological role of the OJ protein CFAP77 in axonemal A- and B-tubule connections in mammals but also combines in situ structural biology and knockout mice to reveal the underlying structural/molecular mechanism involved.

The central apparatus (CA) within the sperm axoneme is vital for sperm motility, yet its molecular architecture and functional mechanisms remain incompletely understood. Combining cryo-electron tomography and AlphaFold2, we resolved the in-cell structure of mouse sperm CA at a subnanometer resolution and built a near-complete atomic model. Our analysis identified 39 CA-associated proteins, including eight previously unreported components. By presenting the full-length structures of CFAP47 and HYDIN, we elucidate their molecular roles in tethering the C1 and C2 microtubules within the CA. Specifically, HYDIN forms a semicircular chain that encircles C1 and C2, with its N-terminal half driving the C1–C2 connection and its C-terminal half providing axial support in C2. CFAP47, the core structural component of the bridge, binds C1 through its N-terminal domains, interacts with HYDIN via its central CFAP47-ring, and anchors to C2 through its C-terminal region. The significantly reduced sperm motility and impaired CA structure observed in Cfap47-knockout mice confirmed the important role of CFAP47. Furthermore, genetic analysis of infertile Chinese men with asthenozoospermia identified previously unreported mutations in the CFAP47. The CA structural model elucidates the pathogenic mechanisms of these mutations, establishing a direct link between CFAP47 dysfunction and impaired sperm motility. Therefore, our study provides mechanistic insights into CA-related fertility disorders.

The assembly and physiological function of cilia and flagella depend on the stable association of A and B tubules, which form axonemal microtubule doublets (DMTs). CFAP77 encodes a core outer junction (OJ) protein within DMTs that is conserved across species and cell types. However, whether and how CFAP77 mediates the connection of B tubules to A tubules of DMTs in mammalian cilia/flagella are unknown. In this study, Cfap77 -KO mice were generated to reveal that CFAP77 is essential for sperm progressive motility and male fertility. Loss of CFAP77 led to opened B tubules specifically at the OJ regions of axonemal DMTs as revealed by conventional transmission electron microscopy. Cryo-electron tomography further resolved the in situ structure of sperm axonemal DMTs from Cfap77 -KO mice, which exhibited a loss of large filamentous density corresponding to the CFAP77-CCDC105-TEX43 ternary subcomplex at the OJ regions. Additionally, sperm proteomic analysis confirmed that CFAP77 knockout led to the complete loss of this ternary complex. Our work not only explores the physiological role of OJ protein CFAP77 on axonemal A-B tubule connection in mammals but also combines in situ structural biology and knockout mice to reveal the underlying structural/molecular mechanism.

Oligo‐astheno‐teratozoospermia (OAT) is a common cause of male infertility, but the genetic basis of most OAT cases is still unknown. Here, one homozygous loss‐of‐function (LOF) variant in TDRD6, c.G1825T/p.Gly609X, was identified in an infertile patient with severe OAT by whole‐exome sequencing (WES) and Sanger confirmation. Furthermore, Tdrd6‐mutant mice (p.Gly615X; equivalent to p.Gly609X in human TDRD6) were generated. Remarkably, the Tdrd6‐mutated mice mimicked the severe OAT symptoms of the patient. In addition, the architecture of chromatoid bodies (CBs) were disrupted in round spermatids from Tdrd6‐mutant mice, leading to blocked spermatogenesis in the round spermatids. The assembly of PIWIL1, TDRD1, TDRD7 and DDX25 in CBs was disturbed in the Tdrd6‐mutant mice. Applying immunoprecipitation‐mass spectrometry (IP‐MS), we identified some TDRD6‐interacting partners, including CB proteins TDRD7, MAEL and PCBP1. Moreover, we described the assisted reproductive technology (ART) outcomes of the infertile patient and his partner. Altogether, our findings provide necessary evidences to support the idea that the homozygous LOF variant in TDRD6 induces male infertility with severe OAT, suggesting that TDRD6 could be a useful genetic diagnostic target for male infertility.

In motile cilia or flagella, the axoneme typically exhibits a “9+2” configuration, with the central apparatus (CA) consisting of extensively modified microtubules C1 and C2 to regulate ciliary motility. How C1 and C2 are interconnected asymmetrically remains unknown due to the lack of complete structural model of CA. Here, we utilized the in situ cryo-electron tomography approach to solve the in-cell structure of intact mouse sperm CA at sub-nanometer resolution and built its near-complete atomic model with the aid of AlphaFold2. We identified 39 different CA-associated proteins with 8 never reported from isolated specimen. We assigned the long chain-like ASH-containing proteins CFAP47 and HYDIN responsible for connecting C1 and C2. Sperm from Cfap47 -knockout mice displayed a hollowing bridge in the CA structure, correlating with its reduced progressive motility. Our findings elucidate the molecular mechanisms of CA components in ciliary motility, and the implications of their mutations in human ciliopathies.

IQ motif-containing proteins can be recognized by calmodulin (CaM) and are essential for many biological processes. However, the role of IQ motif-containing proteins in spermatogenesis is largely unknown. In this study, we identified a loss-of-function mutation in the novel gene IQ motif-containing H (IQCH) in a Chinese family with male infertility characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. To verify the function of IQCH, Iqch knockout (KO) mice were generated via CRISPR-Cas9 technology. As expected, the Iqch KO male mice exhibited impaired fertility, which was related to deficient acrosome activity and abnormal structures of the axoneme and mitochondria, mirroring the patient phenotypes. Mechanistically, IQCH can bind to CaM and subsequently regulate the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis. Overall, this study revealed the function of IQCH, expanded the role of IQ motif-containing proteins in reproductive processes, and provided important guidance for genetic counseling and genetic diagnosis of male infertility.

IQ motif-containing proteins can be recognized by calmodulin (CaM) and are essential for many biological processes. However, the role of IQ motif-containing proteins in spermatogenesis is largely unknown. In this study, we identified a loss-of-function mutation in the novel gene IQ motif-containing H (IQCH) in a Chinese family with male infertility characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. To verify the function of IQCH, Iqch knockout (KO) mice were generated via CRISPR-Cas9 technology. As expected, the Iqch KO male mice exhibited impaired fertility, which was related to deficient acrosome activity and abnormal structures of the axoneme and mitochondria, mirroring the patient phenotypes. Mechanistically, IQCH can bind to CaM and subsequently regulate the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis. Overall, this study revealed the function of IQCH, expanded the role of IQ motif-containing proteins in reproductive processes, and provided important guidance for genetic counseling and genetic diagnosis of male infertility.

Dear Editor,

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm nucleus. The physiological roles of PT in sperm biology and the clinical relevance of variants of PT proteins to male infertility are still largely unknown. We reveal that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts with itself, several other PT proteins, the inner acrosomal membrane (IAM) protein SPACA1, and the nuclear envelope (NE) protein FAM209 to form an ‘IAM–cylicins–NE’ sandwich structure, anchoring the acrosome to the nucleus. WES (whole exome sequencing) of more than 500 Chinese infertile men with sperm head deformities was performed and a CYLC1 variant was identified in 19 patients. Cylc1-mutant mice carrying this variant also exhibited sperm acrosome/head deformities and reduced fertility, indicating that this CYLC1 variant most likely affects human male reproduction. Furthermore, the outcomes of assisted reproduction were reported for patients harbouring the CYLC1 variant. Our findings demonstrate a critical role of cylicin-1 in the sperm acrosome–nucleus connection and suggest CYLC1 variants as potential risk factors for human male fertility.

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm nucleus. The physiological roles of PT in sperm biology and the clinical relevance of variants of PT proteins to male infertility are still largely unknown. We reveal that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts with itself, several other PT proteins, the inner acrosomal membrane (IAM) protein SPACA1, and the nuclear envelope (NE) protein FAM209 to form an ‘IAM-cylicins-NE’ sandwich structure, anchoring the acrosome to the nucleus. WES of more than 500 Chinese infertile men with sperm head deformities was performed and a CYLC1 variant was identified in 19 patients. Cylc1-mutant mice carrying this variant also exhibited sperm acrosome/head deformities and reduced fertility, indicating that this CYLC1 variant most likely affects human male reproduction. Furthermore, the outcomes of assisted reproduction were reported for patients harbouring the CYLC1 variant. Our findings demonstrate a critical role of cylicin-1 in the sperm acrosome-nucleus connection and suggest CYLC1 variants as potential risk factors for human male fertility.