Animals

All mice in this study were housed in a standard animal facility (20–22 °C; 50–70% humidity; 12 h light/dark cycle) with free food and water access. The Institutional Animal Care and Use Committees of Nanjing Medical University approved this study (Approval No. IACUC-2004020), and all experiments were performed as per the Guide for the Care and Use of Laboratory Animals and institutional guidelines.

Human participants

All patients affected by MMAF recruited from the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Anhui Medical University. The included patients presented isolated infertility without other cilia pathological symptoms, such as primary ciliary dyskinesia. The probands in our study presented neither abnormality in chromosome number nor microdeletions of the azoospermia factor region on the Y chromosome. This study was approved by the Ethics Committees of the Suzhou Hospital Affiliated to Nanjing Medical University (Approval No. 2016002) and the First Affiliated Hospital of Anhui Medical University (Approval No. P2020-12-36). Signed informed consents were obtained from all subjects participating in the study.

Cell lines

HEK293T cells were cultured in high-glucose DMEM (Gibco, MA, USA) containing 10% FBS (Gibco) and 1 × penicillin–streptomycin (Invitrogen, MA, USA) at 37℃ in a humid atmosphere with 5% CO2 condition. The transient transfections of HEK293T cells were performed by using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s protocol.

Bacterial strains

E. coli DH5α competent cells (Tsingke, Beijing, China) were used for molecular cloning, and E. coli strain Rosetta (DE3) bacterial strain (Tiangen, Beijing, China) was used for protein expressing.

Semen analysis and sperm morphological analysis

After 2 to 7 days of sexual abstinence, semen samples were obtained by masturbating, and analyzed according to the 5th World Health Organization (WHO) guidelines, in source laboratories. The analysis of semen volume, sperm concentration and motility were repeated twice. Hematoxylin and eosin (H&E) staining was used to evaluate sperm morphology, which divided into six categories: absent, short, bent, coiled flagella and flagella of irregular caliber. Each subject must be examined at least 200 sperm to assess the percentage of abnormal sperm morphology.

Whole-exome sequencing and Sanger sequencing

Genomic DNA was extracted from blood samples using the DNeasy Blood & Tissue Kit (Qiagen, Duesseldorf, Germany). Whole-exome sequencing of samples was prepared using IDT xGen® Exome Research Panel V1.0 (Integrated DNA Technologies, IA, USA). The quantity of the sequencing library was assessed by a Qubit 2.0 fluorometer (Thermo Fisher Scientific, MA, USA). The quality and size of libraries were measured by Agilent 2100 Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, CA, USA). For next-generation sequencing, the qualified libraries were subjected to 2 × 150-bp paired-ends sequencing on the Illumina NovaSeq platform (Illumina, CA, USA). FASTQ files were aligned to the human reference genome (hg19/GRCh37) by BWA (v0.7.13). Variants (single nucleotide variants and indels) were genotyped from recalibrated BAM files by GATK 4.0 and annotated using ANNOVAR against multiple databases, including HGVS variant description, population frequency, disease or phenotype and variant functional prediction. Variants were classified as pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, or benign following the American College of Medical Genetics (ACMG) guidelines. Copy number variations were detected by DNAcopy R-package 5, filtered and classified by ACMG guidelines and manually checked by using the Integrative Genomics Viewer. CFAP65 variants identified via this approach were confirmed by Sanger sequencing.

Antibodies

The specific antibodies for CFAP65 and CFAP70 were produced as follows. In brief, the murine CFAP65 (amino acids 1458-1619) and CFAP70 (amino acids 386-505) was subcloned into a pET-28a expression vector coding for six N-terminally located histamine residues and expressed in BL21 (DE3) pLysS competent cells (Invitrogen) according to the manufacturer’s instructions. The expressed recombinant protein was then purified using Ni–NTA resin (TransGen Biotech, Beijing, China) under denaturing conditions. Polyclonal antibodies were produced in a ICR mouse by injecting recombinant protein and boosting with one half of the initial amount at 2 and 3 weeks after the primary immunization.

Mouse anti-Ac-Tub (T6793), anti-α-Tubulin (T9026), anti-Flag (F1804) and rabbit anti-DNAH5 (HPA037470), anti-HA (H6908) were from Sigma-Aldrich (MO, USA). Rabbit anti SLC2A3 (20403-1-AP), anti-Beta-Actin (81115-1-RR), anti-Beta-Tubulin (10094-1-AP), anti-MYCBP (12022–1-AP), anti-SPAG16 (16883-1-AP), anti-HYDIN (24741-1-AP), anti-NME5 (12923-1-AP), anti-RSPH9 (23253-1-AP), anti-TNP1 (17178-1-AP), Anti-CEP164 Antibody (22227-1-AP), Anti-Centrin Antibody (12794-1-AP) were obtained from Proteintech (Wuhan, China). Rabbit anti-SPATA4 (A7608) was from Abclonal (Wuhan, China). Rabbit anti-Ac-Tub (5335) was purchased from Cell Signaling Technology (MA, USA). Mouse anti-MYCBPAP (sc-390415) was from Santa Cruz Biotechnology (TX, USA). Mouse anti-AKAP4 (611564) was from BD Biosciences (CA, USA). Mouse anti-DRC1 and rabbit anti-IQUB, anti-PPRM2 were self-prepared.

Generation of Cfap65 knockout mice

Cfap65 knockout mice were generated using CRISPR/Cas9 genome editing. We selected one sgRNA (5′-TTTTTGGGAAGATGCCAGGG-3′) to generate a frame-shift mutation (c.5056_5062delinsAA, p.Gly1686Lysfs*4) in exon 31 of Cfap65. The Cas9 plasmid (Addgene, MA, USA) was linearized with AgeI, and then purified using a MinElute PCR Purification Kit (QIAGEN). After in vitro transcription using a mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen), Cas9 mRNA was purified with a RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Mouse zygotes were co-injected with Cas9 mRNA (50 ng/μl) and sgRNA (20 ng/μl) and finally transferred into pseudopregnant females. In seven days old, newborn mice were tagged by cutting their toes, and then the toe DNA was extracted with the Mouse Direct PCR Kit (Bimake, Shanghai, China). PCR amplification was conducted with specific primers (Fw#1 and Rv#1, see Table S1) using Taq Plus DNA Polymerase (Vazyme, Nanjing, China) under following conditions: 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 25 s; and a final step of 72 °C for 5 min. PCR products were subjected to Sanger sequencing.

Histological analysis

For histological analysis, the samples were fixed in modified Davidson’s fluid (MDF), dehydrated through a graded ethanol series, embedded in paraffin, sectioned (5 μm thickness) and stained with hematoxylin and PAS regents.

Immunoblot analysis

The proteins of mice testes tissues were extracted using lysis buffer (50 mM Tris–HCl pH 8.2, 75 mM NaCl, 8 M urea) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The denatured proteins were separated on 10% SDS–polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, CA, USA) that were blocked for 2 h with 5% non-fat milk in TBS at room temperature, followed by overnight incubation with appropriate primary antibodies at 4℃. Blots were then washed thrice with TBST, probed for 2 h at room temperature with appropriate secondary antibodies and protein bands were then detected with the high-sig ECL western blotting substrate (Tanon, Shanghai, China).

Immunofluorescence (IF)

For testis suspension and epididymal sperm, the samples were fixed with 4% PFA for 40 min, followed by smeared onto polylysine-coated slides, and air-dried. For whole mount staining procedure for the lateral ventricle wall, we refers to previously published literature [31]. For paraffin section, the samples underwent deparaffinization, rehydration, and antigen repair (immerged in in sodium citrate buffer, heated for 10 min). Then the slides were washed thrice with PBS and blocked with 3% bovine serum albumin for 2 h at room temperature (RT). The slides were then incubated with primary antibodies overnight at 4 ℃. Following secondary antibody staining for 2 h at RT, Hoechst 33,342 counterstaining was performed for 5 min. Slides were then rinsed with PBS and mounted using glycerol prior to imaging with a LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany) and TCS SP8X confocal microscope (Leica, Weztlar, Germany).

TUNEL assay

Mouse testes were collected and fixed in MDF for up to 24 h and stored in 70% ethanol. Then the samples were dehydrated through a graded ethanol series and embedded in paraffin. Tissue sections were prepared and mounted on glass slides. Apoptotic cells in testis were detected using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Vazyme) according to the manufacturer’s instructions.

Plasmid construction

Full-length cDNA encoding Mouse CFAP65, CFAP70, CFAP20, MYCBPAP, and MYCBP were amplified by PCR with oligos carrying the recombinant sites and cloned into PCAG vector in which a 3 × Flag or 3 × HA or 3 × Myc tag was introduced prior to the multicloning site. The primers used to amplify each gene are listed in Table S2. Finally, these plasmids were confirmed by sequencing.

Immunoprecipitation

Lipofectamine 2000 (Invitrogen) was used to transfect HEK293T cells with plasmids. At two days post-transfection, cells were lysed for 40 min using RIPA buffer containing 1 × complete EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland) at 4 °C, after which samples were spun down for 20 min at 12,000 × g. Supernatants from these lysates were then precleared for 1 h with 10 μL Protein A/G magnetic beads (MCE, NJ, USA) at 4 °C, after which they were combined with anti-HA/Flag-tag antibodies overnight at 4 °C. They were then mixed with 50 μL of Protein A/G magnetic beads for 3 h at 4 °C. Beads were washed thrice with RIPA buffer, boiled for 10 min in 1 × SDS loading buffer, and proteins were then subjected to SDS-PAGE analysis.

Fractionation of spermatozoa

Spermatozoa were suspended in 1% TritonX-100 lysis buffer (50 mM NaCl, 20 mM Tris–HCl, pH 7.5, protease inhibitor mixture) and incubated for 2 h at 4°C. The sample was centrifuged at 15,000×g for 10 min to separate the Triton-soluble fraction (supernatant) and the Triton-resistant fraction (pellet). The pellet was resuspended in 1% SDS lysis buffer (75 mM NaCl, 24 mM EDTA, pH 6.0) and incubated for 1 h at RT. The sample was centrifuged at 15,000×g for 10 min to separate SDS-soluble fraction (supernatant) and SDS-resistant fraction (pellet). The pellet was dissolved in sample buffer and boiled for 10 min.

Transmission electron microscopy (TEM)

The lateral ventricles, sperm and tracheas from Cfap65+/+ and Cfap65−/− mice were fixed with 2.5% glutaraldehyde overnight, post-fixed with 2% OsO4 and embedded in Araldite. Ultrathin Sects. (80 nm) were then stained with uranyl acetate and lead citrate. The samples were imaged by JEM 1010 (JEOL, Tokyo, Japan) and FEI Tecnai G2 (FEI, OR, USA) transmission electron microscopes.

Scanning electron microscopy (SEM)

The lateral ventricles, sperm and tracheas from Cfap65+/+ and Cfap65−/− mice were fixed with 2.5% phosphate-buffered glutaraldehyde for 2 h at 4 °C. Samples were next washed in PBS, dehydrated via an ascending gradient of cold 30%, 50%, 70%, 80%, 90% and 100% ethanol, and dried at critical point using a Leica EM CPD300 Critical Point Dryer (Leica). Samples were then attached to specimen holders and coated with gold particles using an ion sputter coater (EM ACE200, Leica). A Helios G4CX scanning electron microscope (Thermo Fisher Scientific) was used to image photos.

Analysis of tracheal cilia length

Adult mouse tracheas from Cfap65+/+ and Cfap65−/− mice (N = 3 for each group) removed by dissection, and then multiciliated cells were isolated, stained by Ac-tubulin and Hoechst. A TCS SP8X confocal microscope was used to image cells and the cilia length was determined using LAS X (Leica) software by measuring the length of the ciliary tuft of each cell and averaging the measurements (at least 20 cells analyzed in each group).

Assessment of ciliary motility

For ependymal cilia, the brains from mice were dissected, the left and right hemispheres were separated, and then sliced as thin as possible along the sagittal plane. For tracheal cilia, tracheas from mice were dissected, added to high-glucose DMEM containing 10% FBS and 10 mM DTT, opened on the dorsal side, and cut into 5 mm tissue fragments under a stereoscopic microscope. These tissues were then transferred to a confocal dish (BIOFIL, Guangzhou, China) and a Scotch tape spacer was used to facilitate their imaging under a 40 × objective (CFI S Plan Flour ELWD NAMC) with an inverted microscope (Eclipse Ti2-U, Nikon Tokyo, Japan). Movies were recorded for 10 s at frame rates of 30 fps.

To analyze cilia-generated flow in the mouse ependyma and trachea, after adding FluoSpheres Carboxylate-Modified Microspheres beads (1:200, Thermo Fisher Scientific) into the ependyma and trachea explants, the bead flow was recorded at 23 fps for 10 s at 24–27 °C using fluorescent microscopy. ImageJ [32] was used to analyze the trajectories of fluorescent microspheres near the ciliary tuft.

IP-mass spectrometry (IP-MS)

CFAP65 was immunoprecipitated from mouse testis with CFAP65 antibody described above, and IgG antibody was used as negative control. Eluates were precipitated with five volumes of −20 °C pre-chilled acetone followed by trypsin digestion. Trypsin peptide mixtures were injected automatically and loaded into the analytical column (Acclaim PepMap C18, 75 μm × 25 cm; Thermo Fisher Scientific). Before subsequent analysis, the eluted peptides were separated by linear gradient. LC–MS/MS analysis was performed on EASY-nanoLC 1000 system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) by a nano spray ion source. The raw data is accessible from the ProteomeXchange Consortium via the dataset identifier PXD055389.

Ultrastructure expansion microscopy (U-ExM)

Spermatozoa from caudal epididymis of Cfap65+/+ mice were incubated with 1ml DPBS for 10 min at 37 ℃, then attached to 12 mm coverslips coated with poly-L-lysine by centrifuging for 5 min at 300g. The U-ExM experiment was performed as detailed previously. Briefly, samples were crosslinked in 1ml Formaldehyde/acrylamide (FA/AA) mix (1.4% FA, 2% AA in PBS) for 5h at 37 ℃. For gel polymerization, 3 μl monomer solution (23% sodium acrylate, 10% acrylamide, 0.1% N,N′- methylenbisacrylamide, 0.5% TEMED, 0.5% APS in PBS) was dropped onto the parafilm and coverslips were placed on the drop, with samples facing the solution. The gelation process is done on ice for 5 min, followed by incubation for 1 h at 37 °C. Gels were detached from coverslips after incubation in fresh denaturation buffer (200 mM SDS, 200 mM NaCl, 50 mM Tris in ddH2O, pH 9.0) for 15 min at RT, next transferred into 1.5 ml microcentrifuge tube filled with denaturation buffer and incubated for 90 min at 95 °C. Denatured samples were then incubated in ddH2O overnight for first round of expansion. The next day, gels were first washed twice with PBS to remove excess water, and then respectively incubated with primary antibody and secondary antibody in PBS- BSA 2% for 3 h at 37 °C. After three washes with PBS-Tween20 0.1%, gels were placed in ddH2O for second round of expansion. For gels imaging, gels were cut into appropriate size and transferred to a confocal dish coated with poly-L-lysine, imaged by a TCS SP8X confocal microscope (Leica) using a 63 × oil objective.

Intracytoplasmic sperminjection (ICSI) of Cfap65 −/− mice

Two-month-old wild-type female mice were superovulated by injecting 5–7.5 IU of pregnant mare serum gonadotropin (PMSG), followed by 5–7.5 IU of human chorionic gonadotropin (hCG) 48 h later. For ICSI, oocytes were obtained from superovulated females, and sperm heads were injected into oocytes through using a Piezo manipulator. Then, the injected oocytes were cultured in KSOM medium at 37°C under 5% CO2. Two-cell embryos and blastocysts were counted 20 h and 96 h later, respectively.

Prediction of mouse C2a projection structure

To obtain C2a projection structure model of Mus musculus, each model of mouse c2a-associated protein was predicted by AlphaFold2 [33] or SWISS MODEL [34] and docked into the electron density maps of Mouse CA (EMD-27745) in Chimera [35], manually adjusted according to Chlamydomonas cryo-EM structure of CA (EMD-25631).

Quantification and statistical analysis

The difference between the treatment group and the control group was analyzed by unpaired two-tailed t-tests using Microsoft Excel or GraphPad Prism 9.0. All data are presented as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.