A total of 323 Chinese infertile men with ASZ were recruited from August 2019 to June 2024 at the Reproductive Medicine Center of the Women’s and Children’s Hospital of Chongqing Medical University. All somatic chromosome karyotypes were normal (46,XY), and no Y-chromosome microdeletions were found. In this study, the ethical approval (No.: (2023) Ethics Review (Research) 030) was obtained from the Ethics Committee of Chongqing Health Center for Women and Children. An informed consent form was signed before the collection of peripheral blood and semen.
WES, Sanger sequencing and mutation analysisAs previously reported [29], genomic DNA was isolated from peripheral blood samples of the patients using the QIAamp DNA Blood Mini Kit (69504, QIAGEN, Dusseldorf, Germany) and then WES was performed on the Illumina HiSeq X system according to the manufacturer’s instructions. The clean reads were mapped to the human reference sequence (GRCh37) and performed functional annotation using ANNOVAR. Next, PolyPhen-2, SIFT, MutationTaster, and CADD were used for functional prediction. The mutations identified by WES were validated by Sanger sequencing with specific PCR primers (Table S1).
Semen analysis and papanicolaou stainingSemen samples were collected via masturbation from patients after 2–7 days of abstinence and incubated at 37℃ for 30 min for liquefaction. Spermatozoa of mice were released from cauda epididymides and incubated in human tubal fluid media (MR-070, Sigma-Aldrich, Mo, USA) at 37℃ for 30 min. Next, a computer-assisted sperm analysis system (CASA, GSA-180, Jiangsu Rich Life Science Instrument Co.Ltd., Nanjing, China) was applied according to the 6th edition of the WHO guidelines [30]. For sperm morphology, spermatozoa were smeared on slides and stained with Papanicolaou staining according to the manufacturer’s protocol (Cariad Medical Technology Co.Ltd., Zhuhai, China) [29]. For flagellar waveform tracing, single frames of sperm high-speed microscopy video (120 fps) were exported and the flagellar waveforms with each ten frames were traced using Adobe Photoshop software.
Animal model and reverse transcription PCR (RT-PCR)The animal experiments were approved by the Experimental Animal Management and Ethics Committee of Chongqing Health Center for Women and Children (No.: 2023028). All the mice were housed in a pathogen-free environment with a room temperature range of 20℃ to 24℃ under a 12 h light/dark cycle, 50-70% humidity, and free access to food and water. For gene editing, 4-week-old C57BL/6 female mice were super-ovulated and mated to collect zygotes. Then the zygotes were microinjected with CRISPR/Cas9 mRNA and single-guide RNAs (sgRNAs) targeting the exons 2–6 of Iqub (ENSMUST00000052277.5), which were predicted to affect all the transcripts of the IQUB gene as well as the functional domains of the IQUB protein, and cultured in K + simplex optimized medium (KSOM, MR-106, Sigma-Aldrich, MO, USA) to reach 2-cell stage. The embryos were transferred into the oviducts of pseudopregnant female mice to obtain positive F0 mice, which were bred to obtain stable F1 generation. For RT-PCR, total RNA of mouse tissues was extracted with MiniBEST universal RNA extraction Kit (9767, Takara, Osaka, Japan) and converted to complementary DNA (cDNA) using PrimeScript™ RT reagent Kit (RR047A, Takara, Osaka, Japan) according to the manufacturer’s instructions. The sequence of primers used in genotype identification and RT-PCR and sgRNAs were listed in Table S1.
Electron microscopyScanning electron microscopy (SEM) and TEM were performed as previously described [29]. Briefly, semen samples from humans and mice were collected, washed, and fixed in 2.5% phosphate-buffered glutaraldehyde at 4 ℃ overnight. For SEM, the samples were dehydrated using progressive ethanol concentrations (35%, 50%, 75%, 90%, 95%, and 100%), dried using a CO2 critical-point dryer (Eiko HCP-2, Hitachi, Tokyo, Japan), sputter coated by an ionic sprayer meter (ACE200, Leica), and finally observed under the SEM (Nova NanoSEM 450, FEI, Hillsboro, OR, USA) at an accelerating voltage of 5 kV. For TEM, the samples were post fixed in 1% buffered OsO4 and embedded in Epon 812 (90529-77-4, SPI, West Chester, PA, USA) after dehydration. The ultrathin (70 nm) sections were double stained with lead citrate and uranyl acetate, and observed by a TEM (TECNAI-10, Philips, Eindhoven, The Netherlands) at an accelerating voltage of 80 kV. On the longitudinal sections of TEM, we measured and plotted the gray values of two or three 96-nm-repeats axonemal units through ImageJ software.
Immunofluorescence and Hematoxylin and eosin (H&E) stainingSpermatozoa were washed, fixed in 4% paraformaldehyde, then permeabilized with 0.5% Triton X-100 (X100, Sigma-Aldrich, MO, USA), blocked with 3% bovine serum albumin (BSA, B2064, Sigma-Aldrich, MO, USA), and finally incubated with primary antibodies overnight at 4℃, respectively. On next day, the samples were washed and incubated with the secondary antibodies, and the nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, P0131, Beyotime, Shanghai, China). Finally, images were captured under a confocal laser scanning microscope (TCS SP8, Leica, Wetzlar, Germany). The used antibodies were listed in Table S2. Testicular and epididymal tissues from 9-week-old male mice were fixed with animal testicular tissue fixation solution (G1121, Servicebio, Hubei, China) for 24 h. After dehydration by ethanol, they were embedded in paraffin and sectioned at 5 μm. The sections were stained with H&E, and images were captured using a digital biopsy scanner (Pannoramic 250, 3DHISTECH, Hungary).
Tandem mass tag mass spectrometry (TMT-MS)Spermatozoa samples were analyzed with a standard protocol of TMT-MS for proteomic analysis. Briefly, the sample was sonicated on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer. The supernatant was collected after centrifugation, and the concentration was determined with BCA kit (P0010, Beyotime, Shanghai, China). Protein samples were trypsinized and purified, and peptides were reconstituted in 0.5 M TEAB and processed using the TMT kit according to the manufacturer’s protocol. The sample was fractionated into fractions by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length) and subsequently analyzed in Orbitrap Exploris 480 with a nano-electrospray ion source. The raw data were processed using Domain Annotation (InterProScan), GO Annotation (http://www.ebi.ac.uk/GOA/), and KEGG Pathway Annotation (KEGG online service tools KAAS mapper).
Co-immunoprecipitation (Co-IP) and Western blottingHEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C under 5% CO2. A full-length of Iqub, Calm, Rsph14, Ak8 and Morn3 cDNAs cloned in pCDNA3.1-FLAG or pCDNA3.1-HA vectors were constructed by Youbo Biotechnology (Zhengzhou, China). Lipofectamine™ 8000 Transfection Reagent (C0533, Beyotime, Shanghai, China) was used for transfecting and the transfected HEK293T cells or mouse sperm were lysed with RIPA buffer (P0013B, Beyotime, Shanghai, China) to obtain protein extract. For Co-IP, cell lysates were treated with IP buffer (P2181, Beyotime, Shanghai, China), and then incubated with BeyoMagTM anti-Flag magnetic beads (P2181, Beyotime, Shanghai, China) overnight at 4 °C. The beads were washed and then the immunoprecipitated proteins were eluted from the beads by FLAG peptide. Then the proteins were electrophoresed in SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (1620177, BIO-RAD, CA, USA), followed by blocking with skimmed milk and subsequent incubation with primary and secondary antibodies. Finally, the enhanced chemiluminescence reagent (G2020, Wuhan, Servicebio Wuhan, China) and a chemiluminescence imaging system (FluorChem M, Protein Simple, CA, USA) were used for imaging. The related antibodies were listed in Table S2.
Radial spoke 1 (RS1) structure remodelingWe retrieved the RS1 structure of Chlamydomonas reinhardtii from the Protein Data Bank (PDB) (accession codes 7JTK and 7JTS) and performed a BLAST search to identify homologous proteins in Homo sapiens and Mus musculus. This enabled the selection of candidate proteins for homology modeling based on sequence similarity, with all homologous proteins showing over 20% sequence identity (Table S3). To predict the three-dimensional structures of RS1 in humans and mice, we used AlphaFold3 (https://alphafold.com), a state-of-the-art deep learning model known for its high accuracy in protein structure prediction [31]. For the AlphaFold3 predictions, we utilized the default settings outlined in the AlphaFold3 documentation (v3.0), focusing on the latest version. The predicted structures were then aligned, altered, and fitted using PyMol (http://www.pymol.org) to optimize the models. We assessed the reliability of the predicted structure using pIDDT scores, which represent confidence in each residue, flagging those with scores below 50 as potentially uncertain. During this process, we removed amino acid residues with pIDDT scores lower than 50, as these residues were predicted with low confidence and could contribute to structural inaccuracies. Most of the predicted models exhibited pTM scores greater than 0.5, indicating a high level of structural confidence. For reproducibility, we adhered to the guidelines provided in the AlphaFold3 GitHub repository (https://github.com/deepmind/alphafold), where the detailed protocol and all necessary configuration files for running AlphaFold3 are available. The full computational environment, including version numbers of dependencies, can be found in the supplementary materials of the referenced AlphaFold3 publication.
ICSI proceduresThe female mice (ICR, 4-week-old) were intraperitoneally injected with 5 IU Pregnant Mare Serum Gonadotropin (PMSG, Ningbo Sansheng, Ningbo, China) followed with 5 IU human Chorionic Gonadotropin (hCG, Ningbo Sansheng, Ningbo, China) 48 h later for superovulation. 13–14 h after hCG administration, oocytes at metaphase II (MII) stage were collected from the oviducts and incubated in M2 medium (MR-015, Sigma-Aldrich, MO, USA). The single spermatozoa collected from cauda epididymides of wild-type or KO male mice was microinjected into a MII oocyte under a NIKON inverted microscope (Tokyo, Japan) through a Piezo system (PrimeTech, Osaka, Japan). The injected oocytes were then cultured in KSOM at 37℃ in an atmosphere of 5% CO2 in air for development into blastocyst.
Statistical analysisStatistical analyses were conducted using GraphPad Prism software. Statistical significance of the differences between two groups was measured by Student’s t-test with paired, two-tailed distribution. All data were presented as the mean ± SEM and P < 0.05 were considered statistically significant.
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