Study participants

A cohort of 30 Chinese men with MMAF (short, absent, coiled, bent, or irregular flagella) or asthenozoospermia (total sperm motility < 40% and no obvious MMAF phenotype) was enrolled from the Department of Andrology/Sichuan Human Sperm Bank, West China Second University Hospital. All enrolled patients exhibited primary infertility; those with primary ciliary dyskinesia were excluded. Karyotypic analyses of all the enrolled patients revealed normal karyotypes (46XY), hormone levels, bilateral testicular size distributions, and secondary sex characteristics. The patients and healthy controls provided informed consent, and the Ethics Committee of the West China Second University Hospital approved the study. Blood and semen samples were collected from the patients.

Semen analysis

In line with the World Health Organization (WHO) guidelines (fifth edition), human sperm samples were collected by masturbation into sterile semen cups after abstinence for two to seven days. Sperm samples were incubated at 37 °C until liquefaction and were evaluated within 1 h. During routine clinical diagnosis, standard parameters, including semen volume, concentration, motility, progressive motility and morphology were assessed. More than 200 spermatozoa from each individual were counted to evaluate the proportion of morphologically abnormal sperm.

Whole-exome sequencing and Sanger sequencing

DNA was extracted from whole peripheral blood of all patients using a DNeasy Blood and Tissue kit (QIAGEN, Dusseldorf, Germany). Exonic sequences were isolated and enriched using SureSelectXT Human All Exon Kit (Agilent Technologies). Samples were sequenced on an Illumina HiSeq X-TEN platform. Read mapping (Burrows–Wheeler Aligner, BWA v0.7.17), calling (Genome Analysis Toolkit, v4.1), genotyping, and annotation (ANNOVAR and VEP) were performed as part of the sequencing analyses, as detailed previously [11,12,13,14].

Antibodies

Rabbit LRRC48 (DRC3) antibody fluorescein isothiocyanate (FITC), which detects the N-terminus of DRC3, was obtained from Universal Biotech Co., Ltd. (Shanghai, China). Alpha tubulin monoclonal antibody (AF594), mice HA, MYC, FLAG, Strep-tag monoclonal antibodies, β-tubulin, and actin antibodies were obtained from Proteintech (Wuhan, China) [15, 16].

Plasmids

Human DRC3, DRC4, and DRC5 coding sequences were obtained from National Center for Biotechnology Information, and a mutated form of DRC3 (DRC3 MU) was designed based on the mutation identified in the patient. We constructed an MYC tag at the N-terminus of the DRC3 wild type (WT) and mutated form (MU), FLAG tag and HA tag at the N-terminus of DRC4 and DRC5 respectively. DRC1, 2, 7, and 8 were added to the Strep-tag at the N-terminus. All genes were cloned into the pcDNA3.0 vector with the HindIII-XhoI restriction enzyme site, and all plasmids were synthesized by Zhejiang Youkang Biotechnology Co., Ltd. (Zhejiang, China).

Papanicolaou staining

Papanicolaou staining was used to stain the sperm after the semen was liquefied. We counted more than 200 sperm to calculate the proportion of sperm with normal morphology.

Protein extraction and western blot

MYC tag DRC3 WT; MYC tag DRC3 MU; FLAG tag DRC4; HA tag DRC5; and Strep tag DRC1, 2, 7, and 8 were transfected into HEK 293 suspension (293 F) cells, and the cells were collected after 72 h. Cells were lysed using phosphate-buffered saline (PBS) lysis buffer (PBS buffer, pH 7.5, 1% Triton X-100). Immunoprecipitation was performed using protein A/G magnetic beads following the manufacturer’s instructions. Each immunoprecipitation solution contained 1 mL of cell lysate and 1 μL of tag antibody. After overnight incubation at 4 °C, the resin was washed five times with 1 mL cold lysis buffer. In each group, 50 μL of magnetic beads suspension was used for western blot. The samples were heated at 100 °C for 10 min, subjected to SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked in 5% fat-free milk in Tris-buffered saline at 25 °C for 2 h and then incubated overnight in the relevant primary antibodies at 4 °C. After washing with TBST (1◊Tris-buffered saline, 0.1% Tween® 20 detergent), the membranes were incubated with the corresponding secondary antibodies for 2 h at 25 °C and rinsed with TBST. The protein blots were visualized using a chemiluminescence imaging system (BOX Chemi XPQ).

Scanning and transmission electron microscopy

We used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to analyze spermatozoa surface and ultrastructural abnormalities in detail. Samples for SEM were washed with PBS, smeared on a coverslip, and then put into a six-well plate. After the samples had dried, they were fixed with 3% glutaraldehyde at 4 °C. For TEM, samples were first rinsed with PBS and fixed with 0.5% glutaraldehyde at 4 °C for 10 min. After centrifugation at 25 °C and 1500 rpm for 15 min, the supernatant was discarded, and 3% glutaraldehyde was added for fixation. All samples were tested and photographed at Chengdu Lilai Biotechnology (Chengdu, China).

Immunofluorescence

Sperm samples were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 10 min. Then, following blocking with 5% skim milk powder in Tris-buffered saline buffer for 1 h, the samples were incubated overnight with fluorescence conjugated antibodies at 4 °C and washed with TBST. The sperm nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) at 37 °C for 5 min. Images were captured using an LSM 800 confocal microscope (Carl Zeiss AG).

Quantitative real-time PCR

A human MTC Panel II was purchased from Clontech (catalog number: 636743; Takara, Beijing, China) and analyzed using a ChamQ SYBR® qPCR Master Mix kit (Vazyme, Nanjing, China). Thermocycler settings were as follows: 50 °C for 2 min, 95 °C for 5 min, and 40 cycles of 95 °C for 10 s and 60°C for 30 s. The DRC3 primers were as follows: 5’ (upstream) primer, 5’-TTCTGACTTCCCCTTAGCAAC-3’, and 3’ (downstream) primer, 5’-GAGTTCAGGAGTTCAAGACCAG-3’. We chose human G3PDH as the control, the primers were as follows: 5’ (upstream) primer, 5’-TGAAGGTCGGAGTCAACGGATTTGGT-3’, and 3’ (downstream) primer, 5’-CATGTGGGCCATGAGGTCCACCAC-3’.