We recruited three males with primary infertility from a large family (Figure 1A). Each participant completed a detailed questionnaire regarding his reproductive history. According to clinical investigations, all of the affected individuals displayed normal erection and ejaculation, male external genitalia and secondary sexual characteristics. Semen parameters including sperm concentration, motility, and morphology were measured according to the World Health Organization (WHO) criteria for human semen analysis (Boitrelle et al., 2021). Blood samples were collected from all the available family members for genomic DNA extraction. This study was approved by the Institutional Ethical Committee of the University of Science and Technology of China (USTCEC202000003). Each family member provided written consent prior to the collection of biological samples.

Total gDNA from peripheral blood of the patients and their family members was obtained using the QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer’s instructions. Exons were enriched via the Agilent SureSelect Human All Exon V5 Kit. The enriched DNA was sequenced on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA). Sequencing reads (.qseq format) were aligned to the reference human genome (hg19) using Burrows-Wheeler Aligner (BWA) software with default parameters. SAMtool (http://samtools.sourceforge.net/) was used to convert the SAM files from each sample into BAM files. The PCR duplicates were removed with the Picard tool (http://picard.sourceforge.net/). Files were further processed with the Genome Analysis Toolkit (GATK) available from the Broad Institute (http://www.broadinstitute.org/gatk/), followed by realignment through indel realigner and variants (single-nucleotide variants (SNVs) and indels) calling using GATK’s Unified Genotyper (Zubair et al., 2022). PCR amplification and Sanger sequencing were performed to confirm the candidate pathogenic variants from whole-exome sequencing (WES). All primer sequences used for PCR and sequencing are shown in Supplementary Table S3.

The evolutionary conservation of altered amino acid residues was performed with MEGA (Tamura et al., 2013). The effect of pathogenic missense variants on protein structure was predicted by the SWISS-MODEL online tool.

The coding sequences of wild-type (WT) and mutant (MT) DND1 were fused to EGFP. Human embryonic kidney 293T cells (HEK293T) obtained from ATCC (CRL-1573) were cultured in DMEM (VivaCell, C3113-0500) supplemented with 10% fetal bovine serum (FBS, Gibco, 16,000-044) and penicillin-streptomycin (Gibco, 15,140-122). Plasmids harboring WT or MT eGFP-DND1 were transfected into HEK293T cells using Lipofectamine 3000 according to the manufacturer’s instructions (Invitrogen, L3000015). At 36 h post-transfection, plasmid expression was examined under a Nikon ECLIPSE 80i microscope. At 44 h post-transfection, the cells were harvested for Western blotting analysis.

The HEK293T cells were lysed in 4X Bolt LDS Sample Buffer (Invitrogen, B0008) with NuPAGE Antioxidant (Invitrogen, NP0005). The lysates were denatured for 10 min and then were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transferring the proteins to 0.45 μm pore size immobilon-P membranes (Millipore, IPVH00010). Membranes were blocked in TBST buffer (50 mM Tris, pH 7.4, 150 mm NaCl and 0.1% Tween-20) containing 5% nonfat milk for 1 h and incubated with primary antibodies (GFP, Abmart, M20004L; Myc, Abmart, M20002; mCherry, Abcam, AB125096; mCherry, Abmart, AB167453; β-Actin, Abcam, ab8227) at 4°C overnight. Following incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP Donkey anti-rabbit IgG, BioLegend, Inc., 406,401; HRP Goat anti-mouse IgG, BioLegend, Inc., 405,306) for 1 h, the membranes were developed with chemiluminescence substrate by ImageQuant LAS 4000 imaging system (GE Healthcare).

The HEK293T cells were lysed in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA and 0.1% NP-40) supplemented with PMSF, Protease Inhibitor Cocktail and MG132, followed by rotation at 4°C for 20 min. After centrifugation, 50 μL of the supernatant was taken out as the input. Beads (Pierce™ anti c-Myc beads, Thermo scientific, 88,842; Protein A/G Magnetic beads, bimake, B23202) were washed with pre-cooled lysis buffer for three times, then the beads were incubated with supernatant at 4°C for 2–3 h. After washing with lysis buffer for 6 times, the beads were boiled in 2×SDS sample buffer for 10 min. The samples were either analyzed by Western blotting immediately as described above or stored at −80°C.

In this study, we investigated a Pakistani family, having three infertile brothers suffering from NOA and male infertility (Figure 1A). The parents (II:1 and II:2) had three daughters and seven sons. The unmarried sister (III:19, 26 years old) had normal menstrual cycles and the two married sisters had six and five children, respectively. Among the seven brothers, four had children, whereas III:1 (43 years old), III:3 (36 years old) and III:5 (34 years old) were infertile despite being married for 22, 17, and 18 years, respectively.

The patients had no history of drinking, smoking, exposure to toxic chemicals, and any other diseases. The clinical information of each patient is summarized in Table 1. Routine semen analyses revealed that no spermatozoa were detected in their ejaculates, although the semen volume was normal. Ultrasound examination revealed normal vas deferens and small testes in all patients (right: 3.5*1.7*2.8 cm³ and left: 3.6*2.0*2.6 cm³ for III:1; right: 3.8*2.3*2.6 cm³ and left: 4.0*2.3*2.6 cm³ for III:3; right: 3.8*2.3*2.6 cm³ and left: 4.0*2.4*2.7 cm³ for III:5). Additionally, all enrolled patients had normal sex hormone levels and a 46, XY karyotype. Together, azoospermia in those patients is caused by spermatogenic failure rather than obstruction (Esteves, 2015).

To determine the genetic cause of azoospermia in these patients, WES was conducted in the three affected individuals (III:1, III:3, and III:5), their mother (II:1) and the healthy brother (III:13) (Figure 1A) using gDNA extracted from peripheral blood lymphocytes. Analysis of WES data identified three candidate pathogenic variants: PCDHGA8 (c. 862C>T, p. Q288X), ARHGAP26 (c. 2137G>A, p. V713I) and DND1 (c.212A>C, p. E71A), of which only DND1 is a highly testis-expressed gene (Supplementary Table S1) and its homozygous deficiency cause male infertility (Seelow et al., 2009; Zhang et al., 2013; Li et al., 2018; Niimi et al., 2019). Details regarding the exclusion of the two genes can be found in Supplementary Table S2. The autosomal recessive inheritance pattern of the missense variant of DND1 in this family was confirmed by Sanger sequencing (Figure 1C). In addition, the mutation was not present in several general population frequency databases, such as the 1000 Genomes Project and Genome Aggregation Database (gnomAD) (Supplementary Table S1).

DND1 gene is composed of four coding exons and produces 353 amino acids containing two consecutive RNA-binding domains (RBDs) and one double-strand RBD (dsRBD) with five β-subunits and two α-subunits. The variant p. E71A is located in the α-subunit of RBD1 (Figure 1B). Multiple sequence alignment of the DND1 protein showed the amino acid site of variant (p.E71A) is highly conserved across the eutherian species (Figure 2A). We further performed an in silico analysis to predict the structural changes in the DND1 protein (Figure 2B). As shown in Figure 2B (lower panel), disruption of the α-subunit was found in the DND1 mutated protein.

To investigate the functional consequences of the DND1 missense variant, the wildtype (WT) and mutant (MT) DND1 protein with N-terminal EGFP-tags were expressed transiently in HEK293T cells. We found that the E71A mutant exhibited reduced DND1 signal intensity in both the cytoplasm and the nucleus (Figure 3A) and our results are consistent with previous findings, where by using the single guide RNA (sgRNA) and enhanced third generation base editing system (4B2N1) in oocytes and by generating mutant mice targeting Dnd1 gene with base mutation completely depleted PGCs and affected the protein-protein interaction of DND1 (Li et al., 2018). Moreover, the decrease in MT DND1 protein expression was confirmed by immunoblotting (Figure 3B).

Previous studies have shown that DND1 cooperatively functions with NANOS2 in male germ cell development. To test the effects of the missense DND1 variant on the interaction between NANOS2 and DND1, we conducted a co-IP assay in HEK293T cells transfected with Myc-tagged WT DND1, MT DND1 and mCherry-tagged NANOS2. In line with previous studies, Myc-tagged WT DND1 co-precipitated with mCherry-tagged NANOS2. However, the mutated DND1 (E71A) drastically reduced the interaction with NANOS2 (Figure 4A). We further examined whether mutation of DND1 interacts with CNOT1(1-551-aa), a component of the CCR4-NOT (CNOT) deadenylase complex that directly interacts with NANOS2. Similar to other mutants (E59K, V60M, P76L, G82R), DND1 with the E71A mutation almost did not disrupt the interaction with CNOT1 (Figure 4B) (Li et al., 2018). These findings indicated that the E71A mutation decreases protein stability and protein-protein interactions.