Loss-of-function mutations in Ccdc113 cause male infertility in both humans and mice

Abstract

Although genetic factors contribute significantly to male infertility, the underlying mechanisms remain incompletely understood. Coiled-coil domain-containing protein 113 (CCDC113) encodes a coiled-coil domain-containing protein critical for the assembly of cilia and flagella. Using whole-exome sequencing, we identified biallelic CCDC113 mutations in two unrelated families affected by oligoasthenoteratozoospermia. The mutations (c.901A>C; p.K301Q and c.404A>C; p.E135A) cosegregated with infertility phenotypes and were associated with defective sperm flagella. Functional analyses demonstrated that these mutations led to less stable CCDC113 protein and severely disrupted axonemal structures in spermatozoa from our three human patients. We generated Ccdc113 knockout mice, which recapitulated the human infertility phenotypes, including abnormal sperm morphology, impaired motility, and defective spermatogenesis. Importantly, one patient achieved successful pregnancy by intracytoplasmic sperm injection, highlighting the translational potential of genetic diagnostics. These findings suggest that CCDC113 is essential for male fertility and contribute to the understanding of the genetic landscape of infertility, offering novel insights into its diagnosis and management.

INTRODUCTION

Male infertility affects approximately 7% of men worldwide, with genetic factors contributing to its etiology.1,2 Spermatogenesis is a complex developmental process requiring the precise orchestration of multiple cellular and molecular events.3,4 Correct assembly and functionality of the sperm flagellum are crucial for male fertility, with defects in flagellar structure or operation contributing significantly to cases of male infertility globally.5 The mammalian sperm flagellum is a highly specialized motile cilium consisting of several distinct functional and structural domains, namely the connecting piece, midpiece, principal piece, and endpiece.6,7 At its core lies the axoneme, a microtubule-based structure organized in a characteristic “9+2” arrangement, along with various accessory structures that enable and regulate the motility of the flagellum.8

Coiled-coil domain-containing protein 113 (CCDC113) belongs to the coiled-coil domain-containing (CCDC) protein family, which includes approximately 180 members that have diverse roles in cellular processes.9,10 Recent studies have shown that CCDC proteins are crucial for various aspects of reproduction and development. For example, mutations in CCDC103 cause primary ciliary dyskinesia (PCD) with male infertility.11 The CCDC155 protein is essential for telomere-led movement of chromosomes during meiotic prophase, and a deficiency of this protein causes infertility with meiotic arrest in both sexes, highlighting its critical role in human gametogenesis.12 Numerous members of the CCDC family, including CCDC38, CCDC108, CCDC87, CCDC146, CCDC176, CCDC157, CCDC189, and CCDC65, have been implicated in male infertility through diverse mechanisms, from defects in axonemal assembly to dysfunctional meiosis.13,14,15,16,17,18,19 CCDC113 is highly evolutionarily conserved across species, suggesting its fundamental biological importance. Initial studies identified CCDC113 as a component of centriolar satellites in somatic cells, where it plays a role in the formation of primary cilia.20,21 Recent proteomic analysis of mammalian sperm cells has revealed CCDC113 to be a component of the centrosome, further underscoring its significance in the cellular structures essential for microtubule nucleation and organization.20 Moreover, CCDC113 has been shown to connect radial spoke 3 to dynein g and the nexin link, suggesting its involvement in regulation of beating of cilia and coordination of axonemal complexes.22 CCDC113 has also been linked with the tumorigenesis and metastasis of colorectal cancer through the transforming growth factor-beta signaling pathway.23 Recent bioinformatics analyses have shown that CCDC113 is strongly expressed in tissues containing motile cilia, particularly in the testis, suggesting a potential role in male reproduction.22

Recently, Wu et al.24 demonstrated that CCDC113 stabilizes sperm axoneme and head-tail coupling apparatus by interacting with cilia and flagella-associated protein 57 (CFAP57) and cilia and flagella-associated protein 91 (CFAP91), serving as a critical hub for maintaining sperm structural integrity. Their study in mice showed that a deficiency of CCDC113 specifically affected the sperm flagellum without causing the classical symptoms of PCD, such as hydrocephalus or defects in the cilia lining the respiratory tract, suggesting potential tissue-specific functions. However, no mutations in CCDC113 have been reported in cases of human male infertility, and the specific phenotype of human CCDC113 deficiency remains unknown.

In the present study, we identified novel homozygous mutations in CCDC113 that cause male infertility characterized by severe oligoasthenoteratozoospermia (OAT) by whole-exome sequencing in infertile patients from two unrelated families. Using a combination of clinical evaluation, studies in cells, and a Ccdc113 knockout (KO) mouse model, we have confirmed the essential role of CCDC113 in spermatogenesis. We also assessed the clinical presentations of CCDC113-mutated patients, particularly regarding potential systemic ciliopathy manifestations, though comprehensive PCD-related examinations were limited by patient consent. Importantly, successful intracytoplasmic sperm injection (ICSI) in one patient demonstrated the clinical value of genetic diagnosis in CCDC113-associated male infertility, providing immediate translational value for patient management.

PARTICIPANTS AND METHODS

Study participants

The study was approved by the Ethics Committee of the Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital (Chengdu, China; Approval No. 2023-156). Written informed consent was obtained from all the study participants prior to inclusion, including consent for publication of anonymized clinical data and use of biological samples for research purposes. Participants were recruited from the Reproductive Medicine Center, Sichuan Provincial People’s Hospital, between January 2022 and March 2024. The study included two consanguineous families with male infertility. There were two affected brothers in Family 1 and one affected male individual in Family 2. Clinical evaluation included a detailed medical history, including patient self-reporting of any PCD-related symptoms (chronic respiratory symptoms, recurrent sinusitis, bronchiectasis, and situs abnormalities), physical examination, and analysis of semen according to the 6^th edition of World Health Organization (WHO) guidelines.25 All patients underwent karyotype analysis and Y chromosome microdeletion screening. Genetic screening was performed using whole-exome sequencing and included all known male infertility-associated genes.

Analysis of semen

Semen samples were collected from each patient following a period of sexual abstinence and subjected to analysis. The semen parameters were evaluated by trained andrologists in accordance with the 6^th edition of WHO guidelines.25 Sperm morphology was assessed according to the strict Kruger/Tygerberg criteria to ensure thorough and standardized evaluation.25

Whole-exome sequencing and variant analysis

Genomic DNA was extracted from peripheral blood using a standard protocol. Whole-exome sequencing was performed using the HiSeq 6000 platform (Illumina, San Diego, CA, USA). The reads were aligned to the human genome reference assembly (hg19) using the Burrows–Wheeler Aligner. Variant calling and annotation were performed following the standard bioinformatics pipelines. Variants were filtered based on the following: an autosomal recessive inheritance pattern; a minor allele frequency of <0.01 in population databases (gnomAD, 1000 Genomes, and ExAC); pathogenicity predicted by multiple in silico tools (PolyPhen-2, SIFT, and MutationTaster); and expression in the testis according to publicly available databases. Our bioinformatics pipeline included screening for all known male infertility-associated genes with confirmation of no other pathogenic variants therein.

Analysis of single-cell RNA sequencing data

Single-cell RNA sequencing data for the human testis were obtained from the Human Protein Atlas (available from: www.proteinatlas.org, last accessed on 15 September 2024). Normalized expression values for CCDC113 across various cell types during spermatogenesis were extracted and visualized using the integrated Uniform Manifold Approximation and Projection (UMAP) visualization tool provided by the Human Protein Atlas (available from: www.proteinatlas.org, last accessed on 15 September 2024).

RNA extraction and real-time polymerase chain reaction

Total RNAs were prepared using TRIzol reagent (catalog No. 15596-026; Invitrogen, Waltham, MA, USA), and complementary DNAs were obtained by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (catalog No. 4368813; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Maxima SYBR Green qPCR Master Mix (K0252; Thermo Fisher Scientific) was used for reverse transcription polymerase chain reaction (RT-PCR). The gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as an internal control. The following primers were used: Ccdc113-forward, 5’-ACTCTGCTCTCAAAACAGAAACA-3’; Ccdc113-reverse, 5’-GCCTGGATCGTAACTGTGAAA-3’; Gapdh-forward, 5’-AGGTCGGTGTGAACGGATTTG-3’; and Gapdh-reverse, 5’-TGTAGACCATGTAGTTGAGGTCA-3’. Relative amplicon expression was calculated using the 2^−ΔΔCt method.

CCDC113 expression constructs and cell culture

The wild-type CCDC113 coding sequence was amplified from complementary DNA in the human testis using high-fidelity PCR with primers containing BamHI and XhoI restriction sites. The PCR product was digested and ligated into the p3xFLAG-CMV-10 expression vector. The mutant CCDC113 constructs (c.901A>C; p.K301Q and c.404A>C; p.E135A) were generated using the Q5® Site-Directed Mutagenesis Kit (E0554S; New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. All constructs were verified by Sanger sequencing.

HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. For transfection experiments, cells were seeded in 6-well plates at a density of 3 × 105 cells per well 24 h before transfection. When cells reached 70%–80% confluence, they were transfected with 2.5 μg of either wild-type or mutant CCDC113 expression plasmids using Lipofectamine™ 3000 Transfection Reagent (L3000015; Thermo Fisher Scientific) following the manufacturer’s protocol. The DNA to Lipofectamine 3000 ratio was optimized to 1:3. After 6 h of incubation with the transfection mixture, the medium was replaced with fresh complete Dulbecco’s Modified Eagle Medium. Cells were harvested 48 h post-transfection for the analysis of protein expression.

Western blot

The cultured cells were washed twice using pre-cooled phosphate-buffered saline (PBS), and the mouse testes were dissected and rinsed in PBS. The samples were then lysed using radioimmunoprecipitation assay (RIPA) buffer (P0013B; Beyotime Biotechnology, Shanghai, China) containing protease inhibitor cocktail (78425; Thermo Fisher Scientific) and phosphatase inhibitor cocktail (P5726; Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 15 000g for 10 min at 4°C using a refrigerated centrifuge (model 5424R; Eppendorf, Hamburg, Germany), the supernatants were mixed with 1/5 volume of 5× loading buffer and boiled for 10 min.

Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose blotting membranes with a pore size of 0.45 μm (IPVH00010; Millipore, Burlington, MA, USA). The membranes were blocked with 5% nonfat milk dissolved in Tris-buffered saline (TBS)-Tween buffer (50 mmol l⁻¹ Tris, 150 mmol l⁻¹ NaCl, and 0.5% Tween-20, pH 7.4) for 1 h at the room temperature. The following primary antibodies were used: anti-CCDC113 antibody (GTX120455; 1:1000; GeneTex, Irvine, CA, USA), anti-FLAG antibody (20543-1-AP; 1:5000; Proteintech, Rosemont, IL, USA), and anti-GAPDH antibody (60004-1-Ig; 1:10 000; Proteintech). The primary antibodies were diluted in blocking solution and incubated overnight at 4°C. After washing with TBS-Tween three times (10 min each), the membranes were incubated for 1 h at the room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The secondary antibodies used were HRP-conjugated goat anti-mouse IgG (H+L; SA00001-1; 1:10 000; Proteintech) and HRP-conjugated goat anti-rabbit IgG (H+L; SA00001-2; 1:10 000; Proteintech). Chemiluminescent signals were detected using an ImageQuant LAS 4000 system (GE Healthcare, Chicago, IL, USA).

Generation of the Ccdc113-mutant mouse model

Ccdc113 KO mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. We designed single guide RNA (sgRNA) targeting exons 2–6 of mouse Ccdc113. The sgRNA, Cas9 mRNA, and donor template were microinjected into C57BL/6J zygotes. The mice were housed under standard conditions (on a 12-h light/12-h dark cycle at 22°C ± 2°C and 50% ± 10% humidity) with access to food and water ad libitum. All the animal experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.26

Histological analysis and immunofluorescence

Mouse testes were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at a thickness of 5 μm. Sections were stained with hematoxylin and eosin following the standard protocol. Human testis specimens were obtained from obstructive azoospermia patients undergoing testicular sperm extraction (TESE) procedures at Sichuan Provincial People’s Hospital. All samples were collected with written informed consent and approved by the Ethics Committee of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital (Approval No. 2023-156). For immunofluorescence staining, testis sections from both human and mouse samples were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). Sections were blocked with 3% bovine serum albumin and incubated with the following primary antibodies: anti-CCDC113 (GTX120455; 1:200; GeneTex, Irvine, CA, USA), anti-α-Tubulin (66031-1-Ig; 1:500; Proteintech), and fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (FL-1071; 1:200; Vector Laboratories, Newark, CA, USA) overnight at 4°C. After washing, sections were incubated for 1 h at room temperature with the appropriate fluorescently conjugated secondary antibodies. These included goat anti-rabbit IgG (H+L), Alexa Fluor 594 (A-11012; 1:500; Invitrogen) and goat anti-mouse IgG (H+L), Alexa Fluor 488 (A-11001; 1:500; Invitrogen). Nuclei were counterstained with 4’-6-diamidino-2-phenylindole (DAPI; D1306; 1:1000; Invitrogen). Images were captured using an FV3000 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Sperm analysis and computer-assisted sperm analysis

Sperm were collected from the cauda epididymis of adult mice (aged 8–12 weeks) and suspended in human tubal fluid medium. Sperm samples were loaded into 80-μm chambers (Hamilton Thorne, Beverly, MA, USA) for computer-assisted sperm analysis and investigated using a Computer-Assisted Semen Analyzer (version 14.0, Hamilton Thorne). Parameters analyzed included sperm concentration, motility, progressive motility, and swimming speed. At least 1000 sperm cells were analyzed per sample. The analysis was performed using three mice per wild-type genotype and three mice per KO genotype.

Transmission electron microscopy

Human sperm samples were obtained with informed consent following institutional guidelines. Mouse sperm was collected from the cauda epididymis. Samples were fixed in 2.5% glutaraldehyde in 0.1 mol l⁻¹ cacodylate buffer (pH 7.4) overnight at 4°C. After washing, the samples were post-fixed in 1% osmium tetroxide, dehydrated through an ethanol series, and embedded in Epon 812. Ultrathin sections (70 nm) were cut and stained with uranyl acetate and lead citrate. Images were acquired using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV.

Papanicolaou staining of human sperm

Human sperm samples were smeared on glass slides, fixed in 95% ethanol, and stained using the Papanicolaou method in accordance with the 6^th edition of WHO guidelines.25 The staining protocol included Harris hematoxylin (catalog No. 51275; Sigma-Aldrich), Orange G6 (catalog No. HT40116; Sigma-Aldrich), and EA50 (catalog No. HT40316; Sigma-Aldrich). The slides were mounted, and sperm morphology was examined under a light microscope (model BX53; Olympus). At least 200 spermatozoa were evaluated per sample by two trained andrologists working independently using strict Kruger/Tygerberg criteria.

Statistical analyses

Data from each group are expressed as the mean ± standard error of the mean (s.e.m.) and are derived from a minimum of three independent experiments. The Student’s t-test was used for statistical comparisons. One-way analysis of variance was used to compare the three groups. All statistical analyses were performed using GraphPad Prism 10.0.1 (GraphPad Software, Boston, MA, USA). P < 0.05 was considered statistically significant.

RESULTS

Identification and genetic analysis of CCDC113 mutations in infertile patients

We performed whole-exome sequencing to identify the genetic basis of male infertility in two unrelated families with OAT. In Family 1, a consanguineous family, we identified two affected brothers (IV:1 and IV:2) born to parents who were first cousins. Genetic analysis revealed a homozygous missense mutation (c.901A>C; p.K301Q) in CCDC113 (NM_014157.4) that segregated perfectly with the infertility phenotype in this family. The mutation was confirmed by Sanger sequencing in both affected brothers; their unaffected family members were either heterozygous carriers or homozygous for the wild-type allele (Figure 1a). In Family 2, which was not related to Family 1, we identified a different homozygous missense mutation in CCDC113 (c.404A>C; p.E135A) in the affected individual (II:1), as shown in Figure 1a. Genetic screening by whole-exome sequencing confirmed that no other pathogenic or likely pathogenic variants were present in known male infertility-associated genes, including all CCDC family members previously implicated in reproductive disorders. Both identified variants were absent from large population databases, including ExAC and gnomAD (Supplementary Table 1), suggesting their potential pathogenicity. In silico analysis using multiple prediction tools indicated that both variants were likely to be pathogenic: PolyPhen-2 scores were 0.981 (possibly damaging) for p.K301Q and 1.0 (possibly damaging) for p.E135A, while MutationTaster predicted both variants as disease-causing with a probability of 1.0. Furthermore, combined annotation-dependent depletion analysis yielded high pathogenicity scores of 26.5 for p.K301Q and 25.8 for p.E135A (Supplementary Table 1).

Figure 1.

Identification of CCDC113 mutations in patients with oligoasthenoteratozoospermia. (a) Pedigrees of two families carrying CCDC113 mutations. Family 1 included a consanguineous marriage with two affected brothers (IV:1 and IV:2, filled squares). Family 2 also included a consanguineous marriage and had one affected individual (II:1, filled square). Sanger sequencing chromatograms show a homozygous c.901A>C mutation in Family 1 and a c.404A>C mutation in Family 2. WT/WT indicates a wild-type sequence, WT/Mut indicates heterozygous carriers, and Mut/Mut indicates homozygous mutation carriers. The red arrows indicate the mutation sites. (b) Multiple sequence alignment of CCDC113 protein across species indicates evolutionary conservation of the mutated residues (p.E135A and p.K301Q, red arrows). The schematic diagram below shows the domain structure of human CCDC113 protein (amino acids 1–377) with predicted coiled-coil domains (orange boxes). (c) Western blot analysis of Flag-tagged wild-type and mutant (p.K301Q and p.E135A) CCDC113 proteins expressed in HEK293T cells. GAPDH served as a loading control. (d) Quantification of relative expression levels of CCDC113 protein from western blot analysis (the mean of three independent experiments). Data are presented as the mean ± standard error of the mean. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; CCDC113: coiled-coil domain-containing protein 113.

Supplementary Table 1.

Genetic and functional analysis of coiled-coil domain-containing protein 113 variants in patients with oligoasthenoteratozoospermia

Characteristics	Human subjects
	Family 1 Patient IV:1	Family 1 Patient IV:2	Family 2 Patient II:1
Phenotype	Oligoasthenoteratozoospermia	Oligoasthenoteratozoospermia	Oligoasthenoteratozoospermia
cDNA mutation*	c.901A>C	c.901A>C	c.404A>C
Protein alteration	p.K301Q	p.K301Q	p.E135A
Mutation type	Missense	Missense	Missense
Allele frequency
ExAC	0	0	0
gnomAD	0	0	0
Function prediction
PolyPhen-2	Possibly damaging (0.981)	Possibly damaging (0.981)	Possibly damaging (1.0)
Mutation Taster	Disease causing (1.0)	Disease causing (1.0)	Disease causing (1.0)
PROVEAN	Tolerable (−2.1)	Tolerable (−2.1)	Damaging (−5.1)
CADD	Damaging (26.5)	Damaging (26.5)	Damaging (25.8)

^*The GenBank accession number for CCDC113 is NM_014157.4. Values in parentheses represent prediction scores. For PolyPhen-2 and MutationTaster, higher scores indicate greater likelihood of pathogenicity (range 0–1). For PROVEAN, scores ≤−2.5 predict deleterious effects. For CADD, higher scores indicate greater deleteriousness (scaled C-scores). cDNA: complementary DNA; ExAC: Exome Aggregation Consortium; gnomAD: Genome Aggregation Database; CADD: Combined Annotation-dependent Depletion; PROVEAN: Protein Variation Effect Analyzer; CCDC113: Coiled-coil domain-containing protein 113

Sequence alignment analysis revealed that both affected amino acid residues are highly conserved across species, from humans to mice (Figure 1b). The p.K301Q mutation affects a lysine residue within a highly conserved coiled-coil domain, while p.E135A alters a glutamic acid residue in another conserved region of the protein (Figure 1b). The conservation of these residues throughout evolution suggests their functional importance for the activity of the CCDC113 protein. To investigate the impact of these mutations on expression of this protein, we transfected HEK293T cells with Flag-tagged wild-type and mutant (p.K301Q and p.E135A) CCDC113 expression constructs. Western blot analysis revealed that both mutations resulted in protein levels that were markedly lower than those in wild-type CCDC113 (p.K301Q: P = 0.0004; p.E135A: P = 0.0003), as shown in Figure 1c and 1d. These findings suggest that both mutations affect protein stability or expression, potentially compromising the function of CCDC113 in spermatogenesis.

Clinical and phenotypic characterization of patients with CCDC113 mutations

All three patients with CCDC113 mutations underwent systematic clinical evaluation. During their medical history interviews, none of these patients reported symptoms suggestive of PCD, such as chronic respiratory issues, recurrent sinusitis, bronchiectasis, or situs abnormalities. Analyses of semen confirmed a diagnosis of severe OAT in all three patients. As shown in Supplementary Table 2, values for multiple key parameters, including sperm concentration, total sperm count, and progressive motility, were markedly below the lower reference limits cited in the 6^th edition of WHO guidelines.25 The predominant phenotype was characterized by severely impaired motility with multiple flagellar abnormalities, consistent with structural defects in the sperm axoneme. This severe OAT phenotype was confirmed in both affected brothers in Family 1 (IV:1 and IV:2) but could not be confirmed in patient IV:2 who did not consent to morphological and ultrastructural analyses beyond routine clinical assessment.

Supplementary Table 2.

Detailed semen analysis of patients with coiled-coil domain-containing protein 113 mutations

Parameters	WHO reference (2021)	Control (n=3) *	Family 1 Patient IV:1	Family 1 Patient IV:2	Family 2 Patient II:1
Semen parameters
Sperm volume (ml)	≥1.4	3.2±0.7	3.4	2.6	4.7
pH	≥7.2	7.4±0.2	7.3	7.6	7.2
Sperm concentration (106/ml)	≥15	72.5±17.8	5.3	4.7	3.6
Total number (106/ejaculate)	≥39	232.0±67.5	18.0	12.2	16.9
Motility parameters
Progressive motility (PR, %)	≥32	58.5±8.4	12.8	14.6	7.8
Non-progressive motility (NP, %)	-	16.8±4.2	13.5	14.5	9.4
Total motility (PR + NP, %)	≥40	75.3±7.8	26.3	29.1	17.2
Immotile (%)	-	24.7±7.8	73.7	70.9	82.8
Vitality
Viable sperm (%)	≥54	82.5±6.8	47.2	46.8	36.5
Morphology
Normal forms (%)	≥4	13.9±6.3	1.8	1.5	1.2
Defects analysis (%)†
Head defects	-	25.8±6.5	43.7	39.8	47.3
Midpiece defects	-	15.2±4.8	47.3	49.2	53.8
Tail defects	-	38.5±8.5	76.8	81.5	85.2
Morphology indices
TZI	≤1.92	1.38±0.18	2.62	2.57	2.78

^*Control values are mean±s.d. from 3 fertile donors with proven paternity; ^†Defects may overlap; a single spermatozoon may have multiple defects; - indicates no reference value established by WHO 2021 guidelines. All samples analyzed according to WHO 2021 strict Kruger criteria with 200 spermatozoa assessed per sample by two independent observers.²⁵ TZI: teratozoospermia index; s.d.: standard deviation; WHO: World Health Organization

Visual microscopic analysis of spermatozoa from the participating patients revealed multiple structural abnormalities that predominantly affected the flagellum (Figure 2a). A detailed subclassification of tail defects was performed to specifically characterize this flagellar phenotype. Quantitative analysis demonstrated a massive and disproportionate increase in structurally severe abnormalities, specifically, coiled, short, bent, and absent tails, in the available patient samples in comparison with the control samples (Figure 2b). Transmission electron microscopy demonstrated severe ultrastructural defects throughout the sperm flagella (Figure 2c). While control sperm showed the characteristic “9+2” axonemal structure with correctly organized outer doublet microtubules, spermatozoa from the patients were found to have significant structural anomalies in all regions examined. Quantitative analysis revealed greater proportions of axonemal defects in the midpiece, principal piece, and end piece regions in sperm from the patients than those in sperm from controls (all P < 0.0001; Figure 2d).

Figure 2.

Morphological and ultrastructural analysis of spermatozoa from patients with CCDC113 mutation. (a) Light microscopy images of spermatozoa from normal controls (NC) and patients (IV:1 from Family 1 and II:1 from Family 2) showing various morphological abnormalities. Representative images demonstrate multiple morphological abnormalities in the patients’ spermatozoa. Scale bars = 5 µm. (b) Quantitative analysis of specific tail defects based on the assessment of at least 200 spermatozoa per sample. The graph shows a significant increase in coiled, short, bent, and absent tails in patients in comparison with the controls. (c) Transmission electron microscopy analysis of cross-sections of sperm flagella from control and patient samples. Images show the midpiece, principal piece, and end piece regions of the flagella. Samples of control sperm display a normal “9+2” axonemal structure, while those from the patients show various ultrastructural defects. Scale bars = 100 nm. (d) Quantification of abnormal axonemal structures in different regions of sperm flagella. Data are presented as the mean ± standard error of the mean of at least 60 cross-sections per region. CCDC113: coiled-coil domain-containing protein 113.

Despite these severe spermatogenic defects, the patient in Family 2 underwent successful assisted reproductive treatment. Following a long ovarian stimulation protocol, ICSI was performed using spermatozoa that were more morphologically favorable. Twelve of 14 retrieved oocytes were mature (MII), and 10 of these 12 oocytes were successfully fertilized (two pronuclei [2PN]). Eight good-quality embryos were obtained by day 3, and transfer of two embryos resulted in a positive pregnancy outcome (Supplementary Table 3).

Supplementary Table 3.

Clinical features of the couple with intracytoplasmic sperm injection treatment (Family 2 Patient II:1)

Parameters	Result (Patient II:1’s wife)
Age (years)	30
Length of primary infertility history (years)	6
BMI (kg/m2)	21.3
Basal hormones
FSH (IU/l)	6.8
LH (IU/l)	4.2
E2 (pg/ml)	45.6
Progesterone (ng/ml)	0.82
Cycle 1
Protocol	Long
E2 level on the trigger day (pg/ml)	2865
Number of follicles ≥14 mm on the trigger day	12
Number of follicles ≥18 mm on the trigger day	8
Number of oocytes retrieved	14
ICSI progress
Mature oocytes (MII) (n)	12
Fertilized oocytes (2PN) (n)	10
Day 3 good quality embryos (n)	8
Day 3 transfer (n)	2
Pregnancy test	Positive

BMI: body mass index; FSH: follicle-stimulating hormone; LH: luteinizing hormone; E2: estradiol; ICSI: intracytoplasmic sperm injection

Expression pattern and subcellular localization of CCDC113

To investigate the physiological role of CCDC113, we first examined its tissue distribution pattern. Quantitative RT-PCR revealed that Ccdc113 was predominantly expressed in the testis and at markedly higher levels than that in other tissues (Figure 3a). Analysis of single-cell RNA sequencing data from the Human Protein Atlas database revealed stage-specific expression of CCDC113 during spermatogenesis, with the highest expression observed in late-stage spermatids, suggesting its potential role in the development of sperm (Figure 3b).

Figure 3.

Expression pattern and localization of CCDC113. (a) RT-qPCR analysis showing relative expression of Ccdc113 in various mouse tissues. The data are presented as the mean ± standard error of the mean of three independent experiments. (b) Single-cell RNA sequencing analysis of data for the human testis obtained from the Human Protein Atlas showing stage-specific expression of CCDC113 during spermatogenesis. (c) Immunofluorescence analysis of CCDC113 in mouse and human testis sections. DAPI (blue) labels nuclei, CCDC113 (green) shows protein expression, and peanut agglutinin (purple) marks developing spermatids. Merged images show the overlay of all channels. Scale bars = 50 µm. (d) Immunofluorescence analysis of mature mouse and human spermatozoa. DAPI (blue) labels nuclei, CCDC113 (green) shows protein localization along the flagella, and α-Tubulin (purple) marks the axoneme. Scale bars = 5 µm. The right panel shows a quantitative analysis of CCDC113 fluorescence intensity in the midpiece versus principal piece regions of the sperm flagellum. Two hundred spermatozoa were analyzed, and the data are shown as the mean ± standard error of the mean. NS: not statistically significant; CCDC113: coiled-coil domain-containing protein 113; RT-PCR: reverse transcription polymerase chain reaction; DAPI: 4’,6-diamidino-2-phenylindole; PNA: peanut agglutinin.

Immunofluorescence analysis in mouse and human testis sections showed that CCDC113 (green) was specifically expressed in developing spermatids during spermiogenesis (Figure 3c). Using peanut agglutinin as a stage-specific marker for round and elongating spermatids, we observed that expression of CCDC113 was particularly prominent during the elongation phase of spermiogenesis in both species (Figure 3c). Further examination of mature spermatozoa revealed the localization patterns of CCDC113. Coimmunostaining revealed a consistent distribution of CCDC113 along the axoneme in both mouse and human spermatozoa. Quantitative analysis showed no significant difference in fluorescence intensity between the midpiece and principal piece regions (midpiece: P = 0.1616; principal piece: P = 0.2011), suggesting a conserved and general role in maintenance of the flagellum (Figure 3d).

Functional validation in a CCDC113-deficient mouse model

To establish the role of CCDC113 in male infertility, we generated Ccdc113 KO mice using CRISPR/Cas9 technology (Figure 4a). Western blot analysis confirmed the complete absence of CCDC113 protein in KO mice (Figure 4b), and quantitative RT-PCR analysis showed significantly less expression of Ccdc113 mRNA in KO mice than that in wild-type controls (P < 0.0001; Figure 4c).

Figure 4.

Generation and phenotypic characterization of CCDC113-deficient mice revealing its essential role in spermatogenesis. (a) Schematic diagram showing the targeting strategy for generation of the Ccdc113 knockout (KO) mouse model. The yellow boxes indicate exons, and the red arrows indicate gRNA targeting sites. (b) Western blot analysis of CCDC113 protein expression in wild-type (WT), heterozygous (HET), and KO mouse testes. GAPDH served as a loading control. (c) Quantification of relative expression levels of CCDC113 protein from western blot analysis. The data are presented as the mean ± standard error of the mean. (d) Fertility tests revealing complete male infertility but normal female fertility in Ccdc113 KO mice. The data are presented as the mean ± standard error of the mean. (e) Testis-to-body weight ratio in WT and KO mice. The data are presented as the mean ± standard error of the mean. (f) Hematoxylin and eosin staining of testis sections from WT and KO mice showing the organization of the seminiferous tubules. Scale bars = 100 µm. (g) Scanning electron microscopy (SEM) analysis of sperm morphology from WT and KO mice. Scale bars = 10 µm. (h) Transmission electron microscopy (TEM) analysis of cross-sections of sperm flagella from WT and KO mice in the midpiece, principal piece, and end piece regions. Scale bars = 100 nm. NS: not statistically significant; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; CCDC113: coiled-coil domain-containing protein 113; gRNA: guide RNA.

Male Ccdc113 KO mice were viable but infertile, whereas female KO mice were fertile with litter sizes comparable to those of wild-type controls (P < 0.0001 for males; P > 0.05 for females; Figure 4d). Computer-assisted sperm analysis revealed severe motility defects in Ccdc113 KO sperm in comparison with controls, with markedly reduced total motility (mean ± s.e.m.: 4.3% ± 1.7% vs 72.4% ± 3.4%, P < 0.001) and progressive motility (mean ± s.e.m.: 0.9% ± 0.4% vs 68.5% ± 3.0%, P < 0.001). Other motility parameters, including curvilinear velocity, straight-line velocity, and beat-cross frequency, were also significantly impaired (Table 1). Notably, these motility defects closely resembled those observed in the human patients carrying CCDC113 mutations. There was no significant difference in the testis-to-body weight ratio between the KO and wild-type mice (P = 0.1926; Figure 4e). Hematoxylin and eosin staining revealed that the seminiferous tubules were loosely and irregularly arranged in Ccdc113 KO mice but were organized in a compact regular manner in wild-type mice. Elongating spermatids in KO mice also showed an irregular morphology (Figure 4f). Scanning electron microscopy demonstrated that wild-type sperm had a normal flagellar structure with uniform thickness and straight orientation, whereas KO sperm showed various structural defects, including abnormal flagellar bending and irregular tail formation (Figure 4g). Transmission electron microscopy of sperm cross-sections revealed profound ultrastructural defects in Ccdc113 KO sperm. The normal “9+2” microtubule arrangement was severely disrupted across all regions of the flagellum. In the midpiece, we observed disorganized mitochondrial sheaths and loss of axonemal symmetry. The principal piece showed missing or displaced outer doublet microtubules, abnormal radial spokes, and disrupted organization of the fibrous sheath. The end piece frequently showed complete disintegration of the axoneme (Figure 4h).

Table 1.

Computer-assisted sperm analysis of wild-type and coiled-coil domain-containing protein 113 deficient male mice

Parameters	Adult male mice
	WT	KO
Semen parameters
Sperm concentration (106 ml−1)a	35.8±4.9	3.2±1.5
Sperm motility (%)	72.4±3.4	4.3±1.7
Progressive motility (%)	68.5±3.0	0.9±0.4
Sperm locomotion parameters
Curvilinear velocity (VCL; µm s−1)	125.4±8.8	8.3±2.8
Straight-line velocity (VSL; µm s−1)	48.6±5.4	2.3±0.9
Average path velocity (VAP; µm s−1)	62.9±4.9	4.1±1.5
ALH (µm)	1.3±0.2	0.2±0.1
Linearity	0.4±0.1	0.1±0
Wobble (WOB=VAP/VCL)	0.5±0.1	0.2±0.1
Straightness (STR=VSL/VAP)	0.7±0.1	0.3±0.1
BCF (Hz)	5.9±0.5	0.4±0.2

^aEpididymides and vas deferens. Values represent mean±s.e.m. (n=3 mice per group). WT: wild-type; KO: knockout; s.e.m.: standard error of the mean; BCF: beat-cross frequency; ALH: amplitude of lateral head displacement

Collectively, these results demonstrate that a deficiency of CCDC113 leads to male infertility in mice, which is characterized by severely impaired sperm motility, disrupted organization of seminiferous tubules, and a structurally abnormal sperm flagellum. The marked similarity between the phenotypes observed in Ccdc113 KO mice and human patients carrying CCDC113 mutations indicates that CCDC113 has an essential and evolutionarily conserved role in spermatogenesis and male fertility.

DISCUSSION

In this study, we identified biallelic mutations in CCDC113 as a novel genetic cause of male infertility characterized by severe OAT. Our genetic and clinical analyses demonstrated that a deficiency of CCDC113 leads to severe defects in the structure and function of the sperm flagellum in humans. Our findings not only expand the spectrum of genetic causes of male infertility but also provide new insights for clinical management. Identification of two different homozygous missense mutations (p.K301Q and p.E135A) in CCDC113 from unrelated families with consistent phenotypes strongly supports a causative role for these mutations in male infertility. Both mutations affect evolutionarily conserved residues and lead to reduced protein expression. The remarkable similarity between the phenotypes observed in our human patients and Ccdc113-deficient mice further confirms the essential role of CCDC113 in spermatogenesis. Recent research has demonstrated that CCDC113 functions as a structural hub connecting radial spokes, the nexin–dynein regulatory complex (N-DRC), and doublet microtubules in the axoneme through interactions with CFAP57 and CFAP91.24 Consistent with these findings, our ultrastructural analysis demonstrated that CCDC113 deficiency disrupts the organization of the axoneme, leading to severe defects in the structure and function of the sperm flagellum.

The CCDC family is a large group of functionally diverse proteins, with several members essential for motile cilia and male fertility.10 CCDC113 appears to have a unique role in linkage of structures within the axoneme, specifically connecting radial spoke 3 to dynein g and the N-DRC,22,24 thereby differing markedly from the roles of other CCDC proteins involved in male fertility. For example, CCDC155 has a role in movement of chromosomes during prophase I in meiosis, and a deficiency of this protein causes meiotic arrest rather than flagellar defects.12 CCDC146 is specifically required for assembly of the outer dynein arm,27 while CCDC176 stabilizes microtubule doublets 1 and 9.17 CCDC38 and CCDC65 are the components of the N-DRC itself,28,29 whereas CCDC113 serves as a connector between the N-DRC and other axonemal structures.22 CCDC189 affects formation of the sperm flagellum through interactions with CABCOCO1, representing yet another distinct mechanism.19 This functional diversity among CCDC proteins highlights the complex regulatory networks governing assembly of the sperm flagellum, with CCDC113 serving as a critical structural integrator that maintains the integrity of the axoneme through its unique bridging function.

An important clinical observation in this study was the absence of classical symptoms of PCD in patients with CCDC113 mutation. Despite detailed medical history-taking, none of our patients reported the chronic respiratory symptoms, recurrent sinusitis, or bronchiectasis typically associated with PCD. This finding aligns with the recent report by Wu et al.,24 where Ccdc113 KO mice showed no signs of hydrocephalus, situs inversus, or abnormal respiratory ciliogenesis. These observations suggest that CCDC113 may have tissue-specific activity, with a predominant role in reproductive cilia (i.e., the sperm flagellum) rather than in respiratory cilia.24,30 However, our patients refused to undergo detailed PCD-related clinical examinations, which limited our ability to definitively exclude subtle respiratory manifestations. This tissue specificity could be explained by differential expression patterns, compensatory mechanisms in respiratory cilia, or unique structural requirements in sperm flagella that make them particularly vulnerable to CCDC113 deficiency.24

Importantly, successful ICSI in the case in Family 2 demonstrates that careful selection of spermatozoa can support normal embryo development in patients with CCDC113 mutation despite severe motility and morphological defects. This finding has significant clinical implications, in that ICSI can be considered an effective treatment option for these patients provided that spermatozoa are meticulously selected. Given the autosomal recessive inheritance pattern, genetic screening for CCDC113 mutations should be considered in cases of severe OAT to guide treatment planning and genetic counseling. The success with ICSI in our case also offers hope for patients with similar genetic defects, highlighting the value of molecular diagnosis in the management of male infertility. Further studies in larger patient cohorts will help establish genotype–phenotype correlations and better understand how various CCDC113 mutations affect the severity of defective spermatogenesis.

CONCLUSIONS

We identified biallelic mutations in CCDC113 as a novel cause of male infertility characterized by severe OAT. Both mutations affected evolutionarily conserved residues and were associated with reduced protein expression. Importantly, successful ICSI in one of our patients demonstrates that CCDC113-associated infertility can be overcome by assisted reproductive technology. Our findings expand the genetic spectrum of male infertility and have immediate clinical applicability for diagnosis and treatment.

AUTHOR CONTRIBUTIONS

WL and FLL planned the experiments and revised the manuscript. HTY and FLL performed the experiments and prepared a draft of the manuscript. XXZ participated in the collection of clinical samples. FLL performed the statistical analysis. WL and XXZ conceived the project and edited the manuscript. All the authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.