CCDC9 is identified as a novel candidate gene of severe asthenozoospermia

ABSTRACT

Owing to less than 1% of motile spermatozoa in the ejaculated semen, severe asthenozoospermia is a serious threat to the male reproductive health. Herein, we identified a novel homozygous variant in CCDC9 (NC_000019.9: g.47763960C>T, NM_015603.3, NP_056418.1: p. Ser109Leu) in a patient from a consanguineous family. The variant was highly pathogenic and was predicted to be a candidate gene for asthenozoospermia through in silico analysis. The CCDC9 protein levels were significantly low and its morphology and ultrastructure were severely damaged in the spermatozoa containing the novel variant. Therefore, CCDC9 may be a novel pathogenic gene associated with severe asthenozoospermia.

Abbreviations: CCDC9: coiled-coil domain containing 9; AZS: asthenozoospermia; MP: midpiece; MS: mitochondrial sheath; ODF: outer dense fiber; CP: central pair; DMT: doublet microtubule; IDA: inner dynein arm; ODA: outer dynein arm.

KEYWORDS:

• Severe asthenozoospermia
• homozygous variant
• whole exome sequencing
• CCDC9

Introduction

Asthenozoospermia (AZS), also known as asthenospermia, is the most common cause of infertility worldwide and can be detected in up to 40% of infertile male patients (Dirami et al. 2013; Xu et al. 2018). Owing to less than 1% motile spermatozoa in the ejaculated semen, severe asthenozoospermia is a serious challenge in reproductive medicine (Marchini et al. 1991). Severe asthenozoospermia has a complex etiology that includes metabolic defects (Wilton et al. 1992), flagella abnormalities (Collodel et al. 2011; Moretti et al. 2011), genital tract infections (Lores et al. 2018), varicocele (Amer et al. 2015; Mostafa et al. 2016, 2018), unhealthy lifestyle, antisperm antibodies (ASA) (Harrison 1978; Marchini et al. 1991; Dimitrov et al. 1994; Shibahara et al. 2005), and necrozoospermia (Gopalkrishnan et al. 1995). Unfortunately, due to its complex etiology, the exact pathogenesis of severe asthenozoospermia remains largely unclear for a majority of the patients.

Previous studies have demonstrated that genetic variants may be one of the etiologies of asthenozoospermia leading to severe asthenozoospermia (Chemes et al. 1998; Collodel et al. 2011; Xu et al. 2018). Mutations in several genes such as SPAG17 (Xu et al. 2018), ARMC2 (Coutton et al. 2019), AKAP3 and AKAP4 (Baccetti et al. 2005), SEPT4 (Li et al. 2011), CATSPER2 (Zhang et al. 2007), GALNTL5 (Takasaki et al. 2014), and NSUN7 (Khosronezhad et al. 2015) have been reported to be relevant to asthenozoospermia. However, these gene mutations were found only in some sporadic cases. The genetic pathogenesis of most patients with severe asthenospermia remains unclear. Therefore, further exploration is warranted to completely elucidate the pathogenesis of severe asthenospermia.

Herein, we found a homozygous variant of the CCDC9 gene in an infertile patient with severe asthenozoospermia from a consanguineous family for the first time and our data revealed that CCDC9 might be a novel candidate pathogenic gene for severe asthenozoospermia. Our results expand the gene mutation spectrum that causes severe asthenozoospermia and provide a novel basis for clinicians to diagnose and treat patients with severe asthenozoospermia.

Results and discussion

In this study, we examined an infertile patient (Figure 1A II:3), who came from a consanguineous family (Figure 1A). The analysis of sperm motility indicated that there was no forward movement of the spermatozoa, and the immotile spermatozoa accounted for more than 99% of the sperm; therefore, the patient was diagnosed with severe asthenozoospermia. To detect the cause of the motility defect, we performed Papanicolaou staining. Compared to the morphology of normal spermatozoa from the fertile control, the spermatozoa from the patient showed several severe defects in the mitochondrial sheath (MS) and flagella, as indicated by the arrow; the MS was misarranged, and the flagella were short or absent (Figure 1B).

Figure 1. Defects of morphological characteristics in the sperm of the patient. (A) Family tree of the patient with severe asthenozoospermia. The black square represents the proband (II:3). The square or circle with black dots represents the carriers. (B) Morphological analysis of the sperm from the patient with papanicolaou staining. The black arrows represent abnormal sperm flagella, including absent, short, bent flagella. (C-F) Electron microscopic morphological analysis of sperm. (C) Longitudinal section of sperm flagella from control. (D) Longitudinal sections of sperm from the patient with short flagella or absent of and disordered mitochondrial sheath. (E) Cross-section of the sperm mid-piece (MP) from normal control. (F) Cross-section of the sperm MP from the patient showed fuzzy outer dense fibers (ODF), with disappearance of the DMTs and CPs. Multiple images were taken and the representative images were presented. Scale bar, B: 20 μm, C and D: 2 μm, E and F: 200 nm. Abbreviations: MP, mid-piece; MS, mitochondrial sheath; ODF, outer dense fiber; CP, central pair; DMT, doublet microtubule; IDA, inner dynein arm: ODA, outer dynein arm.

Transmission electron microscopy was performed to identify whether there were ultrastructural defects. We detected numerous ultrastructural defects in the MS and the flagellum of the spermatozoa from the patient (Figure 1D,F). The cross section of the mid-piece (MP) ultrastructure of the sperm from normal control is flawless, with two central pairs (CPs) in the center position, nine doublet microtubules (DMTs), and nine outer dense fibers (ODFs) orderly arranged on the outside surrounded by a well-organized mitochondrial sheath (MS) (Figure 1C,E). However, we noticed that all of the sperm flagella of the patient in the MP were severely damaged. Although the fuzzy outer dense fibers (ODFs) remained unaffected, the intact organization ceased to exist, and DMTs and CPs disappeared completely (Figure 1F). These results showed that severe asthenozoospermia in the patient are mainly due to misarranged MS and abnormal flagella.

To determine the pathological origin of severe asthenozoospermia in the patient, we extracted the genomic DNA from whole blood of the patient and subjected it to WES analysis. The results were analyzed using bioinformatics according to the general methods to exclude irrelevant or meaningless variants (detailed in material and methods). The characteristics and rare and potential pathogenic variants in the patient were summarized (Supplemental Tables 1–3). We did not find any gene that had been reported, which is related to sperm flagella development and function in this table. For the candidate genes in the table, CCDC9 was the only gene that may be associated with sperm flagella development and function. Notably, we found the homozygous variant of CCDC9 gene in the patient (Figure 2A). Sanger sequencing was used to validate the variant of CCDC9 gene found in the patient, his elder brother, mother, and father. In detail, the patient carries the following homozygous variant: c.326C>T:p.Ser109Leu (Figure 2A, line 4). Further, we found heterozygous variant c.326C>T:p.Ser109Leu in his father (Figure 2A, line 1) and his mother (Figure 2A, line 2), implying that the homozygous variant in the patient was inherited from his parents. In addition, we found this heterozygous variant c.326C>T:p.Ser109Leu in his unaffected elder brother (Figure 2A, line 3) who has a daughter, suggesting that homozygous variant rather than heterozygous variant at this site is pathogenic.

Table 1. In silico analysis of CCDC9 mutations.

Mutation	Amino acid change	Polyphen-2^a	SIFT^b	ExAC (total)^c	1000G_ALL^d	gnomAD ^e
c.326C>T	p.Ser109Leu	Possibly damaging (0.516)	Deleterious (0.008)	0.0001	0.0002	0.0001743

^a Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/). Prediction Scores range from 0 to 1 with high scores indicating probably or possibly damaging. ^b SIFT (http://sift.jcvi.org/). Scores vary between 0 and 1. Variants with scores close or equal to 0 are predicted to be damaging. ^c Frequency of variation in total of ExAC database. ^d Frequency of variation in total of 1000 Genomes database (A Deep Catalog of Human Genetic Variation). ^e Frequency of variation in total of gnomAD database.

Figure 2. Homozygous mutation of CCDC9 was identified in the patient with severe asthenozoospermia. (A) Sanger sequencing confirmed the homozygous variant in CCDC9 gene of the patient. The red rectangles indicate the variant sites. The patient had the homozygous variant c.326C>T:p.Ser109Leu, while heterozygous mutation c.326C>T:p.Ser109Leu were identified in his father, his mother, and his elder brother. (B) Conservative analysis of the amino acid site affected by the homozygous variant in different species. The variant site and adjacent amino acids in CCDC9 are highly conserved between different species. Abbreviations: CCDC9, coiled-coil domain containing 9 (NC_000019.9, NM_015603.3, NP_056418.1).

The homozygous variant site is located in the coding region of exon 5, and the amino acid influenced by this variant is in the functional domains of CCDC9. In addition, the amino acid encoded by the variant site in CCDC9 is highly conserved between different species (Figure 2B). Furthermore, we used the PolyPhen2 and SIFT databases to assess the effect of the variant. The results suggested that the variant was highly pathogenic as predicted by the bioinformatics analysis (Table 1). The frequency of the homozygous variant in the general population was assessed using ExAC and 1000 Genomes databases, and the results suggest that the homozygous variant is rare (Table 1). The location, pathogenicity, and rarity of the novel variant suggest that the homozygous variant may be the main cause of severe asthenozoospermia for the patient. A genetic summary of characteristic information is available in Supplemental Tables 2 and 3.

The CCDC9 gene is located in the chromosome 19q13.32, and it is approximately 15.974 kb long. This gene has 11 exons encoding 531 amino acids named coiled-coil domain containing 9 (CCDC9). The CCDC9 protein with a weight of 59.703 kDa is a member of the coiled-coil domain-containing (CCDC) protein family. The coiled-coil domain-containing (CCDC) protein family contains conserved coiled-coil motif, which has widely functioned in the regulation of cellular activities, especially performing important roles in molecular recognition and protein refolding (McEvoy 1997; Burkhard et al. 2001). To assess the influence of the homozygous variant on CCDC9 protein expression, we explored CCDC9 protein levels in the spermatozoa of the patient using Western blotting and immunofluorescence assay (Figure 3). The result showed that compared to control, the expression of CCDC9 protein in the sperm of the patient was very weak (Figure 3A). The quantification of the Western blotting result showed that protein expression decreased significantly in the patient’s spermatozoa (Figure 3B). The protein expression level and the localization of CCDC9 were determined with immunofluorescence. The CCDC9 was located along the flagellum in control. The CCDC9 protein was very weak and barely detected compared to control spermatozoa (Figure 3C). These results suggested that the novel homozygous missense variant site might affect the stability of CCDC9 protein and result in significant decrease in the expression level of CCDC9 protein in the patient with severe asthenozoospermia.

Figure 3. CCDC9 protein level in the patient and normal control. (A) CCDC9 protein level was determined using Western blotting. The expression of CCDC9 protein in the sperm of patient was very weak. (B) The density of bands was quantified using ImageJ. CCDC9 protein decreased significantly in the patient. Acetylated-Tubulin was used as the loading control. The results are expressed as the mean SD of the three independent experiments. Data was analyzed with SPSS 18.0 software. **P < 0.01. (C) CCDC9 protein expression (Red) was determined with immunofluorescence assay. Chromatin was labeled with DAPI (Blue), and sperm tails were labeled with Acetylated Tubulin (Green). The CCDC9 was located along the flagellum in control. However, CCDC9 was nearly undetectable along the flagellum in the sperm of the patient. Multiple images were taken and the representative images were presented. Scale bar: 10 μm. Abbreviations: CCDC9, coiled-coil domain containing 9; DAPI: 4ʹ,6-diamidino-2-phenylindole.

In previous studies, several coiled-coil domain-containing genes have been found to take part in male infertility due to its effect on spermatogenesis or sperm functions. Ccdc155 is specially distributed on the telomeres, from the leptotene to the diplotene stage spermatocytes, and it could mediate telomere localization through direct interaction with SUN1 (Morimoto et al. 2012). Ccdc136 protein is richly present in the acrosome of round and elongated spermatids, and the disruption of this gene impairs acrosome formation (Geng et al. 2016). Ccdc87 is a testis-specific gene, and Ccdc87 knockout mice were sub-fertile due to decreased sperm motility and the sperm displaying severe defects in the acrosome and the cell nucleus (Wang et al. 2018). Ccdc39 and Ccdc40 play an important role in the proper arrangement of the flagella and motile cilia, and seriously affect their functions (Merveille et al. 2011; Blanchon et al. 2012; Antony et al. 2013; Abdelhamed et al. 2018). Ccdc172 is localized in the midpiece of the spermatozoa (Yamaguchi et al. 2014) while Ccdc181 is positioned in the sperm flagella (Schwarz et al. 2017), suggesting that they may play an important role in the sperm flagella function. Ccdc103 (Panizzi et al. 2012; King and Patel-King 2015; Shoemark et al. 2018), Ccdc114 (Knowles et al. 2013; Onoufriadis et al. 2013; Wu and Singaraja 2013; Li et al. 2019), and Ccdc151(Alsaadi et al. 2014; Hjeij et al. 2014; Zhang et al. 2019) are localized in the ODAs of the axoneme. Defects including reduced or absent of ODAs and reduced motility of cilia and flagella are observed in these genes mutations. Ccdc65 (Horani et al. 2013; Bower et al. 2018) and Ccdc164 (Wirschell et al. 2013) are the components of the nexin-dynein regulatory complex (N-DRC). Mutations of these genes affect N-DRC, which is essential in motility function of cilia and flagella.

CCDC9 is among the coiled-coil domain-containing (CCDC) proteins, but its function has not been investigated. Here, we first reported a novel homozygous variant of CCDC9 gene in a patient with severe asthenozoospermia (see Supplemental Graphical Abstract). We found heterozygous variant in this site in his father and his mother, implying that the homozygous variant inherited from his parents. In addition, the heterozygous variant was found in his unaffected elder brother, suggesting that homozygous variant rather than heterozygous variant at this site is pathogenic. The CCDC9 protein level significantly reduced in the sperm of this patient, which may be caused by the degradation of the protein due to the change in the structure of the encoded amino acid from the hydrophilic Serine to the hydrophobic Leucine. The p.Ser109Leu amino acid changes will affect the correct folding and three-dimensional conformation of CCDC9 protein, which may lead to decreased stability and eventual degradation of CCDC9 protein. Our results showed that CCDC9 was mainly located along the axoneme of the sperm flagella. Spermatozoa containing the novel homozygous variant had severe damage in the MS and flagella, suggesting that the absence of CCDC9 protein affects the proper arrangement of axoneme. As a result, the intact organization ceased to exist, the DMTs and CPs disappeared completely. These data suggest that the novel homozygous variant in CCDC9 gene is a novel etiology of severe asthenozoospermia in human. Our results provide clinicians with new strategies for diagnosing and treating severe asthenozoospermia.

Materials and methods

Material collection, ethical approval, and laboratory procedures

The proband (32 years of age, Figure 1A, II:3) and his family were recruited for this research from the Affiliated Yantai Yuhuangding Hospital of Qingdao University. The proband had sex 2–3 times per week with normal erection and ejaculation in the past five years after marriage, but his wife did not conceive. The patient has an elder brother (Figure 1A, II:2) who has a daughter (Figure 1A, III:1). The patient exhibited normal secondary sexual characteristics and physical examination results were as follows: height, 175 cm; weight, 73 kg; external genital development, normal; bilateral testicular size, normal; and bilateral spermatic vein, normal. The semen examination results from our hospital were as follows: semen volume, 2.5 mL; semen pH, 7.5; sperm density, 20.5 million/mL; percentage of progressive motility, 0%; percentage of non-progressive motility, 1%; and percentage of immotile sperm, 99%. Sperm morphology showed a normal morphology of 3.4%. Seminal plasma biochemical testing indicated that fructose level, neutral glycosidase activity, and seminal plasma zinc level were normal. The reproductive hormones were within normal ranges (FSH 2.36 mIU/mI, LH 1.85 mIU/ml, T 3.76 ng/ml, E2 24 pg/ml, PRL 7.33 ng/ml). The chromosomal karyotypes of the patient were normal, 46, XY and no microdeletion was found in the Y chromosome. Based on these results, the patient was diagnosed with severe asthenozoospermia.

Five milliliter of peripheral blood was collected from the patient and his family respectively. Control subject was a healthy male of 33 years with normal fertility. Written informed consent was obtained from each participant. This study was approved by the Ethics Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University.

Whole-exome sequencing (WES) and sanger sequencing validation

WES and the raw data processing was carried out as previously described (Sha et al. 2018). Briefly, DNA sequencing was performed on an Illumina Hiseq 2500 platform. The reads were aligned to the human reference sequence (hg19) using Burrows-Wheeler Aligner and sorted by Picard software. Based on the results of the alignment, Single nucleotide variants and InDels were analyzed and quality-filtered using Genome Analysis Toolkit as previously described (McKenna et al. 2010). Then, the candidate variants were annotated by ANNOVAR with SIFT, PolyPhen-2, Mutation aster, and the exome aggregation consortium (ExAC) database. Variants satisfying the following criteria were retained for subsequent analyses: (1) absent or rare (Variants with a minor allele frequency <1% in exome aggregation consortium (ExAC, http://exac.broadinstitute.org/), genome aggregation database (gnomAD, http://gnomad.broadinstitute.org/), 1000 genomes (http://browser.1000genomes.org/index.html), NHLBI ESP6500 (exome variant server, http://evs.gs.washington. edu/EVS/), and the short genetic variations database (dbSNP, http://www.ncbi.nlm. nih.gov/snp/) by considering the rare prevalence of MMAF). (2) Nonsense, frame-shift, splice site variants or missense variants scored as ‘Deleterious’ by SIFT and ‘Possible damaging’ by PolyPhen-2. The original data for all rare and potentially pathogenic variants in the patient is shown in Supplemental Table 1. Among these genes, only CCDC9 is closely associated with sperm flagella development and function. Sanger sequencing was used to validate the variant of CCDC9 gene in the patient, his elder brother, mother, and father.

Papanicolaou staining

Papanicolaou staining was performed according to World Health Organization standards for human semen examination and processing (5th ed.) with modification to confirm morphologic changes in sperm tails as described previously (Sha et al. 2017a).

Transmission electron microscopy (TEM)

Sperm samples were examined following previously published procedure for subcellular structure (Sha et al. 2017a).

Western blot and immunostaining of spermatozoa

Spermatozoa protein was extracted and separated by 10% (w/v) SDS-PAGE. The membrane was incubated with either Anti-CCDC9 or anti-Acetylated Tubulin primary antibody. Immunostaining of the spermatozoa was performed as described previously (Sha et al. 2017b). Briefly, the prepared spermatozoa were fixed in 4% PFA, followed by permeabilization with 0.2% Triton X-100. Slides was incubated with primary antibodies, Anti-CCDC9 and co-stained with anti-Acetylated Tubulin primary antibodies. Antibodies: Anti-CCDC9 (HPA072007, Atlas antibodies, Sweden); Anti-Acetylated Tubulin (66200–1-Ig, Proteintech, USA).

Authors contributions

Designed the study and revised the manuscript: PQ, JC, XW; Analyzed the data: YS, YX; Performed experiments and wrote the manuscript: XW, WL; Collected and analyzed the data: LM, SL, ZJ; Assisted with manuscript writing: XW, ZS. All authors have reviewed and approved the final version of the manuscript.

Acknowledgments

The authors would like to sincerely thank the patient and his family for their interest and cooperation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary Material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the following grants: The open project of Key Laboratory of Male Reproduction and Genetics, National Health and Family Planning Commission [Grant No. KF201704]; Natural Science Foundation of Shandong Province [Grant No. ZR2017LH012]; the National Natural Science Foundation of China [Grant No. 81871200]; the Science Technology Guidance Project of Fujian Province [Grant No. 2017D018].