Introduction

The integrity of spermatozoa is necessary for its migration through the female reproductive tract and successful fertilization, and an intact spermatozoon contains proper sperm head, head-tail coupling apparatus (HTCA), and flagellum (Parker, 2020; Roldan, 2019). Many components have been identified in sperm HTCA or flagellum, and are essential for the sperm integrity (Lehti & Sironen, 2017; Wu et al., 2020). Defects in sperm flagellum and HTCA may lead to reduced sperm motility or aberrant sperm morphology, termed as multiple morphological abnormalities of the sperm flagella (MMAF) or acephalic spermatozoa syndrome (ASS), in turn, which causes male infertility (Sudhakar et al., 2021; Tu et al., 2020).

Sperm flagellum possesses an evolutionarily conserved axonemal structure composed of “9+2” microtubules, specifically, nine peripheral doublet microtubules (DMTs) surrounding two central microtubules known as the central pair (CP) (Inaba & Mizuno, 2016). Axonemal dyneins, radial spokes (RS), and the nexin-dynein regulatory complex (N-DRC), are arranged on DMTs with a 96-nm repeating unit structures (Kumar & Singh, 2021). In axoneme, the N- DRC and RS are required for axonemal integrity, which form a crossbridge between neighboring DMTs and link the DMTs to the central apparatus, respectively (Canty et al., 2021; Ishikawa, 2017; Kumar & Singh, 2021). Recent advances in artificial intelligence, biochemical techniques, and cryo-electron microscopy (cryo-EM) facilitated the analysis of the axonemal structures, and numerous components were detected among RS, N-DRC and DMTs, which may serve as hubs to stabilize the axoneme (Bazan et al., 2021; Leung et al., 2023; Walton et al., 2023; Zhou et al., 2023). Such as, CFAP91 is identified to extend from the base of RS2 through the N-DRC base plate to the RS3, and stabilizes RS2 and RS3 on the DMTs (Bicka et al., 2022; Dymek et al., 2011; Gui et al., 2021). CFAP57 also extends through the N-DRC and interacts with RS3 via its C-terminal region (Ghanaeian et al., 2023). Recent analysis reveals that CCDC96 and CCDC113 could form a complex and extend parallel to the N-DRC and connect the base of RS3 to the tail of dynein g (IDA g) and the N- DRC (Bazan et al., 2021; Ghanaeian et al., 2023). However, the functions of these proteins in sperm flagellum stabilization remain unknown.

Sperm flagellum is tightly anchored to the sperm head through the HTCA, a complex structure based on the centrosome (Wu et al., 2020). This structure consists of two cylindrical microtubule-based centrioles and associated components, including the well-organized segmented columns, along with the capitulum plate and basal plate. The segmented columns and capitulum plate, located below the basal plate, are thought to originate from the dense material emanating from the proximal centriole (Fawcett & Phillips, 1969; Zamboni & Stefanini, 1971). Many proteins have been identified in sperm HTCA, and their mouse models display acephalic spermatozoa syndrome phenotype (Wu et al., 2020). SPATA6 is the first protein identified as a component of the HTCA using the knockout mouse model, and is crucial for the formation of the sperm HTCA (Yuan et al., 2015). Deficiencies in SUN5 (Elkhatib et al., 2017; Fang et al., 2018; Liu et al., 2020; Sha et al., 2018; Shang et al., 2018; Shang et al., 2017; Xiang et al., 2022; Zhang et al., 2021; Zhu et al., 2016) and PMFBP1 (Deng et al., 2022; Liu et al., 2020; Liu et al., 2021; Lu et al., 2021; Nie et al., 2022; Sha et al., 2019; Zhu et al., 2018) have been demonstrated to be associated with ASS in both humans and mice. The centriole-related protein, CENTLEIN, serves as a bona fide linker between SUN5 and PMFBP1 to participates in the HTCA assembly (Zhang et al., 2021). Notably, impaired HTCA often accompanies defects in the sperm flagellum (Hall et al., 2013; Shang et al., 2017; Yuan et al., 2015; Zhang et al., 2021; Zhu et al., 2018), suggesting that the HTCA stabilization may be closely associated with the integrity of sperm flagellum. However, the mechanism that maintains both sperm flagellum and HTCA stabilization remains to be clarified.

Here, we identified an evolutionarily conserved coiled-coil domain-containing (CCDC) protein, CCDC113, and found it could complex with CFAP57 and CFAP91 for the connection of RS, N-DRC and DMTs in axoneme. The knockout of Ccdc113 produced spermatozoa with flagella defects and head-tail linkage detachment, in turn, which caused male infertility. Ultra-structural analyses showed that the depletion of CCDC113 resulted in the destroyed sperm axoneme and HTCA. CCDC113 is localized on the manchette, HTCA and flagellum in elongating and elongated spermatids. Further analysis revealed that CCDC113 is indispensable for the connection of CFAP91 and DRC2 to the DMTs in the sperm axoneme, and CCDC113 interacts with SUN5 and CENTLEIN to stabilize sperm HTCA. All these results suggest that CCDC113 server as a critical hub to maintain the structural integrity of both sperm flagellum and HTCA.

Results

CCDC113 complexes with CFAP57 and CFAP91 for the connection of RS, N-DRC and DMTs

CCDC113 is an evolutionarily conserved coiled-coil domain-containing (CCDC) protein in the ciliated species, and a comparative analysis of the structures of CCDC113 from Tetrahymena thermophila to Homo sapiens showed that CCDC113 orthologs shared structural similarity with each other (Fig. 1A). Recent cryo-EM analysis in the structure of the 96-nm modular repeats of axonemes from the Tetrahymena thermophila cilia and human respiratory cilia revealed that CCDC113 localized the linker region among RS, N-DRC and DMTs (Fig. 1B), indicating it may serve as a structural component connecting RS, N-DRC and DMTs (Bazan et al., 2021; Ghanaeian et al., 2023). To investigate further, we examined the interactions between CCDC113 and its neighboring axoneme-associated proteins, CFAP57 and CFAP91 (Fig. 1B). We transfected HEK293T cells with a GFP-tagged CCDC113 and FLAG-tagged CFAP57 or CFAP91, and then performed anti-FLAG- immunoprecipitations (Fig. 1A). CCDC113 were present in both FLAG-CFAP57 and FLAG- CFAP91 immunoprecipitates (Fig. 1C, D), indicating CCDC113 interacts with both CFAP57 and CFAP91. Given that CFAP91 have been reported to stabilize RS on the DMTs (Bicka et al., 2022; Dymek et al., 2011; Gui et al., 2021) and the cryo-EM analysis shows CCDC113 is closed to DMTs, we speculate CCDC113 may connect RS to DMTs via binding CFAP91 and microtubules. To test it, we detected the interaction between CCDC113 and β-tubulin (TUBB5), and found CCDC113 was present in MYC-TUBB5 immunoprecipitate (Fig. 1E). As CFAP57 extends through the N-DRC and CCDC113 is closed to the N-DRC (Ghanaeian et al., 2023), we further examined the interaction between CCDC113 and N-DRC components that closed to DMTs. The co-immunoprecipitation (co-IP) analysis showed that CCDC113 could bind to DRC1, DRC2, and DRC3 (Fig. 1E-H). Therefore, CCDC113 may be serve as an adaptor protein for the connection of RS, N-DRC and DMTs, and this protein may be a critical hub for axoneme stabilization.

Fig. 1

CCDC113 is required for male fertility

To investigate the physiological functions of CCDC113, we generated a Ccdc113 knockout mouse strain using the CRISPR/Cas9 system (Fig. 2A). Ccdc113^−/−mice were genotyped by genomic DNA sequencing and further confirmed by polymerase chain reaction (PCR). Genotypes were distinguished by a 539 bp band for Ccdc113^+/+ mice, a 461 bp band for Ccdc113^−/−mice, and two bands of 539 bp and 461 bp for Ccdc113^+/– mice (Fig. 2B). Further immunoblotting analysis validated the successful elimination of CCDC113 in total protein extracts from Ccdc113^−/− testes (Fig. 2C). Mice lacking Ccdc113 showed no gross abnormalities in appearance or behavior, and no obvious differences in body weight (Fig. 2H). Symptoms such as hydrocephalus or left-right asymmetry defects were also not found in Ccdc113^−/−mice (Fig. S1A). Additionally, the deficiency of CCDC113 did not affect ciliogenesis in the lung and trachea (Fig. S1B-D). Subsequently, we assessed the fertility of 2- month-old male and female Ccdc113^−/− mice. Ccdc113^−/−female mice were able to generate offspring after mating with wild-type (WT) adult males, which was similar to Ccdc113^+/+ female mice (Fig. 2D). However, Ccdc113^−/− male mice exhibited normal mating behavior with copulatory plugs but failed to produce any offspring after mating with WT adult female mice (Fig. 2E). Thus, the knockout of Ccdc113 results in male infertility.

Fig. 2

Ccdc113 knockout mice produce spermatozoa with flagella defects and head-tail linkage detachment

To further investigate the cause of male infertility, we initially examined Ccdc113^-/- testis at both gross and histological levels. Ccdc113 knockout did not affect either testis size (Fig. 2F, G) or the ratio of testis weight to body weight (Fig. 2H, I). Histological sections stained with hematoxylin-eosin (H&E) revealed that seminiferous tubules of Ccdc113^+/+ mice exhibited a tubular lumen with flagella emerging from the developing spermatids. In contrast, the flagellum staining appeared reduced in Ccdc113^−/− seminiferous tubules (Fig. 2J, red asterisk). Immunofluorescence staining for acetylated tubulin (ac-tubulin), a marker for sperm flagellum (Martinez et al., 2020), further confirmed the defects of flagellum in Ccdc113^−/− mice (Fig. 2K). Next, we examined spermatids at different stages in Ccdc113^−/− testes by using Periodic acid–Schiff (PAS) staining, and found that the acrosome biogenesis and nucleus morphology in Ccdc113^−/− spermatids from step 1 to step 10 were normal compared to Ccdc113^+/+ spermatids. However, abnormal club-shaped heads were observed in Ccdc113^−/− spermatids from step 11 to step 16 (Fig. S2A, black asterisk). In addition, the manchette of Ccdc113^−/− spermatids were more elongated compared to that of Ccdc113^+/+ spermatids (Fig. S2B). Therefore, the disruption of CCDC113 impaired spermiogenesis.

Next, we examined the spermatozoa in the caudal epididymis, and found that the sperm count in the Ccdc113^−/− caudal epididymis was significantly decreased compared with the control group (Fig. 3A, B). The motility of the released spermatozoa from Ccdc113^+/+ and Ccdc113^−/− cauda epididymis showed that the Ccdc113^−/−spermatozoa were completely immobility (Fig. 3C). H&E staining of the caudal epididymis showed fewer hematoxylin-stained sperm heads in the Ccdc113^−/−cauda epididymis compared to that of the Ccdc113^+/+ cauda epididymis. Notably, in contrast to the control group, where the epididymal lumen exhibited linear eosin staining, the Ccdc113^−/− mouse showed a dramatic presence of coiled eosin-stained structures without sperm heads in the epididymal lumen (Fig. 3A, red circles). To determine the morphological characteristics of the spermatozoa, we conducted single-sperm immunofluorescence using an anti-α/β-tubulin antibody to label the sperm flagellum and lectin peanut agglutinin (PNA) to visualize the sperm acrosome (Nakata et al., 2015). We noticed that Ccdc113^−/−spermatozoa showed severe morphological malformations, including sperm head-tail detachment (type 1), abnormal sperm head with curly tail (type 2), normal sperm head with curly tail (type 3) (Fig. 3D, E). Overall, these findings clearly suggest that the deletion of Ccdc113 results in both sperm flagellum deformity and detachment of sperm head-to-tail linkage, and causes a special type of acephalic spermatozoa, which may be responsible for the Ccdc113^−/−male infertility.

Fig. 3

CCDC113 localizes on the sperm neck and flagellum regions

To gain further insights into the functional role of CCDC113 during spermiogenesis, we detected the expression of CCDC113, and found that CCDC113 was predominantly expressed in mouse testis (Fig. 4A). Furthermore, CCDC113 was first detected in testis at postnatal day 7 (P7), and the level of CCDC113 increased continuously from P21 onward, with the highest levels detected in adult testes (Fig. 4B), indicating CCDC113 might be highly expressed during spermiogenesis. Then, we conducted immunofluorescence analysis of CCDC113 in Ccdc113^+/+ and Ccdc113^−/− germ cells to characterize its precise localization during spermatogenesis (Fig. S3). CCDC113 showed punctum single near the nuclei of spermatocyte and round spermatids, and localized on the manchette, sperm neck and flagellum regions in elongating and elongated spermatids (Fig. S3).

Fig. 4.

To further confirm it, we co-stained CCDC113 and α/β-tubulin, which represented the manchette and flagellum in spermatids (Lehti & Sironen, 2016) (Fig. 4D). The immunofluorescence analysis showed that CCDC113 localized on the manchette surrounding the spermatid head from step 9 to step 14, as well as the testicular sperm neck and flagellum (Fig. 4D). Given that CCDC113 was initially identified as a component of centriolar satellites (Firat-Karalar et al., 2014), the punctum single of CCDC113 in spermatocyte and spermatids may localize around the centrosome. To test it, we performed the immunofluorescent staining of CCDC113 and centrosomal protein CENTRIN1/2 in spermatocytes and spermatids, and found the signal of CCDC113 could partially colocalize with CENTRIN1/2 (Fig. 4C). Thus, CCDC113 localizes on the centrosome, manchette, sperm neck and flagellum regions in the developing germ cells.

Next, we examined the localization of CCDC113 in mature spermatozoa, and found that CCDC113 was localized in the sperm neck and flagellum regions (Fig. 4E). Similar localization of CCDC113 could also be detected in human mature spermatozoa (Fig. 4F).

Therefore, the localization of CCDC113 on the sperm neck and flagellum is persistent in mature spermatozoa, which may be essential for the integrity of sperm flagellum and head to tail.

Ccdc113 knockout results in the disorganization of the sperm flagellum structures

To delineate the sperm flagella defects in Ccdc113^−/−mice, we conducted transmission electron microscopy (TEM) examination of longitudinal sections of Ccdc113^−/− spermatozoa. The TEM analysis revealed a significant presence of unremoved cytoplasm, including disrupted mitochondria, damaged axonemes, and large vacuoles in Ccdc113^−/−spermatozoa (Fig. 5A, red asterisks). Cross sections of the principal piece of Ccdc113^−/−spermatozoa further revealed partial loss or unidentifiable “9+2” axonemal structures, along with the disruption of the fibrous sheath and outer dense fibers (Fig. 5A). We further examined the axonemal structure in Ccdc113^−/− testicular spermatids using TEM, and disorganized axonemal microtubules were detected in Ccdc113^−/−testicular spermatids (Fig. 5B). In contrast to the regular positioning of the CP and nine peripheral DMTs in the Ccdc113^+/+spermatid axoneme, the Ccdc113^−/− spermatids exhibited a scattered arrangement of DMTs, and no distinct radial spokes were observed (Fig. 5B, red arrowheads). Diffused axonemal signals could also be observed in Ccdc113^−/− testicular germ cells by using ac-tubulin immunofluorescence staining during spermiogenesis (Fig. 5B, Fig. S2C). These results indicated CCDC113 is essential for the integrity of sperm flagellum.

Fig. 5

CCDC113 has been shown to localize at the base of the RS3 and interact with neighboring axoneme-associated proteins (Fig. 1B-H). Since obvious disorganized “9+2” axonemal structure was detected in the cross-sectioned Ccdc113^−/−flagellar specimen (Fig. 5A, B), we speculated that CCDC113 likely served as an adaptor to connect the neighboring axoneme- associated proteins to DMTs. Pursuing this, we examined the flagellar localization of CFAP91 in Ccdc113^−/− spermatozoa, which is positioned in close proximity to CCDC113 at the root region of RS3, critical for the localizations of calmodulin-associated and spoke-associated complex (CSC) proteins CFAP61 and CFAP251 (Bicka et al., 2022; Meng et al., 2024).

Immunofluorescence results indicated that the absence of Ccdc113 leads to the abnormal distribution of CFAP91 on the axoneme, where CFAP91 could not colocalize with DMTs. (Fig. 5C, white asterisks and 5E). Given that DRC2 serves as the core component of the axonemal N-DRC (Jreijiri et al., 2023) and CCDC113 could bind to DRC2 (Fig 1G), we further detected DRC2 localization in the Ccdc113^+/+ and Ccdc113^−/−spermatozoa. Immunofluorescence analysis showed that DRC2 exhibited distinct signals that were not colocalized with DMTs of Ccdc113^−/−spermatozoa (Fig. 5D, white asterisks and 5F). These findings collectively indicate that CCDC113 is indispensable for the connection of CFAP91 and DRC2 to the DMTs, which is required for structural integrity of the sperm axoneme.

Ccdc113 knockout impairs head-to-tail anchorage of the spermatids

To address how Ccdc113 knockout caused acephalic spermatozoa, we initially examined wherein the flagellum detached from sperm head in Ccdc113^−/− mice. The proportion of decapitated tails in the Ccdc113^−/− caput, corpus and caudal of the epididymis exhibited similar proportions (Fig. 6A), indicating the separation of the sperm head and tail in Ccdc113^−/−mice may occur either within the seminiferous tubules or upon entering the caput of the epididymis. To confirm it, we performed PAS staining to examine the stages of spermiogenesis in Ccdc113^+/+ and Ccdc113^−/−testes (Fig. 6B). We found that Ccdc113^+/+ sperm heads in stages VII-VIII were oriented towards the basal membrane, whereas Ccdc113^−/− sperm heads were oriented towards the tubule lumen during stages VII-VIII (Fig. 6C, arrows), which may be attributed to the separation of sperm heads from flagellum during spermiogenesis. Additionally, mature sperm heads were still observable at stages IX–X in Ccdc113^−/− testes, while mature spermatozoa were released into the lumen of the seminiferous tubule at stage VIII in Ccdc113^+/+testes (Fig. 6B, red asterisk). These results suggest that the sperm head and flagellum might undergo separation during spermiation in the Ccdc113^−/− testes.

Fig. 6

Next, we examined the development of the HTCA in Ccdc113^+/+and Ccdc113^−/− spermatids using TEM. In Ccdc113^+/+ step 9–11 spermatids, the well-defined coupling apparatus consisted of basal plate, capitulum plate, segmented columns, proximal centriole, distal centriole was tightly attached to the sperm head. However, in Ccdc113^−/− step 9–11 spermatids, the abnormal HTCA detached from the sperm head (Fig.6D, red asterisk). Further observation of the HTCA structure revealed the absence of segmented columns and capitulum plate; only dense material surrounding the proximal centriole and basal plate could be detected (Fig. 6D, white arrow). The basal plates were abnormally distant from their native implantation site on the nucleus of Ccdc113^−/− elongating and elongated spermatids (Fig. 6D). Taken together, our results indicate that the disruption of Ccdc113 causes the destroyed coupling apparatus detachment from the sperm head during spermiogenesis, and CCDC113 is required for the integrity of the sperm HTCA.

CCDC113 cooperates with SUN5 and CENTLEIN to stabilize sperm HTCA

To investigate the molecular function of CCDC113 in sperm head-tail linkage, we examined interaction between CCDC113 and reported HTCA-associated proteins, including SUN5, CENTLEIN, PMFBP1, and SPATA6 (Shang et al., 2017; Yuan et al., 2015; Zhang et al., 2021; Zhu et al., 2018). GFP-tagged CCDC113 and FLAG-tagged HTCA-associated proteins were co-transfected into HEK293T cells, followed by the immunoprecipitation with anti-GFP antibody. We found that CCDC113 could interact with SUN5 and CENTLEIN, but not with PMFBP1 and SPATA6 (Fig. 7A-C, F). We further conducted co-immunoprecipitation assays in the reverse direction and verified that both SUN5 and CENTLEIN could bind to CCDC113 (Fig. 7D, E). To further investigate the localization of CCDC113 in the HTCA, we co-stained mature spermatozoa with antibodies against CCDC113 and SUN5, which has been reported to be localized on the root connecting the HTCA to the nuclear envelope (Shang et al., 2017; Zhang et al., 2021). CCDC113 was located below SUN5, showed partial overlap with SUN5 (Fig. 7G). The centriolar protein CENTLEIN was detected to localize on the HTCA and acted as the critical linker protein between SUN5 and PMFBP1 in the elongating and elongated spermatid (Zhang et al., 2021)). Given that CENTLEIN disappears in mature spermatozoa (Zhang et al., 2021), we performed the immunofluorescent staining of CCDC113 and CENTLEIN in elongated spermatids, and found that CCDC113 could partially colocalize with CENTLEIN at the HTCA (Fig. 7H). Thus, CCDC113 could interact with both SUN5 and CENTLEIN, and localize on the sperm HTCA.

Fig. 7

The HTCA localization of CCDC113 may be response for the integrity of HTCA. To test it, we detected the localization of classical HTCA component, SPATA6, in Ccdc113^−/−and Ccdc113^+/+spermatozoa by performing the immunofluorescent staining. We found that SPATA6 was not attached to the implantation fossa of the sperm nucleus (Fig. 7I, J), indicating that CCDC113 is essential for the integrity of sperm HTCA. To further understand the functional relationships among CCDC113, SUN5, and CENTLEIN at the sperm HTCA, we examined the localization of CCDC113 in Sun5^-/- and Centlein^−/− spermatozoa. Compared to the control group, CCDC113 predominantly localized on the decapitated flagellum in both Sun5^-/-and Centlein^−/− spermatozoa (Fig. 7K, L), indicating SUN5 and CENTLEIN are indispensable for the docking of CCDC113 to the implantation site on the sperm head. Taken together, these data demonstrate that CCDC113 cooperate with SUN5 and CENTLEIN to stabilize sperm HTCA and anchor the sperm head to the tail.

Discussion

CCDC113 is a highly evolutionarily-conserved component of motile cilia/flagella. Studies in the model organism Tetrahymena thermophila have revealed that CCDC113 connects RS3 to dynein g and the N-DRC, which plays essential role in cilia motility (Bazan et al., 2021; Ghanaeian et al., 2023). However, the physiological functions of CCDC113 in mammals remain unknown. In this study, we reveal that CCDC113 is indispensable for male fertility, as Ccdc113 knockout mice produce spermatozoa with flagella defects and head-tail linkage detachment (Fig. 3D). CCDC113 is localized on the sperm neck and flagellum regions in the elongating and elongated spermatids. In sperm flagellum, CCDC113 could interact with both CFAP57 and CFAP91, and serve as an adaptor protein for the connection of RS, N-DRC and DMTs to stabilize sperm flagellum. In sperm head to tail connecting piece, CCDC113 could bind to SUN5 and CENTLEIN to stabilize sperm HTCA and anchor the sperm head to the tail. Thus, CCDC113 is essential for the integrity of both sperm axoneme and sperm HTCA.

Recent cryo-EM analysis in the axonemes from Tetrahymena thermophila cilia and human respiratory cilia revealed that CCDC113 localized the linker region among RS3, N-DRC and DMTs (Bazan et al., 2021; Ghanaeian et al., 2023), and we found that CCDC113 indeed interacted with its neighboring axoneme-associated proteins, CFAP57 and CFAP91 (Fig. 1C, D). CFAP57 has been identified as the adaptor protein responsible for assembling dynein g and d, and it can interact with both N-DRC and RS3 (Ghanaeian et al., 2023; Ma et al., 2023). Previous studies demonstrated that CFAP91 extended from the base of RS2 through the N- DRC base plate to RS3, playing a crucial role in stabilizing and localizing RS2 and RS3 on the DMT (Bicka et al., 2022; Dymek et al., 2011; Gui et al., 2021). The CFAP91 ortholog, FAP91, interacts with three N-DRC subunits (DRC1, DRC2, and DRC4), participating in the docking of the N-DRC in Chlamydomonas (Gui et al., 2021). In humans, pathogenic mutations in CFAP91 and DRC2 disrupt sperm flagellum structure, result in MMAF (Jreijiri et al., 2023; Martinez et al., 2020). TEM and immunostaining experiments in spermatozoa showed severe CP and radial spokes defects in CFAP91 mutant patients (Martinez et al., 2020). In this study, we found that the absence of CCDC113 results in severe axonemal disorganization characterized by defective RSs, scattered DMTs, and misplaced CP. Further analysis demonstrated that the deficiency of CCDC113 disrupts the localization of CFAP91 and DRC2 on DMTs. Thus, CCDC113 may function as an adaptor protein to stabilize CFAP91 and DRC2 on the DMT, which facilitates the docking of RS and N-DRC to DMTs, thereby maintaining the integrity of sperm axoneme.

Recent analysis has revealed that certain centrosomal proteins play crucial roles in the assembly and maintenance of sperm HTCA (Avasthi et al., 2013; Liska et al., 2009; Zhang et al., 2021). In early round spermatids, the centriole pair initially localizes to the caudal nuclear pole and expands the electron-dense material, part of which shows striation (Wu et al., 2020). Along with the development of spermatids, the dense material around the centrioles gradually transforms into a well-organized structure, clearly identified as basal plate, capitulum plate and segmented columns (Dooher & Bennett, 1973; Wu et al., 2020). CCDC113 was initially identified through the isolation of centriolar proteins from bovine sperm (Firat-Karalar et al., 2014). During spermiogenesis, CCDC113 could colocalize with CENTRIN1/2 at the centrosome in developing spermatids, and continue to localize on the sperm neck region in elongating and elongated spermatids (Fig. 4C, D). In addition, CCDC113 could bind to HTCA associated centrosomal protein, CENTLEIN, and the disruption of Centlein impaired the attachment of CCDC113 to sperm head. In the Ccdc113^−/−spermatids, the capitulum plate and segmented columns were absent, and the basal plate were detached from the implantation site on the nucleus of Ccdc113^−/− elongating and elongated spermatids during spermiogenesis (Fig. 6D). These observations raise the possibility that CCDC113 might be a HTCA associated centrosomal protein, and play a role in maintaining the structural integrity of the HTCA.

SUN5 is a transmembrane protein located in the nuclear envelope, and acts as the root that connects the HTCA to the sperm nuclear envelope (Shang et al., 2017). CENTLEIN could directly bind to SUN5 and PMFBP1, and works as a linker between SUN5 and PMFBP1 to maintain the integrity of HTCA (Zhang et al., 2021). CCDC113 could interact with SUN5 and CENTLEIN, but not PMFBP1 (Fig. 7A-C), and CCDC113 was in the cytoplasm in Sun5^−/− and Centlein^−/−spermatozoa (Fig. 7L, K). In addition, CCDC113 colocalizes with SUN5 in the HTCA region, and the immunofluorescence staining in spermatozoa shows that SUN5 is closer to the sperm nucleus than CCDC113 (Fig. 7G, H). Therefore, SUN5 and CENTLEIN may be more closed to the sperm nucleus compared with CCDC113.

Overall, we identified CCDC113 as a structural component of both the flagellar axoneme and the HTCA, playing dual roles in stabilizing the sperm axonemal structure and maintaining the structural integrity of HTCA. As Ccdc113^−/−mice did not exhibit other ciliopathies, such as situs inversus, hydrocephalus, and abnormal ciliogenesis of tracheal cilia (Fig. S1A-D), indicating CCDC113 may specially function in spermiogenesis. Given the evolutionary conservation of CCDC113 in humans, we speculate that mutations in CCDC113 may be present in individuals with MMAF or ASS. Men with sperm flagella defects and head-tail linkage detachment may be a new type of ASS.

Materials and Methods

Phylogenetic analysis and structural similarity analysis

Amino acid sequence of CCDC113 of 7 species were downloaded from UniProt. The phylogenetic trees were constructed using MEGA 10.0 (Kumar et al., 2018) with the Neighbor-Joining (NJ) method (Saitou & Nei, 1987). 3D structures of CCDC113 orthologs of 7 species were obtained from AlphaFold Protein Structure Database (Varadi et al., 2022). Structural similarity Z scores were derived through the DALI webserver for all against all structure comparison (Holm & Laakso, 2016).

Animals

The mouse Ccdc113 gene is 44.81 kb and contains 9 exons. Exon 2 to exon 6 of Ccdc113 was chosen as the target site. The knockout mice were generated by CRISPR-Cas9 system from Cyagen Biosciences. The gRNA and Cas9 mRNA were co-injected into fertilized eggs of C57BL/6J mice to generate Ccdc113^+/–mice with a 10215 bp base deletion and 64 bp insertion. The resulting heterozygotes were interbred to obtain offspring, which further were genotyped by genomic DNA sequencing to obtain widetype and homozygote mice. The genotyping primers for knockout were: F1: 5’-TCAAATCATCACACCCTGCCTCT-3’, R: 5’-GCTTGCACTCGGGTGATACATAA-3’, and for WT mice, the specific primers were: F2: 5’-CAGGTTCTCAACACCTACAAAGTA-3’, R: 5’-GCTTGCACTCGGGTGATACATAA-3’.

All the animal experiments were performed according to approved institutional animal care and use committee (IACUC) protocols (# 08-133) of the Institute of Zoology, Chinese Academy of Sciences.

Human adult sperm sample preparation

The sperm donation candidates in this study were healthy young Chinese men. Each participant underwent a thorough medical examination and completed an extensive medical and social questionnaire to ensure the exclusion of individuals with genetic or significant medical issues, as outlined in the Basic Standard and Technical Norms of Human Sperm Bank published by the Chinese Ministry of Health. Individuals who smoked, abused drugs, or were heavy drinkers were also excluded from the study. Those who remained eligible signed informed consent forms for voluntary sperm donation and agreed to reside in Beijing for a minimum of six months. The sperm bank documented each participant’s age, date of birth, and date of semen collection. Ethical approval for this study was granted by the Reproductive Study Ethics Committee of Peking University Third Hospital (2017SZ-035). Semen samples were processed using a 40% density gradient of PureSperm (Nidacon International, Molndal, Sweden) through centrifugation at room temperature (500 × g, 30 min) and washed three times with phosphate-buffered saline (PBS). The obtained spermatozoa were utilized for immunofluorescence staining.

Antibodies

Rabbit anti-CCDC113 generated from Dia-an Biotech (Wuhan, China) was diluted at 1:500 for western blotting and a 1: 25 dilution for immunofluorescence. Mouse anti-α-TUBULIN antibody (AC012, Abclonal) was used at a 1:5000 dilution for western blotting. Mouse anti- GFP antibody (M20004, Abmart) was used at a 1:2000 dilution for western blotting. Rabbit anti-MYC antibody (BE2011, EASYBIO) was used at a 1:2000 dilution for western blotting. Rabbit anti-FLAG antibody (20543-1 AP, Proteintech) was used at a 1:2000 dilution for western blotting. Mouse anti-ac-TUBULIN antibody (T7451, Sigma-Aldrich) was used at a 1:200 dilution for immunofluorescence. Mouse anti-α/β-TUBULIN antibody (ab44928, Abcam) was used at a 1: 100 dilution for immunofluorescence. Rabbit anti-DRC2 antibody (NBP2-84617, Novus) was used at a 1:100 dilution for immunofluorescence. Rabbit anti- CFAP91 antibody (bs-9823R, Bioss) was used at a 1:100 dilution for immunofluorescence. In-house-generated mouse anti-SUN5 antibody targeting the SUN5 SUN domain (aa193–373) was used at a 1:100 dilution for immunofluorescence analysis. Rat anti-CENTLEIN antibody was generated by Absea Biotechnology Ltd (Beijing, China) was diluted at a 1:20 dilution for immunofluorescence. Rabbit anti-SPATA6 antibody (11849 -1 AP, Proteintech) was used at a 1:100 dilution for immunofluorescence. The Alexa Fluor 488 conjugate of lectin PNA (1:400, L21409, Thermo Fisher) was used for immunofluorescence. The secondary antibodies were goat anti-rabbit FITC (1:200, ZF-0311, Zhong Shan Jin Qiao), goat anti-rabbit TRITC (1:200, ZF-0316, Zhong Shan Jin Qiao), goat anti-mouse FITC (1:200, ZF-0312, Zhong Shan Jin Qiao) and goat anti-mouse TRITC (1:200, ZF0313, Zhong Shan Jin Qiao).

Sperm motility and sperm count assays

The cauda epididymis was isolated from 8 weeks of mice. Sperm were released in phosphate- buffered saline (PBS, Gibco, C14190500BT) from the incisions of the cauda epididymis for 10 min at 37 ℃. And then the swim-up suspension was used for the analysis of sperm motility with a microscope through a 20x phase objective. Viewing areas in each chamber were imaged using a CCD camera. The samples were analyzed via computer-assisted semen analysis (CASA) using the Minitube Sperm Vision Digital Semen Evaluation System (12500/1300, Minitube Group, Tiefenbach, Germany) and were also analyzed by a computer- aided semen analysis system (CASA). The incubated sperm number was counted with a hemocytometer.

Histology staining

As previously reported (Wang et al., 2018), the testes and caudal epididymis were dissected after euthanasia, and fixed with Bouin’s fixative for 24 h at 4 ℃, then the testes were dehydrated with graded ethanol and embedded in paraffin. For histological analysis, the 5 μm sections were cut and covered on glass slides. Sections were stained with H&E and PAS for histological analysis after deparaffinization.

Electron microscopy analysis

The cauda epididymis and testis were dissected and fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer at 4℃ overnight. After washing in 0.1 M cacodylate buffer, samples were cut into small pieces of approximately 1 mm³, then immersed in 1% OsO4 for 1 h at 4℃. Samples were dehydrated through a graded acetone series (50%, 60%, 70%, 80%, 90%, 95%, 100%) and embedded in resin (DDSA, NMA, enhancer, 812) for staining. Ultrathin sections were cut and stained with uranyl acetate and lead citrate. Images were acquired and analyzed using a JEM-1400 transmission electron microscope.

Scanning electron microscopy

The trachea was fixed in 2.5% glutaraldehyde solution overnight, and dehydrated in graded ethanol, subjected to drying and coated with gold. The images were acquired and analyzed using SU8010 scanning electron microscope.

Immunofluorescence staining

The testis albuginea was peeled and incubated with collagenase IV and hyaluronidase in PBS for 15 min at 37℃, then washed twice with PBS. Next, fixed with 4% PFA for 5 min, and then coated on slide glass to dry out. The slides were washed with PBS three times and then treated with 0.5% TritonX-100 for 5 min, and blocked with 5% BSA for 30 min. Added the primary antibodies and incubated at 4℃ overnight, followed by incubating with a second antibody and DAPI. The images were taken using LSM880 and SP8 microscopes.

The mouse testis was immediately dissected and fixed with 2% paraformaldehyde in 0.05% PBST (PBS with 0.05% Triton X-100) at room temperature for 5 min. The fixed sample was placed on a slide glass and squashed by placing a cover slip on top and pressing down. The sample was immediately flash frozen in liquid nitrogen, and the slides were stored at −80°C for further immunofluorescence experiments. After removing the coverslips, the slides were washed with PBS three times and then treated with 0.1% Triton X-100 for 10 min, rinsed three times in PBS, and blocked with 5% bovine serum albumin (Amresco, AP0027). The primary antibody was added to the sections and incubated at 4°C overnight, followed by incubation with the secondary antibody. The nuclei were stained with DAPI. The immunofluorescence images were taken immediately using an LSM 780 microscope (Zeiss) or SP8 microscope (Leica).

Spermatozoa were released from the cauda epididymis in PBS at 37°C for 15 min, then were spread on glass slides for morphological observation or immunostaining. After air drying, spermatozoa were fixed in 4% PFA for 5 min at room temperature, and slides were washed with PBS three times and blocked with 5% BSA for 30 min at room temperature. The primary antibodies were added to the sections and incubated at 4°C overnight, followed by incubation with the secondary antibody. The nuclei were stained with DAPI and images were taken using an LSM 880 microscope (Zeiss) or SP8 microscope (Leica).

Immunoprecipitation

Transfected cells were lysed in a lysis buffer (50mM HEPES, PH 7.4, 250mM NaCl, 0.1% NP-40 containing PIC and PMSF) on ice for 30 min and centrifugated at 12000 rpm at 4℃ for 15 min, cell lysates were incubated with primary antibody about 12 hours at 4°C and then incubated with protein A-Sepharose (GE, 17-1279-03) for 3 hours at 4°C, then washed three times with lysed buffer and subjected to immunoblotting analysis.

Statistical analysis

All the experiments were repeated at least three times, and the results are presented as the mean ± SD. The statistical significance of the differences between the mean values for the different genotypes was measured by the Student’s t-test with a paired, 2-tailed distribution. The data were considered significant when the P-value was less than 0.05(*), 0.01(**), 0.001(***) or 0.0001(****).

Acknowledgements

This work was supported by the National Science Fund for Distinguished Young Scholars (Grant No. 81925015), National Natural Science Foundation of China (Grant No. 32230029, 82371615) and National Key Research and Development Program of China (Grant No. 2022YFC2702600).

Author Contributions

B.W. and C.L. performed most of the experiments and wrote the manuscript. Y.Y., Z.Z., and H.J. provided human adult sperm sample and performed some of the immunofluorescence experiments. S.M., Y.M., H.W., and J.L. contributed to the cell experiments and animal breeding. W.L. and C.L. supervised the whole project and revised the manuscript.

Declaration of interests

The authors declare that they have no conflict of interest.