CCDC113 is an evolutionarily conserved axoneme-associated protein.

(A) Multiple species phylogenetic tree of CCDC113. Structural similarity scores (Z scores) of CCDC113 orthologs in Homo sapiens, Mus musculus, Phascolarctos cinereus, Danio rerio, Chlamydomonas reinhardtii and Tetrahymena thermophila were derived through the DALI webserver for pairwise structure comparisons (Holm & Laakso, 2016). (B) Positioning of CCDC113 within the 96-nm repeat of human axoneme (Walton et al., 2023). CCDC113 form a complex with CCDC96, is located at the base of RS3 and adjacent to CFAP91 and CFAP57. CFAP91 originates at the base of RS2 and links the RS3 subunits (CFAP251 and CFAP61). (C-H) Neighboring axoneme-associated proteins were expressed or co-expressed with CCDC113 in HEK293T cells, and the interactions of CCDC113 with CFAP57, CFAP91, TUBB5, DRC1, DRC2 and DRC3 were examined through co-immunoprecipitation. IB, immunoblotting; IP, immunoprecipitation.

Ccdc113 knockout leads to male infertility.

(A) The CRISPR-Cas9 strategy for generating the Ccdc113 knockout mice. (B) Genotyping to identify Ccdc113 knockout mice. (C) Immunoblotting of CCDC113 in Ccdc113+/+and Ccdc113−/− testes. TUBULIN served as the loading control. (D) The average litter size of Ccdc113+/+ and Ccdc113−/− female mice in 2 months (n = 5 independent experiments). Data are presented as mean ± SD. ns: indicates no difference. (E) The average litter size of Ccdc113+/+ and Ccdc113−/− male mice in 2 months (n = 5 independent experiments). Data are presented as mean ± SD. ****P < 0.0001. (F) The size of testes was similar in Ccdc113+/+ and Ccdc113−/− mice. (G) The testis weights of Ccdc113+/+ and Ccdc113−/− male mice (n = 5 independent experiments). Data are presented as mean ± SD. ns: indicates no difference. (H) The body weights of Ccdc113+/+and Ccdc113−/− male mice (n = 5 independent experiments). Data are presented as mean ± SD. ns: indicates no difference. (I) The ratio of testis weight/body weight in Ccdc113+/+and Ccdc113−/− male mice (n = 5 independent experiments). Data are presented as mean ± SD. ns: indicates no difference. (J) H&E staining of testes sections from Ccdc113+/+ and Ccdc113−/− male mice. Red arrowheads indicate the abnormal sperm flagellum in the Ccdc113−/− testis seminiferous tubule. (K) Immunofluorescence of acetylated-tubulin (red) in testes sections from Ccdc113−/− male mice show flagellar defects.

Ccdc113 knockout results in sperm flagella defects and sperm head-tail detachment. (A) H&E staining of the caudal epididymis from Ccdc113+/+and Ccdc113−/− male mice in 2 months. Red circles indicate coiled eosin-stained structures without sperm heads in the epididymal lumen. (B) Analysis of sperm counts in Ccdc113+/+ and Ccdc113−/−male mice (n = 5 independent experiments). Mature spermatozoa were extracted from unilateral cauda epididymis and dispersed in PBS. Sperm counts were measured by hemocytometers. Data are presented as the mean ± SD. ****P < 0.0001. (C) Motile sperm in Ccdc113+/+ and Ccdc113-/-mice (n = 5 independent experiments). Data are presented as mean ± SD. ****P < 0.0001. (D) Ccdc113+/+ and Ccdc113−/− spermatozoa were co-stained with a flagellar marker α/β-tubulin (red) and an acrosomal marker PNA. Nuclei were stained with DAPI (blue). (E) Quantification of different categories of Ccdc113+/+, Ccdc113−/− spermatozoa (n = 3 independent experiments). Data are presented as the mean ± SD.

CCDC113 localizes to the HTCA, manchette and sperm flagellum.

(A) CCDC113 was predominately expressed in testis and slightly in lung. Immunoblotting of CCDC113 was performed in the spleen, intestine, lung, thymus, testis and ovary. Asterisks indicate unspecific bands. TUBULIN served as the loading control. (B) CCDC113 was expressed starting in P7 testes. TUBULIN served as the loading control. Asterisks indicate unspecific bands. (C) Immunofluorescence of CCDC113 (red) and CENTRIN1/2 (green) in developing germ cells. CCDC113 partially colocalize with centriolar protein CENTRIN1/2. (D) Immunofluorescence of CCDC113 (red) and α/β-tubulin (green) in developing germ cells. The manchette was stained with the anti-α/β-tubulin antibody. (E-F) CCDC113 localizes to the HTCA and flagellum in mature mouse spermatozoa and human spermatozoa. Nuclei were stained with DAPI (blue).

CCDC113 is indispensable for the docking of CFAP91 and DRC2 to the DMTs to maintain the structural integrity of the axoneme. (A) Transmission electron microscopy (TEM) analysis of spermatozoa from the cauda epididymidis of Ccdc113+/+and Ccdc113−/− male mice. The flagellar longitudinal sections of Ccdc113−/− spermatozoa revealed unremoved cytoplasm, including disrupted mitochondria, damaged axonemes. and large vacuoles. Asterisks indicate large vacuoles. Cross sections of the principal piece of Ccdc113−/− spermatozoa further revealed partial loss or unidentifiable “9+2” structures, along with the disruption of the fibrous sheath and outer dense fibers. (B) Transmission electron microscopy (TEM) analysis of the axoneme in testicular spermatids from Ccdc113+/+ and Ccdc113−/− male mice. The red arrowheads indicate the absence of significant radial spokes (RSs). MS: mitochondrial sheath, Mi: mitochondrial, AX: axoneme. FS: fibrous sheath. DMT: doublet microtubule, MT: microtubule, CP: central pair, ODF: outer dense fiber, RS: radial spokes. (C) The immunofluorescence analysis for CFAP91 (green) and α/β-tubulin (red) was performed in Ccdc113+/+and Ccdc113−/− spermatozoa. Nuclei were stained with DAPI (blue). White asterisks indicate regions that are not co-located with tubulin. (D) The immunofluorescence analysis for DRC2 (green) and α/β-tubulin (red) was performed in Ccdc113+/+ and Ccdc113−/− spermatozoa. Nuclei were stained with DAPI (blue). White asterisks indicate regions that are not colocated with tubulin. (E-F) Line-scan analysis (white line) using the Image J software.

Ccdc113 knockout spermatids display impaired HTCA. (A) The proportion of decapitated tails in Ccdc113+/+ and Ccdc113−/− corpus, caput and cauda epididymis (n=3 independent experiments). Data are presented as the mean ± SD. ****P < 0.0001. ***P < 0.001. (B) PAS staining of testes sections from Ccdc113+/+ and Ccdc113−/− mice. The green arrows indicate the orientation of the sperm heads in stages V–VIII seminiferous epithelia. Ccdc113−/− sperm head could still be detected in stages IX–X seminiferous epithelia. P: pachytene spermatocyte, spz: spermatozoa, rSt: round spermatid, eSt: elongating spermatid, Z: zygotene spermatocyte, M: meiotic spermatocyte. (C) Ccdc113−/− spermatids lost their head orientation toward the basement membrane during spermiation in stages VII–VIII seminiferous epithelia. L: lumen, B: basement membrane. (D) Defective HTCA formation in Ccdc113−/−spermatids. TEM analyses of the stepwise development of the HTCA were performed in Ccdc113+/+ and Ccdc113−/− testes. In Ccdc113+/+ spermatids, the well-defined coupling apparatus was tightly attached to the sperm head. In Ccdc113−/− spermatids, segmented columns (Scs), the capitulum (Cp) were absent. The red asterisks indicate the distance between the sperm head and HTCA. The white arrows indicate the dense material surrounding the proximal centriole. Nu: nuclear, Bp: basal plate, Cp: capitulum, Sc: segmented column, Pc: proximal centriole, Dc: distal centriole, An: annulus, Ax: axoneme, Rn: redundant nuclear envelope.

CCDC113 interact with SUN5 and CENTLEIN, participating in sperm head-tail linkage.

(A-C, F) HTCA-associated proteins (SUN5, CENTLEIN, PMFBP1, SPATA6) were expressed or co-expressed with CCDC113 in HEK293T cells, and the interactions of CCDC113 with SUN5, CENTLEIN were examined through co-immunoprecipitation. CCDC113 did not interact with PMFBP1 and SPATA6. IB, immunoblotting; IP, immunoprecipitation. (D) SUN5 interacted CCDC113. pCDNA-FLAG-Ccdc113 and pEGFP- GFP-Sun5 were transfected into HEK293T cells. At 48 h after transfection, the cells were collected for immunoprecipitation (IP) with anti-GFP antibody and analyzed with anti-FLAG and anti-GFP antibodies. (E) CENTLEIN interacted CCDC113. pCMV-FLAG-Centlein and pEGFP-GFP-Sun5 were transfected into HEK293T cells. At 48 h after transfection, the cells were collected for immunoprecipitation (IP) with anti-FLAG antibody and analyzed with anti- FLAG and anti-GFP antibodies. (G) Immunofluorescence of CCDC113 (red) and SUN5 (green) in mature spermatozoa. Nuclei were stained with DAPI (blue). (H) Immunofluorescence of CCDC113 (red) and CENTLEIN (green) in testicular step13-step14 spermatid. Nuclei were stained with DAPI (blue). (I) Immunofluorescence analysis for SPATA6 (green) and α/β-tubulin (red) was performed in Ccdc113+/+ and Ccdc113−/− spermatozoa. Nuclei were stained with DAPI (blue). (J) Quantification ratio of SPATA6 on the detached sperm tail (n =3 independent experiments). At least 200 spermatozoa were analyzed for each mouse. (K) Quantification ratio of CCDC113 on the detached sperm tail (n =3 independent experiments). At least 200 spermatozoa were analyzed for each mouse. (L) Immunofluorescence analysis for CCDC113 (red) was performed in wild type (WT), Sun5−/− and Centlein−/− spermatozoa. Nuclei were stained with DAPI (blue).