Identification of two CCDC146 gene variants in MMAF patients

(A) Position of the observed variants in both probands. Electropherograms indicating the homozygous state of the identified variant: variant c.1084C>T is a nonsense mutation, and c. c.2112Del is a single- nucleotide deletion predicted to induce a translational frameshift. (B) Structure of the canonical CCDC146 gene transcript showing the position of the variants and their impact on translation. Variants are annotated according to HGVS recommendations.

Ccdc146 KO mice are infertile and KO sperm exhibit a typical “multiple morphological abnormalities of the flagellum (MMAF)” phenotype. (A) Number of pups produced by wild-type (+/+, WT), CCDC146 heterozygote (+/-) and CCDC146 knock- out (-/-, KO) males (3 males per genotype) after mating with fertile WT females (2 females per male) over a period of three months. (B) Sperm concentration and (C) sperm mobility. (D) Comparison of testis weights (mg). (E) Illustration of WT and KO sperm morphologies stained with Papanicolaou and observed under optic microscopy. Statistical comparisons were based on ordinary one-way ANOVA tests (**** p<0.0001; *** p<0.001, **p<0.01, *p<0.05). Scale bar of images represents 10 µm.

Histological evidence that spermiogenesis is disrupted in Ccdc146 KO males and leads to a strong decrease in sperm concentration in the epididymis.

(A) Transversal sections of WT and KO testis stained with hematoxylin and eosin. The upper images show the sections at low magnification (Scale bars: 50 µm) and the lower images are an enlargement of the dotted square (scale bars: 10 µm). In the KO, spermatid nuclei were very elongated and no flagella were visible within the seminiferous tubule lumen. (B) Transversal sections of WT and KO epididymis stained with hematoxylin and eosin. Despite similar epididymis section diameters in WT and KO testes, KO lumen were filled with round cells and contained few spermatozoa with abnormally- shaped heads and flagella. The upper images show the sections at low magnification (scale bars 50 µm) and the lower images are an enlargement of the dotted square (scale bars 10 µm).

CCDC146 has a centriolar and pericentriolar localization in interphase somatic HEK-293T cells.

HEK-293T cells were immunolabeled for β-tubulin (green), centrin (yellow) and CCDC146 (magenta). DNA was stained with Hoechst (blue). (A) CCDC146 localized to centrioles and/ or to the pericentriolar material in interphase cells. The centrosomes of two cells are shown enlarged in A1 and A2. (B) In serum starved cells with primary cilia, CCDC146 localizes to the basal body of primary cilia. The basal body of two cells are shown enlarged in B1 and B2. CCDC146 is also present as dotted signal resembling the pattern for centriolar satellite proteins. Scale bars on zoomed images: 2 µm.

CCDC146 is a microtubule-associated protein (MAP) associating with microtubule-based structures throughout the cell cycle.

HEK-293T cells were immunolabeled with anti-β-tubulin (green), anti-centrin (yellow), and anti- CCDC146 (magenta) Abs. DNA was stained with Hoechst (blue). In synchronized HEK-293T cells, CCDC146 is observed associated with (A) mother centrioles and their corresponding procentrioles during centriole duplication. (B) In non-synchronized cells, CCDC146 is observed associated with microtubule tips during metaphase, (C) the central spindle during (C) anaphase and (D) telophase, and (E) the midbody during cytokinesis. Images on the right show the enlargement of the dotted square in the left image. Scale bar of zoomed images 2 µm.

CCDC146 is expressed in late spermatocyte and in spermatids; CCDC146 protein is present in spermatids and in epididymal spermatozoa in mouse.

(A) mRNA expression levels of Ccdc146 relative to Actb and Hprt in CCDC146-HA mouse pups’ testes. Extremely low expression was detected at day 9, corresponding to testes containing spermatogonia and Sertoli cells only. Ccdc146 expression was observed from postnatal day 18 (formation of secondary spermatocytes), peaked at day 26 (formation of elongated spermatids), and subsequently decreased from day 35 (formation of spermatozoa), suggesting that Ccdc146 is particularly expressed in elongated spermatids during spermatogenesis. (B) Western blot of HA-tagged and WT sperm showing the specific band (arrow) corresponding to HA-tagged CCDC146 (CCDC146-HA) in epididymal whole sperm. (C) Western blot of HA-tagged testis extracts at different postnatal days. The presence of HA- tagged CCDC146 was revealed by an anti-HA Ab. Faint bands at the expected molecular weight are observed at D26, D35 and in epididymal sperm.

CCDC146 localizes to the flagellum but not to the centrioles of ejaculated human spermatozoa.

(A) Human ejaculated sperm were immunolabeled with Abs recognizing centrin (yellow), CCDC146 (magenta), and β-tubulin (green). DNA was stained with Hoechst (blue). (A1-A3) enlargement of dotted square focused on sperm neck: no colocalization between CCDC146 and centrin. (A4) A faint signal for CCDC146 is present along the length of the sperm flagellum. Scale bar of zoomed images: 1 µm. (B) Human ejaculated sperm observed by expansion microscopy. Sperm were immunolabeled with anti- CCDC146 (magenta) and anti-β-tubulin (green), and DNA was stained with Hoechst (blue). B1 and B5 show strong staining for CCDC146 in the axoneme, B3 showing the localization of the axoneme through tubulin staining. B2, B4 and B6 show enlargements of the dotted square focused on the sperm neck. CCDC146 did not colocalize with the centrioles at the base of the axoneme. The CCDC146 staining observed probably corresponds to non-specific labeling of mitochondria.

CCDC146 localizes to the flagellum of mouse epididymal spermatozoa.

(A) Mouse epididymal spermatozoa observed with conventional IF. CCDC146-HA sperm were labeled with anti-HA #1 (red) and anti-β-tubulin (green) Abs. DNA was stained with Hoechst (blue). The upper image shows the sperm with merged immunostaining, and the lower images, the staining (red, green, and merge) observed in the principal piece of the flagellum (Scale bars 10 µm). (B) Mouse epididymal spermatozoa observed with expansion microscopy. CCDC146-HA sperm were immunolabeled with anti-HA #2 (red) and anti-β-tubulin (green) Abs. DNA was stained with Hoechst (blue). The upper image shows the sperm with merged immunostaining. The lower images show the staining (red, green, and merge) observed in the principal piece of the flagellum. Strong punctiform signals were observed at the level of axonemal breakages induced by the expansion process. White arrows indicate the zones of the micro breaks (Scale bars correspond to 10 µm). (C) Similar experiment performed with a third anti-HA Ab (#3). Scale bars 10 μm. (D) Western blot of whole sperm and flagella fraction extracts. The presence of CCDC146-HA was revealed by an anti-HA Ab.

CCDC146 localizes to the microtubule doublets of the axoneme in human and mouse

(A) Sperm was double-stained with anti-tubulin Ab (green) and anti-CCDC146-Ab (magenta) and observed by expansion microscopy. (B) The position of the CCDC146 signal with respect to the tubulin signal was measured along the entire flagellum. CCDC146 signals were localized to different compartments of the axoneme by the following method: the green signal is quite characteristic with two peaks corresponding to the left and right microtubule doublet, it identifies five axonemal compartments (left outer, left doublet, central pair, right doublet, and right outer). For each CCDC146 signal (magenta dots), the flagellum was perpendicularly cut (example, white bar in A) and the tubulin and CCDC146 signals measured to assign each CCDC146 signal to a localization area. (C) Example of a measurement obtained at the right bar shown in (A). (D) Histogram showing the distribution of CCDC146 labeling in ejaculated human sperm 1, presented in (A). (E) Ejaculated human sperm double- stained with anti-tubulin Ab (green) and anti-CCDC146-Ab (magenta). (F) Histogram showing how CCDC146 labeling distributed in ejaculated human sperm 2. (G) Flagellum of a mouse epididymal spermatozoa observed with expansion microscopy. CCDC146-HA sperm were immunolabeled with anti-HA #2 (red) and anti-β-tubulin (green). The upper image shows the sperm with merged immunostaining, and the lower images, the staining (red, green, and merge) observed in the principal piece of the flagellum. Scale bars 10 µm.

CCDC146 could be a microtubule inner protein in sperm: evidence from sarkosyl treatment

(A) Western blot of WT and CCDC146-HA sperm extract solubilized with N-lauroylsarcosine (sarkosyl), an anionic detergent. Sarkosyl was used at increasing concentrations (0.2, and 0.4%). The presence of CCDC146-HA was detected by an anti-HA Ab. (B) Western blot of WT and CCDC146-HA sperm extracts solubilized with alternative detergents (RIPA, CHAPS, Tris-HCl) and whole sperm extract solubilized in Laemmli. The presence of CCDC146-HA was revealed by an anti-HA Ab. (C) Epididymal CCDC146-HA sperm (C1-C2) and WT sperm (C3-C4), treated with sarkosyl (5 min, 0.2% sarkosyl) or not (NT), were immunostained to reveal the HA-tag (red) and counterstained with Hoechst (blue). (C1) Without treatment, a faint CCDC146 signal (white arrow heads) is observed along the flagellum from CCDC146- HA sperm. (C2) Treatment with sarkosyl enhanced the CCDC146-HA signal along the sperm flagellum. (C3) HA signal in WT non-treated (NT) sperm is present in the midpiece only, suggesting that this signal is not specific. (C4) The HA signal in WT sperm is not enhanced by sarkosyl treatment. Scale bars 10 μm.

The absence of CCDC146 causes defects of the head-tail coupling apparatus in epididymal spermatozoa and duplication and mislocalization of centriole in testicular sperm.

(A) Scanning electron microscopy of WT and Ccdc146 KO epididymal spermatozoa showed aberrant head morphologies and irregular head-tail coupling apparatus (HTCA) linking the sperm head with the flagellum. (B) Testicular spermatozoa from WT (B1) and Ccdc146 KO (B2-B6) mice immunolabeled with anti-β-tubulin (magenta) and anti-centrin (B1-B3) or anti-γ-tubulin (B4-B6) (green) Abs. Centrioles appeared to be normal (B2) in some spermatozoa, separated but partially attached to the head (B3, B4), completely detached from the sperm head (B5) or duplicated (B6).

Absence of elongation of axonemal microtubules at the base of the distal centriole.

(A) In WT spermatids, the proximal centriole (PC) is linked to the base of the compacting nucleus (Nu) through the basal plate (BP) and the capitulum (Ca), and the distal centriole (DC) is embedded in the segmented column (SC). All these sperm-specific cytoskeletal structures make up the HTCA. At the base of the distal centriole, axonemal microtubules (MT) grow. In Ccdc146 KO elongating spermatids, the overall structure of the HTCA is conserved, with the presence of the centrioles and the accessory cytoskeletal structures. However, no axonemal microtubules are visible. (B) The adjunct (Ad) of the proximal centriole is also preserved in Ccdc146 KO spermatids. (C) Serial sections of the HTCA of a Ccdc146 KO spermatid confirm the absence of axonemal microtubules at the base of the distal centriole during spermatid elongation. Scale bars 1 µm.

Analysis of stages of spermatogenesis by IF reveals acrosome formation and manchette elongation defects.

Cross sections of WT and Ccdc146 KO testes showing different stages of mouse spermatogenesis (I, VII-XII). Stages were determined by double immunostaining for β-tubulin (red) and DPY19l2 (green; acrosome localization), and DNA was stained with Hoechst (blue). (A) Very few mature spermatozoa lined the lumen at stage VII-VIII in the KO. Cell orientations appear random from stage IX-X in the KO and the tubules contain more advanced spermatid stages. The acrosome of elongating KO spermatids had become abnormal by stage XII, and the manchette of elongated spermatids at stage I was longer in the KO compared with the WT. (B) Typical spermatids from stages I-XII, showing details of acrosome formation and manchette elongation. Scale bars stages VII-VIII 20 µm, scale bars IX-X, XII and I 10 µm.

TEM of elongating spermatids from Ccdc146 KO male shows ultrastructural defects of the manchette.

(A, B) Ultrastructural analysis of the manchette in WT elongating spermatids shows the normal thin perinuclear ring, anchored below the acrosome and allowing a narrow array of microtubules to anchor. (C-F) In elongating spermatids from CCDC146 KO animals, the perinuclear ring was abnormally broad, usually located on one side of the spermatid (red arrows), creating an asymmetric and wide bundle of microtubules. The resulting manchette was wider and often longer than in WT animals. (E, F) The tubulin nucleation location was sometimes ectopic in the KO and coincided with irregularly shaped sperm heads.

The axonemes of Ccdc146 KO spermatids present multiple defects visible under TEM.

(A1) A longitudinal section of a WT flagellum shows a typical structure of the principal piece, with outer dense fibers (ODF) at the periphery, microtubules (MT) in the center, and mitochondria (Mi) aligned along the flagellum. (B1) A longitudinal section of a Ccdc146 KO flagellum shows a disorganized midpiece, with altered mitochondria, the presence of an amorphous fibrous sheath (FS) and altered microtubules. (B) Longitudinal section of a Ccdc146 KO sperm showing dispersed and non-assembled flagellar material in a cytoplasmic mass. The right-hand image is the enlargement of the red square, showing the presence of an external ring of mitochondria surrounding an outer dense fiber ring devoid of microtubular material. (C) Longitudinal section of another Ccdc146 KO sperm showing a similar abnormal midpiece structure. The right-hand image is the enlargement of the red square, showing the presence of an external ring of mitochondria surrounding an outer dense fiber ring devoid of microtubular material.

Relative mRNA expression levels for human and mouse CCDC146 transcripts.

(A) CCDC146 mRNA levels measured in different tissues/cells in humans using Affymetrix microarrays (data available from the Genevestigator database, https://genevestigator.com). Red rectangles highlight the high expression level in male reproductive organs. (B) Similar data for mice. Data were generated with Genevestigator (Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W and P Zimmermann (2008) Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Advances in Bioinformatics 2008, 420747)

Molecular strategy used to generate Ccdc146 KO mice by CRISPR/Cas9

The exonic structure of mouse Ccdc146 is shown and the coding sequence indicated in black. Exon 2, the first coding sequence, was targeted by an RNA guide (5’-CCT ACA GTT AAC ATT CGG G-3) and the Cas9 induced a deletion of four nucleotides upstream the PAM sequence, as indicated by the red box. Electropherogram presenting the WT and the homozygote deletion are shown.

Increased levels of apoptosis in testes from Ccdc146 KO mice.

The TUNEL assay was used to visualize double-strand DNA breaks, as an indication of the level of apoptosis during WT and KO spermatogenesis. (A) Comparison of the % of tubules per testis cross-section containing at least one TUNEL-positive cell in WT, heterozygote, and Ccdc146 KO animals. Number of sections counted per genotype n=13-20, 3 different mice per genotype. (B) The majority of TUNEL-positive cells in the WT corresponded to pachytene cells undergoing meiosis (B, WT zoomed image) whereas the localization of TUNEL-positive cells in KO was more scattered. (C) Control testis section treated with H202. Statistical comparisons according to ordinary one-way ANOVA test (**** p<0.0001; *** p<0.001, **p<0.01, *p<0.05). Scale bar of zoomed images 50 µm.

CCDC146 does not colocalize with the centriolar satellite marker PCM1.

(A) HEK-293T cells were double immunolabeled for PCM1 (cyan) and CCDC146 (magenta). (A1-A4) Images on the right show the enlargement of the dotted squares in the left image. PCM1 surrounds the CCDC146 signal, but no colocalization is observed, suggesting that CCDC146 is not a centriolar satellite protein. DNA was stained with Hoechst (blue). Scale bars on zoomed images represent 2 µm.

CCDC146 shows a similar localization to the centrosome and to the midbody in primary HFF cells.

(A) Primary human foreskin fibroblast (HFF) cells were triple immunolabeled with anti-β-tubulin (green), anti-centrin (yellow, showing the centrioles) and anti-CCDC146 (magenta). (A1) The right-hand images show the enlargement of the dotted squares in the left-hand image. CCDC146 localized to and around the centrioles. (B) similar staining showing co-labeling of the midbody during cytokinesis by anti-CCDC146 Abs (B2). Scale bar of zoomed images 2 µm.

Molecular strategy used to generate HA-tagged CCDC146 mice by CRISPR/Cas9

The reference CCDC146 mouse transcript is ENSMUST00000115245.7 (GRCm38.p6). Exon 2, the first coding sequence, was targeted by an RNA guide (5’- TAC TTT AGA ACT GTG AAA AAT GG -3’). We used a single-stranded DNA (ssDNA) template to insert the HA sequence (5’-TAC CCA TAC GAT GTT CCA GAT TAC GCT-3’) upstream of the PAM sequence.

Centrioles are identified by anti-POC5 Abs in expanded human ejaculated spermatozoa.

Human control sperm were co-stained, after expansion, with anti- α+β-tubulin (green) and anti-POC5 (magenta) Abs. The centrosomal protein POC5 locates to centrioles at the base of the axoneme. Scale bars 5 μm.

Non-specific midpiece staining in mouse sperm by rabbit secondary antibody.

Mouse sperm were stained with secondary antibodies only. Scale bars 10 µm.

Sperm sarkosyl treatment corroborates the presence of CCDC146 along the mouse flagellum.

CCDC146-HA sperm (A1-A2) and epididymal WT (A3-A4) not treated or treated (5 min, 0.2% sarkosyl), were immunostained with anti-HA Ab (red) and counterstained with Hoechst (blue). (A1) Without treatment, a faint CCDC146-HA signal was observed along the CCDC146-HA principal piece. The strong staining in the midpiece is not specific (see panel B) (A2) After treatment with sarkosyl, the CCDC146- HA signal along the sperm principal piece was enhanced (white arrows), whereas the signal in the midpiece decreased. (A3) WT untreated (NT) sperm exhibited almost no CCDC146-HA signal in the principal piece. The midpiece is however strongly stained (not specific see panel B) (A4) The HA signal is not enhanced in WT principal piece by sarkosyl treatment, suggesting that the enhanced signal observed with sarkosyl on CCDC146-HA sperm is specific. (B) CCDC146-HA sperm were immunolabeled with secondary antibody only. Strong staining is observed on the midpiece, confirming its non-specific nature. Scale bars 10 μm.

Lack of CCDC146 causes centriole duplication and mislocalization in Ccdc146 KO spermatids.

Ultrastructural analysis of centrioles in adult mouse WT (A, B) and Ccdc146 KO (C-H) testis sections. (A) In WT spermatids, the proximal centriole is linked to the base of the compacting nucleus through the basal plate and the capitulum, and the distal centriole is embedded in the segmented column. These sperm-specific cytoskeletal structures make up the head-to-tail coupling apparatus (HTCA). (B) In WT elongated spermatids, the different components of the axonemal structures (microtubules and ODF) were visible downstream the distal centriole. (C) In Ccdc146 KO elongating spermatids, the overall structure of the HTCA was conserved, with the presence of the centrioles and the accessory cytoskeletal structures. However, the HTCAs were often duplicated (C, E, F) and separated from their usual nuclear attachment site (C-F, H), and sometimes misplaced far away from the nucleus (F), likely preventing axoneme elongation. (F) The arrow indicates the misplaced centrioles at the end of the manchette. (G, H) In elongated spermatids with condensed nucleus, malformed and detached centrioles with poorly-assembled or missing flagella compared to the WT (B) can be seen.

Spermatid head shape is aberrant in the absence of CCDC146.

Comparative ultrastructural analysis of the spermatid head in WT (A-D) and Ccdc146 KO (E-H) testis sections. (A, E) Spermatid nuclei at the beginning of elongation. KO spermatid nuclei showing nuclear membrane invaginations and irregular shape that were not present in the WT. The acrosome in the KO sperm appeared intact. (B, F) Spermatid nuclei in elongating spermatids. Whereas nucleus elongation is symmetric in the WT, in the CCDC146-KO, more pronounced head invaginations are observed. (C, G) Elongated spermatids. Although nuclear condensation appeared normal in both WT and KO nuclei, vacuolization is observed in KO nucleus. (D, H) Elongated KO spermatids showed malformed elongated nuclear shapes with frequent invaginations and poorly-assembled flagella (H) compared to the WT (D).