CCDC181 is required for sperm flagellum biogenesis

ABSTRACT


INTRODUCTION
Prevalence of male infertility in the general population approximately ranges between 9% and 15%, according to the available surveys of infertility cases (Barratt et al., 2017).Many defects in sperm development arise from genetic causes, including mutations in structural proteins of the sperm flagella, which are common causes of male infertility (Touré et al., 2021).The manchette is a temporary structure that surrounds the head of elongating spermatids, which is necessary for head shaping and protein delivery during spermatid elongation (O'donnell and O'bryan, 2014).The head shaping and tail assembly involved in the sperm deformation process require intramanchette transport (IMT) and intraflagellar transport (IFT) (Nakayama and Katoh, 2020).Cargo proteins are transported through the manchette to the base of the sperm flagellum by IMT, and into the developing sperm flagellum by IFT (Lehti and Sironen, 2016).
In the present study we demonstrate that the Ccdc181 knockout mouse model represents abnormal manchette and characteristic MMAF manifestations.Additionally, we identified the interacting protein of CCDC181 through immunoprecipitation and mass spectrometry analysis.Collectively, this study demonstrates a specific role for CCDC181 in regulating spermatogenesis with genetic deficiency leading to MMAF and male infertility in mice.

Mouse model
Ccdc181 −/− mice were generated by CRISPR/Cas9 technology (Wang et al., 2013).Briefly, guide RNAs (gRNA1 and gRNA2), targeting exon 2 for generating Ccdc181 knockout mice were transcribed in vitro (Invitrogen, Thermo Fisher Scientific, AM1908).gRNAs were co-injected with Cas9 mRNAs into C57BL/6J zygotes.Genomic DNA samples of founders were extracted from mouse toes and used for detecting the mutations by Sanger sequencing.
The founder mice (F0 generation), heterozygous for the mutation of interest, were backcrossed to wild-type (WT) C57BL/6J mice for at least one generation,

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total tissue RNA was extracted using Trizol reagent, followed by cDNA synthesis using the PrimeScript RT Reagent Kit (TaKaRa, RR047A) according to the manufacturer's protocol.Phanta Flash Super-Fidelity DNA Polymerase (Vazyme #P510) was used for PCR.The PCR reactions were performed under the following conditions: 30 s at 98°C, 38 cycles of 10 s at 98°C, 3 s at 55°C, and 10 s at 72°C.The PCR products were electrophoresed on a 2% agarose gel, followed by Sanger sequencing.Beta-actin (Actb) was used as an internal control.The primers used were as follows: for Ccdc181, forward 5'-GGACTTGGAGTGGTTGATCA-3' and reverse 5'-GCCTCATCCTTTGGAGAGTT-3'.

Fertility test
As we reported (Shi et al., 2023), a fertility test was performed by mating one 8-week-old Ccdc181 −/− male mice with two 8-week-old WT female mice (C57BL/6J) for two months.A total of three Ccdc181 −/− and three WT male mice were tested.The females were monitored for pregnancy, and the litter dates and number of pups were recorded for all litters.

Mouse sperm counts, morphology, and motility
As we reported (Ma et al., 2021a), the sperm number of 8-week-old mice was assessed by cutting the epididymis into pieces in PBS and incubating them for 30 minutes at 37°C to release the sperm.The sperm released from the epididymis was counted using a hemocytometer under a microscope.Sperm motility was determined by placing the epididymis in human tubal fluid containing 10% FBS at 37°C for 15 minutes, collecting the liquid containing sperm, and using computer-assisted semen analysis to assess sperm motility as we described (Zhang et al., 2020).To assess sperm morphology, slides were fixed in 4% PFA for 5 minutes, washed with PBS, and stained with H&E.
At least 200 spermatozoa were examined from each mouse to determine the percentage of morphologically abnormal spermatozoa, as we performed before (Ma et al., 2023a).

Histological analyses of testicular and epididymal tissues
Testicular and epididymal tissues were prepared as we described previously (Liu et al., 2023).Fresh testicular and epididymal tissues were fixed in Bouin's solution or in 4% paraformaldehyde at 4°C overnight, followed by paraffin embedding.Paraffin-embedded tissues were sectioned (5 µm).H&E staining were performed for histological analyses of epididymal and testicular sections, respectively.Images were captured using a microscope (Nikon Eclipse 80i) equipped with a digital camera (Nikon DS-Ri1).

Transmission electron microscopy (TEM) analysis
Samples were prepared as we previously reported (Ma et al., 2021b).Briefly, fresh semen samples were obtained and centrifuged at 400xg for 3 minutes.
After washing with PBS for three times, spermatozoa were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde, 8% glutaraldehyde, and 0.2% picric acid at 4°C for at least overnight.After four washes with 0.1 M PB, samples were post-fixed with 1% OsO 4 and dehydrated, followed by infiltration of acetone and epon resin mixture.Samples were embedded and ultrathin (70 nm) sectioned before staining with uranyl acetate and lead citrate.
The ultrastructure of the samples was examined and captured by Tecnai 10 or 12 Microscope (Philips) at 100 kV or 120 kV, or by H-7650 Microscope (Hitachi) at 100 Kv.

TUNEL assay
TUNEL assay was prepared according to previously described protocols (Xu et al., 2022).Testis sections were deparaffinized in xylene, rehydrated in a graded series of ethanol (100 %, 95 %, 90 %, 80 %, 70 %, 50 % ethanol, and sterile water), and permeabilized with proteinase K (20 mg/mL) in 10 mM Tris-HCl (pH 7.5) for 20 minutes at room temperature.After two washes with PBS, TUNEL reagent mix (In Situ Cell Death Detection Kit, Fluorescein, Roche, 11684795910, 50 mL) was applied to each slide followed by incubation for 1 hour at 37°C according to the manufacturer's protocol.Sections were then washed with PBST three times and mounted in VECTASHIELD mounting medium (H-1000, Vector Laboratories) containing Hoechst 33342 (Invitrogen, H21492).Images were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan) with a scientific complementary metal-oxidesemiconductor camera (Prime BSI, Teledyne Photometrics Inc., USA) and processed with the Olympus cellSens software.

Cell culture and transfection
HEK293T cells were cultured as previously described (Xie et al., 2022) and transfected with P-N1 plasmids expressing CCDC181 protein tagged with Flag using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, L3000015) following the manufacturer's instructions.Cells were harvested for immunoblotting 36 hours after transfection.

Western blotting
Western blotting was performed as we previously described (Zhang et al., 2020).Briefly, cultured cells were lysed using Bolt LDS Sample Buffer (Invitrogen, Thermo Fisher Scientific, B0008) with NuPAGE Antioxidant (Invitrogen, Thermo Fisher Scientific, NP0005) and boiled for 10 minutes.The proteins extracted from testes and sperm were prepared using lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, and 5 mM EDTA, pH 7.5) containing a protease inhibitor cocktail (Solarbio, P6730).After tissue grinding and ultrasonication, the protein lysates were centrifuged at 16,000xg for 20 minutes at 4°C.The supernatant liquid of the extracts was used for immunoblotting.The proteins were separated by SDS-PAGE and electro-transferred onto a nitrocellulose membrane.Then the membrane was blocked in 5% skim milk overnight and then incubated with corresponding primary and secondary antibodies.
After overnight incubation at 4˚C, the agarose beads were washed 6 times in IP buffer, and then the immunocomplexes were dissociated from the beads with elution buffer (0.2 M glysine, 0.15% NP-40, pH 2.3).After validation of the eluted samples by silver staining, the samples were analyzed by mass spectrometry at the National Center for Protein Science Shanghai.The candidate interacting proteins of CCDC181 are listed in Supplementary Table S1.

Homology and phylogenetic analysis
Homologous nucleotide and protein sequences were confirmed using the

CCDC181 is predominantly expressed in the testis
Before exploring the potential molecular functions of CCDC181 in spermatogenesis, we first investigated the expression and localization of CCDC181.We examined its expression pattern in different tissues and found that it was predominantly expressed in the testis (Supplementary Figure S1A).
Further immunoblotting of mouse testis lysates prepared on different days after birth was carried out.The expression of CCDC181 in the testis was observed from postnatal day 20 (PD20) to PD56 (Supplementary Figure S1B).This time course corresponds with the onset of spermiogenesis, suggesting that CCDC181 participates in this process.Furthermore, the expression and localization of the CCDC181 protein were examined in developing mouse spermatids through immunofluorescence.The protein was found to be expressed in spermatogenesis.From step 1 to step 7, a diffuse staining of CCDC181 was detected in the cytoplasm of the spermatids.As the spermatids were elongating from step 8 to step 14, CCDC181 was localized to the manchette.During the final steps of male germ cell development, CCDC181 staining was concentrated in the sperm flagella (Supplementary Figure S1C).
Such signals persisted along the entire sperm flagellum and accumulated at the midpiece of the sperm tail in the cauda epididymis (Supplementary Figure S1D), suggesting that CCDC181 is necessary for spermatid elongation and sperm flagellum development.
To examine the conservation of CCDC181, we analyzed the sequence and structural characteristics of CCDC181.We obtained orthologues of CCDC181 from a wide range of species to assess their conservation, including mammals, reptiles, birds, amphibians, and fish.The phylogenetic tree was constructed using MEGA software.Based on the tree, CCDC181 is evolutionarily conserved from bony fish to human beings (Supplementary Figure S2A).Additionally, only one potential functional domain of CCDC181 (amino acids 337-494) was predicted by the NCBI's Conserved Domain Database (Supplementary Figure S2B), whereas the other parts of CCDC181 remained unknown.Therefore, MEME was applied to predict the conserved motifs of CCDC181 in twelve species.Ten motifs were identified, and the pairwise similarities between various species were high (Supplementary Figure S2C).Previous findings predicted that a region of the murine CCDC181 (amino acids 393-448) interacted with HOOK1 (Schwarz et al., 2017).It is noteworthy that motif 6 (amino acids 404-444), which coincides with the interacting region with HOOK1, is highly conserved across all species.In general, CCDC181 is highly conserved in vertebrate species, suggesting that its function may be also conserved in these species.

The knockout of Ccdc181 leads to male infertility
To investigate the role of CCDC181 during spermiogenesis, Ccdc181 knockout mice were generated using the CRISPR/Cas9 technology.The mutation did not affect the transcription of Ccdc181 (Supplementary Figure S3).Analysis of the Subsequently, the fertility of Ccdc181 male and female knockout mice was examined.Male mice lacking CCDC181 exhibited normal mounting behaviors and produced coital plugs, but failed to produce offspring when mated with WT female mice (Figure 1D).In contrast, Ccdc181 −/− female mice generated offspring of normal litter sizes after mating with WT males.Hence, the disruption of Ccdc181 resulted solely in male infertility, without any impact on the fertility of Ccdc181 −/− female mice.To investigate the cause of male infertility, we inspected the adult Ccdc181 −/− testes at both the gross and histological levels.
Ccdc181 −/− mice showed normal growth and development, with normal testicular sizes and testes/body weight ratios compared to Ccdc181 +/+ mice (Figure 1E-F).However, we examined the spermatozoa released from the caudal epididymis and found that the spermatozoa counts were markedly reduced in Ccdc181 −/− mice (Figure 1G-H).
Notably, although CCDC181 could be detected in motile cilia (Schwarz et al., 2017), homozygous-null mice did not display any obvious signs of primary ciliary dyskinesia.We also examined the cilia in the lung and trachea of Ccdc181 −/− mice and found that the structures were similar to those of Ccdc181 +/+ mice (Supplementary Figure S4), indicating that CCDC181 is specifically required in spermatogenesis.Sertoli cells, respectively (Figure 2B).Further statistical analysis showed that in Ccdc181 −/− mice, the ratios of spermatids to Sertoli cells were significantly decreased from step 15 to step 16 (Figure 2C).Compared to Ccdc181 +/+ mice, the number of step 16 spermatids in Ccdc181 −/− mice was greatly declined.To define whether the elimination of late spermatids was associated with apoptosis, TUNEL assay on mouse testicular sections was performed (Figure 2D).The TUNEL assay revealed overall increases in the proportion of seminiferous tubules containing TUNEL-positive cells, and in the average number of apoptotic cells per seminiferous tubule in Ccdc181 −/− testis sections compared to the control (Figure 2E-F).These results suggest that the defective late spermatids in Ccdc181 −/− mice underwent apoptosis, leading to a significant decline in sperm counts in the epididymis.

CCDC181 deficiency leads to abnormal manchette and acrosome detachment
To further investigate the defects of spermatogenesis in Ccdc181 −/− mice, H&E staining of the semen smears of Ccdc181 +/+ and Ccdc181 −/− mice was performed.The H&E staining results of the semen smears revealed that 50% of the spermatozoa in Ccdc181 −/− cauda epididymis exhibited abnormal head shapes (Figure 3A-B).These misshapen sperm heads were also detected by TEM (Figure 3C).Abnormal sperm head shape is often associated with defects in acrosome and manchette formation (De Boer et al., 2015).To explore the reason for the sperm head deformities, we examined the acrosome structure and manchette formation during spermiogenesis in Ccdc181 −/− mice.
We evaluated the acrosome morphologies of Ccdc181 +/+ and Ccdc181 −/− mice by TEM analysis and immunofluorescence.The elongated nucleus of the Ccdc181 +/+ sperm was tightly covered with a cap-like acrosome, whereas the Ccdc181 −/− sperm exhibited detachment of the acrosome from the nucleus (Supplementary Figure S5A).Additionally, the PNA staining in Ccdc181 +/+ and Ccdc181 −/− mice showed that the sperm from Ccdc181 −/− mice had more absent or aberrant acrosomes compared with Ccdc181 +/+ mice (Supplementary Figure S5 B-C).These findings suggest that CCDC181 deficiency may lead to acrosome detachment.The acroplaxome, consisting of a filamentous actin (Factin) and keratin-containing plate, is thought to anchor the acrosome to the nucleus during sperm head shaping (Kierszenbaum et al., 2003).The

The knockout of Ccdc181 results in a typical MMAF phenotype in male mice
In addition to the abnormal heads, the H&E staining results of semen smears revealed that the spermatozoa from Ccdc181 −/− cauda epididymis exhibited malformed flagella, including coiled, short, bent, absent, and irregular flagella, which are typical features of MMAF (Figure 4A-B).The results of further TEM analysis indicated that, the axoneme was structurally disorganized in

CCDC181 is required for the localization of LRRC46 along flagella
To further investigate the role of CCDC181 during spermatogenesis, we performed anti-CCDC181 immunoprecipitation coupled with quantitative mass spectrometry on testicular lysates of Ccdc181 +/+ mice.A total of 1934 proteins were quantified, of which 113 proteins were identified to be CCDC181associated proteins (IP/IgG > 1.5-fold and IP-IgG > 3).Gene Ontology enrichment analysis of these proteins revealed significantly enriched gene ontology terms in intracellular protein transport and motor proteins (Figure 5A).
These findings are consistent with the above results on defective intramanchette transport and decreased motility of Ccdc181 −/− mice sperm.
Among the candidate interacting proteins, a total of 11 proteins were initially screened out based on the protein function and localization (Supplementary has been linked to male infertility and MMAF phenotypes (Yin et al., 2022).The phenotypes of Lrrc46 knockout mice share many similarities with those observed for Ccdc181 −/− mice, suggesting that LRRC46 and CCDC181 are part of a common pathway.Therefore, we hypothesized that CCDC181 interacts with LRRC46.To confirm this hypothesis, we first tested whether the CCDC181 protein binds to LRRC46 using co-IP in HEK293T cells overexpressing tagged proteins with GFP-empty as a negative control.Protein interaction between CCDC181 and LRRC46 was successfully identified (Figure 5B).We then investigated whether CCDC181 deficiency affected LRRC46 functions.
Immunofluorescence results showed that the LRRC46 signals were severely disrupted in Ccdc181 −/− mice sperm (Figure 5C).These results suggest that CCDC181 interacts with LRRC46 and CCDC181 is essential for the localization of LRRC46.

DISCUSSION
MMAF is the most severe form of asthenoteratozoospermia, but the genetic causes of many MMAF cases are still unknown.It has been reported that many proteins in the CCDC family are required for the proper development or function of spermatozoa (Becker-Heck et al., 2011;Blanchon et al., 2012;Chen et al., 2021;Ge et al., 2024;Geng et al., 2016;Jreijiri et al., 2024;Kaczmarek et al., 2009;Li et al., 2017;Ma et al., 2023b;Sha et al., 2019;Tapia Contreras and Hoyer-Fender, 2019;Wang et al., 2023a;Wang et al., 2018;Wang et al., 2023b;Yamaguchi et al., 2014;Young et al., 2015;Zhang et al., 2022a).This study, for the first time, demonstrates the effects of CCDC181 deficiency on spermatogenesis.The CCDC181 protein shows dynamic localizations in adult mice testes during spermiogenesis (Figure 6).We found that the knockout of Ccdc181 results in a typical MMAF phenotype in male mice, mainly presenting with coiled, curved, short or absent flagella and complete sperm immobility.
Furthermore, we found that CCDC181 is involved in manchette formation and sperm head shaping.Additionally, LRRC46, an MMAF-related protein (Yin et al., 2022), was identified to be an interacting protein of CCDC181 through immunoprecipitation and mass spectrometry analysis.These findings indicate that CCDC181 is essential for spermatogenesis and male fertility in mice, suggesting that CCDC181 may play a role in human male infertility.
During spermiogenesis, the trafficking of proteins to specific cellular regions plays a key role in the development of sperm (Teves et al., 2020).The manchette, composed of microtubules and actin filaments, not only assists with nuclear remodeling but also participates in protein trafficking by intramanchette transport (O'donnell and O'bryan, 2014).HOOK1, which is involved in vesicle cargo transport, can be sequentially seen from the acroplaxome to the manchette and the head-tail coupling apparatus (Kierszenbaum et al., 2011).
The loss of HOOK1 function leads to disruption of the connection between the manchette and the nuclear envelope and subsequently induces abnormal position of the microtubules of the manchette, resulting in abnormal sperm head shape (Mendoza-Lujambio et al., 2002).The cargo-specific binding properties of HOOK1 and its ability to bind motor proteins may contribute to the transport of cargos toward specific locations within the elongating spermatid (Maldonado-Báez et al., 2013).Previous studies have shown that there is an interaction between HOOK1 and CCDC181, with HOOK1 as the carrier for CCDC181 transport (Schwarz et al., 2017).It assumed that CCDC181 does not directly bind to the microtubules of the manchette in spermatids but rather is linked to the manchette by HOOK1 for intramanchette transport to the axoneme of growing sperm flagella.In spermatids from step 8 to step 14, we found that CCDC181 was localized to the manchette.Ccdc181 −/− mice displayed obviously longer manchettes when compared with controls in step 11 spermatids and exhibited an abnormal club-shaped head morphology.These observations indicate that CCDC181 may take part in the protein transport from the Golgi complex towards the sperm tail through the manchette and function through its interaction with HOOK1.
To date, several proteins have been identified that bridge the acrosome and nucleus via acroplaxome-manchette structures and are involved in anchoring the acrosome to the nucleus (Kazarian et al., 2018;Liu et al., 2015;Mendoza-Lujambio et al., 2002;O'donnell et al., 2012).It is recognized that the acroplaxome attaches the developing acrosome to the nucleus during sperm head shaping (Kierszenbaum et al., 2003).The connection between the acrosome and the spermatid nucleus is established via the marginal ring or perinuclear ring.Both the marginal ring of the acroplaxome and the perinuclear ring of the manchette play a role in sperm head shaping (Kierszenbaum and Tres, 2004).The perinuclear ring assembles adjacent to the marginal ring of the acroplaxome.Both HOOK1 and CCDC181 have been observed to localize in the nuclear ring between the manchette and the nucleus (Mendoza-Lujambio et al., 2002;Schwarz et al., 2017).We speculate that the interaction between HOOK1 and CCDC181 may occur at the nuclear ring.CCDC181 deficiency impairs sperm head shaping and causes the acrosome detachment.
Additionally, CCDC181 may also play a role in flagellum protein transport.
Based on the immunoprecipitation-mass spectrometry and co-IP results, it can be inferred that CCDC181 interacts with the MMAF-related protein LRRC46.
LRRC46 is specifically expressed in the testes of adult mice.The knockout of Lrrc46 in mice resulted in typical MMAF phenotypes and a severe axonemal disorganization, which shared high similarities with Ccdc181 −/− mice.Both CCDC181 protein and LRRC46 protein were observed to accumulate at the midpiece of the spermatozoon tail (Yin et al., 2022).The immunofluorescence results demonstrated a deficiency of LRRC46 signals along the flagella in Ccdc181 −/− mice, indicating that CCDC181 is essential for the localization of LRRC46.These findings indicated that they could function in a common pathway during spermiogenesis and the interaction between LRRC46 and CCDC181 may occur at the midpiece.
Although CCDC181 is localized on the motile cilia (Schwarz et al., 2017), our mutant mice did not exhibit primary ciliary dyskinesia.Previous studies have also indicated that sperm flagellar damage is more severe than ciliary damage after mutation of some genes (Zhang et al., 2021a;Zhang et al., 2022b).It is possible that the role of CCDC181 is not as essential in respiratory cilia as it is in sperm flagella.Additionally, the different phenotypes may also result from different mechanisms of axonemal assembly between sperm flagella and respiratory cilia.
In conclusion, our results indicate that CCDC181 is essential for the assembly of sperm tails during spermatogenesis, and contributes to the shaping of sperm heads.These findings highlight the potential roles of CCDC181 in both sperm flagellum biogenesis and manchette formation.Given that CCDC181 is an evolutionarily conserved component of the flagellar proteome in diverse species, it is possible that the mutations of CCDC181 may exist in the infertile patients with MMAF.Thus, further studies should be undertaken to uncover whether and how CCDC181 mutations affect disease presentation.A schematic summary of the dynamic localizations of the CCDC181 protein in adult mouse testes during spermiogenesis.The localization drawing is based on the immunofluorescence staining results.
and the heterozygous offspring (F1 generation and beyond) were intercrossed to produce biallelic Ccdc181 mutant mice (F2 generation and beyond) for conducting experiments.The mice were maintained under specific-pathogenfree conditions in the laboratory animal center of USTC.All experiments involving animals were performed following the guidelines of the institutional Animal Care and Use Committee of USTC.The sequences of gRNAs
cDNA from mutant mice by Sanger sequencing revealed a deletion of 7 bp (c.264_270del) in exon 2 of Ccdc181, resulting in premature termination of translation (p.Ile89Arg, fs*33) (Figure 1A-B).The full-length CCDC181 protein disappeared in the mutant testes indicating the successful preparation of Ccdc181 −/− mice (Figure 1C).
Ccdc181 −/− mice To investigate the causes of decreased sperm counts in Ccdc181 −/− mice, we performed Periodic Acid-Schiff staining and compared the seminiferous tubules between Ccdc181 +/+ and Ccdc181 −/− mice stage by stage.Compared to Ccdc181 +/+ mice, a remarkable decrease in the number of elongating and elongated spermatids were observed in the seminiferous tubules of Ccdc181 −/− mice.The histological analysis revealed a significant decrease in the number of elongated spermatids at step 15 and step 16 (stages V-VIII) in the seminiferous tubules of Ccdc181 −/− mice, suggesting the possible abnormalities occurred during spermiogenesis (Figure 2A).Additionally, spermatids and Sertoli cells were labeled by immunofluorescence staining with peanut agglutinin and anti-SRY-Box Transcription Factor 9, markers of acrosomes and distribution of the acroplaxome was disturbed in Ccdc181 −/− mice, with detached, irregular, fragmented F-actin distribution (Supplementary FigureS5D).These findings indicate that CCDC181 deficiency causes a large amount of loosened acroplaxome structure and acrosome detachment in spermatids.Manchette abnormalities are closely related to the failure of sperm head shaping.The manchette emerges in step 8 spermatids, coincident with the initiation of sperm nucleus elongation, and disintegrates around step 13 to step 14 when the nuclear remodeling completes.Immunofluorescence staining for α-Tubulin and peanut agglutinin showed that the manchette formation was normal in step 8 to step 10 spermatids in Ccdc181 −/− mice.However, Ccdc181 −/− mice displayed significantly longer manchettes compared to controls in step 11 to step 13 spermatids (Figure3D-E).The deficient manchette formation led to the presence of abnormal spermatid heads.During step 11 to step 13, Ccdc181 −/− spermatids displayed an abnormal club-shaped head morphology, whereas Ccdc181 +/+ spermatids displayed normal hook-shaped heads, as confirmed by Periodic Acid-Schiff staining (Figure3F).These findings indicate that the absence of CCDC181 leads to acrosome detachment and impairs manchette formation, leading to severe defects in sperm head shaping.

Ccdc181
−/− sperm flagella, suggesting that the ultrastructure of Ccdc181 −/− sperm flagella were severely impaired (Figure 4C).For validation, immunofluorescence staining was performed for several structural components of the flagella, such as DNAH6, DNAH17, CFAP61, SPAG6 and which represent inner dynein arms, outer dynein arms, radial spokes and central pairs, respectively.Compared to the continuous signals along the entire flagella of Ccdc181 +/+ sperm, the signals of DNAH6, DNAH17, CFAP61, and SPAG6 disappeared in Ccdc181 −/− sperm flagella (Figure 4D-G).Their protein levels were also dramatically reduced in Ccdc181 −/− sperm (Figure 4H-I).Computerassisted semen analysis showed obviously decreased motility of Ccdc181 −/− sperm (Figure 4J), which is likely due to the abnormal morphologies and structures of their flagella.Hence, the deletion of Ccdc181 induces severe defects in sperm flagellum biogenesis, ultimately resulting in a typical MMAF phenotype.

Figure 1
Figure 1 The generation and reproduction of Ccdc181 knockout mice.A: Schematic diagram of the generation of Ccdc181 −/− mice using CRISPR/Cas9 technology.The gRNAs were designed to target exon 2 of the Ccdc181, and a 7 bp-deletion mouse line was obtained.An illustrative representation of the CCDC181 protein structure shows the mutation position.

Figure
Figure 2 CCDC181 deficiency causes a significant decrease in elongated

Figure 3
Figure 3 CCDC181 deficiency impairs manchette formation and sperm head

Figure 4
Figure 4 CCDC181 deficiency results in a typical MMAF phenotype in male

Figure
Figure 5 LRRC46 interacts with CCDC181 and its localization along flagella is

Figure 6 A
Figure 6 A schematic diagram to illustrate the localization of the CCDC181