To the Editor: Infertility is a widespread health issue affecting millions globally, with male factors contributing to about half of cases, many of which remain idiopathic and are thought to have an underlying genetic basis.1 Although many genes associated with male infertility are known in mice, the translation of this information to humans has been slow.2 Without a genetic diagnosis, it is difficult for a clinician to evaluate the potential success of assisted reproductive technology and the reproductive health of their offspring. The coiled-coil domain-containing (CCDC) proteins are suggested to play functional roles in spermatogenesis.3 Compared to the extensive information on CCDC proteins in mouse spermatogenesis, findings related to CCDCs in human fertility remain largely unknown. Recently, it has been reported that the loss of CCDC146 causes male infertility in mice due to defects in sperm flagellum biogenesis.4 However, only two CCDC146 mutations have been identified in two French men with asthenoteratozoospermia.5 Hence, the causative relationship between CCDC146 mutations and human infertility needs further confirmation, especially in Chinese population. In this study, we performed whole-exome sequencing on a large Chinese cohort of 657 unrelated infertile men with asthenoteratozoospermia. Our research adhered to the principles in the Declaration of Helsinki, and ethical approval was acquired from the Ethical Review Board at West China Second University Hospital, Sichuan University (approval number: 2019040). All participants signed an informed consent form. Interestingly, biallelic variants of CCDC146 were initially identified in four affected individuals from unrelated families: a homozygous frameshift mutation of c.778dup in subject, a compound heterozygous mutation of c.640C >T and c.1339dup in subject, a homozygous frameshift mutation of c.1339dup in subject, and a homozygous frameshift mutation of c.296del in subject Figure 1A; Supplementary Table 1, https://links.lww.com/CM9/C806. Furthermore, Sanger sequencing confirmed that the unaffected parents were heterozygous carriers of the variants, these variants were absent in 931 normal controls, and Western blotting analysis of patients’ sperm lysates further demonstrated degradation of the CCDC146 protein Supplementary Figure 1A and 1B, https://links.lww.com/CM9/C806.Figure 1: Deficiency of CCDC146 causes male infertility in humans and mice. (A) Pedigrees of four individuals with asthenoteratozoospermia. The probands are indicated by a black arrow. (B) SEM analysis of spermatozoa from infertile individuals (scale bars, 5 µm). (C) TEM analysis of sperm flagellar ultrastructure from infertile individuals (scale bars, 250 nm). (D) The mixture of scattered mitochondria and MTDs, and sperm heads showed a high presence in patient sperm by TEM analysis (scale bars, 1 μm). (E) SEM analysis of spermatozoa from Ccdc146 KO mice (scale bars, 5 µm). (F) TEM analysis of sperm ultrastructure in Ccdc146 KO mice (scale bars, 500 nm). (G) Venn diagram depicting the 19 overlapping MIPs between the downregulated testicular proteins in Ccdc146 KO mice and CCDC146-interacting proteins. (H) Western blotting analysis of Tektin family proteins in WT and Ccdc146 KO testes. (I) Immunofluorescence staining showed the colocalization of CCDC146 and Tektins in mouse sperm (scale bars, 5 μm). (J) Co-IP assays showed that CCDC146 interacted with Tektins in mouse testes. (K) Two-step Co-IP assays performed in HEK293T cells. BP: Basal plate; Co-IP: Co-immunoprecipitation; M: Mutation; Mito: Mitochondrion; MIPs: Microtubule inner proteins; MTD: Peripheral microtubule doublet; PC: Proximal centriole; SC: Segmented column; SEM: Scanning electron microscope; TEM: Transmission electron microscopy; WT: Wild-type.We next analyzed phenotypic traits in spermatozoa linked to CCDC146 mutations in these patients. Spermatozoa from the four patients showed dramatic decrease in progressive motility, and displayed aberrant tails, including short, coiled, bent or irregular shapes Figure 1B; Supplementary Table 2, https://links.lww.com/CM9/C806. Moreover, transmission electron microscopy (TEM) results revealed an irregular “9 + 2” structure of the sperm flagella, including disordered or missing peripheral microtubule doublets (MTDs) and outer dense fibers (ODFs) and absent central pairs (CPs), as well as the defective connecting piece in sperm from CCDC146 mutation patients Figure 1C. Additionally, a significant number of cross-sections containing aberrant mitochondria, MTDs, and nuclei were reported for the first time in relation to CCDC146 absence Figure 1D. Ccdc146 KO mice was generated to validate the essential role of CCDC146 in spermatogenesis, and found that male Ccdc146 KO mice were completely infertile Supplementary Figure 2A–H, https://links.lww.com/CM9/C806. As expected, the KO male mice resembled the patients’ deformed sperm morphology Figure 1E. However, the KO mice exhibited more severe sperm ultrastructural defects, showing that the flagella were severely defective and dispersed, and only a rare midpiece with irregular “9+2” structure and mitochondria was detected Figure 1F. Interestingly, no obvious abnormalities in ciliary formation were observed in the lungs, trachea, eyes, or brains of KO mice Supplementary Figure 3; Supplementary Table 3, https://links.lww.com/CM9/C806, indicating that the CCDC146 might be indispensable only in flagellar development. To explore the molecular basis of CCDC146 regulating flagellar formation, we conducted proteomics analysis on testes from WT mice and Ccdc146 KO mice. GO analysis showed that the downregulated proteins were related to spermatogenesis Supplementary Figure 4A, https://links.lww.com/CM9/C806. Intriguingly, the most significantly downregulated proteins were MIP proteins which decorate the luminal surfaces of MTD are important for MTD stability Supplementary Figure 4B, https://links.lww.com/CM9/C806.6 To further investigate the interactors of CCDC146, we performed immunoprecipitation–mass spectrometry (IP-MS) analysis using mouse testes. Importantly, the microtubule inner proteins (MIPs) showed obvious enrichment with CCDC146. In particular, 23 MIPs were detected, of which 19 were downregulated in the KO mice Figure 1G. Therefore, we speculated that CCDC146 might interact with MIPs and further regulate their expression to play an essential role in sperm flagellar development. In these downregulated MIPs, TEPP and SMRP1 link a Tektin4 filament to RIBC2 and TEKTIP1, respectively, reinforcing the stability of the microtubule structure.6 Consequently, we confirmed the downregulation of Tektin4 (absence of the available antibodies of TEPP, TEKTIP1 and SMRP1) in KO through Western blotting Figure 1H. In addition, the colocalization and binding of CCDC146 and Tektin4 were observed in mouse sperm and testis Figure 1I, 1J. Moreover, we constructed HA-Tektin4, Flag-Tepp and Myc-Ribc2 overexpression plasmids, and transfected them into HEK293T cells, and validated the TEPP, Tektin4 and RIBC2 complexes by Co-immunoprecipitation (Co-IP) assay Figure 1K. Similarly, we observed that Tektin4 also formed a complex with SMRP1 and TEKTIP1 Figure 1K. In addition to Tektin4/TEPP/RIBC2/TEKTIP1/SMRP1 complex, the reduced expression of other Tektin family members that are critical MIPs in the sperm tail, including Tektin1, Tektin2, Tektin3, and Tektin5, was also observed in KO mice by Western blotting Figure 1H. Moreover, Co-IP assay and immunofluorescence staining validated their interaction with CCDC146 in mouse testis and sperm Figure 1I, 1J. Collectively, CCDC146 might play a role in spermatogenesis by interacting and further mediating the key MIPs, which are essential for sperm development. Notably, poor outcomes were observed in Ccdc146 KO mice subjected to ICSI treatment. Following injection, pronuclei were observed in the majority of embryos in the WT group, whereas embryos derived from Ccdc146 KO sperm exhibited a markedly reduced fertilization rate (8.9%), accompanied by substantially decreased two-cell (6.0%) and blastocyst (0.6%) formation rates Supplementary Figure 5, https://links.lww.com/CM9/C806. These data demonstrated that CCDC146 plays specific biological roles in fertilization and early embryonic development in mice. In conclusion, our findings screened CCDC146 mutations in the Chinese population and identified novel CCDC146 mutations in several unrelated asthenoteratozoospermia men, highlighting the importance of population-specific genetic studies in uncovering novel disease-associated variants. Moreover, we revealed the specific phenotypes associated with CCDC146 deficiency in patients and mice, phenotypes that were not uncovered in the previous study. Mechanistically, it is suggested that MIPs are the key targets of CCDC146 that modulate axonemal organization during spermatogenesis. Our findings therefore expand our knowledge of CCDC146 in male reproduction, which might provide valuable information for the etiology and treatment of male infertility. Acknowledgments We are grateful to Guiping Yuan from Analytical and Testing Center of Sichuan University for her help with transmission electron microscopy images. Funding This study was funded by grants from the National Natural Science Foundation of China (Nos. 81971433, 82271749, and 82301807). Conflict of Interest None.
Ruan et al. (Mon,) studied this question.