Author response:
eLife assessment
This study presents a valuable finding on sperm flagellum and HTCA stabilization. The evidence supporting the authors' claims is incomplete. The work will be of broad interest to cell and reproductive biologists working on cilium and sperm biology.
We thank the Editor and the two referees for their time in carefully reviewing our work, and we are grateful for the helpful guidance about how to improve our study. We will supplement the experiments and provide quantitative data guided by the referees’ comments in the revised manuscript. Additionally, we will polish the manuscript and add further context to help readers understand the significance of this work.
Public Reviews:
Reviewer #1 (Public Review):
In this paper, Wu et al. investigated the physiological roles of CCDC113 in sperm flagellum and HTCA stabilization by using CRISPR/Cas knockouts mouse models, co-IP, and single sperm imaging. They find that CCDC113 localizes in the linker region among radial spokes, the nexin-dynein regulatory complex (N-DRC), and doublet microtubules (DMTs) RS, N-DRC, and DMTs and interacts with axoneme-associated proteins CFAP57 and CFAP91, acting as an adaptor protein that facilitates the linkage between RS, N-DRC, and DMTs within the sperm axoneme. They show the disruption of CCDC113 produced spermatozoa with disorganized sperm flagella and CFAP91, DRC2 could not colocalize with DMTs in Ccdc113-/- spermatozoa. Interestingly, the data also indicate that CCDC113 could localize on the HTCA region, and interact with HTCA-associated proteins. The knockout of Ccdc113 could also produce acephalic spermatozoa. By using Sun5 and Centlein knockout mouse models, the authors further find SUN5 and CENTLEIN are indispensable for the docking of CCDC113 to the implantation site on the sperm head. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. Furthermore, the study's findings offer valuable insights into the physiological and developmental roles of CCDC113 in the male germ line, which can provide insight into impaired sperm development and male infertility. The conclusions of this paper are mostly well supported by data, but some points need to be clarified and discussed.
We thank Reviewer #1 for his or her critical reading and the positive assessment.
(1) In Figure 1, a sperm flagellum protein, which is far away from CCDC113, should be selected as a negative control to exclude artificial effects in co-IP experiments.
We greatly appreciate Reviewer #1’s insightful suggestion. We will include a negative control in the co-IP experiment to eliminate potential artificial effects.
(2) Whether the detachment of sperm head and tail in Ccdc113-/- mice is a secondary effect of the sperm flagellum defects? The author should discuss this point.
Good question. Given that CCDC113 could localized in the sperm neck region, and interact with SUN5 and CENTELIN, CCDC113 may directly function in the sperm head and tail connection. Indeed, PAS staining revealed that Ccdc113–/– sperm heads with abnormal orientation in stages V–VIII seminiferous epithelia (Fig. 6C), and transmission electron microscopy (TEM) analysis further revealed that the disruption of CCDC113 caused the detachment of the destroyed coupling apparatus from the sperm head in step 9–11 spermatids (Fig. 6D). All these results suggest that the detachment of sperm head and tail in Ccdc113–/– mice may be not a secondary effect of the sperm flagellum defects. And we have discuss this point as below:
“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. PAS staining revealed that Ccdc113–/– sperm heads with abnormal orientation in stages V–VIII seminiferous epithelia (Fig. 6C), and transmission electron microscopy (TEM) analysis further revealed that the disruption of CCDC113 caused the detachment of the destroyed coupling apparatus from the sperm head in step 9–11 spermatids (Fig. 6D). All these results suggest that the detachment of sperm head and tail in Ccdc113–/– mice may be not a secondary effect of the sperm flagellum defects.”
(3) Given that some cytoplasm materials could be observed in Ccdc113-/- spermatozoa (Fig. 5A), whether CCDC113 is also essential for cytoplasmic removal?
Good question. Unremoved cytoplasm could be detected in spermatozoa by using transmission electron microscopy (TEM) analysis, including disrupted mitochondria, damaged axonemes, and large vacuoles, indicating cytoplasmic removal defects in Ccdc113–/– mice. We have discussed this point as below:
“Unremoved cytoplasm could be detected in spermatozoa by using transmission electron microscopy (TEM) analysis, including disrupted mitochondria, damaged axonemes, and large vacuoles, indicating cytoplasmic removal defects in Ccdc113–/– mice (Fig. 5A).”
(4) Although CCDC113 could not bind to PMFBP1, the localization of CCDC113 in Pmfbp1-/- spermatozoa should be also detected to clarify the relationship between CCDC113 and SUN5-CENTLEIN-PMFBP1.
We are thankful to Reviewer #1 for this suggestion. We will analyze the localization of CCDC113 in Pmfbp1-/- spermatozoa to clarify the relationship between CCDC113 and SUN5-CENTLEIN-PMFBP1.
Reviewer #2 (Public Review):
Summary:
In the present study, the authors select the coiled-coil protein CCDC113 and revealed its expression in the stages of spermatogenesis in the testis as well as in the different steps of spermiogenesis with expression also mapped in the different parts of the epididymis. Gene deletion led to male infertility in CRISPR-Cas9 KO mice and PAS staining showed defects mapped in the different stages of the seminiferous cycle and through the different steps of spermiogenesis. EM and IF with several markers of testis germ cells and spermatozoa in the epididymis indicated defects in flagella and head-to-tail coupling for flagella as well as acephaly. The authors' co-IP experiments of expressed CCDC113 in HEK293T cells indicated an association with CFAP91 and DRC2 as well as SUN5 and CENTLEIN.
The authors propose that CCDC113 connects CFAP91 and DRC2 to doublet microtubules of the axoneme and CCDC113's association with SUN5 and CENTLEIN to stabilize the sperm flagellum head-to-tail coupling apparatus. Extensive experiments mapping CCDC13 during postnatal development are reported as well as negative co-IP experiments and studies with SUN5 KO mice as well as CENTLEIN KO mice.
Strengths:
The authors provide compelling observations to indicate the relevance of CCDC113 to flagellum formation with potential protein partners. The data are relevant to sperm flagella formation and its coupling to the sperm head.
We are grateful to Reviewer #2 for his or her recognition of the strength of this study.
Weaknesses:
The authors' observations are consistent with the model proposed but the authors' conclusions for the mechanism may require direct demonstration in sperm flagella. The Walton et al paper shows human CCDC96/113 in cilia of human respiratory epithelia. An application of such methodology to the proteins indicated by Wu et al for the sperm axoneme and head-tail coupling apparatus is eagerly awaited as a follow-up study.
We thank Reviewer 2 for his/her kindly help in improving the manuscript. We now understand that directly detection of CCDC113 precise localization in sperm axoneme and head-tail coupling apparatus (HTCA) using cryo-electron microscopy (cryo-EM) could powerfully strengthen our model. Recent advances in cryo-electron microscopy (cryo-EM) have facilitated the analysis of axonemal structures and determined the structures of native axonemal DMTs from mouse, bovine, and human sperm (Leung et al., 2023; Zhou et al., 2023). However, some high-resolution structures of sperm axoneme and HTCA regions, including those involving CCDC113, remain to be detected. Thus, we would like to discuss this point and regard it as an important follow-up study.
References:
Bazan, R., Schröfel, A., Joachimiak, E., Poprzeczko, M., Pigino, G., & Wloga, D. (2021). Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link. PLoS Genet, 17(3), e1009388.
Ghanaeian, A., Majhi, S., McCafferty, C. L., Nami, B., Black, C. S., Yang, S. K., Legal, T., Papoulas, O., Janowska, M., Valente-Paterno, M., Marcotte, E. M., Wloga, D., & Bui, K. H. (2023). Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism. Nat Commun, 14(1), 5741.
Leung, M. R., Zeng, J., Wang, X., Roelofs, M. C., Huang, W., Zenezini Chiozzi, R., Hevler, J. F., Heck, A. J. R., Dutcher, S. K., Brown, A., Zhang, R., & Zeev-Ben-Mordehai, T. (2023). Structural specializations of the sperm tail. Cell, 186(13), 2880-2896.e2817
Walton, T., Gui, M., Velkova, S., Fassad, M. R., Hirst, R. A., Haarman, E., O'Callaghan, C., Bottier, M., Burgoyne, T., Mitchison, H. M., & Brown, A. (2023). Axonemal structures reveal mechanoregulatory and disease mechanisms. Nature, 618(7965), 625-633.
Zhou, L., Liu, H., Liu, S., Yang, X., Dong, Y., Pan, Y., Xiao, Z., Zheng, B., Sun, Y., Huang, P., Zhang, X., Hu, J., Sun, R., Feng, S., Zhu, Y., Liu, M., Gui, M., & Wu, J. (2023). Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility. Cell, 186(13), 2897-2910.e2819.
