ANKRD5: a key component of the axoneme required for sperm motility and male fertility

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal significant structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are well designed and executed.

Weaknesses:

The last section of cryo-ET analysis is not convincing. "ANKRD5 depletion may impair buffering effect between adjacent DMTs in the axoneme".

"In WT sperm, DMTs typically appeared circular, whereas ANKRD5-KO DMTs seemed to be extruded as polygonal. (Fig. S9B,D). ANKRD5-KO DMTs seemed partially open at the junction between the A- and B-tubes (Fig. S9B,D)." In the TEM images of 4E, ANKRD5-KO DMTs look the same as WT. The distortion could result from suboptimal sample preparation, imaging or data processing. Thus, the subsequent analyses and conclusions are not reliable.

This paper still requires significant improvements in writing and language refinement. Here is an example: "While N-DRC is critical for sperm motility, but the existence of additional regulators that coordinate its function remains unclear" - ill-formed sentences.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Weaknesses:

Limited Citations in Introduction: Key references on the role of N-DRC components (e.g.,DRC2, DRC4) in male infertility are missing, which weakens the contextual background.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal any structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are thoughtfully designed and well executed.

Weaknesses:

The cryo-FIB and cryo-ET analyses require further investigation, as detailed below. The molecular mechanism by which the loss of ANKRD5 affects sperm flagellar motility remains unclear. The current conclusion that Ankrd5 knockout reduces axoneme stability is not well-supported. Specifically, are other axonemal proteins diminished in Ankrd5 knockout sperm? Conducting immunofluorescence analyses and revisiting the quantitative proteomics data may help address these questions.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Weaknesses:

(1) Limited Citations in Introduction: Key references on the role of N-DRC components (e.g., DRC1, DRC2, DRC3, DRC5) in male infertility are missing, which weakens the contextual background.

(2) Lack of Functional Insights: While interacting proteins outside the N-DRC complex were identified, their potential roles and interactions with ANKRD5 are not adequately explored or discussed.

(3) Mitochondrial Function Uncertainty: Immunofluorescence suggests possible mitochondrial localization for ANKRD5, but experiments on its role in energy metabolism (e.g., ATP production, ROS) are insufficient, especially given the observed sperm motility defects.

(4) Glycolysis Pathway Impact: Proteomic analysis indicates glycolysis pathway disruptions in Ankrd5-deficient sperm, but the link between these changes and impaired motility is not well explained.

(5) Cryo-ET Data Limitations: The structural analysis of the DMT lacks clarity on how ANKRD5 influences N-DRC or RS3. The low quality of RS3 data hinders the interpretation of ANKRD5's impact on axoneme structure.

(6) Discussion of Findings: The manuscript could benefit from a deeper discussion on the broader implications of ANKRD5's interactions and its role in sperm energy metabolism and motility mechanisms.

Reviewer #1 (Recommendations for the authors):

EMD-35210/35211 are 16-nm maps while the Ankrd5 null map is 8-nm repeat. To generate a difference map, the authors should use maps of the same periodicity.

Thank you for your suggestion. We have replaced the old 16-nm maps with an 8nm map and updated the images (Fig. 7). The 8nm repeats DMT density map we used was obtained by summing two 16nm repeats DMTs that were staggered 8nm apart from each other (EMD-35229). The replacement of the 16nm repeats DMT density map with the 8nm repeats DMT density map has no effect on our scientific findings and experimental conclusions.

"We were able to detect the N-DRC structure in WT sperm, but we failed to find the density of N-DRC adjacent to RS3 in Ankrd5 null sperm". Do the authors imply that the N-DRC is lost in Ankrd5 null sperm? To draw a conclusion, they need to compare the 96-nm map of WT sperm axoneme with that of Ankrd5 null sperm axoneme. Quantitative proteomics shows that the levels of most N-DRC components in Ankrd5 null sperm are comparable with those of WT sperm. Why are the quantitative proteomics results not consistent with the structural observation?

We are very sorry for this improper description. Our original description was not rigorous, which led to misunderstanding. Our original intention is to say that the quality of the density map causes the N-DRC to be difficult to recognize, rather than that the N-DRC has disappeared. In addition, attempts to classify 96nm repeats DMT structure during our data processing failed. In the process of classification, we found that the density of RS was not good. So we changed the picture and the description.

We have changed the description in the text: "During the STA process, many particles were misaligned or deformed in the classification results, revealing various degrees of deformation—particularly affecting the B-tube (Figure 9,Fig. S9E). We could retain only ~10% of the DMT particles to obtain the final density map for ANKRD5-KO sperm (Fig. S9E), whereas ~70% were usable in WT dataset as reported previously [59]. The mutant DMT density map also displayed roughness at its periphery, indicating substantial structural heterogeneity (Fig. S9E). Even after discarding a large fraction of deformed particles, the final density map still showed evident artifacts, implying that although the mutant DMT preserves the fundamental features of both tubes, its shape is highly heterogeneous (Fig. S9E). Furthermore, attempts to classify the 96-nm repeats did not yield a clear density for radial spokes (RSs) (Fig. S9F), indicating that ANKRD5 deficiency may affect the stability of other accessory structures, such as RSs [24-26]. In the raw tomograms, RSs in ANKRD5-KO sperm appeared less regularly arranged than those in WT(Fig. S9A and C)."

Figure S9. The states of DMT particles in sperm of Ankrd5-KO mouse. (A) and (C) Tomogram slices of WT and Ankrd5-KO in Dynamo (The data for WT mouse sperm was EMPIARC-200007). DMT and RS are marked with white dashed lines and white arrows, respectively. (B) and (D) Comparison of DMT particle states between WT and Ankrd5-KO in Dynamo. The visual angles of the DMT particles shown in (B) and (D) show that the DMT fibers within the white box in (A) and (B) are divided equally into 10 slices along the direction of the white arrow, respectively. The DMT particle shapes of WT and Ankrd5-KO are marked by white dashed lines on the right of (B) and (D). The white arrow in (D) identifies the junction of A-tube and B-tube that is suspected to be disconnected. (E) Deformed particles discarded in 3D classification and final aligned DMT artifacts. (F) 3D classification of attempted RS locations.

In the process of obtaining DMT with a period of 8nm, we discarded about 90% of the particles (some were mis-aligned particles and some were deformed particles). Although the final DMT density showed complete A-tube and B-tube, both the particles in our calculation process and the projection of the final structure showed strong particle heterogeneity.

Our results show that in ANKRD5-KO mice, the structure of sperm DMT itself has no apparent effect in tube A and tube B, and we found that DMT in the original tomography were not smooth. We speculate that loss of ANKRD5 may reduce the interaction between N-DRC and neighboring DMTs, resulting in nonuniform force on the axoneme during sperm swimming, which may limit our ability to obtain an average structure of the more dynamic components (RS, N-DRC, ODA, IDA). Therefore, when trying to classify 96nm repeat DMTS, we can only see the density of suspected RS3 and RS2, but it is difficult to obtain the confident 96nm repeat DMT density. It is difficult to further discuss the effects of ANKRD5 on RS3 and N-DRC. To test this conjecture, we further classified the density of suspected RS3, and the results obtained exhibited a variety of mixed states (Fig. S9). To avoid confusion, we have already removed the discussion of RS3 and the related images from the original text.

It's not clear whether N-DRC proteins and ODA, IDA, RS proteins are affected in DMT of Ankrd5 null sperm. Immunofluorescence staining would help to resolve this problem.

Thank you for your suggestion. The levels of N-DRC proteins and ODA, IDA, RS were detected by immunofluorescence, and no difference was found between ANKRD5-null sperm and control. We added figure S6 as a new figure and added the following description in red font on page 7 of the article:

Figure S6. Immunofluorescence results of ANKRD5-null sperm and control. DRC11 serves as a marker protein for N-DRC (nexin-dynein regulatory complex), NME5 as a marker for RS (radial spoke), DNALI1 as a marker for IDA (inner dynein arm), and DNAI1 as a marker for ODA (outer dynein arm).

In addition, ODA and RS were also marked in the figure when we further analyzed the Cryo-ET data (Figure 7 and Figure S9).

Does Ankrd5 express in other cilia cells except for sperm?

We stained mouse respiratory cilia using immunofluorescence and found that the protein was also expressed in mouse respiratory cilia. To support this finding, we added Figure S3 as a new figure and included a description in red font on page 6 of the article.

Page 7, "However, in the process of manual selection of DMT fibers, we found that they were not as smooth as WT particles." This description is too subjective. Please show the data.

Thank you for your suggestion. We have added a supplementary figure showing the difference between mutant samples and WT samples during particle picking (Fig. S9).

Abstract, "These findings establish that ANKRD5 is critical for maintaining axoneme stability, "Page 7, "This suggests that the knockout of Ankrd5 may affect the structural stability of the axoneme," I do not see direct evidence that Ankrd5 KO reduces the axoneme stability.

Our phrasing was not sufficiently precise. These findings suggest that ANKRD5 plays a crucial role in limiting the relative sliding between adjacent microtubule doublets during axoneme bending, rather than directly contributing to the stability of the axoneme. This sentence has already been modified in the abstract and marked in red. We have added the description in the text: "These findings suggest that ANKRD5 may weaken the N-DRC’s "car bumper" role, reducing the buffering effect between adjacent DMTs and thereby destabilizing axoneme structures during intense axoneme motility." and "To further investigate the RS, IDA, and ODA structures of the axonemes, we conducted immunofluorescence assays in both Ankrd5^-/- mice and the control group. No significant differences were detected between the two groups (Fig. S6)."

Page 8, "but our study offers new perspectives for male contraceptive research". Could the authors expand this a bit - how this study may offer new perspectives for male contraceptive research?

We sincerely appreciate the reviewer's insightful feedback regarding the translational potential of our findings. This is indeed a critical aspect that we sought to highlight. In response, we have added a paragraph on page 9 (marked in red) to further emphasize this point. We have added the description in the text: "The potential for male contraceptive development arises from ANKRD5's critical structural role mediated through its ANK domain, which facilitates interaction with the N-DRC complex in sperm flagella. Recent structural evidence suggests the protein's positively charged surface may engage with glutamylated tubulin in adjacent microtubules[41], presenting a druggable interface. Targeted disruption of this interaction through small-molecule inhibitors could transiently impair sperm motility. Sperm function relies more on ANKRD5 than respiratory cilia, so inhibiting ANKRD5 has less impact on the latter. This makes ANKRD5 a promising drug target. This tissue-specific phenotypic uncoupling is not uncommon among axonemal-associated proteins, such as DNAH17 and IQUB[65，66]."

Abstract, "reveals its interaction with TCTE1 and DRC4/GAS8", please provide the alias symbol DRC5 for TCTE1 for clarity.

Thank you for your suggestion, I have revised the abstract by replacing "TCTE1" with "DRC5/TCTE1" to clarify the alias. The changes have been highlighted in red in the manuscript for easy reference.

Introduction, "Fertilization relies on successful spermatogenesis and normal sperm motility (4), which occurs in the testes." Does spermatogenesis or normal sperm motility occur in the testes?

Thank you for pointing out the ambiguity in the sentence. We have revised the sentence in the Introduction and highlighted it in red as follows: Fertilization relies on successful spermatogenesis and normal sperm motility..

Introduction, "The axoneme exhibits a 9+2 microtubule doublet structure". The description is not accurate. The "2" are singlet microtubules.

Thank you for your suggestion. We have revised the sentence to accurately describe the axoneme structure and highlight in red as follows: The axoneme features a 9+2 architecture, comprising nine doublet microtubules encircling a central pair of singlet microtubules, with the N-DRC forming cross-bridges between adjacent doublets.

Page 4, "control sperm successfully fertilized both cumulus-intact eggs". "control" should be a capital "C".

We thank the reviewer for noting this oversight. The correction has been implemented on page 5 with the term highlighted in red (now reading: "Control sperm successfully fertilized both cumulus-intact eggs"), and we have verified capitalization consistency throughout the manuscript.

Page 6, "applied RELION, M, and other software". "other software" is not an appropriate description, please be precise.

We have revised the description as suggested. Specifically, on page 7, the phrase "and other software" has been replaced with "Dynamo and Warp/M," and this change is highlighted in red for clarity.

Reviewer #2 (Recommendations for the authors):

Several components of the N-DRC complex (e.g., DRC1, DRC2, DRC3, DRC5) have been reported to be associated with male infertility in both humans and mice. However, the introduction lacks proper citations for these studies. Adding these references would provide a more comprehensive background for readers.

Thank you for your suggestion to strengthen the comprehensiveness of the research background by incorporating additional literatures. More literatures related to DRC1, DRC2, DRC3, and DRC5 were cited in the background of this paper. We have rewritten and reorganized the language of the last paragraph of the introduction, and the entire paragraph is highlighted in red. The content of the paragraph is as follows:

"It was previously believed that N-DRC comprised 11 protein components[13，18]. However, a new component CCDC153 (DRC12) was found to interact with DRC1[19]. In situ cryoelectron tomography (cryo-ET) has significantly advanced understanding of the N-DRC architecture in Chlamydomonas, demonstrating that DRC1, DRC2/CCDC65, and DRC4/GAS8 constitute its core framework[16], while proteins DRC3/5/6/7/8/11 associate with this framework and engage with other axonemal complexes[20]. Biochemical experiments corroborate these findings and validate this structural model[12，21，22]. The N-DRC functions between the DMTs to convert sliding into axonemal bending motion by restricting the relative sliding of outer microtubule doublets[23，24，25]. Mutations of N-DRC subunits demonstrate that the structural integrity of the N-DRC is crucial for flagellar movements. Mutations in DRC1, DRC2/CCDC65, and DRC4/GAS8 are linked to ciliary motility disorders, causing primary ciliary dyskinesia (PCD)[12，26]. Biallelic truncating mutations in DRC1 induce multiple morphological abnormalities of sperm flagella (MMAF), including outer DMT disassembly, mitochondrial sheath disorganization, and incomplete axonemal structures in human sperm[22，27，28]. Similarly, CCDC65 loss disrupts N-DRC stability, leading to disorganized axonemes, global microtubule dissociation, and complete asthenozoospermia[12，29]. Homozygous frameshift mutations in DRC3 impair N-DRC assembly and intraflagellar transport (IFT), resulting in severe motility defects despite normal sperm morphology[30，31]. TCTE1 knockout mice maintain normal sperm axoneme structure but show impaired glycolysis, leading to reduced ATP levels, lower sperm motility, and male infertility[32]. Both Drc7 and Iqcg (Drc9) knockout mice exhibit disrupted '9+2' axonemal architecture, sperm immotility, and male infertility[21，33]. Drc7 knockout sperm also display head deformities and shortened tails[21]. While N-DRC is critical for sperm motility, but the existence of additional regulators that coordinate its function remains unclear. Our findings indicate that ANKRD5 (Ankyrin repeat domain 5; also known as ANK5 or ANKEF1) interacts with N-DRC structure, serving as an auxiliary element to facilitate collaboration among DRC members. The absence of ANKRD5 results in diminished sperm motility and consequent male infertility."

While many N-DRC components were identified as interacting with ANKRD5, other proteins outside the N-DRC complex were also detected. Notably, GAS8 (DRC4) ranked 165th among the identified proteins. What are the functions of the higher-ranking proteins, and why do they interact with ANKRD5? Discussing their potential roles would enhance the mechanistic understanding of ANKRD5's function.

We thank the reviewer for highlighting the importance of non-N-DRC proteins interacting with ANKRD5 (ANKEF1). Below, we provide a detailed analysis of the roles and interaction mechanisms of the top-ranked non-N-DRC proteins (Krt77, Rab2a, Gm7429) to elucidate their functional relevance to ANKRD5. We have added the following text to page 6 to clarify and highlight this in red:

As for other proteins in the LC-MS results, KRT77 is a classic protein that maintains cytoskeletal stability. It may enhance the physical connection between the N-DRC and adjacent DMTs through interaction with ANKRD5. Recent studies indicate that ANKRD5, a newly identified component in the distal lobe of the N-DRC, has a positively charged surface, which may facilitate binding to glutamylated tubulin on adjacent DMTs[41]. Thus, KRT77 may also regulate its interaction with ANKRD5 via post-translational modifications (PTMs, e.g., phosphorylation), thereby strengthening sperm resistance to shear forces during flagellar movement. Rab family proteins participate in intraflagellar transport and membrane dynamics. RAB2A may promote targeted transport of ANKRD5 or other N-DRC components to axonemal assembly sites by recruiting vesicles, and its GTPase activity might link cellular signals to ANKRD5-mediated axoneme remodeling. However, the observed signals could be false positives due to nonspecific factors such as electrostatic adsorption, high-abundance protein interference, detergent-induced membrane disruption, or protein aggregation tendencies.

The immunofluorescence localization of ANKRD5-Flag appears more aligned with the mitochondrial sheath rather than the axoneme. There is a finer red fluorescent signal extending from the mitochondrial sheath that might correspond to the axoneme. Could this suggest that ANKRD5 has a functional role in the mitochondria? While the authors measured ROS levels, this might not fully clarify whether ANKRD5 is involved in sperm energy metabolism. Considering the motility defects in Ankrd5 knockout mice, further experiments to explore ANKRD5's potential involvement in energy metabolism are necessary.

The increased detection of ANKRD5 in the midpiece region of the sperm axoneme does not necessarily indicate its localization in mitochondria. Immunofluorescence signals of multiple axonemal Nexin-Dynein Regulatory Complex (N-DRC) components (e.g., TCTE1, DRC1, CCDC65, DRC3, GAS8, and DRC7) are also non-uniformly distributed along the entire flagellum[1]. Similar localization patterns are observed in other structural components, such as radial spoke protein NME5[2] and outer dynein arm protein DNAH5[3]. Furthermore, mitochondria are membrane-bound organelles, and ANKRD5 predominantly resides in the SDS-soluble fraction under varying lysis conditions, confirming its association with the axoneme rather than mitochondria. Thus, the spatial distribution of ANKRD5 does not support a functional role in mitochondria. Importantly, we validated intact mitochondrial function through measurements of reactive oxygen species (ROS) levels (Figure S5C, D), ATP content (Figure 6E), and mitochondrial membrane potential (Figure S5A, B).

Proteomic analysis of Ankrd5-deficient sperm revealed disruptions in the glycolysis pathway. While these changes do not appear to affect ATP production, the mechanism by which these disruptions impact sperm motility remains unclear. Further investigation into how glycolysis pathway alterations contribute to impaired motility is warranted.

We appreciate the reviewer's careful consideration of our proteomic data. However, our Gene Set Enrichment Analysis (GSEA) of glycolysis/gluconeogenesis pathways showed no significant enrichment (p-value=0.089, NES=0.708; Fig.6D), which does not meet the statistical thresholds for biological significance (|NES|>1, pvalue<0.05). This observation is further corroborated by our direct ATP measurements showing no difference between genotypes (Fig.6E). We agree that further studies on metabolic regulation could be valuable, but current evidence does not support glycolysis disruption as a primary mechanism for the motility defects observed in Ankrd5-null sperm. This misinterpretation likely arose from the reviewer's overinterpretation of non-significant proteomic trends. We request that this specific claim be excluded from the assessment to avoid misleading readers.

Weaknesses:

Cryo-ET Data Limitations: The structural analysis of the DMT lacks clarity on how ANKRD5 influences NDRC or RS3. The low quality of RS3 data hinders the interpretation of ANKRD5's impact on axoneme structure.

We tried to further calculate the DMT at 96nm period using the present data to analyze the effect of ANKRD5 deletion on RS and N-DRC, however, due to the heterogeneity of the data, we were only able to obtain DMT at 8nm period (we have added a figure in the supplementary material for presentation). And in the process of obtaining DMT with a period of 8nm, we throw away about 90% of the particles (some are misaligned particles, some are deformed particles). Although we were not able to obtain the structure of 96nm repeats DMT, we noticed the enhanced heterogeneity of DMT caused by ANKRD5 knockout, as shown by the 3D classification and other results of the new supplementary images (Fig. S9), and the graphic description was added in the original article.

We have changed the description in the text: "During particle picking of DMT fibers, we observed that transverse sections of axonemal DMT particles from ANKRD5-KO sperm differ markedly from those in WT sperm. Although both A- and B-tubes were visible in both samples, the DMTs in ANKRD5-KO sperm showed a more irregular profile. In WT sperm, DMTs typically appeared circular, whereas ANKRD5-KO DMTs seemed to be extruded as polygonal. (Fig. S9B,D). Notably, ANKRD5-KO DMTs seemed partially open at the junction between the A- and B-tubes (Fig. S9B,D).

During the STA process, many particles were misaligned or deformed in the classification results, revealing various degrees of deformation—particularly affecting the B-tube (Fig. S9E). We could retain only ~10% of the DMT particles to obtain the final density map for ANKRD5-KO sperm (Fig. S9E), whereas ~70% were usable in WT dataset as reported previously [59]. The mutant DMT density map also displayed roughness at its periphery, indicating substantial structural heterogeneity (Fig. S9E). Even after discarding a large fraction of deformed particles, the final density map still showed evident artifacts, implying that although the mutant DMT preserves the fundamental features of both tubes, its shape is highly heterogeneous (Fig. S9E). Furthermore, attempts to classify the 96-nm repeats did not yield a clear density for radial spokes (RSs) (Fig. S9F), indicating that ANKRD5 deficiency may affect the stability of other accessory structures, such as RSs [23，24，25]. In the raw tomograms, RSs in ANKRD5-KO sperm appeared less regularly arranged than those in WT (Fig. S9A and C).

Most recently, following the submission of this work, ANKRD5 was reported to localize at the head of the N-DRC, simultaneously binding DRC11, DRC7, DRC4, and DRC5 [46]. This structural insight agrees with our in vitro findings that ANKRD5 interacts with DRC4 and DRC5 (Fig. 8C-F). However, that study used isolated and purified DMT samples, leaving the precise positioning of ANKRD5 between adjacent axonemal DMTs unconfirmed. We therefore fitted the published structure (PDB entry: 9FQR) into the in situ DMT structure of mouse sperm 96-nm repeats (EMD-27444), revealing that ANKRD5 lies a mere ~3 nm from the adjacent DMT (Fig. 8G). Notably, the N-DRC is often likened to a "car bumper", buffering two neighboring DMTs during vigorous axonemal motion. Given the extensive DMT deformation observed in our cryo-ET data (Fig. S9E), we propose that ANKRD5 contributes to this buffering function at the N-DRC. The loss of ANKRD5 may weaken the "bumper" effect and consequently increase structural damage to adjacent DMTs under intense conditions, while also compromising the stability of associated DMT accessory structures [19，46，60]."

Figure S9. The states of DMT particles in sperm of Ankrd5-KO mouse. (A) and (C) Tomogram slices of WT and Ankrd5-KO in Dynamo (The data for WT mouse sperm was EMPIARC-200007). DMT and RS are marked with white dashed lines and white arrows, respectively. (B) and (D) Comparison of DMT particle states between WT and Ankrd5-KO in Dynamo. The visual angles of the DMT particles shown in (B) and (D) show that the DMT fibers within the white box in (A) and (B) are divided equally into 10 slices along the direction of the white arrow, respectively. The DMT particle shapes of WT and Ankrd5-KO are marked by white dashed lines on the right of (B) and (D). The white arrow in (D) identifies the junction of A-tube and B-tube that is suspected to be disconnected. (E) Deformed particles discarded in 3D classification and final aligned DMT artifacts. (F) 3D classification of attempted RS locations.

Although the loss of ANKRD5 did not affect the density of DMT itself in A Tube and B Tube, we found that DMT particles were not smooth in the original tomogram. We speculate that the loss of ANKRD5, a component of the N-DRC that is close to the neighboring DMT, may reduce the interaction between N-DRC and the neighboring DMT, resulting in uneven force on the axoneme during sperm swimming, which may limit our ability to obtain the average structure of the more dynamic components (RS, N-DRC, ODA, IDA). Therefore, when trying to classify 96nm repeat DMT, we could only see the density of suspected RS3 and RS2, but it was difficult to obtain the complete 96nm repeat DMT density, so that we could not further analyze the effect of ANKRD5 deletion on RS and N-DRC. To test this conjecture, we further classified the density of suspected RS3, and the results obtained exhibited a variety of mixed states (which have been added to the supplementary material). To avoid confusion, we have already removed the discussion of RS3 and the related images from the original text.

The cryo-ET data on the internal structure of the DMT seems to have limited relevance to the N-DRC complex. Additionally, the quality of the RS3 data appears suboptimal, making it difficult to understand how the absence of ANKRD5 influences RS3. Further refinement of the data or alternative approaches may be needed to address this question.

Thank you very much for your suggestions. For the 96 nm periodic DMT, we have conducted multiple rounds of classification, including applying different masks at the positions of ODA, RS, and DMT. We have also tried classifying with both a single reference and multiple references. However, we were unable to obtain a suitable 96 nm periodic DMT. Regarding the heterogeneity of the particles, we have added a discussion in the manuscript. Following your advice, we have reanalyzed the data, but unfortunately, we still could not further optimize the experimental results.

In the process of obtaining the 8 nm periodic DMT, we discarded approximately 90 percent of the particles through multiple rounds of classification and alignment, in order to obtain high-quality 8 nm periodic DMT. We classified the remaining particles and found that the densities of RS3 and RS2 were not in their normal states. RS3 might be a mixture of different states of RS3, which makes it difficult for us to further discuss the effects of ANKRD5 on RS3.

To avoid confusion, we have already removed the discussion of RS3 and the related images from the original text.

Regarding the effects of ANKRD5 deficiency, we speculate that as the head of the N-DRC, its absence might affect the interaction between the N-DRC and the adjacent DMT, thereby influencing the forces experienced by the DMT during sperm movement. The uneven and irregular forces on the nine pairs of DMTs do not affect the structure of the A and B tubes of the DMT itself, but result in some heterogeneity in the peripheral microtubule parts of the DMT particles. We have added a discussion on these hypotheses in the manuscript. In addition, our 3D classification results demonstrate the structural heterogeneity of DMT caused by ANKRD5 knockdown. We have changed the description in the text:"During particle picking of DMT fibers, we observed that transverse sections of axonemal DMT particles from ANKRD5-KO sperm differ markedly from those in WT sperm. Although both A- and B-tubes were visible in both samples, the DMTs in ANKRD5-KO sperm showed a more irregular profile. In WT sperm, DMTs typically appeared circular, whereas ANKRD5-KO DMTs seemed to be extruded as polygonal. (Fig. S9B,D). Notably, ANKRD5-KO DMTs seemed partially open at the junction between the A- and B-tubes (Fig. S9B,D).

During the STA process, many particles were misaligned or deformed in the classification results, revealing various degrees of deformation—particularly affecting the B-tube (Figure 9, Fig. S9E). We could retain only ~10% of the DMT particles to obtain the final density map for ANKRD5-KO sperm (Fig. S9E), whereas ~70% were usable in WT dataset as reported previously [59]. The mutant DMT density map also displayed roughness at its periphery, indicating substantial structural heterogeneity (Fig. S9E). Even after discarding a large fraction of deformed particles, the final density map still showed evident artifacts, implying that although the mutant DMT preserves the fundamental features of both tubes, its shape is highly heterogeneous (Fig. S9E). Furthermore, attempts to classify the 96-nm repeats did not yield a clear density for radial spokes (RSs) (Fig. S9F), indicating that ANKRD5 deficiency may affect the stability of other accessory structures, such as RSs [23，24，25]. In the raw tomograms, RSs in ANKRD5-KO sperm appeared less regularly arranged than those in WT (Fig. S9A and C).

Most recently, following the submission of this work, ANKRD5 was reported to localize at the head of the N-DRC, simultaneously binding DRC11, DRC7, DRC4, and DRC5 [46]. This structural insight agrees with our in vitro findings that ANKRD5 interacts with DRC4 and DRC5 (Fig. 8C-F). However, that study used isolated and purified DMT samples, leaving the precise positioning of ANKRD5 between adjacent axonemal DMTs unconfirmed. We therefore fitted the published structure (PDB entry: 9FQR) into the in situ DMT structure of mouse sperm 96-nm repeats (EMD-27444), revealing that ANKRD5 lies a mere ~3 nm from the adjacent DMT (Fig. 8G). Notably, the N-DRC is often likened to a "car bumper", buffering two neighboring DMTs during vigorous axonemal motion. Given the extensive DMT deformation observed in our cryo-ET data (Fig. S9E), we propose that ANKRD5 contributes to this buffering function at the N-DRC. The loss of ANKRD5 may weaken the "bumper" effect and consequently increase structural damage to adjacent DMTs under intense conditions, while also compromising the stability of associated DMT accessory structures [19，46，60]."

To further enhance the readability of our manuscript, we created a Graphic Abstract to visually illustrate the biological functions of ANKRD5. The figure is placed immediately after the Abstract section and has been designated as Figure 9.