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 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.

Author response:

Thank you for the constructive feedback from the reviewers. We are grateful for their insights and are committed to addressing the key concerns raised in the public reviews through the following revisions:

(1) Validating Axoneme Stability Claims

We have procured new antibodies for DRC11, as well as marker proteins for ODA, IDA, and RS. We will conduct quantitative immunofluorescence staining to validate our claims regarding axoneme stability.

(2) Investigating ANKRD5 Expression in Other Ciliated Cells

We plan to examine the expression of ANKRD5 in mouse respiratory cilia to determine whether it is also expressed in these cells.

(3) Supplementing Key Citations for N-DRC Components

We will add references to published studies on N-DRC components (e.g., DRC1, DRC2, DRC3, DRC5) associated with male infertility in the Introduction to strengthen the background context.

(4) Further Analysis and Validation of ANKRD5 Interactome

We will conduct additional analyses and validation of the interactome of ANKRD5 detected by LC-MS.

(5) Elucidating the Function of ANKRD5 in Mitochondria

We will further investigate the role of ANKRD5 in mitochondrial function.

(6) Investigating Mitochondrial Function and Energy Metabolism

We will further explore the role of ANKRD5 in mitochondrial function and energy metabolism.

(7) Improving Cryo-ET Data Quality and Interpretation

We will attempt to further improve the quality of the STA results and try to calculate the DMT structure with a period of 96 nm. We will also use the WT density map with the same period to generate a difference map.

(8) Expanding Discussion and Correcting Terminology

The Discussion section will be revised to elaborate on the implications of ANKRD5 for male contraceptive research, particularly in targeting sperm motility. We will also correct terminology inaccuracies (e.g., changing "9+2 microtubule doublet" to "9+2 structure") and address formatting issues (e.g., capitalizing "Control").

Response to Reviewer #2 Comment 4:

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.

We will provide a comprehensive point-by-point response, along with detailed experimental data and revised figures, in the resubmitted manuscript. Thank you once again for the opportunity to address the reviewers' concerns. We are confident that these revisions will strengthen our manuscript and contribute to the scientific community.