The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

  1. Miguel Ricardo Leung
  2. Marc C. Roelofs
  3. Ravi Teja Ravi
  4. Paula Maitan
  5. Min Zhang
  6. Heiko Henning
  7. Elizabeth G. Bromfield
  8. Stuart C. Howes
  9. Bart M. Gadella
  10. Hermes Bloomfield-Gadêlha
  11. Tzviya Zeev-Ben-Mordehai

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Abstract

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this study, the authors use focused-ion beam (FIB) milling coupled with cryo-electron tomography and subtomogram averaging to uncover the structure of the elusive proximal and distal centrioles, as well as different regions of the axoneme in the sperm of 3 mammalian species: pig, horse, and mouse. The in-situ tomograms of the sperm neck region beautifully illustrate the morphology of both the proximal centriole, confirming the partial degeneration of mouse sperm, and intriguingly, asymmetry in the microtubule wall of pig sperm. In distal centrioles, the authors show that in all mammalian species, microtubule doublets of the centriole wall are organized around a pair of singlet microtubules. The presented segmentation of the connecting piece is beautiful and nicely shows the connecting piece forming a nine-fold, asymmetric, chamber the centrioles. The authors further use subtomogram averaging to provide the first maps of the mammalian central pair and identify sperm-specific radial spoke-bridging barrel structures. Lastly, the authors perform further subtomogram averaging to show to the connecting site of the outer dense fibers to the microtubule doublet of the proximal principal piece and confirm the presence of the TAILS microtubule inner protein complex (Zabeo et al, 2018) in the singlet microtubules occupying the tip of sperm tails.

    The manuscript provides the clearest insight into flagellar base morphology to date, giving insight into the morphological difference between different mammalian cilia and centriole types. The manuscript is suitable for publication, once the following questions are addressed.

    We are ecstatic that the reviewer shares our enthusiasm for this work. We are particularly grateful that the reviewer appreciates the significance of the unique, and hitherto under-explored biology of the sperm centrioles and the flagellar base.

    **Major Points:**

    How many centrioles and axonemes were used in generating the averages presented in the paper? If too few samples were used, especially in centrioles undergoing dramatic remodeling or degeneration, the reality of MIPs and MAPs being present might be completely affected. For instance, In figure 1d, the authors present a cryoET map of the centriole microtubule triplet. However, centrioles are divided into several regions with different accessory elements. Here, the authors could show the presence of only part of the A-C linker. The A-C linker covers only 40% of the centriole, so does it mean that this centriole is made only of the accessories that characterize the proximal side of the centriole? In the same line, what were the boundaries governing subtomogram extraction? For example, in the distal centriole, were microtubules extracted from just before the start of the transition zone, to the end of the microtubule vaulting, more pronounced at the end of the proximal region? There are known heterogeneities in centriole, as well as flagella, ultrastructure along the proximal distal axis. If no pre-classification was performed for subtomogram longitudinal position along with the centriole and axoneme, structural features may be averaged out, and or present and not reflecting their real longitudinal localization. The classification should be applied here if it was not the case.

    These are all valid points. Because there is no easy way to target the PC/DC when cryo-FIB milling, and because there is only one of each structure in every cell, the chances of catching them in ~150-nm-thin lamellae are slim (not to mention the number of things that can and do go wrong when doing cryo-ET on lamellae). As such, the averages of the PC were generated from 3 tomograms (3 cells) and those of the DC from 2 tomograms (2 cells). We do have more tomograms with the PC/DC, but these were used for segmentation/visual inspection since we only used the best tomograms for averaging. These numbers are not entirely atypical for cryo-FIB datasets; the only other in situ centriole structures are from 5-6 centrioles (from Chlamydomonas, from Le Guennec et al 2020 doi: 10.1126/sciadv.aaz4137 and Klena et al 2020 doi: 10.15252/embj.2020106246).

    To allow readers to adjust their interpretations according to the small number of cells analysed, we explicitly stated the number of animals/cells/tomograms used to generate averages in Table S1. Furthermore, we amended the text to clarify which regions of the centrioles our averages represent. These changes are detailed below:

    (1) proximal centriole

    The lamellae used for averaging PC triplets caught mostly the proximal end of the centriole, and essentially all of the particles come from the most proximal ~ 400 nm. In a sense, this was a form of pre-classification. We now state explicitly that our structure represents only the proximal region and that proximal/distal differences may be identified in the future (see section on distal centriole below). Despite the limited particle number, we are confident in the presence of the MIPs as these are also visible in the raw data (the striations in Fig. 1a, now Fig. 1d, for instance). Page 7, Line 165 was edited accordingly as well as the legend to Fig. 1.

    (2) distal centriole

    The subtomograms used for the DC average were extracted from the region of the distal centriole closest to the base of the axoneme (i.e; the region marked “distal centriole” in Fig. 2h-i). Because the DC doublet average in Fig. 2j was generated from very few particles, we tried to be very conservative when interpreting it. Page 9, Line 216 was edited accordingly likewise the legend to Fig. 2.

    (3) axoneme

    We did attempt to average the axoneme from different regions of flagella (midpiece, proximal principal piece, distal principal piece). This is shown in Fig. 6d-l. The major difference we found was at the doublet-ODF connection. We did not find any striking differences in MIP densities, or in radial spoke densities along the proximodistal axis. As such, the averages in Fig. 5 are from the entire principal piece (but not the midpiece), which we state in the figure legend.

    Because mammalian sperm flagella are very long, it is possible that we missed more subtle differences. We now state this in the Discussion (page 20, line 491):

    **Minor Points:**

    • In line 3, motile cilia are not only used to swim, they can move liquid or mucus for instance.

    Done. Page 3, line 64

    • In line 175, the authors stated " a prominent MIP associated with protofilament A9, was also reported in centrioles isolated from CHO cells (Greenan et al. 2018) and in basal bodies from bovine respiratory epithelia (Greenan et al 2020). Actually, this MIP has been seen in many other centrioles from other species, such as Trichonympha (https://doi.org/10.1016/j.cub.2013.06.061 ), Chlamydomonas, and Paramecium ( DOI: 10.1126/sciadv.aaz4137 ). Citing these studies will reinforce the evolutionary conservation of this MIP and therefore its potential crucial role in the A microtubule.

    We thank the reviewer for pointing out these very important papers, we added them to the manuscript (page 7, lines 175-176).

    • In Line178, the authors stated: "Protofilaments A9 and A10 are proposed to be the location of the seam (Ichikawa et 2017)". High-resolution cryoEM maps confirmed it: https://doi.org/10.1016/j.cell.2019.09.030 . This publication should be cited. Moreover, authors should also refer to this paper when discussing MIPs in the microtubule doublet.

    Done (page 7, lines 178-179 and page 13, line 329).

    We also now cite Ma et al (along with Ichikawa et al 2019 doi: 10.1073/pnas.1911119116 and Khalifa et al 2020 doi: 10.7554/eLife.52760) in the Discussion when alluding to high-resolution structures as a possible means of identifying MIPs (page 19, lines 479).

    • In Line 187-189 the authors stated, "We resolved density of the A-C linker (gold) which is associated with protofilaments C9 and C10." The A-C linker interconnects the triplets of the proximal centriole (Guichard et. al. 2013, Li et. al. 2019, Klena et. al. 2020) with distinct regions binding the C-tubule, as shown by the authors in gold, as well as an A-link, making contact with the A-tubule through various protofilaments in a species-specific manner, but always on protofilament A9. The authors may have identified the A-link, labeled in green, on the outside of protofilament A8/A9 in Figure 1d.

    We thank the reviewer for pointing this out. The position of the olive green density associated with A8/A9 is indeed consistent with the A-link, and this is also now illustrated more clearly in the new version of Fig. 1e (now Fig. 1h, see below). We accordingly edited page 8, lines 187-188.

    • In figure 1e, the authors provide a 9-fold representation of the centriole based on their map. How relevant is this model ? the distance between triplet is inconsistent here, which has not been observed before. Do they use true 3D coordinates to generate this model? The A-C linker, which is only partially reconstructed, does not contact the A microtubule. Is it really the case? did the authors see that the A-link density of the A-C linker has disappeared? If these points are not clearly specified, this representation might be misleading.

    In order to avoid misleading readers, we replaced this panel with a model generated directly by plotting back the averages into their original positions and orientations in the tomogram (new Fig. 1h). This model now shows that the olive green density on A8/A9 is in the right position to form part of the A-C linker (as Reviewer 1 correctly pointed out in their previous point). We have amended the figure legend accordingly. We also described how the plotback was generated in the Materials and Methods section (page 26, line 648).

    As the reviewer points out, the distance between triplets does indeed seem inconsistent in the plotback. This is an interesting observation, but we feel it is a bit too preliminary to discuss in detail here. This can be explored in a follow-up study more focused on sperm centriole geometry.

    • The nomenclature regarding MIPs is sometimes confusing in this manuscript. For example, in lines 228-229 "We then determined the structure of DC doublets, revealing the presence of MIPs distinct from those in the PC." Does this include the gold and turquoise labeled structures in Figure 2j? These densities appear to correspond to the inner scaffold stem in the gold density presented in Figure 2j, and armA, presented in the turquoise density (Li et. al. 2011, Le Guennec et. al. 2020). The presence of this Stem here is important as it correlates with the presence of the molecular player making the inner scaffold (POC5, POC1B, CENTRIN): https://doi.org/10.1038/s41467-018-04678-8

    While we were initially very conservative with interpreting the DC doublet average (as stated above it comes from very few particles), we agree with the reviewer’s assessment that the gold and turquoise densities in Fig. 2j are consistent with the Stem and armA respectively of the inner scaffold. Because the inner scaffold contributes to centriole rigidity, it will be interesting to determine if and how it changes during remodelling of the atypical DC in mammalian sperm. Intriguingly, at least some inner scaffold components (including POC5, POC1B) reorganise into two rods in the mammalian sperm DC (Fishman et al 2018 doi: 10.1038/s41467-018-04678-8). We expanded the section on the DC average (page 9, lines 218-220):

    • The connecting piece is composed of column vaults emanating from the striated columns is compelling and beautiful segmentation data. However, it is important to note how many pig sperm proximal centrioles had immediate-short triplet side contact with the Y-shaped segmented column 9, as well as in how many mouse centrioles have the two electron-dense structures flanking the striated columns.

    Done. Material and Methods Page 25, lines 615-619.

    The resolution of the mammalian central pair is an important development brought by this work. The structural similarity between the central pair of pig and horse is convincing. However, with only 281 subtomograms being averaged for the murine central pair, corresponding to an estimated resolution of 49Å, the absence of the helical MIP of C1 with 8 nm periodicity suggests that there is simply not enough signal to capture it in the average. The same could be said for the smaller MIP displayed in Figure 4 c, panel ii. This point should be clearly stated.

    We agree with the reviewer that the quality of the mouse CPA structure is not on par with the pig and horse CPA structures. We now explicitly state this caveat in the text (pages 11, lines 276-277):

    Another piece of compelling data presented in this study is the attachment of the outer dense fibers to the axoneme of the midpiece and proximal and distal principal pieces. From the classification data presented along the flagellar length, it is clear that the only ODF contact made with the axoneme is at the proximal principle plate. However, this is far from obvious in the native top view images presented. Is it possible to include a zoomed inset of the connection between the A-tubule and ODF connection?

    We are very happy that the reviewer finds this data exciting. As Fig. 6 is quite cluttered as is, we instead tried to better annotate the cross-section views of the axoneme by tracing one doublet-ODF pair in each image (or only a doublet in the case of the distal principal piece). This shows that there is a gap between the doublet and the ODF in the midpiece, and that there is no such gap in the principal piece. We also hope that annotating one doublet-ODF pair helps the reader see that the same pattern holds true for the other doublets/ODFs. The legend to Fig. 6 was changed accordingly.

    Reviewer #1 (Significance (Required)):

    This work is of good quality and provides crucial information on the structure of centriole and axoneme in 3 different species. This work complements well the previous works.

    The audience for this type of study is large as it is of interest to researchers working on centrioles, cilium, and sperm cell architecture.

    We are pleased the reviewer appreciate the quality of our work and see the interest for broad audience.

    My expertise is cryo-tomography and centriole biology

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, Leung et al. used state-of-the-art EM imaging techniques, including FIB cryo-milling, Volta Phase plate, cryo-electron tomography and subtomogram averaging, to study the structure of sperm flagella from three mammalian species, pig, horse and mouse. First, they described two unique centrioles in the sperm, the PC and the DC. They found the PCs are composed of a mixture of triplet and doublet MTs. In contrast, the DCs are composed mainly of doublet and singlet MTs. By using subtomogram averaging, they identified a number of accessory proteins, including many MIPs bound to the MT wall. Many are unique to the mammalian sperm. They further described the connecting piece region of the sperm enclosing the centrioles and found an asymmetric arrangement. Furthermore, the authors presented the structure of sperm axonemes from all three species. These include the DMT and the CPA. Finally, they described the tail region of the sperm and described how the DMTs transitioned to the singlet MTs.

    This is a beautiful piece of work! It is by far the most comprehensive structural study of mammalian sperm cells. These findings will serve as a valuable resource for structure and function analysis of the mammalian flagella in the future. Now the stage is set for identifying the molecular nature of the structures and densities described in this study.

    We thank the reviewer for their positive evaluation! We are very happy that they share our excitement for the work, and that they also see it as “setting the stage” for future studies at the molecular level.

    The manuscript is clearly written. The data analysis is thorough. The conclusions are solid and not overstated. I don't have any major issues for its publication. A number of minor suggestions are listed below. Most are related to the figures and figure legends.

    Figure 1d, the figure legend should mention this is the subtomogram average of PC triplet MTs from pig sperm, though this is mentioned in the text. Also, for convenience, the color codes for the MIPs should be mentioned in the figure legend.

    Done.

    Figure 2J, similarly, the figure legend should mention this is the subtomogram average of DC doublets. It also needs a description of the color codes of the identified MIPs. For the DMT, please indicate the A- and B-tubule, which are colored in light or dark blue.

    Done, except we would prefer not to enumerate the MIPs as we did not name them nor discuss them extensively in the main text as we do not want to over-interpret the MIPs at this point as the average is from relatively small number of particles. However, we did specify that the gold and turquoise densities on the luminal surface are consistent with the inner scaffold. The figure legend was edited accordingly.

    Line 228, "We then determined the structure of DC doublet by subtomogram averaging"

    Done.

    For both Fig 2 and Fig 3. the DC doublets are colored in dark and light blue, please specify which is the A- or B-tubule in the figure legends.

    Done.

    Line 273, need space between "goldenrod"

    We would prefer to keep “goldenrod” spelled as is since this is how the color is referred to in Chimera and ChimeraX.

    Figure 4. need to expand the figure legend. Panels I, ii, iii, iv, are cut-through view of the lumen of CPA microtubules C1 and C2.

    Done.

    Line 338, Interestingly, the RS1 barrel is radially distributed asymmetrically around the axoneme

    Done.

    Figure 5, need color codes for the arrowheads (light pink, pink, magenta) in panels i~n,

    Done.

    Figure 7, (a-c) please use arrowheads to indicate the location of caps in the singlet MT.

    Done.

    Reviewer #2 (Significance (Required)):

    This is a beautiful and significant work - by far the most comprehensive analysis of mammalian sperm structure

    We are thrilled the reviewer appreciate the novelty of our work.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This is a very interesting study that explores the structural diversity of mammalian sperm flagella, in pig, mouse and horse, at high resolution using cryo-FIB milling and cryo-tomography. The study provides the first in situ cryo-EM structure of a mammalian centriole and describes a number of microtubule associated structures, such as MIPs and plugs at the plus-end of microtubules, that were not been reported so far. Additionally, the authors identify several asymmetries in the overall structure of the flagellum in the three species, which have implications for the understanding of the flagellar beat and waveform geometry in sperm, which are discussed by the authors. Although this study does not provide mechanistic novel information on the function of the described structures, it will undoubtedly serve as a reference for future theoretical and empirical work on the role of these structures in shaping the flagellar beat.

    With the exception of a couple of "eclectic word choices" in the Introduction (see detailed feedback in Minor Comments), the manuscript is also well written. Image acquisition and analysis are sound.

    We thank the reviewer for positively evaluating our work. We are glad that they feel our study will “serve as a reference” to inform future studies.

    However, I have some suggestions that should help the authors to strengthen their claims and present their results. The study is in principle suitable to be published, after the following points will be addressed:

    **Major comments:**

    • A major concern is that it is not clear how many animals, sperms and lamellae the authors used to acquire the data presented in the manuscript. This information needs to be provided, because it not uncommon to encounter aberrant flagella, even in a wildtype animal. The authors should state how many animals, and how many flagella per each animal were analyzed, in order to allow the reader to have an opinion on the reliability of their observations.
    • The figures are esthetically pleasing; however, the figures legends should be carefully revised to include necessary information about color codes, image annotations.

    We thank the reviewer for raising these points. We completely agree that the numbers of animals and cells are important pieces of information. As such, we now explicitly state the number of animals/cells/tomograms used for each average in Table S1. For more qualitative observations (such as the relationship between the asymmetry of the pig sperm PC and the Y-shaped segmented columns), we now state in the number of cells and animals in which we see each feature (see detailed response to Reviewer 1).

    **Minor comments:**

    • Line 26. I do not think that the word "menagerie" is properly used in this context.
    • Line 29. The same is true for the word "Bewildering" in this sentence.

    We apologise for our somewhat eclectic word choice. We see the reviewer’s point that unconventional word choice may distract readers, so we replaced these two words with ‘diverse’ and ‘an extensive’, respectively.

    • Line 286 "Our structures of the CPA are the first from any mammalian system, and our structures of the doublets are the first from any mammalian sperm, thus filling crucial gaps in the gallery of axoneme structures." Sentences like this one would fit much better in the Conclusions or at least in the Discussion.

    We thank the reviewer for this suggestion, but we would prefer to keep this sentence where it is, if possible. We think it is useful to tell the audience upfront why these structures are significant, especially since readers who aren’t deep in the field may be bogged down by all the details.

    • Line 377 "Large B-tubule MIPs have so far only been seen in human respiratory cilia (Fig. 5j) and in Trypanosoma (the ponticulus, Fig. 5n), but the morphometry of these MIPs differs from the helical MIPs in mammalian sperm." Please insert the citations for the studies about respiratory cilia and Trypanosoma flagella.

    Done.

    • In Figure 1. What do the stars shown in panel a and a' indicate?

    We indeed failed to specify what the asterisks/stars indicate. They are meant to emphasise that the electron-dense material in the lumen of the PC is continuous with the CP. We have now specified this in the text (page 10, lines 245).

    Given the complexity of the structures that compose the flagellar system of sperms, it would be helpful to add an illustration of the sperm with careful annotation of the centriole structures and the various segments of the flagellum.

    This is an excellent suggestion. To help orient readers, we added three panels to Fig. 1 (Fig. 1a-c) showing low-magnification images of whole sperm cells. We annotated different parts of the flagellum (neck, midpiece, principal piece, endpiece) so that readers can refer back to these panels in case they want to know which part of the cell the averages are from.

    • Figure 2. Explanation of the used color codes is missing. Additionally, the authors should include an explanation for the black and white arrows and for the 2 insets in i.

    Done. For the color code, please see response to Reviewer 2. For the black and white arrows, we edited the figure legend.

    • In "(j) In situ structure of the pig sperm DC with the tubulin backbone in grey and microtubule inner protein densities colored individually" ...it should be written "...sperm DC microtubule doublet..."

    Done.

    • In this figure, but also in every other figure that shows centriole, axoneme, or even microtubule averages it is important to indicate the microtubule polarity. Please add the symbol + and - to indicate microtubule polarity in the figures.

    Done. In order to avoid overcrowding, we only labelled the pig structures as the horse and the mouse structures are always shown in the same orientations as the pig.

    • Figure 3. Additional to the images in a,b, and c, the original tomographic slices (without segmentation) should be shown here, to allow the reader to visualize the structure.

    We now include three additional supplementary movies slicing through the respective tomograms.

    • Figure 7. Scale bars are missing in d-f.

    Done.

    • Scale bars are missing in most Supplementary figures.

    Done.

    • Table S1. The Information about horse and mouse centriole data is missing.

    The reviewer is correct, but this information is missing because we did not average from the horse and the mouse. For the mouse, the triplets were in various stages of degeneration, resulting in heterogeneity that precluded us from averaging. For the horse, we simply did not catch enough centrioles to generate a meaningful structure.

    Reviewer #3 (Significance (Required)):

    This study provides several novel structural insights in to the sperm flagellum structure that have implications for the understanding of the flagellar beat and waveform geometry in sperm. Although this study does not provide mechanistic novel information on the function of the described structures, it will undoubtedly serve as a reference for future theoretical and empirical work on the role of these structures in shaping the flagellar beat.

    Great to see the reviewer appreciate the novelty of our work.

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    Referee #3

    Evidence, reproducibility and clarity

    This is a very interesting study that explores the structural diversity of mammalian sperm flagella, in pig, mouse and horse, at high resolution using cryo-FIB milling and cryo-tomography. The study provides the first in situ cryo-EM structure of a mammalian centriole and describes a number of microtubule associated structures, such as MIPs and plugs at the plus-end of microtubules, that were not been reported so far. Additionally, the authors identify several asymmetries in the overall structure of the flagellum in the three species, which have implications for the understanding of the flagellar beat and waveform geometry in sperm, which are discussed by the authors. Although this study does not provide mechanistic novel information on the function of the described structures, it will undoubtedly serve as a reference for future theoretical and empirical work on the role of these structures in shaping the flagellar beat. With the exception of a couple of "eclectic word choices" in the Introduction (see detailed feedback in Minor Comments), the manuscript is also well written. Image acquisition and analysis are sound.

    However, I have some suggestions that should help the authors to strengthen their claims and present their results. The study is in principle suitable to be published, after the following points will be addressed:

    Major comments:

    • A major concern is that it is not clear how many animals, sperms and lamellae the authors used to acquire the data presented in the manuscript. This information needs to be provided, because it not uncommon to encounter aberrant flagella, even in a wildtype animal. The authors should state how many animals, and how many flagella per each animal were analyzed, in order to allow the reader to have an opinion on the reliability of their observations.
    • The figures are esthetically pleasing; however, the figures legends should be carefully revised to include necessary information about color codes, image annotations.

    Minor comments:

    • Line 26. I do not think that the word "menagerie" is properly used in this context.
    • Line 29. The same is true for the word "Bewildering" in this sentence.
    • Line 286 "Our structures of the CPA are the first from any mammalian system, and our structures of the doublets are the first from any mammalian sperm, thus filling crucial gaps in the gallery of axoneme structures." Sentences like this one would fit much better in the Conclusions or at least in the Discussion.
    • Line 377 "Large B-tubule MIPs have so far only been seen in human respiratory cilia (Fig. 5j) and in Trypanosoma (the ponticulus, Fig. 5n), but the morphometry of these MIPs differs from the helical MIPs in mammalian sperm." Please insert the citations for the studies about respiratory cilia and Trypanosoma flagella.
    • In Figure 1. What do the stars shown in panel a and a' indicate? Given the complexity of the structures that compose the flagellar system of sperms, it would be helpful to add an illustration of the sperm with careful annotation of the centriole structures and the various segments of the flagellum.
    • Figure 2. Explanation of the used color codes is missing. Additionally, the authors should include an explanation for the black and white arrows and for the 2 insets in i.
    • In "(j) In situ structure of the pig sperm DC with the tubulin backbone in grey and microtubule inner protein densities colored individually" ...it should be written "...sperm DC microtubule doublet..."
    • In this figure, but also in every other figure that shows centriole, axoneme, or even microtubule averages it is important to indicate the microtubule polarity. Please add the symbol + and - to indicate microtubule polarity in the figures.
    • Figure 3. Additional to the images in a,b, and c, the original tomographic slices (without segmentation) should be shown here, to allow the reader to visualize the structure.
    • Figure 7. Scale bars are missing in d-f.
    • Scale bars are missing in most Supplementary figures.
    • Table S1. The Information about horse and mouse centriole data is missing.

    Significance

    This study provides several novel structural insights in to the sperm flagellum structure that have implications for the understanding of the flagellar beat and waveform geometry in sperm. Although this study does not provide mechanistic novel information on the function of the described structures, it will undoubtedly serve as a reference for future theoretical and empirical work on the role of these structures in shaping the flagellar beat.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Leung et al. used state-of-the-art EM imaging techniques, including FIB cryo-milling, Volta Phase plate, cryo-electron tomography and subtomogram averaging, to study the structure of sperm flagella from three mammalian species, pig, horse and mouse. First, they described two unique centrioles in the sperm, the PC and the DC. They found the PCs are composed of a mixture of triplet and doublet MTs. In contrast, the DCs are composed mainly of doublet and singlet MTs. By using subtomogram averaging, they identified a number of accessory proteins, including many MIPs bound to the MT wall. Many are unique to the mammalian sperm. They further described the connecting piece region of the sperm enclosing the centrioles and found an asymmetric arrangement. Furthermore, the authors presented the structure of sperm axonemes from all three species. These include the DMT and the CPA. Finally, they described the tail region of the sperm and described how the DMTs transitioned to the singlet MTs.

    This is a beautiful piece of work! It is by far the most comprehensive structural study of mammalian sperm cells. These findings will serve as a valuable resource for structure and function analysis of the mammalian flagella in the future. Now the stage is set for identifying the molecular nature of the structures and densities described in this study.

    The manuscript is clearly written. The data analysis is thorough. The conclusions are solid and not overstated. I don't have any major issues for its publication. A number of minor suggestions are listed below. Most are related to the figures and figure legends.

    Figure 1d, the figure legend should mention this is the subtomogram average of PC triplet MTs from pig sperm, though this is mentioned in the text. Also, for convenience, the color codes for the MIPs should be mentioned in the figure legend.

    Figure 2J, similarly, the figure legend should mention this is the subtomogram average of DC doublets. It also needs a description of the color codes of the identified MIPs. For the DMT, please indicate the A- and B-tubule, which are colored in light or dark blue.

    Line 228, "We then determined the structure of DC doublet by subtomogram averaging"

    For both Fig 2 and Fig 3. the DC doublets are colored in dark and light blue, please specify which is the A- or B-tubule in the figure legends.

    Line 273, need space between "goldenrod"

    Figure 4. need to expand the figure legend. Panels I, ii, iii, iv, are cut-through view of the lumen of CPA microtubules C1 and C2.

    Line 338, Interestingly, the RS1 barrel is radially distributed asymmetrically around the axoneme

    Figure 5, need color codes for the arrowheads (light pink, pink, magenta) in panels i~n,

    Figure 7, (a-c) please use arrowheads to indicate the location of caps in the singlet MT.

    Significance

    This is a beautiful and significant work - by far the most comprehensive analysis of mammalian sperm structure

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this study, the authors use focused-ion beam (FIB) milling coupled with cryo-electron tomography and subtomogram averaging to uncover the structure of the elusive proximal and distal centrioles, as well as different regions of the axoneme in the sperm of 3 mammalian species: pig, horse, and mouse. The in-situ tomograms of the sperm neck region beautifully illustrate the morphology of both the proximal centriole, confirming the partial degeneration of mouse sperm, and intriguingly, asymmetry in the microtubule wall of pig sperm. In distal centrioles, the authors show that in all mammalian species, microtubule doublets of the centriole wall are organized around a pair of singlet microtubules. The presented segmentation of the connecting piece is beautiful and nicely shows the connecting piece forming a nine-fold, asymmetric, chamber the centrioles. The authors further use subtomogram averaging to provide the first maps of the mammalian central pair and identify sperm-specific radial spoke-bridging barrel structures. Lastly, the authors perform further subtomogram averaging to show to the connecting site of the outer dense fibers to the microtubule doublet of the proximal principal piece and confirm the presence of the TAILS microtubule inner protein complex (Zabeo et al, 2018) in the singlet microtubules occupying the tip of sperm tails. The manuscript provides the clearest insight into flagellar base morphology to date, giving insight into the morphological difference between different mammalian cilia and centriole types. The manuscript is suitable for publication, once the following questions are addressed.

    Major Points: How many centrioles and axonemes were used in generating the averages presented in the paper? If too few samples were used, especially in centrioles undergoing dramatic remodeling or degeneration, the reality of MIPs and MAPs being present might be completely affected. For instance, In figure 1d, the authors present a cryoET map of the centriole microtubule triplet. However, centrioles are divided into several regions with different accessory elements. Here, the authors could show the presence of only part of the A-C linker. The A-C linker covers only 40% of the centriole, so does it mean that this centriole is made only of the accessories that characterize the proximal side of the centriole? In the same line, what were the boundaries governing subtomogram extraction? For example, in the distal centriole, were microtubules extracted from just before the start of the transition zone, to the end of the microtubule vaulting, more pronounced at the end of the proximal region? There are known heterogeneities in centriole, as well as flagella, ultrastructure along the proximal distal axis. If no pre-classification was performed for subtomogram longitudinal position along with the centriole and axoneme, structural features may be averaged out, and or present and not reflecting their real longitudinal localization. The classification should be applied here if it was not the case.

    Minor Points:

    • In line 3, motile cilia are not only used to swim, they can move liquid or mucus for instance.
    • In line 175, the authors stated " a prominent MIP associated with protofilament A9, was also reported in centrioles isolated from CHO cells (Greenan et al. 2018) and in basal bodies from bovine respiratory epithelia (Greenan et al 2020). Actually, this MIP has been seen in many other centrioles from other species, such as Trichonympha (https://doi.org/10.1016/j.cub.2013.06.061 ), Chlamydomonas, and Paramecium ( DOI: 10.1126/sciadv.aaz4137 ). Citing these studies will reinforce the evolutionary conservation of this MIP and therefore its potential crucial role in the A microtubule.
    • In Line178, the authors stated: "Protofilaments A9 and A10 are proposed to be the location of the seam (Ichikawa et 2017)". High-resolution cryoEM maps confirmed it: https://doi.org/10.1016/j.cell.2019.09.030 . This publication should be cited. Moreover, authors should also refer to this paper when discussing MIPs in the microtubule doublet.
    • In Line 187-189 the authors stated, "We resolved density of the A-C linker (gold) which is associated with protofilaments C9 and C10." The A-C linker interconnects the triplets of the proximal centriole (Guichard et. al. 2013, Li et. al. 2019, Klena et. al. 2020) with distinct regions binding the C-tubule, as shown by the authors in gold, as well as an A-link, making contact with the A-tubule through various protofilaments in a species-specific manner, but always on protofilament A9. The authors may have identified the A-link, labeled in green, on the outside of protofilament A8/A9 in Figure 1d.
    • In figure 1e, the authors provide a 9-fold representation of the centriole based on their map. How relevant is this model ? the distance between triplet is inconsistent here, which has not been observed before. Do they use true 3D coordinates to generate this model? The A-C linker, which is only partially reconstructed, does not contact the A microtubule. Is it really the case? did the authors see that the A-link density of the A-C linker has disappeared? If these points are not clearly specified, this representation might be misleading.
    • The nomenclature regarding MIPs is sometimes confusing in this manuscript. For example, in lines 228-229 "We then determined the structure of DC doublets, revealing the presence of MIPs distinct from those in the PC." Does this include the gold and turquoise labeled structures in Figure 2j? These densities appear to correspond to the inner scaffold stem in the gold density presented in Figure 2j, and armA, presented in the turquoise density (Li et. al. 2011, Le Guennec et. al. 2020). The presence of this Stem here is important as it correlates with the presence of the molecular player making the inner scaffold (POC5, POC1B, CENTRIN): https://doi.org/10.1038/s41467-018-04678-8
    • The connecting piece is composed of column vaults emanating from the striated columns is compelling and beautiful segmentation data. However, it is important to note how many pig sperm proximal centrioles had immediate-short triplet side contact with the Y-shaped segmented column 9, as well as in how many mouse centrioles have the two electron-dense structures flanking the striated columns.

    The resolution of the mammalian central pair is an important development brought by this work. The structural similarity between the central pair of pig and horse is convincing. However, with only 281 subtomograms being averaged for the murine central pair, corresponding to an estimated resolution of 49Å, the absence of the helical MIP of C1 with 8 nm periodicity suggests that there is simply not enough signal to capture it in the average. The same could be said for the smaller MIP displayed in Figure 4 c, panel ii. This point should be clearly stated.

    Another piece of compelling data presented in this study is the attachment of the outer dense fibers to the axoneme of the midpiece and proximal and distal principal pieces. From the classification data presented along the flagellar length, it is clear that the only ODF contact made with the axoneme is at the proximal principle plate. However, this is far from obvious in the native top view images presented. Is it possible to include a zoomed inset of the connection between the A-tubule and ODF connection?

    Significance

    This work is of good quality and provides crucial information on the structure of centriole and axoneme in 3 different species. This work complements well the previous works. The audience for this type of study is large as it is of interest to researchers working on centrioles, cilium, and sperm cell architecture.

    My expertise is cryo-tomography and centriole biology

  5. Version published to 10.1101/2020.11.18.388975 on bioRxiv