The filipodia-like protrusions of adjacent somatic cells shape the developmental potential of mouse oocytes

  1. Flora Crozet
  2. Gaëlle Letort
  3. Christelle Da Silva
  4. Adrien Eichmuller
  5. Anna Francesca Tortorelli
  6. Morgane Belle
  7. Julien Dumont
  8. Tristan Piolot
  9. Aurélien Dauphin
  10. Fanny Coulpier
  11. Alain Chédotal
  12. Jean-Léon Maître
  13. Marie-Hélène Verlhac
  14. Hugh.J Clarke
  15. Marie-Emilie Terret

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Abstract

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells which surround it. Part of this communication is through filopodialike protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component Myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes, and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

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    Reply to the reviewers

    The authors do not wish to provide a response at this time since they are submitting a Revision plan and not a Full revision.

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    Referee #3

    Evidence, reproducibility and clarity

    The objective of this manuscript was to determine the role of TZPs in mouse oocyte quality. The experimental plan was to compare the phenotypes of global Myo10-/-, oocyte Myo10-/-, and Myo10+/+ follicles. The results indicate that global loss of Myo10 did not prevent oocyte growth, but resulted in lower density of TZPS. Whole ovary image analysis revealed that Myo10-/- follicles actually contained more TZPs than wt, despite the fact that TZP density was decreased in Myo10-/- follicles. In mature knockout females, oocyte growth proceeded, but with impaired oocyte-zona integrity and alterations in gene expression including upregulation of numerous protein encoding genes. Oocytes from Myo10-/- knockout females produced a normal-appearing spindle but exhibited reduced capacity to mature beyond MI. Analysis of ovulated oocytes from mated females revealed an increase in the number of unfertilized and dead oocytes, many of which exhibited gaps between the zona pellucida and the oocyte plasma membrane. Those oocytes that were successfully fertilized exhibited a higher than normal of developmental arrest by the blastocyst stage. Lastly, mating trials revealed that Myo10-/- females were sub-fertile.

    The results are clearly described with high quality imaging to demonstrate phenotypes. The data appear reproducible based on sample size and the number of repetitions. In most cases, statistical analysis demonstrates significance of observed differences.

    Minor comments:

    1. Fig. 2B does not provide statistical evidence that the two data sets differ.
    2. Fig. 6A Was the zona pellucida functional in unfertilized oocytes from Myo10-/- females? That is, were sperm bound to the zona or within the perivitelline space?
    3. The observation that oocytes from Myo10-/- females have more TZPs but lower TZP density raises questions as to how more TZPs (even if less densely spaced) could fail to support oocyte development. Dye diffusion assays comparing the rate of injected dye from Myo10+/+ and Myo10-/- (GV stage) or (maturing) stage oocytes into their attached granulosa cells might reveal an explanation.

    Significance

    The manuscript addresses an important aspect of follicle development using state of the art methodology to test the requirement of Myo10 for successful TZP-oocyte interaction during follicle development. The authors demonstrate significant findings as to the mechanism by which TZPs enable granulosa cell-oocyte contact which is required for transfer of critical components from granulosa cell to oocyte. The requirement of Myo10 in this process in oocyte competence is demonstrated clearly, however the mechanism by which Myo10 ablation causes defective fertilization and development remains unclear. In any case, the results demonstrate new and interesting findings that will be of great interest to basic scientists including oocyte biologists working on diverse animal species. The results could lead to further understanding of TZP-oocyte interaction and reveal ways to improve or restore communication between these two cells.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors have investigated the effect of knocking out the Myosin-X gene (Myo10) on oocytes in mice. The major finding was that transzona processes (TZPs), which are filipodia-like structures that cross the oocyte's extracellular matrix shell (zona pellucida, ZP), were greatly reduced when the gene was globally knocked out. In comparison, an oocyte-specific knockout had no effect on TZPs. Using a machine learning algorithm developed by one of the authors, it was found that characteristics of the ZP were changed, and the oocyte shape was altered in the knockouts. RNAseq showed that many genes were upregulated in oocytes from knockout females. Oocytes from knockouts also failed to complete meiotic maturation at a higher rate and produced embryos that were fertilized less frequently and whose embryos were impaired in reaching the blastocyst stage. Finally, litters per female and pups per litter were lower in knockouts, indicating lower female fertility.

    Major comments:

    Overall, this is a very well done and comprehensive study that indicates a major role for MYO10 in oogenesis and oocyte developmental competence. There are some relatively major issues that should be resolved, however:

    1. An experiment was done to assess the number of follicles per ovary, which is shown in Fig. S3. No significant difference in follicle number (per unit area) was detected. However, there are two problems here. One is that only four repeats were done, and the lack of significance would appear to be driven by only one of the knockout repeats which had a high number of oocytes compared the others. It is possible that there is really not a biologically significant difference between the controls and somatic knockouts, but there are an insufficient number of repeats to determine this (technically, P>0.95 would mean they are the same). Second, it is unclear that the number of follicles per unit area is the relevant parameter for fertility rather than the absolute number of follicles. Both measures should be reported and tested statistically.
    2. A main function of TZPs is to transfer metabolites and other small molecules into the oocyte via Cx37-containing gap junctions. As the authors note, the phenotype here is different from the Cx37 knockout, where oocytes failed to develop. This implies some connectivity remains in Myo10 knockouts, but how much has not been determined. The amount of connectivity should be measured. The techniques are fairly straightforward and involve only microinjection of a fluorophore into the oocyte and measuring the spread into the surrounding somatic cells. This also has implications for the lack of effect on GVBD and resumption of meiosis, since Laurinda Jaffe's group has shown that diffusion of cGMP out though the gap junctions is important in this process.
    3. The TZP-like structures that remain are intriguing, but this was not followed up. They apparently are visible optically but contain neither actin nor membrane. Is it possible that these are tracks left from degenerated TZPs? Electron microscopy might resolve this question and should be considered. In any case, a more extensive discussion is warranted since the data are contradictory, with fluorescence-based methods indicating a decrease in TZPs but optical methods indicating an apparent increase.
    4. The apparent delay in formation of a perivitelline space is interesting. The perivitelline space forms gradually as the ZP detaches from the oocyte independent of meiotic maturation (see, e.g., Richard et al., 2017, J Cell Physiol 232:2436-46). Could this not be a delay in detachment and therefore transient (and dependent on when the assay was performed relative to oocyte isolation)?
    5. While GO analysis was done and shown in Table 1, this is not treated in any depth in the paper. There should be more description of the GO pathways that were upregulated and the implications.

    Minor comments:

    1. The comparisons that were done for whole-body knockout vs. oocyte-specific knockouts were only done by comparing each to its control. There is no direct comparison showing whether the two knockouts differ significantly from each other. The comparisons should be done using ANOVA with appropriate post-hoc tests to test all four groups against each other.
    2. The experiment in which 5-ethynyl uridine incorporation was used to show that global transcription was not increased may not actually be conclusive, since a large amount of RNA synthesized is not mRNA. A global increase in mRNA synthesis could still be occurring but the signal swamped by RNAs such as rRNA and other non-coding RNAs.

    Referees cross-commenting

    It looks like the reviewers basically agree that this is interesting but there are questions remaining about whether cumulus-oocyte coupling is affected (and could explain the phenotype) and why there is an apparent discrepancy between the results for detecting the numbers and densities of TZPs. These should be addressed.

    Significance

    This work has fundamental implications for understanding oocyte development and the role of the surrounding somatic cells in oogenesis and oocyte developmental competence. It also has direct implications for human and animal fertility and assisted reproduction.

    This is a fundamental new set of results that establishes a role for Myo10 and adds to the knowledge about the role of transzonal processes. It is a substantial advance over previously published research.

    The audience will primarily be basic biomedical researchers in the general field of reproductive biology as well as those investigating filipodia and should extend to those interested in translational research in infertility.

    I have direct and extensive expertise in the field of oogenesis in mice.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript by Crozet et al. the authors investigated the contribution of transzonal projections (TZPs) to the oocyte development and acquisition of competence. The results were obtained using two Myo10 knockout mice models: a full knockout for Myo10 (Myo10-/- full) and an oocyte-conditioned knockout (Myo10-/- oo). The major findings due to the global depletion of Myo10 include the decrease in TZP density, discrete morphological alterations in the oocytes, alterations in oocyte gene expression, the inability of the oocytes to complete the first meiotic division (lack of 1PB extrusion), and subfertility in Myo10-/- full females.

    The research topic is interesting and, overall, I consider the manuscript relevant. However, to increase the scientific soundness authors are encouraged to explore the effects of the (partial) interruption of the germ-soma communication on the regulation of meiotic arrest and resumption. This is worth investigating (is optional, but highly recommended) since the lower density of TZPs is associated with an apparent normal meiotic arrest but an abnormal meiotic resumption. At first, the measurement of cGMP and cAMP into oocytes during meiotic arrest and resumption would be a nice try. This will help to shed light on the reasons for the abnormal meiotic progression, indicating if it is the consequence of a direct blockage in the transfer of molecules from follicular cells to the oocyte or an indirect consequence.

    Minor points

    Lines 53-55: The oocyte does not complete two successive meiotic divisions to generate a mature oocyte ready to be fertilized. Instead, meiosis completion only occurs if fertilization of MII-arrested oocytes takes place. Consider rephrasing to communicate the accurate concept.

    Lines 145-153 and Figure S4-F: Authors claim that TZP-deprived oocytes grow up to normal sizes. However, the perimeter of fully grown oocytes is lower in Myo10-/- full oocytes. This is conflicting.

    Referees cross-commenting

    In addition to the comments made by my own, my colleagues both suggested the inclusion of experiments to determine the functionality of the remaining TZP through dye diffusion assays. I concur with them.

    Significance

    The manuscript clearly adds to the existing knowledge. I'm convinced that the findings described here will be of interest for readers from the field of reproductive biology, follicle development, and oocyte biology.

    Authors are encouraged to better frame their findings as to the existing knowledge. There is at least one another knockout model in mice that leads to TZP density reduction (Zhang et al., 2021; Nature Comm., 12:2523). In this paper, the authors show that the TZPs connecting the GCs and the oocyte support proper oocyte development. Also, its removal results in subfertility. These previous findings should be acknowledged in the current manuscript.

    My expertise: researcher in reproductive biology; emphasis on folliculogenesis and oocyte development.

  5. Version published to 10.1101/2022.09.15.508092 on bioRxiv