Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system

  1. Marion Herbette
  2. Xiaolu Wei
  3. Ching-Ho Chang
  4. Amanda M. Larracuente
  5. Benjamin Loppin
  6. Raphaëlle Dubruille

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Abstract

Segregation Distorter ( SD ) is a male meiotic drive system in Drosophila melanogaster . Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD + chromosome. It is well established that distortion results from an interaction between Sd , the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp , on the SD + chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD + sperm elimination remain unclear. Here we show that SD/SD + males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD + spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD + genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD + chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.

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  1. Version published to 10.1371/journal.pgen.1009662
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    Reply to the reviewers

    We thank both reviewers for their comments on our manuscript. Our responses to their specific comments and plan to modify the manuscript are described bellow.

    Response to reviewer #1

    *> Figure 1C is difficult to interpret. Am I supposed to see anything in particular in the two insets? Please provide a descriptive interpretation of the inset and let the reader know if anything in particular is to be noted. *

    We agree that it is a bit difficult to interpret although the goal of these images is to show that spermatogenesis appears globally not disturbed until the histone-to-protamine transition in distorter males. We will change the legend of the figure to clarify this particular point.

    > Figure 1D. Elsewhere, in supplemental S1, they have an image for SD5/CyO. This should be provided here as a control. As presented, the genetics don't prove that the interaction between SD5 and Gla is the cause of the phenotype. As presented in the figure, the effect could be caused by SD5 alone, independent of Gla. In S1, this is not the case - SD5 with CyO doesn't produce the phenotype. Likewise, I think they should provide the SD5/CyO image in S1A in Figure 1C.

    We can add the images of the SD5/CyO genotype (currently in FigS1) in Fig1C (whole testis) and in Fig1D (single cyst). We also suggest to present in this figure the other distorter genotype cn bw/CyO (which is currently in Fig S1). However, because the modified figure is going to be too big, we also suggest to split Figure 1 in two Figures with Figure 2 presenting FISH results including all controls. In this case, we will remove the supplemental figure 1.

    > Figure 1E. These images are the formal proof (especially for Gla/SD5 genotype) that the large Rsp array is on the chromosomes that seem destined for removal from the cyst. However, there is no control. The authors should provide FISH results for the genotypes Gla/CyO and, ideally, also cn1 bw1/Cyo.

    We agree with reviewer #1 and will provide images of the Gla/CyO control and cn1 bw1/Cyo in a new Figure 2 as explained above.

    > Figure 2. Keeping consistent with other figures, can the Gla/SD5 panel be in the middle?

    Yes. We swapped the Gla/SD5 and cn bw/SD-Mad panels.

    *Also, shouldn't there be SD5/CyO in Figures 2, 3 and 4, to demonstrate that the phenotypes are the result of the interaction rather than just SD5? I am OK with providing just the cn 1 bw1/SD-Mad here alone, since it is simply contrasted with Gla/SD5. *

    We agree that it would be better to show also the SD5/CyO controls. However, we chose to show only one control (Gla/CyO) to make the figures easier to read. We thus suggest to provide all images of the SD5/CyO genotype in supplemental figures.

    > Figure 3A. In the scheme, can you provide greater detail as to where F-Actin is expected?

    The scheme was modified to clarify this point.

    > Figure 3B. It is stated that there is a size difference in the nuclei for IC stage and greater variation in ProtB-GFP staining within bundles. Can there be an effort to quantify these observations?

    It would be difficult to quantify ProtB-GFP signal intensity and nuclear size in IC stage cyst because nuclei are very close to each other. The best way would be to squash testes to spread spermatid nuclei but there might be a bias on nuclear size/shape due to the squashing procedure. In addition, on squashed preparations, it is difficult to be sure that the nuclei analyzed and compared belong to the same cyst. We agree that quantification would help to describe the phenotype better but we think that the best read-out of the different SD phenotypes is the quantification of number of abnormally compacted nuclei in seminal vesicles which is provided later in the manuscript.

    *> Figure S4. There doesn't appear to be the same phenotype for Gla/SD-Mad (DAPI, ProtB-GFP) in post-IC stage bundles compared to what is seen in 3C for Gla/SD-5. In particular, in figure 3, the defective nuclei seem to be trailing, but in S4, while the bundle appears disorganized, there doesn't appear to be the trailing nuclei. Is this difference real or is it just the result of a single picture contrast? Some clarification could be helpful. *

    Actually, the images that were shown on Figure 3C for Gla/SD5 post-IC probably show the SD5 nuclei of one cyst (the normal one) and the Rsp nuclei being eliminated from another cyst (these are trailing behind nuclei which are too far to be included in the same image). We thus changed the images for Gla/SD5 for an image which looks like the one shown for *Gla/SD-5 *genotype for clarity.

    We did not mention this observation in the manuscript but we actually see cysts in which abnormally-shaped nuclei are trailing behind the normal nuclei and sometimes IC cones around the abnormally shaped nuclei seem to be stuck close to the normal nuclei which are already individualized. It might be possible that IC progression around abnormal nuclei is slowed down compared to normal nuclei. The difference could not reflect different phenotypes but more likely different states of a dynamic process.

    Response to reviewer #2

    Reviewer #2 had no specific comments.

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    Referee #2

    Evidence, reproducibility and clarity

    SD is a multi-component system, where two major factors Sd (a truncation allele of RanGAP that mislocalizes) and Rsp, a satellite DNA (whose copy number determines sensitivity to RanGAP distorting allele).

    This study by Herbette et al. provide cytological characterization of Drosophila SD (segregation distortor), a male meiotic drive system, focusing on the process of histone-to-protamine transition. By thoroughly studying multiple alleles of SD, they find that the mechanisms by which SD accomplishes segregation distortion are not uniform. In some cases, spermatogenesis is perturbed at the level of protamine incorporation and in other cases, mature sperm can be generated yet they exhibit distorted segregation.

    In one combination Gla/SD5, histone elimination is delayed (never complete), whereas cn bw/SD-Mad exhibit normal timing in histone elimination/protamine incorporation, although these two combinations result in similar, severe degree of distortion. They further show that DNA compaction is incomplete in these SD alleles (again more severe in Gla/SD5 condition) by using dsDNA antibody. Interestingly, defective spermatids in Gla/SD5 combination never progress to sperm maturation and enter seminal vesicle, defective spermatids in cn bw/SD-Mad combination are capable of entering seminal vesicle, but likely fail to fertilize or develop after fertilization, resulting in distortion.

    This is a well-done study and provides important insights into the mechanisms of segregation distortion in the Drosophila melanogaster SD system. The quality of data is high, and I don't have any major concerns on this manuscript. Of course, the exact mechanisms of how SDs drive (i.e. why Rsp(S) alleles fail to condense properly, and how it is related to the Rsp copy number) remains unclear, this study provides a significant step forward to tackle this fascinating phenomenon of segregation distortion.

    Significance

    This study provides important insights into the underlying mechanism of segregation distortion during D. melanogaster spermatogenesis. Segregation distortion is a fascinating phenomenon of significant interest in evolutionary biology. Thorough cytological characterization of spermatogenesis phenotype that leads to segregation distortion provides much needed information, and this study is a significant step forward to understand how meiotic drivers might exploit the system to distort segregation for their advantage.

    Referees cross-commenting

    I think I and reviewer #1 seems to be in good agreement. I don't have anything in particular to add. This is a nice paper.

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    Referee #1

    Evidence, reproducibility and clarity

    This is a very nice paper that combine cytology and genetics to provide insight into the mechanism of segregation distortion in the Drosophila SD system. The conclusions are well supported with multiple different experiments from different in angles. By using different genetic backgrounds - their conclusion that Rsp abundance dictates distinct outcomes is well supported. My primary suggestion is that they include a few more controls and provide some additional quantitative analysis. In some cases, quantitative conclusions are made without sufficient support.

    Specific Comments.

    Figure 1C is difficult to interpret. Am I supposed to see anything in particular in the two insets? Please provide a descriptive interpretation of the inset and let the reader know if anything in particular is to be noted.

    Figure 1D. Elsewhere, in supplemental S1, they have an image for SD5/CyO. This should be provided here as a control. As presented, the genetics don't prove that the interaction between SD5 and Gla is the cause of the phenotype. As presented in the figure, the effect could be caused by SD5 alone, independent of Gla. In S1, this is not the case - SD5 with Cyo doesn't produce the phenotype. Likewise, I think they should provide the SD5/CyO image in S1A in Figure 1C.

    Figure 1E. These images are the formal proof (especially for Gla/SD5 genotype) that the large Rsp array is on the chromosomes that seem destined for removal from the cyst. However, there is no control. The authors should provide FISH results for the genotypes Gla/CyO and, ideally, also cn1 bw1/Cyo.

    Figure 2. Keeping consistent with other figures, can the Gla/SD5 panel be in the middle? Also, shouldn't there be SD5/CyO in Figures 2, 3 and 4, to demonstrate that the phenotypes are the result of the interaction rather than just SD5? I am OK with providing just the cn 1 bw1/SD-Mad here alone, since it is simply contrasted with Gla/SD5.

    Figure 3A. In the scheme, can you provide greater detail as to where F-Actin is expected?

    Figure 3B. It is stated that there is a size difference in the nuclei for IC stage and greater variation in ProtoB-GFP staining within bundles. Can there be an effort to quantify these observations?

    Figure S4. There doesn't appear to be the same phenotype for Gla/SD-Mad (DAPI, protoB-GFP) in post-IC stage bundles compared to what is seen in 3C for Gla/SD-5. In particular, in figure 3, the defective nuclei seem to be trailing, but in S4, while the bundle appears disorganized, there doesn't appear to be the trailing nuclei. Is this difference real or is it just the result of a single picture contrast? Some clarification could be helpful.

    I think this is a nice paper and I enjoyed reading it very much. The combination of the genetics (different RSP alleles from nature and the different X chromosomes) with the cytology provide a very reasonable explanation for why different genotypes seem to yield different effects. It provides some reconciliation among previous studies.

    Significance

    I think it is very significant.

  5. Version published to 10.1101/2021.01.08.425928v2 on bioRxiv
  6. Version published to 10.1101/2021.01.08.425928v1 on bioRxiv