Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work (manuscript ID jdb-3708188) characterizes a novel zebrafish mutant lacking the uracil-DNA glycosylase Unga, showing increased sensitivity to genotoxic stress and male subfertility. In the manuscript, the background appears sufficient, with relevant references to DNA repair pathways, zebrafish as a model, and previous studies on uracil-DNA glycosylases. The research design seems appropriate overall. The methods are generally well described; using AO staining for apoptosis is acceptable, though not specific only for apoptosis. The results are presented clearly through text, figures and statistical analyses. The conclusions appear to be supported by the presented results.

 

 

Major point:

 

Acridine Orange (AO) staining is useful for assessing general cell viability and detecting cell death; however, it is not specific for apoptosis. To clearly distinguish apoptosis from other forms of cell death (e.g., necrosis), a more specific parallel assay—such as Annexin V staining, caspase activation assays, or TUNEL—would be more appropriate. The authors may wish to consider including such an assay, or alternatively, revise the text to refer to “cell death” rather than “apoptosis” where AO staining is the sole method used.

Comments on the Quality of English Language

Minor points:

 

Abstract:

 

Lines 24-25: "have significantly contributed" should be "has significantly contributed" as it refers to "the characterization"

 

 

Introduction:

 

Lines 47-48: "has also became" should be "has also become"

 

 

Materials and Methods:

 

The text uses both "L" and "l" as the symbol for liter. Consistency is key, so whichever form is used (L or l), it should be used throughout the entire document. To avoid confusion with the number "1", the uppercase "L" is often preferred and is widely accepted in scientific writing.

 

 

Results:

 

See the comment for the M&M section.

 

Discussion:

 

Line 405: In the sentence "Zebrafish (Danio rerio) has emerged...", consider changing "has" to "have" as "Zebrafish" is plural. In the same sentence, consider changing "that are used" to "used" for conciseness.

 

Line 447: In "...overall changes in the DNA methylation profile are relatively minor, however, a global compaction of the chromatin ca be observed", "ca" should be "can".

 

 

Supplementary materials:

 

Figure S2 legend: "sequening" should be "sequencing"

Author Response

Comments 1: This work (manuscript ID jdb-3708188) characterizes a novel zebrafish mutant lacking the uracil-DNA glycosylase Unga, showing increased sensitivity to genotoxic stress and male subfertility. In the manuscript, the background appears sufficient, with relevant references to DNA repair pathways, zebrafish as a model, and previous studies on uracil-DNA glycosylases. The research design seems appropriate overall. The methods are generally well described; using AO staining for apoptosis is acceptable, though not specific only for apoptosis. The results are presented clearly through text, figures and statistical analyses. The conclusions appear to be supported by the presented results.

Response 1: We really appreciate both the positive review and constructive comments of Reviewer 1. As detailed below, we have addressed the flagged issues and implemented the requested revisions accordingly. 

Comments 2: Major point:
Acridine Orange (AO) staining is useful for assessing general cell viability and detecting cell death; however, it is not specific for apoptosis. To clearly distinguish apoptosis from other forms of cell death (e.g., necrosis), a more specific parallel assay—such as Annexin V staining, caspase activation assays, or TUNEL—would be more appropriate. The authors may wish to consider including such an assay, or alternatively, revise the text to refer to “cell death” rather than “apoptosis” where AO staining is the sole method used.

Response 2: This is an excellent observation, and indeed, a clear limitation of the method we used. Thanks for pointing this out. To reflect our findings more accurately we have now revised the text throughout section 3.4 (lines 381-415) and the legend of Figure 4 (line 397) accordingly.

Comments 3: Minor points: 
Lines 24-25: "have significantly contributed" should be "has significantly contributed" as it refers to "the characterization"
Lines 47-48: "has also became" should be "has also become"
Line 45: In the sentence "Zebrafish (Danio rerio) has emerged...", consider changing "has" to "have" as "Zebrafish" is plural. In the same sentence, consider changing "that are used" to "used" for conciseness.
Line 447: In "...overall changes in the DNA methylation profile are relatively minor, however, a global compaction of the chromatin ca be observed", "ca" should be "can".
Figure S2 legend: "sequening" should be "sequencing"

Response 3: Thank you for the careful reading and spotting these errors. We have now corrected all the typos as suggested.

Comments 4: The text uses both "L" and "l" as the symbol for liter. Consistency is key, so whichever form is used (L or l), it should be used throughout the entire document. To avoid confusion with the number "1", the uppercase "L" is often preferred and is widely accepted in scientific writing.

Response 4: We duly apologize for not spotting this earlier. We have changed now all abbreviations in the “Materials and Methods” and “Results” sections accordingly, and we use “l” as the symbol for liter.  

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments:

1) Introduction: The background into Unga itself and previous studies on Unga in zebrafish is not well described in the introduction, making the rationale for the study unclear. Please refine the introduction to better directly focus on the preliminary work that form the foundation for hypotheses tested in this study. 

2) Figure 1 appears to be a screenshot and seems to cut off some parts of the screen at the bottom. Please provide a full view of it, or export the results into publication quality JPGs or TIFFs. Also please indicate clearly in the legend that the CAGE-seq data are pre-existing and cite the source within the results and the figure legends.

3) Figure 2: Please provide the gene lists and heatmaps of top expressed genes that were utilized for the GO analysis in 2c.

4) Figure 3b: Visually, it appears the homozygous unga male may be smaller than the wild type. Please perform length and weight assessment of the mutant males and females compared to wild types to show whether size is a significant factor. Where these fish all age-matched?

5) Line 384-385: Please indicate clearly whether scRNAseq data were generated for this study or reanalyzed from a previous study by providing clear citations, in both the results section and the figure legends. If it was generated for this study, please indicate so as well and briefly describe what number of animals were used.

6) Figure 5: Are the sperms of the Unga mutant males undergoing apoptosis? An apoptosis assay, like Acridine Orange, is also needed for the sperms. This experiment is crucial to understand why the male fertility in Unga males is lacking. Did authors check gonads after DEB experiment?

7) These sentences are contradictory and confusing: Line 396-399 "but none of 396
tested parameters, such as concentration, (Figure 5e), total motility (Figure 5f), progressive 397
motility (Figure 5g) or the ratio of immotile sperm (Figure 5h) showed significant statisti- 398
cal difference" but in Discussion the authors claim in Line 430 "However, the motility of these spermatozoa appears to be compromised." Please explain.

8) The major weakness of this study is that there is no clear explanation for the subfertility of Unga mutant males.

9) Line 479: "Our zebrafish data support a nuanced interplay between UDGs, suggesting a conserved evolutionary framework of specialization among uracil-processing enzymes" seems to be overstated, as there is no clear evidence presented within the study.

10) For all figures: please indicate the statistical tests used to identify significant differences in the figure legends.

Minor Comments:

1) Line 47-49: It is unconvincing to directly claim high genetic similarity to humans with zebrafish. Please indicate percentage of similarity, as well as the crucial differences that make zebrafish useful as a tool for some studies but not others.

2) Line 342: Minor typo, it should be "Of note", not "Off note".

Author Response

Comments 1: 1) Introduction: The background into Unga itself and previous studies on Unga in zebrafish is not well described in the introduction, making the rationale for the study unclear. Please refine the introduction to better directly focus on the preliminary work that form the foundation for hypotheses tested in this study. 
Response 1: We are grateful for the overall positive assessment of the reviewer, and also for the thoughtful comments and suggestions. As detailed below, we tried to address all of these excellent points in the revised version.
We have now added to the Introduction to a detailed paragraph on UNGs (lines 94-109), expanded the information that summarizes the findings of the paper describing the morpholino-based knock-down of unga zebrafish Unga, and also included a few sentences to clarify the rationale of creating a unga mutant line (lines 136-142). 

Comments 2: 2) Figure 1 appears to be a screenshot and seems to cut off some parts of the screen at the bottom. Please provide a full view of it, or export the results into publication quality JPGs or TIFFs. Also please indicate clearly in the legend that the CAGE-seq data are pre-existing and cite the source within the results and the figure legends.
Response 2: Figure 1 is a composite figure of the respective genomic regions, each downloaded as a high quality SVG file from the NCBI Genome Data Viewer, using the GRCz12tu assembly. Tracks showing the CAGE-sequencing data are available in the Genome Data Viewer. In the revised Materials and Methods section (lines 208-210) now we provide the links for the two pages that were used for creating this figure: for unga (https://short-link.me/18q2l) and for ungb (https://short-link.me/18q31). The labels of these SVG figures have been lightly edited for better legibility. The figure legend has now been also expanded to state that CAGE-sequencing data was available through the NCBI Genome Data Viewer (lines 313-314).

Comments 3: 3) Figure 2: Please provide the gene lists and heatmaps of top expressed genes that were utilized for the GO analysis in 2c.Response 3: Thank you for this suggestion. Now the complete gene list is included as Table S1 in the Supplementary Material, and tissue- and stage-specific heatmaps of 50 of the most correlated genes (and unga itself) are included as Figures S2 and S3. The revised manuscript has been also amended to reference these novel datasets (line 346) and the full list of genes showing correlated expression, as well as the detailed result of the GO analysis have been added to the linked Zenodo resource.

Comments 4: 4) Figure 3b: Visually, it appears the homozygous unga male may be smaller than the wild type. Please perform length and weight assessment of the mutant males and females compared to wild types to show whether size is a significant factor. Where these fish all age-matched?
Response 4: Thanks for this excellent suggestion. We have now performed the required measurements, and it appears that while the size of unga males is similar to the age-matched wild-type controls, the size of the mutant females is slightly, but significantly reduced. This information is now presented in Figure S5 and has been now added to the revised text as well (lines 157-164 and 369-370).

Comments 5: 5) Line 384-385: Please indicate clearly whether scRNAseq data were generated for this study or reanalyzed from a previous study by providing clear citations, in both the results section and the figure legends. If it was generated for this study, please indicate so as well and briefly describe what number of animals were used.
Response 5: We have amended the Materials and Methods section and the Figure legend to specific that we used the data from the Sposato et al. paper (PMID: 39470160). See lines 216 and 427 in the revised manuscript.

Comments 6: 6) Figure 5: Are the sperms of the Unga mutant males undergoing apoptosis? An apoptosis assay, like Acridine Orange, is also needed for the sperms. This experiment is crucial to understand why the male fertility in Unga males is lacking. Did authors check gonads after DEB experiment?
Response 6: Thanks for this suggestion. We have now performed the suggested experiment and tested the gonads for adult males, but no increase in cell death could be detected with Acridine Orange. A supplementary figure (Figure S6) and summary sentence (line 440) has been added to reflect this. As for DEB treatments, these were performed on embryonic stages, when larval gonads are not developed and the primordial germ cells (PGCs) that are instrumental for gonadal development are still undergoing migration. 

Comments 7: 7) These sentences are contradictory and confusing: Line 396-399 "but none of tested parameters, such as concentration, (Figure 5e), total motility (Figure 5f), progressive motility (Figure 5g) or the ratio of immotile sperm (Figure 5h) showed significant statistical difference" but in Discussion the authors claim in Line 430 "However, the motility of these spermatozoa appears to be compromised." Please explain.
Response 7: We apologize for including statements that are indeed contradictory into the manuscript. In the revised version we have changed the sentence in the Discussion to more accurately describe our findings (lines 477-478).

Comments 8: 8) The major weakness of this study is that there is no clear explanation for the subfertility of Unga mutant males.
Response 8: The reviewer is correct, and we acknowledge this limitation. Nevertheless, while our study cannot provide a definitive answer to this question, we believe it offers an important and valuable observation that the community can use to investigate the underlying mechanism. Additionally, by testing and excluding the most obvious potential causes of the observed subfertility phenotype, we help guide future researchers in formulating and testing novel hypotheses to explain this phenomenon.

Comments 9: 9) Line 479: "Our zebrafish data support a nuanced interplay between UDGs, suggesting a conserved evolutionary framework of specialization among uracil-processing enzymes" seems to be overstated, as there is no clear evidence presented within the study.
Response 9: The reviewer is correct in highlighting this as an issue. To tone down our conclusion, we have now changed the relevant sentence as follows (lines 527-529 in the revised manuscript): „Our reanalysis of existing scRNAseq datasets also suggests a nuanced interplay between UDGs, hinting at the possible specialization of uracil-processing enzymes during spermatogenesis.”

Comments 10: 10) For all figures: please indicate the statistical tests used to identify significant differences in the figure legends.
Response 10: Thanks for pointing this out. The relevant information has been now added to the Figure legends of Figure 3-5.

Comments 11: 1) Line 47-49: It is unconvincing to directly claim high genetic similarity to humans with zebrafish. Please indicate percentage of similarity, as well as the crucial differences that make zebrafish useful as a tool for some studies but not others.
Response 11: We have expanded the relevant paragraph in the revised manuscript to reflect this (see lines 49-54). 

Comments 12: 2) Line 342: Minor typo, it should be "Of note", not "Off note".
Response 12: Thanks for pointing out this typo, it has been corrected in the revised manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is acceptable in the present form.

Author Response

Comment 1: The manuscript is acceptable in the present form.

Response 1: We would like to thank the Reviewer for the positive assessment. 

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewers have sufficiently addressed all major concerns raised in the previous review of the manuscript. Some further minor changes are recommended, as follows:

1) Line 227: Minor typo, "two" appears to be repeated.

2) Figure 3c: please indicate the p-value on the graph.

3) Figure S6c: Please be consistent in use of statistical terminology: use "Mann Whitney test" as the term throughout the manuscript for identical tests.

4) Please switch order of S6 and S7, as S7 is described in the text before S6. Supplemental figures are recommended to be arranged in the order they appear in text.

 

Author Response

Comments 1: 

1) Line 227: Minor typo, "two" appears to be repeated.

2) Figure 3c: please indicate the p-value on the graph.

3) Figure S6c: Please be consistent in use of statistical terminology: use "Mann Whitney test" as the term throughout the manuscript for identical tests.

4) Please switch order of S6 and S7, as S7 is described in the text before S6. Supplemental figures are recommended to be arranged in the order they appear in text.

Response 1: We would like to thank the Reviewer for the careful reading of the manuscript. These issues have been addressed in the revised version.