PONE-D-19-15817

Proteomic characterization and comparison of spermatozoa proteomes between ram (Ovis. aries) and buck (Capra. hircus) using a data-independent acquisition-mass spectrometry (DIA-MS)

PLOS ONE

Dear Dr Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Most concerning to me personally when reading was the reviews was the comment about pseudoreplication. Note it will be important to submit high quality figures in all cases. Plos One provides a guide on how to do this. 

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Academic Editor

PLOS ONE

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.sciencedirect.com/science/article/pii/S0378432016300537?via%3Dihub

https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00369

https://www.sciencedirect.com/science/article/pii/S1874391915000068?via%3Dihub

https://pubs.acs.org/doi/10.1021/acs.jproteome.6b00530

https://www.sciencedirect.com/science/article/pii/S0027510717302014?via%3Dihub

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The main aim of the present manuscript is to characterize sperm proteome in sheep (Hu-sheep breed. Ovis aries) and goat (Anhui white goat. Capra hircus) and identify differences among these proteomes that could help to detect proteins that could be used as potential markers of sperm fertility and functionality and also of sperm cryoresistance. The paper is, in my opinion, very interesting since the topic addressed is of great interest currently for the improvement of reproductive technologies outputs in small ruminants. For performing this study, the authors used 9 rams and 9 bucks for obtaining the ejaculates (one per male) determining three biological replicates (each one composed by three different ejaculate samples) in each species. Spermatozoa from all the ejaculates were obtained by centrifugation and processed for protein analysis by using a data-independent acquisition-mass spectometry approach. A total of 13, 195 peptides corresponding to 2,288 proteins (buck: 2,197 and ram: 91) which 2,109 were identified. Regarding the proteins present in different abundance, a total of 238 were detected, 166 up-regulated and 72-down regulated when sperm proteome of ram and buck were compared. The authors conclude that most of the proteins identified in bucks and rams are related to metabolic pathways. In addition, the authors suggest that the differences found between rams and buck’s sperm proteome should be taken into account for optimizing the results of cryopresevation protocols in these species. Although the results are interesting and could result of high applicability there some aspects that should be clarified by the authors before the manuscript was ready for its publication. The current version of the manuscript is, in my opinion, not relevant enough to be published and therefore, I recommend the publication of this manuscript after major revision.

Please see the specific comments below:

-Title: It seems that the title is incomplete. I would suggest to modify as follows: “Proteomic characterization and comparision of ram (Ovis aries) and buck (Capra hircus) spermatozoa proteome using a data independent acquisition mass spectometry (DIA-MS) approach”. Please, revise it and modify if convenient.

- Abstract: Page 2; line 25: “…in sperm protein proteomes between…”. Please, revise this sentence and modify if convenient.

- Introduction: Page 3; lines 57-58: Please modify this sentence to “ Furthermore, the sperm proteome that have been published…”. In addition, here and all along the manuscript I would suggest to use the word “species” instead of breed since goats and sheeps are two different livestock species. In my opinion the term species would be more appropiate.

- Introduction: Page 4; lines 72-74: The objective should be rewritten, in its present form it is not very clear.

- Material and Methods: Page 4; lines 78-82: Although a figure is included, the expalnation of the experimental design is very poor. An explained description of this experimental design should be included in the text.

- Material and Methods: General comment regarding protein analysis: In my opinion this section is also very poorly explained. The provided information is not informative enough and in addition it is not well organized. I would suggest to deeply modify this part of the manuscript in order to improve the its quality. In addition, ans statistical analysis section should be included.

- Results: Information about differences or repeatability among the three biological replicates should be included.

Results about GO annotation and pathways enrichment should be put together.

- Discussion: The discussion section is in my opinion too superficial and, although in some point the authors make very good reasoning, in general is of low relevance. This section should be revised and rewritten to make it more scientifically relevant. As I have nooted before, the work is interesting and with all the obtained information the authors could perform a very interesting and useful discussion.

- Conclusion: the conclusion is too long and in its present form is, again in my opinion, not adequate. I would recommend to rewritten it in order to make it more concise. In addition, a better justification of the authors for the usefulness of these proteins as potential additives for improving buck sperm cryopreservation ability are necessary

-Figures: Please, provide figures in a higher quality format.

Reviewer #2: In this paper the Authors provide descriptive data concerning the comparison of sperm proteins of ram and buck semen. More than 2 000 proteins were identified and 238 were found to differ in abundance between buck and ram. This finding is interesting and extends our knowledge about sperm proteins in ruminants.

Major critique

1. In my opinion this study is incomplete regarding seminal proteins. Only data for sperm but not seminal plasma proteome are provided. Seminal plasma proteins are found to interact with the surface of spermatozoa, so data both for seminal plasma and spermatozoa are vital for better understanding of sperm physiology.

2. There is a serious probability that pseudoreplicates instead of replicates were used in this study. Pseudoreplication occurs when observational data are pooled prior to statistical analysis and subsamples are incorrectly treated as true replicates for statistical analysis. The authors stated (L102) that semen samples were pooled.

3. Relationship of obtained results to cryopreservation is not provided. Cryopreservation is mentioned as the main justification for this study (L42-51). Cryopreservation experiments were not performed in this study and the importance of the data for cryopreservation is not discussed at all.

4. There is no validation of obtained results with other methods, for example Western blotting.

Specific comments

Introduction:

Line 20, 44 I would change “specie” to “species”

L21 Instead of “sperm protein proteomics” I would use “sperm proteins” or “sperm proteomes” throughout the MS.

L52 other “omics” techniques, especially transcriptomics, are used for studies of spermatozoa as well.

L98 age of animals? Basic description of animals should be provided.

L101 short description of CASA systems should be provided. Semen volume and sperm concentration should be provided.

L104 did you use inhibitor cocktail to prevent proteolysis?

L107 Which buffer did you use to wash the sperm?

L107 time for storage of sperm pellets at -80°C should be provided.

L118 and 131 please explain why you reduced and alkylate proteins two times before and after trypsin digestion

L125 remove “according to Bradford (repetition).

L134 Please explain what you mean by “the combined sample “used for high reverse-phase separation.

L139 10 min

- How did the authors ensure confidence in protein ID with only one unique peptide (Supplementary Tab. S1)?

- What was the exact fold change use for analysis? (Suplementary TableS5 showed Fold Change lower that 2).

L171 please provide the peptide mass tolerance and MS/MS tolerance during database searching

It would be interesting to see data of Table S4 independently for ram and buck.

Please improve the quality of Fig. 2.

Some categories of Fig. 3 seem not to be relevant to sperm physiology, for example “insect hormone synthesis”, “carbon fixation in photosynthetic organisms”,” methane metabolism”, and so on.

Discussion

The Authors did not discuss the obtained results.

It would be meaningful to compare published proteomes with the proteome of ram and buck presented in this study

The authors performed the GO classification and pathway enrichment of DEPs, but they did not discuss these results (Fig. 2A, 2B, Fig.3, table S6, table S7) the obtained results.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-15817R1

Proteomic characterization and comparison of ram (Ovis aries) and buck (Capra hircus) spermatozoa proteome using a data independent acquisition mass spectometry (DIA-MS) approach

PLOS ONE

Dear Dr Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please make sure to handle all remaining issues identified by the reviewer. They are important points. We would appreciate receiving your revised manuscript by Feb 14 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Peter J. Hansen

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed almost all the modifications suggested. However, in the present version of the manuscript there are some aspects that still need to be improved.

The explanation of the experimental design is still very poor and although the figure 1 can help, in my opinion is not enough.

In the current version of the manuscript I have not found the figure legends for the figures provided.

Only legends for figure 1 and 2 are provided in the main text of the manuscript. This is nor the appropriate place and, in addition, the explanations are not adequate being poor and not informative enough.

The quality of some figures is still not good, as for example figure 1 and 2. Please, revise it again and try to improve if possible.

Finally, I recommend the publication of this manuscript after minor revision.

Reviewer #2: The Authors have complied with most of my remarks which led to the improvement of the overall quality and sound of the paper. Therefore, I can now recommend the MS for publication.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Proteomic characterization and comparison of ram (Ovis aries) and buck (Capra hircus) spermatozoa proteome using a data independent acquisition mass spectometry (DIA-MS) approach

PONE-D-19-15817R2

Dear Dr. Zhang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Peter J. Hansen

Academic Editor

PLOS ONE

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