PONE-D-20-10428

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

Dear Dr. Y. Kato,

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.

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,

Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Please refer to the specific comments of each reviewer and try to modify your manuscript accordingly. Please note that at this stage, additional data is expected, as mentioned by the second reviewer, and not just cosmetic changes.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section:

"The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: Vitrolife KK.

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

3. Please amend the manuscript submission data (via Edit Submission) to include author Christian Lessard.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: No

Reviewer #2: Yes

**********

4. 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

**********

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: General comments:

The paper titled “ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model” has carried out experiments that are well-done methodologically, but it is difficult to understand the fundamental reasons that has led the authors to carry out the experiments. The justification for each experiment is not always present, despite being directly related to sperm capacitation. The number of replicates is not shown consistently, as it is absent sometimes or given an interval. There are many studies on sperm capacitation on porcine species, but these authors only refer to theirs or to other species, especially mice, where studies are carried out with sperm from the epididymis that have not been in contact with the seminal plasma and in many times these results are not comparable. Why have not the authors used more bibliographic references of pigs?

In summary, it is a work that could have been better written to make its understanding easier and relying more on authors who have worked on sperm capacitation in pigs.

Specific comments:

Line 185 washed once with ml BTS in a 15 ml centrifuge tube – time and centrifugation force?.

Line 203, should say number of oocytes and replicates used

Line 208, should explain what sperm suspension is

Line 273, should explain what is the pre-capacitated and pre-incubated boar sperm

Line 242, 250, 275, 288 should say the number of replicates,

Line 286 The authors wrote “Capacitation and the AR was assessed using PSA-FITC staining” but capacitation was evaluated by CTC. Specify.

The IVF results should be more extend and shown in a table, it is important to know how many oocytes were used, how many were penetrated, how many sperm penetrated per oocyte, polyspermy rate, asynchronous penetration... And, of course, the number of replicates included.

Reviewer #2: Referee’s comments:

The paper submitted by Kato et al. entitled “ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model” describes the role played by ACRBP on ZP-binding and acrosome reaction in relations with tyrosine phosphorylation and intracellular calcium concentration.

The experiments are clearly described, the results sustain the working hypothesis and the paper is globally well written. I only have a few remarks the authors may consider before the paper can be considered for publication:

MAJOR COMMENTS:

1-/ The fact that antibodies linked on the sperm surface inhibit sperm zona-pellucida binding is not a guarantee that the targeted protein is a key player in the fertilization process, as was proven in past for different candidate (such as the Crisp proteins). The most interesting action o ACRBP shown in this paper may thus be the potential interaction with SERCA. In this regards, even though the authors mention in their conclusion that “Future experiments are necessary to assess how ACRBP promotes the activation of the Ca2+ channels on the sperm surface”, it would strengthen the current work to show the co-localisation of these two proteins during the capacitation process. Maybe the authors could perform simple co-localization IF studies using confocal microscopy or proximity ligation assays? This would not impeach a more mechanistic study as proposed in the conclusion (with co-IP and intracellular calcium measurements maybe?).

2-/ There are differences in the basal states of acrosome reaction between figures 3 and 4. In the “medium only” condition, the ratio is around 40% in figure 3A whereas it barely reaches 20% in figure 4A. In figure 4A, there is a significant effect when anti-ACRBP antibodies are added, which is not commented in the “results” section. There are also huge differences in the percentages of AR when A23187 is added between these two figures (50% less in figure 4C versus 3C). Maybe the conditions are different and I did not get a point, unless, could the authors clarify the reasons of these discrepancies?

3-/ In figure 2A, the difference between “No antibodies” and “Blocking peptide” is around 50% (40% vs. 20%, respectively) with small SEM as indicated by the error bars. There is no significant difference indicated here, this seems curious… Could the authors verify this point?

4-/ Line 430-431, the authors write that “only a few phosphorylated proteins have been formally identified” concerning sperm tyrosine and/or Ser/Thr phosphorylated proteins. Even if the amount of literature is not huge, two papers in mouse spermatozoa report the identification of tyrosine phosphorylated proteins during capacitation (Arcelay et al., 2008 and Chung et al., 2014). Other data are available in different conditions and in different organisms, including Human. If this point needs to be discussed, some more recent and accurate references should be added and commented.

MINOR COMMENTS:

- Line 282 the authors indicate the use of 5mM A23187, is that correct? This concentration seems very high and A23187 is more usually used in the micromolar range.

- Lines 394-395 the authors write that “the additions of 0.1% DMSO in the fertilization medium promoted the parameters of the AR (Fig 4 B, P>0.05)”. Is this a mistake? Nothing seems different from the control situatin in this figure.

Conclusion: Considering the above-mentioned remarks, I propose major revisions before this article can be considered for publication.

**********

6. 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.

PONE-D-20-10428R1

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

PLOS ONE

Dear Dr. Kato,

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 (see below).

Please submit your revised manuscript by Jan 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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

We look forward to receiving your revised manuscript.

Kind regards,

Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear authors,

Your manuscript was returned to the two original reviewers who saw the first draft. Although one of the reviewers appears to be satisfied, the second reviewer is not and I must say that I agree with most of his conclusions/comments. Therefore, I stand by my original decision and return the manuscript to you with a request for "major revision". Please note that this is the last offer to review your manuscript. If it is not sufficiently well done, the following decision will be a rejection.

[Note: HTML markup is below. Please do not edit.]

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: All comments have been addressed

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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: (No Response)

**********

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: (No Response)

Reviewer #2: Referee’s comments:

The paper is a revised version (PONE-D-20-10428R1) of a work originally submitted by Kato et al. and entitled “ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model”.

The authors tentatively replied to the comments made in the first round of reviewing. After reading the new version of the manuscript and the answers of the authors, I am still puzzled by a certain number of things reported below.

- One first point in my major comments was that the fact that antibodies linked on the sperm surface inhibit sperm zona-pellucida binding is not a guarantee that the targeted protein is a key player in the fertilization process, as was proven in the past for different candidate (such as the Crisps proteins). This point has not been discussed by the authors. Are there studies with ACRBP gene invalidation corroborating the putative ZP-binding function of ACRBP?

- Another comment was that “Maybe the authors could perform simple co-localization IF studies using confocal microscopy or proximity ligation assays?” in order to show the co-localisation of the two studied proteins during the capacitation process. The authors performed IF staining of each protein in two independent experiments. The results only show that each protein is present in the acrosome part. Co-localisation studies usually involve co-staining if you use IF, and confocal microscopy with image analysis to prove that the two signals are indeed co-localized. Proximity ligation assay is another possible option, maybe more difficult to perform. In my opinion the authors did not answer that point in a satisfactory way. If the authors believe that the co-localisation is not a crucial point to sustain the function of these proteins, they need to argue in that sense.

Furthermore, the investigation of the co-localisation has not been considered during the capacitation process, as was mentioned in my first report.

- In their answer concerning the discrepancies in the data relating the percentages of acrosome reacted sperm (see Tables 4 and 5 of the revised manuscript), the authors mention that the season is the cause. Nothing has been added in the text. Maybe this point is an evidence for researchers specialized in pig semen, but this is not the case for others although they may work in the reproductive field.

- I suggested two references concerning the identity of identified tyrosine-phosphorylated proteins during capacitation. The authors used other ones, which is not the problem. My concern is that they still mention “only a few phosphorylated proteins, which are hexokinase in the sperm head and the a-kinase-anchoring protein in the flagellum, have been formally identified [43]”. If they mean in porcine, it needs to be clearly stated, because in the two references I have proposed, other proteins were identified (45 proteins in Chung et al., 2014 using phopsphoproteomics analysis). Maybe the authors consider that these proteins were not “formally identified” but they have to argue that point.

- There are very numerous spelling or grammar errors in the added portions of the text, and even a reference in a place where it should not be (lines 324-327). The authors should be more careful when submitting a paper for review. As an indication, I noticed errors in lines 282, 284, 293, 295, 304,307,309, 320, 321, 324-327, 332.334, 361, 363, 400, legend of table 3, 440, 445 (discrepancy between the text and the table legend), 520, 536-537, 563, 564, 573, 575, 668, and probably others that I missed.

Conclusion: Considering the above-mentioned remarks, I cannot change the “major revisions” status that was proposed in the first review.

**********

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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-10428R2

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

PLOS ONE

Dear Dr. Yoku Kato,

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. See specific points below.

Please submit your revised manuscript ASAP. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Please answer the points brought forward by the reviewer.

[Note: HTML markup is below. Please do not edit.]

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 #2: (No Response)

**********

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 #2: Yes

**********

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

Reviewer #2: I Don't Know

**********

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 #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 #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 #2: Referee’s comments:

The paper is a second revised version (PONE-D-20-10428R2) of a work originally submitted by Kato et al. and entitled “ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model”.

The authors replied to the comments made in the second round of reviewing. After reading the new version of the manuscript and the answers of the authors, I only have minor comments:

- The authors wrote lines 540-541: “Indirect immunofluorescence revealed the presence of SERCA 2 at the acrosome (Fig 1, panel A). Then they wrote lines 543-544: Indirect immunofluorescence showed the presence of ACRBP at the acrosome (Fig 1, panel A).

What exactly is presented in Fig 1? It is indicated that it is SERCA in the figure legend.

- The authors wrote lines 600-602: “Our initial experiment demonstrates that anti-phosphotyrosine and anti-ACRBP antibodies significantly reduced sperm-ZP penetration (Table 1 A and C, P<0.05) and fertilization (Figure 1 B and D, P<0.05)”. It seems that there is no Table 1 "A or C" and no Figure1"D" reporting fertilization data.

- Line 635: “Anti-ACRBP antibodies obviously impeded capacitation (Figure 2, P<0.05).” Here also please correct the figure number mentioned.

- Line 731 please correct “is vital for the biding of sperm to the ZP”.

Conclusion: Considering the above-mentioned remarks, I suggest that the paper is acceptable for publication after correcting the mentioned errors.

**********

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 #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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

PONE-D-20-10428R3

Dear Dr. Kato,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

**********

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 #2: (No Response)

**********

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

Reviewer #2: (No Response)

**********

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 #2: (No Response)

**********

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 #2: (No Response)

**********

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 #2: (No Response)

**********

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 #2: No