Dear Dr Whelan,

Thank you for submitting your revised manuscript entitled "Long-term Cryopreservation of Rat Spermatogonial Stem Cells Causes Defects in Regenerated Spermatogenesis" for consideration as a Research Article by PLOS Biology. I have now discussed your manuscript with the Academic Editor, who is satisfied by the changes made in response to his/her previous concerns, and we would therefore like to send your submission out for external peer review. As a note, while the Academic Editor is satisfied by the new analyses, we will also need the reviewers to be convinced that the conclusions are strongly supported.

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Lucas

Lucas Smith

Associate Editor

PLOS Biology

lsmith@plos.org

Dear Dr Whelan,

Thank you for submitting your manuscript entitled "Long-term Cryopreservation of Rat Spermatogonial Stem Cells Causes Defects in Regenerated Spermatogenesis" for consideration as a Research Article at PLOS Biology. Thank you also for your patience as we completed our editorial process, and please accept my apologies for the delay in providing you with our decision. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

As you will see, the reviewers find your study interesting and worth pursuing for publication, but they also raise several concerns that need to be addressed to improve the manuscript and to clarify some points. The reviewers think that you should consider the potential effects of other factors on the long-term cryopreservation, thus we would like you to discuss in the text these limitations of the study. Reviewer 1 thinks you should test if the SSCs can differentiate into sperm even after long-term storage and that the title should also reflect this. Reviewer 2 also raises several points that you should address.

In light of the reviews (attached below), we are pleased to offer you the opportunity to address the comments from the reviewers in a revised version that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again.

We expect to receive your revised manuscript within 1 month.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

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You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

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d) *Protocols deposition*

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

on behalf of

Lucas Smith

Associate Editor

PLOS Biology

lsmith@plos.org

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Reviewers' comments

Rev. 1:

In this paper, Brinster and colleagues transplanted long-cryopreserved rat SSCs into nude mice to find colonization of those SSCs and the production of sperm. The cryopreservation period was amazingly over 23 years. This length of time is realistic and practical when this procedure will become a clinical procedure for human patients. Thus, it is impressive and informative.

Authors, however, rather focused on a negative side of the results that such a long-preservation would affect spermatogenic competence of the SSCs, leading to defective spermatogenesis. They resorted to scRNA-seq analysis and demonstrated that long frozen sample was distinct from those of fresh and short frozen. Interestingly, the scRNA seq of transplanted samples showed that the transplanted germ cells tended to remain as SSC or spermatogonia, even when fresh sample was transplanted. This may suggest that the rat SSCs in the mouse testis had some difficulty to differentiate. It is therefore interesting to test if this would happen also when mouse SSCs were transplanted to mouse testis.

I'm not fully convinced that long-term cryopreservation, but not short-term, causes spermatogenic defects. Thus, if I'm an author of this study, I would like to change the title even though most of the text remains as it is. The title I prefer is something like these.

Revival of Spermatogenesis after over 20 years of Cryopreservation of Rat Spermatogonial Stem Cells

Resurgence of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells

Major points:

1. It is yet difficult for me to accept the conclusion of this paper. There could be several parameters other than long-term cryopreservation that influence the result of this study. I suspect that the quality of cryoprotectant could be critical. Quality of such reagents might have affected the result of this study.

2. It is not clear how many times the cryopreservation was performed in days of 23 years ago. In other words, how many rats were used for the cryopreservation of long frozen sample?

3. In the results, it is written that stocks of Norwegian rat testis cells that were cryopreserved over 23 years ago was used in this study. On the other hand, in the materials and methods, it is written that cells from Sprague-Dawley rats were analyzed. Does this mean that different strains of rat were used in this study?

Minor points:

3. In Fig. 6B, four colors of each, light yellow, orange, dark red, and dark blue, seems to show fresh, transplanted fresh, transplanted short frozen, and transplanted long frozen, respectively. On the other hand, in 6C, the samples and the colors match in a different way. This is really a minor issue but a bit confusing.

Rev. 2: Sue Hammoud – note that this reviewer has signed her review

This study by Whelan et. al. examines the molecular and functional effects of extended periods of cryopreservation on the ability of rat spermatogonial stem cells to successfully regenerate spermatogenesis. To explore this question, the authors perform a comparative scRNA-seq analysis of isolated fresh germ cell populations vs. germ cell populations isolated after transplantation. This elegant work revealed two key insights about SSC fertility preservation: First, long term crypreservation of SSCs have reduced stem cell transplantation efficiency, reduced stem cell differentiation capacity, as well as increased disorganized tubules in areas where germ cell development is restored. Second, the authors show significant molecular changes in transcriptome of fresh vs. frozen germ cells. This work is exciting and is of significant value to the reproductive biology and medicine community. Some minor concerns were noted:

1) What is the absolute frequency of tubule disorganization when comparing fresh and frozen samples? How do you distinguish between a disorganized tubule vs. sectioning vs. embedding artifact?

2) Can differences in stem cell activity be due to : changes in cryopreservation media composition over time. Can the differences in regenerative ability of short vs. long term frozen samples (Figure 1C) be due to certain components? Is the age of SSCs matched in fresh vs. cryopreserved samples?

3) How does the Rat germ cell differentiation program compare to rat germ cell differentiation occurring in mouse testis? Although Rat spermatogenesis can occur in the mouse testis, no one really has explored molecular genetic differences in the differentiation program. The data is currently available in this manuscript, and only needs to be mined. Adding fresh sample data to figure 5 will be an interesting comparison?

4) In Figure 6B, is it possible that the labels for the yellow and violet are swapped? The conclusion that longterm frozen cells have a higher fraction of undifferentiated cells and lower percentage of differentiated germ cells is not consistent with the data. The columns may be mislabeled.

5) Is the decrease in spermatid generation in long-term cryopreservation due to block in differentiation or increase post meiotic germ cell pruning/death?

Dear Dr Whelan,

Thank you for submitting your revised Research Article entitled "Resurgence of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells" for publication in PLOS Biology. I have now obtained advice from the Academic Editor and discussed the revision with the rest of the team.

Based on the discussions, we will probably accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

In addition, we would like to make a suggestion to improve the title - we know that you have changed it as suggested by one of the reviewers, but we do think that it should be more informative for readers:

"Re-establishment of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells reveals an important impact in differentiation capacity"

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

-  a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

-  a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

-  a track-changes file indicating any changes that you have made to the manuscript. 

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information  

*Published Peer Review History*

Please note that 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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

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. 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

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

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FINANCIAL DISCLOSURE:

Please include grant numbers and the URLs of any funder's website.

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DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. 

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 1C, D; Fig. 2A-C; Fig. 3A, C, D; Fig. 4A, B; Fig. 5A, C; Fig. 5A-C; Fig. S1A-G; Fig. S2A, B; Fig. S3A-D; Fig. S4A, B; Fig. S5A-C and Fig. S6B, C

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

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**In addition, you should make the data you have deposited in the NCBI GEO repository (GSE182438) publicly available, before we proceed with Production.

Dear Dr Whelan,

On behalf of my colleagues and the Academic Editor, Masahito Ikawa, I am pleased to say that we can in principle accept your Research Article entitled "Re-establishment of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells reveals an important impact in differentiation capacity" for publication in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Ines

--

Ines Alvarez-Garcia, PhD 

Senior Editor 

PLOS Biology

ialvarez-garcia@plos.org