PONE-D-22-19509Mouse in vitro spermatogenesis in isolated seminiferous tubulesPLOS ONE

Dear Dr. Ogawa,

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. There are some concerns on the efficiency and extent of spermatogenesis and culturing the tissues in environment that lack the factors that are crucial in an in vivo system. Further, there are a number of issues with methodology. Presentation of the manuscript should be improved significantly, especially in providing strong rationale and citing up to date literature; clear mention of the methodologies used; statistical analyses; appropriately describing the results for easy understanding; and providing the discussion that is relevant to the results observed backed by strong literature survey.

Please submit your revised manuscript by Sep 29 2022 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'.
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• 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: https://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,

Suresh Yenugu

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

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: No

Reviewer #3: Yes

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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: No

Reviewer #3: 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: Comments:

Recapitulation of in vitro spermatogenesis is essential to study the molecular mechanisms and to address infertility. Feng et al. extended their previous studies on in vitro spermatogenesis using the ST culture method using Acr-GFP andH3 mice. The authors tried both minced or individual STs and aggregated STs. Interestingly, they found that aggregates or individual STs having aggregates at both ends show efficient spermatogenesis compared to individual STs. Further, using ST culturing, authors have shown the effect of oxygen concentration on spermatogenesis. This is a motivating extension work; however, some aspects of the study need to be clarified, and more experiments are necessary to describe the manuscript's findings. Also, serious issues with the writing must be addressed. Overall, the paper feels premature and would benefit from some major rewriting. Throughout the article, including the abstract, many vocabularies, grammar, and pose errors make it very hard to follow and interpret.

Major comments:

The abstract is poorly written.

If possible, I would suggest to the authors consider making the video of methods A and B. It will be beneficial to the researchers and readers.

Authors made major conclusions that spermatogenesis can be induced in short pieces of STs in culture. However, only 26% of the short pieces show the +ve signals of GFP and mcherry. Also, they are nearby small clustered regions. Of course, we can see the GFP signals but Mcherry and GFP colocalized signals we see nearby clusters only?

Here the point is with the low efficiency of spermatogenesis in short pieces, how come one can use this technique to study mechanisms of spermatogenesis or any toxicological studies?

Line 465: Numerous studies have described the pivotal roles of these cells in spermatogenesis. Leydig cells produce and secrete androgens and other factors, such as colony-stimulating factor 1 (CSF-1) [31]. The testicular macrophages also produce CSF-1 and enzymes involved in retinoic acid (RA) biosynthesis [32]. Lymphatic endothelial cells secrete fibroblast growth factors (FGFs), which maintain stem cell populations for spermatogenesis [33]. All these factors and others not yet identified could contribute to the promotion and maintenance of spermatogenesis and would be necessary for efficient spermatogenesis in vitro.

As the authors stated ST’s external microenvironment may play a critical role in spermatogenesis. Did the authors try to culture short pieces of STs with some of the factors like RA, CSF-1 or growth factors? In another way did the authors try to co-culture with Leydig cells or other cells required for spermatogenesis to improve the efficiency?

Minor comments:

Fig1G Y axis?

323 expression was observed in 55 out of 90 sa268mples (61.1%) (Fig 3D, E).

Fig 3G ; error bars labelled. Make it uniform or mention it in legends

Fig S1 and 2 methods 0.5% agarose solutions with STs kept in 4deg. How about cold stress or shock on tubules and spermatocytes?

Fig3: What % of aggregates were observed? What do the authors mean occasionally?

Line 327: In 40–55 days, STs from an experiment were examined by whole mount immunochemical staining. In 2 samples of cut-isolated ST, cells positive for both GFP and mCherry were observed out of 41 samples examined

The image shown +ve for GPF and Mcherry in cut isolate is not convincing. Because the +ve signals we see near the cluster.

Line 334: We then stained Leydig cells with anti-HSD3β antibody under whole mount condition. In fact, HSD3β-positive cells were detected abundantly in aggregated STs and only marginally in isolated ST samples (Fig 3I). Please add the quantification

Line 340: In our previous study using rat testis tissues, we found that O2 tension could influence the spermatogenic efficiency in vitro and O2 concentration lower than atmospheric 20% would be favourable : Rewrite

377 that this condition, namely uncut-isolated ST under 10%O2: 10% space

Overlapping scale bars

Make uniformity in scale bars

Fig2:

(B) Even though authors have mentioned that Acr-GFP expression in an ST showed unique oscillatory fluctuation, the authors need to explain the reason for the sharp fluctuation in Acr-GFP expression from Day 24 to Day 31.

Fig3:

(F) The authors examined only 41 cut-isolated STs for both GFP –mCherry expression. What about the rest of the cut-isolated STs as the authors have found quite significant (72 nos) GFP expressing cut-isolated STs?

In lines 318, 319 and 320, they found small clumps of STs which they have considered aggregated STs. The authors need to clarify whether this is a human error or if these clumps are forming on their own. In the latter case, the authors also need to mention the frequency and percentage of finding these clumps.

In addition to this, the authors need to clarify whether they have included these clumped STs in their count for aggregated STs or not.

(I) The authors need to provide statistical analysis for this figure as they have stated that ‘aggregated STs may maintain interstitial cells between each ST, which could be a reason that aggregated STs supported spermatogenesis more favourably.’

Fig. 4

(B) Out of 20%, 15% and 10% concentrations, in 10% O2 GFP expression is more. Whether this is the optimal concentration for spermatogenesis or there is the possibility of more spermatogenesis below 10%? The author needs to clarify.

Reviewer #2: In this study the authors evaluated the effect of oxygen concentration on in vitro spermatogenesis in neonatal Acr-GFP/Histone H3.3.3-mCherry double (Acr/H3) transgenic mice testes, using tissue mass, ST aggregate and ST segment culture systems. Although it seems to be a have short follow up duration s (34 day long), the authors report that the lower oxygen tension is favorable for spermatogenesis and for elongating spermatid production, and they demonstrated a new ST segment culture method providing opportunity for elucidating regulatory mechanism of spermatogenesis. While these data are potentially of interest, the manuscript lacks principal points that require a major irevision in order to ensure the data are presented with more clarity and greater empirical support (including controls and detailed data).

Title:could be intentional in order to correctly orient the readers to the output of the study.

Introduction: lacks current literature and it is written in a broad and unfocused manner with a book chapter style. Therefore the rationale (lack of knowledge in recent literature related to the research question) , the clear description of the problem and the hypothesis or the research question that will solve the problem should be clearly declared .

Materials and Method lacks the study design, the sample size with power analysis, the number of repeats taht is required in order to answer the research question objectively. Several concerns related to methodolody and the proff of concepts are as following.

1. Please provide the detailed information of PDMS ceiling chip which was used for seminiferous tubule culturing method (including size of pillars in WxLxH and distance between the pillars) in M&M section.

2. In M&M section, between lines 121-124, the usage of a thin porous polycarbonate membrane is stated in some cases for pressing testis tissues against the base agarose gel by placing them under the PDMS ceiling to hold the tissue in place. Please clarify the optimization of culture systems by explaining in which cases the membrane was used.

3. The all culture platforms used in study should be clarified in M&M section. Please change the title "Culture method" as "Culture methods" in line 136 and explain each culture methods as subtitles individually. The title of "Soft agarose method" in line 156 should also be under culture methods title as a subtitle.

4. In figures 2A and 2B, there is high background for GFP, that makes the labeling data very unreliable.

5. The terminology for culture systems is not consistent. Each culture system should be named in M&M section and the same names should be used in the remaining parts of the manuscript.

6. In figure 4F, PAS staining micrograph is given only for uncut-isolated ST culture, it should be also given for the other culture platforms for the same culture duration. The oxygen concentration is also should be provided in figure legend.

7. It seems that there are four culture methods as (1) cut-isolated ST (PDMS ceiling), (2) uncut-isolated ST (PDMS ceiling with pillars), (3) ST aggregate (soft agarose), (4) tissue mass culture (air-liquid interphase). In results section, the comparison of these 4 methods should be provided (in figure 1G, 3E, 3H, 4B and 4H). In figure 1G, it should be stated which oxygen concentration is used and what is the culture time for that analysis.

The results lack the answers of the research questions and Discussion part itatement of limitations of the current study. The changes in metabolites (analysis of culture media by LC-MS etc.), the functionality of elongated spermatids in terms of fertilization (ROSI, ICSI) or genetic stability tests should be performed or may be added as limitations.

In discussion section, there are missing references in terms of comparison of the efficiency of new cut-isolated ST and uncut-isolated ST culture systems. The following articles should be discussed in terms of in vitro spermatogenetic process:

- Önen S, Köse S, Yersal N, Korkusuz P. Mesenchymal stem cells promote spermatogonial stem/progenitor cell pool and spermatogenesis in neonatal mice in vitro. Sci Rep. 2022 Jul 7;12(1):11494. doi: 10.1038/s41598-022-15358-5. PMID: 35798781; PMCID: PMC9263145.

- Baert Y, Dvorakova-Hortova K, Margaryan H, Goossens E. Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds. Biofabrication. 2019 Apr 26;11(3):035011. doi: 10.1088/1758-5090/ab1452. PMID: 30921781.

- AbuMadighem A, Shuchat S, Lunenfeld E, Yossifon G, Huleihel M. Testis on a chip-a microfluidic three-dimensional culture system for the development of spermatogenesisin-vitro. Biofabrication. 2022 Apr 20;14(3). doi: 10.1088/1758-5090/ac6126. PMID: 35334473.

Reviewer #3: In this study, the authors developed an optimized method to culture the segment of seminiferous tubule (ST), and they showed that ST culture could reach the elongating spermatids stage. The authors patiently troubleshot the strategies to culture ST. They carefully described the detailed processes of their trial and error to develop an optimized protocol. I found the study valuable because the authors describe how they overcame the technical difficulties. This study would become an important asset in the field. Readers can understand the key process of their optimization. Further, the study demonstrates the impact of oxygen concentration in culture and clarifies the benefit of a hypoxic condition.

1. Some sentences in the abstract sound awkward. Lines 27-19 "Theoretically,”, and the last sentence in Lines 32-33.

2. Supplementary figures can be organized in the order of descriptions in the result section. S2A can be S1.

3. Each result section consists of a single paragraph. These sections can be reorganized into some paragraphs to improve their readability.

4. In Fig.1F, it was shown that the frequencies of GFP-positive cells were variable, but these data are not clearly shown in Figure G. These two data can be better described to clarify the frequency of GFP-positive cells.

5. Fig. S2: Please add labels: Acr-GFP and H3.3-mCherry.

6. Line 316: mislabel “S1 Fig. Method B”.

7. Line 334-336: This is just a comment. Leydig cells are the primary source of testosterone or androgens in males. Testosterone or androgens can be supplemented in future studies.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Attachments

Attachment

Submitted filename: PONE-D-22-19509 comments.docx

PONE-D-22-19509R1Mouse in vitro spermatogenesis in isolated seminiferous tubulesPLOS ONE

Dear Dr. Ogawa,

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. While major improvements are made to the manuscript, it is suggested that the authors provide control data for each and every time point assessed. Ensure that the comments of the reviewers are responded in detail. The manuscript should be corrected for English language.

Please submit your revised manuscript by Dec 30 2022 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: https://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,

Suresh Yenugu

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: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: (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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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: No

Reviewer #3: 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: No

Reviewer #3: 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 done a good job revising their manuscript, which is clearly improved and ready for acceptance. Overall, the authors addressed most of my previous comments.

Reviewer #2: The revised manuscript has been improved to some extent, in terms of quality with reorganized Abstract, Introduction, M&M sections and terminology for culture methods. On the other hand, the reliability of data in terms of time points, oxygen concentrations and sample size are not clear. Following are the comments.

1) The authors might provide a more appropriate title for the manuscript including the culture method used for isolated seminiferous tubules that constitutes the main key word of the study. In the present title, “Mouse in vitro spermatogenesis in isolated seminiferous tubules”, the term of “isolated seminiferous tubules” might point the isolation of seminiferous tubules from an adult mouse. Also, the age of the mouse and effect of oxygen level should be indicated in the title. Here you can find some options as possible titles:

2) The authors are strictly recommended to provide a more reliable data for comparison of groups at same time points in figures 1G, 3E, 3H and 4B. For example, in figure 1G, if the culture time for the analysis is variable from 35 to 77 days, it is not possible to compare the efficacy of cut-isolated ST, uncut-isolated ST and tissue mass culture methods in terms of GFP expression since the progression of the spermatogenesis is changeable in in vitro conditions.The time point, sample size (n=?) and oxygen concentration must be given in legends of figures 1G, 3E, 3H and 4B.

3) All of the comments should be answered one by one on the rebuttal letter; then should address to the revised manuscript's specified lines/paragraphs/pages where the question is clearly answered.

4) The language that is used both on the rebuttal letter and the manuscript definitely requires a major refinement and revision in terms of scientific approach, the medical terminology and grammar.

Reviewer #3: The authors fully addressed my previous comments. It became a solid study. One minor thing I found is that the abstract contains too much details now, I feel. It can be reorganized in a way readers can capture the big picture.

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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

Reviewer #3: 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-22-19509R2In vitro spermatogenesis in isolated seminiferous tubules of immature micePLOS ONE

Dear Dr. Ogawa,

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. A major concern that needs to be addressed is to conduct statistical analyses using similar sample size and time points.

Please submit your revised manuscript by Mar 02 2023 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: https://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,

Suresh Yenugu

Academic Editor

PLOS ONE

[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)

Reviewer #3: 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: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: No

Reviewer #3: 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 #2: Yes

Reviewer #3: 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: No

Reviewer #3: 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: It has been improved to some extent by reorganization of the title, figures and figure legends, and additional information of sample size and oxygen level. On the other hand, the reliability of data in terms of time points and sample size are still not clear.

1) In figure 4B, 20% (n= 36), 15% (n=41) and 10% (n=27) oxygen level is compared in terms of the GFP721 and mCherry positivity of seminiferous tubules. The big difference in sample size between the groups gives rise to thought about potential of bias, and it may cause some possible errors in statistical analyses.

2) In figure 4B, the authors stated that “GFP721 positivity was judged during culture days 20 to 30. The mCherry expression was confirmed on culture day 30 or 36.” Both 6 and 10-day difference in in vitro culture of seminiferous tubules is still excessive in order to collect a reliable data from the experiments for two reasons:

1- The duration of mouse spermatogenesis is approximately 34 days. In Acr-GFP/Histone H3.3.3-mCherry double (Acr/H3) transgenic mice, GFP expression starts from pachytene spermatocytes at stage 4 and mCherry is expressed in spermatids from step 11 onward. Since the content and the size of subgroups of germ cells changes within the 6 and 10-day time interval, it is not suitable to include the data collected from day 20 to 30, and 30 to 36.

2-This time gap may affect the viability of germ cells, and number of labelled cells in the meantime.

The authors are recommended to reperform the statistical analysis by using same time points and sample sizes in the groups.

Reviewer #3: (No Response)

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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 #2: No

Reviewer #3: No

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[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-22-19509R3In vitro spermatogenesis in isolated seminiferous tubules of immature micePLOS ONE

Dear Dr. Ogawa,

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. Minor changes in the conclusion section shall be made as per the suggestion of the reviewer.

Please submit your revised manuscript by Apr 07 2023 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: https://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,

Suresh Yenugu

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.

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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 #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 #2: Yes

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

Reviewer #2: Yes

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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

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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

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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: The authors provided a good explanation for concerns about the validity and reliability of the data due to differences in sample size and time points of the groups compared. On the other hand, those explanations should be clearly stated in the manuscript in order to increase the readability of the publication.

The authors corrected the error made in the statistical analysis and explained the reasons for changes in time interval and sample size used to evaluate the GFP and mCherry expression reliably and rationally. When the explanations are added to the limitation section, the article will be ready for publication in order to be clearly understood by the readers.

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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 #2: No

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[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.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice

PONE-D-22-19509R4

Dear Dr. Ogawa,

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,

Suresh Yenugu

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: Yes

**********

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

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 #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: All of the comments have been addressed efficiently by the authors. The the manuscript is suitable for acceptance of 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 #2: No

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