Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators

Akiko Fujishima; Kazumasa Takahashi; Mayumi Goto; Takeo Hirakawa; Takuya Iwasawa; Kazue Togashi; Eri Maeda; Hiromitsu Shirasawa; Hiroshi Miura; Wataru Sato; Yukiyo Kumazawa; Yukihiro Terada

doi:10.1371/journal.pone.0246337

Peer Review History

Original SubmissionSeptember 23, 2020
4 Nov 2020 Decision Letter - Peter J. Hansen, Editor PONE-D-20-29923 Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators PLOS ONE Dear Dr. Fujishima, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 19 2020 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. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: No ****** 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: No ****** 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: In this manuscript, written by Akiko Fujishima and colleagues, the authors study the location of Na+ and K+ electrolytes during mouse preimplantation development. They quantify the electrolytes presence and changes during development by time-lapse confocal microscopy thanks to specific indicators (CoroNa Green AM and ION Potassium Green-2 AM respectively for Na+ and K+). The study focus between morula and late blastocyst (hatching stage) stages, when first lineages and cavitation appear. They show different intensity of the electrolytes indicators depending of the developmental stages, of the cell type (trophectoderm versus inner cell mass) and in the blastocoel. It is of interest for the community to better know Potassium and Sodium concentration in intracellular blastomeres as well as in the blastocoel in this crucial developmental period. However, I have several caveats in the way the analysis is performed and I can not support publication without major reviewing of the manuscript. 1) All the experiments are performed on frozen IVF embryos. It is quite surprising when both IVF and freezing could impact embryonic development and lead to lower developmental yield compared to natural mating. The authors should argument why they did not use superovulated mice followed by natural mating. 2) The authors should avoid overstatement in their abstract and modify the text accordingly. This study does not “confirm the function of Na+/K+ ATPase described in previous studies” (line 37). 3) The authors claim that they have performed time-lapse imaging between zygote and 2-cell stage (why if not in presence of the electrolyte indicators?) and after thawing but none of these data are provided in the manuscript. If time-lapse data are available during blastocoel formation and hatching, it is important to share the movies as well as the quantification of intensity changes over time during these important processes. 4) How the intensity quantification has been performed should be better explain in the Methods section. What are the three replicates that the authors have performed to set up the optimal measurement conditions and what were their controls? A scheme with their imaging features and step-by-step image analysis would be highly appreciated as most of their results are based on this intensity measurement. How many cells per embryo are analysed? The intensity should be normalized to the size of the cell (or blastocoel) in the Z section where the analysis is performed. 5) The low level of embryonic development in presence of 5 uM ION Potassium Green-2 AM (74%), worsen with added ouabain (42%), is problematic and shows cytotoxicity for blastocyst development. Different concentration of the Potassium electrolyte indicator should be tested to find a more suitable concentration and avoid confounding effect. 6) Line 282, “This result indicates that CoroNa Green AM was excreted into the blastocoel after entering the cell”. This results would be better supported by sharing the performed time-lapse movies and analysing the changes over time for the same embryo. ****** 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: Yes:** Maud Borensztein [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. https://doi.org/10.1371/journal.pone.0246337.r001
Revision 1
19 Nov 2020 Author Response To Reviewer 1 Thank you for the thoughtful and constructive feedback for our manuscript titled “Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators.” 1) All the experiments are performed on frozen IVF embryos. It is quite surprising when both IVF and freezing could impact embryonic development and lead to lower developmental yield compared to natural mating. The authors should argument why they did not use superovulated mice followed by natural mating. Response: Thank you for the valuable suggestion. We used IVF because we could accurately identify the fertilization time, obtain more embryos, and make the culture environment uniform from the time of insemination. We have added the sentence “and maintain a uniform developmental environment throughout the experimental period, starting from the time of insemination.” to our manuscript (Collection of mouse oocytes, zygotes, and embryos in Material and Methods, page 7, lines 106-108). We used frozen embryos to efficiently obtain the embryos in the target developmental stage for observation using confocal laser-scanning microscope. In addition, after thawing, recovery culture was performed until the embryos reached the target stage. The embryos were screened using time-lapse observation, and those that exhibited abnormal development were excluded. Moreover, we think that the effects of vitrification and thawing are not significant in this experiment, considering that the embryos were subjected to sufficient recovery culture and that abnormal embryos were excluded. 2) The authors should avoid overstatement in their abstract and modify the text accordingly. This study does not “confirm the function of Na+/K+ ATPase described in previous studies” (line 37). Response: Thank you for pointing this out. We have rewritten the text as follows: “The changes in electrolyte concentration observed in this study were consistent with the activity of Na+/K+ ATPase reported previously” (Abstract, page 3, lines 37-39) 3) The authors claim that they have performed time-lapse imaging between zygote and 2-cell stage (why if not in presence of the electrolyte indicators?) and after thawing but none of these data are provided in the manuscript. If time-lapse data are available during blastocoel formation and hatching, it is important to share the movies as well as the quantification of intensity changes over time during these important processes. Response: Thank you for your valuable insights. We used time-lapse monitoring from zygote to 2-cell stage to screen for normally developing embryos. We used the Primo Vision time-lapse monitoring system, and not confocal laser-scanning microscopy. Due to the duplication of information pertaining to time-lapse imaging with Primo Vision and confocal-laser scanning microscopy, we have removed the paragraph "Time-lapse imaging" in Material and Methods. We have rewritten the "time-lapse imaging" description confined only to confocal laser-scanning microscopy. However, time-lapse images obtained using confocal laser-scanning microscopy were not used for analysis, because frequent laser irradiation causes fading of the indicator and would be unsuitable for comparing the intensities accurately. In this study, the indicator was added to embryos that had grown to the target stage, and the images obtained through a single scan were used for analysis. 4) How the intensity quantification has been performed should be better explain in the Methods section. What are the three replicates that the authors have performed to set up the optimal measurement conditions and what were their controls? A scheme with their imaging features and step-by-step image analysis would be highly appreciated as most of their results are based on this intensity measurement. How many cells per embryo are analyzed? The intensity should be normalized to the size of the cell (or blastocoel) in the Z section where the analysis is performed Response: We apologize for the lack of clarity. We have added the details of the experimental procedure, and added a figure (Fig 3) to the Materials and Methods, under the subheading Intensity measurement (page 12, 13, line 189-211). We analyzed one cell from each embryo at each developmental stage to measure intracellular intensity, and the detailed procedure is described in the manuscript. We have also added, “The same experiments were repeated at least three times.” to our manuscript (Electrolyte indicator, page 10, line 167, and page 11, line 187). 5) The low level of embryonic development in presence of 5 μM ION Potassium Green-2 AM (74%), worsen with added ouabain (42%), is problematic and shows cytotoxicity for blastocyst development. Different concentration of the Potassium electrolyte indicator should be tested to find a more suitable concentration and avoid confounding effect. Response: Thank you for your valuable comment. The appropriate concentration of ION Potassium Green-2 AM was not mentioned in the manufacturer’s instructions, and there are only a few reports for reference. Therefore, we used the concentration stated in a previous report (Sekar P, Oncotarget. 2018). In order to investigate the characteristics of ION Potassium Green-2 AM, time-lapse imaging was performed. As a result, the optimum loading time was determined. Additionally, in the time-lapse imaging, the intracellular intensity continued to increase, and the intensity remained stable even after washing and long-term culturing in medium without the indicator. It was predicted that ION Potassium Green-2 AM is difficult to be excreted, and this property is believed to be associated with its cytotoxicity. Therefore, we concluded that the effect of ION Potassium Green-2 AM on the embryo could not be eliminated even if the reagent concentration was reduced. We have added the time-lapse movie with ION Potassium Green-2 AM as Supporting Information (S2 Movie). 6) Line 282, “This result indicates that CoroNa Green AM was excreted into the blastocoel after entering the cell”. This results would be better supported by sharing the performed time-lapse movies and analyzing the changes over time for the same embryo. Response: We apologize for the gap in the information. Time-lapse images were not used for data analysis, but for understanding the characteristics of the CoroNa Green AM. During imaging, we found that the TE cell was visible initially followed by the blastocoel cavity. We have added the time-lapse movie with CoroNa Green AM as Supporting information (S1 Movie). Additional corrections The following minor changes have been made: The manuscript has been thoroughly proofread and English language issues have been corrected. https://doi.org/10.1371/journal.pone.0246337.r002
31 Dec 2020 Decision Letter - Peter J. Hansen, Editor PONE-D-20-29923R1 Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators PLOS ONE Dear Dr. Fujishima, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Feb 14 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, Peter J. Hansen 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 #1: (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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Dear authors, Thank you for improving your manuscript and in particular for the additional figure 3 and movies S1 and S2. However, I have still two points which need to be answered, including my former question 4 on intensity normalization. 1) According to your answers, I am surprised of your choice of analysing only one cell per embryo. Could you explain why you chose this strategy, please? The number of cells analysed could have been improved by studying at least all the cells of the same Z section or all the cells in contact with the reagents for the Potassium Green-2 AM. 2) In the first round of review, I have asked about the normalization of the mean intensity. According to your answer, it does not seem that you are normalizing the mean intensity by the size of the cell, which is very important to be able to compare your results. Please, provide the normalised analysis of your results. Best regards ****** 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: Yes:** Maud Borensztein [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. https://doi.org/10.1371/journal.pone.0246337.r003
Revision 2
12 Jan 2021 Author Response Thank you for the thoughtful and constructive feedback on our manuscript titled “Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators.” The manuscript ID is PONE-D-20-29923. To Reviewer 1 1) According to your answers, I am surprised of your choice of analysing only one cell per embryo. Could you explain why you chose this strategy, please? The number of cells analysed could have been improved by studying at least all the cells of the same Z section or all the cells in contact with the reagents for the Potassium Green-2 AM. Response: Thank you for the valuable suggestion. As you pointed out, there was concern that selecting a single cell would bias the selection and would not provide accurate data. Therefore, we selected all the target cells of the same Z section and reanalyzed the data. However, it did not change much from the previous result. Regarding the comparison of CoroNa Green AM fluorescence intracellular intensities, in the previous result only the blastocyst stage showed a significant difference when compared to the morula stage, but in the new analysis, the blastocyst stage and the hatching stage showed significantly lower fluorescence intensities than the morula stage. Additionally, the ouabain and control groups showed significant differences not only in the blastocyst stage, but also in the hatching stage. 2) In the first round of review, I have asked about the normalization of the mean intensity. According to your answer, it does not seem that you are normalizing the mean intensity by the size of the cell, which is very important to be able to compare your results. Please, provide the normalized analysis of your results. Response: The ZEN software 2012 black edition, that was used in this study, measures intensity on a pixel-by-pixel basis, and automatically calculates the mean intensity and the number of pixels. Therefore, these intensities are not an area-dependent value. In addition, we searched for reports that measured fluorescence intensity, but could not find one that had normalized by the size of the measured area [1-4]. Since the used mean intensities are already normalized by the number of pixels (based on the reason mentioned previously), we think that their comparison is a valid approach 1. Oksana Iamshanova, Pascal Mariot, V'yacheslav Lehen'kyi, Natalia Prevarskaya. Comparison of fluorescence probes for intracellular sodium imaging in prostate cancer cell lines. Eur Biophys J.2016;45(7):765-777. doi: 10.1007/s00249-016-1173-7. Epub 2016 Sep 22. 2. Charitha Galva, Pablo Artigas, Craig Gatto. Nuclear Na+/K+-ATPase plays an active role in nucleoplasmic Ca2+ homeostasis. J Cell Sci. 2012;125(Pt 24):6137-47. doi: 10.1242/jcs.114959. 3. Jiantao Zhang, Hua Liu, Jian Sun, Bei Li, Qiang Zhu, Shaoliang Chen, et al. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PLoS One. 2012;7(1):e30355. doi: 10.1371/journal.pone.0030355. 4. Dong-Ha Oh, Eduardo Leidi, Quan Zhang, Sung-Min Hwang, Youzhi Li, Francisco J Quintero, et al. Loss of halophytism by interference with SOS1 expression. Plant Physiol. 2009;151(1):210-22. doi: 10.1104/pp.109.137802. https://doi.org/10.1371/journal.pone.0246337.r004
19 Jan 2021 Decision Letter - Peter J. Hansen, Editor Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators PONE-D-20-29923R2 Dear Dr. Fujishima, 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, Peter J. Hansen 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 #1: 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 #1: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (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: (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: (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) ****** 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: Yes:** Maud Borensztein https://doi.org/10.1371/journal.pone.0246337.r005
Formally Accepted
21 Jan 2021 Acceptance Letter - Peter J. Hansen, Editor PONE-D-20-29923R2 Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators Dear Dr. Fujishima: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Peter J. Hansen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0246337.r006

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