In vitro organ culture protocol for intact urogenital systems supporting gonadal differentiation

Sarah L. Whiteley; Clare E. Holleley; Arthur Georges

doi:10.1371/journal.pone.0327930

Peer Review History

Original SubmissionDecember 11, 2024
Decision Letter - Rajakumar Anbazhagan, Editor PONE-D-24-57396In vitro organ culture protocol for intact urogenital systems supporting gonadal differentiationPLOS ONE Dear Dr. Whiteley, 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 May 04 2025 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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Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Partly ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The current article describes an in vitro organ culture protocol involving agar slab for intact urogenital systems in the reptile model, Pogona vitticeps supporting differentiation of biopotential gonads into either ovaries or testes. The protocol delivered superior results over the existing protocols with respect to integrity of gross organ morphology, showed sex specific gene expression and temperature induction of sex reversal during culture. Overall it is providing a reliable method for studying gonadal differentiation in non-model species. Reviewer #2: This In-vitro organ culture protocol for intact urogenital systems is significant. Unlike traditional genetic studies, which rely on whole-animal experiments, this method allows direct functional analysis of gene-environment interactions. It is substantial for reptiles, where temperature-dependent sex determination (TSD) is a key factor, enabling controlled studies. A step forward, but not without its challenges. These include species-specific adaptations, the need for external supplementation of systemic factors, and potential developmental deviations in vitro. However, the future is promising, with potential improvements such as integrating hormonal signaling, optimizing culture conditions for broader species applicability, and exploring co-culture systems to better mimic in vivo environments. The journey towards a more comprehensive understanding continues. The authors need to discuss the points below to mark. 1. In vivo, gonadal development is influenced by systemic factors such as hormones from the hypothalamus-pituitary-gonadal (HPG) axis. This in vitro system, while valuable, lacks these interactions, which may lead to incomplete or altered differentiation patterns. It's important to acknowledge these limitations and consider external supplementation with hormones or signaling molecules to mimic in vivo conditions fully. 2. Even though the gross morphology of the gonads is preserved, subtle changes in differentiation could still occur due to the artificial culture environment. The absence of vascularization and mechanical forces in vivo may affect the development of specific cell types or tissue organization. The impact of in vitro conditions on long-term gonadal function and reproductive potential remains unclear. 3. The study acknowledges that no in vitro system can fully replicate the biological complexity of in situ development. However, it does not sufficiently discuss the potential impact of missing systemic factors, such as endocrine signals or maternal influences, on gonadal differentiation and gene expression. The absence of these factors in the in vitro system may lead to incomplete or altered differentiation patterns, which could affect the expression of sex-specific genes and the overall reproductive potential of the cultured gonads. 4. The study highlights that Biopore membranes fail to maintain gonad morphology, but the agar slab method does not fully replicate in vivo development. The extent to which these morphological differences affect downstream analyses (e.g., gene expression) is not deeply explored. The expression of sex-specific genes in cultured gonads does not fully match that observed in in situ development, particularly in 36°C-induced sex-reversed females. The study suggests that high temperatures may mask sex-specific gene expression, but alternative explanations should be considered, such as differences in developmental timing or stress responses in vitro. These factors could potentially influence gene expression and should be further investigated. 5. While the study presents morphological and gene expression evidence of sex reversal, inconsistencies in gene expression trends (e.g., up regulation of some male genes in sex-reversed females) raise questions. Additional functional validation (e.g., hormonal assays or long-term differentiation studies) would strengthen the conclusions. 6. The inability to cleanly separate gonads from the mesonephros in Biopore cultures limits the specificity of gene expression data. Even in agar slab cultures, residual mesonephric tissue could influence RNA sequencing results. To address this, the study could further evaluate the influence of residual mesonephric tissue on RNA sequencing results by comparing single-cell RNA sequencing data, which would provide a more detailed and accurate picture of gene expression in cultured gonads. 7. The study does not clearly state the number of biological replicates used for gene expression analysis. Given the complexity of temperature-induced sex reversal, a larger sample size would help determine whether observed trends are biologically meaningful or due to individual variability. 8. While agar slabs performed better than Biopore membranes, other 3D culture techniques, such as extracellular matrix-based hydrogels, might provide better structural support and improve morphological and molecular outcomes. The study does not explore these possibilities. 9. The study optimizes culture conditions based on previous P. vitticeps staging but does not test whether earlier or later developmental stages might yield better results. A more detailed assessment of optimal culture windows could improve the model. ****** 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. 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Revision 1
23 May 2025 Author Response Manuscript PONE-D-24-57396: In vitro organ culture protocol for intact urogenital systems supporting gonadal differentiation Response to reviewers We thank the Editor and the two Reviewers for taking the time to consider our manuscript for publication in PLOS One. The manuscript was well received by Reviewer 1 who didn’t provide any suggestions for the manuscript, stating that the protocol provides “a reliable method for studying gonadal differentiation in non-model species.” We have responded in detail to the comments from Reviewer 2 in BLUE, and actions we have taken to address these comments are in RED. In the amended manuscript the amendments have been made using tracked changes as requested by the editorial team, along with a clean revised version. All referenced line numbers correspond to the tracked changes version of the manuscript. Reviewer 2 Response We appreciate the thoroughness of the response from Reviewer 2. We note that the majority of the comments share a theme in that there are limitations of our present method that are inherent to ex ovo culture systems. It is not possible to ever perfectly replicate the in vivo environment in culture. We acknowledged this in the original submission on lines 187-189, where we stated that “we expect the in vitro cultures will not completely recapitulate the expression patterns of gonads developing in situ, as no culture system can ever capture the biological complexities present in an intact and normally developing embryo.” This includes the absence of any signals that may originate from the hypothalamus (comment 1) and other endocrine signalling factors (comment 3) that may mean that there are “subtle changes” in the organ culture (comment 2). ACTION: In order to address the overarching concerns central to these comments, we have added the following lines to the revised manuscript: “While no in vitro culture system can ever completely recapitulate the environment experienced in vivo, the goal is to develop techniques able to sufficiently capture the biological complexity and overcome obstacles present in the in vivo system.” (lines 57-60). “Although there are limitations inherent to in vitro culture systems, in such instances they present the only viable alternative for experimentation.” (lines 71-72). The other concern raised by Reviewer 2 consistent across three comments was that additional validation may shed more light onto the efficacy of the culture method. This includes hormonal assays (comment 5), single cell RNA-sequencing (comment 6), and matrix-based hydrogels (comment 8). We agree that there are many options available for validating and optimising in vitro culture protocols, as described in lines 241-249 of the original submission. We suggest the utility of whole transcriptome sequencing but also acknowledge other more cost-effective approaches like qPCR, and other morphology based techniques alongside histology, such as immunohistochemistry. One of the major advantages of our method is its simplicity and low cost (mentioned in lines 35-36), making it accessible to all researchers compared to techniques that rely on expensive consumables like biopore membranes and hydrogel matrixes. ACTION: To highlight the current limitations of our presented method, we have added the following lines and suggest additional avenues for optimisation of the technique, we have added the following lines: “Many other sequencing techniques are available for validating expression profiles, for example single cell sequencing, but are often cost prohibitive.” (lines 248-249). “Other assays assessing hormonal function could also be considered depending on research requirements.” (lines 254-255). Other comments from Reviewer 2 1. In vivo, gonadal development is influenced by systemic factors such as hormones from the hypothalamus-pituitary-gonadal (HPG) axis. This in vitro system, while valuable, lacks these interactions, which may lead to incomplete or altered differentiation patterns. It's important to acknowledge these limitations and consider external supplementation with hormones or signaling molecules to mimic in vivo conditions fully. We thank Reviewer 2 for their comment and agree that any in vitro system has inherent limitations and will never be able to fully recapitulate the in vivo environment, as addressed in response to comments above. We also note that the organ culture is not fully separated from the entire HPA (hypothalamus-pituitary-adrenal) axis because the intact UGS is explanted such that the adrenal is still present. This is an advantage to previous methods where the gonad was cultured in isolation, so was completely removed from the whole axis. We also note that there is limited evidence for the role of the HPA axis in reptile sex determination (reviewed in Castelli et al, Biological Reviews, 2020). Any signalling molecules potentially originating from the HPA axis are poorly characterised in reptiles, so it would be difficult to determine any necessary supplementation to the culture medium. Additionally, as outlined in this paper, the gonads of multiple reptile species can differentiate in vitro suggesting that any influence from the HPA axis is not critical to this process. ACTION: To address this suggestion from Reviewer 2, we have added the following statement to lines 90-91: “It also removes the gonads from the hypothalamic-pituitary-adrenal (HPA) axis, which may influence gonad growth and development”. We also added the following on lines 102-103: “While culture of the whole HPA axis is impossible, the culture of the intact urogenital system at least includes the adrenal, which may enhance gonad growth and development in culture.” 4. The study highlights that Biopore membranes fail to maintain gonad morphology, but the agar slab method does not fully replicate in vivo development. The extent to which these morphological differences affect downstream analyses (e.g., gene expression) is not deeply explored. The expression of sex-specific genes in cultured gonads does not fully match that observed in in situ development, particularly in 36°C-induced sex-reversed females. The study suggests that high temperatures may mask sex-specific gene expression, but alternative explanations should be considered, such as differences in developmental timing or stress responses in vitro. These factors could potentially influence gene expression and should be further investigated. Throughout the manuscript we have been careful to never claim that our agar slab method fully replicates the in vivo environment. Indeed, we have drawn attention to the fact this is not possible in several parts of the manuscript, and have made further additions to the revised paper in response to other comments from Reviewer 2 in relation to this. We do agree with Reviewer 2 that temperature complicates the gene expression patterns observed in the 36°C sex reversed females, as stated in the original submission: “This may be because high temperatures mask some of the sex-specific gene expression patterns, and there appears to be differences in the timing of gene expression compared to gonads developing in situ.” (lines 214-216). On lines 241-243 we also stated that: “Even for in situ gonads, expression patterns can be complicated, and with the addition of the temperature influences, and be complicated even further. Because of this, care should be taken if using gene expression profiling in general, but particularly for in vitro organ cultures.” ACTION: To further highlight the complicating effects of temperature, we have added the following: “This is especially important for species with thermosensitive sex determination systems where the influence of temperature on sex differentiation is fundamentally linked.” (lines 244-246). 7. The study does not clearly state the number of biological replicates used for gene expression analysis. Given the complexity of temperature-induced sex reversal, a larger sample size would help determine whether observed trends are biologically meaningful or due to individual variability. ACTION: We have included sample sizes for the different groups on lines 172, 189, 262, 266. The sample sizes are comparable to previous studies, particularly for transcriptomics (see Whiteley et al., 2021, 2022 for examples). 9. The study optimizes culture conditions based on previous P. vitticeps staging but does not test whether earlier or later developmental stages might yield better results. A more detailed assessment of optimal culture windows could improve the model. As stated on lines 133-135 of the original submission, we did test the influence of explanting tissue earlier in development (stage 4). We noted on lines 16-169 that we chose stage 6 to “keep the culture duration to a minimum”. Similarly, we did not explant later in development as previous work on the species showed that differentiation can occur as early as stage 8, and we did not want to risk explantation when the gonad was committed to a sexual fate (Whiteley et al., Scientific Reports, 2018). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0327930.r002
Decision Letter - Rajakumar Anbazhagan, Editor In vitro organ culture protocol for intact urogenital systems supporting gonadal differentiation PONE-D-24-57396R1 Dear Dr. Whiteley, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have well addressed the comments made by reviewers. The authors added sections to acknowledge the limitations of in vitro culture system including their proposed model that those cannot fully recapitulate the environment experienced in vivo. However, the proposed model is an improvement on in vitro culture models in that it cultures the intact urinogenital system and can be an experimental choice for in vitro culture model. Reviewer #2: The authors have addressed the major concerns thoughtfully, particularly regarding the limitations of in vitro systems and the absence of systemic endocrine inputs. The inclusion of adrenal tissue is noted, though it does not fully replicate HPG/HPA axis signaling. The clarifications added to the manuscript are appreciated. Functional validation (e.g., hormonal assays) and advanced culture platforms (e.g., hydrogels) could further enhance the model’s utility. Additionally, assessing a broader developmental window may optimize outcomes. This study represents a promising and accessible approach to investigating reptilian sex determination. With further refinement, the model could offer significant and hopeful insights into the environmental and molecular mechanisms of gonadal development. ****** 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 ******** https://doi.org/10.1371/journal.pone.0327930.r003
Formally Accepted
Acceptance Letter - Rajakumar Anbazhagan, Editor PONE-D-24-57396R1 PLOS ONE Dear Dr. Whiteley, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Rajakumar Anbazhagan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0327930.r004

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