In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch)

Christopher Monson; Giles Goetz; Kristy Forsgren; Penny Swanson; Graham Young

doi:10.1371/journal.pone.0311628

Peer Review History

Original SubmissionSeptember 6, 2023
9 Oct 2023 Decision Letter - Michael Schubert, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-23-28919In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch)PLOS ONE Dear Dr. Monson, 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 comprehensively addresses the points raised during the review process. Please submit your revised manuscript by Nov 23 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, Michael Schubert Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "The authors thank Abby Tillotson, Anna Bute, Fritzie Celino, and Erica Curles for their assistance with fish maintenance and sampling and Mollie Middleton, Jon Dickey, and Shelly Nance for technical support with steroid hormone measurements. This study was supported by National Science Foundation awards NSF-OISE-0914009, NSF IOS-0940765, and NSF IOS-1921746." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This study was supported by National Science Foundation awards NSF-OISE-0914009 awarded to KF, NSF IOS-0940765 awarded to GY and NSF IOS-1921746 awarded to GY. NSF.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: 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 Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors tried to identify the positive effects of 11-KT to early secondary ovarian growth in the coho salmon, the fish ovarian tissues were selected at the cortical alveolus stage by tissue section. Changes in the ovarian follicle transcriptome were tested using RNA-Seq followed by pathway analysis after three days of 11-KT treated. The sample size was good (n= 3 /group * 2 groups), BUT the figures are not readable by its bad resolution, I do not know what caused this problem. "Fish were lethally sampled after 3 days. At that time, fork length, body weight, and gonad weight were measured', I do not think these somatic parameters will change only after 3 days of treatment. "Histological screening eliminated any female that displayed overtly asynchronous ovarian stages or were not at the cortical alveolus stage", it is good, since to the defined phenotype is important to elucidate the genotype, but how to determine "similar cortical alveoli abundance" ? (No asterisks is indicated in figure 1). "De novo assembly"? is there no reference genome from Salmonid to re-mapping the reads or contigs? In "Table 1", what are "Additional reads included in backbone"? "A total of 63,048 of these contigs (99.4%) were annotatable", and "Duplicate contigs were collapsed to the gene-ID level using the average expression value relative to controls", have the authors confirmed the presence of isoforms or redundant genes ? (the 4th-WGD in salmonids is significant on the presence of gene isoforms). "Cluster analysis of differentially expressed contigs in 11-KT treated samples (Fig. 2C) ", there is no figure 2C!? please correct it. From figure 2B, the significant variation on the intra-group FC has been observed, do the authors ever consider this variation to the result interpretation ? is it explained by zero-inflation ? "Duplicate contigs were collapsed .... resulting in 3,853 genes with expression altered by 11-KT (P-adjusted ≤0.1)", how did the authors condense (or reduce dimension) the 3853 gene into figure 3s? is there any probability to confirm the results? Please discuss the results from the IPA analysis, since the Neuregulin Signaling is also significant (z-score=3.18), why the authors ignore it ? the authors only discuss the canonical (classic) reproductive pathways (steroidogenesis, vitellogenesis and lipid uptake and processing, Fsh signaling, and tgfb superfamily members). Reviewer #2: Primary growth, secondary growth, and maturation are the three developmental stages of oocytes in fish. Earlier published studies by the corresponding author suggest that during the late primary stage of ovarian follicle development in coho salmon, androgens are potent drivers of growth, and sustained release pellets of 11-KT implanted in vivo, advanced follicle growth and altered the ovarian transcriptome. Previous studies in coho salmon have shown that at early secondary stage, in vitro and in vivo treatment with 11-KT increased the size of ovarian follicles sooner than E2, but did not lead to formation of cortical alveoli. On the other hand, in vivo or in vitro treatment with E2 promoted the formation of cortical alveoli, increased plasma E2 levels, as well as Fsh transcript levels, indicating entry into secondary growth. Hence, it was concluded that 11-KT could be involved in initiating the entry of the ovarian follicles into secondary growth, since it was more efficient than E2 in increasing the size of ovarian follicles. In this study, the authors have focused on the early secondary growth. Here, sustained release pellets containing 11-KT were implanted in female coho salmon that had ovaries at the cortical alveolus stage. Changes in the ovarian follicle transcriptome were determined after three days using RNA-Seq followed by pathway analysis. The fundamental difference between the previous study and this one is the absence/presence of cortical alveoli. Females with overtly asynchronous ovarian stages were eliminated. All ovaries displayed early to mid-cortical alveolus stage phenotype. Comments Frozen ovarian samples from control, 11-KT, and E2 treated females (N=3 per group) were selected for RNA-Seq analysis. I could not see any mention of E2 treated females anywhere else in the results related to this work. Sequencing resulted in 1.6 billion total reads from 9 samples (Table 1) , , , The Table mentions additional reads included in backbone (1 to 3). Please clarify the source of these reads. It is stated that the presence of cortical alveoli is a histological indicator of entry into secondary growth in coho salmon and that secondary growth is characterized by an increase in ovarian E2 synthesis via FSH signaling. However, in this work, even though plasma 11-KT level increased several folds in treated fish, serum E2 levels were unaltered. RNASeq results too show that 11-KT did not affect the expression of fshr, and reduced the expression of hsd3b, cyp19a1 (enzymes that catalyze steroid and E2 synthesis). RNASeq data shows that 11 KT treatment increased Vgtr, while E2 was shown to repress vtgr expression in previtellogenic follicles of largemouth bass and medaka. In my opinion, these major observations do not support the conclusion that ‘after three days of sex steroid exposure,. . . ., the widespread transcriptomic changes induced by 11-KT are consistent with a role for 11-KT in lipid and vitellogenin uptake and processing, and in Fsh signaling, which are hallmarks of secondary growth, as well as cellular development and other cellular processes, and changes in the extracellular matrix. Hence, I would suggest more pragmatic inference of the data. ******** 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: Yung-Sen Huang Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0311628.r001
Revision 1
22 Jan 2024 Author Response Reviewer #1: The authors tried to identify the positive effects of 11-KT to early secondary ovarian growth in the coho salmon, the fish ovarian tissues were selected at the cortical alveolus stage by tissue section. Changes in the ovarian follicle transcriptome were tested using RNA-Seq followed by pathway analysis after three days of 11-KT treated. The sample size was good (n= 3 /group * 2 groups), BUT the figures are not readable by its bad resolution, I do not know what caused this problem. We refer to the editors if the figures do not match required resolution standards. We are not sure why they may be appearing unreadable in the document viewed by reviewer #1. "Fish were lethally sampled after 3 days. At that time, fork length, body weight, and gonad weight were measured', I do not think these somatic parameters will change only after 3 days of treatment. We agree that these somatic parameters would be highly unlikely to change after just three days of treatment but included these data to indicate that the fish were of the same size and weight and therefore likely to be in the same stage of development since coho salmon are semelparous. "Histological screening eliminated any female that displayed overtly asynchronous ovarian stages or were not at the cortical alveolus stage", it is good, since to the defined phenotype is important to elucidate the genotype, but how to determine "similar cortical alveoli abundance" ? (No asterisks is indicated in figure 1). We did not quantify cortical alveoli abundance but did use observations of cortical alveoli as an indication of developmental stage. We selected females that displayed the most similar morphological phenotypes and that were clearly in the ECA stage of development. "De novo assembly"? is there no reference genome from Salmonid to re-mapping the reads or contigs? The assembly was done prior to the coho salmon genome being published. In "Table 1", what are "Additional reads included in backbone"? We initially sequenced E2 treated females of the same cohort and those reads were included in the initial backbone that the treatments were then compared back to. However, we chose to focus our analysis in this manuscript on the 11-KT treatment group out of a concern for brevity. We added text to the figure legend to clear this up. "A total of 63,048 of these contigs (99.4%) were annotatable", and "Duplicate contigs were collapsed to the gene-ID level using the average expression value relative to controls", have the authors confirmed the presence of isoforms or redundant genes? (the 4th-WGD in salmonids is significant on the presence of gene isoforms). We were able to identify isoforms of many genes when sequence information was available. However, for pathway analyses, we could only use genes recognized by the software and thus were limited in our inclusion of salmonid-specific isoforms to those shared by zebrafish. One of the requirements of the pathway analysis software is the collapse of duplicate entries into single gene-level IDs. In doing this we lost potential isoforms that are not shared by zebrafish or homo sapiens as the case may be. "Cluster analysis of differentially expressed contigs in 11-KT treated samples (Fig. 2C) ", there is no figure 2C!? please correct it. 2C should have been labelled 2B, apologies and thank you for catching that error From figure 2B, the significant variation on the intra-group FC has been observed, do the authors ever consider this variation to the result interpretation? is it explained by zero-inflation ? The authors are unsure what the reviewer is asking with this question. There is indeed differences in the level of differential expression between individual genes in the treatment groups as would be expected in a full-transcriptome analysis. "Duplicate contigs were collapsed .... resulting in 3,853 genes with expression altered by 11-KT (P-adjusted ≤0.1)", how did the authors condense (or reduce dimension) the 3853 gene into figure 3s? is there any probability to confirm the results? In figure 3 we are highlighting specific genes that were altered and that we discuss throughout the manuscript, we are not including all of the genes identified. Table S1 has the complete list of genes along with the Entrez, Uniprot, SwissProt accession numbers and ids. The traditional way of confirming RNA-Seq results is by qPCR, but we believe that there are more variables and thus less reliability in the application of qPCR than in the RNA-Seq the method used here, and so qPCR would be unlikely to give accurate confirmation of expression patterns across all of the genes we discuss. Please discuss the results from the IPA analysis, since the Neuregulin Signaling is also significant (z-score=3.18), why the authors ignore it ? the authors only discuss the canonical (classic) reproductive pathways (steroidogenesis, vitellogenesis and lipid uptake and processing, Fsh signaling, and tgfb superfamily members). Due to the enormity of the dataset and our relative expertise in reproductive development, we chose to focus our analysis on only a subset of the genes and pathways that were significantly altered by the treatment. We agree that this dataset opens many potentially interesting hypotheses. There is abundant evidence that the EGF and EGF-like family members play important roles in development of many tissues, with NRGs playing particular roles in granulosa cell survival, but to our knowledge of the current literature, studies are almost exclusively in mammalian models. We include a minor number of mammalian references where appropriate but attempted to keep our discussion within the teleost literature as much as possible. Reviewer #2: Primary growth, secondary growth, and maturation are the three developmental stages of oocytes in fish. Earlier published studies by the corresponding author suggest that during the late primary stage of ovarian follicle development in coho salmon, androgens are potent drivers of growth, and sustained release pellets of 11-KT implanted in vivo, advanced follicle growth and altered the ovarian transcriptome. Previous studies in coho salmon have shown that at early secondary stage, in vitro and in vivo treatment with 11-KT increased the size of ovarian follicles sooner than E2, but did not lead to formation of cortical alveoli. On the other hand, in vivo or in vitro treatment with E2 promoted the formation of cortical alveoli, increased plasma E2 levels, as well as Fsh transcript levels, indicating entry into secondary growth. Hence, it was concluded that 11-KT could be involved in initiating the entry of the ovarian follicles into secondary growth, since it was more efficient than E2 in increasing the size of ovarian follicles. In this study, the authors have focused on the early secondary growth. Here, sustained release pellets containing 11-KT were implanted in female coho salmon that had ovaries at the cortical alveolus stage. Changes in the ovarian follicle transcriptome were determined after three days using RNA-Seq followed by pathway analysis. The fundamental difference between the previous study and this one is the absence/presence of cortical alveoli. Females with overtly asynchronous ovarian stages were eliminated. All ovaries displayed early to mid-cortical alveolus stage phenotype. Comments Frozen ovarian samples from control, 11-KT, and E2 treated females (N=3 per group) were selected for RNA-Seq analysis. I could not see any mention of E2 treated females anywhere else in the results related to this work. Removed text that mentions E2 treated females from the methods section. Sequencing resulted in 1.6 billion total reads from 9 samples (Table 1) , , , The Table mentions additional reads included in backbone (1 to 3). Please clarify the source of these reads. We initially sequenced E2 treated females of the same cohort and those reads were included in the initial backbone that the treatments were then compared back to. However, we chose to focus our analysis in this manuscript on the 11-KT treatment group out of a concern for brevity. We added text to the figure legend to clear this up It is stated that the presence of cortical alveoli is a histological indicator of entry into secondary growth in coho salmon and that secondary growth is characterized by an increase in ovarian E2 synthesis via FSH signaling. However, in this work, even though plasma 11-KT level increased several folds in treated fish, serum E2 levels were unaltered. RNASeq results too show that 11-KT did not affect the expression of fshr, and reduced the expression of hsd3b, cyp19a1 (enzymes that catalyze steroid and E2 synthesis). RNASeq data shows that 11 KT treatment increased Vgtr, while E2 was shown to repress vtgr expression in previtellogenic follicles of largemouth bass and medaka. In my opinion, these major observations do not support the conclusion that ‘after three days of sex steroid exposure,. . . ., the widespread transcriptomic changes induced by 11-KT are consistent with a role for 11-KT in lipid and vitellogenin uptake and processing, and in Fsh signaling, which are hallmarks of secondary growth, as well as cellular development and other cellular processes, and changes in the extracellular matrix. Hence, I would suggest more pragmatic inference of the data. We respectfully disagree with the reviewer on this point. 11-KT induced changes in late primary growth (previous study cited throughout) included increased expression of fshr and cyp19a1. Combined with these results in early secondary growth, especially alterations of Fsh signaling pathway transcripts, the increase in expression of lipid and vitellogenin uptake genes, and genes in the steroidogenic pathways, as well as the general predictions of the pathway analysis, we believe that androgens likely play an important role in preparing the follicle for the events that occur later in secondary growth, and in particular Fsh-mediated processes. https://doi.org/10.1371/journal.pone.0311628.r002
21 Feb 2024 Decision Letter - Michael Schubert, Editor PONE-D-23-28919R1In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch)PLOS ONE Dear Dr. Monson, 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 Apr 06 2024 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, Michael Schubert Academic Editor PLOS ONE Additional Editor Comments: The reviewer notes severe shortcomings concerning the quality of the manuscript's figures. These shortcomings have to be resolved in full before the manuscript can be considered for publication in 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: 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: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #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: The resolution of the figures is still too low, they are not clear to provide the informations, and even something is missed in the figure 3. ****** 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 ******** [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.0311628.r003
Revision 2
19 Sep 2024 Author Response Reviewer #1: The authors tried to identify the positive effects of 11-KT to early secondary ovarian growth in the coho salmon, the fish ovarian tissues were selected at the cortical alveolus stage by tissue section. Changes in the ovarian follicle transcriptome were tested using RNA-Seq followed by pathway analysis after three days of 11-KT treated. The sample size was good (n= 3 /group * 2 groups), BUT the figures are not readable by its bad resolution, I do not know what caused this problem. We refer to the editors if the figures do not match required resolution standards. We are not sure why they may be appearing unreadable in the document viewed by reviewer #1. "Fish were lethally sampled after 3 days. At that time, fork length, body weight, and gonad weight were measured', I do not think these somatic parameters will change only after 3 days of treatment. We agree that these somatic parameters would be highly unlikely to change after just three days of treatment but included these data to indicate that the fish were of the same size and weight and therefore likely to be in the same stage of development since coho salmon are semelparous. "Histological screening eliminated any female that displayed overtly asynchronous ovarian stages or were not at the cortical alveolus stage", it is good, since to the defined phenotype is important to elucidate the genotype, but how to determine "similar cortical alveoli abundance" ? (No asterisks is indicated in figure 1). We did not quantify cortical alveoli abundance but did use observations of cortical alveoli as an indication of developmental stage. We selected females that displayed the most similar morphological phenotypes and that were clearly in the ECA stage of development. "De novo assembly"? is there no reference genome from Salmonid to re-mapping the reads or contigs? The assembly was done prior to the coho salmon genome being published. In "Table 1", what are "Additional reads included in backbone"? We initially sequenced E2 treated females of the same cohort and those reads were included in the initial backbone that the treatments were then compared back to. However, we chose to focus our analysis in this manuscript on the 11-KT treatment group out of a concern for brevity. We added text to the figure legend to clear this up. "A total of 63,048 of these contigs (99.4%) were annotatable", and "Duplicate contigs were collapsed to the gene-ID level using the average expression value relative to controls", have the authors confirmed the presence of isoforms or redundant genes? (the 4th-WGD in salmonids is significant on the presence of gene isoforms). We were able to identify isoforms of many genes when sequence information was available. However, for pathway analyses, we could only use genes recognized by the software and thus were limited in our inclusion of salmonid-specific isoforms to those shared by zebrafish. One of the requirements of the pathway analysis software is the collapse of duplicate entries into single gene-level IDs. In doing this we lost potential isoforms that are not shared by zebrafish or homo sapiens as the case may be. "Cluster analysis of differentially expressed contigs in 11-KT treated samples (Fig. 2C) ", there is no figure 2C!? please correct it. 2C should have been labelled 2B, apologies and thank you for catching that error From figure 2B, the significant variation on the intra-group FC has been observed, do the authors ever consider this variation to the result interpretation? is it explained by zero-inflation ? The authors are unsure what the reviewer is asking with this question. There is indeed differences in the level of differential expression between individual genes in the treatment groups as would be expected in a full-transcriptome analysis. "Duplicate contigs were collapsed .... resulting in 3,853 genes with expression altered by 11-KT (P-adjusted ≤0.1)", how did the authors condense (or reduce dimension) the 3853 gene into figure 3s? is there any probability to confirm the results? In figure 3 we are highlighting specific genes that were altered and that we discuss throughout the manuscript, we are not including all of the genes identified. Table S1 has the complete list of genes along with the Entrez, Uniprot, SwissProt accession numbers and ids. The traditional way of confirming RNA-Seq results is by qPCR, but we believe that there are more variables and thus less reliability in the application of qPCR than in the RNA-Seq the method used here, and so qPCR would be unlikely to give accurate confirmation of expression patterns across all of the genes we discuss. Please discuss the results from the IPA analysis, since the Neuregulin Signaling is also significant (z-score=3.18), why the authors ignore it ? the authors only discuss the canonical (classic) reproductive pathways (steroidogenesis, vitellogenesis and lipid uptake and processing, Fsh signaling, and tgfb superfamily members). Due to the enormity of the dataset and our relative expertise in reproductive development, we chose to focus our analysis on only a subset of the genes and pathways that were significantly altered by the treatment. We agree that this dataset opens many potentially interesting hypotheses. There is abundant evidence that the EGF and EGF-like family members play important roles in development of many tissues, with NRGs playing particular roles in granulosa cell survival, but to our knowledge of the current literature, studies are almost exclusively in mammalian models. We include a minor number of mammalian references where appropriate but attempted to keep our discussion within the teleost literature as much as possible. Reviewer #2: Primary growth, secondary growth, and maturation are the three developmental stages of oocytes in fish. Earlier published studies by the corresponding author suggest that during the late primary stage of ovarian follicle development in coho salmon, androgens are potent drivers of growth, and sustained release pellets of 11-KT implanted in vivo, advanced follicle growth and altered the ovarian transcriptome. Previous studies in coho salmon have shown that at early secondary stage, in vitro and in vivo treatment with 11-KT increased the size of ovarian follicles sooner than E2, but did not lead to formation of cortical alveoli. On the other hand, in vivo or in vitro treatment with E2 promoted the formation of cortical alveoli, increased plasma E2 levels, as well as Fsh transcript levels, indicating entry into secondary growth. Hence, it was concluded that 11-KT could be involved in initiating the entry of the ovarian follicles into secondary growth, since it was more efficient than E2 in increasing the size of ovarian follicles. In this study, the authors have focused on the early secondary growth. Here, sustained release pellets containing 11-KT were implanted in female coho salmon that had ovaries at the cortical alveolus stage. Changes in the ovarian follicle transcriptome were determined after three days using RNA-Seq followed by pathway analysis. The fundamental difference between the previous study and this one is the absence/presence of cortical alveoli. Females with overtly asynchronous ovarian stages were eliminated. All ovaries displayed early to mid-cortical alveolus stage phenotype. Comments Frozen ovarian samples from control, 11-KT, and E2 treated females (N=3 per group) were selected for RNA-Seq analysis. I could not see any mention of E2 treated females anywhere else in the results related to this work. Removed text that mentions E2 treated females from the methods section. Sequencing resulted in 1.6 billion total reads from 9 samples (Table 1) , , , The Table mentions additional reads included in backbone (1 to 3). Please clarify the source of these reads. We initially sequenced E2 treated females of the same cohort and those reads were included in the initial backbone that the treatments were then compared back to. However, we chose to focus our analysis in this manuscript on the 11-KT treatment group out of a concern for brevity. We added text to the figure legend to clear this up It is stated that the presence of cortical alveoli is a histological indicator of entry into secondary growth in coho salmon and that secondary growth is characterized by an increase in ovarian E2 synthesis via FSH signaling. However, in this work, even though plasma 11-KT level increased several folds in treated fish, serum E2 levels were unaltered. RNASeq results too show that 11-KT did not affect the expression of fshr, and reduced the expression of hsd3b, cyp19a1 (enzymes that catalyze steroid and E2 synthesis). RNASeq data shows that 11 KT treatment increased Vgtr, while E2 was shown to repress vtgr expression in previtellogenic follicles of largemouth bass and medaka. In my opinion, these major observations do not support the conclusion that ‘after three days of sex steroid exposure,. . . ., the widespread transcriptomic changes induced by 11-KT are consistent with a role for 11-KT in lipid and vitellogenin uptake and processing, and in Fsh signaling, which are hallmarks of secondary growth, as well as cellular development and other cellular processes, and changes in the extracellular matrix. Hence, I would suggest more pragmatic inference of the data. We respectfully disagree with the reviewer on this point. 11-KT induced changes in late primary growth (previous study cited throughout) included increased expression of fshr and cyp19a1. Combined with these results in early secondary growth, especially alterations of Fsh signaling pathway transcripts, the increase in expression of lipid and vitellogenin uptake genes, and genes in the steroidogenic pathways, as well as the general predictions of the pathway analysis, we believe that androgens likely play an important role in preparing the follicle for the events that occur later in secondary growth, and in particular Fsh-mediated processes. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0311628.r004
23 Sep 2024 Decision Letter - Michael Schubert, Editor In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch) PONE-D-23-28919R2 Dear Dr. Monson, 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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Michael Schubert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311628.r005
Formally Accepted
30 Sep 2024 Acceptance Letter - Michael Schubert, Editor PONE-D-23-28919R2 PLOS ONE Dear Dr. Monson, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. Michael Schubert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311628.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .