PONE-D-20-20204

MOLECULAR MECHANISMS OF INFERTILITY INDUCED BY ERECTILE DYSFUNCTION DRUGS

PLOS ONE

Dear Dr.Sheweita,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

There are major concerns with the experiments performed (controls, statistical analyses etc.) and the stated conclusion. The title does not reflect the results obtained. For instance, the  Western blot experiments require proper controls with full gel figures before any inference can be derived. There are numerous errors that need to be rectified. Please respond to all the queries raised by the reviewers and conduct the appropriate experiments with proper controls. The conclusion should be supported by your data. 

 If you will need extra time 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Vasu D. Appanna

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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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 manuscript by Sheweita et al. boldly proclaims to have found the molecular mechanisms of infertility caused by erectile dysfunction drugs. However, I am strongly concerned that they have based this statement purely on the results of poorly performed Western blots as there is no substantial evidence in this manuscript to support the title.

Major revisions:

The quality of the language must be improved throughout. I have outlined select examples but this is not a thorough list. For instance, in table 1, "weak" is written instead of "week". In all tables, tadalafil is listed twice. In Figure 1, drugs are listed under their branded name rather than their chemical name. Please stick to one nomenclature. In the introduction,

-"ED is highly prevalent, affecting approximately 50 percent of men between 40 and 70 years of age, around people worldwide" - How many people worldwide?

- Results: "The protein expression of CYP11A1 was markedly down-regulated in testes after

treatment of rabbits with any of these drugs, and such effects were also obtained in livers

except sildenafil induced such protein expression" Check sentence structure

- Discussion: "Quenching of free radicals by SOD and CAT as result sildenafil and vardenafil

treatments could sustain the bioavailability of nitric oxide [NO] for vasodilatation, and this

could be another new possible mechanism of actions of ED drugs since most cases of

ED are associated with oxidative stress" Mechanism of action

Major revisions - Results:

The main conclusion to be drawn from this study, that the mechanism of infertility is decreased cytochrome p450 enzymes, is based on poorly described and poorly performed Western blot analysis. I am assuming each lane is representative of a group of rabbits, but the n value is not described. How much protein was loaded in each lane? The densitometry appears to be performed relative to the control band, which is unacceptable, as this should be performed relative to an acceptable loading control (GAPDH/VDAC, etc.). The statistical analyses section describes that significance was set at p<0.05, but the statistical tests that were performed to assess significance are not mentioned for any of the results.

If the authors choose not to measure actual fertility as an outcome, but rather rely on three Western blots to support their conclusion, the title must be changed as to not overstate the results. Moreover, I would like the Western blots performed over a suitable loading control and I would like to see the full uncropped blots with ladders before I can properly re-review the manuscript.

Reviewer #2: The authors test three drugs and their effects on free radical levels and oxidative stress. Specifically, authors investigate the effects of ED drugs on protein levels of cytochrome P450 isozymes (CYP 11A1, 21A2, and 19C), which are involved in the steroidogenesis, and their effects on spermatocytes and the number of sperms. Although the histopathological study is convincing and interesting, however, so far the data are phenotypical observation, the mechanisms that the authors proposed are not solid supported and my major concerns are about the mechanism.

1. In Figure 1, the protein level was impacted by 10-20%, are this minimal changes essential for downstream function (any other groups show similar changes?)

2. Loading controls are required for Figure 1

3. Are there other proteins that were dominantly impacted by drug treatment other than P450? Authors should provide a negative control, a protein that may participate in oxidative stress but is not impacted by these three drugs to indicate the specific mechanism.

4. Does transcription of proteins in Figure 1 were impacted by drug treatment? what are putative reasons for the decrease of protein level? If over-express these proteins, or modulation the enzyme activity by established chemicals, can we recover the spermatocytes and the number of sperms?

5. If we want to specifically state the function of drugs, certain gene knockdown, enzyme inhibitors, or complementation expression are required. There is no strong evidence to support ED drugs induced a decrease of MDA level and increase of nitric oxide-cGMP level based on current data. As such, the mechanism was over-stated. The authors should provide other putative mechanisms for the phenotypes or more literature evidence.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-20204R1

ERECTILE DYSFUNCTION DRUGS ALTERED THE ACTIVITIES OF ANTIOXIDANT ENZYMES, OXIDATIVE STRESS AND THE PROTEIN EXPRESSIONS OF SOME CYTOCHROME P450 ISOZYMES INVOLVED IN THE STEROIDOGENESIS of STEROID HORMONES

PLOS ONE

Dear Dr. Sheweita,

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. Subsequent to the your responses as outlined below, it is important that the full gel figure of the Western blotting experiments are submitted as supplemental information. Additionally, it is also pertinent to emphasize in the Methods section the significance of B-actin as a loading control. (please refer to Samantha L. Eaton et al.  (2013)Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting https://doi.org/10.1371/journal.pone.0072457

Author response to queries raised on the revised manuscript.

1.The  B-actin blot is the same for each protein of interest, and these are all similar molecular weights. As such, they weren't compared on their respective blots to assure equal loading.

Regarding point”The B-actin blot is the same for each protein of interest. Yes defiantly, we have used one blot for  B-actin which is enough for calculation of band densities of different P450 isozymes since in our previous studies, we have used one B-actin blot [please see these citations:

Sheweita SA, ElHady SA, Hammoda HM. Trigonella stellata reduced the deleterious effects of diabetes mellitus through alleviation of oxidative stress, antioxidant- and drug-metabolizing enzymes activities.J Ethnopharmacol. 2020 Jun 28;256:112821. doi: 10.1016/j.jep.2020.112821. Epub 2020 Apr 3.PMID: 32251758

Regarding point” and these are all similar molecular weights.””Actually all CYP isozymes have not similar molecular weights since the molecular weights of most CYP isozymes are ranged between 48 and 56 KDa. In addition, each antibody reacts with its isozyme without interactions with other P450 isozymes as you have seen our data in the originals of WB.  

Regarding point” As such, they weren't compared on their respective blots to assure equal loading”.  One B-actin blot is enough for the calculation of band densities of different CYP isozymes since equal loading of microsomal proteins included P450 isozymes was performed as I have mentioned in the original and in the revised versions.

 2. The statistical analysis used to get their significance level (p<0.05) needs to be clarified in order to improve the confidence in the data reported.

The importance of statistical analysis is to see if the differences between treatments are statically significant or not. I confirm that there are different levels of significances [ <0.001, <0.01, <0.05] but we set all levels of significance  at <0.05. However, if you want to include other levels of significances in addition to <0.05, we can do it.

3. The title appears to the same as in the original submission despite the concerns of the reviewers.

The old  title was” MOLECULAR MECHANISMS OF INFERTILITY INDUCED BY ERECTILE DYSFUNCTION DRUGS” 

The new title is” ERECTILE DYSFUNCTION DRUGS ALTERED THE ACTIVITIES OF ANTIOXIDANT ENZYMES, OXIDATIVE STRESS AND THE PROTEIN EXPRESSIONS OF SOME CYTOCHROME P₄₅₀ ISOZYMES INVOLVED IN THE STEROIDOGENESIS of STEROID HORMONES” which is a mirror for the content of the manuscript.

Please submit your revised manuscript by October 31, 2020. 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,

Vasu D. Appanna

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #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: I am not confident the authors understand the principles of Western blotting and loading controls, as their B-actin blot is the same for each protein of interest, and these are all similar molecular weights. As such, they weren't compared on their respective blots to assure equal loading.

In addition, I requested the statistical analysis used to get their significance level (p<0.05) and did not receive a response. As such, I do not have confidence in the results of the submitted manuscript and must recommend rejection.

Reviewer #2: Due to the value of their observation, I think it is reasonable to accept the new version. The controls are added, which improves the confidence of the data.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[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.

ERECTILE DYSFUNCTION DRUGS ALTERED THE ACTIVITIES OF ANTIOXIDANT ENZYMES, OXIDATIVE STRESS AND THE PROTEIN EXPRESSIONS OF SOME CYTOCHROME P450 ISOZYMES INVOLVED IN THE STEROIDOGENESIS of STEROID HORMONES

PONE-D-20-20204R2

Dear Dr. Sheweita

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.

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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,

Vasu D. Appanna

Academic Editor

PLOS ONE

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