PONE-D-22-06170L-Arginine Reverses Maternal and Pre-pubertal Codeine Exposure-Induced Sexual Dysfunction via Upregulation of Androgen Receptor Gene and NO/cGMP SignalingPLOS ONE

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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

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

Reviewer #1: Yes

Reviewer #2: N/A

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

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Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: Yes

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: General comments: The manuscript brings an interesting and current subject. The experimental design, although a little confusing, makes it clear that the focus is on male reproduction and the possible programmed effects in the next generations. However, as a reproductive toxicology study, many analyzes should have been performed and were not, such as analyzes of sperm quality and fertility parameters. But the most important are the histopathological and consequently immunohistochemical analyzes for some fundamental receptors, at least for the gonads. These analyzes are of great importance due to the objectives of the study itself and of course, due to the data obtained, they could detail how some changes occurred or what were the impacts of the promoted changes, such as the reduction of hormone levels.

> The authors described all the impacts of codeine on male reproduction and sperm quality and only cite a few impacts on female reproduction. Subsequently, the experimental design shows that females were treated for 8 weeks from the beginning. These females, after get pregnancy, were no longer treated. Male pups that were subdivided and treated from the 4th postnatal week onwards. I would like to understand why females are not treated during pregnancy (best window for fetal reprogramming) and why not more emphasis on the impacts of codeine and L-arginine on females reproductive systhem.

> The males were euthanized 24 hours after performing sexual behavior. This protocol directly impacted the sperm reserves of these animals and even if they all performed sexual behavior, any change found in the ejaculatory activity could impact directly on the sperm reserves of these animals, which would not mean a reduction in production or acceleration of sperm transit time, but an increase in ejaculatory activity for example. Why wasn't 15 days waiting (ideal period) for the recovery of the sperm reserves of these animals and sperm quality analyzes performed, in addition to the proposed biochemical analyzes?

> The fertility test after sexual behavior was evaluated after birth. Correct? Why did the authors not perform the euthanasia of the pregnant females to obtain all the data, such as pre and post-implantation losses and also possible malformations, since it is known that females when they give birth, discard by eating malformed or stillborn pups, losing these data.

> Why were histological analyzes of the testes and epididymis not performed, at least, since the authors discussed about fetal programming and for toxicological assays, histophatology is one of the most important assays?

>I believe that a schematic design of the entire experimental protocol adopted would be interesting to clarify the periods and what was analyzed in each generation. I suggest that the authors add a figure 01 to the material and methods, detailing the analyzes and periods performed for the entire experimental protocol (all generations used).

> Figure 05 - this figure would be much more interesting in the form of a table, with the absolute values of each parameter.

> The authors discuss epigenetics effects, however, specific parameters were not evaluated in relation to this result, both in F1 and F2 generations.

> Figure 07 - The reduction in mRNA expression for AR is another fact that reinforces the need for analysis of sperm quality and histopathology of the gonads in this present study.

Reviewer #2: Comments：

PONE-D-22-06170: L-Arginine Reverses Maternal and Pre-pubertal Codeine Exposure-Induced Sexual Dysfunction via Upregulation of Androgen Receptor Gene and NO/cGMP Signaling.

The study evaluated the effect of maternal codeine exposure and prepubertal codeine and arginine treatments on F1 male sexual function and fertility indices, as well as the outcome of F2 progenies. In addition, the epigenetic programming mechanism was also explored, which is related to decreased dopamine analysis, decreased testosterone level, and inhibition of local GR expression in testis.In addition, when the male offspring of F1 generation were mated to F2 generation, pathological changes such as shortened fetal body length, weight loss and reduced number of live fetuses were observed.And these changes were reversed when given arginine.This study is helpful to guide the healthy life of male and to guide eugenics.In my opinion, the manuscript is innovative to a certain extent and needs to be revised accordingly. Specific opinions are as follows:

1、 It is important that the authors write this discussion without discussing the reality of codeine exposure during pregnancy or adolescence, the basis for modeling and the corresponding timing and duration of administration are also very important.

2、 When explaining the mechanism of pathological changes, the author uses AR as the research target, so what is the specific signal pathway that AR participates in this project? The author did not carry out corresponding detection, should not fill?

3、 AR is expressed in a variety of testicular cells, such as Sertoli cells, Leydig cells and epithelial cells. What kind of cells does codeine mainly affect AR? Are you considering further validation in cells?

4、 The result part of this paper is too monotonous, so the result part of the manuscript should be enriched, such as adding some morphological changes of testis, sperm quality and other related indicators;

5、 Why does this paper focus on dopamine? Is it due to the biological effects of codeine? The author needs to describe the corresponding reasons for selection in the discussion.

6、There are many irregularities in writing in the manuscript:

（1） The notation is not standardized, and no specific explanation is given for each small figure

（2） For the reference section, a unified format is required

（3） In the WB results, the sensation is not produced on the same experiment. It should be replaced.

7、There are many grammatical errors in the full text, which make it difficult to read the manuscript. Please check it.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments.docx

L-Arginine Reverses Maternal and Pre-pubertal Codeine Exposure-Induced Sexual Dysfunction via Upregulation of Androgen Receptor Gene and NO/cGMP Signaling

PONE-D-22-06170R1

Dear Dr. Ajayi,

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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Christopher Torrens

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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: (No Response)

Reviewer #2: (No Response)

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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

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

Reviewer #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

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors responded to the comments and justified many points, however, I still wonder why the testis histology was not performed (it would not be impacted by the sexual behavior, only the epididymis, due to the sperm transit time). In addition, I also suggest changing how statistical differences are expressed. In the tables and figures, the letters are showing the statistical difference comparing to a specific group (A for control group, B for X group...) leaving everything very polluted and confusing. I strongly advise authors to use letters to express statistical differences between groups (A, B if different from A, AB if similar to A and B...). It's cleaner and easier to understand. On the other hand, in the table where there was no statistical difference, there is no need to put the specifications below (table 03)

Reviewer #2: L-Arginine Reverses Maternal and Pre-pubertal Codeine Exposure-Induced Sexual Dysfunction via Upregulation of Androgen Receptor Gene and NO/cGMP Signaling, the article is special work, can be accepted

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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: Yes: no

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