Effect of C-Type Natriuretic Peptide (CNP) on Spermatozoa Maturation in Adult Rat Epididymis

Round 1

Reviewer 1 Report

The authors in this manuscript showed that CNP/NPR-B is extremely highly expressed in male genital ducts (seminal vesicle, prostate and epididymis). CNP/NPR-B has mainly expressed in the caput of epididymis, and their expression were 30 regulated by testosterone. Over the manuscript is well designed and written, however, several grammar errors and typos need to be corrected before publication.

Author Response

Thanks for your kind advices.  We have carefully revised the article and corrected grammar and calligraphy errors and typos, such as italics.

Reviewer 2 Report

The author's research design is good, but the degree of completion is poor, including the writing and drawing of the paper. Many experimental details need to be improved. Secondly, there are many basic problems in the paper, which should not appear.

Comment 1, line 15: In the abstract, it is suggested to delete "objective, method, results and conclusion" unless required by the journal.

Comment 2, line 16,50,102: Please write the full name of "NPR-B" for the first time. To unify the full name of cGMP, after its first appearance, all abbreviations are used.

Comment 3, line 21: Gene in italics, it is recommended to check the full text.

Comment 4: It is recommended to rewrite the abstract. The research background is introduced in a short sentence, and then the research purpose is presented. The conclusion is not a repetition of the results, but what the results tell us.

Comment 5, line 56,59: The reference number is placed after the author.

Comment 6, line 58,60: Use English symbols, please double check the full text format.

Comment 7, line 72: It is suggested to add the introduction. In addition to introducing CNP and its research progress, the necessity of research should also be explained. What is the relationship between CNP and NPR-B? What is the significance of the research?

Comment 8, line 83: The tissues here can be divided into different parts, such as the brain, including the cerebellum, thalamus and pituitary gland, and the epididymis, including the caput, corpus and cauda. Which part is used to extract RNA? Is it representative?

Comment 9, lines 90: 2−ΔΔCT should be used.

Comment 10, lines 94: Primer information table lacks serial number and annealing temperature, it is suggested to supplement and improve.

Comment 11, lines 98: Why did you choose rats at this stage?

Comment 12, lines 103: Were CNP and cGMP proteins detected with the cGMP kit?

Comment 13, lines 120: There are Spaces between numbers and units.

Comment 14, lines 135: Is it immunohistochemistry or immunofluorescence?

Comment 15, lines 142: Is the injection dose any definite?

Comment 16, lines 148: Were the experiments on rats or mice?

Comment 17, lines 149: What is the formula of the medium? How to exclude the factors of medium that cause sperm activity to decline?

Comment 18, lines 158: Are the doses of these inhibitors any definite? Are tests done?

Comment 19, lines 165: What methods were used for statistical analysis? Please explain in the text.

Comment 20, lines 175: Please note the differences in the diagram.

Comment 21, lines 176,183: Double check the full text format, including the case of the letters.

Comment 22, line 185: In “P<0.01”, “P<0.05” and so on, “P” is italicized.

Comment 23, lines 188: The content in the picture is not clear at all. It is suggested to make a new picture.

Comments for author File: Comments.docx

Author Response

Reviewer 2: Thanks for your carefully comments. Here is our reply one by one:

Comment 1, line 15: In the abstract, it is suggested to delete "objective, method, results and conclusion" unless required by the journal.

Answer: Thanks for your suggestion. We have deleted "objective, method, results and conclusion"

Comment 2, line 16,50,102: Please write the full name of "NPR-B" for the first time. To unify the full name of cGMP, after its first appearance, all abbreviations are used.

Answer: The full name of "NPR-B" has been added.

Comment 3, line 21: Gene in italics, it is recommended to check the full text.

Answer: Thanks for your suggestion. We have revised the errors and check the full text.

Comment 4: It is recommended to rewrite the abstract. The research background is introduced in a short sentence, and then the research purpose is presented. The conclusion is not a repetition of the results, but what the results tell us.

Answer: according to your suggestion, a short sentence has added to introduced the research background, and the conclusion were revised.

Comment 5, line 56,59: The reference number is placed after the author.

Answer:  The reference number is replaced.

Comment 6, line 58,60: Use English symbols, please double check the full text format.

Answer: Thanks for your suggestion. We have check text format carefully

Comment 7, line 72: It is suggested to add the introduction. In addition to introducing CNP and its research progress, the necessity of research should also be explained. What is the relationship between CNP and NPR-B? What is the significance of the research?

Answer: Thanks for your suggestion. We added some sentences about the necessity of research.

Comment 8, line 83: The tissues here can be divided into different parts, such as the brain, including the cerebellum, thalamus and pituitary gland, and the epididymis, including the caput, corpus and cauda. Which part is used to extract RNA? Is it representative?

Answer: Thanks for your suggestion. We just take the surface of brain (cerebellum) to test. And we will pay more attention about it.

Comment 9, lines 90: 2−ΔΔCT should be used.

Answer: Thanks for your suggestion. 2−ΔΔCT is used.

Comment 10, lines 94: Primer information table lacks serial number and annealing temperature, it is suggested to supplement and improve.

Answer: Thanks for your suggestion. We have added serial number and annealing temperature.

Comment 11, lines 98: Why did you choose rats at this stage?

Answer: We refer to other literatures on castration experiment. At this time, mice are in the early stage of sexual maturity. Because the rats at this stage are in the sexual maturity.

Comment 12, lines 103: Were CNP and cGMP proteins detected with the cGMP kit?

Answer: No, we miss the CNP kit and revise the error.

Comment 13, lines 120: There are Spaces between numbers and units.

Answer: Thanks for your carefully review. We have deleted the spaces.

Comment 14, lines 135: Is it immunohistochemistry or immunofluorescence?

Answer: Thanks for your carefully review. It is immunofluorescence and has been revised.

Comment 15, lines 142: Is the injection dose any definite?

Answer: We refer to other literatures on castration experiment and the results showed it is effective.

Comment 16, lines 148: Were the experiments on rats or mice?

Answer: the experiments were performed on rats. We revised the errors.

Comment 17, lines 149: What is the formula of the medium? How to exclude the factors of medium that cause sperm activity to decline?

Answer: We used the HFT as sperm medium, which is used for sperm.

Comment 18, lines 158: Are the doses of these inhibitors any definite? Are tests done?

Answer: the doses of these inhibitors were used in our previous study [reference 14,15] and proved to be effective.

Comment 19, lines 165: What methods were used for statistical analysis? Please explain in the text.

Answer: Thanks for your suggestion. We added the methods used statistical analysis.

Comment 20, lines 175: Please note the differences in the diagram.

Answer: Thanks for your suggestion. We have noted the differences in the diagram. There are some diagrams (such as difference in different organ or different days) without differences because there is no control group and it is hard to compare.

Comment 21, lines 176,183: Double check the full text format, including the case of the letters.

Answer: Thanks for your suggestion. We have checked throughout the article carefully

Comment 22, line 185: In “P<0.01”, “P<0.05” and so on, “P” is italicized.

Answer: Thanks for your suggestion. We have revised it.

Comment 23, lines 188: The content in the picture is not clear at all. It is suggested to make a new picture.

Answer: Thanks for your suggestion. We have revised the picture to make it clear.

Reviewer 3 Report

The present investigation talks about “the expression level of CNP/NPR-B in various rat tissues and its location in the epididymis and the effect of CNP on sperm motility and maturation to expose the important role of CNP in the process epididymal sperm maturation" This research provides interesting information, however, it is necessary to make some changes before its final publication. 

INTRODUCTION

Line 41. change the word “transport ion”

Line 47. Delete space “Furthermore,   genetic”

MATERIAL AND METHODS

General comments: I suggest restructuring this section and tidying it up. I mean, mention the experimental design, for example, mention how the groups were divided, etc...

 Specific comments:

Line 75.- In the "Animals" section, I suggest adding the number of animals, weight, age at slaughter of the males, as well as the breed to which they belonged.

Line 77.- Add space “and12 h”

Line 78-80.- Add reference “They were treated according to the guidelines of the Animal Research Committee and approved by the Centre of Experimental Animals of Huazhong University of Science and Technology.”

Line 84.- Delete space “10-12w   male”.

Line 91.- Delete space “Table 1.   The”

Line 137.- I suggest changing this section "Establishment of castrated rat model" after the section "Animals".

Line 147.- This section “Mobility and maturation of epididymal sperm treated with CNP in vitro.” Mencionar el procesamiento del semen, al momento de extraer el semen, que diluyente, a que temperatura, etc. Also, what scale did you use to measure sperm motility? Also, I suggest changing "Mobility" to "Motility".

Line 148.- Reference under which guide the animals were slaughtered. “The mice were killed by neck dislocation, and the epididymis was separated.”

Line 168.- In the section "Statistical analysis" I mentioned what tests were used to determine the normality of the data and homogeneity of variances. Also, what statistical tests did you use for each variable?

RESULTS

Specific comments:

Line 176.- Figure 1. Add to the figure the statistical differences in the figure. As well as indicate the level of significance in the figure.

Line 183.- Change the literal “b” for “B” in the sentence “(Fig2. A and b).”

Line 189.- Figure 2 the figures "C, D, E, F" are very small, the content of the figures is not appreciated. I recommend increasing the size of literals and figures.

Line 203.- Figure 3 highlight in the figure where the significant differences are found.

Line 206.- Homogenize the word "mobility" by "motility".

Line 217.- Figure 4 increase the size of the figures and literals.

Line 224.- This phrase “After incubated with CNP 10 * (- 6) nmol/L for 6 hours, the gene expressions of sperm maturation related factors were detected.” It must go in materials and methods.

DISCUSSION

General comments:

I recommend that the discussion be in agreement as the results were reported. I mean, Expression of CNP/NPR-B in rat different tissues and in epididymis at different time after birth, Expression of CNP/NPR-B in different segments of rat epididymis, The expression of CNP, NPR-B in castrated mice model, The effect of CNP on mobility of the epididymis sperm in vitro and The effect of CNP on maturation related factors of the epididymis sperm in vitro.

Specific comments:

Line 299.- increase more on the role of testosterone and CNP/NPR-B expression

Line 333. I suggest that Figure 5 if the model is in mice use an image of mice and not humans.

CONCLUSIONS

I recommend changing the word "In summary" to "Conclusion" as mentioned in the Abstract. In addition to mentioning more about its possible implications and importance of this research.

Author Response

Review 3

 Thanks for your carefully comments. Here is our reply one by one:

INTRODUCTION

Line 41. change the word “transport ion”

Answer: Thanks for your suggestion. We have deleted the word.

Line 47. Delete space “Furthermore,   genetic”

Answer: Thanks for your suggestion. We have deleted the space.

MATERIAL AND METHODS

General comments: I suggest restructuring this section and tidying it up. I mean, mention the experimental design, for example, mention how the groups were divided, etc...

 Specific comments:

Line 75.- In the "Animals" section, I suggest adding the number of animals, weight, age at slaughter of the males, as well as the breed to which they belonged.

Answer: Thanks for your suggestion. We have added the weight and age in the animal section. The number and breed have been provided in the section “Establishment of castrated rat model”

Line 77.- Add space “and12 h”

Answer: Thanks for your suggestion. We have added space “and12 h”

Line 78-80.- Add reference “They were treated according to the guidelines of the Animal Research Committee and approved by the Centre of Experimental Animals of Huazhong University of Science and Technology.”

Answer: Thanks for your suggestion. We have added reference

Line 84.- Delete space “10-12w   male”.

Answer: Thanks for your suggestion. We have deleted the space.

Line 91.- Delete space “Table 1.   The”

Answer: Thanks for your suggestion. We have deleted the space.

Line 137.- I suggest changing this section "Establishment of castrated rat model" after the section "Animals".

Answer: Thanks for your suggestion. We have put this section "Establishment of castrated rat model" after the section "Animals".

Line 147.- This section “Mobility and maturation of epididymal sperm treated with CNP in vitro.” Mencionar el procesamiento del semen, al momento de extraer el semen, que diluyente, a que temperatura, etc. Also, what scale did you use to measure sperm motility? Also, I suggest changing "Mobility" to "Motility".

Answer: Thanks for your suggestion. We have added the some details (such as temperature, medium) in this part and changing "Mobility" to "Motility".

Line 148.- Reference under which guide the animals were slaughtered. “The mice were killed by neck dislocation, and the epididymis was separated.”

Answer: Thanks for your suggestion. We have added reference.

Line 168.- In the section "Statistical analysis" I mentioned what tests were used to determine the normality of the data and homogeneity of variances. Also, what statistical tests did you use for each variable?

Answer: Thanks for your suggestion. We have added the methods of statistical analysis.

RESULTS

Specific comments:

Line 176.- Figure 1. Add to the figure the statistical differences in the figure. As well as indicate the level of significance in the figure.

Answer: Thanks for your suggestion. It is hard to make differences because there is no control group. Moreover, we just want to show the trends in this part and hope it is OK.

Line 183.- Change the literal “b” for “B” in the sentence “(Fig2. A and b).”

Answer: Thanks for your careful review. We have changed it.

Line 189.- Figure 2 the figures "C, D, E, F" are very small, the content of the figures is not appreciated. I recommend increasing the size of literals and figures.

Answer: Thanks for your careful review. We have revised the picture 2.

Line 203.- Figure 3 highlight in the figure where the significant differences are found.

Answer: Thanks for your suggestion. In this part, we just want to show the trends as well as picture 1 and hope it is OK.

Line 206.- Homogenize the word "mobility" by "motility".

Answer: Thanks for your careful review. We use motility" and changed the word "mobility"

Line 217.- Figure 4 increase the size of the figures and literals.

Answer: Thanks for your suggestion. We have revised the picture 4.

Line 224.- This phrase “After incubated with CNP 10 * (- 6) nmol/L for 6 hours, the gene expressions of sperm maturation related factors were detected.” It must go in materials and methods.

Answer: Thanks for your suggestion. We have put the phrase in materials and methods.

DISCUSSION

General comments:

I recommend that the discussion be in agreement as the results were reported. I mean, Expression of CNP/NPR-B in rat different tissues and in epididymis at different time after birth, Expression of CNP/NPR-B in different segments of rat epididymis, The expression of CNP, NPR-B in castrated mice model, The effect of CNP on mobility of the epididymis sperm in vitro and The effect of CNP on maturation related factors of the epididymis sperm in vitro.

Specific comments:

Line 299.- increase more on the role of testosterone and CNP/NPR-B expression.

Answer: Thanks for your suggestion. We added two literates about the he role of testosterone and CNP/NPR-B expression.

Line 333. I suggest that Figure 5 if the model is in mice use an image of mice and not humans.

CONCLUSIONS

 Answer: Thanks for your suggestion. We have changed the image of human to image of mice in picture 5.

I recommend changing the word "In summary" to "Conclusion" as mentioned in the Abstract. In addition to mentioning more about its possible implications and importance of this research.

  Answer: Thanks for your suggestion. We have changed the word "In summary" to "Conclusion" and added its possible implications and importance of this research.

Reviewer 4 Report

The manuscript entitled “Effect of C-type Natriuretic peptide (CNP) on spermatozoa maturation in adult rat epididymis.” summarized the CNP/NPR-B were detected in various tissues of rats and epididymis at different stages after birth.  Generally, the manuscript is well written. This paper has several weaknesses and needs improvement before publication.

This manuscript has major language problems. There are too many for me to modify them all. Authors are strongly encouraged to seek a native English speaker who may assist you modifying the document.

Comments:

1.       Why did the authors choose this technology instead of other GWAS? Please provide the reason.

2.       What cell type was utilized for RNA extraction

3.     How much RNA was used in PCR? Was it one-step or 2-step PCR?

4.     The abstract is not particularly informative and would benefit from more background.

5.     Summarize the abstract, focus on the main findings and mention the small conclusion in at the end of abstract

6.     In the Introduction focus on the objectives and insert a few new reference and relevant findings

7.     In material and method sections, references are missing.

8.     Most of the references mentioned are old and I suggest adding recent references, and the manuscript should be edited accordingly.

9.     I suggest the cite following paper in introduction part For more information you can read below reference

RNA-Seq reveals the potential molecular mechanisms of bovine KLF6 gene in the regulation of adipogenesis. International Journal of Biological Macromolecules, 195, 198-206.

10.  Material and method needs to clarifying and summarizing- some detailed needs

11.  The subtitles in the material and method needs to summarizing Ethical approval and references must be mentioned in M&M

12.  In the result section, the results were written very poorly. It should be written again and try to avoid a brief introduction in the starting of every result.

In conclusion, the research presented is interesting, well planned and carried out. The manuscript can still be improve revise by a native English speaker. Nevertheless, I believe that this work deserves publication in after the inclusion of corrections.

Author Response

Reviewer 4: Thanks for your carefully comments. Here is our reply one by one: 

Comments:

1. Why did the authors choose this technology instead of other GWAS? Please provide the reason.

Answer: Thanks for your comment. There are many experiments in our study. Do you mean the rat model? We will choose  GWAS in our future study according to your suggestion.

2. What cell type was utilized for RNA extraction

Answer: Thanks for your comment. We use different tissues or different part of epididymis for RNA extraction.

3. How much RNA was used in PCR? Was it one-step or 2-step PCR?

Answer: 1 µg total RNA was used in PCR and it was 2-step PCR?

4. The abstract is not particularly informative and would benefit from more background.

 Answer: Thanks for your suggestion. We have improved our abstract.

5. Summarize the abstract, focus on the main findings and mention the small conclusion in at the end of abstract

Answer: Thanks for your suggestion. We have added the small conclusion in at the end of abstract.

6. In the Introduction focus on the objectives and insert a few new reference and relevant findings

 Answer: Thanks for your suggestion. We have inserted a few new reference and relevant findings.

7. In material and method sections, references are missing.

 Answer: Thanks for your suggestion. We have provided the missing references.

8. Most of the references mentioned are old and I suggest adding recent references, and the manuscript should be edited accordingly.

 Answer: Thanks for your suggestion. We have added the some recent references.

9. I suggest the cite following paper in introduction part For more information you can read below reference

RNA-Seq reveals the potential molecular mechanisms of bovine KLF6 gene in the regulation of adipogenesis. International Journal of Biological Macromolecules, 195, 198-206.

 Answer: Thanks for your suggestion. We added reference “Yang L, Lei L, Zhao Q, Gong Y, Guan G, Huang S.C-Type Natriuretic Peptide/Natriuretic Peptide Receptor 2 Is Involved in Cell Proliferation and Testosterone Production in Mouse Leydig Cells. World J Mens Health. 2019 May;37(2):186-198.”

10. Material and method needs to clarifying and summarizing- some detailed needs

 Answer: Thanks for your suggestion. We have improved the part of “Material and method”

11. The subtitles in the material and method needs to summarizing Ethical approval and references must be mentioned in M&M

 Answer: Thanks for your suggestion. We have added the Ethical approval and references.

12. In the result section, the results were written very poorly. It should be written again and try to avoid a brief introduction in the starting of every result.

Answer: Thanks for your suggestion. We revised our result section.

Round 2

Reviewer 2 Report

The author's reply has been received. It can be seen that the author has improved the article, but some problems still exist and there are still many details. For example: (1) Line 1 does not need a period, and no author information is seen 5. (2) The gene in the picture is not italicized. (3) "P" in line 160 is still unitalicized. (4) The icon format is not unified. (5) The text in the picture is too small to affect reading, and why is there no significant difference marked in Figure 1? Why is half the font missing in Figure 4A? (6) Double parentheses appear on line 222. These details need to be carefully corrected by the author.

Author Response

Dear reviewer:

Thanks a lot for your comments, we have revised our article and polished our English expression carefully from beginning to end.

(1) Line 1 does not need a period, and no author information is seen 5.

Answer: Thanks for your suggestion. We have revised the errors.

(2) The gene in the picture is not italicized.

Answer: Thanks for your suggestion. The gene in the picture is italicized as your advice.

 (3) "P" in line 160 is still unitalicized.

Answer: Thanks for your suggestion. We have revised the error and "P" in line 160 is italicized.

 (4) The icon format is not unified.

Answer: Thanks for your suggestion. We have revised the icon format. I hope to fit your request.

(5) The text in the picture is too small to affect reading, and why is there no significant difference marked in Figure 1? Why is half the font missing in Figure 4A?

Answer: Thanks for your suggestion. We have revised our picture (such as Figure 4) and enlarge some text in the figure. We don’t mark the significant in Figure 1, because there is no control group and it is hard to compare. Moreover, we just want to express the trends.

(6) Double parentheses appear on line 222. These details need to be carefully corrected by the author.

Answer: Thanks for your comment. We have deleted excessive parentheses.

Reviewer 4 Report

I appreciate your efforts to respond to my review comments.

Author Response

Dear editor:

Thanks for your comments. We have modified our article carefully.

Round 3

Reviewer 2 Report

I have received the reply from the author, who is advised to read the first two review comments carefully. (1) The first line "Article" does not need a period, but does not need a period after the title. (2) There is no "5" in author information, and "3" needs to be lowercase. It is suggested to further improve the details. (3) The illustrations are not clear enough and the font is fuzzy, which affects reading. Suggest beautifying the picture or redrawing it. (4) The "P" in line 160 is still not italicized.

Author Response

Dear reviewer:

Thanks a lot for your careful comments. We have used the “Track Changes” function, so that any changes can be easily viewed.

(1) The first line "Article" does not need a period, but does not need a period after the title.

Answer: Thanks for your suggestion. We have deleted the period after “article” and title.

(2) There is no "5" in author information, and "3" needs to be lowercase. It is suggested to further improve the details.

Answer: Thanks for your suggestion. We have revised “5” to “3”

 (3) The illustrations are not clear enough and the font is fuzzy, which affects reading. Suggest beautifying the picture or redrawing it.

Answer: Thanks for your suggestion. We have revised our picture and hope to OK

(4) The "P" in line 160 is still not italicized.

Answer: Thanks for your careful comment. . The "P" in line 160 is italicized.

Author Response File: Author Response.pdf

Round 4

Reviewer 2 Report

• We have received the reply from the author, and we can see that the author has made a lot of improvements.