Sperm Phosphoproteome: Unraveling Male Infertility

Round 1

Reviewer 1 Report

Respected editor,

I would like to thank you for inviting me to examine the review article titled as ‘Sperm phosphoproteome: unraveling male infertility’. In this paper, the authors discussed about the usefulness of phosphoproteomics in sperm that aid in understanding of the main causes or molecular mechanisms of male infertility. Here they have covered several of previously published articles. My vote is to accept the article after some minor modifications that are needed in the text and figures. I have stated my comments and concerns below.

1. Page 1, simple summary: In the last part, the author should discuss that the phosphoproteomics data should be supported by some other assay with spermatozoa which will aid us to explain the molecular details. They may suggest something to increase the sensitivity of this assay here as well as in discussion (Last section).
2. This may be something to be done by the publishing editor of this journal, but this article has a lot of abbreviations and there should be a list of them with their full forms at the beginning of the paper.
3. Page 2, 2^nd paragraph, line 56-57: Non-coding RNA may play a role in gene silencing or heterochromatinization. The authors should discuss this here.
4. Page 2, 2^nd paragraph, line 58-59: ‘Thus, post-translational modifications (PTM) of their already present proteins’. Authors should rephrase the sentence to improve the language of this section.
5. Page 3, section 2, line 110-118: The statements with Topoisomerase 2A need to be rephrased to make the clear. The specific role Topo2A in this scenario is not clear to me.
6. Page 4, Line 154: ‘Protein misfolding’ should be ‘misfolded protein’.
7. Page 4, line 183-last paragraph: The authors should reframe the language.
8. Figure 1 B: The heading should be like ‘Cell lysis and protein extraction’.
9. Page 9, line 372: ‘Wip1 seems an important regulator….’ should be ‘Wip1 seems to be an important regulator…’.
10. Page 9, line 405-407: Authors should reframe the sentence ‘Based on these…. men and animals’.
11. Page 11, figure 2: Authors can use different colors of lines to represent the strength of data support.
12. Page 11, line 455-456: Authors should modify the sentence to-‘ In fact, human sperm subpopulations with low and high motility statistically differ in up to 119 sperm phosphoproteins’.
13. Page 12: Authors should replace the terms ‘up phosphorylation and down phosphorylation’ to ‘hyperphosphorylation and hypophosphorylation’.
14. When the authors discuss about some phosphorylations being associated or linked with the phenotypes of low motility or defective morphology etc, they should suggest whether they are the actual or direct cause of the phenotypes or the result of some other upstream regulations.
15. The sections of 4, 5 and 6 should be highlight of this review and should be more detailed than the previous sections. The earlier sections can be concise if there is a word limit for this paper format.

Author Response

Thank you very much for your suggestion and critical review of our manuscript.

1. Page 1, simple summary: In the last part, the author should discuss that the phosphoproteomics data should be supported by some other assay with spermatozoa which will aid us to explain the molecular details. They may suggest something to increase the sensitivity of this assay here as well as in discussion (Last section). A simple summary has been added following the suggestion of de reviewer.
2. This may be something to be done by the publishing editor of this journal, but this article has a lot of abbreviations and there should be a list of them with their full forms at the beginning of the paper. A table of abbreviations has been prepared in case the editor considers their inclusion necessary.
3. Page 2, 2^nd paragraph, line 56-57: Non-coding RNA may play a role in gene silencing or heterochromatinization. The authors should discuss this here. A discussion about non-coding RNA has been included.
4. Page 2, 2^nd paragraph, line 58-59: ‘Thus, post-translational modifications (PTM) of their already present proteins’. Authors should rephrase the sentence to improve the language of this section. As suggested by the reviewer the sentence has been rephrased.
5. Page 3, section 2, line 110-118: The statements with Topoisomerase 2A need to be rephrased to make the clear. The specific role Topo2A in this scenario is not clear to me. As suggested by the reviewer we have clarified the specific role of Topoisomerase 2A.
6. Page 4, Line 154: ‘Protein misfolding’ should be ‘misfolded protein’. The change suggested by the reviewer has been made.
7. Page 4, line 183-last paragraph: The authors should reframe the language. The change suggested by the reviewer has been made.
8. Figure 1 B: The heading should be like ‘Cell lysis and protein extraction’. The change suggested by the reviewer has been made.
9. Page 9, line 372: ‘Wip1 seems an important regulator….’ should be ‘Wip1 seems to be an important regulator…’. The change suggested by the reviewer has been made.
10. Page 9, line 405-407: Authors should reframe the sentence ‘Based on these…. men and animals’. As suggested by the reviewer the sentence has been reframe.
11. Page 11, figure 2: Authors can use different colors of lines to represent the strength of data support. We have added different colors in figure 2 as suggested by the reviewer.
12. Page 11, line 455-456: Authors should modify the sentence to-‘ In fact, human sperm subpopulations with low and high motility statistically differ in up to 119 sperm phosphoproteins’. Sentence has been modified.
13. Page 12: Authors should replace the terms ‘up phosphorylation and down phosphorylation’ to ‘hyperphosphorylation and hypophosphorylation’. As suggested by the reviewer both terms have been changed throughout the full manuscript.
14. When the authors discuss about some phosphorylations being associated or linked with the phenotypes of low motility or defective morphology etc, they should suggest whether they are the actual or direct cause of the phenotypes or the result of some other upstream regulations.  The reviewer's suggestion is very interesting, however, at present, it is unclear whether some phosphorylations are the actual or direct cause of low motility phenotypes or defective morphology, or whether they are the result of some other prior regulation. . Therefore, both possibilities can be possible and one cannot be ruled out by the other.
15. The sections of 4, 5 and 6 should be highlight of this review and should be more detailed than the previous sections. The earlier sections can be concise if there is a word limit for this paper format. As the reviewer has suggested, sections 1, 2 and 3 have been summarized.

Reviewer 2 Report

Since transcriptional and translational activities are nearly silent in spermatozoa, the post-translational modifications are essential for spermatozoa to acquire functionality. This manuscript emphasized the roles of phosphorylation in sperm fertilization and detailed the application of the phosphoproteomic technique in unraveling male infertility.

Major:

1. The authors may consider adding a table to summarize the use of phosphoproteomics in spermatozoa biological processes;

Minor:

1. Line 119: please check the typo in this line.
2. Line 230-233: …the most widely studied post-translational modification of sperm proteins…?
3. Line 393: please check the typo in this line.
4. The author may consider providing more detail for figure 2: how and why to generate this figure.
5. Line 534: please check the typo in this line.

Author Response

Thank you very much for your suggestion and critical review of our manuscript.

Major:

1. The authors may consider adding a table to summarize the use of phosphoproteomics in spermatozoa biological processes. As suggested by the reviewer we have added a table summarizing the use of phosphoproteomics in human spermatozoa biological processes in different phosphoproteomics studies (Tanle 1).

Minor:

1. Line 119: please check the typo in this line. As indicated by the reviewer the typographical error has been corrected.
2. Line 230-233: …the most widely studied post-translational modification of sperm proteins…? As suggested by the reviewer the sentence has been rephrased.
3. Line 393: please check the typo in this line. As indicated by the reviewer the typographical error has been corrected.
4. The author may consider providing more detail for figure 2: how and why to generate this figure. As suggested by reviewer, a more extensive description of figure 2 has been included in the figure legend and in the text.
5. Line 534: please check the typo in this line. As indicated by the reviewer the typographical error has been corrected.