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Reviewer is satisfied by your revision and states that the quality of the manuscript has been greatly improved. Therefore, I am pleased to accept you revised manuscript for publication in PeerJ.
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
The author has dispelled all my doubts, and the quality of the manuscript has been greatly improved. I think it can be considered for acceptance.
no comment
no comment
no comment
Please address the concerns of both reviewers and revise the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
This study examines the role of histone acetyltransferase Hat1 in mouse spermatogenesis, analyzing its expression patterns and potential involvement in chromatin organization. However, the current experimental approach provides limited mechanistic insight into Hat1's specific functions during germ cell development.
Major Concerns:
Figure 1:
The analysis of Hat1 mRNA and protein expression using whole testis samples from 3-week and 8-week-old mice is meaningless. The mouse testis contains multiple cell types (Sertoli cells, Leydig cells, and germ cells at various developmental stages). To properly evaluate Hat1 expression patterns, the authors should isolate specific germ cell populations using established methods (PMID: 30061742, 33257503).
Figure 2:
The quantification of Hat1 protein intensity by immunofluorescence intensity is methodologically incorrect. During spermiogenesis, the dramatic chromatin condensation and cellular compaction will artificially increase fluorescence intensity per unit area regardless of actual protein abundance. Western blot quantification of isolated cell populations would be more appropriate. In addition, the authors could consult existing mouse spermatogenesis proteome datasets that may contain quantitative Hat1 protein expression data across different germ cell types.
Figures 3-7:
The purely computational analyses of existing datasets without experimental validation were not valuable.
To study the Hat1 protein interactions, co-immunoprecipitation (Co-IP) experiments should be performed.
To study the Hat1 function in vivo, the generation of conditional knockout mice should be performed.
No novelty and no impact on the field.
Advance Summary and Potential Significance to Field:
This study focuses on the role of Hat1 during mouse spermatogenesis. Through methods such as RT-qPCR, Western blot, immunofluorescence localization, and bioinformatics, the spatiotemporal dynamics, regulatory network, and molecular mechanisms of Hat1 in mouse spermatogenesis were investigated. It was found that Hat1 mRNA and protein are highly expressed in the testes of 8-week-old mice, with differential expression in various male germ cells. Bioinformatics analysis revealed its involvement in multiple processes of chromatin organization, playing a significant role in spermatogenesis.
From the perspective of chromatin organization, the study investigates the role of Hat1 in mouse spermatogenesis, analyzing its spatiotemporal dynamics and regulatory network to provide new insights into the mechanisms of spermatogenesis. It also uncovers a potential novel mechanism for Hat1 in the protection of sperm DNA. However, there are still several research shortcomings. Although Hat1's potential role in mouse spermatogenesis has been identified, its specific functions remain unclear, and further functional validation experiments are lacking. Additionally, the article lacks logical coherence, the figures are not aesthetically pleasing, and the expressions are unclear, all of which require careful organization and revision for improvement.
Major comments:
1. Materials and Methods: Lines 107-220 are overly verbose; please revise to make them more concise.
2. Please provide the manufacturer and catalog number for the antibodies Hat1 and Ddx4 used in this study.
3. Standardize the font and font size of all figures according to the magazine's requirements to enhance their visual appeal.
4. Figure :
（1）Figure 1: Unify the numbering of all figures in the upper left corner (as Figure 1 is currently at the bottom, which is not aesthetically pleasing), and label the protein size in Figure 1B.
（2）Figure 2: First, adjust the figure to make it more comfortable for the reader, remove the AA and BB labels, and standardize them as A, B, C~. Please include a scale bar in all figures.
（3）What is the purpose of DDX4 staining in Figure 2? As a marker gene for germ cells, if it is intended to co-stain with Hat1 to demonstrate Hat1 expression in germ cells, please merge it with Hat1-positive cells. Additionally, the DDX4-positive cells in the Leydig cells of the testis in Figure 2 do not align with previous studies, indicating that DDX4 staining was not successful. Please revalidate this.
（4）In adult testicular tissue, spermatogonia are difficult to identify. The cells labeled in Figure 2A might be Sertoli cells. The authors are advised to use the spermatogonia marker antibody LIN28 or other antibodies to co-stain with HAT1 to determine this cell type before commencing semi-quantitative statistical analysis. The y-axis in Figure 2B should be revised to "fluorescence intensity."
（5）Figure 4 is the most significant result of this paper. It can be re-summarized by cell type to make it clearer and more visually appealing.
（6）Is the expression level of all genes in Figure 5 obtained from RNA-seq data? If so, the y-axis should represent Log2FoldChange, and please also label the error bars for each gene. Please ensure the expression is standardized.
5. The description in the results section of this article is overly simplistic, whereas the materials and methods, as well as the discussion, are particularly extensive. Additionally, the discussion contains an excessive amount of descriptions that should belong to the results section. The author is requested to refine the content of both the discussion and the results sections.
6. Is Hat1 a protein specifically expressed in mouse testes? Does it have expression or significant roles in other tissues of the mouse?
7. The abstract of this article lacks coherence and is overly verbose; the author is requested to revise it.
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