Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel
, Qiu Yan 1,2, Jie Wang 1,2, Yong Zhang 1,2,3,* and Xingxu Zhao 1,2,3,*
Round 1
Reviewer 1 Report
Comments to the Authors of manuscript number: cimb-1826551 entitled “Cloning, Molecular Characterization and Expression Patterns of HSL in HPG axis at different ages testicle tissues of the Bactrian camels”.
The study seems to be interesting, but there is many gaps, which should be clarified. Authors have to inform how many animals were used in the study. N should be given for each age.
1. L 16 – the full name of the axis should be given
2. L 18 – font
3. L 20 – testis are gonads and belong to the HPG axis
4. L 20-21 – what is 2W and 2Y?
5. L 22, 27, 33, 176– the space. The whole text should be checked
6. L 26 – unclear
7. L 28-29 – it has no sense – 2W higher then 2Y, but both are two-week-old
8. L 31 – which exactly?
9. L 40, 44- small font, the text should be checked
10. L 52 – the abbreviation should be explained, it does not matter that in the abstract it is introduced
11. L 55 – fatty acids?
12. L 55-57 – this sentence is unclear
13. L 80-81 – the description of animals in abstract differs from that
14. L 81- Was the tissue collected only from 6-year-old animals? What about others? How many males were in each group? It should be presented.
15. L 146 – the whole HPG?
16. L 147 – before embedding into paraffin there is other actions
17. L 148- Was each slice given on the slide? Or there were made breaks?
18. L 149-150- completely unclear
19. L 149 and L 151 – what and when? There is a chaos.
20. L 153-157 – two nights?
21. Figure 7 – the full description is needed. In this form is unclear e.g. it is unknown if Authors include known interactions, predicted or others like textmining. The name of all nodes should be given. And the date when it was made. What biological processes or effects it presents?
22. Data from the Western blots appears poor, bands are rather diffuse, maybe an image of an entire SDS PAGE gel might have been useful to demonstrate how good the electrophoresis run was, maybe in combination with a zymogram
Author Response
Dear editor：
On behalf of my co-authors, I thank editor and reviewer of “current issues in molecular Biology” very much for giving us a precious opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, Manuscript ID: cimb-1826551”. We have studied reviewer’s comments carefully and have made revision which marked in yellow in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to submit the enclosed manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, which we wish to be considered for publication in “current issues in molecular Biology”. No conflict of interest exits in the submission of this manuscript. We would like to express our great appreciation to you for comments on our paper. Looking forward to hearing from you.
Point 1: L 16–the full name of the axis should be given
Response 1: Thank you for the reviewer’s suggestion, we have revised full name of the axis in manuscript.
Point 2: L 18 – font
Response 2: Thank you for the reviewer’s suggestion we have revised the font in manuscript.
Point 3:L 20–testis are gonads and belong to the HPG axis
Response 3: Thank you for the valuable comments of the reviewers in the manuscript, testis already present in the HPG axis.
Point 4: L 20-21 – what is 2W and 2Y?
Response 4: Thank you for the reviewer’s question, the 2W is represent two-week-old and 2Y is represent two-year-old, which are presented in the Methods and Materials (2.1 Animals and Sample Collection) and have been marked in yellow in the manuscript.
Point 5:L 22, 27, 33, 176– the space. The whole text should be checked.
Response 5: We are very sorry for our negligence of the space in manuscript, we have revised in manuscript.
Point 6:L 26 – unclear
Response 6: We are very sorry for our negligence of writing in the manuscript, musculus should be Mus musculus. Correct writing is “Based on bioinformatics analysis it can be found that the nucleotide and amino acid sequence of bactrian camels, HSL are most similar to Camelus pacos and Camelus Dromedarius, with the lowest sequence similarity to the Mus musculus”. We have revised and marked in yellow in the manuscript.
Point 7:L 28-29–it has no sense–2W higher then 2Y, but both are two-week-old
Response 7: We are very sorry for our incorrect writing in the manuscript, we have revised the manuscript and have been marked in yellow in the manuscript.
Point 8: L 31 – which exactly?
Response 8: thank you for the reviewer question, Immunofluorescence results indicated that HSL protein is not specifically distributed in a specific tissue, it is positive in each tissue (hypothalamus, pituitary, pineal, testis and epididymis), however, the positive level is different. the HSL protein was localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camels testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, and partially expressed in hypothalamic glial cells, pituitary suspensory cells and pineal cells.
Point 9:L 40, 44- small font, the text should be checked
Response 9: We are very sorry for our incorrect writing in the manuscript, we have revised the manuscript and have been marked in yellow in the manuscript.
Point 10:L 52–the abbreviation should be explained, it does not matter that in the abstract it is introduced
Response 10: We are very sorry for our negligence of writing in the manuscript, we have revised the abbreviation in manuscript and have been marked in yellow.
Point 11:L 55–fatty acids?
Response 11: thank you for your comment, bactrian camel adipose tissue contains large amounts of saturated and unsaturated fatty acids.
Point 12:L 55-57–this sentence is unclear
Response 12: Thank you for the valuable comments of the reviewers, the correct writing is “Hormone-sensitive lipase (HSL) is a rate-limiting enzyme of lipolysis, which plays a decisive role in the synthesis and metabolism of fat. It can hydrolyze triglyceride to di-glyceride”, and we have revised and marked in yellow in this manuscript.
Point 13: L 80-81–the description of animals in abstract differs from that
Response 13: Thank you for the valuable comments of the reviewers, we have revised the description of animals in abstract of manuscript.
Point 14:L 81- Was the tissue collected only from 6-year-old animals? What about others? How many males were in each group? It should be presented.
Response 14: Thank you for your comment, the HPG axis tissues are taken only from 6-year-old adult (male, n=3) bactrian camels and testis tissues at different stages of development, namely two-week-old juvenile (2W, n=3), two-year-old pre-sexual maturity (2Y, n=3), four-year-old somatic maturity (4Y, n=3) and six-year-old adulthood (6 Y, n=3), we have revised and marked in yellow in manuscript.
Point 15:L 146–the whole HPG?
Response 15: Thank you for your comment, only adult 6-year-old camel, and we have completed and marked in yellow in the manuscript.
Point 16:L 147–before embedding into paraffin there is other actions
Response 16:We apologize for our oversight on the manuscript, it should be trimmed, dehydrated, and transparent before it can be dipped in wax and embedded. We have completed and marked in yellow in the manuscript.
Point 17: L 148- Was each slice given on the slide? Or there were made breaks?
Response 17:Thank you for your comment, it should be spread out in hot water, then select the intact tissue to attach to the slide.We have completed and marked in yellow in the manuscript.
Point 18:L 149-150- completely unclear
Response 18:I'm sorry for not stating it clearly in the manuscript. It should be dewaxed with xylene, passed through ethanol gradient, and stained with hematoxylin and eosin after entering water.We have completed and marked in yellow in the manuscript.
Point 19:L 149 and L 151 – what and when? There is a chaos.
Response 19:I'm sorry for not stating it clearly in the manuscript.It should be that, after routine dewaxing and hydration, the sections were heated with microwave to repair the section antigens. We have completed and marked in yellow in the manuscript.
Point 20:L 153-157 – two nights?
Response 20: thank you for your comments, two nights is presentient each primary antibody (1st and 2st) needs 4â—¦C overnight incubations.
Point 21:Figure 7 – the full description is needed. In this form is unclear e.g. it is unknown if Authors include known interactions, predicted or others like textmining. The name of all nodes should be given. And the date when it was made. What biological processes or effects it presents?
Response 21:Thanks for the reviewer's suggestion. I have analyzed protein interactions and modified the main related functions and biological processes of proteins in the picture(eg, Testosterone biosynthetic process, Steroid hormone biosynthetic processd, Steroid metabolic process, Cholesterol metabolic process, Primary alcohol metabolic process, Response to steroid hormone and Lipid metabolic process ) and Molecular Function(eg, Cholestenone5-alpha-reductase activity, Insulin-like growth factor receptor binding and Cholesterol transfer activity.Please see figure 7.
Point 22: Data from the Western blots appears poor, bands are rather diffuse, maybe an image of an entire SDS PAGE gel might have been useful to demonstrate how good the electrophoresis run was, maybe in combination with a zymogram
Response 22:Thank the reviewers for their comments. Indeed, the image of Western blots in the article is not very beautiful, but we have tried to repeat the experiment. However, since the species we do is bactrian camel, there is almost no antibody suitable for this species, and it is difficult to make it. Therefore, we have to choose the primary antibodies from other species for the detection of relative expression. Therefore, the images seem to be somewhat dispersed, and the reviewers are also asked to understand.
Reviewer 2 Report
cimb-1826551, Cloning, Molecular Characterization and Expression Patterns of HSL in HPG axis at different ages testicle tissues of the Bactrian camels
The manuscript presents a research that seem to be conducted in a proper manner, but its final objective is very specialized. It would be helpful if the authors would try to expend a little their discussion in order to be useful to a larger group of readers.
The editing of the paper should be checked. There is a big number of mistakes in both editing style and also on terms of grammar. Check the abbreviations. Present each one before it is used. The authors should consider that the abstract and the main manuscript are independent. One should be able to understand one without reading the other. For example, on row 52, present what HSL means. Do not use abbreviation in the title of the paper.
On row 53 “main role is to catalyze diacylglycerides and cholesterol”. It has no meaning. Detail in better words what reaction is catalyzed by this enzyme. What compounds are transformed and what it is the result?
Row 79, it is not clear how many how many camels were selected in each from each age group.
Row 173, the authors should add “predicted”. It should be “and it was predicted to be C897H1442N304O267S5”
Row 181, it is not clear if this is a prediction or an actual chemical analysis. The authors should present clear what is the case. It seems to be a prediction and if this is the case the authors should argue why a chemical analysis was not performed. Explain why the title suggest “Molecular Characterization”.
Row 183, detail the usefulness of these data. What information provides the predicted chemical composition of the enzyme? What would be the predicted catalytic center? Discuss these data in concordance with the GO prediction.
Row 201, detail what method was used and the mathematical parameters in term of cluster method (it looks like Hierarchical). Present the type of clustering, ex: agglomerative and the type of distance, ex; Euclidian. The authors should explain what each number mean in figure 4. The Pearson coefficient? If so, why 100, and not 1?
Row 262-278. The methods for Gene Ontology (GO) are not described and as mentioned before, the abbreviations need to be presented in the manuscript. Also a reference for the method should be added. The section 3.5 is just a prediction and the authors should use a language that should reflect that. Add some probabilities and some error measurement for this predictions. The same for the figure 7 legend. Add the word prediction and present the method and software used.
The authors should also check the discussion section and the conclusion. The words chosen should reflect if a result was confirmed experimentally or if it is a prediction. If available, provide statistical data for each prediction. Add error measurements.
The conclusion section should reflect the objectives declared in the section presented at rows 65-74. On row 337, it should be “is possible to interact”. Is is predicted. The research adds little information on the actual function of the enzyme on the regulatory mechanism steroid hormones. In the actual form the conclusions don’t reflect the results of the paper and the authors should correct them using an objective style.
Author Response
Dear editor：
On behalf of my co-authors, I thank editor and reviewer of “current issues in molecular Biology” very much for giving us a precious opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, Manuscript ID: cimb-1826551”. We have studied reviewer’s comments carefully and have made revision which marked in yellow in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to submit the enclosed manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, which we wish to be considered for publication in “current issues in molecular Biology”. No conflict of interest exits in the submission of this manuscript. We would like to express our great appreciation to you for comments on our paper. Looking forward to hearing from you.
Point 1：The editing of the paper should be checked. There is a big number of mistakes in both editing style and also on terms of grammar. Check the abbreviations. Present each one before it is used. The authors should consider that the abstract and the main manuscript are independent. One should be able to understand one without reading the other. For example, on row 52, present what HSL means. Do not use abbreviation in the title of the paper.
Response 1: We are very sorry for our negligence of writing in the manuscript, we have revised the abbreviation in manuscript and have been marked in yellow.
Point 2：On row 53 “main role is to catalyze diacylglycerides and cholesterol”. It has no meaning. Detail in better words what reaction is catalyzed by this enzyme. What compounds are transformed and what it is the result?
Response 2: Thanks for reviewer’s comments, Hormone sensitive lipase (HSL) is a rate-limiting enzyme of lipolysis, which plays a decisive role in the synthesis and metabolism of fat. It can hydrolyze triglyceride to diglyceride whose main role is to catalyze diacylglycerides and cholesterol, for instance, steroidogenic acute regulatory protein (StAR) is regulated by HSL, and the expression of StAR is a substrate for steroid synthesis, so HSL can be involved in regulate steroid synthesis and have been revised and marked in yellow in this manuscript.
Point 3：Row 79, it is not clear how many how many camels were selected in each from each age group.
Response 3: Thank you for the reviewer’s suggestion, Bactrian camels are divided into four age groups, and there are three camels in each age group (2W n=3, 2Y n=3, 4Y n=3 and 6Y n=3), which are presented in the Methods and Materials (2.1 Animals and Sample Collection) and have been marked in yellow in the manuscript.
Point 4: Row 173, the authors should add “predicted”. It should be “and it was predicted to be C897H1442N304O267S5”
Response 4: Thank you for your comment, we have revised and marked in yellow in manuscript.
Point 5: Row 181, it is not clear if this is a prediction or an actual chemical analysis. The authors should present clear what is the case. It seems to be a prediction and if this is the case the authors should argue why a chemical analysis was not performed. Explain why the title suggest “Molecular Characterization”.
Response 5:Here is the predicted amino acid compositionï¼›The main reason for not conducting chemical analysis is that the secondary structure of a protein can be statistically analyzed by Chou-Fasman method using online software, based on the theory that each amino acid has a different tendency or frequency to appear in various secondary structuresï¼›Because the cDNA sequence, nucleotide arrangement, translated protein sequence of HSL gene were cloned, and the secondary and tertiary structures of protein were predicted. So ' Molecular Characterization ' is used in the title'.
Point 6:Row 183, detail the usefulness of these data. What information provides the predicted chemical composition of the enzyme? What would be the predicted catalytic center? Discuss these data in concordance with the GO prediction.
Response 6:Net-phos 3.1 Serve prediction results showed that the protein had 12 serine (Ser) kinases, 9 threonine (Thr) kinases and 1 tyrosine (Thr) kinase potential phosphorylation sites, and protein phosphorylation was closely related to HSL activation. HSL protein was found to be hydrophobic, and the catalytic region and lipid-binding region of HSL were known to have hydrophobic properties, which was consistent with the predicted results of this study.
Point 7:Row 201, detail what method was used and the mathematical parameters in term of cluster method (it looks like Hierarchical). Present the type of clustering, ex: agglomerative and the type of distance, ex; Euclidian. The authors should explain what each number mean in figure 4. The Pearson coefficient? If so, why 100, and not 1?
Response 7:The number in Figure 4 is called the Bootstrap value which represents the reliability of the branch structure and ranges from 0% to 100%. The larger the value, the more evidence supports the branch. It is generally believed that if the Bootstrap value of a node is greater than 70, the branch is reliable. In particular, in the classification with large similarity such as microorganism, more than 50% is generally considered credible (less than 50% will not be shown). As can be seen from the figure, the feasibility of each species branch is high.
Point 8:Row 262-278. The methods for Gene Ontology (GO) are not described and as mentioned before, the abbreviations need to be presented in the manuscript. Also a reference for the method should be added. The section 3.5 is just a prediction and the authors should use a language that should reflect that. Add some probabilities and some error measurement for this predictions. The same for the figure 7 legend. Add the word prediction and present the method and software used.
Response 8: Thank you for reviewer comment, we have added the described methods for Gene Ontology (GO) in manuscript. In order to better understand the functional and regulatory roles of HSL in male bactrian camels reproductive hormone biosynthesis, the protein-protein interaction (PPI) networks were constructed using the STING v 10.0 the candidate proteins involved in sterol hormone biosynthesis, using STRING v 10.0 database (online,https://www.string-db.org/ accessed on 20 april 2022).（Damian S , Gable A L , Nastou K C , et al. The STRING database in 2021: customizable protein–protein networks, and functional characterization of user-uploaded gene/measurement sets[J]. Nucleic Acids Research, 2020(D1):D）. Due to large number of research have reported that HSL can regulate steroid synthesis efficiency via regulating the substrate StAR protein in the steroid synthesis pathway, HSL can directly regulate the synthesis and secretion of IGFs, which are the main cytokines regulating the secretion of steroid hormones. So, we predicted the relationship between HSL protein of Bactrian camel and steroid synthesis pathway via online software, for future research on the relationship between HSL and steroid synthesis. The relationship between steroid synthesis provides a basis for research on the mechanism of synthesis.
Point 9:The authors should also check the discussion section and the conclusion. The words chosen should reflect if a result was confirmed experimentally or if it is a prediction. If available, provide statistical data for each prediction. Add error measurements.
Response 9: Thank you for reviewer’s comments, we have revised discussion and conclusion in manuscript.
Point 10:The conclusion section should reflect the objectives declared in the section presented at rows 65-74. On row 337, it should be“is possible to interact”. Is predicted. The research adds little information on the actual function of the enzyme on the regulatory mechanism steroid hormones. In the actual form the conclusions don’t reflect the results of the paper and the authors should correct them using an objective style.
Response 10:Thank the reviewers for their valuable comments and suggestions. Studies have shown that guinea pig and human Sertoli cells, haploid germ cells and Leydig cells have the expression of HSL, and the expression activity of HSL in Leydig cells is positively correlated with the level of Guwan hormone, suggesting that it may be related to the formation of androgen ( Wang Yongbo, Chen Lulu. New progress in hormone-sensitive lipase research [J].Chinese Journal of Diabetes ( 1006-6187 ), 2005, 13 ( 2 ) : 3. ). Secondly, KEGG ( Aldosterone synthesis and secretion pathway ) showed that(https://www.kegg.jp/pathway/map=map04925&keyword=HSL) HSL participated in the regulation of steroid hormone synthesis and secretion by participating in the regulation of StAR, CYP11A1, 3βHSD and CYP21A expression levels.Finally, HSL also expressed in the adrenal gland, which mainly regulated the activity of cholesterol kinase. For example, the activity of cholesterol kinase in the adrenal gland of mice with HSL gene deletion was lower than that of normal mice, showing obvious lipid accumulation, resulting in a decrease in the production of adrenocorticotropic hormones. It suggested that HSL provided cholesterol and other precursor substances for steroid hormone synthesis ( LiH, Brochu M, Wang SP, et al., Hormone-sensitive lipase deficiency in mice cause lipid storage in the cortex and impaired corticosterone response to corticotropin stimulation. Endocrinology, 2002, 143 : 3333–3340 ). Moreover, HSL can participate in MAPA, cAMP, insulin and other signaling pathways, and participate in cell proliferation.
Reviewer 3 Report
The manuscript “Cloning, Molecular Characterization and Expression Patterns of HSL in HPG Axis at Different Ages Testicle Tissues of the Bactrian Camels” submitted by Zhang, Zhao and coworkers provides a study of the sequence and structure as well as expression at the mRNA and protein level together with cellular and tissue localization in dependence on the developmental stage. The data presented may be of interest for the readers of Curr. Issues Mol. Biol.
However, the data presented in Figures 2, 3 and 7 are just the results of bioinformative analysis starting with the sequence identified and as such the manuscript should be supplemented with additional experimentation with regard to the function of the differentially expressed HSL (see below).
In conclusion, the following points have to be addressed by the authors since the research or at least its presentation has apparently not been performed with great accurateness.
Major points:
1. Heading: Something is missing, or “testicle tissues” has to be eliminated
2. Abstract: Some abbreviations are missing, such as HPG
3. Abstract: lines 20,21: There are incorrect explanations for 2W, 2Y, 4Y, 6Y
4. Abstract: lines 28, 29: This statement does not make sense and is at variance with the data presented in Results
5. Introduction: lines 53, 54: “whose (HSL) main role is to catalyze …….cholesterol” is incorrect. HSL hydrolyzes cholesterylesters
6. Introduction: lines 55,56: Sentence incomplete or misleading. Where are the unsaturated fatty acids and cholesterol, in the lipid bilayers?
7. Introduction: In general, the motivation for this study has to be presented in greater detail. What is the rationale for assuming that HSL may represent a target for improving the reproductive capabilities of Bactrian camels?
8. Materials and Methods: lines 82,83: What is meant with “Male camels….were selected from Zhangye (author?) to collect testicular tissues”? What criteria were used?
9. Results: 3.3. Figure 5: The expression of HSL at adipose tissues (fat pads) of the epididymis should be analyzed explicitly. In rats, mice and humans epididymal adipocytes represent a major source of HSL.
10. Results: 3.5. Figure 7: The GO functional enrichment should be enriched with at least one of the following experimental data, such as putative phosphorylation (use of phosphorylation site-specific antibodies), lipolytic cleavage specificity (use of immunoprecipitation and in vitro HSL assay), interaction with other proteins (pull-down), each with tissues from different reproductive age.
11. Discussion: lines 283, 284: The data presented do not demonstrate that “phosphorylation of the protein is closely related to HSL activation”. This has to be shown by experimentation (see point 10!)
12. Discussion: lines 300-303: This statement should be more precise: ATGL is a triglyceride hydrolase, exclusively, whereas HSL hydrolyses predominantly diglycerides.
13. Discussion: lines 303-304: On basis of the motivation for this study, the role of HSL in spermatogenesis and synthesis of steroid hormones should be given in greater detail.
14. Discussion: lines 307-309. “there is close association with neutral fatty acids, adenylate cyclase within the epididymis, and phospholipid fatty acid content”. This statement is not clear for me, both at the level of the structure of the sentence and its contents.
15. Discussion: lines 321-322: “cholesterol is transferred from mitochondria to endometrium” What is meant? Please correct or specify!
16. Conclusions: line 331-332: “the first study…….to explore…..reproduction-related function”. This has not been shown in this study but can be demonstrated only by experimentation.
17. Conclusions: Based on the initial motivation for this study, it should be mentioned that future analysis of putative correlation between HSL expression at the HPG axis (or testicular testis) and Bactrian camels reproduction are urgently required.
Minor points:
1. There are many spelling and grammar errors, missing words, misleading statements etc, e.g. line 14 “involved steroid synthesis”, line 31 “there is different positive expression”, line 140 “Shnaghai”, line 216 “reveled”, line 311 “of the our study”, line 318 “Base”
2. Some sentences or parts of them are duplicated: e.g. lines 283, 284; lines 287-290
Author Response
Dear editor：
On behalf of my co-authors, I thank editor and reviewer of “current issues in molecular Biology” very much for giving us a precious opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, Manuscript ID: cimb-1826551”. We have studied reviewer’s comments carefully and have made revision which marked in yellow in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to submit the enclosed manuscript entitled “Cloning, Molecular Characterization and Expression Patterns of hormone-sensitive lipase in HPG axis at different ages testicle tissues of the Bactrian camels”, which we wish to be considered for publication in “current issues in molecular Biology”. No conflict of interest exits in the submission of this manuscript. We would like to express our great appreciation to you for comments on our paper. Looking forward to hearing from you.
Major points:
Point 1: Heading: Something is missing, or “testicle tissues” has to be eliminated
Response 1: Thank you for the reviewer’s suggestion,We have changed the title of the article to :Cloning and molecular characterization of Bactrian camel HSL gene and its expression pattern in reproductive axons and testis of bactrian camel at different ages.
Point 2: Abstract: Some abbreviations are missing, such as HPG
Response 2: Thanks to the reviewer's suggestions, we have revised the abbreviations and have been marked in yellow in the manuscript.
Point 3: Abstract: lines 20,21: There are incorrect explanations for 2W, 2Y, 4Y, 6Y
Response 3: We are very sorry for our incorrect writing in the manuscript, we have revised the manuscript and have been marked in yellow in the manuscript.
Point 4: lines 28, 29: This statement does not make sense and is at variance with the data presented in Results
Response 4: Thanks to the reviewer's suggestion, the correct description here should be The expression of mRNA in the testis increased with age and was the highest in the six-year-old testis(P < 0.01).The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than 2W and 4Y testis tissues (P < 0.01). We have revised the manuscript and have been marked in yellow in the manuscript.
Point 5: Introduction: lines 53, 54: “whose (HSL) main role is to catalyze …….cholesterol” is incorrect. HSL hydrolyzes cholesterylesters
Response 5: Thanks to the reviewer's suggestion, We have revised the manuscript and have been marked in yellow in the manuscript.
Point 6: Sentence incomplete or misleading. Where are the unsaturated fatty acids and cholesterol, in the lipid bilayers?
Response 6: We are very sorry for our incorrect writing in the manuscript, the implication of the manuscript is that testes are rich in fatty acids, cholesterol and lipid bilayers. We have revised the manuscript and have been marked in yellow in the manuscript.
Point 7: Introduction: In general, the motivation for this study has to be presented in greater detail. What is the rationale for assuming that HSL may represent a target for improving the reproductive capabilities of Bactrian camels?
Response 7:Many thanks for the questions raised by the reviewer. Studies have shown that HSL deletion causes lipid deposition in testicular spermatogenic tubules, disorder and vacuolization of spermatogenic epithelium, and significant reduction of spermatozoa. These structural and functional changes eventually lead to infertility in mice. As the most important enzyme in the lipid catabolic pathway, HSL may play a key role in the maintenance of spermatogenic epithelial structure, the secretion of androgens in the testicular mesenchyme and the transition from primordial germ cells to spermatozoa. It is also involved in the production of testosterone in the testicular interstitium and the stability of spermatogenic epithelium. Whether HSL is involved in the functional regulation of the reproductive system of bactrian camels and its physiological role in the reproductive system are also worthy of further study.
Point 8: Materials and Methods: lines 82,83: What is meant with “Male camels….were selected from Zhangye (author?) to collect testicular tissues”? What criteria were used?
Response 8: Thank you for the reviewer’s comment, we are very sorry for our negligence of writing in the manuscript, the right writing is “Male camels samples were selected from Zhangye city (Gansu province, China)” , we have revised and marked in yellow.
Point 9: Results: 3.3. Figure 5: The expression of HSL at adipose tissues (fat pads) of the epididymis should be analyzed explicitly. In rats, mice and humans epididymal adipocytes represent a major source of HSL.
Response 9: Thank you for the reviewer's opinion. We only analyzed the differential expression of the gonad axis and accessory structure of the bactrian camel, and did not consider the expression differences between different species, because different proteins are expressed in different parts and at different times. Differences. Secondly, we did not carefully subdivide the epididymis to explore the expression patterns of the epididymal head, epididymal body and epididymal tail, because the epididymis is the place where sperm is stored and matured, which goes against our original intention of exploring the regulation of steroid synthesis.
Point 10: Results: 3.5. Figure 7: The GO functional enrichment should be enriched with at least one of the following experimental data, such as putative phosphorylation (use of phosphorylation site-specific antibodies), lipolytic cleavage specificity (use of immunoprecipitation and in vitro HSL assay), interaction with other proteins (pull-down), each with tissues from different reproductive age.
Response 10: Thanks to the reviewers for proposing so many classical protein verification methods, but our purpose is to predict that Bactrian camel HSL may interact with proteins in the regulation of steroid synthesis, so test phosphorylation site-specific antibodies, immunoprecipitation and pull-down etc. methods may not be applicable to this manuscript. We will try the method recommended by the reviewer to verify the mechanism of HSL regulating steroid hormone synthesis in the later stage.
Point 11: Discussion: lines 283, 284: The data presented do not demonstrate that “phosphorylation of the protein is closely related to HSL activation”. This has to be shown by experimentation (see point 10!)
Response 11: Thanks to the reviewer for questioning, this sentence has been reported in the relevant literature and proved by experiments(Olsson H,Belfrage P,Phosphorylation and dephosphorylation of hormone-sensitive lipase. Interactions between the regulatory and basal phosphorylation sites.[J] .FEBS Lett, 1988, 232: 78-82.).the demonstration that the rate of phosphorylation of the regulatory phosphorylation site was dependent on the phosphorylation state of the basal site sug-gests a role for this site in the regulation of hormone-sensitive lipase activity. The basal site may take part in the control of lipolysis in an indirect way by increasing the rate of phosphorylation of the regulatory site and thereby activation of the lipase. It can be concluded that protein phosphorylation is related to the activation of hormone-sensitive lipase.
Point 12: Discussion: lines 300-303: This statement should be more precise: ATGL is a triglyceride hydrolase, exclusively, whereas HSL hydrolyses predominantly diglycerides.
Response 12: Thank you for the valuable comments of the reviewers in the manuscript, we have revised and marked it in yellow in the manuscript.
Point 13: Discussion: lines 303-304: On basis of the motivation for this study, the role of HSL in spermatogenesis and synthesis of steroid hormones should be given in greater detail.
Response 13: Thank you for the reviewer’s questions, 1. HSL was found in Sertoli cells and spermatocytes, suggesting that the effect of HSL on testis was mainly concentrated in spermatogenic tubules. Spermatogenesis and development are carried out in spermatogenic tubules, which are mainly provided by Sertoli cell and spermatogenic cells, and regulated by testosterone secreted by testicular interstitial cells and follicle stimulating hormone (FSH) through the blood testosterone barrier. Adenylyl cyclase is abundant in epididymis. Through a series of changes such as head, body and tail transport to obtain the ability of locomotion and fertilization; 2. Studies have shown that an HSL knockout mice, testicular tissue completely lost the cholesterol ester hydrolysis enzyme activity and the accumulation of a large amount of cholesterol ester, significantly reduce weight, testicular sperm production in cortex significantly reduce and appeared vacuolated structure, its composition is most likely due to an HSL knockout and accumulation of lipid droplets, mature sperm in the epididymis not only significantly reduced in number, There was also a marked decline in exercise ability and eventually infertility. There were no significant changes in circulating steroid hormones, and no significant changes in other steroidogenic tissues in the body, suggesting that HSL directly participates in the hydrolysis of neutral cholesteryl ester in testicular tissue, and the reproductive dysfunction caused by HSL deletion is caused by HSL primary, HSL in steroidogenic tissues is the mobilization of free cholesterol for steroid biosynthesis.
Point 14: Discussion: lines 307-309. “there is close association with neutral fatty acids, adenylate cyclase within the epididymis, and phospholipid fatty acid content”. This statement is not clear for me, both at the level of the structure of the sentence and its contents
Response 14: Thank you for the reviewer’s question. What this sentence is trying to say that spermatozoa acquire the ability to fertilize by passing through the epididymis. Neutral fatty acids, epididymal adenylate cyclase and phospholipid fatty acids play important roles in this process.
Point 15: Discussion: lines 321-322: “cholesterol is transferred from mitochondria to endometrium” What is meant? Please correct or specify!
Response 15: Thank you for the valuable comments of the reviewers in the manuscript, we have revised and marked it in yellow in the manuscript.
Point 16: Conclusions: line 331-332: “the first study…….to explore…..reproduction-related function”. This has not been shown in this study but can be demonstrated only by experimentation.
Response 16: Thank you for your valuable comments. Indeed, in this manuscript, we only briefly predicted the molecular characterization of Bactrian camel HSL protein and possibly related proteins, and explored the expression pattern of HSL in the reproductive system, therefore, to provide basis and support research on the mechanism of HSL in the future.
Point 17: Conclusions: Based on the initial motivation for this study, it should be mentioned that future analysis of putative correlation between HSL expression at the HPG axis (or testicular testis) and Bactrian camels reproduction are urgently required.
Response 17: Thank you reviewers for your valuable comments, we will to verify the relevance of HSL protein in testis and reproduction in the future.
Minor point1: There are many spelling and grammar errors, missing words, misleading statements etc, e.g. line 14 “involved steroid synthesis”, line 31 “there is different positive expression”, line 140 “Shnaghai”, line 216 “reveled”, line 311 “of the our study”, line 318 “Base”
Response: We are very sorry for our incorrect writing in the manuscript, we have revised the manuscript and have been marked in yellow in the manuscript.
Minor point2: Some sentences or parts of them are duplicated: e.g. lines 283, 284; lines 287-290
Response: We are very sorry for our incorrect writing in the manuscript, we have revised the manuscript and have been marked in yellow in the manuscript.
Round 2
Reviewer 1 Report
I have no comments
Author Response
Thank the reviewers again for their constructive comments and suggestions on our contributions. We have done our best to reply and improve. We deeply appreciate your recognition of our research work.
Reviewer 2 Report
The authors responded to the comments I provided in the first review, but they failed to incorporate important information in the corrected manuscript.
For example, there is no actual chemical analysis, but a prediction. Why the title suggest “Molecular Characterization”? Provide in the manuscript an explanation why a chemical analysis was not performed. What information provides the predicted chemical composition of the enzyme? What would be the predicted catalytic center? Discuss these data in concordance with the GO prediction and present these data in the manuscript.
The authors should detail what method was used and the mathematical parameters in term of cluster method. Present the type of clustering, ex: agglomerative and the type of distance, ex; Euclidian and they should explain what each number mean in figure 4. They provided an answer for the reviewer, but nothing for the readers. This problem should be corrected.
Again, the conclusion section should reflect the objectives declared in the section presented at the beginning of the paper. The research adds little information on the actual function of the enzyme on the regulatory mechanism steroid hormones. Still the conclusions don’t reflect the results of the paper and the authors should correct them using an objective style.
Author Response
The authors responded to the comments I provided in the first review, but they failed to incorporate important information in the corrected manuscript.
For example, there is no actual chemical analysis, but a prediction. Why the title suggest “Molecular Characterization”? Provide in the manuscript an explanation why a chemical analysis was not performed. What information provides the predicted chemical composition of the enzyme? What would be the predicted catalytic center? Discuss these data in concordance with the GO prediction and present these data in the manuscript.
Respond: Thank you very much for the questions posed by the reviewers. Here is the predicted amino acid compositionï¼›The main reason for not conducting chemical analysis is that the secondary structure of a protein can be statistically analyzed by Chou-Fasman method using online software, based on the theory that each amino acid has a different tendency or frequency to appear in various secondary structuresï¼›Because the cDNA sequence, nucleotide arrangement, translated protein sequence of HSL gene were cloned, and the secondary and tertiary structures of protein were predicted. So ' Molecular Characterization ' is used in the title'. In the result (3.1 Cloning and Sequence Analysis of Bactrian camels HSL CDS). We have revised the manuscript and have been marked in red.
The authors should detail what method was used and the mathematical parameters in term of cluster method. Present the type of clustering, ex: agglomerative and the type of distance, ex; Euclidian and they should explain what each number mean in figure 4. They provided an answer for the reviewer, but nothing for the readers. This problem should be corrected.
Respond: Thank you for the reviewer question, What is used is Neighbor-Joining method based on distance method. The number in Figure 4 is called the Bootstrap value which represents the reliability of the branch structure and ranges from 0% to 100%. The larger the value, the more evidence supports the branch. It is generally believed that if the Bootstrap value of a node is greater than 70, the branch is reliable. In particular, in the classification with large similarity such as microorganism, more than 50% is generally considered credible (less than 50% will not be shown). As can be seen from the figure, the feasibility of each species branch is high.We have improved this problem in the manuscript and marked it red.
Again, the conclusion section should reflect the objectives declared in the section presented at the beginning of the paper. The research adds little information on the actual function of the enzyme on the regulatory mechanism steroid hormones. Still the conclusions don’t reflect the results of the paper and the authors should correct them using an objective style.
Respond: Thank you very much for the questions posed by the reviewers. GO prediction indicates that HSL participates in the regulation of steroid hormone synthesis and secretion by regulating the expression levels of STAR, HSD3B, IGF1and SRD5A1. HSL is also expressed in the adrenal gland, which is mainly involved in the regulation of cholesterol kinase activity. For example, the activity of cholesterol kinase in the adrenal gland of mice with HSL gene deletion is less than that of normal mice, showing obvious lipid accumulation, resulting in a decrease in the production of adrenocorticotropic hormone[35], suggesting that HSL provides cholesterol and other precursors for the synthesis of steroid hormones. For this issue, we have supplemented the discussion section and marked it as red.
Author Response File: 
Author Response.docx
Reviewer 3 Report
Albeit it is a pity that the authors did not perform a single experiment as suggested to confirm the functional relevance of their findings derived from bioinformatic analysis, exclusively, they have addressed some major points of my critizism.
Author Response
Thank the reviewers again for their constructive comments and suggestions on our contributions. We have done our best to reply and improve. We deeply appreciate your recognition of our research work.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
