Paternal Finasteride Treatment Can Influence the Testicular Transcriptome Profile of Male Offspring—Preliminary Study

Round 1

Reviewer 1 Report

The authors investigated the effects of finasteride administration on testis of rat in next generation by means of transcriptome. Though the data seems to be adequate and clear, it is necessary to revise the manuscript for publication due to following reasons.

 

• The authors confound sperm and testicular tissue. Though the data was obtained by testicular transcriptome, the authors discussed only about the sperm and did not mentioned other component like Sertoli cells.

• As the authors mentioned the present results is preliminary and it is not shown if functional receptors are existed in cells. Thus, discussion is based on a lot of supposition not on obtained data.

• Discussion need a reconstruction. 

• I do not think it is necessary to spend a  huge  space to state characteristics/function of each gene in Table 1. 

Author Response

REVIEW #1

Dear Reviewer,

I would like thank you very much for your constructive review and the time you spent on it. Below answers to your suggestions and I hope you will appreciate my answers.

 

• The authors confound sperm and testicular tissue. Though the data was obtained by testicular transcriptome, the authors discussed only about the sperm and did not mentioned other component like Sertoli cells.

Yes, we did not mention about inter alia Sertoli cell, because results of microarrays analysis of genes for example involved in spermatogenesis will be the subject of the next our manuscript. In this new manuscript we will discussed changes in steroid metabolic process, spermatogenesis, fertilization, developmental process involved in reproduction, cell cycle, regulation of oxidative stress-induced intrinsic apoptotic signaling pathway ect.

• As the authors mentioned the present results is preliminary and it is not shown if functional receptors are existed in cells. Thus, discussion is based on a lot of supposition not on obtained data.

Yes, but we think that the obtained results have been well discussed in the light of the available literature, and that the put forward hypotheses have a chance to turn out to be real/true.

• Discussion need a reconstruction.

To the Discussion has been added new information according the suggestion of the second Reviewer.

• I do not think it is necessary to spend a  huge  space to state characteristics/function of each gene in Table 1. 

The table has been shortened.

Reviewer 2 Report

The manuscript titled “Can the finasteride treatment of adult male rats (F0:Fin) influ-2 ence the testicular transcriptome profile of their male offspring 3 (F1:Fin)? Microarray analysis of the top 10 genes with the high-4 est and lowest fold change values in the testis of the F1:Fin gen-5 eration correlated to postnatal age (immature and mature) – 6 preliminary study” the authors study the effect of finasteride treatment on the testicular transcriptome in the male filial generation (F1:Fin) from paternal F0:Fin rats by microarray analyses.

I thing that this work was carefully conducted. The work is well written but in my opinion it lacks some aspects. So I suggest a major revision because the manuscript should be integrated considering the following aspects:

 

• The title is too long
• Acronyms must be written out in full the first time they are used
• Lines 75-76-77. Since the authors also talk about the effects of pollution on semen quality and possible transgenerational effects of finasteride, I recommend reading and quoting the following works : 10.3390/ijms21124198; 10.3390/ijms21186710.

In particular in the first work it is shown that DNA oxidative damage can also be caused by pollutants present in semen. In the second it is shown trasgenerational effects of pollutants on molecular alterations in spermatozoa of men living in high polluted areas.

• what are the mechanisms by which finasteride treatment might produce transgenerational effects?
• Line 89: “range ≤ -1.5 (down-regulated genes) and fold change ≥ 5 (up-regulated genes)) in the 89” there is an extra parenthesis
• why were rats considered at 14 and 90 days? Explain in Materials and Methods
• Improving the quality of figure 1
• lines 117 and 118 are underlined
• Do the authors have any information about the protamine-histone ratio in these rats?
• Line 201: “important role and generally have a negative effect on reproductive function after attainment”. Arranging spaces. There are extra spaces
• Line 326: How the fully mature gonad with sperm production capacity was verified in 3-month-old (90-day-old) rats?
• The authors need to argue a little more about the molecular mechanisms induced by the alterations they found especially for odorant-like signals
• The authors must also argue about the possible correlation between odorant-like signals, epigenetics and protamines.

Author Response

REVIEW #2

 

Dear Reviewer,

I would like thank you very much for your the detailed and constructive review and the time you spent on it. Below answers to your suggestions and I hope you will appreciate my answers.

 

• The title is too long

The title was changed.

• Acronyms must be written out in full the first time they are used

Has been corrected.

• Lines 75-76-77. Since the authors also talk about the effects of pollution on semen quality and possible transgenerational effects of finasteride, I recommend reading and quoting the following works : 10.3390/ijms21124198; 10.3390/ijms21186710.

According these suggestions we added new information within the Introduction (marked in red color).

• what are the mechanisms by which finasteride treatment might produce transgenerational effects?

The mechanism was now added to the manuscript body (marked in red color in the Discussion).

• Line 89: “range ≤ -1.5 (down-regulated genes) and fold change ≥ 5 (up-regulated genes)) in the 89” there is an extra parenthesis

I don’t understand, because in this line is: “…….(DEGs - fold change ≤ -1.5 (down-regulated genes) and fold change ≥ 1.5 (up-regulated genes))….” This an extra parenthesis because of the first one, pointed here in blue color.

• why were rats considered at 14 and 90 days? Explain in Materials and Methods

Already, it is explain in this chapter, please see: “This experimental model of gonad maturity is based on the fact that in the seminiferous epithelium of 7-day-old rats, there are only gonocytes and Sertoli cells, and during maturation (i.e. successive weeks of the experiment), other generations of germ cells occur within the seminiferous epithelium [24, 54], which is why we selected gonads from 14-day-old rats. A completely mature gonad with spermatozoa production ability can be found in 3-month-old rats (90 days old) [24].”

• Improving the quality of figure 1
• lines 117 and 118 are underlined – these underlined text is because some of software error, because the text that I up-loaded in the journal editorial platform had not any underlining
• Do the authors have any information about the protamine-histone ratio in these rats?

Unfortunately, we do not have such determinations, maybe in the future we will perform determinations of the protamination level of sperm chromatin, because we have frozen semen collected from the epididymis. Please see at the end of Discussion (limitation of our study), there have been added new text (in red color).

 

• Line 201: “important role and generally have a negative effect on reproductive function after attainment”. Arranging spaces. There are extra spaces – these extra spaces are probably the result of some of software error
• Line 326: How the fully mature gonad with sperm production capacity was verified in 3-month-old (90-day-old) rats?

First, literature data show that rats reach sexual maturity at the age of seven to ten weeks [https://doi.org/10.1016/j.reprotox.2013.02.003; PMID: 23930179]. Second, in my research work, I observed many times the morphology of the seminiferous epithelium and epididymis of 90-day-old rats; moreover, these animals were used in my reproductive experiments, so pregnancy and childbirth were proof of their fertility [doi: 10.5603/fhc.a2015.0025].

• The authors need to argue a little more about the molecular mechanisms induced by the alterations they found especially for odorant-like signals.

Please see in Discussion, there have been added new text (in red color).

• The authors must also argue about the possible correlation between odorant-like signals, epigenetics and protamine’s.

Because, as I wrote above, we do not have marked/measured the level of protamination of sperm DNA, therefore I believe that it is not necessary in this publication to describe the correlation between the changes in the expression of genes related to odor signaling and protamination, which we exactly do not have.

Round 2

Reviewer 1 Report

• The authors confound sperm and testicular tissue. Though the data was obtained by testicular transcriptome, the authors discussed only about the sperm and did not mentioned other component like Sertoli cells.

Yes, we did not mention about inter alia Sertoli cell, because results of microarrays analysis of genes for example involved in spermatogenesis will be the subject of the next our manuscript...

>Even so, it is possible that the data shown in the present originated form Sertoli cells. Thus, the should explain that. 

 

Author Response

REV_1

 

Thank you for your support and highlighting the weaknesses of the manuscript and recommendations. Since they were very constructive we tried to fulfill them as much as possible. We agree with you opinion about the presence of heterogenic population of cells within the testicular tissue. Therefore, to emphasize this point we wrote in the introduction that the analysis of the testicular transcriptome profile were conducted without distinguishing cellular populations.

In materials and methods we didn't change anything, because seems obvious that if the total RNA was obtained from testis homogenates, all cell types present within the gonad were present within RNA.In the results we wrote:

"It should be noted that the analyzed transcriptome came from testicular tissue that contains different, heterogenic populations of cells (e.g. Sertoli, Leydig, germ cell, etc.). "  and we payed attention that many functions and receptors changed in our transcriptome could also be a consequence of changes in Sertoli cells. We also identified the presence of heterogenic population of cells in transcriptome as the limitation of the study.

 

We believe the manuscript after the revision is within the scope of Current Issues in Molecular Biology

 

Reviewer 2 Report

Accept in present form

Author Response

REV_2

 

Thank you for your support and highlighting the weaknesses of the manuscript and recommendations. Since they were very constructive we tried to fulfill them as much as possible.

 

We believe the manuscript after the revision is within the scope of Current Issues in Molecular Biology