miR-155 Regulates Photoperiod Induced Gonadal Development in Atlantic Salmon (Salmo salar) by Targeting Brain-Derived Neurotrophic Factor

Round 1

Reviewer 1 Report (New Reviewer)

General comments

I have read carefully the ms entitle “miR-155 regulate gonadal development in Atlantic salmos (Salmo salar) by targeting brain-derives neurotrophic factor” and is very novel with a full application in aquaculture to improve the salmon culture. In general, the authors analyze the importance of how photoperiod influences over gonadal development in RAS systems for S. salar, and this study could be applied in the culture of other species of fishes of commercial interest. The role of mir-155 in the control of FSH and GnRH expression is very interesting, as it could bind to the 3'UTR region of BDNF. In addition, the ms is clear, easy to read and well organised.

The manuscript is therefore suitable for publication.

Specific minor comments:

-pag. 3, line 100: change “thrroid” to “thiroid”

-pag. 3, line 129: one of the photoperiods is wrong; change “16L:8D” to “8L:16D”

-Why the condition of photoperiod 24L:0D-8L:16D is named LL-SL? What is the significance of “LL” and the “SL”’

-I don´t understand the sentence of the pag. 3, lines 131 and 132: “For fish in these gradually….per day in the appropiate direction”

-I don´t understand the sentence of the pag. 3, lines 140: This 3 fishes collected during the daytime, were the same fishes that were sampled for euthanization?

-Pag.3, line  148: only you samled 6 fishes (3 male and 3 female) for miR—155 expression analysis? In which stage of growth?. Specify in this section.

-Are there any results of histological analyses?

-the quality of the legend in the axes x and y of the figure1 is not too good

-the legend of the axes of the figure 5a and 5d is too small in comparing with other figures

-Pag. 10, lines 364 and 365: change “In this paper, in order to investigate whether photoperiod have a cumulative effect on development of fish. We set two…” to “In this paper, in order to investigate whether photoperiod have a cumulative effect on development of fish, we set two…”

Author Response

I have read carefully the ms entitle “miR-155 regulate gonadal development in Atlantic salmos (Salmo salar) by targeting brain-derives neurotrophic factor” and is very novel with a full application in aquaculture to improve the salmon culture. In general, the authors analyze the importance of how photoperiod influences over gonadal development in RAS systems for S. salar, and this study could be applied in the culture of other species of fishes of commercial interest. The role of mir-155 in the control of FSH and GnRH expression is very interesting, as it could bind to the 3'UTR region of BDNF. In addition, the ms is clear, easy to read and well organised.

The manuscript is therefore suitable for publication.

Specific minor comments:

-pag. 3, line 100: change “thrroid” to “thiroid”

Response: We are sorry for the mistake, and we revised it in the MS.

-pag. 3, line 129: one of the photoperiods is wrong; change “16L:8D” to “8L:16D”

Response: We are sorry for the mistake, and we revised it in the MS.

-Why the condition of photoperiod 24L:0D-8L:16D is named LL-SL? What is the significance of “LL” and the “SL”’

Response: before the experiment, we were not sure how long the experiment will be last, so we not sure the light will change from 24L to 8L, and we used LL-SL. Furthermore, if we used “24L:0D-8L:16D” to name the group , we think it’s a little long compared to other groups. LL means Long light photoperiod, and SL means Short light. So LL-SL means the photoperiod in this group changing form long light photoperiod to short light photoperiod. And we revised this in the ms.

-I don´t understand the sentence of the pag. 3, lines 131 and 132: “For fish in these gradually….per day in the appropiate direction”

Response: for example, in the LL-SL group, the photoperiod is 24 hour light and 0 hour dark in first day, and it will change to 23h 55min light and 5 min dark in second day, 23h 50 min light and 10 min dark in third day and so on.

-I don´t understand the sentence of the pag. 3, lines 140: This 3 fishes collected during the daytime, were the same fishes that were sampled for euthanization?

Response: We are sorry for that，yes , they are the same fish and we revised it in the ms.

-Pag.3, line  148: only you samled 6 fishes (3 male and 3 female) for miR—155 expression analysis? In which stage of growth?. Specify in this section.

Response: In this part，we supposed that miR-155 might has a sex-specific expression, but in our experiment, we did not find the differential expression between female and male fish, so we did not mention in the ms.

-Are there any results of histological analyses?

Response: we and some explanation for histological in supplemental figure. The histology were just used for us to confirm the development stage of gonad. So we did not analysis it.

-the quality of the legend in the axes x and y of the figure1 is not too good

Response: Thank you for your suggestion, and we revised it in the ms, and we will give vector graph to the editor.

-the legend of the axes of the figure 5a and 5d is too small in comparing with other figures

Response: Thank you for your suggestion, and we revised it in the ms, and we will give vector graph to the editor.

-Pag. 10, lines 364 and 365: change “In this paper, in order to investigate whether photoperiod have a cumulative effect on development of fish. We set two…” to “In this paper, in order to investigate whether photoperiod have a cumulative effect on development of fish, we set two…”

Response: Thank you for your suggestion, and we revised it in the ms.

Reviewer 2 Report (New Reviewer)

The aim of the study was to investigate the impact of photoperiod on gonadal development and somatic growth of Atlantic salmon raised in recirculating aquaculture systems (RAS), with a specific focus on the role that microRNA (miR)-155 plays as a regulator of Atlantic salmon photoperiodic reproduction. The manuscript shows promisor results and solid data regarding the effect of photoperiod on gonadal development. Moreover, the language is of great quality and the sentences are very fluid, easly leading the reader towards to what the authors meant to say. However, before acceptance, some corrections have to be made.

L100: thrroid -> thyroid

L365-367: the repetition of the words changing and change is resulting in a confuse sentence. Please rewrite.

L370-372: the sentences seem to be unintentionally separated.  

The name of the treatments should be reconsidered. The repeated use of numbers and letters as presented may lead the readers to erroneous ideas regarding the treatments or force them to repeatedly return to material and methods section to remember each was each.

There is a huge chance that the effects of the treatments are not really instantaneous, gradually appearing between to observations. Thus, otherwise experimentally confirmed, the word instantaneous should be avoided.

Regarding the conclusion, I personally do not think that the study should be aiming the continuation of the species. The continuation of the species as an individual aim has fallen since the 70’s with the selfish gene theory of Richard Dawkins. Moreover, the study was conducted in RAS which, although experimental, is one of the most artificial forms to fish farming, presenting a great number of variables that are not in the natural environment. However, the data presented in the manuscript are very important for salmon aquaculture and the conclusion should be regarding this matter.

Finally, I really would like to see some of the images of supplementary data in the manuscript, such as the gonads of fig 1.

The language is of great quality and the sentences are very fluid, easly leading the reader towards to what the authors meant to say.

Author Response

The aim of the study was to investigate the impact of photoperiod on gonadal development and somatic growth of Atlantic salmon raised in recirculating aquaculture systems (RAS), with a specific focus on the role that microRNA (miR)-155 plays as a regulator of Atlantic salmon photoperiodic reproduction. The manuscript shows promisor results and solid data regarding the effect of photoperiod on gonadal development. Moreover, the language is of great quality and the sentences are very fluid, easly leading the reader towards to what the authors meant to say. However, before acceptance, some corrections have to be made.

L100: thrroid -> thyroid

Response: we are sorry for the mistake, and we revised it in the ms.

L365-367: the repetition of the words changing and change is resulting in a confuse sentence. Please rewrite.

Response: We are sorry for the mistakes, and we revised it in the ms.

L370-372: the sentences seem to be unintentionally separated.  

 Response: We are sorry for the mistake, and we revised it in the ms.

The name of the treatments should be reconsidered. The repeated use of numbers and letters as presented may lead the readers to erroneous ideas regarding the treatments or force them to repeatedly return to material and methods section to remember each was each.

There is a huge chance that the effects of the treatments are not really instantaneous, gradually appearing between to observations. Thus, otherwise experimentally confirmed, the word instantaneous should be avoided.

Response: Thank you for your suggestion. Before the experiment, we were not sure how long the experiment will be last, so we not sure the light will change from 24L to 8L, and we used LL-SL. Furthermore, if we used “24L:0D-8L:16D” to name the group , we think it’s a little long compared to other groups. And we revised it in the ms, give a explanation to the group name (Line 130-131).

Regarding the conclusion, I personally do not think that the study should be aiming the continuation of the species. The continuation of the species as an individual aim has fallen since the 70’s with the selfish gene theory of Richard Dawkins. Moreover, the study was conducted in RAS which, although experimental, is one of the most artificial forms to fish farming, presenting a great number of variables that are not in the natural environment. However, the data presented in the manuscript are very important for salmon aquaculture and the conclusion should be regarding this matter.

 Response: Thank you for your suggestion, biology response to environment is a complex process, we just give reader an explanation of environmental response based our experiment.

Finally, I really would like to see some of the images of supplementary data in the manuscript, such as the gonads of fig 1.

Response: Thank you for your suggestion, the developmental stage of gonad were determined by morphology and histology, and we add this in the supplementary figure.

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.

Round 1

Reviewer 1 Report

As mentioned in my first review, there is inconsistency in how siRNA is presented in this ms. It is outlined in M&M, and presented in results, but not mentioned in discussion. I think I understand what was done, but this needs clarification.

Author Response

R1: As mentioned in my first review, there is inconsistency in how siRNA is presented in this ms. It is outlined in M&M, and presented in results, but not mentioned in discussion. I think I understand what was done, but this needs clarification.

Response: Thank you for your suggestion, and we revised this in the MS.

Reviewer 2 Report

The authors attempted to clarify the optimal photoperiod condition for salmon aquaculture in the RAS or the role of miR-155 in photoperiod-dependent maturation. To perform it, many experimental groups were prepared, and mRNA silence technique were used. I reviewed 1st manuscript and suggested many points, but most of them were reflected in this manuscript. Again, I list my comments which the authors did not answered in this manuscript. In addition, the authors should mention the criteria how the authors determined the maturation status, not showing the picture.

Major comments (Line numbers are the number of 1st manuscript)

L90-

Much knowledge about the relationship between photoperiod and maturation has been published in salmonids. Please describe them more detailly and then your research objective.

L109

What condition? SW acclimated salmon? When is these fish acclimated in SW? How old? Will these fish mature in this autumn or winter if these fish are reared in natural photoperiod?

L123-

Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

L146

What are miRNA mimics? How did the authors conform the sequence of Atlantic salmon miR-155? Also, please mention miRNA inhibitors, control miRNA and siRNA more detailly.

L147

Please describe concentration or weight with injection volume.

L207-

Please explain maturational stage and how is the stage confirmed, in ‘Materials and methods’

L207-

Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

Figure 3

The authors should explain how the level were measured in ‘Materials and methods’.

L248-

How did the authors identify Atlantic salmon miR-155? At first, the authors should explain the results. Next, please explain the miR-155 mimic and the difference from Atlantic salmon miR-155, and whether ‘miR-155 mimic’ mimic the native salmon miR-155, and how the inhibitor suppresses the function of the mimic and native miR-155.

If ‘inhibitor’ surely inhibits the expression of the native miR-155, you can exhibit that the inhibitor suppresses the expression level of the native miR-155 using the same cDNA template as that of figure 4b. Please show it.

Figure 5

Please re-illustrate fig. 5a because the lines between the bases are poorly connected.

Figure 6

If BDRF siRNA suppress the expression of BDRF, you can show the suppression by western blotting of BDRF as shown in Fig. 5c.

L325-347

Again, I could not understand the author’s argument. Your arguments may be correct, but are they novel knowledge?

L359-362

At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

Photoperiod may influence the expression of miR-155, but I do not think so from only Fig. 3. In addition, please discuss how the expression is influenced by photoperiod.

L364

The authors measured the expression of FSH receptor gene but did not measure FSH level. I would like to know why the authors measured the receptor not FSH and LH. FSH and LH are directly regulated by GnRH, but I do not know whether the expression of receptors in the gonads are regulated by hypothalamic GnRH much less miR-155.

Author Response

R2:

The authors attempted to clarify the optimal photoperiod condition for salmon aquaculture in the RAS or the role of miR-155 in photoperiod-dependent maturation. To perform it, many experimental groups were prepared, and mRNA silence technique were used. I reviewed 1st manuscript and suggested many points, but most of them were reflected in this manuscript. Again, I list my comments which the authors did not answered in this manuscript. In addition, the authors should mention the criteria how the authors determined the maturation status, not showing the picture.

Major comments (Line numbers are the number of 1st manuscript)

L90-

Much knowledge about the relationship between photoperiod and maturation has been published in salmonids. Please describe them more detailly and then your research objective.

 Response: Thank you for your suggestion, and we revised this in the MS.

L109

What condition? SW acclimated salmon? When is these fish acclimated in SW? How old? Will these fish mature in this autumn or winter if these fish are reared in natural photoperiod?

Response: The fish used in our study is reared in breeding workshop of Guoxin Dongfang Rcirculating Aquaculture, and in order to make fish acclimate for the experimental system. So we reared the fish for one month to acclimate the experimental system before experiment. The fish used in this experiment were reared in sea water, and their gonad were stage II, the water condition were list in the materials and methods. All the fish were reared in RAS, so the fish were under artificial light. Thank you for your suggestion, and we revised this in the MS.

L123-

Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

Response: In this paper, the fish were divided into 6 groups, four constant photoperiod groups and two gradual changes photoperiod groups. This two changes photoperiod groups were used to confirm the role of photoperiod. Cause the in change photoperiod groups, the photoperiod could change from long photoperiod to short photoperiod in one group, and the photoperiod could change from short photoperiod to long photoperiod in another changing photoperiod group. So along with the gonadal development, we think the change photoperiod will provide more evidence of the effect of photoperiod on gonadal development. In addition, 0L:24D means all darkness in 24h , the fish cannot find their food in total darkness environment, so  the fish cannot develop normally.

L146

What are miRNA mimics? How did the authors conform the sequence of Atlantic salmon miR-155? Also, please mention miRNA inhibitors, control miRNA and siRNA more detailly.

Response: The miRNA mimics mean nonnatural double-stranded miRNA-like RNA fragments chemically synthesized. In fact, chemically synthesized miRNA called miRNA mimics. Such an RNA fragment is designed to have its 5'-end bearing a partially complementary motif to the selected sequence in the 3'UTR unique to the target gene. Once introduced into cells, this RNA fragment, mimicking an endogenous miRNA, can bind specifically to its target gene and produce posttranscriptional repression, more specifically translational inhibition, of the gene. The sequence of miR-155 in salmon were obtained from miR-base and we mentioned this in the MS. We revised the detail information of miRNA mimic, inhibitor and control miRNA in the MS (MM 2.2)

L147

Please describe concentration or weight with injection volume.

Response: Thank you for your suggestion and we revised this in the MS, Line 154.

L207-

Please explain maturational stage and how is the stage confirmed, in ‘Materials and methods’

Response: Thank you for your suggestion, the maturational stage were confirmed by HE section, this part we have been published in other papers, and we add this as supplements. In this paper, we regard stage IV and stage V as maturational stage.

L207-

Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

Response: Thank you for your suggestion, in our experiment, the gonad were matured at stage IV by HE. When gonad developed into stage V，the gonad has begun to degenerate (supplementary figure). Furthermore, some fish gonad stage were between IV and V, and it’s hard to determine their precise stage. So we sum these two stage together.

Figure 3

The authors should explain how the level were measured in ‘Materials and methods’.

Response: Thank you for your suggestion, we are sorry for the mistake and we revised this in the MS (Line 198).

L248-

How did the authors identify Atlantic salmon miR-155? At first, the authors should explain the results. Next, please explain the miR-155 mimic and the difference from Atlantic salmon miR-155, and whether ‘miR-155 mimic’ mimic the native salmon miR-155, and how the inhibitor suppresses the function of the mimic and native miR-155.

If ‘inhibitor’ surely inhibits the expression of the native miR-155, you can exhibit that the inhibitor suppresses the expression level of the native miR-155 using the same cDNA template as that of figure 4b. Please show it.

Response:    Thank you for your suggestion, the sequence of miR-155 were obtained from miRbase and we list the number of miRbase (MI0026520) in the materials and methods. The mimic miRNA is microRNA chemically synthesized in vitro; miRNA Inhibitors are single-stranded oligonucleotides comprised of 2’-O-methyl residues that confer increased binding affinity to RNA targets and resistance to endonuclease degradation, both miR-155 mimics and inhibitor were synthesized by Tingske.  The efficient inhibitor suppresses the expression of miR-155 were detected before this experiment and we add this in the supplement.

Figure 5

Please re-illustrate fig. 5a because the lines between the bases are poorly connected.

Response: The connection between 3’-UTR and miRNA is not like the connection between primers, the miRNA in vivo is cervical-loop structures, and this connection between miR-155 and 3’-UTR of BDNF is enough for their function. And we are sorry for the missing link in the picture and we revised this in the MS (Fig 5).

Figure 6

If BDRF siRNA suppress the expression of BDRF, you can show the suppression by western blotting of BDRF as shown in Fig. 5c.

Response: Thank you for your suggestion, siRNA is a double-stranded RNA molecule that is non-coding. It is also known as silencing RNA and short interfering RNA, and gene can be silenced by a synthetic siRNA with a complementary sequence. So the sign of siRNA function is reducing the expression of it’s target gene. And the expression of mRNA is the most direct result.

L325-347

Again, I could not understand the author’s argument. Your arguments may be correct, but are they novel knowledge?

Response: In fact, this part we draw the conclusion from previous study and our experiment. And we found that before puberty, long photoperiod could suppress gonadal development, and after puberty, short photoperiod could suppress gonadal development. However, the SL-LL group is contrary to this rule, the fish were treated by short photoperiod before puberty, and long photoperiod after puberty. Then we think that a long photoperiod (24L:0D) being used to suppress gonadal development in Atlantic salmon before puberty, after which this photoperiod is shortened to 18L:6D or 12L:12D following puberty to similarly delay gonadal development. We think we are the first one to use this combined photoperiod in Atlantic salmon reared in RAS.

L359-362

At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

Response: In this paper, we give the evidence that miR-155 could play a role in photoperiodic reproduction. However, the stage III and stage IV were not very clear in some fish, because the gonad stage developed between stage III and stage IV. And we revised this in the MS.

Photoperiod may influence the expression of miR-155, but I do not think so from only Fig. 3. In addition, please discuss how the expression is influenced by photoperiod.

Response: Thank you for your suggestion. In this paper, we think we could provide the evidence that miR-155 play a role in photoperiodic reproduction. And how the expression is influenced by photoperiod need more research，this paper we just confirmed miR-155 could mediated the photoperiodic reproduction by targeting BDNF.

L364

The authors measured the expression of FSH receptor gene but did not measure FSH level. I would like to know why the authors measured the receptor not FSH and LH. FSH and LH are directly regulated by GnRH, but I do not know whether the expression of receptors in the gonads are regulated by hypothalamic GnRH much less miR-155.

Response: In fact, FSH and LH were not only regulated by GnRH directly, GnRH first regulate GnH in hypothalamus, then GnH regulate the release the secretion of FSH/LH. In this paper, we have found miR-155 could regulate GnRH via BDNF, and to confirm the gonadal development also could be regulate by this process，we test the receptor of FSH and LH in Atlantic salmon gonad. In this part we mainly want to know the response in gonad, so we did not detect the FSH and LH in serum. And this part we just want to found the relationship between miR-155 and gonad development.

Round 2

Reviewer 2 Report

Totally, I think these studies were not performed under fully-considered plan. The authors showed that miR155 downregulate GnRH and GTH receptors (Fig.4), and BDNF (Fig. 5), because the inhibitor of miR155 increased mRNA expression of these genes. However, I could not understand whether miR155 mimic normally function or not. In addition, it was shown in this paper that BDNF siRNA suppresses mRNA expression of BDNF. Therefore, the authors cannot conclude miR155 related to gonadal development (GnRH and GTH expression) through the binding to BDNF gene.

In addition, I unsatisfied the response to the question why two types of experimental groups (constant and gradual change) were prepared. Bellows are my questions to the responses.    

1.

(Question)    Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

(Author’s response)   In this paper, the fish were divided into 6 groups, four constant photoperiod groups and two gradual changes photoperiod groups. This two changes photoperiod groups were used to confirm the role of photoperiod. Cause the in change photoperiod groups, the photoperiod could change from long photoperiod to short photoperiod in one group, and the photoperiod could change from short photoperiod to long photoperiod in another changing photoperiod group. So along with the gonadal development, we think the change photoperiod will provide more evidence of the effect of photoperiod on gonadal development. In addition, 0L:24D means all darkness in 24h , the fish cannot find their food in total darkness environment, so  the fish cannot develop normally.

(Review’s question to the author’s response)

   Please mention the difference of the purpose between constant photoperiod groups and gradual change groups, and write them in ‘Materials and methods’ or ‘Discussion’. In responses, the authors described the purpose of two gradual changes photoperiod groups, but not four constant photoperiod groups. In addition, please discuss novel findings clarified by using two types of groups.

2.

(Question)   Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

(Response)   Thank you for your suggestion, in our experiment, the gonad were matured at stage IV by HE. When gonad developed into stage V，the gonad has begun to degenerate (supplementary figure). Furthermore, some fish gonad stage were between IV and V, and it’s hard to determine their precise stage. So we sum these two stage together.

(Review’s question to the author’s response)

   I understood that it’s hard to determine their precise stage in the response. If so, how the stage was determined in Fig. 1? Indeed, you clearly classified the maturational stage in Fig. 3, and the classification among the experimental groups were largely different. In particular, Stage IV and V was not so different between 24L:0D and SL-LL in female, but Stage V was predominant in 24:0D but Stage IV was predominant in SL-LL. I think that the authors should be discuss the meaning.

3.

(Question)   How did the authors identify Atlantic salmon miR-155? At first, the authors should explain the results. Next, please explain the miR-155 mimic and the difference from Atlantic salmon miR-155, and whether ‘miR-155 mimic’ mimic the native salmon miR-155, and how the inhibitor suppresses the function of the mimic and native miR-155.

If ‘inhibitor’ surely inhibits the expression of the native miR-155, you can exhibit that the inhibitor suppresses the expression level of the native miR-155 using the same cDNA template as that of figure 4b. Please show it.

 (Response)  Thank you for your suggestion, the sequence of miR-155 were obtained from miRbase and we list the number of miRbase (MI0026520) in the materials and methods. The mimic miRNA is microRNA chemically synthesized in vitro; miRNA Inhibitors are single-stranded oligonucleotides comprised of 2’-O-methyl residues that confer increased binding affinity to RNA targets and resistance to endonuclease degradation, both miR-155 mimics and inhibitor were synthesized by Tingske.  The efficient inhibitor suppresses the expression of miR-155 were detected before this experiment and we add this in the supplement.

(Review’s question to the author’s response)

   Please mention in text how the results in supplemental Fig. 4 were obtained. In addition, please explain why miR-155 expression increased by the exposure of miR155 mimics. I understood that miR155 inhibitor suppressed the expression of miR155, but did not understand that the mimic normally functions; thus, please explain.   

4.

 (Question)   Figure 6  If BDRF siRNA suppress the expression of BDRF, you can show the suppression by western blotting of BDRF as shown in Fig. 5c.

 (Response)   Thank you for your suggestion, siRNA is a double-stranded RNA molecule that is non-coding. It is also known as silencing RNA and short interfering RNA, and gene can be silenced by a synthetic siRNA with a complementary sequence. So the sign of siRNA function is reducing the expression of it’s target gene. And the expression of mRNA is the most direct result.

(Review’s question to the author’s response)

   I would like to know whether BDRF siRNA suppress the expression of BDRF mRNA or the peptide. If the authors do not show that BDRF siRNA induce the silencing of the mRNA, the authors cannot conclude that the disfunction of BDRF by miR155 suppressed GnRH and melatonin levels.

5.

(Question)   At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

(Response)   In this paper, we give the evidence that miR-155 could play a role in photoperiodic reproduction. However, the stage III and stage IV were not very clear in some fish, because the gonad stage developed between stage III and stage IV. And we revised this in the MS.

(Review’s question to the author’s response)

   If so, as you say that ‘it’s hard to determine their precise stage in the response’, the classification of maturational stage is not almost confirmed in this study. The authors should establish criteria for determination of maturational stage and classify them based on the criteria.

Author Response

Totally, I think these studies were not performed under fully-considered plan. The authors showed that miR155 downregulate GnRH and GTH receptors (Fig.4), and BDNF (Fig. 5), because the inhibitor of miR155 increased mRNA expression of these genes. However, I could not understand whether miR155 mimic normally function or not. In addition, it was shown in this paper that BDNF siRNA suppresses mRNA expression of BDNF. Therefore, the authors cannot conclude miR155 related to gonadal development (GnRH and GTH expression) through the binding to BDNF gene.

In addition, I unsatisfied the response to the question why two types of experimental groups (constant and gradual change) were prepared. Bellows are my questions to the responses.    

1.

(Question)    Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

(Author’s response)   In this paper, the fish were divided into 6 groups, four constant photoperiod groups and two gradual changes photoperiod groups. This two changes photoperiod groups were used to confirm the role of photoperiod. Cause the in change photoperiod groups, the photoperiod could change from long photoperiod to short photoperiod in one group, and the photoperiod could change from short photoperiod to long photoperiod in another changing photoperiod group. So along with the gonadal development, we think the change photoperiod will provide more evidence of the effect of photoperiod on gonadal development. In addition, 0L:24D means all darkness in 24h , the fish cannot find their food in total darkness environment, so the fish cannot develop normally.

(Review’s question to the author’s response)

   Please mention the difference of the purpose between constant photoperiod groups and gradual change groups, and write them in ‘Materials and methods’ or ‘Discussion’. In responses, the authors  groups. In addition, please discuss novel findings clarified by using two types of groups.

 Response:  In this paper, we think the photoperiod might have a cumulative effect on development of fish. So, we set two changing photoperiod group, one changing photoperiod group change the photoperiod from LL to SL, and another changing photoperiod group change the photoperiod from SL to LL. These two groups can give us the information that the effect of photoperiod on fish gonad development might be instantaneous. It’s a interaction between photoperiod and gonad development.

2.

(Question)   Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

(Response)   Thank you for your suggestion, in our experiment, the gonad were matured at stage IV by HE. When gonad developed into stage V，the gonad has begun to degenerate (supplementary figure). Furthermore, some fish gonad stage were between IV and V, and it’s hard to determine their precise stage. So we sum these two stage together.

(Review’s question to the author’s response)

   I understood that it’s hard to determine their precise stage in the response. If so, how the stage was determined in Fig. 1? Indeed, you clearly classified the maturational stage in Fig. 3, and the classification among the experimental groups were largely different. In particular, Stage IV and V was not so different between 24L:0D and SL-LL in female, but Stage V was predominant in 24:0D but Stage IV was predominant in SL-LL. I think that the authors should be discuss the meaning.

Response:  We sum the stage IV and V, because all these two stages for fish are matured. According to our results, we think the development of fish in 24L:0D group is faster than SL-LL group, and we revised this in the discussion.

3.

(Question)   How did the authors identify Atlantic salmon miR-155? At first, the authors should explain the results. Next, please explain the miR-155 mimic and the difference from Atlantic salmon miR-155, and whether ‘miR-155 mimic’ mimic the native salmon miR-155, and how the inhibitor suppresses the function of the mimic and native miR-155.

If ‘inhibitor’ surely inhibits the expression of the native miR-155, you can exhibit that the inhibitor suppresses the expression level of the native miR-155 using the same cDNA template as that of figure 4b. Please show it.

 (Response)  Thank you for your suggestion, the sequence of miR-155 were obtained from miRbase and we list the number of miRbase (MI0026520) in the materials and methods. The mimic miRNA is microRNA chemically synthesized in vitro; miRNA Inhibitors are single-stranded oligonucleotides comprised of 2’-O-methyl residues that confer increased binding affinity to RNA targets and resistance to endonuclease degradation, both miR-155 mimics and inhibitor were synthesized by Tingske.  The efficient inhibitor suppresses the expression of miR-155 were detected before this experiment and we add this in the supplement.

(Review’s question to the author’s response)

   Please mention in text how the results in supplemental Fig. 4 were obtained. In addition, please explain why miR-155 expression increased by the exposure of miR155 mimics. I understood that miR155 inhibitor suppressed the expression of miR155, but did not understand that the mimic normally functions; thus, please explain.   

 Response: Maybe the reviewer have some misunderstanding with miRNA mimics. In fact, miRNA mimics have the same function with miRNA. For example, if we want to verify the function of some gene, we also synthesis the gene in vitro, then transferred the synthesized gene into in vivo. For miRNA， the synthesized miRNA in vitro called miRNA mimics，in this paper, when we put miRNA mimics into fish, they will perform the same function as miRNA in vivo. And when we injected the miR-155 into fish tissue, this could increase the instantaneous content of miR-155.

4.

 (Question)   Figure 6  If BDRF siRNA suppress the expression of BDRF, you can show the suppression by western blotting of BDRF as shown in Fig. 5c.

 (Response)   Thank you for your suggestion, siRNA is a double-stranded RNA molecule that is non-coding. It is also known as silencing RNA and short interfering RNA, and gene can be silenced by a synthetic siRNA with a complementary sequence. So the sign of siRNA function is reducing the expression of its target gene. And the expression of mRNA is the most direct result.

(Review’s question to the author’s response)

   I would like to know whether BDRF siRNA suppress the expression of BDRF mRNA or the peptide. If the authors do not show that BDRF siRNA induce the silencing of the mRNA, the authors cannot conclude that the disfunction of BDRF by miR155 suppressed GnRH and melatonin levels.

 Response: The function of siRNA is silencing or interfering the expression of target mRNA，so BDNF siRNA could decrease the expression of BDNF mRNA. And siRNA were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China), we have detected its silencing effect by a simply PCR, so we did not put it in the paper. We add it in the supplementary figure.

5.

(Question)   At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

(Response)   In this paper, we give the evidence that miR-155 could play a role in photoperiodic reproduction. However, the stage III and stage IV were not very clear in some fish, because the gonad stage developed between stage III and stage IV. And we revised this in the MS.

(Review’s question to the author’s response)

   If so, as you say that ‘it’s hard to determine their precise stage in the response’, the classification of maturational stage is not almost confirmed in this study. The authors should establish criteria for determination of maturational stage and classify them based on the criteria.

 Response: Thank you for your suggestion, until now, H&E is the most precise method to determine the maturational stage. However, the development of fish gonad is continuous, and the gonad stage of several fish is between III and stage IV. And this part we have some wrong statements, and we revised this in last response.

Round 3

Reviewer 2 Report

I unsatisfied two responses: 1) the question why two types of experimental groups (constant and gradual change) were prepared and 2) the classification of gonadal stage. Bellows are my questions and comments to the responses.

In addition, I strongly request that the authors describe line numbers of the revised parts and additional parts in the revised manuscript, which is common sense in the response.

1.

(1st Question)    Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

(1st Author’s response)   In this paper, the fish were divided into 6 groups, four constant photoperiod groups and two gradual changes photoperiod groups. This two changes photoperiod groups were used to confirm the role of photoperiod. Cause the in change photoperiod groups, the photoperiod could change from long photoperiod to short photoperiod in one group, and the photoperiod could change from short photoperiod to long photoperiod in another changing photoperiod group. So along with the gonadal development, we think the change photoperiod will provide more evidence of the effect of photoperiod on gonadal development. In addition, 0L:24D means all darkness in 24h , the fish cannot find their food in total darkness environment, so the fish cannot develop normally.

 (2nd question to 1st author’s response)   Please mention the difference of the purpose between constant photoperiod groups and gradual change groups, and write them in ‘Materials and methods’ or ‘Discussion’. In responses, the authors groups. In addition, please discuss novel findings clarified by using two types of groups.

(2nd Author’s response)   In this paper, we think the photoperiod might have a cumulative effect on development of fish. So, we set two changing photoperiod group, one changing photoperiod group change the photoperiod from LL to SL, and another changing photoperiod group change the photoperiod from SL to LL. These two groups can give us the information that the effect of photoperiod on fish gonad development might be instantaneous. It’s a interaction between photoperiod and gonad development.

3rd question to 2nd author’s response:

I think that the authors have never mentioned consistently the purpose of the constant photoperiod groups. I request repeatedly that the authors mention the differences of purposes between gradual changes and constant. At first please discuss separately the facts obtained from the constant groups and the gradual groups, and then discuss totally the effects of photoperiod on gonadal development from these two experiments.

2.

(1st Question)    Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

(1st Author’s response)   Thank you for your suggestion, in our experiment, the gonad were matured at stage IV by HE. When gonad developed into stage V，the gonad has begun to degenerate (supplementary figure). Furthermore, some fish gonad stage were between IV and V, and it’s hard to determine their precise stage. So we sum these two stage together.

 (2nd question to 1st author’s response)    I understood that it’s hard to determine their precise stage in the response. If so, how the stage was determined in Fig. 1? Indeed, you clearly classified the maturational stage in Fig. 3, and the classification among the experimental groups were largely different. In particular, Stage IV and V was not so different between 24L:0D and SL-LL in female, but Stage V was predominant in 24:0D but Stage IV was predominant in SL-LL. I think that the authors should be discuss the meaning.

(2nd Author’s response)   We sum the stage IV and V, because all these two stages for fish are matured. According to our results, we think the development of fish in 24L:0D group is faster than SL-LL group, and we revised this in the discussion.

3rd question to 2nd author’s response:

As mentioned in previous review, I understood that it’s hard to determine their precise stage in the response. If so, is the classification in Fig.1 unreliable? If it is unreliable, the authors should newly classify the maturational stage according to new criteria to make the reliable figure. However, I do not that Fig.1 is unreliable even if it may contain slight errors, and the maturational stage was clearly different between SL-LL and 24L:0D. This may related to the difference between the constant condition and the gradual condition; thus, I said that “please discuss separately the facts obtained from the constant groups and the gradual groups, and then discuss totally the effects of photoperiod on gonadal development from these two experiments.” in above comments.

3.

(1st Question)    At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

(1st Author’s response)   In this paper, we give the evidence that miR-155 could play a role in photoperiodic reproduction. However, the stage III and stage IV were not very clear in some fish, because the gonad stage developed between stage III and stage IV. And we revised this in the MS.

 (2nd question to 1st author’s response)   If so, as you say that ‘it’s hard to determine their precise stage in the response’, the classification of maturational stage is not almost confirmed in this study. The authors should establish criteria for determination of maturational stage and classify them based on the criteria.

(2nd Author’s response)   Thank you for your suggestion, until now, H&E is the most precise method to determine the maturational stage. However, the development of fish gonad is continuous, and the gonad stage of several fish is between III and stage IV. And this part we have some wrong statements, and we revised this in last response.

3rd question to 2nd author’s response:

If so, the criteria should be modified and the stage should be determined according to the modified criteria. For example, unclear stage is classified to stage IV and so on.

In addition, which sex data are Fig 3? Although I may have commented, Fig 3 should be prepared separately for male and female.

Author Response

Dear editor and reviewers

Thank you for your excellent work for this paper, especially the reviewer give us a lot of important suggestions to improve the quality of our paper. And we responsed the reviewer as follow:

I unsatisfied two responses: 1) the question why two types of experimental groups (constant and gradual change) were prepared and 2) the classification of gonadal stage. Bellows are my questions and comments to the responses.

In addition, I strongly request that the authors describe line numbers of the revised parts and additional parts in the revised manuscript, which is common sense in the response.

1.

(1st Question)    Please explain why the authors prepare two groups: constant photoperiod groups and gradual change groups. I (also readers) would like to particularly know the aims of gradual change groups. The aims should be discussed in ‘Materials and methods’ or ‘Discussion’. In addition, why was not 0L:24D group prepared?

(1st Author’s response)   In this paper, the fish were divided into 6 groups, four constant photoperiod groups and two gradual changes photoperiod groups. This two changes photoperiod groups were used to confirm the role of photoperiod. Cause the in change photoperiod groups, the photoperiod could change from long photoperiod to short photoperiod in one group, and the photoperiod could change from short photoperiod to long photoperiod in another changing photoperiod group. So along with the gonadal development, we think the change photoperiod will provide more evidence of the effect of photoperiod on gonadal development. In addition, 0L:24D means all darkness in 24h , the fish cannot find their food in total darkness environment, so the fish cannot develop normally.

 (2nd question to 1st author’s response)   Please mention the difference of the purpose between constant photoperiod groups and gradual change groups, and write them in ‘Materials and methods’ or ‘Discussion’. In responses, the authors groups. In addition, please discuss novel findings clarified by using two types of groups.

(2nd Author’s response)   In this paper, we think the photoperiod might have a cumulative effect on development of fish. So, we set two changing photoperiod group, one changing photoperiod group change the photoperiod from LL to SL, and another changing photoperiod group change the photoperiod from SL to LL. These two groups can give us the information that the effect of photoperiod on fish gonad development might be instantaneous. It’s a interaction between photoperiod and gonad development.

3rd question to 2nd author’s response:

I think that the authors have never mentioned consistently the purpose of the constant photoperiod groups. I request repeatedly that the authors mention the differences of purposes between gradual changes and constant. At first please discuss separately the facts obtained from the constant groups and the gradual groups, and then discuss totally the effects of photoperiod on gonadal development from these two experiments.

 Response: Thank you for suggestion, and we revised this in the MS (Line 364-375 ).

2.

(1st Question)    Why did the authors explain the sum of stage IV and V? I think that the authors should treat independently IV and V. Indeed, the sum are highest in SL-LL both in male and female, but stage IV is much more predominant in SL-LL compared with that in other groups.

(1st Author’s response)   Thank you for your suggestion, in our experiment, the gonad were matured at stage IV by HE. When gonad developed into stage V，the gonad has begun to degenerate (supplementary figure). Furthermore, some fish gonad stage were between IV and V, and it’s hard to determine their precise stage. So we sum these two stage together.

 (2nd question to 1st author’s response)    I understood that it’s hard to determine their precise stage in the response. If so, how the stage was determined in Fig. 1? Indeed, you clearly classified the maturational stage in Fig. 3, and the classification among the experimental groups were largely different. In particular, Stage IV and V was not so different between 24L:0D and SL-LL in female, but Stage V was predominant in 24:0D but Stage IV was predominant in SL-LL. I think that the authors should be discuss the meaning.

(2nd Author’s response)   We sum the stage IV and V, because all these two stages for fish are matured. According to our results, we think the development of fish in 24L:0D group is faster than SL-LL group, and we revised this in the discussion.

3rd question to 2nd author’s response:

As mentioned in previous review, I understood that it’s hard to determine their precise stage in the response. If so, is the classification in Fig.1 unreliable? If it is unreliable, the authors should newly classify the maturational stage according to new criteria to make the reliable figure. However, I do not that Fig.1 is unreliable even if it may contain slight errors, and the maturational stage was clearly different between SL-LL and 24L:0D. This may related to the difference between the constant condition and the gradual condition; thus, I said that “please discuss separately the facts obtained from the constant groups and the gradual groups, and then discuss totally the effects of photoperiod on gonadal development from these two experiments.” in above comments.

  Response: Thank you for your suggestion, in fact the only little fish develop to between the stage IV and stage V. In this paper, we judged female salmon by the diameter of fish egg, if the diameter of female fish egg greater than 8mm, we determined them as stage V. For male fish, we judge them by secondary spermatocytes and spermatozoa in H&E section. If the proportion of spermatozoa growing to 80% in male gonadal tissue, we determined the male fish gonad as stage V. Furthermore, both stage IV and stage V are all regarded as mature gonad. And we discussed the separately the facts obtained from the constant groups and the gradual groups in the MS (Line 364-375).

3.

(1st Question)    At later time points, the expression of miR-155 was higher in LL-SL as you said, but it was highest in 8L-16D. As mentioned above, the detailed data, including all stages and each sex, should be shown in Fig. 3 and then the relationship between the expression and maturational stage should be discussed.

(1st Author’s response)   In this paper, we give the evidence that miR-155 could play a role in photoperiodic reproduction. However, the stage III and stage IV were not very clear in some fish, because the gonad stage developed between stage III and stage IV. And we revised this in the MS.

 (2nd question to 1st author’s response)   If so, as you say that ‘it’s hard to determine their precise stage in the response’, the classification of maturational stage is not almost confirmed in this study. The authors should establish criteria for determination of maturational stage and classify them based on the criteria.

(2nd Author’s response)   Thank you for your suggestion, until now, H&E is the most precise method to determine the maturational stage. However, the development of fish gonad is continuous, and the gonad stage of several fish is between III and stage IV. And this part we have some wrong statements, and we revised this in last response.

3rd question to 2nd author’s response:

If so, the criteria should be modified and the stage should be determined according to the modified criteria. For example, unclear stage is classified to stage IV and so on.

In addition, which sex data are Fig 3? Although I may have commented, Fig 3 should be prepared separately for male and female.

Response: Thank you for your suggestion.  Back to the first round of response, we think we made some misunderstandings of reviewer’s comments. The highest miR-155 were found in 8L-16D, here, we make a wrong statement in our first version, and we revised this in the MS (Line 257). And the gonad stage in this part were clear, the fish with unclear gonad stage were not used in this part. In previous response, we misunderstand the reviewer’s comments, and we are very sorry for that. In addition, the fish used in this part were mixed gender. We separately for male and female in growth and development part, because we want to see whether photoperiod has a sex-dependent effect on fish growth and development. The results in first part showed that photoperiod did not have significant sex-dependent effect. Then the fish used in fig.3 were mixed with 3 female and 3 male fish (we revised this in the MS, Line 147-148), and they also did not show sex-dependent characteristic, so, we mixed them in one picture.

Round 4

Reviewer 2 Report

I have one comment to author’s response as mentioned below.

3rd question to 2nd author’s response:

If so, the criteria should be modified and the stage should be determined according to the modified criteria. For example, unclear stage is classified to stage IV and so on. In addition, which sex data are Fig 3? Although I may have commented, Fig 3 should be prepared separately for male and female.

Response: Thank you for your suggestion. Back to the first round of response, we think we made some misunderstandings of reviewer’s comments. The highest miR-155 were found in 8L-16D, here, we make a wrong statement in our first version, and we revised this in the MS (Line 257). And the gonad stage in this part were clear, the fish with unclear gonad stage were not used in this part. In previous response, we misunderstand the reviewer’s comments, and we are very sorry for that. In addition, the fish used in this part were mixed gender. We separately for male and female in growth and development part, because we want to see whether photoperiod has a sex-dependent effect on fish growth and development. The results in first part showed that photoperiod did not have significant sex-dependent effect. Then the fish used in fig.3 were mixed with 3 female and 3 male fish (we revised this in the MS, Line 147-148), and they also did not show sex-dependent characteristic, so, we mixed them in one picture

Question:

Is Stage V (the gonad stage in Fig.3b) clear? In addition, the ovary is not the same organ as the testis, and the authors should prepare figures separately for male ang female, which is common sense in biological research.

Author Response

Dear editor and reviewer, we revised the MS according to the reviewer's suggestion, and we are very thankful for reviewer's suggestion.

Question:

Is Stage V (the gonad stage in Fig.3b) clear? In addition, the ovary is not the same organ as the testis, and the authors should prepare figures separately for male ang female, which is common sense in biological research.

Response: Yes，we misunderstand your first question, and we are very sorry for this, the stage of gonad in Fig.3 is clear. Thank you for your suggestion, and we revised this in the MS, we revised the figure 3 separately for male and female.

Round 5

Reviewer 2 Report

Previous question (Is Stage V (the gonad stage in Fig.3b) clear?) means as followed. You said that as both Stage IV and V was matured, these groups were summed in 3.1 (line 222-229). Thus, I asked why miR-155 levels in Fig.3 were not evaluated using sum of Stage IV and V.

In addition, I felt strongly that newly-made Figs.3 was made by copy and paste of previous Figs.3. Do you think that these figs are different each other. I cannot believe your data. You are too dishonest. If you think that my feeling is wrong, please show me low data.

Comments for author File: Comments.pdf

Author Response

Dear reviewer:

 I think we might have some misunderstanding in the purpose of fig 3, and we think this also could be confused by reader, so we revised the MS according to your suggestion.

Previous question (Is Stage V (the gonad stage in Fig.3b) clear?) means as followed. You said that as both Stage IV and V was matured, these groups were summed in 3.1 (line 222-229). Thus, I asked why miR-155 levels in Fig.3 were not evaluated using sum of Stage IV and V.

In addition, I felt strongly that newly-made Figs.3 was made by copy and paste of previous Figs.3. Do you think that these figs are different each other. I cannot believe your data. You are too dishonest. If you think that my feeling is wrong, please show me low data.

Response: Firstly, the purpose of in fig.3 is to show that the expression pattern of miR-155 in Atlantic salmon could be influenced by photoperiod. So, we detected the miR-155 expression in Atlantic salmon exposed to different photoperiod treatments, during the early phases of the experiment and during the later phases of the experiment. In this part, we just want to see the relationship between photoperiod and miR-155. During experimental period, the development of fish were not not perfectly synchronized. Such as, in the end phases of our experiment, some fish gonad were stage IV and some fish gonad were stage V and some fish were still at stage II or stage III ( In fact some fish stop developing in our experiment ). We think the fish which developed to slow were not suitable in this part (their endocrine might be not normal), so we picked matured fish ( according H&E section) in this part. But the main purpose of this figure were to show the relationship between photoperiod and expression of miR-155, and we re-read our MS about of fig.3, and we found that our explanation might make reader misunderstand our purpose, we only hope reader could understand the relationship between photoperiod and expression pattern of miR-155 from fig.3  but our explanation might mislead reader to gonad development and we revised it in the MS（L249， L 254）.

  Secondly, we have said the differences of expression of miR-155 were not significantly between female and male, so we mixed them together in our previous version. In fact, we redraw the fig 3, but the differences between female and male were not showed clearly in the bar graph. We list a table to show the data.

 Expression of miR-155 in female fish during early phase of experiment

Expression of miR-155 in female fish during later phase of experiment

Expression of miR-155 in male fish during early phase of experiment

Expression of miR-155 in male fish during later phase of experiment

Round 6

Reviewer 2 Report

I checked the raw data for Fig. 3 as shown in the attached file. I am absolutely astonished by the high similarity between female and male in all groups and all maturational status. For example, in 24L:0D group of stage II and III, sample 1 was 1.23 (female) and 1.27 (male), sample 2 was 1.45 (f) and 1.47 (m), and sample 3 was 1.38 (f) and 1.36 (m). Other samples were also very, very, very similar (almost the same) between sample No. in female and male. As results, mean values and figures were almost the same between females and male. I never had such an experience in the whole course of my professional career; so, if raw data are not creations, I appreciate showing very, very, very beautiful data. However, a few standard deviations in the figures do not correspond to raw data (please see attached file). Totally, I cannot believe your data.

Comments for author File: Comments.pdf