Effects of IGF1 and IGF2 on In Vitro Ovarian Follicle Maturation in Rainbow Trout, Oncorhynchus mykiss

Round 1

Reviewer 1 Report

Intro:

Well written and structured

Methods:

Sample sizes are smaller to begin with and a bit concerning that intended replicates were dropped following incubation. Individual fish were also noted to have slightly variable stages/sizes of follicles prior to incubation steps. Understood that larger sample sizes are always complicated to achieve, but alright. 

Methods are practical but it would have been beneficial and more interesting to build off GVBD findings with additional techniques proposed in the author's own discussion (histology, mrna, etc)

Results:

Eliminate the introductory/instructional statement on ln 169-171

Basic but clear

Discussion:

Author has a strong understanding of the literature and conclusions are ok. Consider ways to shorten the section if possible. The length does not reflect the scope of methods.

Overall:

The study is clean and basic. It would benefit from some additional techniques to make it a stronger paper on the topic of IGFs and is a little long in the discussion based on the scope of work done. Overall, it was written well and that was appreciated. Ok for eventual publication.

Author Response

Reviewer 1

• Comment: Methods: Sample sizes are smaller to begin with and a bit concerning that intended replicates were dropped following incubation. Individual fish were also noted to have slightly variable stages/sizes of follicles prior to incubation steps. Understood that larger sample sizes are always complicated to achieve, but alright.

Response: Methods:  In my experience with similar studies, it is rare to find as many replicates as we included in our studies.  Using 3 replicate wells with 25 follicles per well to compare among treatments seems pretty standard, rarely is the experiment then repeated with tissues from 6 different fish split into separate trial dates (Experiment 1).  Experiment 2 was repeated with tissues from 34 fish with six fish for each of six trial dates, including 8 which were MIS competent and 26 were incompetent.  We consider the variation in follicle size and stage among the fish not only as a positive but necessary, as long as all treatments being compared were tested in tissues from each fish.  Acquisition of competence is a continuum and there is the possibility that IGFs act only during a small window along that continuum. The variation in stage and follicle size reduces the chance we missed that window. We provided detailed justification for why we dropped all the data from 2 fish in exp 2, in one the tissues died so there were no data, and in the other 98% of the follicles matured in response to MIS alone so there was no room to observe a potentiating effect of IGF1 which would therefore provide a false negative response. 

• Comment: Methods are practical, but it would have been beneficial and more interesting to build off GVBD findings with additional techniques proposed in the author’s own discussion (histology, mRNA ect.)

Response: The process of OMC (formation of gap junctions, expression of mPRs) has not been characterized in salmonids so major work would have to be done before we could address those questions for IGFs in this species.  I think our findings support the benefit of further characterizing OMC in salmonids focusing on LH and MIS actions, with IGFs as additional treatments.

• Comment: Results: Eliminate introductory/instructional statements lines 169-171.

Response: Done.

• Comment: Discussion: Author has a strong understanding of the literature and conclusions are ok. Consider ways to shorten the section if possible. The length does not reflect the scope of methods.

Response: Discussion: The physiological mechanisms (or lack thereof) of IGF regulation of OFM in rainbow trout is only one part of the paper.  The variation in IGF actions among species is equally of interest, and the trout being the only fish to date in which IGF does not have these actions is significant and worthy of discussion.  These broader implications are a main reason why we chose to publish in “Fishes” instead of a strictly physiology journal. We are hoping those who may study mechanisms of action of hormones that do regulate OFM in salmonids include IGF as a treatment, we plan to.

Reviewer 2 Report

Manuscript ID: fishes-2481171

Title: Effects of IGF1 and IGF2 on in vitro ovarian follicle maturation in rainbow trout, Oncorhynchus mykiss

This manuscript is of high quality in both terms of writing standards and study design and methodology. The results are high relevance to the scientific community. Therefore, prior to publication, I am suggesting only the minor changes that will be detailed here:

Line 44 - the author states the “MIS in most fish” and perhaps the most is not the most appropriate term, therefore please reconsider changing it.

Table 3 is very informative and essential for the understanding of the study. Therefore, I suggest moving it earlier, perhaps in the first place among tables and figures as knowing the initial state of oocyte maturation would be very beneficial for readers in order to easier understand the study design.

Line 117-118 – please state what was the control medium.

Line 161-171 – these lines are most likely mistaken, so I suggest removing them.

Figure 1. I suggest adding in the Figure title some more lines to describe better the figure (which fish, which state of the oocyte development etc), thus following the self-explanatory pattern of the figs. At present, it is indeed complicated to follow the treatments, especially for separate figs A and B, so it would be good to explain more as well.

Line 386 – it is stated that common carp is a multiple clutch ovulator. To the best of my knowledge, this is not correct. Common carp is a single clutch ovulator and I speak from pure experience of artificial reproduction of this species. Please double check:

https://doi.org/10.1016/j.ygcen.2007.01.007

"The commonalities in reproductive strategy and IGF actions between white perch and white bass, but not striped bass, is consistent with the notion that variation in IGF-I actions is linked to reproductive strategy. Comparison of IGF actions among other teleosts also supports this interpretation. Insulin-like growth factor actions are similar between striped bass and common carp, both single-clutch spawners. In both species IGF-I induces the resumption of meiosis via a steroid-independent pathway, increases production of the MIH, and does not induce OMC (Weber and Sullivan, 2000, Mukherjee et al., 2006). To date, the ability of IGFs to induce OMC has not been demonstrated in a single-clutch spawner. "

And modify the paragraph accordingly.

Line 408 – instead of ïn” please change to “is”

Finally, this is perhaps the manuscript of the highest quality I have evaluated thus far and it was my pleasure reviewing it. Therefore, after these minor comments and requirements, I suggest publishing.

Sincerely.

Author Response

Reviewer 2

• Comment: Line 44: The author states the ‘MIS in most fish’ and perhaps the most is not the most appropriate term, therefore please reconsider changing it.

Response: Line 44: Changed ‘most’ to ‘many species of fish’.

• Comment: Table 3 is very informative and essential for understanding the study. Therefore, I suggest moving it earlier, perhaps in the first pklace among tables and figures as knowing the initial stage of oocyte maturation would be very beneficial for readers in order to easier understand the study design.

Response: The data in Table 3 only pertains to the fish in Experiment 2 (Fig 2), so Table 3 has to come after the data for Experiment 1 (Table 2 & Fig 1). Fortunately, edits to the manuscript allowed me to move Table 2 which has the stage for the fish used in Experiment 1 (and fig 1) to the same page as Fig 1 and directly after Fig 1, making it clearer that the stages of the fish used to obtain the data in Fig 1, are shown in Table 2. 

• Comment: Line 117-118 – please state what was the control medium.

Response: Line 117: Edited text to indicate Control medium was TMM without hormone.

• Comment: Line 161-171 – these lines are most likely mistaken, so I suggest removing them.

Response: The lines were mistakes leftover from the template and were removed.

• Comment: Figure 1. I suggest adding in the figure title some more lines to describe better the figure (which fish, which state of the oocyte development ect.) thus, following the self-explanatory pattern of the figs. At present, it is indeed complicated to follow the treatments, especially for separate figs A and B. so it would be good to explain more as well.

Response: I kept going back and forth as to whether to separate the data from experiment 1 (Fig 1) into an “A” and “B” panel or just group the six fish together.   There was no designed difference between the two trials, just one trial was run earlier in the spawning season than the other and therefore the fish in the first trial were earlier in GVM than the second.  I added the labels ‘Trial 1’ and ‘Trial 2’ to the figure to indicate the difference between A and B was simply technical, and now Fig 1 better matches Table 3 which contains additional data from the same fish from the same experiment. I feel the rest of the information is readily available in Table 3.  I also changed the color of the bars in trial 2 to avoid thinking the colors have meaning other than fish identification. As mentioned earlier, the placement of Fig 1 and Table 3 on the same page also helps to put the story together.  Also note that in the materials and methods (line 136) I state “ Two trials were conducted  on two different days with tissues from three females in each trial.”

• Comment: Line 306 – it is stated that common carp is a multiple clutch ovulatory. To the best of my knowledge this is not correct. Common carp is a single clutch ovulatory and I speak from pure experience of artificial reproduction of this species. Please double check. (then passage from the authors own previous paper).  And modify, the paragraph accordingly.

Response: The reviewer’s identification that I misidentified common carp as a multiple-clutch ovulator is an important and embarrassing catch.  I’m not sure how I made this significant mistake since as the reviewer points out, in a previous paper I identified the common carp as a single-clutch ovulator and pointed out that all fish in which IGF was not able to induce OMC are single-clutch ovulators. I think it came from an editing error when constructing Table 1. This correction caused me to slightly but significantly modify my conclusions since this means IGF had the ability to induce OMC in all asynchronous and multiple-clutch ovulators studied to date, but no single-clutch ovulators, including rainbow trout. Not only did this cause me to revise the paragraph in question, I revised other passages including the last sentence of the Conclusions. So again, I want to thank the reviewer for finding this critical mistake.

• Comment: Line 408- instead of ‘in’ please change to ‘is’.Line 408. ‘in’ changed to ‘is’.

Response: Done, now line ‘397’.