Fisetin Attenuates D-Gal-Induced Ovarian Aging by Modulating Mitophagy via the AMPK/mTOR Pathway

Round 1

Reviewer 1 Report

The paper provides novel and interesting results related to the effect of fisetin on D-galactose-induced ovarian aging, including the mechanisms of action on mouse model.

The paper is well prepared and written. Applied methods are proper, results are presented rather clearly.

I will suggest some improvement:

- add the name of animal model on which the experiments were performed.

- explain all abbreviations when are used for the first time, including the name of genes.

- add in Figures the molecular weight (kDa) of proteins evaluated by Western blot.

- L152-154: add more information (including validation) about ELISA kits used for hormone determination.

- L547: please, move the first sentence to the Introduction or Discussion.

- if possible, add higher magnification of micrographs showing histological and immunohistochemical results.

- please describe precisely how BrdU-positive cells were calculated. They were calculated in any area, in granulosa cells or in theca layer or in entire wall of the follicle? How many follicles were examined? Add follicle size in which proliferating cells were scored.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The revised manuscript “Fisetin Attenuates D-Gal-Induced Ovarian Aging by Modulating Mitophagy via the AMPK/mTOR Pathway” by Juan Dong et al. is an original research work which explores exhaustively the role of Fisetin on induced ovarian aging.

The overall manuscript is novel, well organized, correctly presented and it is distinguished by the large number of methods used to confirm the working hypothesis. The use of in vivo and in vitro methodology stands out in achieving the stated objective.

Particularly, the introduction is clear and collaborate to properly introduce the aim of the work. Some minor observations to include in this section are listed in detailed comments. The material and methods section is exhaustive and, despite the several techniques used, is complete and provides all the elements to the proper interpretation and reproduction of the results obtained. About the results section, is very well organized in sections and includes pertinent figures which include many panels of data presentation. In some opportunities, the number of panels difficulted the figure overall interpretation. The discussion is correct, but some translational interpretation of the result can be included (see in detailed comments), especially considering the achievement of the doses used in women. The conclusion is correct and very well supported by the results obtained, the inclusion of the figure 9 reinforces the text. In addition, the bibliography cited is novel, pertinent and collaborates to correctly establish the conceptual framework.

In conclusion, according to this reviewer criterion, the manuscript can be published in Antioxidants after the consideration of minor changes.

Some minor observations can be mentioned to improve the work presentation:

• About the use of D-Gal. As authors stablished in line 100, D-Gal “closely mimicking natural aging”. Please, include some sentences to clearly present the limitation of the model used and how that can affect the translational interpretation of the results.
• Continuing with the translational relevance of the study, please, introduce information related to how the doses used of fisetin can be achievable in humans.
• In line 105, authors wrote “This study aims to investigate the protective effects of fisetin against ovarian aging in mice”. Because the study is realized after induction of aging, it is not correct to consider the protective effect of fisetin. Please, change the sentence to correlate it precisely with the first sentence of the conclusion “the existing data suggest that fisetin attenuates ovarian aging”.

Author Response

Please see the attachment.

Author Response File: Author Response.docx