Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Round 1

Reviewer 1 Report

• More description about why porcine, compared to another animal model, is better. Why porcine and not human?

• Overall, the methods section needs more work and needs to be more detailed. One example is Lines 72-77. What was the incubation temperature, the C02 values (since placed in a CO2 inclubater), speed of Gravite etc. Also.   

• Line 76-77 “The control group was treated at 1G in the same CO2 incubator”- This does not make it clear if all other conditions were the same. Were they?  Was a Gravite used but at a different angle to simulated 1G, or were plates just placed in the incubator?  This is important information that must be added.   From the diagram, it appears that the plates were just added into the incubator, and grown statically. Is this correct.  If so, it is essential that in the discussion, this caveat is noted. The fact that the cells were grown so differently could be the reason for the differences.

• Line 81- what is the cell culture medium that was used?
• Line 131- what program was used for statistical analyses?

• Lines 137-138: The absorbance value of the pGCs from the SMG group was 0.45 ± 0.03, which was lower than cells in the control group (0.49 ± 03, P < 0.001) (Figure 1A) – please check these statistical results. I am skeptical that these values are statistically significant.  I would say that perhaps the wrong test was used
• Discussion section: I would like to see a few added lines as to what the significance is of altered cell growth and morphology, that was observed in the study, when it comes to the astronaut. Would this lead to changes in reproduction and fertility? hormone balances?, cancer risk?

Author Response

Dear Prof. Dr. Rafael Franco and Editorial board,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of the manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows. The changes of our manuscript are highlighted in yellow font.

Response to Reviewer 1 Comments:

Point 1: More description about why porcine, compared to another animal model, is better. Why porcine and not human?

Response 1:  We had a plan to do a study to evaluate the effect of simulated microgravity on human granulosa cells. However, we encountered many difficulties in obtaining human granulosa cell samples due to some ethical issues. We therefore used a pig granulosa cell model for this study. We have good experience of the study in the proliferation and cytoskeleton of animal ovarian granulosa cells. Moreover, pig is one of the important animal model in the biomedical field, especially studies on transplantation, cloning, gene transfer... thus, we want to study the effect of simulated microgravity on pig ovary granulosa cells.

Point 2: Overall, the methods section needs more work and needs to be more detailed. One example is Lines 72-77. What was the incubation temperature, the C02 values (since placed in a CO2 inclubator), speed of Gravite etc. Also.   

Response 2: We have concluded the culture condition in CO₂ incubator in the method of “Microgravity simulation”. The Gravite has 4 programs for microgravity simulation, as follows:

The MODE C has been recommended for cell culture study, and in this study we also use MODE C for our experiments.

Point 3: Line 76-77 “The control group was treated at 1G in the same CO2 incubator”- This does not make it clear if all other conditions were the same. Were they?  Was a Gravite used but at a different angle to simulated 1G, or were plates just placed in the incubator?  This is important information that must be added.   From the diagram, it appears that the plates were just added into the incubator, and grown statically. Is this correct.  If so, it is essential that in the discussion, this caveat is noted. The fact that the cells were grown so differently could be the reason for the differences.

Response 3: In this study, we have used 2 groups for our experiments, including the SMG group (mode C operation of Gravite) and the control group. As we stated above, the Gravite has 4 modes for microgravity simulation (modes A, B, C, D). If the cell plates were placed in the Gravite and operated by one of four modes, the cell will be subjected to the simulated microgravity condition. When operating, this machine only has the function of generating simulated microgravity conditions. Thus, cell plates of control group were not placed in Gravite, they were placed in the lower tray of the same incubator. The cell plates of SMG group were placed in Gravite at the upper tray in CO₂ incubator. The position of these cell plates were demonstrated in Figure 1C of our manuscript.

Point 4: Line 81- what is the cell culture medium that was used?

Response 4: In this study, the medium DMEM/Ham’s F-12 (DMEM-12-A, Capricorn Scientific, Germany), with 15% FBS (FBS-HI-22B, Capricorn Scientific, Germany) and 1% Pen/Strep (PS-B, Capricorn Scientific, Germany) was used for all cell culture experiments.

Point 5: Line 131- what program was used for statistical analyses?

Response 5: In this study, we used Sigma Plot 11.0 to analyze the data.

Point 6: Lines 137-138: The absorbance value of the pGCs from the SMG group was 0.45 ± 0.03, which was lower than cells in the control group (0.49 ± 03, P < 0.001) (Figure 1A) – please check these statistical results. I am skeptical that these values are statistically significant.  I would say that perhaps the wrong test was used

Response 6: Thank you so much for your comment. This is our mistake in statistical analysis. We have corrected the P value for this experiment.

Point 7: Discussion section: I would like to see a few added lines as to what the significance is of altered cell growth and morphology, that was observed in the study, when it comes to the astronaut. Would this lead to changes in reproduction and fertility? hormone balances?, cancer risk?

Response 7: The study of the effect of microgravity on granulosa cells is still limited. In this study, we only evaluated the effect of simulated microgravity on changes in proliferation and morphology of granulosa cell. We have not yet investigated the functional changes of granulosa cells under simulated microgravity conditions. We are applying for a new project to study the effect of simulated microgravity on functional changes in granulosa, specifically the production of hormones, the interaction between granulosa cells and theca cells , as well as interactions between granulosa cells and oocyte during follicular development. Once the results of these studies are available, we will have more precise discussions about the role of granulosa cells in interacting with other cells under simulated microgravity conditions.

We hope that our corrections could meet your requirements,

Thank you so much.

Reviewer 2 Report

In this manuscript “Simulated microgravity induces the proliferative inhibition and morphological changes in porcine granulosa cells” authors describe how microgravity reduces proliferation of pGCs by altering the expression of proteins involved in cell cycle progression. Moreover, microgravity causes morphological changes in pGCs altering cytoskeleton fibres.

Overall the manuscript is well written and clear and offers several interesting points of discussion and development.

However, I have few points that should be addressed by the authors:

1) On page 5 lines 195-200, authors state: “The fibroblast-like shape was also observed in pGCs under 195 SMG. In addition, the rhomboid-shaped and pebble-like cells were detected in this group 196 (Figure 4C, 4D). This result revealed that the SMG condition induced morphological 197 changes in pGCs.” This morphological change in microgravity generating separate cellular groups, is particularly interesting also in light of another paper (doi: 10.1038/s41526-019-0088-x) detailing the same phenomenon but in another cell line. Could the author comment on that in the Discussion?

Moreover, and linked to that observation: do the authors know what happens to the pGCs cells if returned to normogravity after 72h in microgravity? Do they recover the shape and expression of cell cycle proteins?

2) The attenuated proliferation not due to cell death, observed in pGCs under microgravity could be due or accompanied by the acquisition by stem-like properties? Did the authors check the expression of stemness genes?

Author Response

Dear Prof. Dr. Rafael Franco and Editorial board,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of the manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows. The changes of our manuscript are highlighted in yellow font.

Response to Reviewer 2 Comments:

In this manuscript “Simulated microgravity induces the proliferative inhibition and morphological changes in porcine granulosa cells” authors describe how microgravity reduces proliferation of pGCs by altering the expression of proteins involved in cell cycle progression. Moreover, microgravity causes morphological changes in pGCs altering cytoskeleton fibres.

Overall the manuscript is well written and clear and offers several interesting points of discussion and development.

However, I have few points that should be addressed by the authors:

Point 1: On page 5 lines 195-200, authors state: “The fibroblast-like shape was also observed in pGCs under 195 SMG. In addition, the rhomboid-shaped and pebble-like cells were detected in this group 196 (Figure 4C, 4D). This result revealed that the SMG condition induced morphological 197 changes in pGCs.” This morphological change in microgravity generating separate cellular groups, is particularly interesting also in light of another paper (doi: 10.1038/s41526-019-0088-x) detailing the same phenomenon but in another cell line. Could the author comment on that in the Discussion?

Response 1: Thank you so much for your comment. We have discussed more about the effects of SMG conditions on the cell morphology in “Discussion” as follows:

The previous study reported that MCF7 cells grow as a normal 2D monolayer under static 1g-conditions, while SMG causes the appearance of floating-clump cells and adherent cells [27]. Human adult retinal epithelium cells grow in adherent monolayers during exposure to static 1g-conditions. In contrast, the these cells formed small compact round shape under SMG condition for 5 days and 10 days on the RPM [28]. Moreover, the real microgravity generates the changes in cell geometry and rapid cytoskeletal re-organization in primary human macrophages [29]. These studies revealed that cell morphology can be changed under SMG conditions. In the present work, SMG induced the alteration in pGC morphology, demonstrated by the different shapes such as fibroblast-like shape, rhomboid shape, and pebble-like shape [30].

Point  2: Moreover, and linked to that observation: do the authors know what happens to the pGCs cells if returned to normogravity after 72h in microgravity? Do they recover the shape and expression of cell cycle proteins?

Response 2: In this study, we have not checked what happens to the pGCs cells when they were returned to normal gravity after 72h in microgravity. This is a great idea that we would like to do in next study, along with the evaluation of the expression of stemness markers in pGCs.

Point 3: The attenuated proliferation not due to cell death, observed in pGCs under microgravity could be due or accompanied by the acquisition by stem-like properties? Did the authors check the expression of stemness genes?

Response 3: In this study, we have found that the attenuated proliferation of pGCs due to the alteration of cell cycle, especially the down-regulation of major regulators such as cdk4, cdk6 and cyclin D1. We completely agree with your comment, the decrease in pGC proliferation could be associated with stem-like properties, but we regret that we have not evaluated the stem-like properties of pGCs under simulated microgravity. We would like to thank you so much for your great suggestion. In further research,  we will check the expression of stemness marker such as Oct4, Nanog or Sox-2 in pGCs to clarify whether the attenuated proliferation of pGCs under SMG conditions is accompanied by the acquisition by stem-like properties.

We hope that our corrections could meet your requirements,

Thank you so much.