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Table S8 represents polymorphic markers between pairs of parents including some common polymorphic markers. Hence the total number represented in the table S8 is not additive and not matching with the numbers given in the text. 124 is the total polymorphic markers excluding repetition. 27 ILs were selected based on their agro-morphological similarity with recurrent parent ‘Krishna Hamsa’ and evaluated for background recovery. The list of 27 ILs has been included in the revised manuscript as suggested.
| 2 | 1 |
Correct it It was a pleasure to read this manuscript.
| null | null |
plants11050622_makarova
| 0 |
Mention of appendix at L602 is a typo error and has been removed
| 2 | 1 |
Author Response Point-by-point response to the reviewer's comments are given below The ms is the result of an intensive and years-long work of breeding, that eventually pyramidized several resistance genes and QTLs for abiotic traits into an indian elite rice variety.
| null | null |
plants11050622_makarova
| 0 |
We agree with your comments. The term ‘expression’ has, therefore, been changed to ‘abundance’ or ‘level’ throughout the manuscript.
| 2 | 1 |
The statistical significance of any result would be evaluated using multivariate analysis.
| null | null |
reprodmed3020015_makarova
| 0 |
All experiments were repeated at least two times. We have failed the description regarding the reproducible results in the previously submitted manuscript, and so revised the section of the methods in the revised manuscript (Page 4, lins143-143).
| 2 | 1 |
This result must be demonstrated in a substantively convincing manner.
| null | null |
reprodmed3020015_makarova
| 0 |
We agree that AG-205 is not a specific inhibitor of PGRMC1. Therefore, we examined the effects of siRNA-mediated PGRMC1 knockdown on decidualization. As you mentioned, this issue was not appropriately described in the previously submitted manuscript. The citations have been inserted in the revised manuscript (page 8, lines 258-263).
| 2 | 1 |
This result must be demonstrated in a substantively convincing manner.
| null | null |
reprodmed3020015_makarova
| 0 |
The name of the reagent has been corrected to AG-205 consistently throughout the manuscript.
| 2 | 1 |
“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.” Change to: Data of cell culture are expressed as the mean ± SD from three independent experiments.
| null | null |
reprodmed3020015_makarova
| 0 |
The sentence has been separated according to your suggestion.
| 2 | 1 |
There was no control in PGRMC1 genomic/CRISPR KO cells (not si/miRNA KO) to demonstrate that effects induced by AG-205 were due to PGRMC1.
| null | null |
reprodmed3020015_makarova
| 0 |
The description has been modified as followed (page 2, line 69). ‘Endometrial samples were obtained from Japanese patients with a normal menstrual cycle, ….’.
| 2 | 1 |
Such changes may occur at the translational or post-translational level.
| null | null |
reprodmed3020015_makarova
| 0 |
The primary cultured ESCs we used were confirmed immunocytochemically to be positive for vimentin, a marker for stromal cells, and negative for cytokeratin, a marker for epithelial cells (please see above figure). ESCs at early passages (between 2 and 6 passages) were used for the experiment. In this study, the passaged cells were used without freezing. However, we have confirmed that primary ESCs can be cryopreserved in CELLBANKER, and retain characteristics such as responsiveness to decidual stimuli. These descriptions have been added to the revised manuscript (page 3, lines 96-99). Figure. Immunocytochemistry (ICC) of vimentin and cytokeratin in isolated human ESCs.
| 2 | 1 |
In primary endometrial stem cell (ESC) culture they demonstrate that chemical induction (by db-cAMP/P4) of pseudo-decidualization in culture is accompanied by decreased PGRMC1 protein levels (fig.2).
| null | null |
reprodmed3020015_makarova
| 0 |
The protein samples were separated by SDS-PAGE (5-20% gradient gel) and transferred electrophoretically to polyvinylidene difluoride membranes (8 cm x 8.5 cm) for 60 min at constant current of 128 mA using a semi-dry transfer system (ATTO, Tokyo Japan). These conditions have been added to the Materials and Methods section (Pages 3 and 4, lines 127-130).
| 2 | 1 |
The authors have addressed most of my concerns.
| null | null |
reprodmed3020015_makarova
| 0 |
The catalogue number of the antibodies (PI-1000, PI-2000) were inserted (Page 4, line 134).
| 2 | 1 |
The authors could consider whether an inducible miRNA-resistant PGRMC1 promoter could be designed.
| null | null |
reprodmed3020015_makarova
| 0 |
The immunoreactive bands were visualized using an enhanced chemiluminescence (Western Lightning, PerkinElmer, Inc., Waltham, MA, USA) and analyzed with ImageQuant LAS 500 (semi-auto mode; GE Healthcare Japan, Tokyo Japan) (Page 4, lines 136-137).
| 2 | 1 |
The new text does not conceptually link PTMs with PGRMC1 protein stability, which is the only reason they should be mentioned.
| null | null |
reprodmed3020015_makarova
| 0 |
The densitometry analysis was carried out using the “Gel Plot” plug-in of the Image J software. This plug-in has been described in the Materials and Methods (Page 4, line 139) 12)
| 2 | 1 |
Suggest change to; “Further study is required to determine the relationship between miR-98-mediatd PGRMC1 regulation and pregnancy.
| null | null |
reprodmed3020015_makarova
| 0 |
As you indicated, all data are expressed consistently as means and standard deviation (Figs 1 to 4). In our previously submitted manuscript, we had described carelessly the data of as for figure 5B and C with s.d.
| 2 | 1 |
The work would be worthwhile publishing in a revised format, where it should be a well cited publication.
| null | null |
reprodmed3020015_makarova
| 0 |
Thank you for pointing this out. The mistakes have been corrected (Pages 4,5 and 7, lines 160, 174 and 217).
| 2 | 1 |
Sensitivity of a phenomenon to AG-205 is consistent with possible PGRMC1 involvement, but does not demonstrate PGRMC1 involvement.
| null | null |
reprodmed3020015_makarova
| 0 |
Based upon your suggestions, additional experiments have been performed and added individual Western blotting data from there different ESCs (named #1-3) in Figure 2.
| 2 | 1 |
PGRMC1 protein instability could be the dominant effect involved, with miRNA transcript regulation playing only a minor part.
| null | null |
reprodmed3020015_makarova
| 0 |
Western blotting for PGRMC1 in figures 2, 3, and 4 have been replaced in the data with the whole molecular weight range. As you pointed out, PGRMC1 is known to be modified by sumoylation or ubiquitinylation which results in high molecular weight. The right panel shows the western blotting of PGRMC1. There are some higher molecular bands (white arrowhead) in lysates of ESCs, however, the band’s intensity was not changed by PGRMC1 siRNA treatment. Therefore, we believe that these high molecular bands detected in our experiment were non-specific bands.
| 2 | 1 |
Nine PGRMC1 lysines are known to be ubiquitinated (https://www.phosphosite.org/proteinAction.action?id=5744&showAllSites=true).
| null | null |
reprodmed3020015_makarova
| 0 |
As you pointed out, the possible involvement of posttranslational modification of endometrial PGRMC1 such as sumoylation and ubiquitinylation is interesting. Discussion regarding this possibility has been inserted in the revised manuscript (Page 9, lines 300-307). We will further examine whether posttranslational modification of PGRMC1 could be associated with decidualization in the next study.
| 2 | 1 |
Unfortunately, the authors have been unable to demonstrate the direct involvement of PGRMC1 downregulation in decidualization.
| null | null |
reprodmed3020015_makarova
| 0 |
The mistake has been corrected.
| 2 | 1 |
There is some concern about the specificity of the AG-205 inhibitor here and throughout, which weakens the deductive reasoning and is discussed below.
| null | null |
reprodmed3020015_makarova
| 0 |
All data of cell culture represent the mean ± SD of three independent experiments with at least repeated two times each experiment. The explanation of the experiments has been inserted the method (page 4, lines142-143).
| 2 | 1 |
PGRMC1 levels are also controlled by proteolysis, mediated by the ubiquitin proteasome system as described above.
| null | null |
reprodmed3020015_makarova
| 0 |
Thank you for your valuable advice to prove the significance of PGRMC1 downregulation in decidualization. Salsano et al. (19), reported that overexpression of PGRMC1 in ESCs abrogated the decidual markers expression (page 8. Lines 253-254). We will try to examine the effects of enforced expression of codon-altered PGRMC1 which is resistant for siRNA on decidualization in next study.
| 2 | 1 |
Author Response Responses to Reviewer 1 We appreciate your review of our manuscript and constructive comments and suggestions.
| null | null |
reprodmed3020015_makarova
| 0 |
Based upon your suggestions, we have added the data of PGRMC1 knockdown in figure 4. We confirmed efficient siRNA-mediate PGRMC1 knockdown by western blotting.
| 2 | 1 |
That new experimental result is what elevated the required changes to a major revision.
| null | null |
reprodmed3020015_makarova
| 0 |
The statistical analysis was not appropriate in the former manuscript. The data has been re-evaluated by a two-way ANOVA followed by a Turkey-Kramer multiple comparison test (page 4, line13).
| 2 | 1 |
Author Response Responses to Reviewer 2 We appreciate your review of our manuscript and constructive comments and suggestions.
| null | null |
reprodmed3020015_makarova
| 0 |
As you suggested, we should have presented the data in the same graph. However, the two experiments were performed independently. Therefore, we were not able to combine the results in the graph. Because treatment with P4 alone could not induce decidualization in the experiments, we examined the effect of PGRMC1 knockdown and inhibition on db-cAMP-induced decidualization. Further study would be needed to determine whether PGRMC1 is involved in the P4 action in endometrium.
| 2 | 1 |
The paragraph describes a result showing that the results are independent of P4.
| null | null |
reprodmed3020015_makarova
| 0 |
The description has been corrected and cited the references in the section (page 7, lines 223-224).
| 2 | 1 |
Suggest change to “In the present study, we showed that microRNA-mediated the regulation of PGRMC1 abundance during the process of decidualization.” (if this is what is meant) Lines 271-76.
| null | null |
reprodmed3020015_makarova
| 0 |
New discussion regarding the need for PGRMC1 knockout studies to clarify the role of PGRMC1 in decidualization has been inserted in page 9,
| 2 | 1 |
Enunciate what kind of role(s) PGRMC1 may be involved in (up/down regulation, when, how?).
| null | null |
reprodmed3020015_makarova
| 0 |
The error has been corrected.
| 2 | 1 |
If further mechanism exploration is carried out, the research will be more complete and persuasive.
| null | null |
reprodmed3020015_makarova
| 0 |
Your correction is what we meant to mention. We have replaced this sentence in the revised manuscript. (page9, line 284-286) 27)
| 2 | 1 |
It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1.
| null | null |
reprodmed3020015_makarova
| 0 |
As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, line 295-299).
| 2 | 1 |
The fact that PGRMC1 levels fall does not mean that low PGRMC1 levels are required.
| null | null |
reprodmed3020015_makarova
| 0 |
The report that PGRMC1 mediates H2O2-induced cell death in MCF7 cells has been inserted in discussion (page 10, lines 314-317)
| 2 | 1 |
This result must be demonstrated in a substantively convincing manner.
| null | null |
reprodmed3020015_makarova
| 0 |
We have suggested the possibility that PGRMC1 downregulation may be involved in ESCs senescence during the decidualization (page 10, lines 319-321).
| 2 | 1 |
PGRMC1 (Hpr6.6) increased the rate of cell death (in a non-apoptotic mechanism) in MCF-7 cancer cells in response to H2O2 (PMID: 14523988).
| null | null |
reprodmed3020015_makarova
| 0 |
As you pointed out, we infer that downregulation of PGRMC1 may be necessary for decidualization to proceed. To clarify this point, we further attempted to overexpress PGRMC1 by introducing expression vectors into primary ESCs under various conditions using lipofection and calcium phosphate transfection methods, as you suggested. Unfortunately, for reasons unknown, we were unable to establish ESCs overexpressing PGRMC1. In the present study, we found that not only knockdown of PGRMC1 but also functional inhibition with inhibitors promotes ESC decidualization in vitro. Furthermore, in a previous report, overexpression of PGRMC1 suppressed ESCs differentiation (Ref. 19). These results indicate that decreased PGRMC1 in the endometrium may be associated with accelerated dedifferentiation. As you suggested, we have changed the title significantly because further evidence was not available. We plan to establish a model of miR-resistant PGRMC1 overexpression using several ESCs cell lines to investigate the role of PGRMC1 in decidualization, and if possible, we would be happy to make this our next research topic.
| 4 | 1 |
The authors have addressed most of my concerns.
| null | null |
reprodmed3020015_makarova
| 0 |
Thank you for correcting the sentence. The description has been corrected in accordance with your suggestion (page 4, lines 141-142).
| 4 | 1 |
This is a critical point because the very title claims that PGRMC1 downregulation is required for decidualization.
| null | null |
reprodmed3020015_makarova
| 0 |
The error has been corrected (page 8, line 257).
| 4 | 1 |
PGRMC1 is known to be Sumoylated or ubiquitinylated and then degraded by the proteasome pathway, which lead to higher molecular weight species (PMID: 31067491).
| null | null |
reprodmed3020015_makarova
| 0 |
The description has been modified to the following (page 8, lines 261-266). “Although we cannot exclude the possible non-specific action of AG-205 on ESCs, the effect of AG-205 treatment and PGRMC1 knockdown promoted in vitro decidualization. These findings suggested that PGRMC1 downregulation may promote ESC decidualization during the secretory phase.
| 4 | 1 |
Line 261. mechanisms HAVE not (plural mechanisms) Lines 261-262.
| null | null |
reprodmed3020015_makarova
| 0 |
As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, lines 280-281).
| 4 | 1 |
In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
| null | null |
reprodmed3020015_makarova
| 0 |
As you pointed out, we did not explain the relationship between PGRMC1 stability and post-transcriptional regulation of the protein. We have added the following description (page 9, lines 304-308). ”These reports suggested that PGRMC1 protein levels can be modulated by changes in the rates of transcription, translation, and degradation. Therefore, further studies would be needed to clarify the possible involvement of the post-translational modification and degradation that may regulate PGRMC1 protein stability in ESCs during decidualization.”
| 4 | 1 |
In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes.
| null | null |
reprodmed3020015_makarova
| 0 |
We appreciate your review of our revised manuscript, constructive comments, and suggestions for improving the manuscript. We deeply understand the significance of the experiments using miR-98-resistant PGRMC stably expressing ESCs to directly prove the implication of the miR-mediated PGRMC1 regulation in decidualization. We are transfecting the miR-resistant PGRMC1 expression vector into ESCs cell lines to explore the role of PGRMC1 in decidualization but is not going well. As you advised, lentiviral transduction of the PGRMC1 along with fluorescent protein may enable us to effectively select the overexpressing cells without losing differentiation status in response to decidual stimuli. We have acknowledged this and suggested it as a topic for further research in the “Limitation” section of the revised manuscript following editor’s comment (page 10, lines 336-348). “Limitations: In this study, we proposed that miR-98-mediated PGRMC1 downregulation may be involved in decidualization, but were unable to examine the effects of miR-98-resistant PGRMC1 on differentiation due to technical difficulties establishing PGRMC1-overexpressing ESCs. miR-98 may not be the only miRNA that modulates PGRMC1 expression; therefore, comprehensive analysis of transcriptional, post-transcriptional, and post-translational regulation of endometrial PGRMC1 during decidualization is required. In addition, PGRMC1 can bind to various proteins, including receptors for epidermal growth factor [46] and insulin [47], cytochrome P450 [48], and serpine mRNA-binding protein 1 (SERBP1) [19]. Further, PGRMC1 may interact with proteins associated with endomembrane trafficking/cytoskeleton and mitochondrial functions in decidualizing ESCs [49]. Thus, we plan to investigate the modulation of endometrial PGRMC1 expression and the interaction between PGRMC1 and intracellular proteins during decidualization in the future.”
| 6 | 1 |
This requires stronger evidence than that currently presented.
| null | null |
reprodmed3020015_makarova
| 0 |
Our responses to your comments are as follows. We appreciate your review of our revised manuscript, constructive comments, and suggestions. As you pointed out, we have deleted the proposed model of the study (Figure 6). We have added the “Strength and limitation of this study” and “Conclusions” in the revised manuscript in accordance with the academic editor’s comments.
| 8 | 1 |
This result must be demonstrated in a substantively convincing manner.
| null | null |
reprodmed3020015_makarova
| 0 |
As you mentioned, our description of ESCs isolation may be inappropriate in terms of the patients with genetical differences. ESCs were isolated from histologically normal region in the patients with leiomyoma (age 42-45). It has been reported that ESCs obtained from the eutopic endometrium of the patients with endometriosis showed impaired decidualization. Therefore, we should consider the possible influence of leiomyoma on the decidual response, but we confirmed that the isolated ESCs have differentiated into decidual cells in response to cAMP analogue and progesterone, as you pointed out. The information of the patients has been inserted in the method 2)
| 2 | 1 |
In general, the manuscript refers often to PGRMC1 expression or downregulation.
| null | null |
reprodmed3020015_makarova
| 0 |
Because of the ethical difficulties of isolating ESCs from disease-free healthy women, we routinely used ESCs isolated from the patients in surgical cases. ESC lines are commercially available (T-HESC, ATCC) and maybe useful tool to investigate the role of PGRMC1 in decidualization, as you pointed out. However, the cell lines are also established from the endometrium of leiomyoma and immortalized by transfection with hTERT. Therefore, we believe that ESCs used in this study are no different than the above cells and cell lines in examining the role of PGRMC1.In the future, we plan to use several ESCs and their cell lines to clarify the precise mechanisms of PGRMC1 downregulation and decidualization.
| 2 | 1 |
It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1.
| null | null |
reprodmed3020015_makarova
| 0 |
We appreciate your review of our revised manuscript, constructive comments, and suggestions, which have helped us to improve the manuscript. We are thankful for the time and energy you expended.
| 4 | 1 |
The paragraph describes a result showing that the results are independent of P4.
| null | null |
reprodmed3020015_makarova
| 0 |
The author provided explanations for the questions. The manuscript has been modified accordingly. I think the manuscript could be accepted now.
| 3 | 2 |
Comparison of the miR-98/PGRMC1 abundance in proliferative and secretory phases of endometrial biopsy may contribute to the diagnostic prediction of uterine receptivity for embryo implantation.
| null | null |
reprodmed3020015_makarova
| 0 |
We agree with your comments. The term ‘expression’ has, therefore, been changed to ‘abundance’ or ‘level’ throughout the manuscript.
| 2 | 1 |
This cell culture experiment also seems to be based upon just one primary cell culture, split into replicates.
| null | null |
reprodmed3020015_perova
| 0 |
All experiments were repeated at least two times. We have failed the description regarding the reproducible results in the previously submitted manuscript, and so revised the section of the methods in the revised manuscript (Page 4, lins143-143).
| 2 | 1 |
However, in the first instance the manuscript cannot be published as it stands, and should therefore be rejected now with the stated option of subsequent resubmission only if new evidence is obtained.
| null | null |
reprodmed3020015_perova
| 0 |
We agree that AG-205 is not a specific inhibitor of PGRMC1. Therefore, we examined the effects of siRNA-mediated PGRMC1 knockdown on decidualization. As you mentioned, this issue was not appropriately described in the previously submitted manuscript. The citations have been inserted in the revised manuscript (page 8, lines 258-263).
| 2 | 1 |
Cell culture methods must be much more clearly explained so that the reader can reconstruct each exact experiment.
| null | null |
reprodmed3020015_perova
| 0 |
The name of the reagent has been corrected to AG-205 consistently throughout the manuscript.
| 2 | 1 |
Since this is the main publishable finding, the results as they stand do not merit publication.
| null | null |
reprodmed3020015_perova
| 0 |
The sentence has been separated according to your suggestion.
| 2 | 1 |
This result must be demonstrated in a substantively convincing manner.
| null | null |
reprodmed3020015_perova
| 0 |
The description has been modified as followed (page 2, line 69). ‘Endometrial samples were obtained from Japanese patients with a normal menstrual cycle, ….’.
| 2 | 1 |
The entire set of experiments needs to be replicated to demonstrate reproducibility before it can be considered to publish these results.
| null | null |
reprodmed3020015_perova
| 0 |
The primary cultured ESCs we used were confirmed immunocytochemically to be positive for vimentin, a marker for stromal cells, and negative for cytokeratin, a marker for epithelial cells (please see above figure). ESCs at early passages (between 2 and 6 passages) were used for the experiment. In this study, the passaged cells were used without freezing. However, we have confirmed that primary ESCs can be cryopreserved in CELLBANKER, and retain characteristics such as responsiveness to decidual stimuli. These descriptions have been added to the revised manuscript (page 3, lines 96-99).
| 2 | 1 |
The result must be shown for at least each ESC cell culture (each culture or patient named separately) obtained from each patient to demonstrate that the described results are representative of multiple patients, and not potentially atypical results from an atypical patient.
| null | null |
reprodmed3020015_perova
| 0 |
The protein samples were separated by SDS-PAGE (5-20% gradient gel) and transferred electrophoretically to polyvinylidene difluoride membranes (8 cm x 8.5 cm) for 60 min at constant current of 128 mA using a semi-dry transfer system (ATTO, Tokyo Japan). These conditions have been added to the Materials and Methods section (Pages 3 and 4, lines 127-130).
| 2 | 1 |
And please provide basic information of the patient.
| null | null |
reprodmed3020015_perova
| 0 |
The catalogue number of the antibodies (PI-1000, PI-2000) were inserted (Page 4, line 134).
| 2 | 1 |
This new text should be discussing the possibility of the latter also contributing to the observed effects, as well as the effects of mRNA level that the paper pursues.
| null | null |
reprodmed3020015_perova
| 0 |
The immunoreactive bands were visualized using an enhanced chemiluminescence (Western Lightning, PerkinElmer, Inc., Waltham, MA, USA) and analyzed with ImageQuant LAS 500 (semi-auto mode; GE Healthcare Japan, Tokyo Japan) (Page 4, lines 136-137).
| 2 | 1 |
Translational level changes can be due to changed levels of transcription, to altered mRNA stability (such as that proposed here for miR-98), or to altered efficiency of translation of a steady state mRNA pool.
| null | null |
reprodmed3020015_perova
| 0 |
The densitometry analysis was carried out using the “Gel Plot” plug-in of the Image J software. This plug-in has been described in the Materials and Methods (Page 4, line 139)
| 2 | 1 |
Therefore, a resubmission after major revision is recommended.
| null | null |
reprodmed3020015_perova
| 0 |
As you indicated, all data are expressed consistently as means and standard deviation (Figs 1 to 4). In our previously submitted manuscript, we had described carelessly the data of as for figure 5B and C with s.d.
| 2 | 1 |
The authors mention technical difficulties in establishing ESCs that express miRNA-resistant PGRMC1.
| null | null |
reprodmed3020015_perova
| 0 |
Thank you for pointing this out. The mistakes have been corrected (Pages 4,5 and 7, lines 160, 174 and 217).
| 2 | 1 |
“In the present study, we showed that microRNA-mediated PGRMC1 regulation during the process of decidualization.” This is confusingly worded.
| null | null |
reprodmed3020015_perova
| 0 |
Based upon your suggestions, additional experiments have been performed and added individual Western blotting data from there different ESCs (named #1-3) in Figure 2.
| 2 | 1 |
The authors are strongly advised to change from e.g.
| null | null |
reprodmed3020015_perova
| 0 |
Western blotting for PGRMC1 in figures 2, 3, and 4 have been replaced in the data with the whole molecular weight range. As you pointed out, PGRMC1 is known to be modified by sumoylation or ubiquitinylation which results in high molecular weight. The right panel shows the western blotting of PGRMC1. There are some higher molecular bands (white arrowhead) in lysates of ESCs, however, the band’s intensity was not changed by PGRMC1 siRNA treatment. Therefore, we believe that these high molecular bands detected in our experiment were non-specific bands.
| 2 | 1 |
This could be accomplished by making an expression plasmid encoding a codon-altered PGRMC1 gene, and cotransfecting this in a controlled matrix design with siRNA.
| null | null |
reprodmed3020015_perova
| 0 |
As you pointed out, the possible involvement of posttranslational modification of endometrial PGRMC1 such as sumoylation and ubiquitinylation is interesting. Discussion regarding this possibility has been inserted in the revised manuscript (Page 9, lines 300-307). We will further examine whether posttranslational modification of PGRMC1 could be associated with decidualization in the next study.
| 2 | 1 |
In fact, proteasomal degradation could be much more important that miRNA reduction of the transcript in the reduction of PGRMC1 levels during decidualization.
| null | null |
reprodmed3020015_perova
| 0 |
The mistake has been corrected.
| 2 | 1 |
Our responses to your comments are as follows.
| null | null |
reprodmed3020015_perova
| 0 |
All data of cell culture represent the mean ± SD of three independent experiments with at least repeated two times each experiment. The explanation of the experiments has been inserted the method (page 4, lines142-143).
| 2 | 1 |
The salient point concerning the ubiquitination events is that they are correlated for other proteins with proteasome-mediated degradation.
| null | null |
reprodmed3020015_perova
| 0 |
Thank you for your valuable advice to prove the significance of PGRMC1 downregulation in decidualization. Salsano et al. (19), reported that overexpression of PGRMC1 in ESCs abrogated the decidual markers expression (page 8. Lines 253-254). We will try to examine the effects of enforced expression of codon-altered PGRMC1 which is resistant for siRNA on decidualization in next study.
| 2 | 1 |
Some of the tissue samples are from patients with endometriosis, then according to eutopic endometrium determinism, whether such selection will cause the research results to be inaccurate？ The sample size is small.
| null | null |
reprodmed3020015_perova
| 0 |
Based upon your suggestions, we have added the data of PGRMC1 knockdown in figure 4. We confirmed efficient siRNA-mediate PGRMC1 knockdown by western blotting.
| 2 | 1 |
“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.” Change to: Data of cell culture are expressed as the mean ± SD from three independent experiments.
| null | null |
reprodmed3020015_perova
| 0 |
The statistical analysis was not appropriate in the former manuscript. The data has been re-evaluated by a two-way ANOVA followed by a Turkey-Kramer multiple comparison test (page 4, line13).
| 2 | 1 |
Result errors should not be expressed as SEM, but as standard deviation (s.d.).
| null | null |
reprodmed3020015_perova
| 0 |
As you suggested, we should have presented the data in the same graph. However, the two experiments were performed independently. Therefore, we were not able to combine the results in the graph. Because treatment with P4 alone could not induce decidualization in the experiments, we examined the effect of PGRMC1 knockdown and inhibition on db-cAMP-induced decidualization. Further study would be needed to determine whether PGRMC1 is involved in the P4 action in endometrium.
| 2 | 1 |
The methods could describe that all subjects were ethnically Japanese/Asian (or otherwise).
| null | null |
reprodmed3020015_perova
| 0 |
The description has been corrected and cited the references in the section (page 7, lines 223-224).
| 2 | 1 |
The discussion must qualify the results of Salsano et al.
| null | null |
reprodmed3020015_perova
| 0 |
New discussion regarding the need for PGRMC1 knockout studies to clarify the role of PGRMC1 in decidualization has been inserted in page 9, lines 281-282.
| 2 | 1 |
The authors are strongly advised to change from e.g.
| null | null |
reprodmed3020015_perova
| 0 |
The error has been corrected.
| 2 | 1 |
However, the control structure of comparisons is incorrect.
| null | null |
reprodmed3020015_perova
| 0 |
Your correction is what we meant to mention. We have replaced this sentence in the revised manuscript. (page9, line 284-286)
| 2 | 1 |
I requested a major revision, involving expression of an miRNA-resistant PGRMC1 ORF to convincingly demonstrate the mechanistic involvement of PGRMC1 down-regulation in decidualization, rather than a temporal correlation.
| null | null |
reprodmed3020015_perova
| 0 |
As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, line 295-299).
| 2 | 1 |
Enunciate what kind of role(s) PGRMC1 may be involved in (up/down regulation, when, how?).
| null | null |
reprodmed3020015_perova
| 0 |
The report that PGRMC1 mediates H2O2-induced cell death in MCF7 cells has been inserted in discussion (page 10, lines 314-317)
| 2 | 1 |
Nine PGRMC1 lysines are known to be ubiquitinated (https://www.phosphosite.org/proteinAction.action?id=5744&showAllSites=true).
| null | null |
reprodmed3020015_perova
| 0 |
We have suggested the possibility that PGRMC1 downregulation may be involved in ESCs senescence during the decidualization (page 10, lines 319-321).
| 2 | 1 |
Statistical sample sizes were unfavourably small.
| null | null |
reprodmed3020015_perova
| 0 |
We appreciate your review of our revised manuscript, constructive comments, and suggestions for improving the manuscript. We deeply understand the significance of the experiments using miR-98-resistant PGRMC stably expressing ESCs to directly prove the implication of the miR-mediated PGRMC1 regulation in decidualization. We are transfecting the miR-resistant PGRMC1 expression vector into ESCs cell lines to explore the role of PGRMC1 in decidualization but is not going well. As you advised, lentiviral transduction of the PGRMC1 along with fluorescent protein may enable us to effectively select the overexpressing cells without losing differentiation status in response to decidual stimuli. We have acknowledged this and suggested it as a topic for further research in the “Limitation” section of the revised manuscript following editor’s comment (page 10, lines 336-348). “Limitations: In this study, we proposed that miR-98-mediated PGRMC1 downregulation may be involved in decidualization, but were unable to examine the effects of miR-98-resistant PGRMC1 on differentiation due to technical difficulties establishing PGRMC1-overexpressing ESCs. miR-98 may not be the only miRNA that modulates PGRMC1 expression; therefore, comprehensive analysis of transcriptional, post-transcriptional, and post-translational regulation of endometrial PGRMC1 during decidualization is required. In addition, PGRMC1 can bind to various proteins, including receptors for epidermal growth factor [46] and insulin [47], cytochrome P450 [48], and serpine mRNA-binding protein 1 (SERBP1) [19]. Further, PGRMC1 may interact with proteins associated with endomembrane trafficking/cytoskeleton and mitochondrial functions in decidualizing ESCs [49]. Thus, we plan to investigate the modulation of endometrial PGRMC1 expression and the interaction between PGRMC1 and intracellular proteins during decidualization in the future.”
| 4 | 1 |
What percentage acrylamide were the SDS PAGE gels.
| null | null |
reprodmed3020015_perova
| 0 |
Our responses to your comments are as follows. We appreciate your review of our revised manuscript, constructive comments, and suggestions. As you pointed out, we have deleted the proposed model of the study (Figure 6). We have added the “Strength and limitation of this study” and “Conclusions” in the revised manuscript in accordance with the academic editor’s comments.
| 6 | 1 |
“Data of cell culture are expressed as the mean ± SD from three independent experiments with at least repeated two times.” Change to: Data of cell culture are expressed as the mean ± SD from three independent experiments.
| null | null |
reprodmed3020015_perova
| 0 |
As you mentioned, our description of ESCs isolation may be inappropriate in terms of the patients with genetical differences. ESCs were isolated from histologically normal region in the patients with leiomyoma (age 42-45). It has been reported that ESCs obtained from the eutopic endometrium of the patients with endometriosis showed impaired decidualization. Therefore, we should consider the possible influence of leiomyoma on the decidual response, but we confirmed that the isolated ESCs have differentiated into decidual cells in response to cAMP analogue and progesterone, as you pointed out. The information of the patients has been inserted in the method 2)
| 2 | 1 |
And please provide basic information of the patient.
| null | null |
reprodmed3020015_perova
| 0 |
Because of the ethical difficulties of isolating ESCs from disease-free healthy women, we routinely used ESCs isolated from the patients in surgical cases. ESC lines are commercially available (T-HESC, ATCC) and maybe useful tool to investigate the role of PGRMC1 in decidualization, as you pointed out. However, the cell lines are also established from the endometrium of leiomyoma and immortalized by transfection with hTERT. Therefore, we believe that ESCs used in this study are no different than the above cells and cell lines in examining the role of PGRMC1.In the future, we plan to use several ESCs and their cell lines to clarify the precise mechanisms of PGRMC1 downregulation and decidualization.
| 2 | 1 |
All reported results were observed in at least two independent experiments.
| null | null |
reprodmed3020015_perova
| 0 |
As you pointed out, we infer that downregulation of PGRMC1 may be necessary for decidualization to proceed. To clarify this point, we further attempted to overexpress PGRMC1 by introducing expression vectors into primary ESCs under various conditions using lipofection and calcium phosphate transfection methods, as you suggested. Unfortunately, for reasons unknown, we were unable to establish ESCs overexpressing PGRMC1. In the present study, we found that not only knockdown of PGRMC1 but also functional inhibition with inhibitors promotes ESC decidualization in vitro. Furthermore, in a previous report, overexpression of PGRMC1 suppressed ESCs differentiation (Ref. 19). These results indicate that decreased PGRMC1 in the endometrium may be associated with accelerated dedifferentiation. As you suggested, we have changed the title significantly because further evidence was not available. We plan to establish a model of miR-resistant PGRMC1 overexpression using several ESCs cell lines to investigate the role of PGRMC1 in decidualization, and if possible, we would be happy to make this our next research topic.
| 2 | 1 |
The authors could consider whether an inducible miRNA-resistant PGRMC1 promoter could be designed.
| null | null |
reprodmed3020015_perova
| 0 |
Thank you for correcting the sentence. The description has been corrected in accordance with your suggestion (page 4, lines 141-142).
| 2 | 1 |
Downregulation of PGRMC1 During ESC in vitro Decidualization.” All subsequent section header numbers in results should be modified to accommodate the new section.
| null | null |
reprodmed3020015_perova
| 0 |
The error has been corrected (page 8, line 257).
| 2 | 1 |
It is important to determine whether these mechanisms are both operating.
| null | null |
reprodmed3020015_perova
| 0 |
The description has been modified to the following (page 8, lines 261-266). “Although we cannot exclude the possible non-specific action of AG-205 on ESCs, the effect of AG-205 treatment and PGRMC1 knockdown promoted in vitro decidualization. These findings suggested that PGRMC1 downregulation may promote ESC decidualization during the secretory phase.
| 2 | 1 |
The authors have addressed most of my concerns.
| null | null |
reprodmed3020015_perova
| 0 |
As noted, the sentence has been separated and corrected in accordance with your suggestion (page 9, lines 280-281).
| 2 | 1 |
The author provided explanations for the questions.
| null | null |
reprodmed3020015_perova
| 0 |
As you pointed out, we did not explain the relationship between PGRMC1 stability and post-transcriptional regulation of the protein. We have added the following description (page 9, lines 304-308). ”These reports suggested that PGRMC1 protein levels can be modulated by changes in the rates of transcription, translation, and degradation. Therefore, further studies would be needed to clarify the possible involvement of the post-translational modification and degradation that may regulate PGRMC1 protein stability in ESCs during decidualization.”
| 2 | 1 |
In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes.
| null | null |
reprodmed3020015_perova
| 0 |
The XGM2016 model is parameterized as a spherical harmonic series expansion resolved to degree and order (d/o) 719, which is the maximum resolution supported by the 15′ terrestrial gravity grid and a satellite-only model GOCO05s. For XGM2016, a significant focus is the optimal combination of the new terrestrial data with the latest satellite gravity information. The combination is based on the rigorous solution of a full normal equation system up to the maximum d/o 719. The calculation of the XGM2019 spheroidal harmonic model coefficients up to d/o 719 consists of a weighted least squares adjustment of GOCO06s with the primary 15′ NGA ground gravity dataset. The XGM2016 and XGM2019 models used the 15′ NGA ground gravity dataset, however, the refined GFMs in this study are obtained by combining the GRACE/GOCE-based GGMs and EGM2008 model, The gravity field information of 5′ terrestrial gravity data in EGM2008 is fully utilized. In addition, to consider the influence of higher frequency gravity field signals caused by topography, the RTM is utilized to further compensate for the omission errors in the refined GGMs. In this study, the combination of the satellite-only GGM with the EGM2008 is based on a pure complementation of the spherical harmonic coefficients at a specific degree, We added comparisons for XGM2016 or XGM2019 models in Table 4, we can find that the refined GFMs outperform XGM2016 and XGM2019 as well, the major improvement of the refined GFMs can be attributed to the GOCE data and topography signals. Please refer to page 14, Line 435-457.
| 2 | 1 |
Importantly, even though all local height datums are re- 42 lated to the MSL, the vertical offsets between them may be up to 2 m at global scale [1].
| null | null |
rs14061437_makarova
| 0 |
Thank you for your good question and advice. the high-quality GNSS/levelling-based height anomalies are used to check the refined GGMs for obtaining the optimal combination degrees. Thus, the refined GGMs provide better local quasi-geoid results. We have added descriptions and contents in the revised manuscript. The combination of the satellite-only GGM with the EGM2008 in this study is based on a pure complementation of the spherical harmonic coefficients at a specific degree in this paper, which is different from rigorous combination that is done on the basis of the normal equations and co-variance by a least-squares. The purpose of this pure combination can provide better local quasi-geoid results, it is said that it can obtain the characteristics of spatial "localization" for quasi-geoid. Combining the satellite-only GFM with EGM2008 by applying some sophisticated weighting approach usually combines the maximum degree and order of the satellite-only GFM and the EGM2008. Because the degree errors of the satellite-only GFM increase with the increase in degree and order, the noise starts to dominate the signals at high degree and order. The noise of satellite-only GFM maybe introduce by sophisticated weighting approach. In addition, the rigorous combination based on the sophisticated weighting approach usually needs the full error variance-covariance matrix of the spherical harmonic coefficients, but, the full error variance-covariance matrix of the spherical harmonic coefficients might generally not be available. Although the obtained results are already quite promising, it can be expected that the refined GFMs provide a guidance for determining the quasi-geoid or the geopotential value of the vertical datum in China. However, the combination of the satellite-only GGM with the EGM2008 in this study is based on a pure complementation of the spherical harmonic coefficients at a specific degree. However, such a procedure might cause a spectral gap between both models. In the next step, the rigorous combination or a smooth transition (such as: using hanning window) will be considered to derived the refined GGMs. Please refer to page 17, Line 544-545.
| 2 | 1 |
I have a few minor points (marked in the attached PDF) and two general points, which I like you to address.
| null | null |
rs14061437_makarova
| 0 |
Please refer to page 2, Line 60.
| 2 | 1 |
Please comment on that question quantitatifely and qualitatively.
| null | null |
rs14061437_makarova
| 0 |
Please refer to page 2, Line 63.
| 2 | 1 |
I have a few minor points (marked in the attached PDF) and two general points, which I like you to address.
| null | null |
rs14061437_makarova
| 0 |
Please refer to page 11, Line 359.
| 2 | 1 |
Could you please comment on the effects you introduce by applying such a sharp truncation combination approach.
| null | null |
rs14061437_makarova
| 0 |
Thank you for your advice. We have rewritten the mean values. Please refer to page 16, Line 496.
| 2 | 1 |
What is the main methodological difference and advantage of your method?
| null | null |
rs14061437_makarova
| 0 |
Thank you for your comment. The unified topo data and Equation are used to compute RTM quasi-geoid height. We have added some expressions about the using of unified topo data and equations, which makes us easily misunderstand about the calculation process in the text. Please refer to page 12, Line 402.
| 2 | 1 |
Models Max Min Mean STD EIGEN-6C4 1.007 -1.696 0.048 0.187 GECO 1.579 -1.703 0.041 0.223 SGG-UGM-1 1.003 -1.671 0.052 0.194 SGG-UGM-2 1.003 -1.704 0.051 0.191 XGM2016 1.016 -1.757 -0.020 0.214 XGM2019 1.705 -1.737 0.081 0.213 Point 6: Your method for combining the satellite-only GFM with EGM2008 (sec.
| null | null |
rs14061437_makarova
| 0 |
Thank you. Done. Please refer to page 8, Line 259.
| 2 | 1 |
If you have any information, please don’t hesitate to let us know.
| null | null |
rs14061437_makarova
| 0 |
Thank you for your constructive suggestion. We have moved the discussion part in results to Discussion. The conclusions are recompiled and added according to your suggestions. Please refer to Line 498-550 and Line 551-589.
| 2 | 1 |
43 This is due to the fact that the MSL presents geographical and time-dependent variations, 44 Citation: Lastname, F.; Lastname, F.; Lastname, F. Title.
| null | null |
rs14061437_makarova
| 0 |
Please refer to page 2, Line 60.
| 2 | 1 |
However, the 36 ellipsoidal height is not related to the Earth's gravity field.
| null | null |
rs14061437_perova
| 0 |
Please refer to page 2, Line 63.
| 2 | 1 |
The goal of this paper is to derive 13 the geopotential value for the Chinese height datum (CNHD) in order to realize the height datum 14 unification in China.
| null | null |
rs14061437_perova
| 0 |
Please refer to page 11, Line 359.
| 2 | 1 |
Thus, the refined GGMs provide better local quasi-geoid results.
| null | null |
rs14061437_perova
| 0 |
Thank you for your advice. We have rewritten the mean values. Please refer to page 16, Line 496.
| 2 | 1 |
The refined GFMs are evaluated by using high-quality GNSS/lev- 22 elling data, the results show that the quasi-geoid accuracy of the refined DIR_R6_EGM2008_RTM 23 model in China has the optimal accuracy, and compared with the EGM2008 model and the DIR_R6 24 model, this refined model in China is improved by 9.6 cm and 21.8 cm, and the improvement ranges 25 are 35.7% and 55.8%, respectively.
| null | null |
rs14061437_perova
| 0 |
The XGM2016 model is parameterized as a spherical harmonic series expansion resolved to degree and order (d/o) 719, which is the maximum resolution supported by the 15′ terrestrial gravity grid and a satellite-only model GOCO05s. For XGM2016, a significant focus is the optimal combination of the new terrestrial data with the latest satellite gravity information. The combination is based on the rigorous solution of a full normal equation system up to the maximum d/o 719. The calculation of the XGM2019 spheroidal harmonic model coefficients up to d/o 719 consists of a weighted least squares adjustment of GOCO06s with the primary 15′ NGA ground gravity dataset. The XGM2016 and XGM2019 models used the 15′ NGA ground gravity dataset, however, the refined GFMs in this study are obtained by combining the GRACE/GOCE-based GGMs and EGM2008 model, The gravity field information of 5′ terrestrial gravity data in EGM2008 is fully utilized. In addition, to consider the influence of higher frequency gravity field signals caused by topography, the RTM is utilized to further compensate for the omission errors in the refined GGMs. In this study, the combination of the satellite-only GGM with the EGM2008 is based on a pure complementation of the spherical harmonic coefficients at a specific degree, We added comparisons for XGM2016 or XGM2019 models in Table 4, we can find that the refined GFMs outperform XGM2016 and XGM2019 as well, the major improvement of the refined GFMs can be attributed to the GOCE data and topography signals. Please refer to page 14, Line 435-457. Table 4. Statistics of the height anomaly differences between GNSS/levelling and six higher-degree GFMs. Unit: (m).
| 2 | 1 |
The ellipsoid height relative to a given geo- 35 centric ellipsoid can be obtained quickly and accurately by using GNSS.
| null | null |
rs14061437_perova
| 0 |
Thank you for your good question and advice. the high-quality GNSS/levelling-based height anomalies are used to check the refined GGMs for obtaining the optimal combination degrees. Thus, the refined GGMs provide better local quasi-geoid results. We have added descriptions and contents in the revised manuscript. The combination of the satellite-only GGM with the EGM2008 in this study is based on a pure complementation of the spherical harmonic coefficients at a specific degree in this paper, which is different from rigorous combination that is done on the basis of the normal equations and co-variance by a least-squares. The purpose of this pure combination can provide better local quasi-geoid results, it is said that it can obtain the characteristics of spatial "localization" for quasi-geoid. Combining the satellite-only GFM with EGM2008 by applying some sophisticated weighting approach usually combines the maximum degree and order of the satellite-only GFM and the EGM2008. Because the degree errors of the satellite-only GFM increase with the increase in degree and order, the noise starts to dominate the signals at high degree and order. The noise of satellite-only GFM maybe introduce by sophisticated weighting approach. In addition, the rigorous combination based on the sophisticated weighting approach usually needs the full error variance-covariance matrix of the spherical harmonic coefficients, but, the full error variance-covariance matrix of the spherical harmonic coefficients might generally not be available. Although the obtained results are already quite promising, it can be expected that the refined GFMs provide a guidance for determining the quasi-geoid or the geopotential value of the vertical datum in China. However, the combination of the satellite-only GGM with the EGM2008 in this study is based on a pure complementation of the spherical harmonic coefficients at a specific degree. However, such a procedure might cause a spectral gap between both models. In the next step, the rigorous combination or a smooth transition (such as: using hanning window) will be considered to derived the refined GGMs. Please refer to page 17, Line 544-545.
| 2 | 1 |
We have added descriptions and contents in the revised manuscript.
| null | null |
rs14061437_perova
| 0 |
Thank you for your comment. The unified topo data and Equation are used to compute RTM quasi-geoid height. We have added some expressions about the using of unified topo data and equations, which makes us easily misunderstand about the calculation process in the text. Please refer to page 12, Line 402.
| 2 | 1 |
It has improved the readability, clarity, and quality of our manuscript.
| null | null |
rs14061437_perova
| 0 |
Thank you. Done. Please refer to page 8, Line 259.
| 2 | 1 |
Response to Reviewer 1 Comments: We thank you for your constructive and detailed comments.
| null | null |
rs14061437_perova
| 0 |
Thank you for your constructive suggestion. We have moved the discussion part in results to Discussion. The conclusions are recompiled and added according to your suggestions. Please refer to Line 498-550 and Line 551-589.
| 2 | 1 |
Email: [email protected] 9 4 National Geomatics Center of China, Beijing 100830, China; [email protected]; [email protected] 10 * Correspondence: [email protected];Tel.
| null | null |
rs14061437_perova
| 0 |
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