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  • Junkun Lai1,2,3,4,5,6,7,
  • Xiaoyu Gao1,2,3,4,5,6,7 and
  • Guke Zhang1,2,3,4,5,6,7
  • et al.

Reviewer 1: Fangxiong Shi Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript answered my questions and concerns. I don't have any further comments.

Author Response

Thank you for your suggestion.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript entitled “Cloned pig fetuses have a high placental lysophosphatidylcholine level that inhibits trophoblast cell activity”, Lai et al, the authors investigated placental transcriptome and lipidomic profiles from 30-day-old somatic cell nuclear transfer (SCNT) and artificial insemination (AI)-produced pig fetuses. Based on the results of RNA-seq and lipidomics analyses, the authors state that high levels of lysophosphatidylcholine (LPC) in the placental tissues of cloned pig fetuses are the main cause of their abnormal development. They also conducted in vitro experiments using porcine trophoblast cells and reported that LPC affects cell proliferation and migration in these cells.

  1. The manuscript does not provide the approval number of the animal experiment. In addition, please indicate the exact dates (year, month, and day) when the AI and SCNT procedures were carried out in the five sows for each group.
  2. The authors state that three out of five sows became pregnant after AI and three out of five sows became pregnant after SCNT embryo transfer. Please provide detailed information on the number of fetuses obtained and the fetal measurement data.
  3. In Figure 1, the numbers are given as “AI group: n=9, SCNT group: n=9,” yet in Figure 2A, the AI group shows 3 points and the SCNT group 6 points, while in Figure 4A, the AI group shows 8 points and the SCNT group 8 points. This inconsistency requires clarification.
  4. In Figure 2A, the meaning of “IV4” is not explained. Please specify what this denotes.
  5. In Figure 3, the y-axis is labeled as log2(FC), but the line corresponding to 2 is missing. As a result, it is unclear whether the RNA-seq and qPCR results are consistent, and for some genes it is unclear whether they are upregulated or downregulated.
  6. The resolution of Figure 4 is insufficient for proper interpretation. In addition, the meaning of “NT” and “IV” in Figure 4A is not explained.
  7. In the cell culture experiments, the concentration of the cell suspension used for seeding is provided, but the actual volume of suspension added per well is not described. Therefore, the exact number of cells seeded per well cannot be determined.
  8. In several parts of the manuscript, the names, model numbers, and manufacturers of the instruments and reagents used are missing. Please include this information consistently.
  9. There are multiple instances where spaces before units are missing, subscripts are omitted, or sentences begin with lowercase letters. Please correct these issues.
  10. References for the databases used, along with the dates of access, are not provided. Please add this information.
  11. From the DEG analysis of RNA-seq data, the authors highlighted apoptosis-related genes, but no further analysis of apoptosis was performed. Please explain the reason for this omission.
  12. In the cell culture experiments, LPC concentrations of 0, 25, 50, and 75 µM were tested. Please clarify whether these concentrations are physiologically relevant to the in vivo environment.

Author Response

  1. The manuscript does not provide the approval number of the animal experiment. In addition, please indicate the exact dates (year, month, and day) when the AI and SCNT procedures were carried out in the five sows for each group.

Response: Thank you for your comments. In our revised manuscript, we have added the information about the approval number of our animal experiment protocol, and the dates that the AI and SCNT procedures were carried out in the sows.

 

  1. The authors state that three out of five sows became pregnant after AI and three out of five sows became pregnant after SCNT embryo transfer. Please provide detailed information on the number of fetuses obtained and the fetal measurement data.

Response: The three pregnant sows in the AI group carried 14, 16 and 13 fetuses, respectively, while the three pregnant sows in the SCNT group carried 8, 10, 9 fetuses, respectively. Only randomly selected 3 fetuses from each litter (that means 9 fetuses from each group) were used for this study. This information was added to the Materials and Methods section in the revised manuscript.

 

  1. In Figure 1, the numbers are given as “AI group: n=9, SCNT group: n=9,” yet in Figure 2A, the AI group shows 3 points and the SCNT group 6 points, while in Figure 4A, the AI group shows 8 points and the SCNT group 8 points. This inconsistency requires clarification.

Response: For transcriptomic analysis, usually 3 replicates in each group is enough. However, for metabolic analysis, usually 8 replicates are recommended for each group. Although we used 9 AI samples and 9 SCNT samples for body length analysis (Figure 1A), to decrease the cost for transcriptomic and metabolic analyses, we only used 3 AI samples and 6 SCNT samples for transcriptomic analysis (Figure 2A), and only used 8 AI samples and 8 SCNT samples for metabolic analysis (Figure 4A).  

 

  1. In Figure 2A, the meaning of “IV4” is not explained. Please specify what this denotes.

Response: IV means in vivo fertilization-derived samples of the AI group. We have explained this abbreviation in the revised manuscript.

 

  1. In Figure 3, the y-axis is labeled as log2(FC), but the line corresponding to 2 is missing. As a result, it is unclear whether the RNA-seq and qPCR results are consistent, and for some genes it is unclear whether they are upregulated or downregulated.

Response: Thank you for pointing this out. We have added the line corresponding to 2 in Figure 3.

 

  1. The resolution of Figure 4 is insufficient for proper interpretation. In addition, the meaning of “NT” and “IV” in Figure 4A is not explained.

Response: Thank you for your suggestion. To improve the resolution of Figure 4, we have separated Figure 4D from Figure 4 and set it as Figure 5. We also explained the meaning of “NT” and “IV” in Figure 4.

 

  1. In the cell culture experiments, the concentration of the cell suspension used for seeding is provided, but the actual volume of suspension added per well is not described. Therefore, the exact number of cells seeded per well cannot be determined.

Response: Thank you for your comments. The relevant information has been added to the Materials and Methods section in the revised manuscript.

 

  1. In several parts of the manuscript, the names, model numbers, and manufacturers of the instruments and reagents used are missing. Please include this information consistently.

Response: Thank you for your suggestion. The relevant information has been added to the Materials and Methods section in the revised manuscript.

 

  1. There are multiple instances where spaces before units are missing, subscripts are omitted, or sentences begin with lowercase letters. Please correct these issues.

Response: Thank you for your comments. The issues that you mentioned has been corrected in the revised manuscript.

 

  1. References for the databases used, along with the dates of access, are not provided. Please add this information.

Response: Thank you for your suggestion. The relevant information has been added to the Materials and Methods section in the revised manuscript.

 

  1. From the DEG analysis of RNA-seq data, the authors highlighted apoptosis-related genes, but no further analysis of apoptosis was performed. Please explain the reason for this omission.

Response: Our results RNA-seq results showed DEG between AI and SCNT placentas are enriched in apoptosis-related genes. Our cell analysis results showed that LPC inhibits porcine trophoblast cell proliferation, and a high level of LPC (75 µM) even promote porcine trophoblast cell apoptosis (see Figure 5A). Therefore, our cell analysis results actually confirm the RNA-seq results that the abnormally elevated LPC level in the placenta of AI fetuses may induce apoptosis of trophoblast cells.

 

  1. In the cell culture experiments, LPC concentrations of 0, 25, 50, and 75 µM were tested. Please clarify whether these concentrations are physiologically relevant to the in vivo environment.

Response: The in vitro trophoblast culture condition is different from the in vivo placenta environment. Therefore, it is very hard to determine whether the LPC concentrations that we used for treating in vitro cultured trophoblasts are physiologically relevant to the in vivo placenta environment. However, the LPC level in SCNT placenta is abnormally elevated by 22.7 times as compared to that in AI placenta. Such a high level of LPC in SCNT placenta will highly possibly impair the development of SCNT placenta. Actually, our results indicated that treatment with 25 µM of LPC has a mild effect but treatment with 50 or 75 µM of LPC has a significant toxic effect on trophoblast cell proliferation and migration. A similar result was also found in a reported study (Lysophosphatidylcholine Impairs the Mitochondria Homeostasis Leading to Trophoblast Dysfunction in Gestational Diabetes Mellitus.

Hung SC, Chan TF, Chan HC, Wu CY, Chan ML, Jhuang JY, Tan JQ, Mei JB, Law SH, Ponnusamy VK, Chan HC, Ke LY. Antioxidants (Basel). 2024 Aug 19;13(8):1007. doi: 10.3390/antiox13081007.). 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This studyfound that the lipid metabolism is abnormal in the placenta of SCNT-generated pig fetuses, by comparing the placental transcriptomic and lipidomic profiles between SCNT- and AI-derived pig fetuses. The dysregulated lipid metabolism leads to an abnormally high level of a metabolite, lysophosphatidylcholine (LPC), in the placenta of SCNT-produced pig fetuses, which may cause the low intrauterine developmental competence of porcine SCNT fetuses as the authors showed that LPC can inhibit porcine trophoblast cell proliferation and migration. This study uncovers a main cause of erroneous development of pig SCNT fetuses, and therefore it provides new insights into understanding the regulatory mechanisms of SCNT embryo development, and help to develop new methods to improve the efficiency of pig cloning. I think this manuscript is qualified for being accepted for publication in Journal of Developmental Biology. I have the following suggestion to improve the manuscript.

  1. In the Introduction section, the authors state that “the SCNT-generated embryos have a very low full-term developmental rate, while the SCNT-produced animals have a high neonatal death rate”. Please provide the data range of the full-term developmental rate and the neonatal death rate.
  2. Whether theDuroc sows used for AI and the Duroc sows used as SCNT embryo recipients are similar in genetic background and physiological condition?
  3. Figure 1 shows that each group has 9 samples, why figure 2 shows that only 3 samples in AI group and 6 samples in SCNT groups were subjected to RNA sequencing?
  4. The PCA in figure 2A indicates that one sample in AI group is close to the samples in SCNT group. Please explain this result.
  5. The pictures in figure 4c and 4d are blurry. The resolution of these pictures should be improved.  
  6. The authors showed that LPC can inhibit PTr2 cell proliferation. This means that LPC can promote PTr2 cell apoptosis. I suggest that this point can also be discussed in the first paragraph of the Discussion section.
  7. There are some minor English word and gramma errors. Please go through the whole manuscript carefully to correct them.

Author Response

  1. In the Introduction section, the authors state that “the SCNT-generated embryos have a very low full-term developmental rate, while the SCNT-produced animals have a high neonatal death rate”. Please provide the data range of the full-term developmental rate and the neonatal death rate.

Response: Thank you for your suggestion. The data range has been added to the revised manuscript.

 

  1. Whether the Duroc sows used for AI and the Duroc sows used as SCNT embryo recipients are similar in genetic background and physiological condition?

Response: The Duroc sows used for AI and the Duroc sows used as SCNT embryo recipients have similar genetic background and physiological conditions. We have added this information to the Materials and Methods section of our revised manuscript.

 

  1. Figure 1 shows that each group has 9 samples, why figure 2 shows that only 3 samples in AI group and 6 samples in SCNT groups were subjected to RNA sequencing?

Response: We respectively collected 9 samples (fetus + placenta) from AI and SCNT groups. Considering that the cost of RNA sequencing is quite high for us and usually 3 replicates in each group is enough, we only used 3 samples in AI group and 6 sample in SCNT group for RNA sequencing.

 

  1. The PCA in figure 2A indicates that one sample in AI group is close to the samples in SCNT group. Please explain this result.

Response: Although most placentas of the AI group have a normal development, a small portion of placentas in this group may have an abnormal development. These abnormally developed AI placentas may have a gene expression pattern similar with that of SCNT placentas. This probably is the reason why we see one sample in AI group is close to the samples in SCNT group in the PCA in Figure 2A.

 

  1. The pictures in figure 4c and 4d are blurry. The resolution of these pictures should be improved.  

Response: In revised manuscript, we have improved the resolution of Figure 4c and 4d. 

 

  1. The authors showed that LPC can inhibit PTr2 cell proliferation. This means that LPC can promote PTr2 cell apoptosis. I suggest that this point can also be discussed in the first paragraph of the Discussion section.

Response: Thank you very much for your suggestion. In revised manuscript, we have added discussion about this point to the first paragraph of the Discussion section.

 

  1. There are some minor English word and gramma errors. Please go through the whole manuscript carefully to correct them.

Response: Thank you for your suggestion. We have checked our whole manuscript carefully and corrected all the English word and gramma errors that we could find.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In Figure 3, although the authors stated in their response that a line corresponding to 2 was added, the revised figure does not appear to fully reflect this correction. In the upper panel of Figure 3, the inserted line corresponds to y = 1, while in the lower panel, the line corresponds approximately to y = 7. Therefore, the revision described in the response does not match the actual figure, and it remains unclear whether the modification was made correctly.

Although the authors stated that they improved the resolution of Figure 4 by separating Figure 4D and creating a new Figure 5, the revised figure (Figure 5) still contain portions where the image resolution is insufficient and the text appears blurred. Therefore, the issue regarding figure clarity has not been fully resolved.

The authors infer apoptosis from CCK-8 and EdU assays; however, these assays measure cell proliferation rather than apoptosis. Without direct apoptosis analyses (e.g., Annexin V, TUNEL, or caspase assays), the claim that LPC induces apoptosis is not sufficiently supported.

Author Response

1.In Figure 3, although the authors stated in their response that a line corresponding to 2 was added, the revised figure does not appear to fully reflect this correction. In the upper panel of Figure 3, the inserted line corresponds to y = 1, while in the lower panel, the line corresponds approximately to y = 7. Therefore, the revision described in the response does not match the actual figure, and it remains unclear whether the modification was made correctly.

Response: In Figure 3, y=1 represents the fold change (FC) of gene expression level of SCNT group vs. AI group =2, so Figure 3 shows that all tested 5 genes are up-regulated in SCNT group by more than 2 folds and the qPCR results have the same trend as the RNA-seq results, which means that the RNA-seq results are reliable.

 

2.Although the authors stated that they improved the resolution of Figure 4 by separating Figure 4D and creating a new Figure 5, the revised figure (Figure 5) still contain portions where the image resolution is insufficient and the text appears blurred. Therefore, the issue regarding figure clarity has not been fully resolved.

Response: Thank you for your suggestion. We have further improved the resolution of Figure 5 in the revised manuscript.

 

 

3.The authors infer apoptosis from CCK-8 and EdU assays; however, these assays measure cell proliferation rather than apoptosis. Without direct apoptosis analyses (e.g., Annexin V, TUNEL, or caspase assays), the claim that LPC induces apoptosis is not sufficiently supported.

Response: Although in this study the CCK-8 and EdU assays were used to measure cell proliferation rather than apoptosis. Our results showed that the growth curve of cultured porcine trophoblasts goes down after treating with a high dose (75 µM) of LPC (see Figure 6A). This result suggests that a high concentration of LPC can induce apoptosis of porcine trophoblast cells. In line with this, many studies have reported that LPC can induce apoptosis of couple types of in vitro cultured cells [see the references below]. In addition, our results indicated that the LPC level in pig SCNT placentas was 22.7 times higher than that in AI placentas. Therefore, we speculate that the abnormally elevated LPC level in porcine SCNT placentas may induce trophoblast cell apoptosis. These results and information were discussed in the first paragraph of the Discussion section of our revised manuscript.

 

References:

Role of autophagy in lysophosphatidylcholine-induced apoptosis in mouse Leydig cells. Zeng L, Ma B, Yang S, Zhang M, Wang J, Liu M, Chen J. Environ Toxicol. 2022 Nov;37(11):2756-2763. doi: 10.1002/tox.23634. Epub 2022 Aug 3.

 

Role of Autophagy in Lysophosphatidylcholine-Induced Apoptosis of Mouse Ovarian Granulosa Cells. Yang S, Chen J, Ma B, Wang J, Chen J. Int J Mol Sci. 2022 Jan 27;23(3):1479. doi: 10.3390/ijms23031479.

 

17beta-Estradiol Inhibits Lysophosphatidylcholine-Induced Apoptosis in Cultured Vascular Smooth Muscle Cells. Yoon BK, Kang YH, Oh WJ, Roh CR, Kim DK, Kang CD. J Menopausal Med. 2020 Apr;26(1):1-8. doi: 10.6118/jmm.19019.

 

Lysophosphatidylcholine-induced cytotoxicity and protection by heparin in mouse brain bEND.3 endothelial cells. Tsai TY, Leong IL, Cheng KS, Shiao LR, Su TH, Wong KL, Chan P, Leung YM. Fundam Clin Pharmacol. 2019 Feb;33(1):52-62. doi: 10.1111/fcp.12399. Epub 2018 Aug 5.

 

TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells. Wang Y, Wang Y, Li GR.

Oncotarget. 2016 Aug 9;7(32):50937-50951. doi: 10.18632/oncotarget.10853.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Authors answered all of my comments.