DNA Damaged Induced Cell Death in Oocytes

Round 1

Reviewer 1 Report

In this manuscript, entitled “Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility”, Gebel and colleagues have reviewed the molecular mechanisms underlying oocyte quality control in terms of DNA damage and discussed potential therapeutic interventions for the preservation of the oocyte pool by inhibition of apoptosis during chemotherapies. Over all this review does appear to be well-written with well-prepared figures. However, there are some issues that need to be addressed by the authors before publication.

1. Lines 82, please kindly provide more explanation “TAp63” such as “which is a isoform having a transactivation (TA) domain”.
2. Lines 130, please kindly provide what stands for “SAM domain” such as “Sterile alpha motif, a protein module for in protein-protein interactions”.
3. Lines 176, please kindly provide what stands for “ATM” such as “ataxia telangiectasia mutated”.
4. Lines 181, please kindly provide what stands for “CK1” such as “Casein kinase 1”.
5. Figure 3 , If possible, please kindly provide a tetramer in Figure3 although fig 2 has it which is not clear.
6. Lines 237, please kindly provide what stands for “ATR” such as “ataxia telangiectasia and Rad3-related protein”.
7. Line 427, “pubs” is a common word? “pups” is widely used. (Line 430, 433, 455 etc)
8. Line 446-452, please put references.
9. Line 522, please put references for “other studies”.
10. References #30, the doi number seems not correct.

Author Response

We are grateful for the careful review by the referee and have made the requested changes to the manuscript. Please find a point by point response below.

1. Lines 82, please kindly provide more explanation “TAp63” such as “which is a isoform having a transactivation (TA) domain”.

We have added: "These isoforms are created by the use of two different N-terminal promotors that create either proteins containing the full-length transactivation domain (TA-isoforms) or have a truncated transactivation domain (ΔN-isoforms). The different N-termini are combined with several different C-termini created by splicing events [22-24]."

2. Lines 130, please kindly provide what stands for “SAM domain” such as “Sterile alpha motif, a protein module for in protein-protein interactions”.

We changed it to: "While the domain composition of CEP-1 that contains a Sterile Alpha Motif domain (SAM, a domain type implicated in protein-protein interactions)..."

3. Lines 176, please kindly provide what stands for “ATM” such as “ataxia telangiectasia mutated”.

We changed it to: "This triggers the activation of a kinase cascade starting with Ataxia Telangiectasia Mutated kinase (ATM) which phosphorylates..."

4. Lines 181, please kindly provide what stands for “CK1” such as “Casein kinase 1”.

We changed it to: "Instead, S582-phosphorylated TAp63α becomes a target for another kinase, Casein kinase 1 (CK1)."

5. Figure 3 , If possible, please kindly provide a tetramer in Figure3 although fig 2 has it which is not clear.

We have not added an additional tetramer to figure 3 since this figure depicts the transition state from dimer to the tetramer and not the tetramer itself. The full pathway is shown in figure 2. 

6. Lines 237, please kindly provide what stands for “ATR” such as “ataxia telangiectasia and Rad3-related protein”.

We changed it to: "...but also the Ataxia Telangiectasia and Rad3-related kinase (ATR) – Checkpoint kinase 1 (CHK1) route..."

7. Line 427, “pubs” is a common word? “pups” is widely used. (Line 430, 433, 455 etc)

Thanks for seeing this! We changed everywhere pubs to pups. 

8. Line 446-452, please put references.

We added the corresponding reference: "...foci decrease tenfold between P0 and P7, which was interpreted as a sign of DNA repair [33]."

9. Line 522, please put references for “other studies

We added the references: "...the defects seen in blastocyst development differ from the other mouse studies [29,32,71,100] that show no significant effects on surviving oocytes."

10. References #30, the doi number seems not correct

We corrected the DOI.

Reviewer 2 Report

In the review entitled “DNA Damaged Induced Cell Death in Oocytes”, the authors describe the molecular mechanisms of DNA quality control in oocytes and the challenges to save fertility after chemotherapeutic-treatments altering DNA integrity. After a general introduction, the authors describe the evolutionary conserved double-strand break checkpoint occurring in gametes, and the molecular mechanisms leading to apoptosis. They further explain the new updates about the Chromosomal Synapsis Checkpoint in oocyte meiosis. They address the effects of chemotherapy on fertility and describe the latest progress reported in mouse and human. The review ends with a chapter that envisions the suppression of oocyte death with different strategies, to preserve a pool of chemotherapy-treated oocytes.

The review provides concise and precise updates on the latest progress made in the area of oocyte preservation in a context of damaged DNA. Bibliographical works, including molecular mechanism and hypothesis, are abundantly and clearly summarized.

The review is well written. Minor modifications could improve the reading:

• In the legend of figure 2 line 195 “Phosphorylation of Tap63a on S582”
• In the legend of figure 3, the signification of the abbreviations TA2A and TA2B could be added.
• In the legend of figure 4 line 354, 355 and 360 “HORMAD1” instead of HOMRAD

Cite the figures in text in adequate chapter: Ex Cite figure 2 and 3 line 191; figure 4 line 348;  

Comma and following points could be modified to facilitate reading, lines:

• 28 activity; oocyte death;
• 29 development; quality control
• 38 unsuccessful, cells
• 45 importance, germ
• 49 oocytes and fail
• 53 consequence of critical gene silencing for meiotic
• 67 gametes, oocytes align
• 72 chromosomes, lesions are
• 91 DSBs, DNA damage
• 114 [34], and DSBs
• 350 of meiosis,
• 355 DNA repair, Trip13
• 487 is, on one hand,

Sentence lines 226-229 and sentence lines 540-541 (its function) are not clear and should be rewritten.

Line 1009 a remaining point should be erased.

Author Response

We are grateful for the careful review by the referee and have made the requested changes to the manuscript. Please find a point by point response below.

1. In the legend of figure 2 line 195 “Phosphorylation of Tap63a on S582”

We changed it to: "Phosphorylation of TAp63α on S582 by..."

2. In the legend of figure 3, the signification of the abbreviations TA2A and TA2B could be added.

We added in the figure legend: "The segment labeled TA1 forms a helix that in the closed conformation occupies the same site on the oligomerization domain as the second helix of the oligomerization domain in the full tetrameric state [62,64,76]. Only the TA1 peptide sequence acts in the activated state as a transactivation domain, while the two β-strand sequences TA2A and TA2B act as elements of the inhibitory mechanism, thus differing from the bi-partite transactivation domain of p53 and p73 [69]."

3. In the legend of figure 4 line 354, 355 and 360 “HORMAD1” instead of HOMRAD

We corrected it everywhere in the text and figure legend. 

4. Cite the figures in text in adequate chapter: Ex Cite figure 2 and 3 line 191; figure 4 line 348;

We added citation of the figures in the correct order of appearance. 

5. 28 activity; oocyte death;

We changed it.

6. 29 development; quality control

We changed it.

7. 38 unsuccessful, cells

We changed it.

8. 45 importance, germ

We changed it.

9. 49 oocytes and fail

Here we kept the original text as it would otherwise change the meaning.

10. 53 consequence of critical gene silencing for meiotic

We changed it.

11. 67 gametes, oocytes align

We changed it.

12. 72 chromosomes, lesions are

We changed it.

13. 91 DSBs, DNA damage

We changed it.

14. 114 [34], and DSBs

We changed it.

15. 350 of meiosis,

We changed it.

16. 355 DNA repair, Trip13

We changed it.

17. 487 is, on one hand,

We changed it.

18. Sentence lines 226-229 and sentence lines 540-541 (its function) are not clear and should be rewritten.

We have added a full paragraph with references:

"Several human syndromes are caused by mutations in the p63 gene, which are manifested by craniofacial, limb and ectodermal development disorders [83-88]. It is predicted that female patients with p63 mutations will suffer from infertility if the mutations create constitutively active p63 forms (for example by frame shift or nonsense mutations in the C-terminus which delete the TID) that effectively kill all oocytes (Lena et al., NatCom, in press) [77,79-81]. In contrast, mutations in the DBD (causing the Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome [83,84,89]) prevent the binding to DNA which compromises the p63-based quality control function. Mutations in the SAM and TID domains, that cause the Ankyloblepharon-Ectodermal dysplasia-Cleft lip/palate (AEC) syndrome [88], can also lead to the formation of open TAp63α forms that in principle kill affected oocytes. However, AEC-related mutations result in the exposure of aggregation prone peptide sequences followed by aggregation of p63 [90] which compromise the quality control system in oocytes (Lena et al., NatCom, in press). As a result of these two opposing effects (formation of open and tetrameric forms, inactivation through aggregation), premature ovarian insufficiency during early adulthood has been diagnosed in some affected patients [80,81]."

For the second sentence (lines 540-41) we are not sure what the referee has in mind. The text explains two sentences later that no real function is currently known for these treatments: 

"So far, however, none of these investigations is based on a clear molecular mechanism that could explain the proposed protective function of the suggested molecules."

Therefore, we have not made any changes here.

19. Line 1009 a remaining point should be erased.

We are not sure what the referee has seen. All references are imported from EndNote using the same reference style.