PGENETICS-D-26-00149

Oocyte Vitrification Disrupts Zygotic Genome Activation in Embryos by Impairing Maternal Spliceosome Translation and Crxos Splicing

PLOS Genetics

Dear Dr. Zhou,

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Wei-Hsiang Huang

Academic Editor

PLOS Genetics

Pablo Wappner

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Reviewers' comments:

Reviewer #1: Overall Impressions

This study provides a compelling mechanistic link between oocyte vitrification and compromised embryonic development through integrated transcriptome and translatome analyses in mouse oocytes. The authors demonstrate that vitrification specifically disrupts maternal mRNA translation—without affecting global transcriptional output—leading to suppressed expression of spliceosome components, including Phf5a. This perturbation results in persistent alternative splicing defects in 2-cell embryos, notably affecting the zygotic genome activation (ZGA) regulator Crxos (Egam1), where the functional full-length transcript is depleted while a truncated non-coding variant is elevated. Functional assays confirm that Crxos loss in 2-cell embryos impairs both developmental progression and global transcriptional activity, likely through defective RNA Pol II recruitment and elongation at ZGA genes. These findings not only advance our fundamental understanding of post-vitrification embryonic defects but also carry significant translational implications for optimizing cryopreservation protocols in reproductive medicine. The data are robust and well-supported, and the manuscript warrants acceptance after minor revisions.

General Suggestions

1. Please carefully review the manuscript for grammatical issues and improve the clarity of the language throughout the text.

2. Please ensure that all gene names are italicized consistently throughout the manuscript.

3. Please ensure that all figure legends are comprehensive yet concisely presented.

Minor Suggestions and Grammar Corrections

Author Summary

Line 48-50: The sentence structure is slightly awkward. Consider revising to: "Our findings provide a mechanistic basis for optimizing cryopreservation protocols in reproductive medicine."

Introduction

Line 53: The word "dominant" is somewhat subjective and imprecise. Please replace with "a standard" to more objectively reflect its clinical status.

Line 58: Please revise "This developmental compromise poses a significant barrier..." to "This diminished developmental potential (e.g., reduced blastocyst rates) poses a significant barrier..." for greater specificity and impact.

Line 77: Please replace "maternal mRNA" with "maternal mRNAs".

Line 93: Please replace "spliceosome associated mRNAs" with "spliceosome-associated mRNAs".

Line 94: Please replace "failed transcriptional activation" with "the failure of transcriptional activation".

Line 101: Please replace "seek" with "sought".

Results

Line 117: Please replace "before subsequently increasing" with "and subsequently increased".

Line 118: Please replace "fresh group" with "the fresh group".

Line 150: Please replace "subsequent developmental" with "subsequent development".

Line 157: Please replace "stress responsive processes" with "stress-responsive processes".

Line 169-170: Please replace "with skipped exon being the predominant form" with "with skipped exons being the predominant form".

Line 174: Remove the comma before "and".

Line 183-184: Please replace "Crxos's full-length transcript" with "the full-length transcript of Crxos".

Line 185: Please replace "as further validated by PCR" with "as validated by PCR".

Line 196: Please replace "stress related pathways" with "stress-related pathways".

Line 200: Please replace "effectively reduced" with "was found to effectively reduce".

Line 202: Please replace "decreased rates of 2 cell to 4 cell transition" with "decreased rates of transition from 2-cell to 4-cell".

Line 210-211: Please replace "genome wide RNA Pol II binding profiles" with "genome-wide RNA Pol II binding profiles".

Line 212: Please replace "well defined Pol II peaks" with "well-defined Pol II peaks".

Line 245: Please replace "enriched among" with "enriched in".

Line 269-270: Please replace "reduction of maternal spliceosome protein directly disrupts alternative splicing" with "reduction of the maternal spliceosome protein Phf5a directly disrupts alternative splicing".

Discussion

Line 286-287: Please replace "a large number of aberrant alternative splicing genes" with "a large number of genes exhibiting aberrant alternative splicing".

Line 310: Please replace "genome wide mechanism" with "genome-wide mechanism".

Materials and Methods

Line 354-355: Please replace "oocytes were harvested 14 h post-hCG injection" with "oocytes were harvested at 14 h post-hCG injection".

Line 371: Please replace "37.5°C heated stage" with "37.5°C-heated stage".

Reviewer #2: In this manuscript, Qin et al. describe analyses of the effects of oocyte vitrification on gene expression prior to and following zygotic genome activation, to try to identify pathological effects that could contribute to reported reduced IVF success resulting from the vitrification process. They note that indeed oocyte vitrification reduces the rate of developmental progression to blastocyst stage, consistent with prior reports. The authors perform transcriptomic and translatomic measurements to identify potential changes to the gene expression machinery. These experiments can be challenging to interpret if there are global effects, and in fact the raw numbers of differentially expressed genes (DEGs) initially appear to be quite small considering the thousands of genes expressed prior to and post-ZGA. However, the authors do identify some important differentially translated genes such as Phf5a, an important splicing factor, and provide some compelling analyses that suggest a causal role for vitrification in embryonic splicing-associated defects. The overall research question is important given the lack of knowledge of manipulations such as vitrification on oocyte and early embryonic gene expression, and the authors provide important data on potential gene expression defects with important new genomic datasets that may provide quite useful to the field that I believe make this story worthy of publication in PLOS Genetics.

Major points:

“ …the vitrification group showed higher transcriptome-translatome correlation in MII oocytes and 8-cell embryos but lower correlation in at the 2-cell and blastocyst stages (S2B Fig.)

- The differences look very subtle in this figure, given a lack of statistical analysis controlling for multiple testing across various timepoint comparisons it is not apparent if this is a biologically meaningful result.

- If translation utilizing maternally supplied ribosomes/mRNAs was disrupted with consequential effects on ZGA why were there no DEG’s in transcriptome comparisons in post-ZGA stages (8-cell/morula/blastocyst)? It would be helpful to add percentages of genes that were unaffected vs. differential - e.g. out of 12,000 genes detected at the level of transcript abundance and ribosome-association, 1.8% or 218 were significantly differentially expressed. Even at the timepoints with the most DEGs, it would appear that the vast majority of genes were identically expressed between fresh vs. vitrified samples. Why would the effects of vitrification lead to alterations in such a small subset of transcripts? It would seem to me that the fact that the vast majority of genes were not differentially expressed at the level of transcript abundance/translation would support the null hypothesis that the gene expression apparatus of the oocyte/early embryo is largely unaffected by vitrification? Or are only a small fraction of highly expressed genes detected in these analyses, potentially leading to an underestimation of the effects on gene expression?

- Any sense whether global levels of translation were affected? Spike-in or other controls for global effects on gene expression can frequently unmask deficits in translation or RNA stability occurring across the transcriptome that may be otherwise obscured by common normalizations used in genomic analyses (e.g. TPM normalization). How would the analyses changed if accounting for bulk changes in genome-wide transcription as described in Fig. 2E?

- Supplementary tables should be provided with a list of the specific DEG’s described in key figures, such as Fig. 2A.

- In general there are issues with low figure resolution. Figures should be exported at a much higher resolution. For example, Fig. 4C and 4D, 5B etc. are very difficult to read.

- Authors should quantify the RT-PCR experiment shown in Fig. 4B with a statistical analysis of the spliced vs. unspliced form from analyses of the three replicates

- To help interpret the results it would be helpful to be able to compare the decrease for proteins such as Phf5a as measured by T&T-seq vs. the Western blotting by providing additional numbers, e.g. for each protein measured how much was it decreased in this dataset at the levels of translation vs. transcript abundance vs. the net effects on protein levels? This would also help disentangle changes to protein stability vs. synthesis.

- How much knockdown of Phf5a was achieved? It looks like the knockdown was quite subtle in Fig. 6H – is it known if this gene is dosage sensitive where a small degree of knockdown would be predicted to be biologically significant?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Dear Dr Zhou,

We are pleased to inform you that your manuscript entitled "Oocyte vitrification disrupts zygotic genome activation in embryos by impairing maternal spliceosome translation and Crxos  splicing" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Wei-Hsiang Huang

Academic Editor

PLOS Genetics

Pablo Wappner

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I have reviewed the authors' responses to the previous comments and the corresponding revisions. The issues I raised have been well addressed, and the presentation of the manuscript has significantly improved. I agree that the article can be accepted by PLOS Genetics

Reviewer #2: I am satisfied with the authors' responses to my points

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information., and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information., and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information., and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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