Natural degradation of RNA polymerase II is essential for oocyte chromatin reorganization and maternal-to-zygotic transition

Domain: biology
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
The final step of oocyte growth, which reorganizes chromatin from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) configuration is essential for embryonic development after meiotic maturation and fertilization. The underlying mechanisms remain unknown. We identify RNA polymerase II (RNAPII) degradation as the key driver of this process. Inhibitors that trigger RNAPII degradation, but not nucleoside-based transcription inhibitors, induce NSN-to-SN transition in oocytes. By establishing miniTrim-Away for nuclear proteins and using segregase and proteasome inhibitors, we demonstrate that RNAPII degradation is necessary and sufficient for NSN-to-SN transition. Further experiments reveal that RNAPII degradation results in a global collapsing force and a local attractive force required for the transition to SN configuration. Finally, embryos derived from NSN oocytes have aberrant RNAPII levels and localization, and are defective in maternal-to-zygotic transition. Our study elucidates the mechanistic framework of oocyte chromatin reorganization and presents a strategy for inducing fully grown oocyte nuclei.

Introduction
Oogenesis is essential for sexual reproduction in mammals. Growing oocytes have a non-surrounded nucleolus (NSN) chromatin configuration and actively transcribe messenger RNAs (mRNAs), which are used to establish protein and mRNA stores for the next generation. During the final stages of oogenesis, condensed chromatin forms a ring around the nucleolus in a surrounded nucleolus (SN) configuration and transcription ceases. SN oocytes have a higher developmental competence than NSN oocytes, and their nucleus primarily accounts for their differences in the success of embryonic development. Therefore, the reorganization of chromatin from NSN to SN configuration is tied to the developmental potential of mammalian oocytes.

NSN-to-SN transition is accompanied by many characteristic chromatin changes in the nuclear architecture. Compared to NSN nuclei, SN nuclei display a reduction in nucleolar components and ribosomal RNA (rRNA) synthesis, clustering of centromeres and pericentromeric constitutive heterochromatin (chromocenters) around the nucleolus, fewer but larger splicing-related nuclear speckles and altered histone modifications and DNA methylations. How the chromatin is reorganized during this key step of mammalian oocyte development is a long-standing question. Previous studies of knockout mice have reported several proteins that could alter the NSN/SN ratio of isolated oocytes, but whether these proteins are involved in NSN-to-SN transition via directly driving this process or indirectly affecting oocyte growth is unclear. Furthermore, the physiological relevance of these proteins is not known: evidence that their expression or activity changes during NSN-to-SN transition in vivo is lacking. Here, we comprehensively mapped almost 30 nuclear components in NSN and SN oocytes to uncover the underlying molecular differences and to identify candidates that drive the physiological NSN-to-SN transition.

Results
Comprehensive analysis of NSN and SN nuclei
First, we established a workflow for studying NSN and SN oocytes by sorting freshly isolated germinal vesicle (GV) oocytes based on their chromatin configurations, followed by fixation, immunofluorescence staining, high-resolution microscopy, and machine learning-based 3D segmentation analyses. To avoid phototoxicity and DNA damage incurred by the use of UV-excitable Hoechst dyes, we performed live staining of oocyte chromatin using the green-excitable SPY555-DNA or red-excitable 5-SiR-Hoechst dyes, which are routinely used for long-term live imaging of human oocytes. NSN and SN oocytes were sorted with a high accuracy of 99.0% and 100%, respectively. 3D segmentation analysis confirmed that the chromatin volume of NSN and SN oocytes differed by two-fold, consistent with chromatin condensation and detachment from the nuclear envelope during NSN-to-SN transition.

To study the molecular differences between NSN and SN nuclei, we systematically evaluated the localization and staining intensity of 28 common nuclear components. We found that 13 of them localized to chromatin, and the staining intensity for most of them was lower in SN nuclei. Seven nuclear components localized to chromocenters and their staining intensity were all lower in SN nuclei. Nucleolar proteins and the nuclear speckle marker SRSF2 also showed lower staining intensity in SN nuclei, in line with previous reports. These data suggest that most nuclear components are downregulated during NSN-to-SN transition.

RNA polymerase II (RNAPII) inhibitors ectopically induce NSN-to-SN transition in mouse oocytes
NSN-to-SN transition did not spontaneously occur even after prolonged culture of NSN oocytes ex vivo, in line with early studies. In order to identify nuclear components potentially driving NSN-to-SN transition, we used small-molecule inhibition and overexpression approaches to transiently modulate the levels or activities of different nuclear components, thereby mimicking their differences in NSN and SN oocytes. Many of the treatments that led to the desired changes in nuclear components altered the transcriptional activity and/or chromatin compaction in NSN and SN oocytes. In particular, treating NSN oocytes with RNase A, ActD, BRM/BRG1i or GSK-J4 prominently silenced transcription and/or compacted chromatin, whereas CTCF overexpression or DZNep treatment of SN oocytes mildly activated transcription and/or decompacted chromatin. While no treatment was able to reverse NSN-to-SN transition, we found that ActD is able to induce NSN-to-SN transition as evidenced by the ring-like chromatin configuration around the nucleolus and the almost two-fold reduction in chromatin volume.

We also considered the potential contributions of liquid-liquid phase separation (LLPS), which has recently been implicated in driving chromatin reorganization in sperm cells of flowering plants. To test if LLPS drives NSN-to-SN transition, we treated SN oocytes with 1,6-hexanediol, which could disrupt LLPS via weakening hydrophobic interactions between proteins. Although 1,6-hexanediol drastically decompacted chromatin in SN oocytes, the morphology of chromatin did not resemble that of NSN oocytes, as characterized by the excessive fragmentation of chromocenters.

ActD acts on both RNAPI and RNAPII. The RNAPI inhibitor quarfloxin did not phenocopy ActD, implying that NSN-to-SN transition likely involves RNAPII. To test this, we treated NSN oocytes with two widely used RNAPII inhibitors, triptolide and α-amanitin, which covalently bind to the general transcription factor TFIIH to block transcription initiation or impair translocation to block transcription elongation, respectively. As expected, both triptolide and α-amanitin almost completely abolished nascent transcription in NSN oocytes. Importantly, both treatments induced NSN-to-SN transition within 7 hours after treatment, as supported by live-cell imaging. Thus, RNAPII inhibitors can swiftly induce NSN-to-SN transition in mouse oocytes ex vivo.

Induced SN-like nuclei recapitulate epigenetic features, chromatin interactions and developmental potential in SN oocytes
To confirm that RNAPII inhibitors induce NSN-to-SN transition beyond morphological changes, we also examined changes in epigenetic features, chromatin interactions and developmental potential in induced SN-like nuclei. DNA damage marker γH2A.X and pan-histone H4 acetylation (H4ac) staining intensity were increased, whereas DNA methylation, heterochromatin protein HP1β, DNA hydroxymethylation and pan-histone dimethylation (KMe2) staining intensity were reduced in α-amanitin-treated NSN oocytes, similar to SN oocytes. Low-input Hi-C profiling further revealed that the chromatin contact map from α-amanitin-treated NSN oocytes is more similar to SN oocytes as compared to NSN oocytes. Moreover, α-amanitin-treated NSN oocytes and SN oocytes had almost identical contact probability curves with significantly more long-range (>1Mb) contacts than NSN oocytes, in line with previous reports. Remarkably, in vitro fertilization (IVF) after meiotic maturation of α-amanitin-treated NSN oocytes yielded twice as many 2-cell embryos as compared to NSN oocytes. Thus, induced SN-like nuclei recapitulate epigenetic features, chromatin interactions and developmental potential in SN oocytes.

Silencing transcription is insufficient to ectopically induce NSN-to-SN transition
Apart from direct transcriptional inhibition, studies in other systems reported that triptolide and α-amanitin can also trigger RNAPII degradation. We performed immunofluorescence staining with a monoclonal antibody against phospho-serine 2 (pS2) at the C-terminal repeat domain and with a monoclonal antibody against the N-terminal domain of the largest RNAPII subunit RPB1, and found that both phosphorylated and total RNAPII were indeed depleted in triptolide- and α-amanitin-treated NSN oocytes.

To determine if silencing transcription alone is sufficient to induce NSN-to-SN transition, we prematurely terminated transcription by incorporating modified adenosine analogues 8-aminoadenosine (8-AA) and tubercidin/7-deazaadenosine (7-DAA). Both 8-AA and 7-DAA suppressed nascent transcription to a level that was similar to triptolide and α-amanitin treatments, but without degrading RNAPII. Importantly, however, 8-AA and 7-DAA did not induce NSN-to-SN transition. Thus, silencing transcription is insufficient to induce the NSN-to-SN transition ex vivo.

Targeted degradation of RNAPII is sufficient to induce NSN-to-SN transition in mouse and human oocytes
Our data so far suggest that RNAPII degradation might be critical for the NSN-to-SN transition induced by triptolide and α-amanitin beyond transcriptional inhibition. RNAPII is an essential protein and existing oocyte-specific driver lines all express Cre recombinase during early oocyte growth, precluding conditional knockout analysis. We aimed to test this hypothesis by acutely degrading RNAPII in NSN oocytes using Trim-Away, an endogenous protein degradation technique based on Trim21-mediated proteasomal degradation of antibody-antigen complexes. Trim-Away was developed for targeted degradation of cytoplasmic proteins, but RNAPII is a nuclear protein that does not shuttle in and out of the nucleus, so we sought to use the single-chain fragment variable (scFv) for Trim-Away in the nucleus. To this end, we initially tested a scFv developed against pS2 (clone 42B3) by expressing a GFP-fusion protein in oocytes. Although the 42B3-GFP construct did not localize to the nucleus, a 42B3-split GFP construct was able to enter the nucleus and colocalize with pS2 on the chromatin in NSN oocytes. These data show that the size of fusion tag could interfere with the nuclear localization of scFv. Hence, we fused 42B3 with the RING domain of Trim21 (t21R) rather than the Fc domain of IgG to develop a miniTrim-Away approach for targeted degradation of nuclear proteins.

Expression of 42B3-t21R in NSN oocytes depleted not only pS2, but also RNAPII phosphorylated at other residues at the C-terminal repeat domain and unphosphorylated RNAPII. These data suggest that RNAPII dynamically cycles between unphosphorylated and different phosphorylated states, consistent with active transcription in NSN oocytes. NSN oocytes expressing 42B3-t21R for 3 hours underwent a partial NSN-to-SN transition, as supported by the 1.5-fold reduction in chromatin volume. The presence of residual nascent transcription hinted that the partial NSN-to-SN transition could be due to the residual RNAPII, so we extended the expression time from 3 to 16 hours. Remarkably, NSN oocytes expressing 42B3-t21R for 16 hours underwent a full NSN-to-SN transition, as confirmed by the 2-fold reduction in chromatin volume and live-cell imaging.

We also tested whether RNAPII degradation drives NSN-to-SN transition in human oocytes. Because immature human GV oocytes leftover from fertility treatment rarely had fully NSN chromatin configuration, we utilized the ones that had partially compacted chromatin for experiments and specifically used surface area-to-volume (SA/V) ratio instead of volume to quantify the less drastic changes in chromatin. Likewise, 42B3-t21R was able to fully deplete RNAPII in human oocytes, and we found that this treatment can trigger complete NSN-to-SN transition, as supported by the further reduction in chromatin SA/V ratio. Thus, targeted degradation of RNAPII is sufficient to induce NSN-to-SN transition in mouse and human oocytes.

Segregase- and proteasome-mediated degradation of RNAPII is necessary for NSN-to-SN transition
Next, we asked whether RNAPII degradation is physiologically relevant to NSN-to-SN transition in vivo. Previous work reported that the levels of phosphorylated RNAPII are lower in SN oocytes as compared to NSN oocytes, but whether this observation reflects simply dephosphorylation or downregulation at protein level is not clear. To this end, we compared the abundance of phosphorylated as well as of total RNAPII in NSN and SN oocytes by immunofluorescence staining and immunoblotting. We found that phosphorylated RNAPII is not converted to the unphosphorylated form but depleted at protein level, accounting for the substantial reduction in total RNAPII levels in SN oocytes. As orthogonal approaches, we imaged oocytes in follicles at different growth stages on mouse ovary sections and oocytes harvested from ex vivo follicle culture. Both approaches showed that phosphorylated RNAPII was progressively depleted during late stages of follicle growth, concomitant with NSN-to-SN transition. Thus, phosphorylated RNAPII was specifically depleted during NSN-to-SN transition in vivo.

To decipher how RNAPII levels decrease during NSN-to-SN transition, we first examined the transcriptional regulation of Rpb1. Briefly, we performed low-input whole-genome bisulfite sequencing (WGBS), ATAC-seq and fractionated RNA-seq and found that DNA methylation levels, chromatin accessibility, actively transcribing and stored mRNA levels of Rpb1 were not different between NSN and SN oocytes. Consistent with our multiomics analyses, RT-qPCR confirmed that Rpb1 mRNA levels were only marginally reduced in SN oocytes as compared to NSN oocytes. We then examined the translational regulation of Rpb1 by Ribo-seq, poly(A) tail-length assay, RIBOmap and CHX treatment. Rpb1 mRNA did not display a significant difference in ribosome occupancy nor poly(A) tail length. In addition, RIBOmap analysis and CHX treatment confirmed that Rpb1 mRNA was not actively translated in NSN or SN oocytes. Thus, the synthesis of RNAPII protein does not change during NSN-to-SN transition.

To determine whether the depletion of phosphorylated RNAPII is mediated by direct protein degradation during NSN-to-SN transition, we acutely treated ex vivo cultured follicles with inhibitors against segregase and proteasome, two key players of proteolysis in cells. Both segregase inhibitor CB-5083 and proteasome inhibitor MG132 suppressed RNAPII degradation. Strikingly, they also blocked NSN-to-SN transition. Interestingly, however, inhibiting RNAPII phosphorylation with the pan-CDK inhibitor flavopiridol could not prevent RNAPII degradation, suggesting that phosphorylation marks the fraction of RNAPII to be degraded but does not mediate the degradation process.

To investigate whether the degradation of phosphorylated RNAPII is mediated by ubiquitination as previously reported in other systems, we examined the chromatin configuration of GV oocytes from wildtype and conditional knockout (cKO) mice for Ubb, the predominant polyubiquitin gene that encodes 70% of ubiquitin protein and phenocopies proteasome inhibition upon knockout in oocytes. While we do not exclude the possibility that the minor amount of ubiquitin protein remaining in Ubb cKO is sufficient for RNAPII degradation, quantifications revealed no significant difference in their NSN/SN ratio, suggesting that ubiquitination is unlikely to be involved in RNAPII degradation in oocytes. Taken together, RNAPII degradation is necessary for NSN-to-SN transition in vivo.

RNAPII degradation induces NSN-to-SN transition via stripping chromatin-bound RNAPII
To understand how RNAPII degradation induces NSN-to-SN transition, we first compared the levels of RNAPII in oocytes with that in somatic cells, which do not undergo major chromatin reorganization upon RNAPII degradation. Immunoblotting revealed that RNAPII in NSN oocyte is around two- and 12-fold more abundant than that in SN oocyte and somatic cell, respectively, predominantly in the phosphorylated form. High-resolution imaging further revealed that phosphorylated RNAPII is found on chromatin in NSN oocytes, whereas unphosphorylated RNAPII is found in the nucleoplasm in both NSN and SN oocytes, implying that RNAPII degradation primarily removes phosphorylated RNAPII during NSN-to-SN transition.

To profile the chromatin binding patterns of phosphorylated RNAPII prior degradation, we optimized low-input CUT&Tag and found widespread binding of phosphorylated RNAPII to different chromosomes in NSN oocytes, particularly being enriched at the promoter, gene body and intergenic regions. Metagene analyses revealed that genes expressed at higher levels have more RNAPII accumulated at promoters and found within gene bodies, consistent with more release of paused RNAPII for actively transcribed genes. Unexpectedly, different from somatic cells and fertilized eggs, a substantial fraction of phosphorylated RNAPII also accumulated at the 3’ distal intergenic regions, coinciding with the unusual open chromatin and single-stranded DNA signals at the transcription termination site (TTS) that are absent in SN oocytes. Because such enrichment did not result in transcription read-through, this was likely due to paused RNAPII during transcription termination.

To test if RNAPII degradation induces NSN-to-SN transition via clearing RNAPII from chromatin, we sought to reduce the levels of chromatin-bound RNAPII without degrading it. To this end, we treated NSN oocytes with THZ1 and DRB, inhibitors of CDK7 and CDK9 that sequentially phosphorylates RNAPII during active transcription cycle, respectively. As expected, THZ1 was more effective than DRB in inhibiting transcription and reducing phosphorylated RNAPII levels without affecting total RNAPII levels. We found that DRB only partially released chromatin-bound RNAPII concomitant with a partial NSN-to-SN transition, whereas THZ1 fully released chromatin-bound RNAPII concomitant with a full NSN-to-SN transition. Importantly, pooling and plotting control, DRB- and THZ1-treated oocytes together revealed a linear relationship between the levels of chromatin-bound RNAPII and chromatin volume. Taking advantage of DRB as a non-covalent inhibitor, we further investigated whether inducing NSN-to-SN transition alone is sufficient to improve the developmental potential of NSN oocytes. Indeed, significantly more embryos derived from NSN oocytes transiently treated with DRB before meiotic maturation and IVF progressed beyond 2- and 4-cell stages as compared to embryos derived from non-treated NSN oocytes. Taken together, RNAPII degradation strips chromatin-bound RNAPII to induce NSN-to-SN transition.

Removal of chromatin-bound RNAPII induces a global collapsing force and a local attractive force required for NSN-to-SN transition to take place
Studies in other systems reported that chromatin-bound RNAPII could sterically constrain chromatin movement. Indeed, tracking of chromatin structures such as chromocenters revealed an increase in mean square displacement (MSD) upon the induction of NSN-to-SN transition, suggesting that the transition to SN configuration involves an increase in chromatin mobility. To directly test the contributions of chromatin dynamics, we sought to reduce chromatin mobility by overloading histone subunit H2B. Interestingly, upon induction of NSN-to-SN transition, H2B overexpression markedly suppressed the collapse of peripheral chromatin, but without interfering with the formation of the chromatin ring around the nucleolus. This result suggests that the increase in chromatin mobility only contributes in part to the transition to SN configuration, leading us to a model whereby NSN-to-SN transition involves both a collapsing force as a result of the increase in global chromatin dynamics and an attractive force as a result of the nucleolus being converted into an attractive surface for local chromatin attachment.

To demonstrate the presence of the global collapsing force, we designed a histone nanobody-lamin nanobody fusion construct (αHIST-αLMN) to artificially anchor chromatin to the periphery of the nucleus in order to counteract the collapse of chromatin during NSN-to-SN transition. Indeed, expression of αHIST-αLMN suppressed the collapse of peripheral chromatin without interfering with the formation of the chromatin ring around the nucleolus, similar to H2B overexpression. Notably, chromatin detachment from the nuclear envelope alone was insufficient to generate a collapsing force, as evidenced by the insignificant changes in chromatin volume upon acute depletion of lamin by miniTrim-Away. Next, to demonstrate the presence of the local attractive force, we attempted to create an artificial nucleolus but with an inert surface by microinjecting a drop of silicone oil into the oocyte nucleus. Prior to inducing NSN-to-SN transition, chromatin surrounded neither the nucleolus nor the oil droplet. However, after inducing NSN-to-SN transition, chromatin surrounded the nucleolus but not the oil droplet, concomitant with the displacement of nucleolar proteins such as NCL and B23 from the surface of the nucleolus.

To further show that these two forces are independent of each other, we compared the degree of chromatin compaction in EU low (transcriptionally inactive, SN-like) GV oocytes from wildtype and nucleolus-deficient Npm2 cKO mice. Quantifications revealed no significant difference in their chromatin volume, confirming that the absence of nucleolus abolishes the chromatin ring without affecting the collapse of peripheral chromatin that accounts for the major changes in chromatin volume. Thus, removal of chromatin-bound RNAPII increases chromatin dynamics and triggers nucleolar surface remodeling, resulting in a global collapsing force and a local attractive force required for the transition to SN configuration.

Embryos derived from NSN oocytes have aberrant RNAPII levels and localization and are defective in maternal-to-zygotic transition
Most embryos derived from NSN oocytes had poor developmental competence and were arrested early at 1-cell stage, the time when maternal-to-zygotic transition takes place in mice with maternal mRNAs being degraded and replaced by newly transcribed mRNAs during ZGA. We reasoned that the high levels of maternal RNAPII in NSN oocytes may result in aberrant RNAPII levels after meiotic maturation and fertilization, later contributing to defects in ZGA, a process primarily executed by RNAPII and to a lesser extent RNAPI. Indeed, immunoblotting confirmed that the undegraded RNAPII contributes to the higher levels of RNAPII in MII oocytes and 1-cell embryos derived from NSN oocytes, independent of de novo translation of RNAPII during meiotic maturation. Surprisingly, despite the higher levels of RNAPII, immunofluorescence staining revealed lower levels of total and phosphorylated RNAPII in the nuclei, concomitant with lower transcriptional activities in 1-cell embryos derived from NSN oocytes. In addition, we observed smaller chromatin volume and excessive nucleolar fragmentation, indicative of defects and possibly a delay in pronucleus formation in 1-cell embryos derived from NSN oocytes.

We further performed low-input RNA-seq to compare 1-cell embryos derived from NSN and SN oocytes. Notably, 42 out of 223 downregulated genes were minor ZGA transcripts, whereas 52 out of 168 upregulated genes were maternal transcripts, indicative of defects in minor ZGA and maternal mRNA degradation in 1-cell embryos derived from NSN oocytes. Gene ontology (GO) analysis further revealed that remaining downregulated genes are mostly related to RNA splicing and cell division, whereas remaining upregulated genes are mostly related to oocyte development, negative regulation of fertilization and catabolic processes, suggesting additional nuclear and cytoplasmic defects in 1-cell embryos derived from NSN oocytes.

To determine whether the reduced binding of RNAPII to chromatin and hence lower transcriptional activity could be a result of lower chromatin accessibility, we performed ATAC-see but detected no significant difference in chromatin accessibility between 1-cell embryos derived from NSN and SN oocytes. Moreover, trichostatin A (TSA) treatment, which could ectopically enhance chromatin accessibility and increase transcriptional activity, did not rescue the developmental arrest in 1-cell embryos derived from NSN oocytes. These data suggest defects in zygotic factors that promote RNAPII loading into the nucleus and/or binding to chromatin. Taken together, embryos derived from NSN oocytes are defective in maternal-to-zygotic transition, accounting for their arrest at as early as 1-cell stage and poor development beyond.

Methods
Data reporting
No statistical methods were used to predetermine the sample size for experiments. Experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Methods of this research comply with all relevant ethical regulations approved by the National Institute of Biological Sciences, Beijing, Reproductive and Genetic Hospital of CITIC-XIANGYA, and Beijing Perfect Family Hospital.

Preparation and culture of mouse oocytes and follicles
All mice were maintained in a specific pathogen-free environment, including a 12-h light/dark schedule, regulated temperature (approximately 22°C), and 50–60% relative humidity, at the Animal and Plant Center of the National Institute of Biological Sciences, Beijing, according to international animal welfare rules. Standard chow and water were available without restriction. All experiments with mice complied with the ethical guidelines of the Animal Ethics Committee of the National Institute of Biological Sciences, Beijing. Ubb cKO mice were generated by breeding C57BL/6JGpt-Ubbem1Cflox/Gpt (GemPharmatech) with B6.Cg-Tg(Msx2-cre)5Rem/BortJ143 (The Jackson Laboratory), then backcrossing with C57BL/6JGpt-Ubbem1Cflox/Gpt. Npm2 cKO mice were generated by breeding C57BL/6JGpt-Npm2em1Cflox/Gpt (GemPharmatech) with B6.Cg-Tg(Msx2-cre)5Rem/BortJ143 (The Jackson Laboratory), then backcrossing with C57BL/6JGpt-Npm2em1Cflox/Gpt.

Oocytes of around 60-80 μm in diameter were isolated from ovaries of 8- to 10-week-old unprimed FVB/N or C57BL/6J (Charles River Laboratories) or Ubb cKO or Npm2 cKO female mice by puncturing preantral and antral follicles on the surface of ovaries with needles. Cumulus-enclosed GV oocytes were stripped by repeated mouth pipetting and arrested at prophase in homemade phenol red-free M2 supplemented with 250 μM dibutyryl cyclic AMP (dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin oil (ACROS Organics) at 37°C. Generally, around 50 oocytes can be isolated from one 8- to 10-week-old unprimed FVB/N, and around 30 oocytes can be isolated from one 8- to 10-week-old unprimed C57BL/6J female mouse.

Follicles were enzymatically isolated from ovaries of 14-day-old C57BL/6J female mice in L-15 medium (Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS) (Sigma-Aldrich), 1x GlutaMAX (Thermo Fisher Scientific), and 0.1x penicillin G/streptomycin (Sigma-Aldrich) containing 1 mg/ml collagenase A (Roche) for 15 min at 37°C. Intact follicles of around 100-120 μm in diameter with a centered oocyte were cultured in MEM alpha with GlutaMAX and nucleosides (Thermo Fisher Scientific) supplemented with 5% FBS, 0.03 μg/ml ovine follicle stimulating hormone (National Hormone and Peptide Program), 1x insulin/transferrin/sodium selenite (Sigma-Aldrich), and 0.1x penicillin G/streptomycin on 12-mm Transwell-COL collagen-coated 0.4-μm pore polytetrafluoroethylene (PTFE) membrane insert (Corning) at 37°C and 5% CO2. Half of the medium surrounding the insert was replaced every 3 days. After 3 to 9 days of culture, in vitro grown oocytes were denuded in M2 with dbcAMP.

Preparation, culture and IVF of mouse MII oocytes
For in vitro fertilization of IVO MII oocytes, 4-week-old C57BL/6J female mice were superovulated by sequential injection of 0.1 ml CARD HyperOva (Cosmo Bio) and 7.5 IU human chorionic gonadotropin (hCG) (ProSpec) after 48 h. Sperm was collected from caudae epididymides from >12-week-old FVB/N male mice and capacitated for 1 h in 400 μl of homemade phenol red-free HTF. MII oocytes were collected from oviducts 15 h after hCG. IVF was performed in 100 μl of HTF by adding 10 μl of 3.33×107/ml capacitated sperm for 3 h. Fertilized eggs were then washed in 1:1 HTF:EmbryoMax Advanced KSOM Medium (Sigma-Aldrich) for 30 min and further transferred to EmbryoMax Advanced KSOM Medium for long-term culture. MII oocytes, 1-cell embryos (2PN), and early and late 2-cell embryos were collected at 0, 7, 22, and 28 h post fertilization (hpf), respectively.

For in vitro fertilization of MII oocytes matured from GV oocytes, FVB/N GV oocytes with a centered nucleus were first treated with water/100 μM α-amanitin (APExBIO) or DMSO/100 μM DRB (MedChemExpress) in M2 with dbcAMP for 7 h and then released into dbcAMP-free M2 for 13.5 h. Before the addition of sperm, a single hole was created in the zona pellucida of the MII oocytes using XYRCOS (Hamilton Thorne). IVF was performed as described above. After IVF, 1PN (parthenogenetic activation), 3PN (polyspermy), and abnormal embryos were excluded from further experiments.

Preparation and culture of human oocytes
The use of unfertilized human oocytes in this study was approved by the Institutional Review Board (IRB) at the National Institute of Biological Sciences, Beijing under the reference IRB22101001, Reproductive and Genetic Hospital of CITIC-XIANGYA under the reference LL-SC-2017-012 and Beijing Perfect Family Hospital under the reference 2020-09-08.

Oocytes were collected from patients aged 23–35 years who underwent ovarian stimulation for intracytoplasmic sperm injection (ICSI) as part of their assisted reproduction treatment owing to male factor-associated infertility at the Reproductive and Genetic Hospital of CITIC-XIANGYA and Beijing Perfect Family Hospital. Only oocytes that were GV at the time of ICSI and thus unsuitable for the procedure were used in this study. All patients gave informed consent for their surplus oocyte(s) to be used in this study. For oocytes collected at the Reproductive and Genetic Hospital of CITIC-XIANGYA, oocytes were vitrified using previously described homemade vitrification solutions to maximize their survival and polar body extrusion rate after thawing. Briefly, oocytes were initially transferred to 20 μl of G-MOPS PLUS (Vitrolife). 20 μl of G-MOPS PLUS containing 7.5% DMSO and 7.5% ethylene glycol (Sigma-Aldrich) was then added and oocytes were incubated for 3 min at room temperature twice. 240 μl of G-MOPS PLUS containing 7.5% DMSO and 7.5% ethylene glycol were further added and oocytes were incubated for 6 to 10 min at room temperature. Before loading onto Cryotop, oocytes were washed in 300 μl of G-MOPS PLUS containing 15% DMSO, 15% ethylene glycol and 0.5 M D-(+)-trehalose (Sigma-Aldrich) twice. For thawing, Cryotop was inserted into 1 ml of prewarmed G-MOPS PLUS containing 1 M D-(+)-trehalose for 1 min at 37°C. Oocytes were then transferred to 300 μl of G-MOPS PLUS containing 0.5 M D-(+)-trehalose for 3 min at room temperature, 300 μl of G-MOPS PLUS containing 0.25 M D-(+)-trehalose for 5 min at room temperature, and 300 μl of G-MOPS PLUS for 2 min at room temperature. Dead or morphologically abnormal oocytes after thawing were excluded from further experiments, and all other recovered oocytes were transferred to G-MOPS PLUS at 37°C. For oocytes collected at Beijing Perfect Family Hospital, fresh oocytes were directly transferred to and cultured in G-MOPS PLUS at 37°C within 3 to 5 h after retrieval from the ovaries. To select for less mature oocytes, only oocytes that were morphologically normal but did not undergo GV breakdown within 24 h after retrieval from the ovaries were used for experiments.

Drug treatment
All drugs except 1,6-hexanediol were prepared in water for embryo transfer or DMSO (Sigma-Aldrich). Oocytes were treated with 20 μM 8-AA (MedChemExpress), 20 μM A-485 (MedChemExpress), 100 μM α-amanitin, 100 nM ActD (MedChemExpress), 6 μM BRM/BRG1i (MedChemExpress), 10 μM bleomycin (MedChemExpress), 10 μg/ml CHX (Sigma-Aldrich), 100 μM DRB, 10 μM DZNep (MedChemExpress), 25 μg/ml etoposide (MedChemExpress), 100 μM GSK-J4 (MedChemExpress), 500 μM hinokiflavone (MedChemExpress), 10 μM quarfloxin (MedChemExpress), 2.5 μM THZ1 (MedChemExpress), 10 μM triptolide (MedChemExpress), 100 nM TSA (MedChemExpress) or 10 μM 7-DAA (MedChemExpress) in M2 with dbcAMP for 7 h. For 1,6-hexanediol treatment, oocytes were treated with 15% 1,6-hexanediol (Sigma-Aldrich) in M2 with dbcAMP for 10 min. For CHX treatment, oocytes were treated with 10 μg/ml CHX in M2 with dbcAMP for 24 h. For CB-5083, MG132, and flavopiridol treatments, cultured follicles were treated with 5 μM CB-5083 (MedChemExpress), 25 μM MG132 (MedChemExpress), or 5 μM flavopiridol (MedChemExpress) on D14+7 for 48 h. Embryos were treated with 5 nM TSA in EmbryoMax Advanced KSOM Medium for 16 h at 7 hpf.

Expression constructs and mRNA synthesis
To generate constructs for mRNA synthesis, we fused cDNAs with eGFP, mClover3, P2A, NLS(NP), t21R and/or V5 and subcloned them into pGEMHE to obtain 42B3-mClover3, 42B3-mClover3(11)-P2A-NLS-mClover3(1-10) (42B3-split mClover3), 42B3-t21R, mClover3-CTCF, DNMT1-V5, DNMT3A-V5, DNMT3B-V5, DNMT3L-V5, NLS-eGFP-hnRNPU, HP1β, αLMN-t21R, NLS-eGFP-MATR3, PAGAP-t21R, TET1-V5, TET2-V5 and TET3-V5. Histone-Chromobody and Lamin-Chromobody coding sequences (Chromotek) were fused and subcloned into pGEMHE to obtain αHIST-αLMN. pGEMHE-H2B-mClover3 was also used. All mRNAs were synthesized and quantified as previously described.

Microinjection
Mouse oocytes were microinjected with 3.5 to 14 pl of mRNAs as previously described. 42B3-mClover3 mRNA was microinjected at a needle concentration (final concentration in the microinjection needle) of 560 ng/μl, 42B3-split mClover3 mRNA at 560 ng/μl, 42B3-t21R mRNA at 2965.9 ng/μl, mClover3-CTCF mRNA at 3040 ng/μl, DNMT1-V5 mRNA at 572.4 ng/μl, DNMT3A-V5 mRNA at 701 ng/μl, DNMT3B-V5 mRNA at 752.2 ng/μl, DNMT3L-V5 mRNA at 1057 ng/μl, NLS-eGFP-hnRNPU mRNA at 2251.2 ng/μl, H2B-mClover3 mRNA at 1145.8 ng/μl, αHIST-αLMN mRNA at 1584.2 ng/μl, HP1β mRNA at 952.5 ng/μl, αLMN-t21R at 2000 ng/μl, NLS-eGFP-MATR3 mRNA at 1148.1 ng/μl, PAGFP-t21R mRNA at 2000 ng/μl, TET1-V5 mRNA at 801.9 ng/μl, TET2-V5 mRNA at 528.9 ng/μl and TET3-V5 mRNA at 469.3 ng/μl. Oocytes were allowed to express the mRNAs for 3–4 h. Mouse oocytes were also microinjected with 7 pl of RNase A (Sigma) into the cytoplasm or 1 to 2 pl of dimethylsiloxane (Sigma-Aldrich) into the nucleus. Human oocytes were microinjected with 14 pl of 42B3-t21R mRNA at a needle concentration of 2965.9 ng/μl as previously described.

Live staining of GV oocytes for visualizing chromatin configuration
SPY555-DNA (Spirochrome) and 5-SiR-Hoechst were reconstituted in DMSO. For visualizing chromatin configuration, oocytes were stained with SPY555-DNA or 5-SiR-Hoechst for 30 min at 1:500 dilution and 10 μM, respectively. For live imaging, SPY555-DNA or 5-SiR-Hoechst was present throughout imaging at 1:1000 dilution and 50 nM, respectively. For sorting GV oocytes with different chromatin configurations, individual oocytes were transferred to microdrops and manually categorized under an inverted widefield fluorescence microscope equipped with a fluorescent LED. GV oocytes exhibiting dispersed chromatin with a few bright punctate were categorized as NSN oocytes, whereas those displaying highly condensed chromatin with a complete perinucleolar ring were categorized as SN oocytes. Oocytes that could not be unambiguously assigned to either category were excluded from further experiments. NSN and SN oocytes were used for experiments immediately after sorting.

Immunofluorescence staining
Oocytes and embryos were fixed in 100 mM HEPES (pH 7.0, titrated with KOH), 50 mM EGTA (pH 7.0, titrated with KOH), 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.5% triton X-100 for 15 to 60 min at 37°C and washed in phosphate-buffered saline (PBS). For EU and L-AHA labeling, oocytes were pulsed with 1 mM EU (Thermo Fisher Scientific) or 100 μM L-AHA (Click Chemistry Tools) in M2 for 2 h at 37°C before fixation and visualized using Click-iT RNA Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) or AFDye 568 Azide (Click Chemistry Tools) or Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific). For 5mC and 5hmC staining, fixed oocytes were treated with 4 M HCl for 10 min and neutralized with 100 mM Tris-HCl (pH 7.5) for 10 min. All antibody incubations and washings were performed in PBS with 0.5% triton X-100 (PBT) and 5% BSA (PBT-BSA) at 10 μg/ml overnight at 4°C (for primary antibodies) and at 20 μg/ml for 1 h at room temperature (for secondary antibodies). Primary antibodies used were mouse anti-B23 (Santa Cruz Biotechnology #sc-56622), rabbit anti-CTCF (Sigma-Aldrich #07-729-25UL), mouse anti-5mC (Active Motif #39649), rabbit anti-5hmC (Active Motif #39792), rabbit anti-FBL (Abcam #ab166630), goat anti-GFP (Rockland Immunochemicals #600-101-215), mouse anti-γH2A.X (Sigma-Aldrich #05-636), rabbit anti-H3ac (Active Motif #39140), rabbit anti-H4ac (Sigma-Aldrich #06-598), rabbit anti-hnRNPU (Huabio #ET7107-10), rabbit anti-HP1α (Huabio #ET1602-8), mouse anti-HP1β (Sigma-Aldrich #MAB3448), rabbit anti-HP1γ (Huabio #ET1706-02), rabbit anti-H3K9me3 Abcam #ab8898), rabbit anti-KMe2 #1 (Abcam #ab7315), rabbit anti-KMe2 #2 (Abclonal #A5870), rabbit anti-KMe2 #3 (Abclonal #A18296), rabbit anti-KMe3 #1 (Thermo Fisher Scientific #PA5-116816), rabbit anti-KMe3 #2 (Abclonal #A20145), mouse anti-LMNAC (Sigma-Aldrich #SAB4200236), mouse anti-m3G/TMG (MBL #RN019M), rabbit anti-MATR3 (Huabio #ET7106-95), rabbit anti-NCL (Abclonal #A20910), rabbit anti-NuMA (Abcam #ab97585), rabbit anti-POLR1A (CST #24799), rabbit anti-POLR2B (Active motif #61558), rabbit anti-RPB1 (CST #149158S), mouse anti-CTDpY1 (Active Motif #91220), rat anti-CTDpS2 (Active Motif #61999), rabbit anti-CTD pT4 (Abclonal #AP1383), rat anti-CTDpS5 (Active Motif #67102), rat anti-CTDpS7 (Active Motif #61704), mouse anti-SRSF2 (Sigma-Aldrich #S4045), rabbit anti-SMARCA2 (Abclonal #A23291), rabbit anti-SMARCA4 (Abclonal #A19556), rabbit anti-SMC3 (Abcam #Ab128919) and rabbit anti-SMC4 (Sigma-Aldrich #HPA029449). Secondary antibodies used were Alexa Fluor 488-, 594-, or 647-conjugated AffiniPure Fab Fragment anti-goat IgG, anti-rabbit IgG, and anti-rat IgG (Jackson ImmunoResearch Europe), Alexa Fluor 568-conjugated Nano-Secondary anti-mouse IgG1, IgG2a and IgG2b (Proteintech). DNA was stained with Hoechst 33342 (Molecular Probes).

Mouse ovaries were fixed in 100 mM HEPES (pH 7.0, titrated with KOH), 50 mM EGTA (pH 7.0, titrated with KOH), 10 mM MgSO4 and 2% methanol-free formaldehyde for 30 minutes at room temperature. They were then washed in PBS, cryoprotected in 30% sucrose overnight at room temperature, and embedded in OCT. 30 μm-thick cryosections were used for immunostaining. After removing OCT with PBS, ovary sections were blocked with PBT-BSA for 1 hour. Slides were then incubated with primary antibodies at 4°C overnight. Following three 15-minute washes with PBT, slides were incubated with secondary antibodies for 1 hour at room temperature. DNA was stained with Hoechst 33342, and slides were mounted with 50% glycerol.

polyA RNA-FISH
Oocytes were fixed in 100 mM HEPES, 50 mM EGTA, 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.5% triton X-100 for 15 to 60 min at 37°C and washed in PBS. Oocytes were then washed with Wash Buffer A (Biosearch Technologies) containing 10% formamide and incubated with 200 nM Alexa Fluor 647-conjugated oligodT30 (IDT) in hybridization buffer (Biosearch Technologies) supplemented with 10% formamide overnight at 37°C. After probe incubation, oocytes were washed with Wash Buffer A for 30 min at 37°C and stained with Hoechst 33342 in Wash Buffer A for another 30 min at 37°C. Oocytes were further washed with Wash Buffer A and Wash Buffer B (Biosearch Technologies) for 30 min at 37°C and room temperature, respectively, before imaging.

RIBOmap
RIBOmap was performed as previously described. Oocytes were fixed in 100 mM HEPES, 50 mM EGTA, 10 mM MgSO4 and 2% methanol-free formaldehyde for 15 to 60 min at 37°C and washed in PBS. Oocytes were then washed three times with hybridization buffer consisting of 2x SSC, 10% formamide, 0.1% triton X-100, 0.1 mg/ml yeast tRNA (Thermo Fisher Scientific) and 0.2 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific). Hybridization solution was prepared by diluting all probes (Genewiz), including 5 splints and 5 Rpb1-specific padlocks and 4 Rpb1-specific primers, to a final concentration of 50 nM with hybridization buffer. Oocytes were incubated with hybridization solution for 4 to 6 h at 40°C. After hybridization, oocytes were washed twice with PBS with 0.1% triton X-100 and 0.1 U/μl RiboLock RNase Inhibitor (PBSTR) and once with 4x SSC diluted with PBSTR. All washes were performed for 10 min at 37°C. Oocytes were then rinsed with PBSTR and incubated for 1.5 h at room temperature with ligation solution consisting of 0.25 Weiss U/μl T4 DNA ligase (Thermo Fisher Scientific), 1x T4 DNA ligase buffer, 0.5 mg/ml BSA, and 0.4 U/μl RiboLock RNase Inhibitor. After two washes with PBSTR for 5 min each, oocytes were incubated with amplification-labeling mixture consisting of 0.5 U/μl phi29 DNA polymerase (NEB), 1x phi29 DNA polymerase reaction buffer, 250 μM dNTP (NEB), 0.5 mg/ml BSA, 0.4 U/μl RiboLock RNase Inhibitor, and 1 μM labeling probe (5’-/5Cy3/CATACACTAAAGATAACAT-3’) (Genewiz) for 2 h at 30°C. Oocytes were subsequently washed twice with PBSTR with 0.05 mg/ml BSA for 5 min each at room temperature before imaging.

ATAC-see
ATAC-see was performed as previously described after minor modifications. TAMRA-labeled oligonucleotides were resuspended in water to a final concentration of 100 μM, and equimolar of Tn5MErev/Tn5ME-A-TAMRA and Tn5MErev/Tn5ME-B-TAMRA were mixed, denatured for 5 min at 95°C and annealed by slowly cooling down to room temperature. Tn5 transposome was assembled by mixing 0.602 μl of Tn5MErev/Tn5ME-A-TAMRA, 0.602 μl of Tn5MErev/Tn5ME-B-TAMRA, 0.577 μl of 2× dialysis buffer [100 mM HEPES–KOH (pH 7.2), 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100 and 20% glycerol] and 3.221 μl of 50 μM Tn5 (Active Motif #81286) and incubating for 1 h at room temperature.

Embryos were fixed in 100 mM HEPES, 50 mM EGTA, 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.5% triton X-100 for 15 to 60 min at 37°C and washed in PBS. Embryos were then incubated with 4 μl of Tn5 transposome diluted with 10 μl of 2x TD buffer and 6 μl of water for 1 h at 37°C. After the tagmentation reaction, embryos were washed with PBS containing 0.01% SDS and 50 mM EDTA for 15 min at 55°C thrice. Embryos were further washed with PBT-BSA before staining with Hoechst 33342 for 1 h at room temperature.

Confocal and light-sheet microscopy
For confocal imaging, fixed oocytes and embryos were imaged in 2 μl of medium (for live imaging) or PBS with 1% polyvinylpyrrolidone (PVP) and 0.5 mg/ml BSA (for fixed imaging) under paraffin oil in a 35-mm dish with a no. 1.5 coverslip (iBidi). Images were acquired with LSM900 (Zeiss), LSM980 (Zeiss), and Stellaris 5 (Leica) confocal laser scanning microscopes equipped with a 40x LD C-Apochromat 1.1 NA water-immersion objective (Zeiss), 40x C-Apochromat 1.2 NA water-immersion objective (Zeiss), or a 63x Plan-Apochromat 1.4 NA oil-immersion objective (Leica). Hoechst was excited with a 405-nm laser line and detected at 410 to 483 nm. mClover3 and Alexa Fluor 488 were excited with a 488-nm laser line and detected at 493 to 556 nm. Alexa Fluor 568 and Alexa Fluor 594 were excited with a 561-nm laser line and detected at 566 to 635 nm. Alexa Fluor 647 and 5-SiR-Hoechst were excited with a 633- or 640-nm laser line and detected at 645 to 700 nm. For light-sheet imaging, oocytes were imaged in 0.5 ml of medium in four different compartments of a multiwell sample holder (Viventis Microscopy Sàrl). Dual-view images were acquired with LS2 Live (Viventis Microscopy Sàrl) equipped with an environmental incubator box and two 25× 1.1 NA water-dipping objectives (Nikon). SPY555 was excited with a 561-nm laser line and detected with a 523/20 nm–610/25 nm dual band-pass filter. Dual-view images were processed with a fusion script (Viventis Microscopy Sàrl) after acquisition.

Images of the control and experimental groups were acquired under identical imaging conditions on the same microscope. For some images, shot noise was reduced with a Gaussian filter. Care was taken that the imaging conditions (laser power, pixel-dwell time, detector gain, and exposure time) did not cause phototoxicity (for live imaging), photobleaching, and saturation.

Image quantification
To reproducibly segment chromatin (labeled with Hoechst 33342 or SPY555-DNA) or other nuclear structures (labeled with antibodies against different nuclear components), the Machine Learning Segmenter was used for automatic segmentations in Arivis (Zeiss). For each experiment, 3 to 5 z-planes from 5 to 10 cells or time points were manually annotated for foreground and background pixels and subjected to model training under the Fluorescence Fast mode. Suitable smoothing and threshold values were selected to refine the segmentation results before batch applications. Tracking in Arivis was used for tracking segmented chromocenters. Specific parameters used were: Brownian Motion (Centroid) (for Motion type), Center of Geometry (for Centroid), 3 μm (for Max. distance), and 2 (for Max. time gap). Total fluorescence intensity, volume, surface area and MSD of the segmented objects were exported into Excel (Microsoft) and OriginPro (OriginLab) for further processing.

Immunoblotting
40 mouse oocytes or embryos (per lane) were extensively washed in protein-free medium and snap-frozen in 1 μl of protein-free medium in liquid nitrogen. Before thawing, 7 μl of water and 4 μl of 4x NuPAGE LDS sample buffer (Thermo Fisher Scientific) with 100 mM dithiothreitol (DTT) were added. Samples were then thawed at 37°C and snap-frozen in liquid nitrogen twice more before being heated for 5 min at 99°C. For λPP treatment, oocytes were lysed in 8 μl of buffer (1X NEBuffer for protein metallophosphatase and 1 mM MnCl2) by snap-freezing in liquid nitrogen and thawing at 37°C three times, and incubated for 30 min at 37°C after adding 0.16 μl of 400 U/μl λPP. Samples were resolved on a 15-well NuPAGE 8 to 16% Tris-Glycine Plus WedgeWell Gel of 1.0 mm thickness (Thermo Fisher Scientific) with Tris-Glycine SDS Running Buffer (Thermo Fisher Scientific or Beyotime). Proteins were transferred onto a methanol-activated 0.45-μm polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific) with SDS-free Towbin buffer at 200 mA for 2 h on ice. Blots were stained with No-Stain Protein Labeling Reagent (Thermo Fisher Scientific) before blocking. Blocking and antibody incubations were performed in Tris-buffered saline (TBS) with 5% skim milk and 0.1% tween-20. Primary antibodies used were rabbit anti-RPB1 (CST #149158S), mouse anti-CTD pY1 (Active Motif #91220), rat anti-CTD pS2 (Active Motif #61699), rabbit anti-CTD pT4 (CST #26319S) and mouse anti-CTD pS5 (4H8) (Santa Cruz sc-47701). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CST #7074P2), goat anti-rat IgG (CST #7077S) and horse anti-mouse IgG (CST #7076P2). Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and documented with Amersham ImageQuant 500 (Cytiva). Care was taken that the exposure time did not cause saturation.

RT-qPCR
40 mouse oocytes (per sample) were extensively washed in protein-free medium and snap-frozen in 1 μl of protein-free medium in liquid nitrogen. Total RNA was extracted with TRIzol LS Reagent (Thermo Fisher Scientific) in Phasemaker Tube (Thermo Fisher Scientific) and treated with TURBO DNA-free Kit (Thermo Fisher Scientific). Reverse transcription (RT) was performed using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific), and quantitative PCR (qPCR) was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on CFX Opus 96 Real-Time PCR System (Bio-Rad). Endogenous Rpl13a was used as an internal control to calculate the relative transcript level. For amplifying Rpl13a, the following primers were used: 5’-GAGGTCGGGTGGAAGTACCA-3’ and 5’-TGCATCTTGGCCTTTTCCTT-3’. For amplifying Rpb1, the following RT primers were used: 5’-ACATGTGCAGGAAACATGAC-3’ and 5’-CTTCGGGTTATTAGAATCTACAAGC-3’.

Poly(A) Tail-Length Assay
40 mouse oocytes (per sample) were extensively washed in protein-free medium and snap-frozen in 1 μl of protein-free medium in liquid nitrogen. Total RNA was extracted with TRIzol LS Reagent in Phasemaker Tube and treated with TURBO DNA-free Kit. G/I tailing, RT and end-point PCR were performed according to the manual of the Poly(A) Tail-Length Assay Kit (Thermo Fisher Scientific). The poly(A) start site (PAS) fragment was obtained by PCR using the Rpb1-specific forward primer (5’- TTGGTGCCTGCTCTGG-3’) and the Rpb1-specific reverse primer (5’-TGGTCAAAATTAGTAAACTTTATTTCAATTTCAAAAAATAA-CAAA-3’) located immediately upstream of the PAS. The tail fragment was obtained by PCR using the same Rpb1-speicfic forward primer and a 35 nt universal reversal primer provided in the kit. The PCR products were resolved on a 2.5% agarose gel, and the poly(A) tail length was calculated by (size of tail fragment) – (size of PAS fragment) – 35.

Hi-C, data processing and analysis
Chromatin digestion was performed as previously described after minor modifications. 100 mouse oocytes (per sample) were fixed in PBS with 0.05% PVP and 1% methanol-free formaldehyde for 10 min at RT and 2.5 M glycine was added to a final concentration of 0.2 M to quench for 5 min at RT. Oocytes were transferred to 100 μl of PBS with 0.05% PVP and captured by adding 10 μl of washed ConA Beads Pro (Vazyme) for 10 min at RT. Oocytes were lysed in 12.5 μl of Hi-C lysis buffer [10 mM Trim-HCl (pH 8.0), 10 mM NaCl, 0.5% NP-40] supplemented with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Roche) for 15 min on ice. Supernatants were discarded with 2.25 μl remaining, and 0.25 μl of 5% SDS was added before incubating for 10 min at 62°C. 7.25 μl of nuclease-free water and 1.25 μl of 10% Triton X-100 were further added to quench for 15 min at 37°C, and 1.25 μl of 10x NEBuffer 2 (NEB) and 5 U of MboI (NEB) were added to digest chromatin overnight at 37°C. The next day, samples were incubated for 20 min at 62°C to inactivate MboI and then cooled to RT. 2.5 μl of fill-in master mix [7.5 μl of 0.4 mM biotin-14-dATP (Active Motif), 0.3 μl of 10 mM dCTP, 0.3 μl of 10 mM dGTP, 0.3 μl of 10 mM dTTP and 1.6 μl of 5 U/μl DNA polymerase I, Large (Klenow) Fragment (NEB)] was added to fill in restriction fragment overhangs and mark DNA ends with biotin, and samples were incubated for 90 min at 37°C. Ligation master mix [33.2 μl of nuclease-free water, 11 μl of 10x T4 DNA Ligase buffer (NEB), 5 μl of 10% Triton X-100, 0.6 μl of 100x BSA and 0.5 μl of 2000 U/μl T4 DNA Ligase (NEB)] was further added, and samples were incubated for 4 h at RT. After removing supernatants, beads were resuspended with 15.5 μl of nuclease-free water, 1.75 μl of Lambda Exonuclease Buffer (NEB), 0.15 μl of Lambda Exonuclease (NEB) and 0.15 μl of Exonuclease I (NEB)] and incubated for 1 h at 37°C to remove unligated ends. To release DNAs, 42.5 μl of nuclease-free water, 6 μl of 10% SDS and 2.5 μl of 20 mg/ml Proteinase K (Thermo Fisher Scientific) were added, and samples were incubated for 30 min at 55°C. To reverse crosslinks, 6.5 μl of 5 M NaCl was added, and samples were incubated overnight at 68°C. The next day DNAs were extracted with phenol chloroform in Phasemaker Tube, precipitated with ethanol and resuspended in 5.5 μl of nuclease-free water. Library preparation was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme) after minor modifications. DNA fragmentation mix was scaled down to 10 μl. After tagmentation, the biotin pull-down process was begun by washing 12.5 μl of 10 mg/ml Sera-Mag SpeedBeads Neutravidin-Coated Magnetic Particles (Cytiva) or Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) with 40 μl of 1x TWB [5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA, and 0.05% Tween-20]. After resuspension in 25 μl of 2x BB [10 mM Tris-HCl (pH 7.5), 2 M NaCl, and 1 mM EDTA], beads were added to samples and incubated for 15 min at RT. Before PCR, beads were washed with 40 μl of 1x TWB twice and resuspended in 29 μl of nuclease-free water. The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific) and qPCR, analyzed by a Fragment Analyzer System (Agilent) to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System (Illumina).

Reads were processed using HiC-Pro (v3.1.0) with default parameters in the HiC-Pro configuration file to obtain valid contacts. The “.bed” file of restriction fragments from MboI digestion was generated using the “digest_genome” Python script from HiC-Pro. Reads were initially mapped to mm10 (GRCm38.p6) reference genome, and unmapped reads were subsequently cut at restriction sites and re-mapped. After removing PCR duplicates and reads lacking restriction sites, contact matrix files were generated from the final set of valid contacts, which were then normalized by the ICE method. For visualization, the “hicpro2juicbox” shell script was employed to convert valid contacts into “.hic” files, which were then imported into Juicebox (v.2.20). For calculation of contact probability within chromosomes, valid contacts were converted into “.cool” files using Cooler (v0.9.3) and Cooltools (v0.6.1), and the resulting contact probability curves were smoothed and aggregated for plotting.

WGBS, data processing and analysis
30 mouse oocytes (per sample) were lyzed in 20 μl of lysis buffer consisting of 1x M-Digestion Buffer and 1 mg/ml Proteinase K (Zymo Research) for 20 min at 50°C. Bisulfite conversion and library preparation were performed using the Pico Methyl-Seq Library Prep Kit (Zymo Research) after minor modifications. In the amplification with PrepAmp Primer step, PrepAmp primer was diluted from 40 μM to 20 μM. In the library amplification step, 10 cycles were used. The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.

Reads were processed using TrimGalore (v0.6.7) to remove adaptor and low-quality reads with parameters “-q 20, -e 0.1, --length 20, --stringency 1”, and then trimmed reads were mapped to mm10 (GRCm38.p6) reference genome using Bsmap (v2.90) with default parameters. Methylation level of each CpG site was estimated using the function “mcall” of Moabs (v1.3.9.6). Methylation level of each CpG site from multiple replicates was determined using total methylated read count across replicates versus total read count across replicates, and CpG sites with less than 3 reads were discarded.

ATAC-seq, data processing and analysis
Library preparation was performed as previously described after minor modifications. 50 mouse oocytes (per sample) were treated with acidic Tyrode’s solution (Sigma-Aldrich) to remove the zona pellucida and transferred into 500 μl of ATAC-Resuspension Buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl and 3 mM MgCl2] containing 0.1% NP-40, 0.1% tween-20 and 0.01% digitonin to lyze for nuclei using a glass capillary with <50 μm opening. The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.

Reads were processed using TrimGalore (v0.6,7) to remove adaptor and low-quality reads with parameters “-q 20, -e 0.1, --length 20, --stringency 1”, and then trimmed reads were mapped to mm10 (GRCm38.p6) reference genome using Bowtie2 (v2.4.2) with parameters “-L 25, -X 2000, --no-mixed --no-discordant”. Unmapped reads and low mapping quality reads (Q<30) were discarded, and PCR duplicates were removed using the function “MarkDuplicates” from Picard tools (v2.27.2). ATAC-seq bigwig tracks were generated using the function “bamcoverage” from Deeptools (v3.5.1) with parameters “-binSize 50 -normSize 2494787188 -exactScaling -extendReads -normalizeUsing RPKM”. ATAC signal counts in each genomic region were computed using the function “computeMatrix” from Deeptools (v3.5.1) with parameters “-bs 200, --upstream 10000, --downstream 10000”.

RNA-seq, data processing and analysis
Karyoplast and cytoplast were separated by micromanipulation as previously described. 5 μl of Dynabeads MyOne Streptavidin C1 was washed with 20 μl of Dynabead solution A (50 mM NaCl and 0.1 M NaOH) twice and 20 μl of Dynabead solution B (100 mM NaCl). Dynabeads were then resuspended in 5 μl of 2x B&W buffer [10 mM Tris-HCl (pH 7.5), 2 M NaCl, and 1 mM EDTA], and 5 μl of 100 μM biotinylated oligo-dT30VN (Sangon Biotech) was added for 20 min at room temperature. Dynabeads were further washed with 20 μl of 1x B&W buffer four times and resuspended in 10 μl of nuclease-free water. 25 karyoplasts or 3 cytoplasts (per sample) were lyzed in 5 μl of lysis buffer [10 mM Tris-HCl (pH 7.5-8.0), 4% NP-40 and 0.2% SDS], and 0.24 μl of 5 M NaCl and 1 μl of Dynabeads were added for 20 min at room temperature. The supernatant was collected as the polyA-ve fraction, and 1 μl of 105-diluted ERCC RNA Spike-In Mix (Thermo Fisher Scientific) and 6.6 μl of nuclease-free water were added. Dynabeads were resuspended in 5 μl of lysis buffer as the polyA+ve fraction, and 1 μl of 105-diluted ERCC RNA Spike-In Mix and 7.6 μl of nuclease-free water were added. Library preparation was performed using the Ovation SoLo RNA-Seq System (Tecan) after minor modifications. In the primer annealing step, 1 μl of 50 μM random hexamer primer (Thermo Fisher Scientific) was added instead of 2.6 μl of First Strand Primer Mix. The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.

For RNA-seq of 2PN embryos, RNA libraries were constructed using the SMART-Seq Total RNA Pico Input Kit with UMI (Takara), following the manufacturer’s instructions with minor modifications. 3 embryos at 16 hpf were pooled as a sample. During the first PCR amplification step of library preparation, 5 cycles were performed, followed by 25 cycles in the second PCR amplification step. The resulting libraries were purified, quantified using the Qubit 1X dsDNA HS Assay Kit and qPCR, and assessed for quality and successful amplification using a Fragment Analyzer System. Libraries were then sequenced on the NovaSeq X Plus Sequencing System.

Reads were processed using TrimGalore (v0.6.7) to remove adaptor and low-quality reads with parameters “-q 20, -e 0.1, --length 10, --stringency 1”, and the reads with UMI barcode were trimmed with parameters “-q 20, -e 0.1, --length 10, --stringency 1 –clip_R2 14”. Then trimmed reads were mapped to mm10 (GRCm38.p6) reference genome plus ERCC.fasta using STAR (v2.7.10a) with default parameters. Multi-mapping reads were filtered using Samtools (v1.17), and PCR duplicates were removed using the function “MarkDuplicates” from Picard tools (v.2.27.2). Deduplicated reads were mapped to genes using HTseq (v2.0.3) as unstranded samples with default parameters. A gene/ERCC count was considered valid when present in at least five reads in at least two samples. Differentially expressed genes between groups of samples were identified using DESeq2 (v1.38.0). Gene counts of each sample were normalized by defining ERCC genes as the “controlGenes” when estimating the “sizeFactors” with DESeq2. For samples without ERCC, the gene counts of each sample were directly analyzed using the function ‘DEseq’ in DEseq2. The differentially expressed genes were identified based on an FDR cutoff of 0.05 and a log2FoldChange cutoff of 0.05.

For RNA-seq of 2PN embryos, differentially expressed genes were cross-referenced with minor ZGA transcripts (clusters IV, and V) or maternal transcripts (cluster II) from a previous study to calculate the overlap between minor ZGA transcripts and genes downregulated in NSN-derived 2PN embryos, as well as between maternal transcripts and genes upregulated in NSN-derived 2PN embryos. The non-overlapped genes further underwent GO BP enrichment analysis with Metascape, and enrichment results were visualized as dot plots.

Ribo-seq, data processing and analysis
Library preparation was performed as previously described after minor modifications. 50 mouse oocytes (per sample) were lysed in 150 μl of ice-cold lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, and 1 mM DTT] for 5 min on ice, followed by snap-freezing in liquid nitrogen and thawing at 37°C three times. After spinning at 21300 xg for 5 min at 4°C, the supernatant was digested with 0.25 μl of 2 U/μl TURBO DNase (Thermo Fisher Scientific) and 0.2 μl of 1:10-diluted 20 U/μl RNase T1/0.5 U/μl RNase A cocktail (Thermo Fisher Scientific) for 30 min at 37°C. 5 μl of 20 U/μl SUPERase In RNase inhibitor (Thermo Fisher Scientific) and 95 μl of ice-cold lysis buffer were further added, and the mixture was extracted using TRIzol LS Reagent in a Phasemaker Tube. Purified RNAs were resuspended in 5 μl of PNK solution [5 U T4 PNK (NEB) in 1X T4 PNK reaction buffer] and incubated for 10 min at 37°C. After pre-incubation, ATP was added to a final concentration of 10 mM, and the mixture was incubated for another 30 min at 37°C before adding 5.5 μl of Gel Loading Buffer II (Thermo Fisher Scientific). Ribosome-protected fragments (RPFs) corresponding to the 25 to 35 nt band were separated using 6% TBE-Urea Gel (Thermo Fisher Scientific) and subjected to library preparation using NEXTFLEX Small RNA Seq Kit v3 (Perkin Elmer). The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.

Reads were processed using TrimGalore (v0.6.7) to remove adaptor and low-quality reads with parameters “-q 20, --phred33, --stringency 3, --length 20, --max_length 40”. As the NEXTFLEX Small RNA Seq Kit v3 randomly added 4 bases at the 5’ and 3’ end of each read, we clipped these random bases when trimming reads. Reads mapped to rRNA were removed using Bowtie (v1.3.1) with parameters “-v 1, -a, --best, --strata, -q, --phred33-quals”. Remaining reads were mapped to mm10 (GRCm38.p6) reference genome using STAR (v2.7.10a) with parameters “--outFilterType BySJout, --outFilterMismatchNmax 2, --outFilterMultimapNmax 1, --outFilterMatchNmin 16, --alignEndsType EndToEnd, --outSAMattrIHstart 0”. Differentially translated mRNAs were identified using DESeq2 (v1.38.0).

CUT&Tag, data processing and analysis
Library preparation was performed as previously described after minor modifications. 50 to 80 mouse oocytes (per sample) were pre-extracted with ice-cold extraction buffer [25 mM HEPES (pH 7.4), 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose and 0.5% triton X-100] for 10 min on ice and washed three times through ice-cold extraction buffer without triton X-100. Pre-extracted oocytes were directly washed with antibody buffer and incubated with primary antibody without crosslinking. Primary antibodies used were mouse IgG (Beyotime #A7028), mouse anti-CTD pS5 (4H8) (Santa Cruz #sc-47701), rat IgG (Beyotime #A7031), rat anti-CTD pS2 (Active motif #61699). Secondary antibodies used were rabbit anti-mouse (Sigma-Aldrich #06-371) and rabbit anti-rat (Sigma-Aldrich #AP164). In the library amplification step, 16 to 21 cycles were used. The purified libraries were quantified by Qubit 1X dsDNA HS Assay Kit and qPCR, analyzed by a Fragment Analyzer System to confirm successful amplification and quality, and sequenced using Novaseq 6000 Sequencing System.

Reads were processed using TrimGalore (v0.6,7) to remove adaptor and low-quality reads with parameters “-q 20, -e 0.1, --length 20, --stringency 1”, and then trimmed reads were mapped to mm10 (GRCm38.p6) reference genome using Bowtie2 (v2.4.2) with parameters “-L 25, -X 2000, --no-mixed --no-discordant”. Unmapped reads and low mapping quality reads (Q<30) were discarded, and PCR duplicates were removed using the function “MarkDuplicates” from Picard tools (v2.27.2). CUT&Tag bigwig tracks were generated using the function “bamcoverage” from Deeptools (v3.5.1) with parameters “-binSize 50 -normSize 2494787188 -exactScaling -extendReads -normalizeUsing RPKM”. CUT&Tag signals on genomic regions were computed using computeMatrix with parameters “-bs 200, --upstream 10000, --downstream 10000”.

Statistical analysis
Average (mean) and SD were calculated in Excel. Statistical significance is based on unpaired, two-tailed Student’s t test (for absolute values between two groups), one-way ANOVA followed by post-hoc Tukey’s multiple comparison test (for absolute values for multiple groups) and two-tailed Fisher’s exact test (for categorical values) calculated in OriginPro or Prism (GraphPad), assuming normal distribution and similar variance. All box plots show median (horizontal black line), mean (small black squares), 25th and 75th percentiles (boxes), 5th and 95th percentiles (whiskers), and 1st and 99th percentiles (crosses). All data were from at least two independent experiments with at least three biological replicates per experiments. P values are designated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Nonsignificant values are indicated as N.S.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "NCOMMS-25-19027 “Degradation of maternal RNA….”\nThank you for the opportunity to review this manuscript. It addresses a fundamental property in mammalian oocytes at a\nspecific transition in oogenesis when transcriptional silencing presages preparations for maturation, fertilization and zygote\nformation. That property is a reconfiguration of chromatin in the oocyte nucleus known as the GV or germinal vesicle. This\nwork points to degradation of RNApolII as a critical regulator based on experimental manipulations that either promote the\ntransition from NSN to SN or not as elucidate in mouse oocytes and oocytes from ovarian follicles. While preliminary results\nare reported for human oocytes/follicles, the sourcing and manipulation of this material raises serious concerns and detracts\nfrom the overall robustness of the mouse studies.\nA strength of the paper is the array and value of the many approaches taken and the level of analysis and use of truly novel\nmethods to explore the overarching hypothesis being tested.\nA major weakness, unless fully explained, is the sourcing and handling of mouse materials and the lack of appropriate\ncontrols and background information-both from the literature and from the methods section.\nSpecific points are addressed below:\nLine 47: poor selection of background literature on NSN/SN; many refs missing or overlooked (Dev Biol 1991 Vol. 143 Issue\nLine 61: here and in methods , good selection of vital dyes for sorting NSN SN,but where did oocytes come from? Methods\nstates 6-8 week old (2 strains) primed or not? What size follicles were oocytes derived from? And key here is whether naked\nor cumulus enclosed were chosen because it is well known that naked oocytes come from atretic follicles which are\nabundant in this age of mice across strains. Please be specific in how oocytes were chosen for sorting. (see Biol Reprod\nLine 135: since naked oocytes have limited transport ability use of analogues not producing effects become inconclusive as\nintracellular effect has not been demonstrated.So many negative results no NSN to SN) might better reflect a permeability\nissue rather than an effect as described.\nLine 161: reversible cell cycle phosphorylation has been demonstrated in mouse oocytes with functional outcome of meiotic\nresumption or not. Why was this assay not conducted throughout these studies?\nLine 17: No information provided on patients (and might be same PCOS patients they used for cortical strip/follicles)\nhuman\nGVs so drug induced indirect changes are at best a weak suggestion for anything relevant for human oocyte when material\nis uncharacterized and limited to a descriptive and minimal data set. Suggest removing from study.\nLine 194: Quick switch from oocytes to mouse follicles, then human ovarian cortex presumably to evaluate NSN status;\nauthos apparently unaware of differences between mouse and human and provide no details on what makes this material\neven valid for a sophisticated deep dive into the manin hypothesis. Poorly developed section of paper.\nLine 243: comprehensive series of experiments linking RNApol degradation to NSN to SN transition\nLine 271: interesting approach to physical forces attending chromatin compaction; have authors considered anchoring to\nnuclear lamina and what happens if lamin stability compromised?\nLine 300: IVF experiments with GVs are weak and technically problematic as NSN fail at PN and ZGA becomes inferred\nrather than being tested directly. See Methods comments below.\nDiscussion\nLine 317: Wildly exaggerated claim to have “elucidated for all mammalian oocytes”-not so and statement should be\ntempered because thus is ONLY a mouse study and while a good model for NSN to SN, it would not hold for any other\nmammal.\nLine 321: OK to challenge other viewpoints but here that degradation releases chromatin allowing trans stoppage and\nremodeling could also be a cell cycle relevant state as had been well established years ago.\nLine 746: Need information on how oocytes obtained. Were all naked, stripped, arrested in DcAMP for how long before\nmanipulated? And key here, if you did switch to SN, what was maturation rate? M1s, M2s??? Essential to know this is a\nfunctional change and not just a morphological one.\nLine 770: Many methods issues with materials for IVF experiments (??super ovulated yes, but how to compare from follicular\noocytes as not possible to score NSN etc); and you fertiliza GV oocytes (or did you mature them to M2 before ) as hole in\nzona would clearly result in polyspermy since cortical granule positioning would not have occurred; critical information is\nmissing otherwise experiments cannot be interpreted.\nLine 800: were all human GVs vitrified or not..?? were some fresh after retrieval??? And of vitrified, how many survived\nwarming as has been shown to damage GVs with poor survival.\nLine 805: cannot use cortical tissue from PCOS patients and especially a wedge resection; would need a non-PCO ovary\ncortex and do follicle activation to do this study correctly; and enzymatic digestion of human ovaries to get follicles is not\nused because follicles do not survive and GV chromatin becomes irreversibly compacted, precluding this work completely.\nDrop this part as it detracts from the good work.\nFig 2/3 comparison of isolated oocytes and those from follicles are different, as one might expect. Why?\nFig 5 obvious that some epigenetic marks in pronuclei are non specific and precludes claims about SN transition and impact\non development; weak data set.\nFig 6: gammH2AX shows more foci in nsn vrsus sn.How would difference in DDR response as has been reported in mouse\nimpact your results and could this mean DDR is turned off coordinately with chromatin housecleaning you so nicely have\ndemonstrated??", "reviewer_2": "The manuscript entitled \" Degradation of maternal RNA polymerase II is essential for oocyte chromatin reorganization and\nembryonic development in mammals\" led by Wang et al. describes an interesting finding that during oogenesis, the NSN to\nSN transition (reorganization of oocyte chromatin from non-surrounding nucleoli to surrounding nucleoli) is attributed to the\ndegradation of maternal RNAPII and the elimination of chromatin-bound RNAPII. That is, they suggest that this degradation\nsimultaneously induces chromatin reorganization and represses transcription, which is contrary to the traditional view that\nthe NSN to SN transition and transcriptional silencing are two unique features of the final stage of oocyte growth and that\nthese two events are driven by independent mechanisms in mammalian oocytes.\nThe authors searched for common nuclear components between NSN and SN oocyte nuclei and found that actinomycin D,\na transcriptional inhibitor targeting RNA polymerase II, could induce NSN-to-SN transition. They then found that targeted\ndegradation of RNAPII was sufficient to induce the NSN to SN transition in mouse and human oocytes. They further\ndemonstrated that RNAPII degradation during the NSN to SN transition is through the stripping of chromatin-bound RNAPII.\nOverall, the data in this manuscript are clear, sufficient and reliable, and I support its publication in Nature Communications.\nHowever, I would like to share the following suggestions with the authors to consider and improve the manuscript.\n1) I support the observation that RNAPII degradation is required for the NSN to SN transition in vivo, but which mechanism\nregulates the degradation of phosphorylated RNAPII during oocyte maturation from the NSN to the SN stage? Although your\nexperiments in vitro suggest that both separase- and proteasome-mediated RNAPII degradation are required for the NSN to\nSN transition. This needs to be discussed.\n2) Continuing with the above question, although the authors looked at the levels of actively transcribed and stored mRNAs\nsuch as the RNAPII subunit Rpb1, it might be necessary to look at mRNAs transcribed and stored in other RNAPII\ncomponents between the NSN and SN. This would rule out transcriptional differences between RNAPII components in the\nSN and NSN. Or it might be necessary to look at upstream molecular associations that influence RNAPII levels.\n3) The authors challenge the conventional view that removal of chromatin-bound RNAPII leads not only to NSN-to-SN\ntransition but also to global transcriptional silencing in SN oocytes. Therefore, it is necessary to discuss in detail why other\nfactors, such as loss of the cytoplasmic mRNA repressor LSM14B (see Figure 5B in PMID: 37481122; or PMID: 37083226;\nPMID: 37889087; PMID:37578641), also lead to the aberrant NSN-to-SN transition.\n4) The author's last result shows that although the protein WB experiment showed that non-degraded RNAPII led to higher\nRNAPII levels in 1-cell embryos derived from MII oocytes and NSN oocytes, immunofluorescence staining showed lower\nlevels of total RNAPII and phosphorylated RNAPII in the nucleus. At the same time, there was no significant difference in\nchromatin accessibility between embryos derived from NSN and SN oocytes. The author thus concluded that there is a\ndefect in the zygotic factors that promote RNAPII to enter the nucleus and/or bind to chromatin. There is no experimental\nevidence for this, and experimental verification should be provided, or a discussion should be given, otherwise the\nconclusions may need to be weakened.", "reviewer_3": "This manuscript had the stated goal of identifying “candidates that drive the physiological NSN-to-SN transition” that growing\noocytes undergo during antral follicle development. Although it was a reasonable goal, the experiments done did not\naccomplish anything except to document that inhibition of RNA polymerase II-mediated transcription promotes the transition.\nThe authors claim to have demonstrated that reducing RNA polymerase protein levels, not just transcriptional activity, was\nthe factor that induced the transition. I have serious reservations about this claim based on lack of proper controls and\noverinterpretation of results, but even if it were true, the authors have no idea why or how this might be the case. Therefore,\nbest case scenario, this is a minimal advance to the literature. Specific concerns are outlined below.\n1. One key experiment in the manuscript was the targeted degradation of RNA polymerase II in NSN oocytes by a “TRIM-\naway” technique: microinjection of cRNA encoding a genetically encoded antibody fused to the ring domain of TRIM21.\nThe poor experimental design means that this experiment is not possible to interpret for several reasons:\na. The authors microinjected almost 3 ug/ul (pipette concentration) of the cRNA, which is an enormous amount relative to\nstandard cRNA microinjection procedures for numerous other proteins, including what was used in most of their own\noverexpression studies (~500-600 ng/ul). Rather than an appropriate control, which would be an alternate cRNA that\nencoded the ring domain of TRIM21 fused to a genetically encoded antibody targeting a different, non-relevant protein, the\nauthors used injection of water as a control. Microinjection of oocytes with cRNA to induce protein degradation carries with it\nstrong potential for off-target effects due to displacing physiological mRNAs from the translation system as well as causing a\nrelatively large amount of protein to move into the proteasome degradation pathway, which could interfere with physiological\nprotein degradation as well. In our hands, cRNA at >2 ug/uL has clear non-specific toxic effects on mouse oocytes and\nembryos. Why was such a high concentration required? Even if it were necessary, water injection is simply an inadequate\ncontrol.\nb. Figure 4a-c: The immunofluorescence of D14+7 mouse follicles shows clearly that a SN configuration is achieved before\nthere is a significant loss of RNA polymerase protein. This experiment invalidates the authors’ conclusions regarding the\nneed for RNA polymerase II protein elimination to achieve SN.\nc. The human oocyte data conflicts with the authors’ interpretation of their mouse oocyte data. The images of oocytes within\nprimordial, primary, and secondary follicles presumably are just to show that the antibodies being used recognize nuclear\nproteins. What the authors call “early growing oocytes” are already transitioning to SN, and the “late growing” and “fully\ngrown” oocytes have transitioned as well. At the same time, the authors show that there are no changes in total RNA\npolymerase II levels (Figure 4f and 4h), but that the phosphorylated active form (pS2) has decreased (Figure 4g), consistent\nwith a reduction in transcription. These findings suggest that in human oocytes, loss of transcription is associated with the\nSN configuration, not that a loss of RNA polymerase II protein is important.\n2. A second key experiment was to test whether inhibition of RNA polymerase II-based transcription cause the NSN to SN\ntransition. Although alpha-amanitin and triptolide both caused inhibition of transcription, they also both caused degradation\nof RNA polymerase II protein. To separate these two effects, the authors used alternate transcriptional inhibitors that do not\nimpact RNA polymerase II protein levels. Given 7 hours of oocyte incubation time, the transcriptional inhibitors 8-AA and 7-\nDAA did not cause oocytes to convert to SN nuclei. However, there is no information provided regarding how quickly they\nreduce transcription relative to how fast transcription is reduced by the relatively high concentration of alpha-amanitin used\nin these experiments (100 uM vs 26 uM used in many studies). In supplemental video 4, alpha-amanitin had caused the\nchromatin to begin surrounding the nucleolus in 3 hours and triptolide (video 2) by 4 hours. If 8-AA and 7-DAA take longer to\nreduce transcription, then the conclusion that they do not impact the NSN-to-SN transition could be incorrect. Furthermore,\nmy interpretation of both chromatin images shown in Figure S4a-b is that they are in transition to the SN stage, suggesting\nthat transcriptional inhibition alone may be sufficient to induce the transition.\n3. The stated “comprehensive analysis of SN and NSN nuclei” was not well planned or carried out in a robust fashion.\nThese data are unreliable.\na. Sorting of nuclei into NSN vs SN ignores the reality of nuclei in transition. The original paper (referenced by the authors)\ndescribes 4 different chromatin configuration stages, with two being transitory between NSN and SN. The authors do not\ndistinguish these stages with their methodology or indicate what was done with oocytes in transitional stages.\nb. The Method used to define “downregulation” of nuclear components assumes that antigen accessibility to antibody\nstaining does not change during nuclear reorganization. Furthermore, immunofluorescence detection of a punctate\ndistribution of proteins cannot be used to accurately quantify protein levels, particularly when the differences are subtle,\nand the experiment was only done twice! None of the antibodies used were demonstrated to be specific for the targeted proteins.\nc. Using 65 different treatments in oocytes in the absence of documenting that the concentrations used are effective but non-\ntoxic has no meaning. Conclusions cannot be drawn from such studies.\nd. Putting the organic compound 1,6-hexanediol on oocytes appears to have killed the cells (Figure S2g), which again,\ngenerates non-interpretable data. Note the complete lack of evidence of a plasma membrane in the transmitted light image.\n4. The conclusion regarding developmental potential has numerous problems. First, developmental potential is tested by\ndevelopment to the blastocyst stage or else embryo transfer followed by live birth, not development to the 2-cell stage. In\naddition, this conclusion was drawn on the basis of having a total of 16 fertilized eggs. That is an insufficient number, even\nfor just looking at development to the 2-cell stage.\n5. Pages 9-10: It is incomprehensible to this reviewer why the authors would spend the time and effort doing whole genome\nbisulfite sequencing, ATAC-seq, and fractionated RNA-seq to determine whether or not a specific gene is transcribed, rather\nthan just doing a simple RT-qPCR experiment, which they eventually did. Similarly, using Ribo-seq, RIBOmap, and a\npoly(A) tail length assay when a simple cycloheximide experiment would suffice is unjustified. The later CUT&Tag\nexperiments on NSN and SN oocytes to examine binding locations of the pS2 form of RNA polymerase II doesn’t make any\nsense because SN oocytes are not transcriptionally active. Similarly, it is quite unclear how Hi-C profiling added anything to\nthe manuscript. All the unnecessary data simply confuse the reader and do not impact the story being presented.\n6. Lack of robust and reproducible data that was properly analyzed\na. Performing experiments twice is inadequate\nb. The number of oocytes in many of the experiments is too low for robust conclusions\nc. Data analysis is completely inadequate. The authors report using Student ‘s t-test when they are clearly comparing\nmultiple groups. Many experiments lack data analysis at all yet are declared in the text to have differences present.\n7. The Methods as written do not provide sufficient information regarding the experiments that were done. In particular, the\ndescriptions of oocyte collection and microinjection were incomplete. The microinjection was not even described other than\nto reference another paper in which the method was still not described, but it referenced another paper that did a completely\ndifferent method (injecting starfish or sea urchin embryos, not mouse oocytes).", "reviewer_4": "In this study, the authors rigorously analyzed the effect of RNAPII degradation at the final step of oocyte growth, which\ninvolves the NSN-to-SN transition. Interestingly, treatment with RNAPII inhibitors, which lead to RNAPII degradation, was\nsufficient to induce the NSN-to-SN transition. Furthermore, this artificial induction partially rescued the impaired\ndevelopmental potential of NSN oocytes. In addition to these inhibitors, the authors employed miniTrim-Away to selectively\ndegrade RNAPII. Chromatin-bound RNAPII appeared to restrict chromatin mobility, as its degradation resulted in increased\nchromatin dynamics. These changes in chromatin mobility, in conjunction with the attractive forces at the nucleolar surface,\nappeared to play an important role in driving the NSN-to-SN transition.\nThe use of multiple complementary approaches to demonstrate that RNAPII degradation promotes the NSN-to-SN transition\nis highly commendable. However, I believe that some additional work is necessary before this manuscript can be\nconsidered for publication, as detailed below.\nMajor comments:\n1. Fig. 1: The authors tried to indicate the status of SN or NSN by the quantification of chromatin volume. However, as the\nauthors noticed from experiments such as RNase A or 1,6-hexandiol treatment, the chromatin volume does not necessarily\ndiscriminate between SN and NSN. It will be highly helpful if the authors could summarize the phenotype of each treatment\nby providing additional figures or tables.\n2. Fig. 2e: It will be important to examine embryonic development to the blastocyst stage, to understand the developmental\ncompetence of the NSN, a-amanitin treated SN-induced, and SN oocytes.\n3. Fig. S4: The experiments using 8-AA and 7-DAA are important, as these assays suggested that the inhibition of\ntranscription only is not sufficient to induce the NSN-to-SN transition. However, it remained unclear how rapidly each\ntreatment could halt transcription. For instance, if the duration of the transcriptionally inactive state differs between α-\namanitin and 8-AA/7-AA treatments, the interpretation of the data would change accordingly.\n4. Fig. 3: For the experiments using miniTrim-Away, H2O only was used as a control. Obviously, this is not an appropriate\nnegative control. If the authors could perform additional experiments with a more proper control group, the quality of this\nstudy will be improved.\n5. Fig. 4i.j: CB-5083 and MG-132 affect many other proteins and pathways than RNAPII. Thus, the conclusion that the\nartificial stabilization of RNAPII suppresses the NSN-to-SN transition is not sufficiently supported by the provided data.\nI expect that the authors should be able to perform additional experiments to sustain RNAPII expression using more specific\nmethods.\n6. Abstract and line 287: “Nucleolar surface remodeling” is ambiguously described. There is no experimental data showing\nthe nucleolar surface remodeling, as far as I understand. Please explain more precisely. If there is no evidence for it, I feel\nthat the authors should remove such descriptions.\nMinor comments:\n7. Fig. S1: The authors used pan-trimethyl-lysine antibodies. Thus, “KMe3”, but not “HMe3”, will be correct. This will be also\nthe case for “HMe2”.\n8. Line 174: It was not clear what the “growing ones” means. Do they correspond to relatively small growing oocytes?"}, "author_response": "Response to Referees – Nature Communications ID: NCOMMS-25-\n19027\nNatural degradation of RNA polymerase II is essential for oocyte chromatin\nreorganization and maternal-to-zygotic transition\nAuthors: Jing Wang, Wang Li, Jing Guo, Xiaoming Xu, Ge Lin, Bin Li, Chun So\nFirst, we want to thank all referees for their insightful comments and expert suggestions\nthat greatly improved the manuscript.\nReviewer #1:\nThank you for the opportunity to review this manuscript. It addresses a fundamental\nproperty in mammalian oocytes at a specific transition in oogenesis when\ntranscriptional silencing presages preparations for maturation, fertilization and zygote\nformation. That property is a reconfiguration of chromatin in the oocyte nucleus known\nas the GV or germinal vesicle. This work points to degradation of RNApolII as a critical\nregulator based on experimental manipulations that either promote the transition from\nNSN to SN or not as elucidate in mouse oocytes and oocytes from ovarian follicles.\nWhile preliminary results are reported for human oocytes/follicles, the sourcing and\nmanipulation of this material raises serious concerns and detracts from the overall\nrobustness of the mouse studies.\nA strength of the paper is the array and value of the many approaches taken and the\nlevel of analysis and use of truly novel methods to explore the overarching hypothesis\nbeing tested.\nA major weakness, unless fully explained, is the sourcing and handling of mouse\nmaterials and the lack of appropriate controls and background information-both from\nthe literature and from the methods section.\nWe are grateful to the reviewer for his/her enthusiastic comments about our work and\nhope that the revised version of our manuscript could fully address his/her concerns.\nSpecific points are addressed below:\nLine 47: poor selection of background literature on NSN/SN; many refs missing or\noverlooked (Dev Biol 1991 Vol. 143 Issue 1 Pages 162-72,Accession Number: 1985016;\nDev Biol 1992 Vol. 152 Issue 1 Pages 62-74,Accession Number: 1378414; Dev Biol\nWe thank the reviewer for asking us to include additional background literature on\nNSN/SN chromatin configuration. We apologize for omitting these early literature\nowing to the citation limit from previous submissions. We now included the literature\nsuggested by this reviewer as citations 23, 24, 25 and additional background literature\nas citations 1, 4, 6-17, 23-25, 28-31, 33-45, 56-58.\nLine 61: here and in methods, good selection of vital dyes for sorting NSN SN,but where\ndid oocytes come from? Methods states 6-8 week old (2 strains) primed or not? What\nsize follicles were oocytes derived from? And key here is whether naked or cumulus\nenclosed were chosen because it is well known that naked oocytes come from atretic\nfollicles which are abundant in this age of mice across strains. Please be specific in\nhow oocytes were chosen for sorting. (see Biol Reprod 2003 Vol. 68 Issue 3 Pages 812-\n821, DOI: 10.1095/biolreprod.102.008656).\nWe thank the reviewer for asking us to provide additional details for oocyte isolation.\nWe now provided additional details regarding oocyte isolation in the respective\nsentences.\nPrevious submission:\nOocytes were isolated from ovaries of 8- to 10-week-old FVB/N or C57BL/6J (Charles\nRiver Laboratories) or Npm2 KO female mice. GV oocytes were arrested at prophase\nin homemade phenol red-free M2 supplemented with 250 μM dibutyryl cyclic AMP\n(dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin oil (ACROS Organics)\nat 37 °C.\nRevised:\nOocytes of around 60-80 μm in diameter were isolated from ovaries of 8- to 10-week-\nold unprimed FVB/N or C57BL/6J (Charles River Laboratories) or Ubb cKO or Npm2\ncKO female mice by puncturing preantral and antral follicles on the surface of ovaries\nwith needles. Cumulus-enclosed GV oocytes were stripped by repeated mouth pipetting\nand arrested at prophase in homemade phenol red-free M2 supplemented with 250 μM\ndibutyryl cyclic AMP (dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin\noil (ACROS Organics) at 37 °C.\nLine 135: since naked oocytes have limited transport ability use of analogues not\nproducing effects become inconclusive as intracellular effect has not been\ndemonstrated.So many negative results no NSN to SN) might better reflect a\npermeability issue rather than an effect as described.\nWe thank the reviewer for asking us to clarify the respective sentence. Here we were\nreferring to the fact that many of the almost 65 small molecule inhibitors tested did not\nlead to the desired changes in nuclear components (e.g., supposingly a histone\nmethyltransferase inhibition did not reduce the level of KMe2 or KMe3), rather than\nnot leading to NSN-to-SN transition. We did agree with the reviewer that the small\nmolecule inhibitors not working may be related to their limited permeability. We now\nrewrote the respective sentence to avoid confusion and the associated table legend to\ntone down our conclusion.\nPrevious submission:\nOf almost 65 different treatments (Table S1), only 19 of them led to the desired changes\nof nuclear components (Extended Data Fig. 2d). Many of these treatments altered the\ntranscriptional activity and/or chromatin compaction in NSN and SN oocytes (Fig. 1e\nand Extended Data Fig. 2e).\nTable S1 List of tested small-molecule inhibitors that did not work in this study.\nRevised:\nMany of the treatments that led to the desired changes in nuclear components altered\nthe transcriptional activity and/or chromatin compaction in NSN and SN oocytes (Fig.\n1e and Extended Data Fig. 2d,e).\nTable S1 List of small-molecule inhibitors tested that did not lead to desired changes in\nnuclear components in oocytes.\nLine 161: reversible cell cycle phosphorylation has been demonstrated in mouse\noocytes with functional outcome of meiotic resumption or not. Why was this assay not\nconducted throughout these studies?\nWe thank the reviewer for the suggestion. We believe the reviewer referred to the fact\nthat cyclin-dependent kinase inhibitors have been used to reversibly control meiotic\nresumption in oocytes, and can be used to investigate the phosphorylation of RNAPII.\nIndeed, we have used flavopiridol (pan-CDK inhibitor, in Fig. 4i), THZ1 (CDK7\ninhibitor, in Extended Data Fig. 9a), and DRB (CDK9 inhibitor, in Extended Data Fig.\n9a) for later in the text. In addition, we now performed additional experiments to further\nevaluate the developmental potential of induced SN oocytes by transiently treating\nNSN oocytes with DRB, followed by DRB washout before in vitro maturation and in\nvitro fertilization (Extended Data Fig. 9a and Response Fig. 1).\nResponse Fig. 1 Manual scoring of developmental outcomes of NSN, DRB-\ntreated NSN, SN, and DRB-treated SN oocytes after IVM and IVF. (Fisher’s exact\ntest for 2-cell embryos from NSN and DRB-treated NSN oocytes, P = 0.0047;\nFisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN oocytes, P =\n0.0496; Fisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN\noocytes, P = 0.3741;)\nLine 17: No information provided on patients (and might be same PCOS patients they\nused for cortical strip/follicles) human GVs so drug induced indirect changes are at\nbest a weak suggestion for anything relevant for human oocyte when material is\nuncharacterized and limited to a descriptive and minimal data set. Suggest removing\nfrom study.\nWe thank the reviewer for asking us to provide additional details regarding the\ncollection of human GV oocytes. We would like to clarify that the human GV oocytes\nwere collected independently of the cortical strip/follicles from PCOS patients, and\nwere from couples undergoing ICSI with an age of 23-35 years old, and owing to male\nfactor-associated infertility. We now provided additional details in the respective\nsentence. Also, as later suggested by this reviewer, we now removed the data associated\nwith cortical strip/follicles from PCOS patients.\nPrevious submission:\nOocytes were collected from patients who underwent ovarian stimulation for\nintracytoplasmic sperm injection (ICSI) as part of their assisted reproduction treatment\nat the Reproductive and Genetic Hospital of CITIC-XIANGYA and Beijing Perfect\nFamily Hospital. Only oocytes that were GV at the time of ICSI and thus unsuitable for\nthe procedure were used in this study.\nRevised:\nOocytes were collected from patients with an age of 23-35 years old who underwent\novarian stimulation for intracytoplasmic sperm injection (ICSI) as part of their assisted\nreproduction treatment owing to male factor-associated infertility at the Reproductive\nand Genetic Hospital of CITIC-XIANGYA and Beijing Perfect Family Hospital. Only\noocytes that were GV at the time of ICSI and thus unsuitable for the procedure were\nused in this study.\nLine 194: Quick switch from oocytes to mouse follicles, then human ovarian cortex\npresumably to evaluate NSN status; authos apparently unaware of differences between\nmouse and human and provide no details on what makes this material even valid for a\nsophisticated deep dive into the manin hypothesis. Poorly developed section of paper.\nWe thank the reviewer for the suggestion. As later suggested also by this reviewer to\nfocus our attention on mouse in this study, we now removed the corresponding section\non human ovarian cortex owing to concerns associated with PCOS materials and\nprovided new immunostaining data (Fig. 4a-c) on mouse ovarian sections to further\nstrengthen our findings in mouse oocytes and follicles.\nResponse Fig. 2 (new Fig. 4a-c) a, Immunofluorescence images of oocytes from\nfollicles at different growth stages on mouse ovary sections. Green, pS2;\nmagenta, RPB1; blue, chromatin (Hoechst). Yellow dotted circles highlight the\nnuclei. b, c, Automated quantification of total intensity of pS2 and RPB1 in\noocytes from follicles at different growth stages on mouse ovary sections.\nPrevious submission:\nAs an orthogonal approach, we cultured mouse ovarian follicles ex vivo and harvested\noocytes at different growth stages. This approach showed that phosphorylated RNAPII\nwas loaded during the early growth stages, but then progressively depleted during the\nlate growth stages (Fig. 4a-d), concomitant with NSN-to-SN transition (Fig. 4a,e). We\nalso examined human follicles of different growth stages from ovarian cortical tissues\nand found similar changes in phosphorylated RNAPII levels (Fig. 4f-h), suggesting that\nphosphorylated RNAPII was specifically depleted during NSN-to-SN transition in vivo\nin mouse and human.\nRevised:\nAs orthogonal approaches, we imaged oocytes in follicles at different growth stages on\nmouse ovary sections and oocytes harvested from ex vivo follicle culture. Both\napproaches showed that phosphorylated RNAPII was progressively depleted during\nlate stages of follicle growth (Fig. 4a-g), concomitant with NSN-to-SN transition (Fig.\n4a,d,h). Thus, phosphorylated RNAPII was specifically depleted during NSN-to-SN\ntransition in vivo.\nLine 243: comprehensive series of experiments linking RNApol degradation to NSN to\nSN transition\nLine 271: interesting approach to physical forces attending chromatin compaction;\nhave authors considered anchoring to nuclear lamina and what happens if lamin\nstability is compromised?\nWe thank the reviewer for the enthusiastic comments regarding our experimental design.\nTo address the potential contributions of chromatin attaching to/detaching from nuclear\nlamina to NSN-to-SN transition as suggested by the reviewer, we now performed\nminiTrim-Away to acutely deplete lamin and examined its effects on NSN chromatin\n(Extended Data Fig. 10e-h). Interestingly, compromising lamin stability/integrity had\nno impact on the chromatin configuration.\nResponse Fig. 3 (new Extended Data Fig. 10e-h) e, Immunofluorescence images\nof NSN oocytes expressing PAGFP-t21R or αLMN-t21R. f, g, h, Automated\nquantification of total intensity of LMNAC, EU and chromatin volume in NSN\noocytes expressing PAGFP-t21R or αLMN-t21R.\nRevised:\nNotably, chromatin detachment from the nuclear envelope alone was insufficient to\ngenerate such a collapsing force, as evidenced by the insignificant changes in chromatin\nvolume upon acute depletion of lamin by miniTrim-Away (Extended Data Fig. 10e-h).\nLine 300: IVF experiments with GVs are weak and technically problematic as NSN fail\nat PN and ZGA becomes inferred rather than being tested directly. See Methods\ncomments below.\nWe thank the reviewer for raising this concern regarding the inference of ZGA defects\nbased on EU staining. To address this, we now performed RNA-seq to systematically\ncompare 1-cell embryos derived from NSN and SN oocytes (Fig. 6i,j). We found that a\nsignificant portion of downregulated mRNAs in NSN-2PN were ZGA transcripts,\nwhereas upregulated mRNAs in NSN-2PN were mostly maternal transcripts, indicating\ndefects in both ZGA and maternal mRNA degradation.\nResponse Fig. 4 (new Fig. 6i,j) i, Volcano plots showing the differently expressed\ntranscripts between 1-cell embryos derived from NSN and SN oocytes. j, Euler\ndiagrams showing the overlap of ZGA genes with downregulated genes and the\noverlap of maternal genes with upregulated genes in 1-cell embryos derived from\nNSN oocytes.\nRevised:\nWe further performed low-input RNA-seq to compare 1-cell embryos derived from\nNSN and SN oocytes (Fig. 6i). Notably, a significant portion of downregulated mRNAs\nwere ZGA transcripts, whereas upregulated mRNAs were mostly maternal transcripts,\nindicative of defects in ZGA and maternal mRNA degradation in 1-cell embryos\nderived from NSN oocytes (Fig. 6i,j).\nDiscussion\nLine 317: Wildly exaggerated claim to have “elucidated for all mammalian oocytes”-\nnot so and statement should be tempered because thus is ONLY a mouse study and while\na good model for NSN to SN, it would not hold for any other mammal.\nWe thank the reviewer for raising this concern. We now modified the title of the\nmanuscript and rewrote the respective sentence to avoid exaggeration.\nPrevious submission:\nDegradation of maternal RNA polymerase II is essential for oocyte chromatin\nreorganization and embryonic development in mammals\nOur data elucidate the previously unknown mechanism and significance of chromatin\nreorganization in mammalian oocytes…\nRevised:\nNatural degradation of RNA polymerase II is essential for oocyte chromatin\nreorganization and maternal-to-zygotic transition in mice\nOur data elucidate the previously unknown mechanism and significance of chromatin\nreorganization in growing mouse oocytes…\nLine 321: OK to challenge other viewpoints but here that degradation releases\nchromatin allowing trans stoppage and remodeling could also be a cell cycle relevant\nstate as had been well established years ago.\nWe thank the reviewer for this comment. However, we respectfully disagree that\nRNAPII degradation and associated transcriptional silencing and chromatin remodeling\nwere downstream of cell cycle regulation because RNAPII degradation occurred\nindependently of its phosphorylation status (Fig. 4i-k).\nLine 746: Need information on how oocytes obtained. Were all naked, stripped, arrested\nin DcAMP for how long before manipulated? And key here, if you did switch to SN,\nwhat was maturation rate? M1s, M2s??? Essential to know this is a functional change\nand not just a morphological one.\nWe thank the reviewer for asking us to clarify how oocytes were obtained. Cumulus-\nenclosed GV oocytes were isolated from preantral and antral follicles and stripped\nbefore being arrested in dbcAMP. Immediately after staining with fluorescent DNA\ndyes, GV oocytes were sorted under microscope and used straight for manipulations\nsuch as drug treatment or microinjection. We now provided additional details regarding\noocyte handling in the respective sentences.\nPrevious submission:\nOocytes were isolated from ovaries of 8- to 10-week-old FVB/N or C57BL/6J (Charles\nRiver Laboratories) or Npm2 KO female mice. GV oocytes were arrested at prophase\nin homemade phenol red-free M2 supplemented with 250 μM dibutyryl cyclic AMP\n(dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin oil (ACROS Organics)\nat 37 °C.\nRevised:\nOocytes of around 60-80 μm in diameter were isolated from ovaries of 8- to 10-week-\nold unprimed FVB/N or C57BL/6J (Charles River Laboratories) or Ubb cKO or Npm2\ncKO female mice by puncturing preantral and antral follicles on the surface of ovaries\nwith needles. Cumulus-enclosed GV oocytes were stripped by repeated mouth pipetting\nand arrested at prophase in homemade phenol red-free M2 supplemented with 250 μM\ndibutyryl cyclic AMP (dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin\noil (ACROS Organics) at 37 °C.\nFor sorting GV oocytes with different chromatin configurations, individual oocytes\nwere transferred to microdrops and manually categorized under an inverted widefield\nfluorescence microscope equipped with a fluorescent LED. GV oocytes exhibiting\ndispersed chromatin with a few bright punctate were categorized as NSN oocytes,\nwhereas those displaying highly condensed chromatin with a complete perinucleolar\nring were categorized as SN oocytes. Oocytes that could not be unambiguously\nassigned to either category were excluded from further experiments. NSN and SN\noocytes were used for experiments immediately after sorting.\nWe agree with the reviewer that it is essential to know if the induced NSN-to-SN\ntransition represents a functional change rather than just a morphological one. Thus, we\nperformed HiC to systematically compare chromatin interactions in NSN, induced SN\nand SN oocytes, and in vitro maturation and in vitro fertilization in the previous\nsubmission (Fig. 2c-e). To further evaluate the differences in developmental potential\nbetween NSN, induced SN and SN oocytes, as suggested by another reviewer, we now\nprovided additional data to show that embryos derived from induced SN oocytes had\nsignificantly higher 2-cell and 4-cell rate (Extended Data Fig. 9a and Response Fig. 1).\nLine 770: Many methods issues with materials for IVF experiments (??super ovulated\nyes, but how to compare from follicular oocytes as not possible to score NSN etc);\nand you fertiliza GV oocytes (or did you mature them to M2 before ) as hole in\nzona would clearly result in polyspermy since cortical granule positioning would not\nhave occurred; critical information is missing otherwise experiments cannot be interpreted.\nWe thank for the reviewer for raising this concern regarding the IVF experiments. As\nthe reviewer correctly pointed out, it would not be possible to distinguish MIIs from\nNSN/SN oocytes if we proceeded with superovulation. Therefore, we first matured GV\noocytes to MII oocytes before performing IVF. Performing IVF on in vitro matured MII\noocytes was indeed very technically challenging owing to their low fertilization rate,\nso we initially tested a number of optimizations, such as including fetuin or FBS during\nin vitro maturation to alleviate zona hardening, but none of the optimizations\nreproducibly improved the fertilization rate. In the end, we figured out that removing\nthe zona pellucida was essential for reproducible IVF in these in vitro matured oocytes,\nbut to minimize polyspermy (Response Fig. 5), we opted for zona drilling rather than\ncomplete zona removal with acidic Tyrode solution or protease. Also, we excluded all\n1PN, 3PN and abnormal embryos from further experiments. We now provided\nadditional details regarding the IVF experiments in the respective sentences.\nResponse Fig. 5 Manual scoring of IVF of 1PN (parthenogenetic activation),\n2PN, 3PN (polyspermy) and abnormal embryos.\nPrevious submission:\nFor in vitro fertilization of GV oocytes, FVB/N oocytes with a centered nucleus were\nfirst treated with water or 100 μM α-amanitin (APExBIO) in M2 with dbcAMP for 7 h\nand then released into dbcAMP-free M2 for 13.5 h. Before the addition of sperm, a\nsingle hole was created in the zona pellucida of the MII oocytes using XYRCOS\n(Hamilton Thorne). IVF was performed as described above.\nRevised:\nFor in vitro fertilization of in vitro matured MII oocytes derived from GV oocytes,\nFVB/N GV oocytes with a centered nucleus were first treated with water/100 μM α-\namanitin (APExBIO) or DMSO/DRB (MedChemExpress) in M2 with dbcAMP for 7 h\nand then released into dbcAMP-free M2 for 13.5 h. Before the addition of sperm, a\nsingle hole was created in the zona pellucida of the MII oocytes using XYRCOS\n(Hamilton Thorne). IVF was performed as described above. After IVF, 1PN\n(parthenogenetic activation), 3PN (polyspermy) and abnormal embryos were excluded\nfrom further experiments.\nLine 800: were all human GVs vitrified or not..?? were some fresh after retrieval???\nAnd of vitrified, how many survived warming as has been shown to damage GVs with\npoor survival.\nWe thank the reviewer for raising this concern regarding the vitrification and thawing\nof human GV oocytes. In this study, human GV oocytes collected at a local clinic\n(Beijing Perfect Family Hospital) were fresh, whereas those collected at a remote clinic\n(Reproductive and Genetic Hospital of CITIC-XIANGYA) were vitrified for transport\nreason. As the reviewer correctly pointed out, human GV oocytes vitrified with\nconventional kits such as the one from Kitazato had poor survival. Thus, we utilized an\noptimized set of homemade vitrification solutions (previously reported in So et al.,\nScience 2022) that allow high survival and maturation rate of human GV oocytes after\nthawing (routinely >70%, n > 300 so far). Also, we excluded dead or morphologically\nabnormal oocytes after thawing from further experiments. We now provided additional\ndetails regarding the human GV oocytes in the respective sentence.\nPrevious submission:\nTo maximize the survival and developmental rate of vitrified-thawed GV oocytes,\noocytes were vitrified by transferring to…Recovered oocytes were transferred to G-\nMOPS PLUS at 37 °C. Fresh oocytes were directly transferred to and cultured in G-\nMOPS PLUS at 37 °C within 3 to 5 h after retrieval from ovaries.\nRevised:\nFor oocytes collected at Reproductive and Genetic Hospital of CITIC-XIANGYA, to\nmaximize the survival and developmental rate of vitrified-thawed GV oocytes, oocytes\nwere vitrified by transferring to…Dead or morphologically abnormal oocytes after\nthawing were excluded from further experiments, and all other recovered oocytes were\ntransferred to G-MOPS PLUS at 37 °C. For oocytes collected at Beijing Perfect Family\nHospital, fresh oocytes were directly transferred to and cultured in G-MOPS PLUS at\n37 °C within 3 to 5 h after retrieval from ovaries.\nLine 805: cannot use cortical tissue from PCOS patients and especially a wedge\nresection; would need a non-PCO ovary cortex and do follicle activation to do this\nstudy correctly; and enzymatic digestion of human ovaries to get follicles is not\nused because follicles do not survive and GV chromatin becomes irreversibly compacted,\nprecluding this work completely. Drop this part as it detracts from the good work.\nWe thank the reviewer for the suggestion. As suggested earlier also by this reviewer to\nfocus our attention on mouse in this study, we now removed the corresponding section\non human ovarian cortex owing to the concerns associated with PCOS materials.\nFig 2/3 comparison of isolated oocytes and those from follicles are different, as one\nmight expect. Why?\nWe thank the reviewer for the comment. Since we did not show data on follicles in Fig.\n2/3, we believe the reviewer refers to the comparison of isolated oocytes and those from\nfollicles in Fig. 3/4. As the reviewer pointed out above, such differences might originate\nfrom (i) the use of human cortical tissue from PCOS patients and/or (ii) artefacts\nassociated with enzymatic release of follicles. To this end, we now (i) removed the\ncorresponding section on human ovarian cortex and (ii) provided new immunostaining\ndata (Fig. 4a-c) on mouse ovarian sections to further strengthen our findings in oocytes\nand mouse follicles.\nResponse Fig. 2 (new Fig. 4a-c) a, Immunofluorescence images of oocytes from\nfollicles at different growth stages on mouse ovary sections. Green, pS2;\nmagenta, RPB1; blue, chromatin (Hoechst). Yellow dotted circles highlight the\nnuclei. b, c, Automated quantification of total intensity of pS2 and RPB1 in\noocytes from follicles at different growth stages on mouse ovary sections.\nFig 5 obvious that some epigenetic marks in pronuclei are non specific and precludes\nclaims about SN transition and impact on development; weak data set.\nWe thank the reviewer for the comment. We believe that the reviewer referred to the\noriginal H3K9me3 staining pattern of NSN-2PN and SN-2PN obtained using a well-\ncited antibody from Merck, which was only used in this single experiment throughout\nthe entire study. We now repeated the staining and quantifications using another well-\ncited antibody from Abcam (Fig. 6g,h) and included 2PN derived from in vivo ovulated\neggs (IVO-2PN) as a control.\nResponse Fig. 4 (new Fig. 6g,h) g, Immunofluorescence images of male and\nfemale pronuclei of 1-cell embryos derived from NSN, SN and IVO oocytes.\nGreen, H3K9me3; magenta, chromatin (Hoechst). h, Manual scoring of the\nnumber of nucleolus in the male and female pronuclei of 1-cell embryos derived\nfrom NSN, SN and IVO oocytes. (Fisher’s exact test for male pronuclei from 1-cell\nembryos derived from NSN and SN oocytes, P = 0.0001; Fisher’s exact test for\nfemale pronuclei from 1-cell embryos derived from NSN and SN oocytes, P =\n0.6201)\nFig 6: gammH2AX shows more foci in nsn vrsus sn.How would difference in DDR\nresponse as has been reported in mouse impact your results and could this mean DDR\nis turned off coordinately with chromatin housecleaning you so nicely have\ndemonstrated??\nWe thank the reviewer for the suggestion. The seemingly gamma H2A.X foci in NSN\noocytes were indeed weak background staining from secondary antibody, whereas the\nmuch brighter dots in SN oocytes were true signal. To avoid confusion, we now adjusted\nthe display settings and replaced the images in Extended Data Fig. 1b.\nResponse Fig. 7 (new Extended Data Fig. 1b) b, Immunofluorescence images of\ndifferent nuclear components (gamma H2A.X) localizing to chromatin in NSN\nand SN oocytes. Green, protein of interest (gamma H2A.X); magenta, chromatin\n(Hoechst).\nWe believe that DDR is irrelevant to NSN-to-SN transition, as treating NSN oocytes\nwith both topoisomerase-dependent and -independent DDR inducers etoposide and\nbleomycin had no impact on chromatin configuration (Fig. 1e). To address whether\nDDR is turned off together with transcription as suggested by the reviewer, we now\ntreated SN oocytes with etoposide and bleomycin as well. Indeed, the signal of gamma\nH2AX increased after treatment, suggesting that DDR is not turned off together with\ntranscription (Response Fig. 8).\nResponse Fig. 8 a, Immunofluorescence images of SN oocytes treated with\nbleomycin and etoposide. Green, gamma H2A.X; magenta, EU; blue, chromatin\n(Hoechst). b, Automated quantification of total intensity of gamma H2A.X in SN\noocytes treated with bleomycin and etoposide.\nReviewer #2:\nThe manuscript entitled \" Degradation of maternal RNA polymerase II is essential for\noocyte chromatin reorganization and embryonic development in mammals\" led by\nWang et al. describes an interesting finding that during oogenesis, the NSN to\nSN transition (reorganization of oocyte chromatin from non-surrounding nucleoli to\nsurrounding nucleoli) is attributed to the degradation of maternal RNAPII and the\nelimination of chromatin-bound RNAPII. That is, they suggest that this degradation\nsimultaneously induces chromatin reorganization and represses transcription, which\nis contrary to the traditional view that the NSN to SN transition and transcriptional\nsilencing are two unique features of the final stage of oocyte growth and that\nthese two events are driven by independent mechanisms in mammalian oocytes.\nThe authors searched for common nuclear components between NSN and SN oocyte\nnuclei and found that actinomycin D, a transcriptional inhibitor targeting RNA\npolymerase II, could induce NSN-to-SN transition. They then found that targeted\ndegradation of RNAPII was sufficient to induce the NSN to SN transition in mouse\nand human oocytes. They further demonstrated that RNAPII degradation during the\nNSN to SN transition is through the stripping of chromatin-bound RNAPII. Overall,\nthe data in this manuscript are clear, sufficient and reliable, and I support its\npublication in Nature Communications. However, I would like to share the following\nsuggestions with the authors to consider and improve the manuscript.\nWe are grateful to the reviewer for his/her very positive comments about our work\nand hope that the revised version of our manuscript could fully address his/her\nconcerns.\n1) I support the observation that RNAPII degradation is required for the NSN to\nSN transition in vivo, but which mechanism regulates the degradation of phosphorylated\nRNAPII during oocyte maturation from the NSN to the SN stage? Although your\nexperiments in vitro suggest that both separase- and proteasome-mediated RNAPII\ndegradation are required for the NSN to SN transition. This needs to be discussed.\nWe thank the reviewer for the comment on how RNAPII was degraded during oocyte\ngrowth from the NSN to the SN stage. Because NSN-to-SN transition took place\nasynchronously within a short period of time (~4 days in vitro), it was technically\nchallenging to harvest significantly number of oocytes in “intermediate” state (also\n“intermediate” state did not seem to continue transition to SN ex vivo) and perform\nomics profiling or IP-MS to compare them with NSN/SN oocytes in order to identify\nthe pathway involved. Thus, beyond our original experiments with separase and\nproteasome inhibitors, we now included additional data on Ubb cKO mice to further\ninvestigate whether RNAPII degradation is mediated by ubiquitination as reported in\nother processes in other systems before. Interestingly, however, we found that oocytes\nfrom Ubb cKO mice had similar NSN/SN ratio and unaltered RNAPII degradation in\nSN oocytes (Fig. 4l,m), suggesting that RNAPII degradation is likely independent of\nubiquitination.\nResponse Fig. 9 (new Fig. 4l, m) l, Immunofluorescence images of NSN and SN\noocytes from wildtype and Ubb cKO mice. m, Manual scoring of chromatin\nconfigurations in oocytes from wildtype and Ubb cKO mice (Fisher’s exact test, P\n= 0.1296).\nRevised:\nTo investigate whether the degradation of phosphorylated RNAPII is mediated by\nubiquitination as previously reported in other systems, we examined the chromatin\nconfiguration of GV oocytes from wildtype and conditional knockout (cKO) mice for\nUbb, the predominant polyubiquitin gene in oocytes. Quantifications revealed no\nsignificant difference in their NSN/SN ratio (Fig. 4l, m), suggesting that ubiquitination\nis unlikely to be involved in RNAPII degradation.\n2) Continuing with the above question, although the authors looked at the levels of\nactively transcribed and stored mRNAs such as the RNAPII subunit Rpb1, it might be\nnecessary to look at mRNAs transcribed and stored in other RNAPII components\nbetween the NSN and SN. This would rule out transcriptional differences between\nRNAPII components in the SN and NSN. Or it might be necessary to look at upstream\nmolecular associations that influence RNAPII levels.\nWe thank the reviewer for the suggestion. As suggested by the reviewer, we now\nexamined other RNAPII components in our multiomics dataset and found no significant\ntranscriptional or translational differences between NSN and SN oocytes (Response Fig.\n10). Also, we now performed immunostaining of the only other major/large RNAPII\nsubunit RPB2 in NSN and SN oocytes and found similar downregulation at protein\nlevel as for RPB1 (Extended Data Fig. 6g,h).\nResponse Fig. 10 IGV browser views showing DNA methylation levels, chromatin\naccessibility, nuclear RNA-seq (as a proxy for actively transcribing RNAs),\ncytoplasmic RNA-seq (as a proxy for stored RNAs), Ribo-seq signals for Rpb1 in\nNSN and SN oocytes: a, Polr2b; b, Polr2c; c, Polr2d; d, Polr2e; e, Polr2f; f,\nPolr2g; g, Polr2h; h, Polr2i; i, Polr2j;j Polr2l.\nResponse Fig. 11 (new Extended Data Fig. 6g,h) g, Immunofluorescence images\nof NSN and SN oocytes. Green, RPB2; magenta, chromatin (Hoechst). h,\nAutomated quantification of total intensity of RPB2 in NSN and SN oocytes.\n3) The authors challenge the conventional view that removal of chromatin-bound\nRNAPII leads not only to NSN-to-SN transition but also to global transcriptional\nsilencing in SN oocytes. Therefore, it is necessary to discuss in detail why other factors,\nsuch as loss of the cytoplasmic mRNA repressor LSM14B (see Figure 5B in PMID:\n37481122; or PMID: 37083226; PMID: 37889087; PMID:37578641), also lead to the\naberrant NSN-to-SN transition.\nWe thank the reviewer for the suggestion. We now included additional discussion on\nhow previously reported factors could indirectly lead to aberrant NSN-to-SN transition.\nRevised:\nInterestingly, previous studies knocking out a number of proteins such as MSY2/YBX2,\nMATER, EPAB, RPS26 and LSM14B, which are cytoplasmic factors that have no\napparent interactions with RNAPII, could alter the NSN/SN ratio of isolated oocytes.\nThese proteins may have exerted their effects via regulating the stability and/or\ntranslation of machineries in the upstream degradation pathway, thus indirectly\naffecting RNAPII degradation.\n4) The author's last result shows that although the protein WB experiment showed that\nnon-degraded RNAPII led to higher RNAPII levels in 1-cell embryos derived from MII\noocytes and NSN oocytes, immunofluorescence staining showed lower levels of total\nRNAPII and phosphorylated RNAPII in the nucleus. At the same time, there was no\nsignificant difference in chromatin accessibility between embryos derived from NSN\nand SN oocytes. The author thus concluded that there is a defect in the zygotic factors\nthat promote RNAPII to enter the nucleus and/or bind to chromatin. There is no\nexperimental evidence for this, and experimental verification should be provided, or a\ndiscussion should be given, otherwise the conclusions may need to be weakened.\nWe thank the reviewer for the suggestion. To strengthen our claims related to ZGA\ndefects, we now performed RNA-seq to systematically compare 1-cell embryos derived\nfrom NSN and SN oocytes (Fig. 6i,j). We found that a significant portion of\ndownregulated mRNAs in NSN-2PN were ZGA transcripts, whereas upregulated\nmRNAs in NSN-2PN were mostly maternal transcripts, indicating defects in both ZGA\nand maternal mRNA degradation. Also, we now rewrote the respective sentence related\nto tone down the link between ZGA defects and potential defects in zygotic factors that\npromote RNAPII loading into the nucleus and/or binding to chromatin\nResponse Fig. 4 (new Fig. 6i,j) i, Volcano plots showing the differently expressed\ntranscripts between 1-cell embryos derived from NSN and SN oocytes. j, Euler\ndiagrams showing the overlap of ZGA genes with downregulated genes and the\noverlap of maternal genes with upregulated genes in 1-cell embryos derived from\nNSN oocytes.\nPrevious submission:\n…Taken together, embryos derived from NSN oocytes are defective in ZGA and\npronucleus formation, likely due to defects in zygotic factors that promote RNAPII\nloading into the nucleus and/or binding to chromatin.\nRevised:\nWe further performed low-input RNA-seq to compare 1-cell embryos derived from\nNSN and SN oocytes (Fig. 6i). Notably, a significant portion of downregulated mRNAs\nwere ZGA transcripts, whereas upregulated mRNAs were mostly maternal transcripts,\nindicative of defects in ZGA and maternal mRNA degradation in 1-cell embryos\nderived from NSN oocytes (Fig. 6i,j)…These data suggest additional defects in zygotic\nfactors that promote RNAPII loading into the nucleus and/or binding to chromatin.\nTaken together, embryos derived from NSN oocytes are defective in pronucleus\nformation, ZGA, and maternal mRNA degradation, accounting for their arrest at as\nearly as 1-cell stage and poor development beyond.\nReviewer #3:\nThis manuscript had the stated goal of identifying “candidates that drive the\nphysiological NSN-to-SN transition” that growing oocytes undergo during antral\nfollicle development. Although it was a reasonable goal, the experiments done did not\naccomplish anything except to document that inhibition of RNA polymerase II-mediated\ntranscription promotes the transition. The authors claim to have demonstrated that\nreducing RNA polymerase protein levels, not just transcriptional activity, was the factor\nthat induced the transition. I have serious reservations about this claim based on lack\nof proper controls and overinterpretation of results, but even if it were true, the authors\nhave no idea why or how this might be the case. Therefore, best case scenario, this is a\nminimal advance to the literature. Specific concerns are outlined below.\nWe are grateful to the reviewer for his/her critical comments about our work and hope\nthat the revised version of our manuscript could fully address his/her concerns.\n1. One key experiment in the manuscript was the targeted degradation of RNA\npolymerase II in NSN oocytes by a “TRIM-away” technique: microinjection of cRNA\nencoding a genetically encoded antibody fused to the ring domain of TRIM21. The poor\nexperimental design means that this experiment is not possible to interpret for several\nreasons:\na. The authors microinjected almost 3 ug/ul (pipette concentration) of the cRNA, which\nis an enormous amount relative to standard cRNA microinjection procedures for\nnumerous other proteins, including what was used in most of their own overexpression\nstudies (~500-600 ng/ul). Rather than an appropriate control, which would be an\nalternate cRNA that encoded the ring domain of TRIM21 fused to a genetically encoded\nantibody targeting a different, non-relevant protein, the authors used injection of water\nas a control. Microinjection of oocytes with cRNA to induce protein degradation carries\nwith it strong potential for off-target effects due to displacing physiological mRNAs\nfrom the translation system as well as causing a relatively large amount of protein to\nmove into the proteasome degradation pathway, which could interfere with\nphysiological protein degradation as well. In our hands, cRNA at >2 ug/uL has clear\nnon-specific toxic effects on mouse oocytes and embryos. Why was such a high\nconcentration required? Even if it were necessary, water injection is simply an\ninadequate control.\nWe thank the reviewer for raising this concern regarding injected cRNA concentration.\nWe previously found that the non-specific toxic effects of cRNA that the reviewer\nreferred to originated from the leftover free nucleotides (i.e. ATP, GTP…) and uncapped\n(i.e. 5’-phosphate) cRNA, thus we adopted a modified cRNA preparation protocol that\nincorporates an additional column cleanup and a dephosphorylation step as described\nin our previous Nature Protocols paper. We now replaced the previous citation with this\nin the section related to mRNA synthesis and quantification.\nThe reason why we are choosing such a high cRNA concentration for 42B3-t21R was\nthat the degradation of RNAPII was less rapid and efficient, likely owing to the lower\navailability of proteasome in the nucleus when compared to the cytoplasm. To address\nthe reviewer’s concern regarding potential toxicity, we now repeated the key RNAPII\nTrim-Away experiment with a control miniTrim-Away construct composed of PA-GFP\n(with similar size of scFv/nanobody and to avoid remnant GFP signal interfering with\nstaining) and t21R, and observed no impact of PAGFP-t21R on chromatin configuration\n(Extended Data Fig. 5e-i).\nResponse Fig. 12 (new Extended Data Fig. 5e-i) e, Immunofluorescence images of\nNSN oocytes expressing PAGFP-t21R or 42B3-t21R for 16 hours. f, g, h, i,\nAutomated quantification of total intensity of EU, pS2, RPB1 and chromatin\nvolume in NSN oocytes expressing PAGFP-t21R or 42B3-t21R for 16 hours.\nb. Figure 4a-c: The immunofluorescence of D14+7 mouse follicles shows clearly that\na SN configuration is achieved before there is a significant loss of RNA polymerase\nprotein. This experiment invalidates the authors’ conclusions regarding the need for\nRNA polymerase II protein elimination to achieve SN.\nWe thank the reviewer for raising this concern and apologize for the confusion\nassociated with former panel Fig. 4a-c. Owing to the former image display settings, the\noocyte in D14+7 mouse follicles appeared to have a SN configuration but indeed\nadopted an intermediate chromatin configuration with both a perinucleolar ring and\nuncompacted peripheral chromatin. We now readjusted the image display settings to\nclarify this (Fig. 4d-h).\nResponse Fig. 13 (new Fig. 4d-h) d, Immunofluorescence images of oocytes\nharvested from mouse secondary follicles cultured for different days. Green,\npS2; magenta, RPB1; blue, chromatin (Hoechst). e, f. g, h, Automated\nquantification of total intensity of pS2, RPB1, chromatin-bound RPB1 and\nchromatin volume in growing oocytes harvested from mouse secondary follicles\ncultured for different days.\nAlso, we would like to point out that NSN-to-SN transition occurred asynchronously in\nex vivo cultured mouse follicle (Fig. 4e,f), as evidenced by the dispersed pS2 and RPB1\nintensity distribution for D14+5 and D14+7 (during transition) but clustered pS2 and\nRPB1 intensity distribution for D14+9 (transition complete). Therefore, SN\nconfiguration was not achieved before there was a significant loss of RNAPII.\nc. The human oocyte data conflicts with the authors’ interpretation of their mouse\noocyte data. The images of oocytes within primordial, primary, and secondary follicles\npresumably are just to show that the antibodies being used recognize nuclear proteins.\nWhat the authors call “early growing oocytes” are already transitioning to SN, and the\n“late growing” and “fully grown” oocytes have transitioned as well. At the same time,\nthe authors show that there are no changes in total RNA polymerase II levels (Figure\n4f and 4h), but that the phosphorylated active form (pS2) has decreased (Figure 4g),\nconsistent with a reduction in transcription. These findings suggest that in human\noocytes, loss of transcription is associated with the SN configuration, not that a loss of\nRNA polymerase II protein is important.\nWe thank the reviewer for the comment. We would like to clarify that the images of\nhuman oocytes in primordial to secondary follicles and those of early growing to fully\ngrown human oocytes were not acquired in the same experiment, and we thus did not\nattempt to perform any intensity comparison. And as pointed by another reviewer, the\norigin of human follicles from PCOS patient material and the dissociation method\nmight have significant impact on the validity of conclusions related to in vivo NSN-to-\nSN transition in human. Therefore, we now removed the data associated with cortical\nstrip/follicles from PCOS patients and focused the attention of this study on mice.\n2. A second key experiment was to test whether inhibition of RNA polymerase II-based\ntranscription cause the NSN to SN transition. Although alpha-amanitin and triptolide\nboth caused inhibition of transcription, they also both caused degradation of RNA\npolymerase II protein. To separate these two effects, the authors used alternate\ntranscriptional inhibitors that do not impact RNA polymerase II protein levels. Given 7\nhours of oocyte incubation time, the transcriptional inhibitors 8-AA and 7-DAA did not\ncause oocytes to convert to SN nuclei. However, there is no information provided\nregarding how quickly they reduce transcription relative to how fast transcription is\nreduced by the relatively high concentration of alpha-amanitin used in these\nexperiments (100 uM vs 26 uM used in many studies). In supplemental video 4, alpha-\namanitin had caused the chromatin to begin surrounding the nucleolus in 3 hours and\ntriptolide (video 2) by 4 hours. If 8-AA and 7-DAA take longer to reduce transcription,\nthen the conclusion that they do not impact the NSN-to-SN transition could be incorrect.\nFurthermore, my interpretation of both chromatin images shown in Figure S4a-b is that\nthey are in transition to the SN stage, suggesting that transcriptional inhibition alone\nmay be sufficient to induce the transition.\nWe thank the reviewer for raising this concern regarding the potential difference in time\nof action for alpha-amanitin/triptolide and 8-AA/7-DAA. We now repeated 8-AA and\n7-DAA inhibition experiments but with a longer incubation time (extending from 7\nhours to 16 hours). Extended inhibition did result in some degree of chromatin\ncompaction but did not eventually lead to NSN-to-SN transition (Response Fig. 14).\nWe did not attempt inhibition beyond 16 hours because by then even the control oocytes\nstarted to undergo abnormal morphological changes.\nResponse Fig. 14 a, Immunofluorescence images of NSN oocytes treated with 8-\nAA or 7-DAA. b, c, d, e, quantification of total intensity of EU, pS2, RPB1 and\nchromatin volume in NSN oocytes treated with 8-AA or 7-DAA.\n3. The stated “comprehensive analysis of SN and NSN nuclei” was not well planned or\ncarried out in a robust fashion. These data are unreliable.\na. Sorting of nuclei into NSN vs SN ignores the reality of nuclei in transition. The\noriginal paper (referenced by the authors) describes 4 different chromatin\nconfiguration stages, with two being transitory between NSN and SN. The authors do\nnot distinguish these stages with their methodology or indicate what was done with\noocytes in transitional stages.\nWe thank the reviewer for raising this concern regarding the sorting of NSN/SN oocytes.\nAs the reviewer correctly pointed out, we did observe a relatively minor portion (79/692)\nof GV oocytes displaying an intermediate chromatin configuration during sorting and\nexcluded them for further experiments to avoid misinterpretation. We now provide\nadditional details regarding NSN/SN sorting in the respective sentences.\nRevised:\nFor sorting GV oocytes with different chromatin configurations, individual oocytes\nwere transferred to microdrops and manually categorized under an inverted widefield\nfluorescence microscope equipped with a fluorescent LED. GV oocytes exhibiting\ndispersed chromatin with a few bright punctate were categorized as NSN oocytes,\nwhereas those displaying highly condensed chromatin with a complete perinucleolar\nring were categorized as SN oocytes. Oocytes that could not be unambiguously\nassigned to either category were excluded from further experiments. NSN and SN\noocytes were used for experiments immediately after sorting.\nb. The Method used to define “downregulation” of nuclear components assumes that\nantigen accessibility to antibody staining does not change during nuclear\nreorganization. Furthermore, immunofluorescence detection of a punctate distribution\nof proteins cannot be used to accurately quantify protein levels, particularly when the\ndifferences are subtle, and the experiment was only done twice! None of the antibodies\nused were demonstrated to be specific for the targeted proteins.\nWe thank the reviewer for raising the comment regarding the use of terms\n“downregulation”. We totally agree with the reviewer that antigen accessibility may be\naffected by for instance transition to SN configuration owing to chromatin compaction.\nHowever, using the identical method, we did find that not all detected proteins\ndisplayed a reduction in total fluorescence intensity but unchanged or even an increase\nin total fluorescence intensity. Thus, we believe that NSN and SN configurations did\nnot have a major impact on antigen accessibility. We now rewrote the respective\nsentences to refer to the lower staining intensity and avoid the use of terms\n“downregulation”.\nPrevious submission:\nTo study the molecular differences between NSN and SN nuclei, we systematically\nevaluated the localization and abundance of 28 common nuclear components (Fig. 1d\nand Extended Data Fig. 1a-d). We found that 13 of these nuclear components localized\nto chromatin, and most of them were downregulated in SN nuclei. Seven nuclear\ncomponents localized to chromocenters and were all downregulated in SN nuclei.\nNucleolar proteins and the nuclear speckle marker SRSF2 were downregulated in SN\nnuclei, in line with previous reports. Thus, most nuclear components are downregulated\nduring NSN-to-SN transition.\nRevised:\nTo study the molecular differences between NSN and SN nuclei, we systematically\nevaluated the localization and staining intensity of 28 common nuclear components\n(Fig. 1d and Extended Data Fig. 1a-d). We found that 13 of them localized to chromatin,\nand the staining intensity for most of them were lesser in SN nuclei. Seven nuclear\ncomponents localized to chromocenters and their staining intensity were all lower in\nSN nuclei. Nucleolar proteins and the nuclear speckle marker SRSF2 also showed lower\nstaining intensity in SN nuclei, in line with previous reports. These data suggest that\nmost nuclear components are downregulated during NSN-to-SN transition.\nRegarding the reviewer’s concern on punctate distribution having subtle differences\nand might be difficult to quantify, we would like to point out that this was exactly why\nwe performed 3D imaging and machine learning-based segmentation rather than\ntraditional 2D imaging and intensity-based segmentation, which allowed us to quantify\nall signals within the nuclei and quantify challenging targets despite their lower signal-\nto-noise ratio. Also, we would like to highlight that the goal of the IF screen was to\nidentify nuclear components with drastic differences between NSN and SN oocytes,\nwhich we believed are more likely to be involved in NSN-to-SN transition.\nRegarding the reviewer’s comment on experimental replication, we would like to\nclarify that while all data were from at least two independent experiments, we had at\nleast three biological replicates per experiments. Except for the IF screen, which already\ninvolved 542 NSN oocytes and 557 SN oocytes, we performed at least three\nindependent experiments for all other experiments in the study.\nRegarding the reviewer’s comment on antibody specificity, we would like to highlight\nthat most of the antibodies that we chose had previously been used in studies in other\nsystems and wherever possible already in mouse oocytes. Only when we failed to obtain\na signal or expected nuclear localization for a previously cited antibody, we used new\nantibodies such as the case of MATR3 and hnRNPU antibodies. In these cases, we\nconfirmed the specificity of antibody by comparing the detected pattern with the\nlocalization in overexpression experiments (Extended Data Fig. 2).\nc. Using 65 different treatments in oocytes in the absence of documenting that the\nconcentrations used are effective but non-toxic has no meaning. Conclusions cannot be\ndrawn from such studies.\nWe thank the reviewer for asking us for clarification. Here we were referring to the fact\nthat many of the almost 65 small molecule inhibitors tested did not lead to the desired\nchanges in nuclear components (e.g. supposingly a histone methyltransferase inhibition\ndid not reduce the level of KMe2 or KMe3). For the 19 inhibitors that we found to be\neffective, we indeed confirmed their effectiveness by the expected changes in the\nrespective nuclear components. We now rewrote the respective sentence and the\nassociated table legend to avoid further confusion.\nPrevious submission:\nOf almost 65 different treatments (Table S1), only 19 of them led to the desired changes\nof nuclear components (Extended Data Fig. 2d). Many of these treatments altered the\ntranscriptional activity and/or chromatin compaction in NSN and SN oocytes (Fig. 1e\nand Extended Data Fig. 2e).\nTable S1 List of tested small-molecule inhibitors that did not work in this study.\nRevised:\nMany of the treatments that led to the desired changes in nuclear components altered\nthe transcriptional activity and/or chromatin compaction in NSN and SN oocytes (Fig.\n1e and Extended Data Fig. 2d,e).\nTable S1 List of small-molecule inhibitors tested that did not lead to desired changes in\nnuclear components in oocytes.\nd. Putting the organic compound 1,6-hexanediol on oocytes appears to have killed the\ncells (Figure S2g), which again, generates non-interpretable data. Note the complete\nlack of evidence of a plasma membrane in the transmitted light image.\nWe thank the reviewer for raising this concern related to 1,6-hexanediol. The\ntransmitted light images that we showed indeed covered only the nuclei of the oocyte,\nso there is no plasma membrane. The oocytes were not killed by 1,6-hexanediol\ntreatment. We apologize for the reviewer confusing the nuclear envelope as the plasma\nmembrane. The absence of nuclear envelope after 1,6-hexanediol treatment was indeed\nexpected because it could disrupt the liquid-liquid phase separation of the nuclear pore\ncomplex. To avoid further confusion, we now highlighted the nuclear envelope with a\nyellow dotted circle (Extended Data Fig. 2g).\nResponse Fig. 15 (new Extended Data Fig. 2g) g, Immunofluorescence images of\nSN oocytes treated with 1, 6-hexanediol. Gray, transmitted light; blue, chromatin\n(Hoechst). Yellow dotted circles highlight the nuclei.\n4. The conclusion regarding developmental potential has numerous problems. First,\ndevelopmental potential is tested by development to the blastocyst stage or else embryo\ntransfer followed by live birth, not development to the 2-cell stage. In addition, this\nconclusion was drawn on the basis of having a total of 16 fertilized eggs. That is an\ninsufficient number, even for just looking at development to the 2-cell stage.\nWe thank the reviewer for raising this concern related to the way that we evaluate\ndevelopmental potential. We apologize for the relatively low cell number previously\nfor this experiment owing to the low overall fertilization rate for in vitro matured\noocytes. To address this reviewer’s concern, (i) we now increased the n number for the\nNSN, induced SN and SN groups to 56, 37 and 116 (Fig. 2e); (ii) owing to other poor\nwashout efficiency of alpha-amanitin, we now evaluated developmental potential\nindependently by transiently treating NSN oocytes with DRB (to induce SN chromatin\nconfiguration), followed by DRB washout before in vitro maturation and in vitro\nfertilization (Extended Data Fig. 9a and Response Fig. 1).\nResponse Fig. 16 (new Fig. 2e) e, Manual scoring of developmental outcomes of\nNSN, α-amanitin-treated NSN and SN oocytes after IVM and IVF. (Fisher’s\nexact test for 2-cell embryos from NSN and α-amanitin-treated NSN oocytes, P =\n0.0106)\nResponse Fig. 1 Manual scoring of developmental outcomes of NSN, DRB-\ntreated NSN, SN, and DRB-treated SN oocytes after IVM and IVF. (Fisher’s exact\ntest for 2-cell embryos from NSN and DRB-treated NSN oocytes, P = 0.0047;\nFisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN oocytes, P =\n0.0496; Fisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN\noocytes, P = 0.3741;)\n5. Pages 9-10: It is incomprehensible to this reviewer why the authors would spend the\ntime and effort doing whole genome bisulfite sequencing, ATAC-seq, and fractionated\nRNA-seq to determine whether or not a specific gene is transcribed, rather than just\ndoing a simple RT-qPCR experiment, which they eventually did. Similarly, using Ribo-\nseq, RIBOmap, and a poly(A) tail length assay when a simple cycloheximide experiment\nwould suffice is unjustified. The later CUT&Tag experiments on NSN and SN oocytes\nto examine binding locations of the pS2 form of RNA polymerase II doesn’t make any\nsense because SN oocytes are not transcriptionally active. Similarly, it is quite unclear\nhow Hi-C profiling added anything to the manuscript. All the unnecessary data simply\nconfuse the reader and do not impact the story being presented.\nWe thank the reviewer for the comment. We started with a multiomics approach rather\nthan a targeted approach because we would like to examine the potential involvement\nof other proteins or RNAPII subunits, as requested by another reviewer above. Also,\nwe would like to clarify that we did not attempt to profile pS2 in SN oocyte for the\nsame reason pointed out by the reviewer, and the HiC profiling experiment was to\nconfirm NSN-to-SN transition beyond morphological changes as appreciated by\nanother reviewer above.\n6. Lack of robust and reproducible data that was properly analyzed\na. Performing experiments twice is inadequate\nb. The number of oocytes in many of the experiments is too low for robust conclusions\nc. Data analysis is completely inadequate. The authors report using Student ‘s t-test\nwhen they are clearly comparing multiple groups. Many experiments lack data analysis\nat all yet are declared in the text to have differences present.\nRegarding the reviewer’s comment on experimental replication, again we would like to\nclarify that while all data were from at least two independent experiments, we had at\nleast three biological replicates per experiments. Except for the IF screen, which already\ninvolved 542 NSN oocytes and 557 SN oocytes, we performed at least three\nindependent experiments for all other experiments in the study. Unlike other systems,\noocytes are non-dividing and could not be retrieved in large number. Considering one\ncould only retrieve ~40 NSN+SN (NSN:SN≈1:2) oocytes from a single unprimed\nmouse, balancing the considerations from animal welfare and statistical point of view,\nwe respectfully believe that the data size is sufficient for drawing conclusions.\nRegarding the use of Student’s t-test when comparing multiple groups, we thank the\nreviewer for spotting this out and now reanalyzed the respective figures (Fig. 4e-h, j,\n5h, l, m, Extended Data Fig. 9b-f) by one-way ANOVA followed by post-hoc Tukey’s\nmultiple comparison test. For categorical analysis, owing to space limitation in the\nfigure panel, all of the statistical analysis were provided in the figure legends.\nPrevious submission:\nAverage (mean) and SD were calculated in Excel. Statistical significance is based on\nunpaired, two-tailed Student’s t test (for absolute values) and two-tailed Fisher’s exact\ntest (for categorical values) calculated in OriginPro or Prism (GraphPad), assuming\nnormal distribution and similar variance.\nRevised:\nAverage (mean) and SD were calculated in Excel. Statistical significance is based on\nunpaired, two-tailed Student’s t test (for absolute values between two groups), one-way\nANOVA followed by post-hoc Tukey’s multiple comparison test (for absolute values\nfor multiple groups) and two-tailed Fisher’s exact test (for categorical values)\ncalculated in OriginPro or Prism (GraphPad), assuming normal distribution and similar\nvariance.\n7. The Methods as written do not provide sufficient information regarding the\nexperiments that were done. In particular, the descriptions of oocyte collection and\nmicroinjection were incomplete. The microinjection was not even described other than\nto reference another paper in which the method was still not described, but it referenced\nanother paper that did a completely different method (injecting starfish or sea urchin\nembryos, not mouse oocytes).\nWe thank the reviewer for asking additional details related to oocyte isolation and\nmicroinjection. We would like to clarify that our microinjection method was indeed\nmodified from the setup used to inject starfish and sea urchin oocytes/embryos from\nthe Jaffe lab, which was described in details also in our Nature Protocols paper. We now\nreplaced the previous citation with this in the respective sentence, and provided\nadditional details in the section related to oocyte isolation.\nPrevious submission:\nOocytes were isolated from ovaries of 8- to 10-week-old FVB/N or C57BL/6J (Charles\nRiver Laboratories) or Npm2 KO female mice. GV oocytes were arrested at prophase\nin homemade phenol red-free M2 supplemented with 250 μM dibutyryl cyclic AMP\n(dbcAMP) (Sigma-Aldrich or MedChemExpress) under paraffin oil (ACROS Organics)\nat 37 oC.\nSPY555-DNA (Spirochrome) and 5-SiR-Hoechst were reconstituted in DMSO. For\nvisualizing chromatin configuration, oocytes were stained with SPY555-DNA or 5-\nSiR-Hoechst for 30 min at 1:500 dilution and 10 μM, respectively. For live imaging,\nSPY555-DNA or 5-SiR-Hoechst was present throughout imaging at 1:1000 dilution\nand 50 nM, respectively.\nRevised:\nOocytes of around 60-80 μm in diameter were isolated from ovaries of 8- to 10-week-\nold unprimed FVB/N or C57BL/6J (Charles River Laboratories) or Ubb cKO or Npm2\ncKO female mice by puncturing preantral and antral follicles on the surface of ovaries\nwith needles. Cumulus-enclosed GV oocytes were stripped by repeated mouth pipetting\nand GV oocytes were arrested at prophase in homemade phenol red-free M2\nsupplemented with 250 μM dibutyryl cyclic AMP (dbcAMP) (Sigma-Aldrich or\nMedChemExpress) under paraffin oil (ACROS Organics) at 37 oC.\nSPY555-DNA (Spirochrome) and 5-SiR-Hoechst were reconstituted in DMSO. For\nvisualizing chromatin configuration, oocytes were stained with SPY555-DNA or 5-\nSiR-Hoechst for 30 min at 1:500 dilution and 10 μM, respectively. For live imaging,\nSPY555-DNA or 5-SiR-Hoechst was present throughout imaging at 1:1000 dilution\nand 50 nM, respectively. For sorting GV oocytes with different chromatin\nconfigurations, individual oocytes were transferred to microdrops and manually\ncategorized under an inverted widefield fluorescence microscope equipped with a\nfluorescent LED. GV oocytes exhibiting dispersed chromatin with a few bright punctate\nwere categorized as NSN oocytes, whereas those displaying highly condensed\nchromatin with a complete perinucleolar ring were categorized as SN oocytes. Oocytes\nthat could not be unambiguously assigned to either category were excluded from further\nexperiments. NSN and SN oocytes were used for experiments immediately after sorting.\nReviewer #4:\nIn this study, the authors rigorously analyzed the effect of RNAPII degradation at the\nfinal step of oocyte growth, which involves the NSN-to-SN transition. Interestingly,\ntreatment with RNAPII inhibitors, which lead to RNAPII degradation, was sufficient to\ninduce the NSN-to-SN transition. Furthermore, this artificial induction partially\nrescued the impaired developmental potential of NSN oocytes. In addition to these\ninhibitors, the authors employed miniTrim-Away to selectively degrade RNAPII.\nChromatin-bound RNAPII appeared to restrict chromatin mobility, as its degradation\nresulted in increased chromatin dynamics. These changes in chromatin mobility, in\nconjunction with the attractive forces at the nucleolar surface, appeared to play an\nimportant role in driving the NSN-to-SN transition.\nThe use of multiple complementary approaches to demonstrate that RNAPII\ndegradation promotes the NSN-to-SN transition is highly commendable. However, I\nbelieve that some additional work is necessary before this manuscript can be\nconsidered for publication, as detailed below.\nWe are grateful to the reviewer for his/her very positive comments about our work and\nhope that the revised version of our manuscript could fully address his/her concerns.\nMajor comments:\n1. Fig. 1: The authors tried to indicate the status of SN or NSN by the quantification of\nchromatin volume. However, as the authors noticed from experiments such as RNase A\nor 1,6-hexandiol treatment, the chromatin volume does not necessarily discriminate\nbetween SN and NSN. It will be highly helpful if the authors could summarize the\nphenotype of each treatment by providing additional figures or tables.\nWe thank the reviewer for the suggestion. We now summarized the phenotype of each\ntreatment in the new Table S2.\nResponse Table 1 (Table S2) Summary of phenotypes of treatments that lead to\ndesired changes in nuclear components in oocytes.\nNo. Drug/mRNA Concentration Oocyte Readout\nname tested\nchromatin was abnormally\ncompacted.\nNSN-to-SN transition was\ninduced.\nchromatin was slightly\ncompacted.\nchromatin was slightly\ncompacted.\nChromatin was slightly\ncompacted.\nChromatin remained NSN state.\nChromatin remained NSN state.\nChromatin remained NSN state.\nChromatin remained NSN state.\nChromatin was slightly\ncompacted.\n11 1,6-hexanediol 15% SN Nucleolus was disrupted;\nChromatin was abnormally\ndecompacted.\nchromatin was slightly\ndecompacted.\nchromatin was slightly\ndecompacted.\nChromatin remained SN state.\n16 hnRNPU 2251.2 ng/μl SN Chromatin remained SN state.\nChromatin remained SN state.\nChromatin was slightly\ncompacted.\ncompacted.\ncompacted.\nNSN-to-SN transition was\ninduced.\n22 α-amanitin 100 μM NSN Transcription was silenced;\nNSN-to-SN transition was\ninduced.\n2. Fig. 2e: It will be important to examine embryonic development to the blastocyst\nstage, to understand the developmental competence of the NSN, a-amanitin treated SN-\ninduced, and SN oocytes.\nWe thank the reviewer for the suggestion. Owing to other poor washout efficiency of\nalpha-amanitin, we now evaluated developmental potential at later stages\nindependently by transiently treating NSN oocytes with DRB (to induce SN chromatin\nconfiguration), followed by DRB washout before in vitro maturation and in vitro\nfertilization (Extended Data Fig. 9a and Response Fig. 1).\nResponse Fig. 1 Manual scoring of developmental outcomes of NSN, DRB-\ntreated NSN, SN, and DRB-treated SN oocytes after IVM and IVF. (Fisher’s exact\ntest for 2-cell embryos from NSN and DRB-treated NSN oocytes, P = 0.0047;\nFisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN oocytes, P =\n0.0496; Fisher’s exact test for 4-cell embryos from NSN and DRB-treated NSN\noocytes, P = 0.3741;)\n3. Fig. S4: The experiments using 8-AA and 7-DAA are important, as these assays\nsuggested that the inhibition of transcription only is not sufficient to induce the NSN-\nto-SN transition. However, it remained unclear how rapidly each treatment could halt\ntranscription. For instance, if the duration of the transcriptionally inactive state differs\nbetween α-amanitin and 8-AA/7-AA treatments, the interpretation of the data would\nchange accordingly.\nWe thank the reviewer for raising this concern regarding the potential difference in time\nof action for alpha-amanitin/triptolide and 8-AA/7-DAA. We now repeated 8-AA and\n7-DAA inhibition experiments but with a longer incubation time (extending from 7\nhours to 16 hours). Extended inhibition did result in some degree of chromatin\ncompaction but did not eventually lead to NSN-to-SN transition (Response Fig. 14).\nWe did not attempt inhibition beyond 16 hours because by then even the control oocytes\nstarted to undergo abnormal morphological changes.\nResponse Fig. 14 a, Immunofluorescence images of NSN oocytes treated with 8-\nAA or 7-DAA. b, c, d, e, quantification of total intensity of EU, pS2, RPB1 and\nchromatin volume in NSN oocytes treated with 8-AA or 7-DAA.\n4. Fig. 3: For the experiments using miniTrim-Away, H2O only was used as a control.\nObviously, this is not an appropriate negative control. If the authors could perform\nadditional experiments with a more proper control group, the quality of this study will\nbe improved.\nWe thank the reviewer for raising this concern regarding the control for miniTrim-Away\nexperiment. We now repeated the key RNAPII Trim-Away experiment with a control\nminiTrim-Away construct composed of PA-GFP (with similar size of scFv/nanobody\nand to avoid remnant GFP signal interfering with staining) and t21R, and observed no\nimpact of PAGFP-t21R on chromatin configuration (Extended Data Fig. 5e-i).\nResponse Fig. 12 (new Extended Data Fig. 5e-i) e, Immunofluorescence images of\nNSN oocytes expressing PAGFP-t21R or 42B3-t21R for 16 hours. f, g, h, i,\nAutomated quantification of total intensity of EU, pS2, RPB1 and chromatin\nvolume in NSN oocytes expressing PAGFP-t21R or 42B3-t21R for 16 hours.\n5. Fig. 4i.j: CB-5083 and MG-132 affect many other proteins and pathways than\nRNAPII. Thus, the conclusion that the artificial stabilization of RNAPII suppresses the\nNSN-to-SN transition is not sufficiently supported by the provided data. I expect that\nthe authors should be able to perform additional experiments to sustain RNAPII\nexpression using more specific methods.\nWe thank the reviewer for asking for additional experiments to strengthen our claim\nthat RNAPII degradation drives NSN-to-SN transition. The degradation of RNAPII\nreported in other systems so far were all mediated by ubiquitination. Thus we did\ngenerate Ubb cKO mice to investigate whether this was also the case in oocytes.\nInterestingly, however, we found that oocytes from Ubb cKO mice had similar NSN/SN\nratio and unaltered RNAPII degradation in SN oocytes (Fig. 4l,m), suggesting that\nRNAPII degradation is likely independent of ubiquitination. Owing to the fact that\nNSN-to-SN transition took place asynchronously within a short period of time (~4 days\nin vitro), it was technically challenging to harvest significantly number of oocytes in\n“intermediate” state (also “intermediate” state did not seem to continue transition to SN\nex vivo) and perform omics profiling or IP-MS to compare them with NSN/SN oocytes\nin order to identify the pathway involved. Therefore, we preliminarily investigated\nRNAPII degradation using segregase and proteasome inhibitors.\nResponse Fig. 9 (new Fig. 4l, m) l, Immunofluorescence images of NSN and SN\noocytes from wildtype and Ubb cKO mice. m, Manual scoring of chromatin\nconfigurations in oocytes from wildtype and Ubb cKO mice (Fisher’s exact test, P\n= 0.1296).\nRevised:\nTo investigate whether the degradation of phosphorylated RNAPII is mediated by\nubiquitination as previously reported in other systems, we examined the chromatin\nconfiguration of GV oocytes from wildtype and conditional knockout (cKO) mice for\nUbb, the predominant polyubiquitin gene in oocytes Quantifications revealed no\nsignificant difference in their NSN/SN ratio (Fig. 4l, m), suggesting that ubiquitination\nis unlikely to be involved in RNAPII degradation.\n6. Abstract and line 287: “Nucleolar surface remodeling” is ambiguously described.\nThere is no experimental data showing the nucleolar surface remodeling, as far as I\nunderstand. Please explain more precisely. If there is no evidence for it, I feel that the\nauthors should remove such descriptions.\nWe thank the reviewer for the suggestion. We referred “nucleolar surface remodeling”\nto the relocalization of nucleolar surface protein such as B23 that happened at the same\ntime as chromatin became attached to the nucleolus during NSN-to-SN transition. To\nstrengthen this claim, we now provided additional quantifications of B23 and another\nnucleolar surface protein NCL before and after inducing NSN-to-SN transition\n(Extended Data Fig. 10i-k) and rewrote the respective sentences to improve clarity.\nResponse Fig. 17 (new Extended Data Fig. 10i-k) i, Immunofluorescence images\nof NSN oocytes treated with α-amanitin. Green, NCL; magenta, B23; chromatin\n(Hoechst). j, k, Automated quantification of total intensity of B23 and NCL in\nNSN oocytes treated with α-amanitin.\nPrevious submission:\n…leading us to a model whereby NSN-to-SN transition involves both a global\ncollapsing force as a result of the increase in chromatin dynamics and a local attractive\nforce as a result of the nucleolus converting into an attractive surface for chromatin to\nattach.\nHowever, after inducing NSN-to-SN transition, chromatin surrounded the nucleolus but\nnot the oil droplet, concomitant with changes in nucleolar proteins such as B23 (Fig.\n5n).\nRevised:\n…leading us to a model whereby NSN-to-SN transition involves both a collapsing force\nas a result of the increase in global chromatin dynamics and an attractive force as a\nresult of the nucleolus being converted into an attractive surface for local chromatin\nattachment.\nHowever, after inducing NSN-to-SN transition, chromatin surrounded the nucleolus but\nnot the oil droplet, concomitant with the displacement of nucleolar proteins such as\nNCL and B23 from the surface of the nucleolus (Fig. 5n and Extended Data Fig. 10i-\nk).\nMinor comments:\n7. Fig. S1: The authors used pan-trimethyl-lysine antibodies. Thus, “KMe3”, but not\n“HMe3”, will be correct. This will be also the case for “HMe2”.\nWe thank the reviewer for the suggestion. We now changed “HMe3” to “KMe3” and\n“HMe2” to “KMe2”.\n8. Line 174: It was not clear what the “growing ones” means. Do they correspond to\nrelatively small growing oocytes?\nWe thank the reviewer for the suggestion. We referred the “growing ones” to less mature\nhuman oocytes with partially compacted chromatin. We now rewrote the respective\nsentence to improve clarity.\nPrevious submission:\nBecause human oocytes with an NSN configuration were rarely found (1/52) in\nimmature GV oocytes leftover from fertility treatment, we utilized the growing ones\nthat had partially degraded phosphorylated RNAPII and partially compacted chromatin\n(Fig. 3g) and specifically used surface area-to-volume (SA/V) ratio instead of volume\nto quantify the less drastic changes in the chromatin (Fig. 3h).\nRevised:\nBecause immature human GV oocytes leftover from fertility treatment rarely had fully\nNSN chromatin configuration (1/52), we utilized the ones that had partially compacted\nchromatin for experiments (Fig. 3g) and specifically used surface area-to-volume\n(SA/V) ratio instead of volume to quantify the less drastic changes in the chromatin\n(Fig. 3h)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This reviewer commends the authors having undertaken an extensive revision due to the misleading writing and lack of granularity in their original submission. Specifically, they have come to realize that this is a mouse paper despite attempting to retain the GV (vit and fresh from different sources) while removing the cortical strip data as requested. It should not go unnoticed that some human data remains even though the Title and summary statement define the work as being done in the mouse as the only model system. In fact there is little recognition of the fact that no other mammalian species has been studied to test the central hypothesis here, one that is materially strengthened by the much improved revised manuscript.\nThe remaining human GV data is itself still very weak and claims that IVM of >70% to \"improve\" viability and maturation seem greatly exaggerated and continue to detract from the strong mouse data set.\nsee...author comment below\nLine 800: were all human GVs vitrified or not..?? were some fresh after retrieval??? And of vitrified, how many survived warming as has been shown to damage GVs with poor survival. We thank the reviewer for raising this concern regarding the vitrification and thawing of human GV oocytes. In this study, human GV oocytes collected at a local clinic (Beijing Perfect Family Hospital) were fresh, whereas those collected at a remote clinic (Reproductive and Genetic Hospital of CITIC-XIANGYA) were vitrified for transport reason. As the reviewer correctly pointed out, human GV oocytes vitrified with conventional kits such as the one from Kitazato had poor survival. Thus, we utilized an optimized set of homemade vitrification solutions (previously reported in So et al., Science 2022) that allow high survival and maturation rate of human GV oocytes after thawing (routinely >70%, n > 300 so far). Also, we excluded dead or morphologically abnormal oocytes after thawing from further experiments. We now provided additional details regarding the human GV oocytes in the respective sentence.\nIn conclusion I find this manuscript acceptable and trust the authors and editors will weigh in on any remaining concerns.\nDavid Albertini", "reviewer_2": "This reviewer thanks the authors for their responses to the previous suggestions, which I think have satisfied my concerns.", "reviewer_3": "Overall, the manuscript reads dramatically better than it did previously. The take home message regarding the need for degradation of RNA polymerase II for oocyte chromatin remodeling is well supported. I have remaining concerns regarding interpretation of experiments related to the mechanism of RNA polymerase II degradation and zygotic genome activation, as detailed below. I believe that these concerns can be readily addressed with text edits, and regardless of the outcome of any additional analyses, resulting changes will not take away any impact from this very nice and exciting story.\nPage 10, lines 21-23: Based on the lack of a difference in NSN/SN ratio in oocytes lacking UBB protein, the authors conclude that “ubiquitination is unlikely to be involved in RNAPII degradation”. I think it is far more likely that UBC compensates for the loss of UBB in the conditional knockout oocytes and that ubiquitination is still the mechanism, particularly given that Ubc mRNA is expressed at a relatively high level (though not as high as Ubb) in publicly available oocyte RNAseq data. Otherwise, the authors are suggesting a completely novel degradation mechanism in oocytes that is distinct from ubiquitination, which, as they point out, is the mechanism in “other systems”.\nI have several concerns with the authors’ methods for exploring the impact of NSN status on zygotic genome activation and on their interpretation of their RNAseq data.\n1. Zygotic genome activation in the mouse begins with a “minor wave” in the 1C to early 2C stages and then continues as a “major wave” in late 2C stage embryos. The minor wave is characterized by transcription of some genes by RNA polymerase II but also requires transcription of rRNAs by RNA polymerase I (see PMID: 25087892). The statement on Page 14, line 20: “…ZGA, a process executed by RNAPII” is inaccurate. Note that in Figure 6b, the appearance of the EU staining in the NSN-2PN embryo is consistent with active transcription of ribosomal RNAs that localize to the pronuclei.\n2. Figure 6g: Pronucleus size increases with time after fertilization. In the NSN-2PN image of the male pronucleus, it is likely based on the position of the pronucleus at the edge of the embryo that fertilization occurred very recently. Unless the authors wish to document fertilization timing and then examine chromatin volume at a specific time following sperm-egg fusion, it is not possible to compare the chromatin volume across groups. Other studies frequently use the relative size and positions of the pronuclei to “stage” the 1-cell embryos, but here the potential impact of the NSN cytoplasm on pronucleus formation and/or migration would prevent that method from being accurate. What if the NSN oocytes do not fertilize as quickly as the SN oocytes, and therefore do not undergo sperm nuclear decondensation and subsequent pronuclear (and nucleolar) maturation at the same time as the SN oocytes? In the absence of fertilization timing information, comparisons of chromatin volume and nucleolar morphology cannot be made accurately.\n3. While there is a well-established lack of developmental competence in NSN oocyte-derived embryos, it makes no sense to compare downregulated transcripts from pronuclear stage embryos to the 7012 ZGA transcripts identified from a pool of 4500 2-cell embryos (Abe et al., PNAS 2018, PMID: 29967139) and attribute the problem to a failure of ZGA. Instead, the NSN pronuclear stage transcripts should be compared to well documented minor ZGA transcripts, of which there are far fewer – 65 or so (see PMIDs: 27309802 and 37459895). Without this more appropriate type of analysis showing strong overlap with minor ZGA transcripts, the inclusion of “zygotic genome activation (ZGA)” in the subtitle (page 14, line 13), “ZGA” in the final sentence of the Results (page 15, lines 16-18), and “zygotic genome activation” in line 16 of the abstract are unsupported and should be removed. A more careful assessment of maternal RNAs may also be warranted.", "reviewer_4": "I would like to thank the authors for the comprehensive revision. As the authors responded appropriately to my original comments, I support the publication of this paper in Nature Communications."}, "author_response": "Response to Referees – Nature Communications ID: NCOMMS-25-\n19027A-Z\nNatural degradation of RNA polymerase II is essential for oocyte chromatin\nreorganization and maternal-to-zygotic transition\nAuthors: Jing Wang, Wang Li, Jing Guo, Xiaoming Xu, Ge Lin, Bin Li, Chun So\nFirst, we want to thank all referees for supporting the publication of this manuscript.\nReviewer #1:\nThis reviewer commends the authors having undertaken an extensive revision due to\nthe misleading writing and lack of granularity in their original submission. Specifically,\nthey have come to realize that this is a mouse paper despite attempting to retain the GV\n(vit and fresh from different sources) while removing the cortical strip data as requested.\nIt should not go unnoticed that some human data remains even though the Title and\nsummary statement define the work as being done in the mouse as the only model system.\nIn fact there is little recognition of the fact that no other mammalian species has been\nstudied to test the central hypothesis here, one that is materially strengthened by the\nmuch improved revised manuscript.\nThe remaining human GV data is itself still very weak and claims that IVM of >70% to\n\"improve\" viability and maturation seem greatly exaggerated and continue to detract\nfrom the strong mouse data set.\nWe thank the reviewer for further comments on the data related to human GVs. We\ndecided to keep the human GV data because we obtained similar results for both fresh\nand vitrified-thawed human GVs. Thus, the use of vitrified-thawed GVs should not\nweaken the reliability of our data. Also, we did not intend to claim our polar body\nextrusion (i.e. nuclear maturation) rate as in vitro maturation (IVM) rate because we\ndid not attempt to further evaluate cytoplasmic maturation by fertilization of the\nresulting MII. We now rewrote the respective sentence in the materials and methods\nsection to be more precise.\nPrevious submission:\nFor oocytes collected at Reproductive and Genetic Hospital of CITIC-XIANGYA, to\nmaximize the survival and developmental rate of vitrified-thawed GV oocytes, oocytes\nwere vitrified by transferring to 20 μl of G-MOPS PLUS (Vitrolife)…\nRevised:\nFor oocytes collected at the Reproductive and Genetic Hospital of CITIC-XIANGYA,\noocytes were vitrified using previously described homemade vitrification solutions to\nmaximize their survival and polar body extrusion rate after thawing. Briefly, oocytes\nwere initially transferred to 20 of G-MOPS PLUS (Vitrolife)…\nsee...author comment below\nLine 800: were all human GVs vitrified or not..?? were some fresh after retrieval???\nAnd of vitrified, how many survived warming as has been shown to damage GVs with\npoor survival. We thank the reviewer for raising this concern regarding the vitrification\nand thawing of human GV oocytes. In this study, human GV oocytes collected at a local\nclinic (Beijing Perfect Family Hospital) were fresh, whereas those collected at a remote\nclinic (Reproductive and Genetic Hospital of CITIC-XIANGYA) were vitrified for\ntransport reason. As the reviewer correctly pointed out, human GV oocytes vitrified\nwith conventional kits such as the one from Kitazato had poor survival. Thus, we\nutilized an optimized set of homemade vitrification solutions (previously reported in So\net al., Science 2022) that allow high survival and maturation rate of human GV oocytes\nafter thawing (routinely >70%, n > 300 so far). Also, we excluded dead or\nmorphologically abnormal oocytes after thawing from further experiments. We now\nprovided additional details regarding the human GV oocytes in the respective sentence.\nIn conclusion I find this manuscript acceptable and trust the authors and editors will\nweigh in on any remaining concerns.\nDavid Albertini\nReviewer #2:\nThis reviewer thanks the authors for their responses to the previous suggestions, which\nI think have satisfied my concerns.\nReviewer #3 :\nOverall, the manuscript reads dramatically better than it did previously. The take home\nmessage regarding the need for degradation of RNA polymerase II for oocyte chromatin\nremodeling is well supported. I have remaining concerns regarding interpretation of\nexperiments related to the mechanism of RNA polymerase II degradation and zygotic\ngenome activation, as detailed below. I believe that these concerns can be readily\naddressed with text edits, and regardless of the outcome of any additional analyses,\nresulting changes will not take away any impact from this very nice and exciting story.\nPage 10, lines 21-23: Based on the lack of a difference in NSN/SN ratio in oocytes\nlacking UBB protein, the authors conclude that “ubiquitination is unlikely to be\ninvolved in RNAPII degradation”. I think it is far more likely that UBC compensates\nfor the loss of UBB in the conditional knockout oocytes and that ubiquitination is still\nthe mechanism, particularly given that Ubc mRNA is expressed at a relatively high\nlevel (though not as high as Ubb) in publicly available oocyte RNAseq data.\nOtherwise, the authors are suggesting a completely novel degradation mechanism in\noocytes that is distinct from ubiquitination, which, as they point out, is the mechanism\nin “other systems”.\nWe thank the reviewer for further comments on the data related to Ubb cKO oocytes.\nAccording to a previous study (KY Ryu et al., Mol Cell Biol 2008), Ubb encodes for\n~70% of ubiquitin protein in oocytes and its knockout phenocopies proteasome\ninhibition in oocytes. Thus, it is unlikely that Ubc expression is sufficient to compensate\nfor the loss of Ubb.\nHowever, as the reviewer correctly stated, we could not entirely exclude the possibility\nthat the remaining ~30% of ubiquitin protein is sufficient for RNAPII degradation.\nTherefore, we now rewrote the respective sentence to discuss such possibility.\nPrevious submission:\n…we examined the chromatin configuration of GV oocytes from wildtype and\nconditional knockout (cKO) mice for Ubb, the predominant polyubiquitin gene in\noocytes. Quantifications revealed no significant difference in their NSN/SN ratio…\nRevised:\n… we examined the chromatin configuration of GV oocytes from wildtype and\nconditional knockout (cKO) mice for Ubb, the predominant polyubiquitin gene that\nencodes 70% of ubiquitin protein and phenocopies proteasome inhibition upon\nknockout in oocytes. While we do not exclude the possibility that the minor amount of\nubiquitin protein remaining in Ubb cKO is sufficient for RNAPII degradation,\nquantifications revealed no significant difference in their NSN/SN ratio…\nI have several concerns with the authors’ methods for exploring the impact of NSN\nstatus on zygotic genome activation and on their interpretation of their RNAseq data.\n1. Zygotic genome activation in the mouse begins with a “minor wave” in the 1C to\nearly 2C stages and then continues as a “major wave” in late 2C stage embryos. The\nminor wave is characterized by transcription of some genes by RNA polymerase II but\nalso requires transcription of rRNAs by RNA polymerase I (see PMID: 25087892).\nThe statement on Page 14, line 20: “…ZGA, a process executed by RNAPII” is\ninaccurate. Note that in Figure 6b, the appearance of the EU staining in the NSN-\n2PN embryo is consistent with active transcription of ribosomal RNAs that localize to\nthe pronuclei.\nWe thank the reviewer for further comments on the contributions of both RNAPI and\nRNAPII to ZGA. We now correct the respective sentence as suggested by the reviewer.\nPrevious submission:\n…, later contributing to defects in ZGA, a process executed by RNAPII.\nRevised:\n…, later contributing to defects in ZGA, a process primarily executed by RNAPII and\nto a lesser extent RNAPI.\n2. Figure 6g: Pronucleus size increases with time after fertilization. In the NSN-2PN\nimage of the male pronucleus, it is likely based on the position of the pronucleus at\nthe edge of the embryo that fertilization occurred very recently. Unless the authors\nwish to document fertilization timing and then examine chromatin volume at a specific\ntime following sperm-egg fusion, it is not possible to compare the chromatin volume\nacross groups. Other studies frequently use the relative size and positions of the\npronuclei to “stage” the 1-cell embryos, but here the potential impact of the NSN\ncytoplasm on pronucleus formation and/or migration would prevent that method from\nbeing accurate. What if the NSN oocytes do not fertilize as quickly as the SN oocytes,\nand therefore do not undergo sperm nuclear decondensation and subsequent\npronuclear (and nucleolar) maturation at the same time as the SN oocytes? In the\nabsence of fertilization timing information, comparisons of chromatin volume and\nnucleolar morphology cannot be made accurately.\nWe thank the reviewer for further technical comments on the experiments related to\nembryos derived from NSN and SN oocytes. As the reviewer correctly pointed out, the\nphenotypes that we observed could also be explained by delays in embryo development\ntiming.\nHowever, we believe this is unlikely the case because these differences persisted in late\n2PN embryos and most embryos derived from NSN oocytes did not further progress\nbeyond PN stage. Nevertheless, we now rewrote the respective sentence to discuss such\npossibility.\nPrevious submission:\n…, indicative of defects in pronucleus formation in 1-cell embryos derived from NSN\noocytes.\nRevised:\n…, indicative of defects and possibly a delay in pronucleus formation in 1-cell embryos\nderived from NSN oocytes.\n3. While there is a well-established lack of developmental competence in NSN oocyte-\nderived embryos, it makes no sense to compare downregulated transcripts from\npronuclear stage embryos to the 7012 ZGA transcripts identified from a pool of 4500\n2-cell embryos (Abe et al., PNAS 2018, PMID: 29967139) and attribute the problem\nto a failure of ZGA. Instead, the NSN pronuclear stage transcripts should be\ncompared to well documented minor ZGA transcripts, of which there are far fewer –\n65 or so (see PMIDs: 27309802 and 37459895). Without this more appropriate type\nof analysis showing strong overlap with minor ZGA transcripts, the inclusion of\n“zygotic genome activation (ZGA)” in the subtitle (page 14, line 13), “ZGA” in the\nfinal sentence of the Results (page 15, lines 16-18), and “zygotic genome activation”\nin line 16 of the abstract are unsupported and should be removed. A more careful\nassessment of maternal RNAs may also be warranted.\nWe thank the reviewer for further technical comments on the analysis of 2PN RNA-seq\ndata. According to a previous study that performed RNA-seq analysis on precisely\ntimed mouse embryos generated by ICSI (Asami et al., Cell Rep 2023), there were 1777\ngenes upregulated as early as within 4 h post fertilization and 2290 downregulated\ngenes that include many canonical maternal transcripts. Thus, the suggested list of 65\nminor ZGA transcripts that resulted from very stringent log2FC and p-value cut-offs\n(Wu et al., Nature 2016; Ji et al. Nature 2023) may not be appropriate for our analysis\neither.\nHowever, we did agree with the reviewer that our formerly defined ~7000 ZGA\ntranscripts and ~6000 maternal transcripts based on clusters I/IV/V and II/III from Abe\net al. PNAS 2018 may be over-inclusive. Therefore, we now redefined minor ZGA\ntranscripts as clusters IV/V and maternal transcripts as cluster II from Abe et al. PNAS\n2018, resulting in ~3600 and ~1100 genes which are much closer to the numbers from\nAsami et al., Cell Rep 2023 (Fig. 6j). Also, we now performed gene ontology analysis\non the remaining differentially expressed genes that fall into neither minor ZGA\ntranscripts nor maternal transcripts, and found additional nuclear and cytoplasmic\ndefects in embryos derived from NSN oocytes (Fig. 6k).\nResponse Fig. 18 (new Fig. 6j, k) j, Euler diagrams showing the overlap between\ndownregulated genes and minor ZGA transcripts and overlap between\nupregulated genes and maternal transcripts. k, GO analysis of downregulated and\nupregulated genes not overlapping with minor ZGA transcripts and maternal\ntranscripts.\nGiven the complex nature of defects in embryos derived from NSN oocytes, we now\nrewrote the respective sentence to tone down the contributions of (minor) ZGA and\nmaternal mRNA degradation as suggested by the reviewer.\nPrevious submission:\n…, are defective in pronucleus formation, zygotic genome activation and maternal\nmRNA degradation.\nEmbryos derived from NSN oocytes have aberrant RNAPII levels and localization\nand are defective in pronucleus formation, zygotic genome activation (ZGA) and\nmaternal mRNA degradation\nNotably, a significant portion of downregulated mRNAs were ZGA transcripts,\nwhereas upregulated mRNAs were mostly maternal transcripts,…\nTaken together, embryos derived from NSN oocytes are defective in pronucleus\nformation, ZGA, and maternal mRNA degradation,…\nRevised:\n…, are defective in maternal-to-zygotic transition.\nEmbryos derived from NSN oocytes have aberrant RNAPII levels and localization\nand are defective in maternal-to-zygotic transition\nNotably, 42 out of 223 downregulated genes were minor ZGA transcripts, whereas 52\nout of 168 upregulated genes were maternal transcripts,…\nTaken together, embryos derived from NSN oocytes are defective in maternal-to-\nzygotic transition,…\nReviewer #4:\nI would like to thank the authors for the comprehensive revision. As the authors\nresponded appropriately to my original comments, I support the publication of this\npaper in Nature Communications."}]

Decision letter