DDX5 orchestrates RNA homeostasis to ensure oocyte developmental competence

Domain: biology
Published: 2026-03-11
Editorial outcome: minor_revision

Abstract
Oocyte development requires tight regulation of transcription and RNA metabolism, which is coordinated by RNA-binding proteins, whose roles in mammalian oogenesis remain incompletely understood. Here, we identify the DEAD-box RNA helicase DDX5 as a key regulator of RNA homeostasis in oocytes. Oocyte-specific deletion of DDX5 leads to female sterility, which is characterized by defective chromatin remodeling, meiotic arrest, increased aneuploidy, and fertilization failure. Mechanistically, DDX5 maintains RNA homeostasis through three interconnected processes: (1) promoting transcription via interaction with RNA polymerase II in nonsurrounded nucleolus-stage germinal vesicle oocytes; (2) clearing retrotransposon RNAs to safeguard transcriptome integrity; and (3) supporting maternal mRNA storage by coordinating nuclear export, mitochondrial organization, and mitochondria-associated ribonucleoprotein domain assembly. Our study establishes DDX5 as a master regulator that integrates transcriptional and post-transcriptional programs to ensure oocyte competence and fertility.

Introduction
Oocyte development is a highly specialized and tightly regulated process essential for female fertility and early embryonic development1. In mammals, oocytes initiate their growth within primordial follicles and progress through a prolonged developmental timeline as the follicle develops before acquiring meiotic and developmental competence2-4. During this process, particularly at the germinal vesicle (GV) stage, oocytes exhibit robust transcriptional activity and accumulate large amounts of maternal RNAs and proteins5-7. Concurrently, the chromatin remodeling drives the transition from a transcriptionally active, nonsurrounded nucleolus (NSN) configuration to a condensed, transcriptionally silent surrounded nucleolus (SN) state, representing a key preparatory step for meiotic resumption8-13. These changes are tightly coupled to transcriptional and post-transcriptional programs that prepare the oocyte for successful maturation, fertilization, and subsequent embryogenesis14-18.

To better understand the molecular underpinnings of oocyte development, we generated a high-resolution transcriptional atlas that captures the dynamic gene expression programs associated with the acquisition of developmental competence19. However, growing oocytes face unique challenges in maintaining RNA homeostasis, including producing maternal transcripts for future embryogenesis, removing aberrant RNAs, and preserving untranslated mRNAs from premature translation5,20. Emerging evidence, including our proteomic studies21,22, highlights the central role of RNA-binding proteins (RBPs) in orchestrating these processes23,24. While RBPs such as MTR425, SRSF326, and MARF127 contribute to RNA surveillance and retrotransposon RNA control, others including ZAR1, YBX2, DDX6, LSM14B, and EIF4ENIF1 facilitate maternal mRNA storage through the cytoplasmic mitochondria-associated ribonucleoprotein domain (MARDO)5,28-30. Despite these advances, the current catalog of functionally characterized RBPs in oocytes remains incomplete. Many key RBPs have yet to be identified, and their potentially multifaceted roles in shaping oocyte transcriptome dynamics remain largely unexplored.

DEAD-box RNA helicases are a conserved family of RBPs known to regulate various aspects of RNA metabolism31-34. DDX5, a core member of this family, has been implicated in transcriptional coactivation35-37, mRNA splicing38-40, ribosome biogenesis41,42, and microRNA processing43-45. However, these functions have been characterized primarily in cancer cell models31,46,47. Recent studies by our group and others have revealed unique roles for DDX5 in the establishment and maintenance of stem cell pluripotency and spermatogenesis45,48-51. Considering that DDX5 is highly expressed in oocytes, its potential role in female reproduction remains to be elucidated.

In this study, we generated oocyte-specific Ddx5 conditional knockout (cKO) mice to investigate the role of DDX5 in mammalian oocyte development. We found that female mice lacking DDX5 in oocytes exhibit complete sterility due to oocyte chromatin remodeling defects, meiotic arrest, an increased incidence of aneuploidy, and fertilization failure. Mechanistically, we revealed that DDX5 safeguards oocyte RNA homeostasis through three coordinated functions. First, DDX5 promotes global transcription at the GV stage by interacting with RNA polymerase II; its loss causes RNA depletion and widespread transcriptome dysregulation. Second, during oocyte growth, DDX5 is indispensable for the clearance of retrotransposon-derived RNAs, particularly from LTR and LINE elements, by binding and promoting their degradation. Third, DDX5 ensures proper mRNA storage by coordinating nuclear export, mitochondrial organization, and the MARDO assembly. Its absence leads to premature translation and accelerated mRNA depletion. Together, our findings establish DDX5 as a master integrator of the transcriptional and post-transcriptional programs required for oocyte developmental competence and female fertility.

Results
DDX5 oocyte-specific ablation causes female sterility

DDX5 displays dynamic, stage-specific localization during oocyte maturation, shifting from a predominantly nuclear distribution in NSN-configured GV oocytes to nuclear clearance upon SN transition and finally to cytoplasmic accumulation at the MII stage (Supplementary Fig. 1a). This spatiotemporal pattern suggests distinct phase-dependent functions in regulating oocyte competence. To investigate the functional role of DDX5, we generated oocyte-specific conditional knockout (cKO) mice using a multistep breeding strategy (Fig. 1a and Supplementary Fig. 1b) and confirmed complete Ddx5 deletion at both the genomic and protein levels (Fig. 1b and Supplementary Fig. 1c–e).

Given the tight coupling between oocyte maturation and follicular development, we first analyzed ovarian ontogeny in Ddx5f/f; Gdf9-Cre females. The ovarian morphology appeared largely normal at postnatal Days 11.5 and 17.5, corresponding to the emergence of secondary and early antral follicles, respectively (Fig. 1c–d). However, by 3 weeks of age, when antral follicle growth accelerated, the cKO ovaries exhibited significant growth retardation, including reduced size and a lack of preovulatory follicles after PMSG stimulation (Fig. 1d–f). In addition, DDX5-deficient oocytes failed to achieve functional recovery with the appearance of preovulatory follicles in adulthood, as they were unable to form pronuclei following both natural fertilization (NF) and in vitro fertilization (IVF) (Fig. 1g), resulting in a complete lack of two-cell embryos (Fig. 1h–i and Supplementary Fig. 1f). Consequently, Ddx5f/f; Gdf9-Cre females were completely sterile, a phenotype further confirmed in Ddx5f/f; Zp3-Cre mice (Fig. 1j).

Together, these findings establish that DDX5 is an essential regulator of oocyte developmental competence and female fertility.

DDX5 is essential for meiotic resumption in oocytes

Given the observed fertilization failure in DDX5-deficient oocytes, we next evaluated the quality of GV oocytes. We first observed that DDX5-deficient GV oocytes presented a progressively darker appearance with advancing development (Fig. 2a), a phenotype that may be associated with apoptosis (Fig. 2b–c). We then assessed the meiotic progression in vitro and in vivo. Upon in vitro culture of GV-stage oocytes, approximately 80% of the oocytes from Ddx5f/f females underwent GVBD and formed morphologically normal spindles within 8 hours. In contrast, approximately 80% of the DDX5-deficient oocytes remained arrested at the GV stage, whereas the remaining oocytes resumed meiosis but presented pronounced spindle abnormalities, including disorganized radial microtubule arrays (Fig. 2d–e). These findings were confirmed in vivo, where oocytes retrieved from cKO females after PMSG/hCG stimulation exhibited meiotic arrest at the GV stage (Fig. 2f, g). Although a subset (23%) progressed to MII, these oocytes displayed severe chromosomal aneuploidy (Fig. 2h–i), indicating a broad defect in meiotic progression.

Together, these results demonstrate that DDX5 is essential for proper meiotic resumption and explain the failure of pronucleus formation in DDX5-deficient oocytes.

DDX5 deficiency disrupts chromatin remodeling in GV oocytes

Chromatin remodeling is a key prerequisite for establishing meiotic competence in oocytes. To assess this process, we quantified the acquisition of the SN configuration in GV oocytes collected from 3- and 6-week-old mice following PMSG stimulation. In control oocytes, the proportion of SN oocytes increased from 39.9% at 3 weeks to 51.4% at 6 weeks (Fig. 3a–b), reflecting the expected age-dependent maturation. In sharp contrast, DDX5-deficient oocytes consistently failed to acquire a proper SN configuration characterized by a condensed chromatin ring surrounding the nucleolus. Instead, they displayed an aberrant, partially condensed chromatin pattern, which we defined as Failed-SN (FSN). Notably, the proportion of FSN oocytes increased from 23.7% at 3 weeks to 47.2% at 6 weeks (Fig. 3a–b), indicating that chromatin abnormalities worsen as ovarian maturation progresses.

To characterize the chromatin remodeling defects further, we performed ATAC-seq analysis of GV oocytes and reported a striking genome-wide increase in chromatin accessibility after Ddx5 depletion. Specifically, compared with control oocytes, DDX5-deficient oocytes presented 26,363 unique accessible regions (Fig. 3c), resulting in uniform chromatin opening without regional bias (promoter: 29.7% vs. 30.8%; proximal: 19.9% vs. 19.1%; distal: 50.4% vs. 50.1%) (Fig. 3d–f). Moreover, chromatin regions that are normally accessible in control oocytes exhibited further increased accessibility in DDX5-deficient oocytes (Fig. 3g). This molecular finding provides a mechanistic explanation for the failure of SN transition, directly linking nuclear architectural defects to abnormal chromatin organization at the genome-scale.

In addition, we found that DDX5 deficiency led to a significant increase in accessibility near transcription start sites (TSSs) (Fig. 3h). Interestingly, unlike the positive correlation between promoter accessibility and gene expression in control oocytes (Fig. 3i), this relationship was largely uncoupled in DDX5-deficient oocytes. Only 596 differentially expressed genes (DEGs) were significantly upregulated in association with increased promoter openness (fold change>2), representing the transcriptome subset most responsive to chromatin state changes (Fig. 3j). This uncoupling indicates a disruption in the normal coordination between chromatin accessibility and transcriptional activity. The expression of the remaining DEGs could not be explained by local accessibility changes alone, indicating the involvement of additional regulatory mechanisms.

DDX5 orchestrates Pol II to ensure transcription initiation

Given the observed uncoupling between promoter accessibility and transcriptional output in DDX5-deficient oocytes, we next investigated additional layers of transcriptional regulation. Here, we performed total RNA sequencing (rRNA-depleted) on oocytes at three GV substages—representing a critical developmental window characterized by active transcription that supports oocyte growth and early embryogenesis—as well as at the MII stage (identified by polar body extrusion) (Fig. 4a). The data analysis confirmed the expected ERCC spike-in ratios and revealed high reproducibility across samples (Supplementary Fig. 2a–b). Intriguingly, although DDX5-deficient oocytes exhibited characteristic EAF-LAF accumulation followed by degradation similar to that of the control, they consistently presented a significant reduction in total RNA levels at all stages examined (Fig. 4b and Supplementary Fig. 2c). Furthermore, this reduction was confirmed at the mRNA level in DDX5-deficient GV oocytes by fluorescence in situ hybridization (FISH) (Fig. 4c, d).

EU incorporation assays revealed that the reduced RNA levels in DDX5-deficient oocytes resulted from impaired transcriptional activity (Fig. 4e). To investigate this mechanism, we examined RNA polymerase II (Pol II), the core enzyme for mRNA synthesis, and revealed a functional link between DDX5 and the transcription machine. Immunofluorescence and proximity ligation assays (PLA) revealed strong colocalization and interaction between DDX5 and Pol II in transcriptionally active GV-stage oocytes (Fig. 4f–g and Supplementary Fig. 2d). Importantly, this interaction was further validated by co-immunoprecipitation (co-IP) in HEK293T cells and indicated that RNA may contribute to its formation or maintenance (Supplementary Fig. 2e–f). In DDX5-deficient oocytes, this interaction was disrupted, leading to a significant decrease in Ser5 phosphorylation of Pol II (Fig. 4h), suggesting that transcription initiation was inhibited, whereas the total protein level of Pol II remained unchanged (Supplementary Fig. 2g–h). These findings indicate that DDX5 facilitates transcription initiation by interacting with Pol II, thereby promoting active mRNA transcription. This mechanism may partially explain the observed uncoupling between promoter accessibility and gene expression.

DDX5 deficiency induces global transcriptome disturbances

Given the transcriptional impairment in DDX5-deficient oocytes, we next examined the downstream consequences on gene expression. ERCC-normalized RNA sequencing revealed a cumulative increase in DEGs during oocyte maturation, with the majority being downregulated (Fig. 5a–b and Supplementary Fig. 3a). Notably, compared with those in EAF GV oocytes, 2135 genes, including those involved in mitochondrial organization, cell cycle regulation, and protein metabolism, were further downregulated at the cKO PreF GV stage, whereas 692 further upregulated genes were enriched in pathways related to Pol II-dependent transcription, Wnt signaling, and apoptosis (Fig. 5c and Supplementary Fig. 3b).

Gene Ontology (GO) analysis of severely affected Pre GV oocytes revealed that abnormally upregulated genes were significantly enriched in ribonucleoprotein complex biogenesis, protein–RNA complex organization, ncRNA processing, and apoptotic pathways, whereas downregulated genes were strongly associated with ciliary motility, organelle positioning, and chromosome segregation processes (Supplementary Fig. 3c–d).

We also evaluated the transcriptome at the developmental scale. In control oocytes, gene expression followed a tightly regulated trajectory from EAF GV to MII, ensuring transcriptional homeostasis (Supplementary Fig. 3e). In contrast, DDX5-deficient oocytes deviated markedly from this program. Fuzzy clustering revealed four functionally distinct gene modules exhibiting stage-dependent dynamics in control oocytes (Fig. 5d): Wnt signaling and metabolic processes (Cluster 1), chromatin remodeling and organelle reorganization (Cluster 2), translation and energy production (Cluster 3), and mRNA processing and cell cycle regulation (Cluster 4). Strikingly, loss of DDX5 led to widespread dysregulation of all these modules. Cluster 1 genes were aberrantly upregulated, whereas genes in Clusters 2–4 were insufficiently expressed or prematurely silenced (Fig. 5d). This collective disruption demonstrates the essential role of DDX5 in orchestrating the stage-specific transcriptome transitions required for oocyte developmental competence.

DDX5 mediates retrotransposon RNA clearance during oocyte maturation

In addition to regulating protein-coding genes, we further revealed that DDX5 plays an essential role in regulating retrotransposon- derived RNAs, thereby contributing to transcriptome surveillance during oocyte growth. Comparative analysis of repeat element expression between Ddx5f/f EAF and PreF GV oocytes revealed that GV growth is normally accompanied by widespread clearance of repeat-derived RNAs (n=439), with retrotransposons—particularly LTR and LINE elements—being most prominently downregulated (Fig. 6a–b). In contrast, only 64 repeat RNAs, mainly tRNAs, were upregulated during this transition (Supplementary Fig. 4a).

However, this clearance process was markedly disrupted in DDX5-deficient oocytes (Supplementary Fig. 4b). As a result, numerous repeat-derived RNAs, especially retrotransposons, presented increased accumulation during GV development (Fig. 6c and Supplementary Fig. 4c). Notably, 125 of the 155 repeats (80.6%) that were aberrantly upregulated in DDX5-deficient PreF GV oocytes were normally downregulated during the control GV maturation process (Fig. 6d–e), underscoring a failure in retrotransposon RNA clearance upon DDX5 loss.

Mechanistically, low-input RIP-seq revealed that DDX5 associates with retrotransposon transcripts in GV oocytes, binding 32% of expressed retrotransposons compared with only 9.2% of expressed retrotransposons in mESCs (Fig. 6f). This selective interaction enables DDX5 to target these repeat RNAs to promote their degradation, as evidenced by the significant and sustained upregulation of the DDX5-bound repeat RNAs upon DDX5 depletion at both the PreF-GV and MII stage (Fig. 6g). Consequently, Failure to eliminate these transcripts in DDX5-deficient oocytes led to their aberrant accumulation and the ectopic activation of nearby genes (Fig. 6h and Supplementary Fig. 4d).

Together, these findings identify DDX5 as a critical factor for retrotransposon RNA clearance during oocyte growth, highlighting its essential role in preserving transcriptome integrity and ensuring transcriptional homeostasis in GV-stage oocytes.

DDX5 deficiency leads to decreased mRNA storage efficiency

In the post-transcriptional regulation of oocytes, efficient storage of untranslated mRNAs in MARDOs is also critical for supporting subsequent oocyte and embryonic development5. Here, we evaluated the mRNA storage capacity of GV oocytes and found that DDX5 deficiency compromises the mRNA storage process at multiple levels.

First, DDX5 depletion markedly impaired the nuclear export of mRNAs, as evidenced by the abnormal nuclear accumulation of mRNAs in both NSN and FSN GV oocytes (Fig. 7a), thereby weakening the prerequisite for cytoplasmic storage.

Second, loss of DDX5 disrupts the rearrangement of mitochondria, which is a key structural component of the MARDO. In DDX5-deficient oocytes, mitochondria failed to exhibit the typical perinuclear clustering observed in controls and instead accumulated in the subcortical region (Fig. 7b–c and Supplementary Fig. 5a–c), potentially disrupting the local energy supply necessary for GVBD (Supplementary Fig. 5d). In support of this finding, GSEA analysis showed that the expression of genes involved in mitochondrial localization was gradually downregulated (Fig. 7d), providing molecular evidence for the observed mitochondrial mislocalization. Functionally, this spatial mislocalization of mitochondria was accompanied by increased levels of intracellular reactive oxygen species (ROS) (Fig. 7e–f), which may underlie the increase in apoptosis.

Third, we focused on MARDO formation because the mitochondrial membrane potential, which is a key driver of MARDO assembly, was significantly reduced (Fig. 7g and Supplementary Fig. 5e), and most MARDO-related genes, including Zar1, Ybx2, Eif4enif1, and Lsm14b, were downregulated (Fig. 7h and Supplementary Fig. 5f–i). Although immunofluorescence staining indicated that MARDOs could still be recruited, as evidenced by the similar localization of DDX6 and mitochondria, their distribution pattern was altered concomitant with the redistribution of mitochondria, resulting in subcortical aggregation (Fig. 7i). However, this altered MARDO was inefficient in storing mRNA, as indicated by the failure of the subcortical mRNA signal to increase as significantly as that of mitochondria (Fig. 7j). The loss of storage capacity led to premature translation of unprotected mRNAs, which was reflected by significantly increased translation activity in DDX5-deficient oocytes (Fig. 7k–l), thereby accelerating mRNA reduction.

Together, these findings demonstrate that DDX5 is essential for maintaining the integrity of the mRNA storage system in GV oocytes, through the coordinated regulation of nuclear export, mitochondrial organization, and MARDO formation.

Methods
Ethical Statement
All experimental procedures involving animals were approved by the Tongji University Biological Research Ethics Committee and were conducted in strict accordance with the institutional Guide for the Care and Use of Laboratory Animals of Tongji University. All mice were euthanized by cervical dislocation.

Generation of Ddx5 cKO mice
The Ddx5 cKO mice were originally generated by crossing Ddx5f/f females55 with Gdf9-Cre or Zp3-Cre males (Supplementary Fig. 1b). The experiments were mainly performed using Ddx5f/f; Gdf9-Cre mice, and key results were also verified in Ddx5f/f; Zp3-Cre mice.

In vitro fertilization (IVF)
Adult female mice were superovulated by intraperitoneal injections of 6 IU PMSG (Ningbo Sansheng) followed by 6 IU hCG (Ningbo Sansheng) 48 hours later. Cumulus-oocyte complexes were collected from the ampulla of the oviducts 13-15 hours post-hCG administration, treated with 0.6 mg/mL hyaluronidase (Sigma-Aldrich) in HCZB for 3-5 minutes to remove granulosa cells, and washed thoroughly. The denuded oocytes were subjected to zona pellucida drilling to facilitate sperm entry. For sperm preparation, the cauda epididymis was isolated from adult male mice, punctured with a needle, and gently compressed to release sperm into 500 μL of pre-equilibrated G-IVF medium (Vitrolife). The sperm suspension was incubated at 37°C under 5% CO₂ for 20 minutes to allow capacitation. The upper layer containing motile sperm was co-incubated with oocytes in G-IVF droplets under mineral oil for 4 hours at 37°C, 5% CO₂. Fertilized zygotes were subsequently washed and cultured in G1 medium (Vitrolife) at 37°C, 5% CO₂. Embryonic development was assessed every 24 hours until the blastocyst stage.

GV oocyte collection
GV oocytes were collected by isolating ovaries from female mice and immediately mincing them in M2 medium (Sigma-Aldrich) containing 10 μM milrinone (Sigma-Aldrich). Oocytes were initially retrieved using a wide-bore glass capillary connected to a mouth pipette, then transferred to a fresh medium droplet where surrounding granulosa cells were removed by repeated pipetting with a finer capillary (inner diameter approximating oocyte size). Denuded oocytes were either used directly for downstream experiments or cultured in M2 medium droplets under mineral oil at 37 °C with 5% CO₂.

Immunofluorescence staining (IF)
Oocytes were processed in 96-well U-bottom plates, with all solution changes performed using a mouth pipette. The staining procedure included the following steps: fixation with 4% paraformaldehyde (PFA; Servicebio) overnight, washing with TBST (Epizyme), permeabilization with 0.5% Triton X-100 (Sigma-Aldrich) in TBST for 30 minutes at room temperature, and blocking with 2.5% bovine serum albumin (BSA; Sigma-Aldrich) for 1 hour at room temperature. Primary antibodies diluted in 2.5% BSA were incubated with oocytes overnight at 4 °C, followed by washes in TBST. Secondary antibodies, also diluted in 2.5% BSA, were applied for 1 hour at room temperature in the dark, followed by additional TBST washes. Nuclei were counterstained with DAPI (Beyotime). Stained oocytes were then mounted in 3–4 μL CZB medium droplets under mineral oil on glass-bottom dishes and imaged using a Zeiss LSM880 confocal microscope. Antibody information is provided in Supplementary Data5.

Western blot (WB)
A total of 100 fully denuded oocytes were transferred into 10 μL of RIPA lysis buffer (Beyotime) containing PMSF and protease inhibitor cocktail (PIC) using a mouth pipette. The lysate was vortexed and incubated at 4 °C for 1 hour to ensure complete lysis. After incubation, 2.5 μL of 5× SDS-PAGE protein loading buffer (Epizyme) was added, vortexed, and then heat-denatured at 100 °C for 10 minutes. The samples were centrifuged at 20,000 g for 5 minutes at 4 °C, and the supernatant was carefully collected for subsequent WB analysis. A 5% stacking gel and 10% separating gel were prepared using the Color PAGE Gel Rapid Preparation Kit (Epizyme), following the manufacturer’s instructions. Protein samples and markers were loaded, and electrophoresis was performed at 80 V until the dye front reached the separating gel, followed by 120 V until complete separation. Proteins were transferred onto activated PVDF membranes (Merck Millipore) at 200 mA for 2 hours in pre-chilled transfer buffer (Epizyme). Membranes were blocked with Protein-Free Rapid Blocking Buffer (Epizyme) for 30 minutes at room temperature, followed by incubation with primary antibodies overnight at 4 °C. After washing, membranes were incubated with secondary antibodies for 1 hour at room temperature. Protein bands were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged with a Bio-Rad system for quantitative analysis. Antibody information is listed in Supplementary Data 5.

TUNEL assay
GV oocytes were collected from adult female mice 44–48 hours after PMSG stimulation. Oocyte apoptosis was assessed using the One Step TUNEL Apoptosis Assay Kit (Beyotime), following the manufacturer’s instructions. Briefly, oocytes were fixed in 4% PFA for 20 minutes at room temperature, followed by three washes with TBST. Permeabilization was performed with 0.5% Triton X-100 in TBST for 30 minutes at room temperature. Oocytes were then incubated in the TUNEL reaction mixture for 1 hour at 37 °C in the dark. After thorough TBST washes, oocytes were imaged using a Zeiss LSM880 confocal microscope. Fluorescence signals were quantified in NSN GV oocytes.

Chromatin spreading
MII oocytes were transferred to acidic Tyrode’s solution (Sigma-Aldrich) to remove the zona pellucida, followed by removal of the first polar body. The zona-free oocytes were placed onto slides containing fixative solution (1% PFA, 3 mM DTT, and 0.15% Triton X-100 in ddH₂O, pH 9.2–9.3) and allowed to lyse and spread at room temperature. After air-drying, slides were washed three times with PBS, blocked with 3% BSA in PBS for 1 hour at room temperature, and incubated overnight at 4 °C with CREST antibody diluted in blocking buffer. Following three PBS washes, samples were incubated with secondary antibody and DAPI (both in 3% BSA-PBS) for 1 hour at room temperature in the dark. Finally, after additional PBS washes, slides were mounted with anti-fade medium and imaged using a Zeiss LSM880 confocal microscope. Antibody information is provided in Supplementary Data 5.

mRNA fluorescence in situ hybridization (mRNA FISH)
GV oocytes were collected from adult female mice 44–48 hours after PMSG stimulation. Oocytes were fixed in 4% PFA for 20 minutes at room temperature, washed three times with PBS, and permeabilized in freshly prepared 70% ethanol for 20 minutes at room temperature. After equilibration in Wash Buffer A (Biosearch Technologies) for 20 minutes at room temperature, hybridization was performed overnight at 37 °C in the dark using 500 nM oligo-dT30-Cy5 probes (synthesized by GenePharma) diluted in freshly prepared Hybridization Buffer (Biosearch Technologies). Following hybridization, oocytes were washed in Wash Buffer A for 30 minutes at 37 °C, stained with DAPI in fresh Wash Buffer A for 30 minutes at 37 °C, and then washed in Wash Buffer B (Biosearch Technologies) for 30 minutes at 37 °C. Imaging was performed using a Zeiss LSM880 confocal microscope.

EU
GV oocytes were collected from 3-week-old female mice 44–48 hours after PMSG stimulation and used to assess transcriptional activity using the Click-iT RNA Alexa Fluor 488 Imaging Kit (Invitrogen), following the manufacturer's instructions. Briefly, oocytes were cultured in M16 medium containing 1 mM 5-ethynyl uridine (5-EU) and 10 μM milrinone for 2 hours at 37 °C in a 5% CO₂ incubator. After incubation, oocytes were fixed in 4% PFA for 20 minutes at room temperature, washed with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 30 minutes. The samples were then incubated in freshly prepared Click-iT reaction cocktail for 30 minutes at room temperature in the dark, followed by a 5-minute rinse in Click-iT reaction rinse buffer. Nuclei were stained with DAPI for 20 minutes at room temperature in the dark. Finally, oocytes were washed and imaged using a Zeiss LSM880 confocal microscope. Fluorescence signals in NSN-stage GV oocytes were quantified.

Proximity Ligation Assay (PLA)
GV oocytes were collected from P17.5 female mice. PLA was performed using the Duolink In Situ Proximity Ligation Assay Starter Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, oocytes were fixed in 4% PFA for 20 minutes at room temperature, washed with TBST, and permeabilized in 1% Triton X-100 in TBST for 1 hour at room temperature. After TBST washes, oocytes were blocked with 3% BSA/TBST for 1 hour at room temperature. Primary antibodies diluted in 3% BSA/TBST were applied and incubated overnight at 4°C. The next day, oocytes were washed with 1× Wash Buffer A, followed by incubation with PLA probes at 37 °C for 1 hour. After further washes with Wash Buffer A, ligation was performed at 37 °C for 30 minutes. Amplification was then carried out at 37 °C for 100 minutes in the dark. Finally, oocytes were sequentially washed with 1× and 0.01× Wash Buffer B, stained with DAPI, and imaged using a Zeiss LSM880 confocal microscope.

ATP measurement
GV oocytes were collected from adult female mice 44–48 hours after PMSG stimulation. ATP content in oocytes was measured using an Enhanced ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, groups of five GV oocytes, with the zona pellucida removed by Pronase E (Sigma-Aldrich) treatment, were transferred into 20 μL of ATP lysis buffer. After lysis, samples were centrifuged at 18,000 g for 5 minutes at 4 °C, and the supernatant was collected and kept on ice until analysis. A 96-well black plate (Costar) was prepared by adding 100 μL of ATP working solution to each well, followed by incubation at room temperature for 5 minutes to eliminate background luminescence. Subsequently, 20 μL of each oocyte lysate was added to the corresponding wells, mixed quickly by pipetting, and luminescence was recorded immediately using a luminometer. A standard curve was generated in parallel using serial dilutions of ATP standards to ensure accurate quantification and data reliability.

Measurement of Intracellular Reactive Oxygen Species (ROS)
GV oocytes were collected from adult female mice 44–48 hours after PMSG stimulation. Intracellular ROS levels in oocytes were measured using the ROS Assay Kit with CM-H2DCFDA (Beyotime) according to the manufacturer’s instructions. GV oocytes were isolated and immediately transferred into HCZB medium droplets containing 10 μM Milrinone and CM-H2DCFDA (diluted 1:1000 from stock). Oocytes were incubated at 37 °C in a 5% CO₂ incubator for 30 minutes. Following incubation, oocytes were washed three times in fresh HCZB medium to remove excess dye and then immediately imaged using a Zeiss LSM880 confocal microscope.

Measurement of mitochondrial membrane potential
GV oocytes were collected from adult female mice 44–48 hours after PMSG stimulation. Mitochondrial membrane potential in oocytes was assessed using the Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime) according to the manufacturer’s instructions. GV oocytes were isolated and immediately transferred into HCZB medium droplets containing JC-1 dye and JC-1 staining buffer. Oocytes were incubated at 37 °C in a 5% CO₂ incubator for 20 minutes. After incubation, excess dye was removed by washing the oocytes in HCZB washing solution supplemented with JC-1 staining buffer on ice. The oocytes were then immediately imaged using a fluorescence microscope. JC-1 monomers emit green fluorescence, whereas JC-1 aggregates emit red fluorescence. A decrease in the red/green fluorescence ratio indicates a reduction in mitochondrial membrane potential.

Protein synthesis assay
Protein synthesis activity in oocytes was assessed using the Click-iT Plus OPP Protein Synthesis Assay Kit (Invitrogen) following the manufacturer’s instructions. GV oocytes were incubated in HCZB medium supplemented with 20 μM Click-iT OPP and 10 μM Milrinone for 30 minutes at 37 °C in a 5% CO₂ incubator. After incubation, oocytes were washed with PBS and fixed in 4% PFA for 15 minutes at room temperature. Permeabilization was then performed using 0.5% Triton X-100 in PBS for 30 minutes, followed by PBS washes. Oocytes were incubated in the Click-iT Plus OPP reaction cocktail for 30 minutes at room temperature in the dark. After staining, oocytes were washed with the Click-iT reaction rinse buffer and stained with HCS NuclearMask Blue to label DNA. Fluorescence imaging was conducted using a Zeiss LSM880 confocal microscope.

Co-immunoprecipitation
HEK293T cells overexpressing FLAG-tagged full-length RPB1 and HA-tagged full-length DDX5 were lysed in ice-cold lysis buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.2% NP-40, 10% glycerol, supplemented with 1× protease inhibitor cocktail) for 1 h with agitation. Where indicated, RNase A digestion was performed by incubating the lysate at 37°C for 45 min prior to immunoprecipitation. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C. The supernatant was incubated with the specified antibody or control IgG for 3 hours at 4°C, followed by incubation with protein A/G magnetic beads (Smart Lifesciences) for an additional 2 h. Beads were washed four times with wash buffer (1× PBS containing 0.3% Tween-20), resuspended in SDS loading buffer, and boiled for 10 min. Samples were resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies.

Generation of DDX5 knockout mouse embryonic stem cell Line
The Ddx5f/f mouse embryonic stem cell (mESC) line was derived from blastocysts obtained by mating Ddx5f/f male and female mice. To induce DDX5 deletion, the mESCs were transduced with a Cre recombinase-expressing vector, leading to the excision of floxed Ddx5 alleles (Supplementary Fig. 6a).

EdU incorporation assay
Mouse embryonic stem cells (mESCs) were incubated in culture medium containing 10 μM EdU for 40 minutes at 37 °C in a 5% CO₂ incubator. After incubation, cells were harvested using 0.05% Trypsin-EDTA, pelleted by centrifugation, and fixed in 4% PFA for 15 minutes at room temperature. Cells were then washed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15 minutes at room temperature. After additional PBS washes, cells were incubated with the Click-iT™ reaction cocktail in the dark at room temperature for 30 minutes. Nuclei were stained with Hoechst 33342, and after a final PBS wash, samples were analyzed by flow cytometry (Beckman) to assess EdU incorporation.

ATAC-seq
ATAC-seq libraries were prepared using the Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme) according to the manufacturer’s instructions. Briefly, oocytes were lysed to release chromatin, and accessible chromatin regions were simultaneously fragmented and tagged using Tn5 transposase. The resulting DNA fragments were purified using ATAC DNA Extract Beads, followed by PCR amplification and library cleanup. Two biological replicates were prepared for each group. Sequencing was performed on an Illumina NovaSeq platform (150 bp paired-end reads) by Nanjing Jiangbei New District Biomedicine Public Service Platform Co., Ltd.

Total RNA-seq for oocytes
Oocytes at distinct stages were collected for total RNA-seq. Specifically, EAF GV oocytes were isolated from 17.5-day-old mice, while LAF GV oocytes were obtained from 22.5-day-old mice. PreF GV oocytes were collected from adult mice following PMSG stimulation. MII oocytes were retrieved from adult mice after sequential PMSG and hCG stimulation and identified by the first polar body. In addition, Total RNA sequencing was also performed on GV oocytes from 3-week-old mice stimulated with PMSG for combined analysis with ATAC-seq. Three biological replicates were prepared for each group. Collected oocytes were lysed in TRIzol reagent (Takara), and ERCC Spike-In RNA (Thermo Fisher Scientific) was also added to the TRIzol reagent before RNA extraction. Total RNA was isolated by chloroform extraction, followed by isopropanol precipitation. To facilitate RNA precipitation, 1/10 volume of 3 M NaAc and 1 µL glycogen were added to the aqueous phase. The RNA pellet was washed twice with 75% ethanol and resuspended in nuclease-free water. Purified RNA was used for library preparation using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) according to the manufacturer's instructions. Briefly, fragmented total RNA was first reverse-transcribed into cDNA, followed by limited-cycle PCR to add Illumina adapters. After purification, ribosomal cDNA was depleted, and the remaining fragments were further amplified using universal primers. Final libraries were purified and sequenced on an Illumina NovaSeq platform (150 bp paired-end reads) at Nanjing Jiangbei New District Biomedicine Public Service Platform Co., Ltd.

Total RNA-seq for mESCs
Total RNA was extracted and libraries were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) (Roche) according to the manufacturer’s instructions. Briefly, rRNA was first removed, followed by RNA fragmentation, reverse transcription, and cDNA amplification. Final libraries were purified and sequenced on an Illumina NovaSeq platform (150 bp paired-end reads) at Nanjing Jiangbei New District Biomedicine Public Service Platform Co., Ltd.

RNA-Seq analysis
Raw sequencing data were processed using Cutadapt (v4.6) to remove sequencing adapters, low-quality reads, and short fragments. The cleaned reads were then aligned to the mouse reference genome (mm10) and ERCC spike-in sequences using HISAT2 (v2.2.1) with the parameters --no-mixed, --no-discordant, and --no-unal. The aligned reads were subsequently filtered for low mapping quality (MAPQ > 20) and sorted using SAMtools (v1.18). Gene-level and ERCC quantification were performed with featureCounts (v2.0.6) using the parameters -p -B -C -t exon -g gene_id. Sample-wise normalization of gene expression levels was conducted using DESeq2 based on ERCC spike-in counts, followed by differential expression analysis to identify genes with a fold change > 2 and adjusted p-value < 0.05. For further refinement of the differentially expressed gene list, gene expression levels (FPKM) were calculated based on counts obtained from DESeq2 (v1.42.1), retaining differential expression genes with FPKM > 1 in at least one sample. Similarly, repeat element quantification and differential expression analysis were performed using featureCounts and DESeq2. Functional enrichment analysis was carried out using clusterProfiler (v4.10.1).

ATAC-Seq analysis
Sequencing adapters, low-quality reads, and short fragments were removed from the raw sequencing data using Cutadapt (v4.6). The cleaned reads were then aligned to the mouse reference genome (mm10) using Bowtie2 (v2.5.3) with the parameters --no-mixed, --no-discordant, --no-unal, and --very-sensitive. Aligned reads were filtered for low mapping quality (MAPQ > 20) and sorted using SAMtools (v1.18). PCR duplicates were removed using Sambamba (v1.0.1). After merging biological replicates, BAM files were converted to BigWig format for visualization using bamCoverage (v3.5.5). Peak calling to identify accessible chromatin regions was performed with MACS2 (v2.2.9.1) with default parameters. Peak annotation was conducted using annotatePeaks.pl from the HOMER suite (v4.11). In addition, ATAC-seq signal intensities across regions of interest were quantified using computeMatrix and multiBigwigSummary from the deepTools package (v3.5.5).

Statistics & Reproducibility
All results were confirmed by at least two independent experiments. When comparing oocytes of different genotypes, other variables, especially mouse age, were strictly controlled. During the experiment, samples were obtained from at least three mice in both the experimental and control groups to minimize individual differences. Sample sizes were chosen based on prior experience and conventions in the field. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Details of statistical analyses, including the specific tests used, exact sample sizes (n), replicates and definitions of error bars, are provided in the figure legends and Source Data file. GraphPad Prism (v8.4.3) was used for plotting and statistical analyses.

Data availability
The raw sequencing data generated in this study have been deposited in the Genome Sequence Archive (GSA) at the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA028435 [https://ngdc.cncb.ac.cn/gsa/browse/CRA028435]. The information of differentially expressed genes (or repeats) and chromatin accessibility regions were provided in the Supplementary Data files. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al “DDX5 orchestras…”\nWang et al. explore the role of DDX5 in oocyte development by designing a mouse model specifically targeting DDX5. The\nresulting mice are completely sterile. In a series of experiments, they describe the phenotypic alterations observed in DDX5-\ndeficient oocytes and conduct further investigations to uncover the mechanisms underlying this sterility. DDX5 is found to\ninteract directly with RNA polymerase II and with a substantial proportion of retrotransposons, and other RNAs, and to\ninfluence nuclear mRNA export and MARDO assembly. The characterization of oocyte development, along with most\nmolecular analyses, is conducted with high quality, yielding intriguing results. The impact of DDX5 targeting on the\nchromatin landscape, as well as the differences between oocytes and mouse embryonic stem cells (mESCs), is particularly\nfascinating and relevant, especially in light of various epigenetic profiling studies also distinguishing the oocyte from any\nother cells. However, there are some drawbacks to consider. As DDX5 is a known RNA helicase, the results do not clarify\nwhether the observed (molecular) phenotypes are associated with other activities of DDX5 or provide any mechanistic\ninsights.\nMinor Comments:\nThe generation of targeted mice, verification of the knockout, and characterization of oocyte maturation are all carried out\nextremely well and are quite interesting. Did the authors verify the normal expression of DDX5 in neighbouring ovarian\ntissues or cells, such as follicular and cumulus cells?\nThe authors conclude that DDX5 interacts directly with RNA polymerase II. However, it is unclear whether the assays\nconducted can distinguish between direct interaction with RNA polymerase II and mere co-localization due to DDX5’s\ninteraction with a broad range of RNAs (or other RNA binding proteins). The manuscript uses various terms for direct\ninteraction, including direct binding, physical interaction, direct association, and direct functional link. I assume these terms\nare intended to convey the same meaning.\nWhile most genes exhibit downregulation, there are also numerous upregulated genes. I assume the expression of\nindividual genes was normalized to the total amount of RNA?\nDDX5 has been previously shown to be an RNA helicase and to interact with several other proteins, including various RNA\nbinding and RNA modifying enzymes. It may be beneficial for the authors to discuss these findings and their potential\nrelevance to the molecular phenotypes observed.\nOverall, the phenotypic and molecular characterization of DDX5-targeted oocytes is impressive and presents notable data.", "reviewer_2": "This manuscript presents a comprehensive investigation into the critical role of the RNA helicase DDX5 in mammalian\noocyte development. The authors employ a combination of in vivo models, high-throughput sequencing (ATAC-seq, RNA-\nseq, RIP-seq), and detailed cellular and molecular assays to demonstrate that DDX5 is a master regulator essential for\nfemale fertility in mice. The identification of DDX5 as a crucial factor in clearing retrotransposon-derived RNAs (LTR, LINE)\nis especially interesting because this might be a vital quality control mechanism to maintain transcriptome integrity and\ngenomic stability during oogenesis. Other interesting findings include DDX5 loss disrupts nuclear mRNA export,\nmitochondrial organization, and the assembly/function of the mitochondria-associated ribonucleoprotein domain (MARDO),\nleading to premature translation and mRNA depletion. The work contain some novel findings and will be of significant\ninterest to the fields of reproductive biology, RNA biology, and developmental genetics. However, there are several\nquestions and points that the authors need to address before the manuscript is ready for publication:\n1. How does DDX5 mediate retrotransposon RNA degradation? This is an interesting and important finding in this study but\nhas not been clearly determined. Although the authors showed some downregulated repeats in WT oocyte overlap with the\nupregulated repeats in Ddx5 mutant, the effect of degradation could be indirectly. In Fig 6f, the author showed RIP-seq\nresults of DDX5 direct binding sites/transcripts. Are these DDX5-bound transcripts downregulated in the Ddx5 mutant? This\nmight tell us whether DDX5 is likely to be a direct participant of retrotransposon RNA degradation.\n2. In Fig. 3c, 16.8% and 15.8% seemed mislabeled.\n3. The authors described the cKO ovaries exhibited significant growth detardation by 3 weeks of age in Fig 1d-f. In the same\nfigures, the authors also showed week 6 data but the PreF follicle number seemed similar. Could the authors explain why\nthe PreF returned to the control level in the mutant?"}, "author_response": "Point-by-point Responses\nWe sincerely thank both reviewers for their constructive comments on our\nmanuscript. Their feedback has been invaluable in improving the paper's quality. All\nauthors have carefully discussed each comment. In response to the reviewers'\nsuggestions, we have revised the manuscript to meet the standards of Nature\nCommunications. Point-by-point responses to each comment are provided below. To\nbest address the comments, we have organized all new data into Response Figures R1–\nR4, most of which have been incorporated into the revised manuscript. Most concerns\nhave been fully addressed through new experiments, re-analysis, figure revision, and\nmanuscript text updates.\nReviewer #1:\nWang et al. explore the role of DDX5 in oocyte development by designing a mouse\nmodel specifically targeting DDX5. The resulting mice are completely sterile. In a\nseries of experiments, they describe the phenotypic alterations observed in DDX5-\ndeficient oocytes and conduct further investigations to uncover the mechanisms\nunderlying this sterility. DDX5 is found to interact directly with RNA polymerase II\nand with a substantial proportion of retrotransposons, and other RNAs, and to influence\nnuclear mRNA export and MARDO assembly. The characterization of oocyte\ndevelopment, along with most molecular analyses, is conducted with high quality,\nyielding intriguing results. The impact of DDX5 targeting on the chromatin landscape,\nas well as the differences between oocytes and mouse embryonic stem cells (mESCs),\nare particularly fascinating and relevant, especially in light of various epigenetic\nprofiling studies that also distinguish the other cells. However, there are some\ndrawbacks to consider. As DDX5 is a known RNA helicase, the results do not clarify\nwhether the observed (molecular) phenotypes are associated with other activities of\nDDX5 or provide any mechanistic insights. Overall, the phenotypic and molecular\ncharacterization of DDX5-targeted oocytes is impressive and presents notable data.\nMinor Comments:\n1. The generation of targeted mice, verification of the knockout, and characterization\nof oocyte maturation are all carried out extremely well and are quite interesting. Did\nthe authors verify the normal expression of DDX5 in neighbouring ovarian tissues or\ncells, such as follicular and cumulus cells?\nWe appreciate the reviewer's attention to the specificity of our model. Analysis of\npublic scRNA-seq data from wild-type ovaries (GSE294534) confirmed that DDX5 is\nexpressed in oocytes as well as in other ovarian cell types1, including granulosa cells,\ntheca cells, and stromal cells (Fig. R1a). Therefore, it was essential to verify whether\nDDX5 expression remains normal in neighboring cell types in our cKO mice.\nTo test this, we isolated follicles (with oocytes removed) from both cKO and\ncontrol mice and performed qPCR analysis. The results showed comparable DDX5\nexpression levels in the Ddx5f/f and Ddx5f/f; Gdf9-Cre females (Fig. R1b),\ndemonstrating that our DDX5 KO model is highly oocyte-specific. This conclusion is\nfurther supported by our IHC results presented in the main Figure. 1b (Fig. R1c).\na. Violin plot depicting DDX5 expression across distinct ovarian cell types.\nb. Relative Ddx5 mRNA levels in oocyte-depleted follicles from control (Ddx5f/f) and cKO\n(Ddx5f/f; Gdf9-Cre) mice, measured by qPCR using two independent primer sets targeting Ddx5.\nData are presented as mean ± SEM.\nc. Immunohistochemical detection of DDX5 protein in ovarian sections. Scale bar, 10 µm.\n2. The authors conclude that DDX5 interacts directly with RNA polymerase II.\nHowever, it is unclear whether the assays conducted can distinguish between direct\ninteraction with RNA polymerase II and mere co-localization due to DDX5’s\ninteraction with a broad range of RNAs (or other RNA binding proteins). The\nmanuscript uses various terms for direct interaction, including direct binding, physical\ninteraction, direct association, and direct functional link. I assume these terms are\nintended to convey the same meaning.\nWe thank the reviewer for raising this critical point. We agree that relying on PLA\nexperiments and protein co-localization to claim a direct interaction between DDX5\nand Pol II is insufficient. Accordingly, we have thoroughly revised the manuscript and\nremoved all the inappropriate statements.\nTo further clarify the interaction between DDX5 and Pol II, we performed co-\nimmunoprecipitation (co-IP) experiments in HEK293T cells co-overexpressing HA-\ntagged DDX5 and FLAG-tagged Pol II (using the largest subunit, RPB1). The results\nconfirm that DDX5 and Pol II do interact and RNA is essential for this interaction (Fig.\nR2a-b).\nIn summary, we provide additional evidence supporting an RNA-dependent\ninteraction between DDX5 and RNA polymerase II, offering stronger mechanistic\ninsight into the role of DDX5 in transcriptional regulation. We have integrated the co-\nIP results into Supplementary Fig. 2e-f of the revised manuscript.\na. Co-IP (using anti-HA) and Western blot analysis (for HA and Flag) of HA-DDX5 and Flag-Pol\nII from HEK293T cells treated with (+) or without (-) RNase. Input and IgG-IP controls are\nshown.\nb. Co-IP (using anti-FLAG) and Western blot analysis (for HA and Flag) of HA-DDX5 and Flag-\nPol II from HEK293T cells treated with (+) or without (-) RNase. Input and IgG-IP controls are\nshown.\n3. While most genes exhibit downregulation, there are also numerous upregulated\ngenes. I assume the expression of individual genes was normalized to the total amount\nof RNA?\nYes. All gene expression values were normalized to ERCC-based total RNA\namounts before differential expression analysis.\n4. DDX5 has been previously shown to be an RNA helicase and to interact with\nseveral other proteins, including various RNA binding and RNA modifying enzymes.\nIt may be beneficial for the authors to discuss these findings and their potential\nrelevance to the molecular phenotypes observed.\nWe thank the reviewer for this insightful suggestion. We fully agree that a\ndiscussion of DDX5’s established role as an RNA helicase and its broader interaction\nnetwork is essential for contextualizing our findings. Accordingly, we have expanded\nthe Discussion section of our revised manuscript to address this point.\nDDX5 is a well-characterized RNA helicase known to associate with a wide array\nof RNA-binding and RNA-modifying proteins. Integrating these prior observations\nwith our current results, we propose a mechanistic model wherein DDX5 contributes\nto retrotransposon RNA clearance in oocytes. Our analysis of public IP-MS data from\novarian tissue revealed that DDX5 interacts with several components of the RNA\nexosome complex2, including core subunits and cofactors such as MTR4, DIS3, LRP1,\nZC3H18, and SRRT, suggesting that DDX5 may function upstream of the exosome\npathway. We hypothesize that DDX5 leverages its helicase activity to unwind\nstructured retrotransposon transcripts, thereby “priming” them for subsequent\ndegradation by the exosome, akin to the established role of MTR4. This model is further\nsupported by published datasets demonstrating that loss of the RNase-active subunits\nEXOSC10 or DIS3 results in the accumulation of 590 retrotransposon RNAs (Fig.\nR3a)3,4. Notably, 119 of the 155 retrotransposons upregulated upon DDX5 depletion\noverlap with this set (Fig. R3b), strongly suggesting a shared target pool reliant on both\nDDX5 and the exosome machinery for degradation.\nTaken together, these findings support a working hypothesis in which DDX5\nfunctions as a critical upstream auxiliary factor that facilitates exosome-mediated\nretrotransposon RNA decay. We have incorporated this expanded discussion into the\nrevised manuscript to better contextualize the molecular phenotypes observed and to\noffer a testable framework for future mechanistic investigations.\na. Pie Chart showing the distribution of upregulated repeats in EXOSC10 or DIS3-deficient\noocytes across different repeat families.\nb. Venn diagram showing the overlap between upregulated repeats in EXOSC10 or DIS3-deficient\noocytes and upregulated repeats in DDX5-deficient oocytes. P-values were calculated using a\nhypergeometric test.\nReviewer #2:\nThis manuscript presents a comprehensive investigation into the critical role of the\nRNA helicase DDX5 in mammalian oocyte development. The authors employ a\ncombination of in vivo models, high-throughput sequencing (ATAC-seq, RNA-seq,\nRIP-seq), and detailed cellular and molecular assays to demonstrate that DDX5 is a\nmaster regulator essential for female fertility in mice. The identification of DDX5 as a\ncrucial factor in clearing retrotransposon-derived RNAs (LTR, LINE) is especially\ninteresting because this might be a vital quality control mechanism to maintain\ntranscriptome integrity and genomic stability during oogenesis. Other interesting\nfindings include DDX5 loss disrupts nuclear mRNA export, mitochondrial organization,\nand the assembly/function of the mitochondria-associated ribonucleoprotein domain\n(MARDO), leading to premature translation and mRNA depletion. The work contains\nsome novel findings and will be of significant interest to the fields of reproductive\nbiology, RNA biology, and developmental genetics. However, there are several\nquestions and points that the authors need to address before the manuscript is ready for\npublication:\n1. How does DDX5 mediate retrotransposon RNA degradation? This is an interesting\nand important finding in this study but has not been clearly determined. Although the\nauthors showed some downregulated repeats in WT oocyte overlap with the\nupregulated repeats in Ddx5 mutant, the effect of degradation could be indirectly.\nIn Fig. 6f, the author showed RIP-seq results of DDX5 direct binding sites/transcripts.\nAre these DDX5-bound transcripts upregulated in the Ddx5 mutant? This might tell us\nwhether DDX5 is likely to be a direct participant of retrotransposon RNA degradation.\nWe thank the reviewer for this important question. To directly assess whether\nDDX5 promotes retrotransposon RNA degradation, we focused on repeats that were\nphysically bound by DDX5, as identified in our RIP-seq data. Upon DDX5 depletion,\nthese DDX5-bound repeat RNAs exhibited consistent and significant upregulation in\nKO PreF GV oocytes, a trend that persisted through the MII stage (Fig. R4a). These\nresults provide compelling evidence that DDX5 directly binds and facilitates the\ndegradation of specific retrotransposon RNAs.\nAlthough the oocyte system imposes certain limitations on mechanistic dissection,\nour analysis of public IP-MS data from ovarian tissue yielded valuable insights1: DDX5\ninteracts with multiple components of the RNA exosome, including core subunits and\ncofactors such as MTR4, DIS3, LRP1, ZC3H18, and SRRT. This interaction network\nsuggests that DDX5 may function upstream of the exosome, leveraging its helicase\nactivity to resolve secondary structures in retrotransposon RNAs and thereby \"prime\"\nthem for exosome-mediated degradation—mirroring the known role of MTR4.\nSupporting this model, published datasets demonstrate that loss of the RNase-active\nexosome subunits EXOSC10 or DIS3 leads to the accumulation of 590 retrotransposon\nRNAs (Fig. R4b)3,4. Notably, 119 of the 155 retrotransposons upregulated upon DDX5\ndepletion overlap with this exosome-sensitive set (Fig. R4c), strongly suggesting a\nshared pool of targets that depend on both DDX5 and the exosome machinery for\nclearance.\nTogether, these findings support a model in which DDX5 acts as a critical\nupstream auxiliary factor that facilitates exosome-dependent degradation of\nretrotransposon RNAs. We have integrated these new results into Fig. 6g and\nSupplementary Fig. 4g–h of the revised manuscript and incorporated the proposed\nhypothesis into the Discussion section to provide a mechanistic framework for future\ninvestigation.\na. Box plots comparing the relative expression levels of DDX5-bound repeats (identified by RIP-\nseq) in oocytes from the indicated genotypes and developmental stages (PreF GV and MII). ns,\nnot significant; *P < 0.05, **P < 0.01; two-tailed unpaired Student’s t-test.\nb. Pie Chart showing the distribution of upregulated repeats in EXOSC10 or DIS3-deficient\noocytes across different repeat families.\nc. Venn diagram showing the overlap between upregulated repeats in EXOSC10 or DIS3-deficient\noocytes and upregulated repeats in DDX5-deficient oocytes. P-values were calculated using a\nhypergeometric test.\n2. In Fig. 3d, 16.8% and 15.8% seemed mislabeled.\nWe sincerely apologize for the confusion caused by our labeling on the figure. In\nthe original Fig. 3d, the y‑axis represents the number of peaks, and the percentages\nindicate the proportion of each of the three peak categories within the respective\ngenotype. The percentages did not sum to 100% because distal peaks located farther\nthan 100 kb were excluded from that display.\nTo avoid misunderstanding and to present the data more clearly, we have now\nrecalculated the percentages using peaks <100 kb as the denominator. The distribution\nof peaks across categories is now shown separately for improved readability (Fig. R5a-\nb). We believe these adjustments make the presentation more accurate and hope the\nrevised figures can resolve the concern.\nWe have integrated the new figures into Fig. 3d-e of the revised manuscript.\na. Number of ATAC-seq peaks in different genomic regions.\nb. Proportions of ATAC-seq peaks located in different genomic regions.\n3. The authors described the cKO ovaries exhibited significant growth detardation by\n3 weeks of age in Fig 1d-f. In the same figures, the authors also showed week 6 data\nbut the PreF follicle number seemed similar. Could the authors explain why the PreF\nreturned to the control level in the mutant?\nWe appreciate the reviewer’s thoughtful observation. DDX5 is highly expressed\nin transcriptionally active NSN-stage GV oocytes, and its loss likely perturbs early\ntranscriptional programs as well as the accumulation of maternal mRNAs and proteins.\nThese early molecular defects impair oocyte competence during the initial wave of\nfollicular activation, leading to impaired ovarian growth observed at 3 weeks of age.\nRegarding the observation that PreF follicle numbers appear comparable to\ncontrols at 6 weeks, we speculate this reflects a delayed compensatory response.\nNotably, we observed marked upregulation of Ddx17, a functional homolog of Ddx5,\nin KO oocytes (Fig. R6), which may partially compensate for DDX5 deficiency and\nsupport follicular development at later stages. Additional adaptive mechanisms—such\nas the activation of other RNA-binding proteins, induction of stress-response pathways,\nor improved endocrine support during adulthood—may also contribute to this partial\nphenotypic recovery. These possibilities merit further investigation.\nImportantly, despite the apparent recovery in PreF follicle numbers, oocyte\ndevelopal competence remained severely compromised as shown in Fig. 2. This\nfinding suggests that the observed compensation may be incomplete and underscores\nthe unique and non-redundant role of DDX5 in ensuring oocyte quality and\ndevelopmental potential.\nRelative Ddx17 expression levels in oocytes from 3‑week‑old Ddx5f/f and Ddx5f/f;Gdf9‑Cre\nmice. Data are presented as mean ± SEM derived from RNA‑seq analysis. ***P < 0.001; two-\ntailed unpaired Welch’s t-test.\nReference\nBiol 23, e3003193 (2025). https://doi.org/10.1371/journal.pbio.3003193\nof microfilament-associated protein radixin. Theriogenology 240, 117401 (2025).\nhttps://doi.org/10.1016/j.theriogenology.2025.117401\ntranscription in exosome RNase-depleted oocytes. Cell Rep 42, 113247 (2023).\nhttps://doi.org/10.1016/j.celrep.2023.113247\ntransition in mouse oocytes. Nucleic Acids Res 48, 5349-5365 (2020).\nhttps://doi.org/10.1093/nar/gkaa249"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed the reviewers' concerns, providing clear revisions in the text and discussion and\nthey have also added substantial new experimental data where. I therefore recommend acceptance for publication.", "reviewer_2": "The authors have addressed my questions and concerns,and I think the munuscript is ready for publication."}, "author_response": ""}]

Decision letter