{"paper_id":"3f86ce52-81cd-4d6e-b4c0-eebbe84189f5","body_text":"Stella safeguards proteostasis via regulating UBE2D3 translation in the maternal-to-zygotic transition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stella safeguards proteostasis via regulating UBE2D3 translation in the maternal-to-zygotic transition Guorui Zhang, Minjie Ren, Guangyi Sun, Yilong Zhao, Na Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8687106/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Ubiquitin-proteasome-mediated protein degradation is essential for maternal-to-zygotic transition, yet the maternal factors that safeguard ubiquitin-proteasome function remain unclear. Here, using a Stella loss-of-function mouse model, we demonstrate that Stella is required for global protein ubiquitination during early embryogenesis. Stella deficiency in oocytes leads to reduced ubiquitinated protein levels, impaired zygotic genome activation, and early developmental arrest. Proteomic and functional analysis identify UBE2D3 as the key E2 enzyme whose insufficiency underlies the ubiquitination defect. Mechanistically, Stella preserves YBX3 stability, thereby enabling YBX3-dependent translation of Ube2d3 mRNA. Consequently, Stella depletion destabilizes YBX3, diminishes UBE2D3 synthesis, and disrupts protein ubiquitination. Notably, overexpression of Ybx3 or Ube2d3 partially restores ubiquitinated protein levels, zygotic genome activation, and embryo development. These findings identify a previously unrecognized Stella-YBX3-UBE2D3 axis that ensures efficient maternal protein ubiquitination and zygotic genome activation, revealing a cytoplasmic mechanism by which maternal Stella governs proteostasis during maternal-to-zygotic transition. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Early embryonic development is a complex and tightly regulated process initially driven by maternally inherited RNA and protein stored in oocytes[1]. As development proceeds, these maternal reserves are degraded and replaced by embryonically synthesized molecules. The timely degradation of maternal RNAs and proteins, together with the activation of zygotic genome (ZGA), are essential for establishing totipotency during the maternal-to-zygotic transition (MZT) [2, 3]. Although the maternal factors that regulate maternal mRNA decay are relatively well characterized [4], the factors governing maternal protein degradation remain poorly understood. Protein clearance is thought to be more selective than mRNA turnover and follows precise, protein-specific temporal regulation. This process is primarily mediated by the ubiquitin-proteasome system (UPS), and disruption of this degradation results in developmental arrest or severe delays during early cleavage stages[5–7]. Despite its essential role in the MZT, the maternal factors that orchestrate UPS-dependent proteolysis have remained largely unknown. Stella, also known as PGC7 or Dppa3, is a maternal factor indispensable for early development. Stella-deficient females are infertile due to developmental arrest before implantation[8]. Extensive studies have established Stella as a key epigenetic safeguard that preserves DNA methylation integrity in both gametes and embryos. During oogenesis, Stella prevents aberrant de novo methylation by sequestering DNMT1 and UHRF1 in the cytoplasm[9, 10], whereas after fertilization, it protects the maternal pronucleus from TET3-mediated 5mC to 5hmC oxidation through binding to H3K9me2-marked chromatin[11, 12]. In Stella-deficient zygotes, the maternal genome exhibits excessive 5hmC accumulation and γH2AX enrichment, leading to chromosomal instability and embryonic arrest[13]. Moreover, hypermethylation in Stella-deficient oocytes persists into the two-cell stage and disrupts ZGA[9], and the activation of endogenous retroviruses (ERVs), a hallmark of MZT, is markedly reduced[13]. While these findings firmly define Stella’s role in safeguarding the epigenome, the observation that embryos derived from wild-type spindle/knockout cytoplasm oocytes show markedly reduced developmental competence[9] points to an additional, yet poorly defined, cytoplasmic role of Stella. Mass spectrometry-based proteomic analysis has identified 291 Stella-interacting proteins enriched in pathways related to translation, RNA processing, and cell cycle regulation[14], suggesting that Stella may exert various cellular functions through specific protein-protein interactions. Consistently, Stella has been reported to interact with UHRF1 and Nanog, thereby protecting them from proteasome-mediated degradation and supporting the self-renewal of embryonic stem cells[15]. The interaction between Stella and UHRF1 also occurs in oocytes, where the loss of UHRF1 in Stella knockout oocytes can be rescued by the proteasome inhibitor MG132[9]. Furthermore, cytoplasmic proteasomal cleavage of Stella has been implicated in intracellular trafficking and early embryo development in mice[16]. Despite these insights, it remains unclear whether Stella modulates the function of the UPS during the MZT. Here, using a Stella loss-of-function mouse model, we demonstrate that Stella is required for global ubiquitination during early embryogenesis. By integrating proteomics and functional analysis, we identify a translational regulatory circuit in which Stella stabilizes YBX3 to promote Ube2d3 translation and maintain the ubiquitination landscape essential for ZGA and early development. These findings uncover a previously unrecognized cytoplasmic pathway through which maternal Stella controls developmental competence during MZT. RESULTS Impaired Protein ubiquitination and zygotic gene activation in Stella-deficient embryos Maternal protein clearance and zygotic genome activation are essential events governing early embryogenesis[17], yet Stella’s role in these processes remains unresolved. To address this, we generated Stella mutant mice using CRISPR/Cas9. The resulting allele carries a 153-bp deletion in exon 2 that removes 51 amino acids (hereafter referred to as Stella △ mice; Fig. S1 A), and loss of full-length Stella was confirmed by western blotting (Fig. S1 B). Embryos derived from Stella △ oocytes exhibited severely impaired preimplantation development and rarely progressed to the blastocyst stage, consistent with previous reports[8] (Fig. S1 C-D). Ubiquitin-mediated protein degradation, triggered by substrate-specific ubiquitination, is a major mechanism driving maternal protein clearance[18]. To monitor ubiquitination dynamics, we collected oocytes at the GV and MII stages, as well as embryos at defined hours post-fertilization (hpf). As shown in Fig. 1 A, ubiquitinated proteins accumulated progressively from GV to MII stages, peaked at 9-hpf zygotes, and declined thereafter. To investigate whether this process is perturbed in the absence of Stella, we examined MII oocytes and 9-hpf zygotes from Stella △ mice and found markedly reduced ubiquitinated protein levels compared with WT (Fig. 1 B). Treatment with the proteasome inhibitor MG132 increased ubiquitinated protein levels in both WT and Stella △ oocytes; however, Stella △ oocytes still exhibited substantially lower levels (Fig. 1 C), and proteasome activity was comparable between genotypes (Fig. 1 D), indicating that the defect originates from impaired ubiquitination rather than enhanced proteasome-mediated degradation. Given that maternal protein clearance is prerequisite for ZGA, we assessed nascent transcription using EU incorporation assays. Stella △ two-cell embryos exhibited markedly reduced transcription compared with WT, indicative of defective ZGA (Fig. 1 E-F). Together, these results indicate that Stella loss compromises protein ubiquitination and ZGA, which likely contribute to the developmental arrest observed in Stella-deficient embryos. Downregulation of ubiquitination enzymes in Stella △ oocytes To identify factors responsible for the reduced ubiquitination in Stella △ oocytes, we performed liquid chromatography-tandem mass spectrometry (LC-MS) analysis on MII oocytes from WT and Stella △ mice (Fig. 2 A). Among the 6,185 proteins detected, 450 proteins were differentially expressed (fold change > 1.5 and P < 0.05; Supplementary Table 1), with 426 upregulated and 24 downregulated in Stella △ oocytes (Fig. 2 B). The overall protein upregulation is consistent with impaired ubiquitin-mediated turnover in Stella △ oocytes. Because Stella has been reported to interact with and stabilize specific proteins by preventing their degradation[15], we reasoned that the proteins that normally interact with Stella might be decreased in its absence. Indeed, two known Stella interactors, UHRF1[15] and RanBP5[12], were significantly reduced in Stella △ oocytes (Fig. 2 C). We therefore focused on the 24 downregulated proteins, and identified two ubiquitination-related enzymes of particular interest: UHRF1, an E3 ligase essential for embryogenesis[19], and UBE2D3, an E2 conjugating enzyme previously implicated in protein ubiquitination during oocyte maturation[20] (Fig. 2 C). Western blotting further confirmed an ~ 70% reduction in UBE2D3 and a 60% reduction in UHRF1 in Stella △ oocytes compared to WT oocytes (Fig. 2 D-E). Together, our findings identify two downregulated ubiquitinating enzymes, UHRF1 and UBE2D3, providing strong candidates for dissecting how maternal Stella regulates protein ubiquitination in oocytes. UBE2D3 is required for protein ubiquitination and early embryo development To assess the contribution of UHRF1 and UBE2D3 to global protein ubiquitination in early embryos, we performed siRNA-mediated knockdown in MII oocytes prior to IVF (Fig. 3 A). UHRF1 knockdown did not alter ubiquitination in early embryos, indicating that its role in embryogenesis is independent of global ubiquitin-mediated degradation (Fig. S2 ). In contrast, UBE2D3 knockdown resulted in a marked reduction in global ubiquitination (Fig. 3 B-C). We next examined the developmental potential of UBE2D3-KD embryos. Approximately 50% of UBE2D3-KD embryos arrested at the 4-cell stage, and only ~ 25% reached the blastocyst stage, compared with 83% in controls (Fig. 3 D-E). EU incorporation assays further showed a pronounced decrease in nascent RNA synthesis in 2-cell UBE2D3-KD embryos (Fig. 3 F-G), indicating compromised zygotic genome activation. These findings identify UBE2D3 as a key E2 conjugating enzyme required for robust ubiquitination and ZGA during early embryogenesis, positioning it as a potential downstream effector of maternal Stella. Stella modulates protein ubiquitination through UBE2D3 To determine whether Stella directly regulates protein ubiquitination via UBE2D3, we manipulated Stella expression in transcriptionally silent GV oocytes and analyzed protein ubiquitination with or without UBE2D3 (Fig. 4 A). The efficiencies of endogenous Stella knockdown via siRNA injection and exogenous Stella overexpression via mRNA injection were confirmed by immunoblotting (Fig. 4 B). Immunoblotting analysis revealed that global protein ubiquitination was markedly decreased upon Stella knockdown, whereas Stella overexpression led to a pronounced increase (Fig. 4 C-D). Importantly, depletion of UBE2D3 markedly blunted the ubiquitination increase induced by Stella overexpression (Fig. 4 E-F), suggesting that UBE2D3 acts downstream of Stella in this regulatory pathway. Consistently, manipulating Stella expression altered UBE2D3 protein abundance: Stella knockdown significantly decreased UBE2D3 protein levels (Fig. 4 G), whereas Stella overexpression increased them (Fig. 4 H). Together, these findings establish UBE2D3 as the key downstream effector through which Stella controls global protein ubiquitination. Stella promotes Ube2d3 translation by stabilizing YBX3 Because GV oocytes are transcriptionally quiescent, we next examined whether Stella regulates UBE2D3 protein levels through post-transcriptional mechanisms (Fig. 5 A). To this end, we knocked down Stella in GV oocytes and assessed Ube2d3 mRNA abundance and UBE2D3 protein stability (Fig. 5 B). Stella knockdown did not alter Ube2d3 mRNA levels (Fig. 5 C), nor did it affect UBE2D3 protein turnover (Fig. 5 D), suggesting a translational mechanism. To test this, we constructed an eGFP reporter fused to the Ube2d3 coding sequence and microinjected the in vitro-transcribed mRNAs into control and Stella-KD GV oocytes, together with a mCherry transcript as an internal normalization control (Fig. 5 E). Quantification of eGFP-UBE2D3 and mCherry fluorescence revealed that Stella knockdown markedly reduced the translational output of the eGFP-UBE2D3 reporter (Fig. 5 E-F), demonstrating that Stella promotes Ube2d3 translation. To identify mediators of this regulatory process, we examined Y-box (YBX) family proteins, as YBX1, YBX2, and YBX3 participate in mRNA translation[21], and YBX1 was previously shown to interact with Stella to orchestrate translational regulation in oocytes[22]. We first assessed whether YBX1 contributes to UBE2D3 regulation; however, YBX1 knockdown did not alter UBE2D3 protein abundance (Fig. S3 A-B). In contrast, proteomic profiling revealed a marked reduction of YBX3 in Stella △ oocytes (Fig. 2 C), which we validated by western blotting (Fig. S3 C-D). To determine whether YBX3 regulates UBE2D3 expression, we depleted YBX3 in WT oocytes (Fig. 6 A). YBX3 knockdown led to a significant reduction in UBE2D3 protein levels without affecting Ube2d3 mRNA abundance (Fig. 6 B-D). Moreover, YBX3 depletion markedly impaired translation of both MYC-UBE2D3 and eGFP-UBE2D3 reporters (Fig. 5 E and Fig. 6 E), supporting a role for YBX3 in promoting Ube2d3 translation. In addition, YBX3 depletion also diminished global protein ubiquitination, phenocopying the defects observed in Stella-deficient oocytes (Fig. 6 F). We next investigated whether Stella regulates YBX3 protein stability. To assess this, we inhibited de novo protein synthesis with cycloheximide (CHX) following Stella knockdown and monitored YBX3 degradation over time (Fig. 6 G). In Stella-depleted oocytes, YBX3 levels began to decline rapidly after 3 hours of culture and dropped to ~ 30% of the initial level by 12 hours, compared with ~ 50% remaining in control oocytes (Fig. 6 H). However, Ybx3 mRNA levels were unaffected in Stella-KD oocytes (Fig. 6 I). This pronounced destabilization was attributed to enhanced proteasome-mediated degradation, as treatment of Stella-deficient oocytes with the proteasome inhibitor MG132 restored YBX3 protein abundance (Fig. 6 J). To determine whether Stella acts directly on YBX3, we performed co-immunoprecipitation assays. Stella and YBX3 robustly co-precipitated (Fig. 6 K-L), providing biochemical evidence for a physical interaction between the two proteins. Together, these findings support a model in which Stella binds to YBX3 and protects it from proteasomal degradation, thereby maintaining sufficient YBX3 protein to sustain Ube2d3 mRNA translation. Injection of Ybx3 mRNA or Ube2d3 mRNA rescues ubiquitination and development of Stella-deficient embryos The results described above suggested that Stella regulates early embryo development through YBX3-UBE2D3 pathway. To further confirm the functional relevance of this regulatory axis, MII oocytes derived from Stella △ females were microinjected with Stella , Ybx3 , or Ube2d3 mRNA and subsequently subjected to IVF. After fertilization, protein ubiquitination, embryonic gene transcription, and embryo development were assessed (Fig. 7 A). Immunostaining revealed that injection of either Ybx3 or Ube2d3 mRNA greatly increased global protein ubiquitination in Stella-deficient embryos, with both treatments achieving a greater degree of restoration than Stella mRNA overexpression (Fig. 7 B-C). Moreover, increasing YBX3 expression improved embryonic gene activation to levels comparable to those achieved by Stella overexpression and more effectively than UBE2D3 overexpression (Fig. 7 D-E). Notably, while overexpression of all three genes significantly improved the developmental potential of Stella-deficient embryos, YBX3 overexpression produced the most robust rescue (Fig. 7 F-G). Together, these findings demonstrate that Stella regulates protein ubiquitination and early embryonic development through the YBX3-UBE2D3 axis, and the more effective rescue by YBX3 suggests it may further promote embryogenesis through additional mechanisms beyond ubiquitination. Discussion Our study identifies a previously unrecognized Stella-YBX3-UBE2D3 pathway that ensures efficient maternal protein ubiquitination and zygotic genome activation during early embryogenesis. Through integrated proteomic and functional analysis, we demonstrate the cytoplasmic mechanism by which maternal Stella governs early developmental competence. Stella as a central regulator of maternal protein ubiquitination Stella was one of the first reported mammalian maternal effect genes together with Mater , Zar1 and Npm2 [8, 23–25]. Although Stella is widely expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells, loss of function studies in mice showed that it is dispensable for germ cell specification but essential for normal early development. Stella has been proposed to protect maternal pronuclei from TET3 mediated active DNA demethylation and to safeguard the oocyte methylome by preventing aberrant de novo methylation[9, 12]. However, embryos depleted of maternal effect proteins known to regulate epigenetic typically exhibit developmental defects post-implantation, implying that the mechanisms underlying the regulation of early embryo development by mammalian Stella have not been fully investigated. Maternal protein degradation and zygotic genome activation, occurring during MZT, are the first essential events for the development of preimplantation embryos[5]. UPS-dependent proteolysis is a selective degradation system in which the destruction of proteins is initiated by attaching multiple ubiquitin molecules to the target protein that is subsequently degraded by the 26S proteasome complex. Both proteasome function and ubiquitination are indispensable for successful MZT, and perturbation of either process adversely affects this transition. For example, transient inhibition of proteasome activity with MG132 delays the onset of zygotic transcription in otherwise normally developing 2-cell embryos and even induces developmental arrest at the 1-cell stage[26]. In parallel, ubiquitination pathways are equally critical, as RNF114-mediated ubiquitination and degradation of TAB1 is necessary for MZT[27]. Despite these advances, the upstream maternal factors that ensure UPS function have remained unclear. In this study, we found that ubiquitinated proteins were markedly reduced in Stella △ oocytes and embryos, and this defect stemmed from impaired ubiquitination rather than altered proteasome activity (Fig. 1 ). Proteomic profiling reveled widespread protein accumulation, consistent with impaired ubiquitin-mediated turnover (Fig. 2 ). By manipulating Stella expression in transcriptional silent GV oocytes, we demonstrated directly that Stella is required to sustain protein ubiquitination (Fig. 4 ). Collectively, these findings position Stella as a central maternal-effect factor that preserves UPS function, uncovering a previously unrecognized role for Stella in coordinating the proteolytic landscape of the MZT. A translational regulatory network involving Stella, YBX3, and UBE2D3 Ubiquitination is a tightly regulated process mediated by a three-step enzymatic cascade involving E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Among these, E2 enzymes are central because they determine the type of ubiquitin modification that occurs[28]. For example, K48-linked chains target proteins for proteasomal degradation, whereas the K63-linked ubiquitination serves as a molecular platform for protein/protein interaction[29]. Previously, we showed that UBE2D3, the most abundant E2 transcript in oocytes, is required for global protein ubiquitination in oocytes[20]. UBE2D3 is predicted to be involved in protein K48-linked ubiquitination and ubiquitin-dependent protein catabolic process. In this study, UBE2D3 was significantly downregulated in Stella-deficient oocytes (Fig. 2 ), and its depletion alone recapitulated the ubiquitination defects, impaired ZGA, and early developmental arrest observed in Stella-deficient embryos, establishing UBE2D3 as a key downstream mediator of Stella function (Fig. 3 ). Stella has been reported to interact with and stabilize specific proteins by preventing their proteasomal degradation[15]. However, among the 291 proteins identified as Stella interactors, UBE2D3 was not detected[14]. Consistent with this, loss of Stella did not affect UBE2D3 protein stability but instead markedly suppressed the translational activity of Ube2d3 mRNA (Fig. 5 ). Although Stella was recently reported to associate with the RNA-binding protein YBX1 to regulate mRNA translation, YBX1 knockdown did not alter UBE2D3 abundance (Fig. S3 ). Here, we identify YBX3 as the downstream regulator through which Stella controls Ube2d3 expression. This conclusion is supported by several findings: (1) YBX3 and YBX1 share high sequence homology and overlapping RNA-binding functions; (2) YBX3 directly regulates the translational activity of Ube2d3 mRNA (Fig. 5 E); (3) Stella binds to and stabilizes YBX3 (Fig. 6 K-L); and (4) YBX3 overexpression partially rescues the developmental defects of Stella-deficient embryos. Together, these results delineate the cytoplasmic mechanistic framework by which maternal Stella controls embryo development. Notably, global reductions in translational activity were observed following YBX3 depletion, indicating that YBX3 functions more broadly than solely promoting Ube2d3 translation. Consistent with this, YBX3 overexpression produced a stronger rescue than Ube2d3 overexpression (Fig. 7 ), suggesting that dysregulation of additional YBX3-dependent targets also contributes to the developmental defects of Stella-deficient embryos. In summary, we identify a translational regulatory circuit in which Stella stabilizes YBX3 to promote Ube2d3 translation and maintain the ubiquitination landscape essential for ZGA and early development. This pathway provides a mechanistic framework for how maternal Stella orchestrates proteostasis to safeguard the oocyte-to-embryo transition. MATERIALS AND METHODS Mice All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Welfare and Ethics Committee of Nanjing Medical University. Stella homozygous mutant mice ( Stella △ ) were generated using the CRISPR/Cas9 system as previously described[10]. Two sgRNAs targeting exon 2 of the Stella gene (listed in Supplementary Table 2) were used to introduce a 153-bp deletion, resulting in the loss of 51 amino acids in the Stella protein. Homozygous mutant males were crossed with heterozygous females for subsequent experiments. All mice were maintained in a specific pathogen-free (SPF) facility under a 12 h light/12 h dark cycle at 20–26°C and 40–70% humidity. Antibodies The following antibodies were used in this study: rabbit monoclonal anti-STELLA (Abcam, ab13495), rabbit monoclonal anti-Ubiquitin (Abcam, ab134953), rabbit polyclonal anti-UBE2D3 (Proteintech, 11677-1-AP), rabbit polyclonal anti-DYKDDDDK tag (Proteintech, 20543-1-AP), Zonab polyclonal antibody (Thermo Fisher Scientific, PA5-85619), mouse polyclonal anti-UHRF1 (Santa Cruz Biotechnology, sc-373750), mouse polyclonal anti-α-Tubulin (Proteintech, HRP-60031), mouse polyclonal anti-β-Actin (Proteintech, HRP-60008), mouse monoclonal anti-GAPDH (Beyotime, AF00006), and mouse monoclonal anti-MYC tag (Abcam, ab18185). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Proteintech (SA00001-1 and SA00001-2). Oocyte collection and culture GV oocytes were obtained from the ovarian follicles of adult female mice 46–48 h after pregnant mare serum gonadotropin (PMSG, 5 IU; Ningbo Hormone Product Co.) injection. For in vitro maturation, GV oocytes were cultured in M16 medium (Nanjing Luanchuang Co., China) under mineral oil at 37°C in a 5% CO 2 incubator. For MII oocytes collection, female mice were injected with PMSG, followed 48 h later by human chorionic gonadotropin (hCG, 5 IU; Ningbo Hormone Product Co.) injection. Cumulus-oocyte complexes (COCs) were isolated from oviduct ampulla, and denuded MII oocytes were obtained by removing the cumulus mass in medium containing 0.5 mg/mL hyaluronidase (Sigma) at 37°C. In vitro fertilization and embryo culture IVF assays were performed as previously described[10] with minor modifications. Sperm were collected from the cauda epididymis of adult ICR males (10–20 weeks old) and capacitated for 1 h in HTF medium (Nanjing Luanchuang Co., China) supplemented with 10 mg/mL BSA. Capacitated sperm were then added to HTF drops containing denuded oocytes as described previously[30]. After co-incubation at 37°C, presumptive zygotes were washed to remove excess sperm and cultured in KSOM medium (Nanjing Luanchuang Co., China) under mineral oil at 37°C in a humidified atmosphere of 5% CO₂ until the blastocyst stage. Plasmid Construction and cRNA Synthesis Total RNA was extracted from 50 oocytes using the ArcturusPicoPure RNA Isolation Kit (KIT0204; Applied Biosystems), and cDNA was generated using the Quantitect Reverse Transcription kit (Qiagen; 205311). Plasmid construction and cRNA synthesis were performed as described previously[31]. Purified PCR products were digested with EcoRI and XbaI (NEB) and cloned into the pCS2 + vector with Myc tags, or alternativel digested with NheI and KpnI (NEB) and cloned into the eGFP-tag pEGFP vector. For in vitro cRNA synthesis, plasmids were linearized with NotI and transcribed using the SP6 mMESSAGE mMACHINE kit (AM1340, Thermo Fisher) according to the manufacturer’s instructions. The cRNA was then purified using the RNeasy Micro Kit (74004; Qiagen). The mCherry plasmid (Cat#: D2711) were linearized by XbaI. Capped cRNAs were synthesized with T7 mMESSAGE mMACHINE ULTRA Kit (Ambion, CA, USA). Synthesized cRNA was aliquoted and stored at -80°C. The related primer sequences can be found in Supplementary Table 2. Knockdown and Overexpression Experiments Microinjections were performed using an inverted microscope (Eclipse Ti-S, Nikon) equipped with a micromanipulator (Narishige). siRNAs targeting Stella, Ube2d3, Ybx3, or Ybx1 (GenePharma) were prepared as 20 µM stock solutions. For knockdown, 2.5 pL of 1 mM siRNA or negative control siRNA was injected into GV-stage oocytes. For overexpression, 10 pL of cRNA (10 ng/µL) was microinjected into GV oocytes; PBS injection served as a control. After injection, oocytes were maintained in M2 medium containing 2.5 µM milrinone for 24 h to allow siRNA/cRNA function. For embryonic knockdown or overexpression, 5–10 pL of siRNA or cRNA was injected into MII oocytes before IVF. Zygotes were cultured in KSOM medium under mineral oil at 37°C in 5% CO₂. Related siRNA sequences are provided in Supplementary Table 2. Live-cell imaging For live-cell imaging, mRNAs encoding for eGFP-UBE2D3 and mCherry were microinjected into GV oocytes and released from milrinone after 6 h. Oocytes were washed three times and transferred to a live cell-imaging dish, covered with mineral oil. Fluorescent imaging was performed using a Zeiss Laser Scanning Confocal Microscope (LSM 700; Zeiss). Semiquantitative analysis of fluorescence intensity was carried out using ImageJ (NIH). EU incorporation assays EU incorporation assays were performing using the Click-iT RNA Imaging kits (Invitrogen, C10329). Two-cell embryos were cultured in KSOM medium containing 1 mM EU for 3 h and fixed overnight at 4°C in 4% paraformaldehyde. Embryos were permeabilized with 0.5% Triton X-100 for 15 min, washed, and incubated in the Click-iT reaction mixture for 30–60 min in the dark. Nuclei were stained with Hoechst 33342 (1:1000). Images were acquired using a confocal microscope, and EU signal was quantified by calculating average fluorescence intensity after background subtraction. qRT-PCR analysis Total RNA from oocytes was extracted using the RNeasy Micro Kit (Qiagen). cDNA synthesis and qPCR were performed using the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit (Qiagen) on a QuantStudio™ 7 Real-Time PCR System (Applied Biosystems). Relative mRNA levels were calculated using the 2^-ΔΔCt method with Gapdh as the internal control. Primer sequences are provided in Supplementary Table 2. Western Blotting A total of 80–100 oocytes were lysed in 2× Laemmli sample buffer containing protease inhibitor, and denatured at 95°C for 5 min. Proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (Stella antibody, 1:1,000; Myc antibody, 1:1,000; UBE2D3 antibody, 1:1,000; Ubiquitin antibody, 1:1,000; YBX3 antibody, 1:500). After washes in TBST for three times, the membranes were incubated with HRP-conjugated secondary antibodies. Then, the protein bands were visualized using an ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont, United Kingdom). After washing, membranes were incubated with HRP-conjugated secondary antibodies and visualized using an ECL Plus detection system (GE Healthcare). TUBULIN, GAPDH, or ACTIN served as loading controls where appropriate. Band intensities were quantified using ImageJ. Cell culture, plasmid transfection, and immunoprecipitation. HEK293 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Hyclone) and 1% penicillin-streptomycin at 37°C in 5% CO₂. Cells were seeded in 100-mm dishes and transfected at 50–60% confluence with Stella-Flag and Ybx3-Myc expression plasmids using Lipofectamine 2000 (Invitrogen). Forty-eight hours post-transfection, cells were lysed in buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 0.5% NP-40; protease/phosphatase inhibitors). Lysates were centrifuged at 12,000 × g for 10 min at 4°C and incubated with anti-Flag or anti-Myc magnetic beads for 4 h. After washing, bound proteins were eluted by boiling in SDS sample buffer and analyzed by western blotting. Proteasome activity assay Proteasome activity in oocytes was quantified using a fluorometric proteasome activity assay kit (Abcam, ab107921) according to the manufacturer’s instructions. The assay measures the release of the fluorescent molecule 7-amino-4-methylcoumarin (AMC) from a labeled peptide substrate upon proteolytic cleavage. To distinguish proteasomal activity from non-specific protease activity, 50 µM MG132 was included as an inhibitor control. Oocyte lysates were incubated with the substrate at 37°C in the dark, and fluorescence was recorded at excitation/emission wavelengths of 350/440 nm after 60 and 90 min of incubation. Proteasome activity was calculated from the increase in fluorescence relative to MG132-treated controls using an AMC standard curve, where one unit of activity corresponds to the release of 1.0 nmol AMC per minute at 37°C. Proteomic profiling Proteomic profiling of oocytes was conducted as previously described with minor modifications[32]. For TMT-based quantitative proteomics, MII oocytes (n = 330 per replicate, 3 biological replicates per group) were lysed in a urea buffer (8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, 1%EDTA-free protease inhibitor). After reduction, alkylation, and trypsin digestion, peptides were labeled with the TMT6plex™ kit, mixed equally, and fractionated by high-pH reversed-phase (HP-RP) chromatography on an ACQUITY® UPLC M-class system (BEH C18 column, 300 µm × 150 mm, 1.7 µm; Waters). Peptides from TMT samples were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to a Proxeon Easy-nLC 1200 system. Peptide separation was performed on an analytical C18 column (75 µm × 160 mm, 1.9 µm, Dr. Maisch) with a 95 min linear gradient at 300 nL/min. The Orbitrap was operated at a resolution of 60,000 for MS1 and 15,000 for MS2. Additional MS parameters were as described previously[32, 33]. Raw data were processed with MaxQuant (v1.6.5.0) against the UniProt mouse proteome database. Carbamidomethylation (C) was set as a fixed modification, and oxidation (M) and acetylation (protein N-terminus) as variable modifications. Trypsin/P specificity was applied with up to two missed cleavages. A 1% false discovery rate (FDR) was applied at both peptide and protein levels. For TMT quantification, protein intensities were calculated using the reporter ion MS2 method. Proteins identified with ≥ 1 unique peptide and FDR ≤ 1% were retained for downstream analysis. Statistical Analysis All experiments were performed with at least three biological replicates. Data are presented as mean ± SD unless otherwise indicated. Statistical significance was assessed using Student’s t-test for two-group comparisons or one-way ANOVA for multiple groups (GraphPad Prism 8). A P -value < 0.05 was considered statistically significant. Declarations ACKNOWLEDEGMENTS This work was supported by the National Natural Science Foundation of China (NO. 82271689 to L.H.; NO. 82495190 and 82221005 to QW.), Science Foundation of Jiangsu Province (BK20230058 to L.H.). COMPETING INTERESTS The authors declare no competing interests. AUTHOR CONTRIBUTION L.H. and Q.W. conceived the projects. G.Z., M.R., and G.S. performed the majority of experiments. Y.Z. and N.Z. assisted with animal model construction. L.H. and G.Z. designed the experiments and analyzed data. L.H. and G.Z. wrote the manuscript, and Q.W. revised it. DATA AVAILABILITY All data generated or analyzed during this study are included in this published article and its supplementary information files. References Qi, L., et al., miR-370 is stage-specifically expressed during mouse embryonic development and regulates Dnmt3a. FEBS Lett, 2013. 587(6): p. 775-81. Schier, A.F., The maternal-zygotic transition: death and birth of RNAs. Science, 2007. 316(5823): p. 406-7. Zhou, L.Q. and J. Dean, Reprogramming the genome to totipotency in mouse embryos. Trends Cell Biol, 2015. 25(2): p. 82-91. Yu, C., et al., BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes. Nat Struct Mol Biol, 2016. 23(5): p. 387-94. Higuchi, C., et al., Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development. J Reprod Dev, 2018. 64(1): p. 65-74. Hamazaki, J., et al., Rpn10-mediated degradation of ubiquitinated proteins is essential for mouse development. Mol Cell Biol, 2007. 27(19): p. 6629-38. Sakao, Y., et al., Mouse proteasomal ATPases Psmc3 and Psmc4: genomic organization and gene targeting. Genomics, 2000. 67(1): p. 1-7. Payer, B., et al., Stella is a maternal effect gene required for normal early development in mice. Curr Biol, 2003. 13(23): p. 2110-7. Li, Y., et al., Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1. Nature, 2018. 564(7734): p. 136-140. Han, L., et al., Differential roles of Stella in the modulation of DNA methylation during oocyte and zygotic development. Cell Discov, 2019. 5: p. 9. Nakamura, T., et al., PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature, 2012. 486(7403): p. 415-9. Nakamura, T., et al., PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol, 2007. 9(1): p. 64-71. Nakatani, T., et al., Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation. EMBO Rep, 2015. 16(5): p. 582-9. Liu, H., et al., Comprehensive Proteomic Analysis of PGC7-Interacting Proteins. J Proteome Res, 2017. 16(9): p. 3113-3123. Zhao, S., et al., Dppa3 facilitates self-renewal of embryonic stem cells by stabilization of pluripotent factors. Stem Cell Res Ther, 2022. 13(1): p. 169. Shin, S.W., et al., Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice. Nat Commun, 2017. 8(1): p. 1643. Li, L., P. Zheng, and J. Dean, Maternal control of early mouse development. Development, 2010. 137(6): p. 859-70. Verlhac, M.H., M.E. Terret, and L. Pintard, Control of the oocyte-to-embryo transition by the ubiquitin-proteolytic system in mouse and C. elegans. Curr Opin Cell Biol, 2010. 22(6): p. 758-63. Cao, Y., et al., Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos. Faseb j, 2019. 33(7): p. 8294-8305. Zhang, G., et al., UBE2D3 functions in mouse oocyte meiotic maturation. Faseb j, 2025. 39(3): p. e70375. Mordovkina, D., et al., Y-Box Binding Proteins in mRNP Assembly, Translation, and Stability Control. Biomolecules, 2020. 10(4). Liu, Y., et al., PGC7 regulates maternal mRNA translation via AKT1-YBX1 interactions in mouse oocytes. Cell Commun Signal, 2024. 22(1): p. 604. Wu, X., et al., Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat Genet, 2003. 33(2): p. 187-91. Tong, Z.B., et al., Mater, a maternal effect gene required for early embryonic development in mice. Nat Genet, 2000. 26(3): p. 267-8. Burns, K.H., et al., Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos. Science, 2003. 300(5619): p. 633-6. Shin, S.W., et al., Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression. J Reprod Dev, 2010. 56(6): p. 655-63. Yang, Y., et al., The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition. EMBO Rep, 2017. 18(2): p. 205-216. Stewart, M.D., et al., E2 enzymes: more than just middle men. Cell Res, 2016. 26(4): p. 423-40. Li, L., et al., UBE2V1 governs aging induced protein aggregation and developmental defects in oocytes and embryos. Commun Biol, 2025. 8(1): p. 769. Zhang, Z., et al., Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations. Reproduction, 2019. 158(2): p. 123-133. Zeng, J., et al., SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation. Aging Cell, 2018. 17(4): p. e12789. Li, L., et al., Characterization of Metabolic Patterns in Mouse Oocytes during Meiotic Maturation. Mol Cell, 2020. 80(3): p. 525-540.e9. Cheah, M.A., et al., MBN 2016 Aesthetic Breast Meeting BIA-ALCL Consensus Conference Report. Plast Reconstr Surg, 2018. 142(6): p. 971e-972e. Supplementary Files FigS1.png FigS2.png FigS3.png SupplementaryFigurelegends.pdf SupplementaryTable2.pdf SupplementaryTable1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 11 Mar, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers invited by journal 24 Feb, 2026 Editor assigned by journal 29 Jan, 2026 First submitted to journal 28 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8687106\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":596201073,\"identity\":\"8756c44e-67ab-46aa-88e6-a21c0c1854f0\",\"order_by\":0,\"name\":\"Guorui Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guorui\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":596201074,\"identity\":\"dff6241a-ed7c-4dd8-8732-8d2582a99be1\",\"order_by\":1,\"name\":\"Minjie Ren\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Minjie\",\"middleName\":\"\",\"lastName\":\"Ren\",\"suffix\":\"\"},{\"id\":596201075,\"identity\":\"116897a1-89a5-4fa2-870d-d716e3adeabe\",\"order_by\":2,\"name\":\"Guangyi Sun\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guangyi\",\"middleName\":\"\",\"lastName\":\"Sun\",\"suffix\":\"\"},{\"id\":596201076,\"identity\":\"d79f313a-3c43-4eca-8d6d-2eb1cd4d21d5\",\"order_by\":3,\"name\":\"Yilong Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yilong\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":596201077,\"identity\":\"a264c43d-ad7e-458d-a5f9-de7d564e2a0e\",\"order_by\":4,\"name\":\"Na Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Northern Jiangsu People's Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Na\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":596201078,\"identity\":\"b7fca2f3-fdb2-405a-b28d-5913fff07b00\",\"order_by\":5,\"name\":\"Qiang Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qiang\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":596201079,\"identity\":\"f8917d13-abe5-478d-8672-3ddcdf4f6cbc\",\"order_by\":6,\"name\":\"longsen Han\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYBACA3YGxgcMPBA2kVqYGZgNSNbCJgFjE6fFnJn9WXWBzLbEBvbmbRIMNXcIa7Fs5jG7PYPndmIDz7EyCYZjz4hw2GEetts8IC0SOWYSjA2HidHC/qwYrEX+DdFaGMyYIbbwEK2Fx1gaqMW4jSet2CLhGDFajrc//Mzbc1u2n/3wxhsfaojQAgaMPQwMbCBGApEagOAH8UpHwSgYBaNgBAIAh5EzVOU5cOoAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0009-0000-4878-3997\",\"institution\":\"Nanjing Medical University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"longsen\",\"middleName\":\"\",\"lastName\":\"Han\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-01-24 13:39:24\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8687106/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8687106/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":103515684,\"identity\":\"5a7125f2-8329-454d-8da4-d11983a8a397\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:34\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":352098,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStella deficiency impairs protein ubiquitination and zygotic gene activation in early embryos\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Western blot analysis of ubiquitinated protein levels at different developmental stages (oocyte at the GV and MII stages, and early embryos collected at indicated hours post-fertilization) in wild-type (WT) oocytes/embryos.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot analysis of ubiquitinated proteins in MII oocyte and zygotes from WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Western blot analysis of ubiquitinated proteins in MII stage oocytes from WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice treated with or without the proteasome inhibitor MG132. MII oocytes were collected 12 h after hCG injection and cultured for 4 h in M2 medium supplemented with or without MG132. GAPDH served as a loading control. Protein lysates from 80 oocytes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Proteasome activity in MII oocytes from WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) EU staining showing transcription levels in WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e zygotes. Representative fluorescence images show EU (green) and Hoechst (blue). Scale bar, 20 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Quantification of mean EU signal intensity. Data represent mean ± SD from three independent experiments. Statistical analysis was performed using two-tailed Student’s \\u003cem\\u003et\\u003c/em\\u003e test (n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/916784b574ccda0acd52fcc1.jpg\"},{\"id\":103515707,\"identity\":\"45d8c9da-87b6-4645-a0c2-94f55c3490ad\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:44\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":304953,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIdentification of differentially expressed ubiquitination-related enzymes in Stella-deficient oocytes\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Workflow of the proteomic analysis used to identify differentially expressed proteins (DEPs) between WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e MII oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Circular representation of the proportion of down- and up-regulated proteins.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Heat map of the 24 down-regulated proteins in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△ \\u003c/em\\u003e\\u003c/sup\\u003eoocytes compared with WTs. Proteins reported to interact with Stella or associated with ubiquitination are highlighted in red.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Western blot validation of UBE2D3 and UHRF1 protein levels in WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e MII oocytes. A total of 80 oocytes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Quantification of protein abundance shown in (D). Data represent mean ± SD. Statistical significance was determined using two-tailed Student’s \\u003cem\\u003et\\u003c/em\\u003e test (n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/b4ee0d8c64bbeeb3bbb1b647.jpg\"},{\"id\":103516216,\"identity\":\"3b8343ac-8d6d-4a9c-a548-510154e139bd\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:27:38\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":369119,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eUBE2D3 is required for global protein ubiquitination and zygotic genome activation during early embryogenesis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic of UBE2D3 and UHRF1 knockdown experiments in mouse embryos.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot validation of UBE2D3 knockdown efficiency in zygotes.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Western blot analysis of ubiquitinated proteins in control and UBE2D3-knockdown (UBE2D3-KD) zygotes. TUBULIN served as a loading control. Protein lysates from 80 oocytes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Representative bright-field images of E1.5, E2.5, and E3.5 embryos from control and UBE2D3-KD groups. Scale bar, 100 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Quantification of on-time developmental rates.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Representative EU staining images of zygotes from control and UBE2D3-KD oocytes. Scale bar, 20 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(G) Quantification of mean EU signal intensity in (F). Data represent mean ± SD. Statistical significance for (E) and (G) was determined using two-tailed Student’s \\u003cem\\u003et\\u003c/em\\u003e test (n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/2fdcbe5acc0e304391581e45.jpg\"},{\"id\":103515770,\"identity\":\"cce2f1e9-56f8-42d8-953b-a21e40d9590f\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:55\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":354054,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStella regulates protein ubiquitination via UBE2D3 in oocytes\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic of Stella overexpression (Stella-OE), Stella knockdown (Stella-KD), and UBE2D3 knockdown experiments in GV oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot validation of Stella-KD and Stella-OE efficiency.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Western blot analysis of ubiquitinated proteins in Stella-KD and Stella-OE oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Quantification of relative ubiquitinated protein levels in (C).\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Western blot analysis of ubiquitinated proteins in oocytes injected with \\u003cem\\u003eStella\\u003c/em\\u003e mRNA alone or co-injected with \\u003cem\\u003eUbe2d3\\u003c/em\\u003e siRNA.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Quantification of relative ubiquitinated protein levels.\\u003c/p\\u003e\\n\\u003cp\\u003e(G, H) Western blot analysis of UBE2D3 protein levels in control, Stella-KD, and Stella-OE oocytes. Protein lysates from 80 oocytes were loaded per lane. TUBULIN and ACTIN served as loading controls as appropriate.\\u003c/p\\u003e\\n\\u003cp\\u003eData represent mean ± SD from three biological replicates. Statistical analysis was performed using one-way ANOVA followed by post hoc multiple comparisons for (D) and (F). n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/e4e3a6168b7cea62fd18b9cc.jpg\"},{\"id\":103515810,\"identity\":\"5fa9785b-17cb-47db-838a-afdcf2ed7015\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:25:07\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":379218,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStella regulates \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eUbe2d3\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e mRNA translation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic illustrating potential post-transcriptional mechanisms by which Stella regulates UBE2D3.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Workflow for analyzing \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA and protein protein stability following Stella knockdown. GV oocytes were injected with Stella or control siRNA, maintained in milrinone for 24 h, released from milrinone, treated with CHX, and collected at the indicated time points for analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) qRT-PCR analysis of relative mRNA levels of \\u003cem\\u003eStella\\u003c/em\\u003e and \\u003cem\\u003eUbe2d3\\u003c/em\\u003ein control and Stella-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Western blot analysis of UBE2D3 stability after CHX treatment in control and Stella-KD oocytes (top), with quantification (bottom). Protein lysates from 60 oocytes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Schematic of the translational reporter assay using \\u003cem\\u003eUbe2d3-CDS\\u003c/em\\u003e-eGFP and mCherry reporters in control, Stella-KD, and Ybx3-KD oocytes cultured in milrinone for 6 h (top). Representative fluorescence images are shown (bottom). Scale bar, 100 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Quantification of mean eGFP fluorescence intensity. Data represent mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA followed by multiple comparisons for (F), and two-tailed Student’s \\u003cem\\u003et\\u003c/em\\u003e test for (C). n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/7d291291de3c93ec042bacdc.jpg\"},{\"id\":103516139,\"identity\":\"350b9535-5525-4ba3-8dea-c709289ab4d8\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:26:57\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":462993,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStella interacts with and stabilizes YBX3 protein to regulates\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003e Ube2d3 \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003etranslation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic of YBX3 knockdown assays in GV oocytes for subsequent analysis of mRNA and protein expression, and protein ubiquitination.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot validation of YBX3 knockdown efficiency.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) qRT-PCR analysis of \\u003cem\\u003eYbx3\\u003c/em\\u003e and \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA levels in control and YBX3-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Western blot analysis of UBE2D3 levels in control and YBX3-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Western blot analysis of translation of exogenous\\u003cem\\u003e Myc-Ube2d3\\u003c/em\\u003ecRNA in control and YBX3-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Western blot analysis of ubiquitinated proteins in control and YBX3-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(G) Schematic illustrating analysis of YBX3 protein stability following Stella knockdown.\\u003c/p\\u003e\\n\\u003cp\\u003e(H) Western blot analysis of YBX3 protein levels in control and Stella-KD oocytes at the indicated time points after CHX treatment (top), with quantification (bottom).\\u003c/p\\u003e\\n\\u003cp\\u003e(I) qRT-PCR analysis of \\u003cem\\u003eYbx3\\u003c/em\\u003e mRNA levels in control and Stella-KD oocytes.\\u003c/p\\u003e\\n\\u003cp\\u003e(J) Western blot analysis of YBX3 protein levels in Stella-KD oocytes treated with or without MG132. Protein lysates from 60-80 oocytes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(K-L) Co-immunoprecipitation analysis demonstrating Stella-YBX3 interaction in HEK293 cells expressing tagged proteins.\\u003c/p\\u003e\\n\\u003cp\\u003eData represent mean ± SD from at least three independent experiments. n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/855745cdf357a4fe04fe7cc6.jpg\"},{\"id\":103516896,\"identity\":\"a6da26f2-bc99-4f70-81a8-1315a7891d2f\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:30:39\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":512164,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eYbx3\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e or \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eUbe2d3\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e mRNA injection partially rescues defects observed in Stella-deficient embryos\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic of rescue experiments in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e embryos. MII oocytes from \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e females were microinjected with \\u003cem\\u003eStella\\u003c/em\\u003e, \\u003cem\\u003eYbx3\\u003c/em\\u003e, or \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA, followed by in vitro fertilization and embryo culture.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot analysis of ubiquitinated proteins in WT, control (\\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e), and mRNA rescued groups. TUBULIN served as a loading control. Protein lysates from 80 zygotes were loaded per sample.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Quantification of relative ubiquitinated protein levels in (B).\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Representative images of EU staining in WT, \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e, and mRNA-injected zygotes. Scale bar, 20 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Quantification of mean EU signal intensity in (D).\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Representative bright-field images of E4.5 embryos from WT (n = 52), \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e (n = 47),\\u003cem\\u003e Stella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e+\\u003cem\\u003eStella\\u003c/em\\u003e (n = 39), \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e+\\u003cem\\u003eUbe2d3\\u003c/em\\u003e (n = 43), and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e+\\u003cem\\u003eYbx3\\u003c/em\\u003e (n = 42) groups. Scale bar, 100 μm.\\u003c/p\\u003e\\n\\u003cp\\u003e(G) Quantification of on-time developmental rates in (F).\\u003c/p\\u003e\\n\\u003cp\\u003eData represent mean ± SD. Statistical significance for (C), (E), and (G) was determined using one-way ANOVA followed by multiple comparisons. n.s., not significant; *, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ****, \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"fig7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/a84af41e082bb4994add65b5.jpg\"},{\"id\":103517996,\"identity\":\"26820c8c-a42c-4b5c-8112-02eb32908c09\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:34:58\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3870063,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/c2642c95-dd36-4a52-8c20-cc090435a978.pdf\"},{\"id\":103515682,\"identity\":\"ea117055-3b0e-43a3-bae5-863c9a76353e\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:34\",\"extension\":\"png\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":916975,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigS1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/4908df98b016fce79932b990.png\"},{\"id\":103515569,\"identity\":\"4f67d303-3384-4668-8954-9a97bedfaf82\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:10\",\"extension\":\"png\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":437820,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigS2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/e42a382ad9fc0046fbcae8bf.png\"},{\"id\":103515564,\"identity\":\"71d72ee9-b3d2-4820-bd5e-d446e7e46f1e\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:09\",\"extension\":\"png\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":452500,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigS3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/89e6d5d73ddb1443303d9eaa.png\"},{\"id\":103515674,\"identity\":\"cf277f1d-038c-4d3d-9942-26025808a2e4\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:24:29\",\"extension\":\"pdf\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":149600,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFigurelegends.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/fb2010c3929bc63a42801b92.pdf\"},{\"id\":103516151,\"identity\":\"57da0786-0d9e-41f7-b944-ce3a1ba5655c\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:27:03\",\"extension\":\"pdf\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":153770,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable2.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/c7aed9bfdd6cc0b74102870d.pdf\"},{\"id\":103516421,\"identity\":\"ba0f3759-c324-4976-9a82-bdee0cb51ae4\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:28:28\",\"extension\":\"pdf\",\"order_by\":10,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":377009,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8687106/v1/f53b9ceb52174246c66fae3c.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Stella safeguards proteostasis via regulating UBE2D3 translation in the maternal-to-zygotic transition\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eEarly embryonic development is a complex and tightly regulated process initially driven by maternally inherited RNA and protein stored in oocytes[1]. As development proceeds, these maternal reserves are degraded and replaced by embryonically synthesized molecules. The timely degradation of maternal RNAs and proteins, together with the activation of zygotic genome (ZGA), are essential for establishing totipotency during the maternal-to-zygotic transition (MZT) [2, 3]. Although the maternal factors that regulate maternal mRNA decay are relatively well characterized [4], the factors governing maternal protein degradation remain poorly understood. Protein clearance is thought to be more selective than mRNA turnover and follows precise, protein-specific temporal regulation. This process is primarily mediated by the ubiquitin-proteasome system (UPS), and disruption of this degradation results in developmental arrest or severe delays during early cleavage stages[5\\u0026ndash;7]. Despite its essential role in the MZT, the maternal factors that orchestrate UPS-dependent proteolysis have remained largely unknown.\\u003c/p\\u003e \\u003cp\\u003eStella, also known as PGC7 or Dppa3, is a maternal factor indispensable for early development. Stella-deficient females are infertile due to developmental arrest before implantation[8]. Extensive studies have established Stella as a key epigenetic safeguard that preserves DNA methylation integrity in both gametes and embryos. During oogenesis, Stella prevents aberrant de novo methylation by sequestering DNMT1 and UHRF1 in the cytoplasm[9, 10], whereas after fertilization, it protects the maternal pronucleus from TET3-mediated 5mC to 5hmC oxidation through binding to H3K9me2-marked chromatin[11, 12]. In Stella-deficient zygotes, the maternal genome exhibits excessive 5hmC accumulation and γH2AX enrichment, leading to chromosomal instability and embryonic arrest[13]. Moreover, hypermethylation in Stella-deficient oocytes persists into the two-cell stage and disrupts ZGA[9], and the activation of endogenous retroviruses (ERVs), a hallmark of MZT, is markedly reduced[13]. While these findings firmly define Stella\\u0026rsquo;s role in safeguarding the epigenome, the observation that embryos derived from wild-type spindle/knockout cytoplasm oocytes show markedly reduced developmental competence[9] points to an additional, yet poorly defined, cytoplasmic role of Stella.\\u003c/p\\u003e \\u003cp\\u003eMass spectrometry-based proteomic analysis has identified 291 Stella-interacting proteins enriched in pathways related to translation, RNA processing, and cell cycle regulation[14], suggesting that Stella may exert various cellular functions through specific protein-protein interactions. Consistently, Stella has been reported to interact with UHRF1 and Nanog, thereby protecting them from proteasome-mediated degradation and supporting the self-renewal of embryonic stem cells[15]. The interaction between Stella and UHRF1 also occurs in oocytes, where the loss of UHRF1 in Stella knockout oocytes can be rescued by the proteasome inhibitor MG132[9]. Furthermore, cytoplasmic proteasomal cleavage of Stella has been implicated in intracellular trafficking and early embryo development in mice[16]. Despite these insights, it remains unclear whether Stella modulates the function of the UPS during the MZT.\\u003c/p\\u003e \\u003cp\\u003eHere, using a Stella loss-of-function mouse model, we demonstrate that Stella is required for global ubiquitination during early embryogenesis. By integrating proteomics and functional analysis, we identify a translational regulatory circuit in which Stella stabilizes YBX3 to promote \\u003cem\\u003eUbe2d3\\u003c/em\\u003e translation and maintain the ubiquitination landscape essential for ZGA and early development. These findings uncover a previously unrecognized cytoplasmic pathway through which maternal Stella controls developmental competence during MZT.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImpaired Protein ubiquitination and zygotic gene activation in Stella-deficient embryos\\u003c/h2\\u003e \\u003cp\\u003eMaternal protein clearance and zygotic genome activation are essential events governing early embryogenesis[17], yet Stella\\u0026rsquo;s role in these processes remains unresolved. To address this, we generated Stella mutant mice using CRISPR/Cas9. The resulting allele carries a 153-bp deletion in exon 2 that removes 51 amino acids (hereafter referred to as \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice; Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA), and loss of full-length Stella was confirmed by western blotting (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB). Embryos derived from \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes exhibited severely impaired preimplantation development and rarely progressed to the blastocyst stage, consistent with previous reports[8] (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eC-D).\\u003c/p\\u003e \\u003cp\\u003eUbiquitin-mediated protein degradation, triggered by substrate-specific ubiquitination, is a major mechanism driving maternal protein clearance[18]. To monitor ubiquitination dynamics, we collected oocytes at the GV and MII stages, as well as embryos at defined hours post-fertilization (hpf). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, ubiquitinated proteins accumulated progressively from GV to MII stages, peaked at 9-hpf zygotes, and declined thereafter. To investigate whether this process is perturbed in the absence of Stella, we examined MII oocytes and 9-hpf zygotes from \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice and found markedly reduced ubiquitinated protein levels compared with WT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). Treatment with the proteasome inhibitor MG132 increased ubiquitinated protein levels in both WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes; however, \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes still exhibited substantially lower levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC), and proteasome activity was comparable between genotypes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), indicating that the defect originates from impaired ubiquitination rather than enhanced proteasome-mediated degradation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eGiven that maternal protein clearance is prerequisite for ZGA, we assessed nascent transcription using EU incorporation assays. \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e two-cell embryos exhibited markedly reduced transcription compared with WT, indicative of defective ZGA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE-F). Together, these results indicate that Stella loss compromises protein ubiquitination and ZGA, which likely contribute to the developmental arrest observed in Stella-deficient embryos.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eDownregulation of ubiquitination enzymes in\\u003c/b\\u003e \\u003cb\\u003eStella\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e△\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eoocytes\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo identify factors responsible for the reduced ubiquitination in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes, we performed liquid chromatography-tandem mass spectrometry (LC-MS) analysis on MII oocytes from WT and \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Among the 6,185 proteins detected, 450 proteins were differentially expressed (fold change\\u0026thinsp;\\u0026gt;\\u0026thinsp;1.5 and \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Supplementary Table\\u0026nbsp;1), with 426 upregulated and 24 downregulated in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The overall protein upregulation is consistent with impaired ubiquitin-mediated turnover in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eBecause Stella has been reported to interact with and stabilize specific proteins by preventing their degradation[15], we reasoned that the proteins that normally interact with Stella might be decreased in its absence. Indeed, two known Stella interactors, UHRF1[15] and RanBP5[12], were significantly reduced in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). We therefore focused on the 24 downregulated proteins, and identified two ubiquitination-related enzymes of particular interest: UHRF1, an E3 ligase essential for embryogenesis[19], and UBE2D3, an E2 conjugating enzyme previously implicated in protein ubiquitination during oocyte maturation[20] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Western blotting further confirmed an ~\\u0026thinsp;70% reduction in UBE2D3 and a 60% reduction in UHRF1 in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes compared to WT oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD-E). Together, our findings identify two downregulated ubiquitinating enzymes, UHRF1 and UBE2D3, providing strong candidates for dissecting how maternal Stella regulates protein ubiquitination in oocytes.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eUBE2D3 is required for protein ubiquitination and early embryo development\\u003c/h3\\u003e\\n\\u003cp\\u003eTo assess the contribution of UHRF1 and UBE2D3 to global protein ubiquitination in early embryos, we performed siRNA-mediated knockdown in MII oocytes prior to IVF (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). UHRF1 knockdown did not alter ubiquitination in early embryos, indicating that its role in embryogenesis is independent of global ubiquitin-mediated degradation (Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn contrast, UBE2D3 knockdown resulted in a marked reduction in global ubiquitination (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB-C). We next examined the developmental potential of UBE2D3-KD embryos. Approximately 50% of UBE2D3-KD embryos arrested at the 4-cell stage, and only\\u0026thinsp;~\\u0026thinsp;25% reached the blastocyst stage, compared with 83% in controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD-E). EU incorporation assays further showed a pronounced decrease in nascent RNA synthesis in 2-cell UBE2D3-KD embryos (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF-G), indicating compromised zygotic genome activation. These findings identify UBE2D3 as a key E2 conjugating enzyme required for robust ubiquitination and ZGA during early embryogenesis, positioning it as a potential downstream effector of maternal Stella.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003c/h3\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003e\\u003cb\\u003eStella modulates protein ubiquitination through UBE2D3\\u003c/b\\u003e\\u003c/div\\u003e \\u003cp\\u003eTo determine whether Stella directly regulates protein ubiquitination via UBE2D3, we manipulated Stella expression in transcriptionally silent GV oocytes and analyzed protein ubiquitination with or without UBE2D3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The efficiencies of endogenous Stella knockdown via siRNA injection and exogenous Stella overexpression via mRNA injection were confirmed by immunoblotting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). Immunoblotting analysis revealed that global protein ubiquitination was markedly decreased upon Stella knockdown, whereas Stella overexpression led to a pronounced increase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC-D). Importantly, depletion of UBE2D3 markedly blunted the ubiquitination increase induced by Stella overexpression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE-F), suggesting that UBE2D3 acts downstream of Stella in this regulatory pathway. Consistently, manipulating Stella expression altered UBE2D3 protein abundance: Stella knockdown significantly decreased UBE2D3 protein levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG), whereas Stella overexpression increased them (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eH). Together, these findings establish UBE2D3 as the key downstream effector through which Stella controls global protein ubiquitination.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eStella promotes\\u003c/b\\u003e \\u003cb\\u003eUbe2d3\\u003c/b\\u003e \\u003cb\\u003etranslation by stabilizing YBX3\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eBecause GV oocytes are transcriptionally quiescent, we next examined whether Stella regulates UBE2D3 protein levels through post-transcriptional mechanisms (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). To this end, we knocked down Stella in GV oocytes and assessed \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA abundance and UBE2D3 protein stability (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). Stella knockdown did not alter \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC), nor did it affect UBE2D3 protein turnover (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD), suggesting a translational mechanism. To test this, we constructed an eGFP reporter fused to the \\u003cem\\u003eUbe2d3\\u003c/em\\u003e coding sequence and microinjected the in vitro-transcribed mRNAs into control and Stella-KD GV oocytes, together with a mCherry transcript as an internal normalization control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). Quantification of eGFP-UBE2D3 and mCherry fluorescence revealed that Stella knockdown markedly reduced the translational output of the eGFP-UBE2D3 reporter (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE-F), demonstrating that Stella promotes \\u003cem\\u003eUbe2d3\\u003c/em\\u003e translation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo identify mediators of this regulatory process, we examined Y-box (YBX) family proteins, as YBX1, YBX2, and YBX3 participate in mRNA translation[21], and YBX1 was previously shown to interact with Stella to orchestrate translational regulation in oocytes[22]. We first assessed whether YBX1 contributes to UBE2D3 regulation; however, YBX1 knockdown did not alter UBE2D3 protein abundance (Fig. \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003eA-B). In contrast, proteomic profiling revealed a marked reduction of YBX3 in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC), which we validated by western blotting (Fig. \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003eC-D). To determine whether YBX3 regulates UBE2D3 expression, we depleted YBX3 in WT oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). YBX3 knockdown led to a significant reduction in UBE2D3 protein levels without affecting \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA abundance (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-D). Moreover, YBX3 depletion markedly impaired translation of both MYC-UBE2D3 and eGFP-UBE2D3 reporters (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE), supporting a role for YBX3 in promoting \\u003cem\\u003eUbe2d3\\u003c/em\\u003e translation. In addition, YBX3 depletion also diminished global protein ubiquitination, phenocopying the defects observed in Stella-deficient oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe next investigated whether Stella regulates YBX3 protein stability. To assess this, we inhibited de novo protein synthesis with cycloheximide (CHX) following Stella knockdown and monitored YBX3 degradation over time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eG). In Stella-depleted oocytes, YBX3 levels began to decline rapidly after 3 hours of culture and dropped to ~\\u0026thinsp;30% of the initial level by 12 hours, compared with ~\\u0026thinsp;50% remaining in control oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eH). However, \\u003cem\\u003eYbx3\\u003c/em\\u003e mRNA levels were unaffected in Stella-KD oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eI). This pronounced destabilization was attributed to enhanced proteasome-mediated degradation, as treatment of Stella-deficient oocytes with the proteasome inhibitor MG132 restored YBX3 protein abundance (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eJ). To determine whether Stella acts directly on YBX3, we performed co-immunoprecipitation assays. Stella and YBX3 robustly co-precipitated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eK-L), providing biochemical evidence for a physical interaction between the two proteins. Together, these findings support a model in which Stella binds to YBX3 and protects it from proteasomal degradation, thereby maintaining sufficient YBX3 protein to sustain \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA translation.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eInjection of\\u003c/b\\u003e \\u003cb\\u003eYbx3\\u003c/b\\u003e \\u003cb\\u003emRNA or\\u003c/b\\u003e \\u003cb\\u003eUbe2d3\\u003c/b\\u003e \\u003cb\\u003emRNA rescues ubiquitination and development of Stella-deficient embryos\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe results described above suggested that Stella regulates early embryo development through YBX3-UBE2D3 pathway. To further confirm the functional relevance of this regulatory axis, MII oocytes derived from \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e females were microinjected with \\u003cem\\u003eStella\\u003c/em\\u003e, \\u003cem\\u003eYbx3\\u003c/em\\u003e, or \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA and subsequently subjected to IVF. After fertilization, protein ubiquitination, embryonic gene transcription, and embryo development were assessed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eImmunostaining revealed that injection of either \\u003cem\\u003eYbx3\\u003c/em\\u003e or \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA greatly increased global protein ubiquitination in Stella-deficient embryos, with both treatments achieving a greater degree of restoration than \\u003cem\\u003eStella\\u003c/em\\u003e mRNA overexpression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB-C). Moreover, increasing YBX3 expression improved embryonic gene activation to levels comparable to those achieved by Stella overexpression and more effectively than UBE2D3 overexpression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD-E). Notably, while overexpression of all three genes significantly improved the developmental potential of Stella-deficient embryos, YBX3 overexpression produced the most robust rescue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eF-G).\\u003c/p\\u003e \\u003cp\\u003eTogether, these findings demonstrate that Stella regulates protein ubiquitination and early embryonic development through the YBX3-UBE2D3 axis, and the more effective rescue by YBX3 suggests it may further promote embryogenesis through additional mechanisms beyond ubiquitination.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eOur study identifies a previously unrecognized Stella-YBX3-UBE2D3 pathway that ensures efficient maternal protein ubiquitination and zygotic genome activation during early embryogenesis. Through integrated proteomic and functional analysis, we demonstrate the cytoplasmic mechanism by which maternal Stella governs early developmental competence.\\u003c/p\\u003e\\n\\u003ch3\\u003eStella as a central regulator of maternal protein ubiquitination\\u003c/h3\\u003e\\n\\u003cp\\u003e \\u003cem\\u003eStella\\u003c/em\\u003e was one of the first reported mammalian maternal effect genes together with \\u003cem\\u003eMater\\u003c/em\\u003e, \\u003cem\\u003eZar1\\u003c/em\\u003e and \\u003cem\\u003eNpm2\\u003c/em\\u003e[8, 23\\u0026ndash;25]. Although Stella is widely expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells, loss of function studies in mice showed that it is dispensable for germ cell specification but essential for normal early development. Stella has been proposed to protect maternal pronuclei from TET3 mediated active DNA demethylation and to safeguard the oocyte methylome by preventing aberrant de novo methylation[9, 12]. However, embryos depleted of maternal effect proteins known to regulate epigenetic typically exhibit developmental defects post-implantation, implying that the mechanisms underlying the regulation of early embryo development by mammalian Stella have not been fully investigated.\\u003c/p\\u003e \\u003cp\\u003eMaternal protein degradation and zygotic genome activation, occurring during MZT, are the first essential events for the development of preimplantation embryos[5]. UPS-dependent proteolysis is a selective degradation system in which the destruction of proteins is initiated by attaching multiple ubiquitin molecules to the target protein that is subsequently degraded by the 26S proteasome complex. Both proteasome function and ubiquitination are indispensable for successful MZT, and perturbation of either process adversely affects this transition. For example, transient inhibition of proteasome activity with MG132 delays the onset of zygotic transcription in otherwise normally developing 2-cell embryos and even induces developmental arrest at the 1-cell stage[26]. In parallel, ubiquitination pathways are equally critical, as RNF114-mediated ubiquitination and degradation of TAB1 is necessary for MZT[27]. Despite these advances, the upstream maternal factors that ensure UPS function have remained unclear. In this study, we found that ubiquitinated proteins were markedly reduced in \\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e oocytes and embryos, and this defect stemmed from impaired ubiquitination rather than altered proteasome activity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Proteomic profiling reveled widespread protein accumulation, consistent with impaired ubiquitin-mediated turnover (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). By manipulating Stella expression in transcriptional silent GV oocytes, we demonstrated directly that Stella is required to sustain protein ubiquitination (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Collectively, these findings position Stella as a central maternal-effect factor that preserves UPS function, uncovering a previously unrecognized role for Stella in coordinating the proteolytic landscape of the MZT.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eA translational regulatory network involving Stella, YBX3, and UBE2D3\\u003c/h2\\u003e \\u003cp\\u003eUbiquitination is a tightly regulated process mediated by a three-step enzymatic cascade involving E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Among these, E2 enzymes are central because they determine the type of ubiquitin modification that occurs[28]. For example, K48-linked chains target proteins for proteasomal degradation, whereas the K63-linked ubiquitination serves as a molecular platform for protein/protein interaction[29]. Previously, we showed that UBE2D3, the most abundant E2 transcript in oocytes, is required for global protein ubiquitination in oocytes[20]. UBE2D3 is predicted to be involved in protein K48-linked ubiquitination and ubiquitin-dependent protein catabolic process. In this study, UBE2D3 was significantly downregulated in Stella-deficient oocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), and its depletion alone recapitulated the ubiquitination defects, impaired ZGA, and early developmental arrest observed in Stella-deficient embryos, establishing UBE2D3 as a key downstream mediator of Stella function (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eStella has been reported to interact with and stabilize specific proteins by preventing their proteasomal degradation[15]. However, among the 291 proteins identified as Stella interactors, UBE2D3 was not detected[14]. Consistent with this, loss of Stella did not affect UBE2D3 protein stability but instead markedly suppressed the translational activity of \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Although Stella was recently reported to associate with the RNA-binding protein YBX1 to regulate mRNA translation, YBX1 knockdown did not alter UBE2D3 abundance (Fig. \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e). Here, we identify YBX3 as the downstream regulator through which Stella controls \\u003cem\\u003eUbe2d3\\u003c/em\\u003e expression. This conclusion is supported by several findings: (1) YBX3 and YBX1 share high sequence homology and overlapping RNA-binding functions; (2) YBX3 directly regulates the translational activity of \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE); (3) Stella binds to and stabilizes YBX3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eK-L); and (4) YBX3 overexpression partially rescues the developmental defects of Stella-deficient embryos. Together, these results delineate the cytoplasmic mechanistic framework by which maternal Stella controls embryo development.\\u003c/p\\u003e \\u003cp\\u003eNotably, global reductions in translational activity were observed following YBX3 depletion, indicating that YBX3 functions more broadly than solely promoting \\u003cem\\u003eUbe2d3\\u003c/em\\u003e translation. Consistent with this, YBX3 overexpression produced a stronger rescue than \\u003cem\\u003eUbe2d3\\u003c/em\\u003e overexpression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e), suggesting that dysregulation of additional YBX3-dependent targets also contributes to the developmental defects of Stella-deficient embryos.\\u003c/p\\u003e \\u003cp\\u003eIn summary, we identify a translational regulatory circuit in which Stella stabilizes YBX3 to promote \\u003cem\\u003eUbe2d3\\u003c/em\\u003e translation and maintain the ubiquitination landscape essential for ZGA and early development. This pathway provides a mechanistic framework for how maternal Stella orchestrates proteostasis to safeguard the oocyte-to-embryo transition.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"MATERIALS AND METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMice\\u003c/h2\\u003e \\u003cp\\u003e All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Welfare and Ethics Committee of Nanjing Medical University. Stella homozygous mutant mice (\\u003cem\\u003eStella\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e△\\u003c/em\\u003e\\u003c/sup\\u003e) were generated using the CRISPR/Cas9 system as previously described[10]. Two sgRNAs targeting exon 2 of the \\u003cem\\u003eStella\\u003c/em\\u003e gene (listed in Supplementary Table\\u0026nbsp;2) were used to introduce a 153-bp deletion, resulting in the loss of 51 amino acids in the Stella protein. Homozygous mutant males were crossed with heterozygous females for subsequent experiments. All mice were maintained in a specific pathogen-free (SPF) facility under a 12 h light/12 h dark cycle at 20\\u0026ndash;26\\u0026deg;C and 40\\u0026ndash;70% humidity.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAntibodies\\u003c/h2\\u003e \\u003cp\\u003eThe following antibodies were used in this study: rabbit monoclonal anti-STELLA (Abcam, ab13495), rabbit monoclonal anti-Ubiquitin (Abcam, ab134953), rabbit polyclonal anti-UBE2D3 (Proteintech, 11677-1-AP), rabbit polyclonal anti-DYKDDDDK tag (Proteintech, 20543-1-AP), Zonab polyclonal antibody (Thermo Fisher Scientific, PA5-85619), mouse polyclonal anti-UHRF1 (Santa Cruz Biotechnology, sc-373750), mouse polyclonal anti-α-Tubulin (Proteintech, HRP-60031), mouse polyclonal anti-β-Actin (Proteintech, HRP-60008), mouse monoclonal anti-GAPDH (Beyotime, AF00006), and mouse monoclonal anti-MYC tag (Abcam, ab18185). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Proteintech (SA00001-1 and SA00001-2).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eOocyte collection and culture\\u003c/h2\\u003e \\u003cp\\u003eGV oocytes were obtained from the ovarian follicles of adult female mice 46\\u0026ndash;48 h after pregnant mare serum gonadotropin (PMSG, 5 IU; Ningbo Hormone Product Co.) injection. For in vitro maturation, GV oocytes were cultured in M16 medium (Nanjing Luanchuang Co., China) under mineral oil at 37\\u0026deg;C in a 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e incubator.\\u003c/p\\u003e \\u003cp\\u003eFor MII oocytes collection, female mice were injected with PMSG, followed 48 h later by human chorionic gonadotropin (hCG, 5 IU; Ningbo Hormone Product Co.) injection. Cumulus-oocyte complexes (COCs) were isolated from oviduct ampulla, and denuded MII oocytes were obtained by removing the cumulus mass in medium containing 0.5 mg/mL hyaluronidase (Sigma) at 37\\u0026deg;C.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIn vitro fertilization and embryo culture\\u003c/h2\\u003e \\u003cp\\u003eIVF assays were performed as previously described[10] with minor modifications. Sperm were collected from the cauda epididymis of adult ICR males (10\\u0026ndash;20 weeks old) and capacitated for 1 h in HTF medium (Nanjing Luanchuang Co., China) supplemented with 10 mg/mL BSA. Capacitated sperm were then added to HTF drops containing denuded oocytes as described previously[30]. After co-incubation at 37\\u0026deg;C, presumptive zygotes were washed to remove excess sperm and cultured in KSOM medium (Nanjing Luanchuang Co., China) under mineral oil at 37\\u0026deg;C in a humidified atmosphere of 5% CO₂ until the blastocyst stage.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePlasmid Construction and cRNA Synthesis\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from 50 oocytes using the ArcturusPicoPure RNA Isolation Kit (KIT0204; Applied Biosystems), and cDNA was generated using the Quantitect Reverse Transcription kit (Qiagen; 205311). Plasmid construction and cRNA synthesis were performed as described previously[31]. Purified PCR products were digested with EcoRI and XbaI (NEB) and cloned into the pCS2\\u0026thinsp;+\\u0026thinsp;vector with Myc tags, or alternativel digested with NheI and KpnI (NEB) and cloned into the eGFP-tag pEGFP vector.\\u003c/p\\u003e \\u003cp\\u003eFor in vitro cRNA synthesis, plasmids were linearized with NotI and transcribed using the SP6 mMESSAGE mMACHINE kit (AM1340, Thermo Fisher) according to the manufacturer\\u0026rsquo;s instructions. The cRNA was then purified using the RNeasy Micro Kit (74004; Qiagen). The mCherry plasmid (Cat#: D2711) were linearized by XbaI. Capped cRNAs were synthesized with T7 mMESSAGE mMACHINE ULTRA Kit (Ambion, CA, USA). Synthesized cRNA was aliquoted and stored at -80\\u0026deg;C. The related primer sequences can be found in Supplementary Table\\u0026nbsp;2.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eKnockdown and Overexpression Experiments\\u003c/h2\\u003e \\u003cp\\u003eMicroinjections were performed using an inverted microscope (Eclipse Ti-S, Nikon) equipped with a micromanipulator (Narishige). siRNAs targeting \\u003cem\\u003eStella, Ube2d3, Ybx3, or Ybx1\\u003c/em\\u003e (GenePharma) were prepared as 20 \\u0026micro;M stock solutions. For knockdown, 2.5 pL of 1 mM siRNA or negative control siRNA was injected into GV-stage oocytes. For overexpression, 10 pL of cRNA (10 ng/\\u0026micro;L) was microinjected into GV oocytes; PBS injection served as a control.\\u003c/p\\u003e \\u003cp\\u003eAfter injection, oocytes were maintained in M2 medium containing 2.5 \\u0026micro;M milrinone for 24 h to allow siRNA/cRNA function. For embryonic knockdown or overexpression, 5\\u0026ndash;10 pL of siRNA or cRNA was injected into MII oocytes before IVF. Zygotes were cultured in KSOM medium under mineral oil at 37\\u0026deg;C in 5% CO₂. Related siRNA sequences are provided in Supplementary Table\\u0026nbsp;2.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLive-cell imaging\\u003c/h2\\u003e \\u003cp\\u003eFor live-cell imaging, mRNAs encoding for eGFP-UBE2D3 and mCherry were microinjected into GV oocytes and released from milrinone after 6 h. Oocytes were washed three times and transferred to a live cell-imaging dish, covered with mineral oil. Fluorescent imaging was performed using a Zeiss Laser Scanning Confocal Microscope (LSM 700; Zeiss). Semiquantitative analysis of fluorescence intensity was carried out using ImageJ (NIH).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEU incorporation assays\\u003c/h2\\u003e \\u003cp\\u003eEU incorporation assays were performing using the Click-iT RNA Imaging kits (Invitrogen, C10329). Two-cell embryos were cultured in KSOM medium containing 1 mM EU for 3 h and fixed overnight at 4\\u0026deg;C in 4% paraformaldehyde. Embryos were permeabilized with 0.5% Triton X-100 for 15 min, washed, and incubated in the Click-iT reaction mixture for 30\\u0026ndash;60 min in the dark. Nuclei were stained with Hoechst 33342 (1:1000). Images were acquired using a confocal microscope, and EU signal was quantified by calculating average fluorescence intensity after background subtraction.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eqRT-PCR analysis\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA from oocytes was extracted using the RNeasy Micro Kit (Qiagen). cDNA synthesis and qPCR were performed using the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit (Qiagen) on a QuantStudio\\u0026trade; 7 Real-Time PCR System (Applied Biosystems). Relative mRNA levels were calculated using the 2^-ΔΔCt method with Gapdh as the internal control. Primer sequences are provided in Supplementary Table\\u0026nbsp;2.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern Blotting\\u003c/h2\\u003e \\u003cp\\u003eA total of 80\\u0026ndash;100 oocytes were lysed in 2\\u0026times; Laemmli sample buffer containing protease inhibitor, and denatured at 95\\u0026deg;C for 5 min. Proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4\\u0026deg;C with primary antibodies (Stella antibody, 1:1,000; Myc antibody, 1:1,000; UBE2D3 antibody, 1:1,000; Ubiquitin antibody, 1:1,000; YBX3 antibody, 1:500). After washes in TBST for three times, the membranes were incubated with HRP-conjugated secondary antibodies. Then, the protein bands were visualized using an ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont, United Kingdom). After washing, membranes were incubated with HRP-conjugated secondary antibodies and visualized using an ECL Plus detection system (GE Healthcare). TUBULIN, GAPDH, or ACTIN served as loading controls where appropriate. Band intensities were quantified using ImageJ.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eCell culture, plasmid transfection, and immunoprecipitation.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eHEK293 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Hyclone) and 1% penicillin-streptomycin at 37\\u0026deg;C in 5% CO₂. Cells were seeded in 100-mm dishes and transfected at 50\\u0026ndash;60% confluence with Stella-Flag and Ybx3-Myc expression plasmids using Lipofectamine 2000 (Invitrogen).\\u003c/p\\u003e \\u003cp\\u003eForty-eight hours post-transfection, cells were lysed in buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 0.5% NP-40; protease/phosphatase inhibitors). Lysates were centrifuged at 12,000 \\u0026times; g for 10 min at 4\\u0026deg;C and incubated with anti-Flag or anti-Myc magnetic beads for 4 h. After washing, bound proteins were eluted by boiling in SDS sample buffer and analyzed by western blotting.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProteasome activity assay\\u003c/h2\\u003e \\u003cp\\u003eProteasome activity in oocytes was quantified using a fluorometric proteasome activity assay kit (Abcam, ab107921) according to the manufacturer\\u0026rsquo;s instructions. The assay measures the release of the fluorescent molecule 7-amino-4-methylcoumarin (AMC) from a labeled peptide substrate upon proteolytic cleavage. To distinguish proteasomal activity from non-specific protease activity, 50 \\u0026micro;M MG132 was included as an inhibitor control. Oocyte lysates were incubated with the substrate at 37\\u0026deg;C in the dark, and fluorescence was recorded at excitation/emission wavelengths of 350/440 nm after 60 and 90 min of incubation. Proteasome activity was calculated from the increase in fluorescence relative to MG132-treated controls using an AMC standard curve, where one unit of activity corresponds to the release of 1.0 nmol AMC per minute at 37\\u0026deg;C.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProteomic profiling\\u003c/h2\\u003e \\u003cp\\u003eProteomic profiling of oocytes was conducted as previously described with minor modifications[32]. For TMT-based quantitative proteomics, MII oocytes (n\\u0026thinsp;=\\u0026thinsp;330 per replicate, 3 biological replicates per group) were lysed in a urea buffer (8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, 1%EDTA-free protease inhibitor). After reduction, alkylation, and trypsin digestion, peptides were labeled with the TMT6plex\\u0026trade; kit, mixed equally, and fractionated by high-pH reversed-phase (HP-RP) chromatography on an ACQUITY\\u0026reg; UPLC M-class system (BEH C18 column, 300 \\u0026micro;m \\u0026times; 150 mm, 1.7 \\u0026micro;m; Waters).\\u003c/p\\u003e \\u003cp\\u003ePeptides from TMT samples were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to a Proxeon Easy-nLC 1200 system. Peptide separation was performed on an analytical C18 column (75 \\u0026micro;m \\u0026times; 160 mm, 1.9 \\u0026micro;m, Dr. Maisch) with a 95 min linear gradient at 300 nL/min. The Orbitrap was operated at a resolution of 60,000 for MS1 and 15,000 for MS2. Additional MS parameters were as described previously[32, 33].\\u003c/p\\u003e \\u003cp\\u003eRaw data were processed with MaxQuant (v1.6.5.0) against the UniProt mouse proteome database. Carbamidomethylation (C) was set as a fixed modification, and oxidation (M) and acetylation (protein N-terminus) as variable modifications. Trypsin/P specificity was applied with up to two missed cleavages. A 1% false discovery rate (FDR) was applied at both peptide and protein levels. For TMT quantification, protein intensities were calculated using the reporter ion MS2 method. Proteins identified with \\u0026ge;\\u0026thinsp;1 unique peptide and FDR\\u0026thinsp;\\u0026le;\\u0026thinsp;1% were retained for downstream analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eAll experiments were performed with at least three biological replicates. Data are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD unless otherwise indicated. Statistical significance was assessed using Student\\u0026rsquo;s t-test for two-group comparisons or one-way ANOVA for multiple groups (GraphPad Prism 8). A \\u003cem\\u003eP\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eACKNOWLEDEGMENTS\\u003c/h2\\u003e \\u003cp\\u003eThis work was supported by the National Natural Science Foundation of China (NO. 82271689 to L.H.; NO. 82495190 and 82221005 to QW.), Science Foundation of Jiangsu Province (BK20230058 to L.H.).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\\u003ch2\\u003eCOMPETING INTERESTS\\u003c/h2\\u003e \\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAUTHOR CONTRIBUTION\\u003c/h2\\u003e \\u003cp\\u003eL.H. and Q.W. conceived the projects. G.Z., M.R., and G.S. performed the majority of experiments. Y.Z. and N.Z. assisted with animal model construction. L.H. and G.Z. designed the experiments and analyzed data. L.H. and G.Z. wrote the manuscript, and Q.W. revised it.\\u003c/p\\u003e\\u003ch2\\u003eDATA AVAILABILITY\\u003c/h2\\u003e \\u003cp\\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eQi, L., et al., miR-370 is stage-specifically expressed during mouse embryonic development and regulates Dnmt3a. FEBS Lett, 2013. 587(6): p. 775-81.\\u003c/li\\u003e\\n\\u003cli\\u003eSchier, A.F., The maternal-zygotic transition: death and birth of RNAs. Science, 2007. 316(5823): p. 406-7.\\u003c/li\\u003e\\n\\u003cli\\u003eZhou, L.Q. and J. Dean, Reprogramming the genome to totipotency in mouse embryos. Trends Cell Biol, 2015. 25(2): p. 82-91.\\u003c/li\\u003e\\n\\u003cli\\u003eYu, C., et al., BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes. Nat Struct Mol Biol, 2016. 23(5): p. 387-94.\\u003c/li\\u003e\\n\\u003cli\\u003eHiguchi, C., et al., Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development. J Reprod Dev, 2018. 64(1): p. 65-74.\\u003c/li\\u003e\\n\\u003cli\\u003eHamazaki, J., et al., Rpn10-mediated degradation of ubiquitinated proteins is essential for mouse development. Mol Cell Biol, 2007. 27(19): p. 6629-38.\\u003c/li\\u003e\\n\\u003cli\\u003eSakao, Y., et al., Mouse proteasomal ATPases Psmc3 and Psmc4: genomic organization and gene targeting. Genomics, 2000. 67(1): p. 1-7.\\u003c/li\\u003e\\n\\u003cli\\u003ePayer, B., et al., Stella is a maternal effect gene required for normal early development in mice. Curr Biol, 2003. 13(23): p. 2110-7.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, Y., et al., Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1. Nature, 2018. 564(7734): p. 136-140.\\u003c/li\\u003e\\n\\u003cli\\u003eHan, L., et al., Differential roles of Stella in the modulation of DNA methylation during oocyte and zygotic development. Cell Discov, 2019. 5: p. 9.\\u003c/li\\u003e\\n\\u003cli\\u003eNakamura, T., et al., PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature, 2012. 486(7403): p. 415-9.\\u003c/li\\u003e\\n\\u003cli\\u003eNakamura, T., et al., PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol, 2007. 9(1): p. 64-71.\\u003c/li\\u003e\\n\\u003cli\\u003eNakatani, T., et al., Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced \\u0026gamma;H2AX accumulation. EMBO Rep, 2015. 16(5): p. 582-9.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, H., et al., Comprehensive Proteomic Analysis of PGC7-Interacting Proteins. J Proteome Res, 2017. 16(9): p. 3113-3123.\\u003c/li\\u003e\\n\\u003cli\\u003eZhao, S., et al., Dppa3 facilitates self-renewal of embryonic stem cells by stabilization of pluripotent factors. Stem Cell Res Ther, 2022. 13(1): p. 169.\\u003c/li\\u003e\\n\\u003cli\\u003eShin, S.W., et al., Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice. Nat Commun, 2017. 8(1): p. 1643.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, L., P. Zheng, and J. Dean, Maternal control of early mouse development. Development, 2010. 137(6): p. 859-70.\\u003c/li\\u003e\\n\\u003cli\\u003eVerlhac, M.H., M.E. Terret, and L. Pintard, Control of the oocyte-to-embryo transition by the ubiquitin-proteolytic system in mouse and C. elegans. Curr Opin Cell Biol, 2010. 22(6): p. 758-63.\\u003c/li\\u003e\\n\\u003cli\\u003eCao, Y., et al., Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos. Faseb j, 2019. 33(7): p. 8294-8305.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang, G., et al., UBE2D3 functions in mouse oocyte meiotic maturation. Faseb j, 2025. 39(3): p. e70375.\\u003c/li\\u003e\\n\\u003cli\\u003eMordovkina, D., et al., Y-Box Binding Proteins in mRNP Assembly, Translation, and Stability Control. Biomolecules, 2020. 10(4).\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Y., et al., PGC7 regulates maternal mRNA translation via AKT1-YBX1 interactions in mouse oocytes. Cell Commun Signal, 2024. 22(1): p. 604.\\u003c/li\\u003e\\n\\u003cli\\u003eWu, X., et al., Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat Genet, 2003. 33(2): p. 187-91.\\u003c/li\\u003e\\n\\u003cli\\u003eTong, Z.B., et al., Mater, a maternal effect gene required for early embryonic development in mice. Nat Genet, 2000. 26(3): p. 267-8.\\u003c/li\\u003e\\n\\u003cli\\u003eBurns, K.H., et al., Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos. Science, 2003. 300(5619): p. 633-6.\\u003c/li\\u003e\\n\\u003cli\\u003eShin, S.W., et al., Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression. J Reprod Dev, 2010. 56(6): p. 655-63.\\u003c/li\\u003e\\n\\u003cli\\u003eYang, Y., et al., The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition. EMBO Rep, 2017. 18(2): p. 205-216.\\u003c/li\\u003e\\n\\u003cli\\u003eStewart, M.D., et al., E2 enzymes: more than just middle men. Cell Res, 2016. 26(4): p. 423-40.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, L., et al., UBE2V1 governs aging induced protein aggregation and developmental defects in oocytes and embryos. Commun Biol, 2025. 8(1): p. 769.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang, Z., et al., Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations. Reproduction, 2019. 158(2): p. 123-133.\\u003c/li\\u003e\\n\\u003cli\\u003eZeng, J., et al., SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation. Aging Cell, 2018. 17(4): p. e12789.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, L., et al., Characterization of Metabolic Patterns in Mouse Oocytes during Meiotic Maturation. Mol Cell, 2020. 80(3): p. 525-540.e9.\\u003c/li\\u003e\\n\\u003cli\\u003eCheah, M.A., et al., MBN 2016 Aesthetic Breast Meeting BIA-ALCL Consensus Conference Report. Plast Reconstr Surg, 2018. 142(6): p. 971e-972e.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cellular-and-molecular-life-sciences\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"life\",\"sideBox\":\"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)\",\"snPcode\":\"18\",\"submissionUrl\":\"https://www.editorialmanager.com/life/default2.aspx\",\"title\":\"Cellular and Molecular Life Sciences\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8687106/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8687106/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eUbiquitin-proteasome-mediated protein degradation is essential for maternal-to-zygotic transition, yet the maternal factors that safeguard ubiquitin-proteasome function remain unclear. Here, using a Stella loss-of-function mouse model, we demonstrate that Stella is required for global protein ubiquitination during early embryogenesis. Stella deficiency in oocytes leads to reduced ubiquitinated protein levels, impaired zygotic genome activation, and early developmental arrest. Proteomic and functional analysis identify UBE2D3 as the key E2 enzyme whose insufficiency underlies the ubiquitination defect. Mechanistically, Stella preserves YBX3 stability, thereby enabling YBX3-dependent translation of \\u003cem\\u003eUbe2d3\\u003c/em\\u003e mRNA. Consequently, Stella depletion destabilizes YBX3, diminishes UBE2D3 synthesis, and disrupts protein ubiquitination. Notably, overexpression of \\u003cem\\u003eYbx3\\u003c/em\\u003e or \\u003cem\\u003eUbe2d3\\u003c/em\\u003e partially restores ubiquitinated protein levels, zygotic genome activation, and embryo development. These findings identify a previously unrecognized Stella-YBX3-UBE2D3 axis that ensures efficient maternal protein ubiquitination and zygotic genome activation, revealing a cytoplasmic mechanism by which maternal Stella governs proteostasis during maternal-to-zygotic transition.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Stella safeguards proteostasis via regulating UBE2D3 translation in the maternal-to-zygotic transition\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-26 13:51:36\",\"doi\":\"10.21203/rs.3.rs-8687106/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major Revision\",\"date\":\"2026-03-11T11:53:47+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2026-02-24T10:39:02+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-24T10:02:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-01-29T12:07:59+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cellular and Molecular Life Sciences\",\"date\":\"2026-01-28T06:57:29+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cellular-and-molecular-life-sciences\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"life\",\"sideBox\":\"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)\",\"snPcode\":\"18\",\"submissionUrl\":\"https://www.editorialmanager.com/life/default2.aspx\",\"title\":\"Cellular and Molecular Life Sciences\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"004dd697-4531-41e1-b18c-fd78d8d04113\",\"owner\":[],\"postedDate\":\"February 26th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-15T09:41:20+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-26 13:51:36\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8687106\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8687106\",\"identity\":\"rs-8687106\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}