Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development

Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development Huihui Xie, Tiandong Xia, Bochao Zhang, Jiankun Cui, Fuqiang Pan, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3872230/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ezrin is a membrane-cytoskeleton linker, involved in cell polarization, cell migration, cell division, signal transduction and some other cellular activities that play an important role in oocyte maturation, fertilization and early embryonic development. The phosphorylation of Thr567 is an important way to activate ezrin, it has been proved that p-ezrin Thr567 is expressed in oocytes and pre-implantation embryos in mouse. However, little is known about the impact of inhibiting ezrin Thr567 phosphorylation on oocyte maturation, fertilization and early embryonic development. Methods NSC668394 is a small molecule that specifically inhibits the phosphorylation of ezrin Thr567. Here, we investigated the effects of inhibiting ezrin Thr567 phosphorylation with NSC668394 on the mouse oocyte maturation, fertilization, and early embryo development. Conclusion The results show that adding NSC668394 to the in vitro culture medium significantly lowed mouse embryos development competence after 8-cell stage ( P < 0.05). Further experiments revealed that inhibiting ezrin Thr567 phosphorylation during in vitro maturation or in vitro fertilization not only decreased the maturation rate and fertilization rate of mouse oocytes, but also reduced early embryos development competence after 8-cell stage. Microinjection of mRNA encoding ezrin T567D mutant partially rescued the developmental defects of mouse oocytes, fertilization, and early embryonic development caused by NSC668394. These results indicate that ezrin Thr567 phosphorylation plays an important role in mouse oocyte maturation, fertilization and early embryo development. Ezrin phosphorylation mouse oocyte early embryo Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction ERM (ezrin/radixin/moesin) is an evolutionarily highly conserved family of proteins, consisting of a FERM domain at the amino terminus, a C-ERMAD (ERM-association domains) domain at the carboxyl terminus, and an α-helical structure in the middle that connects the two domains.[ 1 ] They cross-link membrane proteins to actin microfilaments directly or indirectly through FERM domains[ 2 – 4 ], and localized in plasma membrane protrusions (such as microvilli, pseudopodia and contractile fibers), intercellular junctions, and mitotic division furrows[ 5 ], involved in the formation of cell surface structures and the maintenance of cell polarity[ 6 ], cell migration, intercellular adhesion[ 5 ], cell division and signal transduction[ 4 ]. As a member of the ERM family, the C-terminal domain of ezrin can interact with the N-terminal domain[ 7 ]. This intramolecular or intermolecular head-to-tail connection shields its interaction with the cell membrane and the actin microfilament skeleton, leaving it in an inactive state and localized in the cytoplasm[ 8 ]. Phosphorylation is an important way to regulate protein activity. Ezrin has multiple phosphorylation sites, such as Ser66[ 9 – 11 ]、Thr213[ 12 ]、Thr332[ 13 , 14 ]、Thr567[ 15 – 17 ]、Tyr145、Tyr353 and Tyr477[ 18 ]. Phosphorylation of Thr567 is currently considered a sign of ezrin activation and is also the most studied phosphorylation site[ 19 ]. Oocyte maturation, fertilization, and early embryonic development are accompanied by cell surface remodeling[ 20 , 21 ], cell division[ 22 , 23 ], cell polarization[ 24 , 25 ], intercellular junction formation, cell adhesion[ 26 , 27 ] cell migration [ 28 , 29 ] and other activities[ 30 – 32 ]. Studies have shown that ERM proteins are expressed in oocytes and early embryos. Knocking down any one of ERM proteins alone cannot inhibit in vitro fertilization of zona-free eggs of mouse, but knocking down ERM comprehensively can significantly reduce the fertilization index of mouse zona-free oocytes [ 33 ]. Another study found that inhibiting the expression of ezrin significantly increased the cleavage rate and blastocyst development rate of bovine somatic cell nuclear transfer embryos[ 34 ]. These studies show that ERM proteins, especially ezrin, are involved in regulating oocyte fertilization and early embryonic development. Louvet S found that ezrin is expressed in mouse oocytes and pre-implantation embryos accompanying dynamic phosphorylation modifications throughout the development[ 35 ]. Overexpression of Ez-T567D, a mutant that mimics the phosphorylation state of ezrin Thr567 in mouse 2-cell embryos inhibited the embryonic compaction and blastocyst formation, while overexpression of non-phosphorylated mutant Ez-T567A did not affect embryo compaction, but interfered with the formation of the blastocyst cavity. These results suggest that ezrin Thr567 phosphorylation is involved in regulating early embryonic development[ 36 ]. Further studies found that atypical PKC (aPKC) phosphorylates ezrin Thr567 during embryonic densification[ 37 ]. However, it is still unclear at which stage of oocyte and early embryo development the phosphorylation of ezrin Thr567 mainly occurs, and what effect inhibiting the phosphorylation of ezrin Thr567 will have on oocyte maturation, fertilization and development potential. NSC668394 is a small molecule compound that specifically inhibits the phosphorylation of ezrin Thr567[ 16 , 38 – 40 ]. In this study, we used NSC668394 to inhibit the phosphorylation of ezrin Thr567 during the in vitro maturation, in vitro fertilization and early embryo culture of mouse oocytes. We found that inhibiting the phosphorylation of ezrin Thr567 not only affected oocyte maturation and fertilization, but also affected its developmental potential, and when a mutant that mimics ezrin Thr567 phosphorylation is expressed at the same time, its maturation, fertilization and development are successfully rescued. 2 Materials and Methods 2.1 Experimental Animals and Ethical Standards Six to eight week-old ICR mice were provided ad libitum access to food and water and maintained under controlled environmental conditions of temperature (23℃ to 26℃), natural light. All animal experiments were conducted in accordance with the regulations of the People's Republic of China on the Management of Laboratory Animals and were approved by the Animal Care and Use Committee of Anhui Agricultural University. 2.2 Sperm preparation Healthy ICR male mice with normal reproductive ability were euthanized, and both epididymal tails were aseptically removed, minced, and placed in capacitation medium (M2050, Easycheck) to release sperm. The mixture was then incubated at 37°C with 5% CO 2 for 30–60 minutes to achieve capacitation. 2.3 Oocyte collection, in vitro maturation, in vitro fertilization and embryo in vitro culture Collection of GV-stage, MII-stage oocytes, and pronuclear-stage embryos: As described in literatures[ 41 , 42 ], in brief, 6–8 week-old female ICR mice were injected intraperitoneally with 10 IU of Pregnant Mare Serum Gonadotropin. (PMSG, Ningbo Sansheng Pharmaceutical Co., Ltd.). Forty-eight hours later, the mice were euthanized and ovarian follicles with a cavity were punctured to release cumulus-oocyte complexes (COCs) containing GV-stage oocytes, or the mice were injected intraperitoneally with 10 IU of PMSG, followed by injection of Human Chorionic Gonadotrophin (HCG, Ningbo Sansheng Pharmaceutical Co., Ltd.) and 13–16 hours later to collect MII-stage oocytes from the ampulla of the oviduct. Alternatively, the mice were placed together with males immediately after HCG injection, and pronuclear-stage embryos were collected 18 hours later[ 43 ]. The collected oocytes or pronuclear-stage embryos were transferred into M2 medium (M7167, Sigma) containing 0.3% hyaluronidase (H3506, Solarbio), and the cumulus cells were removed by pipetting. In Vitro Maturation (IVM): The GV-stage oocytes were transferred to TCM199 medium (Sigma) containing 10% fetal calf serum (FCS, Gibico), and cultured in an incubator at 37°C, 5% CO 2 , and 95% humidity for 20 hours. The maturation rate was calculated based on the extrusion of the first polar body. In vitro fertilization (IVF): Matured oocytes were incubated in HTF medium (MR-070-D, Milipore) containing 10µL of capacitated sperm suspension obtained as described above at 37°C with 5% CO 2 for 4–6 hours[ 42 , 44 ]. The fertilization rate was calculated based on the formation of pronuclear and the extrusion of the second polar body. Embryos IVC: The prokaryotic embryos were transferred into K + Simplex Optimised Medium (KSOM, MR-020P-5F, Milipore) and cultured in an incubator at 37°C, 5% CO 2 , and 95% humidity. 2.4 Immunofluorescence staining As described in reference[ 45 ], oocytes or embryos were fixed and permeabilized, and then incubated in primary antibody for Ezrin (#3145, CST, 1:100) or p-Ezrin Thr567 (EP886Y, Abcam, 1:100) at 4°C overnight followed by incubation with FITC-labeled secondary antibody (HS111, TransGen Biotech, diluted at a 1:100 ratio in 1% BSA) for 1 hour and DAPI (D8200, Roche, diluted at a 1:10000 ratio) for 10 minutes at room temperature. After washing 3 times, samples were mounted on a slide and covered with a coverslip, and then were imaged under an inverted fluorescence microscope (DP72 model, Olympus) [ 45 ]. 2.5 In vitro transcription and microinjection According to the instructions of the TR101-01 kit (Vazyme), mRNA encoding EGFP-Ezrin T567D was transcribed in vitro and purified, and the concentration was adjusted to 0.5 µg/µL. The eggs to be injected were transferred into M2 medium, and the EGFP-Ezrin T567D mRNA solution was injected into each egg at a volume of 5–10 pL per egg. 2.6 Statistical analysis Each experiment was repeated three or more times, and data were presented as mean ± SEM (standard error of the mean). Data analysis was performed using SPSS statistics 17.0, using one-way analysis of variance (ANOVA) with a significance level of P < 0.05 indicating statistical significance. 3 Results 3.1 Expression and localization of Ezrin and p-Ezrin Thr567 in mouse oocytes and preimplantation embryos To investigate the expression and localization pattern of Ezrin and p-Ezrin Thr567 in mouse oocytes and preimplantation embryos, GV-stage, MⅡ-stage oocytes and early embryos at different stages were collected for immunofluorescence analysis. All samples were imaged using the same exposure time under a fluorescence microscope. Figure 1 shows the expression and localization of ezrin in mouse oocyte and preimplantation embryos at different developmental stages. In GV stage oocytes, 4-cell stage, 8-cell stage and morula, ezrin is distributed in both the cytoplasm and nucleus, and the fluorescence intensity in the nucleus is significantly higher than that in the cytoplasm, while in MⅡ stage oocytes, prokaryotic embryo and 2-cell stage embryo the fluorescence signal of ezrin gradually weakens and is evenly distributed within the cells. In blastocyst, ezrin is mainly located in the cell cortex area ( Fig. 1 ). From GV stage oocyte to morula, p-Ezrin Thr567 is primarily localized in the cell cortex area and the intercellular junctions, and in 4-cell stage embryo and blastocyst p-ezrin Thr567 is also distributed in nucleus ( Fig. 2 ). Figure 1: Localization of Ezrin in mouse oocytes and early embryos at different developmental stages. Figure 2. Localization of p-Ezrin Thr567 in mouse oocytes and early embryos at different developmental stages. 3.2 NSC668394 inhibits the phosphorylation of Ez-T567 and influences the development of early embryos. To test the inhibitory effect of NSC668394 on ezrin Thr567 phosphorylation in mouse embryos, prokaryotic embryos were cultured in KSOM medium containing different concentration (0, 2.5, 5, and 10 µM) of NSC668394 and fixed at 2-cell stage for immunofluorescence staining. Immunofluorescence images were captured with the same exposure time. The results showed that 2.5 µM of NSC668394 remarkably inhibited ezrin Thr567 phosphorylation in the embryos. As the concentration of NSC668394 increased, there was a trend of further reduction in p-Ezrin-Thr567 levels (Fig. 3). Corresponding to the reduction of p-ezrin Thr567 in embryos, the rates of 8-cell, morula and blastocyst were significantly decreased ( Table 1 ). Table 1 Effects of the addition of NSC to embryo culture medium on early embryonic development. groups prokaryotic stage 2-cell (2-cell rate /%) 4-cell (2-cell rate / %) 8-cell (8-cell rate / %) Morula (Morula rate / %) Blastocyst (Blastocyst rate/ %) 0.1%DMSO 188 181 (96.36 ± 3.33) a 174 (92.61 ± 4.02) a 148 (88.95 ± 2.24) a 159 (84.78 ± 3.41) a 154 (88.14 ± 3.98) a 0µM NSC 201 193 (96.05 ± 0.53) a 185 (92.02 ± 0.72) a 181 (90.13 ± 1.47) a 174 (86.58 ± 2.03) a 169 (84.06 ± 4.19) a 2.5µM NSC 207 197 (95.19 ± 0.53) a 180 (86.94 ± 1.43) a 172 (83.09 ± 1.96) b 129 (62.38 ± 3.67) b 122 (58.92 ± 0.51) b 5µM NSC 188 181 (95.97 ± 4.38) a 165 (87.55 ± 5.81) a 151 (80.13 ± 4.51) b 129 (68.24 ± 7.14) b 125 (66.14 ± 6.43) b 10µM NSC 193 182 (94.14 ± 2.87) a 26 (12.87 ± 11.31) b 3 (1.47 ± 1.45) c 0 0 c 0 0 c Note: The 2-cell rate, 4-cell rate, 8-cell rate, morula rate and blastocyst rate were calculated as the number of 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts. out of the total number of prokaryotic embryos cultured in vitro × 100%, respectively. Different superscript letters in the same column indicate significant difference ( P < 0.05). Figure 3. NSC668394 inhibited ezrin Thr567 phosphorylation in early embryos. When cultured in KSOM containing 2.5 µM NSC668394 the expression of p-Ezrin Thr567 were significantly reduced. As the concentration of NSC668394 increased, p-ezrin Thr567 was further decreased but still localized in the cell cortex region. Scale bar, 50 µm. Table 1 . Effects of the addition of NSC to embryo culture medium on early embryonic development. Note The 2-cell rate, 4-cell rate, 8-cell rate, morula rate and blastocyst rate were calculated as the number of 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts. out of the total number of prokaryotic embryos cultured in vitro × 100%, respectively. Different superscript letters in the same column indicate significant difference ( P < 0.05). 3.3 Inhibiting the phosphorylation of ezrin-Thr567 in mouse GV-stage oocytes affects the maturation, fertilization and developmental potential of eggs. Considering the expression of p-ezrin Thr567 in both GV and MII stage oocytes in mice, we hypothesize that interfering with the phosphorylation of ezrin at Thr567 during oocyte maturation would also affect its maturation, fertilization and developmental potential. To verify our hypothesis, GV stage oocytes of mouse were cultured in IVM medium containing 0, 2.5, 5, or 10 µM NSC668394 respectively, and then the maturation rate, fertilization rate of oocytes and embryonic development rate of each group at different stages were calculated. As shown in Fig. 4, 2.5 µM NSC668394 dramatically decreased the expression of p-ezrin Thr567 in oocytes and significantly reduced their competence of maturation, fertilization and development ( P < 0.05). When the concentration of NSC668394 reached 10 µM, the oocyte maturation rate did not decrease further. However, the developmental potential of early embryos decreased further dramatically, and no zygote developed to the 8-cell stage (Table 2 ). Table 2 Effect of NSC addition to IVM medium on oocyte maturation and early embryonic developmental potential. Groups GV MII (Maturity rate /%) Fertilized (Fertilization rate / %) 4-cell (4- cell rate / %) 8-cell (8- cell rate / %) Morula (Morula rate / %) Blastocyst (Blastocyst rate/ %) 0.1%DMSO 153 119 (77.24 ± 1.69) a 75 (63.37 ± 3.09) a 74 (99.19 ± 1.61) a 72 (98.39 ± 3.23) a 67 (92.21 ± 8.20) a 66 (91.40 ± 9.73) a 0µM NSC 162 130 (80.17 ± 2.04) a 81 (62.14 ± 1.07) a 77 (94.09 ± 4.57) a 77 (94.09 ± 4.57) a 74 (91.67 ± 2.47) a 74 (91.67 ± 2.47) a 2.5µM NSC 155 75 (48.91 ± 2.47) b 35 (45.92 ± 3.27) b 34 (98.21 ± 3.57) a 33 (97.32 ± 4.16) a 17 (48.21 ± 3.57) b 17 (48.21 ± 3.57) b 5µM NSC 151 93 (61.11 ± 3.82) c 54 (56.52 ± 6.11) c 51 (94.54 ± 4.36) a 50 (93.50 ± 4.45) a 24 (44.59 ± 6.28) b 24 44.59 ± 6.28) b 10µM NSC 155 95 (61.17 ± 1.07) c 34 (35.78 ± 2.36) d 4 (11.14 ± 7.63) a 0 0 b 0 0 c 0 0 c Note: Maturation rate was calculated as the number of MII-stage oocytes out of the number of GV-stage oocytes cultured in vitro × 100%; Fertilization rate was calculated as the number of cleaved embryos out of the number of oocytes exposed to sperm × 100%; 4-cell rate, 8-cell rate, morula formation rate and blastocyst formation rate were calculated as the number of 4-cell embryos, 8-cell embryos, morula and blastocysts out of the number of cleaved embryos × 100%, respectively. Different superscript letters in the same column indicate significant difference ( P < 0.05). (the same below). Figure 4. NSC668394 inhibited phosphorylation of Ezrin Thr567 in oocytes. When cultured in medium containing 2.5 µM NSC668394, the fluorescence intensity of p-Ezrin (Thr567) in mouse oocytes decreased remarkably compared with that in the control group. Scale bar, 50 µm. Table 2 . Effect of NSC addition to IVM medium on oocyte maturation and early embryonic developmental potential. Note Maturation rate was calculated as the number of MII-stage oocytes out of the number of GV-stage oocytes cultured in vitro × 100%; Fertilization rate was calculated as the number of cleaved embryos out of the number of oocytes exposed to sperm × 100%; 4-cell rate, 8-cell rate, morula formation rate and blastocyst formation rate were calculated as the number of 4-cell embryos, 8-cell embryos, morula and blastocysts out of the number of cleaved embryos × 100%, respectively. Different superscript letters in the same column indicate significant difference ( P < 0.05). (the same below). 3.4 Interfering with the phosphorylation of ezrin at Thr567 during IVF inhibits fertilization and subsequent embryonic developmental potential. Since inhibiting the phosphorylation of ezrin at Thr567 during oocytes IVM or embryos IVC can affect mouse embryonic development. So, would reducing the expression of p-ezrin Thr567 during fertilization affect the competence of fertility and development of mouse oocytes? To test our speculation, recovered MII oocytes were fertilized in vitro with capacitated sperm in IVF medium containing different concentrations (0, 2.5, 5, and 10µM) of NSC668394, then transferred to KSOM medium without NSC668394 for further culture. The results showed that inhibiting the phosphorylation of ezrin Thr567 in oocytes during in vitro fertilization significantly reduced the fertilization rate, and the rates of morula and blastocyst formation ( P < 0.05). Specifically, 10.0 µM NSC668394 also arrested fertilized eggs at the 2-cell stage (Table 3 ). Table 3 Effects of NSC668394 in IVF medium on early embryo development. Groups MII Fertilized (Fertilization rate / %) 4-cell (4- cell rate / %) 8-cell (8- cell rate / %) Morula (Morula rate / %) Blastocyst (Blastocyst rate/ %) 0.1%DMSO 198 155 (78.35 ± 2.37) a 152 (98.06 ± 1.89) a 148 (95.55 ± 4.31) a 141 (90.98 ± 6.68) a 137 (88.42 ± 8.57) a 0µM NSC 198 160 (80.90 ± 2.89) a 156 (97.51 ± 2.89) a 154 (96.25 ± 5.01) a 147 (91.84 ± 2.96) a 139 (86.85 ± 6.59) a 2.5µM NSC 200 108 (53.95 ± 3.83) b 102 (94.22 ± 3.42) a 98 (90.05 ± 10.43) a 53 (48.94 ± 2.22) b 49 (44.91 ± 6.95) b 5µM NSC 201 119 (59.29 ± 4.46) b 113 (94.95 ± 4.30) a 108 (90.62 ± 4.33) a 67 (56.58 ± 7.28) b 62 (52.16 ± 1.64) b 10µM NSC 201 68 (33.80 ± 2.83) c 10 (15.00 ± 13.23) b 0 0 b 0 0 c 0 0 c 3.5 Microinjection of ezrin-T567D mRNA partly rescued the competence of maturation, fertilization and developmental potential of oocytes treated with NSC668394 To confirm the decline of maturation, fertilization, and the developmental competence of oocytes or embryos treated with NSC668394 was caused by the inhibition of ezrin Thr567 phosphorylation, rescue experiments were conducted. When GV oocytes, MII oocytes and prokaryotic embryos treated with 2.5 µM NSC668394 during in vitro maturation, in vitro fertilization, or in vitro culture were microinjected with ezrin-T567D mRNA, the maturation rate and the fertilization rate of oocytes, and the developmental potential of early embryos were partially restored (Fig. 5). Figure 5. Microinjection of Ezrin-T567D mRNA partially rescued the reduction of the maturation rate and the fertilization rate of oocytes and the developmental potential of early embryos caused by NSC668394. (A) The development rate of different stage of embryos that treated with 2.5 µM NSC668394 during in vitro culture and microinjected with ezrin T567D mRNA at prokaryotic stage. (B) The maturation rate, fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 µM NSC668394 during in vitro maturation and microinjected with ezrin T567D mRNA at GV stage. (C) The fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 µM NSC668394 during in vitro fertilization and microinjected with ezrin T567D mRNA at MII stage. Note: Different lowercase letters indicate significant differences ( P < 0.05). 4 Discussion Ezrin is a multi-domain protein mainly composed of ERM association domains (ERMADs) located at the N- and C-termini, and a central connecting region. The N- and C-ERMADs can bind to each other to keep the protein in an inactive state. Phosphorylation of Thr567 can release the binding between N- and C-ERMADs, activate the protein, and participate in the reshaping of cell surface structures[ 33 , 46 ]. Overexpression of a phosphomimetic or non-phosphorylatable mutant of ezrin Thr567 in mouse embryos both inhibited compaction of morulae and the formation of blastocoel[ 47 ]. However, the impact of inhibiting ezrin Thr567 phosphorylation on mouse oocyte maturation, fertilization, and pre-implantation embryonic development remains unclear. In this study, we found that adding NSC668394, a small molecule inhibitor specifically targeting ezrin Thr567 phosphorylation, to the culture medium can inhibit the phosphorylation of ezrin Thr567 in mouse oocytes and early embryos (Fig. 3, Fig. 4), and block oocyte maturation, fertilization, and early embryonic development. The maturation of oocytes involves a long and complex process, including the reorganization of the cytoplasmic membrane, changes in cortical tension[ 48 ], deformation of surface microvilli[ 35 ], establishment of oocyte polarity[ 49 ], migration, assembly, and localization of the spindle apparatus[ 50 ], as well as meiotic division and extrusion of polar bodies, ultimately leading to the acquisition of the ability to be fertilized and develop into an embryo. All these processes are regulated by the cytoskeletal network composed of actin microfilaments. Studies have shown that the ERM protein family is involved in mediating oocyte maturation[ 51 , 52 ]. The effect of ezrin on oocyte development may be regulated by the phosphorylation of ezrin Thr567, and there is currently limited research on the role of ezrin T567 phosphorylation in oocyte maturation. NSC668394 specifically inhibits the phosphorylation of ezrin T567[ 19 ]. Here, we found that the treatment with NSC668394 only during the oocyte maturation not only significantly reduced the in vitro maturation rate of oocytes but also affected the i n vitro fertilization and early embryo development potential. There are two possibie explanations for this result, one is that the inhibition of ezrin T567 phosphorylation by NSC668394 may be persistent or irreversible, and the other is that the abnormalities in oocytes caused by the inhibition of ezrin T567 phosphorylation affected the competence of fertilization and subsequent embryonic development. Since generally the phosphorylation and dephosphorylation of proteins are highly dynamic, we prefer the later explanation. Fertilization involves complex membrane and cortical cytoskeleton remodeling process, including acrosome outer membrane fuses with sperm plasm membrane, sperm membrane binds to the microvilli of the oocyte and fuses with oocyte membrane. Previous studies have shown that ezrin phosphorylation is also involved in the process of sperm acquisition and regulates the acrosome reaction[ 53 ]. Ezrin is involved in the formation and maintenance of cell skeletons and microvilli, and small molecule inhibitors of ezrin Thr567 phosphorylation affect the development of microvilli, which in turn affects sperm acquisition and entry into the ooplasm. Furthermore, ezrin activation allows it to bind to its target proteins and form an F-actin-ezrin-membrane protein complex, which plays an important role in the acrosome reaction and fertilization. NSC668394 treatment blocked the activation of Ezrin by inhibiting ezrin Thr567 phosphorylation, and indirectly inhibiting the formation of the F-actin-ezrin-membrane protein complex, which affects oocyte in vitro fertilization. Even if some oocytes completed fertilization successfully, since the microstructure had been interfered, the developmental potential of early embryos was affected. Embryonic development is often accompanied by cell polarization, cell division, and cell migration. As mentioned previously, ezrin is involved in cell polarization and embryonic compaction[ 54 ]. Guang et al.[ 55 ] found that ezrin accumulates excessively in the contractile ring and cleavage furrow during cell division. Therefore, we speculate that ezrin Thr567 phosphorylation may be involved in early embryonic development. The results show that NSC668394 inhibits embryonic compaction significantly (Table 1 ), which is similar to previous studies suggesting that ezrin phosphorylation is involved in regulating embryonic compaction[ 37 , 56 ]. Treating zebrafish embryos with10µM NSC668394 resulted in a unique optokinetic response phenotype[ 38 ]. The optokinetic response phenotype persisted even after washing away the small molecule compound, suggesting that defects caused by NSC668394 through inhibiting ezrin Thr567 phosphorylation seems to be persistent or irreversible. Since ezrin T567D protein mimics the conformation of p-ezrin T567, so it can excise the function of p-ezrin Thr567 to rescue oocyte maturation, fertilization and early embryonic development which reduced by NSC668394 through inhibiting ezrin T567 phosphorylation. However, microinjection of ezrin T567D mRNA did not completely restored the maturation rate, fertilization rate and the dev-elopment potential affected by NSC668394, suggesting mouse oocytes and emb-ryos require dynamic phosphorylation-dephosphorylation rather than sustained phosphorylation of ezrin. 5. Conclusion Here we investigated the impact of ezrin Thr567 phosphorylation on the development of mouse preimplantation embryos by specifically inhibiting ezrin Thr567 phosphorylation with NSC668394 and rescuing with expression of ezrin T567D mutant. Inhibiting the phosphorylation of ezrin Thr567 during in vitro maturation or in vitro fertilization of mouse oocytes not only affected their maturation and fertilization, but also reduced the development competence of preimplantation embryos. Microinjection of mRNA encoding ezrin T567D mutant can partially rescue the developmental defects of mouse oocytes, fertilization, and early embryonic development caused by NSC668394. Declarations Data availability All the data in this paper are authentic and reliable, and the animal models involved are reproducible. Ethics declarations Ethics approval and consent to participate All animal experiments were conducted in accordance with the regulations of the People's Republic of China on the Management of Laboratory Animals and were approved by the Animal Care and Use Committee of Anhui Agricultural University. Consent for publication Not applicable Author contributions Huihui Xie, Bochao Zhang and Jiankun Cui participated in some experiments, performed statistical analysis and drafted the manuscript. Tiandong Xia and Wei Qian performed most experiments. Qing Yi, Mengdi Han, Hongyan Liao, Yuke Jia, Meng Cao and Yanqiuhong Li collect the oocytes and in vitro mature, Fuqiang Pan participated some sample selection, Yunsheng Li and Fugui Fang participated in the design of the experiments. Yunhai Zhang and Yinghui Ling involved in revising the manuscript. Ya Liu conceived of this study. All authors have read this manuscript and approved its publication. Declaration of competing interest The authors have no conflict of interest to disclose. Acknowledgments We are especially grateful to all participants in this study. 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Equatorial Assembly of the Cell-Division Actomyosin Ring in the Absence of Cytokinetic Spatial Cues. Curr Biol. 2018;28. Pan M-H, Wang F, Lu Y, Tang F, Duan X, Zhang Y, et al. FHOD1 regulates cytoplasmic actin-based spindle migration for mouse oocyte asymmetric cell division. J Cell Physiol. 2018;233:2270–8. Anani S, Bhat S, Honma-Yamanaka N, Krawchuk D, Yamanaka Y. Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo. Development. 2014;141:2813–24. Korotkevich E, Niwayama R, Courtois A, Friese S, Berger N, Buchholz F et al. The Apical Domain Is Required and Sufficient for the First Lineage Segregation in the Mouse Embryo. Dev Cell. 2017;40. Ciesielski HM, Nishida H, Takano T, Fukuhara A, Otani T, Ikegawa Y, et al. Erebosis, a new cell death mechanism during homeostatic turnover of gut enterocytes. PLoS Biol. 2022;20:e3001586. La Verde G, Artiola V, Panzetta V, Pugliese M, Netti PA, Fusco S. Cytoskeleton Response to Ionizing Radiation: A Brief Review on Adhesion and Migration Effects. Biomedicines. 2021;9. Vicente-Manzanares M, Webb DJ, Horwitz AR. Cell migration at a glance. J Cell Sci. 2005;118:4917–9. Friedl P, Wolf K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol. 2010;188:11–9. Gaeta IM, Meenderink LM, Postema MM, Cencer CS, Tyska MJ. Direct visualization of epithelial microvilli biogenesis. Curr Biol. 2021;31. Orbach R, Su X. Surfing on Membrane Waves: Microvilli, Curved Membranes, and Immune Signaling. Front Immunol. 2020;11:2187. Fitz GN, Weck ML, Bodnya C, Perkins OL, Tyska MJ. Protrusion growth driven by myosin-generated force. Dev Cell. 2023;58. Cohen J, Wang L, Marques S, Ialy-Radio C, Barbaux S, Lefèvre B, et al. Oocyte ERM and EWI Proteins Are Involved in Mouse Fertilization. Front Cell Dev Biol. 2022;10:863729. Wu Y, Zuo Z, Wang Z, Liu H, Zhou Q, Ren S et al. Bta-miR-183 targets ezrin to regulate microvilli formation and improve early development of bovine embryos. Reproduction. 2023. Louvet S, Aghion J, Santa-Maria A, Mangeat P, Maro B. Ezrin becomes restricted to outer cells following asymmetrical division in the preimplantation mouse embryo. Dev Biol. 1996;177:568–79. Dard N, Louvet-Vallée S, Santa-Maria A, Maro B. Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo. Dev Biol. 2004;271:87–97. Liu H, Wu Z, Shi X, Li W, Liu C, Wang D, et al. Atypical PKC, regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cell embryocompaction. Dev Biol. 2013;375:13–22. Bulut G, Hong SH, Chen K, Beauchamp EM, Rahim S, Kosturko GW, et al. Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells. Oncogene. 2012;31:269–81. Paige M, Kosturko G, Bulut G, Miessau M, Rahim S, Toretsky JA, et al. Design, synthesis and biological evaluation of ezrin inhibitors targeting metastatic osteosarcoma. Bioorg Med Chem. 2014;22:478–87. Rouven Brückner B, Pietuch A, Nehls S, Rother J, Janshoff A. Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells. Sci Rep. 2015;5:14700. Xiang Y, Zhou C, Zeng Y, Guo Q, Huang J, Wu T, et al. NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro. Front Cell Dev Biol. 2021;9:704341. Zhang D, Yu F, Li H, Wang Q, Wang M, Qian H, et al. AgNPs reduce reproductive capability of female mouse for their toxic effects on mouse early embryo development. Hum Exp Toxicol. 2022;41:9603271221080235. Zhang D, Jing H, Dou C, Zhang L, Wu X, Wu Q, et al. Supplement of Betaine into Embryo Culture Medium Can Rescue Injury Effect of Ethanol on Mouse Embryo Development. Sci Rep. 2018;8:1761. Pedersen HS, Liu Y, Foldager L, Callesen H, Larsen K, Sørensen MT. Calibration of sperm concentration for in vitro fertilization in a mouse reprotoxicity model. Toxicol In Vitro. 2019;55:58–61. Yu T, Qi X, Zhang L, Ning W, Gao D, Xu T, et al. Dynamic reprogramming and function of RNA N6-methyladenosine modification during porcine early embryonic development. Zygote (Cambridge England). 2021;29:417–26. Austermann J, Nazmi AR, Müller-Tidow C, Gerke V. Characterization of the Ca2+ -regulated ezrin-S100P interaction and its role in tumor cell migration. J Biol Chem. 2008;283:29331–40. Dard N, Louvet S, Santa-Maria A, Aghion J, Martin M, Mangeat P, et al. In vivo functional analysis of ezrin during mouse blastocyst formation. Dev Biol. 2001;233:161–73. Uray IP, Uray K. Mechanotransduction at the Plasma Membrane-Cytoskeleton Interface. Int J Mol Sci. 2021;22. Kirillova A, Smitz JEJ, Sukhikh GT, Mazunin I. The Role of Mitochondria in Oocyte Maturation. Cells. 2021;10. Nunes V, Ferreira JG. From the cytoskeleton to the nucleus: An integrated view on early spindle assembly. Semin Cell Dev Biol. 2021;117:42–51. Speck O, Hughes SC, Noren NK, Kulikauskas RM, Fehon RG. Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature. 2003;421:83–7. Larson SM, Lee HJ, Hung P-h, Matthews LM, Robinson DN, Evans JP. Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins. Mol Biol Cell. 2010;21:3182–92. Huta Y, Nitzan Y, Breitbart H. Ezrin protects bovine spermatozoa from spontaneous acrosome reaction. Theriogenology. 2020;151:119–27. Humięcka M, Szpila M, Kłoś P, Maleszewski M, Szczepańska K. Mouse blastomeres acquire ability to divide asymmetrically before compaction. PLoS ONE. 2017;12:e0175032. Yang G, Hiruma S, Kitamura A, Kinjo M, Mishra M, Uehara R. Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis. Exp Cell Res. 2021;403:112600. Ezoe K, Miki T, Ohata K, Fujiwara N, Yabuuchi A, Kobayashi T, et al. Prolactin receptor expression and its role in trophoblast outgrowth in human embryos. Reprod Biomed Online. 2021;42:699–707. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-3872230","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268707199,"identity":"f7e0cd40-1e4f-4b41-94b0-0b21c3c04c60","order_by":0,"name":"Huihui Xie","email":"","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huihui","middleName":"","lastName":"Xie","suffix":""},{"id":268707200,"identity":"2bdbc572-0ac2-49cb-bc13-9acac643f772","order_by":1,"name":"Tiandong Xia","email":"","orcid":"","institution":"Hefei PreceDo Pharmaceuticals Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tiandong","middleName":"","lastName":"Xia","suffix":""},{"id":268707201,"identity":"1a499729-3608-4f72-85e9-d9385dd2585f","order_by":2,"name":"Bochao Zhang","email":"","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bochao","middleName":"","lastName":"Zhang","suffix":""},{"id":268707202,"identity":"e17efe21-54b2-4289-948f-fe14562ab634","order_by":3,"name":"Jiankun Cui","email":"","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fugui","middleName":"","lastName":"Fang","suffix":""},{"id":268707213,"identity":"49f544d3-a0af-4815-98df-837d43ff0a87","order_by":14,"name":"Yunhai Zhang","email":"","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunhai","middleName":"","lastName":"Zhang","suffix":""},{"id":268707214,"identity":"4084e706-bc67-4bd3-9b81-af761c6d5299","order_by":15,"name":"Yinghui Ling","email":"","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yinghui","middleName":"","lastName":"Ling","suffix":""},{"id":268707215,"identity":"3c549d32-716f-440f-8313-7a59033d648e","order_by":16,"name":"Ya Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACPmYQWcGQAObxEKOFDazlDElaQARjG0la2HnMJH7Os8vjn5HA+OBtG4O8OWGH8ZhJ9m5LLpa4kcBsOLeNwXBnAxFaJHi3MSc23Ehgk+YFutDgADG2/J1Tnzj/RgL7b6K1SPM2HE7cALSFmUgtbMXWMseOJ24887BZcs45CcMNhLTw8x/eePNNTXXivOPJBz+8KbORJ2gLELBIQGjGBiAhQVg9EDB/IErZKBgFo2AUjFwAAM9ZOESR4hpjAAAAAElFTkSuQmCC","orcid":"","institution":"Department of veterinary medicine, College of Animal Science and Technology, Anhui Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-17 08:00:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3872230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3872230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50111115,"identity":"e102b578-7452-40c9-9f17-9a29e1cd81cb","added_by":"auto","created_at":"2024-01-24 17:21:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10724499,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization of Ezrin in mouse oocytes and early embryos at different developmental stages.\u003c/p\u003e","description":"","filename":"figure1inpngformat.png","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/3b4a490cb1ef06682b5190f9.png"},{"id":50111313,"identity":"db96a1e6-169f-42ba-816a-dbbcddfbfbc4","added_by":"auto","created_at":"2024-01-24 17:29:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8627181,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization of p-Ezrin Thr567 in mouse oocytes and early embryos at different developmental stages.\u003c/p\u003e","description":"","filename":"figure2inpngformat.png","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/b18bbce01aa54fe0db43d994.png"},{"id":50111112,"identity":"43bdd515-52bb-4c50-8e61-7d880f5e5625","added_by":"auto","created_at":"2024-01-24 17:21:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1021682,"visible":true,"origin":"","legend":"\u003cp\u003eNSC668394 inhibited ezrin Thr567 phosphorylation in early embryos. When cultured in KSOM containing 2.5 μM NSC668394 the expression of p-Ezrin Thr567 were significantly reduced. As the concentration of NSC668394 increased, p-ezrin Thr567 was further decreased but still localized in the cell cortex region. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"figure3inTIFFformat.png","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/4d4c8a92cede5b94e317277a.png"},{"id":50111312,"identity":"b8fddeb0-b59f-4a79-97cc-2e92b8b971e7","added_by":"auto","created_at":"2024-01-24 17:29:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376127,"visible":true,"origin":"","legend":"\u003cp\u003eNSC668394 inhibited phosphorylation of Ezrin Thr567 in oocytes. When cultured in medium containing 2.5 μM NSC668394, the fluorescence intensity of p-Ezrin (Thr567) in mouse oocytes decreased remarkably compared with that in the control group. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"figure4inTIFFformat.png","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/76c91e997af2a457529fc29d.png"},{"id":50111111,"identity":"2f2930a2-f07f-4c47-9121-7d62a5c01bfa","added_by":"auto","created_at":"2024-01-24 17:21:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1437459,"visible":true,"origin":"","legend":"\u003cp\u003eMicroinjection of Ezrin-T567D mRNA partially rescued the reduction of the maturation rate and the fertilization rate of oocytes and the developmental potential of early embryos caused by NSC668394. (A) The development rate of different stage of embryos that treated with 2.5 μM NSC668394 during\u003cem\u003e in vitro \u003c/em\u003eculture and microinjected with ezrin T567D mRNA at prokaryotic stage.(B) The maturation rate, fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 μM NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e maturation and microinjected with ezrin T567D mRNA at GV stage. (C) The fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 μM NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e fertilization and microinjected with ezrin T567D mRNA at MII stage. Note：Different lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"figure5inTIFFformat.png","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/a5a693498a683d49d7e7d95c.png"},{"id":51262901,"identity":"435be824-cbed-4837-8643-a8fc6af2543d","added_by":"auto","created_at":"2024-02-17 10:58:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3349130,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3872230/v1/fb6c6047-9483-49ec-b135-bde42385dfde.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eERM (ezrin/radixin/moesin) is an evolutionarily highly conserved family of proteins, consisting of a FERM domain at the amino terminus, a C-ERMAD (ERM-association domains) domain at the carboxyl terminus, and an α-helical structure in the middle that connects the two domains.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] They cross-link membrane proteins to actin microfilaments directly or indirectly through FERM domains[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and localized in plasma membrane protrusions (such as microvilli, pseudopodia and contractile fibers), intercellular junctions, and mitotic division furrows[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], involved in the formation of cell surface structures and the maintenance of cell polarity[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], cell migration, intercellular adhesion[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], cell division and signal transduction[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a member of the ERM family, the C-terminal domain of ezrin can interact with the N-terminal domain[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This intramolecular or intermolecular head-to-tail connection shields its interaction with the cell membrane and the actin microfilament skeleton, leaving it in an inactive state and localized in the cytoplasm[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Phosphorylation is an important way to regulate protein activity. Ezrin has multiple phosphorylation sites, such as Ser66[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]、Thr213[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]、Thr332[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]、Thr567[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]、Tyr145、Tyr353 and Tyr477[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Phosphorylation of Thr567 is currently considered a sign of ezrin activation and is also the most studied phosphorylation site[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOocyte maturation, fertilization, and early embryonic development are accompanied by cell surface remodeling[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], cell division[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], cell polarization[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], intercellular junction formation, cell adhesion[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] cell migration [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and other activities[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Studies have shown that ERM proteins are expressed in oocytes and early embryos. Knocking down any one of ERM proteins alone cannot inhibit \u003cem\u003ein vitro\u003c/em\u003e fertilization of zona-free eggs of mouse, but knocking down ERM comprehensively can significantly reduce the fertilization index of mouse zona-free oocytes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Another study found that inhibiting the expression of ezrin significantly increased the cleavage rate and blastocyst development rate of bovine somatic cell nuclear transfer embryos[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These studies show that ERM proteins, especially ezrin, are involved in regulating oocyte fertilization and early embryonic development. Louvet S found that ezrin is expressed in mouse oocytes and pre-implantation embryos accompanying dynamic phosphorylation modifications throughout the development[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Overexpression of Ez-T567D, a mutant that mimics the phosphorylation state of ezrin Thr567 in mouse 2-cell embryos inhibited the embryonic compaction and blastocyst formation, while overexpression of non-phosphorylated mutant Ez-T567A did not affect embryo compaction, but interfered with the formation of the blastocyst cavity. These results suggest that ezrin Thr567 phosphorylation is involved in regulating early embryonic development[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Further studies found that atypical PKC (aPKC) phosphorylates ezrin Thr567 during embryonic densification[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, it is still unclear at which stage of oocyte and early embryo development the phosphorylation of ezrin Thr567 mainly occurs, and what effect inhibiting the phosphorylation of ezrin Thr567 will have on oocyte maturation, fertilization and development potential.\u003c/p\u003e \u003cp\u003eNSC668394 is a small molecule compound that specifically inhibits the phosphorylation of ezrin Thr567[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we used NSC668394 to inhibit the phosphorylation of ezrin Thr567 during the \u003cem\u003ein vitro\u003c/em\u003e maturation, \u003cem\u003ein vitro\u003c/em\u003e fertilization and early embryo culture of mouse oocytes. We found that inhibiting the phosphorylation of ezrin Thr567 not only affected oocyte maturation and fertilization, but also affected its developmental potential, and when a mutant that mimics ezrin Thr567 phosphorylation is expressed at the same time, its maturation, fertilization and development are successfully rescued.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental Animals and Ethical Standards\u003c/h2\u003e \u003cp\u003eSix to eight week-old ICR mice were provided ad libitum access to food and water and maintained under controlled environmental conditions of temperature (23℃ to 26℃), natural light. All animal experiments were conducted in accordance with the regulations of the People's Republic of China on the Management of Laboratory Animals and were approved by the Animal Care and Use Committee of Anhui Agricultural University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sperm preparation\u003c/h2\u003e \u003cp\u003eHealthy ICR male mice with normal reproductive ability were euthanized, and both epididymal tails were aseptically removed, minced, and placed in capacitation medium (M2050, Easycheck) to release sperm. The mixture was then incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 30\u0026ndash;60 minutes to achieve capacitation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Oocyte collection, in vitro maturation, in vitro fertilization and embryo in vitro culture\u003c/h2\u003e \u003cp\u003eCollection of GV-stage, MII-stage oocytes, and pronuclear-stage embryos: As described in literatures[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], in brief, 6\u0026ndash;8 week-old female ICR mice were injected intraperitoneally with 10 IU of Pregnant Mare Serum Gonadotropin. (PMSG, Ningbo Sansheng Pharmaceutical Co., Ltd.). Forty-eight hours later, the mice were euthanized and ovarian follicles with a cavity were punctured to release cumulus-oocyte complexes (COCs) containing GV-stage oocytes, or the mice were injected intraperitoneally with 10 IU of PMSG, followed by injection of Human Chorionic Gonadotrophin (HCG, Ningbo Sansheng Pharmaceutical Co., Ltd.) and 13\u0026ndash;16 hours later to collect MII-stage oocytes from the ampulla of the oviduct. Alternatively, the mice were placed together with males immediately after HCG injection, and pronuclear-stage embryos were collected 18 hours later[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The collected oocytes or pronuclear-stage embryos were transferred into M2 medium (M7167, Sigma) containing 0.3% hyaluronidase (H3506, Solarbio), and the cumulus cells were removed by pipetting.\u003c/p\u003e \u003cp\u003eIn Vitro Maturation (IVM): The GV-stage oocytes were transferred to TCM199 medium (Sigma) containing 10% fetal calf serum (FCS, Gibico), and cultured in an incubator at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% humidity for 20 hours. The maturation rate was calculated based on the extrusion of the first polar body.\u003c/p\u003e \u003cp\u003eIn vitro fertilization (IVF): Matured oocytes were incubated in HTF medium (MR-070-D, Milipore) containing 10\u0026micro;L of capacitated sperm suspension obtained as described above at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 4\u0026ndash;6 hours[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The fertilization rate was calculated based on the formation of pronuclear and the extrusion of the second polar body.\u003c/p\u003e \u003cp\u003eEmbryos IVC: The prokaryotic embryos were transferred into K\u003csup\u003e+\u003c/sup\u003e Simplex Optimised Medium (KSOM, MR-020P-5F, Milipore) and cultured in an incubator at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% humidity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eAs described in reference[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], oocytes or embryos were fixed and permeabilized, and then incubated in primary antibody for Ezrin (#3145, CST, 1:100) or p-Ezrin Thr567 (EP886Y, Abcam, 1:100) at 4\u0026deg;C overnight followed by incubation with FITC-labeled secondary antibody (HS111, TransGen Biotech, diluted at a 1:100 ratio in 1% BSA) for 1 hour and DAPI (D8200, Roche, diluted at a 1:10000 ratio) for 10 minutes at room temperature. After washing 3 times, samples were mounted on a slide and covered with a coverslip, and then were imaged under an inverted fluorescence microscope (DP72 model, Olympus) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In vitro transcription and microinjection\u003c/h2\u003e \u003cp\u003eAccording to the instructions of the TR101-01 kit (Vazyme), mRNA encoding EGFP-Ezrin T567D was transcribed in vitro and purified, and the concentration was adjusted to 0.5 \u0026micro;g/\u0026micro;L. The eggs to be injected were transferred into M2 medium, and the EGFP-Ezrin T567D mRNA solution was injected into each egg at a volume of 5\u0026ndash;10 pL per egg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was repeated three or more times, and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). Data analysis was performed using SPSS statistics 17.0, using one-way analysis of variance (ANOVA) with a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Expression and localization of Ezrin and p-Ezrin Thr567 in mouse oocytes and preimplantation embryos\u003c/h2\u003e \u003cp\u003eTo investigate the expression and localization pattern of Ezrin and p-Ezrin Thr567 in mouse oocytes and preimplantation embryos, GV-stage, MⅡ-stage oocytes and early embryos at different stages were collected for immunofluorescence analysis. All samples were imaged using the same exposure time under a fluorescence microscope. Figure\u0026nbsp;1 shows the expression and localization of ezrin in mouse oocyte and preimplantation embryos at different developmental stages. In GV stage oocytes, 4-cell stage, 8-cell stage and morula, ezrin is distributed in both the cytoplasm and nucleus, and the fluorescence intensity in the nucleus is significantly higher than that in the cytoplasm, while in MⅡ stage oocytes, prokaryotic embryo and 2-cell stage embryo the fluorescence signal of ezrin gradually weakens and is evenly distributed within the cells. In blastocyst, ezrin is mainly located in the cell cortex area ( Fig.\u0026nbsp;1 ). From GV stage oocyte to morula, p-Ezrin Thr567 is primarily localized in the cell cortex area and the intercellular junctions, and in 4-cell stage embryo and blastocyst p-ezrin Thr567 is also distributed in nucleus ( Fig.\u0026nbsp;2 ).\u003c/p\u003e \u003cp\u003eFigure 1: Localization of Ezrin in mouse oocytes and early embryos at different developmental stages.\u003c/p\u003e \u003cp\u003eFigure 2. Localization of p-Ezrin Thr567 in mouse oocytes and early embryos at different developmental stages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 NSC668394 inhibits the phosphorylation of Ez-T567 and influences the development of early embryos.\u003c/h2\u003e \u003cp\u003eTo test the inhibitory effect of NSC668394 on ezrin Thr567 phosphorylation in mouse embryos, prokaryotic embryos were cultured in KSOM medium containing different concentration (0, 2.5, 5, and 10 \u0026micro;M) of NSC668394 and fixed at 2-cell stage for immunofluorescence staining. Immunofluorescence images were captured with the same exposure time. The results showed that 2.5 \u0026micro;M of NSC668394 remarkably inhibited ezrin Thr567 phosphorylation in the embryos. As the concentration of NSC668394 increased, there was a trend of further reduction in p-Ezrin-Thr567 levels (Fig.\u0026nbsp;3). Corresponding to the reduction of p-ezrin Thr567 in embryos, the rates of 8-cell, morula and blastocyst were significantly decreased ( Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of the addition of NSC to embryo culture medium on early embryonic development.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003egroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eprokaryotic stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-cell\u003c/p\u003e \u003cp\u003e(2-cell rate /%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4-cell\u003c/p\u003e \u003cp\u003e(2-cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8-cell\u003c/p\u003e \u003cp\u003e(8-cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMorula\u003c/p\u003e \u003cp\u003e(Morula rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBlastocyst\u003c/p\u003e \u003cp\u003e(Blastocyst rate/ %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1%DMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181\u003c/p\u003e \u003cp\u003e(96.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e174\u003c/p\u003e \u003cp\u003e(92.61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.02) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e148\u003c/p\u003e \u003cp\u003e(88.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e159\u003c/p\u003e \u003cp\u003e(84.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e154\u003c/p\u003e \u003cp\u003e(88.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.98) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e193\u003c/p\u003e \u003cp\u003e(96.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185\u003c/p\u003e \u003cp\u003e(92.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e181\u003c/p\u003e \u003cp\u003e(90.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e174\u003c/p\u003e \u003cp\u003e(86.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e169\u003c/p\u003e \u003cp\u003e(84.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e197\u003c/p\u003e \u003cp\u003e(95.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e180\u003c/p\u003e \u003cp\u003e(86.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e172\u003c/p\u003e \u003cp\u003e(83.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e129\u003c/p\u003e \u003cp\u003e(62.38\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e122\u003c/p\u003e \u003cp\u003e(58.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181\u003c/p\u003e \u003cp\u003e(95.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e165\u003c/p\u003e \u003cp\u003e(87.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e151\u003c/p\u003e \u003cp\u003e(80.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e129\u003c/p\u003e \u003cp\u003e(68.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.14) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e125\u003c/p\u003e \u003cp\u003e(66.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e182\u003c/p\u003e \u003cp\u003e(94.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003cp\u003e(12.87\u0026thinsp;\u0026plusmn;\u0026thinsp;11.31)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e(1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: The 2-cell rate, 4-cell rate, 8-cell rate, morula rate and blastocyst rate were calculated as the number of 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts. out of the total number of prokaryotic embryos cultured \u003cem\u003ein vitro\u003c/em\u003e \u0026times; 100%, respectively. Different superscript letters in the same column indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure 3. NSC668394 inhibited ezrin Thr567 phosphorylation in early embryos. When cultured in KSOM containing 2.5 \u0026micro;M NSC668394 the expression of p-Ezrin Thr567 were significantly reduced. As the concentration of NSC668394 increased, p-ezrin Thr567 was further decreased but still localized in the cell cortex region. Scale bar, 50 \u0026micro;m.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Effects of the addition of NSC to embryo culture medium on early embryonic development.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eThe 2-cell rate, 4-cell rate, 8-cell rate, morula rate and blastocyst rate were calculated as the number of 2-cell embryos, 4-cell embryos, 8-cell embryos, morula and blastocysts. out of the total number of prokaryotic embryos cultured \u003cem\u003ein vitro\u003c/em\u003e \u0026times; 100%, respectively. Different superscript letters in the same column indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Inhibiting the phosphorylation of ezrin-Thr567 in mouse GV-stage oocytes affects the maturation, fertilization and developmental potential of eggs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConsidering the expression of p-ezrin Thr567 in both GV and MII stage oocytes in mice, we hypothesize that interfering with the phosphorylation of ezrin at Thr567 during oocyte maturation would also affect its maturation, fertilization and developmental potential. To verify our hypothesis, GV stage oocytes of mouse were cultured in IVM medium containing 0, 2.5, 5, or 10 \u0026micro;M NSC668394 respectively, and then the maturation rate, fertilization rate of oocytes and embryonic development rate of each group at different stages were calculated. As shown in Fig.\u0026nbsp;4, 2.5 \u0026micro;M NSC668394 dramatically decreased the expression of p-ezrin Thr567 in oocytes and significantly reduced their competence of maturation, fertilization and development (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When the concentration of NSC668394 reached 10 \u0026micro;M, the oocyte maturation rate did not decrease further. However, the developmental potential of early embryos decreased further dramatically, and no zygote developed to the 8-cell stage (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of NSC addition to IVM medium on oocyte maturation and early embryonic developmental potential.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMII\u003c/p\u003e \u003cp\u003e(Maturity rate /%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFertilized\u003c/p\u003e \u003cp\u003e(Fertilization rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4-cell\u003c/p\u003e \u003cp\u003e(4- cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8-cell\u003c/p\u003e \u003cp\u003e(8- cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMorula\u003c/p\u003e \u003cp\u003e(Morula rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBlastocyst\u003c/p\u003e \u003cp\u003e(Blastocyst rate/ %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1%DMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e119\u003c/p\u003e \u003cp\u003e(77.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e(63.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74\u003c/p\u003e \u003cp\u003e(99.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e72\u003c/p\u003e \u003cp\u003e(98.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e67\u003c/p\u003e \u003cp\u003e(92.21\u0026thinsp;\u0026plusmn;\u0026thinsp;8.20) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e66\u003c/p\u003e \u003cp\u003e(91.40\u0026thinsp;\u0026plusmn;\u0026thinsp;9.73) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e130\u003c/p\u003e \u003cp\u003e(80.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003cp\u003e(62.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003cp\u003e(94.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e77\u003c/p\u003e \u003cp\u003e(94.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e74\u003c/p\u003e \u003cp\u003e(91.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e74\u003c/p\u003e \u003cp\u003e(91.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e(48.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e(45.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e(98.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003cp\u003e(97.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.16) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003cp\u003e(48.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003cp\u003e(48.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e93\u003c/p\u003e \u003cp\u003e(61.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003cp\u003e(56.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51\u003c/p\u003e \u003cp\u003e(94.54\u0026thinsp;\u0026plusmn;\u0026thinsp;4.36) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003cp\u003e(93.50\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003cp\u003e(44.59\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e24\u003c/p\u003e \u003cp\u003e44.59\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003cp\u003e(61.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e(35.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36) \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e(11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;7.63) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Maturation rate was calculated as the number of MII-stage oocytes out of the number of GV-stage oocytes cultured \u003cem\u003ein vitro\u003c/em\u003e \u0026times; 100%; Fertilization rate was calculated as the number of cleaved embryos out of the number of oocytes exposed to sperm \u0026times; 100%; 4-cell rate, 8-cell rate, morula formation rate and blastocyst formation rate were calculated as the number of 4-cell embryos, 8-cell embryos, morula and blastocysts out of the number of cleaved embryos \u0026times; 100%, respectively. Different superscript letters in the same column indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). (the same below).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure 4. NSC668394 inhibited phosphorylation of Ezrin Thr567 in oocytes. When cultured in medium containing 2.5 \u0026micro;M NSC668394, the fluorescence intensity of p-Ezrin (Thr567) in mouse oocytes decreased remarkably compared with that in the control group. Scale bar, 50 \u0026micro;m.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Effect of NSC addition to IVM medium on oocyte maturation and early embryonic developmental potential.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eMaturation rate was calculated as the number of MII-stage oocytes out of the number of GV-stage oocytes cultured \u003cem\u003ein vitro\u003c/em\u003e \u0026times; 100%; Fertilization rate was calculated as the number of cleaved embryos out of the number of oocytes exposed to sperm \u0026times; 100%; 4-cell rate, 8-cell rate, morula formation rate and blastocyst formation rate were calculated as the number of 4-cell embryos, 8-cell embryos, morula and blastocysts out of the number of cleaved embryos \u0026times; 100%, respectively. Different superscript letters in the same column indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). (the same below).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Interfering with the phosphorylation of ezrin at Thr567 during IVF inhibits fertilization and subsequent embryonic developmental potential.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince inhibiting the phosphorylation of ezrin at Thr567 during oocytes IVM or embryos IVC can affect mouse embryonic development. So, would reducing the expression of p-ezrin Thr567 during fertilization affect the competence of fertility and development of mouse oocytes? To test our speculation, recovered MII oocytes were fertilized \u003cem\u003ein vitro\u003c/em\u003e with capacitated sperm in IVF medium containing different concentrations (0, 2.5, 5, and 10\u0026micro;M) of NSC668394, then transferred to KSOM medium without NSC668394 for further culture. The results showed that inhibiting the phosphorylation of ezrin Thr567 in oocytes during \u003cem\u003ein vitro\u003c/em\u003e fertilization significantly reduced the fertilization rate, and the rates of morula and blastocyst formation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, 10.0 \u0026micro;M NSC668394 also arrested fertilized eggs at the 2-cell stage (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of NSC668394 in IVF medium on early embryo development.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMII\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFertilized\u003c/p\u003e \u003cp\u003e(Fertilization rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4-cell\u003c/p\u003e \u003cp\u003e(4- cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8-cell\u003c/p\u003e \u003cp\u003e(8- cell rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMorula\u003c/p\u003e \u003cp\u003e(Morula rate / %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBlastocyst\u003c/p\u003e \u003cp\u003e(Blastocyst rate/ %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1%DMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e155\u003c/p\u003e \u003cp\u003e(78.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e152\u003c/p\u003e \u003cp\u003e(98.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e148\u003c/p\u003e \u003cp\u003e(95.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.31) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e141\u003c/p\u003e \u003cp\u003e(90.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e137\u003c/p\u003e \u003cp\u003e(88.42\u0026thinsp;\u0026plusmn;\u0026thinsp;8.57) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160\u003c/p\u003e \u003cp\u003e(80.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e156\u003c/p\u003e \u003cp\u003e(97.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e154\u003c/p\u003e \u003cp\u003e(96.25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.01) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e147\u003c/p\u003e \u003cp\u003e(91.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e139\u003c/p\u003e \u003cp\u003e(86.85\u0026thinsp;\u0026plusmn;\u0026thinsp;6.59) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e108\u003c/p\u003e \u003cp\u003e(53.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003cp\u003e(94.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98\u003c/p\u003e \u003cp\u003e(90.05\u0026thinsp;\u0026plusmn;\u0026thinsp;10.43) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53\u003c/p\u003e \u003cp\u003e(48.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e49\u003c/p\u003e \u003cp\u003e(44.91\u0026thinsp;\u0026plusmn;\u0026thinsp;6.95) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e119\u003c/p\u003e \u003cp\u003e(59.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e113\u003c/p\u003e \u003cp\u003e(94.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e108\u003c/p\u003e \u003cp\u003e(90.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.33) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e67\u003c/p\u003e \u003cp\u003e(56.58\u0026thinsp;\u0026plusmn;\u0026thinsp;7.28) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e62\u003c/p\u003e \u003cp\u003e(52.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u0026micro;M NSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003cp\u003e(33.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e(15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;13.23)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Microinjection of ezrin-T567D mRNA partly rescued the competence of maturation, fertilization and developmental potential of oocytes treated with NSC668394\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm the decline of maturation, fertilization, and the developmental competence of oocytes or embryos treated with NSC668394 was caused by the inhibition of ezrin Thr567 phosphorylation, rescue experiments were conducted. When GV oocytes, MII oocytes and prokaryotic embryos treated with 2.5 \u0026micro;M NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e maturation, \u003cem\u003ein vitro\u003c/em\u003e fertilization, or \u003cem\u003ein vitro\u003c/em\u003e culture were microinjected with ezrin-T567D mRNA, the maturation rate and the fertilization rate of oocytes, and the developmental potential of early embryos were partially restored (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eFigure 5. Microinjection of Ezrin-T567D mRNA partially rescued the reduction of the maturation rate and the fertilization rate of oocytes and the developmental potential of early embryos caused by NSC668394. (A) The development rate of different stage of embryos that treated with 2.5 \u0026micro;M NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e culture and microinjected with ezrin T567D mRNA at prokaryotic stage. (B) The maturation rate, fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 \u0026micro;M NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e maturation and microinjected with ezrin T567D mRNA at GV stage. (C) The fertilization rate and embryonic developmental rate of oocytes that treated with 2.5 \u0026micro;M NSC668394 during \u003cem\u003ein vitro\u003c/em\u003e fertilization and microinjected with ezrin T567D mRNA at MII stage. Note: Different lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eEzrin is a multi-domain protein mainly composed of ERM association domains (ERMADs) located at the N- and C-termini, and a central connecting region. The N- and C-ERMADs can bind to each other to keep the protein in an inactive state. Phosphorylation of Thr567 can release the binding between N- and C-ERMADs, activate the protein, and participate in the reshaping of cell surface structures[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Overexpression of a phosphomimetic or non-phosphorylatable mutant of ezrin Thr567 in mouse embryos both inhibited compaction of morulae and the formation of blastocoel[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, the impact of inhibiting ezrin Thr567 phosphorylation on mouse oocyte maturation, fertilization, and pre-implantation embryonic development remains unclear. In this study, we found that adding NSC668394, a small molecule inhibitor specifically targeting ezrin Thr567 phosphorylation, to the culture medium can inhibit the phosphorylation of ezrin Thr567 in mouse oocytes and early embryos (Fig.\u0026nbsp;3, Fig.\u0026nbsp;4), and block oocyte maturation, fertilization, and early embryonic development.\u003c/p\u003e \u003cp\u003eThe maturation of oocytes involves a long and complex process, including the reorganization of the cytoplasmic membrane, changes in cortical tension[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], deformation of surface microvilli[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], establishment of oocyte polarity[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], migration, assembly, and localization of the spindle apparatus[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], as well as meiotic division and extrusion of polar bodies, ultimately leading to the acquisition of the ability to be fertilized and develop into an embryo. All these processes are regulated by the cytoskeletal network composed of actin microfilaments. Studies have shown that the ERM protein family is involved in mediating oocyte maturation[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The effect of ezrin on oocyte development may be regulated by the phosphorylation of ezrin Thr567, and there is currently limited research on the role of ezrin T567 phosphorylation in oocyte maturation.\u003c/p\u003e \u003cp\u003eNSC668394 specifically inhibits the phosphorylation of ezrin T567[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Here, we found that the treatment with NSC668394 only during the oocyte maturation not only significantly reduced the in vitro maturation rate of oocytes but also affected the i\u003cem\u003en vitro\u003c/em\u003e fertilization and early embryo development potential. There are two possibie explanations for this result, one is that the inhibition of ezrin T567 phosphorylation by NSC668394 may be persistent or irreversible, and the other is that the abnormalities in oocytes caused by the inhibition of ezrin T567 phosphorylation affected the competence of fertilization and subsequent embryonic development. Since generally the phosphorylation and dephosphorylation of proteins are highly dynamic, we prefer the later explanation.\u003c/p\u003e \u003cp\u003eFertilization involves complex membrane and cortical cytoskeleton remodeling process, including acrosome outer membrane fuses with sperm plasm membrane, sperm membrane binds to the microvilli of the oocyte and fuses with oocyte membrane. Previous studies have shown that ezrin phosphorylation is also involved in the process of sperm acquisition and regulates the acrosome reaction[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Ezrin is involved in the formation and maintenance of cell skeletons and microvilli, and small molecule inhibitors of ezrin Thr567 phosphorylation affect the development of microvilli, which in turn affects sperm acquisition and entry into the ooplasm. Furthermore, ezrin activation allows it to bind to its target proteins and form an F-actin-ezrin-membrane protein complex, which plays an important role in the acrosome reaction and fertilization. NSC668394 treatment blocked the activation of Ezrin by inhibiting ezrin Thr567 phosphorylation, and indirectly inhibiting the formation of the F-actin-ezrin-membrane protein complex, which affects oocyte \u003cem\u003ein vitro\u003c/em\u003e fertilization. Even if some oocytes completed fertilization successfully, since the microstructure had been interfered, the developmental potential of early embryos was affected.\u003c/p\u003e \u003cp\u003eEmbryonic development is often accompanied by cell polarization, cell division, and cell migration. As mentioned previously, ezrin is involved in cell polarization and embryonic compaction[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Guang et al.[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] found that ezrin accumulates excessively in the contractile ring and cleavage furrow during cell division. Therefore, we speculate that ezrin Thr567 phosphorylation may be involved in early embryonic development. The results show that NSC668394 inhibits embryonic compaction significantly (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which is similar to previous studies suggesting that ezrin phosphorylation is involved in regulating embryonic compaction[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Treating zebrafish embryos with10\u0026micro;M NSC668394 resulted in a unique optokinetic response phenotype[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The optokinetic response phenotype persisted even after washing away the small molecule compound, suggesting that defects caused by NSC668394 through inhibiting ezrin Thr567 phosphorylation seems to be persistent or irreversible.\u003c/p\u003e \u003cp\u003eSince ezrin T567D protein mimics the conformation of p-ezrin T567, so it can excise the function of p-ezrin Thr567 to rescue oocyte maturation, fertilization and early embryonic development which reduced by NSC668394 through inhibiting ezrin T567 phosphorylation. However, microinjection of ezrin T567D mRNA did not completely restored the maturation rate, fertilization rate and the dev-elopment potential affected by NSC668394, suggesting mouse oocytes and emb-ryos require dynamic phosphorylation-dephosphorylation rather than sustained phosphorylation of ezrin.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eHere we investigated the impact of ezrin Thr567 phosphorylation on the development of mouse preimplantation embryos by specifically inhibiting ezrin Thr567 phosphorylation with NSC668394 and rescuing with expression of ezrin T567D mutant.\u003c/p\u003e \u003cp\u003eInhibiting the phosphorylation of ezrin Thr567 during \u003cem\u003ein vitro\u003c/em\u003e maturation or \u003cem\u003ein vitro\u003c/em\u003e fertilization of mouse oocytes not only affected their maturation and fertilization, but also reduced the development competence of preimplantation embryos.\u003c/p\u003e \u003cp\u003eMicroinjection of mRNA encoding ezrin T567D mutant can partially rescue the developmental defects of mouse oocytes, fertilization, and early embryonic development caused by NSC668394.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll the data in this paper are authentic and reliable, and the animal models involved are reproducible.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the regulations of the People\u0026apos;s Republic of China on the Management of Laboratory Animals and were approved by the Animal Care and Use Committee of Anhui Agricultural University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eHuihui Xie, Bochao Zhang and Jiankun Cui participated in some experiments, performed statistical analysis and drafted the manuscript. Tiandong Xia and Wei Qian performed most experiments. Qing Yi, Mengdi Han, Hongyan Liao, Yuke Jia, Meng Cao and Yanqiuhong Li collect the oocytes and \u003cem\u003ein vitro\u003c/em\u003e mature, Fuqiang Pan\u0026nbsp;participated some sample selection,\u0026nbsp;Yunsheng Li\u0026nbsp;and Fugui Fang participated in the design of the experiments. Yunhai Zhang and Yinghui Ling involved in revising the manuscript. Ya Liu conceived of this study. All authors have read this manuscript and approved its publication.\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interest\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to disclose.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe are especially grateful to all participants in this study.\u003c/p\u003e\n\u003cp\u003eFounding\u003c/p\u003e\n\u003cp\u003eThis work was supportd by Major Science and Technology Project of Anhui Province (202103b06020023), National Natural Science and Technology Foundation of China (31972629 and 32272881) and Anhui Key Research and Development Program（2023z04020003）.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChishti AH, Kim AC, Marfatia SM, Lutchman M, Hanspal M, Jindal H, et al. The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. 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Reprod Biomed Online. 2021;42:699\u0026ndash;707.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ezrin, phosphorylation, mouse, oocyte, early embryo","lastPublishedDoi":"10.21203/rs.3.rs-3872230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3872230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEzrin is a membrane-cytoskeleton linker, involved in cell polarization, cell migration, cell division, signal transduction and some other cellular activities that play an important role in oocyte maturation, fertilization and early embryonic development. The phosphorylation of Thr567 is an important way to activate ezrin, it has been proved that p-ezrin Thr567 is expressed in oocytes and pre-implantation embryos in mouse. However, little is known about the impact of inhibiting ezrin Thr567 phosphorylation on oocyte maturation, fertilization and early embryonic development.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNSC668394 is a small molecule that specifically inhibits the phosphorylation of ezrin Thr567. Here, we investigated the effects of inhibiting ezrin Thr567 phosphorylation with NSC668394 on the mouse oocyte maturation, fertilization, and early embryo development.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results show that adding NSC668394 to the \u003cem\u003ein vitro\u003c/em\u003e culture medium significantly lowed mouse embryos development competence after 8-cell stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further experiments revealed that inhibiting ezrin Thr567 phosphorylation during \u003cem\u003ein vitro\u003c/em\u003e maturation or \u003cem\u003ein vitro\u003c/em\u003e fertilization not only decreased the maturation rate and fertilization rate of mouse oocytes, but also reduced early embryos development competence after 8-cell stage. Microinjection of mRNA encoding ezrin T567D mutant partially rescued the developmental defects of mouse oocytes, fertilization, and early embryonic development caused by NSC668394. These results indicate that ezrin Thr567 phosphorylation plays an important role in mouse oocyte maturation, fertilization and early embryo development.\u003c/p\u003e","manuscriptTitle":"Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-24 17:21:11","doi":"10.21203/rs.3.rs-3872230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c4fd0cb-8622-40b1-871e-051cf703280b","owner":[],"postedDate":"January 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-17T10:49:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-24 17:21:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3872230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3872230","identity":"rs-3872230","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}