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FIGURE 1. Growth and fertility defects in female mice carrying the ENU-induced nonsense mutation of Eml1. (A) Comparison of body weight between wildtype and Eml1-mutant (Mutant) female mice at the age of 3 and 7 weeks. (B) Microphotographs showing the ovulated cumulus-oocyte complexes by Wildtype and Mutant females. Scale bars indicate 10 μm. (C) Fecundity (pups/litter) of WT (n = 3) and Mutant (n = 3) female mice during the 2 month fertility test period. ***P < 0.005, compared with the Wildtype. (D) Assessment of spindle morphology and chromosome alignment in the Wildtype and Mutant ovulated oocytes. The left panel shows the representative micrographs of the IF stained oocytes. Spindles are stained in green, chromosomes and F-actins are stained in blue and red, respectively. Scale bars indicate 20 μm. The right bar graph shows the percentage of the oocytes with abnormal spindles and/or misaligned chromosomes. **P < 0.05, compared with the Wildtype.
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FIGURE 2. Stable expression and specific localization of EML1 in mouse oocyte during meiotic progression. (A) Western Blot (WB) analysis of the expression of EML1 and ACTB (internal control) protein in oocytes at different stages of maturation. Lysate of 200 oocytes that were collected at 0, 8, and 14 h of IVM corresponding to GV-, MI-, and MII- stage, respectively, was load in each lane. (B) Confocal micrographs showing the dynamic co-localization of EML1 protein with meiotic spindles in oocytes at various stages (i.e., GV, Pro-MI, MI, AI, TI, and MII) of maturation. EML1 and α-tubulin were stained in red and green, respectively, while chromosomes were stained in blue. The far left panel shows the whole-oocyte view of the staining. The magnified view of the boxed area within the oocyte at each stage is listed on the right side. Scale Bar = 20 μm. (C) Confocal micrographs demonstrating the changes of localization of EML1 and tubulin in MI- stage oocytes treated with microtubule-interfering drugs. MI- oocytes were treated with 20 μg/ml nocodazole for 10 and 15 min, respectively, or with 10 μM Taxol for 45 min, and then processed for IF analysis of EML1 and α-tubulin localization. EML1, α-tubulin, and chromosomes are shown in red, green, and blue, respectively. The first row indicates the whole-oocyte view of the staining. The magnified view of the boxed area within the oocyte is shown in the second row. Scale Bar = 20 μm.
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FIGURE 3. Knockdown of EML1 expression in oocytes causes the delay of meiotic progression. (A) Schematic illustration of the experimental design for examining the effect of EML1 knockdown on oocyte meiotic maturation. (B) WB analysis of the efficiency of knocking down EML1 in oocytes by EML1-morpholino oligos (MO). Lysate from 200 oocytes was loaded in each lane. ACTB serves as internal control. Representative gel image is shown in the left panel, whereas quantification of the WB result is shown in the right panel. *P < 0.05, compared with the Control-MO. (C) Effect of knockdown of EML1 in oocytes on the kinetics of GVB (left graph) and PBE (right graph). *P < 0.05, compared with the EML1-MO groups.
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FIGURE 4. Knockdown of EML1 in oocytes impairs MI- spindle assembly and chromosome alignment. (A) Oocytes microinjected with the MOs were first maintained at GV-stage in milrinone-containing for 24 h, and then transferred to milrinone-free medium for IVM. After 8-h IVM, the oocytes were subjected to IF staining of spindles and chromosomes. The left panel is the representative micrographs of the IF staining, with the α-tubulin and chromosomes stained in green and blue, respectively. Arrows point to misaligned chromosomes. Scale Bar = 20 μm. The right bar graph shows the quantification of the percentage of the oocyte with normal spindles as revealed by the IF analysis. **P < 0.01, compared with the Control-MO group. (B) Geometric analysis of the MI- spindles in oocytes that were treated with Control-MO and EML1-MO and EML1-MO. Spindle (stained in green) length and width were measured as illustrated in the left micrographs, and plotted into dot graphs as shown in the right panels. *P < 0.05, ***P < 0.005. (C) Geometric analysis of chromosome alignment in MI- oocytes that were treated with Control-MO and EML1-MO. Chromosome displacement was measured according to the illustration in the left panel, and the quantification was shown in the right graph. The spindle was shown in green and chromosomes in blue. *P < 0.05, ***P < 0.005.
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FIGURE 5. Knockdown of EML1 in oocytes disrupts the normal localization of a MTOC-associated proteins to the spindle pole. (A) Representative images of MO-injected oocytes labeled with anti-γ-Tubulin (red) and anti-α-Tubulin (green). DNA was counterstained with Hochest 33342 (blue). (B). Representative images of MO-injected oocytes labeled with anti-Pericentrin (red) and anti-α-Tubulin (green). DNA was counterstained with Hochest 33342 (blue). In both (A,B), the far left panel indicate the whole-oocyte view of the staining. The magnified images of the boxed areas within the oocyte are listed on the right side. Scale Bar = 20 μm. (C) Quantification of the percentage of oocytes with correct spindle pole Pericentrin (PCNT). **P < 0.01, compared with the Control-MO group.
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FIGURE 6. Increased kinetochore-microtubule attachment error and abnormal activation of spindle assembly checkpoint in EML1-Knocked down MI-stage oocytes. (A) Assessment of kinetochore–microtubule attachments in MO-injected oocytes that were matured in vitro for 8 h by IF staining. Microtubules, kinetochores, and chromosomes were stained in green, red, and blue, respectively. Representative images of end-on (1), unattached (2), and merotelic (3) attachments are shown. A total of 349 and 247 attachments were assessed, respectively, in the Control-MO and EML1-MO treated oocytes. **P < 0.01, ns denotes no significant difference. compared with the Control-MO and EML1-MO group. (B) Assessment of the activation of SAC in MO-injected oocytes that were matured in vitro for 8 h by IF staining of BubR1. Oocytes that were normally matured in vitro for 3.5 h served as positive control. BubR1 and chromosomes are stained in green and blue, respectively. ***P < 0.005, compared with the EML1-MO group.
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FIGURE 7. Knockdown of EML1 causes the spontaneous formation of pronucleus and failure to enter into the second metaphase by the matured oocytes after completion of the first meiotic division. Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 24 h, and then transferred to maturation medium to allow for IVM. Oocytes that had undergone GVB within 2 h of IVM were selected and further cultured for additional 14 h. At the end of the culture, PBE and formation of pronucleus in oocytes were assessed. (A) Scoring PBE in cultured oocyte under light microscope. Representative bright field images of cultured oocytes are shown in the left panel, and the quantification of the rate of PBE are shown in the right bar graph. Red arrows denote pronucleus (PN), black arrows indicate PB1. Scale Bar = 10 μm. ns: non-significant. (B) Examining the formation of PN by fluorescent staining of tubulin (green) and DNA (blue) in the matured oocytes. The left panel is the representative images of the stained oocytes, with the magnified view of the circled area are listed on the right side. The right bar graph shows the quantification of the percentage of oocytes that have both the emitted PB1 and the PN. Scale Bar = 20 μm. ***P < 0.005, compared with the Control-MO group. ns denote no significant difference between the two groups compared.
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FIGURE 8. Depletion of EML1 in oocytes impairs the activation of MPF. Dynamic changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the oocytes during the period of oocytes meiosis resumption (A,B) and MI-to-MII transition (C,D) were assessed by Western Blot analysis, with the level of ACTB used as internal control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown in panels (A,C), and the quantification of the Western Blot results are shown in panels (B,D). *P < 0.05, compared with the Control-MO group.
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FIGURE 9. Activation of MPF by inhibitors of WEE1/2 kinases rescues the defects of meiotic progression in EML1-knocked down oocytes. (A) Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 20 h, and then transferred to maturation medium with (for EML1-MO group) or without (for Control-MO group) the supplementation of the inhibitor of WEE1/2 kinases, PD166285, to allow for maturation. Oocyte GVB was scored during the culture. *P < 0.05, compared with the Control-MO group. (B) Western Blot analysis of the changes in the levels of CCNB1, pCDK1-Y15, and CDK1 in the EML1-knocked down oocytes that were treated with PD166285. ACTB was used as the loading control. Lysate from 50 oocytes was loaded in each lane. The representative gel images of three independent experiments are shown. (C–E) Assessment of the effect of PD166285 on the progression of meiosis to MII in EML1-knocked down oocytes. Oocytes microinjected with Control-MO and EML1-MO were first maintained at GV stage for 24 h. Then the EML1-MO oocytes were split into two groups, one of which was transferred to maturation medium supplemented with PD16628S and directly cultured for 20 h, while the other one was first transferred to maturation medium without PD166285 for 14 h, and then in the PD166285-supplemented medium for another 4 h culture. PBE, PN formation, and spindle morphology were then scored and analyzed. Representative images of the oocytes with the microtubules labeled with anti-tubulin antibody (green) and DNA stained with Hochest (blue) are shown in panel (C). Scale Bar = 20 μm. Quantification of the percentages of oocytes with PB1 and PN, and those with normal MII-spindles are shown in panels (D,E), respectively. *P < 0.05, **P < 0.01, ***P < 0.005, compared with the EML1-MO group. ns denote no significant difference between the two groups compared.
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FIGURE 10. Potential interacting partners identified for EML1. (A) Gene enrichment analysis of the interacting proteins of EML1 identified by IP-MS. (B) List of IP-MS identified EML1interacting proteins that are involved in spindle morphogenesis and actin polymerization (left table), and the validation of the presence of NUDC and EML4 in the EML1-IP product by WB analysis (right panel). (C) WB validation of the interaction between EML1 and NUDC. Expression of 3DDK-tagged EML1 and mKate-tagged NUDC proteins was achieved by simultaneously transfecting HEK293T cells with the corresponding plasmids. Co-IP and WB analysis were then carried out with the anti-DDK and anti-mKate antibodies. (D) IF staining images showing the co-localization of NUDC with EML1 on the spindles of MI-stage oocytes. (E) Knockdown of EML1 did not affect the expression levels of NUDC in the oocyte as indicated by the WB analysis in the top panel, but dislodged the localization of NUDC from the spindles in oocytes (bottom IF images). ns, denote no significant difference between the two groups compared. Scale Bar = 20 μm.
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