The expression of <i>Egr3</i> transcripts and proteins in mouse tissues and cultured cells.

<p>(A) An RT-PCR analysis of <i>Egr3</i> expression in various tissues. Expression was compared to that of ribosomal protein L7 (<i>rpl7</i>) or <i>Gapdh</i>. Oocytes at PI (130 oocytes) or MI stage (120 oocytes) were pooled and subjected to Egr3 RT-PCR and nested PCR. Cu, cumulus cells; -, no RT. (B) Western blot analysis of Egr3 in various tissue and cell types. Upper panel: 293 T cells transfected with CMV-GFP plasmid (GFP), empty plasmid (-), pcDNA3.1/myc-his-NM plasmid (NM), or pcDNA3.1/myc-his-BC plasmid (BC) were analyzed using Western blots probed with anti-Egr3 antibody (Santa Cruz). Fifty micrograms of each cell lysates were run on a 10% SDS-PAGE gel. Bottom panel: MI oocyte (268 GV oocytes cultured for 8 h in vitro), PI oocyte (112 oocytes), ovary, brain, and MCF7 cell (with or without estrogen treatment) lysates were analyzed by Western blotting. Two hundred micrograms of tissue extract was loaded in each lane of a 12% SDS-PAGE gel. The blots were stripped and tubulin was detected using an anti-α-tubulin antibody.</p

Immunofluorescence staining of Egr3 and tubulin proteins in MI oocytes.

<p>PI oocytes were cultured in vitro for 8% formaldehyde, and subjected to immunofluorescence staining with anti-Egr3 (Santa Cruz antibody in upper panel and Abcam antibody in low panel), anti-α-tubulin, or anti-γ-tubulin (Sigma-Aldrich) antibody. All the antibodies were used at 2 μg/ml. DNA was counter-stained with TO-PRO-3-iodide. Red scale bar, 30 μm; white scale bar, 5 μm. Green, Egr3; red, α-tubulin or γ-tubulin; blue, DNA. The arrows indicate the localization of Egr3 protein near γ-tubulin-positive MTOCs.</p

Immunofluorescence staining of Egr3 in preimplantation mouse embryos.

<p>Embryos at different developmental stages were fixed in 4% paraformaldehyde and subjected to immunofluorescence staining with anti-Egr3 (Santa Cruz) and anti-α-tubulin antibodies. The arrows indicate mitotic spindles in the blastomeres. Green, Egr3; red, α-tubulin; blue, DNA.</p

Egr3 localization is associated with microtubule organization in mouse oocytes.

<p>(A) MI oocytes were treated with taxol (microtubule stabilizer, 1 μM) or nocodazole (microtubule depolymerizer, 10 μg/ml) for 1–2 h and were subjected to immunofluorescence staining with anti-Egr3 antibody (Santa Cruz). Red scale bar, 30 μm; white scale bar, 10 μm. Green, Egr3; red, γ-tubulin; blue, DNA. (B) Localization of Egr3 is shown in MI oocyte treated with CytoD (actin depolymerizer, 10 μM) and in <i>Fmn2</i>-/- oocyte. <i>Fmn2</i>-/- oocyte was co-stained with anti-Egr3 (Santa Cruz) and anti-γ-tubulin antibodies. Red scale bar, 30 μm. Green, Egr3; red, γ-tubulin; blue, DNA. (C) The localization of Egr3 and microtubule at thawing of vitrified oocytes. Vitrified MII oocytes were stored in LN₂ for 2 weeks. Oocytes were taken out from LN₂, incubated in decreasing concentrations of sucrose, and then fixed immediately. These oocytes were subjected to immunofluorescence staining with anti-Egr3 (Abcam) and anti-α-tubulin antibodies. Arrows indicate the growing arrays of microtubules at the site of Egr3 accumulation. Green, Egr3; red, microtubule (MT).</p

Microtubule interaction assay using in vitro translated Egr3 isoforms.

<p>To examine whether polymerized microtubules interact directly with Egr3 protein, we performed a microtubule-interaction assay. Tubulin from the bovine brain (5 μg/μl) was polymerized at 37°C and diluted to 0.2 μg/μl. Four microliters of in vitro translated lysates (NM or BC) and 50 μl of diluted MTs were incubated at room temperature for 30 min. The reaction was centrifuged at 100,000×g for 30 min to pellet the polymerized MTs. The supernatant (S) and pellet (P) were run on 10% SDS-PAGE and Western blotting was performed with anti-Egr3 or anti-Myc antibody which detects the epitope in the pcDNA3.1/myc-his vector. The antibodies were used at 1∶5000. The blots were stripped and tubulins were detected with anti-tubulin antibody. The data show that the presence of microtubules does not affect the quantity of Egr3 products in the supernatant and pellet, suggesting that there is no direct interaction between microtubules and Egr3.</p

Subcellular localization of Egr3 protein in meiotic spindle of mouse oocytes.

<p>(A) Egr3 localization in a mouse oocyte within a growing follicle. Immunofluorescence staining of Egr3 was performed on ovarian cryosection fixed in acetone. The rabbit polyclonal anti-Egr3 antibody (Santa Cruz) was used at 2 μg/ml. Primary antibody was probed with goat anti-rabbit IgG-Alexa Fluor 488 antibody. The DNA was counter-stained with TO-PRO-3-iodide. White scale bar, 100 μm. Mock control, rabbit IgG. Green, Egr3; red, DNA. (B) Egr3 is localized to the meiotic spindle of mouse oocytes at all stages of maturation. Germinal vesicle (GV) stage oocytes were obtained by ovary puncture and were cultured in M16. Oocytes were fixed in 3.7% formaldehyde and were subjected to immunofluorescence staining with anti-Egr3 antibody (Santa Cruz). Oocytes in the left panel are shown at 60X and enlarged images of chromosome-containing areas are shown in the right panel. Mock control, rabbit IgG. PI, prophase I; PMI, prometaphase I (cultured for 3 h); MI, metaphase I (cultured for 8 h); MII, metaphase II (cultured for 12 h). Red scale bar, 30 μm; white scale bar, 10 μm. Green, Egr3; blue, DNA.</p

mDia2 is localized to spindle poles of MII oocytes.

<p>Immunofluorescence staining of mDia2 and pericentrin in mouse MII oocytes. Oocytes were stained with goat anti-mDia2 and mouse monoclonal anti-pericentrin antibodies. Overlapped distribution of mDia2 and pericentrin generates yellow fluorescence (overlay). Primary antibodies were used at 2 µg/ml. DNA is counter-stained with TO-PRO-3-iodide. Green, mDia2; red, pericentrin; blue, DNA.</p

Localization of mDia2 and pericentrin in vitrified-warmed oocytes.

<p>MII oocytes were vitrified and stored in LN₂ for 4 weeks. After thawing, oocytes were subjected to immunofluorescence staining with anti-mDia2 and anti-pericentrin antibodies. Overlapped distribution of mDia2 and pericentrin generates yellow fluorescence (overlay). DNA is counter-stained with TO-PRO-3-iodide. Green, mDia2; red, pericentrin; blue, DNA. The bottom panel shows the magnified region of the chromosome-spindle complex. DNA is not visible in high magnification images because it is out of focus.</p

Co-staining of mDia2 and tubulin in MII oocytes and vitrification solution-treated oocytes.

<p>A, Immunofluorescence staining of mDia2 and tubulin in fresh MII oocytes. mDia2, green; α-tubulin, red. B, Immunofluorescence staining of mDia2 and tubulin in oocytes treated with equilibration and vitrification solutions. Note the shrinkage of the ooplasm because of osmosis. mDia2, green; α-tubulin, red.</p

Localization of mDia2 and pericentrin at the time of thawing.

<p>Vitrified MII oocytes were stored in LN₂ for 2 weeks. Oocytes were taken out from LN₂, incubated in decreasing concentrations of sucrose, and then fixed immediately (0 h). Some oocytes were incubated in 37 C for recovery for 1 h after thawing (1 h). These oocytes were subjected to immunofluorescence staining with anti-mDia2 and anti-pericentrin antibodies. Overlapped distribution of mDia2 and pericentrin generates yellow fluorescence (overlay). DNA is counter-stained with TO-PRO-3-iodide. Green, mDia2; red, pericentrin; blue, DNA. The right panel shows the magnified region of the chromosome-spindle complex.</p