Figure 1.
mDia2 is localized to spindle poles of MII oocytes.
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.
Figure 2.
Localization of mDia2 and pericentrin in vitrified-warmed oocytes.
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.
Figure 3.
Localization of mDia2 and pericentrin at the time of thawing.
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.
Figure 4.
Co-staining of mDia2 and tubulin in MII oocytes and vitrification solution-treated oocytes.
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.
Figure 5.
Localization of mDia2 and tubulin at the time of thawing.
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 3 h after thawing (3 h). These oocytes were subjected to immunofluorescence staining with anti-mDia2 and anti-α-tubulin antibodies. Green, mDia2; red, α-tubulin. In the oocyte at 0 h, mDia2 localization at spindle poles is not visible because the image is focused to MTOCs with emanating microtubules. Goat IgG was used instead of anti-mDia2 antibody in double immunofluorescence staining as a negative control (bottom right).