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FIGURE 1:. Structure of the intranuclear actin filaments. Eleven oocytes derived from four different females were examined. The nucleus of a midsagittal (animal–vegetal) cryosection of a full-grown stage VI oocyte was stained with Alexa 488–phalloidin (A) and double stained with Alexa 488–phalloidin and anti-lamin antibody (B). Arrows indicate the actin filaments surrounding the nucleus. (C) Enlarged image of the nucleus by assembling three shots, which had to be taken to cover one section, in a composite plate. The vegetal region (D) and animal region (E) are further enlarged. (F) Comparison of the actin filament mesh size between the vegetal (Veg) and animal (An) sides. The area of the space surrounded by actin filaments (the mesh hole) was measured over a set range by ImageJ software. Twelve oocytes from nine different females were measured. Relative mesh size at the animal side. Bars, 100 μm (A, B), 50 μm (C–E). An, animal pole; Vg, vegetal pole.
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FIGURE 2:. Staining of actin filaments and lamin during oocyte maturation. Representative confocal microscopy images of midsagittal sections of oocytes pretreated with progesterone (A), at the relative time point of 0.8 (just before GVBD; B), and just after GVBD (C) were double stained with Alexa 488âphalloidin (F-actin) and anti-lamin antibody (lamin). Nine oocytes from three different females were examined. Arrows indicate the cytoplasmic actin filaments surrounding the nucleus. Differential interference contrast (DIC; D, F) and lamin-staining images (E, G, H) of maturing oocytes at WMS formation (the relative time point of 1.0; D, E) and 2 h after WMS formation (FâH). Bars, 100 μm (A, E, G), 50 μm (H).
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FIGURE 3:. Staining of actin filaments and microtubules during oocyte maturation. Nine oocytes from three different females were examined. Midsagittal sections of oocytes pretreated with progesterone (A), at the relative time point of 0.8 (just before GVBD; B), just after GVBD (C), and at the relative time point of 1.0 (D) were double stained with TMR–phalloidin (F-actin) and anti-tubulin antibody (MT). Merged and DIC images are also shown. Arrowheads indicate the cytoplasmic actin filament bundles. Arrows in B and in C and D indicate microtubule bundles and the MTOC-TMA, respectively. Bar, 100 μm.
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FIGURE 4:. Localization of XSSH and XAC during oocyte maturation. Midsagittal sections of full-grown oocytes (A, B) were stained with anti-XSSH antibody (A) and anti-XAC antibody (B). For the negative control, the cryosection of the oocyte at the relative time point of 0.8 was stained with Alexa 488âlabeled secondary antibody against rabbit IgG alone (C). Midsagittal sections of maturing oocytes just after GVBD (DâI) and at the relative time point of 1.0 (JâM) were double stained with anti-XSSH antibody (D, J) and anti-tubulin antibody (E, K) or with anti-XAC antibody (G, L) and anti-tubulin antibody (H, M). At the relative time point of 1.0 (at the same time point as in J and L), the fluorescence intensity of either XSSH (F) or XAC (I) staining from the animal to the vegetal direction at the center of the nuclear region (shown with the white line in insets) was examined in different oocytes (six for XSSH and eight for XAC) using ImageJ software. The abscissa of the graphs is the number of pixels from the animal side, and the ordinate represents the fluorescence intensity (arbitrary units). Arrows indicate position of the base of the MTOC-TMA. The asterisks (D, G) indicate the nuclear region. Bars, 100 μm. Images are representative staining from at least 12 oocytes from four different females.
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FIGURE 5:. Effects of injection of anti-XSSH antibody on assembly of the MTOC-TMA. Midsagittal sections of oocytes just before (A) or after (B) GVBD, injected with buffer alone (control) or with 10 mg/ml of 1:1 mixture of anti-XSSH IgG and anti-XSSH IgG-NLS (anti-XSSH), were double stained with TMRâphalloidin (F-actin) and anti-tubulin antibody (MT). (A) Assembly of the cytoplasmic actin filaments (arrowheads) and microtubule bundles (arrows) at the basal region of nuclei is clearly visible in the control but faint at the basal region of nuclei in antibody-injected oocytes (asterisk). On the other hand, microtubule bundles are evident at the animal side of the nuclei of antibody-injected oocytes. (B) The cytoplasmic actin filaments (arrowhead) are apparent at the base of the MTOC-TMA (arrow) in the control, whereas the intranuclear actin filaments (asterisks) clearly remained in a globular shape and the MTOC-TMA is faint at the basal region of the nuclei in antibody-injected oocytes. Merged images are also shown. Bar, 100 μm. Images in A and B are representative staining of 11 and 17 oocytes from five females, respectively.
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FIGURE 6:. Effects of phalloidin injection on intranuclear actin filaments and assembly of the MTOC-TMA. Midsagittal sections of immature oocytes (AâD) and oocytes after GVBD (EâG), injected with vehicle alone (control; A, C, E) or with 10 mM phalloidin (B, D, F, G), were double stained with anti-lamin (red) and anti-tubulin (green) antibodies (A, B, F) or anti-actin (red) and anti-tubulin (green) antibodies (Câ E and G). Merged images and DIC images are shown. Inset in D, anti-actin staining alone. Insets in G, images stained by anti-tubulin (green) and anti-actin (red) antibodies. Arrows in B and F indicate the nuclear periphery stained by anti-lamin antibody. Asterisks in B and D indicate the yolk-free region. Arrow in D represents the cytoplasmic actin filaments surrounding the nucleus. Arrowhead in F indicates the unstained space where actin filaments might be present as shown by the arrowhead in G. (H) Maximum diameter of the nuclei of oocytes injected with vehicle alone (Cont, n = 6), 10 mM phalloidin (Phall; 0 h, n = 4; GVBD, n = 11), or anti-XSSH antibody (α-XSSH; 0 h, n = 4; GVBD, n = 5) before progesterone treatment (0 h) or at GVBD. The gross area was measured from each section (the section that had maximum area of nuclei was selected from serial sections) by ImageJ software, and the maximum diameter was calculated as a perfect circle.
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FIGURE 7:. Effects of injection of 2.5 mg/ml chick S3A-cofilin (A, B) and treatment of 20 μg/ml nocodazole (C, D) on disassembly of intranuclear actin filaments and assembly of the MTOC-TMA. Midsagittal sections of S3A-cofilinâinjected oocytes immediately before (A) or immediately after (B) GVBD were double stained with TMR-phalloidin (F-actin) and anti-tubulin antibody (MT). (A) Assembly of the cytoplasmic actin filaments and microtubule bundles (arrows) at the basal region of the nuclei is clearly visible in S3A-cofilinâinjected oocytes. Twelve oocytes from six different females were examined. (B) Assembly of the MTOC-TMA is affected by S3A-cofilin injection. The TMA itself is well developed, but actin staining disappears from the base of the split region of the MTOC-TMA (arrowhead). Atypical microtubule structures have formed at the animal side as they surround the nuclear region (arrows). A faint staining of actin filaments (indicated by arrow in the F-actin panel) is also visible around the animal side of the residual nuclear actin filaments. Six oocytes from three different females were examined. Midsagittal sections of 20 μg/ml nocodazoleâtreated oocytes immediately after GVBD were double stained with anti-lamin and anti-tubulin antibodies (C) or TMR-phalloidin and anti-tubulin antibody (D). Merged and DIC images are also shown. Arrows indicate bleb-like protrusions characteristic of nocodazole-treated oocyte nuclei. Disassembly of the intranuclear actin filaments from the vegetal side is retarded. Twelve oocytes from three different females were examined.
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FIGURE 8:. Effects of injection of anti-XSSH antibody (AâH) and S3A-cofilin (I, J) on assembly of meiotic spindles. Cryosections were triple stained with DAPI (blue), anti-microtubule antibody (green), and TMR-phalloidin (red; A, B, G inset) or double stained with DAPI (blue) and anti-microtubule antibody (green; CâG, I, J). (A) Control oocytes formed the metaphase I spindle (exactly prometaphase I) at the animal cortex (bar, 100 μm). The spindle is enlarged in the inset (bar, 20 μm). (B) Malformation of the metaphase I spindle is observed in the antibody-injected oocytes (bar, 100 μm). Arrowheads indicate disrupted microtubule bundles associated with chromosomes. Inset, enlarged image of one of the bundles (bar, 20 μm). (CâE) Representative images of metaphase II spindles oriented vertically to the cortex in the control oocytes. The spindle in C (bar, 100 μm) is enlarged in D (bar, 20 μm). Another example of metaphase II spindles is shown in E (bar, 20 μm). (F) Four examples of metaphase II spindles formed in the antibody-injected oocytes (bar, 20 μm). (G) An example of metaphase II spindles (arrow) formed at the center of the antibody-injected oocytes without anchoring to the cortex (bar, 200 μm). Inset, enlarged image of the spindle (bar, 20 μm). (H) Ratio of metaphase II spindles formed at the cortex (blue) to spindles formed at the center of oocytes (orange) in control (n = 9) and anti-XSSH antibodyâinjected oocytes (n = 10). (I, J) Metaphase I spindles formed at the cortex in a control oocyte (I) and formed in the yolk-free region without anchoring to the cortex in S3A-cofilinâinjected oocytes (J; three examples are shown). Bar, 100 μm.
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FIGURE 9:. Summary of organization of microtubules and actin filaments during oocyte maturation in Xenopus oocytes. (A) Stage VI oocytes form dense intranuclear actin filament networks (red) and cytoplasmic actin filament bundles surrounding the nuclei (not shown). Purple line represents nuclear envelopes (lamin). (B) As maturation begins, microtubules and cytoplasmic actin filaments (green and yellow, respectively) concentrate to the perinuclear cytoplasm (yolk-free zone, gray) at the vegetal side of the nuclei in a process that is dependent on XAC-mediated actin dynamics; this process is inhibited by anti-XSSH antibody injection. (C) When GVBD occurs from the vegetal side of the nuclei, the nucleus begins to shrink, the MTOC-TMA (green) develops well, and the cytoplasmic actin filaments (yellow) remain at its base. The intranuclear actin filaments then begin to disassemble from the vegetal side of the nuclei (orange). Although the TMA elongates to the animal side, it never enters the animal region, where the residual intranuclear actin filaments (red) are still present. (D) Disassembly of intranuclear actin filaments leads to the migration of the MTOC-TMA to the animal side and subsequent formation of the first meiotic spindles. The gray region represents the yolk-free zone.
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