Cytoskeletal
organization in stage 6 oocytes...
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Microtubules: Estimates of the tubulin pool indicate that a single stage VI oocyte contains sufficient tubulin to assemble 1.5 - 2.0 kilometers of MT, and that nearly 20% is present in polymer, corresponding to 300 meters of MT per oocyte! Confocal microscopy suggests that a single oocyte contains as many as 0.5 - 1 million individual MTs! In the animal hemisphere, MTs appear to radiate from the GV towards the cortex, often in loosely-associated bundles within yolk-free tracts of cytoplasm, called "radii"(JPEG: 90 KB; AVI or QT: 2.3 MB). Many of the MTs in these radial bundles stain brightly with antibodies to acetylated a-tubulin (JPEG1: 89 KB; AVI or QT: 2.3 MB), suggesting that they are stable or non-dynamic. These acetylated MTs appear to terminate at, or very near, the animal cortex, as shown in these stereo images (JPEG: 89 KB). Recently, we have shown that XMAP230 is associated with acetylated MTs in stage VI oocytes, and that XMAP230 is required for the organization and stabilization of MTs in stage VI oocytes. |
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The MT array of the vegetal hemisphere appears less organized, as individual MTs wend their way around the larger yolk platelets found in the vegetal hemisphere (JPEG: 83 KB; in stereo, JPEG: 43 kB; AVI or QT: 2.3 MB). Fewer acetylated MTs are present in the vegetal hemisphere (JPEG: 68 KB; AVI or QT: 2.1 MB). MTs and acetylated MTs also surround the GV (acetylated MTs in stereo, JPEG: 43 KB), and are highly concentrated in the yolk-free cap of cytoplasm at the base of the GV (JPEG: 72 KB). |
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Antibodies to g-tubulin reveal that this centrosomal protein is concentrated in perinuclear cytoplasm (JPEG: 18 KB) and cortex (JPEG: 14 KB; JPEG: 40 KB) of stage VI oocytes. Interestingly, the distribution of g-tubulin in the cortex of VI oocytes is highly polarized along the animal-vegetal axis. In the vegetal cortex, g-tubulin is found in numerous, brightly-stained spots, often arranged into short linear arrays (JPEG: 54 KB). In the animal cortex, g-tubulin is more evenly distributed in small, less brightlystained, foci (JPEG: 47 KB). The A-V polarity of cortical g-tubulin suggests that this centrosomal protein may play an important role in organizing the polarized MT array of stage VI Xenopus oocytes. |
| The concentration of g-tubulin in the oocyte cortex implies that some oocyte MTs have their minus-ends anchored in the oocyte cortex. Hook-decoration and electron microscopy to revealed that the majority of MTs in stage VI oocytes are oriented with their minus-ends out, an orientation opposite that found in most somatic cells (manuscript in preparation). | |
| Cold- or nocodazole-induced disassembly of the oocyte MT array results in rapid displacement of the GV from its normal position in the animal hemisphere (JPEG: 29 KB), indicating that MTs play an important role in the positioning and anchoring the oocyte nucleus. Treatment of stage VI oocytes with nocodazole also disrupts the A-V asymmetry in the distribution of cortical keratin filaments (JPEG: XX KB), suggesting that keratin organization is dependent upon MTs. | |
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F-actin: The GV of stage VI oocytes is brightly-stained with rhodamine-phalloidin (JPEG: 33 KB), indicating that F-actin is highly concentrated in the oocyte nucleus. Actin cables are prominent in the perinuclear cytoplasm surrounding the intensely-stained GV and in the yolk-free cap of cytoplasm at the base of the GV (JPEG: 58 KB). In the animal hemisphere,
actin cables are concentrated in the yolk-free tracts of cytoplasm
that extend from the perinuclear region to the cortex (JPEG:
63 KB). In deeper regions of the vegetal hemisphere, actin cables
form a complex, 3-D network extending throughout the cytoplasm (JPEG:
63 KB). Grazing views of the animal hemisphere of fluorescent phalloidin-stained
oocytes reveal macrovilli of surrounding follicle cells (JPEG:
68 KB), dense networks of microvilli on the oocyte surface (JPEG:
78KB), and a network of subcortical actin cables (JPEG:
54 KB; seen in stereo from the inside, JPEG:
48 KB). Sub-cortical actin cables are also apparent below the vegetal
cortex (JPEG:
41 KB). |
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Keratin filaments: By stage VI, Xenopus oocytes contain highly polarized networks of cortical and cytoplasmic KFs. A-V polarity of the cortical KF network is evident in the apparent mesh size of the KF filament network in the vegetal and animal cortex: the vegetal KF network has a coarser mesh than that in the animal cortex (JPEG: 98 KB, in stereo, JPEG: 116 KB). Electron micrographs suggest that the anastomosing network of KFs in the cortex of stage VI oocytes actually consists of bundles of KFs. Cross sections (JPEG: 73 KB; AVI or QT: 1.7 MB) reveal that the KF network in the animal cortex is about twice as thick, extending deeper into the sub-cortical cytoplasm, as that in the vegetal cortex. Cross-sections also reveal the A-V asymmetry in the organization of cytoplasmic KFs: KFs in the animal hemisphere exhibit a more radial organization than those of the vegetal hemisphere. 3-D reconstructions of the KFs of the animal hemipshere reveal the radial filaments, and a network of transverse interconnections (JPEG: 43 KB). A meshwork of KFs also surrounds the GV and extends throughout the perinuclear cap yolkfree cytoplasm (JPEG: 47 KB; stereo pairs, JPEG: 116 KB). |
| Cytoskeletal
organization in stage 6 oocytes. Although
the KF network of stage VI oocytes appears complex, the number of radial
KFs (estimated to be ~12,000) is far exceeded by the number of MTs (estmated
to be ~0.8 - 1.0 million). Disruption of the KF network by injection
of monoclonal antibodies (JPEG:
54 KB) has no apparent affect on cytoplasmic organization (including
the position of the GV, the distribution of yolk and pigment, and the
organization of other cytoskeletal elements). Thus, the role of KFs
during oogenesis remains uncertain. However, evidence suggests
that the organization and polarization of KFs in stage VI oocytes are
dependent upon both F-actin and microtubules (Gard
et al., 1997) .
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