F-actin organization in Xenopus oocytes...
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ck-sidebar  Oogonia: Individual interphase oogonia were not readily identifiable in our examination of fluorescent-phalloidin-stained ovaries from juvenile frogs. However, the absence of cytoplasmic cables or GV-staining during stage 0 (JPEG: 43 KB) suggests that F-actin is restricted to the cortex of oogonia, as it is during stage 0 of oogenesis.
actin-sidebar  Stage 0: Confocal microscopy of stage 0 oocytes stained with fluorescent phalloidin reveals that F-actin is restricted to the oocyte cortex. Nests of stage 0 oocytes can be recognized as clusters of oval or pear-shaped cells with prominent staining of the cortical cytoplasm  (JPEG: 44 KB).
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 Stage I: The cortex of early stage I oocytes stains brightly with fluorescent phalloidin, indicating a concentration of f-actin in the cortical cytoplasm. In addition, a network of cytoplasmic actin cables first appears during early stage I (JPEG: 55 kB).These cables are sparsely scattered in the cytoplasm, and do not appear to be associated with discrete organizing centers. The complexity and density of this network increases during oocyte growth and differentiation. Assembly of the cytoplasmic network of actin cables is accompanied by the appearance of F-actin in the oocyte nucleus, or GV. Initially, F-actin appears in patches surrounding nuclear inclusions, such as nucleoli.

By mid-late stage I, the cortex and GV both stain brightly with fluorescent phalloidin (JPEG: 52 kB), and a dense network of actin cables extends throughout the cytoplasm (JPEG: 68 kB; stereo JPEG; AVI or QT: 1.5 MB). Actin cables are associated with the mitochondrial mass, which also exhibits a diffuse staining (JPEG: 69 kB).
S2actnlm Stages II-III: The GV of stage II-III oocytes is brightly stained by fluorescent phalloidin (JPEG: 62 KB), though significant attenuation of the signal is apparent due to the accumulation of yolk. Cortical, subcortical, and cytoplasmic actin cables are also apparent during these stages of oogenesis (not shown).
St4_actin low Stages IV-V: During stages IV-VI, substantial concentration of Factin is seen in the GV (JPEG: 15 KB). In addition,  a complex network of actin cables extends throughout the subcortical (JPEG: 19 KB)
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 Stage VI: 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).
Disruption of F-actin with cytochalasin B results in displacement, rotation, and distortion of the GV, suggesting that F-actin plays a role in anchoring the GV and maintaining GV morphology. In addition, cytochalasin disrupts MT (JPEG XX KB) and KF organization (JPEG: XX KB) in stage VI oocytes, suggesting a hierarchy of interactions between cytoskeletal elements.
Oocyte maturation: Actin is the most difficult of three cytoskeletal networks to visualize. Thus, we know less of the reorganization of actin distribution during maturation. However, the effects of cytochalasin B on oocyte maturation suggest that Factin is required for assembly of the transient MTOC and microtubule array (MTOC-TMA complex, or the "sun"), and for the anchoring and rotation of the meiotic spindles.
sun sidebar Maturation of Xenopus oocytes is accompanied by the assembly of a novel, disc-shaped MTOC and transient MT array (the MTOC-TMA) that serves as the immediate precursor of the first meiotic spindle . Treatment of Xenopus oocytes with cytochalasin B (CB: 10-50 mg/ml, beginning 0-3 hrs prior to addition of progesterone) during progesterone-induced maturation disrupted the organization of this MTOC-TMA complex. In many cases, the MTOC-TMA complex appeared severely distended or stretched (JPEG: 28 KB), and in the most extreme cases, appeared split into multiple parts (JPEG: 20 KB). Time-lapse observation of CB-treated oocytes during maturation reveals a dramatic churning of the cytoplasm (AVI or QT: XX MB), which may rip the MTOC-TMA apart.
Rhodamine-phalloidin revealed a concentration of F-actin in a disc-shaped structure (JPEG: 29 KB), believed to represent the base (MTOC) of the MTOC-TMA complex. These observations usggest that F-actin plays a critical role in the assembly or maintenance of MTOC-TMA structure.Treatment with CB did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism.
cb_spndl_80 Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2, indicating that spindle assembly is not dependent upon Factin. However, multiple M1 spindles  (JPEG: 30 KB) were observed in many CB-treated oocytes, presumably resulting from the splitting of the MTOC-TMA complex and dispersion of the chromosomes in the animal cortex.
monaster 80 The incidence of monaster assembly (JPEG: 19KB; JPEG: 13KB) during early M1 was also significantly increased by CB treatment (table: 68KB), suggesting that F-actin dependent interactions between the nascent spindle and the oocyte cortex may play a role in the timing of spindle elongation.
spindle sidebar CB-treated oocytes in late M1 often contained transversely-oriented metaphase (JPEG: 15KB), anaphase, or  telophase spindles (JPEG: 24KB) indicating that cortical Factin is required for the anchoring and rotation of the meiotic spindles. Interestingly, M1 spindles in CB-treated oocytes often exhibited extensive arrays of astral MTs emanating from both spindles poles (JPEG: 26KB). Extensive polar asters are not commonly observed features of meiotic spindles in normal oocytes. The extensive asters of CB-treated spindles may result from the inability of astral MTs to make proper connections to the oocyte cortex.  Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment (JPEG), further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles.
m2 80 Failure of the M1 spindle to rotate into an axial orientation also blocked cytokinesis, and subsequently resulted in the assembly of twin spindles during M2 (JPEG: 28KB). Together, these observations suggest that there is an intimate association between the nascent meiotic spindles and the oocyte cortex that is dependent upon cortical Factin.
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