MT organization during oocyte maturation:
assembly of the first meiotic spindle...
A dramatic reorganization of the microtubule cytoskeleton accompanies progesterone-induced maturation of Xenopus oocytes, culminating in the assembly of the meiotic spindles. However, unlike most somatic cells, extensive networks of cytoplasmic MTs exist throughout M1 and M2.
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1. Assembly and migration of the MTOC-TMA. Germinal vesicle breakdown (GVBD) begins at the basal (vegetal) surface of the GV, coincident with assembly of a novel, disc-shaped microtubule-organizing center (MTOC) near the base of the GV. MTs extend from this MTOC into the nucleoplasm, forming a transient MT array (TMA) (JPEG: 27KB). Assembly of the MTOC-TMA complex occurs simultaneously with, or slightly precedes, formation of the white maturation spot at the animal pole of the maturing egg.

Within twenty minutes, the MTOC-TMA complex translocates 400-500 um, from its site of assembly near the basal surface of the GV to the animal pole (JPEG: XX KB; JPEG: 20KB). The molecular mechanisms underlying this rapid (20-25 um/min) and directed migration remain unknown. However, the formation of ectopic spindles in oocytes in which the GV has been displaced by cooling and inversion suggests that the MTOC-TMA complex is not specifically targeted to the animal pole. The effects of cytochalasin B suggest that F-actin is required for assembly or maintenance of the MTOC-TMA, but not for its migration.

The MTOC-TMA is highly polarized, with numerous, long MTs extending from its apical side into the nucleoplasm, and fewer, shorter MTs extending from its basal surface into the cytoplasm (mid-sun, JPEG: XX KB; late-sun, JPEG: 24KB). The appearance of the MTOC-TMA complex rising towards the animal pole has led to an informal nomenclature used in the Gard lab: it is simply referred to as "the sun."

During its translocation, the MTOC-TMA complex collects the condensed meiotic chromosomes and transports them to the animal cortex (JPEG: 32KB; JPEG: 30KB).  The mechanism by which the chromosomes become associated with the MTOC-TMA complex is not known. However, the appearance of the MTOC-TMA complex as it rises towards the animal pole, with MTs "sweeping" the nucleoplasm ahead of it, leads us to believe that chromosomes may be captured by dynamic MTs, much in the same way chromosomes are captured by MTs emanating from the poles of the mitotic spindle in somatic cells.
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2. Assembly and rotation of the first meiotic (M1) spindle. Once the MTOC-TMA complex arrives at the animal pole, it serves as the immediate precursor of the first meiotic spindle. Assembly of the first meiotic spindle proceeds by a pathway which includes four distinctive stages: (1) compaction of the complex to form a condensed aggregate of MTs and chromosomes; (2) establishment of the bipolar spindle axis, to form a short M1 spindle; (3) elongation of the spindle during prometaphase in an orientation transverse to the A-V axis (and therefore parallel to the oocyte surface); and (4) anchoring of one spindle pole to the cortex, followed by rotation into alignment with the A-V axis.

During the compaction stage, little or no bipolar organization is evident (JPEG: KB; JPEG: 14 KB), although remnants of the MTOC-TMA organization are often apparent. Staining with antibodies to g-tubulin reveals that this centrosomal protein is concentrated around the individual chromosomes (JPEG: 21 KB), consistent with previous observations that chromosomes play a major role in promoting the assembly and organization meiotic spindles in other species.
  3. Formation of the bipolar spindle axis gives rise to a short spindle with numerous overlapping MTs (JPEG: 31KB). During this stage, g-tubulin is observed throughout the spindle, making it unlikely that the distribution of this protein is solely responsible for organization of the spindle poles. Establishment of the bipolar organization may result from the interaction and bundling of parallel MTs by MT-dependent motors. Interestingly, the initial bipolar spindle axis invariably forms parallel to the oocyte surface, suggesting some interaction between the nascent spindle and the oocyte cortex. Additional evidence for such a regulatory interaction between the cortex and spindle comes from the increased formation of monasters when the normal spindle/cortex interaction is disrupted by cytochalasin B or ectopic spindle assembly.
m1 sidebar2 4. The bipolar spindle then elongates in an orientation parallel to the oocyte cortex (JPEG: 23 KB; AVI or QT: 250KB). The orientation of the spindle axis and elongation appears to bear no relation to the major axis during the compaction phase. Dual labeling with anti-tubulin and chromatin dyes revealed that elongation occurs during prometaphase (JPEG: 13KB). Nearly normal appearing spindles are observed in cytochalasin B-treated oocytes, suggesting that spindle elongation does not depend upon F-actin or anchoring to the cortex. Rather, the presence of numerous, overlapping MTs in the central spindle suggests that spindle elongation during prometaphase occurs by active sliding of anti-parallel MTs, in a manner similar to that observed in anaphase B spindle elongation in some species.  As the spindle elongates, g-tubulin becomes concentrated at the spindle poles (JPEG: 22KB).
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5. The transversely-aligned M1 spindle next anchors to the cortex and rotates into alignment with the A-V axis during or just before metaphase (JPEG: 20KB; JPEG: 21KB). Anchoring and rotation of the spindle is inhibited by cytochalasin B, suggesting that cortical F-actin is required for these processes. Ectopic spindles assembled near the vegetal cortex are also unable to anchor and rotate, providing evidence for a functional polarization of the oocyte cortex.
polar body sidebar Finally, extrusion of the first polar body coincides with completion of anaphase (JPEG: 25KB).
MT organization during oocyte maturation:
assembly of the second meiotic spindle...
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Assembly of the second meiotic spindle follows a pathway (JPEG: 15KB) similar to that observed in M1. Unlike a typical interphase in a somatic cell, the condensed M1 chromosomes never fully de-condense in meiotic interphase (JPEG: 13KB), and there is no evidence that a nuclear envelope forms around them. Spindle assembly begins with formation of a compact, unpolarized aggregate of MTs (JPEG: 13KB) and condensed meiotic chromosomes, which then re-organize to form a short, bipolar spindle (JPEG: 21KB). Much as in M1, the M2 spindle elongates during prometaphase (JPEG: 18KB; AVI or QT: 300KB), anchors to the cortex, and rotates into axial alignment (JPEG: 23KB; AVI or QT: 350KB). Time-lapse confocal microscopy revealed that M2 spindles migrate as much as 50 um during rotation, consistent with a model in which long astral MTs anchor to the cortex and both pull and rotate the spindle into axial alignment. The meiotic cell cycle then arrests during second meiotic metaphase (JPEG: 30KB).
A complex and dynamic network of cytoplasmic MTs exists throughout the maturation of Xenopus oocytes.
 

Coincident with GVBD and assembly of the MTOC-TMA complex, the cytoplasmic MT array of the stage VI oocyte is extensively remodeled. The extensive network of stable, acetylated, MTs is rapidly disassembled. A wave of disassembly appears to begin near the base of the GV, and spreads outward. In the animal hemisphere, disassembly spreads around the GV, often resulting in the appearance of a transient cap of MTs between the remnant of the apical surface of the GV and the animal cortex (JPEG). Disassembly also spreads outward through the vegetal hemisphere towards the vegetal cortex (JPEG). Activation of M-phase MT severing factors may play a role in this remodeling process.

As the MTOC-TMA complex migrates to the animal cortex (through the now released nucleoplasm), an extensive network of cytoplasmic MTs is assembled in the animal hemisphere (JPEG), apparently nucleated by a dispersed MTOC near the region previously occupied by the basal surface of the GV (JPEG). The MTOC-TMA migrates through a MT-free cylinder surrounded by this network of cytoplasmic MTs (JPEG). Unlike the MT array of stage VI oocytes, MTs of this M-phase network are not acetylated, suggesting that they are highly dynamic (JPEG).

A complex network of MT aggregates/asters is commonly observed in the vegetal hemisphere during the early stages of maturation (JPEG). Cross-sections of the oocyte equator often reveal an intricate pattern of MTs loosely organized into concentric rings and spokes (JPEG). It is not clear whether these MTs are remnants of the oocyte MT array, or whether they represent de novo assembly.

The cytoplasmic MT network in the animal hemisphere persists throughout M1 (JPEG: 60KB) and M2 (JPEG: 65KB). Interestingly, spindle assembly occurs in a small, hemispherical region devoid of cytoplasmic MTs. Although MTs are more numerous in the animal hemisphere and cortex, substantial numbers of MTs are also present in the vegetal cortex throughout maturation. These observations indicate that the assembly and dynamics of MTs during oocyte maturation  is regulated both temporally and spatially.
Actin organization during 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.
Keratin filament organization
during oocyte maturation...
As in most somatic cells, the cytoplasmic intermediate filament network of Xenopus oocytes is dramatically remodeled as oocytes enter Mphase during oocyte maturation (Klymkowsky et al., 1987; Klymkowsky and Maynell, 1989). Oocyte maturation is accompanied by the disassembly of oocyte keratin filaments (KFs) into soluble oligomers (Klymkowsky et al., 1991). KF disassembly is induced by MPF, but also requires ongoing protein synthesis (Klymkowsky et al., 1987; Klymkowsky and Maynell, 1989).
kfs cortical Confocal microscopy revealed that disassembly of the cortical KF network begins in the animal hemisphere. The KF network of the animal cortex is largely disassembled within 10 minutes of appearance of the white maturation spot (JPEG: 119KB). In contrast, disassembly of the KFs of the vegetal cortex is not apparent until later, but is usually completed within 60 minutes of WSF ( JPEG: 42KB ).
kf cytoplasmic sidebar The radial array of cytoplasmic KFs is also rapidly disassembled during maturation. Confocal microscopy reveals numerous short KF fragments (JPEG: 46KB), consistent with previous suggestions that disassembly occurs via filament severing (Klymkowsky et al, 1991).
kf sun sidebar As maturation proceeds, short KFs or oligomers become associated with the transient MTOC-TMA that serves as the precursor of the first meiotic spindle (JPEG: 63KB). This association suggests that KFs may play a structural role in the assembly or organization of this complex. However, nearly complete elimination of these KFs by the injection of specific monoclonal antibodies had no effect on the structure of the MTOCTMA complex or subsequent assembly of the meiotic spindles (JPEG: 39KB). The role(s) of KFs during oocyte maturation, if any, thus remains uncertain.