Keratin filament organization in Xenopus oocytes... |
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Oogonia: Interphase
oogonia were not readily identifiable in our examination of ovaries
from juvenile frogs by confocal immunofluorescence microscopy. However,
KFs are conspicuously absent from mitotic oogonia (JPEG:
40 KB; AVI or QT)
and stage 0 oocytes lack KFs (see below), suggesting that they appear
later in oogenesis. |
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Stage
0: KFs
are conspicuously absent from the cytoplasm of stage 0 (post-mitotic)
oocytes. Nests of small stage 0 oocytes (JPEG:
56 KB), and larger individual stage 0 oocytes (JPEG:
45 KB; AVI or QT: 0.6
MB), are surrounded by follicle cells which stain brightly with
antibodies to keratins (counter-stained with a DNA dye to reveal
oocyte nuclei). |
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Stage
I: Cytoplasmic
keratin filaments do not become apparent until mid-stage I of oogenesis.
KFs are first seen in the perinuclear cytoplasm, and associated
with one or more perinuclear mitochondrial aggregates in mid-stage
I oocytes from juvenile frogs (JPEG:
86 kB). By mid-stage I, KFs surround the GV, surround and penetrate
the mitochondrial mass (JPEG:
68 kB; AVI or QT 1.1
MB), and link the GV and mitochondrial mass to the cortex (JPG, AVI or QT 1.8
MB). By late stage I-early stage II, a complex meshwork of KFs
extends throughout the cytoplasm (JPEG:
94 kB). |
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Stages
II - III: The
KF network is further elaborated throughout stages II-III of oogenesis.
By late stage I - stage II, oocytes contain a complex, but poorly-ordered
network of KFs extending throughout the oocyte cytoplasm (JPEG:
54 KB; QT or AVI: KB). During late stages II - III, the KFs adopt
a more radial organization, extending from a poorly organized perinuclear
network towards the KF network in the subcortical cytoplasm (JPEG:
115 KB; JPEG:
83 KB; AVI or QT: KB). |
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Stages
IV-V: The
transition from stage III to stage IV of oogenesis is marked by
the polarization of the distribution of pigment in the oocyte cortex.
However, no polarity is evident in the of KFs at this stage. Extensive
interlinked, radially-organized networks of KFs are apparent in
the animal (JPEG:
47 kB; AVI or QT: 2.0 MB), equatorial (JPEG:
46 kB; AVI or QT: 2.0 MB), or vegetal (JPEG:
43 kB; AVI or QT: 2.0 MB) cytoplasm (all three stereo images, JPEG:
133 KB). The cortical and cytoplasmic KF network of Xenopus oocytes
becomes visibly polarized during late stage IV - stage V of oogenesis
(JPEG:
65 KB). Polarization of the cortical KF network occurs in an animal
to vegetal direction, as the KF network of the animal cortex first
adopts the final thickness and mesh size seen later in stage VI.
The network of KFs in the vegetal cortex remains thickened during
stage IV, adopting its final thickness and mesh size during late
stage V-VI. |
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Stage VI: 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). |
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, the organization and
polarization of KFs in stage VI oocytes are dependent upon both F-actin
and microtubules.
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| 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). | |
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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 ). |
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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). |
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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. |
References: Gard, D.L., and Klymkowsky,
M. W. (1998). Intermediate filament organization during oogenesis
and early development in the clawed frog, Xenopus laevis. In: Subcellular
Biochemistry: Intermediate Filaments (H. Herrmann and J.R. Harris, eds).
Plenum Press. pp 35-69. |
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