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Abstract
In Xenopus, one of the most dramatic events during oocyte maturation is the breakdown of the oocyte's asymmetrically organized system of cytokeratin-type intermediate filaments. Following oocyte maturation in vitro, we found that (1) the breakdown of cytokeratin filament organization proceeds in an animal to vegetal direction, (2) cytokeratin filament breakdown occurs normally in enucleated oocytes and so is independent of nuclear components, and (3) the injection of maturation-promoting factor (MPF) induces the breakdown of cytokeratin filaments. While the MPF-induced breakdown of the nuclear envelope is independent of new protein synthesis, the MPF-induced breakdown of cytokeratin filament organization requires the translation of maternal mRNAs. These results strongly suggest that the factors regulating cytokeratin reorganization in the oocyte are distinct from those involved in the breakdown of the nuclear envelope.
FIG. 1. Cytokeratin filament breakdown in in vitro matured oocytes. A late stage oocyte was stained in whole-mount with a combination of
the antilamin antibody 14a9 and antinuclear pore antibody RLl and immunoperoxidase-conjugated secondary antibody (a). Oocytes were taken
either prior to (Oh) or 4 hr after (4h) exposure to progesterone. Both the nuclear envelope (N) and the cytoplasmic structures stained by the
antinuclear pore antibody RLl (AL) disappear in a global manner. Cytokeratin filament organization could be visualized in cortical whole mount
immunocytochemistry with the monoclonal anticytokeratin antibody lh5. In oocytes cultured in the absence of progesterone (b, c) the
asymmetry of cytokeratin organization is maintained (b-animal hemisphere; c-vegetal hemisphere/oocyte cultured for 6 hr). In progesterone-
treated oocytes (d-g) cytokeratin filament organization breaks down in a clear animal to vegetal direction. At 4 hr after exposure to
progesterone, the cytokeratin filaments in the animal hemisphere (d) have largely disappeared, leaving only some amorphous aggregates of
anticytokeratin reactivity, whereas the cytokeratin filaments in the vegetal hemisphere (e) remain largely intact. By 6 hr after exposure to
progesterone, cytokeratin filament organization has disappeared in both animal (f) and vegetal hemispheres (g). Bar in f marks 10 pm for b-g.
FIG. 2. Cytokeratin filament breakdown in enucleated oocytes. Enucleated oocytes were allowed to heal for 1 hr in modified Ringers and then
were exposed to either progesterone (2 rg/ml), to induce maturation, or to 0.04% DMSO. Only oocytes from which the nucleus was extruded
intact (a) (extruded nuclei marked nut) were used. After 6 hr the oocytes were fixed and stained with the anticytokeratin antibody lh5.
Cytokeratin filament organization appeared normal in enucleated oocytes exposed to DMSO (b), whereas cytokeratin filament organization
was disrupted in enucleated oocytes exposed to progesterone (d); the extent of the disruption was similar to that observed in nucleated oocytes
exposed to progesterone for the same amount of time (c). Images in b-d are of the vegetal hemisphere; bar in a marks 100 pm, bar in c marks
10 um.
FIG 3. Cytokeratin filament breakdown in MPF-injected oocytes. A preparation of MPF was obtained from J. L. Maller (UC Health Sciences
Center, Denver, CO). This preparation was prepared from progesterone-treated oocytes as described in (Lohka et al, 1988). The breakdown of
the oocytenucleus occurred within 1 to 2 hr of the injection of the MPF-containing preparation into oocytes. The nuclear envelope (NE) and
presumptive annulate lamellae (small arrows) were readily visualized in uninjected or control-injected oocytes stained in whole-mount with
RLl (a). The presence of cycloheximide (b) had no effect on the MPF-induced breakdown of the nuclear envelope and the presumptive annulate
lamellae (b). In b, oocytes were fixed and stained 4 hr after injection with MPF. Injection with the buffer used to prepare the MPF had no effect
on nuclear integrity (not shown). The cycloheximide insensitivity of the MPF effect on the nuclear envelope and the presumptive annulate
lamellae distinguishes it from progesterone-induced maturation. The injection of MPF induced the breakdown of the cytokeratin filament
system, which was generally complete in the vegetal hemisphere by 4 hr after injection (c). However, in cycloheximide-treated oocytes, the
cytokeratin filament system of MPF-injected oocytes remained intact, with no evidence of breakdown (d). Bar in c marks 10 pm.
