Gard DL (1991), Organization, nucleation, and acetylation of mi...

XB-ART-25082

Dev Biol 1991 Feb 01;1432:346-62. doi: 10.1016/0012-1606(91)90085-h.

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Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy.

Gard DL .

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Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.

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Species referenced: Xenopus laevis
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	FIG. 1. The DMlA monoclonal antibody is specific for Xe?lo;mcs o(- tubulin. (Lane A) SDS-PAGE of 35 pg of soluble cytoplasmic protein from stage VI oocytes, stained with coomassie blue. (Lane B) SDSPAGE Western blot of an identical lane, stained with DMlA monoclonal antibody against n-tubulin. (Lane C) SDS-PAGE Western blot of 100 ng of phosphocellulose-purified bovine brain tubulin, stained with DMlA.

	FIG. 3. Microtubule organization during stages II-IV. (A) Numerous cytoplasmic inclusions (arrows) in the cortex identify this as an early stage II oocyte. Microtubules are apparent throughout the cytoplasm, with no apparent organizing center (60X obj; bar is 10 pm). Many nucleoli are apparent in this grazing view of the GV. (B) A stage III oocyte (0.4 mm diameter) showing brightly stained cytoplasm surrounding the symmetrically located GV, a brightly stained cortex, and a fibrous array extending between (10X obj; bar is 100 pm). (C) Closer examination of the perinuclear cytoplasm, revealing a granular matrix with numerous microtubules (Y, yolk platelets; 60x obj; bar is 10 pm). (D) Numerous microtubules are apparent in the peripheral and cortical cytoplasm of this stage III oocyte (60X obj; bar is 10 pm). (E) In stage IV (0.6 mm diameter), the GV is asymmetrically located toward the animal pole (A). A fibrous array extends toward the animal pole (A) from the brightly stained perinuclcar cytoplasm, which is becoming localized toward the vegetal surface of the GV. Arrowheads denote the layer of pigment in the animal cortex (10x obj; bar is 100 pm). (F) Closer examination reveals the radial organization of microtubules and microtubule bundles near the animal cortex. The dark zone underlying the cortex (arrowheads) is due to laser scattering and absorption by pigment granules. A thin layer of residual follicle cells (f) is visible (60X obj; bar is 25 pm). (G) A tangential section 1-2 pm below the surface of a stage IV oocyte reveals a nonuniform distribution of microtubules in the lateral cortex (60X obj; bar is 10 pm).
	FIG. 4. Polarized microtubule distribution in stage VI oocytes. (AI Low magnification oocytc reveals the GV located near the animal pole. A brightly stained cap of cytoplasm is apposed to the vegetal surface of the GV. A fibrous network radiates from this material, surrounds the GV, and extends to the animal and lateral cortex. Arrowheads denote the pigmented layer in the animal cortex (10x ohj; bar is 100 pm). (B) Higher magnification of the perinuclear cap reveals an amorphous matrix with numerous emhcdded microtubules. A bright band of microtubules is seen just under the margin of the GV (black arrows). The amorphous matrix and microtubules extend into the nuclear clefts (white arrows; 60x obj; bar is 25 pm). (C) Grazing sections of the vegetal surface of the GV reveal a meshwork of microtubules, which extends into the clefts of the convoluted nuclear surface (60x obj; bar is 25 pm). (D) Numerous microtubules and microtubule bundles are apparent in the animal hemisphere. Arrowheads denote the pigment layer, which obscures the microtubules in the animal cortex (60x obj; bar is 25 pm). (E) A dense microtuhule network is interspersed with the large yolk platelets of the vegetal hemisphere (60X ohj; bar is 25 pm). (F) A tangential section of the lateral-animal hemisphere, about 10 pm beneath the oocyte surface (60X obj; bar is 10 pm
	FIG. 5. Microtubules in stage I oocgtes do not recover from cold-induced tiepolymerization. (A) No cytoplasmic or perinuclear microtubules are apparent after 2 hr of incubation at 4°C. (60x obj; bar is 25 pm). (B) Microtubules associated with the mitochondrial mass are completely depolymerized after 2 hr at 4°C. (60x obj; bar is 10 pm). (C) No regrowth of microtubules is apparent in stage I oocytes, 1 hr after return to room temperature (60x obj; bar is 10 pm). (D) Limited microtubule assembly is apparent in early stage II oocytes, after 2 hr at 4”C, and after 1 hr of recovery at room temperature (60~ obj; bar is 10 pm). (E) Complete recovery of follicle cell microtubules is apparent after 10 min of recovery at room temperature. Perinuclear MTOCs (N, nucleus) are recognizable in several follicle cells (arrows; 60x obj; bar is 10 Fm). (F) Extensive microtuhule assembly is apparent in this stage I oocyte after 45 min of recovery in 90% D,O at room temperature (60x obj: bar is 10 pm).
	FIG. 6. The GV functions as a microtubule nucleation site in stage VI oocytes. (A) No microtubules are apparent in the vegetal region of this oocyte after 45 min at 4°C. (60x obj; bar is 10 bm). (B) No microtubules are apparent in the perinuclear cap of this oocyte after 45 min at 4°C. (60x obj; bar is 10 pm). (C) Microtubule nucleation from the animal surface of the GV is apparent in this oocyte, 1 min after return to room temperature (60x obj; bar is 10 pm). (D) Microtubule nucleation from the vegetal surface of the GV is apparent in this oocyte, 2.5 min after return to room temperature (60x obj; bar is 20 pm). (E) Extensive microtubule assembly is apparent in the animal hemisphere of this oocyte, 5 min after return to room temperature. Arrowheads denote the pigment layer in the animal cortex (60x obj; bar is 20 pm). (F) Rapid recovery of microtubules is also apparent in the vegetal hemisphere of this oocyte, 5 min after return to room temperature (60x obj; bar is 25 pm
	FIG. 7. Microtubule regrowth is limited in enucleated stage VI oocytes. (A) One hour after enucleation, microtubules and microtubule bundles radiate from a brightly stained region of yolk-free cytoplasm (40x obj; bar is 50 pm). (B) Enlargement from A, showing the dense network of microtubules in the yolk-free cytoplasmic mass (60x oI)j; bar is 10 pm). (C) Numerous radially oriented microtubules are apparent in the animal hemisphere of this enucleatcd oocyte (60X obj; bar is 12.5 nm). (D) A less ordered microtubule array in the vegetal hemisphere of an enucleated oocgte (60X obj; bar is 12.5 pm). (E) No microtuhules are evident in the vegetal hemisphere of this enucleated oocyte after 60 min at 4”C, and 30 min recovery at room temperature (60X ohj; bar is 12.5 pm). (F, G) Extensive reassembly of microtubules in the animal (F) and vegetal (G) hemispheres of an enucleated oocgte after 30 min recovery in 90% D,G (60x obj; bars are 10 pm ).
	FIG. 8. Xenopus oocytes contain acetylated microtubules. (A) The 6-llB-1 antibody to acetylated a-tubulin stains microtubules in the cortical and perinuclear cytoplasm of this stage I oocyte (the mitochondrial mass is out of the plane of section; 60X obj; bar is 25 wrn). (B) Acetylated microtubules are also found associated with the mitochondrial mass of this stage I oocyte (60x obj; bar is 10 wm). (C) Numerous acetylated microtubules, organized into radially oriented bundles, are apparent in the animal cortex of this stage VI oocyte (60X obj; bar is 25 Fm). (D) Microtubules of the perinuclear cap stain with 6-llB-1. However, the surrounding matrix material stains very little (60x obj; bar is 10 pm).