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Stage VI Xenopus oocytes contain an extensive network of cytoplasmic microtubules (MTs), with no evidence of a functional centrosome. Recently, Stearns et al. (1991) demonstrated that Xenopus eggs contain a substantial pool of the centrosomal protein gamma-tubulin (gamma-Tb). For this report, I have used confocal immunofluorescence microscopy to examine the distribution of gamma-Tb during the later stages of oogenesis in Xenopus laevis. gamma-Tb was apparent surrounding the germinal vesicle (GV) of stage VI oocytes, consistent with previous results suggesting that the GV serves as an microtubule organizing center in later oogenesis. Surprisingly, gamma-Tb was also concentrated in the cortex of stage VI oocytes, and the distribution of cortical gamma-Tb was polarized along the animal-vegetal (A-V) axis. In the vegetal cortex, gamma-Tb was observed in brightly stained foci, often organized into short linear arrays. In the animal hemisphere, gamma-Tb was more evenly distributed as small cortical foci. Dual immunofluorescence microscopy revealed that gamma-Tb in the vegetal hemisphere was associated with MTs in the cortical cytoplasm. The distribution of gamma-Tb was not significantly affected by either cold or nocodazole, but was partially disrupted by cytochalasin B. gamma-Tb thus may serve as a link between the oocyte MT network and cortical actin. Finally, polarization of the distribution of cortical gamma-Tb temporally coincides with formation of the A-V axis and polarization of the oocyte MT cytoskeleton during stage IV of oogenesis. These observations raise a number of questions regarding the organization and orientation of MTs during Xenopus oogenesis and the role of gamma-Tb in the polarization of the oocyte cytoskeleton during A-V axis formation.
FIG. 1. XGAM antiserum stains centrosomes in Xenom~s ovarian follicle cells. Dual-channel confocal microscopy was used to compare the
staining pattern of XGAM (A) and DM1A (B) antiserum in ovarian follicle cells. XGAM stained a perinuclear focus in interphase follicle cells
(arrows in A), located at the center of the microtubule array stained with DMlA (arrows in B). Faint cytoplasmic staining in A results from
.:rossover between the channels and superposition of the underlying oocyte cortex (see text). Scale bar is 10 I'm.
FIG. 2. 'Y·Tubulin is concentrated in the perinuclear and cortical cytoplasm of stage VI Xenopus oocytes. (A, B) Stage vr oocytes stained with
XGAM at low magnifications exhibited a perinuclear rim of -y-Tb specific fluorescence (al'rows in A) that was not apparent in oooytes stained
with normal rabbit serum (B). (C) A prominent rim of X GAM-specific fluorescence was apparent in the vegetal hemisphere of stage VI oocytes
stained with XGAM (large arrows; small arrows delineate the animal cortex, and A denot~s the animal pole). (D, E) Grazing optical sections of
the vegetal cortex (D) reveal a complex pattern of XGAM·stained foci, often organized into short linear arra~·s (arrows). This pattern was not
observed in oooytes stained with normal rabhit serum (E). (F, G) A thin shell o{ XGAM staining was apparent in cross sections of the vegetal
cortex (F) and in grazing sections in which the vegetal cortex was partially peeled away (G). Scale bar is 100 11m in A and B. 250 ,.min C, 25 I'm in
D and F, and 10 11m in E and G.
FIG. 3. The d istribution of y·tubulin in the oocyte cortex is polari~ed along the A-V axis of stage VI, but not stage lll, oocytes. (A, B)
Aggregates of "Y-Tb are ar1parent in the vegetal cortex of bleached stage VI oocytes (A) while -y-Tb is more evenly distributed as smaller, faintly
stained foci in the animal cortex (B). Images were collected with identical gain and were photographed and printed identically. (C) An albino
stage HI oocyte (approx 650 I'm in diameter) does not exhibit the hright cortical stain observed in stage VI oocytes. GV denotes the germinal
vesicle. (D. E) Grazing sections of opposing regions of the cortex from a bleached st;Jge III oocyte (550-600 ~''" in diameter) stained with XG AM
anti:;erum revealed comparable staining patterns. Images in D and E were collected from a single oocyte at identical gain and were photographed
and pr inted identically. Scale bars are 10 I'm in A, B, D, and E and 100 11m in C.
FIG. 4. Polarization of cortical -y-tubulin coincides with formation of the A-V axis in stage IV m>eytes. (A) A low-magnification image of a
stage IV oocyte (approx 900 ,um in diameter) reveals a bright rim of staining in the vegetal cortex (arrows). (B, C) Grazing optical sections of the
vegetal cortex (B) of a bleached stage IV oocyte (800-900 pm diameter) stained with XGAM antiserum revealed prominent aggregates of -y-Tb in
the vegetal hemisphere. -y-Tb in the animal cortex of the same oocyte (C) was present as small foci, similar to those observed in the animal
cortex of stage VI oooytes (compare with Fig. 3B). (D, E) A-V polarization of "Y·Tb in the cortex of stage IV oocytes is also apparent in oblique
cross sections ofthe vegetal (D) and animal (E} cortex of oocytes stained with XGAM antiserum. The apparent thickness ofthe -y-Tb layer in the
vegetal cortex (D) results from the oblique angle of sectioning. Arrows in E denote eentrosomes in the overlying follicle layer. Scale ban are 100
,.m in A, 25 I'm in Band C, and 10 11m in D and E.
FIG. 5. -y-Tubulin is associated with microtubules in the cortical cytoplasm. (A) Optical cross sections of stage VI oocytes double stained with
DM1A ( red channel) and XGAM (green channel) revealed concentrations of -y-Th in the vegetal cortex where microtubules are closely apposed
to the oocyte surface. Note the lack of significant y-Tb Auorescence associated with microtubules in the oocyte cs'ioplasm. (B) Grazing sections
of the veget;tl cortex of stap:e VI oooytes double stained with 6-11B·1 (acetylated " -tubulin) (red channel) a nd XGAM antis~ra (green channel)
revealed aggregates(){ -y·Tb (arrows) along acetylated (stable) microtubules. Scale bars arc 10 pm.
FIG. 6. )'-Tubulin in the vegetal cortex is partially disrupted by cytochalasin. (A, B) No significant differences were apparent in the distribution
of )'-Tb in the vegetal cortex of control (A) and nocodazole-treated (10 ~-tglml for 4 hr) stage VI oocytes (B). (C, D) In contrast, ,-Tb appears
aggregated and less evenly distributed in a grazing section (C) of the vegetal cortex of a cytochalasin B-treated oocyte (25 p,g/ml for 5 hr). Cross
sections (D) of cytochalasin-treated oocytes revealed disruption of the cortical layer of )'-Tb by CB (compare to Fig. 2F ), with ,-Tb foci in the
subcortical cytoplasm (arrows). Scale bars are 10 ~-tm.
