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A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA.
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In an effort to understand how polarity is established in Xenopus oocytes, we have analyzed the process of localization of the maternal mRNA, Vg1. In fully grown oocytes, Vg1 mRNA is tightly localized at the vegetal cortex. Biochemical fractionation shows that the mRNA is preferentially associated with a detergent-insoluble subcellular fraction. The use of cytoskeletal inhibitors suggests that (1) microtubules are involved in the translocation of the message to the vegetal hemisphere and (2) microfilaments are important for the anchoring of the message at the cortex. Furthermore, immunohistochemistry reveals that a cytoplasmic microtubule array exists during translocation. These results suggest a role for the cytoskeleton in localizing information in the oocyte.
Fig. 1. Specific association of Vgl mRNA with the
detergent-insoluble fraction of extracts. (A) Schematic
representation of the distribution of Vgl mRNA in oocytes
and eggs. Vgl mRNA (dark shading in picture) is initially
distributed homogeneously in early stage oocytes (stage II),
undergoes a process of localization in middle stage oocytes
(early stage IV), and culminates in a tight cortical shell by
the end of oogenesis (stage VI; Melton, 1987). Unfertilized
eggs have Vgl mRNA distributed in a broad band in the
vegetal hemisphere along the cortex (egg; Weeks and
Melton, 1987). (B) Northern blot analysis of RNA
detergent-extracted from oocytes and unfertilized eggs.
Oocytes or eggs were homogenized and fractionated into a
soluble fraction and an insoluble pellet by centrifugation, as
described in Materials and methods. RNA was then
purified from the pellet (P, lanes 1,4, and 7), the soluble
fraction (S, lanes 2, 5, and 8) or total, unfractionated
extract (T, lanes 3, 6, 9) and analyzed by Northern blot
hybridization with both a fibronectin (fb) and Vgl probe.
Two oocyte-equivalents of RNA was run in each lane.
Lanes 1-3, from stage II oocytes; lanes 4-6, from stage VI
oocytes; and lanes 7-9, from unfertilized eggs.
Fig. 2. Vgl mRNA distribution in drug-treated,
late-stage oocytes. In situ hybridization using a
Vgl probe reveals the localization of the
message in oocytes incubated overnight in
saline (A), progesterone (B), nocodazole (C),
or cytochalasin B (D). The scale bar indicates
500/tm. The germinal vesicle (gv) is indicated in
those sections where it is present.
Fig. 3. Specific release of Vgl mRNA into the soluble
fraction of detergent extracts by treating with cytochalasin
B. Stage VI oocytes cultured overnight in saline with no
drug treatment (control; lanes 1-3), with nocodazole
(nocod; lanes 4-6), or with cytochalasin B (cytoB; lanes
7-9) were homogenized and their RNA analyzed as in
Fig. 1. RNA purified from the insoluble pellet was run in
lanes 1, 4, and 7, from the soluble fraction in lanes 2, 5,
and 8, and from total extracts in lanes 3, 6, and 9.
Fig. 4. The effects of cytoskeletal inhibitors on the
translocation of Vgl mRNA in middle stage oocytes. Late
stage III oocytes (0.5-0.6 mm in diameter) were cultured in
vitro for 5 days and then assayed for Vgl mRNA
distribution by in situ hybridization. Oocytes were grown
either in medium without serum (A) or in medium with
serum, either with no drug (B) or in conjunction with
cytochalasin B (C) or nocodazole (D). The scale bar
represents 200 um.
Fig. 5. The specific effect of depolymerizing microtubules
on the translocation of Vgl mRNA in middle stage oocytes.
Late stage III oocytes were cultured in vitro in medium
containing serum and either the potent microtubule
depolymerizing agent tubulozole-C (tub-C) or its inactive
trans isomer tubulozole-T (tub-T) and assayed as in Fig. 4.
The scale bar represents 200 um.
Fig. 6. Whole-mount immunohistochemistry of /3-tubulin in
oocytes. Oocytes were defolliculated, fixed and stained
using an anti-/3-tubulin antibody according to the procedure
of Dent et al. (1989). (A) Untreated oocytes of the
indicated stages were stained. The staining in stages II and
III is radially symmetric. The stage VI oocyte is shown
viewed from the animal hemisphere. (B) Stage VI oocytes
were incubated in either cytochalasin B (cyto B) or
nocodazole (nocod) overnight in saline and then processed
as above. Both are shown as viewed from the animal
hemisphere. The punctate staining of the nocodazoletreated
oocyte, evidence for the disruption of the
microtubule array, is detectable only in the animal
hemisphere. The scale bar for A and B represents 500 fan.
(C) A lateral view of an untreated stage VI oocyte at
slightly higher magnification showing the tubulin array
emanating from around the gv evident only in the animal
hemisphere. The scale bar represents 500UM.
Fig. 7. A two-step model for the localization of Vgl
mRNA in oocytes. The diagram represents the simplest
interpretation of the data presented in the paper. Vgl
mRNA distribution (indicated by shading) becomes
progressively restricted to the vegetal cortex during normal
oogenesis (indicated by the horizontal arrows). Microtubule
inhibitors, such as nocodazole or colchicine, block the first
part of this process, which we have termed translocation,
and result in the prevention of any noticeable migration of
Vgl mRNA. Microfilament inhibitors, such as cytochalasin
B, prevent the second step of the localization, which we
have called anchoring, and result in the release, in late
stage oocytes, of the Vgl mRNA from its tight cortical shell
into a broad band along the cortex. (In middle stage
oocytes when the translocation process is occurring,
cytochalasin B prevents anchoring at the cortex as well and
results in the ectopic accumulation of Vgl mRNA in the
cytoplasm above the cortex; see Fig. 4C).
