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The maternal mRNA Vg1 is localized to the vegetal pole during oogenesis in Xenopus. We have cultured oocytes in vitro to begin to understand how this localization occurs. Endogenous Vg1 mRNA undergoes localization when oocytes are cultured in vitro, and synthetic Vg1 mRNA injected into such oocytes is localized in the same fashion. Vg1 mRNA is associated with a detergent-insoluble fraction from homogenized oocytes, suggesting a possible cytoskeletal association. The use of cytoskeletal inhibitors reveals a two-step process for localizing Vg1 mRNA. Microtubule inhibitors such as nocodazole and colchicine inhibit the localization of Vg1 mRNA in late stage III/early stage IV oocytes, but have no effect on Vg1 mRNA once it is localized. The microfilament inhibitor cytochalasin B, however, has little effect on the translocation of Vg1 mRNA in middle-stage oocytes but causes a release of the message in late-stage oocytes. We propose a model for the localization of Vg1 mRNA in which translocation of the message to the vegetal cortex is achieved via cytoplasmic microtubules and the anchoring of the message at the cortex involves cortical microfilaments.
Fig. 1. Localization of endogenous VgJ
mRNA in cultured oocytes. Oocytes were
grown in Leibowitz medium supplemented
with vitellogenin-containing frog serum as
previously described (Yisraeli and Melton,
1988). After the indicated time in culture,
the oocytes were fixed and hybridized with
a Vgl probe made to the coding region
(Melton, 1987; Yisraeli and Melton, 1988).
In these dark-field photographs, silver
autoradiographic grains appear white.
Fig. 2. Localization of exogenous in vitro synthesized Vgl
mRNA injected into oocytes. Capped, radioactively labeled
Vgl and globin transcripts were synthesized as described
(Krieg and-Melton, 1987). Oocytes injected with message
were cultured as above for 5 days, time 0, the initial site of
injection; J days, oocytes injected with the indicated
message fixed after 5 days in culture.
Fig. 3. Attachment of Vgl mRNA to the detergent insoluble
pellet of extracts. Stage V/VI oocytes were
homogenized in the indicated buffer at room temperature.
After removing an aliquot and extracting the RNA (total
RNA), the homogenate was centrifuged and RNA was
prepared separately from the supernatant (soluble) and
insoluble (pellet) fractions and analyzed by Northern blot
hybridization. The positions of fibronectin and Vgl
messages on the blot are indicated at right. The RNA from
two oocytes prepared in this way was run in each lane.
Fig. 4. Effect of cytoskeletal inhibitors on Vgl mRNA in
late-stage oocytes. Stage V/VI oocytes were incubated in
saline (untreated), cytochalasin B (25ug/mL, cvtochalasin
B), or nocodazole (10ug/mL, nocodazole) overnight at
20°C and then analyzed by in situ hybridization for the
localization of Vgl mRNA. In addition, detergent extracts
of the oocytes were performed for each treatment and the
corresponding Northern blots are shown below each
section, p, pellet; s, soluble fraction.
Fig. 5. Effect of cytoskeletal inhibitors on Vgl mRNA in
middle-stage oocytes. Late stage III oocytes were cultured
for 5 days in the presence of medium and serum alone
(+serum), medium and serum with cytochalasin B (+serum
+cytochalasin 6), and medium and serum with nocodazole
(+serum +nocodazole). In situ hybridization visualized the
location of the Vgl mRNA in the oocyte sections. At left is
a section from an oocyte cultured in medium alone for 5
days, showing no localization whatsoever (-serum).
Fig. 6. A two-step model for the localization of Vgl
mRNA in oocytes. The horizontal arrows indicate the
normal process of oogenesis, with the black dots and black
shading indicating the location of Vgl mRNA and its
progressive localization. The larger, vertical arrows show
where the various cytoskeletal inhibitors are thought to
interrupt this process and point to the results of treating
oocytes with these drugs. Thus, translocation of the Vgl
mRNA to the vegetal cortex is thought to be associated
with microtubules and the anchoring of the message is
thought to involve microfilaments.
