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Previous studies demonstrated that there were two pathways, the messenger transport organizer (METRO) or early and the Vg1 or late, which function during stages 1 to 3 of oogenesis for the localization of RNAs at the vegetal cortex of Xenopus oocytes. In the present study we analyzed the properties of the METRO pathway, which localizes Xlsirt, Xcat2, and Xwnt11 RNAs to a specific region of the vegetal cortex during stage 1 of oogenesis. A combination of methodologies involving both fixed material and living oocytes was used to analyze RNA localization. We show that in early diplotene pre-stage 1 oocytes (25-50 microm in diameter) both endogenous and injected exogenous METRO RNAs translocated to multiple mitochondrial aggregates (pre-mitochondrial clouds) that surround the germinal vesicle (GV). However, by early stage 1 (diplotene oocytes, 50-200 microm), all three of the RNAs discriminated between the different clouds and translocated exclusively within the METRO of a single mitochondrial cloud. Therefore, in stage 1 diplotene oocytes there is a unique mechanism causing a change in the intrinsic property of the mitochondrial clouds which designates one of them as the RNA transport vehicle. During translocation through the cytoplasm Xlsirt and Xcat2 RNAs were detected associated with cytoplasmic particles of different morphologies. Additionally, we also found that the translocation of RNAs through the early or METRO pathway, unlike that of the late pathway, occurred in the absence of intact microtubule and actin microfilament cytoskeletal elements. This supports a cytoskeletal-independent model for localization of RNAs through the METRO pathway.
FIG. 1. Xlsirt, Xcat2, and Xwnt11 were detected within all of the precloud mitochondrial aggregates in early diplotene oocytes. (a)
Unstained living prestage 1 oocytes (25 mm in diameter) showing the distribution of the pre-mitochondrial clouds surrounding the GV
(arrows); (b) isolated GV showing pre-mitochondrial cloud tightly juxtaposed to the nuclear membrane; both a and b were photographed
as live unfixed material. Oocytes in c âf were embedded in paraffin and sectioned. (c) In situ hybridization of early diplotene oocytes (25â
50 mm in diameter) using the Xcat2 probe. Arrows point at the pre-mitochondrial clouds showing the accumulation of Xcat2 mRNA; (d)
hybridization of Xlsirt probe to early diplotene oocytes (filled arrow points to high level of accumulation in one precloud while open
arrow points to low level of accumulation); (e) hybridization of Xwnt11 probe to early diplotene stage oocytes (filled arrow points to high
level of accumulation in one precloud while open arrow points to low level of accumulation); (f) hybridization of Vg1 probe to early
diplotene stage oocytes (arrow points to unstained pre-mitochondrial cloud). Each bar represents 10 mm.
FIG. 2. Localization of Xcat2, Xlsirt, and Xwnt11 in a single METRO-containing cloud in stage 1 oocytes. (a) Pre-stage 1 oocytes showing
the decrease of Xcat2 mRNA in all but one of the pre-mitochondrial clouds (arrows); (b) stage 1 oocyte showing accumulation of Xcat2
mRNA in the METRO region of the dominant cloud (solid arrow) while other clouds do not contain Xcat2 mRNA (open arrow); (c) stage
1 oocyte containing two clouds in close proximity, both of which retain the Xcat2 mRNA; (d) group of early stage 1 oocytes showing
localization of Xlsirt to one of the clouds; (e) Xwnt11 mRNA localized to one of the clouds in stage 1 oocyte. Solid arrows point to
mitochondrial clouds containing Xlsirt or Xwnt11 RNA; (f) stage 1 oocyte hybridized with Vg1 probe. The arrow points to the METROcontaining
mitochondrial cloud that lacks Vg1 mRNA. Each bar represents 25 mm.
FIG. 3. Exogenous Xlsirt and Xcat2 form distinct cytoplasmic particles when translocating through the cytoplasm. (a) Confocal microscopy
image of fluoroscein-labeled Xlsirt (appear green) and Texas red-labeled Xcat2 (appears red) in the cytoplasm within 1 hr following injection
into stage 1 oocyte. The area of overlap between the two RNAs appears yellow. (b) Segregation of the two different RNAs into differenttype
particles 5 hr after injection into stage 1 oocytes.
FIG. 4. Confocal analysis of the localization of exogenous Xlsirt and Xcat2 RNAs within living oocytes shows the RNAs colocalizing
in all clouds in pre-stage 1 oocytes but are restricted to a single cloud in stage 1 oocytes. Fluoroscein-labeled Xlsirt (green) and Texas redlabeled
Xcat2 RNAs were co-injected into pre-stage 1 early diplotene oocytes (A) or into stage 1 diplotene oocytes (B) and examined after
16 hr for the localization of the RNAs.
FIG. 5. Exogenous Xlsirt RNA translocates to all preclouds when injected into pre-stage 1 oocytes but accumulates in the METRO region
of one cloud in stage 1 oocytes. (a) Pre-stage 1 oocytes that were injected with digoxigenin-labeled Xlsirt RNA and cultured for 2 days.
The oocytes were stained and the injected Xlsirt RNA was detected within all of the precloud structures (arrowheads). (b) Another example
of a pre-stage 1 oocyte injected with exogenous Xlsirt RNA. (c) Control oocytes that were not injected but went through the staining
reaction. (d) Early stage 1 oocyte injected with Xlsirt RNA showing the distribution of the transcripts in the METRO region of one of the
cloud. (e) Control early stage 1 oocyte injected with exogenous Vg1 RNA showing the distribution of Vg1 throughout the cytoplasm. The
arrow points to the cloud where Vg1 was excluded. Bars represent 25 mm.
FIG. 6. Co-injected Vg1 and Xlsirt translocate to the vegetal cortex in living stage 2 oocytes. Texas red-labeled Vg1 (open arrow) and
fluoroscein-labeled Xlsirt (filled arrow) were co-injected into stage 2 oocytes. The oocytes were cultured for 36 hr and visualized by
confocal microscopy. The Xlsirt RNA (green) has already reached the cortex while the Vg1 RNA (red) is localizing at the cortex in the
region overlapping the localized Xlsirt (yellow).
FIG. 7. The effect of nocodazole treatment on the localization of injected Xlsirt in the METRO. Oocytes were injected with Xlsirt RNA
and cultured for 48 hr (a), in the presence of 5 mg/ml nocodazole (b) or in the presence of 25 mg/ml cytochalasin B (c). The RNA was
visualized using the color reaction with antidigoxigenin alkaline phosphatase.
FIG. 8. Effectiveness of nocodazole and cytochalasin B treatment on the integrity of microtubules and microfilaments. Stage 3 oocytes
were treated with either nocodazole or cytochalasin B as above and were analyzed for the effects on microtubules (b) or actin microfilaments
(d). (a and c) Control untreated oocytes showing the presence of intact microtubules and actin microfilaments, respectively.
FIG. 9. The organization of the early or METRO pathway in early diplotene oogenesis. In early diplotene oocytes, mitochondrial aggregates
(preclouds) surround the GV. Xcat2, Xlsirt, and Xwnt11 RNAs accumulate indiscriminately within all of the precloud structures (1).
During the period from pre-stage 1 to stage 1 there is a change in the intrinsic property of one of the clouds that designates it as the
messenger transport organizer-containing cloud. Thus, the second step involves the accumulation of RNAs within the METRO structure
of one of the clouds. Xcat2, Xwnt11, and Xlsirt transcripts are lost in all of the other clouds. The third step is the transport of the RNAs
to the vegetal cortex in the METRO containing cloud. Exogenous Xlsirt RNA injected into pre-stage 1 oocytes was targeted to all of the
clouds; however, when injected into older oocytes they localize to the METRO-containing cloud. This suggests that there are changes
that occur within the clouds that produce a mature transport vehicle into which the RNAs accumulate. Additionally, it also indicates
that the METRO RNAs also possess a cis-acting signal directing them to the proper subcellular compartment within the METRO.
