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In Xenopus species, the early stages of oogenesis take place in the developing tadpoleovary when the oocytes are in a period critical for the organization of the germ plasm (believed to be a determinant of germ-cell fate) and the initial stages of localization of RNAs involved in germ plasm functions. We constructed a cDNA library from the ovaries of stage 64 Xenopus tadpoles with the idea that it will be enriched for oogonia and pre-stage I and stage I oocytes and thus, RNAs involved in oocyte development and germ plasm formation and function. From this cDNA library, we cloned a new maternal localized mRNA which we named centroid. This RNA codes for the protein belonging to the DEAD-box RNA helicase family. Some of the members of this protein family are components of the messenger ribonucleoprotein (mRNP) particles stored in the germ plasm in oocytes of Xenopus, Drosophila and Caenorhabditis species and are believed to play a role in translational activation of stored mRNPs and sorting of mRNPs into the germ plasm. We found that centroid mRNA is localized in Xenopus oocytes by a combination of early and late pathways, a pattern of localization that is very similar to the intermediate pathway localization of fatvg mRNA, another germ-plasm-localized RNA in Xenopus oocytes. Also, centroid mRNA is present in the mitochondrial cloud and in the germ plasm at the surface of germinal granules. This suggests that centroid is involved in the regulation of germ plasm-stored mRNPs and/or germ plasm function.
Fig. 3. Centroid mRNA localization in Xenopus oocytes. (A-C) Whole mount in situ hybridization showing localization of centroid mRNA (arrows) in the mitochondrial cloud in a stage 1 oocyte (A) and in the apex of the vegetal cortex in a stage II oocyte (B) and a stage III oocyte (C). (D- J) Sections of whole mount in situ hybridization showing localization of centroid mRNA in oocytes at different stages. (D) In a pre-stage I oocyte, centroid mRNA is uniformly dispersed in the cytoplasm but excluded from the mitochondrial cloud (long arrow) and secondary clouds (short arrows). (E,F) In a stage I oocyte, centroid mRNA (arrow) is located in the center of the mitochondrial cloud. Panel (F) shows the high magnification of the mitochondrial cloud (white sphere) with centrally located centroid mRNA (arrow). (G,H) In late stage I/early stage II and stage II oocytes, centroid mRNA is limited to the vegetal tip of the mitochondrial cloud (co- localizing with the germ plasm [long arrow]) and is excluded from the apical part of the mitochondrial cloud (short arrows). (I-K) Stage III, early stage IV and stage IV oocytes showing localization of centroid mRNA in the islands of a dispersing mitochondrial cloud (arrows). n, nucleus. Scale barsareequalto56μmin(A),90μmin(B,G),100μmin(C),70μmin(D), 75 μm in (E), 65 μm in (F), 86 μm in (H), 80 μm in (I) and 100 μm in (J,K).
Fig. 4. Electron microscopy analysis of centroid mRNA localization in the germ plasm of Xenopus oocytes. (A) Fragment of the mitochondrial cloud in a pre-stage I oocyte hybridized with a centroid anti-sense RNA probe, labeled with nanogold and silver-enhanced showing the mitochondrial cement (long arrows) located between the mitochondria. Centroid mRNA (short arrows) is present in the mitochondrial cloud but excluded from the mitochondrial cement. (B,C) Fragment of the mitochondrial cloud from stage 2 oocytes showing centroid mRNA (short arrows) located in the vicinity (B) and at the periphery (C) of the germinal granules (long arrows). m, mitochondria.
ddx59 (DEAD-box helicase 59) gene expression in Xenopus laevis embryos, oocyte stage III, as assayed by in situ hybridization, transverse section, animal pole up.