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Figure 2. Ultrastructural distribution of Exu. Electron micrographs (a–e) and schematic drawings (a′–e′) of ultrathin Lowicryl sections on which Exu was indirectly immunolabeled by 15-nm colloidal gold particles. (a and a′) Nurse cell, stage 9. Exu is highly enriched in the sponge body in comparison to the surrounding mitochondria and cytoplasm. The nuage particles surrounded by the sponge body contain no Exu. (b and b′) Nurse cell, stage 10. Exu is accumulated in the elongated sponge body, which extends from the nucleus into the cytoplasm. Nuage particles next to the nucleus are partly surrounded by higher amounts of Exu present in small sponge bodies. (c and c′) Nurse cell, stage 10. A large sponge body is present at the apical border of the nurse cell. A small fragment of the overlying follicle cell can be seen at the upper right edge of the micrograph. The gap between these cells is due to the embedding. (d and d′) Oocyte, stage 8. Sponge bodies with high concentrations of Exu are present in the ooplasm. However, the central region of the oocyte where the yolk granules first accumulate (yolk nucleus) contains only very little Exu or sponge bodies. (e and e′) Oocyte, stage 10B. Thick parallel bundles of microtubules run at this stage in the cortical layer of the oocyte. No sponge bodies are present, but Exu is equally distributed between the large yolk granules. No accumulation of Exu on the microtubules can be observed either. g, Golgi vesicles; l, lipid droplet; m, mitochondria (hatched); mt, microtubule; na, nuage (middle gray); nc, nurse cell; nu, nucleus; ooc, oocyte; sb, sponge body (light gray); y, yolk granule (dark gray); yn, yolk nucleus. Each black dot in the drawing corresponds to a 15-nm gold particle in the micrograph. Bar, 1 μm.
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Figure 3. Ultrastructural distribution of the sponge bodies. Electron micrographs (a–d) and schematic drawings (a′–d′) of ultrathin Epon sections of glutaraldehyde- and osmiumtetroxide-fixed wild-type follicles. (a and a′) Nurse cell, stage 9. The sponge body consists of electron-translucent, elongated elements that are interspersed between an electron-dense amorphous mass. It hardly contains ribosomes in contrast to the surrounding cytoplasm. Small vesicles are present within the sponge body. The sponge body is situated close to the nucleus and is surrounded by mitochondria. (b and b′) Nurse cell, stage 9–10. Adjacent to the flattened follicle cells, sponge bodies are accumulated in the apical regions of the nurse cell. ER tubules and Golgi vesicles are present between the sponge body clusters. (c and c′) Oocyte, stage 9–10. The large sponge body can be distinguished from the surrounding ooplasm by the accumulation of elongated elements in an electron-dense matter, excluding ribosomes and other organelles. (d and d′) Oocyte, stage 10A. Most sponge bodies have disappeared by this stage; however, some small clusters are left in the cytoplasm, which is densely packed with yolk granules, ER tubules, mitochondria, and Golgi vesicles. er, endoplasmic reticulum; fc, follicle cells; g, Golgi complex; l, lipid droplet; m, mitochondria (hatched); na, nuage (middle gray); nc, nurse cell; nu, nucleus; ooc, oocyte; sb, sponge body (light gray); y, yolk granule (dark gray). Bar, 1 μm.
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Figure 4. Ultrastructure of sponge bodies after extraction. Micrographs (a and b) and schematic drawings (a′ and b′) of ultrathin Epon sections of wild-type follicles that were only fixed with osmiumtetroxide, not with aldehyde, for better visualizing the membranes in the sponge bodies. (a and a′) Nurse cell, stage 9. The sponge body contains ER-like cisternae. No amorphous matter is visible between these due to the strong extraction of the cytoplasm. However the electron-dense nuage particle contained in the sponge body is not affected by this procedure. (b and b′) oocyte, stage 9. The lumen of the cisternae of the sponge body in the oocyte appears larger than that in the nurse cells. As in the nurse cells no amorphous matter is left after the extraction. g, Golgi complex; l, lipid droplet; m, mitochondria; na, nuage; nc, nurse cell; nu, nucleus; sb, sponge body; y, yolk granule. Bar, 1 μm.
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Figure 5. Sponge bodies are present in ring canals. Micrograph and schematic drawing of a stage 10 follicle that was treated as described for Fig. 3. The cytoplasmic bridge, connecting a nurse cell (to the left) with a yolk-containing oocyte, includes a sponge body as well as other cytoplasmic organelles (ER and lipid droplets). A multivesicular body containing electron-dense, yolk-like material is present in close proximity to the ring canal. ir, inner ring; m, mitochondria; mvb, multivesicular body; sb, sponge body; y, yolk. Bar, 1 μm.
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Figure 6. Cytoplasm of previtellogenic oocyte. Micrograph and schematic drawing of a stage 5 oocyte that was treated as described for Fig. 3. The microtubule network of the oocyte (ooc) is organized by the centrioles (ce) at the posterior pole adjacent to the follicle cells (fc). The cytoplasm is packed with short microtubules (mt), ER-like tubules (er), ribosomes, and electron-dense granule (am, amorphous matter; some of the granules were marked by arrows in the micrograph). The appearance of these granules resembles the amorphous matter in the sponge bodies. nu, nucleus. Bar, 1 μm.
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Figure 7. Sponge bodies in exuVL/exuVL ovaries. Micrograph and schematic drawing of a stage 9 exuVL/exuVL nurse cell that was treated as described for Fig. 3. No obvious difference in the morphology of sponge bodies that do not contain Exu can be observed compared with sponge bodies in wild-type follicles by electron microscopy. As in wild-type follicles, these sponge bodies exclude ribosomes and consist of electron-translucent elements in an amorphous matter with some small vesicles in between. er, endoplasmic reticulum; g, Golgi; m, mitochondria; nc, nurse cell; sb, sponge body. Bar, 1 μm.
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Figure 8. RNA staining of wild-type stage 9 nurse cells. (a) Electron micrograph of an ultrathin Epon section with normal contrast. DNA-rich regions of heterochromatin in the nucleus (nu) are darkly stained (arrowheads), whereas the extended euchromatin and sponge bodies show little contrast (arrows) in comparison to the ribosomes in the cytoplasm. (b) Modified Bernhard staining of an ultrathin Epon section. Note the dark contrast of the RNA rich regions (arrows) of the nucleus (nu) and the sponge bodies in contrast to the bright regions of the heterochromatin (arrowheads). m, mitochondria. Bars, 1 μm.
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Figure 9. Patchy bcd mRNA localization in the nurse cells. bcd mRNA in a whole wild-type stage 10 follicle was fluorescently labeled by in situ hybridization. In the nurse cells, a patchy distribution of bcd mRNA is observed at the apical side of the cells and around the nuclei (arrows). The nurse cells adjacent to the oocyte are depleted of most of the bcd mRNA (as it was described by Wang and Hazelrigg [1994] for the Exu–GFP fusion protein).
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Figure 10. Comparison between Exu (a–c) and α-tubulin (d–f) distribution in wild-type follicles. Ultrathin Lowicryl sections were immunofluorescently labeled with either anti-Exu serum (a–c) or anti– α-tubulin antibodies (d–f) and observed by fluorescent light microscopy. Follicle cells harbor much higher amounts of microtubules than the germ line cells, but no Exu is present in the follicle cells. (a and d) Stage 4 and 6. High amounts of α-tubulin are found at the borders of the nurse cells and in the oocyte (arrows), but the punctate Exu staining is not enriched at these sites. (b and e) Stage 9. Many sponge bodies (b, bright dots) are present but not accumulated at the borders of the nurse cells or in the oocyte (arrows) where tubulin is enriched. (c and f) Stage 10. Hardly any microtubules are labeled, but sponge bodies labeled by Exu (c, bright dots) are accumulated in apical patches in the nurse cells. In the oocyte, Exu is not accumulated along the microtubules running parallel to the anterior border of the oocyte (arrows). Bar, 50 μm.
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Figure 11. Subcellular localization of sponge bodies versus microtubules. Micrograph and schematic drawing of a wild-type stage 8 oocyte that was treated as described for Fig. 3. Microtubule bundles (mt) run across the cytoplasm. Although sponge bodies (sb) are present close by, no microtubules are present in these structures. m, mitochondria; y, yolk. Bar, 1 μm.
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Figure 12. Exu is not accumulated on the thick microtubules forming in cappuccino oocytes. Adjacent ultrathin Lowicryl sections of capuRK/ capuRK follicles were immunofluorescently stained for Exu (a) or α-tubulin (b). Unusual thick bundles of microtubules run in the cortical cytoplasm of the stage 10A oocyte. However, in the same oocyte Exu is equally distributed. The same pattern can be observed in spire follicles. Bar, 50 μm.
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Figure 13. Ultrastructure of sponge bodies in ovaries after taxol or colchicine treatment. Micrographs of stage 9–10 nurse cells of flies that have been fed with cytoskeletal drugs for several days. The follicles were treated as described for Fig. 3. (a) Taxol; (b) colchicine. er, endoplasmic reticulum; g, Golgi; m, mitochondria; na, nuage; nu, nucleus; sb, sponge body. Bars, 1 μm.
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Figure 14. Effects of taxol or colchicine on localization of maternal factors. Ovaries of flies fed without any drug (a, d, and g), with taxol (b, e, and h) or colchicine (c, f, and i) were stained for bcd mRNA (a–c), Exu (d–f) or α-tubulin (g–i). (a–c) bcd mRNA in situ hybridization on whole ovaries. After taxol treatment (b) no striking difference is to be seen compared with wild type (a), however after colchicine treatment (c) the localization of bcd mRNA is completely abolished. Note the central position of the oocyte nucleus in c, which can serve as a control for the MT destabilization. (d–f) Ultrathin Lowicryl sections of ovaries treated with taxol (e) or colchicine (f) were immunofluorescently labeled for Exu and compared with the wild-type staining (d). Neither treatment eliminates the punctate distribution pattern of Exu; however, in colchicine-treated follicles the staining seems to be preferentially close to the nucleus. The size difference of the sponge bodies observed here does not exceed the variance in the size of sponge bodies observed in different wild-type follicles. (g–i) Adjacent ultrathin Lowicryl sections of d–f stained for α-tubulin. After taxol treatment (h), clear microtubules are visible while the staining is diffuse and strongly reduced after colchicine treatment (i) compared with the wild-type staining (g). (The vitelline membrane in i is stained by cross-reaction of the secondary antibody.)
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Figure 15. Vasa distribution in nuage particles. Micrographs (a and b) and schematic drawings (a′ and b′) of follicles embedded in Lowicryl. Vasa protein was indirectly immunolabeled by 10-nm colloidal gold particles. (a) Nurse cell, stage 9. Vasa is accumulated in the nuage (na), which is present next to the nuclear membrane (nu, nucleus) and delaminating into the cytoplasm. The electron-dense, compact nuage particles are surrounded by sponge bodies (sb). (b) Oocyte, stage 9. Small Vasa- labeled nuage particles (na) are found at low frequency in the sponge bodies (sb) in the anterior cytoplasm of the oocyte. m, mitochondria; y, yolk. Bars, 1 μm.
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Figure 16. Transient posterior localization of Exu. Micrograph and schematic drawing of an ultrathin Lowicryl section of the posterior pole of a stage 10 oocyte that was immunogold-labeled for Exu (15-nm gold). The gold particles are accumulated in the posterior cytoplasm next to the vitelline membrane (vm), whereas the cytoplasm between the large yolk granules (y) is hardly labeled for Exu. The polar granules (p) at the posterior pole are free of Exu. m, mitochondria. Bar, 1 μm.
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