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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02413

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The meiotic spindle of the Drosophila oocyte: the role of Centrosomin and the central aster

Department of Evolutionary Biology, University of Siena, Via A.Moro 4, I-53100 Siena, Italy

View larger version (103K): [in a new window]   Fig. 1. Spindle organization during meiosis I-early meiosis II. Projected series of optical sections of oocytes stained with propidium iodide (orange) and an anti-ß-tubulin (green) antibody. (A) Early anaphase I: peripheral MTs intersect at the spindle equator to form discrete clusters (arrowheads) or extend toward the cell cortex (arrow). (B) Late anaphase: the spindle has elongated and the poles appear broad and frayed (arrows), MT clustering at the spindle equator is more pronounced (arrowheads). (B1) A single optical section of the spindle in B at the level of the equator showing the overlapping of some antiparallel MTs (arrowheads). (C) Telophase: a distinct tubulin accumulation is present midway along the spindle where the opposite MT bundles overlap (arrow). (D) Early prophase II: the peripheral MT network expands and some MTs contact the oocyte surface (small arrowheads); several astral arrays of MTs appear within the equator of the spindle and outside its boundaries (arrows) and a distinct tubulin cluster bulges out the spindle midzone (large arrowhead). (D1): single optical section of the spindle in D showing that the equatorial tubulin cluster (arrow) is connected by thin bundles to the peripheral MT network (arrowheads). Bars, 2 µm in A-D, D1 and 0.7 µm in B1.

View larger version (113K): [in a new window]   Fig. 2. Organization of the meiosis II spindle. Projected series of optical sections of oocytes stained with anti-ß-tubulin antibody. (A) Late prophase: the peripheral MT network is dismantled, but small asters are still observed outside the spindle (arrows), the belt-shaped tubulin cluster is prominent and protruding at the spindle equator (large arrowhead); `minispindles' (small arrowheads) are focused at the outer poles. (B) Prometaphase: the interior MTs become focused on separate bundles of different size at the spindle equator (arrowheads), where short MTs spread out to form a loose cloud (arrow). (C) Late prometaphase: the interior MT bundles partially coalesce in three to four distinct bundles (arrowheads) that are focused at the spindle equator where MTs form an extensive astral array (arrow), the central aster. (D) Metaphase: the twin spindles have separated and the central aster (arrows) becomes clearly evident and enlarges during (E) anaphase and (F) telophase. Bar, 2 µm.

View larger version (88K): [in a new window]   Fig. 3. Localization of Asp protein during female meiosis. In all panels MTs are stained green and Asp, orange. (A) Anaphase of meiosis I: Asp is present at the spindle poles. (B) Late anaphase of meiosis I: Asp is localized at the poles and at the spindle equator (arrow). (C) Telophase of meiosis I: Asp localizes in a ring-like structure (arrow) around the accumulation at the central spindle (arrowheads). (D) Early prophase: Asp staining is evident at the spindle poles, at the central spindle, and it is also found at the foci of the sparse asters (arrowheads). (E) Prometaphase and (F) metaphase: Asp remains localized at the outer spindle poles and within the central region of the spindle at the site where the inner poles are forming (arrowheads); a strong labeling is also observed within the individualizing spindles where the central aster is developing (arrow). (G) Anaphase II: Asp accumulates at the spindle poles and at the minus ends of the MTs that organize the central aster (arrow), where a feeble ring-like staining remains. (H) Telophase II: the spindle poles are strongly stained by the anti-Asp antibody, whereas a weak labeling is found within the central aster (arrow). Bar, 3 µm.

View larger version (120K): [in a new window]   Fig. 4. Localization of PAV-KLP in the female meiotic spindle. Oocytes were stained with anti-ß-tubulin (green) and anti-PAV-KLP (orange) antibodies. First meiosis: (A) anaphase, (B) late anaphase, (C) telophase: PAV-KLP accumulates at the middle of the spindle (arrow), at the presumptive midzone. Second meiosis: (D) prophase, (E) metaphase: the staining at the spindle midzone is no longer visible, but PAV-KLP localizes on a discrete ring-like structure at the spindle equator (arrows). (F) Anaphase: PAV-KLP concentrates at the focus of the central aster (arrow) and at the midzone of the twin spindles (arrowheads). Bar, 2 µm.

View larger version (110K): [in a new window]   Fig. 5. Localization of CNN at the female meiotic spindle. Projected series of optical sections of oocytes stained with antibodies against ß-tubulin (green) and CNN (orange). In each panel the inset shows, in monochrome, the localization of CNN protein. (A) Anaphase I: CNN accumulates on distinct bundles at the central spindle (arrow). (B) Prophase II: the protein accumulates at the middle of the spindle in a punctate ring-like structure (arrowheads) that surrounds a faint localization (asterisk) on the interior MT bundles; punctate aggregates of the protein are also found at the center of the MT asters (arrows). (C) Prometaphase II: the CNN localization at the middle of the spindle decreases in intensity (asterisk), although a thin ring-like fluorescent structure is still visible (arrowheads); the protein has also accumulated at the focus of the cytoplasmic asters (arrows). (D) Metaphase II, (E) anaphase II: CNN staining is no longer found within the spindles, but a distinct accumulation is visible at the focus of the central aster (arrows). (F) CNN aggregates are also observed within the sperm aster, at the sites of MT clustering (arrows). Bar, 2 µm.

View larger version (105K): [in a new window]   Fig. 6. Female meiosis in cnn mutants. Oocytes derived from cnn mothers were fixed and stained for tubulin. (A) Anaphase I: the meiotic spindle is organized as in wild type. (B) Prometaphase II: the peripheral MT network has dismantled and the inner poles of the twin spindles start to organize. (C) Early metaphase II: a faint central aster (arrow) is visible between the twin spindles. (D) Early anaphase II and (E,F) telophase II: the central aster becomes hardly detectable (arrows), however, a distinct sperm aster is present (arrowhead, F), although diminutive compared to that in the wild-type. (G) Example of twin anaphase II spindles not properly spaced and rather abnormal in shape. Bars, 2.5 µm in A-E,G and 3.5 µm in F.

View larger version (176K): [in a new window]   Fig. 7. PAV-KLP localization in cnn mutants. The merged images show PAV-KLP (orange) and MT (green) staining. (A) Anaphase I, (B) telophase of the first meiosis, (C) metaphase, (D) late anaphase of the second meiosis. PAV-KLP strongly accumulates at the central spindle during first meiosis (arrows), but the protein is barely detectable or absent within the focus of the faint central aster during the second meiosis (arrowheads); however, a discrete PAV-KLP labeling is found at the midzone of the twin meiotic spindles (small arrows). Bar, 2 µm.

View larger version (53K): [in a new window]   Fig. 8. Diagram of the main features of female spindle morphogenesis during transition from meiosis I to meiosis II. (A) Anaphase I. (B) Telophase I. (C) Prophase II. (D) Prometaphase II. (E) Metaphase II.

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