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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02193

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Centrosome destined to decay in starfish oocytes

Yukako Shirato^1,2,*, Miwa Tamura^1,2, Mitsuki Yoneda³ and Shin-ichi Nemoto^1,2,

¹ Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo, Tokyo 112-8610, Japan.
² Tateyama Marine Laboratory, Marine and Coastal Research Center, Ochanomizu University, Koh-yatsu, Umi-no-Hoshi, Tateyama, Chiba 294-0301, Japan.
³ Takiyama 5-7-7, Higashikurume, Tokyo 203-0033, Japan.

View larger version (10K): [in a new window]   Fig. 1. Centrosomal behaviour during normal meiosis of a starfish oocyte, based on the experimental results by Tamura and Nemoto (Tamura and Nemoto, 2001) and Uetake et al. (Uetake et al. 2002). (A) Fully-grown immature oocyte with the germinal vesicle (GV). (B) Metaphase I. (C) The first polar body (PB1) extruded. (D) Metaphase II. (E) The second polar body (PB2) extruded. The pronucleus (PN) formed. Solid rectangles are reproductive centrioles. Open rectangles are centrioles destined to decay after completion of meiosis. Gray rectangles in A and B are centrioles to be characterized in the present study.

View larger version (33K): [in a new window]   Fig. 2. Experimental protocol. (A) Immature oocyte. (B) The immature oocyte is bisected manually with a fine glass needle into a GV-containing and a non-nucleate fragment. (C) The GV-containing fragment is enucleated with a micropipette. This fragment is used as a centrosome donor. (D) Actinase-treated immature oocyte that is treated with 1-methyladenine (1-MeAde) to induce maturation. (E) Mature egg bearing both PB1 and PB2. (F) Conjugate of a non-nucleate fragment and a mature egg. Scale bar: 50 µm.

View larger version (69K): [in a new window]   Fig. 3. Immunofluorescent staining with an anti--tubulin antibody of a non-nucleate fragment. (A) Whole immature oocyte. Two spots (arrowheads) stained by the anti--tubulin antibody are located between the GV and the animal pole. (B) Enucleated fragment. Two spots (arrowheads) are stained by the anti--tubulin antibody. Scale bar: 25 µm.

View larger version (142K): [in a new window]   Fig. 4. Development of a conjugate at 22°C. The time (minutes) after activation is given in the upper right corner of each image. (A) The first cleavage. (B) The furrow regresses, and the conjugate remains as a single cell. (C) The second cleavage. Multiple furrows appear. (D) Four blastomeres form at the second cleavage. (E) The third cleavage forms eight blastomeres. Scale bar: 50 µm.

View larger version (159K): [in a new window]   Fig. 5. Nuclear events and the formation of mitotic asters in a Pattern 1 conjugate. The time (in minutes) after activation is given in the upper right corner of each image. Black arrows indicate bipolar spindles. White arrowheads indicate nuclei. (A) Pronucleus (PN) formation. (B) The first mitotic cycle. A bipolar spindle develops. (C) Two nuclei form. (D) The second mitotic cycle. Two bipolar spindles develop. (E) Four nuclei form. (F) The third mitotic cycle, forming four bipolar spindles. (G) Eight nuclei form. (H) The fourth mitotic cycle, forming eight bipolar spindles. (I) Sixteen nuclei form. (A,C,E,G,I) Differential interference-contrast microscopy; (B,D,F,H) polarization microscopy.

View larger version (124K): [in a new window]   Fig. 6. Nuclear events and the formation of mitotic asters in a Pattern 2 conjugate. The time (in minutes) after activation is given in the upper right corner of each image. Black arrows indicate monopolar or bipolar spindles; white arrowheads indicate nuclei; black arrowheads indicate single asters. (A) Pronucleus (PN) formation. (B) The first mitosis. One monopolar spindle and one aster develop [see Tamura and Nemoto (Tamura and Nemoto, 2001) for clearer pictures of monopolar spindles]. (C) One nucleus forms. (D) The second mitotic cycle. One bipolar spindle and two asters develop. (E) Two nuclei form. (F) The third mitotic cycle, forming two bipolar spindles and four asters. (G) Four nuclei formed. (a,c,e,g) Differential interference-contrast microscopy; (b,d,f) polarization microscopy.

View larger version (175K): [in a new window]   Fig. 7. Nuclear events and the formation of mitotic asters in a Pattern 3 conjugate. The time (in minutes) after activation is given in the upper right corner of each image. White arrows indicate `aster-like structures'; white arrowheads indicate nuclei; black arrowheads indicate single asters. (A) A pronucleus (PN) forms. (B) The first mitotic cycle. An `aster-like structure' developed at a site where the PN had been located. Two asters emerge, away from the pronuclear site. (C) One nucleus reforms. (D) The second mitotic cycle. Four asters and a single `aster-like structure' develop. (E) One nucleus reforms. (F) The third mitotic cycle forms eight asters and an `aster-like structure'. (G) One nucleus reforms. (H) The fourth mitotic cycle, forms 16 asters and an `aster-like structure'. (I) One nucleus reforms. (a,c,e,g,i) Differential interference-contrast microscopy; (b,d,f,h) polarization microscopy.

View larger version (22K): [in a new window]   Fig. 8. Schemata of nuclear events and the formation of mitotic asters after activation in the conjugates.

View larger version (208K): [in a new window]   Fig. 9. Electron micrographs of a non-activated conjugate with two single asters. Each of the asters (A,B) contains one centriole at the center. The arrows point to the center of the respective aster. Numerals in the upper right corner of each frame indicate the number of the serial thin section (each 0.15 µm thick).

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