Wylie CC et al. (1986), Cytoskeletal changes during oogenesis and early...

XB-ART-28872

J Cell Sci Suppl 1986 Jan 01;5:329-41. doi: 10.1242/jcs.1986.supplement_5.21.

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Cytoskeletal changes during oogenesis and early development of Xenopus laevis.

Wylie CC , Heasman J , Parke JM , Anderton B , Tang P .

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The frog oocyte is well known for studies on the control of gene expression, but has been used much less in studies on the cytoskeleton. However, frog oocytes are very large single cells, whose cytoplasmic movements and asymmetries are fundamental to the correct development of the subsequent embryo. One particular example of asymmetrically distributed cytoplasm is germ plasm, thought to be important in the formation of the germ line. Data are presented that show that germ plasm is highly concentrated mass of cytoskeletal elements, which include tubulin, and an intermediate filament protein of molecular weight 55 X 10(3). The distribution of these molecules has been studied during oogenesis and during early post-fertilization development. The implications of these findings are discussed.

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	Fig. 1. A. Dissecting microscope view of the various stages of oogenesis seen in a piece of adult ovary. The mitochondrial clouds of the stage I oocytes are shown (arrowhead). Bar, 200 ^m. B. Differential interference contrast (Nomarski) micrograph of a single living pre-vitellogenic oocyte, showing the mitochondrial cloud (arrowhead). Bar, 25 fim. C. Nomarski image of an early vitellogenic living oocyte (stage II) to show the mitochondrial cloud breaking down and moving peripherally. Bar, 25 fim. B,C. From Heasman et al. (1984); A, unpublished observation.
	Fig. 2. Immunofluorescence images using anti-intermediate filament antibodies of various stages in the life-cycle of germ plasm in Xenopus. A. Mitochondrial cloud in previtellogenic (stage I) oocyte. B. Mitochondrial cloud fragmenting and moving to future vegetal pole. C. Mitochondrial cloud fragments remain in cortical cytoplasm of vegetal hemisphere of full-grown oocyte. D. Diagram to show orientation of sections shown in E -G . These are grazing sections of the vegetal pole of fertilized egg (E), four-cell stage (F) and 32-cell stage (G) to show stages in the aggregation of germ plasm. The germ plasm surrounds the nucleus of the cells that inherit it at the gastrula stage (H). Bars, 50fim for all micrographs. A. From Wylie et al. (1985); C, from Godsave et al. (1984a); the rest, unpublished observations.
	Fig. 3. C26 and C41 monoclonal antibodies give the same pattern of immunofluorescent staining in oocytes, and react with the same major band on immuno-blots. A. Low-power image of whole section of full-grown oocyte, a , animal pole; v, vegetal pole; n, oocyte nucleus. B,C. C41 stains radial strands of cytoplasm in animal pole (B), and islands of germ plasm in cortex of vegetal pole (C). The solid outermost line of staining in B and C is due to the surrounding follicle cells. Bars, 100 jxm (A); 50 /im (B,C). D. Immunoblot of Triton extracts of oocytes separated by SDS-PAGE. Primary antibodies were anti- IFA (tracks a-d), anti-/3-tubulin (Mr 53X103, track e), non-immune monoclonal supernatant (track f). Secondary antibody in each case was gold-conjugated goat anti-mouse immunoglobulin G (IgG) (Janssen), followed by silver enhancement. E. Immunoblot of Triton-extracted oocyte separated by SDS-PAGE and reacted with LE65 (track a), C41 (track b), C26 (track c) and anti-/3-tubulin (track d). C26 and C41 react with the same major band of slightly heavier molecular weight than /J-tubulin (approx. 55X103). LE65 reacts with a major band at approx. 44X103Mr and two minor bands. Methodology as for D (C. C. Wylie & Peter Tang, unpublished observations).
	Fig. 4. C26 and C41 have the same staining reaction inXenopus cells in culture. C41 also stains intermediate filament bundles in stage II oocytes. Cells of a Xenopus cell line (XL177) were stained with anti-actin (A) and C26 (B). C26 stains the intermediate filament pattern, as does C41 (not shown). Triton extracts of stage II oocytes are shown in C and D. Whole extracted oocytes were stained unfixed in C41 neat supernatant (C), or non-immune monoclonal supernatant from X-63 cells (D). They were then reacted with 4nm gold-conjugated goat anti-mouse IgG (Janssen) before fixation and processing for electron microscopy. Sections are shown without further heavy-metal staining. C41 reacts with intermediate filament bundles in the Triton extracts. Bars, 20,um (A,B); 100 nm (C,D). (C. C. Wylie, unpublished data.)
	Fig. 5. C26 staining of egg and early embryo cytoplasm, compared with anti-/3-tubulin. Germ plasm is stained at the 32-cell stage by both C26 (A) and anti-^-tubulin (B). C26 stains a radially arranged irregular pattern of phase-dense filaments extending outwards from the nuclear area of each blastomere (C,D). Anti-/3-tubulin stains straighter radially arranged microtubules centred on either asters or possibly microtubule-organizing centres (E,F). Bar, 25 fim.