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Dynamics of tubulin structures in Xenopus laevis oogenesis.
Palecek J
,
Habrová V
,
Nedvídek J
,
Romanovský A
.
Abstract
The distribution of tubulin and/or tubulin-containing structures was examined in separate classes of Xenopus laevis oocytes and in germinal vesicles isolated from them. Although a monoclonal antibody has been used, the technique applied on paraffin sections does not allow clear-cut definition of the state of tubulin present (monomeric, dimeric or polymerized form); however, the probable existence of assembled microtubules is indicated by supplementary techniques, i.e. histology and immunoperoxidase staining. Immunofluorescence reveals maximum tubulin concentration in the Balbiani body and in a ring-shaped formation around the nucleus in young oocytes. The Balbiani body disintegrates in the course of vitellogenesis, granules formed from its periphery migrate into the cytoplasm and gradually fill the entire cytoplasm as radial cords. In the ring-shaped formation around the nucleus strongly fluorescent cords and fibres are formed, particularly on the future vegetal-half-facing part of the nucleus. Reorganization of tubulin may be related to the establishment of a structure directing two-way shifts (1) of cytoplasmic organelles from the Balbiani body to the cytoplasm, and (2) of yolk proteins containing endosomes derived from the endocytically active oolemma to the yolk platelets. A distinct fluorescent fibrillar network is found inside the isolated germinal vesicles, near the nucleus membrane. Peripheral nucleoli, often present in nuclear membrane protuberances, seem to be surrounded by this material, which is oriented along the surface, and as a basket towards the inside of the nucleus. It is assumed that the structures may participate in the transport of nucleoli from the nucleus to the cytoplasm via the nuclear envelope.
Figs. 1-4 Stage-I, -III, -IV and -VI oocytes. Aniline blue-orange G-azocarmine staining.
Stage in Fig. 1 corresponds to those in Fig. 7, in Fig. 2 to that in Fig. 9, in Fig. 3 to
that in Fig. 11 and in Fig. 4 to that in Fig. 12. Arrows, Balbiani body; arrowheads,
circumnuclear ring. Magnification x 1000.
Fig. 5 Comparison of overall intensity of tubulin-positive fluorescence in a group of
oocytes of different stages. Magnification 500 x.
Fig. 6 Stage-V oocyte. Tubulin-positive fibrils within the cytoplasm oriented mainly
towards the future animal cell surface (arrowheads). Magnification x500.
Fig. 7 Stage-I oocytes. Intensive fluorescence of the Balbiani body (arrow) from which
fibrillar structures emerge and concentrate around the nucleus. Additional irregularly
localized fibres in the cytoplasm (arrowhead). Magnification x 1000.
Fig. 8 Stage-II oocyte. Beginning of migration of tubulin-positive material from the
Balbiani body (arrow); gradual increase of tubulin in the vicinity of the germinal vesicle.
Magnification x 1000.
Fig. 9 Stage-Ill oocyte. Continuing disintegration of tubulin-positive material of the
Balbiani body (arrow). Fibrillar structures begin to appear near the plasma membrane.
Magnification x 1000.
Fig. 10 Stage-Ill to -IV oocyte. The Balbiani body has disintegrated. A layer of tubulinpositive
material is formed around the germinal vesicle (arrowheads).
Magnification x 1000.
Fig. 11 Stage-IV oocyte. Circum-nuclear ring of tubulin-positive material (arrowheads)
is closed around the germinal vesicle. Fibrillar structures growing out towards the
plasma membrane fill the entire cytoplasm of the oocyte. Magnification x 800.
Fig. 12 Stage-VI oocyte. Tubulin-positive fibrillar structures on the future vegetal side
of germinal vesicle form a basket. Note the fluorescence around nucleoli, B, near
nuclear membrane (arrow). Magnification x 1000.
Fig. 13 Isolated germinal vesicle. Tubulin forms a fine fibrillar network in nuclear
envelope protuberances (arrow); the network is oriented along the germinal vesicle
surface and towards the karyoplasm. Nucleoli (arrowheads) were simultaneously
stained by 50jum fluorescent probe ANS (l-anilinonaphtalene-8-sulphonate).
Magnification x 4000.
Fig. 14 Isolated germinal vesicle. Detail of tubulin-positive structures in nuclear membrane
protuberances (arrow). Nucleoli were not stained by ANS. Magnification x 8000.
Fig. 15 Western blots of one-dimensional polyacrylamide gels. Xenopus laevis oocytes
polypeptides and purified pig brain tubulin were resolved, on 7-5 % one-dimensional gel
transferred and blotted with a monoclonal antitubulin antibody TU-01. Lines A, B are
total Xenopus laevis oocytes polypeptides and line C is the brain tubulin.
