Dynamics of tubulin structures in Xenopus laevis oogenesis

The significance in early embryogenesis of microtubules, microfilaments and intermediate filaments representing cytoskeleton and cytomusculature has become evident. They play a crucial role both in the egg/sperm interaction, cortical reaction, cytokinesis, and in morphogenetic processes such as gastrulation, neurulation and succeeding events (see Burgess & Schroeder, 1979; Cohen, 1979; Weatherbee, 1981 ; for reviews). The amphibian oocyte shows an apparent radial symmetry about the animal-vegetal axis. This symmetry is reflected by the internal arrangement of organelles; it seems very probable that cytoskeletal structures are responsible for the spatial distribution of e.g. yolk, cortical and pigment granules, lipid droplets or mitochondria (Ball & Singer, 1982; Gall, Picheral & Gounon, 1983). Interaction of egg and sperm triggers a number of temporally linked processes such as the cortical reaction, migration of pronuclei, grey crescent formation, dorsoventral polarity determination, and cytokinesis (Elinson, 1980; Gerhart, Ubbels, Hara & Kirschner, 1981; Kirschner, Gerhart, Hara & Ubbels, 1980; Ubbels, Mácha, Palecek & Koster, 1983). All these events, both intracytoplasmic and cortical, involve a precisely organized and collaborating contractile system and a stable supporting matrix (Gall et al. 1983).

Different techniques have been applied to demonstrate microtubular proteins in eggs and embryos of many species. In the majority of cases they are already present in unfertilized eggs in unpolymerized form as a pool and remain at relatively constant amount throughout development of the embryos, e.g. in sea urchin (Harris, Osborn & Weber, 1980; Schatten & Schatten, 1981), Spisula solidissima (Suprenant & Rebhun, 1984), Chaetopteruspergamentaceus (Eckberg & Yuan-Hsu Kang, 1981), Drosophila melanogaster (Green, Brandis, Turner & Raff, 1975).

Cytoskeletal proteins are present in amphibian eggs and embryos as well. Tubulin was detected and characterized in oocytes and eggs of Xenopus laevis (Dumont & Wallace, 1972; Pestell, 1975; Palecek, Ubbels & Mácha, 1982), Rana pipiens (Smith & Ecker, 1968), Pleurodeles waltlii (Gounon & Collenot, 1974; Moreau & Gounon, 1977), Discoglossus pictus (Campanella & Gabbiani, 1980), and the axolotl (Raff, 1977; Raff & Raff, 1978). Accumulation of unpolymerized tubulin takes place in parallel with vitellogenesis and its amount reflects the size of the oocyte. The oocyte tubulin has usually been found to be very similar to somatic tubulin and no interspecific differences have been established (Dales, 1972). Comparing isolated tubulins of eggs and tissues of pigmented and albino axolotls Raff (1977) found only minute differences in electrophoretic, and in particular in colchicine-binding properties. De novo synthesis of tubulin in sea urchin oocytes represents about 4 % of the total protein synthesis (Cognetti, Di Liegro & Cavar-retta, 1977). This endogenously synthesized monomeric tubulin (Raff & Raff, 1978; Raff et al. 1975) is used later during activation and cleavage of the egg (Cohen & Rebhun, 1970). By analogy with yolk accumulation a participation of the nongerm cells in tubulin synthesis might be suspected. However, by using defolliculated eggs of Pleurodeles it was shown that follicular cells take no part in tubulin synthesis, although another mode of transfer was not excluded (Moreau & Gounon, 1977).

Although free assembly-competent tubulin is present in abundance in unfertilized eggs, comprising about 1 % of total soluble proteins in sea urchins and Xenopus (Coffe, Foucault, Raymond & Pudles, 1983; Pestell, 1975), it was not clearly demonstrated in the form of cytoskeletal microtubules until recently (Otto & Schroeder, 1984). At the same time the dynamics of microtubule assembly in vivo is not very clear. It is assumed that in vivo tubulin is stimulated to polymerize after activation of the egg by the penetrating sperm. Microtubule-organizing centres are activated at this stage, and they may be seen to stimulate microtubule formation even after transfer to unfertilized oocytes (Heidemann & Kirschner, 1978; Raff, 1979). Formation of the meiotic spindle may also be induced hormonally in oocytes via plasma membrane receptors (Hirai, Le Gascogne & Baulieu, 1983). Compartmentalization of free tubulin in the cell takes place mainly by means of association with membrane and other lipidic complexes (Caron & Berlin, 1979; Klausner et al., 1981). Free oocyte tubulin may polymerize in vitro forming micro- or macrotubular complexes (Kuriyama, 1977; Suprenant & Rebhun, 1984).

Tubulin was detected by immunofluorescence (Campanella & Gabbiani, 1980) in the periphery of coelomic, uterine, unfertilized and fertilized eggs of Discoglossus pictus. However, the authors were unable to detect microtubules in electron microscopy preparations. In Xenopus laevis, after the disintegration of the nuclear membrane during maturation of the egg, a fibrillar network lying near the basal part of the nucleus has been observed (Brachet, Hanocq & Van Gansen, 1970; Ubbels, personal communication). Electron microscopy (Huchon, Crozet, Canteno & Ozon, 1981) confirmed that this network is made up of bundles of microtubules. It was assumed that they play a role in the assembly of the chromosomes and the control of their migration toward the animal pole of the egg.

In this study we have examined the dynamics of tubulin-containing structures in growing oocytes of Xenopus laevis by means of immunofluorescence. Histological sections were then stained by classical techniques and compared with pictures obtained after immunofluorescence. To elucidate the possible role of microtubules in the nuclear membrane, germinal vesicles were isolated and observed after immunofluorescent staining.

Fragments of Xenopus laevis ovaries, containing oocytes of all classes I-VI, according to Dumont (1972), were fixed in Bouin-Hollande fixative for 24 hours, embedded in paraffin wax and 4 to 6 pm sections were prepared by standard procedures.

Germinal vesicles were isolated according to Ford & Gurdon (1977) in modified Barth saline, buffered with HEPES (MBS-H).

Isolated germinal vesicles were processed by a technique described previously (Paleček & Hašek,1984). They were washed for 20 seconds with potassium phosphate buffer, pH 7·0, complemented with 0-lM-NaCl, 1 mM-EGTA, 1 mM-MgCh and 10% dimethylsulphoxide, and extracted for 1 min in 0·15% TRITON-X-100 in the above solution without dimethysulphoxide (EB -extraction buffer). After washing in EB for 10 min they were fixed in 0·3 % glutaraldehyde, washed in phosphate-buffered saline (PBS-pH 7·2) and treated with 1 mg NaBIL per ml of PBS for 8 min. After additional washing in PBS they were incubated with monoclonal antitubulin serum TU-01 (Viklický, Dráber, HaSek & Bártek, 1982) for 1 hour at 37 °C in a dark moist chamber, washed in PBS and postincubated in SWAM FITC (Sevac, Prague, ČSSR) for 45 min at 37 °C in a dark moist chamber. After final washing in PBS they were mounted in 50 % glycerol in PBS (pH 7·4) and observed in a fluorescence microscope (Fluoval, Carl Zeiss, Jena; maximum excitation at 405 nm).

Sections of oocytes were deparaffinized and transferred through alcohols to PBS. Monoclonal antitubulin serum TU-01 was applied following identical procedures as above. For the details see Palecek & Romanovsky (1985).

After immunofluorescence, sections were stained successively with 1 % azocarmine in 1 % acetic acid for 10 min, 10 % orange G 4-2·5 % aniline blue in 1 % acetic acid for 5 min and 0·5 % aniline blue in water for 7 min, dehydrated and mounted. Staining revealed areas where specific immunofluorescence was present as blue, contrasting with dark orange yolk granules.

The samples for one-dimensional gels were prepared by the method originally described by Laemli (1970). The ovarian oocytes were isolated, equal amounts of 2% SDS and 0·5% mercaptoethanol added and immediately heated in a boiling waterbath for 1 min; homogenized and centrifuged. One-dimensional slab gels were run using standard procedures (Laemli, 1970). Gels were calibrated with standards of known relative molecular mass. Western transfer analysis was run according to the method described by Towbin, Staehelin & Gordon (1979) using a monoclonal antitubulin antibody TU-01 (Fig. 15).

In small oocytes (stage 1) extrachromosomal nucleoli, the mitochondrial mass (Balbiani body) and a sparse fibrillar meshwork arranged close to the nucleus are stained blue after histological staining with aniline blue (Fig. 1). Vitellogenic oocytes (stage-II and later) exhibit distinct blue staining of a layer located around the nucleus and disintegrating Balbiani body (Fig. 2). As oogenesis progresses the circular layer grows thicker and radial bundles connected with this layer are formed in the cytoplasm. Blue staining remains in the vicinity of the nuclear membrane and its invaginations (Fig. 3). In stage-VI oocytes a fine network of blue fibres may be observed close to the area of the nuclear membrane facing the future vegetal half of the egg (Fig. 4).

The general picture of tubulin-containing structures in oocytes of different sizes and in the stage-V oocyte, as revealed by immunofluorescence, is shown in Fig. 5 and 6. In stage-I to -II oocytes the mitochondrial mass (Balbiani body), connected with a fine fibrillar ring-shaped formation around the nucleus, exhibits a strong positive reaction (Figs 5,7). At the beginning of vitellogenesis the mitochondrial mass disintegrates; granules are formed from its periphery, which migrate throughout the cytoplasm. Part of the mitochondrial mass close to the nucleus remains compact. Clusters of strongly tubulin-positive material are formed near the nuclear membrane, which gradually contribute to the ring-shaped strutcture situated around the nucleus (Figs 5, 8, 9,10).

In oocytes of stage-III to -IV cytoplasm, radially oriented bundles exhibiting strong fluorescence are formed, beginning in the area of the Balbiani body (Fig. 9), and gradually filling the entire periphery of the oocyte. In the ring-shaped formation around the nucleus an extremely strongly fluorescent bundle appears (Fig. 10, 11).

In stage-V to -VI oocytes radial bundles are distinct in the future animal half cytoplasm but less distinct in the future vegetal half (Fig. 6). A basket of fine fibres surrounds the part of the nucleus facing the future vegetal half of the oocyte; fibres continue as finger-like processes towards the upper part of the nucleus (Fig. 12). The full-grown oocyte shows additional strong fluorescence inside the germinal vesicle near the nuclear membrane in sections. Peripheral nucleoli are surrounded by brightly fluorescing material (Fig. 12).

The surface of the isolated germinal vesicle of full-grown oocytes exhibits numerous protuberances; inside some of these structures nucleoli are visible. When immunofluorescence is applied the protuberances reveal the presence of tubulin-containing fibrillar structures oriented both along the surface and towards the inside of the nucleus. Apart from the fluorescence of structures present within the protuberances, bright fluorescence may be observed encircling protruding nucleoli (Figs. 13, 14).

Clear changes occur in the distribution and localization of tubulin-containing structures in growing oocytes of Xenopus laevis. On the basis of the technique used it is not possible to decide with certainty whether and where tubulin is present in the form of a free pool or in polymerized form. In pre vitellogenic oocytes the major amount of tubulin is concentrated in the Balbiani body. According to several authors this structure contains mitochondria and membranes of the endoplasmic reticulum and the Golgi apparatus (Raven, 1961-for review; Guraya, 1979). Billet & Adam (1976), using electron microscopy, observed a mitochondrial cloud formed by mitochondria and fibrils in bundles. The affinity of tubulin for lipidic complexes is well known from experiments on invertebrate eggs (Caron & Berlin, 1979; Klausner et al. 1981), while high-resolution electron microscopy has confirmed that, particularly at cell division, mitochondria make connections with microtubules (Nakamura & Ueda, 1982). In the course of vitellogenesis mitochondria are shifted from the Balbiani body to the cell surface while others stay around the nucleus (Raven, 1961; Tourte, Mignotte & Mounolou, 1984). The reorganization of tubulin observed by us may be related to their directed movements or to movements of other membrane structures detected in the Balbiani body. Disintegration of the Balbiani body is distinctly polarized; tubulin-containing structures localized at the nuclear membrane remain compact and gradually form a ring-like structure around the nucleus. Those directed to the cell surface are disengaged as granules which move to the cortical region of the oocyte. The basic phases of this process take place at stages 1—III, according to Dumont’s (1972) classification.

The tubulin-positive band in stage-III and -IV oocytes appears as a finely fibrillar or unstructured layer. At early stages it contains remnants of the disintegrated Balbiani body. Its position within the oocyte corresponds to the area positive for RNA, where ribosomes are mainly concentrated (Brachet, 1967-for review). In this space yolk reserves are gradually deposited, i.e. yolk granules are localized in the area of the tubulin-positive band. Interspaces among the yolk granules are penetrated by tubulin cords, which are particularly prominent in the future animal half of the oocyte.

As vitellogenesis progresses growth and rearrangements of the cell content take place. Yolk protein is bound by the endocytically active oolemma and transported via endosomes to yolk platelets which grow in size (Wallace & Jared, 1968; Brummet & Dumont, 1977). The transport pathway of the endosomes is probably marked by tubulin-containing structures, which are formed, as vitellogenesis progresses, from the pool present in the tubulin-positive band. This band gradually disappears as a continuous layer and radial tubulin-positive cords take its place, showing a picture typical for stage-V to -VI oocytes. Whether these changes represent rearrangement of structures already present, de novo synthesis, or synthesis from the pool of mono- or dimers present in the ring-like layer is not clear.

Changes in the localization of fluorescent material are very similar to those which were observed after the histological staining described above or after the immunoperoxidase reaction (unpublished). The tubulin-containing structures that we describe in this paper are apparently part of a cytoskeletal complex which is taking part in the restructuring of the cytoplasmic space in connection with its growth and vitellogenesis. Although our technique does not allow us to be sure that it is assembled tubulin that undergoes the observed changes, it is plausible to suppose its existence. Moreover, in preliminary experiments (unpublished) using the PAP reaction, distinct fibrillar structures are found in many places where fluorescence shows tubulin to be present.

Immunofluorescence has also confirmed the presence of a distinctly fibrillar network arranged at the surface of the nuclear membrane (Palecek, Habrová & Nedvidek, 1984). Protuberances of the membrane often show a network in the form of a basket, inside which nucleoli are sometimes visible. It seems probable that these tubulin-containing structures participate in the transport of nucleoli from the germinal vesicle to the cytoplasm of the oocyte, which occurs via the nuclear envelope well before its disintegration at the end of the growth phase (Habrová, 1975).

The authors thank Mrs M. Nohýnkovà for technical assistance, Dr G. A. Ubbels and Dr J. Faber (Utrecht) for reading the manuscript and fruitful discussions.