Xenopus laevis fertilized eggs have been treated with wheat germ agglutinin (WGA) before the onset of the first cleavage, at the stripe stage and during groove deepening. The ultrastructure of the animal cortex of the arrested embryos has been compared with that of the same region of control embryos at different stages of first furrow formation and of cytochalasin B-treated embryos. The outer side of the plasma membrane of WGA-treated embryos is covered with a coat which is different from the diffuse material observed in either control or cytochalasin B-treated embryos and which is distributed in patches in the groove region. Narrow indentations of the plasma membrane in the cortex of WGA-treated eggs have been observed, particularly in the blocked or regressed groove. In WGA-treated eggs, a few bundles of microfilaments are located under the plasma membrane at the animal pole, but they are never arrayed in a continuous layer as in the control eggs. In the latter, many microtubules are located in close proximity to the microfilament layer at the beginning of cleavage, but they are only occasionally observed in the same region of WGA-treated eggs.
It is concluded that the binding of WGA molecules to their receptors on the surface of the Xenopus zygote interferes with the alignment of microfilaments in the furrow region and provokes the disorganization of the aligned microfilaments once the cleavage has begun. Internalization of portions of the nascent membrane in the groove could play an important part in the arrest of cleavage.
At the onset of cleavage furrow formation, in particular in amphibian eggs (Selman & Perry, 1970), a band of microfilaments forms under the cell membrane under the influence of the mitotic apparatus (Rappaport, 1971). The contraction of these microfilaments produces a surface constriction at the animal pole and its subsequent propagation leads to the separation of the cytoplasm into 2 blastomeres by growth of new membranes and junction formation.
Lectins (agglutinins) are proteins, generally extracted from plants, which bind to carbohydrate residues. They have been extensively used for studying the distribution and the mobility of specific carbohydrate group-containing glycoproteins on the cell surface. These proteins include receptors for other molecules, hormones for instance. Concanavalin A, one of the most widely used lectins induces the arrest of cytokinesis and regression of a partially formed furrow in embryos of the echiuroid worm Urechis caupo (Das & Sisken, 1975). The same lectin inhibits the development of the amphibian embryo (Moran, 1974). Tencer (1978a, b) has recently shown that wheat germ and soybean agglutinins interfere with the first cleavage formation in Xenopus laevis eggs. According to the dose of lectin used and (or) the time of treatment, the formation of the furrow is inhibited, or the initiated furrow is arrested and eventually regresses.
It seemed interesting to look at the ultrastructural organization of the cell membrane and associated structures in the furrow region at the animal pole, during the first cleavage of normal and lectin-treated embryos with a particular interest for the cytoskeleton. Wheat germ agglutinin, specific for IV-acetyl-D-glucosamine residues, which effectively binds to receptors on the Xenopus zygote surface as shown by fluorescence microscopy (Tencer 1978a, b), and blocks cleavage of the egg, was used during this study.
MATERIALS AND METHODS
Xenopus laevis eggs were artificially fertilized. Collection of the eggs from hormonally stimulated females, removal of the jelly with mercaptoethanol and of the fertilization membrane with watch-maker’s forceps were carried out as described by Tencer (1978a, b). Eggs devoid of jelly and fertilization membrane were incubated in half-strength Niu and Twitty solution (Niu & Twitty, 1953). They were placed in wheat germ agglutinin (WGA) (50 or 100 μg/ml), about 1h after fertilization, before the appearance of the furrow; the blocked embryos were fixed 7–20 min later when the higher dose was used and 35-60 min later in the other case. Other eggs were treated with WGA (50 μg/ml) for 25–30 min, from the stripe stage onwards and fixed after regression of the furrow. Finally, eggs were treated with WGA (100 μg/ml) at the beginning of first furrow formation (various stages of groove deepening) and the arrested embryos fixed 25 min later. The specificity of the treatment was previously controlled by addition of 0-2 M W-acetyl glucosamine (Tencer, 1978a, b). Control eggs at different stages of first furrow formation, from single stripe to just completed furrow (according to the terminology of Bluemink (1971b) and Singal & Sanders (1974)) were also fixed.
The fixative was 2.5% glutaraldehyde in o-i M Sorensen phosphate buffer pH 7.4, used for 2 h at room temperature. After 3 washes in buffer, the eggs were fixed again in 1% OsO4 for 2 h at room temperature. The furrow region of control and WGA-treated eggs was often dissected under a binocular lens. The eggs were then fixed overnight in glutaraldehyde and the fragments postfixed in OsO4 as above. In one experiment, a few eggs were placed, approximately 1 h after fertilization, in a cytochalasin B solution (10 μg/ml) containing 1% dimethyl sulphoxide and fixed 35 min later when furrow regression had begun to take place. After dehydration in ethanol, the material was flat embedded in Epon. Ultrathin sections were observed in an AEI EM6B electron microscope, after floating them for 20 min on uranyl acetate and 10 min on lead citrate solutions.
Single stripe stage
The first furrow normally forms about 1.5 h after fertilization. Grazing tangential sections to the egg surface at the animal pole show a continuous layer of parallel arrays of microfilaments under the plasma membrane.
Shallow groove stage
Transverse stress folds are observed on both sides of the groove. Microfilaments cut transversally appear as dots 6–8 nm in diameter and form a continuous layer about 500 nm thick (Fig. 1). Numerous microtubules, about 20 nm in diameter, are scattered in the animal cortex of the furrow region. Some of them are in very close proximity to the microfilament layer and run fairly parallel to the egg surface (Fig. 1). Occasionally, bundles of 4-5 units are held together within an electron-dense material. The thin coat which covers the outer layer of the plasma membrane only consists of diffuse material (Fig. 1).
In frontal sections of flattened embryos, parallel arrays of microfilaments are visible beneath the tip of the enlarged furrow. Beyond that region, only a few bundles of filaments are observed. Many of the microtubules in the cortex of the furrow region are oblique to the surface and come very close to the filament layer. Some of them are embedded in an electron-dense matrix and are probably mid-body elements. Golgi bodies with associated cisternae and vesicles containing a fibrous material are frequently observed in the cortical cytoplasm.
WGA (50 and 100 μg/ml) added before stripe stage. With both concentrations, the furrow of the embryos is just outlined. The outer side of the plasma membrane is covered with a rather evenly distributed coat about 100 nm thick (Fig. 2). Contacts through this coat are frequently observed between surface folds. In addition, the plasma membrane forms some narrow and deep indentations into the cortex of the embryo, which either meander (Fig. 3), form a fork or bifurcate (Fig. 4) inside the cytoplasm. In cross-sections, they appear as isolated vesicles whose 2 trilaminar membranes are separated by a gap of about 45 nm which is formed by the electrondense material of the coat (Fig. 4). Filamentous material is often associated with the cytoplasmic side of these indentations and the inner side of the neighbouring plasma membrane. Small bundles of microfilaments are observed under the plasma membrane in frontal sections of the stripe region, but they do not form a continuous layer even when the eggs are treated with WGA (100 μg/ml) and fixed as soon as the furrow is outlined (7–20 min of treatment). Some short indentations of the plasma membrane are formed in eggs treated with the lectin (100 μg/ml) for 15–20 min before fixation, but not after shorter treatments.
WGA (50 and 100 μg/ml) added at the outset or during the formation of the first furrow
As has been previously described (Tencer, 1978 a, b), cleavage can continue for a short time in the presence of WGA (50 μg/ml). However, the formation of the furrow soon stops and the groove regresses. Addition of WGA (100 μg/ml) during groove formation results in arrest of development after a few minutes, regardless of the stage of furrow deepening. This is followed by regression of the furrow. The ultrastructural description of the cortex in the furrow region is valid for embryos treated with either concentration of the lectin, with lipid droplets, small yolk platelets and pigment granules being less frequently observed in that region. Golgi bodies with cisternae containing a flocculent material are scattered in the cytoplasm which contains a large quantity of β-glycogen particles and chains. The latter are occasionally encircled by flat vesicles with flocculent contents (Fig. 5) and it seems likely that these vesicles can fuse laterally so that their inner side forms a continuous membrane around the glycogen mass which is surrounded by an empty space limited by a second unilaminar membrane. Sometimes, the inner membrane is not visible (Fig. 7). Glycogen particles mixed with a flocculent material may also be dispersed in the space limited by the outer membrane. A mass of glycogen limited by a single membrane is sometimes secreted outside the cell by an apocrine process. In particular, we have observed large empty spaces formed by partially fused vacuoles at the tip of the regressed furrow.
In the furrow region, the outer side of the plasma membrane shows patches of a material with the same appearance as that seen over the whole surface of the embryo (Fig. 6). Small bundles of filaments are occasionally observed under the plasma membrane. The most characteristic feature of the cortex in the furrow region is the presence of double membranes at an angle to the plasma membrane. These structures, which apparently originate from indentations of the trilaminar plasma membrane at places where a coat patch is present, lead to tightly applied membranes instead of membranes separated by a continuous gap as found in other regions of the cortex of WGA-treated embryos. In addition, these structures are much more numerous and are frequently located side by side (Fig. 7). The 2 trilaminar membranes may be separated for some distance by a gap of about 6 nm, but they are more frequently tightly applied against each other, giving the appearance of pentalaminar structures (Fig. 7). The double membranes twist and turn into the cytoplasm and may be in contact with the surface in several places. These trilaminar membranes are also in contact and apparently fuse with the membrane of glycogen-containing vesicles, where such a structural pattern is not discernible (Fig. 7). Indeed, frontal sections of the furrow region show that these indentations form a network near the surface of the egg (Fig. 8).
In frontal sections of the animal hemisphere of embryos blocked during groove deepening, small, rather vague bundles of microfilaments are observed under the plasma membrane along the bottom of the groove. Tangential sections of the just-outlined second furrow show microfilaments under the plasma membrane.
Cytochalasin B-treated embryos
As in control embryos, a very thin coat consisting of very short filaments or a diffuse material is observed on the outer side of the plasma membrane. Bundles of microfilaments oriented in different directions are observed in frontal sections of the regressed furrow, but only a few microtubules are located in their proximity.
Fluorescent WGA binds all over the surface of the Xenopus zygote (Tencer, 19786). At the beginning of the first cleavage, a coat is observed by electron microscopy over the entire surface of lectin-treated embryos, except in the furrow groove where it is distributed in patches. This coat, which is different from the short filaments observed in control or cytochalasin B-treated eggs, has a different aspect in soybean agglutinin-treated eggs (unpublished results). In our experiments, eggs devoid of their envelopes were incubated in Niu & Twitty solution before lectin treatment. Nevertheless, a crosslinking of bound WGA with extracellular material on the egg surface cannot be excluded. This coat does apparently not result from a crosslinking action of the fixative on lectin molecules since repeated washes before fixation of lectin-treated eggs do not change its aspect. Our ultrastructural observations thus favour an even distribution of the WGA receptor sites on the surface of the Xenopus zygote. The nascent membrane in the furrow region probably has a chemical constitution different from that of the bulk of the membrane (de Laat, Bluemink & Van der Saag, 1978). In particular, fewer receptors for WGA exist there.
Furrow formation in amphibian eggs is the result of several events, the first of which is a surface constriction. The presence of parallel actin-like filaments in the cortical cytoplasm beneath the tip of the groove has led to the hypothesis that they function as a contractile ring and are thus responsible for the surface constriction initiated at the animal pole (Arnold, 1969; Selman & Perry, 1970). This band of microfilaments is probably formed by the organization of filament bundles and meshwork of the oocyte cortex (Franke et al. 1976) and its contraction is regulated by calcium (Gingell, 1970; Schroeder & Strickland, 1974). Some clusters of filaments are located under the plasma membrane of WGA-treated embryos beneath the stripe or at the tip of the blocked furrow. However, we have never observed a continuous layer of filaments in these embryos as in the controls. Such arrays of microfilaments are also absent from cytochalasin B-treated eggs. Alignment of microfilaments in the furrow region could be preceded by displacement and reorganization of their anchorage sites. WGA molecules bound to the egg surface could hinder these movements through a crosslinking of the anchorage sites, or through a transmembrane effect modifying the mobility of the membrane proteins (Edelman, 1976). When the embryos are treated after the onset of furrow formation, the microfilaments are already arrayed and can contract in a coordinated manner. This organization is apparently lost as a consequence of lectin treatment. Our results are in contrast to the observations of Eisenman, Das & Alfert (1977), who found no difference in the structure of the furrow with respect to the microfilament band between concanavalin A-treated and control embryos of Urechis caupo. But their results, obtained with another lectin, are not really comparable with ours since in the echiuroid eggs, the initiated furrow undergoes complete and sudden regression.
Our ultrastructural observations reveal another anomaly which could play an important part in the blocking phenomenon: bound lectin molecules apparently induce spatial movements of the cell surface which lead to narrow indentations of the plasma membrane into the cortex. In the partially regressed or blocked furrow surface of WGA-treated eggs, such indentations are much more frequently observed. In this case, the gap between the 2 membranes is very narrow and apparently does not even exist in many places. It is possible that the fluidity of the new membrane is higher than it is outside the furrow region. Areas of the nascent membrane where new lectin receptors have appeared could become internalized by a zip movement of the surface. Uncoupling of the microfilaments from their initial anchorage sites, followed by a displacement of these sites, is perhaps necessary for furrow groove deepening. This could be inhibited by WGA binding to the surface of the embryo. Indeed, when a wound is made in the regressed furrow surface of WGA-treated eggs, filaments attached to the internalized trilaminar membranes apparently remain associated with them after their organization into a cortical ring (see the companion paper: Geuskens & Tencer, 1979).
At the beginning of a normal first cleavage, a close association between microtubules and the microfilament layer has been observed by us in the furrow region. Such a location of microtubules in the peripheral cytoplasm has also been observed in the egg and in the first 2 blastomeres of Limnaea (Morrill, Perkins & Nasti, 1967). We have not observed many microtubules in close proximity to microfilament bundles in WGA- or cytochalasin B-treated eggs whereas in the initiated furrow region of control embryos microtubules are sometimes embedded in an electron-dense material. These structures seem to be equivalent to the mid-bodies described in mitotic cells and cleaving eggs (Selman & Perry, 1970; Kalt, 1971 ; Singal & Sanders,1974).
After surface constriction, ingrowth of new membrane by precursor insertion is necessary for furrow formation. The process of new membrane growth by precursor or vesicle insertion into the first furrow surface has been discussed in several papers (Selman & Perry, 1970; Kalt, 1971; Bluemink & de Laat, 1973; Arnold, 1974; Singal & Sanders, 1974), but the mechanism of surface expansion in the groove is still unclear. The new membrane which is added to the forming blastomere surface during the^secretion of β-glycogen and other products into the extracellular space contributes’to the growth of the furrow (Kalt, 1971). We have rarely observed large glycogen vesicles in the vicinity of the first furrow in control embryos. To our knowledge, their formation by fusion of vesicles with flocculent contents around a glycogen area, as observed here in WGA-treated eggs, has not been reported in the literature at this early stage of development. The presence of numerous and large glycogen vesicles in the cortex of eggs arrested at the first cleavage stage could result from an abnormal membrane metabolism or from the anticipation of a mechanism which normally occurs on a large scale only later during development. Numerous partially fused vacuoles are observed at the tip of the blocked furrow in WGA-treated eggs, suggesting that, like cytochalasin B (Bluemink, 1971a), WGA interferes with the process of membrane ingrowth.
In conclusion, we interpret our observations in the following way: WGA binding to the egg surface impedes the alignment of microfilaments under the plasma membrane at the animal pole when it is added before furrow formation and it provokes either directly or indirectly the disorganization of these filaments when added during furrow formation. A secondary effect of the lectin on the organization of the microtubules cannot however be excluded. The internalization of the lectin-receptor complexes of the nascent membrane in the furrow region could also play a part in the blocking phenomenon. On the other hand, since we have observed the presence of microfilaments bound to the internalized membranes (see also the companion paper), we are tempted to think that the binding of microfilaments to the membrane could be increased by the binding of WGA to its receptors in such a way that filament detachment and shift would be hindered. Finally, the lack of precursor insertion into the new membrane could hinder new surface formation and consequently contribute to the inhibition of the groove deepening.
Warm thanks are due to Dr J. Osborn for his help with the English version. M. Geuskens is ‘Maître de recherches’ of the Belgian National Fund for Scientific Research.