Contractions in surface structures of Xenopus eggs have been induced by application of the calcium ionophore A23187 or calcium ion. Local applications have shown that the contractile structure is present in both animal and vegetal hemispheres. It is, however, much stronger in the animal hemisphere and pigment embedded in it there defines the animal half.

The injection of cytochalasin B (CB) into whole cells or the application of the antibiotic to half cells cannot prevent the induced contractions. By experimental means, the contraction of a deeper, pigment-containing structure can be uncoupled from a thin, more superficial and relatively pigment-free layer on the egg surface. By this means it has been possible to establish that the CB-resistant contraction is due, at least partially, to a structurally distinguishable layer subjacent to the outer egg cortex. Scanning and transmission electron microscopy demonstrate a dense grainy matrix near the egg surface in which pigment granules but little yolk are embedded. This structure is much thicker in the pigmented hemisphere.

The presence of calcium ions in an isolation medium are shown to cause a loosening or dissolution of the structural connections between the dense, contractile structure near the surface and the cytoskeleton of the endoplasm.

The program for early development in amphibian eggs includes many contractile events which take place at the surface. These events include the engulfment of the fertilizing sperm, exocytosis of cortical granules, surface contractions leading to formation of a grey crescent and associated with the orientation of the first cleavage furrow. (For reviews see Elinson, 1980; Kirschner, Gerhart, Hara & Ubbels, 1980.) In addition, the movement of pigment towards the point of sperm entry, establishment of the grey crescent, movement of an area of yolk-free cytoplasm and movements of the cortex relative to the endoplasmic mass are all associated with the early establishment of the embryonic axes (Kirschner et al. 1980; Ubbels, 1977). Because of the presence of a superficial layer of pigment in the animal hemisphere, many of these outer movements have been described in terms of the movements of the surface pigment.

Surface contractions can also be induced by experimental means. For example, a puncture wound in the egg surface induces the accumulation of pigment around the hole and its subsequent closing (Bluemink, 1972; Gingell, 1970; Holtfreter, 1943a; Luckenbill, 1971; Merriam & Christensen, 1983). Similarly, surface pigment can be caused to move towards a point of local application of polyions (Gingell, 1970) or the calcium ionophore A23187 (Schroeder & Strickland, 1974). If the entire egg is immersed in A23187, an isometric contraction of the entire surface occurs (Schroeder & Strickland, 1974). In their entirety, these reports show clearly that surface contractions occur naturally in the program of development or can be experimentally induced and that they seem to be triggered by, or need, calcium.

Recently, observations on the sensitivity of cortical contractions in Xenopus eggs to cytochalasin B (CB) have been reported. These studies have shown that both induced cortical contractions (Merriam, Sauterer & Christensen, 1983) and a natural cortical contraction (Christensen & Merriam, 1983) are insensitive to CB while wound healing is sensitive to the antibiotic (Merriam & Christensen, 1983). These observations suggested that at least two types of cortical contractions, based on sensitivity to CB, occur in or near the surface of the amphibian egg-

In this report we describe experiments to study the distribution of the CB-insensitive contractile system in the cortical region of Xenopus eggs. We show that it is a coherent system over the entire egg surface, that it is subjacent to a more superficial layer and that it is much thicker and stronger in the animal hemisphere. The pigment of the animal hemisphere is embedded in it and it is thus the system which moves surface pigment and has been implicated in many developmental events.

Eggs of Xenopus laevis were obtained from mature females by the injection of 600 i.u. of human chorionic gonadotropin the previous day and evening. Eggs were stripped into 0·1-strength amphibian Ringer’s solution and dejellied in 35mM-beta mecaptoethanol brought to pH 8·9 with NaOH. After washing, dejellied eggs were maintained in 0·1 Ringer’s util use. Vitelline membranes were dissected off by means of fine watchmakers’ forceps under a disseting microscope.

Contraction medium consisted of Steinberg’s solution, buffered to pH 6·5 with Tris/NaH2PO4 and with the calcium concentration brought up to 1 mM-CaCl2. Cytochalasin B (CB) and the ionophore A23187 were purchased from Sigma Chemical Co., St. Louis, Mo. Both were put up into stock solutions at 10 mg/ml. CB stock was in dimethylsulfoxide (DMSO) and A23187 was in ethanol/DMSO (3/1). Control samples always contained the same concentration of solvent without the active agent. The biological activity of the CB preparation was checked in every experiment by observing the behaviour of a puncture wound in an egg maintained in a CB-containing contraction medium. If the wound failed to close but instead grew larger, the CB was considered active (see Merriam & Christensen, 1983).

Microinjections were performed with micropipettes possessing sharpened tips of 5-10μm outside diameter. About 80 nl were injected into each egg using a carrier solution consisting of 88mM-NaCl; ImM-KCl; 15 mM-Tris-HCl, pH 7·2. The amount of CB stock added to the carrier solution was calculated to make the intracellular CB concentration 10μg/ml of cell water, assuming half of the cell volume to be free water. Contraction medium served as a healing medium after injection.

Contractile events were observed in a dissecting microscope and moved to a Leitz macrolens photographic system for photomicrography. Epi-illumination was used throughout.

Eggs were prepared for scanning electron microscopy (SEM) by a dry fracture procedure (Leverah, Merriam & Sauterer, 1980). Eggs were fixed in 3% glutaraldehyde, buffered to pH7·4 with 0·1 M-phosphate buffer. After washing and dehydration in a water/acetone series, they were split open by the use of fine needles. The half cells were then dried by the critical point method and coated with gold/palladium (60/40) for observation of the fracture face by SEM. The instrument used was a JOEL JSMO 35C.

For transmission electron microscopy eggs were fixed 10 min in 2·5 % glutaral-dehyde, buffered to pH7-2 by 0·05 M-cacodylate, at room temperature. After a brief wash, they were postfixed for 45 min at 0°C in 1% OsO4 at pH 7·2 in 0·05-M S-collidine buffer. The cells were then washed, dehydrated, embedded in DE resin and sectioned. Lead acetate staining was utilized to enhance contrast.

(a) The distribution of surface contractile elements over the egg

We first studied the effect of CB on surface contractions induced by A23187. To circumvent the low permeability of these eggs to CB, we performed two experiments in which CB was directly microinjected into the animal hemisphere of eggs before the challenge with A23187. One of the experiments, the results of which are presented in Table 1, was done with a 3-8 min interval between injection and challenge. Pictures of injected eggs may be seen in Fig. 1. Both of these experiments showed clearly that CB within the cell at sufficient concentration and activity to prevent the appearance of a cleavage furrow in control eggs had no effect on the A23187-induced contractions.

Table 1.

Effect of injected CB on A23187-induced cortical contractions*

Effect of injected CB on A23187-induced cortical contractions*
Effect of injected CB on A23187-induced cortical contractions*
Fig. 1A.

Dejellied eggs after injection of CB but before addition of A23187. Mag. ×25. Line represents 0·5 mm.

Fig. 1B. Same eggs as Fig. 1A after contraction induced by A23187. Leakage of yolk occurs because wound healing is inhibited by CB. Note the contraction of the pigmented areas.

Fig. 1A.

Dejellied eggs after injection of CB but before addition of A23187. Mag. ×25. Line represents 0·5 mm.

Fig. 1B. Same eggs as Fig. 1A after contraction induced by A23187. Leakage of yolk occurs because wound healing is inhibited by CB. Note the contraction of the pigmented areas.

We were interested in possible shape changes in the pigmented cortex of the animal hemisphere caused by these induced contractions. Accordingly, we induced general contractions with both A23187 and ethanol, fixed the contracted eggs in buffered glutaraldehyde and sliced them open. In Fig. 2A the contracted shapes may be compared with the relaxed state. In Fig. 2B and C it can be seen that contraction leads to both a reduction in area of the pigmented cortex and a marked increase in its thickness.

Fig. 2A.

Eggs showing induced contractions: Left cell not contracted; middle cell with the pigmented ‘nipple’ and stretched vegetal hemisphere induced by 4% ethanol in 0·9M-sucrose; right cell with the contracted pigmented area induced by A23187. Mag. ×13. Line represents 1·0mm.

Fig. 2B. The cut surface of a non-contracted egg, sliced in half. Note the thin layer of pigment of the animal hemisphere at the top. Mag. ×24. Line represents 0·5 mm.

Fig. 2C. Same as Fig. 2B except the egg was induced to contract with A23187 before fixation and cutting. Note that the pigment of the animal hemisphere is reduced in surface area but is much thicker.

Fig. 2A.

Eggs showing induced contractions: Left cell not contracted; middle cell with the pigmented ‘nipple’ and stretched vegetal hemisphere induced by 4% ethanol in 0·9M-sucrose; right cell with the contracted pigmented area induced by A23187. Mag. ×13. Line represents 1·0mm.

Fig. 2B. The cut surface of a non-contracted egg, sliced in half. Note the thin layer of pigment of the animal hemisphere at the top. Mag. ×24. Line represents 0·5 mm.

Fig. 2C. Same as Fig. 2B except the egg was induced to contract with A23187 before fixation and cutting. Note that the pigment of the animal hemisphere is reduced in surface area but is much thicker.

In the experiments using induced cortical contractions, the powerful contraction of the pigmented hemisphere predominated. We were curious to know if the cortex of the unpigmented hemisphere also contained a structure capable of contracting in response to A23187. To explore this question, eggs were wedged into glass capillaries whose inside diameters were slightly less than the diameter of the cells. A23187 was then administered only to the unpigmented surface and changes in the distribution of the pigmented cortex were photographed. The results of a typical experiment may be seen in Fig. 3. Initially, the non-pigmented cortex contracted, pulling the pigmented cortex towards the vegetal pole in an epiboly-like movement. Then, presumably as the ionophore entered the cell more fully, contraction of the more powerful pigmented cortex occurred and the pigmented structure was reduced in area as usual. From these experiments we learned that both hemispheres of the egg contain a surface structure capable of contracting in response to A23187, that the responding entities form a structurally coherent system capable of transmitting a pull over the entire surface of the egg and that the structure in the pigmented hemisphere produces a stronger contraction.

Fig. 3.

An egg confined within a glass capillary tube with A23187 administered only to the vegetal hemisphere at the bottom. From left to right, photographs of the same cell at 0min, Imin, 3 min and 5 min after addition of the ionophore. Note the extension of the pigmented surface downward at 1 and 3 min and again upwards at 5 min. The numbers represent the area of the observed pigmented surface as % of total observed surface. Mag. ×28. Line represents 0·5 mm.

Fig. 3.

An egg confined within a glass capillary tube with A23187 administered only to the vegetal hemisphere at the bottom. From left to right, photographs of the same cell at 0min, Imin, 3 min and 5 min after addition of the ionophore. Note the extension of the pigmented surface downward at 1 and 3 min and again upwards at 5 min. The numbers represent the area of the observed pigmented surface as % of total observed surface. Mag. ×28. Line represents 0·5 mm.

It seemed desirable to develop an ‘open’ system in which these surface contractions could be induced in an aqueous medium in which the soluble constituents could be systematically explored. To achieve this, the vitelline membrane was removed and eggs were cut in half with forceps at the equator between the dark and light hemispheres. The half cells were then placed, cut side down, in an isolation medium containing ethyleneglycol-bis-(B-aminoethyl ether)N,N’-tetraacetic acid (EGTA). Upon addition of calcium to the medium to micromolar concentrations of free ion, the surface of both animal and vegetal hemispheres underwent an isometric contraction during 1-4 min. Outlines of each cortex could be drawn with the aid of a camera lucida both before and after contraction. Planimetry of the outlines enabled a quantitative estimate to be made of contractions occurring in both hemispheres. Photographs of the contraction of typical half cells can be seen in Fig. 4. The effects of CB in the isolation medium were studied to see if the antibiotic could inhibit the induced contractions in this more accessible system. The results are summarized in Table 2.

Table 2.

The effect of CB on calcium-induced contraction of pigmented and unpigmented cortices in an open-cell system

The effect of CB on calcium-induced contraction of pigmented and unpigmented cortices in an open-cell system
The effect of CB on calcium-induced contraction of pigmented and unpigmented cortices in an open-cell system
Fig. 4A.

A normal egg cut in half with the cut surfaces downward. The pigmented animal hemisphere is on the left and the vegetal hemisphere on the right. The half cells are relaxed in the absence of calcium. Mag. ×25. Line represents 0·5 mm.

Fig. 4B. The same half cells as in 4A in the contracted state after the addition of calcium.

Fig. 4A.

A normal egg cut in half with the cut surfaces downward. The pigmented animal hemisphere is on the left and the vegetal hemisphere on the right. The half cells are relaxed in the absence of calcium. Mag. ×25. Line represents 0·5 mm.

Fig. 4B. The same half cells as in 4A in the contracted state after the addition of calcium.

We learn several things from these data. It is the surface structures which are contracting because the underlying endoplasmic mass is left behind and exposed to view. Both pigmented and unpigmented hemisphere surfaces can contract in the presence of CB, in the same medium which supplies the calcium trigger. CB does have an inhibitory effect on the extent of contraction in both hemispheres and the percent of inhibition tends to be greater in the unpigmented surface.

(b) The layering of cortical contractile elements relative to the egg surface

An obvious question which arose during these studies concerned the location of the contracting entity on the surface of these eggs. Previous work had shown that both CB-resistant and CB-sensitive contractile systems could be found there. Is the CB-resistant structure a separate structural layer, whose contraction could be uncoupled from the CB-sensitive one? Or are both integrated into a single structure?

We have found that it is possible to uncouple the contractions of a deeper, pigmented structure from a more superficial, non-pigmented layer. This has been accomplished in the open, half cell contracting system by allowing the half cells to sit in the EGTA-containing medium for about 1·0–1·5 min before adding the calcium ion trigger. In the eggs of some animals, but not all, only a deeper, pigmented layer contracts, leaving a non-pigmented, superficial layer still covering the endoplasmic mass. This effect can be seen in Fig. 5A and B. The superficial layer protects the pigmented layer from disturbance by forceps when probed from above. When CB is added to the isolation medium 1·5-2·0min before the calcium trigger, the same contraction occurs (Fig. 6A and B). If eggs from the same frog are cut in half and the calcium trigger added immediately, both inner and outer layers contracted together as usual (see Fig. 4).

Fig. 5A.

Three pigmented animal hemispheres in the half-cell state with cut sides down. The picture was taken about Imin after the addition of calcium. The arrowheads mark the edges of the outer layers and the arrows show the contracting edges of the underlying pigmented layers. Mag. ×18. Line represents 0·5 mm.

Fig. 5B. Same half cells as in Fig. 5A about 3 min after the addition of calcium. Note the further reduction in area of the underlying pigmented layers relative to the area of the upper, unpigmented layers.

Fig. 5A.

Three pigmented animal hemispheres in the half-cell state with cut sides down. The picture was taken about Imin after the addition of calcium. The arrowheads mark the edges of the outer layers and the arrows show the contracting edges of the underlying pigmented layers. Mag. ×18. Line represents 0·5 mm.

Fig. 5B. Same half cells as in Fig. 5A about 3 min after the addition of calcium. Note the further reduction in area of the underlying pigmented layers relative to the area of the upper, unpigmented layers.

Fig. 6A.

Three pigmented animal hemispheres in the half-cell state. They were incubated 1·5 min in the absence of calcium but in the presence of CB at 10 μg/ml. Then the calcium trigger was added. The picture was taken about 0·5 min after addition of the calcium. Mag. ×18. Line represents 0·5 mm.

Fig. 6B. Same half cells as in Fig. 6A about 3 min after the addition of the calcium trigger. Note the contraction in area of the underlying pigmented area relative to the upper layer.

Fig. 6A.

Three pigmented animal hemispheres in the half-cell state. They were incubated 1·5 min in the absence of calcium but in the presence of CB at 10 μg/ml. Then the calcium trigger was added. The picture was taken about 0·5 min after addition of the calcium. Mag. ×18. Line represents 0·5 mm.

Fig. 6B. Same half cells as in Fig. 6A about 3 min after the addition of the calcium trigger. Note the contraction in area of the underlying pigmented area relative to the upper layer.

This experiment showed that the contracting structure on the surface of at least the half egg can be resolved into an outer, pigment-poor (or free) layer and an underlying pigmented layer which can shear past each other. It demonstrated also that the inner, pigmented layer is capable of contraction in the presence of CB.

To confirm this important observation in whole eggs we adopted a means of marking the surface of eggs. A puncture wound was made in the surface near the equator between pigmented and unpigmented hemispheres. The wound was allowed to heal in the presence of calcium ions, leaving a visible surface scar. The cells were then observed during the contractions induced by A23187. A typical result can be seen in Fig. 7. As the pigmented cortex contracted in area the surface wounds moved towards the animal pole at the same rate, maintaining their position relative to the edge of the pigmented area. This showed that the surface layer and underlying pigment were moving together.

Fig. 7A.

Normal cells with healed wounds (arrows). Mag. ×18. Line represents 0·5 mm.

Fig. 7B. Same eggs as Fig. 7A after contraction of the pigmented cortex, induced by A23187. Note that wounds have moved towards the animal pole to the same extent as the pigmented cortex.

Fig. 7A.

Normal cells with healed wounds (arrows). Mag. ×18. Line represents 0·5 mm.

Fig. 7B. Same eggs as Fig. 7A after contraction of the pigmented cortex, induced by A23187. Note that wounds have moved towards the animal pole to the same extent as the pigmented cortex.

In an attempt to uncouple the contracting pigmented structure from the overlying unpigmented layer, we added CB to the medium at a concentration of 20 μg/ml for 5 min before the A23187 challenge. Variable results were obtained in different batches of eggs but in most cases the pigmented area contracted into a uniformly pigmented smaller area or most pigment moved into more localized blotches within the original pigmented area. In the majority of cases, however, the surface marker did not move synchronously with the pigment! The movement of pigment, independently of the superficial marker, may be seen in Fig. 8. This experiment showed that in intact egg cells also, it is possible to uncouple the contractions of the deeper, pigmented layer from a more superficial layer on the surface.

Fig. 8A & 8C.

Normal eggs with healed wounds (arrows). Mag. ×24. Line represents 0·5 mm.

Fig. 8B & 8D. Same eggs as A and C after pre treatment with CB and then an induction to contract with A23187. Note the non-movement of the surface wounds but the movement of the deeper pigmented cortex relative to the wounds.

Fig. 8A & 8C.

Normal eggs with healed wounds (arrows). Mag. ×24. Line represents 0·5 mm.

Fig. 8B & 8D. Same eggs as A and C after pre treatment with CB and then an induction to contract with A23187. Note the non-movement of the surface wounds but the movement of the deeper pigmented cortex relative to the wounds.

(c) The relationship between the contractile surface structure and the underlying endoplasm

In the course of these experiments some observations were made which bear upon the relationship between the contractile structures of the egg surface and the underlying endoplasm. Thus, if an egg was cut in half in a buffered isolation medium, the endoplasm could be dispersed by directing a stream of medium over it, leaving behind a tough and coherent surface cytoplasm. The structure was thick in the animal hemisphere and very thin in the vegetal hemisphere. If the isolation medium was free of calcium, the washed residual structure was thick and laden with embedded yolk. The isolated structure may be seen in Fig. 9A. In such a medium the yolky endoplasm slowly changed from an easily dispersible state to a rubbery cohesive state.

Fig. 9A.

The isolated animal hemisphere cortex from a single egg. The isolation medium contained no calcium. Note the presence of much yolky cytoplasm as compared with Fig. 9B. Mag. ×35. Line represents 0·5 mm.

Fig. 9B. The isolated animal hemisphere cortex from a single egg. The isolation medium contained 0·10mM-calcium. Note the thin, clean appearance as compared with Fig. 9A. Mag. ×35.

Fig. 9A.

The isolated animal hemisphere cortex from a single egg. The isolation medium contained no calcium. Note the presence of much yolky cytoplasm as compared with Fig. 9B. Mag. ×35. Line represents 0·5 mm.

Fig. 9B. The isolated animal hemisphere cortex from a single egg. The isolation medium contained 0·10mM-calcium. Note the thin, clean appearance as compared with Fig. 9A. Mag. ×35.

If calcium was present in the isolation medium, however, the bisected cell surface would contract. After or during contraction the surface structure cleaned more easily, leaving a thin, largely yolk-free but pigment-containing residual structure. A surface structure isolated in the presence of calcium is shown in Fig. 9B, cleaned in the same way and photographed at the same magnification. In the calcium-containing medium, the consistency of the endoplasm slowly became grainy in texture and more easily dispersible.

(d) Morphological observations

In an attempt to visualize these cortical contractile structures we have developed a procedure for viewing a fracture face of the egg cytoplasm with the scanning electron microscope (Leverah et al. 1980). Images from such a preparation are shown in Fig. 10. Note the thick matrix near the surface of the animal hemisphere from which most yolk is excluded. Pigment and cortical granules can be made out embedded in its mass. The dense matrix cannot be seen in the vegetal hemisphere by this technique (Fig. 10B).

Fig. 10A.

An egg was fixed in glutaraldehyde and fractured open (see Materials and Methods). The surface of the animal hemisphere is at the top of the fracture face (arrow). Note the dense cytoplasmic matrix which includes pigment and cortical granules but largely excludes yolk platelets. Mag. ×1500. Line represents 10μm.

Fig. 10B. The same ‘dry fracture’ procedure as used in Fig. 10A. In this picture the cell surface (arrow) is near the vegetal pole of the egg. Note the almost complete lack of the dense, subcortical matrix as compared with the matrix of the animal hemisphere in Fig. 10A. Yolk platelets are found right up to the plasmalemma. Mag. ×1500.

Fig. 10A.

An egg was fixed in glutaraldehyde and fractured open (see Materials and Methods). The surface of the animal hemisphere is at the top of the fracture face (arrow). Note the dense cytoplasmic matrix which includes pigment and cortical granules but largely excludes yolk platelets. Mag. ×1500. Line represents 10μm.

Fig. 10B. The same ‘dry fracture’ procedure as used in Fig. 10A. In this picture the cell surface (arrow) is near the vegetal pole of the egg. Note the almost complete lack of the dense, subcortical matrix as compared with the matrix of the animal hemisphere in Fig. 10A. Yolk platelets are found right up to the plasmalemma. Mag. ×1500.

Thin sections normal to the surface in the animal hemisphere do not allow an identification of the dense surface matrix per se in the electron microscope. As shown in Fig. 11 its structure shows the inclusion of many granules, a membranous reticulum and embedded yolk and pigment. Just under the plasmalemma, however, the thin hypolemmal layer may be seen. Its texture and complete lack of larger inclusions allow its morphological identification.

Fig. 11A.

Thin section of the cytoplasm, cut normal to the surface of the animal hemisphere. Note the morphologically distinguishable hypolemma layer with numerous protrusions near the cell surface (arrowhead). Note also the dense cytoplasm, containing numerous pigment granules (P) but few yolk platelets (Y) under the hypolemma layer. Mag. ×11500. Line represents 1μm.

Fig. 11B. Thin section of the same egg as seen in Fig. 11A at higher magnification. The textural difference between the hypolemma (arrowhead) and subcortical areas of the cortex can be seen. Mag. ×23000. Line represents 0·5 μm.

Fig. 11A.

Thin section of the cytoplasm, cut normal to the surface of the animal hemisphere. Note the morphologically distinguishable hypolemma layer with numerous protrusions near the cell surface (arrowhead). Note also the dense cytoplasm, containing numerous pigment granules (P) but few yolk platelets (Y) under the hypolemma layer. Mag. ×11500. Line represents 1μm.

Fig. 11B. Thin section of the same egg as seen in Fig. 11A at higher magnification. The textural difference between the hypolemma (arrowhead) and subcortical areas of the cortex can be seen. Mag. ×23000. Line represents 0·5 μm.

Previous work in this laboratory has shown that surface contractions in the Xenopus egg can be divided into two classes, based on their sensitivity to CB (Merriam et al. 1983; Merriam & Christensen, 1983; Christensen & Merriam, 1983). In this report we demonstrate that the surface of the egg contains two layers. A thin, pigment-poor superficial layer normally moves with a thicker, pigmented underlying layer during induced surface contractions. Its movement, however, can be experimentally uncoupled so that it remains in place while the pigmented layer contracts. The uncoupling was achieved in both a half-cell system and in intact cells.

In the half-cell system a short pre-incubation period in the isolation medium, before addition of the calcium trigger, sometimes caused the uncoupling. The imperfect reproducibility of this event in different batches of eggs probably derives from the fact that the two layers are structurally coupled in vivo and that the strength of this coupling undoubtedly varies in different batches of eggs. We interpret the result to mean one of two things. Either both layers are contractile and the pre-incubation causes a preferential loss of a necessary, diffusible component in the outer layer, or only the inner layer is contractile under these conditions and the pre-incubation simply causes a structural uncoupling between the two.

Similarly, in the whole cell, the uncoupling of the contraction of the inner, pigmented layer from the outer layer by exogenous CB could be interpreted in the same ways. Either CB inhibits the contraction of the outer layer or causes its structural uncoupling from the subjacent structure, or both.

Our evidence that the inner, pigmented layer is not sensitive to CB is compelling. Neither exogenous nor injected CB can prevent the isometric contractions induced by A23187 or by ethanol (Merriam et al. 1983), even when internal concentration and activity were sufficient to block cytokinesis. The half cell contraction system strongly supported this finding. Incubating half cells for 1·5 min in the presence of CB before inducing contraction by adding calcium to the same medium, still allowed the contraction of the inner layer.

The effects of exogenous CB on induced cortical contractions, observed in these studies, requires some comment. It has been shown that exogenous CB does not enter amphibian eggs to inhibit cleavage furrow formation until after the production of new membrane in the furrow (Bluemink, 1971; Bluemink & De Laat, 1973; Hammer, Sheridan & Estensen, 1971; De Laat, Luchtel & Bluemink, 1973; Selman, Jacob & Perry, 1976). Our results indicate that exogenous CB affects the nature of induced surface contractions before cleavage furrow formation. This difference could be due to several things. We used a high concentration of CB (20μg/ml). It is possible also that the presence of the ionophore, used in these experiments, could facilitate the entry of the CB molecule. In general, we have noticed that eggs show abnormal pigment mottling earlier when CB is in their medium than do controls.

Contractions induced by A23187 are largely reversible (Gingell, 1970; Schroeder & Strickland, 1974) and lead to condensation of a thin extended structure into a thicker, less extended form. This type of network contraction has some structural similarities to the contraction of actin-based gels, produced in extracts of Xenopus oocytes, when ATP is present (Clark & Merriam, 1978).

The relationship of the contractile structures of the egg’s surface to the endoplasm is of interest. Changes in their interaction are involved in the formation of the grey crescent and the early establishment of embryonic symmetry (Elinson, 1980; Gerhart et al. 1981; Kirschner et al. 1980; Ubbels, 1977). Our observations suggest that calcium-induced surface contractions are accompanied by a concomitant loosening of structural connections between the cortical networks and the cytoskeleton of the endoplasm.

Our data are consistent with the idea that the superficial, non-pigmented layer, which can be uncoupled from a contracting deeper layer, is the hypolemma of Dollander (1962) and Gingell (1970) and the ‘cortical material’ of Bluemink (1970). Since the layer is connected to the interiors of surface projections and microvilli, and since it is the layer in which the contractile ring forms for cytokinesis in both eggs and somatic cells, it is probably the eggs’ version of the more universal cortex of eukaryote cells.

The inner, contractile layer which contains the pigment of the animal hemisphere and which we have shown here is insensitive to CB in its contractions, is probably the ‘surface coat’ of Holtfreter (1943a), the ‘vesicular ergastoplasm’ of Pasteels (1964), the ‘subcortical material’ of Bluemink (1970) and the ‘subcortical, pigment-containing structure’ of Merriam et al. (1983). From the work of these and other authors it is likely that this inner contractile layer is involved in embryonic symmetry development, epibolic movements of the early embryo and possibly the changes of cell shape involved in gastrulation (Holt-freter, 1943b; Perry & Waddington, 1966). Until its distribution in other cells and its biological functions are better understood, we suggest the neutral term ‘subcortical matrix’ as a useful designation.

We are grateful for the help of Dr John Bluemink of the Hubrecht Laboratorium, Utrecht, in obtaining the electron micrographs of Fig. 11. We also benefited from many discussions with Drs Bluemink and G. A. Ubbels. This work was supported by grant PCM 7824591 from the National Science Foundation, U.S.A.

Bluemink
,
J. G.
(
1970
).
The first cleavage of the amphibian egg. An electron microscope study of the onset of cytokinesis in the egg of Amblystoma mexicanum
.
J. Ultrastruct. Res
.
32
,
142
166
.
Bluemink
,
J. G.
(
1971
).
Cytokinesis and cytochalasin-induced furrow regression in the first cleavage zygote of Xenopus laevis
.
Z. Zellforsch. Mikrosk. Anat
.
121
,
102
126
.
Bluemink
,
J. G.
(
1972
).
Cortical wound healing in the amphibian egg: An electron microscopical study
.
J. Ultrastruct. Res
.
41
,
95
114
.
Bluemink
,
J. G.
&
De Laat
,
S. W.
(
1973
).
New membrane formation during cytokinesis in normal and cytochalasin B-treated eggs of Xenopus laevis
.
J. Cell Biol
.
59
,
89
108
.
Christensen
,
K.
&
Merriam
,
R. W.
(
1983
).
Insensitivity to cytochalasin B of surface contractions keyed to cleavage in the Xenopus egg
.
J. Embryol. exp. Morph
.
72
,
143
151
.
Clark
,
T. G.
&
Merriam
,
R. W.
(
1978
).
Actin in Xenopus oocytes. I. Polymerization and gelation in vitro
.
J. Cell Biol
.
77
,
427
438
.
De Laat
,
S. W.
,
Luchtel
,
D.
&
Bluemink
,
J. G.
(
1973
).
The action of cytochalasin B during cell cleavage in Xenopus laevis-. Dependence on cell membrane permeability
.
Devi Biol
.
31
,
163
177
.
Dollander
,
A.
(
1962
).
Organization corticale de 1’oeuf d’amphibian
.
Arch. Anat. Histol. Embryol
.
44
,
93
103
.
Elinson
,
R. P.
(
1980
).
The amphibian egg cortex in fertilization and early development
.
In: The Cell Surface: Mediator of Developmental Processes
, (eds
S.
Subtelny
&
N. K.
Wessels
), pp.
217
234
.
New York
:
Academic Press
.
Gerhart
,
J.
,
Ubbels
,
G.
,
Black
,
S.
,
Hara
,
K.
&
Kirschner
,
M.
(
1981
).
A reinvestigation of the role of the grey crescent in axis formation in Xenopus laevis
.
Nature
292
,
511
516
.
Gingell
,
D.
(
1970
).
Contractile responses at the surface of an amphibian egg
.
J. Embryol. exp. Morph
.
23
,
583
609
.
Hammer
,
M. G.
,
Sheridan
,
J. D.
&
Estensen
,
R. D.
(
1971
).
Cytochalasin B. II. Selective inhibition of cytokinesis in Xenopus laevis eggs
.
Proc. Soc. exp. Biol. Med
.
136
,
1158
1162
.
Holtfreter
,
J.
(
1943a
).
Properties and functions of the surface coat in amphibian embryos
.
J. exp. Zool
.
93
,
251
323
.
Holtfreter
,
J.
(
1943b
).
A study of the mechanics of gastrulation
.
J. exp. Zool
.
94
,
261
317
.
Kirschner
,
M.
,
Gerhart
,
J. C.
,
Hara
,
K.
&
Ubbels
,
G. A.
(
1980
).
In: The Cell Surface; Mediator of Developmental Processes
(eds
S.
Subtelny
&
N. K.
Wessells
)
Proc. 38th Symp. Soc. devl Biol. Vancouver, B.C
., pp.
187
216
.
New York
:
Academic Press
.
Leverah
,
L
,
Merriam
,
R. W.
&
Sauterer
,
R.
(
1980
).
The amphibian egg cortex as revealed by dry fracture and scanning electron microscopy
.
J. Cell Biol
.
87
,
90a
.
Luckenbill
,
L. M.
(
1971
).
Dense material associated with wound closure in the axolotl egg (A. Mexicanum)
.
Expl Cell Res
.
66
,
263
267
.
Merriam
,
R. W.
&
Christensen
,
K.
(
1983
).
A contractile ring-like mechanism in wound healing and soluble factors affecting structural stability in the cortex of Xenopus eggs and oocytes
.
J. Embryol. exp. Morph
.
75
,
11
20
.
Merriam
,
R. W.
,
Sauterer
,
R. A.
&
Christensen
,
K.
(
1983
).
A subcortical, pigment-containing structure in Xenopus eggs with contractile properties
.
Devi Biol
.
95
,
439
446
.
Pasteels
,
J. J.
(
1964
).
The morphogenetic role of the cortex of the amphibian egg
.
Adv. Morphogen
.
3
,
363
388
.
Perry
,
M. M.
&
Waddington
,
C. H.
(
1966
).
Ultrastructure of the blastopore in the newt
.
J. Embryol. exp. Morph
.
15
,
317
330
.
Schroeder
,
T. E.
&
Strickland
,
D. L.
(
1974
).
Ionophore A23187, calcium and contractility in frog eggs
.
Expl Cell Res
.
83
,
139
142
.
Selman
,
G. G.
,
Jacob
,
J.
&
Perry
,
M. M.
(
1976
).
The permeability to cytochalasin B of the new unpigmented surface in the first cleavage furrow of the newt’s egg
.
J. Embryol. exp. Morph
.
36
,
321
341
.
Ubbels
,
G. A.
(
1977
).
Symmetrization of the fertilized egg of Xenopus laevis
.
Mem. Soc. Zool. France
41
,
103
116
.