J. Embryol. exp. Morph. 76, 51-65 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
51
Localization of a pigment-containing structure near
the surface of Xenopus eggs which contracts in
response to calcium
By R. W. MERRIAM 1 AND R. A. SAUTERER
From the Department of Neurobiology and Behavior, State University of New
York at Stony Brook
SUMMARY
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.
INTRODUCTION
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 yolkfree 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.
1
Author's address: Department of Neurobiology and Behavior, State University of New
York at Stony Brook, Stony Brook, New York 11794, U.S.A.
52
R. W. MERRIAM AND R. A. SAUTERER
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
eggIn this report we describe experiments to study the distribution of the CBinsensitive 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.
MATERIALS AND METHODS
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 pH8-9 with NaOH. After washing,
dejellied eggs were maintained in 0-1 Ringer's until use. Vitelline membranes
were dissected off by means of fine watchmakers' forceps under a dissecting
microscope.
Contraction medium consisted of Steinberg's solution, buffered to pH 6-5 with
Tris/NathPC^ and with the calcium concentration brought up to 1 mM-CaCk.
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
Localization of a contractile structure near the egg surface
53
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 5-10/im outside diameter. About 80 nl were injected into each egg using a
carrier solution consisting of 88mM-NaCl; lmM-KCl; 15 mM-Tris-HCl, pH7-2.
The amount of CB stock added to the carrier solution was calculated to make the
intracellular CB concentration lOjUg/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 % glutaraldehyde, 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% OsC>4 at pH7-2 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.
RESULTS
(a) The distribution of surf ace 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.
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
54
R. W. MERRIAM AND R. A. SAUTERER
Table 1. Effect of injected CB on A23187-induced cortical contractions*
Injected
No.
fertilized
eggs
not injected
DMSO
CB
57
12
20
not injected
DMSO
CB
25
11
13
A23187induced
contraction
—
25
10
10
%
contracted
—
100
91
80
No.
cleaved
%
cleaved
54
8
4
94
66
20
:
:
* Contraction with these eggs consisted only of reduction of the area of the pigmented
cortex.
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.
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
Fig. 1A. Dejellied eggs after injection of CB but before addition of A23187. Mag.
x25. Line represents 0-5 mm.
Fig. IB. 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. 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. x l 3 . Line represents 1-0 mm.
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. x24. 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. 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 Omin, lmin, 3min and 5min after addition of the ionophore. Note the
extension of the pigmented surface downward at 1 and 3 min and again upwards at
5min. The numbers represent the area of the observed pigmented surface as % of
total observed surface. Mag. x28. Line represents 0-5 mm.
Localization of a contractile structure near the egg surface
55
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.
1A
Figs 1-3
56
R. W. MERRIAM AND R. A. SAUTERER
Table 2. The effect of CB on calcium-induced contraction of pigmented and
unpigmented cortices in an open-cell system
Frog
No.
Cortex
No.
eggs
1
pigmented
unpigmented
2
2
2
pigmented
unpigmented
3
pigmented
unpigmented
8
8
5
5
4
pigmented
unpigmented
5
pigmented
unpigmented
*% rhintrr
12
12
3
3
% change (M ± S.E.M.)*
NoCB
With CB$
-64
-44
-62 ± 7-2
- 2 3 ± 7-9
-24 ± 10-9
- 3 ± 2-8
-56 ± 4-6
-36 ± 6-4
-74 ±13-0
-73 ± 3-0
% inhibition
byCBf
-44
-11
-34 ± 5-4
- 6 ± 5-7
31
75
45
74
-
-
-38
-28
-46
-42
± 5-1
± 5-0
± 6-3
±14-5
32
22
36
42
initial a r e a
~ contracted area w i n n
initial are*i
*CI J was used at a coiicentration of 10jUg/ml.
(no CB) - % change (•
t% inhibition by CB = % change %
change (no CB)
/
Y
inn
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
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. x25. Line represents 0-5 mm.
Fig. 4B. The same half cells as in 4A in the contracted state after the addition of
calcium.
Fig. 5A. Three pigmented animal hemispheres in the half-cell state with cut sides
down. The picture was taken about lmin 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. xl8. 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 lOjUg/ml.
Then the calcium trigger was added. The picture was taken about 0-5 min after
addition of the calcium. Mag. xl8. 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.
Localization of a contractile structure near the egg surface
57
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
• v
4A
l£w—
Figs 4-6
58
R. W. MERRIAM AND R. A. SAUTERER
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.
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
Fig. 7A. Normal cells with healed wounds (arrows). Mag. xl8. Line represents
05mm.
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. 8A & 8C. Normal eggs with healed wounds (arrows). Mag. x24. Line
represents 0-5 mm.
Fig. 8B & 8D. Same eggs as A and C after pretreatment 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. 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. x35. Line represents 0-5 mm.
Fig. 9B. The isolated animal hemisphere cortex from a single egg. The isolation
medium contained 0-lOmM-calcium. Note the thin, clean appearance as compared
with Fig. 9A. Mag. x35.
Localization of a contractile structure near the egg surface
59
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).
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
9A
9B
Figs 7-9
60
R. W. MERRIAM AND R. A. SAUTERER
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.
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 jUg/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.
(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.
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
Localization of a contractile structure near the egg surface
61
•^CL1!
.
* .
*
*
*
#
^^;>*
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. X1500. Line represents 10/mi.
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.
X1500.
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. x 11500. Line represents 1 jum.
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. X23000. Line represents 0-5 /an.
EMB76
62
R. W. MERRIAM AND R. A. SAUTERER
developed a procedure for viewing a fracture face of the egg cytoplasm with the
scanning electron microscope (Leverah etal. 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).
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.
DISCUSSION
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
Localization of a contractile structure near the egg surface
63
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 (20jUg/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 (Holtfreter, 19436; Perry & Waddington, 1966). Until its distribution in other cells and
64
R. W. MERRIAM AND R. A. SAUTERER
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.
REFERENCES
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. /. 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. /. 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. 11, 427-438.
DELAAT, 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 l'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. /. 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.
/. exp. Zool. 93, 251-323.
HOLTFRETER, J. (1943b). A study of the mechanics of gastrulation. /. 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, J., MERRIAM, R. W. & SAUTERER, R. (1980). The amphibian egg cortex as revealed
by dry fracture and scanning electron microscopy. /. 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. /. Embryol. exp. Morph. 75, 11-20.
MERRIAM, R. W., SAUTERER, R. A. & CHRISTENSEN, K. (1983). A subcortical, pigmentcontaining 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.
BLUEMINK,
Localization of a contractile structure near the egg surface
PERRY, M.
65
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.
(Accepted 16 March 1983)