/. Embryo/, exp. Morph. Vol. 36, 2, pp. 321-341, 1976
Printed in Great Britain
321
The permeability to cytochalasin B
of the new unpigmented surface in the first
cleavage farrow of the newt's egg
By G. G. SELMAN 1 , J. JACOB 1 AND M. M. PERRY 2
From the Institute of Animal Genetics, University of Edinburgh
SUMMARY
Two to 10 /ig/ml cytochalasin B (CB) caused retraction of the first cleavage furrow in
Triturus eggs, a spreading of the unpigmented surface from the furrow region and a flattening
of the whole egg. CB appears to act against the contractility of the microfilamentous band
at mid-cleavage so as to relax the furrow and also to weaken unpigmented surface to allow the
egg to flatten. Uncleaved eggs and the initial formation of the cleavage groove were unaffected
by CB. A fully-retracted first cleavage furrow reformed itself on transfer of the egg to normal
medium but only at the time of second cleavage. Initiation of second cleavage depended upon
there being sufficient of the original pigmented surface on the animal hemisphere.
Tritium-labelled CB of high specific activity was prepared and used to study its ability to
penetrate the surface of newt eggs during cleavage. Scintillation counting of whole eggs
showed that CB was not taken into the newt egg until mid-cleavage (about 17 min after the
double stripe stage) when new surface began to spread in the cleavage furrow. Fixation in
glutaraldehyde and osmium tetroxide retained radioactivity in the egg, but more CB was
retained afterfixationin osmium tetroxide alone than after doublefixation.Most of the retained
radioactivity was in yolk platelets. Autoradiographs were prepared of sectioned eggs which
had been fixed at late cleavage after [3H]CB had flattened the furrow. These showed that CB
entered the egg through the unpigmented surface which formed in the furrow but it could not
enter through the pigmented surface. The impermeability of the pigmented surface explains
the observations that CB does not prevent initial furrowing at cleavage. Once inside the egg
CB is transported slowly. CB penetrates to a limited extent beneath the pigmented surface
from its border with the unpigmented surface in the first cleavage furrow and this seems
insufficient in some circumstances to suppress the contractile phase of second cleavage.
INTRODUCTION
Previous studies of normal first cleavage in the amphibian egg have indicated
that the initial dipping inwards of the pigmented animal surface to form a groove
is probably due to contraction of a band of mutually parallel actin-like filaments
which at cleavage has been observed immediately below the plasma membrane
and along the bottom of the groove (Selman & Perry, 1970; Bluemink, 1970;
1
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh, EH9 3JN,
U.K.
2
Author's address: Agricultural Research Council's Poultry Research Centre, West Mains
Road, Edinburgh, EH9 3JS, U.K.
322
G. G. SELMAN, J. JACOB AND M. M. PERRY
Perry, John & Thomas, 1971). At the next stage of cleavage there is a progressive
increase of new unpigmented surface at the bottom of the furrow as it grows
through the egg towards the vegetal pole (Selman & Waddington, 1955; Selman
& Perry, 1970; Bluemink, 1971c; Bluemink & de Latt, 1973).
One of the characteristic properties of the lipid-soluble drug cytochalasin B
(CB) is that it can cause regression of the cleavage furrow without affecting
mitosis, so that binucleate cells are produced (Carter, 1967, 1972). Karfunkel
(1972) showed that CB inhibited neurulation in chick embryos and Spooner &
Wessels (1972) demonstrated the reversible inhibition of morphogenesis in
embryonic mouse salivary glands with the drug. In both these studies, the electron
microscope observations gave support to the hypothesis of Wessels et al. (1971)
that CB acts by inhibiting a contractile function mediated by systems of microfilaments. Spooner, Yamada & Wessels (1971) showed that CB may affect cell
locomotion in a similar manner. The microfilament hypothesis has also been
invoked to explain the action of CB on cleavage, but here there is a difficulty that
conflicting observations have been made when cells were cultured in the presence
of the drug from the time of the preceding nuclear division. In these circumstances Schroeder (1970, 1972) reported that the cleavage furrow never formed
in Arabacia eggs and HeLa cells. On the other hand Carter (1967, 1972) and
Krishan (1971) using Earle's strain of mouse fibroblasts and Bluemink (1971 b, c)
and Hammer, Sheridan & Estensen (1971) for Xenopus eggs, observed that the
furrow forms and subsequently regresses. In these cases only the late stage of the
cleavage shows sensitivity to CB and the microfilaments responsible for the
initial furrowing might be insensitive to the drug. However, it should be
remembered that when these observations were made (and the present observations began) nobody knew the extent to which CB could penetrate a cell.
CB has also been reported to act upon cells in ways which seem unrelated to
the functions of microfilaments but which implicate the cell surface as the
probable site of action. Thus the transport of hexose sugars into mammalian
cells (Estensen & Plagemann, 1972; Kletzien, Perdue & Springer, 1972; Mizel
& Wilson, 1972) was shown to be reversibly inhibited by low concentrations of
CB and Schaeffer, Schaeffer & Brick (1973) showed that inter-cell adhesion
between gastrula cells of Ranapipiens and their electrophoretic mobility could be
reduced by the drug. For chick and mammalian tissue there is experimental
evidence which indicates that the action of CB on morphogenesis and locomotion is not an indirect affect of glucose depletion (Warner & Perdue, 1972;
Taylor & Wessells, 1973; Yamada & Wessels, 1973).
Previous studies of the effect of CB on the cleavage of the amphibian egg have
used Xenopus laevis (Bluemink, 1971 a, b and c; Estensen, Rosenberg & Sheridan,
1971; Hammer^ al. 1971; De Laat, Luchtel & Bluemink, 1973; Bluemink &
De Laat, 1973; De Laat & Bluemink, 1974). The present work describes observations by low power light microscopy on Triturus alpestris whose cleavage is
similar but which also shows some features different from Xenopus. For example,
Cyiochalasin B and cleavage
323
Triturus has a far longer interval between successive cleavages (about 100 min at
18 °C) and unpigmented surface is temporarily exposed in the furrow on the
animal surface when cleavage occurs within the vitelline membrane. The effects
of CB on cleavage also show some different features which will be emphasised
in this paper. The observations can be interpreted within the framework of our
present understanding of cell cleavage in the amphibian egg and the known
biological properties of cytochalasin B.
METHODS
Observation on live eggs
Uncleaved eggs of Triturus alpestris (Laurenti) were collected in pond water,
decapsulated with a pair of sharp needles and examined by binocular microscope.
A stock solution of 1 mg cytochalasin B (CB) in 1 ml dimethyl sulphoxide (DMSO)
was prepared and diluted with 1/10 strength phosphate-buffered Holtfreter's
saline pH 7-1 to obtain the required concentration of CB. Thus 10/tg/ml CB
contained also 1 % DMSO and lower concentrations of CB contained proportionally less DMSO. The cleavages were monitored by taking photographs at
recorded intervals after the double stripe stage (Bluemink, 1970) at the onset of
first cleavage. All photographs are of live eggs, either with or without their
vitelline membrane. Some eggs were allowed to develop inside a small glasswalled box, two sides of which were binocular prisms designed to reflect light
through a right angle. This allowed the egg to be photographed alternately from
above or in side view with a fixed vertical microscope and camera.
For experiments with [3H]CB, the vitelline membrane was first carefully
removed with fine forceps from uncleaved newt eggs in 1 ml of Holtfreter's
saline and then after about 10 min 1 ml of the stock solution of [3H]CB was
added to a final concentration of 10 /tg/ml [3H]CB and 1 % DMSO.
Preparation of [*H]cystochalasin B
Samples of CB labelled with tritium at high specific activity were prepared and
purified by the method of Lin, Santi & Spudich (1974) in which cytochalasin A
(CA) in alcoholic solution is treated with a near-equivalent quantity of sodium
borohydride of high specific activity so that a hydrogen atom becomes tritiated
in the C-4 position of the lactone ring. A labelled product having the same RF
value as CB manufactured by I.C.I. Ltd was separated by chromatography from
any remaining CA and from undesired products such as 2,3-dihydro CB and
y-lactone which have different RF values. After purification, the mass of the
final product was estimated from the height of its ultraviolet absorption peak
at about 213 nm when dissolved in absolute ethanol using an absolute ethanol
blank. The yield of CB ranged between 17 % and 21 % based on the mass of CA
used. The specific activity of the CB product was 1-OCi/mM compared with
324
G. G. SELMAN, J. JACOB AND M. M. PERRY
a theoretical value of 1-47 Ci/mM. Subsequently in 1975, tritium-labelled CB
became commercially available.
To a dried sample of [3H]CB, dimethyl sulphoxide (DMSO) was added
followed by Holtfreter's saline to give a stock solution of 20 /^g/ml [3H]CB and
2 % DMSO in Holtfreter's saline buffered to pH 7-1.
Scintillation counting
A scintillant mixture in 2:3 2-methoxyethanol: toluene was used in glass vials.
Counting efficiencies were determined using an external source and a quench
curve prepared with quenched standards. To measure the activity in whole eggs
(after fixation in glutaraldehyde with Alcian blue followed by washing out in
three buffer rinses) each egg was dropped into a scintillation vial with 0-5 ml
Protosol (New England Nuclear); the capped vial was warmed to 50 °C for 4 h
to assist digestion of the egg and then 10 ml of scintillation fluid was added
followed by refrigeration and counting.
Processing material for sectioning
Triturus eggs were fixed by the addition of a solution of 1 % Alcian blue in
2-5 % glutaraldehyde, buffered to pH 6-5 with 0-05 M sodium cacodylate-HCl
(two changes for 1 h at room temperature). They were rinsed in three changes
of 0-05 M sodium cacodylate buffer, pH 7-2, for 12 min each and then post-fixed
for 2 h in 1 % osmium tetroxide containing 0-1 % ruthenium red with 0-03 M
cacodylate buffer, pH 7-2. The dyes Alcian blue and ruthenium red were added
to the fixatives to preserve polysaccharide material in the surface coat of the egg
outside the plasma membrane. After post-fixation the eggs were rinsed twice in
0-05 M cacodylate buffer (15 min each), dehydrated through 70% and 90%
ethanol (10 min each) and three changes of absolute alcohol (30 min each),
passed through two changes of propylene oxide, infiltrated with an EponAraldite mixture and embedded in disc-shaped blocks.
For all autoradiographic experiments the eggs were fixed at a late cleavage
stage after unpigmented surface had spread in the furrow region and the
[3H]CB had caused the furrow at the animal pole to flatten. Most eggs (e.g. B in
Fig. 11) were double fixed (as above) but some (A in Fig. 11) were fixed directly
by osmium tetroxide with ruthenium red, so that unsaturated lipids might be
better preserved. In this way possible binding sites for CB at the cell membrane
might be detected provided the washing procedures were not too severe. Certain
double-fixed eggs were subjected to more stringent conditions at the time of
fixation in glutaraldehyde-Alcian blue, in order to test the possiblity that
[3H]CB might be carried into the egg by the fixative. Three eggs (e.g. C in Fig. 11)
were washed three times in Holtfreter's saline in the 1-2 min before fixation;
two more eggs (E) were washed three times in unlabelled CB before fixation and
two more eggs (D) were washed three times in fresh fixative during the first
minute of fixation.
Cytochalasin B and cleavage
325
A utoradiography
The eggs were sectioned transversely to the plane of the cleavage furrow at
a thickness of 1 /tm. The sections were placed on glass slides and coated with
Ilford L4 emulsion by dipping. After various exposures (9-27 weeks in the main
experiment) the preparations were developed with Kodak D19B and stained with
basic fuchsin.
Autoradiographs were scanned by light microscopy at a magnification of
x 1500 using an oil-immersion objective. A rectangular area of the image space,
26 [ircv by 2-6 /.om, delineated by an eyepiece graticule, was positioned near the
animal pole of the egg with the long side of the rectangle against the egg surface
and the silver grains within this area were counted. The entire section was then
scanned by moving the area rectalinearly and counting at intervals of 13 jLtm
when moving parallel to the animal-vegetal axis and at intervals of 26 /on when
moving sideways, i.e. perpendicular to this axis. Values for the grains counted
within the defined area were plotted against distance measured from the animal
surface towards the vegetal surface and smooth curves were drawn to give a good
fit (e.g. Fig. 12). Families of such curves were then used to construct charts
which showed the variation in grain density over the whole area of the section, by
drawing lines to connect points of equal grain density (e.g. Figs. 9, 10).
RESULTS
Observation on live material
Control experiments showed that 0-5% and 1% DMSO in 1/10 strength
Holtfreter's solution had no effect on normal cleavage of the newt's egg that
could be discerned by viewing the live egg by low power microscopy.
When cleaved eggs in their vitelline membranes were placed in 2/tg/ml to
10/*g/ml CB, subsequent observation showed that the early stages of the
cleavages were normal. Thus the initial surface contraction took place in the
direction of a double line of pigmentation at the animal pole and normal transverse wrinkles could usually be seen (Selman & Perry, 1970). A cleavage groove
then formed as the egg's surface dipped in normally across the pigmented animal
hemisphere (Fig. 1 A) after which unpigmented surface started to appear at the
bottom of the groove on both sides of the line which marked its deepest extent
(Fig. 1B). Within 8 min of the first deposition of white surface in the furrow it
began to flatten beginning at the animal pole and this flattening process continued
until the furrow had disappeared (Fig. 1C). Recession of the furrow was
completed within 5-8 min. Viewed from above, the white surface appeared as
a narrow band where the furrow had been (Fig. 1D) but while the egg remained
in CB solution the width of the band progressively increased (compare Fig. 1D
with Fig. 2) and in side-view the whole animal surface of the egg was shown to be
abnormally flattened (Fig. 3). At the time second cleavage was due, nearly all
326
G. G. SELMAN, J. JACOB AND M. M. PERRY
Fig. 1. Retraction of the first cleavage furrow in the presence of 10/tg/ml CB. This
egg had been in 10/tg/ml CB since A\ h before cleavage. The egg is within its vitelline membrane. Scale 1 mm.
(A) Side-view, 13 min after the double stripe stage at the start of cleavage. The furrow
has dipped inwards across the animal surface.
(B) View from above, 17 min after the start of cleavage. New unpigmented surface
can be sesn in the furrow.
(C) Side-view, 20 min after the start of cleavage. The furrow has retracted
completely.
(D) View from above, 24 min after the start of cleavage. Unpigmented surface forms
a band where the furrow had been.
Fig. 2
Fig. 3
Fig. 2. Two eggs in their vitelline membranes viewed from above, 32 min after being
put into 10 /jg/mlCB at the start of first cleavage. Both show a considerable spread of
unpigmented white surface from the retracted furrow. Scale 1 mm.
Fig. 3. Side-view of an egg in its vitelline membrane with a white flattened surface
after retraction of the first cleavage furrow in 1 /*g/ml CB. The adjacent egg (half
shown) shows normal curvature. It is a 4-blastomere stage with no unpigmented
surface exposed and it developed normally to blastula. Scale 1 mm.
327
Cytochalasin B and cleavage
Fig. 4. Side-views of an egg placed on an agar surface with the vitelline membrane
removed, in 5 /tg/ml CB since 39 min before the start of first cleavage. Irregularity
of the egg's ventral surface is due to the roughness of the agar. Scale i mm.
(A) 15 min after the double stripe stage at the start of cleavage. The egg is at its
maximum height and shows furrowing its animal surface.
(B) 7 min after A, the furrow has retracted, the egg had flattened slightly and
white surface is exposed in the furrow region.
(C) 12 min after B, the whole egg has flattened considerably and about half the
upper surface of the egg is unpigmented.
Normal
A
10
If
\
of egg
60
o
°
\
-
^ 05 -
V
1 1st cleavage
CB
T 2nd cleavage
i
50
100
150
Time (min)
Fig. 5. The crosses show the fall in height of the egg illustrated in Fig. 4 as the
unpigmented surface spreads in the presence of CB. On the same time scale is shown
the normal rise and fall in height of another egg under normal conditions. The start
of cleavage (double-stripe stage) is arrowed and the attached line shows the duration of the furrowing process.
328
G. G. SELMAN, J. JACOB AND M. M. PERRY
the animal hemisphere was covered with unpigmented surface. Under these
conditions second cleavage did not take place, in contrast to the situation in
Xenopus eggs given similar treatment (Bluemink, 1971Z?) where second cleavage
invariably followed. For newt eggs this sequence of events has been observed
many times with different eggs and the results were found to be identical for
eggs placed in CB solution during the early cleavage stage and for eggs which had
been exposed to CB for as long as 5 h before the first cleavage began (i.e.
from soon after the time of fertilization).
When eggs within their vitelline membranes were exposed to 1 /*g/ml CB,
a flattening of the first cleavage furrow occurred in only two cases out of ten,
while the remaining eight gave normal cleavage and developed to blastulae.
The flattening of the cleavage furrow in these two cases took longer (60 and
87 min from the start of cleavage to a flat furrow) than for eggs in CB at the
higher concentrations for which flattening took 20-30 min. Therefore 2/*g/ml
was the lowest concentration of CB at which the furrow flattening was consistently obtained.
The vitelline membrane of some uncleaved eggs was removed with fine
forceps using full-strength Holtfreter's saline; this was subsequently replaced by
1/10 strength Holtfreter's saline and then by CB at a concentration of 5 /^g/ml.
One such egg was photographed alternately from above and from the side. The
side-views (e.g. Fig. 4) allowed the height of the egg during cleavage to be
estimated from measurements made on the prints. By plotting this height
against time, the greater flattening of the egg in CB was recorded (Fig. 5) and
compared with the rise and fall in height of a normal egg during the cleavage
FIGURE 6
Fig. 6. Recovery after furrow retraction. An egg in its vitelline membrane viewed
from above was put into 5 /*g/ml CB at 10 min before the start of first cleavage.
Scale 1 mm.
(A) 10 min after the start of first cleavage. The egg has formed a furrow normally in
CB.
(B) 16 min after the start of first cleavage. New unpigmented surface has started to
form in the furrow. 6 min after this photograph the CB was replaced by washing out
in 1 /10 strength Holtfreter's saline.
(C) 24 min after the start of first cleavage. The furrow has fully retracted but under
the normal conditions the unpigmented surface did not spread further (compare C
and D).
(D) 84 min after the start of first cleavage, the double stripe stage of second
cleavage is shown and the right-hand blastomere has just begun to furrow.
(E) 9 min after D. Unpigmented surface has begun to form in thee scond furrow but
furrowing has also started along the line of the former first cleavage.
(F) 11 min after D. Furrowing continues along both cleavage planes.
(G) 13 min after D. Furrows are clearly formed along both cleavage planes.
Unpigmented surface is formed normally in the second cleavage furrow.
(H) 43 min after D. The normal 4-blastomere stage after recovery from the effects
ofCB.
Cytochaiasin B and cleavage
329
Fig. 7. CB at second cleavage. The egg with its vitelline membrane removed had a normal first cleavage in 1/10 strength Holtfreter's
saline. Scale 1 mm.
(A) 83 min after the start of first cleavage. 7 min after this photograph, CB was added to a concentration of 5/*g/ml.
(B) 10 min after A. The CB has caused the first cleavage furrow to begin to regress.
(C) 7 min after B. The double stripe stage of second cleavage (first visible 3 min previously) has clearly formed on the pigmented
surface of the left-hand blastomere. The white pigmented surface from the retracted first cleavage furrow has greatly spread and
the egg continues to flatten.
(D) 7 min after C. A furrow has just managed to form on the left-hand blastomere (which alone is shown) at the pigmented edge of
the egg. The right-hand blastomere formed no furrow as unpigmented surface spread.
o
o
w
a
r
GO
O
O
o
Cytochalasin B and cleavage
331
cycle (Selman & Waddington, 1955). CB did not cause flattening of an egg
before first cleavage.
Since newt eggs continuously exposed to 2-10 /tg/ml CB during first cleavage
and afterwards do not show a second cleavage, it is important to test the reversibility of the abnormal flattening of the first furrow so that it is clear we are not
observing changes which are merely degenerative. Therefore several eggs were
exposed to 5 /*g/ml CB solution until regression of the cleavage furrow was
clearly observed and then the solution was replaced by normal 1/10 strength
Holtfreter. The effect of this was that the furrow remained flattened (Fig. 6C,
D) but the band of unpigmented surface no longer increased in width. Subsequently second cleavage took place normally beginning at the normal time.
However, 7-10 min after the second cleavage groove had dipped inwards at the
animal pole in the plane of second cleavage, the first cleavage furrow also dipped
in once more in its plane (Fig. 6E-G) and the two furrows seemed to reach
completion at the vegetal pole at about the same time to give a normal 4blastomere stage (Fig. 6H) and subsequently a normal 8-blastomere stage.
Therefore the regression of the first cleavage furrow caused by CB could be
reversed, but the manner of its reversal was unexpected.
A normal 2-blastomere stage in its vitelline membrane just before second
cleavage often had no unpigmented surface exposed to the outside on its animal
surface. Such an egg was exposed to 10 /*g/ml CB. New white surface was first
observed in the second furrow 10 min after second cleavage began and this
furrow flattened in another 10 min, while the first furrow remained unaffected.
Therefore the effect of CB on the second cleavage can resemble its effect on
first cleavage but at second cleavage the events take place more
quickly.
When third and later cleavages within the vitelline membrane were exposed to
5 /tg/ml CB there was often no effect. This may be because these cleavages
normally take place without unpigmented surface being exposed to the outside.
To make these observations, great care was required to avoid any damage or
disturbance to the eggs because the normal healing reaction seemed to be
inhibited by the CB.
It had been noticed that CB produced abnormal effects at stages when
unpigmented surface was exposed externally. Some experiments were therefore
made with newt eggs, after removal of the vitelline membrane, exposing them
to 5 /*g/ml CB at a stage just before second cleavage when a considerable amount
of unpigmented surface is exposed in the first cleavage furrow (Fig. 7 A). There
was immediate regression of the first cleavage furrow (Fig. 7B) and white
surface increased rapidly in area as the egg flattened but nevertheless a second
cleavage furrow was clearly initiated (Fig. 7C) and dipped inwards within the
pigmented part of the egg adjacent to the white area before the egg degenerated
(Fig. 7D). If the unpigmented surface is permeable to CB and the pigmented
surface is impermeable, then either the contractile elements which initiate
332
G. G. SELMAN, J. JACOB AND M. M. PERRY
7
b
•I
3
0
10
20
30
Fixation time (min after start of first cleavage)
40
Fig. 8. A graph to show the activity of single eggs plotted against their time of
fixation measured in minutes after the double-stripe stage at the start of first
cleavage.
second cleavage are insensitive to CB or alternatively the drug is transported
very slowly once it is inside the egg.
The biological activity of the samples of [3H]CB was tested by exposure of
eggs to 5/^g/ml [3H]CB. Identical furrow recession, spread of unpigmented
surface and egg flattened was observed as described above for CB from I.C.I.
Ltd. When explanted Triturus tissues were cultured with [3H]CB, the cells
showed a tendency to dissociate and tissue flexure was modified as described by
Perry, Selman & Jacob (1976).
Observations with [*H]CB on whole eggs
To measure the penetration and retention of [3H]CB uncleaved eggs were
exposed to a radioactive solution at 10/tg/ml concentration for recorded
periods of time after which some of the eggs were at first cleavage. The eggs were
rixed in glutaraldehyde, rinsed three times in buffer solution and their individual
Cytochalasin B and cleavage
333
radioactivities were then measured. The Alcian blue contained in the fixative
was in each case barely perceptible after the last rinse; thus the uniformity of the
rinsing procedure was confirmed. The radioactivity of whole eggs which had
been fixed after the appearance of new surface in the cleavage furrow was
considerably greater than that of uncleaved eggs or eggs fixed at an earlier stage
of cleavage. When the activity of single eggs was plotted against their time of
fixation measured from the appearance of the double stripe stage (Fig. 8),
a steady rise of activity was noticed, starting from about 18 min. This time
agrees fairly well with that which elapses between the double stripe stage and the
start of deposition of new unpigmented surface (12-17 min). The egg with the
greatest activity was the one with the greatest area of white surface on its
flattened animal surface at the time of fixation.
The results suggest that an appreciable proportion of the [3H]CB which
entered the eggs at mid-cleavage was retained after the initial fixation and
buffer rinses. Further tests were made to see if the activity, initially retained,
was washed out at a later stage of the process which led to embedding and
section cutting. This was checked by counting a 40 /d aliquot at each step from
the liquid containing a late cleavage egg. After the second buffer rinse following
the initial fixation the activity of the washing fluid was only just above background level. It reached background level at the osmium tetroxide with ruthenium red stage; it stayed there during the ethanol dehydration stages and there
was one barely significant rise above background for the first change of propylene
oxide. These results suggest that labelled CB is retained by the late cleavage egg
up to the embedding stage. It was thus feasible to study the distribution of [3H]CB
within the egg by autoradiography of fixed and sectioned material.
Autoradiography of sectioned material
The first autoradiographic experiments were carried out using 10/on/ml of
3
[ H]CB of very low specific activity (0-28 mCi/mM). No silver grains above
background level were found on the autoradiographs even after 9 months
exposure, but these autoradiographs clearly demonstrated that the dyes Alcian
blue and ruthenium red used in the preparation of the material did not give
rise to spurious grain counts.
On repeating the autoradiographic experiment with the [3H]CB of higher
specific activity (1 Ci/mM) in half strength Holtfreter's saline, all sectioned eggs
gave definite autoradiographic response following exposure of 9-27 weeks.
All the autoradiographs showed qualitatively similar patterns of grain distribution (Figs. 9, 10), irrespective of the type of fixation or the stringency of the
washing conditions at the time of fixation. Thus there was a high grain density
immediately below the unpigmented surface of the flattened furrow and the
density gradually diminished towards the vegetal surface of the egg. The grain
density for parts of the egg immediately below the pigmented surface also
diminished with distance measured horizontally away from the junction of
22
EMB 36
334
G. G. SELMAN, J. JACOB AND M. M. PERRY
Fig. 9 A scale drawing of an autoradiographic section cut perpendicularly to the
direction of the furrow and at the animal surface in the mid-furrow region of an egg
(Bl) fixed 35 min after the double-stripe stage offirstcleavage when the furrow had
flattened. Double fixation was employed. The original heavily-pigmented surface is
indicated by a thick line to distinguish it from the unpigmented surface indicated by
a thinner line. Over the area of the section, lines of equal grain density (at 1, 2,4 and
8 units) have been plotted to indicate the grain distribution. The unit of grain density
was 1 grain per area of 67 /*m2. Autoradiographic exposure was 12 weeks. The scale
is 100 /tm.
Fig. 10. A scale drawing of an autoradiographic section cut as described for Fig. 9
from an egg (A2) fixed directly by osmium tetroxide 27 min after the double-stripe
stage. Pigmented surface is indicated as in Fig. 9 and lines of equal grain density have
been plotted using the same unit. Autoradiographic exposure 12 weeks; scale 100//.m.
unpigmented surface with the pigmented surface, so much so in regions remote
from the retracted furrow that the grain densities reached zero.
There was no correlation between the observed densities and the time that the
eggs had been in the [3H]CB before they reached the cleavage stage. Differences in
grain density were, however, found between eggs fixed under different conditions
but subjected to the same autoradiographic exposure. These differences are
shown by a series of curves (Fig. 11) drawn by plotting grain density against
depth from the unpigmented surface of the egg in the former mid-furrow region.
Cytochalasin B and cleavage
335
These are curves of best fit and two such curves are also shown (Fig. 12)
together with the actual points through which the lines were drawn so as to
indicate the observed variation of the counts which arose partly because most of
the silver grains lay over yolk platelets which were non-randomly distributed. A
rough estimate showed that about five times as many silver grains were associated
with platelets compared to cytoplasm without platelets, although it must be
pointed out that the high density of yolk made it quite difficult to find areas,
2-6/tm square and free from platelets, for making a proper comparison.
It was evident that autoradiographs from eggs fixed directly in osmium
tetroxide-ruthenium red showed higher grain counts, by a factor 2-3, than
similar autoradiographs from double-fixed material (Fig. 11). However, this
direct method of fixation was not entirely satisfactory even for light microscopy
because small blisters and cracks appeared on the egg surface. The higher grain
counts observed after direct fixation were not due to local increases round
cracked regions. In all double-fixed material preservation was excellent and
autoradiographs from such material subjected to washing routines of greater
stringency showed grain counts lower by a factor of about one half (compare
eggs C-E with B^Bg in Fig. 4). However, in view of the variation found between
Bl5 B2 and B3 which were similarly treated, the differences between C, D and E
are probably not significant. The finding that eggs C and E retained substantial
radioactivity (about half that of eggs B) in spite of their removal from radioactive
medium before fixation indicates that at least half the activity found in eggs B
could not have been carried in at the time of fixation. In any case, there is no
reason to suppose that any radioactivity was carried into an egg with the
fixative across the pigmented surface, because no silver grains were found
below the pigmented surface remote from the furrow.
In these experiments no radioactivity was found associated with the surface
layers of the egg which could be distinguished from radioactivity associated
with small yolk platelets situated immediately below the surface of the
egg. No residual furrow with associated filamentous material was found in
these eggs when they were examined by light and electron microscopy, probably
because they were fixed at a time just after such structures had dispersed
following treatment with CB.
DISCUSSION
These observations showed that CB had no effect on newt eggs before first
cleavage or at early furrow formation, but soon after the new unpigmented
surface was first laid down, the drug caused the furrow to retract. This is consistent with the idea that only the unpigmented surface, laid down in the furrow at
mid-cleavage is permeable to CB while the surface external to the egg before
cleavage is impermeable to CB. This idea is supported by the results obtained
using tritium-labelled CB, both from scintillation counting of whole eggs
and autoradiography of sections. Indeed the charts (Figs. 9,10) which show the
336
G. G. SELMAN, J. JACOB AND M. M. PERRY
6-
o
100
200
Depth below surface at mid-furrow (ftm)
300
Fig. 11. Graphs to show the variation of autoradiographic grain density with depth
below the animal surface of individual eggs fixed and washed in various ways. The
curves are drawn from counts made on autoradiographs like those of Figs. 9 and 10.
Eggs Ai and A2 were fixed directly in osmium tetroxide; eggs Bl5 B2 and B3 were
double fixed; egg C was washed three times in Holtfreter's saline before glutaraldehyde fixation: egg D was washed three times in fresh glutaraldehyde at fixation and
egg E was washed three times in unlabelled CB just before glutaraldehyde fixation.
The autoradiographic exposure was 15 weeks in each case.
distribution of radioactivity in the cleavage stage eggs at the time of fixation can
be regarded as a representation of the relative concentrations of CB that have
entered the egg through the permeable surface from the medium outside. The
flow of CB fans out somewhat once it is inside the egg. In papers which describe
microinjection experiments, De Laat et al. (1973) and Luchtel, Bluemink &
De Laat (1976) conclude that externally-applied CB can only enter the Xenopus
Cytochalasin B and cleavage
337
100
200
300
Depth below surface at mid-furrow
Fig. 12. Two of the curves from Fig. 11 for eggs Ax and Bi are reproduced together
with the actual values for grain density from the individual grain counts, so that the
the variation can be seen.
egg when its permeability increases at mid-cleavage, but Bluemink (1971Z?) had
earlier rejected the idea because in Xenopus continously exposed to CB the drug
did not block the contractile phase of second cleavage after it had affected
first cleavage.
When a stage is reached at which CB can enter the newt egg through the new
surface, the observations on furrow retraction are consistent with the working
hypothesis that CB inhibits a contractile function based upon the band of
338
G. G. SELMAN, J. JACOB AND M. M. PERRY
microfilaments which Selman & Perry (1970) found immediately below new
surface in the lowest part of the furrow.
After furrow retraction, CB caused a steady spread of a band of unpigmented
surface on the animal hemisphere of the egg. Bluemink (1971a) suggested the
spreading may be looked upon as a normal growth of surface which, in the
circumstance of furrow derrangement caused by CB, takes place on the top of
the egg instead of in its normal position in the interblastomeric region of the
furrow. However, it is difficult to reconcile this idea with the observation that, on
returning the egg to normal solution, the spreading of unpigmented surface
immediately stopped. It seems simpler and more consistent with observation to
assume that CB acts directly on the newly-assembled cell surface to weaken and
so allow it to be stretched by the weight of the egg that the whole egg becomes
flattened.
For Xenopus, Bluemink (1971c) has suggested that CB interferes with the
formation of adherent cell junctions between the daughter blastomeres so that
the new membrane grows outward instead of inwards. This argument does not
seem applicable to the first cleavage of newt, with or without the vitelline
membrane, or to Xenopus without the vitelline membrane, where cleavage takes
place with new surface forming first at the bottom of an open groove and with
the junctions seemingly quite ineffective. In newt, close interblastomeric junctions are derived from ridges which only come together after the furrow is complete (Selman & Perry, 1970).
In cleavage of the amphibian egg, the contractile phase which causes the
furrow to dip inwards is always preceded by a double stripe stage in which two
parallel lines of pigment cross the animal surface where the furrow will form
(Fig. 6D and Bluemink, 1970). When newt eggs are continuously exposed to CB,
the band of unpigmented surface which formed at first cleavage has so expanded
in area by the time of second cleavage that most of the egg's upper surface is
unpigmented so that a double stripe cannot form in it. The formation of the
double stripe may be associated with the assembly of the contractile band of
microfilaments which normally takes place in cell cortex protected from CB by
impermeable surface. This view is supported by the observation of parallel
microfilaments in the region of the double stripe by Bluemink (1970) for
Ambystoma and Perry (unpublished observations) for Triturus. In Xenopus
where the interval between cleavages is only one third as long as in T. alpestris,
the unpigmented surface can expand to cover only a small part of the animal
hemisphere before second cleavage is due. In this case there is plenty of impermeable pigmented surface for second cleavage to be initiated by a double stripe
stage so that groove formation may follow. Remarkably the present observations
on the newt egg show that a regressed first cleavage furrow can, in the absence of
CB, be reinitiated only at the time of second cleavage by the influence of the
second furrow so that the first furrow also dips inwards in its former position
and then grows normally through the egg to the vegetal surface.
Cytochalasin B and cleavage
339
When the effects of CB on the cleavage of amphibian eggs are considered, the
impermeability of the pigmented surface is an entirely sufficient explanation for
the observation that high concentrations of CB do not prevent the initial furrow
formation. Furthermore Luchtel et ah (1976) showed that microinjection of CB
into Xenopus eggs can derange and inactivate the microfilmentous system which
causes the initial furrowing, although normally the intact pigment surface protects
it. The reports of Schroeder (1970, 1972), using Arbacia eggs and HeLa cells,
that CB can entirely suppress furrowing, may be explained if these materials
show a sufficiently high permeability to CB before the stage of furrow formation.
It is not known if the kind of explanation proposed here for amphibian eggs
could be extended to cleavage in other forms, for which CB does not prevent
the initial furrowing.
From the charts showing the distribution of radioactivity in sectioned eggs
(Figs. 9,10) it is possible to estimate roughly the rate at which the concentration
of [3H]CB decreases with distance measured horizontally under the pigmented
surface from its border with the unpigmented area. The concentration is reduced
to 50 % in a distance of 50-150 jtim (say 100 /mi), i.e. to about 17 % in 250 /im.
This could be just sufficient to explain the cases in which 5 /^g/ml CB fails to
prevent the contractile phase of second cleavage when it occurs about 250 /im
from the edge of the unpigmented surface of the first cleavage furrow (Fig. 7 and
Bluemink, 1971 b) and the time of exposure to CB is simliar.
These findings which show the dividing egg to have two surfaces with dissimilar
permeability properties, do run in concordance with former ideas about the
general impermeability of the outer pigmented surface of amphibian eggs and
embryos (Holtfreter, 1943) as well as with the measurements of Takahashi &
Ito (1968) and De Laat & Bluemink (1974) for example which show that new
surface has a higher ionic permeability than pre-existing cell surface.
Autoradiographic studies are commonly made to study the biosynthesis of
macromolecules which are precipitated by the fixative, whereas the excess of
labelled precursor molecule is washed out at fixation. However, in the present
case the fixatives are thought to act differently. They probably change a situation
in which [3H]CB is loosely associated with a variety of organelles and inclusions
in the cytoplasm of the living cell to a state in which a proportion of the molecules are stably bound by fixation so that they are not washed out in the solutions
employed before embedding. However, it is not easy to explain why the sectioned
Triturus eggs after direct fixation should have twice the activity of sections
observed after double fixation. In this connexion some thought was given to the
possible role of small breaks in the egg surface which are produced after direct
fixation and are first observed after the osmium tetroxide-ruthenium red has
been washed out. However, the autoradiographic results gave no support to this
idea; the grain distribution pattern did not alter with the method of fixation. CB
is lipid-soluble and lipids are widely distributed in organelles and inclusions,
including the yolk platelets (Wallace, 1963). It is known that osmium tetroxide
340
G. G. SELMAN, J. JACOB AND M. M. PERRY
generally gives better preservation of lipids than glutaraldehyde alone, but
Williams (1969) quotes figures for lipid retention in double-fixed material which
are similar to those for osmium tetroxide alone. In this work both kinds of
fixation preserved lipid droplets but they had little associated radioactivity and
there was little evidence for extraction of labelled material after double fixation.
We thank Professor P. Chibon of Grenoble University for sending newts. We are indebted
to Dr D. C. Aldridge of I.C.I. Ltd, for small samples of 2,3-dihydro CB and y-lactone and
to Dr S. B. Carter, also of I.C.I. Ltd, who kindly supplied a sample of tritium-labelled CB
of low specific activity. We thank Dr D. C. Warrell of the Radiochemical Centre, Amersham
and Dr D. Leaver of the Chemistry Department, University of Edinburgh for helpful advice
about the borohydride reaction. This work was supported by a grant from the Medical
Research Council.
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