J. Embryol. exp. Morph. Vol. 61, pp. 331-345, 1981
Printed in Great Britain © Company of Biologists Limited 1981
331
Morphogenetic analysis of changing cell associations
following release of 2-cell and 4-cell mouse
embryos from cleavage arrest
By S. J. KIMBER 1 AND M. A. H. SURANI 1
From the A.R.C. Institute of Animal Physiology,
Cambridge
SUMMARY
Two-cell and four-cell mouse embryos were cultured in Cytochalasin D (CD) for 40-48 h.
They were fixed for light and electron microscopy at various times after washing off the
CD. Cleavage-arrested embiyos in CD had well separated blastomeres but by 1 h from
washing the embryos had compacted, in most cases without undergoing cell division. By 2 h
after release from arrest one blastomere of the 2-cell arrested embryos had become crescent
shaped and at 4-5 h the crescent-shaped blastomere had started to spread over the surface
of the other rounded blastomere. This process continued until by 16-24 h from explantation
to fresh medium one blastomere had almost completely engulfed the other. A similar
process occurred in 4-cell arrested and released embryos. At this stage the embryos had
accumulated fluid and become blastocyst-like vesicles. In 20% of 2-cell and 4-cell embryos
one or two blastomeres underwent one cell division after release from arrest. Serial sections
of these embryos lead to the conclusion that one or both progeny of the first cell to divide
tended to be engulfed by the later dividing or non-dividing cell(s). These results are discussed
in relation to the differentiation of ICM and trophectoderm in blastocysts.
INTRODUCTION
After three to four mitotic divisions early mouse embryos undergo compaction with the formation of tight and gap junctions between blastomeres
(Ducibella & Anderson, 1975; Ducibella, Albertini, Anderson & Biggers, 1975;
Magnuson, Demsey & Stackpole, 1977). Changes in the cell surface (Calarco
& Epstein, 1973; Edidin, 1976) and in the distribution of microtubules and
microfilaments (Ducibella & Anderson, 1975; Ducibella, Ukena, Karnovsky
& Anderson, 1977) also occur. After the 32-cell stage, the embryo begins to
accumulate fluid becoming a blastocyst with the formation of the inner cell
mass (ICM) and outer trophectoderm.
Compaction and blastulation are Ca2+ dependent (Ducibella & Anderson,
1975, 1979) and can be prevented by inhibitors affecting the cytoskeleton
(Granholm, Brenner & Rector, 1979; Surani, Barton & Burling, 1980) and
protein glycosylation (Surani, 1979) and also by antibodies against cell surface
1
Author's address: A.R.C. Institute of Animal Physiology, 307, Huntingdon Road,
Cambridge, CB3 0JQ, U.K.
332
S. J. KIMBER AND M. A. H. SURANI
molecules (Kemler, Babinet, Eisen & Jacob, 1977; Ducibella, 1979; Johnson
et at. 1979).
Cytochalasin D (CD) is believed to exert its effect on cells primarily by
inhibiting the polymerization of microfilament actin (Bray, 1979; Brown &
Spudich, 1979; Lin & Lin, 1979; Lin, Tobin, Grumet & Lin, 1980). Cytochalasin
allows chromosomal replication to proceed while arresting cytoplasmic division.
It can therefore be used for the examination of processes of differentiation
which depend on developmental age, nuclear:cytoplasmic ratio and number
of chromosome replications, but which do not depend on number of cell
divisions or cell interactions during early cleavage. The early differentiation
of mouse embryos in the formation of the blastocyst seems to be such
a process (Tarkowski & Wroblewska, 1967; Snow, 1973; Smith & McLaren,
1977; Granholm et at. 1979; Surani et at. 1980). Experiments using the
cytochalasins on the embryos of ascidians (Whittaker, 1973, 1979; Satoh,
1979), amphibians (Cooke, 1973), nematodes (Laufer, Bazzicalupo & Wood,
1980) and mammals (Surani et at. 1980) indicate that cytoplasmic differentiation
and morphogenesis can proceed in the absence of cell division. Thus cleavagearrested embryos provide us with a simplified model with which to investigate
the influence of cell interactions and cell position in development.
We have previously reported the formation of blastocyst-like structures by
2-cell mouse embryos released from arrest in CD (Surani et al. 1980). In this
study we have carried out a cytological examination of cleavage-arrested
embryos following their release from arrest and during their development into
blastocyst-like structures. Our observations reveal rapid changes in cellular
interactions and provide further evidence for the probable mechanism of
formation of the ICM and trophectoderm.
MATERIALS AND METHODS
Animals
Embryos were obtained from an outbred strain of CFLP mice (Anglia
Laboratories Ltd. U.K.) which were superovulated using 5 i.u. pregnant mare's
serum (PMS) followed 42-48 h later by 5 i.u. human chorionic gonadotrophin
(HCG) (Intervet, Milton). One or two females (approximately 6 weeks old)
were caged with each F x male (C57BL/CBA) and checked the following
morning for copulation plugs. The day of the vaginal plug was counted as
day 1 of pregnancy.
Recovery of embryos
Both 2-cell and 4-cell mouse embryos were flushed from the oviducts between
2 and 5 p.m. on day 2 of pregnancy about 45-48 h post HCG. All 2-cell and
all 4-cell embryos were pooled and divided between CD-containing and control
medium.
Morphogenesis of embryos released from cleavage arrest
333
Culture medium
Embryos were cultured in Brinster's medium (Brinster, 1970) to which
4 mg/ml bovine serum albumen was added.
A stock solution of CD (Sigma, London) was prepared in 100% dimethyl
sulphoxide at 2 mg/ml and stored at - 20 °C. Two-cell embryos were incubated
in Brinster's medium containing 0-5 or 1-0/tg/mlCD for 19-21 or 44-48 h
after which time they were approximately 68 or 90 h post HCG. By these
times control embryos were uncompacted 8-cell stage and fully compacted
morulae to early blastocysts respectively. Four-cell embryos were incubated
in medium containing l-0/*g/ml CD for 41 h at which time control embryos
were early blastocysts (about 90 h post HCG).
At the end of the period in CD a group of embryos was removed from
both the control and experimental groups for immediate fixation. The remaining
embryos in CD-containing medium were washed ten times in fresh Brinster's
medium and groups were fixed after 0-25-0-5 h, 1-2 h, 4-5 h and 24 h (2-cell
arrested) or 2-5 h, 7 h and 24 h (4-cell arrested). A few control embryos were
fixed at the same time as each experimental group.
Preparation for light and electron microscopy
Embryos were fixed in 3 % glutaraldehyde, 0-5% paraformaldehyde in
0-05% sodium cacodylate buffer or 0-1 M phosphate buffer (pH 7-3). The
osmolarity of the buffer was corrected to 270-290 m-osmole with sucrose.
Embryos were post fixed in 1 % OsO4, block stained with 0-5 or 1 % uranyl
acetate, dehydrated in a graded series of ethanols and embedded in Spurr's
(1969) medium. Serial sections, 0-5 and 1-0 [im thick, were cut on an LKB or
Reichert ultramicrotome and stained with 1 % toluidine blue for light microscopy. Sections 50-100 nm thick were stained with a saturated solution of
uranyl acetate in 50% ethanol followed by lead citrate (Reynolds, 1963). Thick
sections were viewed and photographed using a Zeiss microscope and camera
attachment. Thin sections were examined in a Philips EM300 or an AEI
electron microscope.
RESULTS
Observations made on 2-cell cytochalasin-arrested and released embryos are
summarized in Table 1. Cytochalasin-arrested embryos have well separated
blastomeres (Fig. 1) and most become binucleate (Snow, 1973; Surani et ah
1980) as has been found for other cytochalasin-arrested cells (e.g. Carter,
1967). The nuclei of cytochalasin-arrested blastomeres have a much greater
volume than those of normal blastomeres at the 2-cell and 4-cell stage. They
are commonly oval in shape with usually four darkly staining spheroidal
nucleoli.
Figure 2 shows a 2-cell embryo fixed 1 h after the start of washing in Brinster's
65
44-48
Time in
CD(h)
114-118
24
110
12
106-110
16
13
JO
27
95-99
90-94
4-5
1-2
0
Nos.
embryos
Incubation
observed
time after
Age at
at
washing fixation post particular
(h)
HCG (h)
stage
Two well separated rounded blastomeres,
binucleate, commonly with 3 or 4 condensed
nucleoli. Microvilli in patches
Adjacent cell membranes of two blastomeres
closer together, reduction in intercellular
space. One blastomere particularly flattened
on surface adjacent to second
Embryos compacted. Junctions between
adjacent blastomeres. One blastomere
beginning to become crescent shaped and
send out processes round second
(i) One of the two blastomeres extends
round the second with an increase in
area of cell contact. Second blastomere
remains rounded while being engulfed
by first
(ii) Blastomeres divided: 3 or 4 cells, 1 or
2 remain rounded, others spread over
outside to surround them
One cell completely or almost completely
engulfed by others(s). Vacuoles present in
cytoplasm. Presence of extracellular cavity
in some embryos
Embryo formed vesicle. One cell often
completely engulfed and present in inside
position when cells had divided. Some
vesicles expanded
50% formed irregular vesicles with cells
containing fluid filled vacuoles
Appearance
Expanded blastocysts
Early blastocysts
Late compacted morulae
Appearance of concurrent
controls
Table 1. Morphology of cytochalasin D-arrested and released 2-cell embryos from plastic-embedded and sectioned material
25
c
as
>
2
S
m
OJ
Morphogenesis of embryos released from cleavage arrest
335
medium following 44-48 h in CD (90-94 h post HCG). The blastomeres are
found to be in much closer association than in the CD-arrested 2-cell embryos.
By 1-2 h after explantation to fresh medium embryos viewed in the dissecting
microscope showed a morphological appearance similar to normal compacted
embryos.
At an early stage after washing off the cytochalasin, adhaerens junctions
and possible gap junctions and focal tight junctions were found between
blastomeres and they continued to be found at all subsequent stages (Fig. 6).
Two hours after release from CD arrest one of the two blastomeres had
invariably become slightly crescent shaped. At 4-5 h after washing the crescentshaped blastomere had extended cytoplasmic processes around the adjacent
second blastomere (Fig. 5). The second blastomere remained rounded. By this
stage the area of contact between the two cells had increased due to the
spreading of one blastomere over the surface of the second. The number of
presumptive focal tight and gap junctions had correspondingly increased.
Between 5 and 16 h after release from CD arrest vacuoles appeared in the
cytoplasm of one or both blastomeres. The cytoplasmic processes of one
blastomere continue to extend around the second so that 16—24 h after the
start of washing one blastomere was sometimes almost totally engulfed by
the other (Fig. 3). By 16 h after washing an extracellular cavity was found
within the embryos which increased in size after 24 h and the washed vesiculated
embryos resembled blastocysts (Fig. 4). They still contained only two cells in
many cases (see below) and neither cell was solely restricted to the inside of
the vesicle.
An understanding of the process of engulfment and vesicle formation was
arrived at by the examination of a number of embryos at different stages in
their formation (Table 1). The number of embryos which showed various
stages in the process of engulfment (recorded in the table) amounted to 90%
of those sectioned between 1 and 16 h after release from arrest.
Most of the 2-cell embryos washed after 44-48 h cytochalasin D incubation
did not undergo cytoplasmic division. However, in about 20 % of the embryos
one or both of the blastomeres divided once and in these embryos also some
blastomeres were engulfed by others (Fig. 7). In 11 of the embryos between
1 and \6 h from the start of washing some cells were arrested at metaphase or
had divided following release from arrest. In seven of these cases serial semithin sections indicated that one or both of the progeny of the first cell to divide
tended to be engulfed by the progeny of the later dividing cell, or by the cell
which did not divide (Fig. 9). In two of the remaining embryos both of the
2-cell blastomeres had divided whilst in the other two engulfment had not
proceeded far enough. In vesicles formed from CD-arrested embryos where
cell divisions did occur after explanting to fresh medium an inside cell was
often found (Fig. 8). Probable gap and focal tight junctions were found between
the cells forming the walls of the vesicles.
336
S. J. KIMBER AND M. A. H. SURANI
MV
MV
•fc*
10 pm
10pm
3
10 urn
Morphogenesis of embryos released from cleavage arrest
337
After embryos were incubated in CD for 41 h from the 4-cell stage they
underwent similar morphological processes on release from CD as did 2-cell
arrested embryos (Table 2). By 2-5 h from the onset of washing the embryos
had compacted (Fig. 10) and one cell was commonly placed more centrally
with the others spread over much of its surface. In embryos fixed 7 h after
washing the outer blastomeres had the crescent shape seen in 2-cell embryos
after washing off CD (Figs 11, 12). Their shape v/as less attenuated than in
the 4-5 h and 16 h washed 2-cells. This is because in the 4-cell arrested and
washed embryos there were three or more cells to cover the surface of the
inner one. In some of the embryos at this stage one or two of the cells had
divided or were in the process of doing so (Fig. 11). Again it seems that one
or both of the progeny of the dividing cell tended to take up a more central
position. Even at 7 h from release some embryos contained a small extracellular cavity (Fig. 1) and by 24 h the embryos were uniformly expanded
vesicles.
In a further experiment, 2-cell and 4-cell embryos were incubated in Cytochalasin D for 65 h (~ 110-115 h post HCG). Tov/ards the end of the period
in CD-containing medium they started to pump fluid and formed irregular
vesicles with large fluid-containing vacuoles in the cytoplasm.
DISCUSSION
Biochemical similarities have been found between 2-cell cleavage-arrested
embryos and their concurrent controls (Surani et al. 1980; Pratt, Chakraborty
& Surani, 1981). After embryos are released from CD arrest they compact
rapidly, some blastomeres show active spreading over the surface of others and
junctions are formed before the embryos become blastocyst-like vesicles. Thus
the programme of morphogenesis is able to catch up with that of biochemical
differentiation after the two have become out of phase.
Based on previous proposals (Steinberg, 1964, 1970; Sternfeld, 1979) a
blastomere which is engulfed would be expected to have a more adhesive
surface over which less adhesive blastomeres could spread. Since there is a
FIGURES
1-4
Fig. 1. Two-cell embryo arrested in cytochalasin D for 44 h. The blastomeres are
not in close contact. MV, microvilli; N, nucleus; Z, zona pellucida.
Fig. 2. Two-cell embryo arrested in CD for 44 h and incubated for 1 h in fresh
medium. The cells adhere closely. MV, patch of microvilli.
Fig. 3. Two-cell embryo arrested in CD for 44 h and incubated for 16 h in fresh
medium. Note the cell process (P) of the outer cell (OC) which is extended around
the inner cell (1C). The inner cell is pumping fluid to form a vesicle.
Fig. 4. Two-cell embryo arrested in CD for 44 h and incubated for 24 h in fresh
medium. The embryo has formed an expanded vesicle which was indented during
preparation for microscopy. C, cavity.
338
S. J. KIMBER AND M. A. H. SURANI
"- I **A
10 Mm
Morphogenesis of embryos released from cleavage arrest
339
critical period in development after which compaction cannot occur (Johnson
et al. 1979) it is probable that cytoplasmic differentiation proceeds to the phase
of increased cell adhesion over a defined period. Differentiation may reach the
'adhesive membrane' phase in the prospective inner cell of the CD arrested
and released 2-cell embryo in advance of the prospective outer cell.
It appears that the cell spreading seen in arrested and released embryos is
an exaggerated form of a process found in normal embryos in which, due to
a change in cell-cell adhesion, the outer cells of the normal compacting morula
spread on one another (Ducibella & Anderson, 1975; Ducibella et al. 1977). As
in the spreading shown here, spreading of the outer cells of normal compacting
embryos involves a redistribution of microvilli (Calarco & Epstein, 1973;
Ducibella & Anderson, 1975; Ducibella et al 1977) which may be involved in
adhesion as in other systems (Lin & Wallach, 1974; Ukena & Karnovsky,
1976). Inhibition of the synthesis of glycoproteins, v/hich presumably mediate
cell adhesion, has been shown to prevent compaction, and blastocyst formation
(Surani & Kimber, 1981; Surani, Kimber & Handyside, in preparation). A coordinate response from the underlying cytoskeleton must also be involved in
the spreading process (Ducibella et al. 1977).
Differences in the adhesive properties and cytoskeletal mobilization between
cells may also be responsible for the relocation of isolated inner cell masses
(ECM) and embryonal carcinoma (ec) cells predominantly towards the inside
of the embryo when these cells are aggregated with 8-cell embryos (Rossant,
1975; Stewart, 1980; Stewart & Kimber, unpublished). In analogy with CDarrest embryos, normal 8-cell blastomeres send out cell processes to engulf
ec cells (Stewart, 1980; Kimber & Stewart, unpublished). Small cell size does
not appear to determine that a cell is engulfed since in 2-cell arrested embryos
both cells are approximately equal in size, and not all types of ec cell are
engulfed (Stewart, 1980).
F I G U R E S 5-8
Fig. 5. Electron micrograph showing an early stage in the spreading of one blastomere over the second. Two-cell embryo arrested in CD for 44 h and incubated
for 4 h in fresh medium. MV, microvilli; P, cell process.
Fig. 6. Electron micrograph showing probable gap junction (GJ) as well as
adhaerens junctions (AJ) between blastomeres of a 2-cell embryo after 44 h in
CD followed by 23 h in fresh medium.
Fig. 7. Two-cell embryo which has undergone cell division after being explanted
from CD to fresh medium. The outer cell (OC) is in the process of division while
the inner cell (IC) is one of the progeny of the earlier dividing blastomere. 48 h CD
arrest 16 h in fresh medium. P, process of outer cell.
Fig. 8. Electron micrograph showing part of a vesicle formed after 44 h arrest
in CD from the 2-cell stage and 24 h after explanting to fresh medium. The cells
divided after washing and one cell (IC) is occupying a position inside the vesicle.
C, cavity.
340
S. J. KIMBER AND M. A. H. SURANI
• /'Si
10
10 pm
9
10
•Am*..
12
Morphogenesis of embryos released from cleavage arrest
341
Cell division occurs in about 20% of embryos released from CD arrest.
Sections of seven embryos indicated that the earlier dividing cell(s) tended to
be engulfed by the later or non-dividing cell(s). The engulfed cells became
localized on the inside in a similar position in the vesicle to the ICM of a normal
blastocyst. It is relevant to note that in normal embryos the first blastomere
to divide tends to contribute more cells to the ICM than later dividing blastomeres (Barlow, Owen & Graham, 1972; Kelly, Mulnard & Graham, 1978).
It has been shown that whether cells become ICM cells or trophectoderm
probably depends partly on cell position (Tarkowski & Wroblewska, 1967;
Hillman, Sherman & Graham, 1972; Rossant, 1975) and partly on their interaction with other cells (Barlow et al. 1972; Graham & Deussen, 1978; Kelly
et al. 1978; Graham & Lehtonen, 1979) at the time of compaction. In this
respect we have previously shown that vesicles formed after prolonged cleavage
arrest in CD give rise only to trophectoderm derivatives (Surani et al. 1980).
However, such vesicles synthesise all the polypeptides detected in intact blastocysts (Pratt, Chakraborty & Surani, 1981) indicating that some or all the
arrested blastomeres synthesise polypeptide detected only in the ICM of
normal embryos. The intracellular polarity of blastomeres observed at the
8-cell stage (Ducibella et al. 1977; Johnson, Pratt & Handyside, 1981) led to
one explanation of this. In the absence of compaction and the development
of intercellular communication it is suggested that each blastomere recreates
the radial polarity of an entire embryo and therefore expresses properties of
both ICM and trophectoderm (Johnson et al. 1981). Alternatively we suggest
that, irrespective of polarization, each blastomere may be developmentally
programmed to produce both sets of polypeptides as is probably the case in
the cleavage-arrested embryos. The normal divergence of biochemical differentiation in the embryonic cell populations may only occur after compaction
due to the influence of microenvironment and cell contact over a period of
hours and depending on cell position. Definitive ICM and trophectoderm
would then be formed.
FIGURES
9-12
Fig. 9. A three-cell embryo resulting from the division of one blastomere of a
2-cell embryo. 48 h arrest in CD 16 h in fresh medium. A and B, progeny of the
divided cell.
Fig. 10. A four-cell embryo arrested for 41 h in CD followed by 1\ h in fresh
medium. The inner cell (IC) is in the process of cell division.
Fig. 11. Four-cell embryos arrested for 41 h in CD and explanted to fresh medium
for 2\ h. In the embryo on the left cell division is underway in one blastomere
(arrowed). The orientation of the cleavage furrow appears to determine that one
of the progeny cells will end up inside the embryo.
Fig. 12. Embryo arrested for 41 h in CD from the 4-cell stage and incubated for
1\ h in fresh medium. One cell (IC) is surrounded by the other cells. A cavity (C)
has just started to appear.
65
41
8
8
8
97
114
115
7
24
0
6
—
2*
12
90
0
Nos.
Incubation
embryos
time after
Age at observed at
Time in washing fixation post particular
CD(h)
(h)
HCG(h)
stage
Compacted 4-6 cells, one cell often
almost surrounded by others
(i) Outer blastomeres crescent shape
surrounding inner blastomere
(ii) Some embryos with small extracellular cavity
Expanded vesicles
Irregular vesicles
Four well separated blastomeres, binucleate. Densely staining spheroidal
nucleoli
Appearance
Fully expanded blastocysts
Early blastocysts
Appearance of concurrent
controls
Table 2. Morphology of cytochalasin D-arrested and released 4-cell embryos from plastic-embedded and sectioned material
73
G
x
>
a
2
P
to
Morphogenesis of embryos released from cleavage arrest
343
We therefore consider that the cell spreading seen after CD treatment and
subsequent release is an exaggerated form of the cell behaviour found in the
normal process of compaction and ICM formation. This suggests that division
order, differential cell adhesiveness and directional deployment of the cytoskeleton are the central process in differentiation of ICM and trophectoderm.
The last two may perhaps be the motive forces behind these processes. Cell
position is presumably dependent on these factors and probably determines
the subsequent differentiation of the cell types.
We would like to thank Mrs Sheila Barton for her expert help during the course of this
work. S.J.K. is in receipt of a Training Fellowship from the Medical Research Council.
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(Received 24 June 1980, revised 18 September 1980)