/ . Embryol. exp. Morph. Vol. 31, 1, pp. 229-234,1974
Printed in Great Britain
229
Size regulation in chimaeric mouse embryos
By MIA BUEHR AND ANNE McLAREN 1
From the Department of Genetics and the Agricultural Research Council Unit
of Animal Genetics, University of Edinburgh
SUMMARY
The size of the inner cell mass and egg cylinder was estimated in mouse embryos derived
from the aggregation of two 8-cell stages and in control 'single' embryos. The comparison
suggests that growth regulation in the 'double' embryos takes place fairly rapidly, after the
formation of the egg cylinder but before the appearance of the pro-amniotic cavity.
INTRODUCTION
When mouse embryos at early stages are aggregated and cultured in vitro, they
form a morula and, eventually, a blastocyst which is roughly proportional in
size to the number of embryos which contributed to it (Tarkowski, 1961; Mintz,
1965). Cell counts have shown that blastocysts derived from two 8-cell stages
contain twice the number of cells of normal blastocysts (Bowman & McLaren,
unpublished observations). However, when such a blastocyst is transferred to
a 'foster-mother' and allowed to develop, the chimaeric embryo derived from
it is normal in size when examined at 16y days (Bowman & McLaren, 1970) or
at 10-14 days post coitum (Tarkowski, 1961). The developing embryo evidently
undergoes some rather precise process of size regulation. No published data
exist as to when or how this process occurs, though Mintz (1970) states that
regulation takes place 'soon after implantation'. The same author (1971)
suggests that 'the basis for this size regulation may lie in origin of the embryo
itself from a small fixed number of cells (possibly as few as three) in the inner
cell mass, regardless of the total cell number in the blastocyst'. It is the purpose
of this study to investigate more fully the phenomenon of size regulation in
chimaeric mice.
MATERIALS AND METHODS
All mice used were of the random-bred Q strain. The techniques used to
aggregate and culture embryos are described in Bowman & McLaren (1970).
Most of the embryos (the exceptions are noted as such in the results) were
cultured free of the zona pellucida for 45-50 h as 'double' embryos, made from
two aggregated 8-cell stages, or 'single' embryos, from one 8-cell stage. At a
1
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh EH9 3JN,
U.K.
230
M. BUEHR AND A. McLAREN
Table 1. Criteria used to 'stage' embryos
Stage
Mean
gestational
age (days p.c.)
1
2
Cultured
Cultured
3
46
4
5-5
5
5-8
6-6
6-9
7-6
6
7
8
Description
Blastocyst, endoderm differentiation beginning
Egg cylinder formation beginning, some distal
endoderm
Egg cylinder partly formed, proximal endoderm
cuboidal and vacuolated
Egg cylinder fully formed, embryonic ectoderm
distinct from extra-embryonic
Pro-amniotic cavity beginning to form
Cavity extending into extra-embryonic ectoderm
Exocoelom beginning to form
Allantois seen
late morula or blastocyst stage they were transferred to the uteri of pseudopregnant females, each female receiving 'double' embryos in one uterine horn,
and' single' embryos in the other. This design was adopted because the increased
precision of within-female comparisons was judged to outweigh the greater
variance introduced by the occasional migration of embryos between uterine
horns (McLaren & Michie, 1954).
The recipient females (after injection of Pontamine sky blue to show implantation sites) were killed at various times post coitum (p.c.) and the uteri dissected
out and fixed in Bouin's fluid. Implantation sites were cut out, embedded in
paraffin, serially sectioned at 8 or 10 (ivn, and stained with haematoxylin and
eosin. Nine 'double' and three 'single' embryos appeared structurally abnormal
when examined under the microscope and were discarded. Some 'double'
and 'single' blastocysts were fixed in Bouin's fluid immediately after culture
developmental stages 1 and 2 in Table 2). These were embedded in agar, and
subsequently treated as the other material.
The volume of the inner cell mass or egg cylinder (according to stage) of each
specimen was estimated by microscopic examination with a graticule fitted with
a modified Chalkley grid. The use of this grid as a method of volume estimation
is described by Curtis (1960). On some early stages, cell counts of the inner cell
mass were also made. Trophoblast and its derivatives were not included in the
measurements. The volume of each embryo was estimated three times; the mean
volume was used in the final calculations.
When the embryos were examined it became obvious that the gestational age
of the foster-mother was not always a reliable indicator of an embryo's stage
of development; i.e. embryos from different foster-mothers of the same gestational age were often at different stages of development. A sequence of developmental stages was therefore drawn up (Table 1) which made it possible to
1
2
3
3
4
5
5
5
5J6~
6
7
8
—
—
4-7
4-7*
5-4
5-7
5-7
5-9*
60*
6-7
6-7*
7-7
7-7
1
1
4t
1
4t
3f
4
5
3
2
1
2
1
No. of
embryos
9-5
20-4
111-3
—
—
—
—
—
—
—
—
—
—
Mean
cell no.
'Single'
1-39
2-40
9-53
114
22-4
1611
119-8
114-3
2343
764-5
6900
2014-8
17562-4
Mean vol.
(/tm 3 xl0 4 )
0
8
3
3
3
2
2
3
2
1
1
1
1
No. of
embryos
—
47-3
243-7
—
—
—
—
—
—
—
—
—
—
Mean
cell no.
'Double'
—
5-95
20-6
20-6
89-7
148-2
174-3
140-5
237-7
7501
4601
1783-9
17120-4
Mean vol.
(/mi 3 xl0 4 )
—
+ 0-412
+ 0-335
+ 0-273
+ 0-597
-0020
+ 0153
+ 0051
-0012
-0008
-0174
-0053
-0011
Log. vol.
difference
('double'
-'single')
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
< 001
N.S.
—
< 0001
< 005
P
* Pooled values for all embryos of the given developmental stage which were 'unpaired', i.e. did not permit a between-horn comparison. Statistical
evaluation in these cases was based on litter means, to allow for between-female variation.
t Embryos derived from transfer of uterine blastocysts with zona pellucida intact.
Developmental
stage
Mean
gestational
age of
foster-mothers
(days p.c.)
Table 2. Size of inner cell mass or egg cylinder in 'single' and 'double' embryos
to
232
M. BUEHR AND A. McLAREN
10
20
30
40
Log 'embryo volume' of 'single' embryos (//m3x 10 4)
Fig. 1. Relative size o f single' and 'double' embryos. • , Foster-litter-mates, from
the same foster-mother. O, Embryos from different foster-mothers, grouped by stage.
D, Embryos taken directly from culture. Solid line = expected regression line if
'double' are the same size as 'single' embryos. Broken line = expected regression
line if 'double' are twice as large as 'single' embryos.
compare the size of embryos from different foster-litters. 'Staging' of embryos
was always done before their volumes were measured.
Since the early embryos on which cell counts as well as volume estimations
were done showed a satisfactory correlation between the two indices (see
Table 2), and since cell counts were not practicable for more advanced stages
of development, volume estimations have been taken as the standard index of
comparison.
RESULTS
The volume estimations are listed in Table 2, together with the gestational
age of the foster-mothers and the developmental stage of the embryos. Some
embryos were measured which were derived from the transfer of 3^-day p.c.
uterine (i.e. non-cultured) blastocysts with intact zonae; these are included in
the Table, since they did not differ in size from embryos of the same stage
derived from the transfer of zona-free cultured blastocysts.
In Fig. 1 the relative size of' double' and ' single' embryos is compared, using
logarithmic units. Where possible, each point represents a single pregnant
Chimaeric mouse embryos
233
mouse, with 'double' embryos in one uterine horn and 'single' in the other.
Some females, however, had embryos implanted in one uterine horn only; in
these cases, the comparison was made between the mean values for all 'double'
embryos of a given stage and all 'single' embryos of the same stage. The same
approach was used for preimplantation stages.
If 'double' and 'single' embryos were the same size, the points would be
expected to fall on a 45° diagonal (Fig. 1, solid line). If 'double' embryos
were twice the size of 'single' embryos, the points would be expected to fall on
a parallel line 0-301 units (i.e. Iog102) above the diagonal (broken line). It will be
seen that from about stage 5 onwards, there is no significant difference in size
between 'double' and 'single' embryos, but that before this stage the 'doubles'
are at least twice the size of the 'singles'. Levels of statistical significance for
individual differences are indicated in Table 2.
DISCUSSION
According to the terminology used in this paper (Table 1), endoderm differentiation begins at stage 1, and by stage 2 a layer of distal endoderm is forming.
By stage 4 the proximal endoderm has become cuboidal and vacuolated, and the
embryonic ectoderm has become distinct from the extra-embryonic ectoderm.
The pro-amniotic cavity first appears at stage 5, spreads into the extra-embryonic
ectoderm by stage 6, at stage 7 the exocoelom is seen, and at stage 8 the allantois
is developing.
Implantation in the mouse begins at 4-0 days p.c, when the embryos are at
stage 1. Fig. 1 suggests that growth regulation in 'double' embryos takes place
shortly before stage 5, at a time when the basic structure of the embryo is being
laid down. Since the egg cylinder at stage 4 is significantly larger in 'double'
than in 'single' embryos, the differentiated tissues which constitute the egg
cylinder presumably contain a correspondingly greater number of cells in the
former, though direct counts of cell numbers in these tissues proved impracticable. Subsequent to this stage, growth of the egg cylinder appears to be less
rapid in 'double' than in 'single' embryos, so that by the time the pro-amniotic
cavity forms, regulation is more or less complete. Our results suggest that the
process takes place rapidly, since the average age of stage 4 and 5 embryos is
respectively 5-5 and 5-8 days p.c.
The mechanism of growth regulation remains obscure. The inner cell mass
gives rise not only to the foetus, but also to the yolk sac, chorion, amnion and
allantois. In a 'single' blastocyst, the number of cells whose descendants contribute to the foetus could be as low as three (Mintz, 1971), but seems more likely
to be 10-12. One possible method of growth regulation would be that this
number is fixed in absolute terms, whatever the size of the inner cell mass. Our
results are hard to reconcile with such a mechanism. Since a 'double' inner cell
mass develops into an enlarged but morphologically normal egg cylinder, with
234
M. BUEHR AND A. McLAREN
no subsequent disproportion between foetus and extra-embryonic membranes,
it seems more likely that the number of cells giving rise to the foetus represents
a certain proportion of the inner cell mass, and that the regulatory process does
not take place until after the formation of the egg cylinder. Our limited data
support the concept of regulation by retardation of growth rather than by
acceleration of differentiation, since 'double' embryos appeared to have reached
the same developmental stage as 'singles' of the same chronological age, carried
by the same foster-mother.
Three out of the four 'pre-regulation' points in Fig. 1 actually lie above the
'twofold' line, i.e. the volume difference is greater than 0-301 logarithmic units
(Table 1), suggesting that the inner cell mass of 'double' embryos is initially
more than twice as big as that of 'single' embryos. This is not unexpected, for
the following reason. The trophoblast and inner cell mass are thought to derive
from those cells of the embryos that are respectively on the outside and on the
inside after about four cleavage divisions of the fertilized egg, i.e. at about the
16-cell stage in a normal embryo (Graham, 1971). The equivalent stage after
aggregation of two embryos will have about 32 cells, and simple geometry
requires that a higher proportion of these be on the inside. For example, if a
16-cell sphere has 13 cells outside and 3 inside (Barlow, Owen & Graham, 1972),
a 32-cell sphere might be expected to have only 21 (13x2$) cells outside and
11 inside, i.e. the inner cell mass should be about three times as big in 'double'
as in 'single' embryos. Thus the tendency for the early points in Fig. 1 to lie
above the 'twofold' line is probably real.
We thank the Ford Foundation for financial support.
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BOWMAN,
(Received 29 June 1973, revised 14 September 1973)