/. Embryol. exp. Morph. Vol. 33, 4, pp. 979-990, 1975
979
Printed in Great Britain
Investigation of the determinative state of the
mouse inner cell mass
I. Aggregation of isolated inner cell masses with morulae
By J. ROSSANT 1
From the Department of Zoology,
University of Oxford
SUMMARY
Inner cell masses (ICMs) were dissected from 3£- and 4|-day blastocysts and cultured in
contact with 2^-day morulae. Blastocysts and morulae were homozygous for different
electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of
ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro
and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed
that most were chimaeric. However, donor ICM-type isozyme was only detected in the
embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic
fraction.
Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to
'outside' conditions as experienced by developing trophoblast cells. This is evidence that
ICM cells, although not overtly differentiated, are determined by 3£ days.
INTRODUCTION
There are two cell populations, morphologically and physiologically distinct,
in the 3^-day mouse blastocyst: the inner cell mass (ICM) and the trophoblast
cells. ICM cells give rise to the embryo and certain extra-embryonic tissues, while
the trophoblast cells form the ectoplacental cone and trophoblastic giant cells
of the later conceptus (Snell & Stevens, 1966; Gardner, Papaioannou & Barton,
1973). However, it is not yet clear whether the fate of both cell types is irreversibly fixed by the blastocyst stage. By 3£ days, trophoblast cells have acquired
several specific properties (Gardner, 1972), but ICM cells still resemble those of
earlier cleavage stages in several ways:
(a) Neither ICMs nor morulae show any signs of cavitation.
(b) ICMs and morulae cannot induce decidualisation of the uterus (Gardner,
1972; Kaufman & Gardner, 1974).
(c) Paired ICMs aggregate readily in culture (Gardner, 1971, 1972) as do
paired morulae (Tarkowski, 1961; Mintz, 19626).
1
Author's address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K.
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33
980
J. ROSSANT
However, this lack of overt differentiation does not necessarily imply that
ICM cells are still labile. When ICMs are isolated from mouse and rat blastocysts and injected into mouse blastocysts, they only colonize the presumptive
ICM derivatives of the host embryos and never contribute to the trophoblast
(Gardner & Johnson, 1973, 1975; Gardner, 1975). However, the failure of
ICM cells to produce trophoblast in this situation does not prove that they are
determined, since they were placed in their normal environment. The present
experiments were designed to provide a more critical analysis of the determinative state of the ICM by studying its development after isolation or
transplantation to a different embryonic site.
Conditions suitable for testing whether ICM cells can form trophoblast are
suggested by blastomere aggregation experiments, which have shown that outside blastomeres in aggregates tend to contribute progeny to the trophoblast
and trophoblast derivatives and inside blastomeres to the ICM and ICM
derivatives (Hillman, Sherman & Graham, 1972). Thus, on this 'inside-outside'
hypothesis, exposure of ICM cells to 'outside' conditions might be expected to
induce them to form trophoblast, if they are still naive. This problem was
approached in two ways. One was to study the further development of ICMs
whose surface cells had been exposed to the outside by simply isolating them
from blastocysts. The results of these experiments will be discussed in a succeeding paper (Rossant, 1975). The other approach, described in this paper,
was to attempt to aggregate ICMs with morulae, thus exposing donor ICM cells
to 'outside' conditions prior to normal host trophoblast formation.
In the initial experiments, single 3^-day ICMs were paired with 2^-day
morulae to test whether aggregation was possible. In later experiments, the
number of ICM cells exposed to the outside was increased by aggregating either
single 4 f day ICMs (30-50 cells) or paired 3^-day ICMs (26-32 cells) with each
morula.
MATERIALS AND METHODS
Recovery of embryos from donor females
Blastocysts and morulae were obtained, after superovulation or natural
mating, from mice homozygous for different alleles of the gene for glucose
phosphate isomerase (GPI). Gpi-lh/Gpi-lh ICM donor blastocysts were obtained
from an inbred strain carrying the alleles Yellow (A?) and extreme non-agouti
(ae). Morulae were obtained from random-bred CFLP mice (Carworth Europe)
of genetic constitution Gpi-l^jGpi-1*. Donor blastocysts were recovered by
flushing the uteri between 11.00 and 15.00 on the 4th day after mating (3^-day
blastocysts) and between 10.00 and 12.00 on the 5th day after mating (4^-day
blastocysts). Eight to sixteen cell morulae were obtained by flushing the oviducts
between 14.00 and 19.00 on the 3rd day after mating (2^-day morulae).
PBI medium (Whittingham & Wales, 1969) containing 10 % foetal calf serum
Determination in the mouse inner cell mass. I
981
was used for recovery, storage, microsurgery and transfer of embryos, and Ml6
medium was used for culture (Whittingham, 1971).
Micromanipulation and culture in vitro
Donor ICMs were dissected from 3^-day and 4^-day blastocysts using a
Leitz micromanipulator assembly, as described by Gardner (1972). The zonae
pellucidae were removed from the host morulae by a 0-5 % solution of Pronase
(Calbiochem, Grade B) in phosphate-buffered saline (Mintz, 1962a). Each
morula was then placed in a drop of Ml6 under light liquid paraffin together
with one or two 3^-day ICMs or one 4^-day ICM. ICMs and morulae were
brought into contact at 37 °C using a blunt, siliconized glass needle. The culture
dishes were then transferred to an incubator at 37 °C and gassed with a mixture
of 5 % CO 2 in air. Contact of ICMs and morulae was checked after 30 min to
lh.
Some ICM/morula pairs were cultured until the blastocyst stage (24-36 h)
to observe and photograph the process of aggregation. However, when transfer
to pseudopregnant recipients was to be performed, the time in culture was
kept to the minimum required for aggregation (2-4 h), since it has been shown
that the longer mouse eggs are kept in culture, the lower their viability after
transfer (Bowman & McLaren, 1970). In only one series of experiments were
the embryos cultured overnight before transfer.
Cell counts
Six blastocysts which had developed from ICM/morula aggregates were
fixed and processed histologically. Three such blastocysts and two control
blastocysts cultured from morulae were fixed in ants' cocoons, embedded in
wax and sectioned serially at 4/an (Mintz, 1971). The other three experimental
blastocysts were fixed in Dalton's fluid (Dalton, 1955) and embedded in Araldite
resin. Two control blastocysts were also fixed in this way. Thick sections
(c. 2 /.im) were cut from these, using a Huxley ultramicrotome and the sections
were stained with a 50:50 mixture of 1 % methylene blue and 1 % Azur II in
1 % borax. Cell counts were made from drawings of the serial sections on
transparent paper.
Transfer to pseudopregnant recipients
Recipient mice were used on the 3rd day of pseudopregnancy. ICM/morula
aggregates were transferred to one uterine horn and, wherever possible, control
morulae were transferred to the contralateral horn. In one experimental series,
the control horn contained paired morulae, which had been aggregated in vitro
under the same conditions used for ICM/morula aggregation.
6l-2
982
J. ROSSANT
Analysis of postimplantation development
In the initial experiments, recipients were killed at 9^ or 10£ days of pregnancy
and any implants in the experimental horns were dissected into embryonic,
extra-embryonic (amnion, chorion, allantois and yolk sac), and trophoblastic
(ectoplacental cone and trophoblastic giant cells) fractions. Conceptuses of this
age provided ample tissue for analysis, but contamination of the trophoblastic
fraction with extra-embryonic membranes was apparent during dissection. Thus,
in later experiments, recipients were killed at 8^ days, when separation of
trophoblast and membranes seemed complete. The dissected samples were
treated and analysed electrophoretically for GPI by the method of Chapman
(Chapman, Whitten & Ruddle, 1971). A single recipient was killed near term
(18^ days) enabling analysis of many different tissues of the conceptuses.
RESULTS
Observations on aggregation in vitro
Successful aggregation of single 3^-day ICMs with 2^-day morulae was
observed in vitro (Fig. 1A-E). Aggregation was achieved within 2-4 h (Fig. 1C)
and continued culture usually resulted in blastocysts with single, large ICMs
(Fig. IE). A high proportion of aggregates developed to the blastocyst stage if
left in culture (Table 1). However, the proportion of 4^-day ICMs aggregating
successfully with morulae was small. Altogether, 88 out of 124 (71 %) single
or pairs of 3^-day ICMs aggregated with morulae, while only 40 out of 119
(33 %) 4 f day ICMs did so.
Table 1. Blastocyst formation in ICM/morula aggregates cultured in vitro
Type of aggregate
One 3fday ICM/morula
Two 3i-day ICMs/morula
One 4^-day ICM/morula
No. paired
No. aggregated
No. of blastocysts
39
7
4
30
7
2
26
5
2
Cell counts
The blastocysts from ICM/morula aggregates appeared to have larger ICMs
than control blastocysts (Fig. IE, F) and cell counts confirmed this visual
impression (Table 2).
Fig. 1. Stages in aggregation of ICMs and morulae in vitro. (A) 3^-day ICM (left)
and morula (right) in culture; (B) 1 h after initial contact, aggregation beginning;
(C) 3 | h after contact; (D) 18 h after contact, cavitation beginning; (E) 36 h after
contact, blastocyst formed; (F) blastocyst formed from a control morula after 36 h
in culture.
Determination in the mouse inner cell mass. I
U.
03,
983
984
J. ROSSANT
Table 2. Results of cell counts on blastocysts derivedfrom ICMjmorula aggregates
Type of blastocyst
No. of
ICM cells
No. of
trophoblast cells
Total blastocyst
cell no.
27
25
25
41
44
45
68
69
70
29
34
40
59
56
54
88
90
94
13
14
13
15
47
51
51
48
60
65
64
63
One 3^-day ICM/morula aggregate
1
2
3
Two 3^-day ICMs/morula aggregate
1
2
3
Control
1
2
3
4
Implantation rates
The overall rates of implantation and embryonic development of ICM/
morula aggregates were consistently lower than those of control morulae
(Table 3). Also, the proportion of recipients which did not become pregnant
was larger in all the experimental transfers than in the control series. The most
valid comparison of implantation rates is probably made by considering only
pregnant females with ICM/morula aggregates in one horn and control morulae
in the contralateral horn (Table 4). The difference between experimental and
control implantation rates is then less striking, although still apparent for two
3^-day ICMs/morula and one 4^-day ICM/morula aggregates. The implantation rate of 4^-day ICM/morula aggregates is also lower than that of morula/
morula aggregates (Table 5). However, the number of recipients is too small to
test whether the differences between experimental and control implantation
rates are statistically significant.
GPI activity in conceptuses developed from ICMjmorula aggregates
One S\-day ICMjmorula aggregates
Eight conceptuses were analysed for GPI and four showed GPI activity of
both ICM donor type and host morula type (Table 6, Fig. 2, Q_ 4 ). In all four
chimaeras, the contribution of ICM-type isozyme to the embryonic and extraembryonic fractions was approximately equal to the contribution from the
morula. However, in two out of four, no ICM-type isozyme could be detected
in the trophoblastic fraction. In the other two (C 2 and C J a weak GPI-1B
activity was detected in the trophoblastic fraction.
33
53
27
38
22
82
8
7
7
8
6
17
12
9
14
13
9
18
17
17
23
23
22
22
No.
transferred
No. of
recipients
No. of
embryos
8
7
5
8
4
17
No. of
decidua
11
9
11
13
6
18
20
76
30
82
4^-day ICM/morula aggregates
Morula/morula aggregates
Type of embryo transferred
No. of
recipients
4
4
No.
transferred
No. of
embryos
1
11
No. of
decidua
41
84
Mean %
decidualisation
5
57
Mean %
embryo formation
21
32
49
38
69
52
51
57
Mean %
decidualisation
per recipient
15
77
13
23
22
41
Embryonic
development
Mean %
embryo formation
per recipient
Decidualisation
No. of
embryos
No. of
decidua
ON OO
Table 5. Implantation rates of 4\-day ICM/morula aggregates and morulalmorula aggregates
(Only pregnant females considered, where controls in contralateral horns.)
One 3^-day ICM/morula aggregates
Control morulae
Two 3^-day ICMs/morula aggregates
Control morulae
One 4^-day ICM/morula aggregates
Control morulae
Type of embryo transferred
8
4
13
9
8
4
36
17
52
34
40
22
No. of pregnant
recipients
Table 4. Implantation rates of ICM/morula aggregates
(Only pregnant females considered, where controls in contralateral horns.)
One 3i-day ICM/morula aggregates
Control morulae
Two 3^-day ICMs/morula aggregates
Control morulae
One 4^-day ICM/morula aggregates
Control morulae
Type of embryo transferred
No. of
recipients
No.
transferred
Table 3. Implantation rates of ICMjmorula aggregates
vo
oo
mass
ON ON
c 13 t
C 18 J
C10t
cBt
Q
C2
C3
Q
Q
Q
Q
C8*
Conceptus
code number
8*
Day analysed
Embryo
A + B3:l
A + B 1:1
A + B 1:1
A + B 1:1
A
A + B 1:1
A + B 1:2
B
A + B 1:2
A + B 1:1
B
A + B 1:3
A + B 1:1
A + B1:1
A + B2:l
Stage or
somite no.
20s
30 + s
30 + s
30 + s
20 + s
20 +s
20 + s
Near term
Resorption
Resorption
20 + s
10 + s
Resorption
7s
7s
A + B 1:1
A+ B1
A + B 1:
A + B 1:
A + B2:
A + B 2:1
B + trace A
A + B 1:3
B + trace A
A
A
A
A
A + B 15:1
A
A + B 15:1
A
A
A
A + B 10:1
A
A
A
Extra-embryonic Ectoplacental cone
membranes
and giant cells
C14
The numbers next to the GPI analysis for each fraction are visual estimates of the proportion of A to B isozyme.
* C8 was killed at 18£ days and many organs analysed. None contained GPI-1 A isozyme.
t C9, C10, and C i3 were just resorbing so that separation into embryonic and trophoblastic fractions was still possible.
% C u and C12 were twin implants sharing ectoplacental cone and trophoblastic giant cells.
One 4^-day ICM/morula aggregates
Two 3^-day ICMs/morula aggregates
One 3^-day ICM/morula aggregates
Experimental series
GPI analysis
Table 6. Distribution of GPI isozymes in chimaeric conceptuses derived from ICMjmorula aggregates
O
in
ON
oo
Determination in the mouse inner cell mass. I
(A)
EX
EX
EX
C,
(A)
(A)
E EX
E E X
987
C3
T
E
T
EX
E
T^ J E E X
T
t
E
T
T
(B)
(B)
(A)
Fig. 2. Electrophoretograms of conceptuses derived from ICM/morula aggregates.
E, Embryonic fraction; EX, extra-embryonic fraction; T, trophoblastic fraction.
(A) Gpi-laIGpi-la mouse standard; (B) Gpi-lb\Gpi-lb mouse standard. Numbers
correspond to conceptus code numbers in Table 6. No photographs available for CX1,
C12, C 1 3 .
Two 3%-day ICMsjmorula aggregates
Ten implanted embryos or resorbing embryos were analysed electrophoretically and nine were found to be chimaeric (Table 6, C 5 _ 13 ; Fig. 2, C5_10). A
variety of patterns of GPI activity was observed in the embryonic and extraembryonic fractions, ranging from C8, where all of the tissues of the embryo
and its extra-embryonic membranes showed only ICM-type activity, to C 5 ,
where the only contribution of GPI-IB isozyme was a minor one to the extraembryonic fraction. However, no ICM donor isozyme was detected in the
trophoblastic fraction of any embryo analysed at 8£ days. ICM-type GPI
activity was detected in the trophoblastic fraction of C8, which was analysed
at 18idays.
988
J. ROSSANT
One 4\-day ICM/morula aggregates
The GPI activity of seven conceptuses was analysed and two were found to
be chimaeric (Table 6, Fig. 2, C14_15). Both were too small to separate embryonic
and extra-embryonic fractions but the contribution of ICM-type isozyme to the
two egg-cylinders was quite large. There was no GPI-IB isozyme detectable in
the trophoblastic fraction of either conceptus.
DISCUSSION
Isolated 3^-day and 4^-day mouse ICMs are able to aggregate with 2^-day
morulae to form blastocysts in vitro and morphologically normal foetuses in
utero. Most (15/25) showed GPI activity of both donor ICM and host morula
type. However, in all but three such chimaeras, GPI of ICM type could only be
detected in the embryo and extra-embryonic membranes, and not in the ectoplacental cone and trophoblastic giant cells (Table 6, Fig. 2). The weak contribution of ICM-type isozyme to the trophoblastic fraction in three conceptuses
(C2, C4, C8) was almost certainly due to contamination with extra-embryonic
membranes. C 2 and C 4 were among three chimaeras analysed at 10^- days,
when clean dissection of the trophoblast was difficult, since union of the chorion
and allantois with the ectoplacental cone had already occurred (Snell & Stevens,
1966). C 8 was analysed at 18^ days when the chorioallantoic placenta is fully
formed, making contamination with extra-embryonic membranes inevitable.
In all other chimaeras analysed, the separation of trophoblast and extraembryonic membranes seemed complete.
Thus, it appears that placing ICM cells on the outside of morulae cannot
induce them to form trophoblast, although in blastomere aggregates outside
cells tend to give rise to trophoblast and trophoblastic derivatives (Hillman et al.
1972). Therefore, the present experiments suggest that the ICM cells of the
mouse blastocyst, although not overtly differentiated, are determined by 3^days.
At present, the possibility that some donor ICM cells contribute to the mural
trophoblast surrounding the blastocoelic cavity cannot be excluded. Mural
trophoblast cells cease dividing and start transforming into primary giant cells
early on the 5th day of pregnancy (Dickson, 1966). Thus, donor ICM cell
progeny would only be present in large enough numbers to be detectable by
GPI analysis if they contributed to the proliferating polar trophoblast overlying
the inner cell mass. However, if ICM cells do form trophoblast, the likelihood
of 11 conceptuses showing no donor ICM isozyme in the trophoblastic fraction
(Table 6, Fig. 2) is remote, unless the ICM cells preferentially form mural
trophoblast. Confirmation that no trophoblast cells are formed thus requires
a marker that can detect individual cells. Use of [3H]thymidine-labelled ICMs
would be questionable because of its toxicity to ICM cells (Snow, 1973).
Determination in the mouse inner cell mass. I
989
Aggregation of rat ICMs with mouse morulae may provide a suitable marker
since the distribution of individual rat and mouse cells in interspecific chimaeras
can be detected by immunofluorescence (Gardner & Johnson, 1973).
Formation of chimaeric foetuses after aggregation of ICMs and morulae is
further evidence of the capacity for regulation possessed by pre-implantation
mouse embryos, since the cells differ considerably in age. Regulation for asynchrony in cell age has been shown previously for cleavage stages, where embryos
which are 10 or 12 h asynchronous ^zr aggregate and form normal blastocysts
(Mulnard, 1971; Stern & Wilson, 1972). At the blastocyst stage, chimaeras can
be formed after injection of 4^-day ICMs into 3^-day blastocysts (Gardner, 1971).
However, the formation of chimaeric embryos after aggregation of 4^-day
ICMs and 2^-day morulae is the first unequivocal example of regulation for
2 days asynchrony in embryonic cell age. ICM/morula aggregation also demonstrates for the first time that cells from the blastocyst stage can aggregate with
pre-blastocyst cells and form normal embryos.
The low rates of implantation of ICM/morula aggregates, particularly 4^-day
ICM/morula aggregates, also remain to be explained (Tables 3,4). Cell aggregation itself does not seem to reduce implantation since morula/morula aggregates
implanted at a much higher rate than 4^-day ICM/morula aggregates (Table 5).
Another possibility is that the increased size of the ICM in ICM/morula aggregates might reduce the ratio of mural to proliferating polar trophoblast. Since
attachment is believed to begin in the abembryonic mural trophoblast (Boyd &
Hamilton, 1952), such a reduction might affect the efficiency of implantation. It
is also possible that the particularly low rate of implantation of 4^-day ICM/
morula aggregates is connected with the difficulty in achieving aggregation of
these stages. Tight junctions occur between the endoderm cells of the 4^-day
ICM (Enders, 1971) and these may interfere with cell aggregation. Thus, some
cells might remain on the outside and adversely affect implantation in some way.
I wish to thank Dr R. L. Gardner, Dr V. E. Papaioannou and Mrs S. C. Barton. The
author is in receipt of an M.R.C. Research Studentship and the work was supported by the
Ford Foundation and the Medical Research Council.
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ENDERS,
(Received 28 October 1974)