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/. Embryol. exp Morph. 86, 311-336 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
Developmental variability within and between mouse
expanding blastocysts and their ICMs
JULIA C. CHISHOLM, MARTIN H. JOHNSON, PAUL D. WARREN,
TOM P. FLEMING AND SUSAN J. PICKERING
Department of Anatomy, Downing Street, Cambridge, CB2 3DY, U.K.
SUMMARY
We have attempted to reduce the developmental heterogeneity amongst populations of mouse
blastocysts by synchronizing embryos to the first visible signs of blastocoel formation. Using
embryos timed in this way, we have examined the extent of variation of inside and outside cell
number and of inside cell size, nuclear DNA content and developmental potential, between and
within embryos of a similar age postcavitation.
The overall impression gained is one of wide heterogeneity in inside:outside cell number ratios
and in cell cycling and its relation to cavitation among embryos of similar age postcavitation.
However, the simplest explanation of our results suggests that cavitation generally begins at a
time when most outside cells are in their sixth developmental cell cycle and that outside cells, as
a population, are a little ahead of inside cells in their cell cycling. Additionally we present
evidence that, within at least some individual inner cell masses (ICM), there is intraembryo
variation in the time at which inside cell developmental potential becomes restricted.
INTRODUCTION
The developmental fate of cells in the early mouse embryo is influenced both by
their inherited phenotypic characteristics and by relative cell position (reviewed
Johnson, 1985a). Phenotypic and positional differences among cells are first
evident in the 16-cell morula which contains distinct subpopulations of outer, polar
and inner, apolar cells (Barlow, Owen & Graham, 1972; Handyside, 1981; Reeve
& Ziomek, 1981; Johnson & Ziomek, 1981; Ziomek & Johnson, 1981; Kimber,
Surani & Barton, 1982; Randle, 1982; MacQueen & Johnson, 1983). Cells occupying an inside position in the morula contribute substantially to the ICM, and outside
cells to the trophectoderm (TE), of the early blastocyst (Tarkowski & Wroblewska,
1967; Hillman, Sherman & Graham, 1972; Wilson, Bolton & Cutler, 1972; Kelly,
Mulnard & Graham, 1978; Ziomek & Johnson, 1982; Balakier & Pedersen, 1982;
Surani & Handyside, 1983). However, these two sets of lineage relationships are
not fixed, and cellular traffic between lineages can and does occur, the extent of the
traffic being regulated by simple and definable cellular mechanisms (see Surani &
Barton, 1984; and Johnson, I9%5a,b for discussion). Whilst the mechanisms by
which regulation of lineage can operate have become clearer, as a result of experimental manipulation and relocation of individual cells or cell clusters, the extent to
Key words: mouse, trophectoderm, ICM, blastocyst, commitment, cell cycle.
312
J. C. CHISHOLM AND OTHERS
which such regulation occurs in situ is less well understood. This deficiency arises
in part from the absence of suitable lineage markers for non-invasive labelling of
single cells in situ and partly because of the wide variation in the estimates of
numbers of inside and outside cells within individual embryos of 16-cells or more.
Analysis of 16-cell embryos for the number of inside cells has provided estimates
as diverse as rarely more than two (Barlow et al. 1972; Graham & Lehtonen, 1979)
and rarely less than four but never more than eight (Handyside, 1981; Johnson &
Ziomek, 1981; Reeve, 1981; Bafakier & Pedersen, 1982; Surani & Barton, 1984).
Analysis of embryos with approximately 32-cells has provided estimates of on
average 14 inside cells (Handyside, 1978), 6-7 inside cells (Barlow et al. 1972;
Randle, 1982) and 13-16 inside cells (Copp, 1979; Surani & Barton, 1984). One
problem with these various studies is that different techniques were used to evaluate
cell position, and different strains of mice and relatively crude methods of embryonic staging were employed at a time when there is considerable developmental
heterogeneity between embryos (Smith & McLaren, 1977). In this study we (i)
examine the extent of mterembryo variability in cell number at the late
morula-early blastocyst stage, (ii) apply four techniques for evaluating the variability of inside and outside cell numbers at relatively homogeneous stages of blastocyst
formation in individual embryos from two mouse embryo strains, (iii) make
estimates of the size heterogeneity within the populations of inside cells at some of
the stages analysed, and (iv) examine variability in the DNA content and developmental potential of cells from individual ICMs taken from relatively homogeneous
populations of blastocysts.
MATERIALS AND METHODS
Embryo recovery and staging
Embryos were derived from superovulated 3- to 5-week-old HC-CFLP or MF1 females
(Hacking & Churchill Ltd or Olac Ltd; 5 i.u. PMS followed after 45-50 h by 5 i.u. hCG, Intervet,
and mating with HC-CFLP males). Embryos were recovered as 8- to 16-cell stages at 62-72 h
post hCG byflushingoviducts with phosphate-buffered medium 1 or Hepes-buffered medium 2
containing ^rngml" 1 bovine serum albumin (PB1+BSA or M2+BSA; Whittingham & Wales,
1969; Fulton & Whittingham, 1978). Embryos were cultured in individual drops of medium 16
plus 4 mg-ml"1 BSA (M16+BSA; Whittingham, 1971) under oil in Sterilin culture dishes at 37 °C
and 5 % CO2 in air and were scored individually for the earliest signs of blastocoel formation,
every 60 or 90 mins between 72 and 100 h post hCG on a Wild inverted phase-contrast microscope
at x 60 or x 100. Nascent blastocysts that had formed since the previous inspection were pooled
and designated as 0 h old. Pooled groups of blastocysts aged 0 to 12 h were used for the analyses.
In some experiments, the extent of cavitation of each embryo was noted during each hourly
scoring. Five categories were used: Stage 1, uncavitated morula; 2, blastocoel occupying less than
half volume of embryo; 3, blastocoel occupying about half volume of embryo; 4, blastocoel
occupying greater than half volume; 5, well-expanded blastocyst (see Fig. 2).
Embryo manipulations
(i) Removal of the zona pellucida was achieved by brief exposure to prewarmed acid Tyrode's
solution, pH 2-5, +4mg-ml" 1 polyvinylpyrrolidone (Nicolson, Yanagimachi & Yanagimachi,
1975).
Variability of mouse early development
313
(ii) Immunosurgery was carried out as described by Handyside (1978). Rabbit antimouse
species antiserum diluted 1/10 with M2 was prewarmed at 37 °C prior to incubation of early
blastocysts (3 min) or expanded blastocysts (10 min). Embryos were washed through three large
volumes of M2+BSA before exposure to guinea-pig complement (Difco) diluted 1:3 with
phosphate-buffered saline, absorbed with agarose (Cohen & Schlesinger, 1970), stored at —70 °C
and thawed immediately prior to use. After 5-7 min embryos were transferred to fresh M2+BSA
and left for a further 20-30 min at 37 °C before removal of outer cells by a flame-polished
micropipette with an internal diameter slightly larger than the ICM.
(iii) Decompaction of ICMs was achieved either by exposure to Ca2+-free M16 containing
6mgml 1 BSA for 20 min (Pratt, Ziomek, Reeve & Johnson, 1982) or M16+BSA containing
cytochalasin D (CCD; Sigma) at OS^g-ml"1 for 20 min.
In vitro culture of ICMs
ICMs or ICM aggregates were cultured individually in 25 jtd drops of RPMI 1640 (Flow)
supplemented with 10 % foetal calf serum (under oil, at 37 °C in 5 % CO2 in air). Falcon plastic
tissue culture dishes were used. ICM cultures were examined using phase contrast optics on a
Wild inverted microscope.
Cell counting techniques
Four techniques were used to evaluate numbers of cells at each stage.
(i) Giemsa staining (Tarkowski, 1966). Whole embryos (with or without the zona pellucida
intact), or inner cell masses (ICM) from them, were exposed to 0-9 % sodium citrate for 10 or 1
min respectively before transfer to acetone-cleaned slides. Fixation was achieved with a drop of
ethanol:acetic acid (3:1) and air drying. Giemsa stain (5 %) was millipored immediately before
use and samples allowed to stain for 10 min prior to counting of nuclei. Mitotic figures were
counted as one cell. This approach allows direct measurement of inside cell numbers and total
cell numbers. Average outside cell numbers can be obtained by subtraction. Some ICMs were
spread and fixed as if for Giemsa staining, but stained by the Feulgen reaction for microdensitometry (see below).
(ii) DAPI staining (4, 6-diamidino-2-phenylindole; Reeve & Kelly, 1983). Zona-intact embryos were subjected to immunosurgical procedures up to and including exposure to complement, and then exposed to M16+BSA+DAPI (100/ig-ml"1) for 30 min at 37 °C to stain nuclei
of dead trophectoderm and live ICM cells which were retained within the zona. Embryos were
then rinsed and transferred individually to separate 10 [A drops of PB1+BSA on a multiwell slide
(Baird & Tatlock). The zona pellucida was then removed and the ICM shelled out with a flamepolished micropipette. The dead trophectoderm cells were allowed to dry down on the slide,
while the ICM was transferred to a lOjul drop of 0-9% Na citrate for 15 min before being
disaggregated into small cell clumps and air dried. Dried samples were fixed with 70 % ethanol
prior to counting of thefluorescentnuclei on a Zeiss universal microscope equipped with incident
source HBO 200 and filter set 48 77 05.
(iii) Disaggregation of inside cell clusters. After immunosurgical isolation, clusters of inside
cells were decompacted as described earlier and disaggregated in a small individual drop of
decompacting medium. Cells were then counted. This technique allows direct enumeration of
inside cells only.
(iv) Fixation and serial section. Intact embryos, within the zona pellucida, were processed for
transmission electron microscopy (see below) and embedded in capsules in groups of
approximately six in Spurr's resin. Serial thick sections were cut (2/im thickness), mounted on
slides and stained with 1 % toluidine blue/borax. Samples were viewed on a Zeiss universal
microscope with a drawing tube attachment and outlines of cells and nuclei recorded; the following criteria were useful in identifying cells as belonging to the inside or outside, namely cytosolic
density, distribution of secondary lysosomes, and cell shape (Fleming, Warren, Chisholm &
Johnson, 1984). Numbers of inside, outside and total cells were recorded from three-dimensional
reconstructions.
314
J. C. CHISHOLM AND OTHERS
Microdensitometry
ICM nuclei were stained by the Feulgen reaction (Pearse, 1972; Bolton, Oades & Johnson,
1984), together with an air-dried mouse liver smear. Nuclear DNA content was assayed on a
Vickers M85 integrating microdensitometer.
Scanning electron microscopy
The procedure of Johnson & Ziomek (1982) was used. All reagents were filtered immediately
prior to use. ICMs, or cells therefrom, werefixedin 6 % glutaraldehyde in O-lM-cacodylate buffer
(pH7-3; osmolality 730 mOsmol) for 1 h at room temperature, stored in O-lM-cacodylate for a
maximum of 24 h before transfer in 1 % glutaraldehyde to clean glass coverslips that had been
coated with poly-L-lysine (Sigma type IB; 1 mg-ml"1 fresh solution) for 15 min followed by two
washes in O-lM-cacodylate buffer and immersion in buffer in wells of a 24-well Linbro (Flow)
tissue-culture dish. After dehydration through alcohols (30 min in 30 % and 50 %; overnight in
70 %; 15 min in 80 %, 90 %, 95 % and 100 %) samples were dried from 100 % ethanol via CO2
in a Polaron E3000 critical-point drying apparatus. Coverslips were mounted on stubs with
double-sided Sellotape, coated with a 50/^11 layer of gold in a Polaron E5000 Diode sputtering
system and viewed on a Cambridge Stereoscan 600 electron microscope. The diameter of individual blastomeres was assessed by averaging the greatest and least diameters measured from
photographs of non-tilted samples. Grossly distorted blastomeres were not measured. Volumes
were calculated from the mean diameters on the assumption that each blastomere was spherical.
Transmission electron microscopy
ICMs or ICM aggregates (after 48 or 72 h of culture in vitro) were fixed in 3 % glutaraldehyde
in 0-1 M-cacodylate buffer for 30 min. After washing in cacodylate buffer they were postfixed in
similarly buffered 1 % osmium tetroxide (30min), washed in distilled water, dehydrated in a
graded ethanol series, and embedded in Spurr's resin. Ultrathin sections were cut on an LKB
Ultrotome III, mounted on copper grids, stained with uranyl acetate and lead citrate and viewed
using an AEI6B electron microscope at 80 kV.
Photography
Phase-contrast photographs were taken on 35 mm Ilford Pan F film.
RESULTS
1. Staging of blastocysts
Over 90 % of 8- to 16-cell embryos, that were flushed between 62 and 72 h post
hCG and cultured overnight, proceeded to cavitation. Typically about 60 % of HCCFLP-derived embryos cavitated between 83 h and 93 h post hCG whereas about
50 % of MF1 derived embryos cavitated between 90 and 98 h post hCG (Figs.
1A,B). There is therefore a variation in excess of 10 h in the time at which cavitation
is initiated within each strain, a result confirming that reported by Smith &
McLaren (1977) for naturally ovulating mice, and there is also variation between
strains in the absolute time of these ranges. In addition hourly scoring of the extent
of cavitation of blastocysts showed that the rate of blastocoel expansion varied
between embryos (see Fig. 2B). Cavitation was detected in 0 h and 1 h blastocysts
as a small, oval translucent region located at the abembryonic pole and bound
distally by attenuated processes of mural TE and occasionally split into two cavities
Variability of mouse early development
100
315
100
A
80
60
8 60
140
•B 40
3
3
E
^
B
U 20
20
0
83 84-5 86 87-5 89 90-5 92 93-5
hpost-hCG
0
90 91 92 93 94 95 96 97 98
hpost-hCG
Fig. 1. (A) Percentage of HC-CFLP embryos showing some indication of cavitation at
various times post-hCG. Results are cumulative and are derived from 981 embryos in
four typical experiments.
(B) Similar plot for 483 MF1 embryos in three experiments.
within a single blastocyst (Fig. 2A). By 11 to 12 h, blastocysts were well expanded,
with the ICM appearing as a disc-shaped cell cluster at the embryonic pole (Fig.
2C). At intermediate times the extent of blastocoel expansion varied among blastocysts within each age group (Fig. 2B). In a few embryos, the cavity collapsed after
its initial formation and these embryos were excluded from analysis.
2. Interembryo variation in relation to time post hCG and time post cavitation
In an effort to reduce interembryo variation we examined the value of synchronizing embryos obtained from superovulated mice to the earliest signs of
Fig. 2. Examples of expanding blastocysts aged (A) 0, (B) 6 and (C) 12 h after cavitation
was first detected. 0 h blastocysts are all at stage 2,12 h blastocysts at stage 5, whilst 6 h
blastocysts vary from stage 2 to 4 in their degree of expansion (see Materials & Methods
for explanation of staging). For light and electron photomicrographs of sections of these
staged embryos see Fleming et al. (1984). Scale bar = 100 fjm.
316
J. C. CHISHOLM AND OTHERS
Table 1. Mean cell numbers in morulae, newly cavitated embryos and blastocysts at
different times post hCG {For explanation of the experimental procedure see text)
Mean cell number ± s .D. (number scored)
Time
(h post
hCG)
91-5
93-0
94-5
96-0
% embryos cavitated in
group fixed for cell
counts (total embryo
number)
31-2
(141)
59-3
(145)
59-7
(149)
70-8
(144)
Morulae
Newly
cavitated
embryos*
Blastocysts
Total
embryo
population
29-9 ± 8-7
(87)
32-2 ± 8-0
(54)
31-2 ±7-9
(54)
30-3 ± 7-2
(39)
34-3 ± 3-7
(12)
34-7 ±7-9
(16)
32-6 ±4-3
(20)
34-4 ±9-6
(14)
41-3 ±10-6
(24)
46-9 ± 10-7
(61)
45-8 ±11-1
(68)
51-2 ±9-7
(82)
32-5 ±9-8
(123)
39-3 ±11-7
(131)
38-4 ±11-6
(142)
43-5 ±13-3
(135)
* Summation of data for all newly cavitated embryos regardless of age in h post hCG gives a
mean cell number of 33-9 ± 6-8 (n = 62); this value differs significantly (t-test, P < 0-01) from the
values of total embryo populations at 93-0, 94-5 and 96-0 h post hCG.
cavitation as a means of reducing developmental heterogeneity between embryos
(cf. results from Smith & McLaren who used naturally mated mice for the same
purpose, 1977). MF1 embryos were recovered at 68h post hCG as 8- to 16-cell
morulae, randomized into four groups and cultured until about 20-25% of the total
embryo population had undergone cavitation. Each group of embryos was then
sorted into subgroups containing those with and those without a blastocoel (blastocysts versus morulae, Table 1). After 90 min the morula subgroups were
examined for newly cavitated embryos. Newly cavitated embryos, morulae and
blastocysts in group 1 were fixed for counting cells by Giemsa staining. In the
remaining groups, newly cavitated embryos were transferred to their blastocyst
subgroups. At consecutive 90 min intervals thereafter the same procedure was
repeated, so that groups 2-4 were fixed serially as morula, newly cavitated embryo
and blastocyst subgroups. Mean cell numbers for each embryo subgroup are given
in Table 1. Although overall mean cell number increased with time post hCG, this
increase was not distributed evenly among different subgroups of embryos. Mean
cell numbers in the morula and newly cavitated subgroups were remarkably constant over the period 91-5 to 96 h post hCG, whereas the substantial increase in
blastocyst cell numbers was sufficient to account for the overall rise in mean cell
number in the total embryo population (Table 1). The range in cell number within
each subgroup was large, but frequency distributions demonstrated that 50-60 %
of embryos in the morula subgroups and 80 % of all newly cavitated embryos had
between 28 and 35 cells inclusive (data not shown). By contrast, embryos in the
blastocyst subgroups showed a fairly even distribution over a broad range of cell
number, never having fewer than 24 cells and rarely less than 30. We have therefore
—
31-0
Calculated
Mean of
means
i
n
t
i
n
t
i
12h blastocyst
o
9-5
9-0 ±3-4
21-5
—
—
66
30-5
—
10-5
12-0 ±4-6
21-0
—
—
22
43-5
—
14-0
13-9 ±2-3
29-5
—
—
44
52-0
—
19-5
22-3 ±4-5
33-5
—
57(49,66 20-8(13, 36-3(35,
4 61,55) 21,25,24) 44,36,30)
—
9
—
20-3 ±4-8
(28-5)* 33 47-8 ±3-9
—
o
15-6(17, 29-6(28,
17,13)
32,29)
—
13
—
14-5 ±2-4
(20-1)* 1143-0 + 5-4
—
o
4 33(32,25, 12(9,8, 21(23,17,
45(45,
34,41)
15,17)
19,24)
3 49,42)
15
—
8-3 ±2-2
8 28-4 ±2-5
—
t
6h blastocyst
n, total no. embryos (or ICMs); t, mean total cell no. of embryos; i, mean inside cell no.; o, mean outside cell no.; All ± S.D.
* Outer cell number obtained by subtraction.
—
n
34-7 ±6-4 10-8 ±2-4 23-9 ±6-2
—
(20-0)*
o
2h blastocyst
—
(27-5)*
.
29-3 ±3-9 8-0 ±1-6 21-1 + 3-0 32 30-5 ± 6-2 9-1 ±2-7 21-4 ±4-5 19 43-1 ±7-3 13-0 ±3-8 30-1 ±5-5 32 50-8 ±8-8 14-3 ±5-5 36-5 ±8-2
i
Oh blastocyst
—
9-8 ±2-2
29-8 ±3-7
—
t
(iv) Disaggregation
of inside cell
clusters
58
Technique of
evaluation of
cell
numbers
n
(i) Giemsa
staining
21
20
(ii) DAPI
staining
25
(iii) Serial
sectioning
10
Stage of embryo and age (in h post-cavitation)
Table 2. Mean cell numbers in expanding blastocysts and their constituent subpopulations in embryos examined at 90 minute
intervals for cavitation
^
OJ
<
S.
>§
-3
^
^
^^.
^
g
^
^
§
v^-
S;
^
2.
318
J. C. CHISHOLM AND OTHERS
used the earliest signs of cavitation, rather than hours post hCG, as a synchronizing
device in all subsequent experiments. Embryos (or ICMs from them) are hence
described as being aged 0 to 12 h post cavitation.
3. Number and distribution of cells from different stage embryos
The mean values for total cell numbers and for inside and outside cell numbers
were obtained from a total of 18 experiments using HC-CFLP mice and are recorded in Table 2. Values for individual Oh blastocysts analysed by the two
techniques that permit evaluation of both inside and outside numbers in the same
embryo (DAPI staining and serial sections) are recorded in Table 3.
Table 3. Cell number and distribution in individual Oh CFLP blastocysts analysed
by DAPI staining or serial section (the latter marked with an asterisk). Embryos were
inspected at 90 minute intervals for cavitation
Total cell No.
49*
42
40*
38*
36
35*
35
33*
33*
33*
31*
31
31
31
30
30
29
29*
29
28
28
28
28
27
27
27
27
26*
26
25
25
25
No. of cells inside
(No. of mitotic cells)
No. of cells outside
(No. of mitotic cells)
14(1)
12
11
8
7
13
10
14
8
8
11
10
9
9
8
7
10
9(2)
7
8
8
8
8
8
7
7
7
12
6
7
7
5
35(3)
30
29(1)
30(3)
29
22(2)
25
19(1)
25(1)
25
20
21
22
22
22
23
19
20(2)
22
20
20
20
20
19
20
20
20
14
20
18
18
20
Variability of mouse early development
319
70-
Disaggregated ICMs
E 12h
6-05
8-8
121
14-85
Diameter (linear units)
Fig. 3. Plots of cell number against cell diameter expressed in arbitrary linear units.
Values in (A) and (B) are taken from an experiment in which ICMs from (A) 2 h and
(B) 12 h blastocysts were decompacted in CCD and any exposed cells measured. Values
in (C-E) are from an experiment in which ICMs from Oh (C), 2h (D) and 12h (E)
blastocysts were disaggregated to single cells and their diameters measured.
4. Size distribution of inside cells from different stage embryos
The diameters of individual inside cells were estimated in two types of preparation. In one experiment, intact CCD-decompacted clusters of inside cells were
examined on the SEM, and the diameters of any exposed cells were measured. The
distribution of diameters is plotted in Fig. 3A,B. In a second experiment, single
cells derived by decompaction and disaggregation of inside cell clusters were
examined on the SEM and the diameters of all non-distorted cells were measured.
The distribution of diameters that were recorded is plotted in Fig. 3C,D,E. In
neither technique were all cells measured, and it cannot be assumed that losses were
random. The distribution of values along each horizontal axis is therefore more
useful than the relative heights of peaks as measured on the vertical axis. Also,
there is considerable shrinkage of cells during preparation that varies with each set
of experimental runs, and so values are expressed in arbitrary linear units (lu).
12
11
8
16
13
20
17
13
10
13
10
18
1
2
3
4
5
6
7
8
9
10
11
12
8
18
11
13
7
21
4
8
11
10
10
10
F
15
11
7
20
23
20
10
12
5
10
12
10
D
* Calcul--* « - t o t a l n o - m i t o t i c c e l l s
total cell no.
G
No.:[CMs counted
Age of
ICM
(h)
11-6 ±2-7
12-9 ±3-2
16-1 ±6-2
16-0 ±5-4
15-3 ±4-5
16-3 ±6-0
16-4 ±4-0
18-4 ±3-8
17-4 ±4-6
20-2 ±4-0
20-6 ±5-0
19-8 ±3-9
G
XXX
V l i v l l
Ut^w
&X
UUU
•
/fs in each a&e proun
'XJ
12-1 ±1-3
13-1 ±2-5
15-1 ±7-1
15-8 ±4-6
14-6 ±4-3
14-6 ±4-6
15-3 ±6-8
14-8 ±3-9
17-7 ±4-0
16-6 ±4-6
18-5 ±5-3
19-9 ±3-0
F
Mean cell number ± S.D.
11-6 ±2-5
11-7 ±3-7
14-3 ±3-0
15-4 ±5-1
15-9 ±4-6
18-0 ±5-0
17-9 ±4-2
18-3 ±4-5
19-0 ±5-5
18-0 ±3-1
17-4 ±3-6
19-8 ±3-7
D
8-17
7-18
11-28
9-26
9-25
7-28
9-23
12-24
9-25
15-29
11-29
13-28
G
8-16
8-17
6-26
8-27
10-23
8-26
5-24
10-20
11-24
10-26
10-28
14-26
F
D
8-16
7-18
10-19
8-25
8-24
9-25
12-24
11-27
13-27
14-23
11-23
15-28
Range in cell no.
0-7
7-8
3-1
1-8
4-5
0-3
1-1
0-8
0-6
0-8
2-4
2-5
G
01
0-8
1-2
4-4
0-0
2-3
1-6
0-0
3-1
3-6
6-5
3-5
F
Mitotic index*
Table 4. Mean ICM cell numbers in timed MFIblastocysts estimated by Giemsa (G) orFeulgen (F) staining, or by disaggregation
in calcium-free M16+BSA (D)
O
O
H
X
w
>
r
O
K
a
o
o
Variability of mouse early development
321
From Fig. 3, it can be seen that there is a reduction in cell size (assuming cell
shape to be spherical) in older ICMs. At 0 and 2 h, diameters are distributed almost
unimodally over a range equivalent to volumes of 300-800 hi3 (median value =
500), with evidence in Figs. 3A and C of a second cluster of larger volumes ranging
from 1000-1900 lu3 (median value = 1200 lu3). At 12 h, volumes are distributed
over the range 100-300 lu3 (median value = 200), with evidence of a second cluster
over the range 350-500 lu3 (median value = 450).
5. DNA content of cells from individual ICMs
ICMs were isolated from MF1 blastocysts at hourly intervals from 0 to 12 h after the
earliest signs of cavitation (60 min inspection regime) for more detailed analysis of
cell numbers and DNA content. Number estimates were made by disaggregation and
by both Giemsa and Feulgen staining and results are shown in Table 4. Considerable
agreement was found between counts made by all techniques at each time point.
All three techniques gave a mean ICM cell number of 11-12 at 1 h postcavitation.
Between 1 h and 3 h postcavitation mean cell number increased by 2-4 whereas it
was roughly constant at around 14-16 cells over the period 3-5 h postcavitation (by
disaggregation) and 3-7 h postcavitation (by Giemsa and Feulgen staining). By 12 h
postcavitation ICMs contained about 20 cells on average. However, the range in
cell number among ICMs within each age group was extremely large (Table 4).
Mitotic figures were recorded in stained preparations and mitotic indices (percentage of cells in mitosis) were calculated for each ICM age group (Table 4). In
general, mitotic indices were low and individual ICMs rarely showed more than one
or two mitotic figures.
In ICMs spread by the Tarkowski method, the majority of cells could be assayed
for nuclear DNA content. However, every nucleus could be assayed in very few
ICMs due to insufficient nuclear separation. Standard 2C and 4C DNA values were
obtained from the liver smear in each experiment. Results showed that in most
ICMs, the DNA content of individual nuclei ranged from 2C to 4C, including
intermediate values. Since all phases of the cell cycle are present from the 2-cell
stage onwards in the mouse embryo (Barlow et al. 1972), each ICM probably
contains cells in Gi, S and G2 phases of the cell cycle. Thus cells within each ICM
were cycling asynchronously.
6. Developmental potential of ICMs
In this study, ICMs were cultured singly and were examined at 24 h, 48 h and 72 h
after immunosurgical isolation for evidence of trophectoderm or extraembryonic
endoderm-like features. Uncommitted ICM cells which differentiate into trophectoderm in vitro are expected to show signs of fluid accumulation and formation of
a blastocyst-like vesicle within 24-48 h of the start of culture (Handyside, 1978),
whereas endoderm formation should only become apparent by 48-72 h. Thus, both
in vitro morphology and the timing of morphological changes may help in assessing
the developmental potential of ICMs. Results are shown in Table 5 and Fig. 4.
21
23
24
34
30
41
31
42
45
39
35
36
2
2
1
—
1
2
5
4
5
5
3
7
Degenerate*
1
1
1
1
2
—
1
1
1
—
No
differentiation
—
—
—
2
—
—
—
2
1
2
—
Some fluid
accumulation f
19
20
22
30
26
33
20
30
• 25
26
18
19
Trophectodermal
features^
—
—
1
—
1
—
—
2
1
1
4
—
—
—
1
5
1
4
7
3
5
4
—
—
1
—
—
3
2
2
1
2
1
—
. —
1
, —
—
—
1
1
1
2
—
2
2
2
2
3
1
4
1
4
—
1
Endodermal
Solid
Mixed
fluid
embryoid Endodermal1
Attached
accumulation monolayer character
vesicle
body
* ICMs which showed no signs of development before degenerating.
t ICMs in which fluid accumulation could not clearly be classified as either trophectodermal or endodermal in nature.
t Includes mixed character structures.
1
2
3
4
5
6
7
8
9
10
11
12
Age of
ICM
(hours
post
Number
cavitation) counted
Table 5. Development of isolated ICMs from timed MFI blastocysts. ICMs were cultured in vitro for 72 h and were scored by
light microscopy at 24, 48 and 72 h after isolation for signs of development.
C/3
H
X
w
w
O
X
r
C/3
o
n
K
323
Variability of mouse early development
100
so
60
40
20
-•
W
•
i
1
1
1
1
1
1
2 3
4 5 6 7
8 9
10
Age of ICM on isolation in h post-cavitation
r-
11
12
Fig. 4. Percentage of MF1 ICMs of different ages postcavitation forming trophectoderm (o; includes mixed character structures) or endoderm alone (•) during in vitro
culture. Data summarized from Table 5. ICMs which degenerated before showing any
signs of development are excluded from total ICM numbers when calculating percentages.
The majority of ICMs scored as showing trophectodermal development formed
blastocyst-like vesicles (Fig. 5A) although some gave 'non-integrated' forms (Fig.
5B; Handyside, 1978) or showed only a few vesiculating cells. Trophectoderm
formation was usually visible by 24 or 48 h after isolation although it was first
observed at 72 h in small number of ICMs. Outgrowth of trophoblast giant cells by
vesiculating ICMs was not observed during the 72 h culture period. The majority
of ICMs in each ICM age group exhibited trophectodermal differentiation by cells
occupying an outside position, although the proportion of ICMs which generated
trophectoderm fell with increasing blastocyst age (Fig. 4). Nevertheless 69% of
viable ICMs from 12 h blastocysts generated some trophectodermal cells on isolation and only 31 % generated endoderm alone.
Amongst those ICMs classified at the light microscope level as showing endodermal differentiation there was considerable variability of phenotype. These forms
fell broadly into four categories: a) structures showing an outer "rind" of cells
surrounding an inner solid core, with variable degrees of intra- and extracellular
fluid accumulation (endodermal vesicles; Fig. 5C); b) structures lacking a clear
outer rind of cells, but nevertheless showing extracellular fluid accumulation which
was judged to be endodermal rather than trophectodermal in nature both by morphology and the time (48-72 h) of fluid appearance (endodermal fluid accumulation, Fig. 5D). These ICMs occasionally gave rise to endodermal vesicles if culture
324
J. C. CHISHOLM AND OTHERS
was extended to 5-6 days; c) solid embryoid structures, usually showing cell rounding or proliferation, and frequently attached to the culture dish by cells which
outgrew within 24 or 48 h of isolation (Fig. 5E); d) a few ICMs spread out to form
a monolayer of cells, usually within 24 or 48 h of isolation (Fig. 5F). It is unclear
whether this form of development represented trophectodermal or endodermal
differentiation. Occasional cells in the outgrowths showed enlarged nuclei (Fig. 5F)
although Solter & Knowles (1975) report outgrowth of polygonal endoderm-like
cells from isolated ICMs. In this study the very few ICMs which formed monolayer
outgrowths were scored as showing endodermal development (Table 5).
B
5A
I
F
G
Fig. 5. Morphology of isolated ICMs differentiating in vitro
(A) Blastocyst-like vesicle.
(B) Trophectoderm generation of a 'non-integrated' form.
(C) Endodermal vesicle. A 'rind' of endodermal cells surrounds a solid core of embryonic ectoderm.
(D) Endodermal fluid accumulation.
(E) Solid embryoid structure, attached to culture dish by outgrowing cells.
(F) ICM spread over culture dish to form a monolayer of cells.
(G) Mixed character development by a single ICM.
Scale bar = 40 jwm.
Variability of mouse early development
325
A small proportion of ICMs appeared to show evidence of mixed differentiation
(at the light microscope level), developing both trophectoderm and endoderm-like
cells in outside positions (Fig. 5G). Generally, in these ICMs vesiculating trophectodermal cells were first noted at 48 h or later postisolation and by 72 h solid
6A
B
Fig. 6. (A) Part of trophectoderm cell from 4h ICM after 72 h in culture. The cell is
elongate, enclosing a blastocoel-like cavity (b), and possesses few microvilli
(arrowheads). These are typical features of trophectoderm cells (Nadijcka & Hillman,
1974). Additionally the cell contains much amorphous cytoplasmic material (am) an
observation common in trophectoderm generated by ICMs cultured in vitro for 20 h or
more from the start of blastocoel formation (Fleming et al. 1984). Bar = 2jum.
(B) Endodermal cell from 12 h ICM after 48 h in culture. The cell has a microvillous
apical surface (mv), numerous apical vesicles (v) with evidence of endocytotic activity,
and is joined to a neighbouring cell by an apicolateral junctional complex (/). The rough
endoplasmic reticulum is dilated by accumulation of a dense intracisternal material
(arrows). These features characterize visceral endoderm (Enders, Given & Schlafke,
1978).
326
J. C. CHISHOLM AND OTHERS
Table 6. Comparison of light microscope {LM) and transmission electron
microscope (TEM) classification of cell types differentiating in outside positions in
isolated ICMs during in vitro culture
TEM Classification
LM classification
Trophectoderm *
Endodermt
Mixed trophectoderm and
endoderm
No. of
ICMs
Trophectoderm
only
12
3
11
3
Endoderm
only
Mixed
trophectoderm
and endoderm
1
3
2
1
*Nine of these ICMs showed trophectoderm-like vesiculation within 48 h of isolation. The
remainder showed limited vesiculation at 72h and were difficult to score, often looking similar
to 'mixed-character' structures (line 3). Vesiculating trophectoderm cells frequently collapsed by
72 h of ICM culture and resembled outer cells of the solid embryoid structures which lacked a
clear 'rind' of endodermal cells.
t All three ICMs showed an outer 'rind' of endoderm.
endodermal or occasionally endodermal vesicle structures were also present. These
mixed character structures were observed to develop from ICMs at any age between
2 and 12 h (with the exception of 11 h ICMs; Table 5). We confirmed this result in
a small number of aggregates made by pairing an early (2 h) ICM with that of a late
(12 h) ICM in which cells are likely to be predominantly uncommitted and committed respectively to the ICM lineage and might therefore generate a mixed
population of trophectoderm and endoderm cells in outside positions in the
aggregate.
To check the proposed classification of ICM development made by morphological observations at the light microscope (LM) level, some ICMs and ICM
aggregates were scored by LM at 24 h intervals, fixed after 48 h or 72 h in culture
and processed for transmission electron microscopy (TEM). Trophectodermal and
endodermal cell types in cultured ICMs are known to resemble the trophectoderm
and extraembryonic endoderm cell types respectively of intact embryos in their
ultrastructural features (Hogan & Tilly, 1978) and are thus clearly distinguishable
from one another. Extraembryonic endodermal cells may further be classified as
Fig. 7. 12 h ICM showing mixed character development after 48 h in vitro. (A) Both
trophectoderm (i) and visceral endoderm (e) cells are present in outside locations in the
cultured ICM. Bar = 5 ^m.
(B) Junctional region between trophectoderm and endoderm cells, outlined in 7A. The
trophectoderm (t) cell is elongate, with its basolateral membrane (arrows) in close
contact with an inside cell (ic). It lacks apical microvilli and contains amorphous
cytoplasmic material (am; see Fig. 6A). It is joined by an apical junctional complex (/)
to a visceral endoderm-like cell (e) which, though it lacks a distinct microvillous pole,
contains apical vacuoles and dilated endoplasmic reticulum (arrowheads). Bar = 1 jum.
Variability of mouse early development
B
327
328
J. C. CHISHOLM AND OTHERS
either visceral or parietal by their ultrastructure (Hogan & Tilly, 1981). Electron
micrographs of typical trophectoderm and endoderm cells generated by cultured
ICMs are shown in Fig 6.
A comparison between LM and TEM classification of ICM development is
shown in Table 6. While it is clear that there is reasonably good agreement between
LM and TEM classification, there is also some discrepancy in the results. From
Table 6 it is apparent that: 1) all ICMs classified by LM as showing only trophectoderm cells without endoderm gave evidence of some trophectoderm at the TEM
level, although it was noted that by 72 h many vesiculating structures had collapsed
on themselves, with subsequent cell rounding and LM morphology not dissimilar
from endoderm; 2) endodermal vesicles showing an outer layer of cells surrounding
a solid inner core always possessed an outer layer of endoderm cells (mostly visceral
endoderm-like) as expected; 3) evidence was obtained in two ICMs for both
trophectoderm and endoderm cells occupying outside positions (Fig. 7), although
this did not correlate well with LM classification of mixed-type structures. Both of
the ICMs which were judged to be mixed in structure by LM but by TEM showed
only endoderm-like cells (Table 6, line 3), contained highly attenuated parietal
endoderm-like cells enclosing a cavity. These vesiculated endoderm cells may have
been mistakenly identified as late-vesiculating trophectoderm. TEM evidence of
mixed development was also obtained for aggregated pairs of ICMs from early and
expanded blastocysts (Fig. 8).
Interpretation of these results must take into account the fact that a section of an
ICM at any one level allows identification of cell type for only a proportion of cells
of that ICM. In this study, ICMs were sectioned at 5 /im and 8 jum for examination,
but it remains impossible to examine every cell unless serial sections are made.
Nevertheless, from these results we conclude that overt appearance of trophectoderm in LM is a good indicator of the actual presence of trophectoderm cells
although they may also be present in situations where there is no vesiculation or the
cavity has collapsed and they will therefore not be seen by LM. Thus it is likely that
the proportion of ICMs of each age generating trophectoderm is slightly higher and
that generating endoderm slightly lower than Table 5 and Fig. 4 suggest. A more
rigorous test for the presence of trophectoderm in ICMs would be giant cell outgrowth in vitro (Hogan & Tilly, 1978) or the initiation of a decidual response when
replaced in utero (Rossant & Lis, 1979). Additionally, we have shown that both
trophectodermal and extraembryonic endodermal cell types can coexist in outside
positions in an isolated ICM and in ICM aggregates, although this may occur less
frequently than expected from LM classification.
Fig. 8. Embryoid body derived from the aggregation of a 2 h and 12 h ICM and cultured
for 48h before fixation. (A) Trophectoderm (t) and visceral endoderm (e) phenotypes
are present in the outside cell layer. Bar = 5 /im. (B) Region of contact between trophectoderm (t) and endoderm (e) cells from a section adjacent to that shown in (A). For
details of morphology see legend to Fig. 7. Bar =
Variability of mouse early development
329
330
J. C. CHISHOLM AND OTHERS
DISCUSSION
We have confirmed previous observations (McLaren & Bowman, 1973; Smith &
McLaren, 1977; Copp, 1979; Rossant & Lis, 1979) that the developmental
heterogeneity among expanding blastocysts is considerable, and show that it is in
excess of 10 h and probably of the order 15 h. Most of this heterogeneity can be
accounted for by the interembryo developmental variability evident at the 8-cell
stage, with only a small component of variability being introduced later. Thus,
there may be as much as 10 h asynchrony among embryos at first cleavage, of which
2-3 h results from an inherent variability in the length of the first cell cycle and the
remainder from differences in the timing of ovulation and/or fertilization of
oocytes in vivo (Bolton et al. 1984; Howlett & Bolton, 1985). During the second
cell cycle, lasting about 18 h, a further increase in asynchrony of about 2h is
introduced among the population of 2-cell blastomeres, with a further increase of
about an hour by the 4-8 cell transition (Smith & Johnson, 1985). Indeed, our
results suggest that between the generation of 8-cell blastomeres and cavitation
(around 32 cells) developmental heterogeneity increases by only 2-3 h further in a
total period of around 30h, i.e. by about l h per cell cycle. This finding that
heterogeneity increases relatively slowly is in accordance with the observation that
mean cell cycle time between the 4- and 16-cell stages is independent of the length
of the cycle in the progenitor cells at the 2-cell stage, showing that a short second
cell cycle is not apparently an heritable feature (Kelly et al. 1978). We attempted
to reduce heterogeneity for experimental purposes by examining embryos at
regular intervals, and collecting those that had commenced cavitation. This
approach should synchronize embryos to the time, within the limits of the inspection interval, at which at least some of their constituent outer cells form an effective
zonular tight junctional complex and engage in vectorial transfer of fluid (Ansell &
Snow, 1974). Although such an approach might reduce heterogeneity between
embryos, it will not eliminate it, since within embryos (a) there is likely to be
heterogeneity within the population of outside cells and some outside cells may
transport and retain fluid earlier than others (see Surani & Barton, 1984; Fleming
et al. 1984), (b) the rate of inside cell development, presumably also itself
heterogeneous, may not necessarily be coupled closely to the rate of outside cell
development, and (c) cells can cross lineages and may therefore contribute to
fluctuating cell ratios if not to total cell numbers. It is probable that the variation
in total cell number of the embryos found at any given age postcavitation arises
partly for these reasons, although a varying incidence of cell death (Copp, 1979) and
loss of cells during manipulation will also account for some variation. Overall, the
impression obtained from Tables 2, 3 and 4 is of very considerable variation in the
number and distribution of cells within an embryo even when these embryos are
synchronized to the same developmental event. Since we do not know whether all
embryos are equally viable, regardless of their cellular constitution, it is impossible
to assess whether the extremely atypical embryos represent failures of an attempted
Variability of mouse early development
331
regulation towards a norm condition that the majority of embryos have achieved
successfully (Johnson & Ziomek, 1983; Surani & Barton, 1984).
With these considerations in mind, and despite the considerable inter-embryo
variation, the agreement in Table 2 of mean values arrived at independently by the
four different techniques used is remarkably good. It is evident that at each point
the highest total cell number was always recorded in the serially sectioned zonaenclosed embryos. This result indicates that use of the other three manipulative
techniques, however carefully performed, is likely to result in some cell losses.
Comparison of the four techniques for inside and outside mean cell numbers hints
that the loss might be slightly greater for the former than the latter. The only major
discrepancy in Table 2 is within the 12 h blastocyst group, in which the mean
number of inside cells is low by DAPI staining and the total (and therefore also
outside) cell number is low by Giemsa staining. Undercounting of DAPI-stained
inside cells at later stages could be due to the greater difficulty in disaggregation of
decompacted 12 h ICMs to single cells. Fluorescent nuclei that overlap are difficult
to distinguish. Similarly, the larger total number of nuclei in Giemsa-stained preparations of whole blastocysts could easily lead to underestimates due to an increased
chance of superimposition. Additionally, it is possible that the cells undergo mitosis
at around 12 h postcavitation, in which case slight interexperiment variations in
timing might give large differences in the results observed.
The 0 h and 2 h groups (Table 2) both contain embryos with a mean value of just
under 32 cells distributed in an inside:outside ratio of just less than 1:2. These mean
values, and the variation about them, are similar to those reported by Smith &
McLaren (1977) for nascent blastocysts and agree fairly well with our counts of 0 h
total cell numbers in MFI embryos (Table 1). The distribution of inside cell volumes
at these stages is almost unimodal, with some evidence of a minor second peak of
cells of approximately double the volume. Taken together, these data suggest that
most ICMs at Oh and 2h contain cells that are in their sixth developmental cell
cycle, with some inside cells in at least some ICMs still in their fifth cell cycle.
Presumably those cells in the fifth cell cycle are more likely to be in ICMs derived
from blastocysts with less than 32 cells. As is shown in Table 3, most such ICMs
contain 7-10 cells, and this range shifts to 9-14 by the 32-cell stage. As reported in
the Introduction, estimates of inside cell numbers at the 16-cell stage range between
1 and 8; 9-14 inside cells at the 32-cell stage could be achieved by a simple doubling
of 5-7 inside cells, but a substantial contribution to the inside cell population by
outer cells could be required were the lower estimates of 1 or 2 inside cells at the
16-cell stage to be correct.
Comparative values for outside cell numbers at 0 h and 2 h reveal a shift from
around 20 to greater than 20. Since the time of visible evidence of vectorial transport and accumulation of fluid by outer cells seems to occur at about 20-24 h after
the beginning of the fifth cell cycle (Ziomek, Johnson & Handyside, 1982), and
since the fifth cell cycle is 11-14 h long (MacQueen & Johnson, 1983), presumably
most TE cells in nascent blastocysts are in their sixth cell cycle with some more
332
J. C. CHISHOLM AND OTHERS
advanced outer cells entering the seventh cell cycle as blastocyst expansion
progresses. This conclusion is supported by the observation that only embryos in
which presumptive outer cells have entered their sixth cell cycle will form a blastocoel (Smith & McLaren, 1977; Braude, 1979), and by the incidence of mitoses in
the outside cell population of more advanced embryos (Table 3). Thus the simplest
explanation for the numerology of Oh and 2h blastocysts is that outer cells, as a
population, are a little ahead developmentally of inner cells. Direct evidence that
the fifth cell cycle is on average 2 h shorter for isolated outer cells than for isolated
inner cells has been presented (MacQueen & Johnson, 1983), but indirect evidence
for cells in situ indicated the opposite (Barlow etal. 1972). Surani & Barton (1984)
have suggested recently that in situ inside cells may act to extend physically the
outside trophectoderm cells so that the division of the latter is delayed, or even
inhibited. This suggestion might resolve the apparent conflict within published cell
cycle data reported above. However, it seems unlikely that delay or inhibition
occurs on a large scale as outside cell numbers continue to increase (Table 2), and
there is no evidence that this increase is due to an extensive contribution from the
ICM (Fleming et al. 1984), indeed mitoses are evident within the trophectoderm
throughout expansion of the blastocyst. However, it is clear from Table 2 that when
Oh and 12h embryos are compared, no technique demonstrates that a doubling of
outside cell number has occurred. There are several possible explanations for this
relative deficiency. It is unlikely that consistent losses of outside cells during
analysis are involved since all techniques indicate their underrepresentation. It is
possible that outside cells have a longer sixth cell cycle than inside cells; however
the evidence on this point is contradictory, one study supporting this idea (Barlow
et al. 1972) and one other suggesting the reverse, i.e. that the inside cells have a
prolonged S phase (Kimura & Kato, 1980). It is also possible that some outside cells
either die and are removed, or contribute to the ICM during expansion; however,
there is no evidence for this (Copp, 1979). Finally, it is possible that there is
heterogeneity among outside cells, some, possibly mural trophectoderm, undergoing mitotic slowing due to their extreme stretching, whilst others, like polar
trophectoderm, proliferate (Copp, 1979; Surani & Barton, 1984).
In contrast, the number of inside cells has doubled by 12 h postcavitation
(Tables 2 and 4) and their modal volume has halved (Fig. 3), an observation
most easily explained if all or most inside cells were "breeding true to lineage"
and that most had entered their seventh cell cycle. It is clear from the detailed
analysis of ICM cell numbers in Table 4 that ICM cells are not entering the
seventh cell cycle synchronously. There is a suggestion of a plateau in ICM cell
numbers at 14-16 cells over the period 3-6 h postcavitation which might
represent the completion of the fifth cell cycle by the slowest cells and the period
of Gi of the sixth cell cycle for the fastest cells. However, the values observed
for total cell number, mitotic indices and DNA C-values for individual embryos
combine to present an impression of great heterogeneity spanning almost the
length of a full cell cycle.
Variability of mouse early development
333
The finding that some ICMs or ICM aggregates showed both trophectodermal
and endodermal differentiation by cells occupying outside positions demonstrates
that it is possible for both cell types to be expressed together during in vitro ICM
culture and to form an integrated, mixed epithelium. This phenomenon would be
expected to result from coexistence in the ICM or ICM aggregate of committed cells
(which would then generate endoderm) and uncommitted cells (which can generate
trophectoderm) that become exposed to an atypical outside position on ICM isolation. The apparent asynchrony in the timing of cell commitment in some ICMs,
coupled with cell cycle asynchrony in most ICMs, is compatible with but not proof
of a relationship between the cell cycle and commitment.
Previous reports on ICM morphology during in vitro culture do not record mixed
character development, although recently Nichols & Gardner (1984) have demonstrated that both parietal endoderm and trophectoderm cells can arise from the
population of outside cells during outgrowth of a single ICM in vitro. However, the
usual formation of blastocyst-like vesicles by ICMs does not necessarily preclude
frequent coexistence of committed and uncommitted cells within a single ICM, as
it is likely that both the ratio of uncommitted to committed cells and the relative
positions of such cells within the ICM are crucial factors influencing the type of
structure formed by a cultured ICM or ICM aggregate. Trophectoderm cells or
outside cells tend to surround and enclose inside cells (Rossant, 1975; Johnson &
Ziomek, 1983) and this capacity to envelop is acquired as an extremely early event
during the transformation of uncommitted ICM cells into trophectoderm (Fleming
et al. 1984). Thus, on ICM isolation, those uncommitted cells which become
exposed on the surface of the ICM will almost immediately surround and enclose
all committed cells, and also any uncommitted cells occupying internal positions
within the ICM which thus become unable to express their potential for trophectoderm formation. A blastocyst-like vesicle will therefore form in which all committed cells become inside cells. Only where the proportion of uncommitted cells
is low, or where few are exposed in an outside position, might they fail to enclose
all committed cells. Unenclosed committed cells will then express their potential for
endoderm formation alongside the trophectoderm cells generated by uncommitted
cells, thus giving rise to a structure of mixed character.
The developmental age at which commitment of inside cells takes place is
seemingly later than was indicated by other work. Previous estimates of 14 (Spindle, 1978) or 16-19 (Rossant & Lis, 1979) inside cells at the time of commitment
suggested that it took place when all cells had on average completed their fifth
cleavage division and some their sixth. In this study however, the mean ICM cell
number at 12 h postcavitation was about 20 and yet 69 % of ICMs apparently
generated some trophectoderm on isolation (Fig. 4). This finding suggests that, if
commitment is cell cycle related, it takes place late in the sixth or early in the
seventh developmental cycle. Since 12 h blastocysts were always expanded, commitment was occurring to a large extent after blastocoel expansion and not during
the process of expansion, as suggested by Handyside (1978). Our results are similar
334
J. C. CHISHOLM AND OTHERS
to those of Hogan & Tilly (1978) who found that 50 % of ICMs from expanded
blastocysts generated trophectoderm in vitro.
Owing to the evident heterogeneity between cavitation time and inside cell
development, and between cell cycles within individual ICMs, it will be necessary
to find more accurate methods of timing and/or synchronizing inside cell cycling if
the relationship between cell cycle and cell commitment is to be investigated effectively.
We wish to thank Ken Thurley, Raith Overhill, Tim Crane, Roger Liles and Gin Tampkins for
their technical help, Dr M. Bennett for use of the microdensitometer at the Plant Breeding
Institute, Cambridge and our research colleagues for their critical comment on the manuscript.
The work was supported by grants from the C.R.C. and M.R.C. to M. H. Johnson, by a grant
from Christ's College to P. D. Warren, and by a grant from the Nuffield Foundation to J. C.
Chisholm.
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{Accepted 21 November 1984)