/. Embryol. exp. Morph. Vol. 51, pp. 109-120, 1979
Printed in Great Britain © Company of Biologists Limited 1979
109
Interaction between inner cell mass and
trophectoderm of the mouse blastocyst
II. The fate of the polar trophectoderm
By A. J. COPP 1
From the Department of Zoology, Oxford
SUMMARY
Selective labelling of polar trophectoderm cells in early mouse blastocysts has allowed the
fate of polar cells to be followed during in vitro and in vivo blastocyst development. Results
show that there is cell movement from polar to mural regions as blastocysts grow. This
indicates that trophectoderm cells directly opposite the inner cell mass are the oldest mural
cells. However, after implantation polar cells invaginate into the blastocoelic cavity and
contribute to the extra-embryonic ectoderm. It is suggested that the morphogenetic changes
occurring in the mouse embryo at implantation result from the maintenance of a balance
between (a) regional differences in rates of cellular proliferation, and (b) mechanical constraints
on the direction in which growth can occur.
INTRODUCTION
It was predicted in a previous paper, on the basis of cellular proliferation
rates in the various blastocyst regions, that there is cell movement from polar
to mural trophectoderm as blastocysts develop (Copp, 1978; see also Gardner &
Papaioannou, 1975). This paper reports experiments in which the fate of polar
trophectoderm cells was followed, both before and after implantation, using
melanin granules as a marker. Trophectoderm cells readily phagocytose melanin
granules, and retain them at least until implantation has occurred, apparently
with no subsequent transfer of granules between cells (Gardner, 1975). Furthermore, melanin granules are easily visualized in standard histological preparations. Therefore, apart from the problem of dilution as labelled cells continue
to divide, melanin granule labelling fulfills most of the requirements for a useful
biological marker (Weston, 1967).
MATERIALS AND METHODS
Blastocysts were flushed from the uteri of pregnant CFLP females between
3 days 14 h and 3 days 16 h after the estimated time of ovulation (see Copp,
1978), and were stored in PB1-medium (PB1, Whittingham & Wales, 1969)
1
Author's address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K.
8
EMB 51
110
A. J. COPP
100
Fig. 1. Partially polar-herniated blastocyst before labelling.
Fig. 2. Completely polar-herniated blastocyst after labelling.
plus 10 % heat-inactivated foetal calf serum (FCS). Any blastocysts which had
collapsed were allowed to re-expand in PB1 + FCS at 37 °C. Each expanded
blastocyst was held by suction and a slit was made in the zona pellucida over
the polar trophectoderm using a pair of straight needles controlled by a Leitz
micromanipulator assembly (Gardner, 1978). Operated blastocysts were
cultured at 37 °C in PB1 + FCS under paraffin oil (Boots, U.K. Ltd) until
blastocoelic expansion caused herniation of the inner cell mass (ICM) plus
covering polar trophectoderm through the slit in the zona pellucida. When part
of the polar region had emerged, herniation was arrested by removing blastocysts to PB1 + FCS at room temperature. A suspension of melanin granules
was prepared by teasing apart retinae from pigmented mice of various strains in
alpha modification of Eagle's medium supplemented with 30 fim adenosine,
guanosine, cytidine and uridine, and 10 /an thymidine (a, Flow Labs) plus
10 % foetal calf serum. Partially herniated blastocysts (Fig. 1) were cultured in
hanging drops of this suspension for 1 h at 37 °C, in an atmosphere of 5 % CO2
in air, after which most embryos showed complete polar-herniation (Fig. 2).
Any blastocysts which had collapsed or showed mural herniation were rejected.
All others were washed thoroughly in a + FCS to remove loosely adhering
granules and their zonae were removed in acid Tyrode's solution, pH 2-5
(Nicolson, Yanagimachi & Yanagimachi, 1975).
In order to study the fate of polar trophectoderm cells before implantation,
labelled blastocysts were cultured in a + FCS, in bacteriological plastic dishes
(Sterilin) under 5 % CO2 in air. After either 1 or 24 h of culture, blastocysts
were fixed and prepared for analysis by serial reconstruction as described
previously (Copp, 1978). Cell numbers were determined by counting nuclei in
both the polar and ICM regions and in the mural subregions. Since cell outlines
were not always easy to see, the number of labelled cells was estimated
Fate of mouse polar trophectoderm
111
indirectly. The distribution of melanin granules was usually clumped, and it was
considered that each 'clump' of melanin granules corresponded to a labelled
cell. A 'clump' was defined as one or more granules occurring within a volume
of cytoplasm which extended around each nucleus in any direction for a distance
equal to the average length of a nucleus in that particular region. However, it
was decided that 'clumps' should be confined within regional boundaries and
therefore they were usually more extensive in certain directions than others
(e.g. elongated, narrow 'clumps' in the polar region). In this way, 'clumps'
were intended to resemble cells in both size and shape (Fig. 3). The 'clump':cell
number ratio gave an estimated labelling index for each blastocyst region. In
order to control for a possible time-dependent spreading of melanin granules
in all directions throughout the blastocyst, mural-herniated blastocysts were
also labelled and analysed in the same way (Figs. 4 and 5). It was not possible
to control precisely whether the proximal or distal mural subregion herniated,
so all mural-herniated blastocysts were pooled for analysis.
The fate of polar trophectoderm cells after implantation was followed by
transferring polar-labelled blastocysts to the uteri of pseudopregnant recipients
3 days 17 h after the estimated time of mating to sterile males. Labelled blastocysts were transferred to one uterine horn of each recipient female, and the
contralateral horn received an equal number of unlabelled polar-herniated
blastocysts. Recipients were killed 38 h later and uteri containing implantation
sites were prepared for histological examination as described previously (Copp,
1978). Embryos had reached a very early egg-cylinder stage in which the polar
trophectoderm was multilayered (Fig. 6). Extra-embryonic ectoderm was
clearly forming in all embryos but none showed an accumulation of polar cells
above the level of origin of the primary giant cells, indicating that the ectoplacental cone had not yet developed. Extra-embryonic ectodermal cells could
not be reliably disinguished from more superficial polar cells, and so the whole
polar region was analysed together. It could be distinguished from the embryonic
ectoderm on the basis of: (a) its greater intensity of cytoplasmic and nuclear
staining; (b) the shape and orientation of its nuclei, and (c) a space between the
two regions. In addition, primary trophoblastic giant cells, visceral and parietal
endoderm were recognizable. Serial sections were scored for the presence or
absence of each tissue and for the presence of one or more labelled cells within
them.
RESULTS
Pre-imp Ian tat ion developmen t
Twenty-four polar-labelled and 19 mural-labelled blastocysts were analysed.
The number of melanin granules per blastocyst ranged from 28 to 648, except
for a single polar-labelled blastocyst which contained four granules. This was
felt to be an abnormally low level of labelling and the blastocyst was excluded
from the granule-distribution analysis. The average number of granules per
8-2
112
A. J. COPP
A
Fig. 3. Drawings of serial sections of polar-labelled blastocysts fixed after (A) 1 h,
and (B) 24 h of culture. Nuclei are drawn in outline. Dashed outlines indicate nuclei
which were only faintly visible. Dots represent melanin granules, rectangles enclose
'clumps'. Note that 'clumps' usually extend to adjacent sections.
113
Fate of mouse polar trophectoderm
100 Him
Fig. 4. Partially mural-herniated blastocyst before labelling.
Fig. 5. Completely mural-herniated blastocyst after labelling.
Polar region
Primary giant
cells
Embryonic
ectoderm
Visceral
endoderm
Parietal
endoderm
Fig. 6. (A) Section and (B) drawing of an early egg-cylinder developed from a
polar-labelled blastocyst transferred to the uterus of a pseudopregnant recipient.
Arrows indicate melanin granules.
embryo did not differ significantly between polar-labelled blastocysts cultured
for 1 and 24 h and the same was true for mural-labelled blastocysts (Table 1).
This indicates that there is no substantial loss of melanin granules from blastocysts during 24 h of culture. Since there appears to be no transfer of granules
between cells (Gardner, 1975), any alteration in the relative numbers of granules
in different blastocyst regions must therefore indicate a redistribution of granulecontaining cells, or their progeny, as development proceeds.
Table 2 shows the distribution of melanin granule ' clumps' in blastocysts
after 1 and 24 h of culture. These results indicate that, for polar-labelled
114
A. J. COPP
Table 1. Numbers of melanin granules in polar- and mural-labelled
blastocysts after 1 and 24 h of culture
Region
labelled
Hours of
culture
Number of
blastocysts
Average
number of
melanin granules
per blastocyst
Polar
1,
24
x1
24
...
11
13
s9
10
..^v,
112-6
171-3
322-3J
ji.^.
216-6
Mural
P
I,
1?4
\' 1 - 2 5
>
0-()5
> 01
* Student's ^-values from 'comparison of two means' tests.
blastocysts: (1) there is an increase in the proportion of labelled cells in the
proximal mural subregion after 24 h of development; (2) there is no comparable
increase in distal and mural labelling; (3) there is no fall in the proportion of
labelled polar cells, showing that all polar daughter cells inherit granules at
least during the next 24 h of development. However, there is some dilution of
label during this period since the average number of melanin granules per polar
'clump' fell from 8-6 after 1 h, to 5-9 after 24 h of culture; and (4) ICM labelling
is negligible. In addition, the failure of polar labelling indices to reach 100 %
after 1 h of culture may indicate that a ' clump' is equivalent, on average, to
more than one labelled cell. This inaccuracy applies equally to all blastocyst
regions and so does not affect the conclusion of this experiment. The low levels
of labelling observed in 'unlabelled' trophectodermal regions after 1 h of culture
presumably represents either the penetration of granules between embryo and
zona pellucida at the time of labelling, or secondary attachment of loose
granules soon afterwards. Material resembling melanin granules could also be
deposited during the histological procedure.
Table 2 shows for mural-labelled blastocysts: (1) there is no time-dependent
spread of label from mural to polar regions; (2) as development proceeds there
is a fall in the proportion of labelled cells in the proximal mural subregion; and
(3) there is no change in the level of distal mural labelling after 24 h culture.
However, an influx of cells into this subregion during blastocyst development
cannot be ruled out since a number of blastocysts were labelled initially in the
distal mural subregion (see Materials and Methods). The marginally significant
difference between levels of labelling in the polar regions of blastocysts 1 and
24 h after mural-labelling can probably be attributed to a single blastocyst,.
fixed after 1 h, which had a very high labelling index (66 %) in this region.
These results demonstrate a movement of cells from polar to proximal mural
trophectoderm during mouse blastocyst development. Furthermore, they are
consistent with a movement of cells, during the same period, from proximal to
distal mural subregions.
12
9
10
1
24
1
24
Polar
Mural
11
Hours of
culture
Region
labelled
< 0001
'
14 54
2U
46
85
36T
159
49
> 005
> 005
41-71
5411
13-57
10-69
/o
°/
Polar
>005
10-37
81-58
75-49
/o
5
3-42
146
005 > P > 0025
135
14
117
155
124
152
No. 'clumps'
No. cells
* P-values from ^ 2 'comparison of two proportions' tests.
TP,
447
-«
i
< 0001*
12-24
^
294
%
No. 'clumps'
No. cells
A
A
Number of No. 'clumps'
No. cells
blastocysts
Distal
Proximal
A
Mural
42l
8
2
236
3
299
7
464
> 005
> 005
No. 'clumps'
No. cells
ICM
1-90
0-85
1-51
100
/o
Table 2. Distribution of melanin granule ''clumps'' in polar- and mural-labelled blastocysts after 1 and 24 h of culture
>
I"
now. fo
3
•*?
vj
phectt
116
A. J. COPP
68
20
1
1
2
1
19
10
0
0
E
C
Primary giant
cells
I
E
C
Polar
region
0
E
C
Embryonic
ectoderm
7777?
E
C
Visceral
endoderm
E
C
Parietal
endoderm
Fig. 7. The distribution of melanin granules in egg-cylinders developed from
polar-labelled blastocysts (E) and from unlabelled polar-herniated blastocysts (C).
Bars represent the average number of sections in which each tissue appeared. Hatched
regions represent the proportion of these sections in which the tissue was labelled.
Numbers above bars indicate the total number of melanin granules counted.
Post-implantation blastocyst development
Five early egg-cylinders developed from polar-labelled blastocysts and five
control embryos developed from unlabelled polar-herniated blastocysts were
analysed. Figure 7 shows, for each tissue, the average number of sections in
which the tissue appeared, and the proportion of these sections which contained
one or more labelled cells. The actual number of labelled cells was very low, and
for instance, represented only a small fraction of the actively proliferating polar
trophectoderm. This illustrates the limitations of melanin granules as a marker
for post-implantation trophoblastic development when rapid dilution of granules
occurs. Nevertheless, the primary trophoblastic giant cells and multilayered
polar trophectoderm were consistently labelled, and granule-containing cells
occurred frequently deep within the newly forming extra-embryonic ectoderm
(which was included in the 'polar trophectoderm' fraction) as well as more
superficially in the mesometrial part of the polar region (Fig. 6). Control levels
of labelling were very low, as expected, and probably represent material,
resembling melanin granules, which occurs endogenously in the uterus or was
deposited during the histological procedure.
Fate of mouse polar trophectoderm
111
These results indicate that, after implantation, polar cells begin to invaginate
into the blastocoelic cavity and contribute to the developing extra-embryonic
ectoderm.
DISCUSSION
The results presented in this paper demonstrate that, during mouse blastocyst
development, there is a shift of cells from polar to proximal mural trophectoderm, without any significant contribution to the distal subregion of later
blastocysts. In addition, cells appear to leave the proximal mural subregion,
during the same period of development, and these presumably enter the distal
mural subregion. This evidence provides support for the idea that trophectoderm cells directly opposite the 1CM are the oldest mural cells (Gardner &
Papaioannou, 1975; Copp, 1978). If continued mural cell division depends
directly on the length of time that cells have been out of contact with the ICM,
this can explain why mural cells directly opposite the ICM are the first to cease
division and begin giant cell transformation.
The analysis of polar-labelled blastocysts transferred to recipient uteri shows
that, after implantation, polar cells invaginate into the blastocoelic cavity, and
contribute to the extra-embryonic ectoderm. A number of previous experiments
have indicated a trophectodermal origin for this tissue (Gardner & Johnson,
1975; Rossant & Ofer, 1977) but the present experiment provides the first
direct demonstration of this.
The results presented here, and elsewhere (Gardner & Papaioannou, 1975;
Copp, 1978) provide the basis for a model (Fig. 8) which attempts to explain the
morphogenetic events leading to egg-cylinder formation. Before implantation,
while the blastocyst is free floating in the uterine lumen, there is cell movement
from polar to mural trophectoderm (Fig. 8a). At the time of attachment to
the uterine epithelium, however, mechanical constraints are imposed on the
blastocyst so that further polar to mural cell movement is prevented. Continued
polar cell division leads to an accumulation of cells over the ICM. An increase
in volume of the polar region is inevitable and will occur in the direction of least
physical resistance. Careful histological analysis of this period of development
indicates that, at the mesometrial end of the implantation crypt, the walls of the
uterine lumen are tightly apposed (see Fig. 3 in Reinius, 1965) and so mesometrial growth would require the walls to be forced apart. On the other hand,
anti-mesometrially the blastocoel presents no such mechanical barrier to growth.
It is suggested, therefore, that growth occurs initially in an anti-mesometrial
direction (Fig. 86), and this idea is supported by the observation that very early
egg-cylinders have small ectoplacental cones but well developed extra-embryonic
ectodermal regions (see Fig. 6, and also figure 12.7 in Snell & Stevens, 1966).
The lateral walls of the uterine luminal crypt, with covering primary giant cells
and parietal endoderm, are closely apposed to the sides of the growing eggcylinder (see figure 4 in Reinius, 1965). Gaps between visceral and parietal
118
A. J. COPP
Fig. 8. Model to explain egg-cylinder morphogenesis in the mouse.
See text for explanation.
endoderm normally seen in wax sections are probably artifactual. Consequently,
growth of the egg-cylinder will be predominantly anti-mesometrial and not
lateral. Once the anti-mesometrial tip of the egg-cylinder comes into contact with
the relatively slowly expanding Reichert's membrane, its further growth in this
direction is restricted. The path of least resistance to polar growth, now, is in a
mesometrial direction since the ectoplacental cone can develop by forcing apart
the apposed walls of the uterine lumen (Fig. 8 c). This series of morphogenetic
events is completed within about 12 h of the onset of implantation, with proamniotic cavity formation occurring mainly during the third phase (Fig. 8 c)
of the sequence.
This model visualises in vivo egg-cylinder morphogenesis as resulting inevitably
from the maintenance of a balance between regional differences in cellular
proliferation rates and mechanical constraints on the direction in which growth
may occur. However, it has been reported that approximately normal eggcylinder development may occur, in the absence of uterine mechanical constraints, after blastocyst outgrowth in vitro (Hsu, Baskar, Stevens & Rash, 1974;
Pienkowski, Solter & Koprowski, 1974; Wiley & Pedersen, 1977). Mechanical
constraints may, nevertheless, operate in such systems if there is an increase in
the strength of adhesion between trophoblast giant cells and their substratum
soon after the onset of blastocyst outgrowth. This could result in a redirection
of polar cell movement so that polar cells accumulate beneath the ICM and
egg-cylinder formation results. Studies of in vitro egg-cylinder morphogenesis
have indicated that polar cells cease to move into the trophoblast giant
cell outgrowth shortly before egg-cylinder formation, as predicted by this
hypothesis (A. J. Copp, in preparation).
Fate of mouse polar trophectoderm
119
In other developing systems it has been suggested that morphogenesis may
involve differential rates of cellular proliferation and mechanical constraints on
the directions of growth. For instance, developing chicken lens epithelial cells
initially elongate and subsequently the placode epithelium invaginates to form
the lens. It has been proposed that these events are the results of continued cell
proliferation within the placode and a restriction on the lateral spreading of the
increasing placode cell population due to firm contact between the lens epithelium and the underlying optic vesicle (Zwaan & Hendrix, 1973). Similar
explanations have been suggested for the evaginating thyroid gland, in which
lateral spreading of thyroid cells is prevented by local mechanical constraints
(Hilfer, 1973), and for the developing pancreas where a limitation on increase
in surface area of the rudiment occurs despite its continued cell number increase
(Pictet, Clark, Williams & Rutter, 1972). In addition, regional differences in
cellular proliferation rates have been noted in the branching salivary rudiment
(Bernfield, Banerjee & Cohn, 1972) and elongation of tubular glands in the
chick oviduct is blocked by inhibitors of cell division (Wrenn, 1971). Finally, it
should be noted that even in systems where regional differences in mitotic
activity do not occur (e.g. the primordial lung, Wessels, 1970) localized mechanical constraints on cell movements accompanied by continued general cell
division could nevertheless lead to the morphogenetic events observed.
I would like to thank Professor R. L. Gardner, Dr V. E. Papaioannou and Miss R.
Beddington for valuable discussion. This work was supported by a Christopher Welch
Scholarship and by the Medical Research Council.
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