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/ . Embryol. exp. Morph. Vol. 28, 2, pp. 255-261, 1972
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255
Experimental studies on the organization of the
preimplantation mouse embryo
II. Reaggregation of disaggregated embryos
By M. SUSAN STERN
From the Department of Zoology,
University College of North Wales, Bangor, U.K.
SUMMARY
The reaggregation and subsequent development of a range of disaggregated embryos has
been examined:
1. Following complete dissociation embryos from 8-cell to late blastocyst reaggregated and
developed to form morphologically normal blastocysts, even when blastomeres of two different
developmental stages were present in the reaggregate.
2. Dissociated mid-blastocysts could also reaggregate to form blastocysts but more
commonly they produced vesiculated masses, as did disaggregates of Jate blastocysts.
3. Successful fusion of pairs of mid or late blastocysts, with full cell numbers, was achieved
following partial dissociation.
These results are discussed in relation to blastocyst formation. It is suggested that even if
the embryo derives polarity from the oocyte it is not functionally essential to normal development in view of the remarkable regulative capacity of the egg.
INTRODUCTION
The preimplantation mouse embryo possesses a considerable degree of
organizational lability. It can regulate for removal of cells (Tarkowski, 1959 a, b;
Tarkowski & Wroblewska, 1967; Lin, 1969; Gardner, 1971), for increase in cell
number up to the early blastocyst (Tarkowski, 1961; Mintz, 1962, 1965), and for
differences in developmental age as when asynchronous pairs of eggs fuse (Mulnard, 1971; Stern & Wilson, 1972). Moreover, the production of egg fusions with
the peripheral cytoplasm of one member of each pair vitally labelled has suggested
that the fate of the presumptive trophoblast may not be determined until after
the early blastocyst stage (Stern & Wilson, 1972).
A detailed study of reaggregation and subsequent development of a range of
disaggregated mouse embryos (8-cell to late blastocyst) is presented. Similar
experiments have recently been reported by Lin & Florence (1970) but they
obtained only a few irregular blastocysts from reaggregates of isolated blastomeres of 4- and 8-cell eggs.
256
M. S. STERN
c
Fig. 1. (A) Mixed disaggregate of 8-cell and late morula. (B) is (A) after 5 h in culture.
(C) is (A) after 19 h in culture. (D) is (A) after 32 h in culture. (E) Blastocyst
developed from (A) after 45 h in culture. (F) Blastocyst developed from a mixed
disaggregate similar to (A).
Reaggregation of disaggregated embryos
257
Table 1. Reaggregation of disaggregates
Developmental stage
completely disaggregated*
Eight-cell
Early morula
Late morula
Early blastocyst
Mid-blastocyst \
Late blastocyst /
Early morula+ 4-cell
Late morula + 8-cell
No. of cells present
at the start of the
culture period
No. of reaggregates
producing blastocysts
8-22
15-32
18-25f
9-23 f
See text
Not counted
30-50
8/8
12/13
2/2
7/9
3
5/6
12/16
* Early morula = 16-cell. Late morula = ca. 32 cells and no cavitation. EarJy blastocyst =
cavitation just commencing. Mid-blastocyst = cavity occupies half the volume of the egg.
Late blastocyst = cavity occupies most of the volume of the egg.
f The low number of cells present in the initial disaggregates of later stages reflects the
technical difficulties in controlling the number of cells in each microdrop. However, since
blastocyst formation occurred regularly on these occasions development can be said to be
comparable to that which occurs when the full cell number is present.
MATERIALS AND METHODS
Spontaneously ovulated eggs, 8-cell to late blastocyst just prior to zona loss,
were recovered from randomly bred Q strain mice and the zona pellucida was
removed withpronase (see Stern & Wilson, 1972 for details). Zona-free eggs of the
same developmental stage were pooled and then disaggregated on an agar surface
(2 % in calcium- and magnesium-free Dulbecco) under liquid paraffin by gentle
pipetting in 0-02 % versene (in calcium- and magnesium-free Dulbecco, Tarkowski & Wroblewska, 1967). Following several washings in Earles balanced
salt solution (BSS) supplemented with bovine serum albumin and a final wash
in BSS supplemented with 30 % heat-inactivated calf serum, groups of cells
treated in this way, of the same or differing stages, were cultured at 37 °C in
microdrops of the serum-containing medium under liquid paraffin previously
equilibrated with 10 % carbon dioxide in air. All manipulations prior to culture
were performed at room temperature.
RESULTS
Complete disruption of organization, at least up to the early blastocyst stage,
appears to have little effect on subsequent blastocyst formation (Figs. 1F, 2-4),
even when the blastomeres from two different developmental stages are present
in the reaggregate (Fig. 1F) (Table 1). Moreover, the process of reaggregation
was similar in all cases. The pooled, isolated blastomeres which were sufficiently
close together reassociated to form an increasingly more compact mass of
cells (Fig. 1A-D) and, with the accumulation of fluid, produced a blastocyst
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M. S. STERN
Reaggregation of disaggregated embryos
259
(Fig. IE, F). This took between 24 and 48 h, depending on the age of the
blastomeres at the start of reaggregation.
Middle and late blastocysts proved to be exceedingly resistant to disaggregation, as has already been observed by Gardner (1971). The response, also, was
very variable; even after 4 h exposure to versene some neither collapsed nor
dissociated. Only three disaggregates of mid-blastocysts were obtained and
subsequently grown to blastocyst. More commonly, pooled blastomeres of these
late stages failed to form integrated units and produced vesiculated masses
(Fig. 6). However, when disaggregation was incomplete such that the cells of the
embryo were very rounded but still loosely associated, then conjoined pairs of
mid or late blastocysts, with full cell numbers, sometimes fused (Fig. 5, one of
five similar examples of late stage fusion). In addition to fusion, late preimplantation embryos treated in this way were found to regulate for removal of
large numbers of cells. Blastocysts with discrete inner cell masses developed
from disaggregates containing as few as 1/4 the number of cells normally present
(32-64) at that stage (Fig. 4).
DISCUSSION
There have been numerous studies concerning the factors involved in cellular
aggregation and segregation of experimentally disaggregated embryonic cells
(for review see Trinkaus, 1969), but few of these have been on the embryo prior
to gastrulation (Giudice & Mutolo, 1970; Lucy & Curtis, 1959; Curtis, 1962;
Yokoyo, 1966; Patricolo, 1967; Lin & Florence, 1970).
The experiments described here, on the pregastrular mouse embryo, have
shown that even after the beginning of blastocyst formation complete disaggregates are capable of reconstituting themselves. However, since it is impossible to
distinguish, in the dissociated state, cells of the inner cell mass from those of the
trophoblast and as at the present time there is no way of marking substantial
numbers of these cells differentially, their relationship and fate during reaggregation could not be followed.
FIGURES
2-7
Fig. 2. Blastocyst developed subsequent to reaggregation of 20 blastomeres isolated at
the 8-cell stage. Two cells were not included in the main reaggregating mass and
formed a vesicle.
Fig. 3. Blastocyst developed from a complete disaggregate of a late morula.
Fig. 4. Blastocyst developed subsequent to reaggregation of ten blastomeres isolated
at the early blastocyst stage.
Fig. 5. Blastocyst developed from a conjoined pair of mid-blastocysts following
partial dissociation.
Fig. 6. Vesiculated mass developed from 22 blastomeres isolated at the late blastocyst
stage.
Fig. 7. Untreated egg cultured from the 4-cell stage in vitro and showing an excluded
cell which has formed a vesicle.
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M. S. STERN
In a previous study (Stern & Wilson, 1972) it was observed that only 42 % of
asynchronous conjoined pairs of eggs produced chimaeric blastocysts. This
compares with 77 % (17/22) in the present study where conjugants were disaggregated prior to fusion. The lower success rate with the untreated eggs may
have been the result of differences in surface properties between each pair. One
such difference could be the presence, in the older member, of tight junctions
between trophoblast cells (Enders & Schlafke, 1965) which are formed by the
late morula stage (Calarco & Brown, 1969) and could preclude intermingling of
the cells unless disrupted by complete disaggregation. Once this initial barrier is
broken down the asynchronous embryos can fuse together in an integrated
manner. In some cases cells were excluded from the reaggregate, either because
they failed to make contact with the main reaggregating mass or because they
differed significantly from their neighbours. However, the exclusion of some cells
is not peculiar to reaggregates since this phenomenon is sometimes observed in
eggs simply cultured after zona removal with pronase, or even, occasionally, in
untreated eggs (Fig. 7).
Although it has been demonstrated that mouse blastocysts can regulate for the
removal of substantial amounts of material from the inner cell mass (Lin, 1969)
and trophoblast (Gardner, 1971), blastocyst fusion has never been reported.
The reason for success in the present study may be attributable to the breakdown of cell contacts as a result of exposure to versene, making possible the
establishment of new contacts with a closely adjacent egg. De novo appearance
of desmosomes, in addition to realignment of those already existing, characterizes reaggregating cells derived from early chick blastoderms (Overton, 1962).
Hence lack of fusion between untreated blastocyst pairs is a consequence not
simply of differentiative changes in the nature of individual cells but also of
purely physical conditions resulting from the trophoblast cells being tightly
bound together. The inability of trophoblastic vesicles to fuse (Gardner, 1971)
could be accounted for on the same basis.
Reaggregating cells formed either blastocysts or irregular vesciculated masses
(Fig. 6). This suggests that the fluid necessary for blastocyst cavitation can only
be discharged correctly if the cells are in a compact group and close together. If
fluid secretion starts before reaggregation has progressed sufficiently and the cells
are not close together then they appear to accumulate the fluid within themselves
and are not able to discharge it. Hence, the physical properties of these cells
may change such that the establishment of proper contacts with neighbouring
cells become impossible, and reaggregation ceases.
In the present study the process of disaggregation ensured that the spatial
relations between the cells of the preimplantation embryo were completely upset.
The use of pooled embryos ensured that the reaggregates consisted of a varied
mixture of blastomeres, so it is improbable that cells in the reaggregate took up
positions corresponding to their original intrinsic polarities. None the less,
disaggregates of very late morulae and mixed disaggregates formed apparently
Reaggregation of disaggregated embryos
261
morphologically normal blastocysts. This clearly implies that any polarity
derived from the oocyte is not functionally essential to normal development and
that epigenesis, with its regulative functions, can provide all the diversification
needed.
I am grateful to Dr I. B. Wilson for guidance and advice during the course of this work
and preparation of this manuscript and to Dr E. J. Jenkinson for helpful discussion. This
work was carried out during the tenure of an S.R.C. research studentship.
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(Manuscript received 17 December 1971, revised 12 May 1972)