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 late 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.

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.

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.

Complete disruption of organization, at least up to the early blastocyst stage, appears to have little effect on subsequent blastocyst formation (Figs. 1F, 24), 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 (Fig. 1E, F). This took between 24 and 48 h, depending on the age of the blastomeres at the start of reaggregation.

Table 1

Reaggregation of disaggregates

Reaggregation of disaggregates
Reaggregation of disaggregates
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).

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).

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. 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. 3

Blastocyst developed from a complete disaggregate of a late morula.

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 pre-implantation 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).

Fig. 4

Blastocyst developed subsequent to reaggregation of ten blastomeres isolated at the early blastocyst stage.

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. 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. 6

Vesiculated mass developed from 22 blastomeres isolated at the late blastocyst stage.

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.

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).

Fig. 7

Untreated egg cultured from the 4-cell stage in vitro and showing an excluded cell which has formed a vesicle.

Fig. 7

Untreated egg cultured from the 4-cell stage in vitro and showing an excluded cell which has formed a vesicle.

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 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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