/ . Embryol exp. Morph. Vol. 71, pp. 215-221, 1982
Printed in Great Britain © Company of Biologists Limited 1982
215
A method for obtaining chimaeric
mouse blastocysts with two separate inner cell
masses: a preliminary report
ByANDRZEJ K. TARKOWSKI1 AND MARIE WOJEWODZKA1
From the Department of Embryology, Institute of Zoology,
University of Warsaw
SUMMARY
Pairs of zona-free mouse blastocysts aggregated in the presence of inactivated Sendai virus
and subsequently cultured in vitro will fuse to form a chimaeric blastocyst with one common
blastocoelic cavity. Depending on the relative position of the inner cell masses in the apposed
'parental' blastocysts, the resulting chimaeric blastocyst contains either a single inner cell
mass (ICM) of dual origin or two discrete ICMs each originating from one embryo. In the
present experiments, fusion between the two aggregated blastocysts occurred in 23 % of the
pairs and 64% of these chimaeric blastocysts contained two ICMs. Blastocysts of the latter
type could potentially give rise to pairs of embryos which as regards the topography of the
foetal membrane would resemble spontaneous identical twins, although they would be
genetically dissimilar. Possible applications of the described method are discussed.
INTRODUCTION
The spontaneous separation of the inner mass cells of a mammalian blastocyst
into two discrete cell groups is believed to be one of the mechanisms by which
identical twins may originate. In the mouse this mechanism appears to operate
very rarely, because so far only one pair of twin embryos have been described
which could have arisen in this way (Bodemann, 1935). Experimental separation
of the inner cell mass into two groups of cells has not yet been achieved.
We describe here a method for producing chimaeric blastocysts with two
separate inner cell masses, each originating from a separate embryo. The method
consists of aggregating two blastocysts in the presence of inactivated Sendai
virus; the virus probably causes fusion between trophectoderm cells of the
apposed blastocysts and the blastocoelic cavities of the two embryos coalesce.
Blastocysts thus produced could potentially give rise to a pair of embryos which
as regards the topography of foetal membranes would resemble spontaneous
identical twins, although they would be genetically dissimilar. Blastocysts with
two genetically different inner cell masses have also been occasionally produced
1
Authors' address: Department of Embryology, Institute of Zoology, University of
Warsaw, 00-927 Warsaw 64, Poland.
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A. K. TARKOWSKI AND M. WOJEWODZKA
as a result of microsurgical transplantation of mouse or rat inner cell mass into
a mouse blastocyst (Gardner, 1971), but only a few pairs of embryos contained
within the same trophoblastic shell have been reported (Gardner & Johnson,
1973, 1975; Papaioannou & Gardner, 1979).
MATERIALS AND METHODS
Early and medium blastocysts were obtained from spontaneously ovulating
CBA-T6T6 and A inbred females and F1(CBA x C57B1), PO and Swiss albino
outbred females by flushing the uteri on the fourth day of pregnancy (day of
vaginal plug = first day). After the zona pellucida had been removed with
0-5% pronase in phosphate-buffered saline (PBS) (Mintz, 1962), the embryos
were washed in cold PBS (solution A of Dulbecco & Vogt, 1954) and then
exposed to /?-propiolactone inactivated Sendai virus in PBS for 1-5 mins at
4 °C (Tarkowski & Balakier, 1980). The concentration of virus varied between
375 and 1500 HAU. Shortly before being removed from the virus suspension,
blastocysts were aligned in pairs with their long axes parallel but the inner cell
masses pointing in opposite directions, and then pushed together with a glass
microneedle or squeezed together with watchmaker forceps. In a number of
pairs, however, the blastocysts rotated during squeezing and their relative
orientation changed, so that the two inner cell masses were eventually situated
close to each other. Pairs of adhering blastocysts were subsequently thoroughly
washed in PBS and cultured for 24 or 48 h in drops of Whitten's medium
(Whitten, 1971) under liquid paraffin at 37 °C in the atmosphere of 5% CO2
in air.
In two experiments single blastocysts after being exposed to virus were
aggregated in PBS with PHA (100/tg/ml).
Chimaeric blastocysts with two inner cell masses and single control blastocysts
(originating from pairs which had separated during the culture period) were
transplanted to the uterus of Swiss albino females on the 4th day of pseudopregnancy. The recipient females were killed on the 10th to 15th day and their
uteri examined under the dissecting microscope.
RESULTS
Of 121 pairs of aggregated blastocysts, 28 united to form single chimaeric
blastocyst (23-1 %). The efficiency of fusion varied greatly from experiment to
experiment and we were not able to correlate it with experimental variables,
such as the concentration of the virus, duration of treatment, etc. In the most
successful experiment 6 of 13 pairs of blastocysts fused while in another none of
18 pairs did so. The proportion of blastocysts which fused after phytohaemaglutinin treatment (7/28) did not differ significantly from other experiments.
Among 28 chimaeric blastocysts 18 had two separate inner cell masses
Chimaeric blastocysts with two ICMs
Figs 1-4. All to the same magnification (x 300). Microphotography of chimaeric
blastocysts taken 17-20 h after placing the aggregated pairs of blastocysts in
culture drops.
Fig. 1. Perfectly oval blastocyst with the two separate ICMs situated at the opposite poles.
Figs 2, 3. Blastocysts with two separate ICMs situated far apart from each other
(in the blastocyst shown in Fig. 2, the ICM in the left half is out of focus). Note the
presence of a constriction along the meridian separating the two 'parental' blastocysts.
Fig. 4. Blastocyst in which the two ICMs lie closely together (the one on the left is
slightly out of focus) but they are still two discrete groups of cells. 24 h later the
dual origin of one single ICM could no longer be recognised. The original constriction between the two 'parental' blastocysts is only visible at the abembryonic pole.
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A. K. TARKOWSKI AND M. WOJEWODZKA
(Figs. 1-3) and 10 had one common inner cell mass (Fig. 4). When fusion
occurred between early blastocysts the resulting chimaeric blastocyst was from
the beginning round or oval, the only evidence of its dual origin being the
presence of two inner cell masses (ICMs) (Fig. 1). When at the time of fusion
the blastocoelic cavities were large, the chimaeric blastocyst could have
retained for a considerable time a slight indentation along the meridian separating the two original blastocysts (Figs. 2, 3).
We have not made extensive observations on the tempo of the fusion process
as judged by the formation of the common blastocoelic cavity, because manipulations were usually carried out in the early afternoon and the aggregated pairs
were left undisturbed in culture and observed again early next morning. By this
time all but one fusion had been completed but it was not known precisely when
fusion occurred. Detailed observations carried out on two pairs of blastocysts
showed, however, that the time of coalescence of the two cavities may vary
considerably:
(1) 2 h and 20 min after aggregation the trophectoderm separating the two
blastocysts already appeared to be a single cell layer and after 3 h 45 min the
two cavities had completely joined together.
(2) 15 h 30 min after aggregation the cavities remained still separated and
began to coalesce two hours later, i.e. as late as 17 h 30 min after aggregation.
In all other unfused pairs, the two blastocysts either fell apart in culture
drops without any intervention or, even if they seemed firmly attached together,
they could be very easily separated by pipetting.
In the majority of cases the formation of chimaeric blastocysts with either
one or two separate inner cell masses could be predicted on the basis of the
relative position of ICMs in the 'parental' blastocysts at the beginning of
culture. As explained in Materials and Methods, the blastocysts sometimes
rotated during squeezing so that the two ICMs were situated side by side or the
blastocysts were attached to each other by the embryonic poles. In all these
cases a large single inner cell mass was formed; in one or two cases the dual
origin of the common ICM could still be recognised at the first inspection
(Fig. 4) but could no longer be detected after a few more hours of culture. In
blastocysts with two ICMs their relative position had never changed between
the first and the last observation, i.e. over a period lasting in some experiments
up to 24 h.
Four blastocysts with two ICMs were transplanted to three recipients but
three failed to implant and only one resorption was found at the time of autopsy
on the 10th day. 73 control blastocysts originating from unfused pairs were
transplanted to eight recipients; implantation occurred in seven females which
contained altogether 14 healthy embryos and 9 resorptions.
Chimaeric blastocysts with two ICMs
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DISCUSSION
Aggregation of mouse blastocysts in the presence of inactivated Sendai virus
causes the formation of chimaeric blastocysts with one common or two separate
ICMs. In the series of experiments described in this paper, fusion occurred in
23 % of pairs and nearly two thirds of chimaeric blastocysts had separate ICMs.
It seems to us that the efficiency of this technique, both as regards the overall
incidence of fusion and the incidence of blastocysts with two rather than one
ICM can be greatly increased. The two most important factors appear to be the
quality of the virus preparation which can be judged by the very close and firm
adhesion of the two blastocysts and the precision in orienting the blastocysts
during aggregation. Even with the relatively low efficiency of our experiments
(14-9 % of chimaeric blastocysts with two separate ICMs) the advantages of the
technique are that it is very simple and does not require sophisticated microsurgery. The elegant technique of transplanting an ICM of one blastocyst into
the blastocoel of another blastocyst, developed by Gardner (for references see
Introduction), while being very effective in producing a single chimaeric ICM,
only occasionally leads to the formation of a blastocyst with two separate ICMs.
The postimplantation survival of single control blastocysts from unfused
pairs, although relatively low (14/73), shows that the procedure itself is compatible with further embryogenesis. As only four blastocysts with two ICMs
were transplanted and three of these have not implanted, no conclusions can be
drawn as regards their developmental potential.
There is no doubt that the essential element of the described technique of
blastocyst fusion is the use of virus. It is a general experience of all students of
aggregation chimaeras that once the trophectoderm is formed the two embryos
put in contact will not integrate into one blastocyst and easily fall apart (cf. also
Gardner & Johnson, 1972). Even if the adhesion of the two blastocysts is
increased by treating them with PHA (Naruse, 1981) or CCA or WGA
(Tarkowski, unpublished observations) the two partners continue to develop
independently. It seems likely that the effect of virus consists in fusion of two or
more trophectoderm cells from the adhering blastocysts so that in the first step
the barrier between the blastocoelic cavities becomes a single cell layer. In the
next step the fused trophectoderm cell(s) retract, leading to the coalescence of
the two cavities. It is conceivable that the speed of this process may depend on
the number of cells undergoing fusion.
In this connection it is worth remembering that simple aggregation of two
cleaving embryos never leads to the formation of a blastocyst with two discrete
inner cell masses. Development of two embryos from a chimaeric blastocyst
produced by aggregation of two cleaving eggs has been observed only twice
(Tarkowski, 1961) and even in these cases the blastocysts were perfectly normal
and contained a single ICM. It appears therefore that in these two exceptional
cases 'twinning' must have occurred by secondary splitting of the originally
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A. K. TARKOWSKI AND M. WOJEWODZKA
single ICM after the blastocysts had been transplanted to the uterus. In the case
of one of these two pairs of embryos it was possible to prove in addition that
the cells originating from one component of the chimaeric blastocyst have
contributed to both embryos (pigmented cells in the outer layer of the retina).
This observation strengthens the conclusion that both embryos have developed
from the originally single ICM. A few other cases of two early egg cylinders
contained inside common Reichert membrane have been observed after
transplantation of blastocysts originating from four rather than two cleaving
embryos (Tarkowski, 1965 and unpublished observations). These giant blastocysts also contained a single ICM, although the inner mass cells appeared to
spread on the inner surface of trophectoderm over a large surface of the
embryonic hemisphere.
Chimaeric blastocysts with two separate ICMs occupying the opposite poles
can prove useful in studying the factors that determine the orientation of the
implanting blastocyst (Gardner, 1977). Other and probably far more important
applications of blastocysts with two discrete and genetically different ICMs
would become available if the two embryos could successfully develop till term
and form anastomoses between their circulations (blood chimaeras). If, in
addition, the two embryos happen to be of the opposite genetic sex then the
condition comparable to free-martinism in cattle would result.
A.K.T. wishes to express thanks to Dr C. F. Graham from the Department of Zoology,
University of Oxford, in whose laboratory the pilot experiments were carried out. Support of
WHO Small Supplies Programme and the Cancer Research Campaign is kindly acknowledged.
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{Received 24 February 1982, revised 7 May 1982)
EMB 71