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From the Department of Embryology, Institute of Zoology,. University of Warsaw. SUMMARY. Pairs of zona-free mouse blastocysts aggregated in the presence of ...
/ . Embryol exp. Morph. Vol. 71, pp. 215-221, 1982 Printed in Great Britain © Company of Biologists Limited 1982

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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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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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(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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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.

REFERENCES twins in the mouse. Anat. Rec. 62, 291-294. formation and isolation of pure lines with poliomyelitis viruses. /. exp. Med. 99, 167-182. GARDNER, R. L. (1971). Manipulations on the blastocyst. Adv. Biosciences 6, 279-296. GARDNER, R. L. (1977). The problem of intrauterine orientation of the implanting mouse blastocyst. /. Anat. 124, 236 (Abstr.). GARDNER, R. L. & JOHNSON, M. H. (1972). An investigation of inner cell mass and trophoblast tissues following their isolation from the mouse blastocyst. /. Embryol. exp. Morph. 28, 279-312. GARDNER, R. L. & JOHNSON, M. H. (1973). Investigation of early mammalian development using interspecific chimaeras between rat and mouse. Nature, New Biology 246, 86-89. GARDNER, R. L. & JOHNSON, M. H. (1975). Investigation of cellular interaction and deployment in the early mammalian embryo using interspecific chimaeras between the rat and mouse. In Cell Patterning, Ciba Foundation Symposium 29 {new series), pp. 183-200. MINTZ, B. (1962). Experimental study of the developing mammalian egg: removal of the zona pellucida. Science, N. Y. 138, 594-595. NARUSE, I. (1981). Development of experimentally fused mouse blastocysts in vitro. Annotationes Zoologicae Japonenses 54, 10-16. y y PAPAIOANNOU, V. & GARDNER, R. L. (1979). Investigation of the lethal yellow A A embryo using mouse chimaeras. /. Embryol. exp. Morph. 52, 153-163. TARKOWSKI, A. K. (1961). Mouse chimaeras developed from fused eggs. Nature, Lond. 190, 857-860. BODEMANN, E. (1935). A case of uniovular DULBECCO, R. & VOGT, M. (1954). Plaque

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A. K. (1965). Embryonic and postnatal development of mouse chimeras. In Preimplantation Stages of Pregnancy, Ciba Foundation Symposium (ed. G. E. W. Wolstenholme & M. O'Connor), pp. 183-193. TARKOWSKI, A. K. & BALAKIER, H. (1980). Nucleo-cytoplasmic interactions in cell hybrids between oocytes, blastomeres and somitic cells. /. Embryol. exp. Morph. 55, 319-330. WHITTEN, W. K. (1971). Nutrient requirements for culture of preimplantation embryos in vitro (ed. G. Raspe). Adv. Biosciences 6, 129-140. TARKOWSKI,

{Received 24 February 1982, revised 7 May 1982)

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