Induced fusion of female gametes and embryonic cells

Cell Differentiation, 16 (1985) 77-82

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Elsevier ScientificPublishersIreland, Ltd. CDF 00297 Review

Induced fusion of female gametes and embryonic cells Sergei G. Vassetzky and Galina G. Sekirina Institute of Deoelopmental Biology, USSR Academy of Sciences, Moscow, and Institute of Experimental Medicine, USSR Academy of Medical Sciences, Leningrad, USSR

(Received2 November1984)

Investigations in which the method of induced cell fusion was used in studying the oocyte maturation, fertilization and early embryogenesis of animals have been reviewed on the basis of published data and the authors' experience. The problems of developing the method of induced cell fusion, its combination with other methods (microsurgery, cell fragmentation, etc.) and the perspectives of its use in embryology are discussed. induced cell fusion; gametes; embryonic cells; oocyte maturation; early embryogenesis

Introduction The method of induced cell fusion (Okada, 1958) is widely used in current biological research and has made possible important advances, particularly in gene mapping on human chromosomes and the analysis of nucleo-cytoplasmic relations and the mitotic cycle (for review see Ringertz and Savage, 1976). Study of the formation of cell hybrids (fusion products) between female gametes, embryonic cells and their fragments, and of their subsequent development, opens new prospects for investigating the controlling mechanisms of oogenesis, embryogenesis, and parthenogenesis.

Basic techniques While working on embryonic cells, it is primarily the same fusion-inducing agents (fusogens) that are used as on somatic cells: Sendai virus inactivated by UV irradiation or fl-propiolactone (Graham, 1971; Ba/akier and Czolowska, 1977;

Soupart, 1978; Tarkowski and Ba|akier, 1980) and polyethylene glycol (Eglitis, 1980; Dyban et al., 1981; Spindle, 1981; Gulyas et al., 1984), as well as lysolecithin (Wakahara, 1980) and polyarginine (Bennett and Mazia, 1981a,b). The fusion of both somatic and embryonic cells under the electric field effect was reported quite recently (Richter et al., 1981; Berg, 1982; Berg et al., 1983). The mechanisms of induced cell fusion are essentially the same for all types of animal cells and have been dealt with in detail in a number of reviews (Ringertz and Savage, 1976; Zelenin et al., 1982; etc.). But the technique of treating the embryonic cells in readiness for their subsequent fusion is different. The eggs and embryos are surrounded by envelopes, and for the contact of the plasma membranes of the respective cells to be established these envelopes should be removed fully and the closest rapprochement of the cells ensured (mechanically, or manually, in an electric field or by specific agglutinating agents: viruses, PHA, etc.). When applying polyethylene glycol or an electric field, the cells of embryos are some-

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78 times treated within the envelope which allows the necessary contact (Lin et al., 1973; Richter et al., 1981; Spindle, 1981; Berg, 1982; Berg et al., 1983). The treatment depends also on the amount of material available. When there are many thousands of cells available (e.g. while working on the oocytes and eggs of marine invertebrates), they may be treated in a suspension, but if there are only a few dozen cells available (e.g. with mammalian gametes and embryonic cells), they are manipulated manually under the microscope. In all cases, the cell hybrids or fusion products are selected and kept under observation to study their formation and subsequent fate.

Nucleocytoplasmic relations The consecutive stages of fusion of starfish oocytes (Skoblina et al., 1982; Sekirina et al., 1983), mouse eggs (Soupart, 1980; Sekirina, in press), and blastomeres (Eglitis, 1980; Spindle, 1981; Sekirina, in press) have been recorded live on microphotographs. The fusion is completed with the formation of a larger cell of regular form. Differences in the color and structure of the cytoplasm of the fusing cells have, in some cases, allowed its distribution during fusion to be followed. In experiments on sea urchin oocytes and the eggs of sea urchins (Bennett and Mazia, 1981a) and starfishes (Skoblina et al., 1982; Sekirina et al., 1983), the boundaries between the cytoplasms of the fusing cells were clearly discernible. A partial segregation of the respective cytoplasms was preserved in the cell hybrids at the 2-cell stage as well (Bennett and Mazia, 1981a). Both the hemispheres of the cell hybrids preserve the state characteristic of the initial cell: the unfertilized halves had a smooth surface and the fertilized halves a microvillar surface (Bennett and Mazia, 1981b). The 'independent state' of the initial cytoplasms was confirmed by cortical reaction studies in the cell hybrids of unfertilized and fertilized sea urchin eggs (Bennett and Mazia, 1981b) and of unfertilized mouse eggs and blastomeres (Szrllosi et al., 1980): numerous cortical granules were found under the plasma membrane in the unfertilized half and were absent in the half derived from the

fertilized egg or blastomere. The mixing of the cytoplasms begins only with mitosis and differences between them gradually disappear: no cortical granules were found under the plasma membrane, and the whole surface was covered by microvilli (Bennett and Mazia, 1981b). Study of nuclear behavior in the experiments on induced fusion of mouse (Balakier, 1978) and starfish oocytes (Skoblina et al., 1982; Sekirina et al., 1983) and of mouse interphase blastomeres (Eglitis, 1980; Sekirina, 1984, in press) has shown that the nuclei do not fuse. The fusion of female pronuclei, similar to the fusion of female and male pronuclei during fertilization, was reported only in experiments on sea urchin eggs (Bennett and Mazia, 1981a; Vassetzky et al., in press). A study of nuclear behavior in the fusion products of mouse embryonic cells at the same (2-cell) and different developmental stages (zygotes and blastomeres of a 16-cell embryo) suggests their tendency for rapprochement: the blastomere nucleus initially located in the periphery moved gradually towards the pronuclei (Sekirina, 1984, in press). A similar picture was observed upon fusion of mouse meiotic oocytes: two initially distinct groups of typical bivalents moved closer to each other and formed a joint metaphase plate (Tarkowski and Ba/akier, 1980). In experiments on mouse embryonic cells at the same (2- and 4-cell) and different developmental stages (zygotes and blastomeres from the morula stage), the chromosomes condensed synchronously (Sekirina, 1984, in press). The synchronous chromosome condensation in the nuclei and the concomitant cytoplasmic changes suggest that the fusion product becomes a united cell morphologically and functionally when it enters mitosis. For mitosis to be synchronized in blastomeres, laser-induced cytoplasmic bridges between the blastomeres were quite sufficient, as was shown in the experiments on nematodes (Schierenberg, 1984). A study of the first mitosis in the cell hybrids of sea urchin eggs (Bennett and Mazia, 1981a) has revealed that there could be one tetrapolar or two centrally located bipolar spindles. As a result, three or four daughter cells could arise, although, as a rule, only two daughter cells were formed. A similar picture was described for medaka blastomeres (Mizukami, 1981). Hence, in spite of

79 the mitosis synchronization, the fusion of the cells can result in a temporal merging of their cytoplasms and nuclei only. When studying nucleo-cytoplasmic relations in experiments on the induced fusion of oocytes, blastomeres and somatic cells, some data were obtained on the cytoplasmic factors of oogenesis and embryogenesis. It was shown that ovarian oocytes resumed meiosis upon fusion with both meiotic oocytes and blastomeres arrested (by colchicine) at the mitotic metaphase (Ba/akier, 1978). These observations led to the conclusion that the meiosis-inducing factor was similar to or identical with the cytoplasmic mitosis-inducing factor. In cell hybrids of mouse and vole (Balakier, 1979) and rabbit and pig embryonic cells (Fulka, 1983), the meiosis-inducing factor proved to be nonspecific, and the germinal vesicle breakdown was determined by the cytoplasm of the species in which the oocyte maturation proceeded at a greater rate (Fulka, 1983). Fusion of the anuclear fragment of the mouse ovarian oocyte with the interphase blastomere resulted in chromosome condensation which proceeded simultaneously with the germinal vesicle breakdown in the nucleate fragment cultivated separately (Balakier and Czolowska, 1977). On the basis of these results, the conclusion was reached that the meiotic maturation in mouse oocytes is induced by a cytoplasmic factor formed or demasked independently of the nucleus (Tarkowski, 1982). In the fusion products of mouse interphase blastomeres and activated (Tarkowski and Balakier, 1980) or fertilized eggs (Sekirina, in press), no condensation of the blastomere chromosomes was observed until the beginning of the first cleavage division. It can therefore be concluded that the factor inducing the immediate chromosome condensation in the interphase nuclei appears in the oocytes with meiosis resumption and disappears in the egg upon its activation. At the same time a factor appears in the activated egg cytoplasm which induces changes in the somatic cell nuclei similar to the sperm head swelling. This was shown upon induced fusion of embryonic and somatic cells of mice (Graham, 1974; Tarkowski and Balakier, 1980) and amphibians (Wakahara, 1980). The regional differences in the plasma membrane fusogenic prop-

erties were shown to change with the egg activation in the experiments on Xenopus eggs (Stewart-Savage and Grey, 1983). Induced fusion has been attempted with mouse zygotes or blastomeres and differentiated somatic cells: spleen and bone marrow (Graham, 1969, 1971, 1972, 1974), lymph nodes and bone marrow (Lin et al., 1973), and transplantable homo- and heterologous cell lines (Baranska and Koprowski, 1970; Bernstein and Mukherjee, 1972). The mutual influence of the respective cytoplasms and nuclei was shown in the cell hybrids of mouse blastomeres and A9 strain cells at the level of nuclear RNA synthesis (Bernstein and Mukherjee, 1972). The experiments on induced fusion of embryonic and somatic cells were designed to obtain the integration of foreign genetic material (similarly with the nuclear transplantation). The fusion of the eggs with the somatic cells did not prevent subsequent egg activation and development, but the cytologic analysis did not reveal any somatic nuclei descendants in such embryos (Baranska and Koprowski, 1970; Graham, 1972). With this in mind, more sophisticated methods are currently used.

Reconstitution of di-, tetra-, and polypioid embryos Application of the induced cell fusion method first produced tetraploid mammalian embryos in experiments on mouse embryonic cells (Graham, 1971). Tetraploid mouse embryos can be reconstituted from the zygotes and isolated blastomeres of 2-cell embryos (Graham, 1971; Dyban et al., 1981), blastomeres of 4-cell embryos (Eglitis, 1980), blastomeres of intact (within zona pellucida) 2-cell embryos (Spindle, 1981; Berg, 1982; Berg et al., 1983). Diploid mouse embryos have been reconstituted from the unfertilized (Soupart, 1978; Dyban et al., 1981) and activated eggs (Gulyas et al., 1984). Polyploid starfish embryos have been reconstituted from oocytes (Skoblina et al., 1982; Vassetzky et al., 1983). The functional and structural unity of the reconstituted embryos was proved by their successful early development. In vitro studies of tetraploid mouse embryos have shown that the treatment with fusogens, the

80 fusion per se, and the chromosome duplication did not prevent their development to the blastocyst stage (Graham, 1971; Eglitis, 1980; Dyban et al., 1981), did not adversely affect the temporal parameters of the blastocoele formation and of the expression of the cell surface stage-specific antigen (Eglitis and Wiley, 1981). The formation of the inner cell mass in such embryos is limited by the cell number: if it is restored to the normal level by the aggregation of several fusion products, morphologically normal blastocysts develop (Graham, 1971; Eglitis, 1980; Dyban et al., 1981). Such blastocysts were successfully implanted into the recipient females, but no complete development was observed (Graham, 1971). It has been shown in experiments on di-tetraploid chimaeric embryos that the postimplantation development is affected adversely by the tetraploid state of the cells (Graham, 1971; Tarkowski, 1972; Lu and Markert, 1980). These data correspond quite well to those obtained on the cytochalasin B-induced tetraploid embryos (Snow, 1973). But the reconstitution of the tetraploid embryos with the induced cell fusion technique allowed the cytochalasin B-induced di-tetraploid mosaicism to be avoided. Cytogenetic analysis of the embryos derived from blastomere fusion has shown that the tetraploid karyotype formed during the first cleavage division was preserved during subsequent development (Sekirina, in press). Study of the metabolism of such embryos (MDH and RNA synthesis) has shown an increase, though not in proportion to their ploidy (Eglitis and Wiley, 1981), i.e. a partial compensation of gene dosage was observed. The reconstitution of diploid mouse embryos from eggs resembles fertilization: the fusion of two mature gametes, completion of meiosis, cortical reaction, the joining of two chromosome sets, and the beginning of development. Therefore this model was called "la f6condation de l'oeuf par l'oeuf" (Soupart, 1978). Soupart (1978, 1980, 1982) believed that the development in such embryos was initiated, as in the fertilized eggs, by the fusion of the plasma membranes. But the fusion of the eggs (Sekirina, in press) or of the eggs with the blastomeres (Tarkowski and Balakier, 1980) did not always result in meiosis resumption, and their

pretreatment (enzymatic denudation, agglutination with PHA, etc. and incubation in fusogen) resulted in their parthenogenetic activation (Sekirina, in press). Hence, embryos reconstituted by the induced fusion of eggs can be considered as diploid parthenogenetic embryos. But, unlike all other parthenogenones, they are not homozygous (see Kaufman, 1983). This permits one of the cardinal problems of mammalian parthenogenesis to be approached using this model: is the lack of complete parthenogenesis in mammals due to homozygotization only? In addition to the above mentioned similarities, there are marked differences between the reconstituted diploid embryos and the fertilized eggs: uniparental chromosome sets, altered nucleo-cytoplasmic ratio, absence of not only genetic, but also extragenetic factors contributed by the sperm. All this makes such a model very promising for the study of the role of different genetic and extragenetic factors in the formation and development of the embryo.

Reconstitution of embryos from cells and cell fragments The induced fusion technique has also been applied for the reconstitution of oocytes (Vassetzky et al., 1984), zygotes and blastomeres (McGrath and Solter, 1983a,b; Sekirina and Arkhangelskaya, 1984; Surani et al., 1984; Sekirina, in press) from the cells and cell fragments obtained either by microsurgery (Tarkowski, 1977; Barton and Surani, 1983) or centrifugation (Dyban et al., 1983). In particular, this allows the role of altered quantitative nucleo-cytoplasmic ratio in oocyte maturation (Vassetzky et al., 1984) or of their qualitative recombination (reciprocal transplantation of pronuclei) in embryogenesis to be studied (McGrath and Solter, 1983a,b, Surani et al., 1984). Such an approach offers great opportunities to study the controlling mechanisms of pre-embryonic and embryonic development. More abundant data have, however, been obtained on the models of embryos reconstituted by the induced fusion of intact cells (oocytes, blastomeres). The variable and original data obtained using

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the method of induced cell fusion in developmental biology, continuous improvement of this method, and its successful combination with the other techniques make further investigation in this area quite hopeful.

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