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/ . Embryo!, exp. Morph. Vol. 34, 3, pp. 633-644, 1975 Printed in Great Britain 633 In vitro study of chorionic and ectoplacental trophoblast differentiation in the mouse ByDANIELE HERNANDEZ-VERDUN1 AND CHANTAL LEGRAND2 From the Laboratoire de Biologie de la Reproduction, Universite Pierre et Marie Curie, Paris SUMMARY Mouse chorioallantoic pre-placental structures alone or in association with the embryo were explanted during the 9th day of gestation (7-somite stage) and cultured in a static medium for 24 to 48 h. From the subsequent morphological study of trophoblast differentiation, using both light and electron microscopy, we draw the following conclusions. 1. The allantoic mesoderm cells migrate inside the trophoblastic population but they do not differentiate a capillary network and trophoblast cells phagocytose the existing foetal erythrocytes. 2. In the absence of allantoic mesoderm, chorionic trophoblast cells remain undifferentiated. 3. The development of the chorionic trophoblast is modified in that chorionic trophoblast cells fail to establish close junctions with ectoplacental trophoblast, and some chorionic cells initiate the formation of multinucleated syncytia. The genesis of these syncytia is discussed. INTRODUCTION In the mouse the placental labyrinth is constituted by the association of ectoplacental and chorionic trophoblast, interposing three different tissue layers between maternal blood and foetal capillary epithelium. For a review of mouse placental structures and trophoblast types in the rodent chorioallantoic placenta, see Duval (1891), Mossman & Fischer (1969). The syncytial nature of the inner and of the intermediate layers has been extensively described (Enders, 1965a; Hernandez-Verdun, 1974a). The existence of 'multinucleated syncytia' or polykaryons raises the problem of their origin and differentiation during normal organogenesis of the mouse labyrinth. These polykaryons appear at the end of the 10th day of gestation when the ectoplacental cells are closely associated with the underlying differentiated chorionic trophoblast (Hernandez-Verdun, 1974a). 1 Author's address: Laboratoire de Pathologie Cellulaire, 21 rue de l'ecole de medecine, 75270 Paris Cedex 06, France. 2 Author's address: Laboratoire de Biologie de la Reproduction, Tour 32, 4 place Jussieu, 75230 Paris Cedex 05, France. 634 D. HERNANDEZ-VERDUN AND C. LEGRAND In vitro, rodent embryos surrounded by their membranes develop for 2-4 days depending on their stage of development at explantation (New, 1973). Embryos capable of growing in culture include the stages, when in utero, the placental labyrinth differentiates. Indeed, when mouse egg-cylinders are explanted at the 8th day of gestation and maintained in culture for a period of 48-72 h trophoblast cells do not give origin to polykaryons (Hernandez-Verdun & Legrand, 1971). The present data refer to the differentiation of trophoblast cells in vitro when egg-cylinders within their membranes or chorioallantoic pre-placental tissues (ectoplacental cone and chorionic plate) were explanted during the 9th day of gestation and cultured in static medium for periods of 24 or 48 h. MATERIALS AND METHODS Mouse egg-cylinders of the Swiss strain were explanted during the morning of the 9th day of gestation, the first day of gestation being determined by the detection, in the morning, of vaginal plugs in females mated the night before. Techniques for embryo culture The egg-cylinders were dissected from the decidual swellings (New & Stein, 1964) and Reichert's membrane was removed as completely as possible (Hernandez-Verdun & Legrand, 1971). After several washings the tissues were cultured in watch-glasses containing 1-5 ml of culture medium. These were closed, sealed with paraffin wax and incubated at 37 °C. The medium was renewed after 24 h. Two types of organotypic culture were used. In the first series the tissues of the chorioallantoic pre-placenta were cultured together with the whole embryos. One hundred and ten egg-cylinders were grown for a period of 24 or 48 h; development continued in 55 %. In the second series the tissues of the chorioallantoic pre-placenta were cultured alone. They were dissected from the eggcylinders by means of a transection across the middle of the exocoelom (Fig. 1). Composition of the culture medium The nutrient medium consisted of Eagle's medium containing 25 % foetal calf serum, 30 % mouse serum and glucose to a final concentration of 2 g/1. The mouse serum was prepared from blood from the mother, from another mouse at the same stage of gestation or from a non-pregnant female. The blood was obtained by cardiac puncture of animals anaesthetized with chloroform. It was centrifuged at 4 °C and the serum decanted. Any contamination by red blood cells was carefully avoided. In the preparation of the media and throughout the whole culture period, sterile procedure was observed. Antibiotics were not used and two persons worked simultaneously to reduce any delay. In vitro differentiation of mouse trophoblast 635 After a culture period varying from 24 to 48 h, the explants were fixed in 1 -5 % glutaraldehyde in cacodylate buffer, then in 1 % osmium tetroxide in the same buffer. They were embedded in Epon and examined under the light microscope (1 /tm-thick section stained in Giemsa solution) then under the electron microscope as ultra-thin sections stained in uranyl acetate and lead citrate. Chorionic trophoblast Ectoplacental trophoblast Giant cell Ovocylinder 9th day t=24h /=0 Fig. 1. Explantation of chorioallantoic pre-placental tissues. Arrows show the transection of egg-cylinder. / = 0, explanted tissues; / = 24 h, same tissues after 24 h culture. RESULTS Culture of the whole egg-cylinder Growth of the embryo. Mouse embryos explanted during the morning of the 9th day of gestation have 6-7 pairs of somites. They exhibit an S-like shape, the head region being convex while the dorsal surface of the trunk is concave. The embryo is attached to the chorioallantoic placenta by the allantois which is anchored in the centre of the chorionic plate (Fig. 2). At that stage no heartbeat is visible and the allantoic circulation is not yet established. Twenty four hours after the beginning of culture, blood islands appeared 636 D. HERNANDEZ-VERDUN AND C. LEGRAND over the whole of the yolk-sac splanchnopleure. In 11 out of 39 instances the vitelline circulation had started. By that time the embryo had begun its rotation so as to assume a C-shape by the 18-somite stage. This rotation was not observed in all embryos and only 9 out of 39 embryos turned completely. The umbilical cord formed, sometimes along its whole length (24/39) (Fig. 4), sometimes only to a small extent (15/39), and the allantoic circulation had started in 10 embryos. After 48 h culture, the umbilical cord was formed in most cases (17/20) but allantoic circulation was observed in only 3 out of 20 embryos. The anterior limb-buds were conspicuous in embryos which had turned. Development of chorioallantoicplacental structures. By the 9th day of gestation the placental structures consist of an ectoplacental cone associated with the chorionic plate (Fig. 3). The ectoplacental cone is composed of two types of trophoblast: the giant cells and the ectoplacental trophoblast. After 24 h growth in vitro, the different trophoblast cell populations segregated; the giant cells were rearranged and grouped in a new mass separated from the ectoplacental trophoblast by hyalin substance derived from the omphalopleure (outer layer of the yolk sac which cannot be completely removed by dissection) (HernandezVerdun & Legrand, 1971). The ectoplacental cells, extending over the greater part of the ectoplacental cone, multiplied (Fig. 6) but were only slightly differentiated and, in contrast with what can be observed in utero, did not develop polykaryons when in contact with the chorionic trophoblast. Though in close contact with the chorionic trophoblast, they exhibited features characteristic of cellular degeneration: irregular distribution of the chromatin in clots at the periphery of the nucleus, segregation of nucleolar constituents and cytoplasmic extrusions bulging out of the cell. These extrusions formed a zone of cellular debris between FIGURES 2-5 Fig. 2 (x 30). Mouse egg-cylinder explanted at 9th day of gestation (7-somite stage). The splanchnopleure has been removed to allow better visualization of the embryo, a.b., Allantoic bud; a.c, amniotic cavity; p., chorioallantoic pre-placental structures. Fig. 3 (x 130). Chorioallantoic pre-placental tissues explanted at 9th day of gestation. 1 fim thick section. Note the presence of omphalopleure (arrow) which cannot be completely removed at explantation. c.t., Chorionic trophoblast; e.t., ectoplacental trophoblast; g.c, giant cell. Fig. 4 (x 30). A similar embryo to Fig. 2, after 24 h growth in culture. Note the presence of blood in one vessel of the umbilical cord (arrow), p., Chorioallantoic preplacental tissues; h., heart. Fig. 5 (x 900). Ectoplacental and chorionic trophoblast after 24 h growth in vitro of an embryo explanted during 9th day of gestation. 1 /tm thick section. Note lack of foetal blood vessels inside the trophoblast tissues despite the presence of foetal capillaries in the mesodermic ampulla, c.t., Chorionic trophoblast; e.t., ectoplacental trophoblast; f.c, foetal capillary; m.a., mesodermic ampulla. In vitro differentiation of mouse trophoblast 637 *5 638 D. HERNANDEZ-VERDUN AND C. LEGRAND FIGURES 6 AND 7 Fig. 6 (x 130). Chorioallantoic pre-placental tissues after 48 h growth in culture. c.t., Chorionic trophoblast; e.t., ectoplacental trophoblast; g.c, giant cell; m., allantoic mesoderm. Fig. 7 (Ax4000, Bx 17000). Area of cytoplasmic fusion between two chorionic trophoblast cells (arrow). A, General view: note foetal erythro-phagocytosis (ph.) by the cells. B, Insert: detail of cellular bridge indicated by an arrow in Fig. 7A. Observe cytoplasmic uninterrupted connexion between the two chorionic cells. Next to this cytoplasmic bridge the small arrow points to a desmosome {d.) between these adjacent cells. In vitro differentiation of mouse trophoblast 639 chorionic and ectoplacental trophoblast, and after 48 h culture resulted in the formation of a 'pseudo-ectoplacental' cavity. The chorionic trophoblast was organized as islands between which cells of allantoic origin were progressively destroyed, whereas in utero the allantoic capillaries constitute the foetal blood system of the placenta. The functional foetal capillaries (containing numerous foetal red blood cells) were confined to the mesoderm adjacent to the chorionic plate (Fig. 5). The cells of mesodermal origin migrated to the anterior part of the chorionic layer but the foetal capillaries never developed in the trophoblastic tissues. After 48 h culture, many foetal red blood cells were found within the chorionic cells, having been phagocytosed by them. The formation of large multinucleated cells or polykaryons was observed in 5 out of 12 instances (Fig. 8). These polykaryons originated in the chorionic trophoblast. They contained numerous indented nuclei each with a conspicuous nucleolus and the nucleocytoplasmic ratio was estimated to be as high as 1/2. These nuclei exhibited no mitotic figures. Around these polykaryons some cells were found with only two or three nuclei, having the appearance of transitional forms between one cell and a 'syncytium'. Partial fusion of cellular membranes involving a cytoplasmic bridge between adjacent cells was observed (Fig. 7 A, B). Chorioailantoic pre-placental structures isolated from the embryo were grown for 24 or 48 h in organotypic culture. By the 9th day of gestation, the allantoic bud had anchored. The ectoplacental cone was therefore cultured in association with the allantoic mesoderm and fragments of the vitelline splanchnopleure (Fig. 1). The cone was attached to the bottom of the watch-glass either by its chorionic side at the level of the mesodermal ampulla or by the mass of the giant cells. After 24 h culture the fragments of vitelline splanchnopleure formed again a vesicle under the chorionic side of the cone (Fig. 1). A high mitotic index was observed in the chorionic trophoblast and these cells remained undifferentiated. They were faintly basophilic and arranged as a parenchyma, which excluded any potential motility. Foetal erythrocytes were found within the cells of the chorionic trophoblast, although phagocytosis was rarely observed and did not compare with the absorption of red cells by the chorionic trophoblast of a complete egg-cylinder at the same stage. After 48 h, a zone of degeneration appeared between the chorionic and the ectoplacental trophoblast. It appears that in an isolated chorioallantoic placenta the allantoic mesoderm cannot grow or re-form (Fig. 1). DISCUSSION Culture of rodent embryos within their membranes has mainly been performed in order to provide easy access to mammalian embryos for experimental investigation. Consequently, the interest of investigators was focused on the development of the embryo itself and the placental structures have been less 640 D. HERNANDEZ-VERDUN AND C. LEGRAND B In vitro differentiation of mouse trophoblast 641 studied. In vitro, the trophoblast does expand, but the functioning of the chorioallantoic placenta is not fully established (New & Daniel, 1969). Problems in culture are encountered when the allantoic circulation becomes functional. This is the period when, in utero, the placental labyrinth and the 'multinucleated syncytia' differentiate, and when the nutritional and respiratory function of the yolk sac is taken over by the allantoic placenta. In this study the embryos were maintained in culture as long as necessary for the allantoic circulation to be established. Within the first 24 h of culture, embryos explanted during day 9 of gestation (7 somites) start to turn, establish vitelline circulation and develop an umbilical cord. They grow for 48 h in vitro with the allantoic circulation! functioning for several hours longer. These results are comparable to those obtained in static medium by New & Stein (1964) and Clarkson, Doering & Runner (1969) on the mouse embryo at the same stage and by New (1966), Payne & Deuchar (1972) and Steele (1972) on the rat embryo at an equivalent stage. From the study of trophoblast differentiation in 9th-day-cultured embryos, several conclusions can be drawn: 1. Three types of trophoblast develop independently in vitro (giant cells, ectoplacental cells and chorionic cells) as discussed in a previous paper (Hernandez-Verdun & Legrand, 1971). 2. Foetal blood vessels fail to differentiate in the chorioallantoic placenta. Foetal erythrocytes that we can nevertheless observe between trophoblast cells are progressively destroyed as a result of phagocytosis by the trophoblast. The phagocytosis of these mesodermal elements in conjunction with the lack of foetal capillary network might explain the failure to establish a stable vascular connexion between placenta and foetus whenever the differentiation of the placental membrane is incomplete at the time of explantation. This fact agrees with the study of New (1967) in which a functional allantoic circulation in embryos explanted before the 22-somite stage could not be obtained. At that early stage (5-10 somites) embryos develop an abnormal 'bypass' vessel which eliminates flow between the allantoic vessels and the aorta. 3. After 48 h of culture, chorionic cells give rise to polykaryons in some eggcylinders, but chorionic trophoblast cells do not undergo the same transformation in isolated placental structures explanted during the 9th day of gestation. The main difference between egg-cylinders in which multinucleated syncytia develop and the placental structures where they do not occur seems to result FIGURE 8 (x5000) A, Chorionic trophoblast polykaryon after 48 h of development in vitro in an embryo explanted at 9th day of gestation. Note the presence of large nucleolus (nu.) in the nucleus (n.) of the syncytium, and signs of active phagocytosis (ph.) in this polykaryon. B, Explicative pattern of Fig. 8A. /., lipids; n., nuclei; nu., nucleolus; ph., phagosomes. 642 D. HERNANDEZ-VERDUN AND C. LEGRAND from the presence or absence of the allantoic mesodermal cells. The allantoic mesoderm appears to induce or maintain a differentiated stage in the chorionic trophoblast. Consequently it seems reasonable to postulate that differentiation of this degree is necessary for the process of polykaryon formation to occur. These results may be compared with normal organogenesis of the labyrinth during development in utero. In that case the existence of zones of incomplete fusion of cytoplasmic membranes among adjacent trophoblastic cells can be clearly demonstrated at the ultrastructural level (Hernandez-Verdun, 19746). These observations favour the assumption that the multinucleated syncytia described in the placental labyrinth of the mouse would result from trophoblastic elements. In the human and non-human Primate placenta a comparable origin has been assigned to the syncytial layer, which would then arise from the Langhans cells (Carter, 1964; Enders, 19656; Kemnitz, 1970). Midgley, Pierce, Deneau & Gosling (1963), Tao & Hertig (1965) and Gerbie, Hathaway & Brewer (1968), using [3Hlthymidine as a cell tracer, have obtained data in support of such a hypothesis. On the other hand, enzyme subunit reassociation tests (Gearhart & Mintz, 1972a; Chapman, Ansell & McLaren, 1972) have demonstrated the absence of glucose-phosphate-isomerase hybrid enzymes in trophoblastic tissues derived from chimaeric embryos in mice. These results differ from those obtained by Baker & Mintz (1969) and Gearhart & Mintz (19726) in muscle polykaryons whose syncytial nature has been clearly established. In that case heterodimers of the enzyme could be demonstrated after fusion of myoblasts of allophenic mice with genotypic differences. Gearhart & Mintz (1972 a) come to the conclusion that 'although syncytial trophoblast may in fact arise by cell fusion, there is no appreciable mixing of the macromolecular contents of fused cells'. In addition to cell fusion, other mechanisms may be involved in the genesis of trophoblastic polykaryons, such as intensive multiplication of nuclei without any cytoplasmic cleavage, nuclear amitotic fragmentation, or phagocytosis. Injections of colchicine and [3H]thymidine testify to the absence of mitosis in the nuclei of multinucleated syncytia in utero (Hernandez-Verdun, 19746) and no mitotic or amitotic figure has ever been observed in vitro. In vivo, the hypothesis of phagocytosis of trophoblastic elements has to be discarded (Hernandez-Verdun, 19746); in vitro, although the great number of lytic bodies might favour such a hypothesis, we would have to assume that the nuclei of phagocytosed cells are alone resistant to the action of lytic enzymes, thus allowing the formation of polykaryons. It therefore seems reasonable to postulate that the cytoplasmic bridges already referred to represent an intermediate stage in the genesis of multinucleated syncytia. Such pictures are comparable to those observed during the genesis of myotubes through fusion of myoblasts (Rash & Fambrough, 1973). In utero as well as in vitro, we observe the formation of polykaryons from trophoblast cells of the labyrinth. In utero, the polykaryons arise from ecto- In vitro differentiation of mouse trophoblast 643 placental trophoblast cells after they have established close junctions with the differentiated chorionic trophoblast. In vitro, the polykaryons occur in chorionic trophoblast cells in which differentiation is found to be abnormal. If fusion of trophoblastic cells is to be postulated as the origin of the syncytium, we are led to formulate a speculative hypothesis which nevertheless would afford a coherent explanation for all the observations made in our work: that of the existence of a 'fusion factor' in the chorionic cells once differentiated. During normal organogenesis in utero this factor could be transferred from the chorionic to the ectoplacental cells at the moment when close junctions are established between them. This 'factor' would not exist in undifferentiated chorionic cells (in the absence of mesoderm). 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