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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.
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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
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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
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*5
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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
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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.
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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). In vitro, when close junctions are
not established between the two cell populations, it could not be transferred
to the ectoplacental cells and consequently could induce fusion among chorionic
cells only.
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{Received 26 March 1975, revised 26 June 1975)