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J. Embryol. exp. Morph. Vol. 32, 2, pp. 365-374, 1974
Printed in Great Britain
365
The differentiation of the chick chorionic
epithelium: an experimental study
By R O B E R T O N A R B A I T Z 1 AND P I E R R E P. T E L L I E R 1
From the Department of Histology and Embryology,
University of Ottawa
SUMMARY
An electron microscopical study of the chorionic epithelium under two different experimental situations was conducted in order to gain knowledge on the mechanisms controlling
the differentiation of 'calcium-absorbing' cells. In chorioallantoic membranes from 10-dayold embryos cultured on a semi-synthetic medium, the chorion became avascular but
'calcium-absorbing' cells differentiated, showing that the differentiation of these cells is
not dependent on the vascular changes which in vivo occur simultaneously.
The number of 'calcium-absorbing' cells was much larger in the expiants cultured on
parathyroid hormone-enriched medium than in controls, indicating that the hormone is
capable of stimulating their differentiation. Since it is known that parathyroid glands are
active at the time in which 'calcium-absorbing' cells normally appear, our results suggest
that the hormone may be the normal inductor of their differentiation.
Finally, a small number of 'calcium-absorbing' cells differentiated in vivo in a portion of
chorion which had been artificially separated from the shell membranes, showing that their
differentiation does not depend on the proximity of the external source of calcium.
INTRODUCTION
The amount of calcium contained in the yolk of the chicken's egg is insufficient for the needs of the developing embryo. During the second half of its
development the embryo obtains from the egg-shell the large amount of mineral
that its skeleton requires (Simkiss, 1961). Absorption of calcium from the shell
occurs through the chorionic epithelium which is in direct contact with the shell
membranes. At the time when this absorption of large quantities of calcium
begins (Johnston & Comar, 1955), very specialized cells known as 'intercalated' (Skalinsky & Kondalenko, 1963), 'calcium-absorbing' (Owczarzak,
1971) or ' villus-cavity' cells (Coleman & Terepka, 1972) appear in the chorion.
They are characterized by a very electron-dense cytoplasm, numerous mitochondria, large sub-apical vacuoles and very long apical microvilli.
It has been proposed (Skalinsky & Kondalenko, 1963; Skalinskii, 1965;
Owczarzak, 1971 ; Narbaitz, 1972) that these cells are involved in the resorption
of calcium from the shell either by secreting agents which aid in dissolving
1
Authors' address: Department of Histology and Embryology, Faculty of Medicine,
University of Ottawa, Ottawa, Ontario, Canada.
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R. NARBAITZ AND P. P. TELLIER
the shell's mineral or by actually transporting the calcium ions. Coleman &
Terepka (1972) accept a possible secretory role of these cells but believe that the
actual transport of calcium ions takes place through the cytoplasm of another
cell type which they term 'capillary-covering' cells. The studies of the last
mentioned authors have been made on the region of the chorion which contacts
the air chamber and which for that reason is not involved in calcium absorption
in the living embryo. This is unfortunate, since Narbaitz (1972) has demonstrated that the cell population in this zone diflfers from the one in the portion of
chorion which normally absorbs the shell mineral. Although it has not been
proved conclusively whether the electron-dense cells with large microvilli secrete
mineral-dissolving agents or have an active role in calcium transport, in the
present paper we shall continue to designate them 'calcium-absorbing' cells as
suggested by Owczarzak (1971).
Since both endocrine glands concerned with the regulation of calcium transportin the adult, i.e. the parathyroid glands and the ultimobranchial bodies already
show signs of activity (Stoeckel & Porte, 1969; Narbaitz, 1971) when the increase
in the utilization of calcium occurs and the 'calcium-absorbing' cells appear, it
is possible that the differentiation of these cells is hormonally controlled.
The in vivo and in vitro experiments here described have been conducted in
order to explore the possible role of the above mentioned factors in the differentiation of 'calcium-absorbing' cells.
MATERIAL AND METHODS
White Leghorn eggs from a commercial source were used in all our experiments.
Organotypic cultures. Since in preliminary experiments using a purely
synthetic medium we failed to obtain adequate survival and differentiation of
the expiants, the following semi-synthetic formula was used in all the experiments here described: (a) 2 x Medium 199, with Hanks's salts, 18 ml; (b) 2%
agar in double-distilled water, 18 ml; (c) chicken serum, 3-5 ml; (d) Penicillin
5000 units, streptomycin 5000 /*g. Two ml of the melted medium were poured
into each Petri dish (Falcon Plastics; 35 x 10 mm).
Hormone-containing medium was prepared by dissolving lyophilized
parathyroid hormone (Sigma Chemical Co., St Louis, Mo. ; prepared according
to Rasmussen, Sze & Young, 1964) in the chicken serum in the above formula.
The final concentration was of 2 USP/parathyroid units per ml of medium.
Eggs were incubated for 10 days and then opened under sterile conditions.
Portions of the shell membrane and the adhering chorioallantoic membrane
were dissected and cut into small 2 x 3 mm rectangular portions. These portions
were explanted with the endoderm towards the medium and the shell membrane
towards the air phase. Cultures were maintained for three or four days at 37 °C
in closed chambers containing air saturated with water vapor. At the end of the
Differentiation
of chick chorionic epithelium
367
culture time, expiants were fixed and processed as described below. A total of
120 expiants cultured on standard medium and 30 cultured on hormonecontaining medium were studied.
Artificial air chamber. Twenty eggs were incubated for 3 days. At that time,
and following the usual procedure for chorioallantoic grafting (Hamburger,
1960), they were positioned horizontally, a small orifice was opened into the
air chamber, a small window was cut on the upper side and the shell membrane
was pierced in order to let air enter; the chorioallantoic membrane immediately
dropped and separated from the shell membrane. Eggs were then carefully
sealed and returned to the incubator where they were kept in the same position
until the fifteenth day of incubation. At that time they were opened and portions
of the chorioallantoic membrane underlying the artificial air chamber were
fixed and processed. Portions of the membrane which had remained attached to
the shell membrane were also fixed and used as controls, together with the
membranes of unoperated embryos of the same age.
Processing of the tissues. All tissues were fixed in half-strength Karnovsky's
fixative (Karnovsky, 1965) for 6 h, washed in 0-1 M cacodylate buffer at pH 7-2
with 0-2 M sucrose, post-fixed with 1 % osmium tetroxide for 2 h, dehydrated
in ethyl alcohol, and embedded in Araldite. 1 jam sections were stained with
toluidine blue for examination with the optical microscope and thin sections
with uranyl acetate and lead citrate according to Reynolds (1963). They were
examined with a Philips 300 electron microscope.
RESULTS
Organ culture. The expiants survived adequately and their four-layered
arrangement (shell membrane, chorion, mesoderm and allantoic endoderm)
persisted unmodified. A small space frequently separated the chorion from the
shell membrane. In a few cases, the endoderm overgrew the expiants and some of
its cells were found on the shell membrane. The chorionic and allantoic epithelia fused with each other at the periphery of the expiants and it was difficult
to distinguish the origin of each cell found in this region with the optical microscope. However, their ultrastructural characteristics were very distinctive and
are described below.
The ultrastructural aspect of the chorion in controls at the age of explantation
is shown in Fig. 1. The chorion is formed by two or three layers of cells. The
most superficial layer is generally formed by flat degenerating cells with very
electron-dense cytoplasm and distorted mitochondria. The cells in the lower
layers have a cytoplasm of lower density, mitochondria with typical cristae and
few profiles of rough endoplasmic reticulum. Lipid droplets are sometimes found
in them. Intra-epithelial capillaries are numerous and always located in the
deeper zone of the epithelium, closer to the basal lamina than to the shell
membrane (Fig. 1).
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R. NARBAITZ AND P. P. TELLIER
Fig. 1. Chorionic epithelium from a 10-day-old control embryo, cap., Capillary;
sh.m., shell membrane.
Fig. 2. Chorionic epithelium from an expiant cultured on control medium for 3 days.
Differentiation
of chick chorionic epithelium
369
After 3 days of culture (Fig. 2) the intra-epithelial capillaries had disappeared
and the chorion was formed by one, two or even three layers of cells. Most of
the cells had a small, irregularly shaped nucleus. The lateral surfaces of the cells
had thin microvilli which interdigitated with those of neighbouring cells; apical
microvilli were rarely found. Very large and numerous mitochondria with
typical cristae and few profiles of rough endoplasmic reticulum were always
observed and dense bodies appeared frequently.
In some of the expiants cultured for 3 days and in most of those cultured for
4 days a second type of cell was found in the chorionic epithelium. These cells
usually had a more electron-dense cytoplasm and a very characteristic tuft of
large apical microvilli (Fig. 3). Transitions between the regular chorionic cell
and this second type were frequently found ; as the cells continued to differentiate
the cytoplasm became more electron-dense and numerous vacuoles appeared in
the sub-apical zone (Fig. 4). The fully differentiated cells were very similar to
the 'calcium-absorbing' cells found in controls (Fig. 6) although no intersinusoidal, cup-shaped cavity was found at their apex. The expiants cultured on
medium containing parathyroid hormone were similar to those cultured on
control medium although in some of them the chorion appeared somewhat
thicker and showed isolated hypertrophic patches three to four cells thick. The
cell types were similar to those found in the previous experiment, but a clear
quantitative difference existed. Thus, while in expiants cultured on control
medium for 3 days, only some of the sections contained isolated 'calciumabsorbing' cells, in the expiants cultured on medium containing parathyroid
hormone, these cells were constantly observed in all sections and it was frequent
to find two or three of them in one single section.
The mesoderm and the allantoic endoderm did not appear to change much
during the culture period. The endoderm was formed by one or two layers of
cells. In some cases, dark degenerating cells were observed. Typical endodermal
cells (Fig. 5) had a flat nucleus, and their cytoplasm contained many mitochondria, profiles of rough endoplasmic reticulum, a very well developed Golgi
apparatus, numerous short microvilli in the lower surface and, most characteristically, numerous rounded granules of moderate electron-density and surrounded
by a membrane. These granules were already present before explantation and
have been repeatedly described (Leeson & Leeson, 1963; Skalinsky & Kondalenko, 1963). These cytological characteristics permitted the distinction
between endodermal and chorionic cells at the periphery of the expiants, where
they were usually found intermingled.
Artificial air chamber. At the optical level no striking differences were found
between the sections of chorioallantoic membrane underlying the artificial
chamber and those of the opposite pole of the egg. Clear differences were
disclosed, however, by the electron microscopical observation. The portion of
chorion which had remained attached to the shell membrane (Fig. 6) was very
similar to the one of unoperated controls. Superficial capillaries were very
24-2
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R. NARBAITZ AND P. P. TELLIER
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Fig. 3. Chorionic epithelium from an explant cultured on control medium for
4 days, d.b., Dense bodies.
Fig. 4. Chorionic epithelium from an expiant cultured on parathormone-enriched
medium for 3 days, v., Vacuoles.
Differentiation of chick chorionic epithelium
371
Fig. 5. Allantoic epithelium from an expiant cultured on control medium for 3 days.
G, Golgi complex; gr., granules.
Fig. 6. Chorionic epithelium from a 15-day-old embryo with an artificial air
chamber (attached portion), cap., Capillary; sh.m., shell membrane.
Fig. 7. Same embryo. Unattached portion of the chorion.
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R. N A R B A I T Z A N D P . P . T E L L I E R
numerous and were only separated from each other by cup-shaped cavities
containing the numerous microvilli of the 'calcium-absorbing' cells. In the
portion of chorion underlying the artificial air chamber, capillaries were less
numerous and no intersinusoidal cavities were present. However, 'calciumabsorbing' cells had differentiated (Fig. 7) and their microvilli floated free in
the air chamber. There were fewer of these cells than in the normal chorion.
In some zones the epithelium was partially covered by cellular debris (Fig. 7).
No signs of keratinization were found.
DISCUSSION
Moscona (1959) succeeded in maintaining in organotypic culture chorioallantoic membranes from 8-day-old chick embryos. He observed that after 3
or 4 days of culture the chorionic epithelium started to undergo keratinization.
These differences in the results may be explained by the fact that, in our case,
the chorioallantoic membranes were explanted together with the overlaying shell
membranes in such a way that the chorionic epithelium was not in direct
contact with the air phase.
Between the 11th and 13th day of incubation, two characteristic changes
occur normally in the chorionic epithelium: (a) an outward migration of the
intra-chorionic blood vessels results in the formation of an extensive network of
superficial intra-epithelial capillaries (Romanoff, 1960; Leeson & Leeson,
1963; Skalinsky & Kondalenko, 1963) and (b) the 'calcium-absorbing' cells
begin to appear in the spaces between the capillaries (Skalinsky & Kondalenko,
1963). In vitro, the migration of blood vessels is not possible and in our cultures
the chorionic epithelium became avascular. However, 'calcium-absorbing'
cells did appear after 3 days of culture, indicating that the differentiation of
these cells, although normally occurring simultaneously with the vascular
changes, is not dependent on them.
In the expiants cultured on parathyroid hormone-enriched medium, there
were notably more 'calcium-absorbing' cells than in expiants cultured on control medium, showing that this hormone is capable of stimulating the differentiation of the cells. As mentioned previously (see Introduction), the chorion
contains another cell type which was termed 'capillary-covering' cell by Coleman & Terepka (1972) and which they suggest plays a role in calcium resorption.
According to Narbaitz (1972) these cells are more numerous in the zone which
contacts the air chamber than in the rest of the chorion. Since their only distinctive characteristic is their particular association with the capillaries, they
would not be recognizable in the cultures, even if present, due to the absence
of capillaries. Therefore we cannot decide if the differentiation of these cells
can also be stimulated by parathyroid hormone.
The formation of an artificial air chamber by dropping the chorioallantoic
membrane is*a technical procedure currently used in both embryological and
Differentiation
of chick chorionic epithelium
373
virological laboratories (Hamburger, 1960). It has also been used as a means of
reducing the absorption of calcium from the shell (Narbaitz, Belanger & Hunt,
1973). This procedure is usually carried out between the 8th and 13th day of
incubation and it usually produces many cytological alterations on the chorionic
epithelium (Ganote, Beaver & Moses, 1964). Moscona (1959) performed the
experiment on 3-day-old embryos and claimed that no histological alterations
in the chorionic epithelium were present when the embryos reached 18 days of
age, provided the sealing of the windows was hermetic. The present electron
microscopical study on similarly treated chorioallantoic membranes shows that
although the cytological changes occurring in the chorionic epithelium are not as
intense as those described by Ganote et al. (1964) after late treatment, they do
exist and consist mainly of a reduction in the number of intra-epithelial capillaries, and of'calcium-absorbing' cells. The fact that 'calcium-absorbing' cells
do appear in the portion of chorion which has had no contact at any time with
the shell membranes indicates that the differentiation of these cells is not
dependent on their proximity to the source of calcium.
Thus, in the present experiments, the only factor which appeared to affect
the differentiation of 'calcium-absorbing' cells was parathyroid hormone.
Since parathyroid glands appear to be active when these cells differentiate
(Narbaitz, 1971) our results suggest that parathyroid hormone may be the
normal inductor of their differentiation. If this assumption proves to be true,
the following sequence of events could be suggested for calcium transport
during the second half of development: massive utilization of calcium by the
skeleton -*- transitory hypocalcemia ->- stimulation of parathyroid secretion ->
differentiation of 'calcium-absorbing' cells -> increased resorption of calcium
from the shell.
The authors wish to thank Dr L. F. Bélanger, who read the manuscript and made important
suggestions, and Mr Vijay Kapal for his technical assistance.
This work was supported by a grant of the Medical Research Council of Canada (MA
4845).
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(Received 15 January 1974, revised 21 March 1974)