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-day-old 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 explants 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.

The amount of calcium contained in the yolk of the chicken’s egg is insuffi-cient 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 ‘inter-calated’ (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 mito-chondria, 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 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 demonstra-ted that the cell population in this zone differs 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 trans-portin theadult,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 differen-tiation of ‘calcium-absorbing’ cells.

White Leghorn eggs from a commercial source were used in all our experi-ments.

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 experi-ments here described: (a) 2 × 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 μg. Two ml of the melted medium were poured into each Petri dish (Falcon Plastics; 35 × 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 × 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 culture time, explants were fixed and processed as described below. A total of 120 explants cultured on standard medium and 30 cultured on hormone-containing 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 μm 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.

Organ culture

The explants 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 explants and some of its cells were found on the shell membrane. The chorionic and allantoic epi-thelia fused with each other at the periphery of the explants and it was difficult to distinguish the origin of each cell found in this region with the optical micro-scope. 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).

Fig. 1.

Chorionic epithelium from a 10-day-old control embryo, cap., Capillary; sh.m., shell membrane.

Fig. 1.

Chorionic epithelium from a 10-day-old control embryo, cap., Capillary; sh.m., shell membrane.

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.

Fig. 2.

Chorionic epithelium from an explant cultured on control medium for 3 days.

Fig. 2.

Chorionic epithelium from an explant cultured on control medium for 3 days.

In some of the explants 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 inter-sinusoidal, cup-shaped cavity was found at their apex. The explants 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 explants cultured on control medium for 3 days, only some of the sections contained isolated ‘calcium-absorbing’ cells, in the explants 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.

Fig. 3.

Chorionic epithelium from an expiant cultured on control medium for 4 days, d.b., Dense bodies.

Fig. 3.

Chorionic epithelium from an expiant 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.

Fig. 4.

Chorionic epithelium from an expiant cultured on parathormone-enriched medium for 3 days, v., Vacuoles.

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 mito-chondria, profiles of rough endoplasmic reticulum, a very well developed Golgi apparatus, numerous short microvilli in the lower surface and, most characterist-ically, 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 & Kon-dalenko, 1963). These cytological characteristics permitted the distinction between endodermal and chorionic cells at the periphery of the expiants, where they were usually found intermingled.

Fig. 5.

Allantoic epithelium from an explant cultured on control medium for 3 days. G, Golgi complex; gr., granules.

Fig. 5.

Allantoic epithelium from an explant cultured on control medium for 3 days. G, Golgi complex; gr., granules.

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 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, ‘calcium-absorbing’ 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.

Fig. 6.

Chorionic epithelium from a 15-day-old embryo with an artificial air chamber (attached portion), cap., Capillary; sh.m., shell membrane.

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.

Fig. 7.

Same embryo. Unattached portion of the chorion.

Moscona (1959) succeeded in maintaining in organotypic culture chorio-allantoic 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 explants cultured on parathyroid hormone-enriched medium, there were notably more ‘calcium-absorbing’ cells than in explants 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 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).

Coleman
,
J. R.
&
Terepka
,
A. R.
(
1972
).
Fine structural changes associated with the onset of calcium, sodium and water transport by the chick chorioallantoic membrane
.
J. Membrane Biol
.
7
,
111
127
.
Ganote
,
C. E.
,
Beaver
,
D. L.
&
Moses
,
H. L.
(
1964
).
Ultrastructure of the chick chorioallantoic membrane and its reaction to inoculation trauma
.
Lab. Invest
.
13
,
1575
1589
.
Hamburger
,
V.
(
1960
).
A Manual of Experimental Embryology, revised edition
.
Chicago
:
Chicago University Press
.
Johnston
,
P. M.
&
Comar
,
C. L.
(
1955
).
Distribution and contribution of calcium from the albumen, yolk and shell to the developing chick embryo
.
Amer. J. Physiol
.,
183
,
365
370
.
Karnovsky
,
M. J.
(
1965
).
A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy
.
J. Cell Biol
.
27
,
137A
138A
.
Leeson
,
T. A.
&
Leeson
,
C. R.
(
1963
).
The chorio-allantois of the chick. Light and electron microscopic observations at various times of incubation
.
J. Anat
.
97
,
585
595
.
Moscona
,
A.
(
1959
).
Squamous metaplasia and keratinization of chorionic epithelium of the chick embryo in egg and in culture
.
Devi Biol
.
1
,
1
23
.
Narbaitz
,
R.
(
1971
).
Submicroscopical aspects of chick embryo parathyroid glands
.
Gen. comp. Endocr
.
19
,
253
258
.
Narbaitz
,
R.
(
1972
).
Cytological and cytochemical study of the chick chorionic epithelium
.
Revue can. Biol
.
31
,
259
267
.
Narbaitz
,
R.
,
Belanger
,
L. F.
&
Hunt
,
B. J.
(
1973
).
Calcium regulation by the chick embryo. An experimental approach
.
Calcified Tissue Abstracts
11
,
238
241
.
Owczarzak
,
A.
(
1971
).
Calcium-absorbing cell of the chick chorio-allantoic membrane. I. Morphology, distribution and cellular interactions
.
Expl Cell Res
.
68
,
113
129
.
Rasmussen
,
H.
,
Sze
,
Y.-L.
&
Young
,
R.
(
1964
).
Further studies on the isolation and characterization of parathyroid polypeptides
.
J. biol. Chem
.
239
,
2852
2857
.
Reynolds
,
E. S.
(
1963
).
The use of lead citrate at high pH as an electron-opaque stain in electron microscopy
.
J. Cell Biol
.
17
,
208
212
.
Romanoff
,
A. L.
(
1960
).
The Avian Embryo: Structural and Functional Organisation
.
New York
:
Macmillan
.
Simkiss
,
K.
(
1961
).
Calcium metabolism and avian reproduction
.
Biol. Rev
.
36
,
321
367
.
Skalinskii
,
E. I.
(
1965
).
Functional morphology of the intercalated cells of the chick embryo chorio-allantois. Dokl. Akad. Nauk SSSR. Translation into English in Dokl (Proc) Acad
.
Sci. U.S.S.R. (Biological Sciences)
161
,
178
181
.
Skalinsky
,
E. I.
&
Kondalenko
,
V. F.
(
1963
).
Electron microscopic studies of the chick chorio-allantois during embryogenesis
.
Acta morph, hung
.
12
,
247
259
.
Stoeckel
,
E.
&
Porte
,
A.
(
1969
).
Etude ultrastructurale des corps ultimobranchiaux du poulet. I. Aspect normal et développement ambryonnaire
.
Z. Zellforsch. mikrosk. Anat
.
94
,
495
512
.