An electron microscopical and biochemical study of the chorionic epithelium from 16-and 17-day-old chick embryos was conducted. Using a combination of sections made either parallel or perpendicular to the chorionic surface, it was confirmed that the cell populations of the polar and equatorial regions of the membrane are different; the former contains a very reduced number of VC cells which are, contrariwise, widely distributed in the latter. This evidence was required for the interpretation of data on carbonic anhydrase activity. This enzymatic activity is known to increase in the chorioallantoic membrane at the time at which shell resorption begins but the enzyme is believed to be localized in other cell types in addition to VC cells. The results of the present experiments show that carbonic anhydrase activity is significantly higher in the region where VC cells are more frequent (equatorial) than in the region where they are rarely found (polar) and thus suggests that a substantial proportion of the activity measured in the whole membrane corresponds to VC cells.

Carbonic anhydrase activity in the equatorial region of the chorioallantoic membrane increased after 4 h and peaked 24 h after the administration of 1,25-DHCC. This time course is similar to the one on previous experiments in which serum calcium concentrations were determined after similar treatment. It is suggested that one of the effects of 1,25-DHCC on its target cells in the membrane (VC cells according to previous evidence) is to increase synthesis and/or activity of carbonic anhydrase.

During the second half of incubation the chick embryo resorbs from the egg shell large amounts of calcium which it utilizes for the mineralization of its skeleton (Johnston & Comar, 1955; Simkiss, 1961; Narbaitz & Jande, 1978). Calcium is absorbed by the vascular network of the chorionic epithelium and at the time in which calcium absorption starts to increase specialized cells with numerous mitochondria, apical vacuoles and long microvilli differentiate in the chorion. These cells have been variously designated as ‘intercalated’ (Skalinsky & Kondalenko, 1963), ‘calcium-absorbing’ (Owczarzak, 1971) or ‘villus-cavity’ cells (Coleman & Terepka, 1972). Coleman & Terepka (1972) also described a second type of chorionic cells which they named ‘capillary-covering’ cells. Since the vascular spaces in the chorion were shown to constitute a single blood sinus and not capillaries, Narbaitz (1977) proposed to replace the name ‘capillarycovering’ by ‘sinus-covering’. For the sake of simplicity we shall use the name VC (villus-cavity) for the first type and SC (sinus-covering) for the second one. The role of these cell types in the process of calcium resorption is under debate (Skalinsky & Kondalenko, 1963; Owczarzak, 1971 ; Narbaitz, 1972; Coleman & Terepka, 1972; Simkiss, 1980)

At the blunt end of the egg the air chamber separates the chorion from the shell so that calcium is not normally absorbed through this polar zone of the membrane. Narbaitz (1972) indicated that the relative frequencies of chorionic cell types were different in the polar and equatorial regions of the chorion. In a later study Narbaitz (1977) did an electron microscopical analysis of sections oriented parallel to the surface of the chorion; in these sections the blood sinus is seen in all its extension and its lumen is only interrupted at regular intervals by ‘strands’ or ‘columns’ of tissue connecting the basal and apical portions of the epithelium. These ‘columns’ contain the apical portions of both VC and SC cells and since numerous columns can be observed simultaneously this particular orientation of the sections allows a simple and efficient method of sampling the cell population in the chorion. In the above mentioned work (Narbaitz, 1977), only the equatorial region was studied and it was established that one to three VC cells are present in most columns; similar studies have not been conducted on the polar region of the chorion. In the present study a thorough sampling of both polar and equatorial regions was carried out using a combination of parallel and cross sections, in order to define with more precision differences in cell populations.

Carbonic anhydrase is probably involved in the process of calcium resorption by the embryo (Owczarzak, 1971 ; Heckey & Owczarzak, 1972; Tuan & Zrike, 1978: Rieder, Gay & Schraer, 1980). In the chorionic epithelium the enzyme appears to be located mainly in VC cells (Heckey & Owczarzak, 1972; Rieder et al. 1980). In the present study comparative determinations of carbonic anhydrase activity in the two regions of the chorioallantoic membrane were conducted in order to find out if the enzymatic activity differed in correlation with the relative frequency of VC cells.

1,25-dihydroxycholecalciferol (1,25-DHCC) is a very active metabolite of vitamin D3 and is considered to be a real steroid hormone (DeLuca, 1978). When injected into the chick embryo it produces hypercalcemia and this has been attributed to an increase in the resorption of calcium from the shell (Narbaitz & Tolnai, 1978) Narbaitz et al (1980) have shown that radioactive 1,25-DHCC is concentrated by chorionic cells which have a spatial distribution similar to that of VC cells. The possibility that the effects of 1,25-DHCC on VC cells might include stimulation of carbonic anhydrase activity must be considered. We here report the results of determinations of enzymatic activity in the equatorial region of the chorioallantoic membrane at various periods of time after administration of a single dose of 1,25-DHCC

Eggs from White Leghorn hens, obtained from a commercial source, were incubated in a forced-air incubator. Embryos were injected on the 15th or 16th day of incubation with a single dose of 230 p-moles 1,25-DHCC in 0·05 ml 95 % ethyl alcohol; injections were made in the yolk sac. Controls received 0·05 ml 95 % ethyl alcohol. The age of injection, the dose and the length of time between injection and sacrifice were selected on the basis of previous experiments (Narbaitz & Tolnai, 1978). Those experiments had shown that 230 p-moles produces hypercalcemia which is detectable 4 h after, reaches a peak 24 h after and is still present 48 h after the injection. At the time of sacrifice the eggs were opened, the chorioallantoic membranes were separated, blotted with filter paper and frozen in liquid nitrogen. Samples were maintained at –30 °C until homogenization. This was carried with a Potter-Elvehjem homogenizer. Samples were homogenized in 0·02 M-Veronal buffer pH 8·0 (250 mg tissue/ml buffer). In some of the experiments 1 mM-dithiothreitol was added to the buffer following the recommendation of Bernstein & Schraer (1972). No differences in the results were observed with or without dithiothreitol and the data obtained in both cases were pooled. Carbonic anhydrase activity in the samples was determined following the electrometric procedure of Wilbur & Anderson (1948). Protein content was determined according to Lowry, Rosebrough, Farr & Randall (1951). Haemoglobin content was determined with the cyanomethemoglobin method as described by Davidsohn & Wells (1962) using the reagent kit supplied by Hycel Inc. (Houston, Texas). Data on haemoglobin concentration were used to calculate the contamination of the samples with carbonic anhydrase of erythrocytic origin, according to Clark (1951). In our experiments contamination was never higher than 1·8 % of the total carbonic anhydrase activity; this being considered insignificant, corrections were not made and the figures in Table 1 represent total activity. In a second series of experiments, carbonic anhydrase activity was determined separately in samples obtained from the polar and equatorial regions of the chorioallantoic membranes.

Table 1.

Carbonic anhydrase activity (Units/mg protein) in chorioallantoic membranes from embryos injected with 1,25-DHCC

Carbonic anhydrase activity (Units/mg protein) in chorioallantoic membranes from embryos injected with 1,25-DHCC
Carbonic anhydrase activity (Units/mg protein) in chorioallantoic membranes from embryos injected with 1,25-DHCC

Electron microscopic studies

Portions of the polar and equatorial regions of the chorioallantoic membranes from 16-day-old chick embryos were fixed together with the attached portion of shell membrane. Samples were obtained from different locations in each of the mentioned regions. Fixation was conducted in half-strength Karnovsky’s (1965) fixative for 6 h. Tissues were then washed in 0·1 M-cacodylate buffer at pH 7·2 containing 0·2 M-sucrose and postfixed in 2% osmium tetroxide for 1 h. They were then dehydrated in ethyl alcohol and embedded in Araldite. Tissues were oriented in the blocks in such a way that sections could be made either parallel to the surface or perpendicular to it (cross sections). In the first case, 1 μm sections were serially obtained until the lumen of the sinus was reached. At this time thin sections were produced for electron microscopical examination. Thick sections were stained in toluidine blue and thin sections with uranyl acetate and lead citrate according to Reynolds (1963).

Ultrastructural data

The analysis of sections oriented parallel to the surface of the chorion, shows that the vascular spaces are constituted, both in the polar and equatorial regions, by a single extensive blood sinus (Figs. 1, 2). Its lumen contains erythrocytes and other blood cells. When the sections are close to the floor of the sinus, parts of its endothelial cells may be included in the section (Fig. 1). At regular intervals the lumen is interrupted by cross sections of the columns. A comparative analysis of parallel (Figs. 1, 2, 3) and cross (Figs. 4, 5, 6) sections permits a better understanding of the structure of the columns. Figures. 4, 5 and 6 show that the columns are constituted by that part of the chorionic cells which lies between two vascular spaces. The dotted lines in these figures, indicate the direction in which the columns are cut when parallel sections are made.

Fig. 1.

Electron micrograph from a parallel section of polar chorion. The lumen of the sinus contains dense erythrocytes and other blood cells. Portions of the endothelium of the floor of the sinus (E) are included in the section. Four columns (C) are included in this section; they all contain SC cells.

Fig. 1.

Electron micrograph from a parallel section of polar chorion. The lumen of the sinus contains dense erythrocytes and other blood cells. Portions of the endothelium of the floor of the sinus (E) are included in the section. Four columns (C) are included in this section; they all contain SC cells.

Fig. 2.

Electron micrograph from a parallel section of equatorial chorion. The four columns (c) in this section contain VC cells.

Fig. 2.

Electron micrograph from a parallel section of equatorial chorion. The four columns (c) in this section contain VC cells.

Fig. 3.

Parallel section through the polar chorion. Note endothelium surrounding the basal lamina (small arrows). The core of the column contains portions of 2 SC cells (large arrow in intercellular space).

Fig. 3.

Parallel section through the polar chorion. Note endothelium surrounding the basal lamina (small arrows). The core of the column contains portions of 2 SC cells (large arrow in intercellular space).

Fig. 4.

Cross section of chorion in the polar region. A SC cell constitutes the core of the column. The dotted line indicates the direction in which a parallel section would cut this column.

Fig. 4.

Cross section of chorion in the polar region. A SC cell constitutes the core of the column. The dotted line indicates the direction in which a parallel section would cut this column.

Fig. 5.

Cross section of equatorial chorion. A SC cell (SC) and a VC cell (VC) are forming part of the column. Note the connexion of the body of the SC cell with the rim of the cavity in the column. The dotted line indicates the direction in which this column would be cut in parrallel sections.

Fig. 5.

Cross section of equatorial chorion. A SC cell (SC) and a VC cell (VC) are forming part of the column. Note the connexion of the body of the SC cell with the rim of the cavity in the column. The dotted line indicates the direction in which this column would be cut in parrallel sections.

Fig. 6.

Cross section of equatorial chorion. A degenerating SC cell (SC) and a VC cell (VC) form part of the column. Dotted line indicates direction in which the column would be cut in parallel sections.

Fig. 6.

Cross section of equatorial chorion. A degenerating SC cell (SC) and a VC cell (VC) form part of the column. Dotted line indicates direction in which the column would be cut in parallel sections.

In both polar and equatorial regions columns are surrounded by endothelium resting on a basal lamina (Figs. 1,2, 3); however, the core of the column is very different in both cases. Thus, in the polar region most of the columns have a solid core formed by parts of 1 to 3 SC cells (Figs. 1, 3). Contrariwise, in the equatorial region, the core of the columns is most frequently occupied by a cavity containing floating microvilli of VC cells (Fig. 2) or the vacuoles of the apical part of these cells (right upper column in Fig. 2). The cavity is always surrounded by a thin rim of cytoplasm (left lower column in Fig. 2). If one compares this column with the one in Fig. 5, it becomes evident that the thin rim surrounding the cavity belongs to SC cells which have their main body in the basal part of the chorion and are connected by the rim to the thin cytoplasmic layer overlying the sinus. SC cells in the equatorial region are thus morphologically different from those in the polar region, in that their main body has been displaced from the intervascular space by the presence of VC cells. In some cases, the SC cells degenerate and become very electron dense (right lower column in Fig. 2 and Fig. 6). This often happens also to VC cells (upper left column in Fig. 2). The types of column described as typical of the polar region, can also be found in the equatorial region but only very infrequently (1−2 % of the columns). Similarly, the types of columns here described as typical of the equatorial region can be, although rarely, found in the polar region, mainly in the peripheral part of it; i.e. where the polar and equatorial regions meet with each other.

From the composition of the columns it can thus be deducted that VC cells are frequent in the equatorial zone (one to three cells in almost all columns) and very infrequent in the polar zone (few and mostly located in the periphery of the zone). Similarly, degenerating cells are only frequent in the equatorial zone. SC cells are present in both zones; however, in the polar zone they occupy the whole column while in the equatorial region they have a different shape and are displaced by the VC cells.

Biochemical data

In normal 16-day-old embryos the carbonic anhydrase activity was significantly lower in the polar region of the chorioallantoic membrane (average 8 embryos: 0·6 U/mg protein ±0-08 S.D.) than in the equatorial region (average 8 embryos: 1·45 U/mg protein ±0·2 S.D.).

Table 1 contains the data on carbonic anhydrase activity in the chorioallantoic membrane of embryos injected with 1,25-DGCC. It can be observed that the enzymatic activity started to increase 4 h after, reached a maximum 24 h after and returned to normal 48 h after the injection.

Although numerous histological and ultrastructural studies on the chick chorion are available in the literature, certain details of its morphological organization remain controversial. The facts that cell types cannot be defined with precision with the light microscope and are distributed unequally in different zones of the chorion explain some of the difficulties encountered. The present results contribute to clarify some of the points under debate. Thus, we have confirmed that VC cells are very frequent in the equatorial zone of the chorion and very infrequent in the polar zone. It was also shown that SC cells are widely distributed both in polar and equatorial regions but that they have a different shape in both cases. In the polar region they conform to the typical description of Coleman & Terepka (1972) presenting a broad apex branching into thin cellular process toward both sides; in the equatorial zone, however, their cell body is displaced and is connected with the corresponding supra-sinusal cell processes by the narrow rim surrounding the cavity in the column. The understanding of the differences in cell populations between polar and equatorial regions is of importance for the interpretation of epithelial functions as described below.

The facts that VC cells differentiate at the time at which resorption of shell mineral becomes active, and that their morphological characteristics (abundance of mitochondria, apical vacuoles and microvilli) are similar to those of oxyntic cells of the stomach and osteoclasts have suggested the idea that they are concerned with the secretion of agents aiding to the solubilization of the shell (Skalinsky & Kondalenko, 1963; Leeson & Leeson, 1963; Owczarzak, 1971). Our present results demonstrating that these cells are very frequent in the zone of chorion concerned with mineral resorption and rare in the region not involved in this resorption adds support to that idea.

VC cells appear to be rich in carbonic anhydrase (Rieder et al. 1980). That this enzyme is involved in the solubilization of the shell is suggested by the fact that its activity in the membrane increases at the time at which resorption begins (Tuan & Zrike, 1978). It could be objected that since there is also carbonic anhydrase in the allantois (Reider et al. 1980), we cannot be sure if the increase in activity in the whole membrane should be attributed to the chorion or to the allantois. Our present results showing that carbonic anhydrase activity varies in correspondence with the frequency of VC cells, being lower in the polar and higher in the equatorial regions, tend to suggest that a significant amount of the total activity in the membrane corresponds to VC cells.

In our experiments showing that carbonic anhydrase activity in the equatorial zone of the membrane increases significantly after 1,25-DHCC administration, a similar objection could be raised. However, the fact that target cells for 1,25-DHCC are known to be in the chorion and not in the allantois (Narbaitz et al. 1980) tends to suggest that this increase corresponds to the former. In addition, the particularities of the time course obtained are of great interest. Narbaitz & Tolnai (1978) showed that after the injection of 1.25-DHCC the concentration of calcium in blood started to increase 4 h after, reached a peak 24 h after and declined, although not completely, 48 h after the injection. In agreement with this, our present experiments show that carbonic anhydrase activity in the chorioallantoic membrane follows a similar time course with its maximum 24 h after the injection. This coincidence strongly suggests that both phenomena are interrelated and that the action of the hormone in the membrane involves stimulation of the synthesis and/or activity of this enzyme. This conclusion is consistent with the concept that, like most steroid hormones, 1,25-DHCC acts on its target tissues by stimulating the synthesis of one or more proteins. In the case of the duodenum from adult animals the hormone is known to stimulate the synthesis of a vitamin D-dependent calcium-binding protein, Ca, Mg-dependent ATPase and perhaps other enzymes (Wasserman & Corradino, 1973). It is possible that in the case of the chorion, the hormone also produces other effects besides increasing carbonic anhydrase activity. Ca, Mg-dependent ATPase activity has also been detected in the chorioallantoic membranes (Saleudin, Kyriakides, Peacock & Simkiss, 1976) and it would be of interest to know if it is regulated by 1,25-DHCC. A calcium-binding protein has also been detected in the membrane but it appears to be vitamin K-rather than vitamin D-dependent (Tuan, Scott & Cohn, 1978).

The mechanism(s) by which carbonic anhydrase would influence calcium resorption is (are) not clear. It probably involves the production of H+ ions necessary for the solubilization of the shell’s calcium carbonate (Coleman & Terepka, 1972; Dawes, 1975). It is not clear if the enzyme is also required for the actual transport of calcium through the epithelium, nor is it known through which cell type does this transport occur.

Numerous physiological experiments (Terepka, Stewart & Merkel, 1969; Coleman, DeWitt, Batt & Terepka, 1970; Crooks & Simkiss, 1975) have indicated that the polar region of the chorioallantoic membrane is capable of active transport of calcium. Our present demonstration that this region of the chorion contains mainly SC cells and very few VC cells would indicate that the former are responsible for the transport demonstrated in those experiments. It would be desirable, however, that similar experiments would be conducted on the region of the membrane that normally is responsible for calcium transport. This is especially so in the case of experiments involving electron probe localization of calcium (Coleman et al. 1970) since the cell population is so different in both regions of the membrane.

Skilled technical assistance was provided by Mr V. Kapal and Miss G. A. Calderwood, Jr.

Bernstein
,
R. S.
&
Schraer
,
R.
(
1972
).
Purification and properties of an avian carbonic anhydrase from the erythrocytes of Gallus domesticas
.
J. biol. Chem
.
247
,
1306
1322
.
Clark
,
A. M.
(
1951
).
Carbonic anhydrase activity during embryonic development
.
J. exp. Biol
.
28
,
332
343
.
Coleman
,
J. R.
,
Dewitt
,
S. M.
,
Batt
,
P.
&
Terepka
,
A. R.
(
1970
).
Electron probe analysis of calcium distribution during active transport in chick chorioallantoic membrane
.
Expl Cell Res
.
63
,
216
220
.
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
.
Crooks
,
R. J.
&
Simkiss
,
K.
(
1975
).
Calcium transport by the chick chorioallantois in vivo
.
Q. J. expl Physiol
.
60
,
55
63
.
Davidsohn
,
I.
&
Wells
,
B. B.
(
1962
).
Todd-Sanford Clinical Diagnosis By Laboratory Methods
, pp.
73
77
.
Philadelphia. W. B. Saunders
.
Dawes
,
C. M.
(
1975
).
Acid-base relationships with the avian egg
.
Biol. Rev
.
50
,
351
371
.
DeLuca
,
H. F.
(
1978
).
The hormonal nature of vitamin D function
.
In Hormones and Cell Regulation
, vol.
2
(eds.
J.
Dumont
&
J.
Nunez
), pp.
249
-
270
. Elsevier/North Holland Biomedical Press.
Heckey
,
R. P.
&
Owczarzak
,
A.
(
1972
).
The chorioallantoic membrane histochemistry and electron microscopy of carbonic anhydrase
.
J. Cell Biol
.
55
,
110a
.
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
.
Leeson
,
T. S.
&
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
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin phenol reagent
.
J. biol. Chem
.
193
,
265
275
.
Narbaitz
,
R.
(
1972
).
Cytological and cytochemical study of the chick chorionic epithelium
.
Rev. Canad. Biol
.
31
,
259
267
.
Narbaitz
,
R.
(
1977
).
Structure of the intra-chorionic blood sinus in the chick embryo
.
J. Anat
.
124
,
347
354
.
Narbaitz
,
R.
&
Jande
,
S. S.
(
1978
).
Ultrastructural observations on the chorionic epithelium parathyroid glands and bones from chick embryos developed in shell-less culture
.
J. Embryol. exp. Morph
.
45
,
1
12
.
Narbaitz
,
R.
&
Tolnai
,
S.
(
1978
).
Effects produced by the administration of high doses of 1, 25-dihydroxycholecalciferol to the chick embryo
.
Calcif. Tiss. Res
.
26
,
221
226
.
Narbaitz
,
R.
,
Stumpf
,
W. E.
,
Sar
,
M.
,
Deluca
,
H. F.
&
Tanaka
,
K.
(
1980
).
Autoradiographic demonstration of target cells for 1, 25-dihydroxycholecalciferol in the chick embryo chorioallantoic membrane, duodenum, and parathyroid glands
.
Gen. Comp. Endocrinol
.
42
,
283
289
.
Owczarzak
,
A.
(
1971
).
Calcium-absorbing cell of the chick chorioallantoic membrane. Morphology, distribution and cellular interactions
.
Expl Cell Res
.
68
,
113
129
.
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
.
Rieder
,
E.
,
Gay
,
C. V.
&
Schraer
,
H.
(
1980
).
Autoradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane
.
Anat. Embyol
.
159
,
17
31
.
Saleudin
,
S. S. M.
,
Kyriakides
,
C. P. M.
,
Peacock
,
A.
&
Simkiss
,
K.
(
1976
).
Physiological and ultrastructural aspects of ion movements across the chorioallantois
.
Comp. Biochem. Physiol
.
54A
,
7
12
.
Simkiss
,
K.
(
1961
).
Calcium metabolism and avian reproduction
.
Biol. Rev
.
36
,
321
367
.
Simkiss
,
K.
(
1980
).
Water and ionic fluexes inside the egg
.
Amer. Zool
.
20
,
385
393
.
Skalinsky
,
E. I.
&
Kondalenko
,
V. F.
(
1963
).
Electron microscopic studies of the chick chorio-allantois during embryogenesis
.
Acta Morph. Hung
.
12
,
247
259
.
Terepka
,
A. R.
,
Stewart
,
M. E.
&
Merkel
,
N.
(
1969
).
Transport functions of the chick chorioallantoic membrane
.
Expl Cell Res
.
58
,
107
117
.
Tuan
,
R. S.
,
Scott
,
W. A.
&
Cohn
,
Z. A.
(
1978
).
Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression
.
J. Cel! Biol
.
77
,
752
761
.
Tuan
,
R. S.
&
Zrike
,
J.
(
1978
).
Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane
.
Biochem. J
.
176
,
67
74
.
Wasserman
,
R. H.
&
Corradino
,
R. A.
(
1973
).
Vitamin D, calcium and protein synthesis
.
Vit. Hormone
31
,
43
103
.
Wilbur
,
K. M.
&
Anderson
,
N. G.
(
1948
).
Electrometric and colorimetric determination of carbonic anhydrase
.
J. biol. Chem
.
176
,
147
153
.