J. Embryol. exp. Morph. Vol. 65, pp. 127-137, 1981
Printed in Great Britain © Company of Biologists Limited 1981
\ 27
Carbonic anhydrase activity in the chick
embryo chorioallantois: regional distribution
and vitamin D regulation*
By ROBERTO NARBAITZ 1 , SAM KACEW AND
LUCJAN SITWELL
From the Departments of Anatomy and Pharmacology,
University of Ottawa
SUMMARY
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.
INTRODUCTION
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
* Supported by grants from Medical Research Council of Canada
Author's address: Department of Anatomy, Faculty of Medicine, University of Ottawa,
Ottawa, Ontario, Canada, KIN 9A9.
1
5-2
128
R. NARBAITZ, S. KACEW AND L. SITWELL
& 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 ah 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 D 3 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
Vitamin D and carbonic anhydrase in chick chorion
129
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
MATERIAL AND MBTHODS
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 Lov/ry, 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.
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 con^ducted in half-strength Karnovsky's (1965) fixative for 6 h. Tissues were then
washed hi 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
130
R. NARBAITZ, S. KACEW AND L. SITWELL
Table 1. Carbonic anhydrase activity {Units/mg protein) in chorioallantoic membranes from embryos injected with 1,25-DHCC
Age of
sacrifice
(days)
Time of
injection
(h before)
Injected with
Ethyl alcohol
1,25-DHCC
(8)2-15±0-26
(8)3-O7±O-33*
(16) 4-23±064**
(16) 1 -57±0-29
(8)l-20±016
(8) l-40±0-16
Figures between brackets indicate number of embryos sampled. Figures represent averages
±S.D.
* Difference with corresponding controls significant; P = 005.
** Difference with corresponding controls significant; P < 0001.
16
16
17
4
24
48
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 jum 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).
RESULTS
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). Jts 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.
Jn 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
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.
Vitamin D and carbonic anhydrase in chick chorion
131
t
i
• E.
^
132
R. NARBAITZ, S. KACEW AND L. SITWELL
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:
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. 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.
Vitamin D and carbonic anhydrase in chick chorion
133
1
134
R. NARBAITZ, S. KACEW AND L. SITWELL
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.
DISCUSSION
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 denned
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
Vitamin D and carbonic anhydrase in chick chorion
135
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 1,25DHCC 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 L25-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 & Conn, 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
136
R. NARBAITZ, S. KACEW AND L. SITWELL
localization of calcium (Coleman et al. 1970) since the cell population is so
different in both regions of the membrane.
Skillpd technical assistance was provided by Mr V. Kapal and Miss G. A. Calderwood, Jr.
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