Regional and developmental variations of blood vessel morphometry in the chick embryo chorioallantoic membrane
1 Department of Zoology, Tel Aviv University, Tel Aviv, Israel
2 Department of Pathology, Tel Aviv University, Tel Aviv, Israel
* Author for correspondence (e-mail: aarah{at}post.tau.ac.il)
Accepted 25 April 2005
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Summary |
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Key words: air cell, blood vessel numerical density, chick embryo, chorioallantoic membrane, morphometry
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Introduction |
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There are 120 pores cm-2 of
17 µm diameter each,
which traverse the 300 µm-thick eggshell of the domestic hen
(Ar and Rahn, 1985
). According
to Rol'nik (1970
), Rizzo
(1899
) was the first to study
the histology of the eggshell. He found that the pores in the blunt, middle
and pointed regions of eggs decline in number from 149 to 131 to 90 pores
cm-2, respectively. Romanoff
(1943
) and Romanoff and
Romanoff (1949
) found that
permeability of the eggshell to an air pressure difference of 200 mmHg (26.7
kPa) ishighly variable but averages higher at the blunt pole than at the small
pole (14.5 and 10.5 ml cm-2 min-1 mmHg-1,
respectively, which are 108.76 and 78.76 ml cm-2 min-1
kPa-1, respectively).
Rokitka and Rahn (1987)
measured water vapor diffusive conductance of eggshells of six avian species
and found an average decline of 88 and 63% of blunt end values, for the middle
and the pointed end, respectively. These relationships corresponded to the
changes in regional eggshell pore density. Romijn
(1950
) estimated that
80%
of the O2 consumed towards the end of the incubation is supplied
through the shell over the air cell area. However, direct measurements show
that
29% of the total O2 consumption comes from the area over
the air cell and that the respiratory gas exchange ratio (RQ) is higher under
the air cell (0.75) compared with the rest of the chorioallantoic membrane
(CAM) (0.68) while the total RQ is 0.7 (Visschedijk,
1968a
,b
).
Seymour and Visschedijk (1988
)
have obtained similar results. Paganelli et al.
(1988
) analysed these ratios
and concluded that they indicate a relative `hyperventilation' over the air
cell area, namely a higher ratio of gas diffusion rate through the shell to
the corresponding CAM blood perfusion.
The CAM is a fusion of the chorion and the allantois, which start to
develop on the 5th day of the 21 incubation days. On the 11th day, it is
completely formed and lies attached under most of the inner eggshell membrane
(Romanoff, 1960;
Rol'nik, 1970
). In parallel,
it begins its role as a respiratory organ. The CAM is rich in blood vessels
and its capillaries migrate towards the inner shell membrane and lie close to
the outer surface from day 14 (Duncker,
1978
).
Dusseau and Hutchins (1988)
followed the vascular density of the CAM with time from day 7 and found an
increase in CAM vascular density index (VDI = intersections per unit area) of
36% and 68% on days 10 and 14, respectively. They found an additional increase
in VDI of
34% on these days in 15% O2. Strick et al.
(1991
), who incubated chicken
eggs in 12, 16, 21, 45 or 70% O2 from day 7 to 14, showed that the
graded exposure to O2 produced a dose-related change in the VDI:
hypoxia increased and hyperoxia decreased VDI. Corona and Warburton
(2000
) covered
25% of
chicken eggshells with beeswax before placing them in an incubator and
measured VDI in the formed hypoxic region of the CAM on day 12 of incubation.
They did not find VDI differences between the hypoxic and the control regions
at this early stage. On the other hand, Wagner-Amos and Seymour
(2003
) demonstrated that
artificially covering half of the shell with wax at the beginning of
incubation reduced vessel density of the CAM under the covered area compared
with untreated eggs.
As far as we know, there are no quantitative histological studies on the VDI, area fraction and CAM thickness under the air cell in comparison with the rest of the CAM. We hypothesized that local eggshell porosities and conductances, which are known to exist between the two zones, would be correlated with the local CAM blood vessel numerical density, area fraction and thickness. Thus, the aim of this research was to compare the blood vessel numerical density, area fraction of blood vessels and local thickness of the chick embryo CAM under the air cell with those of the rest of the CAM at different embryonic ages.
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Materials and methods |
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On days 10, 12, 14, 16, 18 and 20 of the incubation, 914 fertile
eggs were randomly sampled. The eggshell above the air cell was removed and
the CAM, including the shell membrane attached to it, was cut off for further
examination. In addition, five pieces of 5060 mm2 CAM
attached to the shell membranes were taken at random from other locations on
the eggshell. All samples were immediately fixed in 10% buffered formaldehyde
for at least 24 h. Tissues were embedded in paraffin, thick sectioned
(46 µm), stained with hematoxylineosin and observed using an
Olympus BX50 light microscope. Counts and measurements were performed using
common morphometric approaches (Williams,
1977; Russ and DeHoff,
2000
). Blood vessels from 8 µm (lowest limit of detection) in
diameter were counted using the systematic sampling approach
(Russ and DeHoff, 2000
). In
short, the tissue was divided into 510 equally distributed fields.
Measurements were made in the center of each field.
Blood vessel numerical density
Counts of blood vessel numerical density [number per unit area;
NA(v)] were done with a 20x objective and using a
projected calibrated square grid graticule (5x5) inside the 10x
ocular. For each CAM sample (under the air cell and under the rest of the
shell), the blood vessel profile was counted at 10 areas and the results were
averaged for each sample.
Blood vessel area fraction
Counting of area fraction [AA(v)] was done as described
above for blood vessel numerical density using a calibrated square grid
graticule (10x10). Each CAM sample was point counted at five different
locations and the results were averaged. The total number of points hitting
blood vessels [Pi(v)] and the reference tissue
[Pi(t)] were counted. Thus, the area fraction
(Williams, 1977) is:
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Thickness of the CAM
Measurements of the tissue thickness (DCAM) were done
with a calibrated objective of 40x and using a 10x10 calibrated
grid in the 10x ocular. Each CAM sample was measured by the use of the
projected and calibrated grid at five different locations and the results were
averaged.
In paraffin-embedded tissue, material shrinkage is estimated to be 25%
relative to the fresh material (Mandarim-deLacerda et al.,
1985
,
1987
;
Mandarim-de-Lacerda, 2003
). We
assumed that, since all tissues were prepared similarly, tissue shrinkage is
the same in both CAM zones. Thus, shrinkage corrections are unnecessary for
tissue comparisons.
Total egg surface area was calculated from initial fresh egg mass (after
Paganelli et al., 1974). We
assumed that the area of the dome-shaped part of the shell, covering the air
cell (blunt end), equals the area of the inner shell membrane to which the CAM
under it is attached, since initially the shell membranes of the freshly laid
egg adhere to the shell and presumably do not stretch or shrink during
incubation. To calculate this area (Sac), we used the
following equation: Sac=2
Rh, where R
is the radius of a hemisphere (assuming that the blunt side of the egg is a
part of a hemisphere). This R was measured at the widest part of the
egg waist. h is the height of the line normal to the plane of an
imaginary disc (of a radius r) formed by the line of contact at the
edge of the air cell and the CAM and is calculated as:
. A comparison
with measured values of Romijn
(1950
) demonstrated a high
correlation with our calculated values (r2=0.923).
Since the total length of the blood vessels (L) is calculated as
L=2NA(v)V
(Williams, 1977;
Russ and DeHoff, 2000
), and
the volume (V) of a CAM sample is the product of its surface area
(SCAM) multiplied by CAM sample thickness
(DCAM), we may calculate the total length of blood vessels
within the CAM in different zones as L = 2 NA(v)
SCAM DCAM. Thus, the ratio between the
Lac and Lre is:
[NA(v)ac Sac
Dac]/[NA(v)re Sre
Dre].
Calculation of total number of pores
Thirteen eggs were used to calculated the total number of pores in the
different zones. This was done by calculating for each egg the total eggshell
surface area using the initial egg mass according to Paganelli et al.
(1974). The area over the air
cell of the same eggs was calculated as described above. These values were
used to calculate the shell area of the rest of the shell, by subtracting the
area of the shell over the air cell from the total surface area for each egg
on day 16 of the incubation. These values, multiplied by the pore density
taken from Rizzo (1899
), gave
values for the total number of pores in each area.
Statistical methods
The two different zones were statistically compared for the same eggs using
paired Student's t-test. The results for different embryonic ages
were compared using one-way analysis of variance (ANOVA). All data ratios
between the two different zones for every embryonic age were compared using
the KruskalWallis ANOVA median test.
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Results |
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The calculated surface area of the CAM under the air cell (Sac), increased, and that of the rest of the shell (Sre) decreased, from day 10 to 20 (Table 1). The ratio between Sac and Sre increased with embryonic age from 0.13 to 0.25.
Blood vessel numerical density [NA(v)] varied at different embryonic ages and in the two zones (Table 1). The NA(v) under the air cell on each embryonic age tested was significantly higher (range: 102164 vessels mm-2) than the NA(v) of the rest of the CAM (range: 6193 vessels mm-2). NA(v) increased significantly in both zones up to day 16 and then decreased until day 20 (Table 1). The ratio between the NA(v) in the CAMac and CAMre was not significantly different among the embryonic development days. The overall ratio averaged 1.79±0.59 (N=67).
Table 1 also shows that the mean area density of blood vessels [AA(v)], as a percentage of the total CAM area under the air cell, ranged from 32 to 42% with no significant differences among the days. The AA(v) values under the air cell were almost double and significantly different from those under the rest of the shell (1724%).
Although our results may be biased due to the fact that they are based on some model assumptions, e.g. calculation of surface area, tissue shrinkage during histological preparation and the kind of morphometric measurements used, the final comparisons would yield bias-free results, since all techniques used would have the same systematic errors. Therefore, for the purpose of comparisons they are still valid.
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Discussion |
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We consider that the potential for CAM gas exchange can be estimated from
the ratio between the eggshell pore density (determining eggshell diffusive
gas conductance) and CAM blood vessel density (involved in gas transfer by the
blood in the CAM circulation), since both structures participate in this gas
exchange. Regional differences in shell pore density
(Rizzo, 1899; according to
Rol'nik, 1970
) and shell gas
conductance (Rokitka and Rahn,
1987
) have been reported, indicating higher area-specific gas
conductance over the air cell (Table
2). It can be inferred from Simkiss
(1980
) that temperature
fluctuations in the air cell may enhance gas exchange over it in comparison
with the rest of the eggshell. It has been demonstrated that the partial
pressure of oxygen in the air cell of goose eggs is higher than in other areas
under the eggshell (Meir et al.,
1999
). According to Romanoff
(1943
), the
differential-pressure gas permeability of the shell over the air cell is 28%
higher than that of the shell over the small end of the egg. These conductance
differences manifest themselves in the different RQ over the different regions
of the shell (Paganelli et al.,
1988
).
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The CAM reacts differently to total or local hypoxia
(coverage of part of the eggshell) as follows: when the eggshell is entirely
exposed to various O2 atmospheres, there is a dose-dependent change
in the vascular density index of the CAM (VDI) on days 714; in
hyperoxia the VDI decreases, and in hypoxia it increases
(Dusseau and Hutchins, 1988;
Strick et al., 1990
). This
corresponds to an increase in embryonic mass and a decrease in CAM mass in
hyperoxia, and vice versa in hypoxia (Richards et al.,
1991
,
1992
).
When part of the eggshell of chicken eggs was covered at the beginning of
incubation, the CAM VDI under that part showed no changes on day 12, in
comparison to non-covered control eggs
(Corona and Warburton, 2000).
However, the results of Wagner-Amos and Seymour
(2003
) showed a decrease in
CAM VDI under the covered side under similar conditions from day 12 onwards,
indicating suppressed angiogenesis of the circulatory system when oxygen
supply is limited. This may correspond to the fact that at day 12, although
the CAM is complete, the size of the embryo is only 10% of its final size, and
thus the rate of oxygen consumption is very low
(Romanoff, 1967
;
Duncker, 1978
).
The apparent contradiction in the exposure of the CAM to full hypoxia (VDI increases) and partial coverage of the shell (VDI decreases under the covered area) may stem from the fact that, in the latter, compensation in the non-affected CAM areas is possible. However, evidence in the literature is not conclusive and it is premature at this stage to speculate further about the reasons.
In view of the natural regional differences in shell conductance and pore density between the eggshell over the air cell and the rest of the eggshell, we discuss the possibility of an appropriate matching of the CAM VDI under them. In contrast to previous work, the present study focuses on and discusses the two different CAM zones, which presumably are exposed to different oxygen levels during normal incubation.
Strick et al. (1990) found
that VDI and blood vessel length density increased from day 8 until day 14,
after which there were negligible changes until day 18. Similarly, Wagner-Amos
and Seymour (2003
) found that
the density of pre- and post-capillary vessels increased in control eggs,
reaching a maximum on day 14. Table
1 shows that blood vessel numerical density in both CAM areas
[NA(v)] reaches a peak on day 16, which is the time of
maximal growth rate of the embryo (Dietz et
al., 1998
; Romanoff,
1967
). It seems that maximal NA(v) (and VDI)
is reached on days 1416 and, from that time on, other mechanisms, such
as increased CAM blood flow and blood oxygen affinity
(Tazawa, 1980
) and the
movement of the blood capillaries to a position nearer the inner shell
membrane (Duncker, 1978
),
enhance oxygen delivery rate.
It is interesting to note that, on day 14 of incubation, the
DCAM is reduced both under the air cell and the rest of
the shell (Table 1). Similar
results were found in other studies
(Romanoff, 1960;
Ar et al., 1987
). The reason is
not yet known, but day 14 also seems to be a pivotal time in terms of air cell
and blood gas pressures (Tazawa et al.,
1980
; Tazawa,
1980
) and marks the beginning of fast growth
(Romanoff, 1967
).
The values of NA(v) and AA(v) under
the air cell are significantly higher than those of the rest of the CAM at all
embryonic ages checked (Table
1). From day 16 onwards, NA(v) and
AA(v) values under the air cell are almost twice those of
the rest of the CAM. Tazawa and Ono
(1974) found that the blood
vessels show similar area densities for days 1218 of incubation
(33.640.8%). We found comparable values (32.242.1%) and showed
that there is no statistically significant difference in the
AA(v) under the air cell on days 1020 of incubation
(Table 1).
Our data show that the total length of blood vessels (L), the
ratio of the CAM surface area under the air cell to that of the rest of the
CAM (Sac/Sre) and the same ratio for
total length of blood vessels
(Lac/Lre) all increase with embryonic
age and air cell size (Table 3)
and, thus, with the increase of the O2 consumption rates and
embryonic age (Romanoff, 1930,
1967
). The relative increase
in Lac with age, together with the increase in
Sac (Table
1), parallels the increased participation of the eggshell over the
air cell in gas exchange during incubation.
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As Table 3 shows, the total
length of blood vessels under the air cell (Lac) on day 20
is three times that on day 10
(Lac=3.67x104 mm and
1.20x104 mm, respectively). This increase is more than the
increase in Sac. In comparison, the total length of blood
vessels under the rest of the CAM (Lre) on day 16 is
1.64 times that on day 10
(Lre=9.83x104 mm and
5.99x104 mm, respectively). This increase is despite a
decrease in Sre (Table
1). From day 16 onwards, a decrease in Lre to
7.91x104 mm on day 20 is observed. The final
Lre value is only 1.32 times that of day 10.
Romijn and Roos (1938)
found that the `allantoic surface, which lines the floor of the air space'
comprises 1020% of the entire respiratory area of the embryo in the
last days of the incubation. In our study, the ratio
Sac/Sre increases from 0.13 (day 10)
to 0.25 on day 20 (Table 3).
Concurrently, the ratio Lac/Lre
increases from 0.20 to 0.47 (Table
3). This difference can explain the ratio of embryonic
O2 consumption rate of 0.42 between the air cell and the rest of
the eggshell towards the end of the incubation found by Visschedijk
(1968a
) and the relatively
higher CO2 loss rate through it
(Paganelli et al., 1988
).
The total number of pores in the different zones was calculated according
to the egg surface area (Paganelli et al.,
1974) and the relative number of pores in each zone
(Rizzo, 1899
)
(Table 2). The regional length
per pore found for the CAM under the air cell and for the rest of the CAM is
almost the same (1.47 cm pore-1 and 1.42 cm pore-1,
respectively; Table 2). This
may mean that each pore, whether of the air cell or the rest of the shell,
serves a similar length of blood vessels. This corresponds to the almost
constant gas exchange rate per pore and the area of service per pore concept
advanced by Ar and Rahn (1985
)
for bird eggs in general.
The relationships between blood vessel density and pore density in the CAM
indicate optimization of embryonic gas exchange. We suggest that in natural
incubation, the increased gas exchange under the air cell compensates for
covering of the central part of the eggshell by the incubating parent
(20% of the eggshell area: Kendeigh,
1973
; YomTov et al.,
1986
; Handrich,
1989
).
Our results seem to explain, in part, the apparent `paradox of the shell'
(Riddle, 1930; as cited by
Rol'nik, 1970
) where the
living embryo adapts by varying angiogenesis in relation to the shell
permeability to gases. Whether this concept holds for other bird species
remains to be seen.
List of symbols/abbreviations
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Acknowledgments |
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