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Relocation during incubation of endothelial nuclei in the chick chorioallantois
~.~.~0 N P H p~.
ELSEVIER
Respiration Physiology 100 (1995) 171-176
Relocation during incubation of endothelial nuclei in the chick chorioallantois Andrea A. Mayer 1, James Metcalfe*, Michael K. Stock Department of Physiology, L334, Oregon Health Sciences University, Portland, OR 97201, USA (Accepted 21 November 1994)
Abstract
Early in incubation the nuclei of the chick chorioallantoic capillaries are randomly distributed around the capillary lumen; later most of them are located on the portion of the capillary surface opposite the inner shell membrane. This is one of the complex of processes that results in progressive thinning of the diffusion pathway for gases between the external environment and the blood of the embryo. The present study quantified this nuclear "relocation". Our data show a progressive relocation of the endothelial nuclei from the tenth through the sixteenth day at an average rate of 6~o per day. Key words': Birds, chick: Development, chorioallantoic capillaries; Embryonic gas exchange; Endothelium cells, nuclear relocation; Gas exchange
I. Introduction
Oxygen availability has a powerful influence on the growth and development of the embryo of Gallus domesticus (Metcalfe et al., 1984; Stock and Metcalfe, 1987). The capillary meshwork in the chorioallantoic membrane is well-suited to its essential role in the respiration of the avian embryo (Metcalfe and Stock, 1993). It develops from a vascular bed that traverses the mesoderm of the allantoic sac. Beginning on the fifth day of incubation the allantoic mesoderm fuses with the mesoderm of the enveloping
This work was performed in partial fulfillment of the requirements for the Bachelor of Science degree from George Fox College, Newberg, OR. * Corresponding author. Tel: (503)494-8440, Fax: (503)4942383. 0034-5687/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 3 4 - 5 6 8 7 ( 9 5 ) 0 0 1 2 7 - 8
chorion (Hamilton, 1965), and the capillary network expands to eventually encompass the contents of the whole egg, lining the entire inner surface of the inner shell membrane. When the chorioallantois is viewed in cross section near the end of incubation, the capillaries occupy more area than the ectodermal tissue separating them, thus forming "a great blood sinus interrupted by strands of tissue" (Ftllleborn, 1894). In addition to this process of capillary proliferation, which enlarges the surface area for gas exchange, the rate of net gas transport by diffusion between embryonic blood and environmental air is enhanced by a reduction in the diffusion distance. Beginning on about the tenth day of incubation, the capillary network migrates through the ectoderm of the chorion from its mesenchymal site of origin (Danchakoff, 1917; Fuchs and Lindenbaum, 1988). By the fourteenth day the embryo's blood, as it cir-
172
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
culates through the chorioallantoic capillary network, is separated from the air in the interstices of the inner shell membrane by tissue with an average thickness of only 0.5 to 0.7 microns (FitzeGschwind, 1973; Wangensteen and Weibel, 1982; Burton and Palmer, 1992). This barrier to gas diffusion is comprised of a thin layer of ectoderm (Hoshi and Mori, 1971), an incomplete basement membrane (Wangensteen and Weibel, 1982) and an attenuated layer of endothelium; their aggregate thickness is similar to that of the tissue barrier in the human lung (Weibel, 1984). One important characteristic of the process that results in this remarkably thin air-blood barrier has not been systematically studied; at ten days of incubation the nuclei of the endothelial cells appear to be randomly distributed around each capillary's circumference, but near the end of incubation nuclei are found predominantly on the mesenchymal surface of the capillary, away from the air-blood interface. These nuclei vary in shape during the course of incubation (Ausprunk et al., 1974) but they are at least one order of magnitude thicker than the final tissue barrier, so their relocation would appear to have an anatomical advantage by allowing the diffusion barrier to be thin. The purpose of the present report is to describe the temporal characteristics of the change in the position of the endothelial cell nuclei in the chorioallantoic membrane of the chick embryo, as determined morphometrically.
2. Methods Fertile eggs of White Leghorn chickens (Dekalb strain) were obtained from the Poultry Science Department, Oregon State University. Eggs were weighed and incubated, blunt end up, at 37.5 °C and approximately 60~o relative humidity in a forceddraft incubator (Roll-X, Marsh Farms, Garden Grove, CA) which tilted them through a 90 ° arc automatically once each hour. On days 10, 12, 14, 16 and 18 of incubation four eggs, selected to span the range of initial egg weights and found by candling to contain a living embryo, were removed from the incubator. Each egg was weighed, then refrigerated at 4 °C for one hour to induce anesthesia, before sampling the membrane.
On incubation days 14, 16 and 18 one sampling site on each egg was selected using a grid that had been drawn on the shell prior to incubation. The grid divided the egg into equal quadrants longitudinally and four sections parallel to the short axis equator. Using a table of random numbers, one sampling site was selected from the 16 quadrangles that resulted from this procedure. A second sample was taken from the quadrangle directly opposite the first. On days 10 and 12, before the chorioallantoic membrane is firmly attached to the entire inner surface of the egg, only those quadrangles shown by candling to be lined by membrane were eligible for sampling. The egg was cut along its long axis, avoiding the selected sampling sites, the embryo was killed by decapitation and weighed, and the egg was emptied of its contents, leaving the chorioallantoic membrane lining the shell sections. These were immersed in fixative and after thirty minutes of fixation a sample of membrane and its attached shell, measuring approximately 1 cm 2 was taken from each site. These were left in fixative overnight, then the membrane was peeled from the shell, and two samples approximately 2 mm 2 were taken from each, yielding a total of four samples from each egg. The fixative used was that recommended by Wangensteen and Weibel (1982), containing 5 ~o glutaraldehyde, 0.1 M sodium cacodylate, and 0.01 mM calcium chloride in distilled water (final pH of fixative = 7.4). After fixation the samples were placed in buffer at pH 7.4 for 24 h, then postfixed with osmium tetraoxide followed by uranyl acetate, dehydrated in graded alcohols and embedded in epon as described by Wangensteen and Weibel (1982). Thick sections were cut from each block, stained with Azure II and methylene blue as described by Graziadei and Metcalf (1971), and examined at 1000 x magnification using a Zeiss Axiophot light microscope. The image was simultaneously projected on the monitor of a Macintosh Ilci computer using Image 1.22 software (Microsoft Corporation, Bothell, WA). Following the technique of Burton and Palmer (1992), the image was rotated so that the surface of the membrane in apposition to the inner shell membrane was horizontal. Only those capillaries cut in cross section (defined as having a horizontal diameter no greater than twice the vertical diameter) were examined. An imaginary equator was
173
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
ciably rounder and often bulged noticeably into the capillary lumen (Ausprunk et al., 1974). Table 1 shows the number of eggs studied on each day of incubation, the number of endothelial nuclei whose position was determined in each egg and the percentage of those nuclei that were adjacent to the shell membrane. From day 10 to day 18 of incubation the percentage of nuclei adjacent to the shell membrane dropped from 43 ~o to 4 ~ o On average, the percentage of nuclei in the path of gas exchange decreased by 6~o per day from the tenth to the sixteenth day of incubation, as analyzed by linear regression. Fig. 3 is a graph of the results, showing the mean and the standard error of the mean for each day of incubation that was sampled. These data were evaluated by one way analysis of variance. It yielded an F-ratio of 19.79 with a probability of 0.0001. Thereafter Tukey's (Steel and Torrie, 1980) multiple comparisons test was used to evaluate the differences between groups. The value for day 10 differed significantly ( P < 0.05) from that of days 14, 16 and 18 and day 12's value differed significantly from those of days 16 and 18. Chisquare analysis of paired values gave a P < 0 . 0 5
drawn horizontally on each. Nuclei with at least 75 ~'o of their cross-sectional area lying on the half of the capillary circumference adjacent to the shell membrane were counted separately from the remainder and considered to lie in the path of gas exchange. Samples from at least three eggs on each incubation day were examined, and the positions of at least 25 nuclei from each egg were determined.
3. Results In contrast with earlier studies (Romanoff, 1960), at 10 days of incubation the capillaries in the chorioallantois were still located immediately below the chorionic ectoderm, on the surface of the ectoderm farthest from the inner shell membrane, as shown in Fig. 1. The endothelial cells were very fiat, tapering only slightly toward the edges, with only a minimal swelling at the site of each nucleus. By day 18 the morphology had changed dramatically, as shown in Fig. 2. The migration of the chorionic capillaries was complete and they lay adjacent to the inner shell membrane. The endothelial cell nuclei were appre-
M
ktmm
Fig. 1. A cross-section of the inner shell membrane (SM), the epithelium (E) of the chorion, two chorioallantoic capillaries, one containing erythrocytes (R), and the mesenchyme (M) of chorion and allantois at ten days of incubation. Arrows indicate two nuclei of endothelial cells, which at this time are randomly distributed around the capillarycircumference. Note also that the capillaries lie on the mesenchymal surface of the chorioallantoic membrane.
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
174
Fig. 2. The chorioallantoic membrane on the eighteenth day of incubation, labeled as in Fig. 1. Almost all the endothelial nuclei (two are indicated by arrows) lie on that half of the capillary circumferenceopposite the inner shell membrane. Note that the capillaries now lie on the shell membrane surface of the chorioallantois. Table 1 Day of incubation
Number of eggs studied
Total number of nuclei
Percent nuclei adjacent to shell membrane
10 12 14 16 18
3 4 3 3 3
91 122 ill 105 105
43 29 18 6 4
between the data for days 10 and 12 and days 14 and 16. O n this basis we conclude that the change in nuclear position continues through day 16 of incubation. The m e a n value for day 10 (43~0) did not differ significantly from 50~o, so our data do not indicate that the reorientation of the endothelial nuclei from a r a n d o m distribution begins before that time.
50 m 45
4. D i s c u s s i o n
~ 4o ~
35
[
I
0 g
25
~
20
Z
10
08
10
12
14
10
lh
ab
Day of Incubation
Fig. 3. The relationship between duration of incubation and the location of the endothelial nuclei in the chorioallantois. Data are plotted as the mean and standard error of the mean for each day of observation.
In theory the progressive change that we have d e m o n s t r a t e d in the position of the endothelial nuclei of the chorioallantoic capillaries could result either from the m o v e m e n t of these organelles or a change in endothelial cell turnover resulting in the progressive p r e d o m i n a n c e of endothelial cells whose nuclei are almost always located on the portion of the circumference that is away from the shell membranes. The present study c a n n o t discriminate between these alternatives. Displacement oforganelles, either by the active contraction of filaments or microtubules or by the m o v e m e n t of other cellular constituents, is a well-recognized p h e n o m e n o n (Schroer
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
and Sheetz, 1991). On the other hand, Ausprunk et al. (1974) measured the rate of division of endothelial cells in the chorioallantoic capillary membrane. They found a thymidine labeling index of at least 2.8~o after the eleventh day of incubation following a 5-h exposure to the label. Assuming that only the first generation of cells was labeled, this finding is consistent with a cell replacement rate of 13~o per day if growth of the capillary network is neglected. The growth of the capillary mesh has not been completely described. The allantoic membrane reaches its greatest weight by the tenth day of incubation (Romanoff, 1960) and Hamilton (1965) represents the chorioallantois as completely lining the eggshell on the twelfth day. The total vascularized area within the chorioallantois was measured by Burton and Palmer (1992) from the sixth through the tenth day of incubation. On the tenth day the vascular area of eggs incubated under normoxic conditions measured 45.6 cm 2. These workers did not specify the strain of domestic hen from which their eggs were obtained, but if the surface area of the average egg of Gallus d o m e s t i c u s is taken as 68 cm 2 (Romanoff and Romanoff, 1949) their data show that 67 ~o of the surface area of the eggshell has been covered by the tenth day, allowing only an average growth of 4°,4, per day for the remaining eight days before lung breathing begins. This, taken with the cell labeling rate of 13~o per day reported by Ausprunk et al. (1974) and the average 6~o daily rate of nuclear "relocation" found in the present study, is consistent with the hypothesis that the change in nuclear location is due to endothelial cell replacement. In other words, the relocation of nuclei may be due to the progressive replacement of cells whose nuclei are randomly distributed around the circumference of the capillary by cells whose nuclei almost always lie in that portion of the capillary circumference opposite the shell membranes. However, Ausprunk et al. (1974) also attributed the progressive changes in endothelial cell morphology over the course of incubation to m a t u r a t i o n , an interpretation that is more consistent with the preferential survival of endothelial cells whose nuclei are located out of the pathway of gas exchange. Temple and Metcalfe, using carbon monoxide as the test gas, demonstrated that the diffusing capacity of the fertile hen's egg increased progressively
175
from the eighth to the eighteenth day of incubation. Bissonnette and Metcalfe (1978), on the basis of a mathematical analysis, attributed the increase in diffusing capacity mainly to changes in the chorioallantoic membrane (including the capillary wall). These changes include an increase in surface area due to capillary growth and thinning of the barrier separating the embryo's blood from gas in the interstices of the inner shell membrane. The attenuation is achieved partly by migration of the capillaries through the chorionic epithelium and, as quantified in the present study, partly by relocation of the endothelial nuclei out of the pathway of gas diffusion.
Acknowledgement This work was supported by the Medical Research Foundation of Oregon, the Portland Veterans Affairs Medical Center and the Department of Physiology, Oregon Health Sciences University, Portland, OR. The authors express their thanks to Kent Thornburg, Professor of Physiology at Oregon Health Sciences University, for his help and stimulation.
References Ausprunk, D.H., D.R. Knighton and J. Folkman (1974). Differentiation of vascular endothelium in the chick chorioallantois: a structural and radiographic study. Dev. Biol. 38: 237248. Bissonnette, J.M. and J. Metcalfe (1978). Gas exchange of the fertile hen's egg: components of resistance. Respir. Physiol. 34: 209-218. Burton, G.J. and M.E. Palmer (1992). Developmentof the chick chorioallantoic capillary plexus under normoxic and normobaric hypoxic and hyperoxic conditions: a morphometric study. J. Exp. Zool. 262: 291-298. Danchakoff, V. (1917). The position of the respirator)' vascular net in the allantois of the chick. Am. J. Anat. 21: 407-420. Fitze-Gschwind, V. (1973). Zur Entwicklungder ChorioallantoisMembran des HOhnchens. Adv. Anat. Embryol.and Cell Biol. 47: 6-51. Fuchs, A. and E.S. Lindenbaum (1988). The two- and threedimensional structure of the microcirculation of the chick chorioallantoic membrane. Acta Anat. 131: 271-275. Ft~lleborn, F. (1894). Beitr~tgezur Entwicklung der Allantois der VOgel. Berlin, Dissertation. Graziadei, P.P.C. and J.F. Metcalf(1971). Autoradiographicand ultrastructural observations on the frog's olfactory mucosa. Z. Zellforsch. Mikrosk. Anat. 116: 305-318.
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Hamilton, H.L. (1965). Lillie's Development of the Chick: An Introduction to Embryology, Third edition, Holt, Rinehart and Winston. (Editors) New York, pp. 275-282. Hoshi, H. and T. Mori (1971). The fine structure of the chorionic epithelium of chick embryo. Arch. Histol. Jap. 33: 45-58. Metcalfe, J., M.K. Stock and R.L. Ingermann (1984). The effects of oxygen on growth and development of the chick embryo. In: Respiration and Metabolism of Embryonic Vertebrates, edited by R.S. Seymour. Dordrecht, the Netherlands, Dr. W. Junk Publ. pp. 204-230. Metcalfe, J. and M.K. Stock (1993). Current Topic: Oxygen exchange in the chorioallantoic membrane, avian homologue of the mammalian placenta. Placenta 14: 605-613. Romanoff, A.L and A.J. Romanoff (1949). The Avian Egg. John Wiley and Sons, Inc. New York p. 105. Romanoff, A.L. (1960). The Avian Embryo: Structural and Functional Development. New York, The Macmillan Company. p. 1135, p. 1116.
Schroer, T.A. and M.P. Sheetz ( 1991). Functions of microtubulebased motors. Annual Review of Physiology 53: 629-652. Steel, R.G.D. and D.H. Torie (1980) Principles and Procedures of Statistics: A Biometrical Approach. 2nd edition. New York, McGraw-Hill, p. 185. Stock, M.K. and J. Metcalfe (1987). Modulation of growth and metabolism of the chick embryo by a brief (72 h) change in oxygen availability. J. Exp. Zool., Suppl. 1: 351-356. Temple, G.F. and J. Metcalfe (1970). The effects of increased incubator oxygen tension on capillary development in the chick chorioallantois. Respir. PhysioL 9: 216-233. Wangensteen, D. and E.R. Weibel (1982). Morphometric evaluation of chorioallantoic oxygen transport in the chick embryo. Respir. Physiol. 47: 1-20. Weibel, E.R. (1984). The Pathway for Oxygen: Structure and Function in the Mammalian Respiratory System. Cambridge, MA, Harvard University Press, p. 354.
ELSEVIER
Respiration Physiology 100 (1995) 171-176
Relocation during incubation of endothelial nuclei in the chick chorioallantois Andrea A. Mayer 1, James Metcalfe*, Michael K. Stock Department of Physiology, L334, Oregon Health Sciences University, Portland, OR 97201, USA (Accepted 21 November 1994)
Abstract
Early in incubation the nuclei of the chick chorioallantoic capillaries are randomly distributed around the capillary lumen; later most of them are located on the portion of the capillary surface opposite the inner shell membrane. This is one of the complex of processes that results in progressive thinning of the diffusion pathway for gases between the external environment and the blood of the embryo. The present study quantified this nuclear "relocation". Our data show a progressive relocation of the endothelial nuclei from the tenth through the sixteenth day at an average rate of 6~o per day. Key words': Birds, chick: Development, chorioallantoic capillaries; Embryonic gas exchange; Endothelium cells, nuclear relocation; Gas exchange
I. Introduction
Oxygen availability has a powerful influence on the growth and development of the embryo of Gallus domesticus (Metcalfe et al., 1984; Stock and Metcalfe, 1987). The capillary meshwork in the chorioallantoic membrane is well-suited to its essential role in the respiration of the avian embryo (Metcalfe and Stock, 1993). It develops from a vascular bed that traverses the mesoderm of the allantoic sac. Beginning on the fifth day of incubation the allantoic mesoderm fuses with the mesoderm of the enveloping
This work was performed in partial fulfillment of the requirements for the Bachelor of Science degree from George Fox College, Newberg, OR. * Corresponding author. Tel: (503)494-8440, Fax: (503)4942383. 0034-5687/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 3 4 - 5 6 8 7 ( 9 5 ) 0 0 1 2 7 - 8
chorion (Hamilton, 1965), and the capillary network expands to eventually encompass the contents of the whole egg, lining the entire inner surface of the inner shell membrane. When the chorioallantois is viewed in cross section near the end of incubation, the capillaries occupy more area than the ectodermal tissue separating them, thus forming "a great blood sinus interrupted by strands of tissue" (Ftllleborn, 1894). In addition to this process of capillary proliferation, which enlarges the surface area for gas exchange, the rate of net gas transport by diffusion between embryonic blood and environmental air is enhanced by a reduction in the diffusion distance. Beginning on about the tenth day of incubation, the capillary network migrates through the ectoderm of the chorion from its mesenchymal site of origin (Danchakoff, 1917; Fuchs and Lindenbaum, 1988). By the fourteenth day the embryo's blood, as it cir-
172
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
culates through the chorioallantoic capillary network, is separated from the air in the interstices of the inner shell membrane by tissue with an average thickness of only 0.5 to 0.7 microns (FitzeGschwind, 1973; Wangensteen and Weibel, 1982; Burton and Palmer, 1992). This barrier to gas diffusion is comprised of a thin layer of ectoderm (Hoshi and Mori, 1971), an incomplete basement membrane (Wangensteen and Weibel, 1982) and an attenuated layer of endothelium; their aggregate thickness is similar to that of the tissue barrier in the human lung (Weibel, 1984). One important characteristic of the process that results in this remarkably thin air-blood barrier has not been systematically studied; at ten days of incubation the nuclei of the endothelial cells appear to be randomly distributed around each capillary's circumference, but near the end of incubation nuclei are found predominantly on the mesenchymal surface of the capillary, away from the air-blood interface. These nuclei vary in shape during the course of incubation (Ausprunk et al., 1974) but they are at least one order of magnitude thicker than the final tissue barrier, so their relocation would appear to have an anatomical advantage by allowing the diffusion barrier to be thin. The purpose of the present report is to describe the temporal characteristics of the change in the position of the endothelial cell nuclei in the chorioallantoic membrane of the chick embryo, as determined morphometrically.
2. Methods Fertile eggs of White Leghorn chickens (Dekalb strain) were obtained from the Poultry Science Department, Oregon State University. Eggs were weighed and incubated, blunt end up, at 37.5 °C and approximately 60~o relative humidity in a forceddraft incubator (Roll-X, Marsh Farms, Garden Grove, CA) which tilted them through a 90 ° arc automatically once each hour. On days 10, 12, 14, 16 and 18 of incubation four eggs, selected to span the range of initial egg weights and found by candling to contain a living embryo, were removed from the incubator. Each egg was weighed, then refrigerated at 4 °C for one hour to induce anesthesia, before sampling the membrane.
On incubation days 14, 16 and 18 one sampling site on each egg was selected using a grid that had been drawn on the shell prior to incubation. The grid divided the egg into equal quadrants longitudinally and four sections parallel to the short axis equator. Using a table of random numbers, one sampling site was selected from the 16 quadrangles that resulted from this procedure. A second sample was taken from the quadrangle directly opposite the first. On days 10 and 12, before the chorioallantoic membrane is firmly attached to the entire inner surface of the egg, only those quadrangles shown by candling to be lined by membrane were eligible for sampling. The egg was cut along its long axis, avoiding the selected sampling sites, the embryo was killed by decapitation and weighed, and the egg was emptied of its contents, leaving the chorioallantoic membrane lining the shell sections. These were immersed in fixative and after thirty minutes of fixation a sample of membrane and its attached shell, measuring approximately 1 cm 2 was taken from each site. These were left in fixative overnight, then the membrane was peeled from the shell, and two samples approximately 2 mm 2 were taken from each, yielding a total of four samples from each egg. The fixative used was that recommended by Wangensteen and Weibel (1982), containing 5 ~o glutaraldehyde, 0.1 M sodium cacodylate, and 0.01 mM calcium chloride in distilled water (final pH of fixative = 7.4). After fixation the samples were placed in buffer at pH 7.4 for 24 h, then postfixed with osmium tetraoxide followed by uranyl acetate, dehydrated in graded alcohols and embedded in epon as described by Wangensteen and Weibel (1982). Thick sections were cut from each block, stained with Azure II and methylene blue as described by Graziadei and Metcalf (1971), and examined at 1000 x magnification using a Zeiss Axiophot light microscope. The image was simultaneously projected on the monitor of a Macintosh Ilci computer using Image 1.22 software (Microsoft Corporation, Bothell, WA). Following the technique of Burton and Palmer (1992), the image was rotated so that the surface of the membrane in apposition to the inner shell membrane was horizontal. Only those capillaries cut in cross section (defined as having a horizontal diameter no greater than twice the vertical diameter) were examined. An imaginary equator was
173
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
ciably rounder and often bulged noticeably into the capillary lumen (Ausprunk et al., 1974). Table 1 shows the number of eggs studied on each day of incubation, the number of endothelial nuclei whose position was determined in each egg and the percentage of those nuclei that were adjacent to the shell membrane. From day 10 to day 18 of incubation the percentage of nuclei adjacent to the shell membrane dropped from 43 ~o to 4 ~ o On average, the percentage of nuclei in the path of gas exchange decreased by 6~o per day from the tenth to the sixteenth day of incubation, as analyzed by linear regression. Fig. 3 is a graph of the results, showing the mean and the standard error of the mean for each day of incubation that was sampled. These data were evaluated by one way analysis of variance. It yielded an F-ratio of 19.79 with a probability of 0.0001. Thereafter Tukey's (Steel and Torrie, 1980) multiple comparisons test was used to evaluate the differences between groups. The value for day 10 differed significantly ( P < 0.05) from that of days 14, 16 and 18 and day 12's value differed significantly from those of days 16 and 18. Chisquare analysis of paired values gave a P < 0 . 0 5
drawn horizontally on each. Nuclei with at least 75 ~'o of their cross-sectional area lying on the half of the capillary circumference adjacent to the shell membrane were counted separately from the remainder and considered to lie in the path of gas exchange. Samples from at least three eggs on each incubation day were examined, and the positions of at least 25 nuclei from each egg were determined.
3. Results In contrast with earlier studies (Romanoff, 1960), at 10 days of incubation the capillaries in the chorioallantois were still located immediately below the chorionic ectoderm, on the surface of the ectoderm farthest from the inner shell membrane, as shown in Fig. 1. The endothelial cells were very fiat, tapering only slightly toward the edges, with only a minimal swelling at the site of each nucleus. By day 18 the morphology had changed dramatically, as shown in Fig. 2. The migration of the chorionic capillaries was complete and they lay adjacent to the inner shell membrane. The endothelial cell nuclei were appre-
M
ktmm
Fig. 1. A cross-section of the inner shell membrane (SM), the epithelium (E) of the chorion, two chorioallantoic capillaries, one containing erythrocytes (R), and the mesenchyme (M) of chorion and allantois at ten days of incubation. Arrows indicate two nuclei of endothelial cells, which at this time are randomly distributed around the capillarycircumference. Note also that the capillaries lie on the mesenchymal surface of the chorioallantoic membrane.
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
174
Fig. 2. The chorioallantoic membrane on the eighteenth day of incubation, labeled as in Fig. 1. Almost all the endothelial nuclei (two are indicated by arrows) lie on that half of the capillary circumferenceopposite the inner shell membrane. Note that the capillaries now lie on the shell membrane surface of the chorioallantois. Table 1 Day of incubation
Number of eggs studied
Total number of nuclei
Percent nuclei adjacent to shell membrane
10 12 14 16 18
3 4 3 3 3
91 122 ill 105 105
43 29 18 6 4
between the data for days 10 and 12 and days 14 and 16. O n this basis we conclude that the change in nuclear position continues through day 16 of incubation. The m e a n value for day 10 (43~0) did not differ significantly from 50~o, so our data do not indicate that the reorientation of the endothelial nuclei from a r a n d o m distribution begins before that time.
50 m 45
4. D i s c u s s i o n
~ 4o ~
35
[
I
0 g
25
~
20
Z
10
08
10
12
14
10
lh
ab
Day of Incubation
Fig. 3. The relationship between duration of incubation and the location of the endothelial nuclei in the chorioallantois. Data are plotted as the mean and standard error of the mean for each day of observation.
In theory the progressive change that we have d e m o n s t r a t e d in the position of the endothelial nuclei of the chorioallantoic capillaries could result either from the m o v e m e n t of these organelles or a change in endothelial cell turnover resulting in the progressive p r e d o m i n a n c e of endothelial cells whose nuclei are almost always located on the portion of the circumference that is away from the shell membranes. The present study c a n n o t discriminate between these alternatives. Displacement oforganelles, either by the active contraction of filaments or microtubules or by the m o v e m e n t of other cellular constituents, is a well-recognized p h e n o m e n o n (Schroer
A.A. Mayer et al. / Respiration Physiology 100 (1995) 171-176
and Sheetz, 1991). On the other hand, Ausprunk et al. (1974) measured the rate of division of endothelial cells in the chorioallantoic capillary membrane. They found a thymidine labeling index of at least 2.8~o after the eleventh day of incubation following a 5-h exposure to the label. Assuming that only the first generation of cells was labeled, this finding is consistent with a cell replacement rate of 13~o per day if growth of the capillary network is neglected. The growth of the capillary mesh has not been completely described. The allantoic membrane reaches its greatest weight by the tenth day of incubation (Romanoff, 1960) and Hamilton (1965) represents the chorioallantois as completely lining the eggshell on the twelfth day. The total vascularized area within the chorioallantois was measured by Burton and Palmer (1992) from the sixth through the tenth day of incubation. On the tenth day the vascular area of eggs incubated under normoxic conditions measured 45.6 cm 2. These workers did not specify the strain of domestic hen from which their eggs were obtained, but if the surface area of the average egg of Gallus d o m e s t i c u s is taken as 68 cm 2 (Romanoff and Romanoff, 1949) their data show that 67 ~o of the surface area of the eggshell has been covered by the tenth day, allowing only an average growth of 4°,4, per day for the remaining eight days before lung breathing begins. This, taken with the cell labeling rate of 13~o per day reported by Ausprunk et al. (1974) and the average 6~o daily rate of nuclear "relocation" found in the present study, is consistent with the hypothesis that the change in nuclear location is due to endothelial cell replacement. In other words, the relocation of nuclei may be due to the progressive replacement of cells whose nuclei are randomly distributed around the circumference of the capillary by cells whose nuclei almost always lie in that portion of the capillary circumference opposite the shell membranes. However, Ausprunk et al. (1974) also attributed the progressive changes in endothelial cell morphology over the course of incubation to m a t u r a t i o n , an interpretation that is more consistent with the preferential survival of endothelial cells whose nuclei are located out of the pathway of gas exchange. Temple and Metcalfe, using carbon monoxide as the test gas, demonstrated that the diffusing capacity of the fertile hen's egg increased progressively
175
from the eighth to the eighteenth day of incubation. Bissonnette and Metcalfe (1978), on the basis of a mathematical analysis, attributed the increase in diffusing capacity mainly to changes in the chorioallantoic membrane (including the capillary wall). These changes include an increase in surface area due to capillary growth and thinning of the barrier separating the embryo's blood from gas in the interstices of the inner shell membrane. The attenuation is achieved partly by migration of the capillaries through the chorionic epithelium and, as quantified in the present study, partly by relocation of the endothelial nuclei out of the pathway of gas diffusion.
Acknowledgement This work was supported by the Medical Research Foundation of Oregon, the Portland Veterans Affairs Medical Center and the Department of Physiology, Oregon Health Sciences University, Portland, OR. The authors express their thanks to Kent Thornburg, Professor of Physiology at Oregon Health Sciences University, for his help and stimulation.
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