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Cholesterol storage and bile secretion in chorio-allantoic grafts of liver.

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CHOLESTEROL STORAGE AND BILE SECRETION I N
CHORIO-ALLANTOIC GRAFTS O F LIVER
A. J . DALTON
Luborutory of Histology and Embryology, Western Reserve University SchooI
of XeEZidne
o m PL,ATE
(TBREE FIGURES)
INTRODUCTION
But few workers have investigated the problem of cholesterol storage in the liver of the chick embryo. Valentin
(1871) in a generalized study of double refraction in tissues of
the chick embryo found no evidence of it in the livers of
embryos of 7 , 9 and 11 days incubation. Hanes ('12) in a
rather comprehensive study concluded that anistropic droplets
of cholesterol esters first make their appearance in the liver
of the chick embryo on about the fourteenth day of incubation.
Such studies may determine the time during development when
the liver first gives evidence of its function as a storage organ,
but they obviously cannot supply evidence as to the time when
this function is first established. It is possible that the storage
function of the liver may have become established previous
to the time when stored material is first identifiable uiidcr the
microscope. An increase in the concentration of cholesterol
in the blood stream may in part determine the time when this
substance first becomes visible in the liver. To deiermine the
time when the storage function first becomes established, livers
from young embryos have been brought in contact with the
blood stream of older cmbryos by means of the chorio-allantoic
graft.
Sandstrom ( '34) noted the constant occurrence of degenerative changes in grafts of hepatic tissue with a total incubation age of 12 o r more days. She suggested that the inability
431
432
A. J. DALTON
of hepatic tissue to function in grafts leads to its degeneration.
It seemed possible that functional differentiation may have
occurred previous to the time of onset of these degenerative
changes. To investigate this possibility, a series of grafts has
been examined for the presence of exocrine secretory products.
MATERIAL AND METHODS
All embryos used in these experiments were of the Single
Comb White Leghorn breed. Both the donor and host ages
at the time of transplantation were varied so that the grafts
might be grown on the membrane during the period when the
concentration of cholesterol was at its maximum in the
embryonic blood stream. The high point of the concentration
of cholesterol in the blood is reached on the seventeenth day
of incubation (Zorn and Dalton, ’36). One series of grafts was
fixed in 10% formalin. Frozen sections were than made in
preparation for the histochemical analysis. A series of control
embryo livers was treated in a similar fashion. To complete
this analysis, another series of grafts was fixed in Chainpy
fluid followed by post-osmication by the Kolatchev-Nassanov
method.
OBSERVATIONS
Table 1 gives the results of the histochemical analysis of
the droplets present in 10- and 18-day livers and in 10-day
grafts removed from the chorio-allantois of 18-day hosts.
As the table indicates, the droplets present in the graft livers
give reactions identical with the droplets present in the
18-day livers. It should be mentioned that the majority of
these droplets contain regions which give reactions with Nile
blue sulphate and with Smith’s stain. This suggests the
presence of cholesterol, cholesterol esters, and cliolesterolfatty acid mixtures in the same droplet.
Table 2 lists and describes twenty-two of the more satisfactory grafts examined for the presence of anisotropic droplets. No grafts have been included in this table which show
any evidence of degenerative changcs. As the table demonstrates, anisotropic droplets first appear in grafts during the
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Cholesterol-oleic
acid glyecrin
ester
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The sources from which most of the histochemical tests were obtained are Hanes ( '12), Lee ('28) and Malloq and Wright
( '24).
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Fattyarids
cerebrosides
CholestwoIfatty acid
mixtures
True fats
~liolesterol
Cholesterol
esters
Phosphatides
Cerebrosides
Phosopho-
NI
434
A. J. DALTON
seventh day of incubation (B-83), none being present previous
to this time. Barely visible droplets on the eighth day, many
increase in size during the next 24 hours, until on the ninth
day, they approach the volume of the nucleus of the hepatic
cell. The droplets in 10- and 11-day grafts equal the volume
of those in normal 18-day livers. Figure 1 shows the size and
distribution of the anisotropic droplets (changed to crystals
TA
GRAFT
XZMBER
B-101
13-102
B-111
B-112
B-113
R-114
B-77
B-83
B-109
8-115
B-100
B-104
3-105
B-54
€3-58
B-103
:<-lo7
B-47
B-56
B-45
B-46
B-48
DONOR
AGF: IN
HOURS
GRAFT
AGE IN
DAYS
80
72
80
80
80
80
6
6
6
6
60
65
72
72
72
72
72
96
72
72
80
72
72
72
72
96
6
6
6i
63
6%
6%
7
7
7
8
8
8
8
9
9
10
10
10
HOST
AGE IN
DAYS
15
15
15
15
15
15
17
17
16
16
16
16
16
16
17
17
17
17
17
18
18
17
JE
2
DAYS O F
GRAFT O N
MEXBBANE
3
3
3
3
3
3
4
4
4
4
4
4
4
4
5
5
5
6
6
7
7
6
ANISOTaOPID DROPLET8
None
Sone
None
None
None
None
None
Few and minute
Fairly numerous and minute
E’airly numerous and miiiute
Numerous and small
Numerous aiid small
Numerous and small
Numerous and small
Numerous and small
Numerous and small
Numerous and small
Numerous aiid large
Numerous and large
Numerous and very large
Numerous and very large
Numerous and very large
after fixation in 10% formalin) as they appear in the 18-day
l i ~ e r . Figure 2 shows similar bodies in graft B-46, a 10-day
graft on an 18-day host.
A series of livers from embryos varying in age from 9 to
19 days incubation were examined f o r the presence of anisotropic droplets. The first evidence of double refraction in
this series WRS found in 11-day livers, more than 4 days after
it first becomes apparent in grafts of livers on older hosts.
CHOLESTEROL STORAGE AND BILE SECRETION
435
The presence of substances in the bile wliich will reduce
osmic acid makes it possible to demonstrate the presence of
secretory products in the bile canaliculi and in the gall bladders
in liver grafts. Eighteen grafts varying from 8 to 13 days
total age were fixed in Champy fluid and post-osmicated. A11
show the presence of secretory products in the bile canaliculi
and also in the gall bladder when it is present. Figure 3
shows a portion of a section of graft B-20, a graft with a total
age of 11 days, removed from a host of 19 days incubation.
It shows the gall bladder filled with heavily osmicated material. This is evidence of very active secretion on the part
of the liver cells of the graft.
DISCUSSION
Needham (’31) in referring to the work of Valentin (1871)
states “Valentin investigated the doubly refracting droplets
in chick embryos, and stated that none of the tissues of the
3rd, 4th, 5th o r 6th day embryos showed them, but that on
the 7th day a very few appeared. He obtained comparable
results in a study of the embryonic liver of the frog.” Actually
Valentin found no clear-cut evidence of double refraction in
the liver cells of the 7-day embryo. He states “die Anlage der
beiden Leberlappen, . . . . licfcrten kein deiitliches Merkmal
von Doppelbrechung. ” In the 9-day liver Valentin found
‘Faserzellen ’ showing double refraction, but he makes no
mention of the parenchymal cells. Further, in referring to
double refraction in the 11-day embryo, he states “Die A h s e n
der Leher und dcr Nieren fuhrten zu keinen bemerkenswerthen
Ergebnisscn. ” Clearly Valentin found no evidence of anisotropic droplets in the liver cells of the chick embryo up t o the
eleventh day of incubation. From my histochemical study of
the series of normal livers I have concluded that anisotropic
droplets first make their appearance on the elevcnth day of
incubation. This is earlier than the time determined by Hanes
(’la) but substantiates my previous finding (Dalton, ’34).
I n addition to the cholesterol esters identified by Hanes, free
cholesterol and cholesterol-fatty acid mixtures also appear to
436
A.
J. DALTON
be present in the anisotropic droplets. This is true for the
droplets present in the 18-day livers and in the 10-day grafts
on 17- or 18-day hosts. This finding would appear to be
evidence for the esterification of cholesterol by fatty acids in
the liepatic cells, both in graft and in host livers.
In a study of the hepatic tissixe of the chick embryo in chorioallantoic grafts, Sandstrom ('34) found that degeneration of
the hepatic cells occnrred regularly, beginning on the twelfth
day of incubation. She interpreted this phenomenon as being
due t o the inability of the liver cells to function in their abnormal environment. It has been sliown above that liver cells
in grafts are capable of the storage of cholesterol and that they
possibly take part in its esterification. This storage first
becomes evident in grafts during the latter part of the seventh
day. I n addition, I have fonnd products of secretion in the
bile canaliciili and in the gall bladder in liver grafts. The
vacuoles described by Sandstrom and interpreted by her as
evidence of beginning degeneration may well have contained
cholesterol esters previoiis t o fixation. Since embryonic lircr
cells have been shown to be capable of ftinction on the chorioallantoic membrane, it is suggested that the regularly occiirring degeneration of hepatic tissue in grafts is due, indirectly,
to the active functioning of the cells. Considerable degeneration of hepatic tissue follows complete obstruction of the
common bile duct in humans (Lieber and Stewart, '34). Continued bile secretion in grafts in which there is no outlet,
associated with an abnormal and possibly incomplete 01- insuficient blood supply, obviously would lead t o degeneratire
changes. This possibility is supported by the observation of
Sandstrom that degenerative changes occur more S ~ O Wwhen
I~
the gall bladder develops in liver grafts.
SUMMARY
1. Anisotropic droplets first appear in the chick embryo
liver on the eleventh day of incubation.
2. I n cliorio-allantoic grafts of liver to older hosts, anisotropic droplets appear during the latter part of the seventh
day.
CHOLESTEROL STORAGE A N D RILE S E C R E T I O N
437
3. The histochemical study of individual anisotropic droplets, in both hosts and grafts, indicates the presence in them
of free cholesterol, cholesterol esters, and cholesterol-fatty
acid mixtures. This suggests that esterification of cholesterol
occurs in the hepatic cells of both hosts and grafts.
4. Cholesterol esterification and storage, and the presence
of secretory products in the bile canaliculi are considered to
be evidence f o r the functional activity of liver cells in chorioallantoic grafts.
5 . The regularly occurring degeneration of embryonic
hepatic tissue in grafts is thought to be due, in part, to bile
stasis resulting from functional activity in the absence of an
outlet.
LITERATURE CITED
DALTON,A. J. 1934 The ontogenetic history of the mitochondria and the Golgi
netnTork of the hepatic cell of the ehick. Anat. Rec., vol. 58, pp. 321-347.
HANDS,F. N. 1912 Lipoid metabolism in the developing chick and its relation
t o calcification. J. Exp. &fed., 001. 16, pp. 512-526.
LEE, B. 1928 The Microtomist’s Vade-mecum. P. Blakiston’s Soil a d Co.
L I ~ E RM., M. AND H. L. STEWART
1934 Hepatic and bile duct changes from
obstructioii of common bile duct due to pancreatic carcinoma. Arch.
Path, 001. 17, p. 362-380.
MALLQEY, F. B. AND J. H. WRIGHT 1924 Pathological Technique. W. R. Saunders
Go.
NEEDHAM,
J. 1931 Chemical Embryology. Cambridge University Press.
SANDSTROM,
R. H. 1934 The differentiation of hepatic and paiiereatic tissues
of the chick embryo in chorio-allantoic grafts. Physiol. Zool., vol. 7,
pp. 226-246.
~‘ALEN‘MN, G . 1871 Beitrage zur Mikroskopie. Arch. f. mikr. Anat., Bd. 7,
S. 140-156.
ZORN,C. M. A N D A. J. DALTON 1936 Blood chemistry of the ehick embryo during
ontogenesis. Proc. Soc. Exp. €501. a n d &fed., vol. 35, pp. 4 5 1 4 5 3 .
PLATE 1
EXPLANATIQN OF FIGURES
1 Frozen section of the liver of an 18-day host embryo viewed by polarized
light, showing the size and distribution of the anisotropic crystals (cholesterol)
present in the hepatic cells. Formalin; Nile blue sulphate. X 105.
2 Frozen section of the liver g r a f t B-46,a 10-day graft on an 18-day hoet,
viewed by polarized light. Note the relatively large crystals of cholesterol.
Formalin; Smith’s stain. X 105.
3 Bection of liver graft B-20, an 11-day graft on a 19-day host showing a
masa of heavily osmicated material in the gall bladder. Champy ; post-osmication.
X 520.
438
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