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/ . Embryol. exp. Morph. Vol. 18, 2, pp. 289-98, October 1967
With 6 plates
Printed in Great Britain
289
Immunochemical investigations on the origin of
serum albumin in the chick embryo
ByD. ZACCHEO 1 & C. E. GROSSP
From the Institute of Anatomy, University of Genoa
Information concerning physical and chemical properties of serum albumin
during the development of the chick has been collected by different techniques,
e.g. paper, gel and continuous flow electrophoresis, ultracentrifugation and
immunochemical methods. However, the results of the various authors are
not always in full agreement. The main questions deal with the precise stage of
development when serum albumin can first be detected in the circulating
blood, and with the heterogeneity of the molecular types in the group of serum
albumins. Other aspects of the problem concern the possibility of different
origins of serum albumin at various stages and the metabolism of this protein
which probably has peculiar features at each intra- and extra-ovular developmental period (Ivanyi, Hraba & Cerny, 1964).
The stage at which serum albumin can first be detected in the circulating
blood or in embryonic extracts has been indicated as ranging between the 3rd
and the 10th day of incubation (Nace, 1953; Weller & Schechtman, 1962).
These differences are probably related both to the molecular types which have
been investigated and to the techniques which have been employed.
Hepatic synthesis of serum albumin has now been demonstrated in mammals
(Hamashima, Harter & Coons, 1964) and it is also commonly admitted for
birds; in the latter, however, a direct demonstration is still lacking and, in
particular, it is unclear when hepatocyte synthesis of serum albumin begins.
Our research has dealt with two of the above-mentioned problems, i.e.
(1) when can serum albumin first be demonstrated in the serum and (2) when
does the hepatic synthesis of this protein begin? We have used immunochemical
techniques (Ouchterlony test and immunoelectrophoresis), taking as a reference
the serum albumin of the adult. For our embryonic stages we shall speak of
immunochemically adult-like molecules, that is of molecules with, at least, the
same antigenic determinants.
As an additional criterion of the function of the hepatocytes at different
stages of development, the appearance of hepatic glycogen has been investigated
both by chemical and by histochemical methods. Sections of liver of embryos
1
2
Author's address: Institute of Anatomy, Cagliari, Italy.
Author's address: Institute of Anatomy, University of Genoa, Genoa, Italy.
290
D. ZACCHEO & C. E. GROSSI
and of various stages after hatching have also been examined by techniques
for the histochemical demonstration of acid phosphatase and succinic oxidase
activities.
MATERIAL AND METHODS
Preparation of antisera. We have used two types of antiserum. The first,
prepared against total adult chicken serum (anti-TACS), has been supplied
by Behringwerke; the second, against adult chicken serum albumin (anti-CSA),
was prepared in our laboratory from two groups of rabbits. The first group of
five animals received 40 mg of albumin intramuscularly; 5 weeks later, after
administration of an anti-hystaminic drug, 10 mg of the protein was injected
intravenously. A test bleeding was performed 1 week after the last injection
and in the event of a good antibody titre (a-precipitation test), total bleeding
was effected. When the antibody response was insufficient a recall dose was
injected (10 mg of the protein intravenously once a month). A second group of
five animals was immunized by interscapular injection of 70 mg of the protein
in 3 ml, together with an equal amount of complete Freund's adjuvant. One
month later a bleeding test was performed and, in the event of an insufficient
antibody titre, a recall dose, identical to the first, was injected. An antibody
titre of 1/32,768 (a-precipitation test) was considered satisfactory. The sera
of the two groups were separately pooled.
The antigen was prepared from adult chick serum. Globulins were first
precipitated at 50 % saturation of ammonium sulphate; the supernatant was
dialysed for 7 days against repeated changes of phosphate-buffered saline and
concentrated in PVP (M.W. 25000, 2 5 % solution). The protein was then
filtered through Sephadex G-200 (Pharmacia) using phosphate-buffered saline
as eluent and the filtrate collected in 5 ml fractions. The albumin peak was
localized by spectrophotometric reading at 280 m/t. A second concentration
of the protein was thereafter performed by PVP up to 10 mg/ml (micro-Kjeldahl
technique). Anti-CSA and anti-TACS sera, when tested in Ouchterlony plates
against this antigen, gave a single precipitation line.
Collection of chick sera. Blood was collected from vessels of the amniotic sac
of the embryos, from the heart in young chickens and from the carotids in the
adults. Sera were stored at - 2 0 °C (without preservative) and thawed only
once before use.
Amniotic and allantoic fluids were collected from the sacs after isolation of
the embryo with its whole membranes. Both fluids were tested with anti-CSA
and anti-TACS sera.
Preparation of a protein fraction from yolk. For the preparation of protein
extracts we have applied the method of Shepard & Hottle (1949), using yolk
obtained from fresh non-fertile eggs. Yolk was diluted 1:2 with saline, filtered
through gauze and extracted with ether 6 times at 0 °C. The residual material
has been centrifuged at 10000 g for 15 min. This procedure allows the separation
Origin of chick serum albumin
291
of a supernatant containing lipids, an intermediate fluffy layer and, at the
bottom, a clear protein solution. This has been tested with anti-CSA and antiTACS sera.
Fractionation and extraction of liver homogenates. Liver homogenates were
prepared in 0-25 M sucrose at 0 °C using a Potter-Elvehjem type homogenizer
with Teflon pestle. The livers were collected every other day from the 6th day
of incubation up to hatching; in addition, livers from chickens, non-laying hens
and adults were used.
From the homogenate the following fractions have been prepared by centrifugation (at 0 °C): nuclear fraction at 2000 g for 5 min; mitochondria at 3300g
for lOmin;lysosomes at 10 000 g for 25 min; microsomes at 25 000 gfor 120 min.
Since the histochemical tests for acid phosphatase indicate the almost complete
absence of lysosomes in the hepatocytes (as shown in livers of embryos at the
6th day of incubation (Plate 1)), centrifugation to recover the lysosomal fraction
was omitted in this case.
A morphological control of the fractions was not performed; however, the
fractionation of the homogenate was checked through the assay of some
enzymatic activities bound to cytoplasmic organelles. It has thus been shown
that acid phosphatase activity was detectable in lysosomal fractions while it
was lacking in the lighter fractions. On the other hand, only the microsomal
fractions exhibited glucose-6-phosphatase activity, which was never present in
the heavier fractions.
The nuclear as well as the soluble fractions have not been considered in this
study. The mitochondrial, lysosomal and microsomal fractions were washed
once in 0-25M sucrose and then dissolved in £ of the original volume of the
homogenate of 1 % Triton X-100 (Rhom and Haas) in saline. In using Triton
X100 instead of the more commonly used sodium deoxycholate we have
followed the suggestion of Ugazio (1962a, b). In fact, unlike some ionic
substances, a non-ionic detergent avoids the formation of non-specific precipitation lines in the agar gel (Tombs & Weston, 1961). Extraction (for at least
30 min at 0 °C) was followed by centrifugation to sediment the undissolved
particles. The nitrogen content of each solution has been standardized at around
0-40 mg/ml (micro-Kjeldahl). The protein solutions thus prepared were used
for the immunological tests.
In the case of adult chicken, mitochondrial, lysosomal and microsomal
proteins have also been extracted from fractions of kidney homogenates.
Glycogen determination. The chemical determination of the glycogen content
has been accomplished on 5 % homogenates in 0-15 M KC1, 0-1 M KF (Zaccheo,
Orunesu & Grossi, 1963), following the method of Van der Kleji (1951). For
the histochemical demonstration of the polysaccharide, tissue sections prepared
from cold Gendre-fixed material were treated for the PAS-reaction with and
without saliva digestion.
Enzyme-histochemical methods. Succinic oxidase activity (Plate 2) was
292
D. ZACCHEO & C. E. GROSSI
demonstrated by the Nitro-BT technique (Pearse, 1960). An excellent demonstration of acid phosphatase activity was obtained with Gomori-type substrate,
prepared as suggested by Holt & Hicks (1961).
RESULTS
The immunoelectrophoretic patterns obtained with chicken sera tested against
rabbit anti-CSA and anti-TACS sera have shown that serum albumin can be
demonstrated in embryos at the 4th day of incubation (Plate 3). A single
precipitation line, in the albumin zone, characterized the immunopherograms.
It must be noted that, in two specimens of serum from embryos at the 4th day,
albumin showed a different pattern of migration (Plate 3). The identification
of the protein was accomplished by comparing the precipitation line obtained
in Ouchterlony plates by the use of anti-TACS serum with the line which
appears in the albumin-anti-albumin system. The continuity of the two lines
was considered as a reaction of identity. Only sera from 10-day embryos showed
a second precipitation line in p-zone; this may be taken as an indication of
a protein which migrates faster than albumin and can be considered a prealbumin (Plate 3). After the 14th day of incubation, the adult-type pattern of
the precipitation band for albumin is established; the albumin-anti-albumin
line appears in its characteristic boat-shaped form (Plate 4). An inspection of
the series of immunopherograms of sera in different stages of development
shows the gradual establishment of the protein pattern.
The amniotic fluid contains serum proteins from the 4th day of incubation.
The combined use of anti-CSA and anti-TACS shows that, at least in stages
before the 14th day, only serum albumin is present (Plate 5, A). Quite similar
results have been given by the allantoic fluid in which serum albumin has also
been demonstrated (Plate 5, A). However, the intensity of the precipitation
lines has always been very weak.
In protein extracts of yolk from non-fertile eggs we have detected at least
six proteins which immunochemically correspond to those of the adult serum;
among these, there is an albumin with serum-like characters (Plate 5, B).
Serum proteins and, in particular, albumin, were never demonstrated in
extracts of liver mitochondria at any developmental stage. The lysosomal
extract prepared from the 8th day of incubation always gave, in Ouchterlony
plates, a single precipitation line both with anti-CSA and anti-TACS serum
(Plate 5, C). The reaction of identity for the two lines indicates that serum
albumin is present in the lysosomal fraction. The microsomal extract prepared
from liver at the 6th day of incubation did not give any reaction with the immune
sera. On the other hand, starting from the 8th day a precipitation line with the
microsomal extract has been demonstrated and identified as the result of an
albumin-anti-albumin reaction. This line was always weaker than the one
obtained with the lysosomal extract. The immunochemical identity of lysosomal
J. Embryol. exp. Morph., Vol. 18, Part 2
PLATE
Acid phosphatase activity in liver sections of embryos at the 4th day (1), 6th day (2), 8th
day (3), 14th day (4), hatching (5) and of adult chick.
D. ZACCHEO & C. E. GROSSI
facing p. 292
J. Embryo!. exp. Morph., Vol. 18, Part 2
PLATE 2
51HSSSI
Succinic oxidase activity in liver sections of embryos at the 6th day (1), 8th day (2), 12th
day (3), 20th day (4) and of chicken at 21 days (5) and 45 days (6).
D. ZACCHEO & C. E. GROSSI
J. Embryo/, exp. Morph., Vol. 18, Part 2
PLATE 3
4th
4th
8th
10th
12th
14th
Immunoelectrophoretic patterns obtained for chicken sera by the use of anti-TACS sera
during early embryonic stages.
D. ZACCHEO & C. E. GROSSI
/. Embryol. exp. Morph., Vol. 18, Part 2
PLATE 4
16th
20th
Hatching
1st
5th
Adult
Immunoelectrophoretic patterns obtained for chicken sera by the use of anti-TACS sera
during late embryonic stages and after hatching.
D. ZACCHEO & C. E. GROSST
J. Embryol. exp. Morph., Vol. 18, Part 2
PLATE 5
Anti-CSA
•
Amn. •
# All.
Anti#TACS
Amn. •
•
All.
•
Anti-CSA
Embryo 8th
Adult
Anti-CSA
a-TACS
Mt.
a-CSA
a-TACS
Ms.
A. Demonstration of serum albumin in amniotic and allantoic fluids.
B. Tmmunoelectrophoresis of a yolk-protein fraction against anti-TACS serum.
C. Demonstration of serum albumin in lysosomal (Ly.) and microsomal (Ms.) extracts from
liver homogenates at different developmental stages. Mitochondrial extracts (Mt.) are negative.
D. ZACCHEO & C. E. GROSSI
PLATE 6
/. Embryol. exp. Morph., Vol. 18, Part 2
Embryo 8th
Hatching
Adult
Anti-CSA
Anti-TACS
Ms.
Anti-CSA
•
•
•
Kidney Mt.
Anti-CSA
•
#
%
% Kidney Ly.
•
Liver Mt.
Anti-CSA
•
9 Liver Ly.
•
•
•
•
Kidney Ms.
Anti-CSA
# Liver Ms.
A. Reaction of identity for lysosomal (Ly.) and microsomal (Ms.) albumin.
B. A comparison of the results obtained in agar gel diffusion tests for kidney and liver
mitochondrial (Mt.), lysosomal (Ly.) and microsomal (Ms.) extracts.
D. ZACCHEO & c. E. GROSSI
facing p. 293
Origin of chick serum albumin
293
and microsomal albumin can also be demonstrated (Plate 6, A). Hence it can
be concluded that from the 8th day of incubation a molecule immunochemically
corresponding to serum albumin can be demonstrated in liver extracts. Extracts
prepared from kidney of adult chicken never gave any reaction with immune
sera (Plate 6, B).
Glycogen determinations have shown a detectable amount of this polysaccharide only from the 8th day of incubation. The values increase progressively
up to hatching (Text-fig. 1). Histochemical tests confirm these results.
5
6 7
10
13
16
Days of incubation
18
21
5
Text-fig. 1. Biochemical determination of hepatic glycogen in different
developmental stages.
With the histochemical reactions for the succinic oxidase activity, many
mitochondria in the hepatocytes are stained on the 4th day of incubation; the
stained mitochondria increase in number during further development (Plate 2).
A positive reaction for acid phosphatase has been noted in Kupffer's cells
in embryos on the 4th day. A few lysosomes have been demonstrated within
the hepatocytes on the 6th day, but their number undergoes a definite increase
only from the 8th day (Plate 1).
DISCUSSION
Before discussing the results presented in this paper, it seems advisable to
consider the immunochemical techniques which have been employed, especially
in relation to the occurrence of artefacts such as those occurring because of
contamination of cell fractions by serum proteins (Perlmann, Hultin, D'Amelio
294
D. ZACCHEO & C. E. GROSSI
& Morgan, 1959; D'Amelio & Perlmann, 1960; D'Amelio, Mutolo & Piazza,
1963).
The first question is the possibility that the albumin which has been demonstrated in lysosomes and microsomes might have been adsorbed during the
preparation of the homogenates. Further, in view of the fact that lysosomal
proteins have, on the whole, a basic character (de Duve, 1963), we have considered the possibility of an electrostatic interaction with albumin, whose isoelectric point is in the acid range. Perfusion of embryonic livers being practically
impossible, the cell fractions were washed in 0-25 M sucrose before extraction.
An immunological control like that of D'Amelio & Perlmann (1960) could
not be performed because extracts of embryonic liver yield a concentration of
antigen too low for a proper preparation of immune sera. However, the
possibility of an interaction in vitro between lysosomes or microsomes and an
acid serum protein (bovine serum albumin) could be ruled out by immunochemical analysis. Other observations add further to the reliability of our findings.
In the 6-day embryos serum albumin, which is already present in the circulating
blood, could not be detected in the extract of liver microsomes; furthermore,
mitochondrial, lysosomal and microsomal extracts from adult chicken kidney
have constantly been found to be devoid of albumin. Finally, the fact that of
the various serum proteins only albumin has been detected speaks also against
a contamination.
Our results show that a molecule having the same antigenic determinants
as serum albumin is detectable in the circulating blood already on the 4th day
of incubation. As previously noted, the first appearance of albumin in serum
has been recorded within wide limits. Generally those authors who, like Nace
(1953), have used immunochemical techniques (ring-test after Boyd), indicate
an earlier appearance than those who, like Heim & Schechtman (1954) and
Weller & Schechtman (1962), have applied physical methods (paper and continuous flow electrophoresis).
These discrepancies can be attributed both to the different sensitivities of the
methods and to possible modifications which the same type of molecules can
undergo in some of its physical properties during development (Marshall &
Deutsch, 1950; Schjeide & Deutsch, 1953).
As to the origin of serum proteins, Nace (1953) has proposed a theory which
may apply also to albumin. The occurrence of cross-reactions between yolk
and anti-TACS serum and the results of different adsorptions suggest, according
to this author, that in the molecular population of albumin one can distinguish
a vitelline from a non-vitelline fraction, the latter probably being synthesized
within the body of the embryo. The possibility of a transfer of protein molecules
from yolk to the circulating blood has also been accepted by Schechtman (1947).
Circumstantial evidence for the utilization of yolk proteins has been given by
Mclndoe (1960) who has recorded, up to the 14th day of incubation, a gradual
decrease of the protein nitrogen of the yolk. Both Schechtman and Nace, adapt
Origin of chick serum albumin
295
the hypotheses of Weiss (1947) and Tyler (1947) to this problem and suggest
that a transfer of proteins from yolk to serum means something more than
a simple supply to an organism not yet able to synthesize them. They think
that, through the yolk, a transfer of maternal molecules (accumulated during
oogenesis) to the embryo occurs; these molecules could act either as stimulants
for an autonomous synthesis or as templates from which new molecules could
be formed.
Our immunoelectrophoretic investigations on yolk have shown the presence
of at least six proteins, albumin among these, which have immunochemical
characters like the corresponding serum proteins of the adult.
A transfer of serum proteins to the yolk during oogenesis has also been
demonstrated in many animal species by immunohistological techniques
(Knight & Schechtman, 1954; Kaster & Schechtman, 1957; Telfer, 1958,1961;
Glass, 1959 a, b, 1961, 1962, 1963; Mancini et al. 1961, 1962, 1963; Telfer &
Melina, 1963).
In view of the demonstration that yolk proteins can reach the embryonic
serum unchanged (Brierly & Hemmings, 1956) one should accept the immunochemical identity of yolk molecules and serum molecules either before or after
the period when embryonic synthesis begins.
The results lead to the conclusion that the embryonic liver begins to synthesize
albumin between the 6th and 8th days of incubation. This accounts for the
presence of albumin in microsomal extracts.
The presence of serum albumin in lysosomal extracts is more difficult to
interpret. It can be hypothesized that albumin in lysosomes represents a
metabolic fraction which is taken by pinocytosis from serum in the liver cells
to be degraded by the proteolytic enzymes (cathepsins) which are a part of the
lysosomal enzymic complement.
It should be mentioned that a labelled heterologous albumin has been detected,
after parenteral administration, in the lysosomal fraction of mouse liver (Mego
& McQueen, 1965) and that a highly active metabolic system is necessary for
a protein that, like serum albumin, has a rather short half-life (from 1-75 to
2-65 days, according to the determinations of Patterson et al. 1962). If lysosomes
play any role in the metabolism of serum albumin it must be assumed that
before the 8th day of incubation when a lysosomal system has not yet appeared,
a different metabolic pathway is followed. The presence of albumin in the
allantoic and amniotic fluids, the latter also before sero-amniotic junction
occurs, may help in the interpretation of this question.
Experiments on surviving rat liver (Roberts & White, 1949) suggest that
glyconeogenesis from serum albumin may occur in vitro. Livers depleted of
glycogen can synthesize in vitro remarkable amounts of glucose while at the same
time albumin disappears from the medium.
In the liver of the chick embryo between the 6th and 8th days of incubation,
we have shown that the hepatocyte acquires a number of important properties:
296
D. ZACCHEO & C. E. GROSSI
namely, the capacity for albumin synthesis, the differentiation of a lysosomal
system and the beginning of glycogen storage. The latter facts might perhaps
be related to the onset of a new metabolic pathway for serum albumin and
suggest that, in previous ontogenetic periods, the protein derived from yolk
might be employed in a glyconeogenetic cycle.
SUMMARY
1. The origin of serum albumin has been investigated during the development
of the chick.
2. A protein with the antigenic determinants of adult serum albumin can
already be detected in embryos at the 4th day of incubation.
3. Serum albumin can be demonstrated in liver microsomal extracts only
from the 8th day of incubation. In the same period it appears also in liver
lysosomal extracts.
4. Albumin is present in yolk, in the amniotic fluid even before the seroamniotic junction is established, and in the allantoic fluid.
5. It is suggested that, up to the 6th day of incubation, serum albumin is
supplied to the embryo from the yolk-reservoir and that, after the 6th day, it
can also be synthesized in the liver.
6. A correlation has been attempted for the beginning of albumin synthesis
within the hepatocyte, the beginning of glycogen storage and the differentiation
of a lysosomal apparatus. All these occur between the 6th and the 8th day of
incubation.
RESUME
Etudes immunochimiques sur Vorigine de la seralbumine
chez Vembryon de poulet
1. L'origine de la seralbumine a ete essayee pendant le developpement de
l'embryon de poulet.
2. Une proteine avec les determinants antigeniques de la serumalbumine
de l'Adulte peut etre demontree dans le sang a partir du 4eme jour d'incubation.
3. On a pu demontrer la presence de serumalbumine dans les extraits lysosomaux du foie seulement apres 8 jours d'incubation.
4. L'albumine est presente dans le vitellus, dans le liquide amniotique, meme
avant la communication serum-amniotique, et dans le liquide allantoiidien.
5. On a suggere que, jusqu'au 6eme jour d'incubation la serumalbumine
parvient a l'embryon de vitellus et que, apres, cette proteine peut etre synthetisee
dans le foie.
6. On a cherche de trouver une relation entre la synthese hepatique de l'albumine, Paccumulation du glcyogene et la differentiation d'un appareil lysosomal.
Tous ces faits se verifient dans une periode comprise entre le 6eme et le 8eme
jour d'incubation.
Origin of chick serum albumin
297
This investigation has been supported by the Italian Consiglio Nazionale delle Ricerche.
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