/ . Embryol. exp. Morph. Vol. 20, 1, pp. 107-18, August 1968
With 2 plates
Printed in Great Britain
107
Albumen absorption during chick embryogenesis
By P. CARINCI 1 & L. MANZOLI-GUIDOTTI 1
Institute of Histology and General Embryology, University of Bologna
In birds albumen represents a substantial part of the reserve material for their
embryological development (Romanoff & Romanoff, 1949). In Gallus gallus
albumen proteins form 50 % of non-incubated egg protein content (Romanoff &
Romanoff, 1949). During incubation such material is absorbed and utilized, to
a great extent, for the synthesis of embryonal proteins (Fiske & Boyden, 1926;
Rupe & Farmer, 1955). It is not yet well understood how these processes (albumen
absorption and utilization for the energetic and nutritional needs of the embryo)
take place. The present available data indicate that the amount of albumen solids
begins to diminish starting from 11 to 12 days of incubation (Romanoff &
Romanoff, 1933; Rupe & Farmer, 1955). From 13 to 14 days, after the seroamniotic connexion perforates, the protein content of the amniotic fluid shows
a marked increase because of the passage of albumen (Needham, 1931; Romanoff,
1960). During the same development period albumen proteins can be recovered,
in large amounts, in the yolk (Saito, Martin & Cook, 1965; Carinci, Wegelin &
Manzoli-Guidotti, 1966).
Indeed the major part of albumen is very likely absorbed across the yolk and
the amnion. In this connexion the function of the albumen sac is still not clearly
known. This sac, formed starting from the 9th to 10th day of incubation by a
folding of the chorioallantois, is considered by some authors to be responsible
for albumen absorption (Patten, 1950).
During development, the protein composition of the residual albumen remains
substantially unchanged and very similar to that of the non-incubated egg (as
judged by paper and free-boundary electrophoresis) (Marshall & Deutsch, 1950;
Rizzoli, 1956); besides, the immunological behaviour of the individual proteins
is unmodified (Kaminski & Durieux, 1954). The electrophoretic data indicate
also that the relative proportion of different albumen proteins is constant during
incubation (Marshall & Deutsch, 1950), though some quantitative observations
demonstrate a relative diminution in ovalbumin concentration (Hasegawa,
Taguchi & Hasegawa, 1956).
Less is known about the protein composition of amniotic fluid (Kaminski &
Durieux, 1954; Geelhoed & Conklin, 1966).
1
Authors' address: Istituto di Istologia e Embriologia Generale, Universita di Bologna, Via
Belmeloro, Bologna, Italy.
108
P. CARINCI & L. MANZOLI-GUIDOTTI
With regard to the yolk it has been demonstrated that the albumen proteins
can be recovered from the water soluble fraction (WSF) (Carinci & ManzoliGuidotti, 1968).
Up to the present time, however, there is no analysis performed simultaneously
on embryonic fluids to furnish a complete picture of the qualitative and quantitative modes of albumen absorption. In this research we have followed the
albumen absorption, determining at the same time the wet weight, dry weight
and protein content of residual albumen and amniotic fluid, and also estimating
relative proportion of yolk WSF proteins. In addition we have carried out
electrophoretic and ultracentrifugal analysis of the proteins of these embryonic
fluids.
MATERIALS AND METHODS
We have used eggs (60 ± 0-5 g, average weight) laid by a small group of White
Leghorn hens in June-October 1967, provided by the Corticella agricultural
station (Bologna). The eggs were incubated at 38±0-5°C, at 60% relative
humidity and turned twice a day.
Fluids were obtained from fertile non-incubated eggs within a 24 h period of
their being laid (albumen and yolk) and also after 10, 12, 13, 14, 15, 16, 17
and 18 days of incubation (albumen, yolk and amniotic fluid), utilizing at
least 10 eggs per day, and placed according to the embryo morphogenetic
age, evaluated by Hamburger-Hamilton development stages (Hamilton,
1952).
Albumen was removed in toto, including the chalazae, immediately weighed
and diluted with 0-16 M NaCl (1:2, w/w) to obtain an albumen dilution without
ovomucin precipitation (Forsythe & Foster, 1950); then the albumen was homogenized with a Waring blender. From this material globulins were prepared by
repeated precipitations with ammonium sulphate at 45-50 % saturation (Carinci &
Manzoli-Guidotti, 1968); ovomucoid was prepared according to Warner (1954).
At 10 and 12 days of incubation, owing to the difficulty of completely isolating
albumen from yolk sac, quantitative determinations were also carried out on
boiled eggs, as performed by Romanoff & Romanoff (1933). In some cases,
3-5 volumes of distilled water were added to the non-incubated egg albumen.
The resulting precipitate (ovomucin) was centrifuged down and discarded and
the supernatant used for electrophoretic analysis.
Amniotic fluid was obtained by puncturing the amniotic sac, taking care to
avoid any loss or contamination with the other embryonic fluids. Globulins were
prepared as described for albumen.
Yolk was obtained by puncturing the vitelline membrane or the yolk sac and
the WSF was prepared as previously described (Carinci et ah 1966). To prevent
contamination with egg albumen, for the non-incubated eggs, yolks were first
washed with tap water and carefully rolled on filter paper.
For each fluid (albumen, amniotic fluid and yolk) and for each stage of
Albumen absorption
109
incubation we performed at least 10 independent determinations, whose arithmetic averages are recorded on the graphs.
Chemical analyses. The albumen and amniotic fluid dry weights were determined gravimetrically on samples dried at 110 °C to constant weight; albumen
values were corrected for added NaCl.
The total and protein N content were estimated by the micro-Kjeldhal procedure. To determine the protein N content, trichloroacetic acid (15 %) was
added to aliquots of the different fluids to give 5 % final concentration. The
proteins were then centrifuged, washed with 10 % trichloroacetic acid and
analyzed for their N content (protein = N x 6-25).
Electrophoretic analyses. Samples, diluted with 0-16 M-NaCl to 2-5 % protein
concentration, were examined on cellulose polyacetate (Gelmann Sepraphore
III, 1 x 6f in. strips) in tris barbital-sodium barbital buffer, pH 8-8, ionic
strength /i = 0-05, 300 V, 60 min. The strips were stained with amido Schwartz.
The individual proteins on electropherograms have been recognized both by their
behaviour during electrophoresis and by comparison with isolated proteins from
albumen (globulins and ovomucoid). For quantitative estimations strips were
scanned with a Joyce-Loebl Chromoscan densitometer.
Ultracentrifugal analyses. Samples were exhaustively dialysed against 1 M-NaCl
at 4 °C, diluted to 1 % protein concentration and then examined in a Phywe
model U50L analytical ultracentrifuge at 20 °C and 167241g. Calculated sedimentation coefficients (in Svedberg units S), referred to 1 % protein concentration, were corrected for temperature, viscosity and partial specific volume.
RESULTS
1. Albumen
Quantitative analyses. The albumen wet weight after 10 days of incubation is
30-3 % of that of the non-incubated egg; it then does not change until the 12th
day of incubation, and thereafter diminishes steadily. After 17 days of incubation
albumen is still present, in low amounts, in about 10 % of the embryos examined
(Text-fig. 1A).
The albumen dry weight remains nearly unchanged up to 12 days of incubation, then decreases similarly to that of the wet weight (Text-fig. 1B).
The decrease in the residual albumen wet weight in the early stages of development is due mainly to water absorption (Needham, 1931; Romanoff, 1960). The
ratio between dry and wet weight is 37 % after 10 days of incubation, compared
with 11 % in the non-incubated egg.
Our quantitative values show moderate variations in embryos at the same
developmental stages; these become pronounced after 14-16 days of incubation,
at the same time as albumen absorption is taking place.
The total N content is nearly constant up to 12 days of incubation, and then
diminishes like the dry weight (Text-fig. 1C). As proved by determinations on
110
P. CARINCI & L. MANZOLI-GUIDOTTI
TCA precipitates, almost all the Ncontent is present as protein N (Text-fig. 1D).
The % content of N and proteins, calculated on a dry weight basis, does not
show any noticeable variation during the incubation period studied.
A
40 •s
30 |
20
!
10 -
s
s
\
1
N
S.
I
/ /
I
I
B
TT
4 -
>*
3 2 -
1 1
I
/ (
I
I
c
0-6
'So ,. _
~ 0-5
|
0-4
J 0-3
| 02
0-1
i
\
—0
D
4 h
—
.
v
3
2
1 1
0
I
/ /
"
10
I
11
I
i
12 13
i
i
14
15
•—
16
17
Incubation (days)
Text-fig. 1. Average values of albumen wet weight (A), dry weight (B),
N (C) and protein content (D) for the indicated days of incubation.
Electrophoretic analyses. The electrophoretic pattern of the non-incubated egg
albumen, diluted with 0-16 M-NaCl, shows six zones with anodic mobility and
one variable zone with cathodic mobility. The electrophoretic zones are identifiable by their position after migration and by comparison with isolated fractions
as ovalbumin, which is divided into three components, globulins G3 and G2,
conalbumin and lysozyme (Plate 1 a). The subdivision of ovalbumin into three
bands is due to the fact that the phosphorus content is different in each fraction
(Fevold, 1951). Sometimes ovalbumin divides into five bands with a better
resolution than that observed on starch gel electrophoresis (Steven, 1961). By
o
15
am-
14
17
14
21
Electrophoretic patterns of albumen (a, b, c), amniotic fluid {d, e), and yolk water-soluble fraction (/, g, h) for the indicated
days of incubation. See text for experimental details.
10
>
H
W
•§.
S
/. Embryol. exp. Morph., Vol. 20, Part 1
PLATE 2
A
a-0
b-10
c-15
V
d-15
e-17
g-15
h-15
b
*
f-o
i-17
Ultracentrifugal patterns of albumen in toto (a, b, c), amniotic fluid in toto (d, e), albumen
globulins (/; g), and amniotic fluid globulins (/?, /) for the indicated days of incubation. See
text for experimental details.
P. CARINCI & L. MANZOLI-GUIDOTTI
Albumen absorption
111
electrophoresis we did not succeed in separating zones of ovomucoid and
ovomucin, proteins which have been isolated from non-incubated egg albumen
(Warner, 1954).
The qualitative electrophoretic pattern of residual albumen after 10, 12, 13,
14, 15 and 16 days of incubation is the same as that of the non-incubated egg.
Table 1. Relative proportion (%) of protein fractions
Albumen*
(Days of incubation)
Fra ctionsf
0
Amniotic fluid
(Days of incubation)
5
10
14
15
14
15
16
36-5
15-9
3-4
4-8
12-6
f 8-6
118-2
—
32-9
18-5
3-8
60
12-5
5-8|
20-5J
—
33-6
17-5
4-1
6-5
10-2
281
35-6
14-9
4-4
5-8
14-2
251
—
—
i
I
II
III
IV
V
VI
VII
VIII
(OvO
(Ova)
(Ov.)
(Om)
(G.)
(G,M
(Ca)J
(Li)
34-2
18-5
40
—
13-3
290
41-6J
17-4
4-5
—
9-6
26-9
31-9
16-3
4-5
6-4
131
26-8
36-2
15-5
3-4
61
13-2
25-6
37-1
16-8
5-6
40
10-9
25-6
10
—
i-o
—
—
* Diluted with 016 M-NaCl.
t Numbered starting from anode (top) to cathode (bottom). In the parentheses the principal
protein component is indicated.
% Diluted with 3/5 volumes of water.
Table 2. Sedimentation coefficients of protein components
Days of incubation
0
10
15
17
2-6
2-5
2-7
—
31
16-3
30
15-7
3-3
16-2
—
—
—
—
2-6
2-6
—
—
—
—
3-0
151
2-8
160
Total albumen
S
Precipitated albumen*
(fl)S
(b)S
Total amniotic fluid
S
Precipitated amniotic fluid
(a)S
(b)S
S = Svedbergs at 20 °C and 1 % protein concentration, (a) and (b) indicate the
individual peaks of various fractions.
* Previously published (Carinci & Manzoli-Guidotti, 1968).
However during development there appears, on electropherograms, a zone of
ovomucoid, identified by comparison with isolated ovomucoid (Plate Ib, c).
The relative proportion of protein fractions on electrophoresis is given in
Table 1. For the non-incubated egg our values differ from those obtained by
112
P. CARINCI & L. MANZOLI-GUIDOTTI
paper and free-boundary electrophoresis (Fevold, 1951; Rizzoli, 1956). In our
opinion the presence of ovomucin in our electropherograms perhaps accounts
for these differences; the densitometric values of electropherograms of albumen
diluted with H2O (causing ovomucin precipitation) agree with those already
published (Table 1, column 2).
The relative proportion of the various fractions is constant during incubation.
Ultracentrifugal analyses. The ultracentrifugal analysis of non-incubated egg
albumen shows only one peak, with sedimentation coefficient of 2-6 S (Plate 2a).
Only one peak is present during the entire development period. Sedimentation
coefficients are given in Table 2.
2. Amniotic fluid
Quantitative analyses. Amniotic fluid volume is approximately 3 ml after
10 days of incubation; it increases up to 15 days, then decreases slowly up to
17 days and drops quickly on the 18th day, as previously reported (Romanoff,
1967) (Text-fig. 2A).
Amniotic fluid dry weight is very low at 12 days of incubation; it undergoes
a quick increase after 14 days, and then it decreases, at first slowly and then at the
same speed as the reduction in volume (Text-fig. 2B).
After 14 days of incubation about 90 % of the dry weight is protein, as shown
by N analysis of TCA precipitates (Text-fig. 2C).
Electrophoretic analyses. On the 14th day, seven anodic fractions and one
irregular fraction migrating to the cathode can be observed on electropherograms; the qualitative pattern is very similar to that of albumen. The electrophoretic pattern remains unchanged during the whole period examined (Plate
\d,e).
The relative proportion of the different fractions is given in Table 1; it is
nearly the same as that for albumen.
Ultracentrifugal analyses. After 15 days of incubation the amniotic fluid
in toto shows only one peak on ultracentrifugal examination, even after 90 min
centrifugation (Plate 2 c); the sedimentation rate (2-6 S) agrees with that of
the total albumen ultracentrifugal component. Only one peak can be found up
to 17 days of incubation (Table 2).
At the 15th and the 17th day of incubation the globulin shows two peaks after
40 min centrifugation (Plate 2h, /). Their sedimentation rates are given in Table 2.
3. Yolk water-soluble fraction
As previously reported, electrophoretic examination of WSF shows three
bands (a-, /?- and y-livetin) up to 13 days of incubation. Sometimes y-livetin is
divided into two bands (yx- and y2-livetin) (Mok & Common, 1964). Besides an
ovalbumin migration fraction is present (Plate 1/) (Marshall & Deutsch, 1951).
After 14 days the electrophoretic pattern undergoes some changes due to the
Albumen absorption
113
« o
w U
O <D
11 12
13 14 15 16 17 18
Incubation (days)
Text-fig. 2. Average values of amniotic fluid volume (A), dry weight (B)
and protein content (C) for the indicated days of incubation.
Table 3. Yolk WSF. Relative proportion (%) of protein fractions
Days of incubation
t
Fractions*
0
13
14
15
21
I
(a-liv)
II (OvJ
III (Ov2)
IV (Ov3)
V (A-liv, Om)
VI (G3)
VII ( G ^ - l i v )
VIII(Ca,ya-liv)
IX (Li)
15-6
2-5
—
—
470
—
15-4
2-8
—
—
43-2
—
110
101
7-7
—
32-4
—
5-3
40-2
14-2
4-1
3-8
7-6
3-4
38-7
101
41
7-0
110
34-7}
38-6J
38-8}
23-2J-
25-7J
—
—
—
10
—
* Numberec1 starting from anode (top) to cathode (bottom). In parentheses the principal
protein components are indicated.
J E E M 20
114
P. CARINCI & L. MANZOLI-GUIDOTTI
appearance of the albumen proteins (Plate \g, h). We have to call attention to
the fact that, under our experimental conditions, yj-livetin has a mobility slightly
greater than that of conalbumin.
The relative proportion of the different fractions is given in Table 3. In nonincubated eggs /?-livetin is the prevalent fraction; the ovalbumin component
amounts to 2-5 %. The relative proportion of the various components remains
unchanged up to 13 days of incubation. After 14 days ovalbumin is somewhat
less than 20 % of total protein content, after 15 days it increases to 58-5 %. The
increase in proportion of ovalbumin takes place mostly from 14 to 15 days of
incubation. After 15 days, albumen proteins represent at least 6 5 % of total
protein content.
DISCUSSION
As a result of this study we have first to underline the fact that albumen
solids absorption does not take place, in noticeable amounts, before 12 days of
incubation. Indeed during this period dry weight and protein content are
nearly the same as those of the non-incubated egg. Nevertheless, it is possible
that during this period small quantities of albumen are absorbed. Electron
microscopic evidence indicates the presence of albumen in blastoderm ectodermal
cells (Ruggeri, 1967); moreover, proteins with the immunological behaviour of
ovalbumin and conalbumin have been found in embryonic fluids at 5-8 days of
incubation (Kaminski & Durieux, 1954; Kaminski & Durieux, 1956).
Albumen is therefore a reserve protein material which the embryo does not
utilize, in noticeable amounts, before the second incubation phase. In early
development nourishment for the embryo is provided almost completely from
yolk proteins (Rupe & Farmer, 1955; Walter & Mahler, 1958).
The residual albumen protein composition remains substantially unchanged
during incubation and hence is very similar to that of the non-incubated egg.
This agrees with previously reported electrophoretic data. All the fractions are
absorbed in the same relative proportion, so we can exclude any preferential
absorption of ovalbumin.
After 13 days of incubation albumen absorption begins. The absorption
becomes very high after 14 days; at this time a 5-fold increase in amniotic fluid
protein content is observable. Amniotic fluid proteins are identifiable as albumen
proteins by their electrophoretic and ultracentrifugal behaviour. Even the relative proportion of the different fractions is very similar to that of albumen, and
therefore it is clear that albumen passes unchanged into the amniotic sac.
After 14 days of incubation amniotic fluid proteins account for7 0 % of absorbed albumen proteins. On the following day the amniotic fluid protein content
increases; it is impossible however to quantify the absorbed albumen, because
it is very likely that at the same time some proteins are absorbed from amniotic
fluid by the embryo.
After 14 days of incubation albumen proteins are also present in the yolk.
Albumen absorption
115
In this research we have not determined their total amounts, mainly because all
the proteins absorbed into yolk are not present in the WSF, a part being
adsorbed on vitelline granules (as proved for ovalbumin by Martin & Saito
(1967). At least 60 % of WSF proteins, prepared according to our procedure,
consists of albumen proteins. Taking into account the quantitative determination by Saito et al. (1965) on WSF, this explains the absorption of about 530 mg
of albumen proteins after 15 days of incubation. Albumen proteins are recovered
from the yolk in a relative proportion very similar to that of albumen, as shown
by densitometric measurements on electropherograms. We have not observed
reduction in ovalbumin! as compared with ovalbumin2, as found by Saito &
Martin (1966).
The albumen absorption mechanism is under further study in our laboratory.
As for the fate of albumen absorbed into yolk and amniotic sac we can formulate
the following hypotheses:
The albumen proteins absorbed into yolk are likely to be hydrolyzed by yolk
enzymes. Acid-soluble N, mainly due to amino acids, is present in very low
concentration in albumen and amniotic fluid during all the period examined and
in the yolk up to 13 days of incubation, and after 14 days increases remarkably
in the yolk, just when albumen is absorbed (Mclndoe, 1960).
During the last incubation period embryonic protein synthesis becomes very
great, as shown indirectly by conspicuous embryo growth and directly by the
synthesis of specific proteins such as muscle proteins (Csapo & Herrmann, 1951;
Herrmann, White & Cooper, 1957; Carinci & Manzoli, 1964). This synthesis
utilizes amino acids and can be prevented by amino acid analogues (Waddington & Perry, 1958; Carinci, Manzoli & Zaniboni, 1964). These increasing needs
are likely to be met mainly through a supply of amino acids from albumen
proteins hydrolyzed in the yolk.
It is more difficult to explain the significance of albumen absorption into the
amniotic sac. It is known that some amniotic fluid is ingested by the embryo
(stained particles introduced into amniotic sac have been found in the alimentary
channel (Hamilton, 1952). In this way albumen proteins are supplied to the
developing embryo. Secondly, the increase of amniotic protein content would
probably cause a rise in viscosity which could be related to the protective function of this fluid.
SUMMARY
1. Study of albumen absorption during chick embryogenesis was carried out,
determining wet weight, dry weight and protein content of residual albumen and
amniotic fluid, and the relative proportion of yolk water-soluble fraction proteins.
2. Electrophoretic and ultracentrifugal analysis of proteins of these embryonic fluids have been carried out.
3. In early development albumen wet weight decreases sharply, while dry
weight and protein content remain nearly unchanged up to 12 days of incubation
8-2
116
P. CARINCI & L. MANZOLI-GUIDOTTI
and very similar to those of non-incubated egg. They then decrease slowly up to
14 days and more quickly up to 17 days of incubation.
4. During development the qualitative composition and the relative proportion of the residual albumen proteins remain nearly unchanged as judged by
electrophoretic and ultracentrifugal examination.
5. After 13 days of incubation protein content of amniotic fluid increases
rapidly; the recovered proteins are identifiable as albumen proteins by electrophoretic and ultracentrifugal examination.
6. After 14 days of incubation albumen proteins are found in the yolk in
relative proportions very similar to those of albumen.
7. Almost all of the albumen is absorbed across yolk and amnion. The
embryological significance of these processes is discussed.
RIASSUNTO
Riassorbimento deWalbume durante lo sviluppo delVembrione di polio
1. E' stato esaminato il riassorbimento dell'albume durante lo sviluppo
deU'embrione di polio mediante valutazione del peso fresco, peso secco e contenuto proteico dell'albume e del fluido amniotico ed inoltre valutando la
concentrazione relativa delle componenti proteiche della frazione idrosolubile
del vitello.
2. Si e anche eseguita l'analisi qualitativa e quantitativa mediante ultracentrifugazione ed elettroforesi delle proteine di tali fluidi embrionali.
3. Mentre il peso fresco dell'albume diminuisce fortemente nel primo periodo
di incubazione, peso secco e contenuto proteico rimangono pressoche immodificati fino al 12° g, per diminuire lentamente fino al 14° g e rapidamente fino al
17° g di incubazione.
4. Durante lo sviluppo la composizione qualitativa e la concentrazione relativa delle proteine dell'albume residuo rimangono pressoche immodificate e
dello stesso ordine dell'uovo non incubato (analisi all'elettroforesi e all'ultracentrifuga).
5. Dopo il 13° g il contenuto proteico del fluido amniotico aumenta fortemente; le proteine ivi ritrovabili sono identificabili come proteine dell'albume
{analisi all'elettroforesi e aH'ultracentrifuga).
6. Dal 14° g si ritrovano nel vitello e precisamente nella frazione idrosolubile
proteine dell'albume in concentrazione relativa analoga a quella con cui si
ritrovano nell'albume.
7. L'albume e pressoche totalmente riassorbito via vitello e fluido amniotico;
viene discusso il significato embriologico di tale fatto.
Albumen absorption
117
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