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J. Embryo/, exp. Morph. Vol. 24, 1, pp. 13-20, 1970
Printed in Great Britain
13
Immunochemical analysis of the water-soluble
fraction of the chick embryo yolk
By C. E. GROSS!, 1 P. CARINCP AND L. MANZOLI-GUIDOTTF
From the Institute of Anatomy, University of Genoa and the Institute
of Histology and General Embryology, University of Bologna
SUMMARY
By means of immunochemical techniques, the protein components of the water-soluble
fraction (WSF) of the egg yolk have been examined in the unincubated egg and during
incubation.
Anti-ovalbumin and anti-total adult chicken serum antisera have been employed.
Ovalbumin can be detected in the unincubated WSF as well as during incubation; its
concentration seems to increase during incubation.
In the WSF of the unincubated egg, six proteins immunologically related to adult serum
proteins can be detected. They correspond to a-livetin, o^-globulin, /?-livetin, ovotransferrin
(conalbumin) and y-livetin (two components). /?-Livetin disappears after the 14th day of
incubation while the other components can be demonstrated till hatching.
The findings are discussed in relation to the data available in the literature.
INTRODUCTION
The water-soluble fraction (WSF) of the unincubated hen's egg yolk (livetin
fraction) (Plimmer, 1908) shows, in moving boundary electrophoresis, three main
components (a-, /?-, and y-livetin) (Shepard & Bottle, 1949; Martin, Vandegaer
& Cook, 1957); by paper electrophoresis the y-livetin zone can be partially resolved into two bands (yx- and y2-livetin) (Knight & Schechtman, 1954; Mok &
Common, 1964), while, by the use of cellulose polyacetate, a fourth minor component with an ovalbumin migration pattern can be detected (Carinci &
Manzoli-Guidotti, 1968).
Immunoelectrophoretic investigations have demonstrated the immunological
identity of the major protein components of the yolk WSF with serum proteins
(Williams, 1962; Mok & Common, 1964; Zaccheo & Grossi, 1967); namely,
a-livetin corresponds to serum albumin, /?-livetin to serum a2-globulin and ylivetin to serum y-globulin. The identification of the electrophoretic yj-livetin
fraction with serum components is still under discussion (Mok & Common,
1964). In addition, ovotransferrin (conalbumin) and o^-globulin can be detected
1
Author's address: Institute of Anatomy, University of Genoa, Genoa, Italy.
Author's address: Department of Histology and General Embryology, Faculty of Medicine and Surgery, University of Perugia, Perugia, Italy.
3
Author's address: Institute of Histology and General Embryology, University of Bologna,
Bologna, Italy.
2
14
C. E. GROSSI AND OTHERS
in the WSF by means of immunological techniques, while the presence of ovalbumin is still uncertain (Marshall &Deutsch, 1951; Williams, 1962; Stratil, 1967).
During incubation the protein composition of the yolk WSF undergoes some
changes, as shown by electrophoretic and ultracentrifugal analysis; some components disappear while the yolk is enriched by new proteins absorbed from
the egg white (Saito, Martin & Cook, 1965; Saito & Martin, 1966; Carinci,
Wegelin & Manzoli-Guidotti, 1966; Carinci & Manzoli-Guidotti, 1968).
The data in the literature do not show a full agreement about such modifications; therefore we have analysed the protein composition of WSF in various
developmental stages by means of immunochemical methods employing antitotal chicken serum and anti-ovalbumin antisera.
MATERIAL AND METHODS
We have used White Leghorn fertile eggs, provided by the Corticella Agricultural Station (Bologna). The WSF was prepared from unincubated eggs and
after 12, 14, 15 and 21 days of incubation (38 °C, 60% relative humidity),
according to Martin et al. (1957), as previously described (Carinci, Wegelin &
Manzoli-Guidotti, 1966). Globulins were prepared by repeated precipitations
with ammonium sulphate (Carinci & Manzoli-Guidotti, 1968).
The anti-total chicken serum antisera (anti-TACS) have been supplied by
Behringwerke. Anti-ovalbumin antisera have been prepared in our laboratories
by the use of ovalbumin extracted from unincubated eggs and purified according to Warner's technique (Warner, 1954). Rabbit antisera against ovalbumin
have been obtained following different immunization schedules. The first group
of rabbits received subcutaneously 40 mg of antigen in a volume of 2 ml
together with 2 ml of complete Freund's adjuvant. Repeat injections (10 mg of
antigen each) were given intravenously and animals were bled when their serum
reached a precipitation titre of about 1/32000 (a-precipitation test). The second
group of rabbits received 20 mg of ovalbumin intraperitoneally and a series of
subcutaneous repeat injections (10 mg of antigen each) was given until the same
antibody titre was obtained.
Anti-TACS and anti-ovalbumin antisera have been tested against the antigens
by the immunoelectrophoretic microtechnique of Scheidegger (1955) and the
Ouchterlony (1964) double gel immunodiffusion.
The two antisera were always used at a constant litre, while the antigen
solutions were adjusted to the same nitrogen concentration (micro-Kjeldahl).
RESULTS
The purified ovalbumin fraction shows, on immunoelectrophoretic analysis
against the homologous antiserum, only one precipitation line in the ovalbumin
zone (Fig. 1A). Therefore it contains only one antigenic component and it
behaves like a highly purified preparation.
Immunochemical analysis of yolk
15
The anti-ovalbumin antiserum gives a single arc in the ovalbumin zone when
tested against WSF prepared from unincubated eggs (WSF0) (Fig. IB).
The same component can be detected in WSF after 12, 14, 15 and 21 days of
incubation (e.g. Fig. 2 A, B). On the other hand, a slight change can be observed
in the immunoelectrophoretic pattern after 15 and 21 days; the different shape of
the precipitation arc is possibly related to a higher concentration of the antigen
during the latter stages.
Fig. 1. Immunoelectrophoretic pattern of ovalbumin (A) and WSF0 (B) against
anti-ovalbumin antiserum.
Ovalbumin can also be detected in WSF0, WSF12, WSF14, WSF15 and WSF21
by the double diffusion tests (Fig. 3).
Immunoelectrophoretic investigations performed on adult chicken serum
against anti-TACS antiserum show the presence of at least 15 antigenic components (Fig. 4A). The same antiserum when tested against WSF0 forms six
precipitation lines whose electrophoretic migration rate corresponds respectively
to albumin, ^-globulin, a2-globulin, ^-globulin and y-globulin (two overlapping
components which might correspond to IgM and IgG: immunoglobulins M and
G) (Fig. 4B). From earlier work (Williams, 1962; Mok & Common, 1964), the
albumin-like fraction can be identified with a-livetin, the a2-globulin-like
fraction with /Mivetin, and the y-globulin-like fraction with the faster cathodic
migration rate can be identified with y-livetin. In addition, the /?-globulin-like
fraction detected by us should correspond to ovotransferrin (conalbumin),
16
C. E. GROSSI AND OTHERS
A
WSF 12
O
Anti-OvA
I
)
—
^
O
WSF 1S
B
Fig. 2. Immunoelectrophoretic pattern of anti-ovalbumin antiserum against
WSF12 (A) and WSF15 (B).
o
o
0
0 ,
fr
Anti-OvA VJ^
0
WSF 21
y
o
o
WSF 1 S
ft
WSF 0
0
(C^ (<-
WSF 12
•
OvA
o
0
0
o
OvA
Fig. 3. Ouchterlony test. The central holes contain anti-ovalbumin antiserum. In
the peripheral holes are different preparations from WSF and ovalbumin. The holes
labelled with dots contain other antigenic samples.
Lmmunochemical
1
)
analysis of yolk
17
while the y-globulin-like component with the slower cathodic migration rate
might be identified with yi-livetin.
The same electrophoretic pattern shown by WSF0 can be observed with
WSF12 (Fig. 5A) and WSF14. On the contrary, WSF15 when tested against
anti-TACS antiserum shows a different pattern. As previously, a-livetin, a r
globulin, ovotransferrin and the two -/-globulin precipitation lines can be detected, while the arc corresponding to /?-livetin has disappeared (Fig. 5B).
The same finding can be obtained after 21 days of incubation (Fig. 6A).
Fig. 4. Immunoelectrophoretic patterns given by anti-TACS antiserum against
TACS (A) and WSF0 (B).
These observations are confirmed by the Ouchterlony tests.
The ammonium sulphate precipitates prepared from WSF0 (WSFP0), when
tested against anti-TACS antiserum, give three precipitation lines; one is found
in the a2-globulin zone and two in the y-globulin area. The latter might be
respectively referred to as IgM and IgG. Two lines in the y-globulin zone can
also be observed in WSFP15 and WSFP21.
DISCUSSION
The detection by immunological methods of ovalbumin in the yolk extracts
prepared from unincubated eggs confirms previous electrophoretic observations (Carinci & Manzoli-Guidotti, 1968). This finding corresponds with
observations by Marshall & Deutsch (1951) and by Stratil (1967), but not with
those of Williams (1962). Such a disagreement could be explained by the fact that
2
EM B 2 4
18
C. E. GROSSI AND OTHERS
I
Fig. 5. A comparison of the immunoelectrophoretic patterns given by WSF12
(A) and WSF15 (B) against anti-TACS antiserum. In the latter is shown the disappearance of /?-livetin (arrow).
Fig. 6. A, Immunoelectrophoretic pattern given by WSF21 against anti-TACS antiserum. The yff-livetin arc is lacking. B, Globulin components in a fraction prepared
from albumen; they are antigenically related to WSF and serum globulins.
\
Immunochemical analysis of yolk
I
19
the last observation was made only by double immunodiffusion which, especially
in case of complex antigens like WSF, may be faulty because of the masking of
some components.
WSF0 contains, in addition, other proteins which give an immunological reaction identical with that of albumen proteins, i.e. ovotransferrin (conalbumin)
and two globulin fractions, as shown by testing the same anti-TACS antiserum
against WSF and albumen (Fig. 6A, B). It seems appropriate to stress that
ovalbumin can also be detected in the subgerminal fluid (unpublished data),
which represents an early nutrient of the developing embryo (Elias, 1964).
The ovalbumin content of WSF seems to undergo a progressive increase
during incubation, as judged from the evolution of the immunoelectrophoretic
pattern and according to previous quantitative evaluations (Saito et al. 1965;
Carinci et al 1966; Carinci & Manzoli-Guidotti, 1968).
Six proteins immunologically identical with adult homologous serum proteins have now been detected in yolk WSF0. Williams (1962) has found only
four of these and does not record a r globulin or the first y-globulin component.
Mok & Common (1964) have been able to show the o^-globulin fraction. These
different findings can be referred to the different antibody pattern of the tested
anti-TACS antisera; our anti-TACS antiserum has a very wide antibody range
and therefore it is able to detect a higher number of antigenic components.
The paper electrophoretic y^livetin fraction should correspond to ovotransferrin, according to Hui & Common (1966); in our opinion it is possible
that, at least partly, this fraction contains LgM (macroglobulin), even though
we have not been able to demonstrate any macroglobulin in WSF before the
14th day of incubation by means of ultracentrifugal analysis (Carinci & Manzoli-Guidotti, 1968). On the basis of immunological observations we suggest
that a macroglobulin is already detectable in the yolk of unincubated eggs.
The immunoelectrophoretic analysis of the protein pattern in WSF during
incubation has shown that /Mivetin disappears while the other components are
all detectable till hatching.
On the basis of electrophoretic observations Saito & Martin (1966) conclude
that a-livetin disappears from the WSF after the 15th day of incubation; on the
other hand Carinci & Manzoli-Guidotti (1968) have been able to demonstrate
this protein while /?-livetin seemed to disappear after the 15th day. The present
data confirm the observations of the latter authors.
In conclusion, by means of the immunological analysis it has been shown
that (1) ovalbumin is present in the yolk before and during incubation and (2)
/Mivetin disappears from the yolk after the 14th day of incubation, but the
other components remain.
20
C. E. GROSSI AND OTHERS
RIASSUNTO
Anal isi immunochimica della frazione idrosolubile del vitello dell' embrione di polio.
Per mezzo di tecniche immunochimiche e stata studiata la composizione proteica della
frazione idrosolubile del vitello dell'uovo di polio non incubato e durante lo sviluppo dell'embrione. La frazione idrosolubile e stata esaminata in toto e nelle sue componenti globuliniche,
con l'impiego di sieri anti-ovalbumina e anti-sieroproteine totali di polio adulto.
Ovalbumina e immunologicamente dimostrabile nell'uovo non incubato e per tutto il
periodo dello sviluppo; la sua concentrazione sembra aumentare durante l'incubazione.
Nella frazione idrosolubile dell'uovo non incubato si ritrovano sei proteine immunologicamente identiche a proteine del siero adulto; esse corrispondono ad a-livetina, o^-globulina,
/?-livetina, ovotransferrina (conalbumina), y-livetina (due componenti). Durante l'incubazione
si osserva la scomparsa della /?-livetina dopo il 14° giorno mentre le altre componenti
persistono fino alia schiusa.
II significato di questi reperti viene esaminato in relazione con i dati della letteratura.
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(Manuscript received 6 August 1969)