/. Embryol. exp. Morph. Vol. 20, 3, pp. 295-305, November 1968
With 2 plates
Printed in Great Britain
295
Immunoelectrophoresis of egg
and plasma proteins during development of the
house sparrow, Passer domesticus
By FRANCIS M. BUSH1 & CHARLENE A. SEIBERT 2
From the Department of Anatomy, Medical College of Virginia
Differentiation of plasma proteins in avian embryology has been analysed by
electrophoresis (Brandt, Clegg & Andrews, 1951; Schechtman, 1952; Heim &
Schechtman, 1954; Weller & Schechtman, 1962). Information has been obtained
on affinities between plasma and egg proteins by electrophoretic studies (Wise,
Ketterer & Hansen, 1964) and by immunochemical studies (Kaminski &
Durieux, 1956; Williams, 1962a; Stratil, 1967; Zaccheo & Grossi, 1967).
There is a certain orderliness in the appearance of proteins. Embryo specific
proteins, distinct from adult proteins, occur in the developing chicken. These are
considered unique on the basis of electrophoretic mobility and by virtue of their
transitory nature. Examples include some alpha globulins and gamma globulin
found initially by 5-12 days (Kaminski & Durieux, 1956; Weller & Schechtman,
1962), and pre-albumin, present by 8-11 days of incubation (Heim & Schechtman, 1954; Kaminski & Durieux, 1956; Weller & Schechtman, 1962; Wise et al.
1964). Furthermore, the precise time of 'switchover' from embryonic to adult
forms is well established for some classes of proteins.
Adult proteins can also be synthesized by embryos; however, some others
form after hatching. Vitelloid (yolk-like) albumin exists by the 3rd (Nace, 1953)
or 4th (Zaccheo & Grossi, 1967) day of incubation, whereas liver synthesis of
albumin occurs by the 7th embryonic day (Zaccheo & Grossi, 1967). Proteins
having the same immunochemical character throughout the remainder of
morphogenesis are fl2 globulins and gamma globulin, present between 11
and 20 embryonic days (Heim & Schechtman, 1954; Kaminski & Durieux, 1956)
and pre-albumin, alpha and beta! globulins, present between 18 days embryonic
and 2 days post-hatching (Kaminski & Durieux, 1956; Weller & Schechtman,
1962). Whether the /?2 globulin, transferrin, exists earlier than 11 day of
incubation (Wise et al. 1964) is unknown, but conalbumin, which has an identical
protein moiety (Williams, 19626), can be detected by 8 days of incubation
(Kaminski & Durieux, 1956).
1
Author's address: Department of Anatomy, Richmond, Virginia, 23219, U.S.A.
Author's address: Radiation Immunology, Oak Ridge National Laboratory, Oak Ridge,
Tennessee, 37830, U.S.A.
2
296
F. M. BUSH & C. A. SEIBERT
Details of the embryogeny of some altricial species, such as the house sparrow,
Passer domesticus, are fairly well known (Romanoff, 1960), but little is known of
changes in its plasma proteins during ontogeny. Recent studies have shown that
the plasma of newly hatched young has half the proteins found in adult plasma
(Bush, 1965), and non-specific esterase, cholinesterase and lipoprotein increase
and amine oxidase decreases with maturation (Bush, 1967). This present paper
reports the results of continued study on hatchling and adult plasma proteins,
including reactions of their antisera with both embryonic plasma and egg
proteins. Embryo and egg proteins referred to herein are not specifically confined
to these stages, but instead are necessarily immunologically identical with the
hatchling or adult proteins used to evoke the immune sera. This paper also
discusses the degree of antigenic correspondence between house sparrow plasma
proteins and those of chicken and human.
METHODS AND MATERIALS
Collection and preparation of samples. Heparinized blood was obtained by
capillary pipette after cutting embryonic vitelline vessels; by syringe, from
hatchling and adult sparrow hearts and from chicken axial and human median
cubital veins. After centrifugation at 2000 g for 15 min, each plasma sample was
used immediately or stored at - 20 °C without preservative. It was thawed only
when used. Individual samples studied were from 50 females, 50 males, 91
hatchlings, 10 embryos and 5 eggs collected from the same population at
Richmond, Virginia.
Fresh egg white was pipetted away from egg yolk. Yolk was washed three
times with 0-9 % saline without rupturing the vitelline membrane and then
collected by pipette after rupture. Egg white was used undiluted and yolk was
diluted with an equal volume of saline.
Embryonic stages were determined by comparison with chick developmental
stages (Hamilton, 1952); age of hatchlings was assigned by weights and lengths
and of older birds by the degree of skull ossification as used previously (Bush,
1965).
Quantitation of plasma protein. Protein concentration was determined in
duplicate by the biuret method (Colowick & Kaplan, 1957) using bovine serum
albumin (Sigma) as a standard. Values were read at 550 m/i with a Bausch and
Lomb Spectronic 20 colorimeter.
Production of antisera. Antisera against sparrow plasma were produced in
mature rabbits of either sex. Two kinds of antisera were produced. They had
to be prepared with pooled plasmas to elicit a satisfactory antibody response.
One was produced against pooled adult plasmas (anti-A serum) and the other,
against pooled hatchling plasmas (anti-H serum). Three rabbits were immunized
with the adult plasmas and one rabbit with the hatchling plasmas.
Twelve ml of plasma from 20 adults (11 females: 9 males) were used to make
Sparrow plasma proteins
297
each anti-A serum. A total of 50 hatchling plasmas, mean age, 18 days (range:
6-25 days) was used to make the anti-H serum. Antisera were prepared by
modifying a procedure used previously (Proom, 1943). Pooled adult and pooled
hatchling plasmas containing 40 mg protein each were mixed separately with
16 ml of distilled water and 18 ml of 10 % potassium aluminum sulfate and the
pH of the solution adjusted to 6-5 with 5 N sodium hydroxide. After centrifugation at 4000 g for 30 min, the sediment was washed twice with saline and the
final volume made to 20 ml in saline with addition of 0-01 % merthiolate.
The schedule used for injection followed one described previously (Hirschfeld,
1960). Each rabbit was injected intramuscularly with 10 ml of antigen mixture;
14 days later the same quantity was injected and then 1 ml of fresh plasma was
injected intraperitoneally on the 24th day. Blood was collected 10 days after the
final injection and the antisera frozen after addition of 0-01 % merthiolate.
Chicken and human antisera were purchased commercially (Lloyd Brothers:
Cincinnati, Ohio).
Immunoelectrophoresis. Previous results showed that newly hatched sparrow
chicks had so little plasma that the conventional starch gel technique yielded
low resolution of total proteins. Such direct analysis proved even more difficult
using embryonic plasma. Thus we employed the highly sensitive immunoelectrophoretic analysis as described previously (Grabar & Williams, 1953). Microscopic slides coated with 1 % agar (Nobel: Difco) in barbiturate buffer, 0-05
ionic strength, pH 8-6 were used. Samples were electrophoresed with the same
buffer at 25 °C for 4 h at 250 V using LKB equipment, type 3276. After electrophoresis, antiserum was applied, slides were incubated at 4 °C for 36 h, washed,
stained with amidoblack, cleared and then photographed.
RESULTS
Typical immunoelectrophoretic resolution of adult plasma proteins is shown
when reacted with anti-A serum (Text-fig. 1). Mobilities of precipitates are
compared with specific migration rates of human plasma precipitates. Similarity
in mobilities does not imply homologous proteins.
The mean number of precipitates is 15 for either sex (range: female, 12-17;
male, 12-19). Neither sex exhibits any unique precipitate. Albumin, /?2-oan(l
gamma globulins exhibit a characteristic boat-shape (Text-fig. 2; Plate 1).
/?2_2 globulin migrates as tandem intersecting arcs. Double lines of precipitation
occur for some patterns in the ax and /?2 regions indicating additional proteins.
Their presence is not caused by denaturation. Plasmas heated to 100° C form
smudged patterns; only albumin, a ^ , a2_x and a2_3 globulins precipitate.
The hatchling essentially attains its adult complement by 23 days. This is
reflected by the gradual increase in number of precipitates present after hatching.
Pre-albumin, albumin, aL_l3 /?2-o and probably a2_x globulins exist by the time of
hatching. Some minor precipitates can be regarded as provisional, since their
precipitation varies depending upon the plasma sampled.
298
F. M. BUSH & C. A. SEIBERT
Most beta globulins usually react with anti-A serum by 3-5 days after hatching, as shown by the frequency of precipitates in Table 1. Gamma globulin and
pre-albumin precipitate weakly by 5 days and intensely by 11 and 14 days,
respectively. Plasma obtained from hatchlings 7-14 days old exhibits the greatest
Text-fig. 1. Schematic diagram of principal antigens detected in adult house sparrow
plasma (above). Antibody: anti-adult sparrow serum. Schematic diagram of principal
antigens detected in human plasma (below). Antibody: anti-human serum.
Anti-hatchling serum
Anti-adult serum
White
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Text-fig. 2. Schematic diagram of sparrow plasma and egg proteins reacted with
anti-H (left) and anti-A sera (right).
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300
F. M. BUSH & C. A. SEIBERT
variation. Albumin, /?2-o and gamma globuhn assume their adult appearance by
11 days. In this hatching period, /?2-o and /?2_2 globulins precipitate intensely;
a
2-2 globulin typically reacts in later post-hatching development. Probably the
A-o globuhn and the cathodal arc of the /?2_2 globulin are unique to anti-Aserum,
which shows that they are the last proteins synthesized.
40
30
20
10
Text-fig. 3. Total quantity of plasma protein at different developmental stages.
Female plasma forms 12 precipitates and male plasma, 13 precipitates (range:
11-14) when reacted with anti-H serum (Text-fig. 2; Plate 2). Some plasmas lack
distinct a2_2 and /?2_2 globulins. Hatchling plasmas form even fewer precipitates.
Their number increases with advancing age. Gamma and /?2_0 globulins are
boat-shaped by 9 days. As shown in Table 1, ax_3 and /?2-i globulins precipitate
intensely by 7-14 days as does a^g, even until 23 days. a2_r and fi^ globulin
stain intensely after 11 days, whereas most /?2 globulins appear less intense.
Embryos of 8-12 days have plasma proteins that form distinct precipitates;
however, their migration rates are comparable to those of adult plasma when
electrophoresed at the same time. The principal difference is that embryonic arcs
of precipitation are shorter. Albumin, a ^ and /?2-0 globulins react with both
antisera. Pre-albumin is apparent at this time. Most a2 and /?2 globulins are
absent; gamma globulin is undetectable.
Both egg white and yolk precipitate the /?2_0 globulin with both antisera.
Albumin and /?2_2 globulin of yolk stain distinctly with anti-H serum.
Sparrow and chicken plasmas cross-react with antisera produced against each
species' plasma. Albumin, a2_1, /?2_2 and probably a^g globulins are the common
proteins. The mobility of sparrow albumin is slower than the mobility of
/. Embryo!, exp. Morph., Vol. 20, Part 3
PLATE 1
Female
Male
Male, a
Yolk
8-9-day E
Chicken female
Sparrow female
Immunoelectrophoretic pattern obtained for sparrow egg and plasma proteins using anti-A
serum during development and the immunoelectrophoretic patterns for chicken (below)
and sparrow plasmas using anti-chicken serum.
F. M. BUSH & C. A. SEIBERT
facing p. 300
/. Embryol. exp. Morph., Vol. 20, Part 3
PLATE 2
Female
Male
Yolk
White
23-day H
Immunoelectrophoretic pattern obtained for sparrow egg and plasma proteins
using anti-H serum during development.
F. M. BUSH & C. A. SEIBERT
Sparrow plasma proteins
301
chicken albumin. Sparrow and human plasmas fail to cross-react with antisera
produced against their plasmas.
The protein content of sparrow plasma increases progressively after hatching
(Text-fig. 3). The most rapid synthesis occurs from 1 to 10 days after hatching.
Plasmas pooled from five embryos have slightly more protein than is found in
1- to 2-day hatchling plasma. Embryo plasma is yellow from the presence of
yolk carotenoid. This pigment disappears a short time after hatching. Results
of a Mest show no difference (P < 0-05) between the plasma protein content of
mature individuals of either sex.
DISCUSSION
The results of our immunoelectrophoresis of house sparrow plasma proteins
are generally in accordance with changes observed for the chicken. A greater
variety of proteins is synthesized in the early hatching period of the house
sparrow, a disparity explained by the shorter time of incubation. The mean
length of incubation is 12 days for this species (Summers-Smith, 1963) and 21
days for the chicken. Because of the shorter time, a house sparrow embryo that
has incubated for 5 days will have reached a developmental stage comparable
to an 8-day chick embryo. Development of a 7-day hatchling would correspond
to an 18- to 20-day chick embryo. Thus, sparrow embryo plasma still lacks some
essential proteins just before hatching, which makes continual brooding of
hatchlings obligatory. One may infer that precocity is advantageous because
such essential proteins are synthesized by the time of hatching.
Relative mobilities of precipitates show that house sparrow embryos lack
proteins specifically limited to stages examined in this study. The absence of
pre-albumin between the 6th and 13th day after hatching may be interpreted as
the end of an embryonic form followed by synthesis of an adult form having
the same protein moiety. But such an interpretation must be accepted with
reservation because of the degree of variability found throughout the population
(compare pre-albumin in male patterns, Plate 1). Some adult patterns have and
some lack pre-albumin, its presence or absence appearing to depend on the
individual bird as reported previously for the chicken (Williams, 1962a; Stratil,
1967) rather than being associated specifically-with hen plasma (Brandt et al.
1951; Heim & Schechtman, 1954; Kaminski & Durieux, 1956). But there are
several proteins produced only after hatching; for example, most a 2 and /?2
globulins, particularly a2_2 and the cathodal arc of y#2-2 globulins found only
with anti-A serum.
Immunoelectrophoretic patterns differ slightly from individual to individual.
The extent of variability is unrelated to the technique employed. Non-immunized
rabbit serum also fails to react with house sparrow plasma. The number of
precipitates is within the range determined for the chicken (Kaminski &
Durieux, 1956; Croisille, 1962; Williams, 1962a; Peetoom et al. 1963;
Stratil, 1967) and also confirmed for this species in the present study. This
20
JEEM2O
302
F. M. BUSH & C. A. SEIBERT
variability is also consistent with the variability found using starch gel electrophoresis (Bush, 1967). Therefore, these variable patterns reflect genetic differences
in this random sample from the Richmond sparrow population.
The developmental pattern of proteins synthesized in the house sparrow and
in the chicken is comparable with changes reported in plasma proteins for both
the developing rat and the human. The processes of differentiation are fundamentally similar for all of these species. Proteins formed by these birds are
essentially the same general classes present shortly before or shortly after
parturition in these mammal species. Pre-albumin, albumin, alpha globulins, a
major /?2 globulin (probably transferrin) and gamma globulin comprise the
initial complement. Other remarkable similarities include the remainder of
proteins necessary to complete the adult complement. As in the sparrow and in
the chicken, some a2 and beta globulins form in rat plasma 3 days after birth
(Kelleher & Villee, 1962) and in human plasma during the first to third trimesters after birth (Hitzig, 1964).
Sparrow yolk has only four proteins found in adult plasma. This egg yolk
appears to lack gamma globulin, which is prominent in chicken egg yolk.
There is also less variety of alpha and beta globulins in sparrow egg yolk than
has been reported for the chicken (Stratil, 1967). Although nothing is known of
the iron binding properties of egg or plasma proteins in the house sparrow, the
presence of /?2_o globulin in egg white and yolk suggests that these may be a
conalbumin and a transferrin, respectively, on the basis of their identical
precipitation.
Lack of cross-reactivity between sparrow and human plasmas with their
antisera shows that although mobilities may be similar for some proteins, their
moieties are unique to each species. Cross-reactivity of sparrow and chicken
plasmas with their antisera shows that the moieties of albumin and some alpha
and fi2 globulins are also immunologically different between the chicken and the
human.
The protein content of adult plasma is slightly higher than that determined for
young Passer (species ?), mean 2-2 g/100 ml plasma (Lustig & Ernst, 1937).
This difference should not be considered significant because of the variation
observed in chicken plasma protein content. While no systematic study has been
made of the protein content during ontogeny of the chicken, evidence shows that
embryos have the lowest and laying hens, the highest per ml plasma. Embryos of
11 days have 0-86 g/100 ml which increases to 1-08 and 2-40 by 14 and 17 days
respectively (Kaminski & Durieux, 1956). Hatchlings between 2 and 58 days
have from 3-36 to 3-64 g/100 ml (Brandt et ah 1951; Patterson, Youngnen,
Weigle & Dixon, 1962). Greater viariation has been reported for adult plasma,
from 1-96 (Kaminski & Durieux, 1956) to 6-11 g/100 ml (Brandt et al 1951).
Therefore the difference between this altricial species and the chicken is the
slower appearance of proteins during house sparrow embryogenesis. Whether
there are any specifically embryonic proteins at earlier stages cannot be deter-
Sparrow plasma proteins
303
mined from these present data, but the more rapid incubation period coupled
with the relatively labile system after hatching suggests less likelihood of an
earlier 'switchover'. This pattern of differentiation coincides with a slower
'switchover' from embryonic to adult hemoglobins for this species (Bush &
Farrar, 1967) than reported for the chicken (Manwell, Baker & Betz, 1966) and
for the red-winged blackbird, Agelaius phoeniceus, whose two major adult
hemoglobins are present from the beginning of ontogeny (Manwell, Baker,
Roslansky & Foght, 1963). The quantitative increase in protein content for the
house sparrow parallels the quantitative increase in total hemoglobin only to
23 days after hatching. There is a significant increase in hemoglobin between
fledging and adult stages (Bush & Farrar, 1967).
Studies, such as these, of avian plasma proteins make it desirable to investigate further the significance of specifically embryonic plasma proteins, genetic
controls restricting the initial and the final complements and why variability
exists in time of appearance of some classes of proteins such as alpha globulins.
SUMMARY
1. Plasma and egg proteins of the developing house sparrow have been
examined by immunoelectrophoresis.
2. Quantitative and qualitative changes occur in the plasma proteins during
morphogenesis. Albumin, aj_ l5 /?2_0 and /?2-i globulins characteristically occur
after the 8th day of incubation. Pre-albumin is present in embryos, diminishes
soon after hatching and increases by 14 days after hatching. Gamma globulin
forms by the 5th day after hatching; most alpha globulins, by 9 days; and /?x
globulins, by 14 days. /?2 globulins complete the complement by 23 days.
3. Total protein increases over two-fold within the period between hatching
and fledging.
4. Both egg white and yolk form the same /?2-o globulin when reacted with
antisera against sparrow plasma. Yolk forms the albumin precipitate. Both egg
white and yolk seem to lack gamma globulin.
5. The protein moieties of house sparrow and human plasmas are immunologically distinct. Albumin, a^g, ct^ and /?2-2 globulins are not species-specific
for either house sparrow or chicken plasmas.
RESUME
Analyse immunoelectrophoretique des proteines de Vceuf et du plasma
pendant le developpement du moineau domestique, Passer domesticus
1. Ce travail est consacre a une etude immunoelectrophoretique des proteines du plasma et de l'ceuf pendant le developpement du moineau domestique.
2. Pendant le developpement on observe des variations quantitatives et
qualitatives dans les differentes proteines du plasma. L'albumine et les globu-
304
F. M. BUSH & C. A. SEIBERT
e
lines CL-L-1, /?2-0, * A-i s o n t detectables a partir du 8eme jour de Fincubation. La
prealbumine est presente chez l'embryon, diminue peu apres l'eclosion, et
augmente de nouveau vers le Heine jour apres l'eclosion. La gamma-globuline
est detectable au moins a partir du 5eme jour apres l'eclosion. La plupart des
alpha-globulines sont decelables aux environs du 9eme jour; les globulines (ix
sont detectables a partir du 14eme jour et les globulines /?2 font leur apparition
vers le 23eme jour apres l'eclosion.
3. La quantite de proteines totales augmente considerablement (elle est plus
que doublee) pendant la periode qui s'ecoule entre l'eclosion et le moment de
abandonnement du nid.
4. Le serum anti-plasma de moineau revele la presence de la meme /?2_0
globuline dans le blanc d'oeuf et dans le jaune d'oeuf. L'albumine est presente
dans le jaune d'ceuf alors que la gamma-globuline semble absente du jaune
d'oeuf et du blanc d'oeuf.
5. Les proteines du plasma de moineau et du plasma humain sont immunologiquement distinctes. L'etude des plasmas de poulet et de moineau revele
que l'albumine et les globulines ax-3, ct^x et /?2_2 ne sont pas specifiques d'espece.
Our appreciation is expressed to Drs J. I. Townsend, Department of Biology and
Genetics and J. D. Burke, Department of Anatomy, Medical College of Virginia, for
criticisms on this paper and J. R. Price, Medical College of Virginia, for help with photographs and diagrams. We acknowledge the technical assistance of C. G. Sheppek, Medical
College of Virginia and the assistance with the arrangement of photographs by J. Ahlquist,
Department of Biology, Yale University. This work is supported by NIH Grant GM
13649-01.
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{Manuscript received 5 March 1968)