J. Embryol. exp. Morph. Vol. 16, 1, pp. 197-202, August 1966
Printed in Great Britain
197
Ontogenetic changes in the concentration of serum
proteins in chick and mammalian embryos
By CASIMER T. GRABOWSKI 1
From the Laboratory for Quantitative Biology,
Department of Biology, University of Miami
It is generally recognized, on the basis of electrophoretic studies, that the
serum proteins of individuals in the perinatal period of development are somewhat different than those of the adult (Kekwick, 1959; Lehrer & Toben, 1965).
It is also known that the protein content of the serum of perinatal mammals is
low, and gradually rises to the adult level over a period of weeks or months
(McMurray, Roe & Sweet, 1948; Knapp & Routh, 1949; Ewerbeck & Levens,
1950; Potter, 1952; Halliday & Kekwick, 1957). Virtually nothing, however, is
known about the serum proteins of very young vertebrate embryos, presumably
because of technical difficulties involved in obtaining samples suitable for
quantitative study.
In the present study the protein levels of embryonic serum were assayed in
samples obtained from the earliest age possible, shortly after circulation began,
to birth. Ontogenetic patterns of changes in concentration were compared
between the chick, which develops as a cleidoic egg, and the rat, a mammalian
embryo which develops in intimate relationship with the maternal blood stream.
The study was prompted not only by interest in the normal physiology of
embryos, but also by teratological considerations. Abnormal serum proteins
have been described in human mothers who subsequently had abnormal births
(Langman, Van Drunen & Bouman, 1959) and in pregnant rabbits treated with
teratogenic doses of Trypan blue (Langman & Van Drunen, 1959). Qualitative
and quantitative changes in yolk-sac proteins, which apparently reflect changes
in maternal proteins, have been described after exposing mothers to Trypan
blue (Beaudoin & Ferm, 1961). Such observations indicate that the plasma
proteins of the young embryo may mediate or reflect abnormal development,
and additional information is desirable.
MATERIALS AND METHODS
The chick embryos used were of the Kimber strain of White Leghorn. Blood
samples were obtained from 3- and 5-day embryos from vitelline arteries with
1
Author's address: Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables, Florida 33124, U.S.A.
198
C. T. GRABOWSKI
the aid of glass microneedles. At 10 and 15 days, samples were obtained from
chorio-allantoic vessels; at 20 and 25 days, from the heart of lightly anesthetized
chicks. Adult samples were obtained from young roosters, either from the wing
vein or the heart. The rats were of the Holtzman strain. Mothers were quickly
anesthetized with chloroform and maternal blood samples obtained from the
heart. Uterine segments were excised as needed, placed under a stereoscopic
microscope and embryonic blood samples withdrawn from the umbilical
arteries with glass needles, Samples were drawn form newborn rats by heart
puncture of lightly anesthetized individuals. Rabbits were killed by air injection
and samples obtained in the same manner as for rats.
All samples were drawn as rapidly as possible and centrifuged within 25 min. Assays were performed on 5 jul samples with the Beckman Ultramicro
Analytic System, utilizing a modification of the method of Kingsley (1939) and
Gornall, Bardawill & David (1949). Since this is a micromethod, only samples
obtained from the very youngest embryos were pooled. All other assays were
performed on individually drawn samples.
RESULTS
Chick embryos
The protein content of serum of chick embryos between 3 and 10 days old
ranged from 1-1 to l-2g/100ml, approximately one-fourth of the adult level
(Table 1; Fig. 1). From 15 days on, there is a simple, gradual increase in protein
levels. At 25 days (4 days after hatching) the concentration has not quite reached
the adult level.
Table 1. Total protein in serum of chick embryos
Concentration (g/lOOml)
Age
(days)
No. of
determinations
Mean
Range
3
5
10
15
20
5
13
7
7
4
7
13
1-2
11*
1-2
1-5
2-7
3-6
4-2
0-9-1-7
0-8-1-4
0-9-1-6
1-1-2-4
1-9-3-2
2-9-4-0
3-8-5-4
25t
Adult!
A
\
* In an earlier study (Grabowski, 1966) the concentration of proteins in the 5-day chick
was listed as l-4g/100ml. This lower figure is based on additional determinations and a
somewhat improved technique.
f Four days after hatching.
% Young roosters.
Embryonic serum proteins
199
Rat embryos
The youngest rat embryos from which blood samples were obtained were
12* days old. This is about 2 days after circulation begins. The protein concentration at this stage is 0-9 g/100 ml, approximately one-eighth the adult level
8 r
Adult rat —^
I
7 -
l
l
6 -
5
-
4
-
c
'3
o 3
I-I
a
-
2
-
Adult chicken—^
Rat embryos
/
7
•—•—5^
Chick embryos
*r
1 1
1
1
1
1
1
1
1
i
i
i
Rat 12
13
14
i
i
i
I
I
15
16
17
18
I
1
1
1
20
15
10
Chick 3
19
20
21
1
25
22
23
24
Birth
Age (days)
Fig. J. Ontogenetic changes in serum protein concentration of chick and rat embryos.
Table 2. Total protein in serum of rat embryos
Concentration (g/100 ml)
Age
(days)
12**
13*
14*
15*
16*
18*
19*
20*
23*t
Maternal
>Jr»
of
JL>U. U l
c
determinations
Mean
Range
2
7
2
15
5
18
4
5
12
20
0-9
1-5
1-7
1-9
1-9
21
3-6
41
4-5
7-2
0-5-1-3
0-5-1-9
1-5-1-9
0-9-2-7
0-9-2-6
1-6-3-1
2-6-4-5
3-4-4-4
3-3-5-8
5-3-9-0
* The morning that sperm was found in the vagina was considered as day *.
t One day after birth.
200
C. T. GRABOWSKI
of 7-2 g/100 ml (Table 2; Fig. 1). The concentration rises very slowly to 2-1
g/100 ml at 18 days and then increases sharply before birth. In the perinatal
period the protein level is still approximately one-half the adult concentration.
After birth, the rise becomes more gradual (see also Halliday & Kekwick (1957)
who report an increase in plasma proteins of young rats from 3-92 g/100 ml at
12 days to 5-85 g/100 ml at 90 days).
Other mammals
Several protein determinations were made on the serum obtained from 22-day
rabbit embryos, which is approximately the end of the second trimester. At this
time the serum contained 2-5 g/100 ml of protein, compared to a maternal level
of 7-1 g/100 ml. Single determinations of serum proteins of relatively young
human fetuses have been reported as follows: 2-86 g/100 ml at 3 | months
(Potter, 1952); 1-55 g/100 ml at 4 months and 1-95 g/100 ml at 5 months
(Ewerbeck & Levens, 1950). It would appear that the low serum protein levels
reported here for rat embryos are a characteristic of mammalian development.
DISCUSSION
The physiological relationships of chick and mammalian embryos to their
surrounding environments are different. The chick embryo develops within a
closed system except for gaseous interchange. Furthermore, the extra-embryonic
capillaries are surrounded by fluids which are of markedly different composition
from the serum itself (Grabowski, 1963, 1966). The rat embryo develops in
juxtaposition to a maternal environment which presumably is similar to that of
the embryo. A comparison of any characteristic of the embryonic blood of the
two groups is therefore of interest.
With respect to total protein content, we find that both curves start at a
surprisingly low figure of about 1-0 g/100 c.c. Both curves rise very slowly during
the early stages of development, then more rapidly nearing birth. At birth the
concentration of protein in chick serum approaches the adult level more nearly
than it does in the newborn rat. However, protein concentration in the adult
chick is substantially lower than in the adult mammal. In summary, the ontogenetic patterns of changes in protein concentration of the serum of both chick
and rat embryos are similar, despite different adult levels and the different
physiological circumstances of development.
What is the functional significance of these observations? Little can be said
at the present, but one speculation is offered here. Proteins make an important
contribution to the osmotic pressure of serum and, by virtue of their ionbinding capacities, help regulate ionic concentrations. In the early stages of
mammalian development, the embryonic serum is in juxtaposition to maternal
serum containing 4-6 times more protein. Such a concentration differential
can be expected, therefore, to affect fluid and ionic interchange between the
Embryonic serum proteins
201
two blood streams. Fluid disturbances are readily induced in embryos of the
chick (Grabowski, 1964, 1966), rabbit (Jost, 1951), rat(Giroud, Lefebvres, Prost
& Dupuis, 1955), and mouse (Waddington & Carter, 1953) by a variety of
teratogenic agents. These disturbances, in turn, can lead to abnormal development. Is it possible that the plasma proteins are somehow involved in these
manifest fluid problems? What is the relationship of the observed quantitative
and qualitative changes in serum proteins to the abnormal development mentioned in the introduction? Additional studies of the serum proteins of young
embryos, in normal and abnormal circumstances, are needed before these
questions can be more intelligently phrased.
SUMMARY
1. The concentration of serum proteins was determined in chick embryos
between 3 and 25 days old and rat embryos between 12-| and 231 days old. In
both species, the earliest determinations were made on embryos about 2 days
after circulation started.
2. In the chicken the adult level of serum proteins is 4-2 g/100 ml; at 3 days
it is 1-2 g/100 ml and gradually rises to around 3 g/100 ml at hatching. In the
rat the adult level is 7-2 g/100 ml; at 12| days it is 0-9 g/100 ml and gradually
rises to about 4 g/100 ml at birth. Comparison with a few scattered observations
of others suggests that the very low level reported here for the young rat embryos
may be a general mammalian characteristic.
3. The physiological and teratological significance of these data is briefly
discussed.
RESUME
Changements dans la concentration des proteines seriques durant
le developpement des embryons de poulet et de Mammiferes
1. La concentration en proteines du serum est mesuree chez des embryons de
poulet ages de 3 a 25 jours et chez des embryons de rat ages de 12 et demi a
23 jours et demi. Chez des deux especes, les determinations les plus precoces sont
pratiquees sur les embryons environ 2 jours apres le debut de la circulation.
2. Chez les poulets adultes le taux de proteines seriques est de 4,2 g/100 ml;
a 3 jours, il est de 1,2 g/100 ml et augmente graduellement jusqu'a 3 g/100 ml
environ a l'eclosion. Chez le rat adulte le taux est de 7,2 g/100 ml; a 12 jours et
demi il est de 0,9 g/100 ml et augmente graduellement jusqu'a 4 g/100 m
environ a la naissance. Une comparaison avec des observations non systematiques d'autres auteurs suggere que le niveau tres faible trouve ici chez les jeunes
embryons de rat est un caractere general des Mammiferes.
3. La signification physiologique et teratologique de ces faits est discutee
brievement.
202
C. T. GRABOWSKI
This investigation was supported by grant No. CRMS 197 from The National FoundationMarch of Dimes and grant No. HD 00641 from the National Institutes of Child Health and
Human Development, National Institutes of Health.
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(Manuscript received 25 February 1966)