Download The Serum Proteins of the Rat During Development

The Serum Proteins of the Rat During Development
by WERNER G. HEIM1
From the Department of Biology, Wayne State University
INTRODUCTION
T H E morphological changes which occur during late embryonic and early
postnatal life in common laboratory animals are, with one set of exceptions,
now well understood. The exception relates to those changes which require the
use of physiological, biophysical, or biochemical techniques. Outstanding among
these are the changes in the relative concentrations of the serum proteins
(Kekwick, 1959). Only for the chicken (Moore, Shen, & Alexander, 1945;
Marshall & Deutsch, 1950; Heim & Schechtman, 1954; Weller & Schechtman,
1957) has a reasonably complete picture been built up.
In the case of the changes in the relative concentrations of the serum proteins
of the late embryonic and early postnatal rat, the various investigations, including the more extensive ones of Jameson, Alvarez-Tostado, & Lew (1948),
Shmerling & Uspenskaya (1955), and Gurvich & Karsaevskaya (1956), leave a
confusing and contradictory picture due to the use of widely divergent methods
and the limited number of stages investigated.
In view of this, a systematic survey of the serum proteins of the rat, from the
earliest suitable developmental stage to the immediate postnatal stages, was
undertaken with the aid of a highly standardized technique. Sera of adult male
and female rats were also examined as controls and for purposes of comparison.
MATERIALS AND METHODS
All animals used were Sprague-Dawley rats, obtained from the Holtzman
Company, or their descendants, bred in this laboratory. The animals were
between 90 and 200 days old when mated. They were maintained on an ad
libitum diet of Purina rat chow and water supplemented with occasional feedings
of raw pork-liver, hard-boiled egg-slices, or slices of orange. The date of insemination was determined by the finding of spermatozoa in daily vaginal
smears. Pregnancy was assumed to have begun at about 7 a.m. on the morning
on which spermatozoa were found in the smear. In order to maintain proper
timing, all bleeding was done between 7 and 8 a.m. Delivery was assumed to
have occurred, and generally did occur, early during the 23rd day of pregnancy.
This day is designated hereafter as the first day post-partum.
1
Author's address: Department of Biology, Wayne State University, Detroit 2, Michigan, U.S.A.
Contribution No. 56 from the Department of Biology, Wayne State University.
[J. Embryol. exp. Morph. Vol. 9, Part 1, pp. 52-59, March 1961]
W. G. HEIM—RAT SERUM D U R I N G DEVELOPMENT
53
Blood was obtained in one of three ways. In the procedure used with most
foetuses, the mother was anesthesized with ether, her abdominal and uterine
cavities opened, and the foetuses successively removed from their membranes.
A small, canoe-shaped piece of lucite was slipped under the umbilical cord.
After drying off the outside of the cord, one or more vessels within it were
nicked and the blood collected in a siliconed pipette as it escaped into the
plastic boat. In the cases of some of the older foetuses and of the new-born rats,
a deep dorsal incision was made into the neck, care being taken to avoid cutting
into the trachea or oesophagus. The first drop of blood to appear was usually
discarded and the next few drops collected directly into a siliconed centrifuge
tube. The blood from at least four foetuses or neonatal animals from the same
litter was pooled. The immature and adult animals were bled by direct heartpuncture under light ether anesthesia. In all cases, the blood was allowed to clot
for approximately 4 hours in siliconed centrifuge tubes at about 4° C. After
brief centrifugation, the supernatant serum was removed and its refractive index
determined with white light in an Abbe-type refractometer. No dilutions were
made.
Paper electrophoresis was carried out in accordance with the instructions
(Publication RIM-5, 1957) for, and utilizing the equipment of, the Spinco
model R system. The buffer was barbital (5,5-di-ethylbarbituric acid) 2-76 g.;
barbital sodium (2-sodium, 5,5-di-ethylbarbituric acid) 15-40 g.; distilled water
to make one litre. This buffer is 0-075 M in the sodium salt and has a pH of 8-6.
0-010 ml. of adult serum was applied to each paper strip. The volume of serum
from the earlier stages applied to each strip was adjusted so that the amount of
protein, as estimated from the refractive index, was approximately equal to that
in 0-010 ml. of adult protein. This precaution proved necessary because the
resultant pattern is somewhat dependent on the amount of protein applied. A
potential gradient of 2-6 v./cm., resulting in a current flow of 0-31 ma. per strip,
was applied under constant current conditions for 19 hours. After staining with
bromphenol blue, photometric evaluation was performed with the Spinco
Analytrol. Components were delimited by dropping verticals (Tiselius & Kabat,
1939) and the relative concentrations were calculated from the ratio of the area
under each section of the curve to that under the total curve.
RESULTS
Typical patterns obtained from the sera of the various developmental stages
examined are shown in Text-fig. 1. The leading component visible in the patterns
of animals of 17 and 18 days of gestation has an anodic mobility well within the
range shown by adult albumin. An asymmetry on the trailing edge of the gamma
globulin component is evident at all stages. The leading edge of the beta globulin
fraction shows considerable asymmetry in the neonatal and immature stages. No
significant changes in the mobilities of the components were detected.
The percentage composition of the serum proteins at the various develop-
W. G. HEIM—RAT SERUM DURING DEVELOPMENT
54
mental stages examined is given in Table 1 and the changes in these percentages
during development are shown in Text-fig. 2. Albumin constitutes the largest
17 days of gestation
2(
21 days
of gestation
7 days
post-partum
2
\3
18 days of gestation
2 4 - 3 0 days post-partum
A
2/
19 days of gestation
2/
2 0 days of gestation
2 days
post-partum
Adult
male
TEXT-FIG. 1. Typical patterns of sera obtained at various
stages of development. Component 1: fast albumin. Component 2: albumin. Component 3: alpha-1 globulin. Component 4: alpha-2 globulin. Component 5: alpha-3 globulin.
Component 6: beta globulin. Component 7: gamma
globulin.
single component at all stages. The fast albumin fraction loses its identity as a
component distinct from the main mass of albumin between the 18th and 19th
days of gestation. The changes in the relative concentrations of albumin and
55
W. G. HEIM—RAT SERUM DURING DEVELOPMENT
alpha-1 globulin are generally opposite to each other. Gamma globulin does not
begin to approach adult level until after at least one month of postnatal life.
TABLE 1
Relative concentration of the serum protein fractions
Relative concentration in percen
Age
17 days' gestation
18 days' gestation
19 days' gestation
20 days' gestation
21 days' gestation
22 days' gestation
1 day post-partum
2 days post-partum
7 days post-partum
24 30 days postpartum
Adult female
Adult male
Gamma
No. of No. of
Alpha-1 Alpha-2 Alpha-3
Beta
Fast
samples analysis albumin Albumin globulin globulin globulin globulin globulin
10
9
10
10
9
13
12
13
14
35
36
40
39
35
53
60
56
71
7
28
19
71
7
28
11-3*
±2-9
110
±4-3
31-7
±4-3
33-6
±5-2
40-8
±9-4
37-8
±5-0
33-7
±31
42-6
±6-6
52-4
±6-4
48-7
±6-6
56-4
±4-5
58-1
±5-4
49-8
±4-7
38-8
±2-5
15-9
±4-5
171
±2-3
24-2
±7-8
24-4
±7-8
250
±2-1
16-7
±5-5
11-5
±3-6
16 5
±4-2
9-2
±3-9
10-6
±2-3
12-9
±5-4
14-4
±3-2
8-3
1-1
±1-2
70
±2-2
±1-3
71
±1-6
8-5
15-4
7-4
±2-0
15-7
±2-1
15-2
±1-9
16-2
±1-8
±2-1
91
8-3
4-3-0
9-2
±1-7
9-4
±3-4
100
6-7
±1-8
6-3
±1-2
5-6
±1-2
3-7
±0-6
5-6
±0-9
8-2
±3-1
±2-2
6-8
±2-2
±1-3
±11
9-8
±1-7
8-5
±1-6
16 9
±2-8
7-7
±3-3
9-2
±3-5
5-6
±10
4-5
±09
3-4
±0-6
41
±0-5
15-8
±2-0
14-6
±3-5
13-5
±2-3
15 5
±2-7
151
±2-3
15-5
±2-5
16-7
±0-8
5-4
±1-7
5-2
±1-3
5-9
±11
5-7
±1-8
5-3
±1-8'
5-4
±11
70
±13
61
±13
14-7
±3-3
20-4
±1-5
* Percentage± standard deviation.
DISCUSSION
Gurvich & Karsaevskaya (1956) report the presence of a pre-albumin component in the sera of rat embryos weighing from 0-43 to 0-72 g. According to the
data of Stotsenburg (1915), such animals would be between about \6\ and \1\
days of gestation. The average weight for rats of 17 days of gestation was found
to be 0-74±0-05 g. in the present experiment, thus confirming the above age
estimates. It is in this same age-range, and only in this age-range, that the fast
albumin component was detected in the present work. The relative concentration
of the pre-albumin listed by Gurvich & Karsaevskaya (1956) ranges from 16-00
to 21 -0 per cent., whereas the largest average value measured in the present work
was 11-3±2-9 per cent. It is likely, therefore, that the pre-albumin fraction of
these workers and the fast albumin component found in the present work include,
at least in part, the same protein groups. The term 'pre-albumin' refers to a
protein fraction having a greater anodic mobility at a pH above its isoelectric
point than does the family of proteins designated as albumin. Such components
are found, for example, in the sera of chicken embryos and laying hens (Heim
56
W. G. HEIM—RAT SERUM DURING DEVELOPMENT
& Schechtman, 1954). In the present case, however, the leading edge of the component in question does not exhibit a greater mobility than that of the leading
Fast
albumin
% 15io5-
% 60555045403530252015105-
Albumin
Alpha-I globulin
% 105_
Alpha— 3
% 105.
globulin
globulin
Gamma globulin
t
% 30252015105-
Alpha —2 globulin
Beta
Hv\
% 2015105|5_
105AGE =
Days of gestation
Days post-partum
Adult
-\—i—i—i——•—r
17
18 19 20 21 22
I
2
7 24-30/
Female Male
TEXT-FIG. 2. Average percentage composition of electrophoretic
components in rat serum from the 17th day of incubation to the
mature adult. The short vertical lines indicate the range of plus or
minus one standard deviation. The dashed lines connect points
plotted on an interrupted time scale and hence their slope does not
represent a rate of change.
edge of the albumin fraction of the adult or of later foetal stages. Secondly, it
may be seen from Text-fig. 2 that the disappearance of the fast albumin on the
19th day of gestation is accompanied by a nearly proportional increase in the
W. G. HEIM—RAT SERUM DURING DEVELOPMENT
57
relative concentration of the albumin. This suggests that the material previously
recognized as fast albumin does not disappear at all but that it becomes merged
with the main bulk of the albumin group. If the fast albumin material had disappeared, all remaining components would be expected to show a compensatory
increase in relative concentration, proportional to the fraction each constitutes
of the total protein concentration. Actually, only two of the six components
show any significant increase between the 18th and 19th days of development.
Supporting evidence for the hypothesis that the fast albumin fraction becomes
merged with the main body of albumin comes from the great variability in the
relative concentration of albumin in the serum of the animals at the 19th day of
gestation as shown by the high standard deviation value (Table 1). The loss of
distinction between the fast albumin and the albumin may be due to the appearance of a relatively small amount of a new albumin sub-fraction having a
mobility intermediate between those of the fast albumin and the peak of the
main albumin mass. The appearance of new serum proteins during the course of
rat ontogeny has been postulated on immunochemical grounds by Gurvich &
Karsaevskaya (1956).
An asymmetry was found in the trailing region of the gamma-globulin fraction
during all stages of development, including the adult. An asymmetry very similar
in appearance and position was also found by Gurvich & Karsaevskaya (1956),
but only in sera from perinatal animals. These workers have designated one
component of this complex as eta globulin and consider it, on immunochemical
grounds, to be a distinct protein family. Since in the present work, however, this
component or asymmetry appears at all stages of development and always at or
near the point of initial application of the serum, it is suggested that the asymmetry is an artifact due to adsorbed proteins. Gurvich & Karsaevskaya (1956)
have recognized that, in their immunoelectrophoretic procedure, confusion can
arise between an immunological precipitate and an adsorbed protein. Contrary
to the findings of Shmerling & Uspenskaya (1955), gamma globulin could be
demonstrated in the sera from all stages examined.
Shmerling & Uspenskaya (1955) report the presence, in embryos weighing
2 g. or more and in rats one day after birth, of an alpha-2 and an alpha-4
globulin which more or less completely replace the alpha-1 and alpha-3 globulins
found in other developmental stages. They believe the former two components
to be analogous to foetuin (Pedersen, 1947). Some support is lent to this view by
the finding of a considerable degree of asymmetry in the beta-globulin peak of
sera from perinatal and juvenile animals. The observation of these authors that
the sera of suckling young are cloudy is confirmed, although occasionally nearly
clear sera were obtained. Since optical clarity is not a prerequisite for paper
electrophoresis, as it is for the classical Tiselius technique, no defatting process
was used. The present work also confirms the view of these workers that, on the
basis of their mobilities, the fractions of the same denomination in embryonic
and adult sera are electrophoretically identical.
58
W. G. HEIM—RAT SERUM D U R I N G DEVELOPMENT
The observation that the relative concentration of gamma globulin of immature rats remains for an extended period below the levels found in the adult is
in accord with the finding of Halliday & Kekwick (1957).
That the alpha-globulin fraction or series of fractions was found in the present
work but not in that of Jameson, Alvarez-Tostado, & Lew (1948) may be due
to the use of very different buffer systems.
The comparatively constant relative concentration of alpha-2, alpha-3, beta,
and gamma globulins during intra-uterine existence may indicate that the embryonic pools of these components are in rather free communication with the
corresponding, larger, maternal pools. Should this be the case, we would be
presented with the physiologically interesting situation in which the internal
environment of one organism, the foetus, changes in response to the demands
placed upon, and the response pattern of, another organism, namely, the
maternal one. The relations between the serum proteins of mother and foetus
and the possible selective action of the placenta on the exchange of serum
proteins is presently under study.
SUMMARY
1. The sera of rats at various stages of development from the 17th day of
gestation to adulthood were examined by paper electrophoresis.
2. A component distinct from, and of higher mobility than, the bulk of the
albumin was found at the 17th and 18th days of development. However, the
mobility of this component is well within the limits of mobility of adult albumin.
3. Evidence supporting the existence of a component in the beta-globulin
region specific to the perinatal animal is presented.
4. Gamma globulin and an asymmetry associated with it were demonstrable
in all stages examined. The possible relation of this asymmetry to eta globulin
is discussed.
5. Problems concerning the physiology of the foetus arising from the changes
in the relative concentrations of the serum proteins are pointed out.
RESUME
Les proteines du serum du Rat pendant le developpement
1. Les serums de rats ont ete etudies a differents stades du developpement a
partir du 17e jour de la gestation jusqu'a l'age adulte par la methode de l'electrophorese sur papier.
2. Un composant distinct et d'une plus grande mobilite que la plus grande
partie de l'albumine a ce stade a ete mis en evidence aux 17e et 18e jours du
developpement. Cependant, la mobilite de ce composant est bien dans les limites
de mobilite de l'albumine adulte.
3. II y a de serieuses raisons d'admettre l'existence d'un composant specifique
situe dans la region de la beta-globuline, aux environs de la naissance.
W. G. HEIM—RAT SERUM D U R I N G DEVELOPMENT
59
4. A tous les stades etudies, on a demontre l'existence de gamma-globuline
et de l'asymetrie qui lui est associee. La possibility d'une relation de cette
asymetrie avec l'eta globuline est discutee.
5. L'accent est mis sur les problemes concernant la physiologie du foetus en
rapport avec les changements dans les concentrations relatives des proteines du
serum.
ACKNOWLEDGEMENTS
This work was supported by research grants G-4821 and G-10104 from the
National Science Foundation.
I gratefully acknowledge the technical assistance of Miss Frances Maleniak,
Miss Carol Sperling, Miss Judith Krym, and Miss Janie Whittler.
REFERENCES
GURVICH, A. E., & KARSAEVSKAYA, N. G. (1956). Study of serum proteins in ontogenesis by the
electrophoresis-precipitation method. Biokhem. Zh. (U.S.S.R.) 21, 771-8.
HALLIDAY, R., & KEKWICK, R. A. (1957). Electrophoretic analysis of the sera of young rats. Proc.
Roy. Soc.B, 146,431-7.
HEIM, W. G., & SCHECHTMAN, A. M. (1954). Electrophoretic analysis of the serum of the chicken
during development. / . biol. Chem. 209, 241-7.
JAMESON, E., ALVAREZ-TOSTADO, C , & LEW, W. (1948). Electrophoretic changes in the blood serum
proteins in the mother and young of rats during pregnancy and lactation. Stanford med. Bull.
6, 89-91.
KEKWICK, R. A. (1959). The serum proteins of the fetus and young of some mammals. Advanc.
Protein Chem. 14, 231-54.
MARSHALL, M. E., & DEUTSCH, H. F. (1950). Some protein changes in fluids of the developing chick
embryo. /. biol. Chem. 185, 155-61.
MOORE, D. H., SHEN, S. C , & ALEXANDER, C. S. (1945). The plasma of the developing chick and
pig embryo. Proc. Soc. exp. Biol. Med. N.Y. 58, 307-10.
PEDERSEN, K. O. (1947). Ultracentrifugal and electrophoretic studies on fetuin. / . phys. Chem. 51,
164-71.
SHMERLING, ZH. G., & USPENSKAYA, V. D. (1955). Embryonic serum proteins in the rat and rabbit.
Biokhem. Zh. (U.S.S.R.) 20, 31-40.
STOTSENBURG, J. (1915). The growth of the fetus of the albino rat from the thirteenth to the twentysecond day of gestation. Anat. Rec. 9, 667-82.
TISELIUS, A., & KABAT, E. A. (1939). An electrophoretic study of immune sera and purified antibody
preparation. / . exp. Med. 69, 119-31.
WFLLER, E. M., & SCHECHTMAN, A. M. (1957). Chicken serum proteins from the fifth day of development to the adult. Anat. Rec. 128, 638-9.