/. Embryol. exp. Morph. 92, 33-41 (1986)
33
Printed in Great Britain © The Company of Biologists Limited 1986
Albumin and transferrin synthesis in whole rat
embryo cultures
CAROLYN L. WILLIAMS, PAUL K. PRISCOTT, I. T. OLIVER
AND GEORGE C. T. YEOH
The Raine Centre for the Study of Developmental and Perinatal Biology,
Departments of Biochemistry and Physiology, University of Western Australia,
Nedlands, Western Australia 6009
SUMMARY
The uptake of [3H]leucine by the rat yolk sac and embryo and the subsequent synthesis of
albumin and transferrin have been studied in whole embryo culture. Rat embryos of 12 days
gestation were used in all experiments. Isotopically labelled transferrin was detectable in yolksac and embryo tissue extracts. In contrast, [3H]albumin could not be found in either tissue
extract. Levels of radioactive transferrin in the yolk sac of cultured whole conceptuses decreased
during 12 h in cold media. Embryonic transferrin showed an opposite trend in that it increased
over 12 h by nearly 30-fold. In view of these results experiments were conducted in embryos and
yolk sacs cultured in separate bottles. Radioimmunoprecipitation for transferrin revealed that
there was synthesized protein in the yolk sac which then decreased by approximately 30 % after
2h in normal cultured medium. There was no evidence of transferrin synthesis in embryo
extracts over a 12 h period. These results present evidence that the visceral yolk sac is the
primary site of transferrin synthesis in the rat and that the protein is thereafter transported,
intact, to the embryo.
INTRODUCTION
At about 10-5 days gestation in the rat, the liver diverticulum emerges from the
embryonic gut. The rat liver diverticulum in organ culture, will, after 3 days,
release plasma proteins into the medium (Parsa & Flancbaum, 1975). This
suggests that the ability to synthesize plasma proteins is a property which is
acquired by the liver at a very early stage in its development. It is of interest to
determine when plasma-protein-synthesizing cells emerge from the primitive gut
and to learn whether the ability to synthesize the different plasma proteins such as
albumin and transferrin is acquired collectively or sequentially. Furthermore, it is
of interest to determine whether extrahepatic sites for plasma protein synthesis
exist and to assess the relative importance of these sites during embryonic
development.
In a previous study using minced material from rat embryos and membranes,
Yeoh & Morgan (1974) reported that incorporation of [14C]leucine into transferrin
could be detected in foetal membranes but not the foetus at 13 days gestation.
Albumin synthesis was present in preparations from both tissues. These results
Key words: rat embryo, albumin, transferrin, protein.
34
C. L. WILLIAMS AND OTHERS
suggested that the liver may acquire the capacity for albumin synthesis prior to
transferrin synthesis. Furthermore they indicated that extrahepatic sites of
albumin and transferrin synthesis may reside in the extraembryonic membranes.
Indeed, it has been shown in the mouse that transferrin is synthesized in the
visceral yolk sac at an equivalent developmental stage (Janzen, Andrews &
Tamaoki, 1982; Adamson, 1982). Using isolated hepatocytes derived from rat
foetuses of varying gestational age maintained in culture, Yeoh, Wassenberg,
Edkins & Oliver (1979) demonstrated that hepatocytes from as early as 12 days
gestation secreted measurable amounts of both albumin and transferrin into the
medium after 1 day in culture. Taken together these results suggest that at this
stage of development, the foetal liver and hence the whole foetus as well as the
extraembryonic membranes are capable of synthesizing transferrin. Failure to
detect its synthesis in whole embryonic preparations may be due to a rapid
turnover of the protein in that situation. Since albumin is detected in preparations
from the whole embryo as well as extrahepatic membranes it is possible that it is
not broken down as rapidly as transferrin in the whole embryo. In this study,
albumin and transferrin synthesis were examined for by the incorporation of
[3H]leucine into immunoprecipitates obtained with specific antiserum. To ensure
specificity of the product, and to detect possible breakdown products, the
immunoprecipitates were analysed by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE).
MATERIALS AND METHODS
The Wistar Albino strain of rats was used throughout this study and was obtained from the
Animal Resource Centre, Murdoch, WA. Timed matings were determined by the presence of
spermatozoa in vaginal smears (taken as day 0).
Embryos were explanted on day 12 of gestation (36 ± 0-5 somites, Witschi stage 21-22;
Witschi, 1962) and prepared for culture by the method of Cockroft (1973). The culture medium
used contained 75% Modified Eagles minimal essential medium (Flow Laboratories,
Annandale, NSW) and 25% rat serum prepared by the method of Steele & New (1974). This
was supplemented with glutamine (2-4 mM final concentration) and penicillin/streptomycin
(lOOi.u.ml""1 and 100^gml"1 respectively; Grand Island Biological Co., NY). Embryos were
cultured in 60 ml all-glass reagent bottles using the roller bottle method (Priscott, Yeoh &
Oliver, 1984). The cultures were gassed with a mixture of 60% O 2 /5% CO 2 /35% N2 and
maintained at 37 °C. Embryos so cultured for 24 h remained viable and continued to differentiate
normally, having 51-3 ±0-6 somites on completion of experiments. Initial experiments were
conducted on 12-day conceptuses cultured for 24 h in the presence of SjuCiml"1 [3H]leucine
(Radiochemical Centre, Amersham). Results from these indicated only limited amounts of
radiolabelled albumin were present in both embryo and yolk-sac extracts. As a follow up to
these results conceptuses were pulse chased in 25 /iCi ml" 1 [3H]leucine for 2 h then transferred to
normal culture media for time intervals of 0,1,2,4,8 and 12 h. In one experiment, embryos and
yolk sacs were divided and cultured separately for a total of 4h. Yolk sacs and embryos were
immunoprecipitated separately and in groups of four in all experiments.
After culture, tissues were washed three times in balanced salt solution, transferred to 3 ml of
buffer A (20mM-Tris-HCl, pH7-6, 0-14M-NaCl, 5mM-EDTA, 1% Triton X-100) and
sonicated. Samples were then centrifuged at 3000g for 5min. 800 jul aliquots of the remaining
supernatants were immunoprecipitated for albumin or transferrin according to the method of
Gross et al. (1982). Elution was achieved by incubation with 4% SDS, 10 % 2-mercaptoethanol,
20% glycerol and 0-25 M-Tris-HCl pH 6-8 at 90°C for 5min. The eluted proteins were
Albumin and transferrin synthesis in whole rat embryo cultures
35
Gel slice number
Fig. 1. Albumin and transferrin production by 19-day foetal hepatocytes during 16 h of
culture. SDS-PAGE profiles of immunoprecipitated radioactivity using non-immune
rabbit serum (O
O), rabbit anti-rat albumin ( •
• ) , and rabbit anti-rat
transferrin serum (A
A).
immediately subjected to SDS-PAGE (Cashman & Pitot, 1971). Samples were run at a constant
current of ImA/gel for the first hour and then increased to 2 mA/gel. Electrophoresed gels
were frozen and cut into 2 mm slices and the radioactivity counted in individual slices in 5 ml gel
counting solvent.
RESULTS
To determine the reliability of the immunoprecipitation technique 19-day foetal
rat livers were cultured in primary monolayers for 48 h and incubated in the
presence of 5 fiQimX'1 [3H]leucine for a subsequent 16h. SDS gel profiles of the
immunoprecipitates are shown in Fig. 1. Based on these findings this immunoprecipitation protocol was applied to the embryo cultures. Initial experiments
were conducted on whole conceptuses cultured for 24 h in the presence of
5piC\m[~l [3H]leucine. SDS-PAGE profiles (Fig. 2) indicated albumin and transferrin may be present but at very low levels. Throughout all experiments the
position of the immunoprecipitate peak along the gel was reproducible within
±2 mm. Additional peaks in the gels were initially thought to be due to nonspecific binding of synthesized protein to protein A and hence three clearing steps
whereby protein A was added and the extracts centrifuged, prior to the addition of
specific antibody, were incorporated into the protocol. Rabbit anti-rat lgG and
protein A-Sepharose 4B were also tested as a superior alternative immunoadsorbent. Protein A-Sepharose 4B proved the most efficient immunoadsorbent
with the least degree of non-specific binding and was used throughout subsequent
experiments. However it was still necessary to incorporate blanks for each sample.
These contained non-immune rabbit serum, and the radioactivity found in these
was subtracted from the immunoprecipitates containing anti-albumin and antitransferrin respectively. It was possible that the low molecular weight proteins
36
C. L. WILLIAMS AND OTHERS
which coprecipitated with albumin or transferrin represented degraded products
of the respective proteins. To test this, two series of experiments were conducted.
Firstly, pulse-chase experiments were conducted on the whole conceptus. Cultures were preincubated for 4h in normal culture medium, pulsed for 2h in
25//Ciml~ 1 [3H]leucine and then transferred to normal culture media for various
time intervals. SDS-PAGE profiles (Fig. 3) showed an increase in the amount of
radioactive transferrin in the embryo extracts and a corresponding fall in amount
in the yolk-sac extracts during the 12 h in normal media. The presence of the low
molecular weight material that was coprecipitated with specific antiserum was not
evident until after 8 h in normal culture medium where it was most noticeable in
yolk-sac extracts. Immunoprecipitation for albumin in the same tissue extracts
could not be distinguished from background radioactivity. Subsequent cultures
were therefore immunoprecipitated for transferrin only.
To further substantiate the possibility of degradation products a second
experiment was conducted using radioiodinated proteins. 125I-transferrin was
added to the culture media and the whole embryos cultured for 24 h. Culture
media, yolk sac and embryos were immunoprecipitated for transferrin. Only the
native form of transferrin was found in media samples both before and after
culture. However, in yolk-sac extracts there were proteolytic breakdown products
of varying sizes in addition to the native transferrin (Fig. 4). Similar results were
obtained when 125I-albumin was added to the culture media, except that no native
albumin was seen, indicating a more rapid proteolysis for that protein. No
immunoreactive radiolabelled transferrin or albumin was found in the embryo.
To gain further insight into the potential transfer of transferrin from the yolk-sac
membrane, yolk sacs were dissected free of the embryo and each cultured
10
20
Gel slice number
30
40
Fig. 2. Immunoprecipitated radioactivity from 12-day-old embryos cultured in the
presence of [3H]leucine for 24 h. SDS-PAGE profiles of immunoprecipitated radioactivity using rabbit anti-rat albumin (O
O), and rabbit anti-rat transferrin serum
37
Albumin and transferrin synthesis in whole rat embryo cultures
1-0 T
0-5 •
12
Hours after pulse
Fig. 3. Immunoprecipitated transferrin radioactivity from 12-day whole conceptus
culture given a 2h pulse with [3H]leucine followed by a further 12h of culture.
Transferrin-specific radioactivity in separated embryos (O
O) and separated yolk
separately under conditions similar to those aforementioned for 4h (Fig. 5).
Embryos remained viable during this time as judged by the presence of beating
hearts. The extent of development was not determined. Gel profiles showed a
decrease in transferrin levels in the yolk sac, similar to that observed with the
whole conceptus (Fig. 3). However, in the absence of the yolk sac, there was no
accumulation of radioactive transferrin in the embryo. This suggests that the
10 T
Gel slice number
Fig. 4. SDS-PAGE profiles of immunoprecipitates from the yolk sacs of 12-day whole
conceptuses cultured for 24 h in the presence of 125I-transferrin. Immunoprecipitates
from the culture medium at the commencement of the experiment (O
O) and from
extracts from separated yolk sacs at the conclusion of the experiment (
38
C. L. WILLIAMS AND OTHERS
1-0 T
o
X
0-5 ••
E
Q.
•6
0
1
2
Hours after pulse
Fig. 5. Immunoprecipitated transferrin radioactivity from 12-day embryos and yolk
sacs cultured separately during a 2 h pulse with [3H]leucine followed for a further 2 h
of culture. Embryo transferrin-specific radioactivity (O
O), yolk-sac transferrinspecific radioactivity ( •
•).
increasing amounts of transferrin seen in the embryo when the whole conceptus
was cultured were the result of active transport from the yolk sac.
DISCUSSION
Our experiments provide evidence that in the rat the visceral yolk sac is the
primary site of transferrin synthesis and that a proportion of the protein is
subsequently transported to the embryo. In contrast, we could not find evidence
for albumin synthesis in either the embryo or yolk sac at this stage of development.
Previous studies in our laboratories have demonstrated albumin synthesis in 13- to
14-day embryos (Yeoh & Morgan, 1974) and 12-day embryonic hepatocytes after
24h in culture (Yeoh et al. 1979). Furthermore, studies using 11-day liver organ
cultures showed that albumin was secreted into the culture medium in detectable
quantities by the third day, i.e. approximately equivalent to 14 days in vivo (Parsa
& Flancbaum, 1975). Taken together these results suggest that albumin synthesis
commences sometime on day 13.
The pattern of appearance of transferrin, on the other hand, is quite different.
At 12 days gestation there is no embryonic production of the protein. This extends
earlier in gestation the observation made by Yeoh & Morgan (1974) on 13-day
embryonic material. In common with this earlier study definitive evidence of
transferrin synthesis by the visceral yolk sac was obtained. The actual site of
synthesis within the yolk sac was not studied by us, but may be in the sites of
haemopoiesis in the blood islands that are still active at this stage (Fantoni,
Lunadei & Ullu, 1975).
Albumin and transferrin synthesis in whole rat embryo cultures
Unlike any previous study, we have shown that transferrin synthesized in the
yolk sac is exported in significant quantities to the embryo. We suggest that the
most likely pathway is by secretion into the vitelline circulation and thence to the
embryo. Transferrin has a well-documented role as the principal iron-binding
protein of serum (Morgan, 1981) and presumably also has this role in the early
embryonic circulation. It has also been recently shown to be essential for the
differentiation of mouse kidney tubules in vitro (Ekblom, Thesleff, Miettinen &
Saxen, 1981) and it is therefore tempting to speculate that it may be involved as a
specific growth factor in organ differentiation (Levin et al 1984), perhaps together
with other circulating proteins (Priscott, Gough & Barnes, 1983).
We have been careful to validate our protein detection methods and have
confirmed the checks on antibody specificity made previously in this laboratory
(Yeoh & Morgan, 1974; Yeoh et al. 1979). In developing the techniques for use
with the whole-embryo culture method we became aware of greater problems,
with regard to non-specific radioactivity, than we have encountered with hepatocyte cultures. This necessitated SDS-PAGE of immunoprecipitated proteins
and subtraction of radioactive values derived from samples immunoprecipitated
with non-immune serum. We believe this non-specific radioactivity probably
derives from the complexity of the whole conceptus in culture. For example, the
[3H]leucine will be taken up and utilized in a variety of biosynthetic pathways and
is likely, therefore, to be present in a range of metabolic products in addition to the
proteins we examined for specifically. Some of these associated with the immune
complexes in a highly reproducible fashion as evidenced by characteristic patterns
of radioactivity in gels after immunoprecipitation with non-immune serum. This
was particularly evident in embryo preparations from the pulse-chase experiments
where the radioactive peaks, in addition to being in the same positions,
progressively increased in magnitude with time after the pulse. This was not a
feature in the yolk-sac preparations and leads us to postulate that these peaks of
radioactivity represent metabolic products unique to the embryo. Another
confounding factor in the whole embryo culture technique is the proteolytic
potential of the yolk sac for a variety of serum proteins (Freeman, Beck & Lloyd,
1981; Freeman & Lloyd, 1983) including both albumin and transferrin (McArdle &
Priscott, 1984). We have confirmed that exogenous radioiodinated albumin and
transferrin is proteolysed to smaller molecular weight immunoreactive fragments.
Furthermore, we have obtained evidence that albumin is more susceptible to
proteolytic breakdown than is transferrin in the yolk sac. It is thus conceivable that
a low level of synthesis of albumin in the yolk sac would go undetected due to a
rapid breakdown of the newly synthesized protein. However, proteolysis usually
follows uptake from the apical surface of the yolk sac by pinocytosis (Lloyd,
Williams, Moore & Beck, 1976). We have no direct evidence that proteins
synthesized in either the embryo or yolk sac find their way to such sites of
proteolysis from the proximal surface of the yolk sac. It is of interest that little
intact exogenous transferrin reaches the embryo (McArdle & Priscott, 1984) and
yet a high proportion of transferrin synthesized by the yolk sac does cross to the
39
40
C. L. WILLIAMS AND OTHERS
embryo. This suggests two pathways are in operation and may represent an
evolutionary mechanism to ensure the embryo receives an adequate supply of an
essential protein.
The present study demonstrates the usefulness of whole-embryo culture as an
adjunct to other in vitro studies into embryonic development. In principal, the
same approach could be applied to any protein synthesized by the embryo for
which a specific antiserum is available.
We thank Dr H. J. McArdle for the 125I-transferrin and 125I-albumin. This work was
supported by grants from the Raine Medical Research Foundation, the Australian Research
Grants Scheme and the National Health and Medical Research Council.
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{Accepted 6 May 1985)