Download PDF

/. Embryol. exp. Morph. 85, 191-206 (1985)
191
Printed in Great Britain © The Company of Biologists Limited 1985
Synthesis of apolipoproteins, alphafoetoprotein,
albumin, and transferrin by the human foetal yolk
sack and other foetal organs
W-K. SHI, B. HOPKINS, S. THOMPSON, J. K. HEATH,
B. M.' LUKE AND C. F. GRAHAM
Department of-Zoology, South Parks Rd, Oxford OX1 3PS, U.K.
SUMMARY
Fragments of human foetal organs and blood at 5-11 weeks of postfertilization development
were cultured in radioactive protein precursors. The secreted products were characterized by
immunoprecipitation, and by measuring the mobility of the immunoprecipitates on polyacrylamide gels. It was found that secondary human yolk sacks secreted apolipoproteins Al and
B. The work of previous authors on the synthesis of other serum products by this organ and by
the foetal liver and by the foetal intestines was confirmed. Within the yolk sack, the endoderm,
the blood cells, and the outside epithelium reacted with antibodies against apolipoprotein Al
and transferrin. By metabolic labelling of umbilical cord blood, it was found that blood did not
secrete apolipoproteins Al and B. Blood cells could therefore not be a source of these secreted
products.
INTRODUCTION
The rapid metabolism of human foetal testes and adrenals depends on a supply
of lipids which must be delivered to the cell surface bound to apolipoproteins (Carr
et al. 1980; Carr et al. 1983). Further, it is known that complexes of lipid and
apolipoprotein sustain the rapid growth of some human-teratoma-derived cells
which resemble early human embryonic stem cells (Engstrom, Rees & Heath,
1985). We have studied the potential sources of apolipoproteins in early human
development, since an abundant intrinsic supply of these lipid carrier molecules
may be essential for human embryogenesis.
In the adult, the liver and the intestines synthesize almost all the apolipoproteins
which circulate in the blood (rat: Wu & Windmueller, 1979). The same organs
synthesize these proteins in the 16- to 22-week postfertilization human foetus
(Zannis, Kurnit & Breslow, 1982), and there is also evidence that lipoproteins are
synthesized much earlier, at 29 days postfertilization (Gitlin & Biasucci, 1969). We
Major key words: Apolipoproteins, human, embryo, yolk sack.
Minor key words: Gut, stomach, liver, trophoblast, brain, transferrin, alphafoetoprotein.
192
W-K. SHI AND OTHERS
have investigated the synthesis of these molecules in the first 5 to 11 weeks of
postfertilization human development, identifying some of the individual
apolipoprotein molecules, and concentrating our studies on the secondary yolk
sack, because in the mouse embryo the analogous visceral yolk sack is the earlies.t
intrinsic source of apolipoprotein (Shi & Heath, 1984; Meehan et al. 1984).
In humans, the inner cell mass apparently gives rise to the endoderm of the
primary yolk sack during the fifth day of postfertilization development. The
endoderm of the primary yolk sack, which lies beside the epiblast, seems to persist
and form the inner lining of the secondary yolk sack, which is a coherent structure
by the 14th day after fertilization (sequential histology: Luckett, 1978). The
endoderm of the secondary yolk sack has all the features of a secretory tissue
(reviewed by Branca, 1913, and Gonzalez-Crussi, 1979), its cytoplasm is heavily
stained by antibodies against prealbumin, albumin, a^-antitrypsin, alphafoetoprotein, and transferrin (Albrechtsen, Wewer & Wimberley, 1980; Jacobsen,
Jacobsen & Henriksen, 1981). Consequently, it is a likely source of the variety of
serum proteins which the human yolk sack is already known to synthesize in
common with the foetal liver and intestines (Gitlin & Perricelli, 1970; Gitlin,
Perricelli & Gitlin, 1972).
MATERIALS AND METHODS
1. Foetal samples
Foetal organs were collected exactly as described previously (Thompson et al. 1984). The
foetal age was estimated from the date of the reported last menstrual period (LMP). In some
cases, the date of the LMP was reported to lie within the span of a particular week; the mid point
of this week was taken as the LMP date. In rare instances, no date was reported, and the size of
the limbs was used to establish that the age of the embryo fell within the 5- to 11-week
postfertilization range included in this study; a question mark is used to indicate the age of these
samples (O'Rahilly & Gardner, 1975). In all cases, foetal age is expressed as days
postfertilization.
2. Tissue fixation and processing
For histology, the organs werefixedat between 30min and 3 h after foetal aspiration. Routine
histology was performed on formal-saline-fixed material, and this procedure was used on all
liver samples to avoid confusion with clotted blood. For antigen localization, the organs were
fixed for 12-24h at 5°C in 96% (v/v) aqueous ethanol:glacial acetic acid, 99:1 (Engelhardt's
1971 modification of Sainte-Marie's fixative; Sainte-Marie, 1962; Engelhardt, Goussev, Shipova
& Abelev, 1971). The tissues were dehydrated through a graded series of alcohols and
embedded in Paraplast. For scanning electron microscopy, the organs were fixed for 3 h at 4°C
with 2-5 % (w/v) glutaraldehyde in 0-05 M-cacodylate buffer containing, 5 % (w/v) sucrose at
pH 7-4. They were washed in several changes of buffer with sucrose alone over 20 h at 4°C,
postfixed with 1 % (w/v) osmium tetroxide in buffer for 1 h at 4°C, and dehydrated in a series of
ethanols. Following transfer to acetone, they were processed with a Samdri critical-point drier
with liquid CO 2 . They were sputter coated with gold, and examined in a Philips SEM500
microscope.
Apolipoprotein synthesis in human embryos
193
3. Antibodies and antigens
Antibodies were used to both immunoprecipitate metabolically labelled proteins, and to
localize antigens on tissue sections. We purchased rabbit antibodies to the following antigens:
apolipoprotein Al (Seward Laboratories, UAC, Blackfriars Rd, London SE1); human
apolipoproteins Al & B, human transferrin, human albumin, human pre-albumin, and human
alphafoetoprotein (Behring, Hoechst House, Salisbury Rd, Hounslow, Middx). Sheep antirabbit IgG conjugated to peroxidase was obtained from Serotec Ltd (Station Rd, Blackthorn,
Bicester, Oxon OX6 OTP). Rabbit antibodies against human fibronectin were a gift from Dr D.
Turner. Rabbit antibodies to human low-density lipoproteins (LDL) were prepared here (Shi &
Heath, 1984).
To test the specificity of the reaction of some of the antibodies with antigens on tissue sections,
the antibodies were absorbed with various antigens. These included human albumin and
transferrin (Behring), and human apolipoprotein Al which was purified according to the
following protocol. Freshly collected human venous blood was adjusted to a density of
1-063 gm/ml with solid KBr (Hatch & Lees, 1968), and it was centrifuged at 105 OOOg in a 45Ti
rotor attached to a Beckman L8 for 20-22h at 12-15°C (Havel, Eder & Bragdon, 1955). The
bottom fraction was pooled, the density raised to 1-125 gm/ml with solid KBr, and this was spun
for 18 h at 173000g. The top fraction from this spin was dialysed against PBS (solution A of
Dulbecco & Vogt, 1954) for 2-3 days at 5°C in a Spectrapor membrane (cut off Mr 2000), and
delipidized in 25 vols of ethanol:ether (3:1) for 16-24 h at room temperature (Chapman, Mills &
Ledford, 1975). The precipitate was washed in the same ethanol:ether mixture, and then stored
at —20°C until use. This material was run on a 12-5% (w/v) polyacrylamide gel, using the
method of Laemmli (1970). Following staining with Coomassie brilliant blue, the gel was
scanned, and the area under visible peaks was calculated by weighing cut outs of the peak
profiles. Between 89 and 96 % (n = 5) of the absorbance was in a single peak with a mobility of
28xlO3, which is the correct relative molecular mass (Mr) for apolipoprotein Al; in addition
there was a minor peak of absorbance in the region of albumin on the gel, which amounted to
1-2% (n = 5) of the absorbance in peaks on these tracks. The commercial preparations of
transferrin and albumin were analysed similarly, and the proportion of the absorbance at the
correct MT in these two samples amounted to 88-93 % (n = 3) and 77-87% (n = 3) respectively;
in neither case was there any absorbance in the apolipoprotein Al region in these preparations.
4. Metabolic labelling
Labelling was begun within 3-5 h of foetal aspiration. Despite this inevitable delay, we know
that cells in most of the eye specimens from the same samples were viable (Hyldahl, 1984), and
we only excluded two samples from our reported results on grounds of low total metabolic
labelling (two gut fragments weighing 2 and llmgms). Most foetal organs were torn into
fragments of approximately 5 mm3. The stalk was removed from yolk sack samples, which were
torn open but otherwise left intact. In most cases, gut samples were cut open along their length.
The samples were weighed after excess moisture had been removed by dabbing on filter paper.
The weights of the organ fragments are recorded in the legend to Table 1. In most experiments,
the individual samples were placed in lml of serum-less medium containing transferrin, highdensity lipoproteins (HDL), and low-density lipoproteins (LDL) in a basal medium of
Dulbecco's modified Eagles medium diluted 100-fold with methionine-free minimal essential
medium (ECM medium of Heath & Deller, 1983; basal media from Gibco Europe, Paisley,
Scotland). The medium was supplemented with 50-100juCi of [35S]methionine (Amersham
International pic, Amersham, U.K.; specific activity 1310Ci/mmole), and the tissue incubated
in this medium for 16 h, at 37°C in an humid mixture of 5 %(v/v) CO2 in air.
Samples of foetal blood were obtained by blowing PBS through the arteries and vein of the
umbilical cord. To reduce the possibility of cross contamination with cells which might fall off
the allantois and the midgut, the umbilical cord was first severed below the region which
contains these organs. The blood cells were pelleted by centrifugation, and taken up in
0-8-1-5 ml of minimal essential medium (Gibco Europe) containing l/100th of the normal
194
W-K. SHI AND OTHERS
methionine concentration, 0-5% (v/v) FCS, and lOOjuCi/ml of [35S]methionine. At the end of
20 h incubation, the cells were pelleted by centrifugation, and counted with a haemocytometer.
These blood samples contained between 2-7xlO 6 cells. Subsequently, the culture medium was
centrifuged at 15 000 g for 15min, and the supernatant stored at -70°C before immunoprecipitation. In a few experiments, the organ fragments were labelled in [3H]leucine exactly as
described in Thompson et al. 1984.
Radioactive secreted proteins were immunoprecipitated from the culture medium as
described by Shi & Heath, 1984. Briefly, between 1/5 and 1/10 of the medium was incubated for
60 min at 4°C with protein A-Sepharose CL-4B (PAS, Pharmacia Ltd, Midsummer Boulevard,
Milton Keynes, Bucks MK9 3HP, U.K.), which had been precomplexed with a particular
antibody. The PAS:antibody:antigen complexes were removed by centrifugation and the
incubation repeated with a second aliquot of PAS.antibody. The beads from the two incubations
were combined, and pelleted through 10% (w/v) sucrose in PBS which overlaid a cushion of
20% (w/v) sucrose in PBS, at 15000g for 15 min. The pellets were washed once with 0-1%
(w/v) bovine serum albumin, 0-1% (w/v) sodium dodecyl sulphate in PBS, and then boiled for
5 min in Laemmli sample buffer (1970), which contained 50mM-dithiothreitol.
Samples were subjected to one-dimensional slab gel electrophoresis on a 12-5 % (w/v)
polyacrylamide gel containing 0-1 % sodium dodecyl sulphate, using the tris buffer system of
Laemmli. After electrophoresis, the gels were processed for fluorography according to Bonner
6 Laskey (1974). The dried gels were exposed to Fuji RX X-ray film at -70°C for two weeks
before development in Kodak DX 90.
5. Immunolocalization
For immunolocalization, 8jum sections were attached with diluted chick egg albumin to each
of the four wells of a teflon-coated slide (C. A. Hendley Essex Ltd, Oakwood Industrial Estate,
Laughton, Essex IG10 3TZ, U.K.), and stored at -20°C until use. They were dewaxed in
toluene, rehydrated and taken to 70% (v/v) aqueous ethanol. Control experiments, using the
components of the peroxidase reaction alone, snowed that endogenous peroxidase activity was
inhibited with treatment for 30 min with 3 % (w/v) hydrogen peroxide in methanol. All sections
were subsequently taken through this procedure. The slides were washed with 70 % (v/v)
aqueous ethanol for 10min, air dried, washed in PBS, and given three 10min rinses in either
0-5 % (w/v) bovine serum albumin or 0-5 % (w/v) gelatin in PBS. Diluted 50jul aliquots of the
primary antibody were placed in the wells in an humid atmosphere for 1 h at room temperature
(R.T.), and the PBS and 0-5 % BSA in PBS rinses repeated as above. The second antibody was
then added and incubation conducted as above. After a PBS rinse, the sections were incubated
for two 10min periods in aqueous 10mM-Tris, 0-15M-NaCl at pH 7-3 (TBS). The peroxidase
reaction was developed for about 30 s in 0-75% (w/v) diaminobenzidine, 0-03% (w/v)
hydrogen peroxide, 10mM-imidazole in TBS. The reaction was terminated by a rinse in TBS,
and the sections were dehydrated through a graded series of ethanols, cleared in toluene, and
mounted in DPX. To demonstrate that the second layer antibody did not react with endogenous
IgG, some sections were treated with the second antibody alone after the first antibody
treatment had been replaced by incubation in 0-5 % bovine serum albumin in PBS. No brown
peroxidase reaction product developed on these sections. The specificity of the primary
antibodies was checked by absorbing each (except the anti-AFP antibody) in separate tests with
each of the other antigens at concentrations which were sufficient to completely inhibit the
reaction of the homologous antibody. Excess antigen was added to the antibody for 30 min at
37 °C, and the complexes spun down by centrifugation before proceeding to use the supernatant
in the standard protocol as above. In each case, a particular antibody reaction was inhibited by
absorption with its known target antigen, but the reaction was not inhibited by the other
antigens used in this study.
We also attempted to elute and dilute any of the antigens which may have absorbed to cells in
the intact embryo. Before reaction with the antibodies against apolipoprotein Al and
transferrin, foetal fragments were incubated for 19 h in alpha medium lacking nucleosides and
deoxynucleosides (Stanners, Eliceiri & Green, 1971; Gibco Europe), which contained 1%
Apolipoprotein synthesis in human embryos
195
(w/v) bovine serum albumin. Before reaction with the antibodies against AFP and albumin,
foetal fragments were cultured in the medium which was used for metabolic labelling; this
lacked these molecules (see above).
RESULTS
We have confirmed previous anatomical descriptions of the human secondary
yolk sack, and follow older authors in describing the large granulated cells which
line the inside of the yolk sack as endoderm cells (Figs 1, 3). As Hesseldahl &
Larsen (1969) and Hoyes (1969) observed during the 5th-8th weeks of postfertilization development, the layer of endoderm cells is continuous with columns
of similar large cells which extend through the mesenchyme to touch the base of
the outside epithelial layer of the sack (samples aged 35, 42, and 50 days
postfertilization). These columns appear to be associated with pits which indent
the endoderm surface (Fig. 1, arrow and Fig. 3), and they are reminiscent of the
pits which are seen on the external surface of the mouse visceral yolk sack
endoderm (Hogan & Newman, 1984). In older secondary yolk sacks (samples aged
61 and 81 days postfertilization), the endoderm cells form a simpler columnar
epithelium which is completely separated from the outer epithelial layer by the
mesenchyme and blood vessels. We also examined secondary yolk sack stalks and
can confirm that the endoderm cells extend as a partially closed tube down long
regions of the stalk.
The radiolabelled secreted products of a variety of foetal organs were examined,
and the results from weighed samples labelled with [35S]methionine are summarized in Table 1. Human secondary yolk sack, foetal gut, and foetal liver each
synthesized apolipoproteins. In contrast, there was no precipitation of radioactive
material by antibodies to apolipoprotein Al and to LDL when these were tested
on the medium over cultured stomach, trophoblast, adrenal, kidney and brain.
The anti-LDL antibody precipitated major labelled molecules with apparent
relative molecular masses of 28xlO 3 , and >200xl0 3 . The mobilities of these
proteins correspond with the respective known mobilities of apolipoproteins A l ,
and B (Fig. 5, lane D). The identity of the 28xlO 3 and >200xl0 3 molecules was
confirmed by their precipitation by antibodies which had been raised against these
individual apolipoprotein species (Fig. 6, lanes D and I; apolipoprotein B results
not shown). From Table 1 it can be seen that we were rarely able to
immunoprecipitate apolipoprotein A l from the culture medium with the antiapolipoprotein Al antibody; we attribute this difficulty to its low avidity in
comparison with the anti-LDL antiserum. Both antibodies immunoprecipitated
molecules with the mobility of apolipoproteins Al and B from liver culture
medium (Fig. 6, lanes D and E). A similar result was obtained with embryonic gut
(Fig. 6, lanes I and J), but the relative intensity of the apolipoprotein B band was
very low, and again the anti-apolipoprotein Al antiserum only rarely precipitated
detectable material (Table 1). The anti-LDL antibody also immunoprecipitated a
196
W-K. SHI AND OTHERS
Figs 1-4.
Apolipoprotein synthesis in human embryos
197
3
42-44xlO molecule from the medium over cultured yolk sacks; this labelled
molecule was not detected in the liver and it was only once found in the gut
samples.
As expected from the work of previous authors, the synthesis of alphafoetoprotein and transferrin were, with one exception, confined to foetal tissues
which contained endoderm cells (Table 1). In addition, we used [3H]leucinelabelled samples to extend these observations on foetal liver and the yolk sack: we
specifically immunoprecipitated AFP, apolipoprotein A l , transferrin (70, 74, and
75 days postfertilization yolk sacks, one liver of unknown age), albumin (74 days
postfertilization yolk sack, one liver of unknown age), and pre-albumin (same
specimens). We also showed that two of these yolk sacks and this liver sample
synthesized and secreted the 230xlO3 form of fibronectin (results not shown).
We attempted to identify the cell type which synthesized apolipoproteins,
transferrin, albumin, and AFP in the secondary human yolk sack by studying the
distribution of these molecules on tissue sections (see Materials and Methods).
The anti-AFP antibody reaction was concentrated in the cytoplasm of the
endoderm cells in two samples at 50 and 51 days postfertilization, confirming the
results of previous authors (results not shown: see Albrechtsen et al. 1980). In
contrast, the apolipoprotein Al and anti-transferrin antibodies reacted with the
cytoplasm of both the endoderm cells and the outer epithelial cells, and they also
reacted with the periphery of the nucleated erythrocytes (samples at 42,48, and 66
days postfertilization; Figs 7-10). These three samples had been cultured for 19h
in the absence of these serum products, in an attempt to elute and dilute out
molecules which might have absorbed to the cell surface or which might have been
taken up into the cytoplasm of these cells. This treatment reduced the already
weak staining over mesenchyme and the lining of blood vessels, but it did not
affect the relative staining intensity of the endoderm, the outer epithelial cells, and
the erythrocytes. The weak albumin staining showed the same distribution on yolk
sack samples taken at 50, 51, and 54 days postfertilization, and cultured for 20 h in
the medium without albumin before processing. Erythrocytes from an embryo of a
similar age have previously been observed to react with anti-albumin antibodies
(Jacobsen et al. 1981).
Fig. 1. Composite scanning electron microscope view of a fractured fragment of the
human secondary yolk sack, at 50 days postfertilization. The inner endoderm layer is to
the right, and it is possible to see regions where the large (endoderm) cells in this layer
make contact with the outside epithelial cells to the left (arrow). These regions of
contact appear to be associated with pits on the inner endoderm surface (arrow heads).
Blood vessels in the middle mesenchyme layer are marked with asterisks (x200).
Fig. 2. Whole view of a 44-day postfertilization secondary yolk sack, showing an
unusually swollen stalk (s), and blood vessels in the stalk and in the sack (b). The sack
has split open. Scale bar equals 1 mm.
Figs 3 & 4. High-power view of the endoderm layer inner surface with pits (Fig. 3),
and the small epithelial cells on the outside surface of the sack (Fig. 4). Microvilli are
apparent on both sides of the yolk sack. Same specimen as Fig. 1 (magnification of
both, X400).
1/1
>200
50,51 ,51
54,54
3-7-7 •1
1/5
5/5
5/5
6/6
5/6
5/6
28
28
>200
42-44
69
76
Range of weights (mgm)
Ages of individuals
Apolipoprotein Al
Apolipoprotein B
LDL
Transferrin
AFP
O
Antigen
recognized byJ
antibody
Table 1. Secretion of serum proteins by foetal tissues
13-7-33-1
42,61,63
1/1
3/3
3/3
3/3
0/3
3/3
2/3
58,63
64,66,72
14-215
1/5
3/4
1/5
3/5
4/5
2/5
4/5
6-2-23-1
50,54,61
1/3
0/3
0/3
0/3
0/3
0/3
0/3
45-98
?
41,50,54
N.D.
1/4
0/3
0/3
0/3
0/3
0/2
o/i
o/i
10
52
N.D.
8-6
52
N.D.
o/i
0/1
0/1
.0/1
0/1
o/i
o/i
o/i
o/i
o/i
48
N.D.
o/i
o/i
o/i
o/i
o/i
o/i
Relative
Proportion of organs or paired organs from different embryos which secreted labelled
molecular mass
molecules specifically recognized by each antibody
(Xl0 3 )of
precipitated
Yolk sack Liver
Brain
molecule
Gut
Stomach
Kidney
Trophoblast Adrenal
HH
X
H
o
o
>
HH
C/5
<
OO
Apolipoprotein synthesis in human embryos
A
B
C
D
mm- ApoB
200-
92569-
46-1
-
- Apo A1
Fig. 5. Products secreted by human yolk sack. SDS-PAGE slab gel electrophoretic analysis of [35S]methionine-labelled culture medium from a 51-day postfertilization embryo. Lane A: total secreted proteins; Lane B: anti-transferrin
immunoprecipitation; Lane C: anti-AFP immunoprecipitation; Lane D: anti-LDL
immunoprecipitation. In lane D, the major apolipoproteins are identified with
reference to their apparent relative molecular mass (Mr). Some labelled material with
the apparent relative molecular mass of AFP or albumin has also been precipitated by
this antibody. The relative molecular mass markers are myosin: 200xl03; phosphorylase b: 92-5xlO3; bovine serum albumin: 69xlO3; ovalbumin: 46xlO3; and
carbonic anhydrase: 30xl0 3 .
199
200
W-K. SHI AND OTHERS
Gut
Liver
/Wrx10'3
200-
A
B
C
D
E
F
G
H
I
J
/
ApoB
46-
##
30-
M
Apo A1
/ \
143Fig. 6. Secreted products of foetal liver; specimen at 61 days postfertilization. Secreted
products of foetal gut; specimen at 63 days postfertilization. Lanes A and F are total
secreted products. Lanes B and H are anti-AFP immunoprecipitates. Lane C and G are
anti-transferrin immunoprecipitates, although in lane G no transferrin is detectable in
this case. Lanes D and I are anti-apolipoprotein Al immunoprecipitates. Lanes E and J
are anti-LDL immunoprecipitates. The major apolipoproteins are identified by their
apparent molecular weight. Relative molecular mass standards as before, with the
addition of lysozyme at 14-3xlO3.
Apolipoprotein synthesis in human embryos
201
We wished to exclude the possibility that it was the blood cells in these foetal
organ samples which were synthesizing these apolipoproteins; foetal blood has
been previously reported to synthesize /3-lipoproteins which might contain these
apolipoprotein species (Gitlin & Biasucci, 1969). The umbilical cord blood did not
incorporate detectable radioactivity into either apolipoproteins A l or B, as judged
by the failure to immunoprecipitate labelled molecules with the correct mobility
with any of the anti-apolipoprotein antibodies. In the same experiment, and on the
same gel, these molecules were immunoprecipitated from the culture medium
over a yolk sack, when the yolk sack sample contained similar quantities of TCAprecipitable radioactivity.
In addition, one yolk sack stalk was fixed at 54 days postfertilization, and the
endoderm tube was found to react with anti-AFP, anti-transferrin, and antialbumin antibodies.
DISCUSSION
1. Identity of the apolipoproteins
The anti-LDL antibody precipitated labelled molecules with the mobilities of
apolipoproteins A l , and B from almost all secondary yolk sack samples (Table 1).
The identity of synthesized human apolipoproteins Al and B was further
supported by their precipitation with commercial antisera raised to these
individual apolipoprotein species. The coprecipitation of several apolipoprotein
species by both the anti-human LDL and anti-apolipoprotein Al antisera does not
necessarily indicate that all these apolipoproteins are bound together in a single
particle, for at least the anti-LDL antiserum is known to recognize several
different mouse apolipoproteins individually (immunoblotting: Shi & Heath,
1984). Coprecipitation can then be also due to specific recognition of determinants
on a variety of apolipoproteins. Although it is already known that sialo groups can
alter the mobility of the apolipoprotein E secreted by the foetal liver (Zannis et al.
1982); such changes have not been observed with apolipoproteins Al and B, and
we consider that the mobilities of these two apolipoproteins are sufficiently distinct
from that of other apolipoproteins to establish their identity in this study. It should
be noted that we have not distinguished the various forms of apolipoprotein B in
this study (e.g. Milne et al. 1984).
We can not identify the 42-44xlO 3 molecule, which is precipitated by the antiLDL antibody; it is slightly smaller than apolipoprotein AIV (46x 103, Bieseigel &
Utermann, 1979), and slightly larger than the 40xl0 3 form of apolipoprotein E
which is secreted from foetal liver (Zannis et al. 1982).
2. The yolk sack as an early source of apolipoproteins
This study establishes that the human secondary yolk sack is a rich source of
secreted apolipoproteins during the 5—11th weeks of postfertilization development; over this time, the liver and the gut also synthesize and secrete
202
8
W-K. SHI AND OTHERS
10
Figs 7-10.
Apolipoprotein synthesis in human embryos
203
apolipoproteins. There are several reasons for thinking that the yolk sack may be
the sole source of these lipid carrier molecules during the second and third week of
human development. First, the visceral yolk sack is the earliest organ to synthesize
apolipoproteins in mouse development (Shi & Heath, 1984); the synthesis of
apolipoproteins is detected on the 10th day of development, two days after this
mouse tissue starts to synthesize alphafoetoprotein and transferrin (Adamson,
1982; Dziadek & Adamson, 1978; Dziadek & Andrews, 1983). Second, the
secondary human yolk sack which we have studied appears to be the direct cellular
descendant of the primary yolk sack (Luckett, 1978), and the primary human yolk
sack might therefore synthesize apolipoproteins at the earliest stages of
postimplantation development. Third, the liver and the gut of human embryos are
not coherent organs during these two weeks (Moore, 1982). In humans,-the lining
of the gut and the allantois is continuous with that of the yolk sack, and it is already
known that these epithelia are distinct in the third week of postfertilization
development; it is only inside the human yolk sack endoderm cell cytoplasm that
embryonic serum proteins are found at this time (20-day postfertilization embryo,
Jacobsen et al. 1981). The capacity of the gut lining to secrete serum products must
therefore develop later. Fourth, it is a possibility that maternal apolipoproteins
are unable to cross the placenta (Winkel, Snyder, MacDonald & Simpson, 1980).
If these speculations are correct, then an intrinsic source of apolipoproteins from
the human yolk sack may be essential to sustain the rapid cell divisions of early
embryogenesis (e.g. mouse: Solter & Skreb, 1971; Snow, 1977).
In the mouse, it has been established that it is the endoderm layer of the yolk
sack which is the source of apolipoproteins (Shi & Heath, 1984; Meehan et al.
1984). We have failed to establish that this is the case in the human, both because
knowledge of a molecules distribution does not provide information about its site
of synthesis, and because we considered it impractical to cleanly separate the
convoluted endoderm from the other yolk sack tissues. It should be noted that
apolipoprotein synthesis is not always restricted to endoderm cells, and that at
least apolipoprotein E can be synthesized by human blood monocytes, and the
Figs 7-10. Localization of apolipoprotein Al (Fig. 7), and transferrin (Fig. 9) on a
transverse section of a 42-day postfertilization secondary yolk sack. This sack had been
cultured for 19 h in the absence of these serum proteins (see Materials and Methods), e,
endoderm; m, mesenchyme; ep, epithelium; b, blood cells. Scale bar, 50jum.
Fig. 7. Reacted with anti-apolipoprotein Al antibody (1/50), and 1/100 sheep
peroxidase conjugated second layer. The endoderm, blood, and epithelial layer are
strongly stained.
Fig. 8. Two sections away and stained under identical conditions with the first
antibody absorbed with apolipoprotein Al. Photographic procedures identical for both
prints.
Fig. 9. Reacted with anti-transferrin antibody at 1/200, and peroxidase conjugated
second layer at 1/100. The endoderm column and epithelial layer are strongly stained.
Fig. 10. Two sections away and stained under identical conditions with the first
antibody absorbed with transferrin. Photographic procedures kept constant for both
prints.
204
W-K. SHI AND OTHERS
mRNA for this apolipoprotein is present in human adult leucocytes and kidney
(Basu et al. 1982; Wallis et al. 1983). It therefore remains a possibility that the
intensive localization of apolipoproteins in both the endoderm, the outer epithelial
layer, and the nucleated erythrocytes of the human yolk sack does reflect synthesis
of apolipoproteins by all these cell types. However, our inability to detect the
synthesis of apolipoproteins Al and B by foetal blood cells makes it unlikely that
blood cells are a source of these apolipoproteins; it remains a possibility that the
haemopoietic stem cells in the yolk sack and liver synthesize these proteins and
that such cells never enter the foetal circulation.
In general, our observations on the synthesis of the other foetal serum proteins
is consistent with previous authors (Table 1, see Gitlin & Biasucci, 1969; Gitlin &
Perricelli, 1970; Gitlin etal. 1972; Nishi, 1970). On one occasion, labelled AFP was
detected in the culture medium over trophoblast; a similar case of exceptional
AFP synthesis has been observed before in 1/14 placental samples (Gitlin et al.
1972). One stomach sample also secreted AFP; we do not regard this as a reliable
result because we can not exclude the possibility that small fragments of the
neighbouring foetal liver contaminated this specimen.
There was variation in our ability to detect the synthesis and secretion of both
apolipoproteins and the other serum proteins from gut samples; a sample usually
secreted all or secreted none. This variability could not be explained either by the
size of the fragments or their age (Table 1). It is likely that there are local
variations in the secretion of these molecules along the length of the foetal gut; for
in the adult gut apolipoprotein synthesis is inducible (Bisgaier & Glickman, 1983),
and there may be local distinctions along the length of the gut (Green et al. 1982).
The disruption caused by the vacuum abortion procedure made it impossible to
routinely recognize the region of the gut which was incubated with label.
This study extends the list of products which are secreted in common by the
mouse visceral yolk sack and the human secondary yolk sack. These two organs
have a very different anatomical relations with the embryo and the other
extraembryonic membranes in the two species, and it is still unclear how either
iron or lipid reaches these organs, to be picked up by the transferrin and
apolipoprotein carrier molecules which they synthesize and secrete.
CONCLUSION
1. During the 5-11th weeks of postfertilization human development, the
secondary yolk sack is a source of apolipoproteins Al and B.
2. These lipid carrier molecules are also synthesized by the liver and the gut
over this period of embryonic development.
We thank the Blood Transfusion Unit of the John Radcliffe Hospital for fresh venous blood.
The Cancer Research Campaign generously supported these studies. Dr W-K. Shi was on
sabbatical leave from The Shanghai Institute of Cell Biology, Academia Sinica, China,
supported by the Chinese Academy of Sciences: Royal Society exchange scheme, the Henry
Lester Fund, and the British Universities China Committee.
Apolipoprotein synthesis in human embryos
205
REFERENCES
E. D. (1982). The location and synthesis of transferrin in mouse embryos and
teratocarcinoma cells. Devi Biol. 91, 227-234.
ADAMSON,
ALBRECHTSEN, R., WEWER, U. & WIMBERLEY, P. D. (1980). Immunohistochemical demonstration
of an hitherto undescribed localization of hemoglobin A and F in endodermal cells of normal
human yolk sac and sinus tumor. Ada path, microbiol. Scand. Sect. A 88, 175-178.
BASU, S. K., HO, Y. K., BROWN, M. S., BILHEIMER, D. W., ANDERSON, R. G. W. & GOLDSTEIN,
J. L. (1982). Biochemical and genetic studies of apoprotein E secreted by mouse
macrophages and human monocytes. /. biol. Chem. 257, 9788-9795.
BEISIEGEL,U. &UTERMANN, G. (1979). An apolipoprotein homolog of rat apolipoprotein AIV in
human plasma: isolation and partial characterization. Eur. J. Biochem. 93, 601-608.
C. L. & GLICKMAN, R. M. (1983). Intestinal synthesis, secretion, and transport of
apolipoproteins. Ann. Rev. Physiol. 45, 625-636.
3
BONNER, W. M. & LASKEY, R. A. (1974). Afilmdetection method for H-labelled proteins and
nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88.
BRANCA, A. (1913). Recherches sur la structure, revolution et la role de la vesicule ombilicale de
l'homme. /. Anat. Physiol. (Paris) 49, 1-40,171-211, 383-407.
BISGAIER,
CARR, B. R., PARKER, R. C , MILEWICH, L., PORTER, J. C , MACDONALD, P. C. & SIMPSON, E. R.
(1980). The role of low density, high density, and very low density lipoproteins in
steroidogenesis by the human foetal adrenal gland. Endocrinology 106, 1854-1860.
CARR, B. R., PARKER, R. C , OHASHI, M., MACDONALD, P. C. & SIMPSON, E. R. (1983).
Regulation of human foetal testicular secretion of testosterone: low density lipoprotein:cholesterol and cholesterol synthesis de novo as steroid precursor. Am. J. Obstet.
Gynaec. 146, 241-247.
CHAPMAN, M. J., MILLS, G. L. & LEDFORD, J. M. (1975). The distribution and partial
characterisation of the serum apolipoproteins in the guinea pig. Biochem. J. 149, 423-436.
DULBECCO, R. & VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis
viruses./, exp. Med. 99, 167-182.
DZIADEK, M. & ADAMSON, E. D. (1978). Localization and synthesis of alphafetoprotein in
postimplantation mouse embryos. J. Embryol. exp. Morph. 43, 289-313.
DZIADEK, M. A. & ANDREWS, G. K. (1983). Tissue specificity of alpha-fetoprotein messenger
RNA expression during mouse embryogenesis. EMBO. J. 2, 549-554.
ENGELHARDT, N., GOUSSEV, A. I., SHIPOVA, L. J. & ABELEV, G. I. (1971). Immunofluorescent
study of c-foetoprotein in liver and liver tumors. 1. Techniques of a-foetoprotein localization
in tissue sections. Int. J. Cancer 7, 198-206.
ENGSTROM, W., REES, A. R. & HEATH, J. K. (1985). Proliferation of an human embryonal
carcinoma cell line in a defined serum free medium. Interrelationship between growth factor
requirements and membrane receptors. /. Cell Sci. 73 (in press).
GITLIN, D. & BIASUCCI, A. (1969). Development of yG, yA, yM, B l c /B l a , C\ esterase inhibitor,
ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, arrantitrypsin,
orosomucoid, /Mipoprotein, o^-macroglobulin, and prealbumin in the human conceptus.
/. Clin. Invest. 48, 1433-1446.
GITLIN, D. & PERRICELLI, A. (1970). Synthesis of serum albumin, prealbumin, ar-foetoprotein, ar
antitrypsin, and transferrin by human yolk sac. Nature 228, 995-997.
GITLIN, D., PERRICELLI, A. & GITLIN, G. M. (1972). Synthesis of or-foetoprotein, by liver, yolk sac,
and gastrointestinal tract of the human conceptus. Cancer Res. 32, 979-982.
GONZALEZ-CRUSSI, F. (1979). The human yolk sac and yolk sac (endodermal sinus) tumors. A
review. Perspectives in Paediatric Pathology 5,179-215.
GREEN, P. H. R., LEFKO WITCH, J. H., GLICKMAN, R. M., RILEY, J. W., QUINET, E. &BLUM, C. B.
(1982). Apolipoprotein localization and quantitation in the human intestine. Gastroenterology
83, 1223-1230.
HATCH, F. T. & LEES, R. S. (1968). Practical methods for plasma lipoprotein analysis. Adv. Lipid
Res. 6, 2-63.
206
W-K. SHI AND OTHERS
HAVEL, R.
S., EDER, H. A. & BRAGDON, J. H. (1955). The distribution and chemical composition
of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345-1353.
HEATH, J. K. & DELLER, M. J. (1983). Serum free culture of PC13 murine embryonal carcinoma
cells./. CellPhysiol. 115,225-230.
HESSELDAHL, H. & LARSEN, J. F. (1969). Ultrastructure of human yolk sac: endoderm,
mesenchyme, tubules, & mesothelium. Am. J. Anat. 126, 315-336.
HOG AN, B. L. M. & NEWMAN, R. (1984). A scanning electron microscope study of the
extraembryonic endoderm of the 8th day mouse embryo. Differentiation 26, 138-143.
HOYES, A. D. (1969). The human foetal yolk sac. An ultrastructural study of four specimens.
Z. Zellforsch. mikrosk. Anat. 99, 469-490.
HYLDAHL, L. (1984). Primary cell cultures from embryonic corneas. /. Cell Sci. 66, 343-351.
JACOBSEN , G. K., JACOBSEN , M. & HENRIKSEN , O. B. (1981). An immunohistochemical study of a
series of plasma proteins in the early human conceptus. Oncodevelopmental Biol. Med. 2,
399-410.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of bacteriophage
T4. Nature 227, 680-685.
LUCKETT, W. P. (1978). Origin and differentiation of the yolk sac and extraembryonic mesoderm
in presomite human and rhesus monkey embryos. Am. J. Anat. 152, 59-98.
MEEHAN, R. R., BARLOW, D. P., HILL, R. E., HOGAN, B. L. M. & HASTIE, N. D. (1984). Pattern of
serum protein gene expression in murine visceral yolk sac and foetal liver. EM BO. J. 3,
1881-1885.
MILNE, R. W., WEECH, P. K., BLANCHETTE, L., DAVIGNON, J., ALAUPOVIC, P. & MARCEL, Y. L.
(1984). Isolation and characterisation of apolipoprotein B-48 and B-100 very low density
lipoproteins from type III hyperlipoproteinemic subjects. /. Clin. Invest. 73, 816-823.
MOORE, K. L. (1982). The Developing Human. Philadelphia: W. B. Saunders.
NISHI, S. (1970). Isolation and characterization of a human fetal ar-globulin from the sera of
fetuses and a hepatoma patient. Cancer Res. 30, 2507-2513.
O'RAHILLY, R. & GARDNER, E. (1975). The timing and sequence of events in the development of
the limbs of the human embryo. Anat. Embryol. 148, 1-23.
SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. /. Histochem. Cytochem. 10, 250-256.
SHI, W-K. & HEATH, J. K. (1984). Apolipoprotein expression by murine visceral yolk sac
endoderm. /. Embryol. exp. Morph. 81, 143-152.
SOLTER, D. & SKREB, N. (1971). The cell cycle analysis in the mouse egg cylinder. Expl Cell Res.
64, 331-334.
SNOW, M. H. L. (1977). Gastrulation in the mouse: growth and regionalisation of the epiblast.
J. Embryol. exp. Morph. 42, 293-303.
STANNERS, E. P., ELICEIRI, G. & GREEN, H. (1971). Two types of ribosome in mouse-hamster
hybrid cells. Nature, New Biol. 230, 52-54.
THOMPSON, S., STERN, P. L., WEBB, M., WALSH, F. S., ENGSTROM, W., EVANS, E. P., SHI, W-K.,
HOPKINS, B. & GRAHAM, C. F. (1984). Cloned human teratoma cells differentiate into neuron-
like cells and other cell types in retinoic acid. /. Cell Sci. 72, 37-64.
WALLIS, S. C , ROGNE, S., GILL, L., MARKHAM, A., EDGE, M., WOODS, D., WILLIAMSON, R. &
HUMPHRIES, S. (1983). The isolation of cDNA clones for human apolipoprotein E and the
detection of apoE RNA in hepatic and extra-hepatic tissues. EMBO. J. 2, 2369-2373.
C. A., SNYDER, J. M., MACDONALD, P. C. & SIMPSON, E. R. (1980). Regulation of
cholesterol and progesterone synthesis in human placental cells in culture by serum
lipoproteins. Endocrinology 106, 1054-1060.
Wu, A-L. & WINDMUELLER, H. G. (1979). Relative contribution of liver and intestine to
individual plasma lipoproteins in the rat. J. biol. Chem. 254, 7316-7322.
ZANNIS, V. I., KURNIT, D. M. & BRESLOW, J. L. (1982). Hepatic Apo-A-1 and Apo E and
intestinal Apo-A-1 are synthesized in precursor isoprotein forms by organ cultures of human
foetal tissues. J. biol. Chem. 257, 536-544.
WINKEL,
{Accepted 23 August 1984)