It was found that secondary human yolk sacks secreted apolipoproteins Al and ... postfertilization range included in this study; a question mark is used to indicate ...
/. Embryol. exp. Morph. 85, 191-206 (1985)
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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.
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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.
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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
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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
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Figs 1-4.
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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
