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/ . Embryol. exp. Morph. Vol 24, 2, pp. 313-330, 1970
313
Printed in Great Britain
Protein transmission across the
rabbit foetal membranes
By A. E. WILD 1
From the Department of Zoology, University of Southampton
SUMMARY
Fluorescent protein tracing, involving F.I.T.C.-conjugated proteins and the fluorescent
antibody technique, was employed to study the sites and mechanism of transport of a variety
of proteins across the rabbit foetal membranes.
No evidence was found for an intercellular transmission of proteins across the yolk sac
splanchnopleure to the exocoel, but all proteins were shown to become localized in absorptive
vesicles in the yolk-sac endoderm.
Different proteins became similarly localized in absorptive vesicles having different sizes
and profiles. Characteristic broken vesicles were present, and more than one protein was
demonstrated in each absorptive vesicle.
The yolk-sac endoderm was confirmed as the selective site for transmission of proteins to
the foetal circulation, since only proteins readily detected in the foetal serum were present in,
and below, the basement membrane.
The paraplacental chorion was shown to be the site for transmission of proteins to the
exocoel and the process to be one of diffusion.
Unlike normal proteins, F.l.T.C. conjugates readily became localized within macrophages
present in the paraplacental chorion and yolk-sac vascular mesenchyme.
These findings are discussed in the light of differences previously shown to occur between
transmission of proteins to the foetal fluids and to the foetal blood, and in the light of a
current hypothesis to account for selection of proteins by the yolk-sac endoderm.
INTRODUCTION
In a previous study of the protein composition of the rabbit foetal fluids
(Wild, 1965) a paucity of the high molecular weight proteins was found in the
exocoelomic, amniotic and allantoic fluids. A greater proportion of albumin
was also found in these fluids when comparison was made with the foetal and
maternal sera. These findings pointed to a selective transmission of proteins
based on molecular size and similar findings for the amniotic fluid of other
species are consistent with this view (for details see review by Wild, 1966). In
contrast, transmission of proteins to the foetal rabbit circulation is clearly
independent of molecular size since the process favours homologous y-globulin
(Hemmings & Brambell, 1961) and even 19 S y-globulins are transmitted
(Hemmings & Jones, 1962; Kaplan, Catsoulis & Franklin, 1965). A further
1
Author's address: Department of Zoology, The University of Southampton, Southampton,
Hants, SO9 5NH, England.
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A. E. WILD
interesting difference is that y-globulins of different species are transmitted
selectively to the foetal circulation but non-selectively to the fluids (Batty,
Brambell, Hemmings & Oakley, 1954; Kulangara & Schechtman, 1962). Whilst
no doubt exists that the yolk-sac splanchnopleure is the site for transmission of
proteins to the foetal circulation (Brambell, Hemmings & Henderson, 1951) the
site for transmission to the exocoel has been a matter for more speculation.
Kulangara & Schechtman (1962) suggested that proteins reached the exocoel by
diffusion across the yolk sac, which acted as a molecular sieve. Brambell et al.
(1951) could not rule out the paraplacental chorion, and I have also emphasized
its possible role since in in vitro dialysis experiments it was much more permeable
to protein than the yolk sac (Wild, 1965).
In the present investigation use has been made of fluorescent protein tracing
in order to locate the site and elucidate the mechanism of transmission to the
exocoel in vivo, and also to throw light on the selective mechanism involved in
transfer to the foetal vitelline circulation.
MATERIALS AND METHODS
Direct labelling of a variety of proteins including rabbit y-globulins (98 %
pure, Mann Laboratories), human y-globulins (98% pure, Calbiochem), bovine
y-globulins (Armour, Fraction 11), human serum albumin (Mann Laboratories),
and egg albumin (Armour) was carried out according to Nairn (1964) except
that fluorescein isothiocyanate (F.I.T.C.) adsorbed on to celite (Calbiochem)
was used. Conjugation of proteins at a ratio of 20 mg protein: lmg F.I.T.C. was
continued overnight at 4 °C. Non-reacted F.I.T.C. was removed by Sephadex
G-25 chromatography and the conjugates brought back to their original
volumes by ultrafiltration in Visking tubing. Final concentrations of albumins
were 30 mg/ml and y-globulins 10 mg/ml.
For the fluorescent antibody technique, ammonium-sulphate-fractionated,
high titre rabbit antisera to human and bovine y-globulin, and to egg albumin,
were conjugated to F.I.T.C. on celite, and in the case of human y-globulin, also
to Lissamine rhodamine R.B. 200 chloride (R.B. 200 Cl) as described by Nairn
(1964). Non-reacted fluorochromes were removed as previously described.
F.I.T.C.-labelled rabbit anti-human a-2 macroglobulin and R.B. 200 Cl labelled
anti-human serum albumin were obtained commercially (Mann Laboratories).
Immunoelectrophoresis employing the conjugated absorbed antisera revealed
only single lines for all antigens. In the case of the y-globulins this was yG.
For direct tracing, protein conjugates were sterilized by filtration and after
laparotomy injected into the uterine lumen of Dutch rabbits, 26-28 days
pregnant, at a dose of 1 ml/conceptus. At various time intervals, ranging from
15 min to 8 h, rabbits were killed with nembutal and the conceptuses exposed.
After washing with normal saline, areas of yolk sac, paraplacental chorion and
amnion were removed, spread over rectangular windows cut in card, and fixed
Protein transmission
315
for 24 h in 10% formol/saline or 95% ethanol. Where appropriate, exocoelomic
and amniotic fluids and foetal blood were collected and tested for the presence
of heterologous proteins by means of the interfacial ring test and in some cases
immunoelectrophoresis. Subsequent dehydration, clearing and embedding of
the membranes was carried out according to Nairn (1964). After dewaxing in
three changes of xylene, sections (4 /t) were passed through three changes of
100% ethanol and then three changes of phosphate-buffered saline (0-01 M,
pH 7-2), and then mounted in a medium consisting of nine parts glycerol/one
part phosphate-buffered saline. Membranes from conceptuses of non-injected
horns, similarly processed, were used as controls. Sections were examined for
u.v. fluorescence in a Wild M 20 microscope. Photographs were taken in colour
using Kodak High Speed Ektachrome and in black and white using Tri-X.
For tracing by the fluorescent antibody technique, normal human serum was
used as a source of human y-globulin, a-2 macroglobulin and albumin, and
was supplemented with egg albumin at a concentration of 30 mg/ml. Solutions
containing human and bovine y-globulins in normal saline (each at 10 mg/ml)
were also used. Injection and subsequent procedures were as for direct tracing,
except that membranes were fixed in cold 95% ethanol and processed according
to the technique described by Sainte-Marie (1962). In order to reduce nonspecific staining, conjugated antisera were absorbed first with pig liver powder
(Burroughs Wellcome) followed by one or sometimes two absorptions with
foetal membrane powder prepared from chorion, yolk sac and amnion of
conceptuses not exposed to heterologous proteins. Rabbit antibovine y-globulin
serum was found to cross-react slightly with human y-globulin, and to remove
such cross-reacting antibodies, 5 ml batches of conjugated antiserum were
absorbed with 50 mg human y-globulin for 24 h at 4 °C. The precipitate was
then removed by filtration. Absorbed, specific conjugated antisera were applied
to dewaxed sections for 30 min in a saturated environment. Sections were then
washed for 1 h in two changes of phosphate-buffered saline. Blocking tests were
carried out with non-conjugated antisera but the best controls were sections of
membranes not exposed to antigen and which had been similarly processed.
RESULTS
Direct tracing with protein F.T.T.C. conjugates
All protein conjugates, as indicated by specific fluorescence, became localized
in vesicles in the endodermal cells of the yolk-sac splanchnopleure (Figs. 1, 2).
There was no indication that conjugates passed through, or were discharged
into, intercellular spaces. In some experiments, particularly short-term ones,
fluorescence could not be detected in any of the sections of yolk sac examined,
or it was absent from certain areas of the yolk sac, thus giving identical appearance to control tissue. In such cases the conjugates had presumably failed to
spread around the conceptus, or had done so unevenly.
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A. E. W I L D
VHS
A
Protein transmission
317
The vesicles showed a variation in size and in distribution and intensity of
fluorescence. After 15 and 30min exposure to conjugates, the vesicles were
mainly sub-apically located (Fig. 1A, B) and then became more randomly
distributed (Fig. 1C-E; Fig. 2A, C). Fluorescence was distributed throughout
vesicles, or it was confined to the perimeter, or unevenly distributed on the
perimeter of intact and broken vesicles (Fig. 1B, E; Fig. 2 A, C). Broken vesicles
were the largest and usually confined to the supra-nuclear region, whilst smaller
vesicles were more randomly distributed.
Bovine, human, and rabbit y-globulin, human serum and egg albumin conjugates, all showed a similar localization of fluorescence in vesicles which were
similarly distributed within the endodermal cell. However, only with human and
rabbit y-globulin conjugates was fluorescence ever detected in the basement
membrane of the yolk-sac endoderm, in the vascular mesenchyme, or in the
lumen of the vitelline vessels (Fig. 1D, E; Fig. 2 A, B). Vesicles containing rabbit
ABBREVIATIONS ON FIGURES
bb = brush border; bm = basement membrane; bv = broken vesicle; cs = canalicular system; cyt = cytotrophoblast; e = erythrocyte; em = exocoelomic mesothelium; end = endothelium; Imt = loose mesenchymal tissue; m — macrophage;
// = nucleus; tgc = trophoblastic giant cell; vm = vascular mesenchyme; vv =
vitelline vessel; yse = yolk-sac endoderm.
Fig. 1. (A) Yolk-sac splanchnopleure of rabbit no. 20 exposed to rabbit y-globulin
F.l.T.C. conjugate for 30 min. The conjugate is confined to vesicles situated mainly
in the apical regions of the yolk-sac endoderm. x 100. Bright-field fluorescence
1-^ min exposure.
(B) Another area of yolk-sac splanchnopleure of rabbit no. 20 at higher magnification. The conjugate is confined to intact and broken vesicles. Erythrocytes in a
vitelline vessel show autofluorescence. x 500. Dark-field fluorescence, l^min
exposure.
(C) Yolk-sac splanchnopleure of rabbit no. 7 exposed to bovine y-globulin F.l.T.C.
conjugate for 2i h. Vesicles containing conjugate are distributed throughout endodermal cells. The conjugate is absent from the basement membrane but occurs in a
short region of the exocoelomic mesothelium (arrowed) indicating derivation from
the exocoel. x 100. Bright-field fluorescence, \\ min exposure.
(D) Yolk-sac splanchnopleure of rabbit no. 48 exposed to human y-globulin
F.l.T.C. conjugate for 2^h. Note the presence of the conjugate in the basement
membrane and in macrophages in the vascular mesenchyme. x 100. Bright-field
fluorescence, J£ min exposure.
(E) An area of yolk-sac splanchnopleure of rabbit no. 48 at higher magnification.
Note the variation of size of vesicles containing the conjugate in the endoderm. The
largest are broken and fluorescence is less intense in these. A vesicle (arrowed)
appears to lie within the basement membrane, which shows a diffuse distribution of
the conjugate. Other vesicles lie in close apposition to the basement membrane.
Macrophages containing small, intensely fluorescent vesicles lie close to vitelline
vessels. Specific fluorescence indicative of the conjugate is present along the exocoelomic mesothelial border, again indicating a derivation from the exocoel. x 500.
Dark-field fluorescence, !•£• min exposure.
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A. E. WILD
Protein transmission
319
and human y-globulin were often seen closely apposed to the basement membrane and on rare occasions apparently within it (Fig. 1E), but whilst fusion of
vesicles with, or discharge of their contents into the basement membrane seems
the only way of accounting for the observed specific fluorescence, more convincing evidence of such a process was not observed. Diffuse specific fluorescence
was also evident in the vascular mesenchyme and in the endothelium of the
vitelline vessels, and where red blood cells remained in the vessel lumen, it was
frequently seen around them (Fig. 2 A). A further striking feature with rabbit
and human y-globulin conjugates was their localization in macrophages which
frequently bordered the vitelline vessels (Fig. I D , E) or lay in the vascular
mesenchyme in close apposition to the basement membrane (Fig. 2A, B).
Within the macrophages fluorescence was confined to much smaller vesicles
than observed in the endodermal cells. All conjugates were unevenly distributed
along the exocoelomic mesothelium (see Fig. 1C, E) and at least in the case of
bovine y-globulin, human and egg albumin, this represented a derivation from
the exocoelomic fluid.
Absence of fluorescence indicative of bovine y-globulin, human and egg
Fig. 2. (A) Another area of yolk-sac splanchnopleure of rabbit no. 48 (cf. Fig. 1 D,
E) showing presence of the conjugate around erythrocytes in a vitelline vessel. A
macrophage lies in close contact with the basement membrane. Diffuse fluorescence
is present along the brush border of the yolk-sac endoderm. x 500. Dark-field
fluorescence, \% min exposure.
(B) Yolk-sac splanchnopleure of rabbit no. 17 exposed to rabbit y-globulin F.I.T.C.
conjugate for 5 h. Vesicles are few in number in the yolk-sac endoderm but the basement membrane and underlying macrophages show presence of the conjugate,
which is also dispersed through the vascular mesenchyme. x 400. Bright-field fluorescence, 1-^min exposure.
(C) An area of yolk-sac splanchnopleure of rabbit no. 48 in which broken vesicles
are particularly prominent but conjugate is absent from the basement membrane
and vascular mesenchyme. Note the variation in intensity of fluorescence within the
vesicles. x400. Bright-field fluorescence, 1-J-min exposure.
(D) Paraplacental chorion of rabbit no. 7 exposed to bovine y-globulin F.I.T.C.
conjugate for 2\ h. In this field two macrophages, containing numerous, brightly
fluorescing droplets of conjugate, can be seen in the loose mesenchymal tissue. The
basement membrane of the cytotrophoblast and the mesenchymal tissue itself show
much weaker fluorescence. x400. Bright-field fluorescence, l^min exposure.
(E) Paraplacental chorion of rabbit no. 5 exposed to human y-globulin F.I.T.C.
conjugate for 30 min. The conjugate is localized in the basement membrane of the
cytotrophoblast and dispersed throughout the loose mesenchymal tissue and exocoelomic mesothelium. Specificfluorescencealso occurs around the cytotrophoblast
cells in some areas, but not within them, x 100. Bright-field fluorescence, H min
exposure.
(F) Paraplacental chorion of rabbit no. 34 exposed to egg albumin F.I.T.C. conjugate for 3 h. In this field the conjugate is heavily localized in the cellular debris and
surrounds giant cells in an area of giant-cell proliferation. There has been little penetration beyond the cytotrophoblast basement membrane, x 100. Bright-field
fluorescence, H min exposure.
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A. E. WILD
Protein transmission
321
albumin from in and below the basement membrane was generally correlated
with negative precipitin tests for these proteins in the foetal blood, but egg
albumin was sometimes detected in low titre when there was no evidence
for its transmission to the vitelline vessels. However, all conjugates were
readily detected in the exocoelomic fluid and within 15min of injection in
the case of that conceptus nearest the injection site. Entry into the exocoel
was clearly by way of the paraplacental chorion, as evidenced by diffuse
fluorescence around the trophoblastic giant cells and cytotrophoblast, and by
intense fluorescence of the basement membrane of the cytotrophoblast and of
the loose mesenchymal tissue and exocoelomic mesothelium (Fig. 2E, F). All
conjugates, again in the form of small vesicles, were found in macrophages
dispersed through the mesenchymal tissue (Fig. 2D). Such macrophages varied
considerably in density throughout the chorion, but they were more abundant
in longer term experiments and easily observed in living tissue spread as a thin
sheet and exposed to u.v. light (Fig. 3 A). Diffuse specific fluorescence was also
observed in the extracellular ground substance and on the myofibrils present in
the amnion. Again in longer term experiments (after 2 h), all conjugates were
seen as fluorescent droplets in the amniotic epithelium and mesenchymal tissue
(Fig. 3B), and readily observed in spreads of living amnion.
Because egg albumin was sometimes detected in the foetal serum when
specific fluorescence was absent from the basement membrane and vitelline
Fig. 3. (A) Spread of living chorion from rabbit no. 34 showing an area rich in
macrophages in which the conjugate has become localized. Focused through the
exocoelomic mesothelium. x 100. Bright-field fluorescence, J^min exposure.
(B) Amnion of rabbit no. 34 showing intracellular localization of conjugate, x 100.
Bright-field fluorescence, 1-fc min exposure.
(C) Yolk-sac splanchnopleure of rabbit no. 40 exposed for 15 min to normal human
serum supplemented with egg albumin, and treated with rabbit antihuman y-globulin
F.l.T.C.-conjugated antiserum. Specific fluorescence indicative of human y-globulin
is confined to the brush border and subapical vesicles in the endoderm. Fluorescence
is also present along the exocoelomic mesothelium indicating protein derived from
the exocoel. x 100. Bright-field fluorescence, \% min exposure.
(D) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit anti-egg albumin
F.l.T.C.-conjugated antiserum. Weak diffuse specific fluorescence indicative of
protein is present in the subapical canalicular system and concentrated below this
in terminal absorptive vesicles, x 500. Dark-field fluorescence, \\ min exposure.
(E) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit antihuman
y-globulin F.I.T.C. conjugate. Distribution of protein is similar to that seen in (D),
but protein is additionally present on the brush border. x400. Bright-field fluorescence, \\ min exposure.
(F) Yolk-sac splanchnopleure of rabbit no. 39 exposed for 30 min to normal
human serum supplemented with egg albumin, and treated with rabbit antihuman
y-globulin F.l.T.C.-conjugated antiserum. Protein is present in intact and broken
vesicles, and on the surface of erythrocytes (arrowed). The basement membrane
shows no specificfluorescencealthough vesicles lie in close apposition to it. x 500.
Dark-field fluorescence, \\ min exposure.
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A. E. W I L D
Protein transmission
323
vessels, the possibility that protein and fluorescein became dissociated as a result
of enzymic degradation within the vesicles, had to be considered. When F.I.T.C.
alone was injected into the uterine lumen at a concentration of 15mg/ml in
phosphate-buffered saline, and the foetal membranes examined after 3 h
exposure, fluorescence was again detected in vesicles in the yolk-sac endoderm.
These vesicles were much smaller than those observed with the conjugates and
there was no fluorescence in or below the basement membrane. In the chorion
bright diffuse fluorescence was distributed throughout the tissue, but no macrophages were located. Sections of yolk sac exposed to egg albumin conjugates
were also treated with rabbit anti-egg albumin F.I.T.C. This treatment failed
to reveal any further localization of fluorescence, either in or below the basement membrane, or in the vitelline vessels. In order to check whether the
fluorescence observed in the basement membrane, the vascular mesenchyme and
macrophages, was in fact human y-globulin conjugate and not free fluorescein,
sections of yolk sac exposed to human y-globulin conjugate for 2\ h were
treated with rabbit antihuman y-globulin R.B. 200 Cl. This resulted in a
yellowish fluorescence in previously apple-green fluorescing sites and as opposed
to orange-red fluorescence in yolk sac exposed to normal human y-globulin,
indicating that human y-globulin was present as antigen in these sites.
Indirect tracing with the fluorescent antibody technique
All protein antigens (human serum albumin, a-2 macroglobulin and
y-globulin; bovine y-globulin and egg albumin) were again detected within
vesicles in the yolk-sac endoderm, as evidenced by specific fluorescence after
treatment of sections with specific conjugated antisera (see Figs. 3-5). As with
the direct tracing technique, only a non-selective intracellular route of entry was
indicated. This was well illustrated in experiments of 15min duration, after
which time proteins were localized on the brush border, in the subapical region,
Fig. 4. (A) Yolk-sac splanchnopleure of rabbit no. 37 exposed for 5 h to normal
human serum supplemented with egg albumin and treated with rabbit anti-egg
albumin F.I.T.C.-conjugated antiserum. Note the variation in distribution and
intensity of fluorescence within the vesicles. Many lie close to the basement membrane which shows no specific fluorescence, x 500. Dark-field fluorescence, 1$ min
exposure.
(B) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman
y-globulin F.I.T.C.-conjugated antiserum. Note that the vesicles show a similar
variation in intensity and distribution offluorescencewhen compared with those in
Fig. 4 (A), indicating a similar distribution of the two proteins in the endoderm.
The basement membrane, vascular mesenchyme and endothelium all show intense
specific fluorescence, x 400. Bright-field fluorescence, 1^- min exposure.
(C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman
y-globulin F.I.T.C.-conjugated antiserum. In this field broken vesicles are more
evident and intense specific fluorescence indicative of human y-globulin is present
within a vessel. The basement membrane shows no specific fluorescence in this
region, x 500. Dark-field fluorescence, 1 | min exposure.
21
EMB24
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A. E. WILD
and concentrated immediately below this into vesicles (Fig. 3C-E). Greater
non-specific fluorescence was observed with this technique, especially in nuclei
and in erythrocytes, but specific fluorescence within vesicles was never observed
in treated control tissue and blocking tests carried out with non-conjugated
antisera considerably reduced specific fluorescence in experimental material.
Distribution of fluorescence within vesicles was similar to that encountered with
the protein F.I.T.C. conjugates, except that broken vesicles, although present,
were not so clearly defined (Fig. 3F; Fig. 4C). Of those proteins investigated,
Protein transmission
325
human y-globulin was the only one readily detected in the basement membrane,
the vascular mesenchyme, and the vitelline vessels (Fig. 3F; Fig. 4B, C;
Fig. 5 C, E) and could be located in such sites as early as \ h after exposure of
membranes to antigen. Human serum albumin could also be detected in these
sites, but only after longer exposure and with much less intensity of specific
fluorescence. Although vesicles containing egg albumin and bovine y-globulin
lay close to the basement membrane (Fig. 4A; Fig. 5B, D) they were never
detected in or below it, but as in previous long-term experiments with conjugate,
egg albumin was sometimes detected in low titre in the foetal serum.
Except for bovine y-globulin, normal proteins were never observed in macrophages, either in the vascular mesenchyme or in the paraplacental chorion, and
this provided the only striking difference between treatment of conjugated and
normal proteins. Bovine y-globulin was sometimes observed in macrophages in
the paraplacental chorion (Fig. 5D) and was present as droplets in mesenchymal
cells of the amnion. After 15 min exposure, proteins were again detected in the
exocoelomic fluid when they were absent from the foetal serum. Results illustrating this in a typical experiment are shown in Table 1. This rapid entry of
proteins to the exocoel was again correlated with the presence of specific fluorescence indicative of all proteins (except human a-2 macroglobulin) in and
around multinucleate giant cells, around the cytotrophoblast and in its basement
membrane, and dispersed through the loose mesenchymal tissue and exocoelomic
mesothelium (Fig. 5 A). Comparison of litres of bovine and human y-globulin
Fig. 5. (A) Paraplacental chorion of rabbit no. 40 exposed for 15 min to normal
human serum supplemented with egg albumin and treated with rabbit antihuman
y-globulin F.I.T.C.-conjugated antiserum. Specific fluorescence is present in debris
surrounding giant cells, in the cytotrophoblast basement membrane and dispersed
throughout the loose mesenchymal tissue. Nuclei of cytotrophoblast and giant cells
show non-specific fluorescence (arrow), x 100. Bright-field fluorescence, l^min
exposure.
(B) Yolk-sac splanchnopleure of rabbit no. 37 exposed for 5 h to normal human
serum supplemented with egg albumin and treated with rabbit anti-egg albumin
F.i.T.C.-conjugated antiserum. Specific fluorescence is confined to vesicles in the
yolk-sac endoderm. Nuclei in the vascular mesenchyme (arrowed) show non-specific
fluorescence, x 100. Bright-field fluorescence, 1^ min exposure.
(C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman
y-globulin F.T.T.C.-conjugated antiserum. Note intense specific fluorescence in the
vascular mesenchyme and lumen of vitelline vessel, x 100. Bright-field fluorescence,
\\ min exposure.
(D) Yolk-sac splanchnopleure and paraplacental chorion of rabbit no. 51 exposed
for 3 h to a mixture of human and bovine y-globulins and treated with rabbit antibovine y-globulin F.i.T.C.-conjugated antiserum. Specific fluorescence is confined
to vesicles in the yolk-sac endoderm and in the chorion to the loose mesenchymal
tissue and macrophages. x 100. Bright-field fluorescence, 1^ min exposure.
(E) Yolk-sac splanchnopleure of rabbit no. 51 treated with rabbit antihuman
y-globulin F.i.T.C.-conjugated antiserum. Note intense specific fluorescence in the
basement membrane and vascular mesenchyme. x 100. Bright-field fluorescence,
l^min exposure.
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A. E. WILD
in the exocoelomic and amniotic fluids with foetal serum titres, confirmed the
non-selective entry of these proteins to the fluids. Results for a typical experiment are shown in Table 2. Specific fluorescence representing protein derived
from the exocoel was again evident in intermittent areas along the exocoelomic
mesothelium of the yolk sac (Fig. 3C).
Table 1. Transmission of human y-globulin and egg albumin to the fluids and blood
of the single conceptus of rabbit no. 40,15 min after exposing it to 1 ml of normal
human serum supplemented with 3% egg albumin
Foetal fluid
Exocoelomic
Amniotic
Serum
Injected serum
Titre against
rabbit
anti-egg
albumin
128
32
— ve
1048000
Titre against
rabbit
anti-human
y-globulin
8
— ve
— ve
32000
Table 2. Transmission of human and bovine y-globulin to the fluids of the first
conceptus of rabbit no. 51, 3 h after exposing it to a mixture of both proteins
Foetal fluid
Exocoelomic
Amniotic
Serum
Injected solution
Titre against
rabbit
anti-bovine
y-globulin
Titre against
rabbit
anti-human
y-globulin
64
32
— ve
8000
64
32
32
8000
In order to determine whether or not human y-globulin and egg albumin were
localized in the same vesicles, sections of yolk sac exposed to human serum supplemented with egg albumin were treated first with F.I.T.C.-labelled antiserum
to egg albumin and the fluorescence of the same fields compared after subsequent treatment with R.B. 200 Cl-labelled antiserum to human y-globulin.
Vesicles previously fluorescing apple green, gave an intermediate orange-yellow
fluorescence after such treatment, and no fields were found in which apple-green
fluorescing vesicles co-existed with reddish orange vesicles. Such results were
interpreted as indicating that all vesicles contained both proteins.
DISCUSSION
The use of F.I.T.C. conjugates for tracing proteins presents certain limitations
not encountered with the fluorescent antibody technique. The fluorochrome
may alter the way in which protein is taken up into the cell and it may then
Protein transmission
327
become dissociated from protein as a result of enzymic action. The fluorescent
antibody technique, on the other hand, presents problems not encountered with
F.l.T.C. conjugates, namely possible loss of antigenicity and non-specific
staining. Thus findings common to both techniques are better guides to the
mechanisms involved in uptake and transport of proteins than are findings
attributable to one technique alone.
All proteins, whether detected as conjugates or by fluorescent antibodies,
were taken up intracellularly by the yolk-sac endoderm. This finding gives
a visual demonstration that protein uptake is non-selective, a conclusion which
Hemmings (1957) also reached as a result of investigations on the distribution
of iodinated bovine and rabbit y-globulin following injection into the uterine
lumen. There was no evidence of an intercellular route of transmission across
the yolk-sac endoderm, although such a route has been suggested for the rat
yolk sac on purely morphological grounds (Padykula, Deren & Wilson, 1966)
and is implied by Kulangara & Schechtman (1962) in the suggestion that
proteins reach the exocoel by molecular sieving across the yolk sac. However, an
intercellular route of transmission to the exocoel is indicated, but across the
paraplacental chorion. Here little intracellular uptake into the cytotrophoblast
occurred, but there was observed with both techniques an intense staining of the
basement membrane indicative of all proteins except human a-2 macroglobulin.
Proteins appear to pass between cells in certain areas and reach the basement
membrane directly. This route is supported by the localization of specific fluorescence around the cytotrophoblast and by electron microscope investigations
on transmission of ferritin and imferon across the chorion, in which these
electron-dense molecules have been seen in gaps leading to the basement membrane (A. E. Wild & B. S. Slade, in preparation). Larsen & Davies (1962) have
also alluded to the chorion as a possible site for protein transfer, but on circumstantial evidence only.
Davies (1959) also employing the fluorescent antibody technique, reported
detecting bovine y-globulin in the yolk-sac exocoelomic mesothelium and interpreted this as indicating that protein had passed directly across the yolk sac.
The same localization of proteins in the yolk-sac exocoelomic mesothelium was
seen here, but it is clear that such protein is derived from the exocoelomic fluid,
having traversed the chorion and not the yolk sac. Entry to the exocoel and
subsequently the amniotic fluid by this route explains why there is a paucity of
the large molecular weight proteins (Wild, 1965) and why there is no selection
of y-globulins during transmission to these fluids (see Table 2 in this investigation; Batty et al. 1954; Kulangara & Schechtman, 1962) since the process
clearly involves a selective ultrafiltration, as has been suggested for entry to the
human amniotic fluid (Derrington & Soothill, 1961).
It has been reasonably assumed (see Brambell, 1966) that the endodermal
cell is the selective site during transmission of proteins to the rabbit foetal circulation. The results of the present investigation confirm this, for whilst all proteins
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A. E. WILD
investigated were similarly distributed in the endodermal cell, only rabbit and
human y-globulin, and to a lesser extent human serum albumin, were detected
in and below the basement membrane. Other possible sites for selection, such
as the foetal reticulo-endothelial system and yolk-sac vascular mesenchyme and
endothelium, can now be firmly ruled out. The failure to detect egg albumin in
and below the basement membrane on those occasions when it was detected in
the foetal blood, may be related to the low level of transmission of this protein
across the yolk sac or to the existence of other sites of entry. Brambell,
Hemmings & Morris (1964) have suggested that selective attachment of pinocytosed proteins to a finite number of receptors present on the limiting membrane
of an absorptive vesicle, might provide protection from enzymic degradation
after fusion with lysosomes. In some unknown way, after movement through the
cell, protein is then presumed to be liberated from the vesicle into the intercellular
space. Investigations on the ultrastructure of the yolk-sac endoderm (Petry &
Kuhnel, 1965; Padykula et al. 1966; Slade, 1969) suggest that absorptive
vesicles are formed as terminal dilations of the subapical canalicular system.
This region gave a weak diffuse fluorescence in the present experiments, providing pictorial evidence of protein transport through the canalicular system
and concentration in the underlying absorptive vesicles, since fluorescence here
was much increased. From their later distribution in the endodermal cells it is
clear that the vesicles are involved in transport of proteins towards the basement membrane, but whether or not all vesicles are involved is open to question.
Proteins were associated with the perimeter of the vesicles or dispersed throughout them and this difference was not merely a function of the plane of the
section. The characteristic breakages in the large, mainly supranuclear vesicles,
may possibly be artifacts, but they feature prominently in electromicrographs
of the yolk-sac endoderm. Padykula et al. (1966) observed a greater proportion
of 'ruptured' vesicles in 21-day-old, compared to 14-day-old rat yolk-sac endoderm. These vesicles could therefore be part of the normal, intercellular degradation cycle, representing 'dead ends' as far as protein transport is concerned. Of
the available, homologous y-globulin which is present in the yolk sac, only
about 12% is transported to the foetal circulation (Hemmings, 1957) and it is
possible that such protein is confined to the intact vesicles described.
A necessary requisite for the hypothesis proposed by Brambell et al. (1964)
is that more than one protein should be present in the same absorptive vesicle.
In the double tracing experiment described, results were obtained which seem
to confirm this. However, vesicles lying close to the basement membrane might
have been expected to contain only human y-globulin if such protein was being
protected, but this was not the case. Actual fusion of vesicles with the basement
membrane was not readily observed, but it seems a more likely process for discharging proteins than liberation into the intercellular space. When such fusion
does occur the vesicles may then contain only the transmitted protein. Subsequent passage of normal protein from the basement membrane to the lumen of
Protein transmission
329
the vitelline vessel appears to be merely a process of diffusion through the loose
vascular mesenchyme and the endothelia, but F.I.T.C.-conjugated human and
rabbit y-globulin which reaches the vascular mesenchyme is in addition taken
up into macrophages. Luse (1958) mentions detecting colloidal gold and fat in
such cells. All conjugated proteins were also taken up into macrophages in the
paraplacental chorion and into mesenchymal cells in the amnion. This anomalous
treatment of conjugated proteins by macrophages has previously been reported
by Nairn (1964) and is clearly related to the presence of the dye. Such cells
probably have little role to play in the actual transport of proteins across the
foetal membranes, but may further regulate their transmission by pinocytosing
and digesting them.
RESUME
Transfer t de proteines a t ravers les membranes feet ales chez le lapin
Le marquage des proteines par fluorescence, faisant intervenir des proteines conjuguees
F.I.T.C. et la technique d'immuno fluorescence, a ete utilise pour etudier les sites et le
mecanisme de transport de diverses proteines a travers les membranes fcetales du Lapin.
II n'a pas ete possible de mettre en evidence un transfertintercellulaire de proteines a travers
la splanchnopleure de la vesicule vitelline vers le ccelome extraembryonnaire, mais toutes les
proteines se sont localisees dans des vesicules d'absorbtion de Pendoderme de la vesicule
vitelline.
Differentes proteines ne sont localisees de fa?on similaire dans les vesicules d'absorbtion
dont la taille et I'aspect sont differents. Des vesicules eclatees caracteristiques ont ete observees
et Ton a pu mettre en evidence la presence de plusieurs proteines dans chacune des vesicules
absorbantes.
Le role de Pendoderme de la vesicule vitelline en tant que site selectif pour le transfert des
proteines vers la circulation foetale a ete confirme; seules les proteines rapidement decelees
dans le serum foetal sont observees, a Pinterieur et au dessous de la membrane basale.
II est demontre que le chorion paraplacentaire constitue bien le site de transfert des
proteines vers le coelome extraembryonnaire, et que ce transfert s'effectue suivant un processus
de diffusion.
Contrairement aux proteines normales, les conjugues F.I.T.C. se localisent rapidement
parmi les macrophages presents dans le chorion paraplacentaire et dans le mesenchyme
vasculaire de la vesicule vitelline.
Ces resultants sont discutes a la lumiere des travaux montrant que des differences existent
entre le transfert de proteines vers les fluides fcetaux et vers le sang foetal et a la lumiere des
hypotheses courantes rendant compte de la selection des proteines par Pendoderme vitellin.
1 am grateful to Mr M. Childes for his technical assistance and to the Science Research
Council for their financial support.
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(Manuscript received 3 December 1969)