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Transport of horseradish peroxidase across monkey trophoblastic epithelium in coated and uncoated vesicles.

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THE ANATOMICAL RECORD 211:174-183 (1985)
Transport of Horseradish Peroxidase Across
Monkey Trophoblastic Epithelium in Coated and
Uncoated Vesicles
Department o f Human Anatomy, School of Medicine, University of California
Davis, C A 95616
This study used membranous chorion of the macaque monkey placenta to examine uptake and processing of exogenous proteins. Tissue was incubated
with either cationic or anionic horseradish peroxidase. Incubation time was varied
between 5-25 min to follow the endocytic pathways. In spite of some differences in
binding, uptake and processing of the isozymes was similar. In the presence of
tracers at 37°C both horseradish peroxidases were taken up in large (150-175) nm
diameter) coated vesicles. In addition, coated tubules 300-400 nm in length and 50100 nm in diameter were seen in the apical cytoplasm. Studies using ruthenium red
indicated that the coated tubules were derived from long coated invaginations of the
free surface that pinch off into the apical cytoplasm. Often, the tubules bud off small
(85-105 nm diameter) protein-filled coated vesicles which traversed the cytoplasm
and fused with the basal-lateral plasma membrane. In other cases, the tubules or
vesicles lost their clathrin coats and fused to form larger endocytic vesicles which
later fused with phagolysosomes. After longer incubation, larger uncoated vesicles
(endosomes)were seen releasing their contents at the basal-lateral membrane. These
results suggest that multiple transport pathways exist in this epithelium. The first,
involving only coated structures, may function to sort and concentrate specific
ligands important for embryonic development. The second, involving the formation
and translocation of large uncoated vesicles to the basal-lateral membrane, may also
provide nutrients to the embryo. A third pathway directs the protein to phagolysosomes where it is presumably degraded. Degradation products could be used by the
cells of the membranous chorion or may provide nutrients to the embryo.
The membranous chorion of the macaque monkey placenta is composed of a columnar trophoblastic epithelium backed by a relatively avascular connective tissue
(King, 1981).As a result of superficial implantation in
the macaque monkey, the membranous chorion is exposed to the uterine lumen during early gestation. Morphological studies have suggested that this epithelium
may be absorptive, possibly taking up macromolecules
from the uterine lumen (King, 1981). The suggestion is
also supported by the observations that similar cells in
other species have been shown to have a n absorptive or
phagocytic function (for references see Dantzer, 1982;
Burton, 1982; King, 1982a,b; 1984). However, in virtually all cases there is no direct evidence delineating
the intracellular pathway(s) by which material actually
crosses these cells.
In other absorptive epithelia, protein may be routed to
lysosomes andor transported across the cell in clathrincoated or uncoated vesicles (Farquhar, 1983).Because of
our interest in establishing the role of extraembryonic
membranes in early development, we have studied the
processing of absorbed protein in the trophoblastic epithelium of the monkey membranous chorion. We used
two isozymes of horseradish peroxidase (HRP), acid
0 1985 ALAN R. LISS, INC.
phosphatase (AcPase)cytochemistry, and ruthenium red
staining to analyze the pathways of protein uptake and
processing. In addition to a pathway that routes vesicles
to phagolysosomes, we demonstrate two pathways of
transepithelial transport. The first pathway involves
exclusively clathrin-coated structures, while the second
involves larger, uncoated endosomes.
Membranous chorion of macaque monkey (Macaca
mulatta and M. cynomolgus) placentas was obtained
after cesarean section performed at 25 to 40 days of
gestation. Tissue was dissected away from the placental
disc and washed with cold Earle’s Balanced Salt Solution (Gibco Laboratories, Grand Island, NY).
For binding studies, tissue was transferred to Minimal
Essential Medium, pH 7.4 (Gibco), gassed with 0 2 C02(95%-5%) containing 2 mg/ml Type VI or Type VIII
HRP (Sigma Chemical Co., St. Louis, MO) and 5 mg/ml
bovine serum albumin (BSA; Sigma). Isoelectric focusing showed the Type VIII HRP (AHRP) had a PI of 4.5,
with a minor band a t 3.5. The Type VI HRP (CHRP) had
Received May 21, 1984; accepted August 16,1984
(Fig. 1).In addition, a population of moderately electrondense granules distributed throughout the apical cytoplasm was not reactive (Fig. 2). These granules ranged
from 300 to 1,000 nm in diameter and were relatively
homogeneous in content.
The well-developed Golgi apparatus which surrounds
the nucleus exhibited extensive AcPase activity (Fig. 3).
Often, nearly all of the cisternae contained reaction
product, although some cis cisternae remained unlabeled. Although some of the trans-most cisternae exhibited morphological characteristics of GERL (i.e.,
thickened membrane, endoplasmic reticulum with ribosomes on one membrane face), a majority did not have
these attributes. Small 65-75 nm diameter coated vesicles containing reaction product were also seen in the
Golgi region but were not observed in the apical portion
of the cell or in contact with any endocytic vacuoles.
There were also many small coated vesicles in the Golgi
region that were not reactive for AcPase.
Fixation in the presence of ruthenium red resulted in
staining of the glycocalyx of the apical cell surface (Figs.
4-6). Coated pits and long coated invaginations of the
cell surface were stained, with staining varying from
heavy to very light (Figs. 4,5).Areas of glycocalyx which
were opposite clathrin-coated areas often exhibited periodicity in staining (Fig. 4). Unstained coated vesicles
and tubules were also seen in the apical cytoplasm (Fig.
6). The stain did not penetrate between the cells below
the level of the tight junctions.
The glycocalyx at the apical surface was most prominent in association with clathrin-coated areas of the
membrane (Figs. 7-10). This clathrin coat is present on
the cytoplasmic side of surface invaginations as well as
on noninvaginated areas of the membrane (Fig. 10).
Incubation of the tissue with HRP isozymes at 4°C demonstrated a difference in the absorption of the glycoproteins to the cell surface. CHRP was bound to the
glycocalyx over the clathrin-coated areas, whereas uncoated membrane areas, including microvilli, were unlabelled (Fig. 7). AHRP was not absorbed to any area
obviously associated with the free surface, although occasional “coated vesicular profiles,” presumably attached to the surface via a narrow neck, were labeled
(Fig. 8).
After a short incubation (5-10 min) at 37”C, both
isozymes were seen a t the apical surface in clathrinRESULTS
coated pits and vesicles measuring 150-175 nm in diThe ultrastructure of the membranous chorion of the ameter (Figs. 9, 16). There was some variability in the
rhesus monkey placenta early in gestation has been absorption and uptake of both glycoproteins, since even
described in detail elsewhere (King, 1981). Therefore, after the longest incubation (25 rnin), some heavily laonly those features pertinent to this study will be de- beled coated pits and vesicles were adjacent to others
scribed, specifically the vacuolar apparatus which in- that were unlabeled (Fig. 10). In addition to coated vesicludes the endocytic complex and Golgi-lysosomal cles, other clathrin-coated structures with complex
shapes were seen in the apical cytoplasm (Figs. 11-13,
AcPase cytochemistry showed abundant deposition of 16). A majority of these structures contained reaction
reaction product in large vacuoles and multivesicular product, although occasional profiles were observed
bodies in the apical cytoplasm (Figs. 1, 2). These vacu- without reaction product. Of these complex structures,
oles ranged from 0.5 to 2 pm in diameter and often had the most common was a dumbbell-shaped profile 300irregular shapes (Fig. 2). The basal portion of the cell 400 nm in length with rounded ends 85-105 nm in
also contained AcPase-positive granules. However, they diameter (Figs. 11-13, 16). The morphology of these
were often smaller and less abundant than in the apical structures included coated cylindrical profiles (Fig. 161,
cytoplasm. Small, electron-lucent vacuoles approxi- more compact cylinders with bulbed ends (Fig. 111, and
constricted at one end,
mately 500 nm in diameter and a n extensive tubular- structures that were .
vesicular system which contained no reaction product forming a coated vesicular profile almost separated from
were seen h the cortical portion of the apical cytoplasm the cylinder (Fig. 12). In some instances, the cylindrical
a PI of 8.5-8.6, with a minor band at 9.0. BSA was
included in all media because it resulted in improved
morphology. Incubation was carried out for 5 min at
4°C. Tissue was fixed in 3% glutaraldehyde buffered
with 0.1 sodium cacodylate-HC1at 4°C and processed as
described below.
For uptake studies, tissue was placed in medium identical to that of the binding studies but which was maintained at 37°C. Incubations were carried out for 5 to 25
min and then fixed as described above.
All tissues were washed in 0.1 M cacodylate-HC1
buffer, pH 7.3, and incubated in a diaminobenzidine
(DAB)medium which contained 0.5 mg/ml DAB (Sigma)
+ 0.01% H202 in phosphate buffer (pH 6.0). To allow
penetration of substrate, samples were preincubated 15
min in buffer containing only DAB and then incubated
105 min in complete medium. Controls included incubation in DAB medium without H202 and incubation of
tissue not exposed to tracer in complete medium. After
DAB incubation, samples were postfixed in 2% OsO4 in
0.1 M collidine buffer, stained en bloc with 0.5% aqueous
uranyl acetate, dehydrated through a series of cold acetones, and embedded in a n Epon-araldite mixture.
For AcPase cytochemistry, tissue was immediately
fixed (without incubation in tracer) in 2% paraformaldehyde + 2.5% glutaraldehyde or in 1.5% glutaraldehyde in 0.1 M cacodylate-HCl buffer, pH 7.3, a t 4°C for
1hr. Enzyme localization was carried out as previously
described (King and Wilson, 1983)in a medium containing 40 mM Tris maleate + 11.5mM Na-b-glycerophosphate + 2.4 mM Pb(N03)2 + 5% sucrose. After
incubation, tissue was washed in buffer, postfixed in 1%
Os04 buffered with veronal acetate (pH 7.4) and stained
en bloc with 0.5% uranyl acetate in Tris maleate buffer.
Controls included incubation in the absence of substrate
or heating the tissue to 60°C for 15 min prior to incubation in complete medium.
For ruthenium red cytochemistry, tissue was fixed for
5 min at 4°C in 3% glutaraldehyde in 0.1 M cacodylateHC1 buffer (pH 7.3) and then immersed in a solution
containing 0.5% ruthenium red (Polyscience, Inc., Warrington, PA), 2% glutaraldehyde, and 0.067 M cacodylate-HC1 buffer (pH 7.3) for 2 hr at 4°C. Subsequent
processing was basically according to the method of Luft
Fig. 1. AcPase. Apical portion of epithelial cell. Several nonreactive vesicles of the tubulovesicular system are evident (asterisks), as well as a nonreactive multivescular body (MVB)
and a large vacuole (which may be an MVB) filled with reaction product (top). ~20,900.
Fig. 2. AcPase. Large (1.8 pm diameter) lysosome with dense reaction product. The cells also
have a population of moderately electron-dense granules of various sizes that are unreactive
for AcPase (asterisk) x 18,200.
Fig. 3. AcPase. Extensive reaction product in Golgi cisternae. ~20,900
portion of the profile was uncoated on one or both sides;
however, the rounded ends retained their clathrin coat
(Figs. 13, 16). Coated vesicles 85-105 nm in diameter
and filled with reaction product were seen in the apical
cytoplasm (Fig. 16). However, they were distinct from
another class of smaller (65-75 nm diameter) coated
vesicles seen in both the apical cytoplasm and Golgi
region but which did not contain reaction product (Fig.
16).After longer incubation, 85-105 nm diameter HRPfilled coated vesicles were seen below the level of the
tight junction (Fig. 17) and fused with the basal-lateral
plasma membrane (Fig. 18).
In addition to coated structures, uncoated tubular profiles 200-400 nm in length and 50-100 nm in diameter
and filled with reaction product were located near the
apical membrane (Fig. 10). Uncoated tubules were also
fused with endocytic vacuoles and phagolysosomes (Figs.
14, 15; see also Fig. 16, inset). Phagolysosomes were
identified based on correspondence in size and distribution of AcPase-positive vacuoles (see Fig. 2) to similar
vacuoles containing HRP reaction product in the uptake
experiments. Smooth surfaced vesicles 140-175 nm in
diameter, which contained a rim of reaction product,
occurred after short incubation (Figs. 13, 16) and were
often fused with larger endocytic vacuoles (Fig. 13).After
longer incubation (15-25 min), the endocytic vacuoles
surround large phagolysosomes and often fuse with them
(Fig. 15).
Large, smooth surfaced vesicles containing reaction
product were present toward the basal aspect of the cell,
and at 25 min of incubation some were fused with the
basal plasma membrane (Figs. 19,ZO).The basal lamina
contained reaction product in discrete areas that were
associated with a recess in the basal membrane. The
reaction product was most dense adjacent to the basal
cell membrane (Fig. 20). Smooth surfaced vacuoles also
were fused with the lateral membrane and reaction
product was seen in punctate areas of the intercellular
space, with adjacent unlabeled areas (not illustrated).
There was no reaction product in the intercellular space
in or near the tight junctions a t any time. HRP reaction
product occurred in a few connective tissue cells near
the epithelium after the longer incubations, but was not
present after short incubations.
Although the Golgi apparatus was well developed in
these cells, HRP reaction product was not found in the
cisternae or associated vesicles. In addition, the population of moderately electron-dense granules in the apical
cytoplasm never contained reaction product, regardless
of incubation time (Fig. 14). No differences in uptake or
More importantly, it demonstrates two distinct pathways for vesicular transepithelial transport of the absorbed protein-one using coated vesicles and another
using uncoated “endosomelike” vacuoles. Transepithelial transport via two distinct pathways is uncommon,
as most epithelia use either coated vesicles or smooth
vesicles to transport proteins across the cells (see below).
The dual transport pathways may play a role in the
nutrition and development of the embryo.
Surface Binding
Surface binding studies show adsorption of CHRP to
the glycocalyx over clathrin-coated areas, whereas
AHRP is seen only in very deep coated pits. Adsorption
of CHRP to the glycocalyx may be mediated by the
molecular charge of the protein. The cell surface is negatively charged, which can result in preferential adsorption of positively charge molecules to the glycocalyx
(Mehrishi, 1972). If the binding is solely related to
charge, the patchy distribution of the CHRP reaction
product suggests that anionic sites are clustered over
clathrin-coated areas of the membrane. This contrasts
with macrophages, which diffusely bind positively
charged molecules, including CHRP (Thyberg and Stenseth, 1981). The difference in distribution may reflect a
functional difference, as the mechanism of internalization of cationic protein in macrophages involves large
membrane folds rather than vesicles (Thyberg and Stenseth, 1981).
The appearance of AHRP in the deep pits may be a
result of incomplete washing of inaccessible pits, as
binding of native (PI 6-7) HRP is morphologically undetectable in mouse peritoneal macrophages (Thyberg and
Stenseth, 1981).However, charge may play a role in the
adsorption of anionic macromolecules to the cell surface,
as binding sites for negatively charged ligands have
been demonstrated in macrophages (Brown et al., 1980).
Also, other factors, such as carbohydrate residues, may
play a role in adsorption of the glycoprotein to the cell
(Ashwell and Harford, 1982). Carbohydrate-specific
binding sites have been demonstrated (Kawasaki et al,
1978; Stahl et al., 1978), and HRP has been shown to
bind in a specific and saturable manner to many cells
Fig. 4. Ruthenium red. The glycocalyx of the apical surface is stained, (Straus, 1983).
with staining in the coated pit exhibiting periodicity (arrows). x 63,600.
One interesting observation was the presence of coated
Fig. 5. Ruthenium red. This figure illustrates the variation in stain- pits with no detectable reaction product immediately
ing intensity, which may indicate restricted access of the stain to some adjacent to a heavily labeled pit. This phenomenon has
coated structures. The coated tubule (CT)exhibits heavy staining while also been reported in sea urchin egg-coated vesicles
the coated pit (CP) is lightly stained. ~63,600.
(Fisher and Rebhun, 1983). This observation may be
Fig. 6. Ruthenium red. Coated tubule (CT) and vesicle (CV) have no explained by 1)heterogeneity of coated pits; 2) the empty
staining of the glycocalyx, indicating their dissociation from the sur- pit may have a large neck out of the plane of section,
face. ~ 6 3 , 6 0 0 .
which allowed the HRP to be washed out; or 3) the
glycocalyx binding sites may have been loaded with
processing were present at difyerent gestational ages, endogenous protein. Further studies are needed to clarand controls showed no endogenous peroxidase activity ify this observation.
in the cells.
Uptake and Processing
We used two isozymes of HRP and AcPase and ruthenium red cytochemistry to attempt to reconstruct the
pathways of endocytosis and processing of absorbed protein in the columnar epithelium of the monkey membranous chorion. This study confirms the suggestion that
this trophoblastic epithelium is absorptive (King, 1981).
Cells of the membranous chorion appear to take up
and process both HRP isozymes by similar mechanisms,
suggesting that charge does not play a role in their
ultimate fate in this system. Only a few studies have
compared uptake of isozymes of HRP. Endothelial cells
in culture take up cationic HRP more rapidly than neutral or anionic HRP (Davies et al., 1981). Macrophages
Fig. 7. CHRP, 5 min at 4°C. Reaction product is concentrated in the
glycocalyx over clathrin-coated areas. ~ 4 4 , 9 0 0 .
Fig. 8. AHRP, 5 min at 4°C. The glycocalyx opposite some clathrincoated areas is unlabeled (arrowheads), hut an adjacent “deep-coated
pit” is heavily labeled. ~ 4 5 , 0 0 0 .
Fig. 9. AHRP, 10 min at 37°C. Coated pitivesicle filled with reaction
product. Compare diameter with Figure 17. Adjacent unlabeled coated
pithesicle is also apparent (arrow). Bar 200 nm. ~ 6 3 , 6 0 0 .
labeled coated pit adjacent to unlabeled pits. Uncoated tubules ( U T
i.e., lacking a clathrin coat) filled with reaction product are also shown.
Fig. 11. AHRP, 25 min at 37°C. Coated tubule filled with reaction
product. Note bulbous ends. The neck of the tubule near the end
contains less reaction product, possibly preparing for fusion (arrow).
Fig. 10. AHRP, 25 min at 37°C. The glycocalyx is most predominant
opposite clathrin-coated areas (arrow).This figure also shows a heavily
Fig. 12. CHRP, 10 min at 37°C. Coated tubule filled with reaction
product, which seems to he concentrated in the bulbous termini of the
tubule and one end (arrow) may he budding off. Compare diameter of
bulbed end with Figure 17. Bar = 200 nm. X63,600.
show little difference in amount of different HRP isozymes that are internalized, although the mechanism of
uptake differs between isozymes and native HRP and
AHRP are degraded more rapidly than CHRP (Thyberg
and Stenseth, 1981; Stenseth et al., 1983) The limited
number of studies comparing the uptake of HRP isozymes precludes generalizations about absorption rates
and fate of internalized protein.
The absence of uncoated invaginations a t the apical
cell membrane suggests that protein uptake in this epithelium is mediated exclusively by coated vesicles and
tubules. However, uncoated vesicles are observed in the
apical cytoplasm, and AcPase cytochemistry indicates
that these vacuoles are nonlysosomal. They probably
correspond to endosomes or receptosomes found in many
other cell types (Willingham and Pastan, 1980; Helenius
Fig. 14. CHRP, 10 min at 37°C. This figure illustrates the continuity
Fig. 13. AHRP, 10 min at 37°C. Tubule containing reaction product.
The middle portion of the tubule has lost its clathrin coat (large arrow), of an uncoated tubule (UT) with a labeled endosome (El. Two moderbut the ends have retained it (small arrows). Small vesicles sometimes ately electron-dense granules which do not contain reaction product
contained reaction product (arrowhead); alternatively, they may be a are also present (asterisks). x 63,600.
cross section through a tubule. An uncoated endocytic vesicle (V) is
fused with a labeled endosome (E). ~63,600.
Fig. 15. AHRP, 15 min at 37°C. Apical portion of epithelial cell. Note microvilli (top). Large
phagolysosome (PL) is surrounded by endosomes filled with reaction product. Both vesicular
and tubular structures are fused with a phagolysosome (arrows). x 38,400.
et al., 1983).In this system, we define endosomes as acid
hydrolase-negative vacuoles approximately 0.3-0.5 pm
in diameter that contain HRP reaction product. Also
included in this category are the acid hydrolase-negative
tubules 200-400 nm in length and 50-100 nm in diameter. The endosomes may derive directly from coated
pits which never leave the cell surface, as has been
suggested to occur in certain fibroblasts (Willingham
Fig. 16. AHRP, 10 min at 37°C. An example of several steps in the
endocytic pathway. A large coated vesicle (CV) containing reaction
product is seen near the surface, as well as uncoated vesicles (UV) of
comparable diameter. Uncoated tubules (UT), partially coated tubules
(PCT), and completely coated tubules (CT) loaded with reaction product
and Pastan, 1983), or the coated pits may pinch off
forming coated vesicles which subsequently lose their
clathrin coat and fuse with one another. The 150-175
nm coated vesicles seen at the apical surface appear to
be routed exclusively to endosomes, as coated vesicles of
that diameter are never seen at the base of the cells.
Another interesting “organelle” frequently observed
in the present study is the “coated tubule.” The studies
are also seen. An intermediate-sized coated vesicle (arrowhead) is contrasted with small (presumably golgi-derived) coated vesicles which
contain no reaction product (arrows). A labeled phagolysosome (PL) is
also apparelit. x 46,600. Inset, fusion of uncoated tubule with lysosome
(arrow). x 36,400.
Fig. 17. AHRP, 25 min at 37°C. Intermediate-sized coated vesicle
filled with reaction product near the lateral membrane at the base of
the cell. Compare diameter with Figures 9 and 12. ICS, intercellular
space. Bar = 200 nm. ~63,600.
Fig. 19. AHRP, 25 min at 37°C. Large uncoated vacuole filled with
reaction product fused with basal cell membrane. BL, basal lamina.
Fig. 18. AHRP, 25 min at 37°C. Apparent fusion of protein-filled
coated vesicle (arrow)with lateral membrane. Intercellular space near
point of fusion also contains reaction product, but more distal areas do
not. ~63,600.
Fig. 20. AHRP, 25 min at 37°C. Localized area of reaction product in
a recess of the basal cell membrane and basal lamina (BL), suggesting
a more advanced stage of vesicle fusion than that shown in Figure 19.
Reaction product is absent in adjacent areas of BL and in deeper
connective tissue. ~ 4 5 , 0 0 0 .
using ruthenium red suggest that these structures are
derived from elongated coated invaginations of the apical cell surface and a t least some of these structures are
detached from the surface. The fate of the coated tubules
appears to be varied. Although directionality is difficult
to determine using static electron microscopic images,
the fact that the bulbed ends of the coated tubules correspond in size to the protein-filled coated vesicles seen
a t the base of the cells suggests that the termini of the
tubules are the source of the 85-105 nm coated vesicles.
These coated vesicles then traverse the cell and fuse
with the basal-lateral membrane. A class of coated vesicles of this intermediate diameter has been described in
rodent liver, and they may be a distinct shuttle compartment between the Golgi and plasma membrane (Croze
et al., 1982).In the present study, the intermediate-sized
vesicle may be involved in transport of protein across
the epithelium. The observation that coated vesicles may
be involved in transcellular movement of protein across
trophoblastic cells of membranous chorion is consisent
with similar conclusions in other absorptive epithelia
(King and Enders, 1971; Rodewald, 1973; Moxon et al.,
1976; Van Deurs et al., 1978; King, 1982a,b). A slightly
different pathway was described in thyroid follicular
cells by Herzog (1983). He observed coated pits at both
the apical and basal-lateral cell membranes, but pro-
posed an intermediate compartment of small uncoated
The presence of partially coated tubules suggests that
coated tubules also give rise to the uncoated tubules
seen in the apical cytoplasm. The uncoated tubules are
nonlysosomal but are often fused with other endocytic
vesicles. They may represent a n intracellular pathway
leading to degradation of endocytosed ligand. Tubules
that label with protein tracer have been reported in
several epithelia (Graham and Karnovsky, 1966; King
and Enders, 1970; Orlic and Lev, 1973) and tubular
structures have been implicated as sites of intracellular
sorting events in neonatal rat gut, guinea pig yolk sac,
and liver (Abrahamson and Rodewald, 1981; King,
1982b; Geuze et al., 1983). In these systems the tubules
were uncoated or had a n incomplete clathrin coat.
In addition to transepithelial transport in intermediate-sized coated vesicles, protein accumulates in endosomes that migrate to the basal portion of the cell and
release their contents. Although transport via this
mechanism is not common, apparent endosomal transport of intact IgG was demonstrated in neonatal pig gut
(Kraehenbuhl and Campiche, 19691, and IgA is transported across hepatocytes in small, uncoated vesicles
(Reston et al., 1980). The movement of viral proteins
from the apical cell membrane to the basal membrane
embryo. After crossing the membranous chorion, substances could gain access to the embryo by crossing the
amnion or the yolk sac (King, 1980; King and Wilson,
1983). The coated tubule-coated vesicle pathway may
function to sort and concentrate specific ligands important for embryonic development. The endosomal transport pathway may also provide nutrients to the embryo
before the establishment of the definitive placenta and
could function concurrently with the chorio-allantoic
placenta. As in some other epithelia (Maunsbach, 1966;
Van Deurs, 1978), the lysosomal system may have a
major role in processing internalized protein in these
trophoblastic cells. Endosomes fuse with phagolysosomes, presumably resulting in degradation of internalized protein. Low molecular weight degradation products
may diffuse out of the lysosomes to provide nutrients to
the rapidly growing, but relatively avascular, membranous chorion or may diffuse to the embryo. Since this
trophoblastic epithelium is functionally active during
the period of major organogenesis, teratogens gaining
access to the uterine lumen could potentially reach the
embryo via this route, resulting in malformation or embryonic death.
Fig. 21. A summary of the pathways of protein uptake and processing
in the membranous chorion. Large (150--175nm diameter) coated vesicles lose their coats and form endosomes (pathway 1).The endosomes
then fuse with phagolysosomes (pathway la) or fuse with the basallateral membrane (pathway lb). An alternative pathway involves the
formation of coated tubules (pathway 2). The coated tubules bud off
intermediate-sized (85-105 nm diameter) coated vesicles that fuse with
the basal-lateral membrane (pathway 2a). These medium-sized coated
vesicles are distinct from the small (65-75 nm diameter) coated vesicles that are derived from the Golgi apparatus. The portion of the
coated tubule that remains loses its coat and fuses with endosomes or
phagolysosomes (pathway 2b).
in endosomes and nonlysosomal multivesicular bodies
has also been demonstrated (Matlin et al., 1983). This
study demonstrates the previously unreported phenomenon of coated vesicle and endosomal diacytosis operating in tandem. The multiple transport pathways present
in these cells are summarized in Figure 21.
Since these studies were carried out in vitro, some of
the absorbed protein, or protein that was localized in the
intercellular spaces or basal lamina, may have diffused
in from the cut edges of the tissue or from the connective
tissue side of the sample. However, in both the case of
coated vesicle transport and endosomal transport, the
patchy distribution of reaction product in the intercellular space or basal lamina, which is usually associated
with a recess in the plasma membrane, indicates vesicular transport and release rather than a type of diffusion artifact.
Developmental Significance
The transport of proteins or other substances across
the membranous chorion may have important implications for the normal growth and development of the
This investigation was supported by National Institutes of Health grants HD 11658 and RR 00169. We wish
to thank Dr. Andrew G. Hendrickx and the staff of the
Perinatal Biology Unit of the California Primate Research Center for assistance in obtaining the tissue; the
laboratory of Dr. Jerry Hedrick, Department of Biochemistry and Biophysics, for determining the isoelectric points of the isozymes; and Ms. Katherine Wilson
for preparing the drawing. J.M.W. was supported by
NIH training grant HD 07131 through a portion of this
Abrahamson, D.R., and R. Rodewald (1981) Evidence for sorting of
endocytic vesicle contents during the receptor-mediated transport
of IgG across the newborn rat intestine. J. Cell Biol., 91t270-280.
Aswell, G., and J. Harford (1982)Carbohydrate-specific receptors of the
liver. Ann. Rev. Biochem., 51531-554.
Brown, M.S., S.K. Basu, J.R. Falck, Y.K. Ho, and J.L. Goldstein (1980)
The scavenger cell pathway for lipoprotein degradation: Specificity
of the binding site that mediates the uptake of negatively-charged
LDL by macrophages. J. Supramol. Struct. 13r67-81.
Burton, G.J. (1982)Review article. Placental uptake of maternal erythrocytes: A comparative study. Placenta, 3t407-434.
Croze, E.M., D.M. More, J. Kartenbeck, W.W. Franke, and D.J. More
(1982)Three size classes of spiny
- .(clathrin-coated)vesicles in rodent
liver. J. Cell Biol., 95t429a.
Dantzer, V. (1982) Transfer tubules in the porcine placenta. In: Structural and Functional Organization of the Placenta, Bibliotheca
Anatomica, Vol. 22. P. Kaufmann and B.F. King, eds. S. Karger,
Basel, pp. 144-149.
Davies, P.F., H.G. Rennke, and R.S. Cotran (1981)Influence of molecular charge upon the endocytosis and intracellular fate of peroxidase activity in cultured arterial endothelium. J. Cell Sci., 49:6986.
Farquhar, M.G. (1983) Multiple pathways of exocytosis, endocytosis,
and membrane recycling: Validation of a Golgi route. Fed. Proc.,
Fisher, G.W., and L.I. Rebhun 11983) Sea urchin cort,iral p-annlw
exocytosis is followed by a burst of membrane retrieval via uptake
into coated vesicles. Dev. Biol., 99t456-472.
Geuze, H.G., J.W. Slot, G.J.A.M. Strous, H.F. Lodish, and A.L. Schwartz
(1983) Intracellular site of asialoglycoprotein receptor-ligand uncoupling: Double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell, 32277-287.
Graham, R.C., and M.J. Karnovsky (1966)The early stages of absorp-
tion of injected horseradish peroxidase in the proximal tubules of
mouse kidney: Ultrastructural cytochemistry by a new technique.
J. Histochem. Cytochem., 14t291-302.
Helenius, A., I. Mellman, D. Wall, and A. Hubbard (1983) Endosomes.
Trends Biochem. Sci., 8t245-250.
Herzog, V. (1983) Transcytosis in thryoid follicle cells. J. Cell Biol.,
Kawasaki, T., R. Etoh, and I. Yamashina (1978) Isolation and characterization of a mannan-binding protein from rabbit liver. Biochem.
Biophys. Res. Commun., 81r1018-1024.
King, B.F. (1980) Developmental changes in the fine structure of the
rhesus monkey amnion. Am. J. Anat., 157t285-307.
King, B.F. (1981) Developmental changes in the fine structure of the
chorion laeve (smooth chorion) of the rhesus monkey placenta.
Anat. Rec., 200t163-175.
King, B.F. (1982a) Comparative anatomy of the placental barrier. In:
Structural and Functional Organization of the Placenta, Bibliotheca Anatomica, Vol. 22. P. Kaufmann and B.F. King, eds. S. Karger, Basel, pp. 13-28.
King, B.F. (1982b) The role of coated vesicles in selective transfer
across yolk sac epithelium. J. Ultrastruct. Res., 79t273-284.
King, B.F. (1984) The fine structure of the placenta and chorionic
vesicles of the bush baby, Galago crassicaudata Am. J. Anat.,
King, B.F., and A.C. Enders (1970) Protein absorption and transport
by the guinea pig visceral yolk sac placenta. Am. J. Anat., 129t261288.
King, B.F., and A.C. Enders (1971) Protein absorption by the guinea
pig chorioallantoic placenta. Am. J. Anat., 13Ot409-430.
King, B.F., and J.M. Wilson (1983) A fine structural and cytochemical
study of the rhesus monkey yolk sac: Endoderm and mesothelium.
Anat. Rec., 205r143-158.
Kraehenbuhl, J.P., and M.A. Campiche (1969) Early stages of intestinal absorption of specific antibodies in the newborn. An ultrastructural, cytochemical, and immunological study in the pig, rat,
and rabbit. J. Cell Biol., 42345-365.
Luft, J. (1971)Ruthenium red and violet. I1 Fine structural localization
in animal tissues. Anat. Rec., 171:369-416.
Matlin, K., D.F. Bainton, M. Pesonen, D. Louvard, N. Genty, and K.
Simons. (1983) Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of ModinDarby canine kidney cells. I. Morphological evidence. J. Cell Biol.,
Maunsbach, A.B. (1966) Absorption of 1125-labeledhomologous albumin by rat kidney proximal tubule cells. A study of microperfused
single proximal tubules by electron microscopic autoradiography
and histochemistry. J. Ultrastruct. Res., 15t197-241.
Mehrishi, J.N. (1972)Molecular aspects of the mammalian cell surface.
In: Progress in Biophysics and Molecular Biology, Vol. 25. J.A.V.
Butler and D. Noble, eds. Pergamon Press Ltd., Oxford, pp. 1-70.
Moxon, L.A., A.E. Wild, and B.S. Slade (1976) Localisation of proteins
in coated micropinocytotic vesicles during transport across rabbit
yolk sac endoderm. Cell Tissue Res., 171t175-193.
Orlic, D., and R. Lev (1973)Fetal rat intestinal absorption of horseradish peroxidase from swallowed amniotic fluid. J. Cell Biol., 56t106119.
Renston, R.H.. A.L. Jones, W.D. Christiansen. G.T. Hradek. and B.J.
Underdown (1980) Evidence for a vesicular transport mechanism
in hepatocytes for biliary secretion of immunoglobulin A. Science,
208: 1276-1 278
Rodewald, R. (1973) Intestinal transport of antibodies in the newborn
rat. J. Cell Biol., 58t189-211.
Stahl, P.D., J.S. Rodman, M.J. Miller, and P.H. Schlesinger (1978)
Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc.
Natl. Acad. Sci. U.S.A., 75:1399-1403.
Stenseth, K., U. Hedin, and J. Thyberg (1983) Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in
cultured mouse peritoneal macrophages. Eur. J. Cell Biol., 31t1525.
Straus, W. (1983) Mannose-specific binding sites for horseradish peroxidase in various cells in the rat. J. Histochem. Cytochem., 31.7884.
Thyberg, J., and K. Stenseth (1981) Endocytosis of native and cationized horseradish peroxidase by cultured mouse peritoneal macrophages. Variations in cell surface binding and intracellular traffic
and effects of colchicine. Eur. J. Cell Biol., 25t308-318.
Van Deurs, B. (1978) Horseradish peroxidase uptake into the rat choroid plexus epithelium, with special reference to the lysosomal
system. J. Ultrastruct. Reds., 62,155167.
Van Deurs, R., M. Miller, and 0. Amtorp (1978)Uptake of horseradish
peroxidase from CSF into the choroid plexus of the rat, with special
reference to transepithelial transport. Cell Tissue Res., 187t215234.
Willingham, M.C., and I. Pastan (1980)The receptosome: An intermediate organelle of receptor-mediated endocytosis in cultured fibroblasts. Cell, 21t67-77.
Willingham, M.C., and I. Pastan (1983) Formation of receptosomes
from plasma membrane coated pits during endocytosis: Analysis
by serial sections with improved membrane labeling and preservation techniques. Proc. Natl. Acad. Sci. U.S.A., 80t5617-5621.
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uncoated, epithelium, vesicle, monkey, trophoblast, transport, acros, coates, horseradish, peroxidase
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