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Multivesicular bodies and related structures of the syncytiotrophoblast of human term placenta.

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Multivesicular Bodies and Related Structures of the
Syncytiotrophoblast of Human Term Placenta '
B. J. MARTIN AND S. S. SPICER
Institute of Pathobiology, Department of Pathology, Medical University of
South Carolina, 80 Barre Street, Charleston, South Carolina 29401
ABSTRACT
Multivesicular bodies and related structures of the syncytiotrophoblast of human term placenta have been studied ultrastructurally and
cytochemically. Circular profiles of vesicles termed pre-multivesicular bodies (premvb's) were observed often near Golgi complexes. Multivesicular bodies (mvb's
with electron lucent matrices (L-mvb's), mvb's with electron dense matrices
(D-mvb's), and dense bodies were also prevalent organelles of the syncytium.
These organelles all exhibited dialyzed iron reactivity and staining characteristics
suggesting that the organelles were related. Material within Golgi saccules, Golgi
vesicles, vesicles of pre-mvb's, and vesicles of mvb's were all reactive with osmium zinc iodide (OZI). This OZI reactivity further indicated a relationship
between the organelles. The matrix of the mvb's exhibited variable reactivity for
acid phosphatase (AcPase) but such activity was not encountered in Golgi elements of syncytiotrophoblast. It is suggested that the mvb's of human term
placental syncytium are likely formed by aggregation of vesicles of Golgi origin
into pre-mvb's, that the pre-mvb's are then capable of transforming into L-mvbs,
D-mvb's, and finally into dense (residual) bodies. It is also suggested that mvb's
may function in the selective hydrolysis and transport of endocytosed proteins,
including globulins. Additional bodies with intense acid phosphatase activity
were designated elongate bodies. Other distinctive organelles designated dense
cored spheroids were occasionally observed in the syncytioplasm.
The placental syncytiotrophoblast functions as a barrier between the fetal and
maternal vascular systems, as well as in
the transport of nutrients and metabolic
wastes (Grosser, '27; Hagerman and Villee,
'60; Boyd and Hamilton, '70), and is apparently the site of synthesis and secretion
of both protein and steroid hormones
(Villee, '69; Wattenberg, '58; Benirschke
and Driscoll, '67; Pierce and Midgley, '63;
Dreskin, Spicer and Greene, '70). Thus, it
is not surprising that the syncytioplasm is
packed with a diverse population of inclusions and organelles. Among the numerous ultrastructural studies of the human
pIacenta are investigations that have associated certain of the syncytioplasm organelles with a particular function (Tighe,
Garrod and Curran, '67; Dreskin et al., '70;
Burgos and Rodriguez, '66; Sato, '66).
However, none has made a careful attempt
to describe and classify the inclusions and
organelles of the syncytiotrophoblast, a
ANAT. REC., 175: 15-36.
logical prerequisite to understanding their
functional significance. This investigation
is the beginning of an effort of this type
and considers in detail the multivesicular
bodies and related structures of the syncytium of human term placenta, particularly emphasizing morphologicaI and certain cytochemical characteristics of the
multivesicular bodies (mvb's).
MATERIALS AND METHODS
Tissue was excised as quickly as possible
from full term placenta obtained postpartum or by Cesarian section. Some of the
specimens were fixed in 1.5-6.25% glutaraldehyde in 0.1 M cacodylate buffer at
0-5" C and pH 7.4 for 60 minutes or in
Karnovsky's formaldehyde-glutaraldehyde
fixative (Karnovsky, '65) at 0-5" C and
pH 7.4 for 60 minutes, Other specimens
Received Feb. 29, '72. Accepted Oct. 3, '72.
1 Research supported by Public Health
Service
grants AM-I0956 and AM-11028.
15
16
B. J. MARTIN AND S. S. SPICER
were fixed in either Karnovsky’s fixative
or in 3% glutaraldehyde in 0.12 M Millonig’s phosphate buffer (Millonig, ’61) at
room temperature and pH 7.4 for 90 minutes. Following several buffer rinses, selected samples of the fixed tissues were
post-fixed for one hour in either 2% collidine-buffered osmium tetroxide or in 1%
osmium tetroxide in Millonig’s buffer, dehydrated through an ethanol series, transferred to propylene oxide, and embedded in
Epon 812. Ultrathin sections were stained
conventionally with uranyl and lead salts
and examined with an AEI-6B or a Hitachi
HS-8 electron microscope.
Tissue fixed in either glutaraldehyde
or formaldehyde-glutaraldehyde was sectioned at 40 in a cryostat and stained for
three hours with dialyzed iron (DI) solution (Rinehart and Abul-Haj, ’51). This
procedure provides, in addition to staining
of extracellular sites, selective staining of
intracellular acidic mucosubstances at the
ultrastructural level (Wetzel, Wetzel and
Spicer, ’66). Fixed specimens were also
rinsed in 7.5% sucrose, sectioned at 40
in a cryostat and incubated 30, 60 and 90
minutes at 37°C in the Gomori medium
(Barka and Anderson, ’63) for demonstrating acid phosphatase ( AcPase). After
staining with DI or incubation for AcPase
the cryostat sections were post-fixed in 1%
buffered osmium tetroxide for 90 minutes.
Unfixed minced specimens were placed in
an osmium zinc iodide (OZI) solution
(Niebauer, Krawczyk, Kidd and Wilgram,
’69) for 1 to 24 hours at room temperature
or at 0.5”C. Tissue prepared by the latter
three methods was dehydrated in ethanol
and embedded in Epon 822. Ultrathin sections of the material treated by these three
cytochemical techniques were examined
without the usual staining with heavy
metal salts.
RESULTS
Morphological preparations
On the basis of morphology and cytochemical characteristics four basic types
of organelles were identified that appeared
to be mvb’s or to be related to mvb’s.
Pre-rnultiuesicular bodies. Circular profiles of loosely associated vesicles, 0.06 pm
in diameter were observed regularly in the
syncytioplasm (fig. 1). Profiles of these
bodies measured 0.49 -C 0.06 pm (diameter 2 S.E.M.) and the number of peripheral vesicles varied from 12 to 23 averaging 17. The vesicles were associated in
blastula-like configurations which isolated
regions of cytoplasm. The material enclosed within the sphere of vesicles had a
distinctive, fine particulate texture, indicating that this internal space represented a
separate or isolated compartment. Vesicles
identical to those forming the periphery of
the body were regularly observed in its internal matrix (fig. 1); thus the body had
the basic form of a multivesicular body.
Because of the prevalence of these bodies
in the syncytium and their morphological
similarity to multivesicular bodies they
were designated premultivesicular bodies
( pre-mvb’s),
Light multivesicular bodies (L-mvb’s).
These profiles were 0.71 2 0.03 IZmin diameter and were generally round or ellipsoid (fig. 3 ) . They had a single peripheral
membrane that was often irregular and
apparently interrupted by small discontinuities (arrows, fig. 3 ) . The number of
0.06 pm internal vesicles varied from as
few as two to as many as 30. The matrix
varied from lighter than, to about the same
density as the surrounding cytoplasmic
background and consisted of finely particulate material.
These profiles of mvb’s often enclosed
one and occasionally two spherical structures which have been termed nucleoids
(fig. 4 ) . The nucleoids consisted of finely
particulate, homogeneous material with
greater density than the surrounding matrix. They had no limiting membrane, and
measured about 0.23 pm in diameter. The
nucleoids were evident without staining but
became more electron dense after lead
staining. Since the nucleoid was about onethird the diameter of the mvb’s, i t was possibly present in all L-mvb’s but because of
the plane of section, was observed only in
slightly more than 30% of the profiles.
Dense multivesicular bodies (D-mvb’s).
These mvb’s were very similar to L-mvb’s.
Their mean diameter was 0.68 2 0.03 pm
and their profiles were consistently circular in contour (figs. 5, 6 ) . They had a
single, sometimes irregular, peripheral
membrane that often gave the body a serrated appearance. They differed from
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
Lmvbs, however, in having an electron
dense matrix which contrasted with the
translucent internum of the enclosed vesicles (figs, 5, 6). Many of these mvb’s displayed nucleoids (fig. 6), and some may
have contained nucleoids that were not apparent because of the dense matrix.
D-mvbs frequently displayed a “corona” or
zone of surrounding cytoplasm that only
infrequently contained vesicles similar to
the internal vesicles of the mvb (fig. 6 ) .
Dense bodies. Dense bodies differed
considerably from the preceding types of
mvb’s (fig. 7). They were smaller, only
0.39 i 0.02 pm in diameter, and were
often irregular and less frequently circular or ellipsoid in profile. They were limited
by a single membrane which contained a
“halo” region along its inner surface. This
appearance was due to periodic dense lines
that extended radially from the matrix of
the body to the limiting membrane (fig. 8).
These organelles, although usually lacking
distinct internal vesicles, were considered
related to the mvb’s because of occasional
profiles which had the appearance of dense
bodies and, in addition, contained internal
vesicles (fig. 9). The matrix of the dense
bodies was much more dense than the surrounding cytoplasm and often was mottled
with several irregularly shaped dark flecks
(fig. 8 ) . On occasion, internal structures
were observed within the matrix of the
dense bodies that were larger than most of
the flecks and that displayed a periodic
pattern of very electron dense dots (fig.
10). These structures were termed organoids.
Additional types of syncytiotrophoblast
organelles. Certain regions of the syncytium contained dense populations of
fingerlike structures designated elongate
bodies (fig. 23a). The elongate bodies averaged 0.08 pm in diameter, were usually 0.4
to 0.5 pm in length, and were frequently
quite tortuous. They were rarely observed
deep within the syncytium but rather appeared concentrated into a narrow band
about 0.5 pm below the maternal surface
of the syncytium.
Another organelle, termed the dense
cored spheroid, was observed most frequently in the superficial portion of the
syncytioplasm occasionally in close proximity to the Golgi. The dense cored spher-
17
oids were always round in profile, about
0.20 pm in diameter and had a very characteristic translucent rim about 0.01 pm in
width that separated the peripheral membrane from the dense core (fig. 23a).
These spheroids may correspond with
structures thzt have been referred to as
“membrane bound dense structures” (Ashley, ’65, see diagram, fig. 16) or with organelles designated dense “secretory granules’’ (Tighe et al., ’67; see fig. 11). The
spheroids differed from the latter “secretory granules,” however, in having only a
single limiting membrane. This distinctive
structure was not observed in other cell
types of the placenta and is apparently a
unique structure in the syncytioplasm.
Dialyzed iron staining
The maternal surface of placenta has a
very extensive surface coat which stains
intensively with colloidal iron (Tighe et al.,
’67). However, in the present investigation
a dialyzed iron technique was employed
(Wetzel, Wetzel and Spicer, ’66) which
provides intense staining of acidic mucosubstances at the maternal surface (figs.
11-17) as well as selective staining of
these substances at intracellular sites.
Structures with a morphology similar to
that of pre-mvb’s were observed frequently
in the DI stained material. Their mean
diameter was 0.70 2 0.03 pm and they
were conspicuous for the DI staining of
their peripheral vesicles (fig. 11). The internal vesicles of the bodies vaned in number from 3 to 15 and were dispersed randomly throughout the internum of the
body. The membrane of these vesicles
stained variably and usually with less intensity than the peripheral membrane. The
matrix of the bodies consisted of a flocculent material either lighter than, or
equal in density to the surrounding cytoplasm. Numerous profiles classified within
this group exhibited distinct 0.06 pm peripheral vesicles but others had a complex
periphery only somewhat vesicular (fig.
12). The nucleoids observed within the
matrix of these inclusions did not stain
with DI.
Numerous L-mvb’s were observed in the
DI stained preparations. They measured
0.81 2 0.02 pm in diameter and their characteristic discrete peripheral membrane
18
B. J. MARTIN AND
stained with DI (fig. 13). The membrane
of the internal vesicles was usually lightly
stained and the nucleoid remained DI
negative.
The D-mvb’s of this preparation measured 0.60
0.04 pm and their DI staining was identical to that of the L-mvb’s
except that the membrane of the internal
vesicles seemed less reactive or, in many
cases, appeared unreactive (fig. 14).
Organelles classified as dense bodies
measured 0.60 -C 0.03 pm. The DI staining
of their peripheral membrane was similar
to that of the D-mvb’s (fig. 1 5 ) . This staining served further to relate the dense
bodies to the mvb’s. Since the flecks, characteristic of this group, were very electron
dense in the morphologic specimens it was
not possible to determine conclusively their
DI reactivity. However, close observation
suggested an increased electron density
after DI staining. Occasionally bodies were
observed that appeared transitional between dense bodies and the preceding types
of mvb’s since they contained both internal
vesicles and flecks (fig. 16).
Some of the dense bodies contained organoids. These organoid containing dense
bodies measured 0.42
0.04 pm in diameter. Usually the organoids occurred singly
but on rare occasions two or even three
were observed within the same profile (fig.
17). They were generally spherical, about
0.07 p in diameter, and contained the
periodic internal structure already described. Although the organoids had considerable inherent density, their increased
electron opacity after DI staining indicated
that they were DI reactive.
The only other organelles within the
syncytium with DI reactivity were the
dense cored spheroids. The morphologically translucent rim at the inner surface
of the limiting membrane of the spheroid
stained with DI indicating a mucosubstance at this site (fig. 12, arrow).
*
*
Acid phosphatase activity
The acid phosphatase reactivity of
mvb’s in the syncytium of human term
placenta varied from strongly positive to
negative within contiguous regions of the
same preparation (fig. 19). Specimens
such as that of figure 19 were purposely
S. S. SPICER
incubated longer than the optimal time
(e.g., 90 minutes) in order to demonstrate
the nonreactivity of some mvb’s. The mean
diameter of the AcPase positive mvb’s
(0.61 & 0.04 pm) did not differ significantly from that of the AcPase negative
bodies (0.65 r+ 0.03 pm). Since the inherent density of the unreactive bodies was
the same as, or less than, the surrounding
cytoplasm the unreactive bodies might represent L-mvb’s. Whether the acid phosphatase activity differed for L-mvb’s and
D-mvb’s, however, was complicated by the
possibility that cytochemical processing
may well have diminished the inherent
density of the unreactive mvb’s; and the
AcPase reaction product might have obscured the inherent density of the reactive
bodies. Although the nucleoid was possibly
obscured in some of the intensely AcPase
positive mvb’s (fig. 1 9 ) , in many cases it
could be clearly observed as a 0.20
round profile with little or no reactivity
within the otherwise AcPase-positive matrix of the mvb (fig. 20). In optimally incubated sections only the matrix of the
mvb’s revealed AcPase activity and the vesicles appeared unreactive.
Rather infrequently AcPase positive,
ellipsoid structures within the size range of
the dense bodies were observed. The presence of the reaction product precluded the
detailed observation of the internal structure of these bodies necessary for their
positive identification.
Low power observation of the syncytium
revealed definite zones along the villi that
contained dense populations of elongate
bodies. The intense reactivity of these
bodies was emphasized by the frequent
presence of virtually negative mvb’s
(arrows, fig. 23) in the region and by the
intense staining observed after only 30
minutes incubation time. The dense cored
spheroids were AcPase negative (arrowheads, fig. 23).
The Golgi apparatus of the syncytium
was consistently unreactive for AcPase
(arrow, fig. 21) even where observed in
close proximity to intensely reactive bodies
(fig. 21). It was of interest that the Golgi
apparatus within the cytotrophoblast, however, usually exhibited acid phosphatase
activity (fig. 22).
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
19
Osmium zinc iodide staining
The cisternae of the granular reticulum
In certain regions of the syncytiotropho- and nuclear envelopes of cytotrophoblast
blast, material lining the irregular cis- differed from those of the syncytium in
ternae of the endoplasmic reticulum (fig. lacking OZI reactivity. The technique was
24) and the cisternae of the nuclear envel- not selective for the syncytiotrophoblast,
ope (fig. 26) stained with osmium zinc however, since both of the above sites often
iodide (OZI). However, in other regions of stained in fetal endothelial cells and fibrothe same section these sites were often blasts. Golgi elements also stained with
OZI negative. Additional sites that stained OZI in the cytotrophoblast, fetal endotheintensely with OZI included : the periph- lium, and fibroblasts. The characteristic
eral vesicles of the presumed pre-mvb’s lipid droplets of the syncytium were OZI
(fig. 24, large arrows), the internal vesi- negative.
cles of mvb’s (fig. 24; arrows, fig. 25), and
In this investigation the inclusions were
Golgi saccules and assorted vesicles (fig.
classified
according to their morphology;
24a).
The mvb’s observed in the OZI prepara- however, a study of their relative sizes protions measured 0.73 2 0.06 pm in diam- vides interesting information (see table 1).
eter and contained 5 to 30 internal vesicles The often very different preparative proapproximately 0.06 f l in diameter (fig. cedures apparently differentially affected
25). In all morphologic respects they were the sizes of the various organelles and in
thus identical to the L- and D-mvbs but, some cases prevented precise identification
unfortunately, the quality of fixation did of body since the technique obscured some
not allow differentiation between the two of the identifying characteristics, It is
types. The internal vesicles were the only clear from table 1 that the L-mvbs are
structures stained in the mvb’s and, al- largest, that the D-mvb‘s are somewhat
though they usually disclosed very high smaller, and that the pre-mvbs and dense
affinity for OZI (fig. 24), some of the bodies are considerably smaller than either
vesicles within an mvb showed less reactiv- type of mvb. An interesting inconsistency
ity (fig. 25).
in the size correlation is that the pre-mvb’s
Configurations composed of OZI-positive of the DI preparations rank fourth in size
vesicles that were grouped in a blastula- and are thus much larger than the organlike arrangement closely resembled the pre- elles classified as pre-mvb’s from the other
mvb’s in the morphologic preparations preparations. This suggests that as a result
(figs. 24, 26). Like the pre-mvb’s observed of the DI technique some type of larger
in the morphologic preparations, these
configurations consisted of 8-21 (average organelle has been included within this
15) 0.06 pm peripheral vesicles surround- group. These are likely organelles similar
ing an internal matrix. However, the bodies to that of figure 12 which has a complex,
averaged only 0.34 pm in diameter com- but not distinctly vesiculated periphery.
Although variations in the thickness of
pared to 0.50 pm for the pre-mvb’s obthe
syncytiotrophoblast made assessment
served in the morphologic preparations.
The smaller size may result from shrink- difficult, the pre-mvb’s, mvb’s and dense
age in the OZI material. Frequently, the bodies were more often observed nearer
pre-mvb’s of OZI-treated specimens were the maternal than the fetal surface. No
observed in close association with the differences in the distribution of the variOZI-reactive Golgi complex (fig. 26), and ous types of mvb’s were observed in this
their vesicles exhibited the same size, study and, in general, they seemed to have
shape and solid OZI staining as did vesi- no systematic relationship to each other or
cles apparently derived from the Golgi. The to any other organelle in the syncytium.
Golgi saccules and vesicles of the pre-mvb’s There was a tendency for small vesicles to
and mvb’s, all appeared to have a similar occur in close proximity to the mvb’s and
affinity for the reagent since only these the pre-mvb’s often were observed near
structures reacted when the specimens Golgi complexes. The mvb‘s seemed to
were stained in the cold or for a short time occur in both thick and thin regions of the
syncytium.
period.
20
B. J. MARTIN AND S. S. SPICER
TABLE 1
Rank
Tn$
N&y;-er
sured
organelle
Diameter 1
2 S.E.M.
Preparation
Duncan's multiple range test 2
fim
1 L-mvb
2 Mvb's
3
4
5
6
7
8
9
10
11
12
13
14
15
L-mvb
Pre-mvb
D-mvb
Negative
Transitionalmvb
Positive-mvb
Densebody
D-mvb
Pre-mvb
DenseBody
+ Organoid
DenseBody
Pre-mvb
DenseBody
17
33
23
23
23
18
6
DI
021
Morphology
DI
Morphology
AcPase
Morphology
0.81 2 0.02
0.74 & 0.06
0.71 k 0.03
0.70? 0.03
0.68 ? 0.03
0.65 t0.03
0.65 & 0.05
16
16
17
8
12
AcPase
DI
~DI
Morphology
DI
0.61 f 0.04
0.602 0.03
0.60 k 0.04
0.49 2 0.06
0.42k0.04
12
11
10
Morphology
OZI
021
0.39 & 0.02
0.34 2 0.02
0.20 2 0.01
1 An average of the smallest and largest diameter of elliptical profiles was taken as the true diameter of the
organelle.
2 The bar extending down from each type of inclusion, respectively, indicates the other types of inclusions
that are not significantly different in size according to the Duncan multiple range test (Steel and Torrie, '60).
DISCUSSION
The term multivesicular body refers
only to a morphologic structure characterized by a number of vesicles contained
within a membrane-limited body (Sotelo
and Porter, '59), and thus carries no functional implications. This term has been applied to a morphologically heterogeneous
group of structures that probably function
in different ways (Palay, '60; Behnke, '63;
Farquhar and Palade, '62; Gordon, Miller
and Bensch, '65; Robbins, Marcus and
Gonatas, '64; Rosenbluth and Wissig, '64;
Balis and Conen, '64; Droller and Roth,
'66; Holtzman and Dominitz, '68; Friend,
'69; Kilarski and Jasknski, '70; Nunez and
Becker, '70; Bibefield, '71; King and
Enders, '71). For example, many of the
structures contain pleomorphic internal
vesicles and are thought to be autophagic
lysosomes since they contain vesiculated
membranes apparently derived from partially lysed cell organelles (Behnke, '63;
Smith and Farquhar, '66). Some mvb's,
however, contain internal vesicles of uniform size and appearance and are devoid
of materials suggesting autophagic activity. These mvb's are lysosome-like in that
they often exhibit AcPase reactivity; but
they appear to play some specialized role in
cell metabolism other than autophagy.
Some mvb's may function in relation to en-
docytosis and indeed mvb's with similar
morphology have been shown to take up
endocytosed exogenous proteins in a number of cell types (Rosenbluth and Wissig,
'64; Farquhar and Palade, '62; Miller and
Palade, '64; Friend and Farquhar, '67;
Kraehenbuhl and Campiche, '69). The
major population of mvb's in the syncytium of human term placenta appears to
fall within this latter group.
The numerous physiologic activities of
the placental syncytium (Hagerman and
Villee, '60; Benirschke and Driscoll, '67)
make it quite possible that pre-mvb's, mvb's
and dense bodies represent a heterogeneous population of organelles engaged in
separate and independent functions. However, the morphologic similarities of these
structures, their similar patterns of DI
staining, and their OZI reactivity suggests
that they likely represent different states of
an organelle engaged in one basic function.
For example, the results of this investigation may be interpreted as suggesting the
following sequence. Pre-mvb's appear to
develop in the region of the Golgi complex
and establish an internal compartment
separate from the cytoplasm. The vesicles
at the periphery of the pre-mvb may then
coalesce into a single membrane to provide
the characteristic morphology of the
L-mvb's .
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
The nucleoid, which is first seen in
Gmvbs, to our knowledge has not been
described or illustrated previously. It may,
therefore, be peculiar to the mvbs of the
placental syncytium and indicate a unique
physiological activity of the mvb’s in this
site. Sotelo and Porter (’59) have described
a “nucleoid in the mvb’s of rat ovum.
However, this structure is an aggregate of
vesicles and thus is morphologically distinct from the nucleoids of placental mvb’s.
The nucleoid also resembles morphologic d y some of the cores or “nucleoids” of
microbodies (Bruni and Porter, ’65; Hruban
and Recheigl, Jr., ’69). However, the nucleoid-containing structures of placenta
are obviously not microbodies because they
have the internal vesicles characteristic of
mvb’s and exhibit acid phosphatase activity. The smaller and more dense D-mvb’s
could easly result from condensation of the
L-mvb’s.
The relationship of the dense bodies to
the previous forms is more tenuous. The
term dense body (or dense granule) has
been used in a general sense to designate a variety of electron opaque cytoplasmic structures (Daems, Wisse and
Brederoo, ’69; Holtzman, ’69; Hruban and
Recheigl, ’69) and it has been used in placenta by some investigators to indicate virtually all membrane limited bodies of high
density (Ashley, ’65; Tighe et al., ’67;
Wynn, Panigel and MacLennon, ’71). In
this investigation the term is applied
specifically to the one type of body as described. It has been suggested that the
dense bodies in the placental syncytium
are “pure” lysosomes and that they give
rise to mvb’s (Tighe et al., ’67). However,
protein uptake studies of both bat (Enders
and Wimsatt, ’71) and guinea pig (King
and Enders, ’71) chorioallantoic placenta
have shown that endocytosed peroxidase
and ferritin appear in mvb’s within a few
minutes and that after a few hours they
are found predominantly within dense
bodies. These results are more consistent
with the morphological and cytochemical
observations of this investigation and with
them suggest that the dense bodies are
likely a catabolic residuum of mvb’s that
could be termed residual bodies. A specific
relationship between mvb’s and dense
bodies is also suggested by the fact that
21
both types of organelle stain similarly with
DI indicating acid mucosubstance in their
peripheries, and by the fact that they are
the only sites with such reactivity except
for the smaller and clearly different dense
cored spheroids (arrow, fig. 12).
The dense bodies exhibit a peripheral
lucent zone (halo) which is apparently
a unique property of this type lysomal inclusion, as it may be observed in dense
bodies seen in numerous other studies
(Farquhar and Palade, ’62; Smith and
Farquhar, ’66; Tighe et al., ’67; Daems et
al., ’69; Holtzman, ’69; Nunez and Becker,
’70; Biberfield, ’71; Wynn et al., ’71). Although dense bodies are thought to function in the catabolism of endocytosed proteins in a number of cell types (Daems
et al., ’69), the possibility is also to be considered that they could be involved in the
processing of protein for transport.
In light of the recognized capacity of the
placenta to transport immunoglobulin G
(Brambell, Hemmings and Henderson, ’52;
Vahlquist, ’60; Yang, Kleinman, Rosenberg
and Wei, ’71) and the observation in this
investigation that the syncytium of the human term placenta contains an abundance
of large morphologically unique mvb’s it
seems reasonable to suggest that the mvb’s
may be involved in immunoglobulin transport. Further, mvb’s are prevalent in neonate intestinal epithelium and ova (Rhodin,
’63; Sotelo and Porter, ’59; Droller and
Roth, ’66; Kraehenbuhl and Campiche,
’69), both of which are engaged in the
selective transport of proteins. Additional
support for this position comes from experiments with various types of animal
placentae indicating that these tissues take
up certain exogenous proteins, (Ashley,
’65; Sato, ’66; King and Enders, ’70; Enders
and Wimsatt, ’71; King and Enders, ’71),
into vacuoles many of which appear to be
similar to the mvb’s of this study. The view
is also consistent with general suggestions
concerning the possible role of certain
types of lysosomes in selective transport
(deDuve, ’69).
Although Rodewald (’70) concluded
that antibody selectivity occurred at the
cell apical surfaces in the intestine of
neonatal rats, other studies concerning the
transport of globulins from mother to
fetus in rat intestine (Kraehenbuhl and
22
B. J. MARTIN AND S. S. SPICER
Campiche, ’69; Walker et al., ’72) and in
rabbit yolk sac placenta (Brambell, ’66;
Slade and Wild, ’71), suggest that the process involves a nonselective uptake from
the maternal system and a selective release
into the fetal system (Brambell, ’66). The
mechanism by which this selective transport occurs is not understood at the present
time but it is certainly conceivable the lysosoma1structures (e.g., mvb’s in the human
syncytium) may function in this role by
the process of selective degradation.
It is tempting to suggest that the acidic
mucosubstances indicated by the DI staining of the mvbs could, by selective binding, or in some other manner prevent the
hydrolysis of certain globulins. If this were
the case, the mvb’s could select certain
proteins for hydrolysis (e.g., heterologous
globulins) and others for passage intact
(e.g., homologous globulins). This mechanism would then allow the placental maternal surface to endocytose proteins
nonspecifically and within the syncytiotrophoblast the endocytosed proteins that are
not transported could be hydrolyzed to
amino acids. This hypothesis is very similar to that proposed by Leissring and
Anderson (’61) for the selective transport
of proteins in guinea pig intestine since
the pinocytotic vesicles appear to fuse with
and become a part of the mvbs. However,
it differs in suggesting that acidic mucosubstances, a seemingly ubiquitous component of cell plasmalemmas, are significantly involved in protein recognition.
Consistent with numerous other studies
concerning AcPase reactivity of mvb’s
(Gordon et al., ’65; Smith and Farquhar,
’66; Friend and Farquhar, ’67; Holtzman
and Dominitz, ’68), reaction product was
observed within the matrix of the placental mvb’s but was not observed within the
internal vesicles. It is also of interest that
the nucleoids were AcPase negative and
accordingly must comprise an aggregation
of material other than this enzyme. The
considerable variability of mvb’s with respect to their AcPase reactivity suggests
that in some cases the enzyme is either
absent or inhibited or alternatively, that
the substrate cannot penetrate the body in
certain functional states.
The elongate structures that were often
present in large numbers in a stratum
close beneath the maternal surface exhibited intense AcPase activity. They resemble in size and appearance structures
referred to as “tubules” and shown to take
up tracer materials in bat placenta (Enders
and Wimsatt, ’71) and in guinea pig yolk
sac placenta (King and Enders, ’70). Since
the “tubules” and mvbs both were endocytic and appeared to fuse in the guinea
pig placenta it seems possible that the
AcPase positive elongate bodies and mvb’s
of human placenta may function in a similar manner. Alternatively, the elongate
bodies may be involved in autophagic activity such as the disposal of cytoplasmic
membranes resulting from exocytotic activity.
The concentration of the elongate structures in portions of the superficial zone
paralleling the villar surface and their
absence in intervening regions of this
stratum provides additional evidence for
the functional zones that have been suggested for placenta (Burgos and Rodriguez,
’66).
The large amount of AcPase activity in
zones where relatively few Golgi are present and where those present are AcPase
negative, provides an interesting contrast
to many cell types such as leukocytes in
which enzyme activity in the Golgi coincides with genesis of AcPase-rich cytoplasmic granules (Spicer and Hardin, ’69).
The relationship assumes additional interest in light of the AcPase-positive Golgi
of neighboring cytotrophoblast cells which
are precursors of the syncytium.
Lack of knowledge concerning the spec‘ificity of the osmium-zinc iodide technique limits its usefulness; however, its
affinity for mvb’s in rat epididymal tissue
(Friend, ’69) provided a rationale for its
use in this investigation. It is obvious from
the variety of structures and organelles
that stain with the technique (Niebauer,
Krawczyk, Kidd and Wilgram, ’69; Friend,
’69; Friend and Brassil, ’70; Clark and
Ackerman, ’71; Niklowitz, ’71) that it is
not highly specific but more likely stains a
number of different substances which may
or may not contain a common component
(e.g., lipid). Some investigators have suggested an enzymatic basis for the staining
(Clark and Ackerman, ’71), but this is
unlikely since osmium tetroxide greatly
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
inhibits most enzymes and staining was
obtained in the present preparations at
5°C. Notably, the lipid droplets of the
syncytium, believed by some workers to be
involved in steroid hormone production
and secretion did not stain with OZI. Notwithstanding the limitations of the technique, the strong OZI reactivity of the contents of the vesicles of both the pre-mvb’s
and the mvb’s, as well as the vesicles and
saccules of the Golgi, suggests that these
structures may contain the same or similar
materials. Such content of similar material
is consistent with transport of the material
from one structure to the other. In specimens reacted for a brief time or in the cold,
the OZI reactivity was confined to the
Golgi, the pre-mvb’s and the vesicles of
the mvb’s. This similar OZI reactivity and
the consistent association of the pre-mvb’s
with the Golgi, suggests that the vesicles
of pre-mvb’s and those of the mvb’s may
have a common origin in the Golgi
complex.
The consistent OZI reactivity of the nuclear envelope and endoplasmic reticulum
of the syncytium in specimens reacted for
24 hours at 37°C and the absence of such
reactivity from these same sites in the cytotrophoblast provides another contrast between the two cell types and suggests that
only the syncytium is involved in synthesizing the stainable material. Immunohistologic techniques have shown such a
difference also in that human chorionic
gonadotropin (HCG) synthesis occurs in
the syncytiotrophoblast but not in the CYtotrophoblast (Midrrlev and Pierce. ’62:
Mason, Phifer, Spice;, Swallow and Dreskin, ’69). The OZI staining in numerous
profiles of the nuclear envelope and granular reticulum in the syncytiotrophoblast
but not in the cytotrophoblast correlates
well with the ultrastructural immunostaining for HCG observed in the syncytiotrophoblast but not the cytotrophoblast.
(Dreskin et al., ’70). However, the surface
of the syncytiotrophoblast microvilli which
stained for HCG was never OZI positive.
A number of hypotheses have been suggested to explain the formation of mvb’s
(Ericsson, ’64; Novikoff, Essner and
Quintana, ’64; Merker, ’65; Friend, ’69;
Holtzman, ’69; Kilarski and Jasenski, ’70;
Geuskens, ’71), and it is likely that such a
23
heterogeneous group of organelles are
formed by different mechanisms in different tissues. The pre-mvb’s of this study
were often observed in close proximity to
Golgi complexes, and their peripheral vesicles are quite similar to the vesicles of
mvb’s and Golgi complexes. Further, the
peripheral vesicles of pre-mvb’s, mvb vesicles, and Golgi vesicles all stain similarly
with OZI. All of these factors may be interpreted as indicating that vesicles of
Golgi origin aggregate to form the pre-mvb
which then develops into an mvb. The
mvb peripheral membranes probably fuse
with endocytic vacuoles to bring materials
into the mvb, and it seems likely that vesicles of Golgi origin may be incorporated
into the fully mature mvb’s. It is conceivable, however, that the Golgi-like single
saccules occasionally observed enclosing
vesicles (fig. 2) may represent an alternative or possibly an additional mode of mvb
formation in this tissue. For example, these
saccules may completely enclose coated
vesicles, then disruption of the internal
membrane of the saccule with loss of the
coating of the coated vesicles would provide the basic mvb morphology.
ACKNOWLEDGMENTS
The authors are grateful to Ms. Nancy
Smythe for skilled technical assistance and
to Ms. Fran Cameron for ably preparing
the manuscript.
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25
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Abbreviations
N, Nucleus
n, Nucleoid
S, syncytiotrophoblast
M, maternal blood space
PLATE 1
EXPLANATION O F FIGURES
1
A pre-mvb of the syncytium illustrating the peripheral vesicles, the
isolated matrix (X) and internal vesicles. Note the similar vesicles in
close proximity (arrowheads ). Formaldehyde-glutaraldehyde. Uranyl
acetate-lead citrate stain. x 40,000.
2
A folded saccule in the syncytial cytoplasm that partially encloses
coated vesicles (arrow). Note the caviolae (arrowheads) in the
plasmalemma of the syncytial maternal surface. Three percent glutaraldehyde. Uranyl acetate-lead citrate stain. x 30,000.
3 A typical L-mvb. The single peripheral membrane is frequently
interrupted (arrows). Three percent glutaraldehyde. Uranyl acetatelead citrate stain. x 30,000.
4
An L-mvb with a nucleoid (n). A Golgi-like saccule with numerous
budding vesicles may be observed in close proximity to the mvb (X).
Three percent glutaraldehyde. Uranyl acetate-lead citrate stain.
x 25,000.
5
A D-mvb illustrating the very electron dense matrix and serrated periphery of the body. One and one-half percent glutaraldehyde. Uranyl
acetate-lead citrate. x 33,850.
6
A D-mvb which contains a nucleoid ( n ) . The “corona” surrounding
the body is devoid of cytoplasmic structures except for one protrusion
from the mvb and a vesicle in contact with the mvb surface. One and
one-half percent glutaraldehyde. Uranyl acetate-lead citrate stain.
x 20,000.
26
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
B. J. Martin and S. S. Spicer
PLATE 1
27
PLATE 2
EXPLANATION OF FIGURES
7 A dense body located near the maternal surface of the syncytium.
Note the very prominent microtubules oriented parallel to the maternal surface (arrow). One and one-half percent glutaraldehyde.
Uranyl acetate-lead citrate stain. x 55,000.
8
Note the characteristic “halo” at the inner surface of the peripheral
membranes of dense bodies (arrows ). Formaldehyde-glutaraldehyde.
Uranyl acetate-lead citrate stain. x 55,000.
9
A profile of body with the general appearance of a dense body but
with vesicles typical of mvb’s. Note the close association with an
L-mvb. Three percent glutaraldehyde. Uranyl acetate-lead citrate.
X 27,500.
10 The electron dense inclusions of this dense body have the periodic
substructure characteristic of organoids. Three percent glutaraldehyde.
Uranyl acetate-lead citrate stain. x 65,000.
11 Note the DI reactivity of both the peripheral and internal vesicles of
a pre-mvb. A small body that is slightly DI reactive appears attached
or a t least closely associated with the pre-mvb (arrowhead). Vesicles
similar to those of the pre-mvb periphery (arrows) are observed in
close proximity to the pre-mvb. One and one-half percent glutaraldehyde-DI. x 49,500.
12 This body has a complex periphery with a somewhat vesiculated appearance, however, distinct vesicles cannot be observed. Note the DI
reactivity of the normally translucent rim of the dense cord spheroid
(arrow). One and one-half percent glutaraldehyde-DI. x 45,000.
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
B. J. Martin and S. S. Spicer
PLATE 2
29
PLATE 3
EXPLANATION O F FIGURES
13 A nucleoid ( n ) containing L-mvb. Note the intense DI staining of
the peripheral membranes and faint staining of the internal vesicles.
One and one-half percent glutaraldehyde-DI. x 45,000.
14 A D-mvb showing DI staining a t the periphery of the body. The
internal vesicles are virtually unreactive. One and one-half percent
glutaraldehyde-DI. x 50,000.
15 A dense body with its characteristic flecks. Note the DI reactivity
of the peripheral membrane. A small or tangentially sectioned mvb
(X) is associated with the residual body. Formaldehyde-glutarddehyde-DI. x 38,500.
16 Two bodies generally quite similar in appearance, however, one contains predominantly internal vesicles and the other predominantly
flecks. One and one-half percent glutaraldehyde-DI. x 26,700.
17 A profile of a dense body containing two organoids. Note the paracrystalline substructure and apparent DI reactivity of the organoid.
Formaldehyde-glutaraldehyde-DI.x 90,000.
18 In this unstained section an mvb and a dense body appear in close
proximity. When the figure is compared with figures 11 through 17 the
sites of DI-positivity become quite obvious. Some of the internal vesicles of the mvb may be observed but a definite limiting membrance
is not apparent in this micrograph. Three percent glutaraldehyde.
X 45,000.
30
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
B. J. Martin and S. S. Spicer
PLATE 3
31
PLATE 4
EXPLANATION OF FIGURES
19 Acid phosphatase reactivity of the syncytiotrophoblast. The considerable amount of reaction product in the cytoplasmic background resulted from the long incubation time (90 minutes) and emphasizes
the non-reactivity of the mvb at the upper left. Note the intense
AcPase reactivity of the mvb at the lower right. X 31,900.
20
An mvb illustrating the AcPase reactivity of the matrix. The vesicles
and nucleoid are relatively unreactive. Numerous AcPase positive
elongate W i e s are observed near the maternal surface. Three percent glutaraldehyde. AcPase 60 minutes. x 35,500.
21
An AcPase negative Golgi complex is illustrated (arrow) in the syncytioplasm. Note the adjacent AcPase positive elongate body. Six and
one-quarter percent glutaraldehyde. AcPase 60 minutes. x 50,000.
22
A typical AcPase positive Golgi complex of a cytotrophoblast cell
which contrasts with the unreactive Golgi complexes of the syncytium. Six and one-quarter percent glutaraldehyde AcPase. 90 minutes. x 45,000.
23 A region along the maternal surface of the syncytium containing
large numbers of intensely AcPase positive elongate bodies. Note the
two mvb's (arrows) showing little reactivity during the relatively
short incubation time (30 minutes). The dense cored spheroids are
AcPase negative (arrowheads). Six and one-quarter percent glutaraldehyde. AcPase 30 minutes. x 20,000.
23a
32
The elongate bodies (arrows) as observed in the morphologic preparations. Note that the dense cored spheroids (arrowheads), although
within the same size range as the elongate bodies, display the characteristic translucent rim that is not observed in the elongate bodies.
Three percent glutaraldehyde. Uranyl acetate-lead citrate stain.
X 21,300.
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
B. J. Martin and S. S. Spicer
PLATE 4
33
PLATE 5
EXPLANATION OF FIGURES
24
Note the variety of OZI positive structures within the syncytial cytoplasm. Material within the endoplasmic reticulum is OZI positive
and the vesicles of the pre-mvb’s (large arrows) and mvb (small
arrow) stain intensely. OZI 24 hours. x 12,500.
24a
Material reactive with OZI is observed in saccules throughout the
Golgi complex and in numerous nearby vesicles. OZI 24 hours.
X 28,750.
25
Considerable variation in the 021 reactivity of different internal
vesicles within an mvb may be observed at higher magnification.
Note the OZI positive vesicles at the surface of the mvb (arrows).
OZI 24 hours. x 45,000.
26 A premvb in close association with a Golgi complex (G). T h e OZI
reactivity of the syncytial nuclear envelope and its continuity with
the reactive endoplasmic reticulum may be observed. OZI 24 hours.
X 50,000.
34
MULTIVESICULAR BODIES OF THE SYNCYTIOTROPHOBLAST
B. J. Martin and S. S. Spicer
PLATE 5
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