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Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat.

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THE ANATOMICAL RECORD 217:203-219 (1987)
Trophoblast-Decidual Cell Interactions and
Establishment of Maternal Blood Circulation in the
Parietal Yolk Sac Placenta of the Rat
ALERICK 0. WELSH AND ALLEN C. ENDERS
Department of Human Anatomy, University of California, Dauis, CA 95616
ABSTRACT
Implantation sites from rats were studied on days 6, 7, and 8 of
pregnancy to determine the sequence of events in the formation of blood spaces in
the trophoblast that is part of the parietal wall of the yolk sac placenta and to
determine how trophoblast gains access to maternal blood. The maternal blood
flowing through these spaces is the source of nutrients that reach the embryo via
the visceral endoderm. Tissues were prepared for light microscopy, scanning electron
microscopy, and transmission electron microscopy. Trophoblast blood spaces are
derived from the lateral intercellular spaces of trophoblast cells and are present in
a collapsed condition until day 8, when maternal vessels are tapped by trophoblast.
These spaces then contain circulating maternal blood, and trophoblast cells reflect
adaptations for metabolic exchange including thinning of trophoblast covering
Reichert’s membrane and the appearance of numerous fenestrations, with and
without diaphragms, in the areas where trophoblast is attenuated. Between days 6
and 7 decidual cells appear to form a barrier between the maternal circulation and
trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelia1 basal lamina, and then the decidual cells that are juxtaposed
to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of
the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain
access to the maternal vessels by replacing decidual cells or by direct imposition of
trophoblast cell processes between endothelial cells.
The importance of the visceral yolk sac as a functional
placenta in laboratory rodents is well established (Anderson, 1959; Lambson, 1966; Padykula et al., 1966;
Jollie, 1968, 1986; Carpenter, 1980). During early gestation, prior to the formation of the complementary chorioallantoic placenta, the yolk sac placenta is especially
important since it is the only placental structure present. There have also been some studies about the role
that the parietal wall of the yolk sac plays in presenting
materials to the visceral wall of the yolk sac via the yolk
sac cavity (Everett, 1935; Seibel, 1974; Carpenter, 1980;
Parr and Parr, 1986). It has been shown that trophoblast
cells form a network of channels that distributes maternal blood throughout the parietal wall of the yolk sac
placenta (Everett, 1935).These trophoblast blood spaces
are present during midpregnancy (day 8 to day 16 in the
rat) and are in the layer of trophoblast cells composing
the outermost layer of the parietal wall of the yolk sac
(i.e., bilaminar omphalopleure). This layer of trophoblast cells is situated between decidual cells and Reichert’s membrane. Reichert’s membrane probably plays a
significant role as a selective barrier to the movement
of material from maternal blood (flowing through trophoblast blood spaces) to the yolk sac cavity (Jollie, 1968;
Carpenter, 1980). Most studies of parietal yolk sac have
been concerned with middle to late gestation periods,
and early development of the parietal wall of the yolk
0 1987 ALAN R. LISS, INC
sac has not been adequately studied. For example, while
it is generally known that the parietal wall of the yolk
sac ruptures during the later stages of pregnancy (Bridgman, 1948; Jensh et al., 1977; Dickson, 1979), there is
little information in the literature about the formation
of trophoblast blood spaces or even the cellular events
involved in trophoblast cell migration to the maternal
blood supply.
In order to tap maternal vessels, trophoblast cells must
penetrate or displace the uterine luminal epithelium
and the layers of decidual cells separating it from the
maternal vessels. There has been some controversy
about the degree of invasiveness of trophoblast cells
through both the uterine luminal epithelium and the
decidualized uterine stroma. Many studies have favored
the concept that penetration of the luminal epithelium
in rats and mice is a result of programmed cell death
(Finn and Hinchliffe, 1964; Enders and Schlafke, 1967;
Finn and Bredl, 1973; El-Shershaby and Hinchliffe, 1974,
1975) and not a result of cytolytic invasion of trophoblast. It is commonly agreed, however, that trophoblast
is phagocytic (Sherman and Wudl, 1976). El-Shershaby
and Hinchliffe (1975) proposed that uterine luminal penetration in the mouse involves autolysis or self-digestion
accompanied by trophoblast phagocytosis. In their ultraReceived June 19,1986; accepted October 6, 1986.
204
A.O. WELSH AND A.C. ENDERS
structural study they found little evidence of lysosomal
activity in trophoblast cells prior to uterine epithelial
deterioration but did find evidence of autolysosomes in
the uterine epithelial cells. Although a few studies have
focused on the presence of lysosomes and lysosomal enzymes in trophoblast and uterine luminal epithelium
(Christie, 1967; Hall, 1971; Elangovan and Moulton,
1980), to date there have been no ultrastructural cytochemical studies performed in order to localize lysosomal enzymes during normal implantation. Unfortunately the degeneration of the uterine luminal epithelium, its penetration by trophoblast, and the later
stromal cell development have not been studied in light
of more recent concepts of cell death (Wyllie, 1981).
The lytic potential of trophoblast has also been brought
into question with regard to penetration of the uterine
luminal basal lamina. That trophoblast penetration of
the uterine stroma of rats and mice is delayed somewhat
a t the uterine luminal basal lamina has been known for
some time (Enders and Schlafke, 1967; Potts, 1969) and
is generally accepted (Glasser, 1985). Only recently,
however, has it been shown that in the rat, decidual cell
processes rather than trophoblast cell processes penetrate the basal lamina (Schlafke et al., 1985).Here again
the active agents in the disruption of a potential barrier
to trophoblast migration are not well understood but
imply a n active role by maternal tissues.
Once decidual cells have penetrated the basal lamina
they come into direct contact with trophoblast cells.
Although many light microscopic studies have demonstrated the invasive characteristics of rodent trophoblast in a variety of ectopic sites (see review by Sherman
and Wudl, 19761, there have been few morphological
studies dealing with the interactions of trophoblast and
normal decidua. Those that do consider this subject do
so indirectly and derive their information from paraffinembedded material (Kirby and Cowell, 1968; Billington,
1971).
To clarify some of the events of trophoblast blood space
formation, trophoblast penetration of decidua, and tapping of maternal vessels, we have studied rat implantation sites on days 6, 7, and 8 of normal pregnancy.
Previous studies from this laboratory have examined
trophoblast cell-epithelia1 cell interactions (Enders and
Schlafke, 1967), basal lamina penetration (Schlafke et
al., 1985), and the structure of mature decidua (Welsh
and Enders, 1983).
MATERIALS AND METHODS
Primigravid Sprague-Dawley rats used for this study
were maintained on a light cycle of 14-hour light and 10hour dark. Males were placed overnight in cages with
two females, and females with positive vaginal smears
the following morning were considered to be in day 1of
pregnancy. Implantation sites were collected on days 6,
Fig. 2. By day 7 many changes have occurred at the interface of
maternal and embryonic tissues. Trophoblast cells (Tr)overlap one
another extensively with very little extracellular space separating
them (between arrows in 2 and 2a). In Figure 2a, a higher magnification of a region similar to that shown in Figure 2, fenestrations that
first appear on day 7 are seen (arrowheads) in the thin trophoblast
flanges. The decidual cells (Dec) are elongated along the mesometrialantimesometrial axis in Figure 2 and a decidual cell process (Pr)is
penetrating the uterine luminal basal lamina (BL) in Figure 2a. Note
also the presence of collagen (C) in the spaces between decidual cell
processes and between the decidual cell process and the trophoblast
cell. Longitudinal section. Day 7, 2 P.M. Figure 2, ~ 9 , 0 7 0Figure
.
2a,
x 16,600.
Fig. 1 . This electron micrograph illustrates some of the earliest features of trophoblast blood space
formation. Processes on the basal-lateral surfaces of adjacent trophoblast cells (Tr) interdigitate (upper
right) and the lateral intercellular space is somewhat open. Just below the lateral intercellular space
there is an apical junctional complex connecting the trophoblast cells (arrow). At this time trophoblast
cells are in close association with the apical surfaces of uterine luminal epithelial cells (Ep). Longitudinal
section. Day 6 , 2 P.M. x 11,180.
TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT
205
206
A.O. WELSH AND A.C. ENDERS
Fig. 3. This is a higher magnification of a region similar to that in the previous micrograph. The
trophoblast cells (Tr) are separated by a small extracellular space (arrows) and the thin flange of the
trophoblast cell adjacent to Reichert’s membrane (RM) is fenestrated (arrowheads). In the center portion
of the micrograph a process (Pr)of a decidual cell (Dec) is penetrating the residual uterine lumina1 basal
lamina (BL). There is flocculent material of moderate electron density in the extracellular space between
folds and processes of decidual cells. Longitudinal section. Day 7 , 2 P.M. ~20,800.
7, and 8. Although most tissues were collected a t 2 P.M.,
some were collected a t 2 A.M., 8 A.M., and 8 P.M. Animals
were anesthetized with ether, laparotomized, and perfused with Dulbecco’s phosphate-buffered saline (Gibco,
Grand Island, NY) followed by fixative with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.3). Perfusion was performed as previously
described (Welsh and Enders, 1985). Isolated implantation sites were fixed for a total of 1.5-2 hours.
Some sites were carefully trimmed and postfixed in
2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.3,
for 1-2 hours. These were dehydrated in ethanol and
propylene oxide and embedded in Durcupan Araldite
resin. Thick sections (1-2 pm) were stained with Azure
B, and thin sections cut on a Sorvall MT-2 or MT-5000
ultramicrotome were stained with uranyl acetate and
lead citrate. Electron micrograph negatives were taken
with a Zeiss EM-10 or a Philips 410 electron microscope.
207
TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT
variably open at the basal-lateral border (Fig. 1).The
basal surface of trophoblast abuts a basal lamina that is
destined to become part of Reichert’s membrane. Basallateral processes from adjacent trophoblast cells interdigitate with one another in the basal-lateral compartment and also frequently overlap one another. With
further development the basal-lateral juxtaposition of
cells becomes more frequent and the overlapping more
extensive.
Day 7
Fig. 4. Extensive development of decidual cells (arrows)in the region
adjacent to the conceptus appears to promote the separation of maternal blood vessels from the conceptus. Longitudinal section. Day 7, 2
P.M.
X130.
Other sites were cut with a razor blade and torn in a
manner that would expose the conceptus, extraembryonic membranes, and uterine stroma. These were
also postfixed in 2% osmium tetroxide in phosphate
buffer, dehydrated in ethanol, placed in acetone, and
critical-point dried with COZ. Specimens were selected
under a dissecting microscope, mounted on aluminum
stubs, and coated with gold in a metal evaporator, and
photomicrographs were taken with a Philips SEM 501
scanning electron microscope.
OBSERVATIONS
Since the first trophoblast blood spaces are derived
from the lateral intercellular spaces of mural trophoblast, their formation begins with the formation of mural trophoblast of the blastocyst. Early in gestation (days
5 and 6), these lateral spaces are demarcated by junctional complexes on the apical-lateral border and are
At 2 P.M. on day 7 thin flanges from trophoblast cells
may extend for great distances across the apical-lateral
and basal-lateral surfaces of adjacent cells (Fig. 2). In
other areas the shorter flanges or processes mentioned
above persist. There is usually little intercellular space
between adjacent trophoblast cells. By 2 P.M. on day 7
fenestrations appear in trophoblast cell flanges (Figs. 2,
3). These tend to develop earlier and more extensively
in the flanges on the basal surfaces apposed to Reichert’s
membrane. The fenestrations vary in diameter (60-90
nm) and usually appear to have a diaphragm. In places
wider gaps are present and no diaphragm is observed.
Shorter flanges that project into the lateral and basal
compartments also have fenestrations.
By the afternoon of day 7 the luminal epithelium in
the lateral walls of the implantation chamber is gone.
There are still epithelial cells in the mesometrial, antimesometrial, proximal, and distal areas of the implantation chamber.
The antimesometrial decidual cells are well developed
and appear to have segregated the maternal vasculature
from the trophoblast by some distance (Fig. 4). Decidual
cells in the region next to the implantation chamber are
tightly packed with extensive areas of membrane apposition between cells (Fig. 2). Although it appears that
membrane specializations may occur (Figs. 5, 7a), no
attempt was made to characterize them in this study.
Collagen bundles occur often between apposed decidual
cells (Fig. 5). There are lysosomelike granules and granules with a heterogenous matrix (Figs. 3, 5). Golgi elements, vesicles, and other small granules are numerous.
Nuclei are somewhat irregular in outline and contain a
moderate amount of heterochromatin and prominent
nucleoli. On the afternoon of day 7 the decidua appears
healthy with few signs of cellular degeneration except
in the decidual crypt (Welsh and Enders, 1983).
At this time the layer of decidual cells lining the
implantation chamber is polarized. Polar views of the
implantation chamber (i.e., sections cut perpendicular
to the mesometrial-antimesometrial axis of the implantation chamber) demonstrate that the apical surface is
thrown into folds and processes that surround amorphous extracellular matrix or bundles of collagen or
both (Fig. 5). The collagen bundles are oriented along a
mesometrial-antimesometrial axis. Some processes extend through the residual uterine luminal basal lamina
and contact trophoblast cells. Following this penetration
it is common to find collagen fibrils on the luminal side
of the uterine luminal epithelial basal lamina between
decidual cell processes and trophoblast and between decidual cell processes and the basal lamina (Fig. 2a).
There are numerous intermediate filaments and microtubules in these decidual cells, and they are generally
oriented along the mesometrial-antimesometrial axis.
A.O. WELSH AND A.C. ENDERS
208
Fig. 5. When polar sections of the implantation chamber are studied,
a high degree of cellular and extracellular organization becomes evident. In this micrograph, fields of microtubules (MT)and intermediate
filaments (F) are seen in cross section in the decidual cells. Processes
or flanges at decidual cell surfaces facing trophoblast are wrapped
around collagen fibrils that are oriented in a mesometrial-antimeso-
metrial direction. These processes occur primarily at one surface and
give the decidual cells polarity. There is a decidual cell process on the
luminal side of the uterine luminal basal lamina (arrows) indicating
penetration of the basal lamina. Note the increased density in the
regions where trophoblast and decidual cell membranes are closely
apposed. Polar section. Day 7,2 P.M. ~28,700.
These cells often contain lipid droplets and areas of
concentrated glycogen. The mitochondria are of intermediate electron density with lamellar cristae. There
are numerous stacks of rough endoplasmic reticula
@ER).
contain a few maternal blood cells and platelets but are
mostly open spaces (Figs. 6,7,10). In nonperfused specimens the spaces are packed with maternal blood cells or
collapsed. Trophoblast cell bodies may be juxtaposed to
decidual cells or Reichert’s membrane or individual cells
may span the blood space and be juxtaposed to both.
Trophoblast cell processes cover the areas of Reichert’s
membrane and regions of decidual cells not in contact
with trophoblast cell bodies (Fig. 7). Moreover, these
processes tend to cover Reichert’s membrane more completely than the decidual cell surface. Processes are usually thin and fenestrated, and many of the larger
fenestrations do not have diaphragms. Frequently decidual cells form part of the wall of trophoblast blood spaces
(Fig. 7a), although their contribution is never as great
a s that of trophoblast cells. Fenestrated trophoblast
Day 8
Prior to day 8 the plasma membranes of adjacent
trophoblast cells are variably close together, and trophoblast spaces are only potential blood spaces. By the
afternoon of day 8, however, after trophoblast cells tap
the maternal vessels and blood begins flowing through
these spaces, they open up to become large channels
that are limited by the lateral cell membranes of trophoblast cells and trophoblast cell processes. After vascular
perfusion the spaces within the trophoblast layer may
Fig. 6. This light micrograph was prepared from a day 8 implantation site. Note that the embryo occupies only the central portion of the
implantation chamber. The upper box indicates the region shown at a
higher magnification in Figure 6a and the lower box indicates Figure
6b. Figure 6a illustrates the opening of a maternal blood vessel into
the former uterine lumen (arrow). Most of the uterine lumina1 epithelium has been sloughed into the lumen and is degenerating. Figure 6b
shows the presence of trophoblast blood spaces VBS) that have been
cleared of maternal blood during perfusion fixation. The decidual cells
(Dec) in the regions next to the trophoblast (Tr) are more flattened,
darker, and smaller, indicating the onset of apoptosis. The parietal
endoderm (PE) and visceral endoderm N E ) are evident. Longitudinal
section. Day 8 , 2 P.M. Figure 6, ~ 8 0Figure
.
6a, ~ 3 0 0Figure
.
6b, ~ 3 0 0 .
210
A.O. WELSH AND A.C. ENDERS
Fig. 7. This electron micrograph taken from a region similar to that
seen in Figure 6b shows the relationship of decidual cells (Dec),trophoblast (Tr), and parietal endoderm (PE) around a perfused trophoblast
blood space (TBS). Trophoblast processes protrude into the trophoblast
blood space and are thin and fenestrated. Trophoblast processes that
cover Reichert’s membrane are usually thin and fenestrated as well
but there are no substantial breaks in the continuity of the trophoblast. The bracket indicates the region shown in Figure 7a where the
trophoblast is discontinuous over the decidual cells and the decidual
cells participate in the wall of the trophoblast blood space (between
arrows). Longitudinal section. Day 7, 2 P.M. Figure 7, x3,800. Figure
7a, ~ 2 6 , 2 0 0 .
TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT
Fig. 8. This scanning electron micrograph illustrates the trabecular
nature of the trophoblast processes that extend from one side of the
trophoblast blood space (TBS) to the other, connecting the regions of
trophoblast that are juxtaposed to the decidual cells (Dec) and Reichert’s membrane. Parietal endoderm and Reichert’s membrane cannot
211
be seen in this micrograph because the trophoblast is folded back and
covers them (arrows). In this micrograph the visceral endoderm is at
the bottom WE). Figure 8a is a higher magnification of the blood space
in Figure 8. Note the maternal blood cells in the trabecular meshwork.
Longitudinal view. Day 8 , 2 P.M. Figure 8, X 1,750. Figure 8a, X5.700.
212
A.O. WELSH AND A.C. ENDERS
Fig. 9. This scanning electron micrograph gives a polar view of the implantation chamber. The area is
similar to that shown in the previous micrograph except the egg cylinder was lost during processing. The
parietal endoderm (upper left) is clearly visible in Figure 9a. The shape of the trophoblast trabeculae seen
in Figure 9a in conjunction with the information in Figure 8 suggests that the trabeculae have a
mesometrial-antimesometrial orientation. Polar view. Day 8, 2 P.M. Figure 9, X 750. Figure 9a, ~ 2 , 9 0 0 .
flanges that protrude into the blood spaces form incomplete septa and have maternal blood cells on both surfaces when viewed in transmission electron micrographs.
Scanning electron micrographs give the impression
that the flanges seen in thin sections are trabeculae that
form a network that crosses the trophoblast blood space
between the trophoblast covering Reichert’s membrane
and the trophoblast juxtaposed to decidual cells (Figs. 8,
9).
By day 8 trophoblast cells have grown to giant proportions. These cells are electron lucent and contain many
free ribosomes and polyribosomes. Stacks of RER cisternae are abundant in some cells (Fig. 7) but scarce in
others. When scarce, RER cisternae tend to be more
vesicular. The mitochondria are also fairly electron lucent and have few cristae. There are numerous lipid
droplets, and patches of intermediate filaments occur in
the cytoplasm but are most frequently found at the
apical border next to decidual cells. The nuclei are very
large and irregular in outline. Nucleoli are well developed. By day 8 it appears that trophoblast cells are
connected to other trophoblast cells primarily by desmosomes. There is little evidence of typical apical junctional complexes by the afternoon of day 8.
The layer of polarized decidual cells juxtaposed to the
residual basal lamina of the uterine lumina1 epithelium
on day 7 is no longer seen by the afternoon of day 8.
Instead, the cells along the inner wall of the implantation chamber are flattened, elongated along the meso-
metrial-antimesometrial axis (Fig. 61, and have no
apparent apical polarization; that is, they do not have
flanges on the surface toward the trophoblast. Many
decidual cells in proximity to the implantation chamber
and trophoblast are degenerating (Figs. 6, 11, 12). Although there are variations between cells, there are
many similarities in the morphology of their degeneration, which appears to be apoptotic rather than necrotic.
Nuclear chromatin marginates into large dense masses,
nuclei often become deeply indented and may fragment,
and nucleoli appear as dispersed coarse granules (Fig.
11).Decidual cells lose contacts with adjacent cells and
round up. The cytoplasm becomes condensed; that is, the
organelles become densely compacted with little intervening material (Fig. 12). The RER often dilates, and
the cells become smaller and often break up into small
fragments. In many cases the condensing cells or cell
fragments may contain nuclear fragments and usually
a n abundance of intermediate filaments.
When this degeneration occurs in the vicinity of trophoblast cells, as it usually does, processes from trophoblast
cells engulf the dying cells and cell fragments (Fig. 12).
Trophoblast cells sometimes engulf whole decidual cells
as they round up and detach from adjacent cells. Trophoblast cells also phagocytize some erythrocytes but not
others. In addition to these phagosomes, trophoblast cells
also contain inclusions that resemble secondary lysosomes or phagolysosomes, but few dense bodies that
might include a significant lysosomal population are
TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT
213
way as to form a portion of the wall of the blood vessel
p i g . 13). This participation of decidual cells occurs before and after maternal blood begins circulating in
trophoblast blood channels. Endothelial cells that appear osmotically damaged are also fairly common. These
cells are extremely pale, with dispersed organelles and
damaged membranes.
As trophoblast cells engulf decidual cell fragments,
they occupy the area once occupied by decidual cells and
hence become close to the maternal blood vessels. When
connections are observed between maternal blood vessels and trophoblast blood spaces, they usually appear
as a direct transition from trophoblast to endothelium
(Fig. 14).Although decidual cells are not seen participating directly in this coupling, there are spaces between
decidual cells that do contain erythrocytes and could
presumably form blood channels.
At the same time that maternal blood begins flowing
in trophoblast blood spaces, maternal vessels open into
the mesometrial uterine lumen (Fig. 6). The epithelium
is sloughed into the lumen, and degenerating, necrotic,
epithelia1 cells are found mixed with maternal blood
cells. There is no trophoblast in this region at this time.
DISCUSSION
Fig. 10. In this light micrograph of a polar view of the implantation
site the maternal blood vessels are all cut in cross section, a n indication that they have a mesometrial-antimesometrial orientation. The
egg cylinder was lost during tissue processing. On the right, the trophoblast blood space is filled with maternal erythrocytes whereas, on the
left, it is empty. Polar section. Day 8, 2 P.M. X 150.
present. Although trophoblast cells phagocytize effete
decidual cells and occasionally a n erythrocyte, maternal
phagocytic cells do not appear to be activated.
The blood vessels in the implantation region become
oriented in a mesometrial-antimesometrial direction.
This is especially apparent in polar views of the implantation site in which the vessels appear in cross section
(Fig. 10). In the vicinity of the implantation chamber the
blood vessels are composed of continuous, nonfenestrated endothelial cells, often without a clearly distinguishable basal lamina. These differ from the blood
vessels in the deeper stroma and those of mature decidua (Welsh and Enders, 1985). Decidual cells are usually in close proximity to endothelial cells and there is a
material of moderate electron density filling the space
between them. It is common to find decidual cells that
are in contact with endothelial cells, and they often
become interposed between endothelial cells in such a
The results reported here support the position that the
uterus plays a n active role during embryo implantation
and yolk sac placentation in the rat. Although trophoblast cells migrate to the maternal vessels that are the
blood supply for the parietal wall of the yolk sac placenta, they appear to do so because degeneration of the
decidualized stromal cells that intervene permits trophoblast migration to the maternal vessels. This is similar
to the earlier situation when spontaneous or programmed cell death of the uterine luminal epithelium
(El-Shershaby and Hinchliffe, 1975) permits trophoblast
migration to the uterine luminal basal lamina. Kirby
and Cowell (1968) articulated the position that trophoblast cells, while capable of phagocytizing degenerating
decidual cells, might not be able to dispose of healthy
decidual cells. They derived this assumption from work
of Billington (1963) that indicated deciduomata break
down in the central area first, then progressively toward
the outer regions. They presented no morphological data
to indicate the cellular basis of cellular degeneration in
this model.
There have been no thorough electron microscopic
studies of trophoblast migration or invasion of decidual
tissue during normal pregnancy in the rat. Several light
microscopic reports have described the invasiveness of
trophoblast in ectopic sites (Fawcett, 1950; Kirby, 1965;
Porter, 1967). These studies have shown that mouse
trophoblast is highly invasive of nonuterine tissues and
uterine stroma during the estrus cycle, but not invasive
of decidualized uterine stroma. There does not appear to
be a consensus of opinion about the cellular aspects of
invasion in ectopic sites, due, no doubt, to the limits of
observation imposed by the methods used. Porter (19671,
however, used ultrastructural analysis to report active
trophoblast invasion of renal parenchyma and found
that it was accompanied by little necrosis, but there was
no mention made of phagocytosis of kidney tissue by
trophoblast. Some investigators reported that trophoblast cells transplanted to ectopic sites formed a meshwork that was confluent with the blood vessels of the
host (Fawcett, 1950; Porter, 1967).
214
A.O. WELSH AND A.C. ENDERS
Fig. 11. Although decidual cells are usually detached from adjacent
cells and have undergone some fragmentation before being surrounded
by trophoblast processes, in this micrograph trophoblast processes
(arrows) are surrounding a decidual cell (Dec) that is beginning fragmentation (upper right) but is still attached to the underlying cells.
Notice the deep indentations of the nuclear membrane and the dispersed granular nucleolus. A trophoblast blood space ('I'BS) containing
a maternal platelet is at the top of the micrograph. The decidual cells
are elongated and contain an abundance of intermediate filaments.
Longitudinal section. Day 8,8 P.M. x 14,800.
The work presented here shows the initial degeneration of the antimesometrial decidua that is restricted to
the area adjacent to trophoblast. The pattern of cellular
degeneration is often similar to that described by Kerr
et al. (19721, who proposed the term apoptosis to describe
the controlled physiological deletion of cells during em-
bryonic development. They delineated a two-part process that included first the formation of apoptotic bodies
and second their phagocytosis and degeneration within
nearby cells. They contrasted this to necrosis or nonphysiological cell death (Wyllie, 1981) caused by trauma or
noxious substances.
TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT
Fig. 12. This micrograph illustrates the interface of trophoblast and
degenerating decidual cells. Although the decidual cell (Dec) in the
lower right is still attached to the underlying cells, it appears to be
separating from them. At the left there are decidual cell fragments
that have not been phagocytized while the center of the micrograph is
occupied by fragments that have been phagocytized. In the upper left
215
is a trophoblast blood space CTBS). Much of the cytoplasm of the
decidual cells and their fragments (pale areas) is occupied by intermediate filaments. Also some of the cytoplasmic fragments contain nuclear fragments while others do not. Apparent phagolysosomes (PL)
are also present in the trophoblast. Longitudinal section. Day 8 , 8 P.M.
~9,180.
2 16
A.O. WELSH AND A.C. ENDERS
Fig. 13. Decidual cells (Dec) often form part of the walls of maternal blood vessels (MBV) and appear to
have some junctional attachment to endothelial cells (arrows).Day 8 , 2 P.M. ~ 2 3 , 3 5 0 .
In a manner similar to other apoptotic cells, degenerating decidual cells separate from adjacent cells and,
after condensation and fragmentation of the nucleus
and cytoplasm, become small, dense bodies. The cytoplasmic fragments sometimes contain nuclear fragments, but other organelles within the fragments often
appear normal. These free cytoplasmic bodies do not
show ultrastructural evidence of degradation. Most decidual cell apoptotic bodies are found phagocytized by
trophoblast cells although some are free in the intercellular space between decidua and trophoblast. Also,
trophoblast cells sometimes begin phagocytizing individual decidual cells as they round up and detach from
the adjoining decidual cells before there is any sign of
fragmentation.
After they are taken up by other cells, apoptotic bodies
undergo degenerative changes that are independent of
lysosomal enzyme action (Kerr et al., 1972). Lysosomal
enzymes, however, are implicated in the final degradation of the phagosomal contents because preexisting secondary lysosomes of the ingesting cell are depleted and
residual bodies are produced. Trophoblast cells that ingest decidual cell debris do not appear to contain a n
extensive lysosomal system, as their hydrolase enzyme
activity is not high (Christie, 1967). Since cell death in
deciduoma occurs in the absence of trophoblast, one
would expect either a maternal phagocytic response or
some sort of autolytic system where the by-products
would be expelled into the uterine lumen. Unfortunately, there is no ultrastructural information about
this process. In the case of decidual cell breakdown,
lysosomal as well as nonlysosomal events of cellular
breakdown might occur outside of the influence of the
ingesting cells. If decidual cells are the source of lysosomal enzymes that are responsible for the final stages of
cell breakdown, decidual cell death would be autolytic
a s well as apoptotic.
Several in vitro studies have addressed the question
of trophoblast invasiveness. Salomon and Sherman
(1975) favored a cytolytic mechanism to explain the displacement of uterine cell monolayers by mouse trophoblast outgrowths. Glass et al. (1979) found no evidence
TROPHOBLAST,STROMA, AND YOLK SAC DEVELOPMENT
217
Fig. 14. Trophoblast (Tr)appears to tap maternal capillaries (MVB) by first becoming part of the blood
vessel wall (large arrows). It is not clear whether this occurs through a displacement of decidual cells or
is a direct intervention on the part of trophoblast. In this micrograph decidual cell .fragments occur in
trophoblast, and fragmenting decidual cells are seen adjoining trophoblast. In the lower part of the
micrograph, decidual cells (Dec) are in very close proximity (arrows) to endothelial cells (Endo). Longitudinal section. Day 8,8 P.M. ~ 7 , 4 5 0 .
of’cell lysis but proposed that a variety of cultured mouse
cells retract upon contact with mouse trophoblast. In
further studies (Kubo et al., 19811, they found that both
attachment and outgrowth of mouse trophoblast could
be reversibly inhibited by protease inhibitors.
Mouse trophoblast was able to degrade complex radiolabeled extracellular matrix that contained glycoproteins, elastin, and collagen (Glass et al., 1983).Although
this may be important in species where trophoblast is
primarily interacting with uterine matrix, in the rat
and mouse there is little extracellular matrix between
decidual cells at the time trophoblast cells come into
contact with uterine stroma. In these species interaction
of trophoblast cells with extracellular matrix may play
a secondary role to interactions between trophoblast and
decidual cells.
The degeneration of large numbers of decidual cells in
the region juxtaposed to trophoblast appears to be a
programmed and controlled form of cell death that allows remodeling of the implantation chamber without
disrupting the growth and development of the embryo
and the integrity of the uterus. The trophoblast acts on
the decidualized stroma by phagocytizing cellular fragments and fragmenting cells. The decidual cell mem-
branes do not appear to be damaged as would be expected
if the trophoblast were cytolytic. In other tissues where
apoptosis is observed, large numbers of cells can also be
removed without disrupting the basic arrangement of
tissues and their function Wyllie, 1981). Later in gestation, days 13-16, a massive degeneration of the remaining antimesometrial decidua and primary trophoblast
giant cells occurs in the region adjacent to the reformed
uterine lumen (Welsh and Enders, 1983, 1985), but this
appears to be more necrotic than apoptotic.
As the decidual cells lining the implantation chamber
degenerate and are phagocytized by trophoblast, the
latter comes closer to the stromal blood vessels. From
the information presented in this study it appears that
trophoblast somehow occupies the position held by endothelial cells, but it is not clear how trophoblast comes
to occupy this position. Other studies have shown that
decidual cell processes penetrate endothelial basal laminae (O’Shea et al., 1983; Welsh and Enders, 1985; Parr
et al., 1986), and this study clearly shows decidual cells
in conjunction with endothelial cells lining vessels prior
to and during the time of blood circulation in trophoblast
blood spaces. Presumably as these decidual cells degenerate trophoblast could occupy the space once held by
218
A.O. WELSH AND A.C. ENDERS
decidual cells and thus tap maternal vessels. On the
other hand, trophoblast might be capable of a more
direct approach to maternal blood.
The fate of endothelial cells after they come in contact
with trophoblast is not clear. It appears that trophoblast
can form junctions with adjacent endothelial cells, but
how endothelial cells are disposed of is not obvious.
There is no evidence of apoptosis of endothelial cells
although in a few instances there appears to be necrosis;
that is, swollen, pale cells with osmotic damage. Also,
since the implantation chamber is rapidly increasing in
size at this time, mitosis of endothelial cells and growth
of blood vessels may not keep pace with changes in the
decidua, and the endothelium could become discontinuous without extensive cell death.
An incidental observation that argues against invasiveness of rat trophoblast in opening maternal vessels
is found in the mesometrial part of the implantation
chamber on day 8. There is no trophoblast in this area
at this time, yet the uterine lumina1 epithelium and
basal lamina degenerate and maternal vessels become
open to the mesometrial portion of the implantation
chamber. This occurs a t the same time that blood vessels
are tapped by trophoblast in the antimesometrial area.
Epithelial cell death in the mesometrial region differs
from that in the region juxtaposed to trophoblast (unpublished observations). According to guidelines proposed by Kerr et al. (1972), the mesometrial epithelia1
cells appear necrotic whereas the epithelia1 cells in the
region of trophoblast appear apoptotic.
After the maternal vessels are breached, maternal
blood can move into trophoblast blood spaces. The
method of perfusion of the uterus used in this study
demonstrates that there is circulation of blood through
these spaces. Scanning and transmission electron micrographs indicate that the blood spaces are lined by trophoblast lying against Reichert’s membrane and in contact
with decidual cells. The two layers of trophoblast cytoplasm are connected by trabeculae or flanges. These
may give structural support, keeping the two layers of
trophoblast from separating, and may direct blood flow.
The arrangement of trophoblast along Reichert’s
membrane suggests a potential path for substances moving between the maternal circulation and visceral endoderm. Trophoblast is characteristically very thin in
this area with many fenestrations. Carpenter (1980)
studied the permeability of the hamster parietal yolk
sac placenta on day 8 of gestation using horseradish
peroxidase (HRP) and found that trophoblast, Reichert’s
membrane, and parietal endoderm were highly permeable to HRP. He also noted the appearance of both open
and closed fenestrations in the trophoblast lining Reichert’s membrane.
Jollie (1968) used ferritin and Thorotrast to study the
permeability of the rat parietal yolk sac between days
12 and 22 of gestation. He found that the parietal yolk
sac was a selectively permeable barrier to material entering the yolk sac cavity.
While this manuscript was in preparation a study by
Parr and Parr (1986) was published. They intravenously
injected various macromolecular tracers to study the
permeability of the early decidual cell layer that separates the yolk sac from maternal blood vessels. On days
6 and 7 they found that the permeability of the decidual
cell layer decreased as the mass of the tracers increased.
On day 9, however, the tracers had ready access to
Reichert’s membrane and the yolk sac placenta although the permeability of the stromal vessels per se
was decreased at this time. They noted that the decidual
cell layer separating the maternal blood vessels and the
yolk sac placenta was greatly reduced, and that there
was maternal blood adjacent to Reichert’s membrane
and in the uterine lumen mesometrial to the ectoplacental cone, but they did not mention mural trophoblast.
They suggest that the tracers reach the implantation
chamber from blood spaces adjacent to Reichert’s membrane or from blood in the space adjacent to the ectoplacental cone.
The work presented in this paper illustrates the cellular basis for the observations of Parr and Parr (1986).
The decidual cell layer is reduced by degeneration of the
cells, probably apoptotic, and their phagocytosis by
trophoblast cells. The maternal vessels are then tapped
and maternal blood flows through the trophoblast blood
spaces that are adjacent to Reichert’s membrane.
Although this study focused primarily on the development of mural trophoblast, incidental observations suggest that there are also blood channels in the
abembryonic trophoblast later on day 8 but these cells
have no direct contact with maternal vessels. On the
other hand, the superficial layer of polar trophoblast
that is mesometrial to the ectoplacental cone does tap
directly into maternal vessels channeling maternal blood
into the blood spaces in the polar trophoblast.
ACKNOWLEDGMENTS
It is a pleasure to acknowledge the valuable discussions and support of Sandra Schlafke during the course
of this investigation. This research was supported by
grant HD 10342 from the National Institute of Child
Health and Human Development.
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