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Initial vascularisation in the pig placentaI. Demonstration of nonglandular areas by histology and corrosion casts

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THE ANATOMICAL RECORD 238:177-190 (1994)
Initial Vascularisation in the Pig Placenta: 1. Demonstration of
Nonglandular Areas by Histology and Corrosion Casts
Department of Anatomy and Physiology, Royal Veterinary and Agricultural Uniuersity,
Frederiksberg C, Denmark (V.D.); Institute of Veterinary Anatomy, Histology, and
Embryology, Justus-Liebig-University, 0-6300 Giessen, Germany (R.L.)
The vascular interrelationship of the well-established porcine placenta has previously been described from vascular casts and histology, but not its developmental stages. This study was performed using
the same methods on 17 sows of well-known stages of gestation ranging
from 9% to 43 days post coitum (p.c.1. At the precontact stage, days 91/2 to
12% P.c., the subepithelial capillaries formed a wide open meshwork of
variable diameter, 3-14 pm, without any difference between meso- and antimesometrial side. At the early contact and adhesion stages (days 13 to 18
p.c.1, the first increase in vasculature was seen at the mesometrial side close
to the embryonic disc of the very long blastocyst at day 15 P.c., 2 days after
the first contact between trophoblast and maternal epithelium was seen. At
day 18 P.c., the areas with dense capillaries increased markedly at the
mesometrial side with the same parallel organization as seen at day 15 P.c.,
whereas the antimesometrial side still had a relative loose appearance comparable to the previous stages. At the early placental stages (days 20Vz to 23
P.c.), the capillary bed formed smooth folds, which in some areas at day
20% days developed into smaller folds or prerugae. Here the capillaries
changed to convoluted forms with slightly bulbous dilations measuring
about 3 0 3 5 pm in diameter. This developmental progress became more
elaborate at day 23: capillaries of the low ridges of prerugae formed irregular dilations up to 50 pm in some areas. At this stage the parallel arrangement of the capillary meshwork characteristic of the previous stage was
not longer discernable. By days 3 2 4 3 P.c., an increase in microscopic folding was present, and the maternal arterioles could be traced to the top of
the ridges, creating the characteristic vascular architecture needed for an
efficient exchange of oxygen, carbon dioxide, and nutrients of the basically
developed porcine placenta. o 1994 Wiley-Liss, Inc.
Key words: Pig placenta, Vascularisation, Scanning electron microscopy,
During placentation the vasculature on the maternal and fetal sides are in continuous development
though different characteristics exist for each type of
species (Dantzer et al., 1988; Leiser and Koob, 1992).
Development of the placental vascular architecture is
of considerable importance in influencing the exchange of nutrients, oxygen, and carbon dioxide between mother and fetus (Faber and Thornburg, 1983).
Other factors, such as change of the components of the
interhemal membrane and prolonged inanition of the
mother pig, have a lesser effect on fetal growth (Hard
and Anderson, 1982) than does restriction of blood flow
(Molina et al., 1985).
Microvascular architecture and maternal-fetal blood
flow interrelations have been investigated in the pig
model between days 35 and 99 of gestation (Leiser and
Dantzer, 19881, though the vascular morphology during earlier placental development has not hitherto
been described. During porcine gestation uterine blood
flow up to 11 days p.c. was found to be similar to the
non-pregnant state, whereafter it increased on days 12
and 13 (Ford and Christenson, 1979). Blood flow to
those uterine segments in contact with the conceptus
was increased in comparison with those segments not
in contact (Ford et al., 1982).
The ultrastructural anatomy of placentation from
days 10 to 19 (Keys and King, 1990) and from days 13
Received March 4, 1993; accepted September 10, 1993.
Address reprint requests to Dr. Vibeke Dantzer, Department of
Anatomy and Physiology, Royal Veterinary and Agricultural University, Bulowsvej 13, DK-1870 Frederiksberg C, Denmark.
Fig. 1. Scanning electron micrograph of endometrial blood vessel
cast viewed from the luminal side from day 9% p.c. a: Low magnification of the circular endometrial folds formed by an open and loose
capillary network. The bar represents 100 Fm. b Higher magnifica-
tion showing the variable diameter of the capillaries of the open and
irregular network, giving sight to vessels deeper in the endometrium.
The bar represents 50 Fm.
to 26 p.c. (Dantzer, 1985) has previously been described, as has embryonic and uterine development
during early porcine gestation (Stroband et al., 1986;
Stroband and Van der Lende, 1990; Dantzer et al.,
1991). The three-dimensional architecture of the exposed complementary maternal and fetal placental
surfaces from day 20 to day 100 has previously been
published (Dantzer, 19841, though no details of vascular development were described.
In a study of the endothelial cells of the subepithelial
endometrial capillaries from cyclic and pregnant pigs
a t days 10-19 after estrus, Keys and King (1988) described increased numbers of fenestrations of capillaries close to the maternal epithelium at day 13 p.c. compared to day 10 p.c. and cyclic stages. The fenestrations
occurred independently of adherent fetal membranes,
and this observation was supported by a later study
(Laforest and King, 1992) of endometrial capillaries on
days 13 and 15 after estrus or mating. However neither
the location, nor the development of the vascular bed
was described.
This study was undertaken to elucidate the development of the endometrial microvasculature during the
initial stages of placentation. Vascular casts and sections from hard plastic embedding were used from day
9 when tissue is similar to non-pregnant stages (Ford
and Christenson, 1979;Keys et al., 1986), and up to day
43 when formation of interdigitating microvilli is still
progressing at the periphery of the chorionic sac (Friess
et al., 1982). Preliminary observations have previously
been reported (Dantzer and Leiser, 1988; Leiser and
Dantzer, 1990; Dantzer et al., 1991).
Uteri from 17 pregnant Danish Landrace sows were
used. The sows were inseminated at 6 am and at 6 pm
on day 0 , 12 hours after the first sign of oestrus. The
uteri were obtained at 10-11 am on gestational day
9%*, 12%*, 14, 14, 15* (3 animals), 16, 18+ (2 animals), 201/2+, 21, 23+, 26, 32+, 33, and 43+ at an
abattoir 4 min after slaughter. Microperfusion fixation
was performed on placentas from all stages (Leiser and
Dantzer, 19881, using 3% glutaraldehyde in 0.07 M
phosphate buffer (Bjorkman et al., 1981) with 3%PVP
(polyvinyl pyrrolidone). Uterine horns from the 5 earliest stages were either microperfusion fixed andlor im-
mersion fixed by gentle installation of the fixative into p.c. the blastocysts were isolated by flushing one of the
the uterine lumen, as earlier described (Dantzer, 1985). uterine horns in order to verify pregnancy.
The contact areas of blastocysts to the mesometrial
The stages marked with an * and + were also used for
preparation of vascular casts. At day 9% and day 12% side of the endometrium were identified in well fixed
areas by gentle dissection in Ringer chloride solution,
whereafter these areas, and part of the corresponding
antimesometrial side, were cut into 2 mm pieces and
immersion-fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer for a further 3 hours. The pieces were
then rinsed in buffer, postfixed in 1%OsO, in 0.1 M
cacodylate buffer at 5°C for 2 hours, and subsequently
dehydrated and embedded in epon or historesin (Technovit 7100 “Kluzer”) by routine methods. From all
stages 2 p.m epon or historesin sections were stained
with toluidine blue and studied by light microscopy in
order to see variations in the vasculature beneath the
maternal epithelium and its relation to the embryonic
attachment sides from the same part of the horn. From
selected stages up to 100 serial sections were also studied.
Parts of the uterine horns (20 cm in length) of the
animals marked above with an * as well as placental
parts from sows marked with a + were used for blood
vessel casts. The uterine horns of sows are long with
anastomosing rami cornuales from arteria uterina towards the rami uterini from arteria ovarica, as well as
of the arteria vaginalis (Boye, 1956). Anastomosing
branches on each side of the injection sites were therefore clamped in order to obtain efficient rinsing and
subsequent filling of a given uterine or placental part
(Leiser and Dantzer, 1988). For the detailed preparation of vascular casts see Leiser and Kohler (19831,
Dantzer et al. (19881, and Leiser and Dantzer (1988). A
mixture of methylmethacrylate of Batson no. 17 compound (Polyscience, Warrington, PA) and Sevriton (R)
was used as a vessel-filling medium because it produces very accurate vessel casts after polymerization
(Risco and Nopanitaya, 1980).
At the early placental stages there is a very dense
vasculature in the myometrium and the circular mucosal folds, which almost overlap creating an extremely
narrow uterine lumen. The specimens for scanning
electron microscopy therefore required thorough corrosion by potassium hydroxide solution before water rinsing and processing for scanning electron microscopy.
To keep an overview of the surface of these folds, large
casts (3.5 x 4 cm2) were first examined in order to
select specific areas; thereafter specimens were prepared by sectioning the cast after embedding in 20%
gelatine cooled to 5°C (compare with Leiser, 1985).
These much smaller pieces were then corroded by po-
Fig. 2. Light micrograph of the endometrium from the mesometrial
side at day 12% p.c. The capillaries (MC) are related to the uterine
epithelium as seen in cyclic diestrous stage, with some distance both
to the maternal epithelium (ME) and among themselves. The bar
represents 50 pm.
Fig. 3. Scanning electron micrograph of vascular cast, at day 12V2
P.c., from an area comparable to Figure 2. The capillary network still
has an open form and some capillaries show short and dilated segments, which were not seen at the previous stage. The bar represents
10 pm.
Fig. 4. Light micrographs from day 15 p.c. a: The endometrium from
the antimesometrial side with extremely columnar epithelium (ME).
The underlaying capillary bed (MC) has not yet developed and is
comparable to the mesometrial side at day 12% p.c. seen in Figure 2.
The bar represents 50 pm. b Endometrium from the mesometrial side
with the maternal epithelium (ME) in contact with the trophoblast (T)
of the fetal membranes. At this side, the capillaries (MC) have
changed markedly, as they have become larger and closer to each
other. Notice their variable size and oval shape. The connective tissue
is rather poor in cellular elements. The bar represents 50 pm.
Fig. 5. Scanning electron micrograph of endometrial blood vessel
cast from the mesometrial side at day 15 p.c. a: The low magnification
on parts of the top of endometrial folds demonstrates how the capillary network is still mainly loose, but lined with areas where the
network is much denser (**I. The bar represents 0.5 mm. b Higher
magnification of the area marked in Figure 5a showing the transition
from a part with a very well-developed and dense capillary network
(DC) to the slightly more open network seen around (OC). The bar
represents 50 pm.
tassium hydroxide as above before final examination in
the scanning electron microscope.
Corrosion casts
The vascular casts from day 9% p.c. showed a very
widely meshed and irregular network of capillaries,
covering large irregular endometrial folds and arRESULTS
ranged in a circular pattern in the uterine horn (Fig.
Precontact Stage-Days 9% to 12%p.c.
la). The caDillaries had a variable diameter (3 to 14
pm). There-was no prevailing orientation of the capilHistology
laries in relation to the circular endometrial folds. The
The capillaries were seen a t irregular intervals be- network was so open that the larger vessels of the subneath the maternal epithelium a t day 9?hand 12%P.c., jacent lamina propria and submucosa were demonwith a distance of 2-8 epithelial cells (10 to 40 pm) strated through the meshes (Figs. la,b, 14).
between them. In most cases there is some distance
At day 12% P.c., the vasculature showed no major
between the capillaries and the epithelium (Fig. 2). No difference from the previous stage, though the diameapparent morphological differences were found be- ters of the capillaries were within the same range and
tween the mesometrial and antimesometrial sides of looked more voluminous due to local swellings (Fig. 3).
uterine horns. Mast cells with metachromatically At these two early stages, there were no differences in
stained granules are frequently observed in the rather morphology between vessels from the mesometrial and
the antimesometrial side.
cell-rich stroma.
Fig. 6. Scanning electron micrograph of blood vessel casts at the
mesometrial side a t day 15 p.c. a: Detail of a fractured cast seen from
the side. The arterioles (Al) and the venules (V1) reaching the subepithelial capillary bed (top and right) seem not to have any preference
for the origin a t the top or at the base of the smooth endometrial folds.
Early Contact and Adhesion Stages-Days
13 to 18 p.c.
Macroscopic observation
At days 13 and 15 P.c., it was not possible to determine the position of the embryo when inspecting the
intact uterine horn, whereas the dilations representing
the embryos in their amniotic sacs were clearly visible
at days 16 to 18 p.c. The freshly opened uteri had a fine
hyperemic line at the mesometrial side at day 13 P.c.,
most clearly seen close to the embryonic disc. At day 15
p.c. there was a double-hyperemic line.
At day 15 P.c., there was a marked increase in vascularisation of the mesometrial side in contact to the
blastocyst (Fig. 4b). This is in contrast to the antimesometrial side (Fig. 4a), which looked like the previous
stages described above. The capillaries of the mesometrial side were densely packed beneath the epithelium,
Luminal side (L).The bar represents 100 pm. b: Detail of the capillary network from the mesometrial side seen from the uterine lumen.
The capillaries are larger in diameter than at the previous stages and
form a close meshwork with a distinct parallel arrangement. Notice a
few thin anastomoses (triangles). The bar represents 10 pm.
leaving only a small space between the capillary wall
and the base of the maternal epithelium. The diameter
of the capillaries varied from 5 to 30 pm. The largest
capillaries were most numerous, whereas the very
smallest were scattered between them. The larger capillaries were not round but oval, with their long axis
perpendicular to the maternal epithelium (Fig. 4b). Occasionally the endothelium seemed to be close to the
maternal epithelium. At day 18P.c., the capillaries had
become slightly larger than at day 15 P.c., showing less
variation in size, although the oval form was still very
pronounced. A small distance to the maternal epithelium remained for most of the Capillaries.
Corrosion casts
At day 15 P.c., the endometrial capillary network
outlined irregular rather broad folds oriented circularly to the longitudinal axis of the uterus (Fig. 5). At
a: At the mesometrial side the folds become more numerous or sub-
divided as evident on a presumably developing cleft (arrow). The capillaries have increased in diameter compared to the previous stage
and compose a very closed parallelly arranged network with many
connections (*) between the parallel capillaries of almost the same
diameter. The bar represents 100 pm. b At the antimesometrial side
the capillaries still form an open network, which is more developed
than at day 12%P.c., with a tendency to parallel arrangement across
the length of the circular folds (the edge of one is seen to the left). The
bar represents 100 pm.
the mesometrial side, besides the generally loosely
meshed network, there was a marked increase in capillary density. This was often seen as two parallel lines
or areas of more densely packed capillaries on both
sides of a central area with somewhat larger meshes
(Fig. 5a). This represents a transition stage. The
loosely packed network showed very irregular meshes
with a wide variation in the diameter of the capillaries,
which also had a bulbous appearance. The capillaries
in the dense areas formed a parallel pattern perpendicular to the circular endometrial folds (Figs. 5a,b, 6b,
14). The majority of the capillaries here having a diameter of 15-25 p,m were closely related and connected
with many thick as well as few thin branches (Fig. 6b).
The precapillary arterioles and the postcapillary
venules, seen a t the cut edge of the cast (Fig. 6a),
showed no preference in location towards the endometrial folds, and exhibited a smooth surface formed by
the capillary network. The vascular casts of the antimesometrial side a t day 15 p.c. formed a wide open
irregular network, comparable to the earlier stages.
At day 18 P.c., the areas with a dense capillary network had increased markedly at the mesometrial side.
The tight network was clearly organized in the same
parallel way as described for the dense capillary network seen at day 15 p.c. (Figs. 7,8a); the antimesometrial side still had a relatively loose appearance, with
some tendency towards a parallel arrangement of the
Fig. 7.Scanning electron micrograph of cast from the mesometrial
side at day 18p.c. The capillary bed now forms a dense layer outlining
the smooth endometrial folds. The capillaries are oriented in a parallel manner across to the direction of the endometrial folds. The arrow
marks location of arteriole or venule reaching the capillary network.
The bar represents 100 pm.
Fig. 8. Details of the capillary bed of the endometrium at day 18 p.c.
Fig. 9.Light micrograph from day 20% p.c. of gestation. Fetal capillaries (FC)) with hemocytoblasts are seen close to the trophoblast
(T). Maternal capillaries (MC) of the endometrium are very wide and
form impressions (arrows) into the uterine epithelium (ME). The bar
represents 50 pm.
Fig. 10. Overview scanning electron microscopic picture, 2OYz days
P.c., showing the transition from the rather large and smooth endometrial folds on the bottom (arrows) to an increase in surface area by
a finer subdivision of microscopic folds or prerugae (**). The bar represents 0.5 mm.
capillaries (Fig. 8b). The vascular architecture at the
mesometrial side then developed into low smooth microscopic folds (Figs. 7, 8a), oriented in the same direction as the large irregular circular folds mentioned earlier (Fig. 5a, 14).The arterial and venous supply of the
capillary network was not yet visible at either the top
or base of these newly formed microscopic folds.
Fig. 11. Details of the capillary bed from day 20% p.c. a: Mesometrial side with development of prerugae (left). The very dense capillary bed is more convoluted than seen at the previous stages, but a
parallel arrangement is still visible. The capillary diameter has increased during the last two days bulging to “intraepithelial” locations
(compare with the marked impressions in Figure 9). The bar represents 50 pm. b At the antimesometrial side the capillary network of
the smooth endometrial folds has retained the open form. The bar
represents 100 Fm.
Early Placental Stage-Days 20% to 23 px.
At day 20% P.c., the maternal capillaries increased
in diameter at the mesometrial side often making a
slight impression into the maternal epithelium (Fig.
9). At this stage, the fetal side of the placenta in the
same area was similarly vascularised, specially close to
Fig. 12. Demonstration of different degrees of endometrial blood
vessel development on casts at day 23 p.c. a: This detail shows the
transition in capillary development from the pattern seen a t day 20Y2
p.c. (compare with Fig. l l a ) to the more developed stages seen in
Figures 12b,c. Notice the beginning of capillary enlargements (*) at
the top of the prerugae. The bar represents 100 pm. b Low magnification of endometrial cast from a very well-developed area. The capillary network now forming prerugae to distinct parallel ridges with
a bulging appearance, due to dilated loops (*). The bar represents 100
pm. c: Slightly higher magnification demonstrating the pattern of
these dilated capillaries (*) from an area not quite as well developed
as the one seen in Figure 12b. The capillaries at the top of the rugae
provide them with an undulating surface. The bar represents 100 pm.
observed at the mesometrial side with a few capillaries
reaching the same size in diameter, namely 30-35 pm,
as seen at the mesometrial side. When seen from the
myometrial side, the branching of venules and arterioles can be followed, but without any preference between the top of the prerugae or the fossae between
At day 23 P.c., this developmental process could be
followed from a pattern comparable to the previous
stage, but with a further increase in capillary enlargement (Fig. 12a) to other areas, where the process of
vascular development progressed dramatically forming
irregular dilations of the capillaries of the low ridges of
Corrosion casts
prerugae (Fig. 12c). In some areas this pattern became
At day 20Y2 P.c., the capillary network at the me- even more elaborate (Fig. 12b), because the capillaries
sometrial side formed smooth folds, becoming more increased in diameter to about 50 pm; those capillaries
elaborate towards smaller folds, or prerugae, in some only measured about 40 pm in diameter a t the dilated
areas (Fig. 10). In the smooth folds the capillaries cre- parts of the more regular rugae. Hence it was almost
ated a very close meshwork with a parallel arrange- impossible, in those dilated parts (Figs. 12b,c), to disment perpendicular to the longitudinal axis of the cern the parallel pattern seen at the earlier stages of
folds, roughly comparable to the first dense network development.
seen a t day 15 p.c. However in the prerugae, the capinitial Stage of Basic Placental Developmentillaries changed to convoluted forms (Figs. 10, l l a , 14)
Days 32 to 43 p.c.
with slightly bulbous dilations measuring about 30-35
pm in diameter. The interconnections between these Histology
At day 32 P.c., the placenta increased its microscopic
capillaries generally showed the same size, but were
also as small as 5 pm. At the antimesometrial side, the foldings compared to the previous stages, though the
capillary network was still rather loose (Fig. l l b ) . The depth of the fossae was less, than at later stages of
capillaries had a greater variability in diameter than gestation. The capillaries, also called “intraepithelial
the embryo. Small microscopic folds, prerugae, were
now developed in some parts. At the antimesometrial
side, the capillary network was less well developed
than at the mesometrial side, although it showed more
variations as compared with the previous stages.
At day 23 P.c., the microscopic folding had increased
followed by a well-developed capillary network beneath the maternal epithelium at the mesometrial side
close to the embryo. At the fetal side the vascularisation had increased, too. This development did not yet
include the antimesometrial side or paraembryonic
parts of the endometrium.
Flg. 13.Vascular casts from day 32 p.c. a: Endometrial cast viewed
from the luminal side. The ridges (R) have become more pronounced
forming fossae (F) between them, where the moderately dense network of capillaries is clearly seen. The tops of the ridges are undulated
and provided with some dilated capillaries (9.An arteriole (AI) can be
followed running from the myometrial side to the top of a ridge. The
bar represents 50 pm. b The branching of arterioles (Al) and a venule
(V1)can be followed at the myometrial side of the endometrial capillary network. The arteriofes reach to the ridges (not visible here) by
entering between the densely meshed network (arrows) of large capillaries forming the row-shaped fossae. The venule arises at the bottom of a fossa ( + 1. The bar represents 50 pm.
capillaries,” were indented into the epithelium. The
epithelium was thinned on the fetal side at the top and
upper sides of the fetal ridges, and a t the bottom and
sides of the complementary maternal fossae. This development progressed during gestation becoming
clearly discernable 11 days later, a t day 43 p.c. (compare Figs. 2 and 3 in Friess et al., 1982).
a basic principle during the gestation period with a
continuous placental development, as previously described and illustrated from days 43 to 109 (Leiser and
Dantzer, 1988).
Corrosion casts
At day 32 P.c., the capillary network of the endometrium was well developed, with parallel ridges, or
rugae, producing an undulating upper profile (Fig.
13a). Some of the capillaries were dilated at the top of
these undulations to a diameter of 35-40 pm. The diameter of the capillaries in the fossae averaged 22 pm
with some small interconnections of 5 to 8 pm (Fig. 14).
When viewed from the myometrial side, the arterioles
could now be followed towards the top of the ridges
where they continued into the endometrial capillary
network (Fig. 131. The venules from this network arose
close to the bottom of the fossae (Fig. 13b). The distribution of the arterioles to the top, and of the venules
arising a t the base, of the endometrial ridges (first seen
clearly at day 32 p.c. and described above) remained as
This study describes, for the first time, the development and architecture of the endometrial microvasculature of the diffuse folded epitheliochorial pig placenta, both before and during the initial stages of
~ o r p ~Related
o l ~ to~~ ~
e v e l o p ~ of
Vasculature/Conceptus Interrelationship
The initial contact between the long (1-1.5 m) porcine blastocysts and the endometrium is restricted to
the mesometrial side (Perry and Rowlands, 1962). Here
the development of the epithelioc~orial
placenta begins
a t days 13-14 p.c. a t an area close to the embryonic
disc at the mesometrial side (Dantzer, 1985). The contact progresses during initial placentation and subsequent developmental stages from the mesometrial side
to the antimesometrial side and also towards the distal
ends of the blastocyst (Friess et al., 1982; Dantzer,
Schematic drawing of early porcine placental stage-related development of vessel
ktagesl) and
ime schedule
9.5 to 12.5 days p.c.
large irregular
13 to 18 days p.c.
large irregular + decreasing size
jize and form2)
lateral relief)
)f endometrial
C: rather small, variable diameter
CM: open, irregular, loose
irregular, generally large,
strictly parallel orientation
CM: dense, cleftlike openings
:apillaries (C:)
and capillary
meshwork (CM:I3
Refer t o the most developed endometrial areas at the mesometrial side
Fig. 14. Schematic drawing summarizing events during initial placentation in the pig (continued on next page).
1985; Keys and King, 1990; Stroband and Van der
Lende, 1990; Dantzer et al., 1991). The main events
during contact are: (1)protrusion of epithelial proliferations of the endometrium enclosed by chorionic caps
which immobilize and anchor the blastocyst at day 13
P.c., followed by a close apposition between the apical
plasma membrane of maternal epithelium and trophoblast at day 14 p.c. and ( 2 ) the development of interdigitating microvilli-adhesion first seen at day 15 p.c.
of this diffuse epitheliochorial placenta type (Dantzer,
1985).The vasculature at day 15 p.c. forms areas at the
mesometrial side with a marked increase in capillary
density and size. This development in vasculature,
therefore, happens ll/z to 2 days after the anchoring
effect is established and only 1 day after the first occurrence of close apposition, thus indicating a specific
local effect due to the presence of the blastocyst. A comparable delay of endometrial vascular development has
been reported in the rabbit during implantation (Leiser
and Beier, 1988).The subsequent extensions of the vas-
tasted endometrial folds and corresponding capillary meshworks
2 0 . 5 to 23 days p.c.
32 to 43 days p.c.
early: small, numerous, smooth to
irregular (prerugae)
later: small ridges with bulbous tops
narrow ridges (often with bulbous
tops) and caveous fossae in between
2 - top of ridges: bulbous, undulated, largc
convoluted with some parallelity,
large and slightly bulbous dilations
CM: very dense
Scale for 100 pm
- fossae: flattenend, randomly oriented,
2M: dense on ridges, moderately dense
in fossae
Scale for 100 pm
cular development follow the same pattern as seen for or ridges. At day 32 p.c. the basic vascular pattern of
the establishment of the materno-fetal epithelial con- the mature porcine placenta has been created. The vascular development into low ridges described from mactact, but with slight delay.
From day 15 there is an increase in the uterine di- erated tissue exposing the basal lamina of maternal
ameter due to the development of the fetal membranes, epithelium (Dantzer et al., 1991) seems to precede the
and it is at this stage that the increased number of formation of microscopic endometrial folds, when obcapillaries are first seen. There is a remarkable in- served from the uterine or fetal side of exposed placencrease in capillary density from day 15 to day 18 P.c., tal surfaces of the endometrium (Dantzer, 1984). This
becoming much more elaborate at day 20% p.c. At day indicates that the changes in the endometrium, i.e.,
23 P.c., a dynamic change in capillary diameter has formation of the first ridges and rugae, occur as a contaken place together with an increase in the endome- sequence of the vascular development described here.
In an early publication Perry and Rowlands (1962)
trial surface area by the formation of microscopic folds
observed a hyperemic line in the endometrium a t the
mesometrial side at day 13 P.c., similar to where the
initial contact between endometrium and the long blastocysts is first established (Dantzer, 1985). We made
similar macroscopic observations, but in addition we
revealed a double hyperemic line along the mesometrial side a t day 15 and 16 p.c. corresponding to the
observation from overview vascular casts (Fig. 5a).
Here the mesometrial side a t day 15 p.c. often showed
a mid-zone with a slightly looser central network than
the dense capillary network a t each side, thus apparently being identical to the double hyperemic lines. In
parallel with the occurrence of these hyperemic lines, a
fluorescence was first demonstrated using Evans blue
(Keys et al., 1986), and later revealed to be an autofluorescence phenomenon (Keys et al., 1989). This
autof luorescence consists of two types, namely bright
green and red. The green was confined to the endometrium in contact with embryonic membranes and became randomized a t days 16-18 p.c. whereas the red
fluorescence continued to increase in intensity and
area up to day 29. By day 32 p.c. it was gone. Therefore,
the red fluorescence corresponds in position to the lateral limits of direct contact between the embryonic
membranes and the endometrium and seems to proceed
or follow the development of the dense capillary network described in the present investigation. Whether
the fluorescence is due to increased vascular permeability as suggested by Keys and King (19881, or if it
represents a substance which influences vascularisation by other means, remains to be clarified.
Angiogenesis is affected by many different factors
both chemical, mechanical, and their combinations
(Hudlicka and Tyler, 1986). Edema will diminish the
compactness of the tissue to facilitate capillary growth,
creating conditions for faster angiogenesis and threedimensional organisation. During the initial stages of
placentation there are several events which favour this
concept: the porcine endometrium develops different
degrees of edema during the cyclic stages (Leiser et al.,
1988), becoming even more pronounced a t days 13 and
15 p.c. (Laforest and King, 1992) as also observed in the
present study. This physiological edema is induced by
estrogens (Geissinger et al., 1979) and seems to preceed
the development of the dense capillary network, which
develops close to the luminal epithelium at the mesometrial side and close to the embryo a t day 15 P.c., as
described. Mast cell-like granulocytes were seen during estrus (Stroband et al., 1986) and we observed
granula-loaded mast cells in the endometrium a t days
9% and 12Y2 P.c., but only few or not recognisable at
later stages of gestation. Due to the fact that mast cells
may contain the well-known autocoids histamine, prostaglandin D2, and leukotrienes, their containment of
these substances contribute to vascularisation and angiogenesis by inducing vasodilation and increased vascular permeability as well as interacting with endothelial cells, growth factors, and angiogenin (Norrby et al.,
1986; Folkman and Klagsbrun, 1987; Cocchiara et al.,
stages. However, in the present morphological study of
the endometrium there are no major changes in the
capillary architecture between day 9% and day 12%
p.c. of gestation, as both stages are characterized by a
wide irregular network with the same variations in
diameter. According to these authors the blood flow a t
day 14 p.c. decreased markedly to a level slightly
higher than that of day 11p.c. and hence stayed almost
constant up to day 19 P.c.; thereafter a dramatic increase in blood flow was demonstrated up to day 30 p.c.
The increase in total uterine blood flow a t day 11-13
p.c. may be due to myometrial activity during blastocyst migration (Ford et al., 1982; Thilander et al.,
19901, whereas the second increase in the blood flow,
19-30 days P.c., can be related to the development of
the endometrial microvasculature described in the
present investigation. This is because the gradual development of the endometrial capillaries, from the mesometrial side to the antimesometrial side, takes 5
days or more as described above. The first vascular
changes are seen a t day 15 and a t the antimesometrial
side at day 20Y2 p.c. when compared to the earlier
stages indifferent to cyclic stages. Therefore, the increase in uterine blood flow after day 19 p.c. correlates
well with the development in endometrial microvasculature .
Estrogens play, together with the endometrial synthesis of prostaglandins, an important role for the regulation of luteolysis and thereby cycle and placentation
(Zavy et al., 1980; Bazer et al., 1984; Mondschein et al.,
1985). The development in endometrial vasculature
and the variations in blood flow pointed out above correlates also with the synthesis of estrogens by blastocysts. The pig blastocysts synthesize estrogens from
day 10-11 p.c. (Perry et al., 1973; Heap et al., 1979;
Flint et al., 1979; Gadsby et al., 1980; Geisert et al.,
1982; King and Ackerley, 19851, whereafter blastocyst
elongation (Geisert et al., 1982) and spacing within the
two uterine horns takes place (Dziuk et al., 1964;
Dhindsa et al., 1967). This will give an increase in
blood flow to the myometrium but will not influence
endometrial blood flow very much since no changes
were observed in the microvasculature. At day 14 there
is a decline in estrogen production whereafter there is
a second rise of estrogens from day 16 up to day 30 p.c.
(Gadsby et al., 1980; Dantzer and Svenstrup, 1986).
However this synthesis of estrogens is not uniformly
distributed, as in vitro cultures of different segments of
the pig blastocysts from days 14-16-18 p.c. showed
that the region enclosing the embryonic disc had the
highest production of estrogens (Bate and King, 1988).
Estrogens seem therefore to act locally as it is the same
region where the initial contact between the blastocyst
and the endometrium is established (Dantzer, 1984)
and where, with a delay of 1-2 days, the first increase
in capillary density occurs as shown in the present investigation.
Vascular-Stromal Interrelationship
Blood Flow
Total blood flow to the porcine uterus was investigated by Ford and Christenson (1979). It increases 3 to
4-fold by day 12-13 p.c. of gestation compared to cyclic
Interaction between endothelial cells and the extracellular matrix may serve to regulate capillary development and architecture (Ryan and Barnhill, 1983; Yasunaga et al., 1989; Merwin et al., 1990). Experiments
indicate that the extracellular matrix components may
act locally to regulate growth and pattern. Fibroblast
growth factor-stimulated endothelial cells may
"switch between growth, differentiation, and involution modes during angiogenesis by altering the adhesiveness or mechanical integrity of their extracellular
matrix (Ingber and Folkman, 1989). In addition insulin-like growth factor 1 has recently been shown to be
localized in the vessel wall during initial placentation
in the pig (Persson et al., 1993).
Hudlicka and Tyler (1986)referred that intravascular
pressure would not change the capillaries as their diameter is supposed to be rather rigid. However, a
higher blood pressure would increase blood flow and
subsequently capillary growth, because wall tension
and stress seem to be important factors for stimulation
of endothelial growth. This may be implicated in the
development of the vascular architecture of pig placenta as greater curvatures occur where preexisting
vessels bend (Waxman, 1981)or branch. An increase in
both blood flow, and flow-oriented stress will act as a
viscous drag on the luminal surface of the vascular
endothelium, being higher a t the outer sides of vessel
curvatures and around branching points (Nerem, 1981;
Waxman, 1981). This will give greater growth at the
convex curvatures of bendings, which may explain
some of the morphological features seen during initial
vascular development of porcine placentation. When
the architectural changes of the vasculature are considered in this respect the initial growth at day 15 P.c.,
and later the growth a t the top of the ridges as well as
the dilations into sinusoidal structures a t the arterioral side of the capillary bed (Figs. 12, 141, can be understood.
The revealed vascular development may therefore
reflect a local induced interaction between estrogens,
proteins secreted by the blastocyst, endometrial production of prostaglandins, factors for granular depletion of mast cells, and blood flow oriented stress a t the
luminal side of the endothelium: all events which aim
to stimulate angiogenesis and vascular changes during
implantation and early placentation in the pig.
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This study was supported by Grant 13-4224 from the
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histology, cast, vascularisation, initial, area, pig, demonstration, corrosion, placental, nonglandular
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