BIOLOGY OF REPRODUCTION 53, 321-338 (1995)
Striking Changes in the Structure and Organization of
Rat Fetal Membranes Precede Parturition'
Laurie G. Paavola, 3 Emma E. Furth, 4 Violeta Delgado,5 Charles O. Boyd5
Candace C. Jacobs, 3 Hanqin Lei,5 and Jerome F. Strauss III 2,4,5
Department of Anatomy and Cell Biology,3 Temple University School of Medicine, Philadelphia,Pennsylvania
Departments of Pathology and LaboratoryMedicine4 and Obstetricsand Gynecology 5
University of Pennsylvania, School of Medicine, Philadelphia,Pennsylvania
ABSTRACT
Premature rupture of fetal membranes can harm infant and mother. It is unclear whether structural changes predispose these membranes to breaking. We thus assessed rat visceral yolk sac placenta (VYSP) and amnion by light and by transmission electron microscopy
on Days 18-21 of gestation. Light microscope sections were stained for connective tissue (extracellular matrix) components: collagen,
glycoprotein, and glycosaminoglycans/proteoglycans. Some tissue was incubated with chondroitinase ABC. We observed that fetal membranes became increasingly fragile, rupturing readily on Day 21.
On Days 18-20, the two epithelial layers of the capsular VYSP were separated by a well-developed, well-vascularized connective tissue
layer that stained intensely for all matrix components studied; on Day 21, the connective tissue layer was thinner, moderately stained, and
less vascularized. On Days 18-20, the two cellular layers of the amnion were separated by a narrow, compact connective tissue layer that
stained modestly for all matrix components; on Day 21, this area was widened and stained faintly. Transmission electron microscopy
showed that collagen fibrils of the amnion were abundant, closely packed, and well organized on Days 18-20, whereas on Day 21 they
were few in number, widely spaced, and disorganized. Similar changes were present after incubation with chondroitinase ABC. Inaddition,
amniotic epithelial cells were moribund and delaminating, basal laminae were deteriorating or absent, and few cells were at the outer
surface of the amnion. All changes preceded parturition.
We conclude that the structural integrity of rat fetal membranes is impaired before birth through the loss of connective tissue components and cells, changes that presumably underlie membrane rupture. Lastly, the similarity of structural changes in rat and human fetal
membranes point to the potential usefulness of the rat model.
INTRODUCTION
coverings can precipitate early delivery with possible adverse consequences for both mother and fetus [1]. Although
fetal membranes rupture after initiation of labor in most
term human pregnancies [1], prelabor breakdown occurs in
roughly 10% of pregnancies [2]. Moreover, preterm membrane rupture is more likely to be associated with preterm
birth [3, 4].
The precise nature of the structural alterations that predispose fetal membranes to rupture as well as the signals
regulating the onset of such changes remain unknown for
most species, including humans. Recently, however, Malak
and Bell [5, 6] have identified, in normal human term amniochorion obtained immediately after delivery, a restricted
area along the rupture site that displays striking histological
alterations. These authors propose that this area may represent the initial site at which rupture occurs during labor.
Data emerging from their work and that of others [7-9] point
to the possibility that changes in the connective tissue components and tensile strength of fetal membranes may underlie their rupture in preterm and term pregnancies, at least
in humans.
Because of the limited availability of preterm human fetal
membranes, it is clear that there is a need for a nonhuman
model in which changes in such membranes can be analyzed in the periparturition period. The rat may provide
such a system. In this species, after Day 17 of pregnancy,
During gestation in placental mammals, the membranes
enclosing the developing fetus have a number of functions
that are central to the proper development and maintenance
of the fetus and to events associated with parturition. Fetal
membranes and their associated structures are complex
and, depending on the species, can consist of an amnion,
chorion, decidual elements, yolk sac placenta, and/or chorioallantois. Each component serves one or more functions
critical to fetal survival in utero, such as transfer of nutrients,
gaseous exchange, provision of the fluid environment in
which the fetus floats, and protection from pathogenic organisms.
Equally critical is the ability of the fetal membranes to
break down at the end of gestation, permitting the fetus to
reach the external environment. The timing of such events
is important since premature rupture of these protective
Accepted March 28, 1995.
Received February 6, 1995.
'Supported in part by NIH Research Grants HD-06274 and HD-17301, and a grant from
the Rockefeller Foundation. Aspects of sample preparation for transmission electron microscopy were performed at the Electron Microscopic Core of the University of Pennsylvania
Diabetes Center, which is supported by NIH Grant DK-19525. V. Delgado is a visiting investigator from the Universidad Francisco de Miranda, Area Ciencias de la Salud, Coro, Falcon, Venezuela.
2Correspondence:Jerome F. Strauss III, M.D., Ph.D., Department of Obstetrics and Gynecology, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104.
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PAAVOLA ET AL.
the fetal membranes consist of an amnion, a visceral yolk
sac placenta (VYSP), and a chorioallantoic placenta, which
are comparable in large part to the chorion laeve and chorion frondosum/decidua basalis, respectively, of the human
placenta. The availability of a rat model for fetal membrane
studies would also permit effects of intervention and perturbation to be assessed, which is an important consideration.
Much information is available on the development, morphology, and functions of the yolk sac and chorioallantoic
placenta in rats, which has been reviewed by Jollie [101.
Although many studies have focused on the structure and
function of the amnion in primates, relatively little is known
about this membrane in rats. A fine structural investigation
of rodent and primate amnions by Wynn and French [11]
focused on the basic morphology of this membrane near
term.
Perhaps more importantly, however, little information
exists on the structure and organization of rat fetal membranes in the period immediately preceding the onset of
parturition. A single study in which the biochemical and
physical properties of rat fetal membranes were examined
indicated that a loss of collagen in the peripartal period was
paralleled by a decline in membrane strength [12]. This implies that fetal membranes are undergoing important alterations in their architecture, presumably in anticipation of
parturition. That work, however, did not address the issue
of whether the observed changes in collagen content and
fetal membrane strength occurred in the yolk sac placenta,
amnion, or both.
The aim of the current work was to delineate the nature
of the structural alterations in rat fetal membranes. To this
end, we used a morphological approach to analyze the organization, histochemistry, and fine structure of the amnion
and visceral yolk sac placenta in rats at intervals preceding
parturition, including the use of specific light and electron
microscopic stains for various connective components.
In this report, we describe the development of marked
changes in the structure of rat fetal membranes. These
changes occur late in gestation but before the onset of labor
and predispose the membranes to rupture during the birthing process. These alterations are most dramatic in the amnion and involve both epithelial and extracellular matrix
components.
MATERIALS AND METHODS
Animals
Timed pregnant Sprague-Dawley rats were purchased
from Zivic-Miller Breeding Laboratories (Zelienople, PA)
and maintained in the animal facilities according to NIH
guidelines until use. In our laboratory, the animals deliver
between 1300 and 1600 h on Day 21 of gestation.
Light Microscopy
Rats were killed by CO 2 asphyxiation at the following
intervals: 1000 h on Days 18 and 19 of gestation; 1000 h,
1600 h, and 2200 h on Day 20; and 1000 h, 1430 h, 1630 h,
and after initiation of parturition on Day 21. Fetal membranes (VYSP and amnion) were detached from the chorioallantoic placenta and placed in Bouin's solution for 2448 h (after which the fixative was replaced with 70% ethanol) or in 10% neutral buffered formalin.
Fixed tissue was embedded in paraplast, and 7-10-tm
sections were prepared and stained with hematoxylin and
eosin (morphology), Mallory-Azan (connective tissue),
Jones stain (basement membranes, connective tissue), or
periodic acid Schiff (PAS; glycoproteins) before and after
digestion with diastase to remove glycogen. Stained tissue
was photographed with a Leitz (Malvern, PA) microscope
using Ektachrome (Eastman Kodak, Rochester, NY) daylight
film (ASA 100) and a neutral density filter.
TransmissionElectron Microscopy (TEM)
Pregnant rats were anesthetized with 5 mg sodium pentabarbital/g body weight at the intervals noted above, and
the abdomen was opened as described earlier [13]. The
uterus was exposed, and fetal membranes were removed and
placed in 2% paraformaldehyde-2% glutaraldehyde-0.01 M
sodium cacodylate buffer or 1% paraformaldehyde-1% glutaraldehyde-0.01% 2,4,6-trinitrocresol-0.1 M sodium cacodylate buffer (FGC) [14] and secured flat on dental wax (in a 30mm tissue culture dish) with stainless steel pins. After 2-3 h
at room temperature (RT), the fixative was replaced with cold
0.01 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose (buffer). Some fetal membranes were secured flat in
buffer before fixative was added. Other fetal membranes
were immersed directly in fixative without flattening.
To fix fetal membranes in a natural configuration, fetuses
still encased in their membranes were removed from the
uterus and placed in fixative, either with both VYSP and
amnion left intact, or with VYSP removed to expose the
underlying amnion. In other cases, FGC was dripped directly onto previously exposed yolk sac placenta or amnion,
or perfused in situ to the uterus by the method of Paavola
[13, 15]. After perfusion, the uterine wall was removed, and
fetal membranes were placed in fresh FGC for 2-3 h at RT.
After being washed in cold buffer, pieces of immersion- or
perfusion-fixed fetal membranes were excised and postfixed
in Os0 4-0. 67 M sodium cacodylate buffer, stained en bloc with
alcoholic uranyl acetate, and embedded in epoxy resin as previously described [13]. Semithin (1 pm-thick) sections were
cut and stained with toluidine blue for light microscopy. Pale
gold to silver thin sections were prepared, stained with uranyl
acetate and lead citrate, and photographed with aJEOL 100CX
transmission electron microscope.
ALTERATIONS IN RAT FETAL MEMBRANES
323
FIG. 1. Diagram of rat fetal membranes. After Day 17 of gestation, rat fetus issurrounded by VYSP and amnion. VYSP has capsular
and villous portions, each consisting of three layers: simple columnar epithelium facing maternal decidua, modest (capsular) to
well-developed (villous) connective tissue layer containing blood vessels, and simple low cuboidal epithelium facing amnion.
Three-layered amnion has simple epithelium facing amniotic cavity, thin connective tissue layer, and condensationof fibroblastlike cells on surface facing VYSP. This isdifferent from human placenta since amnion in rats is not fused with chorion-like VYSP.
Demonstration of Glycosaminoglycans (GAGs)
To demonstrate GAGs at the TEM level, fetal membranes
were stabilized by placing the fetus still encased in yolk sac
placenta and amnion or in amnion alone into aldehyde fixatives containing ruthenium hexamine trichloride (Polysciences, Inc., Warrington, PA) as detailed by Hunziker et al.
[161 for 3 h at RT or containing 1% cetylpyridinium chloride
for 24 h at RT as described by Malchiodi-Albedi et al. [17]
and Landemore et al. [18]. Ruthenium hexamine trichloride
and cetylpyridinium chloride increase retention in tissue of
water-soluble, sulfated proteoglycans and GAGs. Fixatives
for ruthenium hexamine trichloride were 10% neutral buffered formalin (light microscopy) and 1% paraformaldehyde-1% glutaraldehyde-0.1 M sodium cacodylate buffer or
FGC (TEM); for cetylpyridinium chloride, the fixative was
4% paraformaldehyde in 0.2 M KH2 PO 4-Na 2HPO4. Pieces of
fixed fetal membranes were excised and washed in buffer
at RT, postfixed in 1.33% Os0 4 -0.067 M sodium cacodylate
containing 5% sucrose, and processed as usual for TEM,
except that some tissue was processed without osmium
postfixation; the buffers and postfixative were prepared
with or without ruthenium hexamine trichloride for tissue
fixed in the presence of ruthenium hexamine trichloride.
For light microscopy, the type and distribution of GAGs
in fetal membranes were determined as follows. To distinguish between carboxylated, nonsulfated GAGs and sulfated GAGs, sections of yolk sac placenta or amnion were
fixed in 10% neutral buffered formalin for 4 h, embedded
in paraplast, serially sectioned at 7 lm, and stained according to the critical electrolyte concentration method of Scott
and Dorling [19]. For this, sections were stained overnight
at RT with 1% alcian blue 8 GX prepared in 0.025 M sodium
acetate buffer (pH 5.8) that contained MgC 2 of varying ionic
strengths (0.025 [carboxylic, nonsulfated GAGs, e.g., hyaluronic acid], and 0.3 and 0.5 M [sulfated GAGs, e.g., chondroitin and dermatan sulfate]). Some sections were digested
with 1.5 U chondroitinase ABC (from Proteus vulgaris;
Sigma Chemical Co., St. Louis, MO) in PBS, pH 8.6, at 37.5°C
overnight before being stained with alcian blue as described. At this pH, the main substrate for the enzyme is
chondroitin sulfate/dermatan sulfate.
Enzyme Digestion of FetalMembranes
Immediately after removal of the fetus from the uterus,
some fetal membranes were washed in warm (37 0C) Dulbecco's Modified Eagle's medium with 25 mM HEPES
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PAAVOLA ET AL.
FIG. 2. Gross aspects of rat fetal membranes: a) 1730 h Day 20 of gestation; b) 1000 h Day 21 of gestation. Fetal membranes shown were fixed flat and photographed
using same level of illumination. Capsular VYSP, which fills most of field in (a)and (b) is comparatively thick on Days 18-20 of gestation giving it rather dense appearance
(a)but is thinner on Day 21 resulting in an overall pale look (b). a)Blood vessels are prominent on Days 18-20 (arrows); b) few are obvious on Day 21 (arrow). Avascular
structure in (b) isamnion (double arrows). Fixation, FGC-cacodylate. x 5.
(DMEM; Gibco, Grand Island, NY) to remove blood, incubated at 37°C for 2-24 h in DMEM containing 1 IU chondroitinase ABC (Sigma), 5 mM benzamidine HCL, 0.1 M 6amino-n-caproic acid, 0.05% BSA, and 50 gg/ml gentamicin
0
for 2-24 h at 37 C in a humidified atmosphere with air-5%
CO2 . After being washed three times in warm DMEM containing 5% sucrose, the tissue was fixed by immersion in
aldehydes containing ruthenium hexamine trichloride. For
controls, fetal membranes were incubated and processed as
noted except that chondroitinase ABC was omitted. Enzyme-incubated and control membranes were processed
for TEM as noted above. Semithin (1 ptm) sections were
stained with toluidine blue, and thin sections were stained
as detailed above.
RESULTS
Rat FetalMembranes: Overview
Placentation in the rat is characterized by the formation of
an inverted yolk sac placenta, a chorioallantoic placenta, and
an amnion [101. The structural relationships of these mem-
FIG. 3. Capsular VYSP thins before onset of parturition. a)On Days 18-20 of gestation (Day 20 shown), simple cuboidal epithelium of capsular VYSP (between arrowheads) is firmly attached to underlying connective tissue, which contains prominent blood vessels (arrows). Villous VYSP fills lower half of field. b) By Day 21 of
gestation, capsular VYSP is attenuated (arrowheads), its epithelium may be separated from connective tissue (arrow), and its blood vessels are less conspicuous.
10% neutral buffered formalin fixation, Mallory-Azan. x 275.
FIG. 4. Changes in amnion histochemistry occur before parturition. On Days 1820 of gestation, amnion is narrow, with compact connective tissue layer that is
intensely argyrophilic (a, between arrowheads), PAS-positive after staining of diastase-digested sections, suggesting presence of glycoproteins (c, between arrowheads), and rich in collagen (d, between arrowheads). Dramatic changes are evident
on Day 21, prior to parturition. Amnion is markedly widened (b,d, between arrowheads, and f) and disorganized, due largely to changes in connective tissue layer.
Latter is now meshwork of fine argyrophilic fibers (b), is no longer strongly PASpositive, being either unstained (d, *) or stained a light blue Id, between arrowheads) implying loss of glycoproteins, and is deficient in collagen (f, arrowhead).
Moreover, amniotic epithelium (f, arrows) is widely separated from remaining connective tissue, leaving large clear areas {f, *). All, 10% neutral buffered formalin
fixation. a,b) Jones silver stain, basement membrane/connective tissue black; cells
reddish-purple. c,d) PAS after diastase digestion; glycoproteins pink; cells faintly
blue. e,f) Mallory-Azan: collagen blue; cells reddish purple. Day 19 of gestation
(a,c,e), Day 21 of gestation (b,df). a-b) x350; (c-d) x600; (e-f) x900.
ALTERATIONS IN RAT FETAL MEMBRANES
325
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PAAVOLA ET AL.
branes are illustrated in Figure 1. Briefly, the rat fetus is enclosed by the amnion, which in turn is surrounded by the
visceral and parietal components of the yolk sac placenta.
The parietal portion ruptures on about Day 16 of pregnancy
while the visceral portion persists throughout gestation. The
latter has a villous part, which is limited in extent and located
near the chorioallantois, and a capsular part, which is extensive (Fig. 1). The villous and capsular parts of the VYSP have
three layers (starting at the side facing the amnion): 1) an
attenuated low simple cuboidal epithelium that lies on a thin
basement membrane, 2) a connective tissue layer consisting
of cells, ground substance, and fibers, and 3) a basement
membrane that supports a simple cuboidal-to-columnar secretory epithelium that faces the maternal decidua (Fig. 1).
The amnion has a similar tripartate structure. It is composed
of 1) a simple epithelium and its basement membrane, 2) a
thin connective tissue layer, and 3) an accumulation of cells
on the outer border (facing the VYSP).
The VYSP Weakens as ParturitionApproaches: Gross
Aspects
At the earlier stages examined, the VYSP closely covered
the fetus, whereas late in pregnancy (-Days 20-21) it was
loose and "baggy," especially the capsular portion. Moreover, from Days 18 to 21 of pregnancy, it became increasingly fragile. On Day 18, this membrane was quite tough,
resisting rupture from fairly firm pressure with blunt forceps. In contrast, on Day 21, the day of parturition, it broke
readily upon slight pressure. Gross examination revealed
that, from Day 18 of gestation to the time of impending
parturition, the capsular portion became increasingly attenuated. This change was readily apparent in all specimens
regardless of the mode of fixation and was particularly obvious in fetal membranes that were fixed flat (Fig. 2). Finally, changes in the gross appearance of the villous portion
of the VYSP were not conspicuous.
The Visceral Yolk Sac Thins as Birth Nears: Histology and
Histochemistry
During Days 18-20 of gestation, the villous portion of the
VYSP was thick, with numerous, tall villous-like folds whose
lamina propria contained many thin-walled blood vessels
(Fig. 3a). Over the period studied, the epithelial cells underwent a number of secretory cycles, discharging PAS-positive,
diastase-resistant material into the exocelomic lumen (which
faces the maternal decidua). On the other hand, the capsular
portion of the VYSP was smooth, lacking villous folds, and
relatively thin (Fig. 3a). Its connective tissue layer was about
one-half the thickness of that in the villous portion and was
well vascularized. The secretory cells of the capsular portion
were similar in staining and secretory activity to those of the
villous portion. Staining with Mallory-Azan and with PAS (af-
ter diastase digestion) on Days 18-20 of gestation revealed
that the connective tissue layer and basement membranes of
both villous and capsular portions were rich in collagen (Fig.
3a) and glycoproteins. The extent of the basement membranes in both portions was particularly well seen with the
Jones stain.
It was evident from simple inspection that as parturition
drew near, the capsular VYSP became attenuated. The predominant change was a thinning of the connective tissue
layer (Fig. 3b), which showed decreased staining intensity
with Mallory-Azan (Fig. 3b), PAS reagent, and Jones silver
stain. In addition, the amount of blood contained within the
vessels of the capsular portion appeared to decline as parturition approached (Fig. 3b) compared to that in vessels of
similar areas at earlier stages (Fig. 3a). Also, some areas of
the capsular portion appeared to be denuded of or losing
epithelial cells (Fig. 3b). Although the thickness and staining
intensity of the connective tissue layer in the villous VYSP
appeared to decrease as parturition approached, this decrease was not as marked as that seen in the capsular portion.
Some Glycosaminoglycansin VYSP DecreaseNear Term
Staining of the connective tissue layer in both villous and
capsular VYSP with alcian blue at 0.025 M MgCl 2 was intense
and did not change either over the period of time studied or
upon digestion with chondroitinase ABC. This implies the
sustained presence of a carboxylic GAG, probably hyaluronic acid, in the connective tissue layers of the VYSP over
Days 18-21 of gestation. At higher concentrations of MgC12
(0.3-0.5 M), alcian blue staining of the connective tissue layer
in the villous and capsular VYSP was moderate and appeared
to decline near parturition and was considerably diminished
after enzyme treatment. This implies that sulfated GAGs,
probably chondroitin/dermatan sulfate, were present in
VYSP connective tissue and declined as birth approached.
The Amnion Becomes Looser and More Fragileas Birth
Approaches: Gross Aspects
On Days 18 and 19, the amnion smoothly enclosed the
fetus and was relatively free of folds and/or pouches. In
contrast, by Day 21 of gestation, the amnion loosely invested the fetus. As noted for the VYSP, the amnion became
increasingly frail as parturition approached, resisting rupture upon firm to moderate pressure with blunt forceps at
the earlier stages but breaking upon slight pressure on the
day of parturition.
It is noteworthy that, on Day 21 of pregnancy, a viscous
material was present between the uterus and the fetal membranes, and between the VYSP and the amnion. This material, which was not present on the other days studied, did
not fix well with glutaraldehyde, paraformaldehyde, or
Bouin's solution. It remained in some of the solutions used
ALTERATIONS IN RAT FETAL MEMBRANES
327
FIG. 5. Fine structure of amnion, Days 18-20 of gestation. Entire width of amnion on Day 20 isshown here. From Days 18-20, amnion is thin and has compact but welldeveloped connective tissue layer separating epithelium (arrows) from outer border, which is comprised of alternating layers of fibroblast-like cells (arrowheads) and
collagen bundles. Note that connective tissue layer contains abundant closely spaced collagen bundles that are typically oriented parallel to luminal surface of amnion.
Fixation for all TEMs unless otherwise stated, FGC-cacodylate. x7000.
for tissue processing (e.g., fixative, buffer, etc.). Interestingly, Harkness and Harkness [121 noted an apparently similar material, which they termed "slimy," associated with rat
fetal membranes near the time of parturition.
The Amnion Becomes DisorganizedBefore Birth: Light
Microscopy
Light microscopy revealed that, from 1000 h on Day 18 to
2200 h on Day 20, the amnion was thin and compact, with
an attenuated epithelium, a narrow but dense core of connective tissue, and a layer of flattened cells (Fig. 4, a, c, and
e). The organization of the connective tissue core was particularly obvious after staining with the Jones stain (Fig. 4a);
it was modestly PAS-positive (diastase-resistant; Fig. 4c) and
moderately blue with Mallory-Azan (Fig. 4e). This organization persisted throughout the morning and afternoon of Day
20 of gestation, but by 2200 h on Day 20, limited areas of the
connective tissue layer had a loose, lace-like appearance.
The changes noted in restricted areas at 2200 h on Day
20 of pregnancy were extensive on Day 21 of gestation,
involving the entirety of the amnion. Frequently, the amniotic epithelium appeared to be detached from the underlying connective tissue layer (Fig. 4, b, d, and f). This layer,
which was previously narrow, was now much widened,
with its fibrillar components widely separated from each
other. This was particularly well shown with the Jones and
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PAAVOLA ET AL.
FIG. 6. Fine structure of amnion on Day 21 of gestation, day of parturition. Amnion has morphology strikingly different from that on Days 18-20 of pregnancy. Itis wider
mainly due to increased width of empty-appearing connective tissue layer, and contains far fewer and more widely separated collagen bundles, which now fail to be oriented
to luminal surface. Moreover, cytoplasm of amniotic epithelial cells (arrows) is usually pale, a sign of cell deterioration. Fewer fibroblast-like cells (arrowheads) lie at outer
surface of amnion (facing VYSP). Double arrows, possible macrophage. x7000.
Mallory-Azan stains (Fig. 4, b and 0. Compared to earlier
stages, the connective tissue layers now showed diminished
diastase-resistant PAS staining (Fig. 4d) and were only faintly
blue with Mallory-Azan (Fig. 4f). Some of the cells on the
outer surface of the amnion appeared to be detaching.
Changes in Amnion Fine StructurePrecedeParturition:
Overview
The fine structural appearance of the amnion, including
the epithelium, the connective tissue layer, and the condensation of cells on the outer surface (i.e., that facing the
exocoelom and visceral yolk sac) was similar from Days 1820 of gestation (Fig. 5), resembling that described for this
species by Wynn and French [11]. During this period, we
found the amnion to be -12 gm in thickness.
By Day 21 of gestation, however, striking morphological
changes were evident in the fine structure of all components
of the amnion (described separately below). One notable
change was that this membrane had increased in width
compared to that on Days 18-20 (Fig. 6), now measuring
up to 31 Im (mean = 20 gim; range 11.4-31.4 ilm; n = 18
measurements).
Amniotic Epithelial Cells Become Moribund
On Days 18-20 of gestation, the fetal aspect of the amnion was covered by a flattened, almost squamous epithe-
ALTERATIONS IN RAT FETAL MEMBRANES
329
FIG. 7. Amniotic epithelial cells lose desmosomes as parturition approaches. Numerous well-developed desmosomes bind amniotic epithelial cells to each other
from
Days 18-20, but on Day 21 (shown here) these junctions are involuting. While some desmosomes appear intact (double arrows) and attach two amniotic
epithelial cells
that show signs of impending cell death, many others have been internalized (short arrows), allowing lateral borders of adjacent cells to separate. Signs of cell
dissolution
include swollen, lucent Golgi complex (large arrowheads) and increased prominence of cytoskeletal filaments (small arrowheads). This cell was previously
attached to
another amniotic epithelial cell that isout of view (bottom of photograph). N, nucleus. Fixation, FGC-cacodylate-ruthenium hexamine trichloride; x 17 800.
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PAAVOLA ET AL.
FIG. 8. Cytoplasmic changes in two moribund amniotic epithelial cells on Day 21 of gestation. Cytoplasm of upper cell is extracted and electron-lucent, and contains
autophagic vacuoles (arrowheads), pale swollen mitochondria (*), numerous large clear vacuoles (v), and prominent cytoskeletal filaments. Plasmalemma remains intact
as do multiple desmosomes (arrows) attaching it to underlying cells. Basal lamina under lowermost cell is incomplete (out of view). x 24 400.
lium, although occasional cuboidal cells were present. At
this time, the luminal surfaces of the cells were covered with
numerous long, irregular microvilli and/or deep folds or
cup-shaped, pocket-like depressions (Fig. 5). These cells
contained cisternae of rough endoplasmic reticulum (ER),
free ribosomes, Golgi complex, mitochondria, occasional
lysosomes and autophagic vacuoles, and small (-70-90 nm
in diameter) coated or smooth-surfaced vesicles (especially
abundant along the basal surface of the cell). Adjacent cells
had complex lateral interdigitations and were attached to
each other by numerous well-developed desmosomes.
On Day 21 of pregnancy, relatively few epithelial cells
exhibited a normal fine structure, at least as judged by simple inspection. Even those that appeared viable displayed
changes indicative of deterioration (Fig. 7), including increased electron lucency of the cytoplasm and nucleus, increased prominence of cytoskeletal filaments (Fig. 7), more
autophagic vacuoles (Fig. 8), and changes in organelle morphology such as distended Golgi cisternae (Fig. 7) and pale
and swollen mitochondria (Fig. 8). Many epithelial cells
were clearly moribund, exhibiting a highly electron-lucent,
extracted cytoplasm (Figs. 9a,b and 10d), often enclosed by
a plasma membrane that bore small breaks along its length.
As the cell continued to deteriorate, the nuclear envelope
and nuclear matrix typically persisted, but both heterochromatin and euchromatin were lost (Figs. 9b and 10d), and,
although fibrous components of the cytoskeleton remained
conspicuous, few other organelles persisted (Figs. 9b and
10d). Other cells had undergone massive dissolution, with
only portions of them remaining. In some instances, desmosomes persisted far into cell regression, attaching even
highly moribund cells to each other (Fig. 8). However, it
was clear that desmosomes and hemidesmosomes involuted at all stages of epithelial cell death, characteristically
leaving modified (desmosomal) remnants within the cytoplasm (Fig. 7). Finally, strips of epithelial cells, sometimes
still connected to each other at their lateral margins via desmosomes, were delaminating from underlying layers (Fig.
9b); in some instances the loss of desmosomes gave rise to
two clear-cut cell layers (Fig. 9a).
The Amniotic Epithelium BasalLamina Deteriorates
During Days 18-20 of gestation, the basal lamina (lamina
densa) upon which amniotic epithelial cells rest was a con-
FIG. 9. Dissolution of amniotic epithelium. a) On Day 21 of gestation, large segments of epithelium are deteriorating, and two epithelial cell morphologies, not
seen on Days 18-20, are those that are either highly electron-dense (arrowheads)
or electron-lucent (arrows). These cells have lost their desmosomal attachments,
allowing separation from each other (*). Note that sparse collagen bundles of connective tissue layer are widely dispersed and are no longer oriented to luminal
surface. b) Portion of amniotic epithelium in terminal stages of degeneration (arrowheads) on Day 21. Note nearly complete dissolution of nucleus. Intact portion
of epithelium, which is separated from connective tissue (*), has clinging to its
undersurface fragments of dead epithelial cells (small arrows) still attached by desmosomes. Fibroblast-like cells no longer form more or less continuous border on
outer surface (large arrows). ct, connective tissue. x7000.
ALTERATIONS IN RAT FETAL MEMBRANES
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PAAVOLA ET AL.
ALTERATIONS IN RAT FETAL MEMBRANES
tinuous, delicate structure, -25 nm thick (Fig. 10a). It was
separated from the overlying epithelial cells by the lamina
rara, a pale area of uniform width, -- 50 nm. Numerous delicate filaments extended from the basal lamina through the
lamina rara to contact the basal plasma membranes of the
overlying amniotic epithelial cells (Fig. 10a). Similar fine filaments protruded from the basal lamina into the underlying
connective tissue.
In contrast, on Day 21 of pregnancy the basal lamina was
often attenuated and no longer closely followed the basal
surfaces of the epithelial cells (Fig. 10b,d). Instead, it was
separated from them by gaps of varying widths (Fig. 10b),
displayed discontinuities along its length, or was absent
over considerable lengths (Fig. 10c). Where the epithelium
was detaching from the underlying connective tissue, the
basal lamina was frequently absent (Fig. 9b). Finally, processes arising from epithelial cells penetrated the basal lamina more frequently than at earlier stages (Fig. 10b).
Loss and DisorganizationofAmnion Connective Tissue
Collagen
During Days 18-20 of gestation, the connective tissue
layer consisted primarily of collagen and occasional cells
embedded in an amorphous ground substance; individual
collagen fibrils were remarkably uniform in diameter, -50
nm, and displayed the typical 69-nm banding periodicity
(Fig. 11la). The fibrils occurred singly, in small clusters, and
in bundles ranging in diameter from 0.3 to 1.5 gm and containing 50-100 fibrils. Within a bundle, individual collagen
fibrils were evenly spaced 25-50 nm apart and interconnected by wispy threads of moderately electron-dense material (Fig. 11la). The distribution of collagen fibrils/fibers
across the width of the connective tissue layer was quite
uniform (Fig. 5). Individual fibrils and fiber bundles were
oriented predominantly parallel to each other and to the
luminal surface of the amnion (Fig. 5).
On Day 21 prior to the onset of parturition, the connec-
FIG. 10. Dissolution and loss of basal lamina and advanced cell death. a) Day 18
of gestation. From Days 18-20 amniotic epithelium rests on well-developed, 50-nmthick basal lamina that follows precisely contours of overlying cell (arrowheads).
Fine filaments extend from basal lamina to attach both to epithelial cells and to
underlying connective tissue. Lucent layer (lamina rara) separating basal lamina
from cell is of uniform width. (b-d) Day 21 of gestation, prior to parturition. b) On
Day 21, basal lamina is discontinuous (arrowhead), no longer closely follows contours of basal plasmalemma, and is often separated from overlying cell (*). Moreover, processes from epithelial cell penetrate through but fail to be enveloped by
basal lamina (arrow). c) Frequently, basal lamina has involuted entirely and ismissing over long stretches (arrowheads). d)Amniotic epithelial cell in late stage of cell
death. Although patches of heterochromatin and nucleolus (arrow) persist, nucleus
lacks euchromatin, is highly electron-lucent, and appears to be enclosed by deteriorating nuclear envelope. Most organelles are absent, and pale patchy areas occur
in cytoplasm. Basal lamina isattenuated and incomplete (arrowheads). N,nucleus.
(a) x 43 200; (b-c) x22 400; (d) x 17 800.
333
tive tissue layer had increased in width (Fig. 6) compared
to that at Days 18-20. This change accounted in large part
for the overall increase in thickness of the amnion. At this
time large areas of the connective tissue layer were devoid of
any visible structures (Figs. 6 and 9a,b). Perhaps more striking
was the marked diminution in collagen content; there were
fewer individual fibers and fewer fiber bundles (Figs. 6 and
9a,b). Moreover, the fiber bundles that remained were smaller
in diameter and widely separated from one another (Fig. 1lb).
In addition, the individual collagen fibrils comprising a bundle
were more heterogenous in diameter than those of earlier
stages (Fig. Ilb). Also, small (-25 nm in diameter) fibrils,
which were not noted on Days 18-20, now occurred within
fiber bundles (Fig. 1lb). The periodicity of the collagen banding pattern remained unchanged. Finally, the orientation of
collagen relative to the luminal surface was no longer a striking feature (compare Fig. 5 with Figs. 6 and 9a,b).
Some Glycosaminoglycans/Proteoglycansof the Amnion
Diminish as Birth Nears
As shown by light microscopy, the connective tissue
layer of the amnion stained moderately with alcian blue at
0.025 M MgC12; there was no change in staining intensity
either over gestational time (Days 18-21 of pregnancy) or
after incubation of the sections with chondroitinase ABC
prior to alcian blue treatment. This implies that carboxylated
GAGs, probably hyaluronic acid, were present and appeared not to change in amount over the period studied. At
MgC12 concentrations of 0.3-0.5 M, alcian blue staining of
amnion connective tissue was less than that at 0.025 M
MgCl 2. However, the staining was diminished in the connective tissue layer on Days 20-21 of pregnancy compared
to Days 18-19 and greatly decreased after incubation with
chondroitinase ABC on all days studied. This implies that
sulfated GAGs, probably chondroitin/dermatan sulfates,
were present in amnion connective tissue and underwent a
decrease in amount as parturition approached.
TEM showed that, on Days 18-20 of gestation, cross and
longitudinal sections of collagen fibrils were connected to
each other by fine, wispy thread-like structures (Fig. 1la).
Such threads probably represent GAGs/proteoglycans since
they resemble those described by Landemore et al. [18]. In
contrast, on Day 21 of pregnancy far fewer wispy threads
were observed joining adjacent collagen fibrils (Fig. lib).
These findings also imply a loss of GAGs/proteoglycans
from amnion extracellular matrix.
Loss of Cells and Collagenfrom the Outer Surface of the
Amnion
The surface of the amnion facing the visceral yolk sac
consisted of layers of cells and collagen fibrils/fibers (Fig.
5). The morphology of the main cell type resembled that of
334
PAAVOLA ET AL.
ALTERATIONS IN RAT FETAL MEMBRANES
fibroblasts in ordinary soft connective tissue. Such cells possessed numerous cisternae of rough ER containing a moderately electron-dense material, confirming the observation
of Wynn and French [11]. Together, the fibroblast-like cells
and collagen formed an incomplete layer that comprised
the outermost boundary of the amnion.
On Day 21, changes were evident in this area. The alternating layers of fibroblast-like cells and collagen that were
present at earlier stages were absent. Instead, the outer
boundary was formed by collagen and a discontinuous layer
of fibroblast-like cells (Figs. 6 and 9b). At this time, the lateral
extensions of these cells were often scalloped and separating
from the remaining collagen. Most of the cells that persisted,
however, had a normal fine structural appearance. Lastly, occasional cells were present that had a morphology consistent
with that of ovarian macrophages [20, 21]. Macrophages were
previously identified by immunohistochemistry in rat endometrium, myometrium, spongiotrophoblast, and decidua
basalis [22].
DISCUSSION
We have described for the first time the striking changes
in the morphology and organization of rat fetal membranes
that occur late in gestation. These alterations develop prior
to delivery and probably represent the histological and cellular bases underlying fetal membrane rupture at the time
of birth.
Marked changes in the gross appearance of rat fetal
membranes occur as the time of birth nears. We found that,
on Days 18-20 of gestation, both amnion and VYSP are
smooth, free of folds, and invest the fetus rather closely,
whereas on Day 21, the day of parturition, they are "baggy,"
have folds, and loosely envelope the fetus. As expected,
both membranes become increasingly fragile towards the
end of pregnancy. On Days 18-20, they are sturdy and do
not break upon gentle pressure with forceps, but on Day
21 they rupture readily with slight pressure.
The histological changes occurring in the rat VYSP were
subtle but important. They involve primarily the capsular
part of the yolk sac, the portion that forms the greater extent
FIG. 11. Loss of collagen and GAGs from amnion as birth nears. a) Day 20 of
gestation. From Days 18-20 of pregnancy, connective tissue layer is filled with large
bundles of collagen fibers (between arrowheads) that range from 0.3-1.5 pm in
diameter and contain up to 100 collagen fibrils. Fibrils are uniform 50 nm in diameter, are separated from each other by 25-50 nm, and display 69-nm banding pattern. Delicate material in form of wispy threads, probably representing GAGs/proteoglycans, connects adjacent fibrils. b) On Day 21 of pregnancy, dramatic changes
in amount, structure, and organization of connective tissue layer are apparent.
Fewer collagen bundles are present, and they as well as individual collagen fibers
(arrows) are widely separated from each other. Collagen bundles that remain contain fewer collagen fibrils, and fibrils are no longer of homogenous diameter (between arrowheads); some fibrils are unusually small (double arrows). Fewer wispy
threads connect fibrils of collagen bundles. x 43 200.
335
of the VYSP and covers most of the fetus. Under the conditions of this study, changes in the villous part of the VYSP
were difficult to detect. The most conspicuous changes in
the capsular yolk sac were a thinning of the connective tissue layer, due largely to a decrease in extracellular matrix
components, and an apparent decline in vascularity. These
alterations were present on Day 21, prior to the onset of
labor. Lavery and Miller [8] suggest that similar thinning of
term and preterm human fetal membranes has a role in their
rupture.
Changes in the rat amnion were more dramatic than
those of the VYSP and appeared to involve the entirety of
this structure. Prelabor modifications of the amnion included disorganization and/or loss of some of its constituents, including the deterioration and eventual loss of portions of its epithelium. The ultrastructural changes typically
seen in moribund amniotic epithelial cells appeared to be
consistent with those occurring during necrosis [231. We
found that the nucleoplasm and cytoplasm had a flocculent
appearance, cytoplasmic organelles were swollen, and the
nuclear envelope and plasma membrane displayed interruptions. It is noteworthy that cells comprising various
structures within the reproductive system, including luteal
cells and granulosa cells, often undergo apoptotic cell death
[24-27]. In this regard, it is interesting that amniotic epithelial cells did not display condensation and fragmentation of
the nucleus, morphological features that frequently but not
always accompany death by apoptosis [28]. Clearly, further
work is needed before we can define precisely how these
cells die. Finally, in some but not all human amnions obtained after premature membrane rupture and preterm delivery, Jenkins et al. [29] observed degenerative changes in
amniotic epithelial cell fine structure that were similar to
some of those observed by us in rats.
Equally dramatic was the delamination of portions of the
amniotic epithelium from the underlying connective tissue.
We found that detachment of this epithelium was associated
temporally with the appearance of abnormalities and/or deficits in the basal lamina. On Days 18-20 of pregnancy, the
basal lamina closely followed the contours of the basal plasmalemma of the epithelial cells. It bound them firmly to the
underlying connective tissue via its interaction with collagen [30]. By Day 21, the day of birth, marked changes were
apparent. The basal lamina was typically separated from the
epithelial cells. Even more striking was the absence of this
structure over considerable distances. Also occurring at this
time was a loss of amniotic epithelial cell cohesiveness as
the desmosomes, which normally provide strong cell-cell
attachment, deteriorated. It seems likely that separation of
epithelial cells from each other and of the epithelium from
the connective tissue involved one or more of the following:
loss of desmosomes, loss of basal lamina or its components,
loss of connective tissue collagen, and/or loss or modification of attachment sites, e.g., integrins on amniotic cells. In-
336
PAAVOLA ET AL.
terestingly, human amnions, obtained from full-term pregnancies at elective cesarean section, showed disruption of
the basement membrane in that portion of the membrane
covering the external os; however, detachment of the epithelium was not described [31].
Moreover, the rat amnion increased 3-fold in width as
parturition neared. This change was evident in limited areas
on the evening of Day 20 and involved the entire amnion
by the morning of Day 21. The increased width reflected a
"loosening up" of the connective tissue layer, which in turn
was probably due to the disorganization and loss of key
extracellular matrix components such as collagen and its
associated GAGs/proteoglycans. As shown here, the connective tissue layer of the amnion was compact on Days
18-20 of gestation and was composed of closely spaced
collagen bundles that were oriented largely parallel to the
surface of this membrane. Both its bundles and their constituent fibrils were of uniform size and shape. On Day 21
of pregnancy, however, marked differences were apparent.
The now-widened connective tissue layer appeared empty,
containing few, widely spaced, irregularly shaped collagen
bundles. In addition, the bundles contained fewer fibrils,
some of which were of unusually small diameter. Lastly, on
Day 21 the specific orientation of collagen bundles seen on
Days 18-20 was absent. These structural alterations, which
have not been previously described for rat fetal membranes,
are consistent with the findings of Harkness and Harkness
[12], who reported that weakening of rat fetal membranes
was accompanied by a decline in collagen concentration.
Our histochemical and TEM findings suggested that the
loss of collagen from rat fetal membranes was accompanied
by a decrease in glycoprotein and GAG/proteoglycan content. For example, diastase-resistant PAS staining, and
hence glycoprotein content, was diminished on Day 21
compared to Days 18-20 of gestation. Similarly, alcian blue
staining at higher molarities of MgCl 2 was decreased, and
fewer wispy threads linked adjacent collagen fibrils in the
connective tissue layer of Day 21 amnions compared to
those from Days 18-20 of pregnancy. The wispy threads
had a distribution remarkably like that reported for collagen-associated, sulfated proteoglycans, which were in the
form of "stick-like" structures in spleen fixed with GAGretaining compounds [18]. Similar stick-like structures on
collagen fibrils in connective tissue of rat amnions fixed
with GAG-stabilizing agents were abundant on Days 19-20
of pregnancy but were few in number on Day 21 (Paavola,
unpublished results). Moreover, the viscous material we observed clinging to rat fetal membranes on Day 21 of gestation may well represent the sugar-containing GAGs that
they lose at this time. It is germane that human fetal membranes also suffer a loss of collagen and GAGs as gestation
ends [32-34]. Taken together, the changes in connective tissue collagen and associated GAGs/proteoglycans described
in this and the preceding paragraph probably account for
the increased "bagginess" of rat amnion and VYSP on Day
21 of pregnancy.
The factors responsible for deterioration of the basal lamina, for the loss and disorganization of collagen and GAGs/
proteoglycans in the connective tissue layer of the amnion,
and for the diminution of the connective tissue layer in the
capsular portion of the VYSP remain obscure. However,
work in this laboratory [351 has shown that a marked rise in
matrix metalloproteinase 9 (MMP-9, a gelatinase) mRNA occurred in rat amnion on the evening of Day 20 of pregnancy,
and on Day 21 MMP-9 activity appeared in the amnion [35].
MMP-9 activity was also higher in the capsular VYSP on Day
21 than on Days 18-20 [35]. Since this enzyme can degrade
extracellular matrix components including constituents of
the basal lamina [36] and since its appearance coincided
temporally with the structural changes seen here, it may be
instrumental in bringing about the decline and disorganization of connective tissue elements in rat fetal membranes.
Indeed, alterations in the biomechanical properties (i.e.,
tensile strength) of human chorioamniotic membranes have
been proposed to be mediated by enzyme action [37]. Recently, we have provided evidence that enzymes have a role
in parturition by showing that MMP-9 activity is increased
in human amniochorion at the time of birth [38]. Moreover,
these data are consistent with our observations on changes
in this enzyme in rat amnion [351. Also relevant is our finding that incubation of rat amnion with connective tissuedegrading enzymes produced histological changes in the
connective tissue layer similar to those seen in intact amnions fixed in situ on the day of parturition. Both showed
markedly reduced staining for collagen and GAGs/proteoglycans. Nonetheless, further work is necessary to clarify
the role of amnion-produced MMPs in preparing this membrane for its eventual rupture during labor.
Collagen, mainly types I, III, and IV [391, and associated
GAGs/proteoglycans (chondroitin and dermatan sulfates)
[401 account for the bulk of human fetal membrane matrix
[39], and this is undoubtedly true for rats as well. Collagen
is the key molecule that imparts tensile strength to tissues,
including fetal membranes [39]. Thus, it seems likely that
loss of this molecule from fetal membranes would lead to
their weakening and to an increased likelihood of breaking.
However, a consensus on this issue has not yet been
reached, at least for humans. Al-Zaid and Bou-Resli [41],
Evaldson et al. [42], Halaburt et al. [43], and Yoshida and
Manabe [44] report that neither term human fetal membranes nor those that ruptured prematurely had decreased
amounts of collagen compared to earlier stages. In contrast,
Skinner and coworkers [33, 34] and Bou-Resli et al. [32]
found that total collagen as well as the number of collagen
fibrils decreased as birth neared in full-term and prematurely ruptured human fetal membranes. These disparities
may be reconciled by the recent findings of Malak and Bell
337
ALTERATIONS IN RAT FETAL MEMBRANES
[6] who, using a quantitative structural approach, identified
a zone of highly altered morphology that was of limited
distribution along the rupture site in recently delivered term
human placentas. This zone was marked by disruption of
the connective tissue and by swelling of various layers of
the fetal membranes, including the amnion. The authors
believe this zone to be the initial site at which rupture occurs. This notion gains support from the findings that 1)
disruption of connective tissue collagen was present only in
areas adjacent to the rupture site in human fetal membranes
that ruptured prematurely [32] and 2) decreased amounts of
soluble collagen occurred only in the region of the rupture
site in human chorions obtained at term delivery [9].
Few reports have focused on fetal membrane rupture in
the rat. A single early biochemical and physical study by
Harkness and Harkness [12] showed that a decrease in collagen content of rat fetal membranes was accompanied by
a decline in their physical strength. The approach these researchers used, however, did not permit a determination of
the membrane(s) affected, i.e., VYSP, amnion, or both. We
have corroborated and expanded upon these findings by
documenting at a visual level that collagen is decreased and
disorganized in both amnion and VYSP. We have also
added much new information by identifying temporal and
fine structural aspects of rat fetal membrane deterioration
at the end of gestation.
The nature and source of the signals precipitating the
morphological changes seen in rat fetal membranes in late
pregnancy have not been established. As noted above, the
agents mediating the striking structural changes in the rat
and human could be a matrix metalloproteinase [35, 43].
What brings about the production and/or activation of these
enzymes? Katsura et al. [45] have reported that cytokines,
including interleukin-la and tumor necrosis factor a, increase synthesis of collagenase, at least in cultured human
chorionic cells. Furthermore, these and other cytokines accumulate in amniotic fluid in pregnancies associated with
preterm labor [46]. However, whether or not cytokines are
indeed the factors that set in motion the events leading to
membrane rupture remain unknown. Whatever the nature
of the signals, it is clear that the factors are in place before
active labor begins since both functional and structural alterations precede parturition by hours. On the basis of our
data we propose that, in response to an as yet unknown
event, a signal(s) appears that induces and/or activates fetal
membrane matrix metalloproteinases that, in turn, degrade
membrane components, leading to their disorganization
and weakening. Thus, rupture of rat fetal membranes is not
merely a passive event mediated solely by myometrial contractions during birth, but an active one, resulting from development of structural weaknesses within the membranes
themselves before the onset of active labor.
Lastly, it is important to note that the changes in histo-
logical organization that we have described for rat fetal
membranes bear a marked similarity to those noted by Jenkins et al. [29] and Malak and Bell [6] for term human fetal
membranes. For example, there is a strong resemblance between the swelling and disruption of connective tissue in
the amnion in the "zone of extreme altered morphology"
in humans (Fig. 5; Malak and Bell [6]) and the connective
tissue changes we noted here for rat amnion. Such parallels
raise the possibility that the rat may be a suitable model for
membrane studies, one that would allow the effects of experimental manipulation to be assessed.
In conclusion, our results indicate that dramatic changes
occur in the structure and organization of rat fetal membranes in the prelabor period. Both VYSP and amnion become increasingly fragile and susceptible to breaking as
birth approaches. The decline in extracellular matrix components that impart strength and integrity occur several
hours before the onset of labor and presumably underlie
the ready rupture of the fetal membranes during birth. Such
changes are remarkably similar to those described for human fetal membranes, underscoring the potential usefulness of the rat model.
ACKNOWLEDGMENTS
We would like to express our gratitude to Mr. Robert Smith and Mrs. Neelima Shah for
their invaluable technical assistance in preparing samples for TEM.
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