/ . Embryol exp. Morph. Vol. 39, pp. 9-21, 1977
Printed in Great Britain
Morphologic alterations in
the parietal yolk-sac of the rat from the
12th to the 19th day of gestation
By R.P. JENSH, 1 T. R. KOSZALKA, M.JENSEN,
L. BIDDLE AND R. L. BRENT
From the Department of Anatomy and Stein Research Center, Jefferson
Medical College, Philadelphia
SUMMARY
The rat parietal yolk-sac and its adherent epithelial cells were examined at various stages
of gestation using an en face technique. Specimens were observed at both the light and
electron microscopic level. Diastase pretreatment and PAS-staining were used to determine
the presence of glycogen. As early as the 12th day of gestation the cytoplasm of the parietal
yolk-sac cells contained numerous ribosomes and mitochondria and a large amount of endoplasmic reticulum. The glycogen content of the epithelial cells increased from the 12th day
of gestation and accumulated in large quantities by the 16th day. By the 17th day many cells
exhibited variable degrees of degeneration. Cellular elements of degenerating cells appeared
to be trapped within Reichert's membrane. Contrary to the reports of other investigators, the
present study indicates that the capsular portion of the parietal yolk-sac consisting of
Reichert's membrane and its adherent epithelial cells remained intact until at least the 18th
day of gestation. Some of the unique characteristics of the parietal yolk-sac provide experimental models to study the effects of environmental factors on (1) the synthesis of basement
membranes, (2) the ageing of cells and (.3) the correlation of these histologic changes with the
functions of the parietal yolk-sac.
INTRODUCTION
The role of the rat parietal yolk-sac (PYS) in normal fetal development has
been of increasing interest since it was found that teratogenic kidney and rat
visceral yolk-sac antisera localized in Reichert's membrane (RM) of PYS as
well as in the visceral yolk-sac (Slotnick & Brent, 1966; Brent, Johnson &
Jensen, 1971; Jensen & Brent, 1972; Jensh, Koszalka, Jensen & Brent, 1974;
Jensen, Koszalka & Brent 1975).
Previous investigators have described the yolk-sac placenta of the rat (Everett,
1935; Wislocki & Padykula, 1953; Wislocki & Dempsey, 1955; Jollie, 1964,
1968; Padykula, Deren & Wilson, 1966; Beck, Lloyd & Griffiths, 1967) at
different stages of gestation. Some of these studies have concentrated on yolksac morphology at specific times of gestation or throughout gestation (Everett,
1
Author's address: Department of Anatomy and Stein Research Center, Jefferson Medical
College, 1020 Locust Street, Philadelphia, Pennsylvania 19107, U.S.A.
10
R. P. JENSH AND OTHERS
1935; Wislocki & Dempsey, 1955; Jollie, 1968), while other studies have been
concerned with the histochemical and biochemical nature of the yolk-sac
placenta under normal and experimentally altered states (Wislocki & Padykula,
1953; Padykula et al 1966; Beck et al. 1967).
The importance of PYS in normal rodent development has been previously
discussed, usually in the context of placental formation and function. The objective of the present investigation was to examine the changes in morphology
of the PYS cells and RM from the 12th to the 18th day of rat gestation.
METHODS AND MATERIALS
Pregnant Wistar albino rats were placed in cages with males overnight. The
following morning was considered to be the first day of gestation, if sperm were
found in smears obtained from the vagina. Rats were killed at various stages of
gestation, the uterine wall was dissected away, and the implantation sites (consisting of the visceral and parietal yolk-sacs, amnion, fetus, chorioallantoic
placenta and associated tissues) were isolated and placed in phosphate-buffered
saline (PBS) at pH 7-2. The capsular portion of the PYS was detached and
separated from the chorioallantoic placenta. After the 15th day of gestation,
the dissected PYS was mounted en face on microscope slides with the PYS
cells facing upward, using albumin-glycerin affixative. PYS less than 14 days
old contained trophoblastic giant cells which were firmly attached to RM on
the maternal side and had to be removed before mounting as follows: PYS was
transferred to a Petri dish containing 0-01 M EDTA in PBS and allowed to
remain at 25 °C for about 15-20 min. At the end of this time RM, with its
adherent PYS cells, was separated from the sheet of trophoblastic giant cells
containing many islands of red blood cells. The PYS, now devoid of trophoblast,
was then mounted on microscope slides as described earlier. The PYS was
air-dried for 10-15 min at 40 °C. Half of the slides were stained directly with
Periodic acid-Schiff base (PAS) reagent without prior fixation. The remaining
slides were fixed in Formalin-acetic acid-ethanol for 5 min and then routinely
stained with Mayer's hematoxylin and eosin (H & E) or PAS.
Distribution of glycogen in the PYS cells was determined histochemically
using light microscopy as follows. One drop of the cell suspension obtained
from PYS on the 16th or 17th day of gestation was placed on the microscope
slide, air-dried, fixed in 95 % ethanol, and incubated in malt diastase (1 mg/ml)
at 37° for 30 min. Control slides were incubated in distilled water at 37° for 30
min. Slides were then stained with PAS to demonstrate the presence of glycogen
(Vallance-Owen, 1948).
Electron microscopy
Parietal yolk-sac from the 12th to 16th day of gestation was dissected free
from the surrounding tissue and placed quickly into cold 3 % glutaraldehyde
in 0-1 M phosphate buffer, pH 7-4 (Sabatini, Bensch & Barnett, 1963). EDTA
Parietal yolk-sac of the rat embryo
11
was not utilized during the electron microscopy procedures. After 20 h, the PYS
was rinsed in Sorensen's phosphate buffer and postfixed for 30min in 2 %
osmium tetroxide. Tissue was then dehydrated in graded concentrations of
ethanol and embedded in Epon 812. Tissue blocks were oriented to achieve a
section perpendicular to the epithelial surface of the PYS. Grids were doublestained with lead citrate according to the method of Reynolds (1963), and with
uranyl acetate (Huxley & Zubay, 1961). Thick sections were also cut and stained
with 1-0% toluidine blue-0 according to Trump, Smuckler & Benditt (1961).
The presence and intracellular localization of glycogen were determined in
the PYS in the following manner. On the 16th day of gestation, the PYS was
dissected free from surrounding tissue, flattened and placed in 3 % glutaraldehyde for 20 min. Each PYS was cut into two pieces and washed quickly in
three changes of phosphate buffer. Half of the PYS was incubated for 30 min
at 37 °C with malt diastase (1 mg/ml of distilled water). The other half of the
PYS served as a control and was incubated in phosphate buffer. After incubation
both samples were washed in 0-1 M phosphate buffer and returned to the glutaraldehyde. The tissue samples were then prepared for electron microscopy
as previously described.
RESULTS
Throughout the period of observation (12th to 18th day of gestation), a
pattern of cellular morphological changes occurred in PYS cells which was
consistent with tissue ageing. On the 12th day of gestation PYS cells were
spherical or oval and exhibited well developed endoplasmic reticulum and
mitochondria (Table 1). The cell outlines were regular, and the basal aspects
exhibited smooth attachments to RM. Dead or dying cells were rarely observed
until the 14th day of gestation (Figs. 3 and 4). The spherical cells continued to
exhibit progressively greater amounts of endoplasmic reticulum and mitochondria (Table 1). By the 15th day of gestation dying cells became progressively more apparent. PYS cells were beginning to exhibit invasive processes
into RM.
The basal part of the cell outline of many PYS cells showed significant irregularities with processes projecting into RM by the 15th day. By the 16th and 17th
days of gestation, most of the PYS cells showed evidence of progressive degeneration, with many vesicular spaces in the cytoplasm, little endoplasmic reticulum,
and mitochondria with incomplete, poorly structured cristae (Figs. 11 and 12).
As pregnancy proceeded, many PYS cells died while others maintained their
normal morphologic appearance (Figs. 6 and 8).
Three morphological variants of the PYS cell became apparent as early as
the 16th day, although they became more obvious on the 17th and 18th days of
gestation: (a) apparently normal spherical cells, with processes extending into
RM, (b) cells undergoing autolysis via expansion and breakdown, and (c) cells
undergoing autolysis via condensation (Fig. 8). The embryonic side of RM
12
R. P. JENSH AND OTHERS
Fig. 1. A rat implantation site obtained at the 18th day of gestation. The entire
structure was fixed for one week in 3 % glutaraldehyde at 4 °C. The visceral yolksac (VYS) has shrunk as a result of fixation and is displaced from its original location, thus exposing the parietal yolk-sac (PYS) to full view. At this stage PYS is
completely intact, consists of Reichert's membrane and parietal cells, and is devoid
of trophoblast. x 3.
Fig. 2. The same implantation site as shown on Fig. 1, but with Reichert's membrane (RMj ruptured. A large piece (arrow) of RM is still attached to the periphery of the chorioallantoic placenta (P). It is virtually transparent except for several
areas which presumably contain parietal cells and debris, x 3.
Parietal yolk-sac of the rat embryo
13
Table 1. A summary of the changes in the cells and basement membrane of the
rat parietal yolk-sac occurring from day 12 to 18 of gestation
Age (days)
12
13
14
15
16—17
18
Cell type
description
Cell death
Little or none
Oval or round;
apparent
well developed
ER, ribosomes
and mitochondria; smooth
cellular membrane
Same as above:
Same as above
smooth membrane at surface
ofRM
A few cells
As above
appear to be
dying (ER +
mitochondrial
breakdown etc.)
Some cells exIncrease in cell
hibit autolysis
death; cells show
by dispersion;
progressive inothers by
vasion of procondensation
cesses into RM
Increased; cell
Same as above
debris entrapped in RM
Three cell types:
(a) healthy;
(b) expanding
and dispersing
(dying); (c) condensed, pyknotic
(dying) viable
cells deeply
penetrating RM
Greatly increased
Reichert's
membrane
Presence and
distribution of
glycogen
Regular and
smooth monolaminar;
homogeneous
Little; granules
scattered throughout cytoplasm
Same as above
Increases; granules
throughout cell
Same as above
Same as above
RM is bilaminar
Granules begin to
be massed near
the cell membrane
Bilaminar; on
embryonic side
heterogeneous
due to entrapped
cell debris
More heterogeneous facing
the y.s. cavity;
that part facing
the trophoblast
remained relatively homogeneous
Large masses near
basal area of cell
membranes
Viable cells contain
a large localized
mass of granules
near to basal surface of the cell
(nearest RM)
exhibited large amounts of entrapped particles by the 18th day. Upon isolating
the freshly dissected implantation site, it was found that the entire PYS consisting
of RM, but devoid of trophoblast, was still intact on the 18th day of gestation (Figs. 1 and 2). By the 19th day, the capsular portion of PYS had already
ruptured.
Until the 15th day, RM appeared characteristically homogeneous. After
the 15th day of gestation RM began to exhibit a bilaminar appearance, the
2
EMB 39
14
R. P. JENSH AND OTHERS
Fig. 3. A typical rat parietal yolk-sac cell at the 12th day of gestation. An abundance of rough endoplasmic reticulum and mitochondria are apparent, as are the
individual masses of glycogen dispersed throughout the cytoplasm. Reichert's
membrane (RM) lying between the maternal blood sinuses (MBS) and the yolk-sac
cavity appears homogeneous and electron-dense, x 7000.
Fig. 4. A parietal yolk-sac cell at the 14th day of gestation showing large numbers
of mitochondria and an abundance of rough endoplasmic reticulum. The glycogen
is located primarily at the periphery of the cell, especially in the basal areas next to
Reichert's membrane (RM). (MBS = maternal blood sinus; YSC = yolk-sac
cavity. x7000.
Parietal yolk-sac of the rat embryo
Fig. 5. A parietal yolk-sac cell and Reichert's membrane (RM) on the 16th day of
rat gestation. Glycogen granules can be seen massed primarily at the periphery of
the cell. The cell membrane is becoming more irregular, its projections into Reichert's
membrane (RM) being similar to the dense masses observed in that part of the membrane adjacent to the yolk-sac cavity (YSC). x 7000.
Fig. 6. A section of parietal yolk-sac from a rat on the 16th day of gestation. The
parietal yolk-sac cells are close together and, as in this photograph, are often multilayered. The glycogen is located in distinct masses (arrows). Some cells show signs
of degeneration. Reichert's membrane (RM) now appears bilaminar; the side
adjacent to the yolk-sac cavity containing a large amount of electron-dense debris,
while the side adjacent to the maternal blood sinus (MBS) remains homogeneous.
The cell surfaces are becoming irregular, x 5500.
15
16
R. P. JENSH AND OTHERS
YSC
Fig. 7. One type of terminal stage for a parietal yolk-sac cell, showing cell debris
entrapped on the 17th day of gestation in that part of Reichert's membrane (RM)
adjacent to the yolk-sac cavity (YSC). x 7000.
Fig. 8. Two stages of autolysis of rat parietal yolk-sac cells at the 17th day of gestation. The pale center cell is undergoing autolysis by expansion and breakdown,
while the cell above it is beginning to condense. Cytoplasmic projections can be
seen penetrating Reichert's membrane (RM). The lower cell presents a normal
pattern. x6000.
Parietal yolk-sac of the rat embryo
17
embryonic side becoming progressively more heterogeneous while the maternal
side remained homogeneous (Figs. 5-7). The heterogeneity of the former
appeared to be due to entrapment of cellular debris, which increased with age
(Fig. 7). On the 15th day of gestation, RM underwent a significant change in
structure. As PYS cells died, they autolyzed and the cell remnants appeared to
become entrapped within the embryonic side of RM (Figs. 6 and 7). The endodermal (embryonic) but not the trophoblastic (maternal) side of RM contained
many electron-dense particles within the membrane.
The PAS method was originally used in an attempt to follow histochemically
the intracellular biosynthesis of glycoprotein precursors involved in the synthesis and secretion of RM. Initially it was believed that the PAS-positive
granules which are present in the PYS cells represented such precursors, since
it had been previously reported that these granules were resistant to amylase
digestion (Wislocki & Padykula, 1953). However, our results indicated that by
the 14th day of gestation most of the PAS-positive granules were sensitive to
diastase and probably represent glycogen (Figs. 4, 5 and 9). By the 16th day of
gestation these granules became localized primarily in discrete masses in the
basal portion of the cells (Figs. 9 and 11). The clear areas within a specific cell
represent sites of malt diastase digestion and support the view that many of the
granules are glycogen and not glycoprotein as previously supposed (Fig. 10).
DISCUSSION
In the present study the PYS and isolated PYS cells were observed not only
by the use of transmission electron microscopy, but also by use of flat {en face)
histologic preparations in order to observe relationships obscured in histological cross-sections. The results of the present study are in agreement with the
findings of Pierce, Beals, Sri Ram & Midgley (1963, 1964) and Jollie (1968), in
that RM is neither a condensation of connective-tissue ground substance nor a
secretion by the trophoblastic cells, but is synthesized by PYS cells. Their conclusions were based on Coons' (1958) histochemical techniques using fluorescent
antibodies and by various studies involving the mouse 'parietal yolk cell'
carcinoma. The recent works of Clark et ah (1975) and Minor et ah (1976) on
the incorporation of [14C]proline into RM in vitro proved unequivocally that
basement-membrane collagen in RM is synthesized only by PYS cells in the rat,
and that new basement membrane is deposited on existing RM in contact with
the PYS cells. Furthermore, studies reported by Payne & Deuchar (1972) on the
functions of rat embryonic membranes in vitro indicate that the PYS is necessary
for normal growth of the embryo. Their studies demonstrated that visceral yolksac alone is not adequate for optimal nutrition, since the presence of an intact
PYS resulted in increased amino acid incorporation by the embryo.
In the present study the PYS cells have been shown to contain glycogen
granules (Fig. 9). In addition, malt diastase pretreatment specifically hydrolyzed
18
R. P. JENSH AND OTHERS
Parietal yolk-sac of the rat embryo
19
glycogen, leaving clear areas in the cytoplasm which are seen electron microscopically. Although in earlier studies Wislocki & Padykula (1953) were unable
to demonstrate glycogen in rat PYS cells, later Wislocki & Dempsey (1955)
succeeded in observing glycogen granules using the electron microscope. In the
present study, utilizing both histochemical and electron microscopic techniques,
glycogen has been shown to be present in the PYS cells of the rat as early as the
12th day of gestation. Furthermore, the amount of glycogen appeared to increase within the PYS cell during gestation. The glycogen in PYS cells may play
a role in the maintenance of the fetus, or may act as an energy store for the
metabolic demands of the PYS cells.
Previous studies have indicated that the PYS ruptures by the 15th (Everett,
1935; Padykula et ah 1966) or 16th (Wislocki & Padykula, 1953; Jollie, 1964)
day of gestation. In the present study, however, PYS was removed directly
from living material as late as the 18th day of gestation. In all cases PYS was
shown to be fully intact, since the capsular portion of PYS could be removed in
toto from the 12th to the 18th day of gestation (Figs. 1 and 2). PYS was examined
in its entirety, including both the area anatomically related to the decidua parietalis and that which covers the fetal surface of the chorioallanotic placenta. It is
therefore suggested that the rupturing of RM, particularly that part related
to the decidua parietalis, observed by other authors on the 15th or 16th days of
gestation was most likely a fixation artifact, especially since many earlier studies
involved observations on fixed preparations of the embryo and its extraembryonic membranes.
In the present study changes were observed in the structure of rat PYS cells
with age. Although previous investigators (Wislocki & Padykula, 1953; Jollie,
FIGURES
9-12
Fig. 9. An area of the PYS cell showing an abundance of masses of glycogen (Gl).
The cell seems still metabolically active as evidenced by the presence of many mitochondria (M) and the large amounts of endoplasmic reticulum(ER). (N = nucleus.)
x 30000.
Fig. 10. An electron micrograph of the rat parietal yolk-sac cell treated on the 16th
day of gestation with malt diastase to remove glycogen. The clear areas (arrows)
represent areas where glycogen had been stored. It is evident that hydrolysis was not
completed as particles of glycogen still remain. Exposure to malt diastase was limited in order to maintain the integrity of the cell, x 17000.
Fig. 11. A typical rat PYS yolk-sac cell on the 17th day of gestation. There is an
accumulation of glycogen basally. Much of the cell's cytoplasmic area is of the
same density as Reichert's membrane (RM). Basally, the cell has processes which
extend deeply into the part of Reichert's membrane (RM), facing the yolk-sac cavity
(YSC). x5000.
Fig. 12. Seventeenth-day parietal yolk-sac cell showing the cytoplasmic inclusions
in Reichert's membrane and apparent continuity with RM (arrow). The cell membrane appears discontinuous at magnifications three to four times that of the
present photograph, x 17000.
20
R. P. JENSH AND OTHERS
1964,1968) had observed specific limited changes, these were not discussed as an
ageing phenomenon. The results of the present investigation revealed a population of cells which exhibit progressive ageing within a relatively short time span.
From the 12th to 14th day the PYS cells were all spherical, with well developed
RER and mitochondria, and showing a gradual increase in glycogen with age.
By the 14th day aggregates of glycogen became evident near the cell membrane
of the PYS cells. At this stage RM became bilaminar and its embryonic side
appeared to contain entrapped cellular debris. By the 18th day three morphologic variants of PYS cells could be observed (Table 1). Cell death, as such, was
greatly increased. Although still intact, RM was increasingly heterogeneous on
its embryonic side. Throughout the period of observation the PYS cells exhibited progressive penetration of RM by cellular processes.
In conclusion, the data presented in this study indicate that the PYS cells
undergo an identifiable life-cycle during gestation in the rat and that during that
life-cycle the cells undergo several morphologic changes consistent with the
process of ageing. During their life-time the PYS cells synthesize and secrete a
basement membrane, i.e. RM, and accumulate increasingly large amounts of
glycogen within their cytoplasm. Contrary to previous reports, the capsular
portion of the PYS consisting of RM and its epithelial cells remains intact until
the 18th day, after which time it ruptures. The exact roles of the PYS cells and
RM in development remain to be elucidated.
This research was supported by N1H grant HD-630. The authors gratefully acknowledge
the assistance of Ms Carole Andrews; Mrs Kathryn Robnett and Mrs Betty Ward for typing
the manuscript.
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{Received 22 June 1976, revised 23 November 1976)