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Ultrastructural study of the keratinization of the dorsal epithelium of the tongue of Middendorff's bean goose Anser fabalis middendorffii (Anseres Antidae)

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THE ANATOMICAL RECORD 247:85–101 (1997)
Formation of Reticulated Endoderm, Reichert’s Membrane, and
Amniogenesis in Blastocysts of Captive-Bred, Short-Tailed Fruit Bats,
Carollia perspicillata
NILIMA K. BADWAIK,1 JOHN J. RASWEILER IV,1* AND SERGIO F. OLIVEIRA2
1Department of Obstetrics and Gynecology, Cornell University Medical College,
New York, New York
2Department of Histology and Embryology, Institute of Biomedical Sciences, University of
Sao Paulo, Sao Paulo, Brazil
ABSTRACT
Background and Methods: As part of an effort to develop
the short-tailed fruit bat (Carollia perspicillata) as a new animal model
for the study of interstitial implantation and trophoblast–uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive-bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]).
Results: In most of the early uterine embryos examined on days 16–18
p.c., much of the endoderm appeared as a reticulated meshwork; however,
a unilocular yolk sac was formed prior to the development of any
mesoderm. Early blastocysts of Carollia were also unusual in that endoderm surrounded much of the inner cell mass (ICM), Reichert’s membrane
continued over the dorsal side of the ICM, and basal laminalike material
was observed around many of the endoderm and epiblast cells. A primordial amniotic cavity was formed between days 19 and 26 p.c. by cavitation.
The first mesoderm appeared between days 23 and 26 p.c., concommitant
with the development of an embryonic shield.
Conclusion: The unusual reticulated appearance of early endoderm in
Carollia, which is reminiscent of that seen in early human blastocysts,
may be attributable to constraints imposed on growth of the blastocyst by
the site and mode of implantation, temporary retardation of trophoblastic
invasion by the basal laminae of endometrial epithelial elements, and
endodermal proliferation in anticipation of rapid yolk sac expansion.
Reichert’s membrane appears to play an important role in this species in
tethering the ICM and embryonic shield to the developing placenta prior
to the formation of significant amounts of mesoderm. Anat. Rec. 247:85–101
r 1997 Wiley-Liss, Inc.
Key words: reticulated endoderm, reichert’s membrane, amniogenesis,
embryogenesis, fruit bat
Early blastocysts of the human and chimpanzee are
unusual in possessing a reticulated meshwork that was
initially identified as precociously formed extraembryonic mesoderm (Heuser, 1940; Hertig and Rock, 1941;
Hertig, 1945). Luckett (1974, 1978) subsequently suggested that the reticulated meshwork of cells that fills
much of the blastocyst cavity in the 11-day-old human
and the 10.5-day-old chimpanzee is actually extraembryonic endoderm. Such a meshwork is absent in rhesus
monkey and baboon blastocysts; however, cells appear
between the primitive endoderm and trophoblast of
rhesus monkey blastocysts and progressively develop
mesenchymal characteristics. This finding led Enders
and King (1988) to conclude that endoderm gives rise to
the first extraembryonic mesoderm in the rhesus monkey. They also suggested that the ‘‘endodermal reticur 1997 WILEY-LISS, INC.
lum’’ in the human may actually constitute forming
mesenchymal cells and hence the first extraembryonic
mesoderm.
In the course of studies intended primarily to examine implantation, placental development and trophoblast-uterine interactions in captive-bred, short-tailed
fruit bats (Carollia perspicillata), early blastocysts
were seen to develop a reticulated meshwork much like
that observed at comparable stages in the human.
Although the cells making up this meshwork initially
Received November 6, 1995; accepted August 19, 1996
*Correspondence to: Dr. John J. Rasweiler IV, Department of
Obstetrics and Gynecology, Cornell University Medical College, 1300
York Avenue, New York, NY 10021.
Contract grant sponsor NIH; Contract grant number HD28592.
86
N.K. BADWAIK ET AL.
have some mesenchymal characteristics, they soon give
rise to a unilocular yolk sac lined by an endodermal
monolayer. This transformation occurs prior to amniogenesis and is sharply separated in a temporal sense
from the formation of any mesoderm. Several other
aspects of early blastocysts in this bat are also unusual:
endoderm is not limited to the lining of the yolk sac
but rather surrounds much of the inner cell mass;
Reichert’s membrane is not associated just with the
parietal endoderm (as in rodents) but continues over
the dorsal side of the inner cell mass; and basal
laminalike material is commonly observed on other
surfaces of the endoderm cells and between some of the
epiblast cells.
Early embryonic development in Carollia is also of
interest because the morphological organization of the
blastocyst, mode of initial formation of the yolk sac, and
differentiation of cytotrophoblast within the preplacenta differ significantly from that observed in another
bat (Noctilio albiventris; Rasweiler and Badwaik, 1996)
belonging to the same superfamily. This difference
indicates that, even in relatively closely related mammals, early cell lines can exhibit considerable interspecies diversity in their phenotypic and functional differentiation.
The timing of the developmental events reported
here has assumed considerable importance because
pregnancy in Carollia can be substantially prolonged
under some circumstances both in captivity and the
wild. In captivity, this prolongation is due primarily to
the retardation of conceptus development after implantation is completed (Rasweiler and Badwaik, in press).
MATERIAL AND METHODS
Source of the Animals
All of the C. perspicillata used in this study were
collected on the island of Trinidad in the West Indies.
Animal Maintenance
The animals were kept in rooms with a controlled
light cycle (12 hr light and 12 hr dark), with the dark
phase set to commence at 1600 hr to facilitate feeding
just before its onset. The temperature was maintained
between 24 and 27°C. The bats were housed in bipartite
cages, measuring approximately 170 cm wide 3 81 cm
high 3 77 cm deep, which permitted the animals to fly.
The cages were divided into an open feeding area (92 cm
wide) and a darkened roosting box connected by an
access hole (15 cm high 3 30 cm wide). The bats were
maintained in sexually segregated groups composed of
8–12 males or 10–20 females until the time of breeding.
They were fed a fruit-based liquid diet prepared from
readily available canned and powdered components
(peach nectar, canned peaches, ground monkey chow,
dibasic calcium phosphate, corn oil, an emulsifier, and a
multivitamin preparation). This diet was occasionally
supplemented with small amounts of apple, banana, or
melon as treats. The animals were fed every night
without exception, and the diet was served cold, usually
no more than 60–90 min before the room lights went off,
to minimize microbial growth. Water was provided ad
libitum in chick waterers. Further details of the animal
husbandry procedures may be found elsewhere (Rasweiler and Badwaik, 1996).
During the course of this work, it was discovered that
lab-bred C. perspicillata exhibited substantial variation in gestation length (.2 months). This variation
was more pronounced in wild-caught animals bred
during their first year in captivity than during their
second. Furthermore, most of the developmental delays
occurred after the initiation of implantation (Rasweiler
and Badwaik, in press, unpublished observations). The
extent to which the rate of development may differ
during the preimplantation period and implantation
has not yet been determined. For this reason, it should
be pointed out that the material preserved for light
microscopic examination in this study was obtained
from animals killed at the following intervals after
capture in the wild: 4–9 months, 17 bats; 12–18 months,
22 bats; and 24–27 months, 36 bats. All of the specimens examined with the electron microscope were
obtained from females that had been maintained in
captivity for more than 12 months.
Timing of Reproductive Stages
For breeding purposes, a single male with prominent
testes was added to groups of 10–18 females. On
subsequent mornings, between 0530 and 0900 hr, a
small quantity of distilled water was aspirated in the
vagina of each female with a microeyedropper. The
aspirate was then dried and examined for spermatozoa.
Most females exhibited a single period of spermpositive vaginal aspirates that usually varied from 1 to
5 days in duration (Rasweiler and Badwaik, 1996). In a
few cases, females exhibited two separate periods of
sperm- positive smears. For these females, the probable
time of conception could be identified from an examination of their breeding records and the stage of development of their embryos. Mated females were killed
between 0630 and 1030 hr at different intervals after
the first morning on which spermatozoa were detected
in their vaginal aspirates (day 1 postcoitum [p.c.]). The
animals were killed by administering an intraperitoneal injection of sodium pentobarbital at a dosage of
approximately 90 mg/kg body weight.
Histological Procedures
The reproductive tracts were fixed in Zenker’s fluid
for 8–10.5 hr, immersed in 2.5% aqueous potassium
dichromate for an additional 2 hr, washed overnight in
running tap water, dehydrated through graded ethyl
alcohols, cleared overnight in warm cedar wood oil
(37°C) followed by Histosol or Histoclear II (National
Diagnostics, Atlanta, GA), and embedded in paraffin
wax. The tracts were then serially sectioned (in a
frontal plane) at 6 µm. The histological sections were
stained with hematoxylin and eosin, Weigert’s resorcinfuchsin (Clifford and Taylor, 1973) followed by Masson’s
trichrome procedure (modified from Humason, 1972),
or by the periodic acid-Schiff (PAS) technique and
counterstained with hematoxylin. In the modified Masson’s trichrome procedure, mordanting in iron alum
was omitted, and the nuclei were stained with stabilized iron chloride hematoxylin (Lillie, 1965). Also,
when the sections were washed in running water after
87
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
TABLE 1. Location of the embryo during early pregnancy in the short-tailed fruit
bat, Carollia perspicillata
Day postcoitum
Developmental stage and location
12
13
14
15
16
Oviductal blastocyst (1ZP)
Oviductal blastocyst (2ZP)
Uterine blastocyst close to uterotubal junction
Uterine blastocyst within usual implantation zone
Implanting uterine blastocyst
1
1
1
3
—
—
—
1
5
1
—
1
—
1
3
—
—
—
1
7
—
1
—
—
6
1ZP, zona pellucida present; 2ZP, zona pellucida not present.
being stained with acid fuchsin and ponceau de xylidine, the wash time and water temperature (23°C) were
precisely controlled. Some sections of each tract were
incubated for 1 hr at 37°C in 0.1% a-amylase (1,4a-D-glucan-glucanohydrolase; Sigma Chemical Company, St. Louis, MO) dissolved in a 0.02 M phosphate
buffer (pH 6) to remove any glycogen before being
processed according to the PAS procedure. Parallel
sections were incubated under similar conditions in the
buffer alone and then stained.
OBSERVATIONS
Location of Embryos
The location of the embryos examined histologically
on days 12–16 p.c. is shown in Table 1. From day 17 p.c.
on, all of the blastocysts were found in the uterus. The
first clear evidence of implantation (trophoblastic penetration of the uterine epithelium) was observed in
some animals on day 14 p.c. However, earlier phases of
the process may have been disrupted by shrinkage of
the specimens during histological processing.
Specimens Examined Histologically
Uterine Anatomy and Site of Implantation
The reproductive tracts from 5–8 mated females per
day were examined for embryos on days 12–25. The
tracts from 2–3 mated females per day were examined
on days 26, 28, and 30.
Although Carollia has a simplex uterus, relatively
narrow tubular segments are interposed between the
distal end of each oviduct and the main uterine cavity.
These segments are clearly uterine because they are
lined by endometrium, and some uterine glands open
into their lumina. They appear to be homologous to the
cranial ends of the uterine horns in related species with
bicornuate uteri and have therefore been referred to as
‘‘intramural uterine cornua’’ (Rasweiler, 1979, 1993).
A detailed description of implantation is beyond the
scope of this paper, but a brief summary of the process
is presented because of its possible relevance in understanding some unusual aspects of the early embryogenesis of Carollia. Although implantation usually occurs
within one of the intramural uterine cornua, it sometimes takes place from the main uterine cavity in
immediately adjacent areas of endometrium. Initial
trophoblastic attachment within the intramural uterine cornu is circumferential, and trophoblastic penetration of the endometrium is temporarily retarded when
the basal laminae of the uterine luminal and glandular
epithelia are reached. Much of the trophoblast then
grows along these basal laminae for a while before
passing into the endometrial stroma. Even when the
blastocyst first enters the main uterine cavity, there
appear to be significant spatial constraints during the
implantation process because of closure of the uterine
lumen and rapid engulfment of the embryo by the
decidual reaction. Regardless of the site of initial
attachment, the blastocyst becomes interstitially implanted and covered by a decidua capsularis (Rasweiler,
1979; unpublished observations).
Electron Microscopy
To confirm some of the light microscopic observations,
five blastocysts obtained on days 16 and 17 p.c. were
examined ultrastructurally. To obtain these specimens,
pregnant females were anesthetized between 800 and
1000 hr with an intraperitoneal injection of sodium
pentobarbital at a dosage of approximately 90 mg/kg
body weight. The animals were then perfused via the
left ventricle with approximately 15 ml of lactated
Ringer’s solution followed immediately by cold fixative
consisting of 2.5% glutaraldehyde and 2% formaldehyde (methanol-free) in 0.1M cacodylate buffer (pH 7.4)
containing 2.5 mM CaCl2. The uterus was then removed, immersed in fixative, its maximum diameter
measured, and cut in half in a frontal plane. Implantation sites could be readily identified as rounded hemorrhagic sites. The uterine pieces were immersion fixed
for an additional 24 hr at 4°C, rinsed for 2 hr in three
changes of the same buffer, and then postfixed for 2 hr
at room temperature in 1% osmium tetroxide in 0.1 M
cacodylate buffer (pH 7.4) containing 2.5 mM CaCl2.
The specimens were dehydrated through a graded
series of acetones and embedded in PolyBed 812 (Polysciences, Warrington, PA). Semithin sections were cut
and stained with 1% toluidine blue in 1% aqueous
sodium borate for light microscopic examination. Thin
sections were picked up on copper grids and stained for
30 min with methanolic uranyl acetate followed by 3
min with lead citrate. Sections were then examined
with a JEOL-JEM 100CXII electron microscope at an
accelerating voltage of 80 kV.
Endoderm
The first clear examples of endoderm cells were found
histologically in a blastocyst examined on day 14 p.c.
88
N.K. BADWAIK ET AL.
Figs. 1–4.
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
89
These were located on the ventral surface of the inner
cell mass and also partially lined the interior of the
mural trophoblast. The endoderm cells had smaller
nuclei and darker cytoplasm than did the epiblast cells.
On day 15 p.c., endoderm could be positively identified
in three-quarters of the blastocysts (Fig. 1). In one of
these, a small unilocular yolk sac had developed, and
this sac contained an acidophilic, moderately PAS1,
granular coagulum.
All six of the uterine blastocysts examined on day 16
p.c. contained endoderm, which in most had a mesenchymelike appearance (Fig. 2). The endoderm was most
abundant on the ventral side of the inner cell mass, but
it also extended up and around much of the mass.
Apparent gaps were sometimes noted in the supraembryonic endodermal layer at this stage; however, these
were often quite small (i.e., the approximate size of a
few endoderm cells). Ultrastructural studies would be
required to confirm that endoderm was not still present
but was highly attenuated and/or less differentiated in
such regions. The interstitial spaces within the endodermal meshwork ventral to the inner cell mass contained
a granular coagulum, and in three of six blastocysts
these spaces were dilated to a considerable extent with
fluid. On day 17 p.c. reticulated endoderm was present
but not abundant in one small implanting blastocyst.
The interstitial spaces in the endoderm of this specimen also lacked any granular coagulum. In three other
blastocysts obtained on this day, the endodermal meshwork was more extensive and contained dilated pockets
of fluid (Fig. 3). Another day 17 p.c. specimen had a
unilocular yolk sac cavity lined by an endodermal
monolayer, but some of the endoderm surrounding the
epiblast was still multilayered and mesenchymal in
appearance (Fig. 4).
Several lines of evidence clearly indicated that the
cells on the dorsal and lateral sides of the inner cell
mass were endoderm. In histological sections, they
appeared similar in size and staining characteristics to
the visceral and parietal endoderm cells (Figs. 2–4). At
the ultrastructural level, both the parietal and supraembryonic endoderm cells typically contained many elon-
Fig. 1. Early implanting blastocyst, day 15 p.c. The arrowheads
indicated darker-staining endoderm cells. Reichert’s membrane had
developed between the parietal endoderm and the trophoblast. Masson’s trichrome. 3403.
Fig. 2. Early implanting blastocyst, day 16 p.c. By this stage,
endoderm (E) surrounded much of the epiblast (EP) and had formed a
meshwork on the ventral side of the inner cell mass. Reichert’s
membrane was present between the endoderm and the cytotrophoblast (e.g., at arrowheads). Deposits of PAS1, basal laminalike
material, were also frequently evident between many of the endoderm
cells and epiblast, around some of the endoderm cells, and to a lesser
extent between some epiblast cells. The granular coagulum present in
pockets within the endodermal meshwork was less intensely stained
by the PAS reaction. Amylase-PAS-hematoxylin. 3287.
Figs. 3, 4. Implanting blastocysts, day 17 p.c. The endoderm on the
ventral side of the inner cell mass of one embryo (Fig. 3) contained
several fluid-filled pockets, and that of the other (Fig. 4) had formed a
unilocular yolk sac. These pockets again held flocculent material that
was moderatedly PAS1. In both embryos, endoderm (E) dorsal and
lateral to the epiblast (EP) was often multilayered and mesenchymal
in appearance. Portions of Reichert’s membrane are indicated by
arrowheads. Deposits of basal laminalike material were distributed as
in the blastocyst shown in Figure 2. Fig. 3: Amylase-PAS- hematoxylin, Fig. 4: PAS-hematoxylin. 3287.
Fig. 5. Portion of the parietal wall of the yolk sac in an implanting
blastocyst, day 17 p.c. Reichert’s membrane (R) is evident between the
endoderm (E) and the mural cytotrophoblast (C). In this region,
Reichert’s membrane consists of two layers (inset). The outer layer
appears to be the basal lamina of the cytotrophoblast. The inner layer
may be of endodermal origin because it is present on all surfaces of the
endoderm cells (e.g., at arrowheads) and in the yolk sac cavity (YSC).
Very similar material is present in the cisternae of the rER (arrows) in
the endoderm cells. 39,579. Inset, 322,320.
Fig. 6. Embryonic pole of an implanting blastocyst, day 16 p.c.
Reichert’s membrane (R) passes between the polar cytotrophoblast (C)
at the top of the figure and a layer of supraembryonic endoderm cells
(E). The endoderm cells contain a moderate abundance of rER with
anastomosing cisternae, as is the case with the parietal endoderm
(Fig. 5). Rough ER is much less abundant in the epiblast cells (EP).
Basal laminalike material is also present as a coating on exposed
surfaces of the endoderm and epiblast cells (arrowheads), and larger
masses (arrows) frequently occur between these cells. Apoptotic bodies
(A) are evident, and two of these have been phagocytosed by epiblast
cells. 35,035.
Fig. 7. Embryonic pole of an implanting blastocyst, day 16 p.c.,
showing three examples of well-differentiated supraembryonic endoderm cells (E) with their characteristic content of rER. In general,
much less rER is present in the epiblast cells (EP; cf. Fig. 9). Material
similar to Reichert’s membrane (R) is also present on other surfaces of
the endoderm and epiblast cells (e.g., at arrowheads). C, polar
cytotrophoblast; R, Reichert’s membrane. 36,270.
Fig. 8. Higher power view of the portion of Reichert’s membrane (R)
shown in Figure 7. In this region, Reichert’s membrane does not
appear to consist of two layers, in contrast to what has been observed
between the parietal endoderm and mural trophoblast (cf. Fig. 5). C,
polar cytrotrophoblast; E, endoderm. 319,143.
92
N.K. BADWAIK ET AL.
Fig. 9. Mass of basal laminalike material (arrow) between epiblast cells in an implanting blastocyst, day
16 p.c. This mass is composed of at least two components of different electron densities. A thinner coat of
extracellular material is also present on other portions of the epiblast cells (e.g., at arrowheads). 311,970.
gated, branching cisternae of rough endoplasmic reticulum (rER), whereas epiblast cells contained much less
rER (Figs. 5–9). The endoderm cells in both locations
were closely associated with Reichert’s membrane and
an abundance of extracellular material with similar
histological and ultrastructural characteristics.
Two of the implanting blastocysts examined on day
18 p.c. were quite small. In one, much of the epiblast
was surrounded by a single layer of endodermal cells
with a mesenchymal appearance. In the other, the endoderm was multilayered on the ventral side of the
epiblast. No dilated pockets of fluid were seen between
the endoderm cells of either embryo. Three other day18-p.c. blastocysts were much larger and contained
unilocular yolk sacs. In one case, however, the yolk sac
still had several outpocketings (Fig. 10). In the other
two embryos, some of the endoderm surrounding the
epiblast still appeared mesenchymal.
A multilocular yolk sac was seen only in two blastocysts examined on days 19–21 p.c. (Table 2). All of the
remaining blastocysts examined on those days and on
days 22–25 p.c. had unilocular yolk sacs lined by an
endodermal monolayer (Fig. 11). In two of the specimens examined on days 20 and 21 p.c., however, some of
the endoderm dorsolateral to the epiblast still appeared
mesenchymal and contained small pockets of extracellular fluid (Fig. 12).
Reichert’s Membrane and Extracellular Matrix Material
In three of the blastocysts examined histologically on
day 15 p.c. and in all six uterine blastocysts examined
on day 16 p.c., a prominent acidophilic and PAS1 basal
lamina (Reichert’s membrane) had developed between
the parietal endoderm and the cytotrophoblast (Figs. 1
& 2). This basal lamina continued up between the
endoderm surrounding much of the epiblast and the
cytotrophoblast. At the ultrastructural level, Reichert’s
membrane between the parietal endoderm and mural
trophoblast could be seen to consist of two components
(Fig. 5). One appeared to be the basal lamina of the
cytotrophoblast, and the other was identical to extracellular material coating free surfaces of the endoderm
cells. Clumps of similar material were present in the
yolk sac cavity along with many small, dense granules.
Dorsal to the inner cell mass (Figs. 6–8), Reichert’s
membrane appeared to consist of just one broad, homogeneous layer composed of material similar to that
found on the surface of the endoderm cells. A transition
in Reichert’s membrane to a bipartite structure occurred at about the level of the junction of the parietal
and visceral endoderm cells.
In paraffin sections, extracellular masses of glycoprotein-rich material (i.e., strongly PAS1 after amylase
digestion) were commonly seen along the interface of
the endoderm (visceral and supraembryonic) and the
epiblast (see Fig. 12). In tangential sections of the
endoderm–epiblast interface, much of this basal laminalike material was found to be actually part of a meshwork rather than a continuous layer. This meshwork
extended inward between some of the epiblast cells
(particularly those on the periphery) and outward
between some of the endoderm cells to Reichert’s
membrane. Additional clumps or cords of strongly
PAS1 material were observed between some of the
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
93
Fig. 10. Blastocyst, day 18 p.c. This specimen has a unilocular yolk
sac with outpocketings (*) that, because of the plane of section, appear
to be separate sacs. Some portions of the endoderm (E) dorsal and
lateral to the epiblast (EP) are multilayered and organized into small
sacs. Reichert’s membrane is present between the endoderm and
cytotrophoblast (e.g., at arrowheads). Amylase-PAS-hematoxylin. 3200.
Fig. 11. Implanted blastocyst, day 19 p.c. This embryo possesses a
unilocular yolk sac lined by an endodermal monolayer. Endoderm (E)
also surrounds much, if not all, of the epiblast (EP). Masson’s
trichrome. 3200.
Fig. 12. Implanted blastocyst, day 20 p.c. Prominent, extracellular
accumulations of glycoproteins strongly stained by Schiff’s reagent are
present in many places (e.g., at the arrowheads) between the endoderm and epiblast or around endoderm cells. Reichert’s membrane
(below R) passes dorsal to the inner cell mass. Many of the epiblast
cells seem to have assumed a similar concentric orientation (e.g.,
around asterisk). Amylase-PAS- hematoxylin. 3450.
epiblast cells, even in the center of the inner cell mass.
Masses and cords of basal laminalike material were
found in comparable locations at the ultrastructural
level (Figs. 6, 7, 9). These masses and cords contained of
at least two components differing in electron density
(Fig. 9). In many areas, the epiblast cells also had a thin
coat of extracellular material of variable thickness.
As development progressed, trophoblast-lined clefts
developed on the interior of the preplacenta dorsal and
lateral to the inner cell mass or embryonic shield, and
94
N.K. BADWAIK ET AL.
TABLE 2. Summary of major developmental events during early embryogenesis in the short-tailed fruit bat,
Carollia perspicillata
Number of embryos exhibiting developmental characteristic
Day postcoitum
n
Endodermal
meshwork
Unilocular
yolk sac
Extensive apoptosis
and multiple
cavities
within epiblast
16
17
18
19
20
21
22
23
24
25
26
28
30
6
5
5
4
6
5
3
4
5
5
3
2
2
4
4
2
1
—
1
—
—
—
—
—
—
—
2
1
3
3
6
4
3
4
5
5
3
2
2
—
—
—
—
1
—
1
—
3
1
—
—
—
Large primordial
amniotic cavity
containing
cellular debris
Embryonic
shield present;
formation of
mesoderm
—
—
—
—
—
—
2
1
1
3
1
—
—
—
—
—
—
—
—
—
1
—
1
2
21
22
1In both embryos, mesoderm had begun to grow along the amniotic ectoderm, and part of the yolk sac wall had been converted into a trilaminar
omphalopleure.
2In one embryo, the amnion had been formed, and part of the yolk sac wall had been converted into a trilaminar omphalopleure.
Reichert’s membrane inserted into these clefts (Figs.
13, 17, 19, 20).
Cytotrophoblastic Villi
Cytotrophoblast extended into the preplacenta from
the base of these clefts and elsewhere in the form of villi
(Fig. 20). The cells constituting these villi appeared
relatively undifferentiated, were not disposed in a
highly ordered fashion, and lacked obvious basal laminae. Ultrastructural studies in progress have confirmed that when cytotrophoblast cells initially penetrate portions of the developing placenta of Carollia in
the form of primary villi (i.e., lacking mesoderm) they
lack basal laminae (Badwaik and Rasweiler, unpublished observations).
Amniogenesis and the First Appearance of Mesoderm
Commencing on day 19 p.c., it was common to see
groups of epiblast cells with a similar orientation
pattern, indicative of changing cell shape and possibly
increased motility, that appears to portend the initiation of amniogenesis (Fig. 12).
Amniogenesis in Carollia occurs by cavitation. At
first this involved the apoptosis of scattered cells within
the epiblast, the rearrrangement and polarization of
epiblast cells in the forming embryonic shield, the
formation of bulges and indentations on some portions
of the surface of the epiblast (apparently associated
with morphogenetic cell movements), and the development of small cavities within the epiblast (Figs. 13–16).
The apoptotic epiblast cells generally exhibited cytoplasmic shrinkage, nuclear condensation, and fragmentation, and there was no associated inflammatory reaction (Fig. 16). Many of the epiblast cells bordering on
the small cavities that developed in the embryonic mass
had a radial orientation and had clearly become polarized (Figs. 14–15). These spaces appear very similar to
the primordial amniotic cavities observed in another
bat (Molossus ater; Sansom, 1932; Rasweiler, 1990), the
rhesus monkey, and human (Luckett, 1975; Enders et
al., 1986).
Although some apoptosis was observed within the
epiblast during implantation (Fig. 6), this became much
more extensive when sizeable cavities appeared within
the embryonic mass (Figs. 17–19). These coalesced and
enlarged to form the primordial amniotic cavity. The
timing of amniogenesis exhibited some variation between embryos (Table 2), but for the most part it
commenced after the formation of a unilocular yolk
sac. Amniogenesis was also associated with extensive
disruption of the supraembryonic endoderm (Figs. 13–
15, 19).
Mesoderm formation was first noted when the epiblast of the inner cell mass had been converted into a
curved embryonic shield and amniotic ectoderm (Fig.
19). Mesoderm was present in some specimens examined on days 23–25 p.c. (Fig. 21) and in all examined on
days 26–30 p.c.
DISCUSSION
In most mammals, the primitive endoderm (hypoblast) develops initially on the basal (inner) surface of
the inner cell mass. It then spreads peripherally to line
the interior of the blastocyst cavity, thereby creating
the yolk sac. In Carollia, the primitive endoderm also
differentiates on the surface of the inner cell mass, but
it proliferates to create a meshwork of mesenchymelike
cells in the blastocystic cavity and initially forms a
covering for much, if not all, of the inner cell mass. The
endoderm dorsal and lateral to the inner cell mass also
often has a reticulated appearance. Concomitant with
formation of the endodermal meshwork, a granular
coagulum begins to accumulate in its extracellular
spaces, and a prominent PAS1 basal lamina (Reichert’s
membrane) develops between the peripheral cells of the
meshwork and the cytotrophoblast. Reichert’s membrane is also produced dorsal and lateral to the inner
cell mass.
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
95
Figs. 13, 14. Two sections through the inner cell mass of an
implanted blastocyst, day 22 p.c. Some of the epiblast cells have
become organized as a columnar epithelium (between the arrowheads
in Fig. 13), in what presumably would have become part of the
embryonic shield. Others have become polarized around small cavities
that had formed between other epiblast cells (e.g., at tip of arrow in
Fig. 14). There are also some scattered apoptotic cells within the inner
cell mass. Part of the mass at the top of the figures lacks a covering of
darker-staining endoderm cells (E), apparently due to partial disruption of the supraembryonic endoderm at this stage. Examination of all
sections of this embryo suggests that some of the epiblast has grown
outward (e.g., in the direction of the arrowheads in Fig. 14) to partially
engulf endoderm cells and basal laminalike material originally at the
endoderm–epiblast interface. Reichert’s membrane (R) extends into a
cleft in the preplacenta (P) in Figure 13. Amylase-PAS-hematoxylin.
3274.
Although much of the endoderm in early implanting
Carollia blastocysts has a mesenchymal phenotype,
there is no question about the identity of these cells.
The multilocular yolk sac composed of such cells is soon
converted into a unilocular one lined by an endodermal
monolayer. Furthermore, the first mesoderm does not
appear until several days later, when a large primordial
amniotic cavity has formed and conversion of the
epiblast into an embryonic shield and amniotic ectoderm is nearly complete.
Carollia resembles another bat (Glossophaga) belonging to the same family (the Phyllostomidae) in exhibiting the formation of the yolk sac from a reticulated
endodermal meshwork, but this is not seen in the bat
Noctilio, a member of the closely allied family Noctilionidae. Our comparative studies suggest that there may
Fig. 15. Higher power view of part of the section shown in Figure 14.
The radial arrangement of epiblast cells around small intercellular
spaces (e.g., at arrow) portends imminent development of the primordial amniotic cavity. Only one cluster of endoderm (E) cells is evident
at the top of the figure, on the dorsal surface of the inner cell mass.
Prominent PAS1 cords of basal laminalike material (e.g., at the
arrowheads) can be seen to extend into the epiblast, and these were
found in serial sections to be linked to Reichert’s membrane. AmylasePAS-hematoxylin. 3996.
96
N.K. BADWAIK ET AL.
Fig. 16. Inner cell mass in an implanted blastocyst, day 24 p.c. The
onset of amniogenesis is indicated by the reorganization of a small
group of epiblast cells (next to asterisk) into a columnar epithelium
(presumptive embryonic shield) and the relative abundance of apoptotic cells and bodies (e.g., at arrowheads). The apoptotic cells are
characterized by cytoplasmic shrinkage and nuclear condensation or
fragmentation. Many other epiblast cells are undergoing mitosis (e.g.,
at arrows). E, endoderm; YSC, yolk sac cavity. Amylase-PAShematoxylin. 3566.
be several reasons for this difference. Implantation in
Carollia and Glossophaga usually takes place in a
relatively constricted site (i.e., in one of the intramural
uterine cornua) that is interposed between the end of
each oviduct and the main cavity of a simplex uterus.
Furthermore, during the early phases of implantation
in Carollia, blastocyst expansion appears to be constrained by (1) closure of the uterine lumen, (2) growth
of the decidua over the abembryonic pole of the embryo,
and (3) retardation of trophoblastic invasion of the
endometrial stroma by the basal laminae of the uterine
luminal and glandular epithelial cells (unpublished
observations). These three conditions exist even when
implantation occurs from the main uterine cavity rather
than within an intramural uterine cornu. Although
proliferation of the endoderm continues at this time in
both Carollia and Glossophaga, such spatial constraints at the implantation site may serve to prevent
rapid early expansion of the yolk sac. In contrast, in
Noctilio, implantation occurs at the cranial end of a
uterine horn, and the yolk sac can (and does) expand
into a more spacious uterine lumen (Rasweiler, 1979,
1993; also see Figs. 5 and 7 in Anderson and Wimsatt,
1963). The depth of implantation in Carollia and Glossophaga is ultimately interstitial, whereas in Noctilio it
is only partially interstitial, in part because of early
expansion of the yolk sac. Early proliferation of the
endoderm may be important in Carollia and Glossophaga because it creates a cellular reservoir that can
be used for subsequent rapid growth of the yolk sac. The
large yolk sac that soon develops in all three species
presumably plays an important role in early physiological exchange between the embryo and mother, and it
provides space for further growth of the conceptus.
The development of an endodermal meshwork is
quite rare among mammals. In addition to Carollia and
Glossophaga, this has been reported to occur only in the
human, chimpanzee, and a prosimian primate,
Demidoff’s bushbaby (Galago demidovii). Small patches
of reticulated endoderm also are present in implanted
blastocysts of three other prosimian primates: the
Senegal bushbaby (Galago senegalensis), the slender
loris (Loris tardigradus), and the slow loris (Nycticebus
cougang; Luckett, 1974). The present study provides
additional comparative support for Luckett’s suggestion that an endodermal meshwork forms in early
human and chimpanzee blastocysts because of spatial
constraints imposed by an interstitial mode of implantation. Such a meshwork is not seen in blastocysts of the
rhesus monkey or baboon, which exhibit central implantation. However, Enders and King (1988) obtained
strong evidence that extraembryonic mesoderm can
differentiate from endoderm in the rhesus monkey, and
they go on to suggest that the endodermal reticulum in
the human may actually constitute forming mesenchymal cells and hence the first extraembryonic mesoderm.
Envelopment of much of the inner cell mass by endoderm is unusual but not unique to Carollia. This
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
Fig. 17. Implanted blastocyst, day 20 p.c. Amniogenesis has progressed further in this specimen by apoptosis and the enlargement of
cavities within the epiblast (EP). The arrowheads indicate points at
which Reichert’s membrane is tethered to clefts in the preplacenta (P).
The supraembryonic endoderm (E) appears less organized and not in
the form of an epithelial monolayer like the visceral and parietal
endoderm. YSC, yolk sac cavity. PAS-hematoxylin. 3166.
97
Fig. 18. Implanted blastocyst, day 25 p.c. The inner cell mass has
differentiated into an epiblastic shield (EP) and amniotic ectoderm
(AE). The primordial amniotic cavity (AC) contains a large mass of
epiblast cells, most of which are undergoing apoptosis. In other
sections, this mass was still connected to the shield and developing
amnion; however, in view of the location of the mass and its degenerate
condition, it seems unlikely that most of its cells would have survived
amniogenesis. VE, visceral endoderm; YSC, yolk sac cavity. Masson’s
trichrome. 3487.
98
N.K. BADWAIK ET AL.
Figs. 19–20
BLASTOCYSTS OF THE SHORT-TAILED FRUIT BAT
99
Fig. 21. Section through the caudal end of the embryonic shield of a blastocyst, day 25 p.c. Abundant
mesoderm (M) is present between the shield epiblast (EP) and the visceral endoderm (VE). AC, primordial
amniotic cavity; AE, amniotic ectoderm; P, preplacenta; YSC, yolk sac cavity. Masson’s trichrome. 3242.
envelopment had been reported for another microchiropteran bat (Glossophaga soricina; Rasweiler, 1974) belonging to the same family and for a megachiropteran
bat (Haplonycteris fischeri; Heideman, 1989). It also
may occur in the vampire bat (Desmodus rotundus),
where it was identified as precociously formed extraembryonic mesoderm (Wimsatt, 1954), the disc-winged bat
(Thyroptera tricolor; Wimsatt and Enders, 1980), and
several other megachiropterans (Selenka, 1892; Kohlbrugge, 1913; Keibel, 1922; Moghe, 1956).
In rats and mice, Reichert’s membrane develops
between the parietal endoderm and mural trophoblast
and is generally considered to be secreted by the
endoderm (Hogan et al., 1980, 1982, 1984; Mazariegos
et al., 1987). Recent evidence indicates, however, that
the mural trophoblast may also be involved in its initial
formation (Salamat et al., 1995). Both the endoderm
and cytotrophoblast probably contribute to the formation of Reichert’s membrane in Carollia. At the ultra-
Fig. 19. Implanted blastocyst, day 23 p.c. Cavitation has progressed
still further in this specimen, and the inner cell mass has differentiated into a curved epiblastic shield (EP) and amniotic ectoderm (AE).
Apoptotic epiblast cells containing condensed and fragmented masses
of chromatin are still evident in the primordial amniotic cavity. Some
disorganized supraembryonic endoderm (E) persists, and Reichert’s
membrane extends into two deep clefts (arrowheads) in the preplacenta (P). Some mesoderm (e.g., at arrows) is present between the
epiblast (EP) and visceral endoderm (VE). YSC, yolk sac cavity.
Amylase-PAS-hematoxylin. 3242.
Fig. 20. Higher power view of Reichert’s membrane (R) inserting into
the two clefts in the preplacenta shown in Figure 19. These clefts are
lined by cytotrophoblast (C). The deepest points of penetration by
Reichert’s membrane are indicated by the arrows. Although two
villous projections of the cytotrophoblast (CV) extended much deeper
into the preplacenta, these lacked obvious, central basal laminae.
Syncytiotrophoblast (S) on the periphery of each villus has been
labeled. E, endoderm; MBS, maternal blood space. Amylase-PAShematoxylin. 3521.
structural level, Reichert’s membrane is most intimately associated with the cytotrophoblast of early
blastocysts and, in the parietal wall of the yolk sac,
consists of two layers. One layer appears to be the basal
lamina of the trophoblast. The other layer may be of
endodermal origin because similar material is commonly observed all around the endoderm cells and in
cisternae of their rER. For reasons that are still unclear, the outer layer of Reichert’s membrane (corresponding to what is apparently the basal lamina of the
cytotrophoblast) is not evident where this structure
passes between the inner cell mass and polar trophoblast.
Reichert’s membrane has also been observed dorsal
and lateral to the inner cell mass in other phyllostomid
bats (Glossophaga soricina and Macrotus californicus),
a thyropterid bat (Thyroptera tricolor), and a noctilionid bat (Noctilio albiventris; Rasweiler, 1974; Bleier,
1975; Wimsatt and Enders, 1980; Rasweiler and
Badwaik, 1996). The situation in Noctilio is of additional interest because, during initial development of
Reichert’s membrane dorsal to the inner cell mass, no
endoderm is evident in that region. Therefore, the
supraembryonic portion of Reichert’s membrane in
Noctilio must be a product of the epiblast and/or
cytotrophoblast (Rasweiler and Badwaik, 1996).
Masses and cords of basal laminalike material were
also observed deep within the inner cell mass of Carollia and Noctilio blastocysts (Rasweiler and Badwaik,
1996). This finding raises the possibility that the material is secreted by the epiblast cells. Basal lamina
material has been seen to a more limited extent between epiblast cells in baboon blastocysts (Enders et
al., 1990).
In view of the disposition of Reichert’s membrane in
Carollia and Noctilio and its linkage to a meshwork of
basal laminalike material at the endoderm–epiblast
100
N.K. BADWAIK ET AL.
interface and extending into both layers, this structure
probably plays an important mechanical role in tethering the inner cell mass and the embryonic shield to the
preplacenta. This tethering may be important because,
at least in comparison with primates like the human
and rhesus monkey (Hamilton and Mossman, 1972;
Luckett, 1978; Enders et al., 1986; Enders and King,
1988), these components of the embryo become unusually large in Carollia and Noctilio before mesoderm
appears and contributes to the early development of a
connecting stalk.
In both bats, cytotrophoblast proliferates into the
syncytiotrophoblast of the preplacenta in the form of
villous projections. There are, however, substantial
species differences in the structure of these cytotrophoblastic villi until at least the early primitive streak
stage. In Noctilio, all of the villi consist of a bilayer of
clearly polarized cytotrophoblast cells, with central
basal laminae that stain prominently with the PAS
reaction. These basal laminae appear to be continuous,
or fused, with Reichert’s membrane in the clefts that
develop on the interior of the preplacenta. In Carollia,
Reichert’s membrane appears to end abruptly within
the clefts. Furthermore, the cytotrophoblast cells of villi
extending deep into the preplacenta are much less
regularly arranged and, at least initially, lack basal
laminae. The significance of these differences in cytotrophoblastic development in the preplacentae of these
bats remains to be elucidated.
Comparative studies of Carollia and Noctilio (Rasweiler and Badwaik, 1996) have now established that
their early embryos differ in a number of very significant ways. In Noctilio, a typical inner cell mass only
forms and becomes properly oriented after the initiation of implantation. Their embryos also exhibit differences in the distribution of endoderm, mode of formation of the yolk sac, and the differentiation of
cytotrophoblast in the preplacenta. These differences
are particularly noteworthy because Carollia and Noctilio are generally considered to be closely related,
being assigned to the same superfamily (the Noctilionoidea; Koopman, 1994), and this classification is strongly
supported by reproductive similarities observed between these species (Rasweiler, 1979, 1993).
This study has established that amniogenesis in
Carollia occurs by cavitation. This process involves
both the apoptosis of some cells within the inner cell
mass and the coalescence of spaces that develop between the epiblast cells. This process is very similar to
what has been observed in other phyllostomid bats
(Glossophaga: Hamlett, 1935; Rasweiler, 1974; and
probably Desmodus: Wimsatt, 1954), Noctilio (Rasweiler and Badwaik, 1996), Thyroptera (Wimsatt and
Enders, 1980), and several megachiropteran bats
(Selenka, 1892; Kohlbrugge, 1913; Keibel, 1922; Moghe
1951, 1956; Heideman, 1989). In most other microchiropteran bats, a primordial amniotic cavity is formed
within the inner cell mass. The epiblastic roof of this
space is then lost, creating a trophoepiblastic cavity,
and the definitive amnion is subsequently formed by
folding (Gopalakrishna and Karim, 1979, 1980; Luckett, 1980). Although there are substantial differences
between these two modes of amniogenesis, there are
also some similarities. The black mastiff bat, Molossus
ater, exhibits amniogenesis by the second method;
however, formation of the primordial amniotic cavities
in Molossus involves the radial arrangement and polarization of epiblast cells in the inner cell mass, as has
been observed in Carollia. Furthermore, apoptosis appears to occur in the epiblast at this time in both species
(Sansom, 1932; Rasweiler, 1990).
ACKNOWLEDGMENTS
The technical assistance of Yarka Chvojka, Anita
Piccolie, Andrea O’Neill, and Simeon Williams in different aspects of this work is gratefully acknowledged.
Thanks also go to the staff of the Department of Zoology,
University of the West Indies, St. Augustine, Trinidad
for their generous assistance and the use of departmental facilities during field work required for this study.
This research was supported by departmental funds
and National Institutes of Health grant HD-28592.
Thanks are due to the United States Educational
Foundation in India and the Council for International
Exchange of Scholars for their support and assistance
to N.K. Badwaik as a Visiting Scholar under the
Fulbright Scholar Program and to the Fundacao de
Amparo a Pesquisa do Estado de Sao Paulo for a
fellowship (grant 94/2348-1) provided for S.F. Oliveira.
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