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Ultrastructural differentiation of the endodermal cells of the yolk sac of the bat Tadarida brasiliensis cynocephala.

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Ultrastructural Differentiation of the Endodermal
Cells of the Yolk Sac of the Bat, Tadarida
brasiliensis cynocephala
R. J. STEPHENS AND N. EASTERBROOK
L i f e Sciences Division, Stanford Research Institute,
Menlo Park, California 94025
ABSTRACT
In Tadarida, the endodermal cells that form the yolk sac originally delaminate from the inner cell mass and proliferate to form a complete
lining of the trophoblastic vesicle, creating a bilaminar omphalopleure. These
cells remain squamous until the splanchnic mesoderm migrates in between the
two layers of the omphalopleure, at which time they begin to hypertrophy. The
current study is an analysis of the cytological changes that accompany this hypertrophy as well as additional changes that occur throughout the remainder of the
gestation period. Among the early changes are: ( 1 ) the formation of numerous
microvilli along the apical surface of the cells, (2) the appearance of coated vesicles, also along the apical plasma membrane, (3) the establishment of a system
of absorption tubules in the apical cytoplasm, ( 4 ) an increase in mitochondria,
and ( 5 ) the appearance of glycogen within the channels of the membranous
organelle.
A wave of hematopoietic activity follows the migration of splanchnic mesoderm around the trophoblastic vesicle, and at this time the erythroblasts and embryonic erythrocytes can be seen in a close relationship with the endodermal cells.
Subsequent changes include the enlargement of the membranous organelle and
the appearance of a paracrystalline membranous structure. In addition, the endodermal cells store large quantities of lipid and glycogen that are substantially depleted just before birth.
Certain morphological aspects of the development of the yolk sac in several families of the order Chiroptera have been
described at the light microscope level. Variability in their development is probably related to the type of implantation observed
in each case. Some families, i.e., Phyllostomidae (Hamlett, '35) and Desmodontidae
(Wimsatt, '54), exhibit interstitial implantation, but in Vespertilionidae (Wimsatt, '49, '54) and Emballonuridae (Gopalakrishna, '58), implantation is partially
superficial. In Noctilionidae (Anderson and
Wimsatt, '63) implantation is excentric;
however, in Molossidae it is central and
completely superficial (Stephens, '62). Subsequent development of the yolk sac is similar in those families in which implantation
is either partially or completely superficial,
and contrasts significantly with that in
families in which implantation is interstitial.
ANAT.REC.,169: 207-242.
A histological and histochemical description of the development of the yolk sac in
Tadarida was published in 1962 (Stephens,
'62). More recently we have undertaken an
ultrastructural study of this organ and have
described the existence of a unique cytoplasmic organelle, referred to as the "membranous" organelle (Stephens and Easterbrook, '68, '69), which is located only in the
endodermal cells. The present paper describes the cytological changes that take
place in the endodermal cells of the yolk
sac as they develop from an extremely flat
squamous epithelium in early development
until parturition.
MATERIAL AND METHODS
The material used in this study was
taken from 125 pregnant bats collected in
Received March 2, '70. Accepted Sept. 4, '70.
1 This investigation was supported by the National
Institutes of Health, Child Health and Human Development, grant HD 03391-01.
207
208
R. J. STEPHENS AND N . EASTERBROOK
the vicinity of Gainesville and Tallahassee, gion (figs. 1, 2). In one small area overlyFlorida, during the spring in 1966 and ing the papilla of the fallopian tube, no
1968. Live animals were air-shipped to New syncytium is formed but the cellular troHaven, Connecticut in 1966 and to San phoblast proliferates, giving rise to a thickFrancisco in 1968. Shipments of approxi- ened pad referred to as the "placental pad,"
mately 10 bats each were made at two-week as it is destined to become the discoidal
intervals. Upon arrival, five bats were sacri- placenta (Stephens, '62, '69). The cellular
ficed; the rest were fed for one week on and syncytial trophoblasts, which are part
mealworms prior to sacrifice. This method of the prominent diffuse labyrinthine endogave us animals sacrificed at one-week in- theliochorial placenta established over the
tervals. Although the variation in age from parietal area, are included here in figures
a given sacrifice may exceed two weeks, by 1 and 2 to orient the reader and are the
using additional criteria (i.e., embryonic subject of a companion paper (Stephens
stage, uterine and crown-rump measure- and Cabral, '71 ).
ments), we were able to collect yolk sacs
At first the yolk sac consists of a single
from a complete chronological series of layer of squamous endodermal cells (figs.
stages of gestation.
1, 2). The attenuated ends of these cells
Tissue was fixed in 1% OsOl (buffered are held together by desmosomal attachwith Veronal acetate, pH 7.4) for 45 min- ments and a short zonula occludens is
utes or in 3% glutaraldehyde for two hours usually present (fig. 3). A sparse popula(Sabatini et al., '63). The glutaraldehyde tion of short, rod-shaped mitochondria is
was buffered with 0.1 M cacodylate to pH scattered throughout the cytoplasm and the
7.4. Tissue fixed in glutaraldehyde was rough endoplasmic reticulum (ER) is
subsequently washed repeatedly in 0.1 M similarly distributed (figs. 2, 3, 4). The
cacodylate buffer at pH 7.4 for two hours ground cytoplasm contains numerous free
and then refixed for 45 minutes in 1% 0 ~ 0 , ribosomes (figs. 3, 5 ) and the Golgi combuffered to pH 7.4 with Veronal acetate. All plex is small, consisting of only a few cismaterial was then dehydrated in graded ternae with a few small vesicles in the area
ethyl alcohol and embedded in Maraglas (fig. 5). The membranous organelle, which
(Freeman and Spurlock, '62). Sections has been the subject of two previous papers
were cut on a Porter-Blum MT2 ultramicro- (Stephens and Easterbrook, '68, '69), is
tome, placed on uncoated nickel grids, and usually very small at this early stage, constained with lead citrate (Reynolds, '63) sisting of only a few channels (fig. 4). At
prior to observation with a Philips 200 elec- first there is no glycogen present within
tron microscope.
these channels (fig. 4 ) , but when glycogen
For correlative study with the light mi- first appears within the cell it is seen only
croscope, sections l thick were cut from within the channels of the membranous
the same blocks used for electron micros- organelIe (fig. 5). The plasma membrane
copy. These sections were placed on glass facing the cavity of the yolk sac is relatively
slides and stained with a 1% solution of smooth (figs. 1, 2). An occasional invagitoluidine blue before observation with the nation along its surface appears to be the
light microscope.
beginning of the formation of a coated
vesicle (fig. 4). Small, coated vesicles may
OBSERVATIONS
also be seen within the cytoplasm (fig. 5).
In Tadarida, endodermal cells originally The basal aspect of the endodermal cells is
delaminate from the inner cell mass and very irregular (fig. 2 ) ; occasionally a cytoproliferate to form a complete lining of the plasmic process extends to the surface of
trophoblastic vesicle, creating a bilaminar the cellular trophoblast (fig. l ) , but there
omphalopleure. By the time the embryo has is usually a considerable space between the
reached the neural groove stage, the cavity two tissues that contains a sparse, floccuof the yolk sac has become large and the lent substance (fig. 2). In subsequent deomphalopleuric wall comes in contact with velopment the splanchnic mesoderm mithe endometrium (Stephens, '62). The cel- grates into this space (fig. 6). A thin
lular trophoblast gives rise to a syncytium basement membrane covers the cellular
over the major portion of the parietal re- trophoblast facing the endoderm (fig. 2).
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
Within a short period of time, dramatic
changes occur in the endodermal cells. The
cells lose their squamous appearance and
become cuboidal (figs. 6, 7). The oval nuclei are located toward the basal end of
the cell and possess a slightly irregular
nuclear membrane (fig. 7). Junctional
complexes are seen at the apical ends of
the cells between the lateral membranes
(figs. 7, 19). Functionally, the zonula occludens would prevent absorption from the
cavity of the yolk sac via the intercellular
space. The remainder of the lateral plasma
membrane is very irregular, an interstitial
space is present between the cells, and attenuated cytoplasmic extensions protrude
from the lateral membranes into this space
(fig. 7). Numerous microvilli extend from
the apical surface into the cavity of the
yolk sac, and a system of absorption tubules
develops in the superficial cytoplasm (fig.
7). These tubules become more extensive
in later stages (figs. 17, 21). Numerous
small vesicles and a moderate population of
mitochondria are seen throughout the cytoplasm (fig. 7).
Shortly after the mesoderm migrates into
the space between the endoderm and the
trophoblast (fig. 6 ) , hematopoiesis begins
(figs. 8, 9). A few of the stellate mesodermal cells round up and become erythroblasts, their cytoplasm remains pale and
mitochondria are randomly distributed in
the cytoplasm (fig. 10). In addition, various small vesicles and short stretches of ER
are present (fig. 10). These cells proliferate rapidly, producing a large number of
immature erythrocytes (figs 8, 10). At first
there are no blood vessels in the area, but
they are eventually established and the embryonic, nucleated erythrocytes can be seen
within the vitelline blood vessels (fig. 9 ) .
There is a close association between the
erythroblasts and the irregular basal cytoplasmic extensions of the endodermal cells
(figs. 11, 12); and erythrocytes in various
stages of maturity may be seen in the interstitial space immediately underlying the
endoderm (fig. 12). Frequently the immature erythrocytes push into the space between the lateral plasma membranes of
the endodermal layer and undergo a degree of maturation in a close relationship
with the endoderm (figs. 11,12). When the
erythroblasts are in close contact with the
209
endoderm, the density of their cytoplasm
markedly increases, but both the nuclei and
the mitochondria are retained as long as
hematopoiesis continues in the yolk sac
(figs. 11, 12). The mitochondria become
more elongate, however, during maturation
(cf., figs. 10, 12). At this stage a very sparse
amount of glycogen may be seen scattered
throughout the cytoplasm of the endodermal cells (fig. 12), but the channels of
the small membranous organelle invariably
contain glycogen. A particular point of interest is that a basement membrane is
never observed underlying the epithelium
formed by the endodermal cells (figs. 7,
10, 12, 16, 20).
To include a full consideration of details
involved in hematopoiesis and vitelline
capillary formation would unduly lengthen
this communication, and a subsequent paper is now in preparation on this subject.
The micrographs included here are for purposes of presenting a complete picture of
endodermal maturation. Discussion of the
topic will not be undertaken here.
By the time the embryo has reached the
stage where the neural groove is beginning
to close, the allantois has begun to develop.
As the allantoic circulation displaces the
vitelline circulation from the parietal region, the yolk sac begins to collapse (figs.
13-15). The endodermal cells continue to
hypertrophy, eventually becoming columnar in appearance (figs. 15, 16). As the
yolk sac collapses, the endoderm is thrown
into folds; the lateral sides of these folds
approximate one another and eventually
come together, forming small groups of
cells that pinch off and lose contact with
the central cavity of the yolk sac (figs. 14,
15). These isolated groups of cells are distributed between the prominent vitelline
vessels (fig. 15). The central cavity of the
yolk sac becomes progressively smaller and
is eventually obliterated (fig. 22).
Certain ultrastructural features of the endodermal cells become more pronounced as
the cells become increasingly columnar in
shape. Glycogen up to this stage has been
rather sparse within the cytoplasm, but it
is now observed in patches (fig. 16). Microvilli protrude into the yolk sac cavity and
numerous coated vesicles pinch off from
the apical plasma membrane (figs. 17, 18).
The network of tubules in the apical cyto-
210
R. J. STEPHENS AND N . EASTERBROOK
plasm becomes increasingly prominent as
collapse of the yolk sac continues (figs. 7,
17, 21). As the walls of the yolk sac approach one another, a dense substance appears in the central cavity (fig. 21). Additional cytoplasmic inclusions appear within
the cells, some of which are autophagic
vacuoles (fig. 21); others that are more
electron-lucid are closely associated with
the apical tubular networks (fig. 17). The
lateral plasma membranes are more closely
applied to one another at this point than
they were previously, and additional desmosomes appear between them (fig. 19). The
membranous organelle remains quite small
(fig. 20) and occasionally more than one
is seen in a single cell. Glycogen is always
present in the channels of this structure.
Lipid droplets are also seen more frequently
in the cytoplasm (fig. 20).
The apical ends of the endodermal cells
from the opposite sides of the yolk sac
eventually become closely associated, leaving only a 200-300 A space between the
cells where the former cavity existed (figs.
22,26). At this stage adjacent cells may appear extremely different from one another.
Although most of the endodermal cells contain the complex of organelles described,
an occasional cell contains, in addition, a
large number of spherical dense granules
thought to be small lipid droplets (fig. 27).
These droplets may be observed within
large, autophagic vacuoles (fig. 28). As
the embryo approaches mid-gestation, the
mitochondria increase in number and become more elongate (figs. 1, 6, 16,27, 30).
At this time some of them possess a thinner, elongate portion (figs. 23-25). These
narrow portions contain no cristae but retain the double outer membrane.
The number of individual stacks of Golgi
cisternae increase after mid-gestation (fig.
30) and we have observed as many as ten
separate stacks in a section of a single cell.
Numerous large vesicles appear at the ends
of the cisternae. These vesicles contain a
number of small dense droplets, probably
lipid in nature (fig. 30). Previous work indicates that after mid-gestation the lipid
content of these cells is markedly increased
(Stephens, '62; Stephens and Easterbrook,
'68, '69). The membranous organelle becomes somewhat more prominent at this
time (figs. 29, 30) and lipid droplets may
be observed within this structure (fig. 29).
Microbodies not easily distinguished in
earlier tissue are present after mid-gestation. They possess a single limiting membrane and are homogeneous except for a
central dense area (figs. 34-36).
As maturation of the yolk sac continues,
the groups of endodermal cells become
more widely separated (figs. 31, 37)). The
mesodermal layer becomes deeply infolded
and the width of the organ increases substantially (figs. 31, 37). A prominent network of vitelline vessels develops in the
connective tissue (fig. 32). Glycogen,
which has been present previously in small
amounts in the endodermal cells, now frequently occupies large areas within the
cytoplasm (fig. 33). In two previous papers
we described the relationship of glycogen to
the membranous organelle during the latter half of the gestation period (Stephens
and Easterbrook, '68, '69); in the latter paper we noted that toward the end of gestation the membranous organelle is occasionally observed adjacent to a crystalline-like,
membranous structure with membranes
confluent between the two organelles. Recent observations have produced additional
information on this paracrystalline structure, however, and we shall describe them
here. It was previously thought that this
paracrystalline structure was present only
at the very end of the gestation period, but
in this more intensive study we have observed it occasionally even prior to midgestation. It becomes larger and is more
frequently observed as term is approached.
Initially this structure may possess only a
single unit (fig. 36) consisting of a central
homogeneous region from which radiate
short membranous tubules. These tubules
are observed between adjacent units (figs.
34, 36). The central homogeneous region
possesses small, electron-lucid areas that
are seen in linear register when these units
are cut longitudinally (fig. 34). At the periphery of these structures the narrow tubular elements become enlarged and are continuous with the membranes of the ER (fig.
34).
During this period a rich population of
mitochondria is scattered throughout the
cytoplasm. Several profiles of the Golgi
complex and patches of rough ER are also
present (fig. 35). Characteristically, a num-
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
211
ber of relatively large inclusions are seen the formation of such vesicles and the esin the area of the Golgi complex (fig. 35). tablishment of a system of tubules in the
These structures contain smaller spheres of apical cytoplasm immediately beneath the
material that are very electron-dense (figs. plasma membrane. In addition, a number
35, 36).
of larger vesicles appear within the cytoDuring the latter third of gestation there plasm. A rich population of microvilli is
is a marked increase in the lipid and gly- also formed along the apical plasma memcogen stored within the endodermal cells brane during the early stages of hyper(figs. 38, 39). Lipid droplets range in size trophy; they increase in number as
from a diameter greater than 10 down gestation proceeds. These morphological
to the limit of the light microscope (fig. changes are probably associated with a
38), and large glycogen beds are present functional change in the endodermal cells,
(fig. 39). Both of these storage materials permitting them to absorb material from
are substantially depleted during the last the cavity of the yolk sac and hypertrophy.
few days of gestation.
Although it is likely that the fluid within
the cavity of the yolk sac is dynamic, i t apDISCUSSION
pears evident that eventually the processes
The development of the yolk sac in the of absorption exceed those of replenishbat has been studied in a number of species ment and contribute to the collapse of the
of bats with the light microscope (Anderson cavity.
and Wimsatt, '63; Branca, '23; Gkrard, '28;
Morphological, physiological, and bioGopalakrishna, '58; Hamlett, '34, '35; chemical investigations of the yolk of variStephens, '62; Wimsatt, '45, '49, '54). In ous laboratory animals have been undertakeach case a squamous endoderm begins to en in recent years (Bellairs, '63; Brambell
hypertrophy when the splanchnic meso- and Hemmings, '60; Butt and Wilson,
derm migrates in between the two layers '68; Dempsey, '53; Deren et al., '66a,b);
of the omphalopleure. The extent of the mi- Holdsworth and Wilson, '67; Jollie and
gration of the splanchnic mesoderm around Jollie, '67; Larsen, '63; Luse, '57; Padykula
the yolk sac endoderm and the extent of its et al., '66). Most of these studies have been
vascularization are considerably different performed on rodents during the latter porin various species on which reports are tion of the gestation period when inversion
available. It would appear likely that these of the yolk sac has taken place and the
differences have a considerable effect on visceral endoderm is exposed to the uterine
the functional attributes of the organ. In cavity. From these and other studies, a
Molossidae the mesoderm completely en- considerable volume of knowledge has been
velops the endoderm and is also completely accumulated on the uptake and transport
vascularized (Hamlett, '34; Stephens, '62). of various materials (e.g., antibodies, vitaIn Vespertilionidae, however, a portion of min BIZ, amino acids and sugar) by the
the trilaminar omphalopleure remains yolk sac endoderm of various rodents. At
avascular (Wimsatt, '45). A large area of present, however, these findings cannot be
the yolk sac remains avascular in the noc- applied directly to the yolk sac of the Molostilionid bat Noctilio labialis m i n o r (Ander- sid bats, even though the ultrastructural
son and Wimsatt, '63), a situation that is morphology is similar.
also pronounced in both the PhyllostomaPerhaps the major difference between
tidae (Hamlett, '35) and Desmodontidae the yolk sac of the rodent and that of the
(Wimsatt, '54), where the intra-embryonic Molossid bat is one of developmental mormesoderm does not advance beyond the phology. The inverted yolk sac of the rodent is produced by degeneration of the
margin of the placental disc.
The electron microscope has shown that avascular portion, and the inverted vascuthe early hypertrophy of the endoderm is lar portion of the yolk sac is then in direct
accompanied by increased activity associ- contact with the uterine cavity and its conated with the plasma membrane facing the tents. As a result, the visceral endoderm is
yolk sac cavity. At first a few coated vesi- able to absorb materials directly from this
cles form along the apical plasma mem- location. In Tadarida, on the other hand,
brane. This is followed by an increase in the entire circumference of the yolk sac is
212
R. J. STEPHENS AND N. EASTERBROOK
vascularized by the lateral spread of the
mesoblast and area vasculosa; thus the yolk
sac remains a closed system and the endoderm is never directly exposed to the
uterine contents (Stephens, '62). This major difference in the development of the
yolk sac of the rodent and the bat necessitates further investigation to determine
whether the physiological and biochemical
attributes of these tissues parallel the ultrastructural similarities of their endodermal
cells.
The longer gestation period of the bat
( 11-12 weeks) apparently permits further
development and differentiation to take
place than that observed in the rodents
studied. It also appears to permit formation
of additional functional characteristics in
the yolk sac of the bat, such as the storage
and mobilization of both lipid and glycogen
(Stephens and Easterbrook, '68, '69).
It is clear from ultrastructural studies
that in both the rodents ( Padykula et al.,
'66) and the bat the visceral endoderm
cells become absorptive. As the cells differentiate, microvilli develop along their
apical surface and numerous coated vesicles pinch off from the plasma membrane
between the microvilli. In addition, a network of tubules is present in the apical cytoplasm along with absorption vacuoles.
These characteristics are common to such
well-known absorptive cells as the small intestine and proximal convoluted tubules of
the kidney.
Coated vesicles have received a great
deal of attention over the past several years
since Roth and Porter ('62) first suggested
that these specialized structures were involved in protein uptake by the cell. Since
their original work, numerous other investigators working with a wide variety of tissues have observed these structures in conjunction with the plasma membranes, and
there is a strong probability that they are
indeed related to protein uptake (Ericsson,
'65; Friend and Farquhar, '67; Maunsbach,
'66; Rosenbluth and Wissig, '64; Roth and
Porter, '63, '64; Sedar, '66). The work of
Padykula et al. ('66) and Deren et al.
('66a,b) also suggests that such a mechanism is involved in the movement of macromolecules from the maternal to the fetal
circulation in the inverted yolk sac endoderm of the rat and rabbit. Two macro-
molecules that may be absorbed by the
embryo through this mechanism are maternal antibodies and vitamin BIZ.Similar
structures for protein absorption are observed in the endodermal cells of the bat
yolk sac, but it is impossible to say at this
point if they are directly involved in the
movement of macromolecules from the maternal circulation to the fetus, as appears
to be the case in rodents, because - as described above - the morphology of the
yolk sac and its relationship to the uterine
cavity are quite different. It is not clear
how the macromolecules produced by the
mother would gain entrance into the cavity
of the yolk sac of the bat so that they
could be absorbed by the endoderm. In this
regard, however, it is interesting to note
that Anderson ('59) observed passage of
labeled proteins into the cavity of the yolk
sac of the rat before degeneration of the
parietal portion. Protein appears to be present within the cavity of the yolk sac of the
bat also, and as the sac collapses, the protein becomes more concentrated and is
deeply stained with toluidine blue. It is
probable that this material is being taken
up by the coated vesicles and that this activity contributes to the collapse of the yolk
sac.
During the latter half of the gestation
period, an alternative pathway for the absorption of protein appears to exist in the
bat. Ultrastructural observations on the
discoidal chorioallantoic placenta suggest
that protein uptake may be accomplished
by the trophoblast. Coated vesicles pinch off
from the apical plasma membrane, which
is in direct contact with the maternal blood
(Enders and Wimsatt, '68; Stephens, '69).
Enders and Wimsatt ('68) have also shown
that thorotrast accumulates in multivesicular bodies in the trophoblast although no
thorotrast was observed in the coated vesicles. The work of Friend and Farquhar
('67) would suggest, however, that the
coated vesicles are involved in the accumulation. At this point there is no biochemical
or physiological information available on
the bat to support the possibility of protein
uptake by the chorioallantoic placenta, but
i t appears to be an attractive possibility and
an alternative to absorption via the yolk
sac. Antibodies are known to be transferred
via the chorioallantoic placenta in various
DIFFERENTIATION OF ENDODERMAL CELLS O F THE YOLK SAC
other mammals including man (Dancis
et al., ’61) and the Rhesus monkey (Bangham et al., ’58).
In two previous papers (Stephens and
Easterbrook, ’68, ’69) we have described
and discussed the presence of a new cytoplasmic organelle present in the endodermal cells of the yolk sac during the latter half of the gestation period. It was noted
that this structure, the “membranous organelle,” is always closely associated with
the glycogen and lipid storage material. The
present intensive study of the yolk sac has
revealed that this unique organelle can be
observed early in the establishment of the
endoderm, when the endodermal cells are
squamous, before the splanchnic mesoderm
migrates between the two layers of the
omphalopleure. During this period this
organelle is very small and at first its channels contain n o glycogen; in fact, no glycogen is present in the entire cell at this
stage. When glycogen does appear, it is
first seen -prior to mid-gestation -in the
channels of the membranous organelle. We
interpret this observation as an additional
indication that the membranous organelle
is directly involved in glycogen metabolism
throughout the gestation period. The similarity of this organelle to other structures
reported in the literature has been discussed in our previous papers and thus will
not be repeated here.
We have also reported the presence of a
paracrystalline membranous structure in
the endodermal cells. The membranes of
the membranous organelle and the paracrystalline structure were occasionally confluent (Stephens and Easterbrook, ’68).
Improved electron micrographs of this
structure presented here allow us to speculate a little further about the association of
these two structures. The membranes that
connect the repeating units of the paracrystalline structure have approximately
the same width as those that form the
membranous organelle, about 85 A, and
the rows of electron-lucid areas within the
central homogenous portion of each unit
have approximately the same diameter
(approximately 150 A) as the small perforations between the channels of the membranous organelle. In addition, the peripheral membranes of the paracrystalline
structure are continuous with the ER, as
213
are those of the membranous organelle
(Stephens and Easterbrook, ’68).These features tend to support the hypothesis that
there is a dynamic structural relationship
between these two entities, although at
this point it is not clear how the transformation from one to the other is accomplished.
ACKNOWLEDGMENTS
The authors wish to express their sincere appreciation for the technical assistance of Miss L. Cabral and to Mr. Wilson
Baker and Mr. John Weiss for collecting
and mailing the animals used in this investigation.
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Freeman, J. A., and B. 0. Spurlock 1962 A new
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1935 Notes o n the embryology of a
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1966 Development of structure and function
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-__
1964 Yolk protein uptake i n the oocyte
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1963 Cytochemistry and electron microscopy.
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-___
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285-34 1.
-
PLATES
PLATE 1
E X P L A N A T I O N O F FIGURES
216
1
A light micrograph showing a small portion of the parietal region of
the uterine wall that has been infiltrated by the syncytial trophoblast
(arrows). Maternal capillaries (MC) are still intact. A thin layer of
squamous endodermal cells ( E ) lines the cavity of the yolk sac (YC).
Cellular Trophoblast (CT). x 550.
2
An electron micrograph a t low magnification of a similar area to that
shown in figure 1. The squamous endoderm ( E ) of the yolk sac i s
shown to the left. A considerable tissue space (TS) exists between
the endoderm and the cellular trophoblast (CT). A thin continuous
basement membrane (BM) covers the surface of the cellular trophoblast toward the endoderm. Yolk sac Cavity (YC), Syncytial Trophoblast (ST), Maternal Capillary (MC). x 6,200.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
3 This micrograph shows the desmosomal attachment ( D ) between two
squamous endodermal cells ( E ) . Note the numerous free ribosomes
and sparse ER within the cytoplasm. Yolk sac Cavity (YC). ~20,400.
218
4
A small section of the cytoplasm of a squamous endodermal cell is
shown here. Several channels of the membranous organelle (MO) are
present but there i s no glycogen in these channels or any other portion
of the cell. Note the coated vesicles (arrows) being formed by the
plasma membrane facing toward the cavity of the yolk sac (YC).
Nucleus ( N ) . x 38,000.
5
The membranous organelle (MO) shown here is larger and more mature than that shown in figure 4, but it is from a n embryo in the same
stage of development. Glycogen i s seen as dense granules within the
channels of this structure. At this time, glycogen is not seen in any
other part of the cell. The Golgi complex (Go) and a coated vesicle
(arrow) are seen toward the upper part of the micrograph. Numerous
free ribosomes are present within the ground cytoplasm. Yolk sac
Cavity (YC). x 37,000.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
220
6
This light micrograph shows the early migration of the mesoderm ( M )
into the area between the endoderm ( E ) and cellular trophoblast (CT).
Hypertrophy of the endoderm has begun. Yolk sac Cavity ( Y C ) . x 550.
7
The endoderm shown here is as it generally appears shortly after the
mesoderm migrates into the tissue space beneath it, as shown in
figure 6. The cells have changed from their earlier squamous condition to a cuboidal shape. Numerous microvilli ( M V ) appear on the
apical surfaces and attenuated cytoplasmic extensions also are seen
along the lateral surfaces, protruding into a n irregular intercellular
space (IS) between the cells. Apical functional complexes are observed
between adjacent lateral membranes. The basal surface is irregular
and no basement membrane is observed underlying the endoderni
throughout its existence. x 9,800.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 3
22 1
PLATE 4
EXPLANATION O F FIGURES
8
Numerous embryonic erythroblasts (EB) accumulate between the
endoderm ( E ) and mesoderm ( M ) before the establishment of the
vitelline blood vessels takes place. Some of the erythroblasts are closely
associated with the endodermal cells. Yolk sac Cavity (YC). x 1,200.
9 This light micrograph shows hematopoiesis in the yolk sac after the
vitelline blood vessels are established. Embryonic blood cells can be
seen within the vitelline vessels (VV) and some also remain within
the interstitial space beneath the endoderm (E). x 550.
10 An electron micrograph a t low magnification showing embryonic erythroblasts (EB) within the tissue space between the endoderm ( E )
and vitelline blood vessels (VV). Note that the basal plasma membrane of the endodermal cells is very irregular and no basement membrane is present. The cytoplasm of the erythroblasts contain numerous
free ribosomes and a sparse population of mitochondria are scattered
throughout the cytoplasm. x 8,800.
222
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N . Easterbrook
PLATE 4
PLATE 5
EXPLANATION O F FIGURES
11 As maturation of the erythroblasts takes place they become more
closely associated with the endodermal cells ( E ) and they stain more
deeply with the toluidine blue. x 1,200.
12 The maturing embryonic blood cells (EBC) are more electron-dense
here than in figure 10 and are more closely related to the endodermal
cells (E >.Frequently, cytoplasmic extensions of the endodermal cells
completely surround the erythrocytes. Embryonic erythroblast (EB ).
x 10,000.
224
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 5
225
PLATE 6
EXPLANATION OF FIGURES
13-15
226
This sequence of light micrographs shows the collapse of the yolk
sac and the increasing complexity of the endoderm (E). In figure
13 the endoderm is seen in simple folds. Figure 14 shows how the
crests of these folds come together and form a nest of cells. Finally,
in figure 15 these nests of cells pinch off and separate from the
endoderm lining the main cavity of the yolk sac. A reduction in the
width of the cavity of the yolk sac is also evident. Yolk sac Cavity
(YC), Vitelline Blood Vessels (VV). x 550; x 550; x 550.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 6
227
PLATE 7
EXPLANATION O F FIGURES
228
16
As the yolk sac collapses, the endodermal cells become more columnar
and beds of glycogen ( G ) appear in the cytoplasm. The cytoplasmic
contents become more complex, with a n increase in the ER and mitochondria. The mitochondria are frequently seen in a branched configuration. Yolk sac Cavity ( Y C ) . ~ 6 , 4 0 0 .
17
This micrograph shows the cytoplasmic structures of the apical cytoplasm of the endodermal cells. Microvilli (MV) protrude from the
plasma membrane and coated vesicles form at the surface (arrows).
This feature is more clearly shown in figure 18. The cytoplasm just
beneath the plasma membrane contains a network of absorption
tubules ( T ) as well as a few inclusions (I), which are probably absorption vacuoles. Mitochondria and ER are also present. A centriole
( C ) is seen in the center of the micrograph. ~ 2 1 , 5 0 0 .
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
PLATE 7
R. J. Stephens and N. Easterbrook
229
PLATE 8
EXPLANATION O F FIGURES
18 This micrograph clearly shows the formation of coated vesicles
(arrows) along the apical plasma membrane of the endodermal cells.
x 25,000.
19 Prominent desmosomal attachments ( D ) are seen only between the
lateral plasma membranes of adjacent cells close to the apical surface.
A short zonula occludens ( Z O ) is also present. Note fibular attachment (arrow) between the three desmosomal units shown here.
X 29,000.
230
20
The basal portion of an endodermal cell is shown in this micrograph.
There is no basement membrane underlying this epithelial tissue.
However, a flocculent substance (FS) is seen in the tissue space under
the cells. Glycogen is observed i n the channels of the small membranous organelle ( M O ) and lipid ( L ) may be seen in small droplets
within the cytoplasm. Coated vesicles (arrows) are also observed i n
the basal cytoplasm. x 34,300.
21
A moderately dense precipitate appears in the cavity of the yolk sac
(YC) prior to complete obliteration of the cavity and fusion of the
cells from opposite poles of the yolk sac. Immediately under the apical
plasma membrane is a layer of ground cytoplasm devoid of organelles.
A rich network of absorption tubules (T) appears beneath this layer.
Numerous inclusions ( I ) and mitochondria appear deeper in the cytoplasm. x 21,000.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N . Easterbrook
PLATE 8
PLATE 9
EXPLANATION O F FIGURES
232
22
Eventually the cavity of the yolk sac is completely lost and the
apical plasma membranes come in close contact with one another
(arrow). Nests of endodermal cells ( E ) are seen pinched off from
the central region. Eventually the entire endoderm is broken up
into small isolated groups of cells (see figs. 37, 38). Vitelline blood
vessels (VV). ~ 5 5 0 .
23-25
At this stage, mitochondria with lengthy attenuated portions
(arrows) were observed. Previous investigators have referred to
similar mitochondria1 forms as “club” or “tennis racket” shaped.
There are no cristae in the narrow portion of the mitochondria.
x 39,000.
26
The yolk sac cavity has been completely obliterated and close attachment of the apical plasma membranes i s observed (arrows).
The clear cytoplasm and tubular network (T) remain at the apex
of the cell. However, mitochondria and rough ER are scattered
throughout the remainder of the cytoplasm. x 17,500.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 9
PLATE 10
EXPLANATION OF FI GU R ES
234
27
After the yolk sac collapses, the endodermal cells may differ considerably in their cytoplasmic content. Here we see a portion of cytoplasm
from a cell filled with very small dense droplets, probably lipid.
x 24,000.
28
In this micrograph can be seen an autophagic vacuole containing several small dense lipid granules and a number of cytoplasmic membranes. x 30,000.
29
The membranous organelle has increased in size, and lipid droplets
(L) and glycogen are seen among its membranes. x 38,000.
30
Another prominent feature of the endodermal cells just after midgestation is the increase in the number of stacks of Golgi cisternae
(Go) seen in the cytoplasm. Note the numerous vesicles filled with
dense granules associated with the ends of the cisternae. These structures are present during the period of increased lipid deposition within
the endodermal cells. Inclusions ( I ) , Membranous Organelle (MO).
x 21,000.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N . Easterbrook
PLATE 10
PLATE 11
EXPLANATION O F FIGURES
236
31-32
These light micrographs demonstrate the increased complexity of
the yolk sac about three quarters through gestation. Note the continued isolation of nests of endodermal cells ( E ) and the complex
folding of the mesodermal layer ( M ) in figure 31. Figure 32 shows
a significant hypertrophy of the mesodermal cells. x 125; x 550.
33
As the gestation period increases there is a marked increase in the
glycogen content ( G ) and the cytoplasmic inclusions ( I ) can still
be observed. x 14,000.
34
During the later part of the gestation period a crystalloid structure
( C S ) may be observed within the cytoplasm. The width of the
membranous bridge between the individual units is approximately
85 A and the diameter of the rows of electron-lucid areas (arrows)
within the more homogeneous portion of the crystalloid is approximately 150 A (see also fig. 36). The membranes a t the periphery
of the structure are confluent with the ER. Granules that appear
morphologically similar to microbodies ( M B ) are also seen. Glycogen ( G ) . ~ 4 5 , 0 0 0 .
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N . Easterbrook
PLATE 11
PLATE 12
EXPLANATION OF FIGURES
238
35
The endodermal cells become polygonal as the nests of cells become
progressively isolated. The plasma membranes of adjacent cells show
a marked interdigitation. Mitochondria are scattered throughout the
cytoplasm and numerous inclusions ( I ) containing small dense granules are also observed. Several groups of Golgi membranes and vesicles (Go) may be seen in a single cell. Endoplasmic Reticulum (ER).
x 11,000.
36
This micrograph is a n enlargement of the area indicated in figure 35.
A small crystalloid membranous structure ( C S ) is observed at the
center. Note the inclusions (I) containing small electron-dense granules. A microbody (MB) is seen at the upper right. Endoplasmic
Reticulum (ER). x 33,800.
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterbrook
PLATE 12
PLATE 13
EXPLANATION OF FIGURES
37-38
This tissue was taken toward the end of gestation. Note the complete separation between the small groups of endodermal cells ( E ).
The large dense droplets in figure 38 are lipid. The mesodermal
cells ( M ) are cuboidal and contain a number of small granules,
x 1,200.
39 Portions of four endodermal cells are shown here. They demonstrate the increased glycogen content ( G ) within these cells as the
gestation period nears termination. Both the lipid and glycogen will
be substantially depleted before parturition is accomplished. Inclusions ( I ) , Nucleus ( N ) . x 17,500.
240
DIFFERENTIATION OF ENDODERMAL CELLS OF THE YOLK SAC
R. J. Stephens and N. Easterhrook
PLATE 13
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ultrastructure, endoderm, bat, tadarida, differentiation, cynocephala, sac, brasiliensis, cells, yolk
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