THE AMERICAN JOURNAL OF ANATOMY 166:313-327 (1983)

Ultrastructure of the Placenta and Fetal Membranes of the

Dog: II. The Yolk Sac
SUE Y. LEE, JOHN W. ANDERSON, GRAYSON L. SCOTT, AND HARLAND W.
MOSSMAN
Department of Biological Sciences, Humboldt State Uniuersity, Arcata,
California 95521 (S.Y.L.)and Department of Anatomy (J.W.A., G.L.S.,
H.W.M.) and Zoology Museum (H.W.M.), University of Wisconsin, Madison,
Wisconsin 53706

ABSTRACT Yolk sacs from dogs at 40, 50, and 60 days of gestation were
examined by electron microscopy. Free ribosomes, mitochondria, and rough en-
doplasmic reticulum (rER) are more prominent in both endoderm and mesoth-
elium at 40 and 50 days than at 60 days, suggesting a greater synthetic capacity
a t the earlier stages. Smooth endoplasmic reticulum (sER) and glycogen are also
present in greater amounts in the endoderm in the earlier stages. In the me-
sothelium, however, low amounts of sER and glycogen are consistently present.
Certain possibilities relative to the nature of the synthetic activities in these two
tissues are discussed. Large amounts of smooth-surfaced vesicles were observed
along the basal edges of the 60-day mesothelium; they are indicative of transport
processes occurring at this time. As gestation proceeds, in both endoderm and
mesothelium, the Golgi complex remains well developed, there are more numer-
ous lysosomelike bodies, and bundles of intermediate filaments either increase
or become more diffused. In some endoderm cells a t 60 days, large vacuoles and
dense glycogen deposits were noted. These observations indicate that degenerative
processes are gradually occurring in the endoderm and mesothelium as partu-
rition draws near. Erythropoiesis occurs i n the mesenchyme a t 40 and 50 days.
At 40 days also, segments of endothelium were seen within blood islands, indi-
cating that the endothelial lining of some yolk sac vessels differentiates from
cells located in the interior of such islands.

In the dog, as in other carnivores, a chorio-
vitelline placenta is established in very early
gestation. This is accomplished by the invasion
of vascular mesoderm into the bilaminar om-
phalopleure. Subsequently, the exocoelom ex-
tends into this area and separates the vascular
splanchnopleuric yolk sac from the nonvas-
cular extraembryonic somatopleure or true
chorion except for a threadlike mesodermal at-
tachment a t each end (Fig. 1). The yolk sac
then persists until birth as a large, wrinkled,
highly vascularized sac. A large allantoic sac
eventually expands into the exocoelom and fi-
nally intervenes between the yolk sac and the
the chorion.
Studies of yolk-sac ultrastructure in mam-
mals have been largely carried out on noncar-
nivores. These have dealt with the morphol-
ogical and functional aspects of the epithelia
(Calarco and Moyer, 1966; Dempsey, 1953;
Enders et al., 1976; Hesseldahl and Falck Lar-
sen, 1969; Hoyes, 1969; Karim et al., 1979;
King, 1971; King and Enders, 1970; Padykula
et al., 1966; Stephens and Cabral, 1971; Ste-
phens and Easterbrook, 1971; Tiedemann and
Minuth, 1980a).Other such investigations have
focused on the hemopoietic functions of the
mesenchyme (Fukuda, 1973; Haar and Ack-
erman, 1971; Hesseldahl and Falck Larsen,
1971; Sorensen, 1959, 1961; Stephens and Ca-
bral-Anderson, 1976).
The only carnivore yolk sac in which anal-
ysis of the structure has been carried out thus
far is that of the cat (Tiedemann, 1976, 1977,
1979).The cat yolk sac also persists until birth.
(The gestation period of both cat and dog is
Address reprint requests to Dr S u e Y . Lee. Department ofBiological
Sciences. Humboldt S t a t e University. Arcata. CA 95521.
Received J u n e 7. 1982 Accepted Novernher 15. 1982.

0 1983 ALAN R.LISS, INC.

3 14 S.Y. LEE ET AL.

E xo.

Vit. C.
 ULTRASTRUCTURE O F DOG YOLK SAC
about 63 days.) Ultrastructural and biochem-
ical investigations of the cat yolk sac (Tiede-
mann, 1976; Tiedemann and Minuth, 1980b)
have shown that the endoderm functions in
protein synthesis (with greatest activity a t
midgestation), in triglyceride or cholesterol
metabolism, and in glycogen metabolism. Gly-
cogen metabolism is also a suggested function
of the mesothelium. Definitive erythropoiesis
was described as occurring up to day 45.
In this study which continues the series be-
gun some years ago (Anderson, 1969), we ex-
amined the fine structure of the dog yolk sac
from the latter half of gestation in order to
document in another carnivore any further
morphological clues regarding the functional
basis for its persistence.
MATERIALS AND METHODS
Yolk sacs were removed from pregnant bea-
gles a t 40, 50, and 60 days after copulation.
These were prepared for electron microscopy
by fixation in 2% glutaraldehyde in 0.075 M
or 0.1 M, pH 7.4, phosphate buffer, and further
fixed 30-40 minutes a t 0°C. Following fixation
the tissues were rinsed 30 minutes in the same
buffer, postfixed 1 hour in 1% Os04in the same
buffer, dehydrated rapidly in a graded series
of ethanol, and embedded in Epon 812. For
orientation, semithin sections were cut a t 2 m p
and stained with azure-methylene blue. Thin
sections were cut on a Porter-Blum MT2 mi-
crotome and stained with uranyl acetate fol-
lowed by lead citrate. Two yolk sacs a t 40 days
of gestation, two at 50 days, and one 60-day
yolk sac were examined on a Philips 200 or an
Hitachi 11E electron microscope.
OBSERVATIONS
The yolk sac is composed of three layers. A
single layer of endoderm, varying from low to
tall columnar, lines the vitelline cavity. A sim-
ple mesothelium faces the exocoelom, and
sandwiched between the endoderm and me-
Fig. 1. Diagrammatic sagittal section of a uterine ges-
tation sac and the definitive fetal membranes of a dog to
show the relation of the yolk sac to the other membranes
and the exocoelom. Part of the yolk sac is indicated by heavy
dashed lines. All. C., allantoic cavity; Am.C., amniotic cav-
ity; Exo., exocoelom; Vit. C., yolk sac cavity. The rectangle
indicates an area comparable to Figure 2.
Fig. 2. Section of a fold of the wall of the yolk sac of a
50-day dog fetus, showing a resldual erythroblastic island
(long arrow) and vessels with presumably circulating blood
(short arrows). E, endoderm; M, mesothehum. x 500.
315
sothelium is vascular mesenchyme (Fig. 2).
Depending upon age, several cell types and
various-sized blood spaces and blood vessels
containing mature red blood cells andlor eryth-
ropoietic cells are located in the middle layer.
The cells of all three layers vary structurally
at different stages of gestation.
Endodern
The endoderm a t all stages examined is com-
posed of large dome-shaped cells. Numerous,
short microvilli extend from the apical cell sur-
faces; lateral interdigitations are present; and
from the basal surfaces there are short,
branching processes (Figs. 3, 5-71, Microvilli
are generally shorter in the endoderm than in
the mesothelium at all stages. The basal lam-
ina is thin relative to that of the mesothelium
(Figs. 3, 4). Desmosomes and junctional com-
plexes connect adjoining cells, with the former
being present not only between adjacent flat
surfaces but also on lateral interdigitations of
adjacent cells. At the inner surfaces of the tips
of the microvilli, densely stained material is
often visible and may represent sites of fila-
ment at,tarhment,.Co;tt,e!dvesicles, alt,hoIighnnt,
numerous, are present throughout, being gen-
erally more abundant on the apical and basal
sides of the cells. In all three stages, vacuoles
of various sizes were seen. The Golgi complex
is well developed (Figs. 3 , 5 , 7), and while not
restricted to any cellular region, it is fre-
quently associated with the nucleus.
In addition to the consistent features noted
above, cytoplasmic variation over time was ob-
served (Table 1).Short, tightly packed, bundles
of intermediate filaments are scattered in the
cytoplasm at 40 days. Filaments were also seen
extending into the microvilli and attached to
desmosomes. More numerous filament bundles
were visible a t 50 and especially a t 60 days
(Fig. 5). Although filaments can be found ori-
ented in almost any direction, they tend to lie
parallel to the longest axis of the cell.
Secondary lysosomes and bodies resembling
primary lysosomes occur more frequently in
60-day endoderm than at 40 and 50 days. At
the earlier stages also, secondary lysosomes
are generally smaller (Figs. 3, 5, 7). An occa-
sional multivesicular body was seen at all these
stages.
Mitochondria are abundant in the endoderm
at 40 and 50 days. At these times, they are
distributed throughout the cell. More centrally
located mitochondria are often associated with
rough endoplasmic reticulum (rER) while pe-
ripheral ones tend not to be. In the 60-day yolk
sac, there are generally fewer mitochondria in
3 16 S.Y. LEE ET AL.
 ULTRASTRUCTURE O F DOG YOLK SAC 317
the endoderm, and these tend to be more cen- mately similar proportions and in some in-
trally situated. Also at this stage in some en- stances are continuous. Although sER and rER
dodermal cells, fewer mitochondria are asso- are best developed a t 40 and 50 days, occa-
ciated with rER; within some of these sional stacks of rER are present at all stages
mitochondria are dark amorphous spots pos- examined. At 40 and 50 days also, some of the
sibly suggestive of degenerative change (Fig. cisternae of the sER are expanded. There is a
5). tendency for the rER to be centrally located.
In each of the stages examined, both the Moderate numbers of free ribosomes are pre-
smooth endoplasmic reticulum (sER) and the sent in the 40 and 50-day endoderm with fewer
rER are present in the endoderm in approxi- ribosomes evident a t 60 days (Figs. 3, 5 , 7).
The nuclues is euchromatic and may be either
spherical or irregular in shape at 40 and 60
TABLE 1 . Summary of cytoplasmic changes in the days; at 50 days, the nucleus is consistently
endoderrn and mesothelium of the dog yolk sac spherical.
Large vacuoles were sometimes observed in
40 days 50 days 60 days
the endodermal nuclei of 60-day-old yolk sacs
Endoderm] (Fig. 6).
Intermediate filaments + ++ +++ Glycogen is evenly distributed in all endo-
Primary lysosomelike + + ++ derm cells a t 40 and 50 days (Figs. 3, 7). In
bodies and secondary most endoderm cells at 60 days, glycogen is
lysosomes very much reduced in amount, although still
Mitochondria ++ ++ + evenly distributed (Fig. 5 ) . In a few cells at
sER and rER ++ ++ +
Free ribosomes ++ ++ + this later time, glycogen was observed in large
Large vacuoles in - - + dense clumps associated with large vacuoles
nucleus and large secondary lysosomes (Fig. 6).
Glycogen ++ ++ + Vacuoles of various sizes are present in some
Lipid vacuoles in +
~
+
- +
cytoplasm endoderm cells at all three stages. At 60 days,
Mesothelium' large vacuoles are sometimes found associated
Lengths & numbers of ++ ++ + with aggregations of glycogen, while others may
microvilli & basal be located near a group of secondary lysosomes
processes or within a nucleus. The endoderm cells with
Intermediate filaments ++ ++ +++ clumps of glycogen and large vacuoles also con-
Primary lysosomelike ++ ++ +++ tain fewer mitochondria and more numerous
bodies and secondary
1ysosomes secondary lysosomes than endoderm cells of
Mitochondria ++ ++ + the same stage without such aggregations.
sER + + +
-
Free ribosomes ++ ++ +
Smooth-surfaced - + +++ Mesothelium
vesicles
The mesothelium at all stages examined is
Based on general observations and not on quantitative measure- composed mostly of squamous cells with often
ments. overlapping lateral edges. At 50 days, some of
the mesothelial cells are dome-shaped (Fig. 7).
The basal lamina is thicker than that of the
endoderm and has a reticular appearance, with
numberous basal processes of mesothelial cells
Fig. 3. Endoderm cell from 40-day yolk sac showing short interspersed. Lateral interdigitations are not
microvilli and thin basal lamina. There are more abundant so numerous here as they are between endo-
mitochondria, rough and smooth endoplasmic reticulum, free derm cells. Junctional complexes and desmo-
ribosomes, and glycogen a t 40 and 50 days than at 60 days
(cf. Fig. 5). The Golgi complex is well developed farrow). x somes are present (Fig. 7).Coated vesicles are
10,200. present at all stages examined, while no lipid
vacuoles were seen at any stage. The Golgi
Fig. 4. Mesothelial cell from 40-day yolk sac. Numerous complex is well developed from 40 to 60 days,
free ribosomes, mitochondria, and some rough endoplasmic being located both apically and laterally. While
reticulum are present. The Golgi complex is well developed the numbers of free ribosomes decrease from
(arrows). Compared to endoderm, microvilli are longer and
more numerous, and the basal lamina is thicker. Clear areas 40 to 60 days, rER remains fairly constant,
in some mitochondria are presumed fixation artifacts. x being moderately dilated, distributed through-
10,400. out the cell, and often associated with mito-
318 S.Y. LEE ET AL.

Fig. 5. Endoderm from tiO-day yolk sac with well-devel- structure (arrow). More secondary lysosomes and fewer mi
oped Golgi complex. large secondary lysosomes. abundant tochondria are present than In earlier stages x 18.000
filaments. and mitochondria showing unusual internal
 ULTRASTRUCTURE OF DOG YOLK SAC 3 19

Fig. 6. Occasional endodermal cells from 60-day yolk sac The cytoplasmic vacuoles are associated with dense glyco-
have large vacuoles in the nucleus and in the cytoplasm. gen and secondary lysosomes. x 10,900.
320 S.Y. LEE ET AL.
 ULTRASTRUCTURE OF DOG YOLK SAC
chondria (Figs. 4,7,9).On occasion, very small
groups of glycogen granules were noted.
As with the endoderm, several cytological
features undergo changes from 40 to 60 days
(Table 1).The microvilli and basal processes
in the mesothelium decrease in number and
length from 40 to 60 days.
Short dense bundles of intermediate fila-
ments were seen in the 40-day mesothelium
(Fig. 4).With increasing age, the filaments from
a diffuse network throughout an entire cell (Fig.
9). As in the endoderm, although orientation
of the filaments may be in almost any direc-
tion, many are situated parallel to the long
axis of the cell. Fine filaments were also ob-
served around the nucleus of some 50- and 60-
day mesothelial cells.
All mesothelial nuclei are euchromatic; those
in the 60-day yolk sac have a n irregular profile
in contrast to the generally spherical nuclei at
earlier stages.
Both the numbers and sizes of primary ly-
sosomelike bodies and secondary lysosomes ap-
pear to increase with advancing age (Figs. 4,
7, 9).
Numerous smooth-surfaced vesicles are pre-
sent along the margins of most 60-day me-
sothelial cells, with the highest concentrations
on the basal sides (Fig. 9). Smooth vesicles were
not observed on 40- and most 50-day mesoth-
elium.
Mitochondria are most abundant a t 40 and
50 days, and as stated earlier, are situated near
rER (Figs. 4,7). The sER is not well developed
at any stage examined. Smali amounts of it
are present at 40 and 50 days, and it is very
scanty in the 60-day mesothelium (Fig. 9).
Mesenchyme
The main constituents in the mesenchyme
layer a t 40 and 50 days are embryonic con-
nective-tissue cells, collagen fibrils, erythro-
blastic islands, some macrophages, blood ves-
sels, and blood spaces enclosed partially or
completely by endothelium. The endothelial
Fig. 7. Dome-shaped mesothelium from 50-day yolk sac.
As in 40-day mesothelium, there are numerous free ribo-
somes, mitochondria, and some rough endoplasmic reticu-
lum. The Golgi complex is well developed (arrows). x 11,300.
Fig. 8. Edge of a completely enclosed blood space from
50-day yolk sac with fenestrated endothelium (arrows) and
lacking a typical basal lamina. Between the endothelium
and the endoderm are situated parts of embryonic connec-
tive-tissue cells (CT).x 7,000.
321
basal lamina of these blood spaces is either
lacking or indistinct (Fig. 8). Single erythro-
cytes, surrounded usually by collagen fibrils
and occasionally by a connective tissue cell,
also are observed outside of blood vessels (Fig.
10). These erythrocytes are generally in closer
juxtaposition to the endoderm, and are more
frequently encountered at 50 days than at either
40 or 60 days. In the mesenchymal layer at 60
days, no erythroblastic islands, macrophages,
or incompletely enclosed blood spaces are seen.
At this stage, the basal laminae of these en-
closed blood spaces appear discontinuous and
are thus either lacking or indistinct in certain
places on the endothelium.
Capillaries and/or larger blood vessels lined
by endothelium with a definite basal lamina
are present in the mesenchyme at all stages
examined. Some of these are seen to contain
blood cells, generally mature erythrocytes, and
occasionally a few reticulocytes.
Connective-tissue cells a t all stages exam-
ined are irregular in form and contain various
amounts of glycogen, free ribosomes, expanded
rER, coated vesicles, and Golgi complexes (Fig.
8).Large vacuoles surrounded by glycogen were
observed in a few connective-tissue cells at 40
days. The nucleus may be irregular in profile.
As gestation proceeds, there is a n increase in
the amount of collagen and the size of the in-
terstitial spaces. The glycogen content of the
connective-tissue cells increases, while there
is a considerable reduction i n the numbers of
free ribosomes, rER, mitochondria, and Golgi
complexes.
Erythroblastic islands, a s mentioned previ-
ously, are observed in 40- and 50-day yolk sacs,
but not a t 60 days. Some of the erythroblastic
islands at 40 days and all islands observed a t
50 days are not surrounded completely by en-
dothelium. Although some of these extravas-
cular islands are adjacent to the mesothelium,
most are located closer to the endoderm. These
islands are usually separated from the endo-
derm or mesothelium by connective-tissue cells
and collagen, and the basal lamina of the en-
doderm and mesothelium in these regions al-
ways is intact. At 40 days only, some erythro-
blastic islands are also found within enclosed
blood spaces having a fenestrated endothelium
and no basal lamina.
In these erythroblastic islands, definitive
erythroblasts in various stages of maturation
(basophilic and polychromatophilic erythro-
blasts, normoblasts, and reticulocytes),erythro-
cytes, or megakaryocytes may be identified
(Figs. 10, 11). In addition, macrophages are
322 S.Y. LEE ET AL.
 ULTRASTRUCTURE OF DOG YOLK SAC
present in very close association with these
maturing cells. They are also seen in blood
spaces of 40- and 50-day sacs either lying near
a normoblast in the process of extruding the
nucleus, or having phagocytized a normoblas-
tic nucleus (Fig. 10). Macrophages are not nec-
essarily restricted to erythroblastic islands but
may be found almost anywhere in the mes-
enchyme.
In two different erythroblastic islands a t 40
days, a segment of endothelial cells is observed
situated between two groups of cells partially
enclosing a vascular space. These cells are
probably involved in the initial stages of vas-
culogenesis. No basal lamina is present on these
endothelial cells (Figs. 10, 11).
DISCUSSION
In the dog yolk sac, there are indications that
the endoderm and mesothelium are more syn-
thetically active at 40 and 50 days than at 60
days. The numbers of free ribosomes and mi-
tochondria in both layers are greater at the
earlier stages than near term. These obser-
vations are consistent with decreased growth
toward the end of gestation. Although still
present in fair abundance at 60 days, expanded
rER (with cisternal stacks) has decreased in
the endoderm from the earlier stages. In the
mesothelium however, the amount of rER re-
mains about the same in all three stages. The
Golgi complex in both endoderm and mesoth-
elium remains well developed between 40 and
60 days.
Thus, in the endoderm of the dog yolk sac,
there is a period when the morphological basis
for protein synthesis is present, as has also
been demonstrated in the cat endoderm by Tie-
demann (1976). In the cat endoderm the num-
bers of mitochondria and stacks of rER were
reported to be highest between 25 and 38 days
of gestation. Also found were peak numbers of
free ribosomes on day 40, an increase in gly-
cogen granules to a maximum on day 46, and
well-developed Golgi complexes throughout
gestation. Tiedemann suggested on the basis
of these morphological findings that the great-
est amount of protein synthesis in the cat en-
doderm occurs at midgestation (between days
Fig. 9. In the mesothelial cells of the 60-day yolk sac,
intermediate filaments are distributed throughout, numer-
ous smooth-surfaced vesicles are present along their basal
edges, and the Golgi complex is well developed. More sec-
ondary lysosomes and fewer mitochondria are found a t this
stage than in earlier stages. x 30,000.
323
25 and 38). This correlates with our morpho-
logical findings in the dog that suggest that
the endoderm may have reached its greatest
protein synthetic capacity by day 40. Later,
Tiedemann and Minuth (1980b)demonstrated
biochemically that the cat yolk-sac endoderm
was involved in the production of serum pro-
teins and that this activity was evident near
the end of gestation. These workers attributed
the persistence of the cat yolk sac beyond the
cessation of erythropoiesis (see below) to this
synthetic function.
Except for the occasional endoderm cells a t
day 60 with large glycogen clumps, glycogen
and sER of the dog endoderm are more abun-
dant in the earlier stages. Tiedemann (1976)
observed a peak of sER from 25 to 38 days in
the cat yolk-sac endoderm, and an abundance
of glycogen granules, increasing to a maximum
on day 46. He considered the possibilities that
the abundant sER of the cat yolk-sac endoderm
may be involved in triglyceride or cholesterol
metabolism and in the polymerization of glu-
cose to glycogen. The large amounts of sER
that we have seen in the dog yolk-sac endo-
derm may have a similar role. Ticdcmann (1976)
hypothesized further that because the rER and
sER appear in the cat yolk-sac endoderm be-
fore they appear in the fetal liver, the yolk-sac
endoderm may be a “functional precursor” of
the liver parenchyma. Tiedemann (1979) in fact
later reported the presence of glucose-6-phos-
phatase (which is involved in glycogen mobi-
lization) within the cisternae of the endo-
plasmic reticulum in the cat. It is quite possible
also that the yolk-sac endoderm of the dog may
continue to supplement the hepatic functions
of the fetal liver for the whole of gestation, as
suggested by Tiedemann and Minuth (1980b)
for the cat.
Analysis of yolk-sac fluid in the cat (Tiede-
mann, 1976) indicated that proteins synthe-
sized in the endoderm are not stored in the
yolk-sac fluid but rather are secreted directly
into the fetal circulation. Tiedemann (1977)
suggested that, in the cat yolk sac, fenestrated
blood vessels located in the mesenchyme and
having incomplete basement membranes may
facilitate protein transport. As stated earlier
in this paper, fenestrated blood vessels are also
present in the dog yolk sac. However, their
involvement in the passage of proteins from
the yolk-sac endoderm in the dog remains to
be demonstrated.
The mesothelium of the dog yolk sac appears
to be active in protein synthesis a t 40 and 50
days and less so near term. At the earlier times,
324 S.Y. LEE ET AL
 ULTRASTRUCTURE OF DOG YOLK SAC
there are fair amounts of expanded rER, mi-
tochondria, and free ribosomes. This activity,
however, is probably less than that which oc-
curs in the endoderm, for no cisternal stacks
associated with the rER were ever observed in
the three stages studied.
Of particular interest a t 60 days compared
to earlier stages is the occurrence of abundant
smooth-surfaced vesicles along the basal sur-
face of much of the mesothelium. Similar but
fewer such vesicles are visible along the other
surfaces. Coated vesicles are present near all
surfaces a t all stages. The smooth and coated
vesicles are obviously components of a trans-
port system whose direction of movement is not
yet clear. Since smooth-surfaced vesicles are
more numerous on the basal sides of the cells,
it is tempting to speculate that they may be
involved in the exchange of substances be-
tween the mesenchyme and the mesothelial
tissue itself.
As the dog mesothelium ages, the lengths of
the microvilli and their numbers decrease.
Perhaps the more extensive microvilli in the
earlier stages are significant in the transfer of
small molecules from the exocoelom. This is in
contrast to the cat mesothelium, where Tie-
demann (1979) observed that long microvilli
are retained throughout gestation. Thus, the
mesothelium may function differently in dif-
ferent species, or its function(s) may contin-
ually change in the same organism during the
course of gestation.
We have noted other changes in both the
endoderm and mesothelium as gestation pro-
gresses. In both tissues, there is a n increase in
the number of filaments in the cytoplasm and
in the numbers of primary lysosomelike bodies
and secondary lysosomes, while fewer mito-
chondria and free ribosomes were observed.
With increasing age of the endoderm, some cells
exhibit large vacuoles and dense accumula-
tions of glycogen, while there is a n overall de-
crease in sER. These observed changes are likely
associated with the slowing down of some syn-
thetic activity in these tissues as the end of
Fig. 10 Part ofan erythroblastic island from 40-day yolk
sac showing association of a macrophage with maturing
erythroblasts. A normoblastic nucleus has been phagocy-
tized by the macrophage (N). A segment of endothelium
(arrow) is located between the erythrocyte and the other
cells of the island. x 10.400.
Fig. 11. Segment of fenestrated endothelium (arrow)
within a blood island of 40-day yolk sac. Parts of basophilic
erythroblasts and mature red blood cells are visible. x 12,500.
325
gestation nears. Interestingly, we find that in
both the endoderm and mesothelium, the Golgi
complex remains as well developed at 60 days
as at earlier stages. Likewise the amount of
rER in the mesothelium remains moderately
developed between 40 and 60 days as was also
found in the cat by Tiedemann (1979). The rER
in the endoderm, although less extensive a t 60
days, is still obvious. It is very likely that in
both tissues the continued activity of the rER
and Golgi complex is correlated with the in-
creased numbers of presumed lysosomes noted
at this time. These lysosomes in turn may be
responsible for the observed degradation and
decreased abundance of mitochondria, fewer
free ribosomes, and, in the endoderm, de-
creased sER. These changes suggest a cessa-
tion of yolk-sac activity as parturition ap-
proaches.
Hemopoiesis in the dog yolk sac at 40 and
50 days of gestation is primarily erythropoi-
etic. This is the major type of blood-cell for-
mation observed in the yolk sacs of a number
of other mammals (Tiedemann, 1977; Weiss
and Greep, 1977). (Examination of a 53-day
dog yolk sac by light microscopy revealed some
hemopoiesis still occurring.) We found no evi-
dence of erythropoiesis in the 60-day yolk sac.
Thus, definitive erythropoiesis in the dog yolk
sac is finished a t a slightly later time than has
been reported for the cat yolk sac, which is
around day 45 (Tiedemann, 1977). According
to Tiedemann (1977) also, definitive erythro-
poiesis in the cat is “almost exclusively intra-
vascular.” In the dog, definitive erythropoiesis
may be extravascular as well.
Macrophages and a n occasional megakary-
ocyte were seen in erythroblastic islands in the
dog. Such cells were also observed in blood is-
lands of human and guinea-pig yolk sacs (Fu-
kuda, 1973; Sorensen, 1961). The macrophages
may function in phagocytizing erythroblastic
debris or may transfer substances (possibly fer-
ritin) to the maturing erythroblasts (Sorensen,
1961). They also undoubtedly serve in recy-
cling nucleoplasm of extruded normoblastic
nuclei (Fig. 10).
Tiedemann (1977) reported the presence of
extravascular neutrophilic granulocytes in the
cat yolk sac. We have found occasional gran-
ulocytic myelocytes in the dog yolk sac that
were also extravascular.
A t day 40, segments of endothelium were
seen partially separating groups of erythro-
blastic cells. The endothelium presumably is
derived from differentiating cells in the blood
islands. Such partially enclosed blood spaces
326 S.Y. LEE E T AL.
on day 40 indicate that vasculogenesis is still
taking place in the yolk sac at this time. This
process is apparently completed before day 50,
for only completely enclosed vascular spaces
were observed a t this stage and in the 60-day
yolk sac.
According to Bloom (1965) and Sorensen
(19591, peripheral cells of blood islands flatten
and differentiate into endothelium while the
more centrally located cells form the blood cells.
In this study, we observed segments of endo-
thelium in the 40-day yolk sac situated be-
tween groups of erythroblastic cells. Erythrob-
lastic islands at this time are both intravascular
and extravascular. These observations indi-
cate that, a t least in some yolk-sac vessels, part
of the endothelial lining differentiates from the
more internally located cells of a blood island.
In such a case, some of the potential blood cells
in a blood island become enclosed within a vas-
cular space, and consequently are observable
as an intravascular erythroblastic island. The
potential blood cells that remain outside of the
enclosed blood space thus form an extravas-
cular erythroblastic island. In the 50-day dog
yolk sac all blood spaces observed were com-
pletely enclosed, and all erythroblastic islands
observed were extravascular. No erythroblas-
tic islands were observed in the 60-day yolk
sac. Presumably at 50 days, vasculogenesis is
no longer occurring, but the presence of extra-
vascular blood islands in the mesenchyme sug-
gests their continuing formation. These then
differentiate into blood cells, some of which
presumably migrate into blood vessels. Since
many of the blood spaces in the 40- and 50-day
dog yolk sac lack a complete basal lamina, and
since much of the endothelium is fenestrated,
it is possible that some of the blood cells that
differentiate from extravascular blood islands
may migrate into the vessels through such vas-
cular walls.
In the cat yolk sac, Tiedemann (1977) re-
ported that the endothelium of the blood ves-
sels is heavily fenestrated and that the base-
ment membrane is incomplete. Tiedemann
suggested that such conditions are favorable
for the passage of proteins and blood cells (pre-
sumably differentiated during primitive eryth-
ropoiesis which may be extravascular). Dia-
pedesis of platelets, reticulocytes, and
erythrocytes has been reported to occur in hu-
mans (Hesseldahl and Falck Larsen, 1971).
Within the mesenchyme at both 50 and 60
days, we observed individual isolated eryth-
rocytes in the interstitial spaces. These eryth-
rocytes were often surrounded by collagen fi-
brils. Such cells perhaps differentiated from
extravascular blood islands and not only did
not migrate into vessels but also escaped
phagocytosis.
Thus, morphological correlates of synthesis,
absorption, transport, and erythropoiesis are
found in the dog yolk sac in the latter part of
gestation. These functions subside for the most
part as parturition nears. These findings are
consistent with those in other species and lead
to consideration of this organ as a supplemen-
tary liver during part of gestation. The ques-
tion remains, however, as to why these yolk-
sac functions should persist for a relatively long
period in some mammals and not in others.
ACKNOWLEDGMENTS
The authors extend their appreciation and
indebtedness to: Ms. Sherry Feig and Dr. Den-
nis Walker for technical advice; Miss Lucy
Taylor for the preparation of Figure 1; the De-
partment of Anatomy of the University of Wis-
consin for extending Visiting Fellow privileges
to one of us (S.Y.L.);Ms. Jody Nicole-Garoutte
for typing the manuscript; and the reviewers
of thc manuscript. This work was supported in
part by National Institutes of Health grant HD-
00277 and by Humboldt State University
Foundation grant 2-8.
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