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Histological and histochemical observations on the armadillo uterus during the delayed and post-implantation periods.

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Departmevat of Biology, T h e Eiee Institate, Zouston, Texas
Patterson ('13), in his classical study of the early embryology of the nine-banded armadillo ( D a s y p u s qzovcnaciizctus nzexicanus, Peters), described the general morphology o i the uterus
of this extraordinary mammal. EIamlett ('35) described the
gross uterine changes which accompany implantation. Newf a n g ('47) has briefly considered the embryology of the uterus
of the armadillo.
The present histological study was undcrtaken as a part
of the investigation of the dclay of implantation being0 conducted in this laboratory. I t is felt that this information will
permit some conclusions concerning the relationship of the
endometrium to blastocyst implantation. Additionally, it
will provide a n index of normality against which to compare
the effccts of hormone administration.
Most of the armadillos used in this study were purchased
from a supplier who catches them in a n area fifty to one hundred miles north of Houston. Additional material was obtained from animals caught or shot in the field by the authors.
Aided by a grant from the National Scipnce Foundation.
* Present address: Sam Houston State Teachers College, Huntsville, Texas.
The animals were rarely kept in the laboratory for more than
a few days, except those animals in which implantation was
produced experimentally.
During the last three years, the uteri from over 100
female armadillos killed during the period of delay of
implantation or the first two months of post-implantation developnient have been examined. Twenty-eight uteri were used
for the study of the early post-implantation conditions.
Twelve of these uteri were obtained from normal animals.
Sixteen of the uteri were obtained from animals in which
early implantation was produced by bilateral ovariectomy
(vide i n f r a ) . Thirteen uteri were obtained from animals in
the last two months of pregnancy. The other animals mere
killed during the period of delay of implantation.
Routinely, parasagittal portions through the body and fundie regions of the uteri were fixed in Zenker’s or Carnoy’s
fluid and in 10% formalin. A few uteri, or portions thereof,
were fixed in IZossman’s fluid, 80% alcohol, and other fixatives. Hematoxylin and eosin preparations were made from
sagittal sections, except in a few cases where cross-sections
were made. Polysaccharides were demonstrated by the Periodic Acid-Schiff test, using materials fixed in either Rossman’s
or Carnoy’s fluids. The Sudan black method was used for
lipid demonstration. Cholesterol and/or its esters was shown
by the Schultz method. Alkaline glycerophosphatase was demonstrated by a variation of the Gomori method on paraffin
sections. Two uteri were injected with India ink prior to
fixation to aid in the study of the vascular pattern.
Ruchanan et al. (’56) have shown that if armadillos are
bilaterally ovariectomized during the period of delay of implantation, implantation occurs some 25 days later. a t implantation, the endometria of castrate animals assume the
characteristics of normal implantation, and the products of
conception a r e normal and viable. Since there are no detectable differences in material obtained from thcse animals and
normally implanted animals, no distinction is made between
them in the results reported here.
I n the description which follows, three regions of the armadillo uterus will be distinguished : the fundic, body, and cervical regions. The fundic region extends from the cranial tip of
the uterus to the level of entry of the oviducts. The body of the
uterus extends from the level of the oviducts to approximately the level of the recto-uterine fold. There is no specific
line of demarcation between the body and the cervical region
of the uterus, either internally or externally. The cervix appears externally as an area of constriction of the uterus,
starting somewhat below the attachment of the ligamenta
teres uteri and terminating a t the superficial junction of the
reproductive tract with the urethra (the vesico-uterine ligament).
The uterus of delay of implantation
The armadillo uterus reaches the typical delay condition
a few days following ovulation, and remains essentially unaltered until implantation (four months later in feral animals). Since the uterine morphology is more stable during this
relatively long period than at previous and subsequent periods
of pregnancy, it is taken, for purposes of description, as the
reference point from which variations in the other periods are
The myometrium. The myometriam of the armadillo uterus
may be divided into three layers. Clear muscle layers (determined by fiber direction) are difficult to discern. However,
in parasagittal sections, the outer layers are generally oblique or longitudinal, and the inner layers, more nearly circular. The bundles of smooth muscle are relatively large in
the outermost layer. Relatively smaller bundles of smooth
muscle cells surround the blood vessels which form an area
vasculosa. These vessels are relatively deep as they enter
the uterus from the broad ligaments, but lie in a more superficial position in the midventral and mid-dorsal portions of
the organ. The innermost layer of the myometrium is com-
posed of small bundles of smooth muscle cells which form a
continuous meshwork. I n between these bundles and their
surrounding connective tissue a r e a series of small, numerous
anastomotic sinusoids of a uniform size which communicate
with both the endometrial sinusoids and major veins located
in the area vasculosa. I n consequence, this innermost and
thickest layer has a spongy appearance.
F r o m the level of the ligamenta teres to the junction with
tlie urethra, there is a n increase in the amount of connective
tissue of the myomctrium, especially in the outer layers. Some
of the collagen fibers of the outermost myometrial layer appear to be reflected dorsally into the recto-uterine fold. Tlierc
is a diminution jn width of the innermost muscle layer, also.
The decrease in number of muscle fibers and increase in collagen results in the formation of an inelastic tube of considerably smaller diameter than the body of the uterus.
T h c endometrizcm. The condition reached by the endometrium within ten days after fertilization remains cssentially
unaltered until the time of implantation. The follox-ing observations pertain to the endometrium as it appears during
this time.
The lamina propria. I n the body of the uterus the loose
areolar connective tissue of the lamina propria forms a continuum surrounding the tubular glands (fig. 1). The connective
tissue of the snbluminal region appears to be less dense than
that in the basal region of the endometrium. Where the lamina propria of the endometrium is continuous with the stroma
of the myometrium, small bundles of smooth muscle cells nre
interspersed between the basal twists of the glands.
The stroma of the endometrium is mildly positive after
the P A S procedure, whether or not this procedure has been
preceded by diastase digestion. It is mildly acidophilic at
a pH of 5.7. I n the area of the sinusoids, the stroma displays
a moderate alkaline phosphatase activity.
The vascular pattern of the endometrium is extraordinary.
The arterioles a r e irregularly coiled as they course through
the inner layer of tlie myometrium and enter the endometrium.
I n the endometrium the arterioles branch, become capillaries,
and course toward the luminal surface. Kear the luminal
surface, a few of the capillaries become sinusoidal, but the
majority loop back toward the intermediate zone of the endometrium, mhcre they dilate to form the endometrial sinusoids.
These sinusoids a r e large, extensive and anastomotic. They
consequently divide the endometrium into basal, sinusoidal,
and suprasinusoidal layers. These sinusoids communicate
tlirough the basal region with the sinusoids of the inner layer
of the myometrium.
I n the fundic region, the connective tissue of the lamina
propria forms a relatively sparse stroma supporting the uterine glands. The endometrial sinusoids are extensive in this
region, forming a n anastomotic cap over the entire fundic
area. Although the endometrium is thinner in the fundic region than in the body, the sinusoids a r e as large or largcr.
Consequently, the distance from the lumind surface to the
endothelium of the sinusoids is greatly reduced, and the sinusoidal layer of the endometrium becomes sublurninal.
I n the cervical region of the uterus, the connective tissue
of the lamina propria is more fibrous than in the other regions of the uterus. The stroma constitutes a greater portion
of the total endometrium than does the epithelium. Although
the lamina propria is highly vascular, sinusoids of the type
found so abundantly in the body of the uterus are largely
absent. Those sinusoids which a r e occasionally present are
The epit7ieZium. I n the body of the uterus, the glands are
elongate, straight, and tubular (fig. 5). The liiminal epithelium
varies from simple to stratified columnar but is most commonly pseudostratified columnar. I n the basal layer and to a
lesser extent the sinusoidal layer, the glands may he somewhat
coiled, but in the suprasinusoidal layer of the endometrium
they a r e quite straight. The glands are slightly dilated. The
epithelial cells of the straight neck regions a r e columnar.
The nuclei of these cells a r e oval, and are frequently basally
situated (fig. 3). I n the sinusoidal region, the glandular epi-
thelium is similar to that in the neck region, with the exception
that the cells are niore vacuolated. I n the basal region, the
lumina are smaller, and the epithelial cells tend to be low
columnar. The nuclei of these cells are more spherical than in
other layers of the endometrium.
I n the basal region and the sinusoidal region, small amounts
of glycogen may be demonstrated by the PAS method (fig. 7).
A thin fringe of saliva-resistant mucopolysaccharide was
stained on the luminal surface of many of the gland cells.
This mucopolysaccharide in tissue sections tends to obscure
the cilia which are present on many of the cells, especially
where the oviducts enter. The activity of these cilia can be
observed easily in slices of fresh tissue. Numerous lipid
droplets can be demonstrated in the majority of gland cells.
These droplets are especially abundant in the sinusoidal and
basal regions of the glands (fig. 5). Much of this lipid is positive when tested with the Schultz method. A few finer droplets are frequently present in the epithelium of the more
superficial portions of the glands. Most of the lipid is readily
extracted by acetone.
I n the fundic region, the glands are short and relatively
coiled. The basal and sinusoidal portions of these glands are
similar to the corresponding portions of the glands of the
body. However, the suprasinusoidal portions are largely absent. The luminal epithelium of this region is more commonly
simple o r pseudostratified columnar, rather than stratified.
This epithelium is the surface against which the blastocyst
lies during the four month delay period.
The transition from the body of the uterus t o the cervix
is gradual. The glands in the cervical region are less elongate,
and relatively uncoiled. The epithelium is somewhat less columnar, and there is very little lipid, though glycogen is present in most of the gland cells. The glands are relatively few,
with a consequent increase in lamina propria. At the level of
the vesico-uterine ligament, there is a sharp transition t o a
luminal epithelium of mucus-secreting cells.
Changes i m the u.terus during the course of pregnamcy
The myometrium. After ovulation, the fundic tip of the
uterus becomes rounded, rather than peaked. The vascularity
of the entire myometrium is slightly enhanced. There is
little increase in over-all thickness of the myometrium during
the delay period.
During the latter part of pregnancy, the myometrium becomes greatly attenuated due to the growth of the uterine
contents. As parturition approaches, the myometrium becomes so thin in the fundic region (where the villi have degenerated) that the uterine contents are clearly visible through
the uterine wall. It is interesting to note that uterine rupture
seems quite common (three cases in 60 pregnancies observed),
but does not necessarily interfere with subsequent pregnancies.
Histologically, some hypertrophy and elongation of the
smooth muscle cells is apparent after implantation. Hyperplasia of these elements has not been observed. The smooth
muscle cells of the myometrium contain moderate amounts of
glycogen at all stages of pregnancy; however, the glycogen
content of the smooth muscle cells of the arteries is greatly
enhanced following implantation.
The endometrium. I n the first few days after ovulation,
the endometrium is essentially in the non-pregnant stage. The
connective tissue of the lamina propria is relatively abundant.
The vascular pattern already described for the endometrium
of delayed implantation is present but not yet prominent. The
glands are more coiled at this early stage than during delay,
and bifurcating glands are not uncommon. The glandular
epithelium is columnar, but there is less evidence of crowding
and the cells are low columnar, rather than palisade. Neither
glycogen nor lipid is as abundant as during the delay period.
Moderately numerous glycogen granules are present, however, in the basal and sinusoidal regions of the glands. Small
numbers of lipid granules are also present, especially in the
basal portions of the cells constituting the deeper two-thirds
of the endometrial glands.
The differences observed in the endometrium between the
early post-ovulation period and the rest of the period of delayed implantation are not striking. I n contrast, the onset of
implantation is accompanied by extensive changes. The lamina propria is markedly edematous (fig. 2). The cellular elements are relatively dispersed, and the stroma stains less intensely with both conventional trichrome methods and the
PAS method. The sinusoids become highly dilated, especially
as tlic cell chords and secondary villi of the placenta enter
and extend along tliese sinnsoicls from the initial site of
The epithelial portions of the endometriuni also undcrgo
extensive alterations. Tlie glands dilate Iiroadly and become
mildly coiled. The epithelial cclls of these glands become
highly vacuolated (fig. 4). The nuclei become spherical rather
than oval, and they a r e more centrally located within the cell.
The hypertrophy of these cells results in an increased width
with the consequence that they appear to be low columnar or
cuboidal. Only in the most basal portions of the glands of the
body of the uterus and in the glancls of the cervix do the epithelial cells remain essentially unaltered.
Examination of the lipid and glycogen content of these cells
reveals that while the lipid content diminishes, c y m k l l y in
the suprasinusoidal region (fig. 6 ) , the glyeogcn content is
much higher (fig. 8). Not only do the cells of the basilar and
sinusoidal regions contain glycogen, but also the cells of
the neck regions of the gland and the epithelium of the uterine
lumen. 1,arge numbers of glycogen granules a r e present both
in the basal and apical portions of the cells of the glandular
E s t a b l i s h m e n t of t h e d e f i n i f k e placenta. ,4t implantation,
the trophoblast actively invades the endometrium in a relatively small spot in the fundic region. Following yolk-sac inversion and division of the embryonic shield and amnion, the
4 resultant embryos and their common yolk-sac develop in
the uterine lumen (Patterson, '13). The trophoblast, however,
penetrates the sinusoids. The subsequent development of the
triiger chords and the vascular villi occurs within the endometrial sinusoids.
The result of this unusual pattern of implantation is the
establishment of a point of initial penetration surrounding
which there is a circumf erential junctional zone. Extending
peripherally from this zone is an extensive area where the
villi have penetrated the endonietrial sinusoids and are consequently overlain by the more superficial portions of the
endometrium which forms a partial capsule (but not a true
decidua capsularis) (fig. 9). Apparently, the trophoblast is
actively invasive only during the initial stage of implantation.
Even at this stage it appears to progress through the tissues
by penetration rather than by lysis.
Only a few decidual cells are ever observed in the armadillo
uterus (fig. 12). These are found in the junctional region i n
the early stages of pregnancy. They are usually in juxtaposition to the invasive trophoblast, though they may occasionally be separated from it by the endothelium of the sinusoids.
These decidual cells are large cells containing glycogen
After the establishment of the mature placental pattern,
the villi iiicrease in numbers, and increasingly distend the
endometrial sinusoids. The villi do not penetrate into the
relatively small sinusoids of the internal layer of the myometrium, nor is the endothelium of the sinusoids destroyed.
The lamina propria of the endometrium becomes increasingly
fibrous as pregnancy continues. The endometrial glands also
begin to have a stretched appearance, and become somewhat
disorganized. The alkaline phosphatase activity of the stroma
and the lipid content of the glandular epithelium cells diminishes. Some glycogen granules remain in the epithelial cells
of the endometrium throughout pregnancy.
During the latter portion of pregnancy, the endometrium
is reduced in the placental area to thin arcades over the
multitudinous villi. The blood vessels in the lamina propria
become increasingly distended. Since the villi do not disrupt
the endothelium, except a t the site of initial invasion the
integrity of the maternal vascular system is uninterrupted.
Thus the initial pattern of endometrial circulation is essentially maintained throughout pregnancy, although the functional significance of the pattern alters with the onset of
implantation (figs. 10, 11, 13).
I n the latter quarter of pregnancy, the epithelial cells of
the glands of the decidua Vera undergo enlargement. This
enlargement occludes the lumina of the glands. As a result,
solid cords and strands of cells are formed as far as the cervix.
At parturition the endometrium is torn loose at the sinusoids (fig. 14). The contractive portions of the coiled endometrial vessels and the basal twists of the endometrial glands
remain. Presumably, it is from these latter portions of the
glands that the endometrial epithelium is reconstituted.
The armadillo uterus is ideally suited to a villous hemochorial type of placenta. The blastocyst is at all times in the
fundic region where the sinusoids lie closest t o the surface
and the stroma is scant between the hypertrophied epithelial
cells of the endometrial glands. Once penetration into the
sinusoids has occurred, a continuous series of blood spaces
is made available to the proliferating villi. Unlike the condition in primates where the histolytic action of the placenta
interrupts the blood vessels and blood pools are formed which
are both drained and fed through the basal plate (Boyd, '55;
Ramsey, '55, '57), the blood flow in the endometrial sinusoids
remains largely unidirectional. Neither is the vascular supply
to the endometrium itself greatly disturbed until the sinusoids
have been extensively enlarged by the proliferation of villi.
During the delay period, the blastocyst though unimplanted
is in contact with the luminal epithelium. Despite the ciliary
activity and the presence of a mucopolysaccharide secretion in
the uterine lumen, it is quite possible that the nutrients used
by the blastocyst come from the underlying tissue. Some physiological alterations in the endometrium must occur prior to
implantation, or it would not be possible to produce early im-
plantation by bilateral ovariectomy. Preliminary observations
have shown augmented glycogen levels in ovariectomized armadillos, but morphological changes have not been visualized
prior to implantation in feral females. Further histochemical
investigations of the endometrium now in progress may be
more revealing in this respect.
That there are few decidual cells in the armadillo endometrium would appear to be a corollary of the small amount of
histolytic activity of the placenta. There is neither extensive
trauma of the uterus nor a substantial decidual response by
the uterus. Consequently, products of liistolysis probably
play only a minor part in the nutrition of the developing
As mentioned previously, the inverted yolk-sac lies in the
uterine lumen. It is thus in a position to phagocytize or absorb the products of the endometrial glands. Tlie abundance
of glycogen in the yolk-sac epithelium is probably an indication
of such activity. I t is tempting to speculate that in the early
stages of pregnancy, the yolk-sac epithelium garners products
not readily synthesized by the developing embryos ; while the
villi serve as a region of exchange of more highly soluble
substances. As pregnancy proceeds, the exchange between
the maternal blood stream and the villi becomes increasingly
important and the secretory activity of the endometrium
ceases. Further studies on the histochemical history of the
placenta and on exchange rates may serve to support o r refute this hypothesis.
The smooth muscle bundles of the myometrium of the simplex uterus of the armadillo form three rather ill-defined
layers. The outer largely longitudinal and the inner largely
circular layers are most clearly distinguishable where the
area vasculosa is interposed between them. The innermost
layer of muscle consists of small bundles of smooth muscle
which are oriented in all directions and between which are
numerous anastomotic sinusoids.
Large sinusoids divide the endometrium into basal, sinusoidal and suprasinusoidal layers. During the period of delay
of implantation, the glands are relatively straight and the
glandular epithelium is composed of tall columnar cells with
oval, basally situated nuclei. Glycogen granules and lipid
droplets are moderately abundant in these cells in the basal
and suprasinusoidal regions of the endometrium. Ciliated epithelial cells are also abundant.
At the onset of implantation, the glands hypertrophy and
the lamina propria becomes edematous. The glandular epithelial cells are large and vacuolated. The vacuolation is due
to the high glycogen content. The lipid in the epithelial cells
diminishes slightly.
The chorionic villi penetrate into the endometrial sinusoids
shortly after the initial attachment of the blastocyst. Subsequently, the trophoblast loses its invasiveness. The further
expansion of the placenta occurs through the enlargement of
the endometrial sinusoids. Consequently, the maternal vascular system is uninterrupted and the blood flow in the sinusoids remains largely unidirectional.
Though the villi penetrate into the sinusoids, the foetuses
and the yolk-sac develop in the uterine lumen. Consequently,
the epithelium of the inverted yolk-sac is in a position to absorb or phagocytize the products of the uterine glands, while
the position of the villi is most admirably suited for the exchange of substances between the two blood streams.
BOYD, J. D. 1955 Morphology and physiology of the utero-placental circulation. Transactions of the Second Conference on Gestation. Josiah
Macy, Jr. Foundation, 132-194.
BUCHANAN,G. D., A. C. ESDERSAND R. V. TALMAGE1956 Implantation in
armadillos ovariectomized during the period of delayed implantation.
J. Endocrinol., 14: 121-128.
G. W. D. 1935 Delayed implantation and discontinuous development
in the mammals. Quart. Rev. Biol., 10: 432-447.
hTEwFANG, D. M. 1947 Sex differentiation in the nine-banded armadillo, Dasypus novemcinctus. J. Morph., 81 : 283-316.
J. T. 1913 Polpnbryoriic devclopiiient ill T a l / t s i a n o w t n c i n c l u x .
Ihitl., 2 4 : 559-684.
R.%arsEu, E. hl. 1955 Distribution of arteries and veiiis i n the ni:iiiiiiidinii p l n ceiitn. Tr:irisactioiis of the Seeoiid Conferciice o n Gest:itioii. Josinll
Macy, J r . Fouiidation, 229-251.
_____ 1957 Flow of inateriixl blood through the priiwItc> p1:iceiitn. Annt.
Rec., 1 2 7 : 4T8.
E X P L A N A T I O N 0)’ F I G U K l l S
1 Eii(loint+5uni of dr1:ryed iinp1:uit:itioii.
II ant1 E.
x 160.
~ ~ o s t - i i i i ~ ~ I ~ i i i t : i TT
t i o i:ind
x 150.
Eiidoinrtriuiii froin tlie first inoutli
I+itlomctrium of delayed iniplnntntioii. S o t c crowdiiig of l):is:il, or:il nnclci.
I1 :1nd E.
Eii(1onietriurn f roin the first nioiitli ~)ost-iiiipl:nit:itioii.S o t e tlic syneytial troplioblnst 011 tlie r i g h t and tlic Iiypcrtropliicvl, wciio1:itccl cc~llsof thr. g1andul:lr
c~i)ithcliiiiii. I€ and R. x ,575.
x GO.
Eiiclonictriuni from t h e delay period. Sut1:ui b1:ick.
lhcloiiic~triuri~from tlic first nioiitli post iiiiplaiitation. Tlic blood i s no longer
ywseiit in tlic 1:rrge biiiiisoids in t h i s section. Sud:ni black.
Ehtlonic~tiiuiiifro111 tlic delay pcriotl.
Eiidoiiic~triurnf r o m l a t e in t h e first iiioiith of pregn:mey. X o t e t h e cxteiisioii
of the siiiusoids by the villi :md t h e :ibuiida~iccof g l y c o g c ~ iii
~ i the c~idoinetrinl
g1:iiids. Periodic Acid Schiff. X GO.
Periodic Acid Selliff.
x GO.
E a r l y 1)rcgiiaiic!-. S o t c yolk-s:ic iii tllc uteriiic luiiicii aiid villi iii tlic c n i l o iiictrial siiiusoids. Masson. X 6’7.
Tiiiierinost h y e r of the iiiyoiiietriu~ii. S o t ? v:nious fil~erdircctioiis and ititcrcoiiiiectioii of siiiusoiils. I I i i i i t l E. X 4.70.
11 O p e n i ~ i g of t h e str:iiglit portioii of :in eiitloiiietri:11 ve
S o t e t h e allseiice of :I iiiuscul:rr w:ill :iiitl tlic positioii of tlic o p w i i ~ gj u s t
uiitlcr tlic, cliorioiiie p1:ite. L a t e prcgiiaiicy. H m i l E. x 80.
Deciilual cells iii tlic stroinn near tlie jiuictioiial zoiic. S o t ( %tlic 1:irgc. size
of tlicse cells and t h e vacuo1:itioii of tlir glaiidular cpitlic~liuni. F i r s t iiioiitl~
of p w p m c y . 1% mil E. X 363.
Tlic. voiletl portioii of a iilatcrii:tl vessel j u s t liefore i t ciitcrs the c~iidoiiic~triiiiii.
F r o i i i the satlie uterus as figurcx 11. Periodic Acid-Schiff. x 80.
1-1- Eii(loiiietri;il iiiyoiiietri:iI juiictioii froiii aii :rrnin~lillo killctl
:I few I I I ~ I I L I ~
post-p:ntum. S o t e t h a t oiily the basal twists of t h e ghiids rciiiaiu. I1 :tiid I:..
x 110.
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uterus, period, observations, delayed, armadillo, post, histological, implantation, histochemical
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