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Ultrastructural observations on the shell membrane of the North American opossum (Didelphis virginiana).

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THE ANATOMICAL RECORD 207:335-338 (1983)
Ultrastructural Observations on the Shell Membrane of the
North American Opossum (Didelphis virginiana)
WILLIAM J. KRAUSE AND J. HARRY CUTTS
Department ofAnatomy, School of Medicine, University of MissouriColumbia, Columbia, MO 65212
ABSTRACT
Each developing opossum embryo is surrounded by a shell
membrane which completely separates embryonic and maternal tissues. During the eighth and ninth prenatal days, the embryos together with their
limiting shell membranes float freely within the uterine lumen, surrounded
only by the secretions of the uterus. The shell membrane is transparent,
nonelastic, tough, and capable of extreme deformation. It consists of a mat of
interwoven fibers which vary in external diameter, are electron dense, and
show no apparent substructure. The morphology and arrangement of component fibers are similar throughout the width of the shell membrane.
After ovulation, the metatherian egg remains in the oviduct for about 24 hr before it
enters the uterus; during this short transit
period, the egg is fertilized by a single spermatozoon. Although the sperm are paired,
both in the epididymis and in the female
reproductive tract of Didelphis (Biggers and
Delamater, 1965; Krause and Cutts, 1979)
the sperm-pairs always separate prior to fertilization (McCrady, 1938). A corona radiata
is not present and the metatherian egg initially is surrounded only by a zona pellucida.
Following fertilization, a second layer, consisting primarily of acid mucopolysaccharides, is laid down around the zona pellucida.
This second layer is the product of glandular
secretions of the oviduct (McCrady, 1938;
Tyndale-Biscoe, 1975).As the fertilized ovum
passes along the remainder of the oviduct,
spermatozoa may become embedded within
the substance of this layer. In Dasyurus viw
errinus, a n Australian marsupial, the second
layer may measure 1.5-8.0 pm in thickness.
A similar coat of mucopolysaccharide has
been described about the ovum of the rabbit
(Tyndale-Biscoe, 1975).
The metatherian egg, unlike that of the
eutherian, becomes surrounded by a third
layer, the shell membrane, which also is
thought to be produced by secretory cells in
the distal oviduct (Hartman, 1916; Anderson,
1928). In the Australian form, Trichosurus
vulpecula, the shell membrane is reported to
be keratinous in nature, and nonelastic but
0 1983 ALAN R. LISS, INC.
capable of extreme attenuation (Tyndale-Biscoe, 1975).It is said to be resistant to enzyme
digestion (Hughes, 1977)and apparently does
not act as a barrier for the passage of most
large molecules to and from the surrounding
uterine fluid (Hughes and Shorey, 1971). In
most marsupials it persists between the
forming fetal membranes and the maternal
uterine epithelium until late in gestation.
The ultrastructure of the shell membrane
has not been reported previously in Didelphis virginiana or any other didelphid marsupial. Even in the Australian forms, the
structure of the shell membrane has been
described only in passing, primarily during
the very early stages of development. The
present study reports the light-, scanning-,
and transmission electron microscopic observations of the shell membrane in the North
American opossum late in the gestation
period.
MATERIALS AND METHODS
Fifteen embryos were collected, the approximate ages of which were determined from
timed pregnancies. Wild female opossums,
carrying litters, were stripped of their pouch
young and placed in breeding pens with continuous access to males. The females usually
reenter into estrus within a week after removal of the young and a sperm-positive date
was obtained by examining vaginal smears
~~
Received March 14,1983; accepted June 2, 1983.
336
W.J. KRAUSE AND J.H. CUTTS
a t 24-hr intervals. The embryos were collected during the last third of the eighth, and
first third of the ninth prenatal day. Three
adult female opossums were killed by ether
anesthesia and the uteri removed and
opened. The embryos appeared as clear
spheres floating freely within the uterine lumen, and were recovered intact by tipping
the uterine contents into fixative. The specimens were fixed for 4 hr a t room temperature, in 3.0% glutaraldehyde buffered in 0.1
M phosphate to a pH of 7.4, after which the
tissues were transferred to 0.1 M phosphate
buffer for 2 hr, then osmicated for 2 hr in
1.0% osmium tetroxide in 0.1 M phosphate.
After washing in the phosphate buffer and
dehydrating through a series of graded
ethanol solutions, specimens for scanning
electron microscopy were critically point
dried by liquid COz substitution in a Bomar
critical point dryer, mounted on spinner
stubs, and coated with gold to a depth of 20
nm in a Polaron SEM coating unit. The specimens were viewed in a JOEL-35 scanning
electron microscope operated a t 20 kV.
Tissues for transmission electron microscopy were dehydrated through ethanol solutions, cleared in propylene oxide, and infiltrated with and embedded in Epon 812. Thin
sections of this material, mounted on uncoated grids and stained with uranyl acetate
and lead citrate, were examined in a Phillips
300 electron microscope operated a t 60 kV.
Thick sections (0.3-2.0 pm) of the Eponembedded material were examined by light
microscopy after staining with toluidine blue.
RESULTS
Each uterus contained several transparent, fluid-filled spheres approximately 6.5
mm in diameter, floating freely within the
secretion of the uterine lumen. Following fixation, a developing embryo could be seen on
each sphere by direct observation (Fig. 1).All
three germ layers were present and from five
to eight somites could be seen. The forming
brain consisted of closed cavities but the developing spinal cord remained open.
The shell membrane around each embryonic sphere is transparent during life and
following fixation. During mechanical manipulation it appears nonelastic but is tough
and capable of extreme deformation. When
the external surface of the shell membrane
is examined at low magnification with the
scanning electron microscope, it appears
smooth, but at increased magnification it is
seen to consist of a mat of interwoven
branching fibers that vary in diameter (Fig.
2). With the light microscope, the shell membrane appears as a thin, homogeneous band
with no apparent substructure (Fig. 3). However, ultrastructurally, the shell membrane
consists of numerous, interwoven electrondense fibers (Fig. 4)that appear to be homogeneous and show no evidence of smaller subunits.
DISCUSSION
In marsupial species with short gestation
periods, the shell membrane surrounds the
developing embryo for most of the prenatal
period and, even in those species with a much
longer gestation (macropodids), it persists for
a t least two-thirds of the prenatal period,
completely separating embryonic and maternal tissues (Hughes, 1974; Tyndale-Biscoe,
1975). In Didelphis the shell membrane remains intact through the 10th prenatal day
(Renfree, 1975) and some portions may persist until near term (McCrady, 1938). During
the period when the embryos are surrounded
by a n intact shell membrane, they lie free
within the uterine lumen and are thought to
be nurtured by secretions from the uterus.
The nutrients make their way to the growing
embryo by passing through the surrounding
shell membrane. Renfree (1975) found the
uterine secretions of Didelphis to be rich in
proteins, principally albumins, prealbumins,
and one or two P-globulins. The shell membrane of Trichosurus vulpecula is permeable
to both peroxidase and ferritin, although
some filtration of the latter does occur
(Hughes and Shorey, 1971,1973). Such observations support the idea of free passage of
nutrients and wastes to and from the developing embryo.
In the shell membranes of three Australian marsupials-the bandicoots, Isoodon
macrourus and Perameles nasuta, and the
brush-tailed opossum, Trichosurus vulpecula (Hughes, 1977; Lyne and Hollis, 1977)the electron-dense, fibrous material appears
to be tightly packed rather than in the form
of a series of loose fibers, as observed in
Didelphis. The close packing seen in the
Australian forms may reflect the very early
stages of development examined in these
species. The electron-dense material in the
shell membrane of marsupials appears similar to that present in the egg shells of the
two monotremes, the platypus and echidna
(Hughes et al., 1975; Hughes, 1977).
The shell membranes of the domestic fowl
and some reptilian forms (Solomon and Tippett, 1983) appear similar in structure both
to metatherians and prototherians. The avian
shell membrane consists of a n open mesh-
Fig. 1. A fixed embryo from an opossum uterus taken
early during the ninth post-sperm-positive day. Note that
the developing embryo is part of a n embryonic sphere.
The shell membrane is transparent and, although present, cannot be seen in this photomicrograph. The region
of the farming brain, spinal cord, and somites are visible.
x25.
Fig. 2. The external surface of the shell membrane
consists of a mat of interwoven fibers which vary considerably in diameter. x 11,000.
Fig. 3. The shell membrane appears as a thin homogeneous band (arrows) when viewed in section at the
light microscopic level. Epon 812-toluidine blue. X250.
Fig. 4. At the ultrastructural level the shell membrane is seen to consist of numerous electron-dense fibers without apparent substructure. The external surface
(E) of the shell membrane lies t o the left of the photomicrograph. x 15,000.
338
W.J. KRAUSE AND J.H. CUTTS
work of electron-dense proteinaceous (keratinous) fibers (Simons and Wiertz, 1963;
Bellairs and Boyde, 1969; Kaplan and Siegmund, 1973; Creger et al., 1976) and is the
product of secretions from glands in the oviduct (Breen and Bruyn, 1969; Wyburn et al.,
1973) as is the case both for marsupials and
monotremes. In birds, however, the events
are complicated somewhat in that calcification of the egg shell begins just before the
egg enters the uterus.
Permeability of the shell membranes has
been thought to be of primary importance in
the evolution of viviparity (Hughes, 1977)
since it would permit transfer of nutrients
from the uterine secretions to the embryo
and eliminate dependence upon contained
yolk material. Such a change in the means
by which the embryo is nourished during
development may have permitted the reduction in size of the egg. Similarly, Hughes
(1977) has hypothesized that the loss of the
shell membrane in eutherian species may be
associated with the early invasive properties
of the chorion, thus permitting the intimate
association between maternal and fetal vascular systems during placentation.
ACKNOWLEDGMENTS
This work was supported in part by a grant
from the J.B. Reynolds Foundation.
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