Early Ontogeny

J. Zool. Syst. Evol.
Research 39 (2001) 137158

2001 Blackwell Wissenschafts-Verlag, Berlin
ISSN 09475745
Received on 15 February 2000
Museum fur Naturkunde, Institut fur Systematische Zoologie, Berlin, Germany
Early ontogeny and placentation of the grey short-tailed opossum, Monodelphis

domestica (Didelphidae: Marsupialia): contribution to the reconstruction
of the marsupial morphotype
U. ZELLER and C. FREYER
Abstract
This study provides new ndings on the placenta of Monodelphis domestica and a reconstruction of the marsupial morphotype. To achieve this,
early ontogeny and placentation of the grey short-tailed opossum, M. domestica, from 3 h after copulation until birth (day 15), were studied and
compared with other mammals. Both the ultrastructure and histochemistry of egg membranes, foetal membranes, oviduct and uterus were
examined. The results of this study provide the rst detailed ultrastructural description of a trophoblastic syncytium in a marsupial. In addition,
this is the rst original documentation of an invasive trophectoderm and an inammatory reaction at parturition in M. domestica. These ndings
were compared with literature data and included into the reconstruction of the marsupial morphotype. Based on marsupial phylogeny as
proposed by Luckett (J. Mammal. Evol. 2, 255283, 1994), characters that are consistent at least within didelphids and dasyurids were determined
to be characters of the marsupial morphotype. These characters are a central yolk separated from the peripheral yolk-poor cytoplasm in the
unfertilized oocyte, the presence of a zona pellucida, a mucoid coat and a shell coat, the absence of a corona radiata, oviductal mucoid secretion,
no shell secretion distal to the isthmus of the oviduct, uterine shell secretion, a short tubal passage (1 day at maximum), the apposition of
blastomeres to the zona pellucida prior to intercellular association, the absence of a morula stage, the polarity of the zygotic yolk, the localized
segmentation of deutoplasm (yolk) during the rst cleavage and subsequent extrusion of yolk vesicles during the rst two cleavage stages. With
regard to the marsupial morphotype, the non-polarized yolk distribution in the zygote [Hartman (J. Morphol. 27, 184, 1916); McCrady (Am.
Anat. Mem. 16, 1233, 1938)] is a derived character of Didelphis virginiana. Didelphis virginiana [Hartman (J. Morphol. 27, 184, 1916); Hartman
(J. Morphol. 32, 1139, 1919); McCrady (Am. Anat. Mem. 16, 1233, 1938)] and Didelphis marsupialis (Hill, Q. J. Micr. Sci. 63, 91139, 1918)
share the synapomorphous reduction of deutoplasmolysis to a generalized extrusion of vesicles. The absence of separated yolk and consequently a
cleavage without yolk extrusion (Renfree and Lewis, Reprod. Fert. Dev. 8, 725742, 1996) are apomorphies of macropodids. This is possibly
correlated with the association of blastomeres in early cleavage stages (Renfree and Lewis, Reprod. Fert. Dev. 8, 725742, 1996). A yolk sac
placenta and a vascularized allantochorion can be assumed for part of the ontogeny in the marsupial morphotype, irrespective of the formation of
an allantoic placenta at near term stages. The character polarization of the mode of placentation and parturition needs further investigation.
Key words: Monodelphis domestica Marsupialia early ontogeny evolution morphotype placentation phylogeny reproduction
Abbreviations: bc, blastocyst cavity; bm, blastomeres; CRL, crownrump length; DAB, diaminobenzidine; dpc, days post copulation; em,
endometrium; EM, electron microscopy; ER, endoplasmatic reticulum; ESI, electron spectroscopic imaging; fbc, foetal blood cell; fc, formative
cytoplasm; HC, histochemistry; hpc, hours post copulation; ht, histiotrophe; ic, intercellular capillary; l, lipid droplet; LM, light microscopy; mc,
mucoid coat; me, mesenchyme; mt, mitochondrion; n, nucleus; ne, nucleated erythrocytes; PAS, Periodic Acid Schi's Reagent; pp, pseudopodialike process; pr, propria; r, ribosomes; rer, rER, rough endoplasmatic reticulum; S.E.M., Standard Error of Mean; SEM, scanning electron
microscopy; sl, syncytial knot; sm, shell coat; sp, secretory process; st, sperm tail; te, tubal epithelium; TEM, transmission electron microscopy; tr,
trophoblast; trc, trophoblast cell; trs, trophoblastic syncytium; ue, uterine epithelium; ug, uterine gland; ul, uterine lumen; uv, uterine vessel; vv,
vitelline vessel; y, yolk; yp, yolky cytoplasm; zp, zona pellucida
Introduction
This study is a contribution to the reconstruction of the
morphotype of recent marsupials by providing current ndings, a character polarization and a phylogenetic interpretation.
Marsupialia (Metatheria) and Placentalia (Eutheria) form
the monophyletic Theria, which are the sistergroup of the
Monotremata (Novacek et al. 1988; Shoshani and McKenna
1998; Zeller 1999b). Although marsupials and placentals are
both viviparous (unlike the egg-laying monotremes) their
modes of reproduction dier considerably (see Table 1). The
polarization of most of these characters, as well as their
evolutionary signicance are still unresolved. For the reconstruction of the evolution of reproductive strategies in both
groups, the morphotypes, which are the sum of characters
present in the last common ancestors (cf. Hennig 1950;
Konigsmann 1975), of Theria, Marsupialia, Placentalia and
internal groups must be reconstructed.
The morphotype reconstruction is based on marsupial
systematics as proposed by Luckett (1994). According to
U.S. Copyright Clearance Center Code Statement:
Luckett (1994), marsupials can be divided into the monophyletic

sister-taxa Australidelphia and Ameridelphia. Ameridelphians
comprise all American marsupials except Dromiciops australis,
australidelphians consist of all Australasian marsupials including the South American Dromiciops australis. The monophyly
of these groups is supported by the sperm pairing in the
epididymis of ameridelphians (Temple-Smith 1987) and the
fusion of two facets on the calcaneus (conuent lower ankle joint
pattern, Szalay 1993) in australidelphians. This basal dichotomy
of marsupials is essential for the morphotype reconstruction,
based on character optimization and outgroup comparison. For
character optimization and outgroup comparison the further
division into syndactyls and diprotodonts is useful. The phylogeny of marsupials is represented by the cladogram in Fig. 1.
The dierences in reproductive biology could have had
important inuence on the dierences in extinction rates,
radiation and migration between marsupials and placentals at
the CretaceousTertiary boundary (Archibald 1982; Case and
Woodburne 1986; Caroll 1997). We regard the reconstruction
of the marsupial morphotype as a rst step toward an
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Small, less yolk bodies7,8

1030 lm12,13
Without deutoplasmolysis that releases material as membrane bound
vesicles7,14,15
In most cases in the oviduct; exception: Elephantulus myurus19
In most cases present (when entering the uterus)14, exception: three genera
of insectivora21
Present14,32, (inner cell mass forms later in three genera of insectivora21)
Absence of shell coat, absence of mucoid25; exception in lagomorphs25
and horse9,26
Always involving the allantois15,26,27,29,30,31 Mesodermal villi at the side
of the allantochorion30,32
Large at ovulation, many yolk bodies7,8
Thinner than in placentals2,9 : 1108.611 lm
Including deutoplasmolysis that releases material as membrane-bound

vesicles7,1418
In the uterus2,1416,18
Absent (direct blastocyst formation)2,16,18,20
Absent10,14,16,18,2224
Shell coat, mucoid coat9,25
Seldom involving the allantois2,15,2729, never mesodermal villi

at the side of the allantochorion31
Always longer than intrauterine development: 50 days (Monodelphis,

Cercartetus)540 days (Macropus giganteus, M. fuliginosus)2
Since longer than pregnancy not aected by pregnancy exception: Wallabia bicolor,
Macropus giganteus35,36 aected by lactation2,37
Oocyte
Zona pellucida
Cleavage
Start of cleavage
Morula stage
Entypic condition
of the blastocyst
Egg coats
Placenta
Lactation phase
Oestrous cycle
Renfree (1994); 2 Tyndale-Biscoe and Renfree (1987); 3 Sminthopsis macroura, Selwood and Woolley (1991); 4 Dasykaluta rosamondae, Woolley (1991); 5 Mesocricetus auratus, Lochbrunner (1956);
Elephas maximus, Nowak (1999); 7 Selwood and Sathananthan (1988); 8 Renfree and Lewis (1996); 9 Selwood (2000); 10 Hartman (1916); 11 Selwood et al. (1997); 12 Austin (1961); 13 Bedford (1996); 14 Hill
(1910); 15 Cruz and Pedersen (1991); 16 Selwood (1992); 17 Selwood (1980); 18 Selwood and Young (1983); 19 van der Horst (1942) cited in Tyndale-Biscoe and Renfree (1987); 20 Selwood (1996);
21
Elephantulus myurous, van der Horst (1942); Hemicentetes: Bluntschli (1937); Goetz (1938); Eremitalpa, Gabie 1959 all cited in Mossman (1987); 22 MacCrady (1938); 23 Selwood (1986a); 24 Selwood
(1986b); 25 Hughes (1977); 26 Herrler et al. (1997); 27 Amoroso (1952); 28 Morris (1975); 29 Padykula and Taylor (1976); 30 Luckett (1977); 31 Wooding and Flint (1994); 32 Mossman (1987); 33 Cavia, Nowak
(1999); 34 Pan troglodytes, Nowak (1999); 35 Tyndale-Biscoe (1973), p. 53; 36 Renfree (1980); 37 Heap and Flint (1984).
Usually aected by pregnancy37
Can be shorter than intrauterine development2: 5 days334.5 years34
Long intrauterine development possible2: 1656686 days
Precocial relative to marsupial neonates, never xed to nipple2
Placentals
Always short intrauterine development : 9.5 49.7 days (including sperm storage,
possible arrest of conceptus development)
1,2
Pregnancy
Extremely altricial, active movement to nipple, xed to nipple for 14127 days
neonate
2
Marsupials
Characters of
reproduction
Table 1. Characters of reproduction in placentals and marsupials
138
ZELLER and FREYER
Early ontogeny and placentation of the grey short-tailed opossum
139
acrylate (Kulzer). Specimens embedded in paraplast were sectioned at

7 lm and stained with hematoxylin and eosin (HE), azan or trichrome
(Mason-Goldner). Specimens embedded in methylmethacrylate were
sectioned at 2 lm and stained with HE.
Frozen sections were obtained from tissues shock frozen in liquid N2
1 and later sectioned at 46 lm with a Kryostat (LeicaFrigocut, Leica
Instruments GmbH, Heidelbergerstr. 1719, 69226 Nuloch, Germany) at )25 C.
Histochemistry
Four methacrylate sections of specimen 82 [29 h post copulation (hpc)]
and one frozen section of specimen 135 [7 days post copulation (dpc)]
were stained with Kongo red (Romeis 1968). One frozen section of
specimen 135 was also stained for keratin by Martinotti (Romeis 1968)
to demonstrate the shell coat. Numerous frozen sections of specimen
135 were stained with Sudan red.
Twenty-four methacrylate sections of specimens 45 (22.5 hpc), four
of specimen 85 (29 hpc) and eight of specimen 95 (4dpc) were stained
with PAS. Six paran sections of specimen 45 (22.5 hpc) were stained
with peroxidase-conjugated Ulex-europaeus-lectin (substrate: DAB).
Four frozen sections of specimen 135 were xed in acetone (6 C) for
10 min and treated with antibodies against keratin, laminin, collagen
type IV and bronectin (Table 2); the immunoreaction was visualized
by the usage of biotinylated secondary antibodies and the biotin
2 avidinperoxidase method according to Dako LSAB-Kit (DAKO
Diagnostika GmbH, Hamburg, Germany). In addition, sections were
treated with collagenase (Sigma no. C-0773; Sigma-Aldrich Chemie
3 GmbH, Taufkirchen, Germany). Antibodies against transferrin were
applied to two frozen sections of specimen 132 and to two paran
Fig. 1. Cladogram representing marsupial systematics as proposed by
Luckett (1994). 1, Ameridelphia; 2, Australidelphia; 3, Syndactyla; sections of specimen 28 (12 dpc), followed by the application of Dako
LSAB-Kit.
4, Dasyuroidea; 5, Diprotodontia; 6, most phalangeroids; 7, VombatiControl: sections treated with all chemicals used in immunohistoformes; 8, Macropodoidea
chemistry without the primary antibody did not stain.
evolutionary scenario that explains the mammalian evolution

at the CretaceousTertiary boundary.
Materials and methods
Electron microscopy
For transmission electron microscopy (TEM), specimens were xed by
perfusion through the maternal vascular system with a mixture of
1.5% glutaraldehyde and 1.5% freshly prepared paraformaldehyde in
0.1 M cacodylate buer (pH 7.35; total osmolality 658 mosmol/l).
Specimens of yolk-sac placenta (endometrium with adjacent yolk-sac
wall) prepared for TEM were cut into small pieces and further xed for
1 h with glutaraldehyde/paraformaldehyde in cacodylate buer. Tissue
blocks were rinsed for 1 h in cacodylate buer, post-xed for 2 h with
1% osmium tetroxide by immersion (specimen 194 (14 dpc) with 1%
tannic acid) and embedded in araldite after dehydration with ethanol.
Ultrathin sections (6080 nm) were stained with lead citrate and uranyl
4 acetate and examined with a Zeiss CEM 902 electron microscope (Carl
Zeiss Oberkochen, Germany) operating at 80 kV with an integrated
5 imaging electron energy spectrometer and a Phillips CM 10 (Phillips
Electron Optics, Chatswood, NSW, Australia) operating at 60 kV.
Electron spectroscopic imaging (ESI) without elemental analysis was
performed at energy losses of either 0 eV (èlastic bright eld') or 250
eV (with the carbon signal at minimum). For scanning electron
microscopy (SEM), embryos or pieces of endometrium were dried
according to the critical point method, sputtered with gold palladium
and examined with the Zeiss DSM 960 scanning electron microscope.
Monodelphis domestica was bred in a colony established at the Institute

of Anatomy of the University of Gottingen. This colony was later
moved to the Institute of Zoology of the University of Mainz and then
on to the Institute of Systematic Zoology at the Museum of Natural
History, Berlin, where it is presently located. Estrus of female
Monodelphis is induced by the presence of male pheromones (Fadem
1985; Hinds et al. 1992). Copulation takes place within the estrus
(Baggott et al. 1987; Fadem and Rayve 1985). Ovulation occurs 20
(Mate et al. 1994), 22.5 (n 1, own observation) to 24 h (Baggott and
Moore 1990) after copulation. Fertilization occurs 22 (Mate et al. 1994),
22.5 (n 1, own observation) to 24 h after copulation (Mate et al. 1994).
Mating was monitored by direct observation. All embryos and stages
mentioned in this study are dated according to the time of copulation.
Thus, the rst day of pregnancy coincides with the day of fertilization,
that is, about 24 h after mating. Parturition occurs between the 14th and
15th day after copulation (Fadem et al. 1982; personal observation)
after a 13- to13.5-day period of gestation (Mate et al. 1994).
The consistency between our ndings and descriptions of Mate et al.
(1994) conrmed the normality of embryos described in this study.
Photography
Abnormal stages were not included in the description. In addition, the
chronology of development in M. domestica as described here matches 6 Whole embryos were photographed with a Wild macroscope M 400
the timetable given by Mate et al. (1994).
(Leica), and a Zeiss Axioplan was used for microphotography.
Twenty-seven adult female M. domestica were killed with chloroThe specimens and procedures adopted are summarized in Table 2.
form at dierent times of pregnancy, ranging between a few hours and
15 days after copulation. They were xed by perfusion through the
Taxonomy
vascular system immediately after their death. Gravid uteri, embryos
Species names are used according to the nomenclature of Wilson and
and pieces of yolk-sac placenta were prepared for histochemistry, light
Reeder (1993). If authors cited in this paper used synonyms, the correct
and electron microscopy (EM) according to the following procedures.
name is given in square brackets. Classication of species into higher
taxonomic units also follows the taxonomy of Wilson and Reeder
Light microscopy
(1993). The species included in both the discussion and the morphoSpecimens were either (1) xed with Bouin s and embedded in type reconstruction belong to the higher marsupial taxa as summarized
paraplast or (2) xed with 4% formol and embedded in methylmeth- in Table 3.
140
ZELLER and FREYER
Table 2. Female Monodelphis domestica investigated in this study

Stage
Specimen no.
Methods
3 hpc
4 hpc
22.5 hpc
24 hpc
29 hpc
31 hpc
68
81
45
36
85
118
4
5
6
7
dpc
dpc
dpc
dpc
95
128
102, 145
135
8 dpc
10 dpc
146
101
156
187, 231
8, 46
132
LM, SEM
LM
HC (PAS, Ulex-europaeus-lectin (DAB)), LM
LM, SEM
HC (Kongo red, PAS), LM
LM, immunohistochemistry: Dako EPOS: mouse-antihuman antibodies against keratin,
clone MNF 116, Code No. U7022, against keratin 10, 17, 18 (4556.5 kDa), DAKO EPOS CK10,
DE-K10, Lot 043011, against keratin 10
LM
LM
LM
left uterus: LM
right uterus: LM, HC: Kongo red, Sudan red, Keratin (Martinotti), collagenase (Sigma no. C-0733);
immunohistochemistry:
(1) tissuGnost (Merck): mouse-anti-human antibodies against intermediate keratin laments
(54 kDa), LSAB-Kit (Dako)
(2) Dako EPOS: mouse-anti-human antibodies against keratin, clone MNF 116, Code No. U7022,
against keratin 10, 17, 18 (4556.5 kDa)
(3) Dako Collagen Type IV (CIV 22): mouse-anti-human antibodies against collagen
(Code No. M 785. Lot no. 102, 1 : 100), LSAB-kit
(4) Dako Laminin (4C7): mouse-anti-human antibodies against laminin (Code No. M 638,
Lot no. 121, 1 : 25), LSAB-kit
(5) Dako Fibronectin: rabbit-anti-human antibodies against bronectin (Code No. A 245,
Lot. no. 117, 1 : 400), LSAB-kit
LM
LM
TEM
LM
LM, TEM
Left uterus: HC
Immunohistochemistry: Dako anti-transferrin, rabbit-anti-human antibodies against transferrin
(Code No. A 0621 02, Lot. no. 040 D, 1 : 800), LSAB-kit.
LM, TEM
HC
Immunohistochemistry: Dako anti-transferrin (same as in 11 dpc)
LM, TEM
LM, TEM
LM
LM
11 dpc
12 dpc
28, 44
13 dpc
14 dpc
12
6, 194, 429
677
122
15 dpc
HC, histochemistry; hpc, hours post copulation; dpc, days post copulation; LM, light microscopy; SEM, scanning electron microscopy; TEM,
transmission electron microscopy.
Phylogenetic reconstruction
Reconstruction of morphotypes follows the algorithm for reconstructing ancestral states using parsimony as described by Cunningham et al.
(1998). As pointed out by Cunningham et al. (1998), `the algorithm uses
a ``downpass'' and `ùppass'' traversal to optimize ancestral states using
two rules: Rule 1: if descendant nodes share any states in common,
assign the set of shared states to the ancestor; Rule 2: if no states are
shared in descendant nodes, assign the union of descendant's states to

ancestor.' (p. 362, Box 1). According to the algorithm, the downpass
optimization proceeds `down' the tree towards the root, optimizing
each ancestral node. The uppass optimization proceeds ùp' the tree
away from the root, optimizing each ancestral node. In a nal
optimization, the sets of downpass and uppass reconstruction are
optimized to the state that has the greatest number in both reconstruc-
Taxon
Species
Didelphidae
Dasyuridae
Monodelphis domestica, Didelphis marsupialis, Didelphis virginiana, Philander opossum

Dasyurus viverrinus, Sminthopsis crassicaudata, Sminthopsis macroura, Antechinus
stuartii, Phascogale sp.
Isoodon obesulus, Isoodon macrourus, Perameles nasuta, Perameles gunnii, Echymipera
rufescens, Echymipera kalubu
Vombatus ursinus
Phascolarctos cinereus
Petauroides volans, Pseudocheirus peregrinus
Trichosurus vulpecula
Acrobates pygmeus, Distoechurus pennatus
Macropus eugenii, Petrogale sp., Macropus robustus, Macropus giganteus,
Macropus rufogriseus, Setonix brachyurus
Potorous tridactylus, Bettongia cuniculus
Peramelidae
Vombatidae
Phascolarctidae
Pseudocheiridae
Phalangeridae
Acrobatidae
Macropodidae
Potoroidae
Table 3. Species mentioned in this

study and their relation to higher
marsupial taxa

tions. This procedure implies that there is no constant outgroup.
Instead, the outgroup changes as does the respective ingroup during the
process of optimizing within the group of investigation.
This procedure is conducted using the cladogram by Luckett (1994).
We favour this hypothesis of marsupial phylogeny, because it is based
on total evidence (molecular and morphological data). Since ontogenetic data are often restricted to didelphids and dasyurids, which are
representatives of ameridelphians and australidelphians, respectively,
consistent data within both groups are assumed to be characters of the
marsupial morphotype. Certain of these morphotype hypotheses can
be supported by ndings among additional australidelphians. Inconsistent data within ameridelphians or australidelphians were polarized
using the mentioned method for morphotype reconstruction.
Due to insucient and inconsistent data concerning marsupial
placentation, reconstruction of the allantochorion in the marsupial
morphotype was separately conducted by optimizing with the sistergroup (Placentalia) of marsupials and the outgroup of the Theria
(Monotremata). In some cases, the state of marsupial morphotype
characters (e.g. zona pellucida, mucoid coat) is determined by
outgroup comparison with monotremes.
Results
By 3 hpc, 11 tertiary follicles were present in the right ovary
investigated. The oocytes contain large yolk vacuoles and are
surrounded by follicular epithelial cells. Paired spermatozoa
can be seen in the oviduct on SEM images. One hour later, the
follicle epithelium degenerates.
By 22.5 hpc, the corpora lutea have developed. The eight
oocytes passing each oviduct lack a corona radiata and are
merely surrounded by the zona pellucida, which is PASreactive. They measure 170 lm in diameter. The oocyte
nucleus is located in the peripheral, formative cytoplasm,
which contains minimal yolk (Fig. 2a). The formative, yolkfree cytoplasm encloses the yolky deuteroplasm (Fig. 2a),
which in turn encloses a small central zone that contains little
yolk. Paired spermatozoa are visible inside the oviduct by light
microscopy (LM) and SEM (24 hpc). Fertilization occurs in
the oviduct (Fig. 2a). At this time, the second polar body is
separated o. The surface of the zygote is in close association
with the tubal epithelium (Fig. 2a) and binds the lectin Ulex
europaeus agglutinin (UEA) like oviduct cells, but unlike the
uterine epithelium. Mucoproteid layers covering the surface of
the conceptus are secreted by non-ciliated oviductal cells.
Corpora lutea are already present in the ovary.
By 29 hpc, the zygotes (n 5) have entered the uterus in the
pronuclear stage. Thus, a maximum of 7 h is needed for the
passage down the oviduct. Each zygote is covered by a
concentric, lamellar mucoproteid layer (mucoid coat)
(Fig. 2b), in which spermatozoa are trapped. A secondary
yolk polarity is achieved by polar accumulation of the
formative cytoplasm around the pronuclei (Fig. 2b), which
fuse at 31 hpc. The uterine epithelium secretes the proteinaceous shell coat, which is Kongo red reactive but does not bind
antibodies against keratin.
At the fourth day after copulation, the 16-cell stage (n 1)
has been achieved. The yolk, which is not aected by cleavage,
forms a compact mass opposite the blastomeres (Fig. 2c). This
is due to deutoplasmolysis and apocrine yolk extrusion during
the rst and second cleavage at 4250 hpc and 5868 hpc,
respectively (Fromm and Zeller 1997). The diameter of the
conceptus measures 180 lm inside the zona pellucida and its
entire diameter measures 440 lm. The mucoid coat surrounding the zona pellucida consists of two layers. The thinner outer
one is strong PAS reactive, whereas the thicker inner one is less
141
reactive to PAS. Pseudopodia-shaped processes of mucoid

coat and shell coat project into the niches of the endometrium
(Fig. 2d). The shell coat cannot be stained with PAS.
By 5 dpc, the blastomeres of the conceptus (n 7) contain
large yolk vacuoles. One day later, the zona pellucida is partly
broken down (n 9). The blastomeres containing yolky lipid
droplets in vacuoles form the early unilaminar blastocyst. The
mucoid coat is still visible.
By 7 dpc (bilaminar blastocyst, n 10), the mucoid coat and
yolk have been incorporated into the blastocyst and the
blastomeres are apposed to the peripheral shell coat (Fig. 2e).
At this time, the diameter of the blastocyst measures 340 lm
and almost doubles within one day (670 lm by 8 dpc). At this
stage the pseudopodia-shaped projections are absent. The
embryonic area partly consists of a thickened ectoderm with
underlying endoderm, and it does not form an inner compact
cell mass (Fig. 2e).
The shell coat surrounding the blastocyst cannot be stained
with PAS or Alcian blue and does not bind antibodies against
cytokeratin, laminin or bronectin. It is resistant to collagenase, but can be stained for keratin by Martinotti, and Kongo
red. Keratin was not detectable by immunohistochemistry.
By 10 dpc (embryo, ve to six somites, n 6), the vesicle has
formed a bulging sphere 5 mm in diameter. The embryo has a
neural groove, neural crest with a developing mesenchyme, a
primitive streak, a paraxial mesoderm with ve to six somites,
a lateral plate mesoderm with pericardium, extraembryonic
mesoderm and a yolk sac cavity.
One-third of the yolk sac lying towards the embryonic pole
forms the trilaminar omphalopleure, the abembryonic part is
bilaminar. Ultrastructurally, the trophendoderm (yolk sac
endoderm) consists of at cells connected by cell projections
and tight junctions. Wing-shaped processes of trophendoderm
cells partly appose to the bases of trophectoderm cells in
bilaminar omphalopleure and project with short processes into
the basal folding, whereas extended parts of trophectoderm
(ectoderm of the yolk sac) cells remain uncovered. The cells
have abundant polyribosomes, mitochondria, endoplasmatic
reticula (ER) and lipid droplets.
The trophectoderm (outer wall of the yolk sac) is formed of
columnar to attened distinct cells leaving small intercellular
capillaries for transport in between. Apices are united by
junctional complexes. Exocytotic vesicles secrete into wide
intercellular gaps (about 15 lm in width) between trophendoderm and trophectoderm. The apical surface is densely covered
with microvilli directed to the endometrium. The cells contain
numerous mitochondria, smooth and rough ER, polyribosomes, vacuoles and lipid droplets. Endocytotic complexes and
transport vesicles are present. The cell bases are folded. All this
indicates active transepithelial transport of material.
The cells of the one-layered, high-columnar uterine epithelium are densely covered with microvilli on their luminal
surface, their bases border a capillary net in the propria. A
basal lamina could not be found. The propria contains only few
cells. The endometrial glands consist of a thick pad of coiled
tubular glands, which in some areas extend for 3 mm. They are
devoid of glandular ducts and secrete histiotrophes directly into
the uterine lumen. The myometrium does not exceed 0.2 mm.
The shell coat is still intact and measures 1.8 lm.
By 11 dpc (4 mm total length), the dierentiation of the
frontal half of the embryo is highly advanced following rapid
development. It shows a strong curvature of head and neck, a
dorsally concave lumbar curvature, a foregut with visceral
142
ZELLER and FREYER
Fig. 2. Monodelphis domestica, photomicrographs of early development from tubal zygote to placentation. (a) 22.5 hpc, tubal zygote. The
formative cytoplasm (fc) surrounds the central yolky cytoplasm (yp).
The surface of the zygote is in close association with the tubal
epithelium (te). , sperm tails, scale bar 10 lm. (b) 29 hpc, uterine
zygote. The yolky cytoplasm (yp) is polarized. Pronuclei (arrows) are
located in the formative cytoplasm (fc). arrows, pronuclei; *, shell coat;
circle, artefact; scale bar 20 lm. (c) 4 dpc, 16-cell stage. After yolk
extrusion, the yolk (y) forms a compact mass opposite the blastomeres
(). scale bar 50 lm. (d) 6 dpc. Pseudopodia-shaped processes (pp) of
the mucoid coat (mc) and the shell membrane (asterisk) project into
the niches of the uterine epithelium (ue). bm, blastomeres; s, artefact;
scale bar 50 lm. (e) 7 dpc, bilaminar blastocyst. The embryo
consisting of a thickened ectoderm and underlying endoderm (arrows)
does not form an inner compact cell mass. bc, blastocoel; ul, uterine
lumen; scale bar 50 lm. (f) 12 dpc. The yolk-sac placenta is greatly
expanded with elaborate folds increasing the surface area. em,
endometrium; arrows, yolk sac; , embryo; scale bar 500 lm
clefts and visceral arches, brain plate with eye anlagen, ear
placodes, a heart anlage, and crest-shaped anlagen of upper
extremities. The vitelline duct is still wide open. The neural
tube, somites, notochord, aortae, coelom and nephrogenic
crest can be seen on transverse sections through the embryo.
The cranial and caudal ends of the neural tube are still open
and form neuropores, respectively. Two-thirds of the anterior
end of the embryo are sunk into the yolk sac and covered with
the two-layered proamnion (ecto- and endoderm). The caudal
third of the embryo is still part of the surface of the conceptus.
A small extra-embryonic coelom can be seen between the
trilaminar yolk sac and proamnion.
The yolk sac is trilaminar for over one-third of its extension.
It measures 56 mm in diameter and is greatly expanded with
elaborate folds increasing the surface area (cf. Fig. 2f). It is
closely apposed to the endometrium (Fig. 3a). The endodermal
cells of the bilaminar omphalopleure have long projections
enclosing large intercellular spaces between trophendoderm
and trophectoderm. In the trilaminar yolk sac wall the
endodermal cells are attened and closely connected to each
other. Blood vessels with endothelium and haemocytoblasts
are developed within the mesenchyme of the trilaminar yolk
sac. The sinus terminalis appears at the border between the
bi-and trilaminar omphalopleure.
The trophectoderm mainly consists of distinct cells united by
desmosomes with intercellular transport capillaries in between.
In particular, the cells of the trophectoderm of the bilaminar
omphalopleure are at and contain large vacuoles exceeding
the diameter of the cell nucleus. Ultrastructurally, the trophectoderm is formed in most parts by distinct cells, which are
apically united by junctional complexes (Figs 3b, 4a) with
intercellular transport capillaries in between (Fig. 3b). Some
trophectodermal cells of the trilaminar omphalopleure have
already fused and intercellular borders can not be seen. The
nuclei of those cells are close to each other. The trophectoderm
is densely covered with microvilli (Figs 3a, 4a), and pinocytotic
vesicles are present on its surface. The cells contain numerous
transport vesicles, mitochondria, lipid droplets, rough and
smooth ER, free ribosomes, and big euchromatic nuclei with a
maximum diameter of 21 lm (Figs 3b, 4a). This indicates the
contribution of the trophectoderm to both transport and
synthesis.
The cells of the single-layered, high-columnar uterine
epithelium contain often more than one large euchromatic
nucleus (up to 15 lm in diameter) with prominent nucleoli
143
(Fig. 3a). They are connected by apical junctional complexes,

and are covered with a dense apical border of microvilli of
3 lm length (Fig. 3a). Secretory canaliculi are located
between the cells of the apocrine secreting epithelium. The
cells contain mitochondria, rER, polyribosomes, secretory
granules and abundant lipid droplets (Fig. 4a). The uterine
epithelium mainly serves as a pathway for materials from the
maternal blood to the embryo. The basal plasmalemma is
folded and borders on a capillary net in the propria, which is
situated 0.52 lm beneath the epithelium. A basal lamina
is lacking. The capillary endothelium is not fenestrated. Two
layers of endometrial glands can be distinguished: The
endometrial glands near the myometrium consist of a singlelayered secreting epithelium with basal nuclei and cytoplasm
that is comb shaped due to abundant inclusions. The
endometrial glands near the uterine epithelium have mostly
homogeneous cytoplasm and central nuclei, with inclusions
and a comb-shaped cytoplasm that is restricted to their bases.
The cells of the uterine glands contain ergastoplasm, dictyosomes, mitochondria, lipid droplets and secretory granules.
The surface is covered partly with branching microvilli and
partly with kinocilia. The uterine glands are the sites of
synthesis and secretion of histiotrophes. The propria contains
more free cells (histiocytes, plasmacytes, lymphocytes, polymorphonuclear granulocytes) on its subepithelial side than on
its basal side.
The shell coat, which measures 2.3 lm, is unruptured at this
stage (Figs 3a, 4a). It consists of narrow meshes, through which
all material for the exchange between mother and embryo has
to pass. Shell coats of adjacent embryos do not fuse.
By 12 dpc [5 mm crownrump length (CRL)], the dierentiation of the embryo is more advanced, especially in the facial
and branchial regions as well as the upper extremities. The
large heart supports the yolk sac circulation. The lumbar
curvature is attened out, the caudal amnion expanded and the
embryo has completely sunk into the yolk sac. The exocoelom
is expanded.
Only the yolk sac, which is now vascularized over two-thirds
of its extension, contributes to the placenta (Fig. 2f). The
endodermal cells of the vascular part are at and in close
contact with each other. The yolk sac vessels are the sites of
blood formation by mitotic multiplication of proerythroblasts
and myeloblasts. Immunohistochemistry demonstrates transferrin within the maternal epithelium as well as in the
trophectoderm and the underlaying vascular layer of the
omphalochorion, indicating transferrin-rich histiotrophe passing the meshes of the shell coat (Zeller 1999a).
The trophectoderm still consists in most parts of distinct,
laterally interconnected cells, that are united by apical junctional complexes (compare Fig. 4a). The length of the
microvilli is reduced. The cells contain large vacuoles and big
nuclei mainly arranged in a regular pattern. At some areas of
the trilaminar omphalopleure the nuclei of the trophectoderm
are situated close to each other and between those areas the
trophectoderm free of nuclei is attened. The extent of folding
of the endometrium is increased (Fig. 2f). The length of
microvilli of the uterine epithelium is reduced (1.7 lm).
Macro- and microapocrine secretion occurs. The endometrial
glands secrete histiotrophes (Fig. 3c). The thickness of the
glandular pad is reduced to about 1.5 mm. The former twolayered condition of the glandular pad has been lost. The shell
coat, remnants of which can be seen between trophoblast and
endometrium (Fig. 3c), is ruptured.
144
ZELLER and FREYER
Fig. 3. Monodelphis domestica, (ae) transmission electron micrographs of the yolk-sac placenta from 11 to 14 dpc. (f) photomicrograph
of post-partum endometrium. (a) 11 dpc. The shell coat (sm) separates
uterine epithelium (ue) and trophoblast, which consists of individual
cells (trc) covered with numerous micovilli. l, lipid droplets; me,
mesenchyme; scale bar 10 lm. (b) 11 dpc. The trophoblast consists
of single cells united by junctional complexes (arrows) that are
interspersed with intercellular capillaries (ic). Numerous mitochondria
(mt), rER (rer), ribosomes (r) and lipid droplets (l) are present; scale
bar 10 lm. (c) 12 dpc. Remnants of the shell coat (arrows) lay
between trophoblast (tr) and uterine epithelium (ue). The uterine
epithelium releases secretory processes (sp), which form histiotrophes
(ht); scale bar 10 lm. (d) 14 dpc, non-invasive area of the trophoblastic syncytium (trs). Trophoblast and uterine epithelium are densely
packed with microvilli (thin arrows). The trophoblastic syncytium is
rich in rER (thick arrows), polyribosomes, laments (arrow heads) and
lipid droplets. *, mitochondrium; ue, uterine epithelium; scale
bar 10 lm. (e) 14 dpc, invasive area of the trophoblastic syncytium
(trs). The trophoblast erodes the uterine epithelium (ue) and reaches
uterine vessels (uv). The barrier for foeto-maternal exchange between
vitelline vessels (vv) containing nucleated erythrocytes (ne) and uterine
vessels (emptied by perfusion) is diminished; scale bar 10 lm. (f) 15
dpc, post-partum uterus. The propria (pr) is invaded by leucocytes
(thin arrows), i.e. granulocytes, lymphocytes, monocytes and others.
Indentation (thick arrows) in the uterine epithelium (ue) remain from
invasive areas of the trophoblastic syncytium. ug, uterine gland; scale
bar 30 lm
By 13 dpc (8.5 mm CRL), the neural tube is closed,

peripheral nerves and ganglia, the eye cup, the labyrinth
vesicle, an epithelial nasal sac, a muscular tongue, a chondried endoskeleton with occipital pillar, an axial skeleton,
Meckel's cartilage and the anterior extremity, a gut canal,
liver, heart, lung and mesonephros are dierentiated. The
frontal acropodium is pentarch, whereas the back one is still
paddle-shaped.
The shell coat is completely broken down. Most of the
trophectodermal cells of the trilaminar omphalopleure have
fused to form a trophoblastic syncytium (see below).
By 14 dpc (10 mm CRL), the embryo has a two-layered
head amnion lined by high microvilli on the side of the yolk
sac. This indicates a transport of amniotic uid between yolk
sac and amnion. The exocoelom, which never covers the whole
embryo, is located ventrolaterally to the embryo, from its navel
to the caudal end. The allantois is small.
The trophendoderm of the trilaminar omphalopleure
consists of at, expanded cells united by junctional complexes.
It is covered with short microvilli on the side of the yolk sac
lumen. The yolk sac vessels around the navel are embedded in
large, bulging cells. The trophendodermal cells of the bilaminar omphalopleure are also attened and close to the base of
the trophectodermal cells (Fig. 5d).
Most cells of the trophectoderm of the trilaminar omphalopleure have fused to form a syncytium (Figs 4b, 5a). Cell
borders are rare. The thin basal lamina underlying the single
layered trophectoderm measures about 150 nm. The syncytium
is covered with microvilli and shows pinocytosis, intracellular
vesicles and laments (Fig. 3d), indicating active transepithelial
transport of materials. It is rich in rER, polyribosomes and
lipid droplets (Fig. 3d) and multivesicular bodies. Nuclei are
located in syncytial knots (25 nuclei per knot) (Fig. 4b).
Between the syncytial knots are thin lamellae of syncytial
epithelium (Fig. 4b). Occasionally, cell borders were found
within the syncytial lamellae. Lipid droplets and accumulations
of glycogen are mainly located in the knots, cell organelles are
145
rare. The trophectoderm is in close association with the uterine

epithelium with an intimate interdigitation of trophoblastic and
endometrial microvilli (Fig. 3d). The height of the microvilli
does not exceed 1.6 lm. The invasive trophoblast penetrates
the maternal epithelium at regular intervals, and reaches the
maternal endothelium (Figs 3e, 5a). To the side of the invasive
areas, the trophoblast is intimately interconnected with the
uterine epithelium by newly formed cellular adhesions
(Fig. 5a). The invasive trophoblast displaces the uterine
epithelium at either small (Fig. 3e) or expanded areas (Fig. 5a).
Thus, a mixed epithelio-endothelio-chorial yolk sac placenta is
formed. At the invasive areas, the maternal endothelium, the
trophoblast (with thin basal lamina underneath), interconnected cells of the connective tissue of the yolk sac, the extracellular
matrix and the foetal endothelium form the layers, through
which the foeto-maternal exchange of material and gases takes
place. Owing to the thinning of foetal layers, the barrier
between foetal and maternal blood does in some areas not
exceed 2.5 lm. In these areas, the uterine epithelium is close to
the cells of the foetal connective tissue near the vitelline vessels
(Fig. 4b). Bilaminar parts of the yolk sac are rare, probably due
to fusion of yolk sacs of adjacent embryos and subsequent loss
of these areas (Freyer in prep.). In the bilaminar yolk sac wall,
fusion of trophectodermal cells could not be found (Fig. 5bd).
Thinning of the trophoblast and accumulations of lipid
droplets are similar to the trophoblastic syncytium (Fig. 5b
d). The trophoblast of the bilaminar yolk sac walls were closely
attached to the surface of the maternal epithelium. However,
invasive areas could not be found.
The endometrium is strongly folded. The largest folds form
chambers for single embryos and their foetal appendices. As
pointed out above, the height of the microvilli is reduced. The
thickness of the endometrial glandular pad is reduced to
0.20.4 mm. The thickness of the myometrium remains
unchanged at 0.1 mm.
At the 15th day after copulation, the propria is highly
invaded by polymorphonuclear granulocytes, macrophages
and lymphocytes (Fig. 3f). Granulocytes are also found in the
glandular tubes. This is followed by the parturition of the
placenta.
Discussion
In the present study of the grey short-tailed opossum,
M. domestica, the early ontogeny and placentation from
ovulation to parturition are described. To conrm and
complement previously published detailed studies and timetables of early embryonic development (Harder et al. 1993;
Mate et al. 1994; Selwood et al. 1997), the development of the
oocyte and zygote, the embryonic development, as well as
changes of the embryonic membranes and the endometrium
during ontogeny are considered. Special emphasis is placed on
the ultrastructural changes of the placenta, and the development of structures is interpreted functionally, where possible,
by correlating data obtained from LM, EM, histochemistry
and immunohistochemistry. The following discussion is aimed
at reconstructing the marsupial morphotype based on the
character distribution of own ndings and literature data.
Tubal passage
The entrance of the zygote into the uterus at 29 hpc in
M. domestica relates well to the times given by Mate et al.
146
ZELLER and FREYER
Fig. 4. Monodelphis domestica, transmission electron micrographs of

the yolk-sac placenta. (a) 11 dpc, cytotrophoblast. The trophoblast
consists of individual cells (trc) connected by tight junctions (arrows).
Trophoblast and uterine epithelium (ue) are separated by the shell coat
(sm). fbc, foetal blood cell; me, mesenchyme; n, nucleus of trophoblast
cell; scale bar 10 lm (b) 14 dpc, syncytiotrophoblast. The trophoblast cells fused to form a syncytium (trs). Nuclei (n) are located in
syncytial knots (sl). Only thin lamellae of syncytial epithelium separate
uterine epithelium and vitelline vessels (vv). The trophoblastic syncytium penetrates the uterine epithelium (ue). me, mesenchyme; ne,
nucleated erythrocyte; scale bar 10 lm
(1994) and Selwood et al. (1997) (24 hpc and 30 hpc,

respectively). This short time for passing the oviduct (about
7 h) is equivalent to that found in Macropus eugenii (Macropodidae) with 713 h (Renfree and Lewis 1996) and Antechinus
stuartii (Dasyuridae), in which the zygote enters the uterus
12 h after fertilization (Selwood 1980). In Didelphis virginiana
(Didelphidae), the zygote enters the uterus between 15 and
24 h after ovulation (Hartman 1924; Rodger and Bedford
1982). Thus, a short tubal passage lasting 1 day at maximum
can be regarded as a character of the marsupial morphotype.
Egg coats
147
Schoinobates [Petauroides] volans: Bancroft 1973; A. pygmaeus:

Ward and Renfree 1988), which suggests that it may prevent
polyspermy in addition to the zona reaction (Selwood 1982;
Baggott and Moore 1990). In addition, Hartman (1919)
assumes a nutritive function of the mucoid coat in
D. virginiana. Furthermore, Selwood (2000) suggests that the
mucoid coat as well as the molecules that are trapped in it are
taken up by the trophoblast and may thus be a nutritional
source for the embryo. The mucoid coat is also suggested to
act as an osmotic stabiliser (Selwood 2000). The secretion of
the mucoid coat by oviductal cells as described for several
other marsupial taxa as well as for monotremes (monotremes:
Hughes and Carrick 1978; Trichosurus vulpecula: Hughes 1974;
S. crassicaudata: Roberts et al. 1994), is also observed in
M. domestica (Phillips and Fadem 1987; Baggott and Moore
1990; this article). Thus, mucoid coat secretion by oviductal
cells is a plesiomorphic character of the marsupial morphotype. Accordingly, the oviduct consists of ciliated and nonciliated secretory cells (Phillips and Fadem 1987; Baggott and
Moore 1990; this article). The secretion of mucoid coat
precursors in S. crassicaudata is shown to be restricted to the
oviduct (Roberts et al. 1994). Similar to D. virginiana (Hartman 1928), T. vulpecula (Hughes 1974), D. viverrinus (Hill
1910), Bettongia cuniculus [B. gaimardi] (Kerr 1935) and
A. pygmeus (Ward and Renfree 1988), the mucoid coat in
M. domestica disappears, with the zona pellucida, when the
blastocyst expands.
Zona pellucida and corona radiata

The oocyte of M. domestica is surrounded by the zona
pellucida (Phillips and Fadem 1987; Baggott and Moore 1990;
Selwood et al. 1997; personal observations). Phillips and Shell coat
Fadem (1987) describe the zona pellucida as 23 lm thick Although the marsupial shell coat is suggested to consist of
and lamentous. The zona pellucida, which is also found in keratin, namely ovokeratin (Hughes 1974; Krause 1998), it was
monotremes (Hughes 1984) and eutherians, is present in all impossible to verify the presence of keratin by immunohistomarsupial taxa so far examined (cf. Tyndale-Biscoe and chemistry in M. domestica. This could be due to the absence of
Renfree 1987: Didelphidae, Dasyuridae, Peramelidae, Phalan- keratin, a change of epitopes during condensation or a
geridae, Macropodidae; Ward and Renfree 1988: Acrobatidae) masking eect.
The shell coat is secreted within the uterus in all marsupial
and is, consequently, a plesiomorphic character of the marsupial morphotype. As already summarized by Tyndale-Biscoe 7 taxa so far examined (M. domestica: Baggott and Moore 1990,
and Renfree (1987), the thickness of the zona pellucida ranges this article; T. vulpecula: Hughes 1974, 1977, 1984; Hughes and
from 1 lm in Didelphis marsupialis (McCrady 1938) to Hall 1984; S. crassicaudata: Roberts and Breed 1996;
6.3 1.4 lm in M. eugenii (Hughes and Hall 1984). The A. stuartii: Selwood 1982; personal comm.; D. virginiana:
width of the zona pellucida, like that of the mucoid coat and Hartman 1916; McCrady 1938; Dasyurus, Perameles, Trishell coat, may change by pressures set up by the extrusion of chosurus, Macropus, Petrogale, Phascogale, Acrobates, Phasthe yolk mass and the rst two cleavages as observed in colarctos, Bettongia: Hill 1910; M. eugenii: Tyndale-Biscoe and
A. stuartii (Selwood and Young 1983). The disappearance of Renfree 1987). Thus, at least uterine shell coat secretion can be
the zona pellucida by expansion of the blastocyst as assumed to be a character of the marsupial morphotype.
Inconsistent data exist concerning a possible oviductal shell
we observed in M. domestica (5 dpc) is also shown for
D. virginiana (Hartman 1928) and Acrobates pygmaeus (Ward coat secretion in addition to the uterine shell coat secretion.
and Renfree 1988). Bancroft (1973) also reports the absence of Whereas Hill (1910) reports that the shell coat is laid down in
the zona pellucida at the blastocyst stage of Petauroides volans. the oviduct in Dasyurus, Perameles, Trichosurus, Macropus,
Ovulated eggs of M. domestica (Phillips and Fadem 1987; Petrogale, Phascologale [Phascogale], Acrobates, Phascolarctos
Baggott and Moore 1990; this article), D. virginiana (Krause and Bettongia, the shell coat of M. domestica (this article,
1998), Dasyurus viverrinus (Hill 1910) and Sminthopsis crassi- Baggott and Moore 1996) and T. vulpecula (Hughes 1974,
caudata (Breed and Leigh 1990) lack a corona radiata. Thus, 1977, 1984; Hughes and Hall 1984) was found to be secreted by
the absence of the corona radiata can be assumed for the uterine cells only. As Hughes (1974) points out, although
oocytes recovered from the oviduct of T. vulpecula may possess
marsupial morphotype.
a shell coat, shell glands are restricted to the uterus, suggesting
a retrograde ow of uterine shell secretions. However,
Mucoid coat
The mucoid coat of M. domestica consists of concentric layers Tyndale-Biscoe and Renfree (1987) argue that such a retro(Phillips and Fadem 1987; this article). Spermatozoa are grade ow is impossible in M. eugenii and other macropodids.
entrapped in the mucoid coat (Phillips and Fadem 1987; In S. crassicaudata, as in M. domestica, only uterine embryos
Baggott and Moore 1990; Selwood and Vandeberg 1992; this have a shell coat, whereas those recovered from the oviduct
article). This is also described for other marsupial taxa have no shell coat (Roberts and Breed 1996). Immunohisto(D. viverrinus: Hill 1910; M. eugenii: Renfree and Shaw 1996; chemically, shell coat precursors of S. crassicaudata are found
148
ZELLER and FREYER
Fig. 5. Monodelphis domestica, transmission electron micrographs of

the yolk-sac placenta,14 dpc. (a) The trophoblastic syncytium (trs)
largely invades the maternal mucosa and reaches up to uterine vessels
(uv). Between invasive trophoblast and uterine epithelium (ue) cellular
adhesions are present (arrows, inset).vv, vitelline vessels; me, mesenchyme; ug, uterine gland; scale bar (inset) 2 lm, scale bar 10 lm.
(bd) Bilaminar yolk sac wall. Trophoblastic cells (trc) are clearly
separated from each other and apically united by junctional complexes
(arrows). n, nucleus; l, lipid droplets; ue, uterine epithelium; enc,
trophendodermal cell. (b) scale bar 2.5 lm; (c) scale bar 2.5 lm;
(d) scale bar 5 lm
to be restricted to the anterior region of the uterus, but also to

the utero-tubal junction and adjacent glands (Roberts et al.
1994). In D. virginiana (Hartman 1916; McCrady 1938) and
A. stuartii (Selwood 1982; personal comm.), zygotes recovered
from the `lower part of the oviduct' were covered with shell
coat material. In the `lower oviduct' of D. virginiana, there are
numerous mucosal glands (Andersen 1928); the posibility that
they are shell glands cannot be excluded as the function of
these is unknown (Tyndale-Biscoe and Renfree 1987). It can be
assumed, that the part described as `lower oviduct' in
D. virginiana and A. stuartii corresponds to the utero-tubal
junction that was found to secrete shell coat precursors in
S. crassicaudata. Although Hill (1910) does not mention which
part of the oviduct secretes the shell coat, tubal shell secretion
can be excluded distal to the isthmus of the oviduct in
didelphids (this study; Baggott and Moore 1996), phalangerids
(Hughes 1974, 1977, 1984; Hughes and Hall 1984) and
dasyurids (Roberts and Breed 1996). Based on this distribution, shell secretion distal to the isthmus of the oviduct can be
excluded for the marsupial morphotype. Since the shell coat is
secreted by cells of the utero-tubal junction in dasyurids
(Roberts et al. 1994) and didelphids (McCrady 1938; Hartman
8 1916), this can be assumed for the marsupial morphotype as
well. In conclusion, the shell is secreted by the utero-tubal
junction and the uterus in the marsupial morphotype.
The monotreme and marsupial shell coats have been
suggested to be partly homologous (Hughes 1977). The
monotreme shell coat was found in eggs recovered from the
`lower oviduct', but it is also found to be secreted in the uterus
(Caldwell 1887). The presence of shell coat glands in the lower
third of the Fallopian tube and within the uterus (Hughes
1977) shows that this is due to both tubal and uterine shell coat
secretion. Thus, shell secretion by cells of both uterus and the
proximal oviduct (but not distal to the isthmus) may be a
plesiomorphic character of the marsupial morphotype.
Yolk arrangement and cleavage
Yolk arrangement
Our observations agree with that of Baggott and Moore (1990)
in that the peripheral yolk-poor cytoplasm, which is the
formative cytoplasm, surrounds the central yolky cytoplasm
(deutoplasm) in the unfertilized oocyte of M. domestica. As
this arrangement is also found in unfertilized oocytes of
D. virginiana (Krause 1998), S. crassicaudata (Breed and Leigh
1990) and A. stuartii (Selwood and Young 1983), this can be
concluded to be a character of the marsupial morphotype. In
M. domestica, we additionally observed a central portion of
yolk-poor cytoplasm enclosed in the deutoplasm. The yolk
bodies of compound yolk and lipid yolk in D. virginiana, as
Krause (1998) points out, move toward the peripheral cyto-
149
plasm. This probably leads to the three-layer arrangement of

central and peripheral yolk-free cytoplasm and subperipheral
yolky cytoplasm (Hartman 1916; McCrady 1938).
In the fertilized oocyte (zygote), the yolky cytoplasm is polaroriented in M. domestica (Baggott and Moore 1990; Selwood
and Vandeberg 1992; Breed et al. 1994; Selwood et al. 1997;
Zeller 1999a), D. viverrinus (Hill 1910), S. crassicaudata (Breed
and Leigh 1990; Selwood and VandeBerg 1992), A. stuartii
(Selwood 1982; Selwood and Sathananthan 1988), Phascolarctos cinereus (Caldwell 1887) and T. vulpecula (Frankenberg and
Selwood 1998). Thus, polarity of the zygotic yolk is concluded
to be a character of the marsupial morphotype. The polarized
features in the zygote are suggested to identify the future
embryonic and abembryonic poles (Selwood 1994); the side of
the pronuclei is suggested to be the embryonic hemisphere,
whereas the yolky cytoplasm is located in the abembryonic pole
(Frankenberg and Selwood 1998). Although the yolk mass
becomes eccentric prior to ovulation in dasyurids (D. viverrinus:
Hill 1910; S. crassicaudata: Breed and Leigh 1990) an
P. cinereus (Caldwell 1887), fertilization enhances the prior
polarity (cf. Merry et al. 1995). The zygote of D. virginiana
shows no yolk polarity (McCrady 1938; Hartman 1916), but
lipid droplets can be located more on one pole (Hartman 1916;
McCrady 1938). However, Hartman (1916) and Hill (1918)
agree that àn inherent, if nonvisible, polarity probably does
exist in the unsegmented ovum' (Hill 1918; p. 105). Although the
pronuclei are rst eccentrically located (Hartman 1916), they
later migrate to the centre (Hartman 1916; McCrady 1938). The
non-polarized yolk arrangement of the zygote is a derived
character of D. virginiana with regard to the proposed marsupial
morphotype.
Cleavage
In all marsupials so far examined cleavage starts in the uterus
(Selwood 2000), probably due to the shortness of the tubal
passage. This can also be assumed for the marsupial
morphotype.
In M. domestica, the rst cleavage is meridional and
accompanied by segmentation of most of the yolk and subsequent extrusion of smaller yolk vesicles (Baggott and Moore
1990; Fromm and Zeller 1997; Selwood et al. 1997). The
subsequent two-cell stage (4653 hpc: Selwood et al. 1997) is
characterized by apocrine extrusion of further yolk vesicles
(Fromm and Zeller 1997). The second cleavage is asynchronous, meridional (Selwood et al. 1997; Fromm and Zeller
unpublished) and sometimes latitudinal (Baggott and Moore
1990; Selwood and Vandeberg 1992; Selwood et al. 1997)
depending on the amount of yolk. This cleavage is accompanied by further yolk extrusion (Baggott and Moore 1990;
Selwood and Vandeberg 1992; Selwood et al. 1997; Fromm and
Zeller unpublished). Separated yolk and deutoplasmolysis are
also found in D. virginiana (Hartman 1916, 1928), dasyurids
(D. viverrinus: Hill 1910; A. stuartii: Selwood and Young 1983;
S. crassicaudata: Selwood 1987), phalangerids (T. vulpecula:
Frankenberg and Selwood 1998) and acrobatids (A. pygmaeus:
Ward and Renfree 1988). Consequently, a cleavage including
deutoplasmolysis is a character of the marsupial morphotype.
The absence of any separated yolk in the zygote of macropodids (grey kangaroos, M. eugenii: Renfree and Lewis 1996)
and the correlated cleavage without deutoplasmolysis is a
derived character of this group and may be a synapomorphy
of macropodids. The mode of deutoplasmolysis is the same
in M. domestica (Didelphidae) and dasyurids (Sminthopsis
150
macroura, S. crassicaudata, A. stuartii). In these taxa, most of
the deutoplasm (yolk) is separated o at one pole during the
rst cleavage, lesser quantities are subsequently extruded as
numerous vesicles during the rst two cleavages (Selwood 1982;
Selwood 1987; Baggott and Moore 1990; Selwood and VandeBerg 1992; Fromm and Zeller unpublished). En bloc segmentation of deutoplasm (yolk) during the rst cleavage is also
described for D. viverrinus (Hill 1910). Based on character
distribution, this mode is a character of the marsupial
morphotype. Didelphis virginiana (Hartman 1916, 1919;
McCrady 1938) and D. marsupialis (Hill 1918) show no
localized en bloc segmentation of deutoplasm (yolk). Instead,
deutoplasm is separated o by generalized extrusion of vesicles.
This is a synapomorphy of D. virginiana and D. marsupialis
with regard to the proposed marsupial morphotype.
9 As pointed out by Selwood (1982), yolk quantity, yolk
polarization and the mode of deutoplasmolysis may be
correlated. This correlation is based on the assumption that
oocyte dimension is determined by the amount of yolk. Then,
large oocytes such as those of D. viverrinus (Hill 1910),
S. crassicaudata and S. macroura (Selwood 1987) with 240,
254 and 343 lm diameter, respectively, contain great yolk
quantities. Medium-sized oocytes of A. stuartii (Selwood and
Young 1983) and M. domestica (this investigation) with 158
and 170 lm contain medium amounts of yolk. Small eggs, as
found in D. virginiana (Hartman 1916) and D. marsupialis (Hill
1918) with 135165 and 144 lm, respectively, are poor in yolk.
Early yolk polarization, i.e. before fertilization, was found in
species with yolk-rich oocytes, namely Dasyurus and Sminthopsis. In those species, the morphotype mode of deutoplasmolysis was found. The same applies to species with oocytes with a
medium amount of yolk (A. stuartii, M. domestica). Both types
dier in the time of yolk polarization. In yolk-rich oocytes,
polarization rst occurs in the ovary oocyte (Hill 1910; Breed
and Leigh 1990), whereas in oocytes with a medium amount of
yolk, polarization is only found at the pronuclear stage (this
paper; Selwood 1982, 1992). The yolk-poor oocytes of
D. virginiana and D. marsupialis do not exhibit yolk polarity
and the deutoplasmolysis is reduced to a generalized extrusion of vesicles. The smallest oocytes, 126 lm in diameter
(M. eugenii: Tyndale-Biscoe and Renfree 1987), are found
among macropodids which lack a separated deutoplasm (yolk).
In M. domestica, the asynchronous, third (Baggott and
Moore 1990; Selwood and Vandeberg 1992; Selwood et al.
1997; Fromm and Zeller unpublished) and fourth cleavage
(Baggott and Moore 1990; Selwood and Vandeberg 1992; this
paper) are latitudinal or meridional, the fth cleavage was
found to be latitudinal (Selwood and Vandeberg 1992). The
sequence of meridional and latitudinal cleavages leads to a
blastocyst at 6 dpc (this paper). At this stage, the blastocoel
contains little yolk (this paper; Fromm and Zeller unpublished). We assume that the yolk has been taken up by
blastomeres.
In M. domestica, the cells are closely apposed to the zona
pellucida at the two-cell stage (Fromm and Zeller unpublished)
and eight-cell stage (Baggott and Moore 1990; Selwood et al.
1997; Fromm and Zeller unpublished), whereas no cell contact
is established. Baggott and Moore (1990) report rst intercellular junctions between blastomeres at the 16-cell stage in
M. domestica. Apposition of blastomeres to the zona pellucida
prior to intercellular association and connection by tight
junctions was also found in A. stuartii (third + fourth
cleavage, Selwood and Young 1983), S. crassicaudata
ZELLER and FREYER

(second cleavage, Selwood et al. 1997) and T. vulpecula
(second cleavage, Frankenberg and Selwood 1998). In
M. eugenii, the blastomeres are in close association with one
another during the rst cleavages, but no tight junctions are
present at least until the eight-cell stage is reached (Renfree
and Lewis 1996). This is probably a derived character
of macropodids. This could be correlated with the absence of
separated yolk in the zygote (see above). The apposition of
blastomeres to the zona pellucida prior to intercellular
association, and the consequent absence of a morula stage
(Selwood 1996) is a character of the marsupial morphotype,
since this is present in ameridelphian didelphids, as well as in
australidelphian dasyurids and phalangerids.
Placentation
Our observations agree with that of Harder et al. (1993)
concerning both the close apposition of the trophectoderm to
the uterus at 13 and 14 dpc and the microvillous surface of the
endo- and trophectoderm in M. domestica. Our observed
increase of foldings at 12 dpc is conrmed by the increase of
the index of epithelial surface area from 1.66 at 9 dpc to 6.9 at
13 dpc measured by Harder et al. (1993). We can further
conrm, that M. domestica possesses a choriovitelline placenta
(Harder et al. 1993; Roberts and Breed 1994a) during the
entire development from implantation to birth. The observed
drop in uterine gland density in M. domestica between 9 and 14
dpc (Harder et al. 1993) can be correlated with the decreasing
thickness of the uterine gland pad from 3 mm at 10 dpc,
1.5 mm at 12 dpc to 0.20.4 mm at 14 dpc. This indicates the
shift from histiotrophic to haemotrophic nutrition of the
embryo.
Invasiveness of the placenta
Occurrence and degree of invasion of uterine epithelium by the
trophectoderm of the bilaminar and trilaminar yolk sac varies
among marsupials as summarized in Table 4.
Contrary to earlier reports given by Harder et al. (1993) and
Roberts and Breed (1994a), who report the trophectoderm of
M. domestica to be non-invasive, our electron-microscopic
observations clearly demonstrate that the trophoblastic syncytium of the trilaminar yolk sac penetrates the uterine epithelium and is thus invasive (Fig. 3e, Fig. 4b). An invasive
trophectoderm in a didelphid was only known for Philander
opossum (Enders and Enders 1969).
The variation between invasive and non-invasive trophectoderm among closely related species may be explained by
either interspecic variation or a lack of information. The
latter suggestion is supported by the ndings of an invasive
trophectoderm in M. domestica, which was formerly described
as possessing a non-invasive trophectoderm. Thus, this character needs to be re-examined during the entire ontogeny from
implantation to birth, and by EM.
Formation of an allantochorion and yolk sac placenta
The dierent conditions of the allantois relative to the chorion
found in dierent marsupial taxa were often mentioned and
grouped into four placental types (Sharman 1959; Hughes
1984; Tyndale-Biscoe and Renfree 1987), which are summarized in Table 5.
However, it must be kept in mind that this classication is
an articial grouping of dierent characters, namely the
contact between allantois and chorion, the size of allantois
151
Table 4. Distribution of invasive bilaminar and trilaminar yolk sac areas among marsupials
Taxon
Didelphidae
Dasyuridae
Peramelidae
Phalangeridae
Pseudocheiridae
Acrobatidae
Macropodoidea
Vombatiformes
Species
Monodelphis domestica
Philander opossum
Didelphis virginiana
Didelphis marsupialis
Dasyurus viverrinus
Sminthopsis crassicaudata
Isoodon obesulus
Perameles gunnii
Perameles nasuta
Echymipera rufescens
Echymipera kalubu
Trichosurus vulpecula
Pseudocheirus peregrinus
Petauroides volans
Distoechurus pennatus
Potorous tridactylus
Macropus robustus
Macropus giganteus
Macropus rufogrisea
Setonix brachyurus
Macropus eugenii
Bettongia cuniculus
Phascolarctos cinereus
Vombatus ursinus
Bilaminar (avascular) yolk sac

1
non-invasive
non-invasive3
non-invasive1,4,5
non-invasive1,4,5
invasive6
invasive7,8
non-invasive9,10,11
non-invasive11
non-invasive7,12
non-invasive7
non-invasive13
non-invasive7,12,15
non-invasive12,16
non-invasive14
invasive17
non-invasive12,15
non-invasive7
non-invasive18
non-invasive15
non-invasive15,19
non-invasive20
non-invasive21
invasive7,12,22
invasive7
Trilaminar (vascular) yolk sac

invasive2
invasive3
non-invasive1,4,5
non-invasive1,4,5
non-invasive6
non-invasive8
non-invasive9,10,11
non-invasive11
non-invasive7,12
non-invasive7
non-invasive13
non-invasive7,12,15
non-invasive12,15,16
invasive14
non-invasive17
non-invasive12,15
non-invasive7
non-invasive18
non-invasive15
non-invasive15,19
non-invasive20
invasive21
non-invasive7,22
non-invasive7
Source: 1 Harder et al. (1993); 2 this paper; 3 Enders and Enders (1969); 4 Hartman (1923); 5 McCrady (1938); 6 Hill (1900a); 7 Hughes (1974);
8
Roberts and Breed (1994a); 9 Hill (1895); 10 Hill (1898); 11 Flynn (1923); 12 Hughes and McNally (1968); 13 Hughes et al. (1990); 14 Bancroft
(1973); 15 Sharman (1961); 16 Hughes et al. (1965); 17 Hughes et al. (1987); 18 Chapman (1882); 19 Sharman (1959); 20 Tyndale-Biscoe and Renfree
(1987); 21 Flynn (1930); 22 Caldwell (1884).
and yolk sac, their vascularization and the function of both

allantois and yolk sac. This is the reason why a character
polarization based on these types has failed. For a phylogenetic interpretation of these ndings, a polarization of
homologous characters is needed. To date, the insucient
and inconsistent data concerning the allantochorion in
marsupials can only be polarized by comparison with the
sistergroup (eutherians) of marsupials and the outgroup of
the Theria (monotremes). Since a fusion between chorion and
allantois was found in Dasyuridae, Vombatiformes and
Perameloidea, and this is a character also found in eutherians
(Wooding and Flint 1994) as well as in monotremes (Caldwell

1887), this may be a plesiomorphic character of the marsupial
morphotype. Thus, the absence of a chorioallantois in
didelphids on one side and australidelphian taxa (acrobatids,
macropodids, phalangerids) on the other side is a convergent
reduction. The parsimony of this assumption, however, is
weakened by the absence of an allantochorion in the dasyurid
S. crassicaudata (Roberts and Breed 1994a). In a morphotype
reconstruction using parsimony (see Cunningham et al.
1998), the downpass reconstructed marsupial morphotype
remains ambiguous, but the uppass reconstruction suggests an
Table 5. Types of marsupial placentation according to Tyndale-Biscoe and Renfree (1987)

Type
Description
Taxon
Type I
Allantois remains small and does not fuse with the chorion, only the well vascularized
yolk sac contributes to the placenta
Didelphidae14, Phalangeridae1,
Type II
Allantois reaches the chorion and seems partly to fuse with the chorion, but allantoic
vessels degenerate, later in ontogeny the allantois retreats and degenerates;
assumption: the allantochorion does not form a placenta
Dasyurus viverrinus6
Type III
Allantois fuses with chorion, neither mesodermal nor ectodermal chorionic villi at the
side of the allantochorion, neither invasion of uterine epithelium nor fusion of the
uterine epithelium with the allantochorion, no replacement of the yolk sac placenta;
assumption: yolk sac is the main organ of nutritive absorption, allantochorion
functions principally as a respiratory organ
Phascolarctos cinereus1,710
Vombatus ursinus10
Type IV
Allantois fuses with chorion, no mesodermal villi at the side of the allantochorion,
ectodermal villi at the side of the allantochorion, fusion of uterine epithelium and
trophoblast of the allantochorion, no replacement of yolk sac placenta; assumption:
allantochorion forms a placenta additionally to the yolk sac placenta
Peramelidae1,1116
Macropodidae1, Acrobatidae5
Source: 1 Tyndale-Biscoe and Renfree (1987); 2 Harder et al. (1993); 3 Roberts and Breed (1994a); 4 this paper; 5 Hughes et al. (1987); 6 Hill
(1900a); 7 Caldwell (1884); 8 Semon (1894); 9 Hughes (1974); 10 Hughes (1984); 11 Hill (1895); 12 Hill (1898); 13 Hill (1900b); 14 Flynn (1923);
15
Padykula and Taylor (1976); 16 Padykula and Taylor (1982).
152
ZELLER and FREYER
Fig. 6. Reconstruction of the

allantochorion in the marsupial
morphotype using parsimony as
described by Cunningham et al.
(1998)
allantochorion to be present in the marsupial morphotype

(Fig. 6). The absence of an allantochorion in phalangeroids
(phalangerids, acrobatids) and macropodids may thus be
synapomorphic. Since the allantochorion is vascularized in
monotremes (Caldwell 1887), placentals (Wooding and Flint
1994), peramelids and vombatiforms, it is to date parsimonious to assume a reduction of this vascularization in
D. viverrinus derived from a vascularized allantochorion in
the marsupial morphotype. This preliminary polarization,
however, demands a re-examination of recent ndings, the
examination of the ontogeny from implantation until birth in
further species and the re-evaluation of marsupial systematics,
which are in progress (Freyer, Zeller and Renfree in prep.).
We are aware that our hypothesis appears to contradict with
the interpretation of Luckett (1977; p. 490), who assumes a
`retardation in expansion of the exocoelom and allantois' in
the marsupial morphotype.
Although the chorioallantoic placenta in marsupials never
forms mesodermal villi and never replaces the yolk sac
placenta as that of eutherians (Luckett 1977), it can be
assumed that the allantochorion in vombatiforms and peramelids serves at least respiratory function (Luckett 1977;
Semon 1894; Hughes 1984; Tyndale-Biscoe and Renfree 1987).
A respiratory function of the vascularized allantochorion in
the marsupial stem species seems likely. However, it is
unknown whether this allantochorion formed a placenta
which also allowed the exchange of material.
All marsupials so far examined possess a yolk sac placenta
for most of the time after implantation (cf. Tyndale-Biscoe and
Renfree 1987). Thus, a yolk sac placenta can be assumed for
part of the ontogeny in the marsupial morphotype (Luckett
1977), irrespective of the formation of an allantois placenta at
near-term stages.
Formation of a trophoblastic syncytium

Zeller (1999a) provides the rst evidence of a trophoblastic
syncytium ever observed in a marsupial by EM. Here, we give
a detailed description of the ultrastructure of this syncytium by
presenting original electron micrographs. We assume that the
aggregations of nuclei in plasma accumulations are syncytial
knots rather than plasmodia, i.e. nuclear division. This
assumption is based on the constant nuclear sizes from 11 to
14 dpc (Fig. 4a versus 4b), the absence of evidence on nuclear
divisions at any stage, and the great extension of the syncytial
parts relative to former cell sizes and regular cell arrangements
with tight junctions between single cells (Fig. 4a versus 4b).
Hill (1900) reported a trophoblastic syncytium in D. viverrinus
by LM. As the cell boundaries can hardly be seen by LM, this
description needed to be veried by EM. However, the electron
microscopic ndings in M. domestica presented herein make a
syncytium in D. viverrinus highly probable. There is no report
of fusions between cells of the trophoblast or extended parts of
it in any other marsupial species. If further examinations
conrmed a trophoblastic syncytium to be present in
D. viverrinus, a trophoblastic syncytium could be assumed
for the marsupial morphotype.
The description of trophoblastic giant cells in M. domestica by
Roberts and Breed (1994a) may represent syncytial knots of the
trophoblastic syncytium not identied by the authors. The
question whether the trophoblastic giant cells as observed in
other marsupial species, e.g. S. crassicaudata (Roberts and
Breed 1994a), are syncytical knots can only be answered
through further examination.
Formation of a feto-maternal syncytium
Until now, there has been no evidence for a foeto-maternal
syncytium in M. domestica. A foeto-maternal syncytium is

known for the bilaminar yolk sac of Dasyurus sp. and
P. cinereus (Flynn 1923). In S. crassicaudata, cells of the
trilaminar omphalopleure partly fuse with uterine epithelial
153
cells (Roberts and Breed 1994b). In peramelids (Isoodon

macrourus), the trophoblast of the chorioallantois `may
have fused with maternal homokaryons to create placental
Table 6. Characters of the marsupial morphotype (0) and apomorphic characters (1)
Character
no.
1
3
4
5
Character state 0
(morphotype)
Central yolky cytoplasm
and peripheral yolkpoor cytoplasm in the
unfertilized egg
Zona pellucida
Absence of a corona
radiata
Cell apposition to zona
pellucida prior to
intercellular connection
Mucoid coat
Spermatozoa trapped in
the mucoid coat
Oviductal mucoid
secretion
Shell coat
9a
Uterine shell coat

secretion
9b
No oviductal shell coat

secretion distal to the
isthmus
Short tubal passage
(37 h)
10
11
Separated yolk in zygote
12
Yolk polarity in the

zygote
13
No morula stage
14
Localized segmentation of
deutoplasm at the rst
cleavage and subsequent
extrusion of yolk vesicles
during the rst two
cleavage stages
Taxon
Character state 1
(apomorphy)
Taxon
Didelphidae;
Dasyuridae
Didelphidae;
Dasyuridae;
Macropodidae;
Peramelidae;
Phalangeridae;
Acrobatidae
Didelphidae;
Dasyuridae
Didelphidae;
Dasyuridae;
Phalangeridae
Didelphidae;
Dasyuridae;
Macropodiae;
Pseudocheiridae;
Acrobatidae;
Phalangeridae
Didelphidae;
Dasyuridae;
Acrobatidae;
Pseudocheiridae
Didelphidae;
Dasyuridae;
Phalangeridae
Didelphidae;
Dasyuridae;
Phalangeridae;
Peramelidae;
Macropodidae;
Acrobatidae
Didelphidae;
Dasyuridae;
Phalangeridae;
Peramelidae;
Macropodidae;
Acrobatidae
Didelphidae;
Dasyuridae;
Phalangeridae
Didelphidae;
Dasyuridae;
Macropodidae;
Didelphidae;
Dasyuridae;
Acrobatidae;
Didelphidae;
Dasyuridae;
Phascolarctidae;
Phalangeridae
Didelphidae;
Dasyuridae;
Macropodidae
Monodelphis
domestica;
Dasyuridae;
Acrobatidae
Character not
applicable [?]
Taxon
No separated
yolk in oocyte
Macropodidae
Intercellular association Macropus eugenii

of blastomeres at
early cleavage stages
No separated yolk in
zygote
Macropodidae
Non-polarized yolk
arrangement in the
zygote
Didelphis
virginiana
No separated
yolk in zygote
Macropodidae
No localized
segmentation of
deutoplasm;
generalized extrusion
of yolk vesicles
Didelphis
virginiana;
Didelphis
marsupialis
No separated
yolk in zygote
Macropodidae
154
heterokaryons' (Padykula and Taylor 1982; p.95). This data
base is insucient for a morphotype reconstruction.
Parturition
The timing of parturition in M. domestica determined to be 15
dpc in this study equals that of 15.2 0.3 (n 16) found by
Harder et al. (1993) and almost matches that of 13.514 days
after fertilization found by Moore (1992).
At the time of parturition, the propria of M. domestica was
found to be highly invaded by polymorphonuclear granulocytes,
macrophages and lymphocytes (Fig. 3f). Granulocytes are also
found in the glandular tubes. In a single specimen of S. crassicaudata, which had given birth 37 h before the time of death,
`the nuclei of the trophoblast giant cells had become pyknotic
and there was a massive inltration of granular leucocytes and
lymphocytes into the endometrial stroma' (Roberts and Breed
10 1994a, p.109). In late pregnancy (CRL 2.93.5 mm), scattered
neutrophils, lymphocytes and macrophages were found in the
endometrial stroma of S. crassicaudata, `particularly below the
invasive trophoblast giant cells' (Roberts and Breed 1994a;
p. 108). Although Cruz and Selwood (1993) report that
lymphocytes are clustered in a dense mat in the uterine stroma
adjacent to the basal lamina of the endometrial epithelium in
both pregnant and nonpregnant specimens of A. stuartii, it is
striking that the number of lymphocytes was found to be
signicantly greater in pregnant than non-pregnant animals on
the rst and 15th day after ovulation. In addition, the lymphocyte density decreases in non-pregnant animals, but increases in
pregnant animals from the fourth to the eighth day after
ZELLER and FREYER

ovulation. An inltration of the placenta by polymorphonuclear
leucocytes is also reported for two specimens of Perameles
obesula [Isoodon obesulus] (Flynn 1923). Whether this inammatory reactions is in any way related to birth is still unknown.
Conclusions
This study is a contribution to the reconstruction of the
morphotype of recent marsupials by providing current ndings, a character polarization and a phylogenetic interpretation. This is based on marsupial systematics as proposed by
Luckett (1994).
Our data support most of the previous examinations on
the early ontogeny and placentation of M. domestica. We
describe both the ultrastructure of a trophoblastic syncytium
and an invasive trophoblast for the rst time in M.
domestica by presenting original electron micrographs.
Table 6 summarizes characters that are concluded to be
present in the marsupial morphotype and those that are
derived, based on the character distributions within marsupials (Fig. 7).
To date, polarization of most characters of the marsupial
placenta is impossible due to insucient and incongruent data.
Obviously, a yolk sac placenta is established at early stages of
placentation in the marsupial morphotype. In a preliminary
parsimony analysis, a vascularized allantochorion can be
assumed for the marsupial morphotype. The contribution of
this allantochorion to placentation in the marsupial morphotype has not yet been established.
Fig. 7. Reconstruction of the early

ontogeny in the marsupial morphotype using parsimony as described by Cunningham et al.
(1998). For character coding see
Table 6. Numbers above rectangle
are character numbers; the numbers below rectangle are the character states

The early ontogeny and placentation in the stem species of
recent marsupials may have occurred as follows: When
released from the oviduct, the oocyte was covered by the zona
pellucida and a corona radiata was lacking. The yolky
cytoplasm was centrally located and surrounded by the
formative, yolk-poor cytoplasm. Fertilization occurred in the
oviduct. Prior to or shortly after fertilization, the yolky
cytoplasm migrated to one pole (abembryonic pole) opposite
the formative cytoplasm (embryonic pole). During the passage
down the oviduct, the oocyte became surrounded by the
mucoid coat, which was secreted by non-ciliated oviductal
cells. The remaining spermatozoa were entrapped into the
mucoid coat, preventing polyspermy. The mucoid coat also
prevented direct contact between the oocyte and the tubal
epithelium and may also have functioned as an osmotic
stabilizer. With the assistance of ciliated oviductal cells, the
conceptus reached the uterus within 1 day after ovulation.
There was no cleavage during the tubal passage. Cells of both
the utero-tubal junction and uterus secreted shell coat precursors, which polymerized into the shell coat on the mucoid coat.
The shell coat may have consisted of keratin which diered in
its chemical characteristics to known human keratins. The
meshes of the shell coat could be passed by transferrin,
nutrients and gases, but it also prevented direct contact of the
conceptus with the uterine epithelium. The cleavage of the
zygotes that contained moderate to large amounts of yolk
started in the uterus. Most yolk was separated o at one pole
during the rst cleavage. Lesser quantities were subsequently
extruded during the rst two cleavages. A sequence of
equatorial and latitudinal cleavages lead to the unilaminar
blastocyst. Blastomerezona adhesions preceded blastomere
blastomere connections. Thus, a morula stage was omitted.
Subsequently, the yolk was reabsorbed by the blastomeres
and the blastocyst expanded. The zona pellucida and the
mucoid coat disappeared during this process. The uptake of
mucoid coat and entrapped molecules may have served as a
nutritional source for the embryo. Before implantation the
embryo was nourished by secretions (histiotrophe) of the
uterine glands. Within the last third of pregnancy, the shell
coat broke down and the trophectoderm apposed to the
uterine epithelium. In certain areas the trophoblast may have
penetrated the uterine epithelium. Early at implantation, the
placenta was solely formed by the yolk sac. Histiotrophic
nutrition may have been shifted toward an exchange of
material and gases between foetal and maternal vessels
(haemotrophic nutrition). Later in ontogeny, a vascularized
allantochorion was formed, but did not replace the yolk sac
placenta. It is unknown, whether this allantochorion served
material transfer beside its respiratory function.
We regard the reconstruction of the marsupial morphotype
as a rst step toward an evolutionary (holistic) scenario that
explains mammalian evolution arround the Cretaceous
Tertiary boundary.
155
We thank Mrs. J. Zeller for technical assistance. We are indebted to Dr

M. Ade, Dr S. Frahnert and Dr A. Mess for critical comments and
fruitful discussions. We especially thank Dipl. Biol. P. Giere for his
assistance with linguistic corrections. We thank Dr P. Luckett, Dr L.
Selwood, Dr M. B. Renfree and Dr G. Shaw for critical comments and
fruitful discussions. We especially thank Dr M. B. Renfree for her kind
permission to use her research facilities during Dr Zeller' s stay there
from 316 October 2000.
Zusammenfassung
Fruhe Ontogenie und Plazentation der grauen Hausspitzmausbeutelratte,
Monodelphis domestica (Didelphidae: Marsupialia): Ein Beitrag zur
Rekonstruktion des Grundplans der Marsupialia
Die vorliegende Arbeit beschreibt die fruhe Ontogenese und Plazentation von 3 Stunden nach der Kopulation bis zur Geburt der
Beutelratte Monodelphis domestica. Es wird die Ultrastruktur und
Histochemie der Eihaute, der Fetalmembranen, des Oviductes und des
Uterus beschrieben. Erstmalig wird die Ultrastruktur eines trophoblastischen Syncytiums bei einem Beuteltier beschrieben. Weiterhin
wird ein invasives Trophektoderm und eine Entzundungsreaktion zum
Zeitpunkt der Geburt bei M. domestica festgestellt. Die Befunde dieser
Studie und Literaturdaten werden verglichen und in eine Grundplanrekonstruktion integriert. Merkmale, die mindestens zwischen
Vertretern der Didelphidae und Dasyuridae ubereinstimmen, werden
basierend auf dem phylogenetischen System der Marsupialia nach
Luckett, J. Mammal. Evol. 2, 255283, 1994, fur den Grundplan der
Marsupialia angenommen. Diese Merkmale sind zentral separierter
Dotter und peripheres dotterarmes Zytoplasma in der unbefruchteten
Eizelle, das Vorhandensein von Zona pellucida, Mucoidschicht und
Schalenhaut, das Fehlen einer Corona radiata, die Mucoidsekretion
durch den Oviduct, die Schalensekretion durch den Uterus und nicht
distal der Isthmusregion des Oviductes, eine kurze Tubenwanderung
(maximal einen Tag), die Anlagerung der Blastomeren an die Zona
pellucida vor der interzellularen Verbindung, das Fehlen eines
Morulastadiums, die Dotterpolaritat in der Zygote, die lokale Dotterabtrennung bei der ersten Teilung und die anschlieende Dotterextrusion wahrend der ersten beiden Teilungen. In Bezug auf den
Grundplan der Marsupialia ist die unpolare Dotterverteilung in der
Zygote ein abgeleitetes Merkmal von Didelphis virginiana. Didelphis
virginiana und Didelphis marsupialis teilen als Synapomorphie die
Reduktion der Deutoplasmolyse auf eine generelle Vesikelextrusion.
Das Fehlen separierten Dotters in der Oocyte und die resultierende
Furchung ohne Dotterextrusion [Renfree and Lewis, Reprod. Fert.
Dev. 8, 725742, 1996] ist eine Apomorphie der Macropodidae.
Hiermit hangt moglicherweise die fruhe Zusammenlagerung der
Blastomeren zusammen [Renfree and Lewis, Reprod. Fert. Dev. 8,
725742, 1996]. Ein vaskularisiertes Allantochorion und eine Dottersackplazenta konnen fur einen Teil der Ontogenese im Grundplan der
Marsupialia angenommen werden. Ob das Allantochorion neben der
Respiration auch dem Stoaustausch diente ist unklar. Die Lesrichtung fur den Modus der Plazentation und der Geburt bedarf weiterer
Untersuchungen.
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J. Zool. Syst. Evol.

Research 39 (2001) 137158

Received on 15 February 2000

Museum fur Naturkunde, Institut fur Systematische Zoologie, Berlin, Germany

Early ontogeny and placentation of the grey short-tailed opossum, Monodelphis

U.S. Copyright Clearance Center Code Statement:

Luckett (1994), marsupials can be divided into the monophyletic

Small, less yolk bodies7,8

Large at ovulation, many yolk bodies7,8

Thinner than in placentals2,9 : 1108.611 lm

Including deutoplasmolysis that releases material as membrane-bound

Absent (direct blastocyst formation)2,16,18,20

Shell coat, mucoid coat9,25

Seldom involving the allantois2,15,2729, never mesodermal villi

Always longer than intrauterine development: 50 days (Monodelphis,

Usually aected by pregnancy37

Can be shorter than intrauterine development2: 5 days334.5 years34

Long intrauterine development possible2: 1656686 days

Precocial relative to marsupial neonates, never xed to nipple2

Table 1. Characters of reproduction in placentals and marsupials

Early ontogeny and placentation of the grey short-tailed opossum

acrylate (Kulzer). Specimens embedded in paraplast were sectioned at

evolutionary scenario that explains the mammalian evolution

Materials and methods

Monodelphis domestica was bred in a colony established at the Institute

ZELLER and FREYER

Table 2. Female Monodelphis domestica investigated in this study

shared in descendant nodes, assign the union of descendant's states to

Monodelphis domestica, Didelphis marsupialis, Didelphis virginiana, Philander opossum

Table 3. Species mentioned in this

Early ontogeny and placentation of the grey short-tailed opossum

reactive to PAS. Pseudopodia-shaped processes of mucoid

ZELLER and FREYER

Early ontogeny and placentation of the grey short-tailed opossum

(Fig. 3a). They are connected by apical junctional complexes,

ZELLER and FREYER

Early ontogeny and placentation of the grey short-tailed opossum

By 13 dpc (8.5 mm CRL), the neural tube is closed,

rare. The trophectoderm is in close association with the uterine

ZELLER and FREYER

Early ontogeny and placentation of the grey short-tailed opossum

Fig. 4. Monodelphis domestica, transmission electron micrographs of

(1994) and Selwood et al. (1997) (24 hpc and 30 hpc,

Schoinobates [Petauroides] volans: Bancroft 1973; A. pygmaeus:

Zona pellucida and corona radiata

ZELLER and FREYER

Early ontogeny and placentation of the grey short-tailed opossum

Fig. 5. Monodelphis domestica, transmission electron micrographs of

to be restricted to the anterior region of the uterus, but also to

plasm. This probably leads to the three-layer arrangement of

ZELLER and FREYER

Early ontogeny and placentation of the grey short-tailed opossum

Bilaminar (avascular) yolk sac

Trilaminar (vascular) yolk sac

and yolk sac, their vascularization and the function of both

(Wooding and Flint 1994) as well as in monotremes (Caldwell

Table 5. Types of marsupial placentation according to Tyndale-Biscoe and Renfree (1987)

ZELLER and FREYER

Fig. 6. Reconstruction of the

allantochorion to be present in the marsupial morphotype

Formation of a trophoblastic syncytium

Early ontogeny and placentation of the grey short-tailed opossum

cells (Roberts and Breed 1994b). In peramelids (Isoodon

Uterine shell coat