Placentation in the dasyurid marsupial, Sminthopsis crassicaudata, the fat-tailed dunnart, and notes on placentation of the didelphid, Monodelphis domestica C. T. Roberts and W. G. Breed Department of Anatomy and Histology, University of Adelaide, South Australia 5006, Australia

A light microscope study of the choriovitelline (yolk sac) placenta of the dasyurid marsupial, Sminthopsis crassicaudata, and some comparative observations on that of the didelphid, Monodelphis domestica, were performed. In the former species, the placenta was composed of an invasive bilaminar, avascular, yolk sac and a non-invasive trilaminar, vascular yolk sac. The bilaminar yolk sac placenta had trophoblast giant cells that eroded the maternal epithelium, but there was no evidence of invasion of maternal capillaries; thus, an endotheliochorial placenta was present. In the trilaminar yolk sac placenta, the convoluted chorion followed the contours of the highly folded endometrial epithelium but did not erode it and, therefore, an epitheliochorial placenta was formed. In late pregnancy, the chorio\x=req-\ vitelline placenta of Monodelphis domestica also had two regions, but the fetal trophoblast did not invade the uterine epithelium in either region. Rather, there were discontinuous areas of adhesion between trophoblast giant cells and uterine epithelium in the trilaminar yolk sac placenta and some extensive areas of adhesion in the attenuated bilaminar yolk sac placenta. The yolk sac placenta in M. domestica, unlike that of S. crassicaudata, therefore appears to be epitheliochorial in the vascular and non-vascular regions.

Introduction of vascular and non-vascular regions; most marsupials do not subsequently develop a chorioallantoic placenta. In some Marsupials have a comparatively short gestation that generally species, the trophoblast erodes the uterine epithelium, for occurs within one oestrous cycle and, often, within the luteal example, in the dasyurids Dasyurus viverrinus and Sminthopsis phase. The shortest recorded marsupial pregnancy is that of crassicaudata (Hill, 1900; Hughes, 1974), the didelphid, Philander Sminthopsis macroura which lasts for only 9.5 days (Selwood opossum (Enders and Enders, 1969), as well as in the koala, and Woolley, 1991). The fat-tailed dunnart (Sminthopsis crassi¬ Phascolarctos cinereus, and the feather-tailed glider, Distoechurus caudata) has a gestation period of 13.5 days, although sperma¬ pennatus (Hughes et al, 1987). However, the depth of invasion tozoa may be stored in the isthmus for up to three days before and the area of uterine epithelium penetrated varies among fertilization, so that birth may not occur until up to 16 days these species. In addition, the bandicoots (Peramelidae) develop after mating (Selwood, 1987; Breed et al, 1989). an invasive chorio-allantoic placenta on the final 2 days of In marsupials, early embryos reach the uterus between 12 pregnancy (Padykula and Taylor, 1976, 1982; Hughes et al, and 24 h after fertilization. They are surrounded by a mucoid 1990). coat and shell membrane that are permeable and allow the In the present study of the fat-tailed dunnart, Sminthopsis passage of gases, nutrients and waste products between the crassicaudata, the stage of embryonic development when the embryo and the uterine environment (Hughes and Shorey, shell membrane is shed is described and, subsequently, the time 1973). The embryo, surrounded by its shell membrane, does at which implantation occurs, as well as the microscopic not become attached to the uterine epithelium until about structure of the resultant placenta. Some comparative notes on two-thirds of the way through its comparatively short ges¬ the structure of the placenta of the South American didelphid tation, at which time the shell membrane is lost and implan¬ marsupial, Monodelphis domestica, are also presented. tation proceeds. There have been few studies on marsupials on the embryonic—maternal cell associations at the time of implan¬ Materials and Methods tation, other than to determine whether the trophoblast is invasive (Hughes, 1974). All marsupials, like most eutherians, Sminthopsis crassicaudata individuals were obtained from the develop a choriovitelline or yolk sac placenta that is composed colony housed in the Department of Genetics at the University of Adelaide, and maintained as described by Bennett et al Received 2 March 1993. (1990). Each female was paired with a fertile male, and every Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access Table 1. Stages of embryo development of Sminthopsis crassicaudata killed 11—15 days post-coitum

Spermatozoa found in Time of death urine (days) (days post-coitum) Embryonic development

1 and 2 PC 14.5 Unilaminar blastocysts3 Unknown Unknown Early bilaminar blastocysts 1 PC 14 Degenerating blastocysts 1 PC 13 Degenerating blastocysts 1 and PC 14 Late presomite, no shell membrane, implanting 1 and PC 13 2.9-3.2 mm CRL 1 PC 12 3.2 mm CRL 1PC 12 3.3 mm CRL 1 PC 12 3.0-3.5 mm CRL I PC 13.8 Neonates < 7 h old, 4.0-4.2 mm CRL

aIn this species, birth may occur between 13.5 and 16 days post-coitum; this variation is largely due to storage of spermatozoa in the isthmus for up to 3 days between mating and ovulation. PC: post-coitum; CRL: crown-rump length. morning, urine samples from each female were placed on Results microscope slides and examined for the presence of spermato¬ zoa cornified cells. The first that and vaginal epithelial day Placental structure in Sminthopsis crassicaudata spermatozoa were present in the smear was designated day 1 post-coitum and the first day of pregnancy was assumed to be Selection of animals. Of the 69 females from which vaginal the last day that spermatozoa were present in the smear, cell were obtained, 25 were found to have after this samples only although sperm storage may have occurred time in spermatozoa in the urine. The embryonic developmental some females (Selwood, 1987). Daily smears were examined stages found in animals killed 11—15 days post-coitum is from 69 females for between 4 and 29 days over a period of shown (Table 1). The apparent inconsistency of the number 4 months. of days post-coitum and the stage of embryonic develop¬ Fourteen killed an of sodium females, by i.p. injection ment was due to the variable time that spermatozoa are pentobarbitone (Nembutal, Bomac Laboratories, Sydney) stored in the isthmus of the oviduct before ovulation and 11—15 days post-coitum, gave a range of gestational ages. Uteri fertilization. from one animal of unknown that was gestational age being As the pregnancy rate was not as high as expected early used in a were also obtained. separate study in the study, females were checked for signs of pregnancy by after one uterus was dissected into Immediately death, abdominal palpation. Fourteen females were killed 11—15 and the of and crown- fixative, stage embryonic development days post-coitum, nine of which were found to be pregnant. when recorded. The other uterus was rump length, applicable, The other 11 females that had sperm-positive smears were slit to to enter the uterine and either allow fixative lumen, not killed as no evidence of pregnancy was found upon processed whole, or dissected, into separate sites of embryonic abdominal palpation. Histological sections showed that two attachment. Uteri and embryos were fixed in 3% glutaralde- of the killed, pregnant animals had degenerating blastocysts hyde : 3% made up in 0.2 mol phosphate not to term. Of 25 females with 1 paraformaldehyde that would have proceeded in a series of cleared buffer 1 ~ alcohols, (pH 7.4), dehydrated spermatozoa in the urine, the pregnancy rate was therefore in Safsolv (Ajax Chemicals, Adelaide) and embedded in paraffin only 28%. wax. Later, 7 µ serial sections were cut and stained with either haematoxylin and eosin, periodic acid : Schiff (PAS) or alcian blue at pH 1.0 or 2.5 (Drury and Wallington, 1980). Nonpregnant animals. At the time of death, 11-15 days Some tissue was post-fixed in 1% osmium tetroxide in phos¬ after spermatozoa had been found in the urine, the uteri of five phate buffer, dehydrated in a series of alcohols, cleared in animals were about 2 mm in diameter. Histology revealed that propylene oxide and embedded in TAAB TK3 resin (Taab the luminal epithelium was simple, columnar and unfolded and Laboratories, Berkshire). Subsequently, 0.5 pm thick plastic overlay a stroma with little extracellular matrix in which many sections were cut and stained with 0.025% toluidine blue in glands occurred. The endometrial, glandular epithelium gener¬ 0.5% sodium tetraborate. ally had a similar appearance to that lining the lumen, although, Uteri of a single pregnant Monodelphis domestica were occasionally, glands were found that were widely dilated with obtained from the laboratory of G. Schatten (University of a simple, squamous epithelium. Wisconsin, Madison). This individual was estimated to be at Secretion from a few of the glands was PAS-positive and about day 13 of the 14.5 day pregnancy. Immediately after amylase resistant. A different secretion, which was PAS- death, the uteri were removed and fixed in 10% phosphate negative and alcian blue-positive at pH 2.5 but negative at buffered formalin. After arrival at Adelaide, the tissue was pH 1.0, was found at the surface of part of the uterine lumen processed and stained as above for light microscopy. and in a few of the glands. Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access sectioning (Fig. 2a). Embryos were at the late presomite-early somite stage (Fig. 2d). Convoluted ribbon-like fragments of shell membrane lay adjacent to the endometrium (Fig. 2a). At this time, the embryo was still a part of the wall of the chorionic vesicle and had a pro-amniotic head-fold, such that the future head was submerged beneath the surface of the vesicle. Blood islands, with primitive erythroblasts, had formed in the extraembryonic mesoderm in an annular region adjacent to the embryo (Fig. 2e). In the yolk sac, trophoblast cells showed regional differences in size. In the region of the bilaminar yolk sac they were large and cuboidal (Fig. 2b). In the trilaminar region, near to the maternal epithelium, they were large and vacuolated above the extraembryonic mesoderm (Fig. 2c), but, when trophoblast cells, which were closely apposed to the mesoderm below, were some distance away from the endometrium, they were tall and columnar cells at the Fig. 1. Unilaminar blastocyst (B) of a fat-tailed dunnart lying in (Fig. 2e). Trophoblast abembryonic shallow depression of endometrium closely apposed to the uterine pole of the yolk sac were attenuated and resembled the epithelium (E). Gland (G). Scale bar represents 100 pm. Haematoxylin squamous endoderm. and eosin stained. Late pregnancy stages crown-rump length 2.9—3.5 mm. After the development of the placenta, the simple, cuboidal, uterine, luminal epithelium had lost the cobblestone appearance. The Unilaminar Four unilaminar blastocyst stage. expanded, yolk sac was composed of three regions: (1) the unattached, blastocysts, approximately 750 pm in diameter and surrounded bilaminar omphalopleure (yolk sac), (2) the attached, bilaminar, mucoid a were by a thin layer and shell membrane, flushed avascular, yolk sac placenta, and (3) the attached trilaminar, from one of the other uterus. Paraffin-embedded sections uterus vascular, yolk sac placenta. In region 1, the lateral walls of yolk from the same female showed five collapsed embryos lying in sacs of neighbouring embryos fused and were lost late in of the columnar shallow depressions simple epithelium (Fig. 1). pregnancy, so that up to five embryos occupied the same The underlying endometrial stroma was highly glandular. chorionic cavity. Region 1 was composed of two simple, epithelial cell layers bilaminar Bilaminar blastocyst stage. Eight early blastocysts, where squamous trophoblast overlay a squamous endoderm approximately 1.8 mm in diameter, were found completely that lined the yolk sac cavity. This part of the chorionic vesicle enveloped by folds of intact luminal epithelium in 'pockets' of was not closely apposed to the uterine epithelium. the endometrium of one uterus. These blastocysts, still sur¬ In region 2 (the bilaminar yolk sac placenta), epithelial rounded by a shell membrane, were flushed out of the uteri penetration continued even on the last day of pregnancy, and with fixative. uterine epithelial cells became surrounded by trophoblast giant The endometrial stroma close to the blastocysts was cells (Fig. 3a, b). The nuclei of trophoblast giant cells were up oedematous. The simple, columnar, luminal epithelium had a to 70 pm in diameter and presumably polyploid. The uterine thin coat of PAS-positive, amylase resistant material which was epithelium was replaced on one side of many endometrial folds also found in the basal cytoplasm of many luminal epithelial by trophoblast giant cells of the avascular yolk sac but it cells. The secretion of the glands was generally eosinophilic remained intact on the other side (Fig. 3a). The cytoplasm of with a few glands having PAS-positive, amylase resistant the trophoblast giant cells was pale and extremely vacuolated. secretion. All secretions were alcian blue-negative. In the bilaminar yolk sac placenta, trophoblast giant cells of up to 160 pm in diameter replaced the maternal epithelium (Fig. 4) Late presumile embryos. The uteri in one animal were but did not appear to have invaded the maternal capillaries. An dorsoventrally flattened and measured about 13 mm in diam¬ endotheliochorial placenta had apparently formed, although the eter and 8 mm thick. Superficially, the endometrium was folded boundary between fetal and maternal tissue was not easily into aglandular, thin tongues of tissue composed of epithelium seen. with a thin layer of vascularized stroma. However, below In region 3, the convoluted chorion followed the contours these folds the endometrium was very glandular (Fig. 2a). of the endometrium. Trophoblast cells in the trilaminar yolk The epithelium was of a simple cuboidal type and its cell sac placenta were usually, but not always, much smaller apices bulged into the lumen giving its surface a cobblestone (15-30 pm) than the trophoblast giant cells of the bilaminar appearance (Fig. 2b, c). Many capillaries were found immedi¬ yolk sac placenta and were not invasive; thus, an epithelio¬ ately below the basement membrane of the epithelium. The chorial placenta had developed (Fig. 5a, b). The distance myometrium was very thin (Fig. 2a). between fetal and maternal blood in the trilaminar yolk sac Chorionic vesicles flushed from the uteri had lost their shell placenta was less than 10 pm as maternal stromal, fetal membranes which lay in clumps in the uterine lumen, visible mesodermal and trophoblast nuclei near the placental capillaries with the dissecting microscope. The yolk sac had an irregular were displaced. At the junction of the trilaminar and bilaminar ellipsoid shape and measured about 4 mm by 2 mm after yolk sac placentae a large blood vessel, the sinus terminalis, Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access Fig. 2. (a) Yolk sac of late presomite embryo, of a fat-tailed dunnart, which is still a part of the wall of the vesicle. Embryo (arrowhead), shell membrane (SM), endometrium (End), myometrium (M), yolk sac cavity (YSC). Scale bar represents 500 pm. (b) Higher power photomicrograph of bilaminar yolk sac (box 1 in (a)) shows that the uterine luminal epithelium (E) has a cobblestone appearance and trophoblast cells (T) are large and cuboidal. Scale bar represents 50 pm. (c) Trophoblast cells (T) are large and vacuolated and adjacent to the maternal epithelium (E). Scale bar represents 25 pm. (d) Enlargement of embryo shown in (a) at the late presomite:early somite stage. Neuroectoderm (arrowhead); embryonic endoderm (IE). Scale bar represents 100 pm. (e) Higher power photomicrograph of the extraembryonic trilaminar region (box 2 in (a)); blood islands with primitive erythroblasts (arrows) are present in the mesoderm adjacent to the embryo; trophoblast above them (T) is tall and columnar. Scale bar represents 50 pm. Haematoxylin and eosin stained. was present. Below the placenta in the endometrial stroma, Late in development (embryonic manus paddle-shaped) the there were scattered neutrophils, lymphocytes and macro¬ allantois formed an evaginated diverticulum that approached, phages, particularly below the invasive trophoblast giant cells. but did not reach, the chorion. The wall of the allantois was Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access Fig. 4. Bilaminar yolk sac placenta (BYS) of a fat-tailed dunnart appears to merge with folds of endometrium; trophoblast giant cells (TGCs) (arrowheads) have invaded the endometrial stroma. Scale bar represents 50 pm. Stained with haematoxylin and eosin.

properties as the trophoblast giant cells. The gland lumina were PAS and alcian blue negative at this stage.

After parturition. One animal had given birth 3—7 h before the time of death and had 10 neonates (crown—rump length 4.2 mm; weight 15 mg) attached to the 10 teats. Drops of milk in the stomachs of the neonates could be seen through the still moist skin of their abdomens. The young had gaping mouths and were firmly attached to the teats. The digits of the neonatal manus had claws, while the pes was still paddle-shaped, but scalloped where the future digits would develop. The eyespot was pigmented. The uterine epithelium of the mother, when present, was and and its luminal was Fig. 3. (a) Implantation of embryo of fat-tailed dunnart has taken simple cuboidal, surface coated with material that was and alcian place and groups of maternal epithelial cells (arrows) on one side of PAS-positive, amylase resistant an endometrial fold are surrounded by trophoblast giant cells blue-positive (at pH 1.0 and 2.5). The secretion within the (TGC), while the epithelium on the other side of the fold remains endometrial glands was PAS-positive but alcian blue-negative. intact (E). Extraembryonic endoderm (En). Scale bar represents 25 pm. The nuclei of the trophoblast giant cells had become pyknotic (b) Vacuolated uterine epithelial cells (E) surrounded by TGCs and there was a massive infiltration of granular leucocytes and are maternal are found degenerate; capillaries (arrowhead) also into the endometrial stroma. It was unclear between lymphocytes the trophoblast and extraembryonic endoderm (En). Scale bar whether the remains of the placenta would subsequently have represents 50 pm. Stained with toluidine blue. been shed or resorbed, as there was an additional embryo found in the median pseudovagina. composed of vascular mesenchyme. At the forelimb digit stage, the and remained within the allantois had become smaller, folds Placenta of domestica of the yolk sac. Monodelphis Trophoblast giant cells in the bilaminar and trilaminar yolk The most mature embryo found in the uteri of the grey sac placentae contained granules in their apical cytoplasm that short-tailed opossum had a crown—rump length of 7 mm, the were PAS-positive, amylase resistant and were positive with manus had five distinct digits, the pes was paddle-shaped and alcian blue at pH 2.5 (and at pH 1.0 in one animal) (Fig. 6a, b). the eyespot was pigmented. The endometrium was very folded Similar material was found on the cell surface of trophoblast and glandular, with a simple columnar epithelium that had a giant cells and on the apical surface of the uterine epithelium, smooth luminal surface (Fig. 7a). Remnants of the shell mem¬ especially at the interface between fetal and maternal tissues, brane lay between maternal and fetal tissues in patches across although this material was found on the surface of the luminal the surface of the yolk sac, especially in the avascular region epithelium even away from sites of intimate contact. The (Fig. 7a), but in many places it had already been shed. cytoplasm of small trophoblast cells (about 15 pm in diameter) The placenta of Monodelphis domestica was also chorio- in the unattached bilaminar yolk sac had the same staining vitelline. There are three regions of the yolk sac at this stage of Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access Fig. 6. (a) Cytoplasm of trophoblast giant cells in the bilaminar yolk sac placenta (arrowheads) contains PAS-positive and amylase resistant granules. Similar material coats uterine luminal epithelial cells and fills the luminal space (L) between closely adjacent folds of endometrium. Scale bar represents 50 pm. (b) The plasmalemma of trophoblast giant cells is coated with PAS-positive material (arrows). It is also at the interface between fetal and maternal tissues in the trilaminar yolk sac (TYS) (arrowheads). Scale bar represents 50 pm. Stained with PAS.

near the sinus terminalis were columnar and were about 50 by 25 pm in diameter. The cytoplasm of the trophoblast giant cells had many vacuoles, some of which contained eosinophilic granules. Fig. 5. (a) Trilaminar yolk sac (TYS) from a fat-tailed dunnart follows The allantois was present at this stage, although it remained contours but does not erode the of folded endometrium epithelium close to the embryo and was enveloped by folds of the yolk (arrowheads). Scale bar 50 (b) cells are represents pm. Trophoblast sac. It had thin walls and was comparatively avascular. smaller in the TYS than in the bilaminar yolk sac usually although Histochemical staining showed that the uterine occasional cells (arrowhead) are found adjacent to epithelium trophoblast giant and the endometrial were PAS- and alcian blue- fetal Nucleated fetal erythrocytes (arrows) fill the extensive glands capillaries. The between the endoderm and in capillary network within the TYS. Yolk sac cavity (YSC). Scale bar negative. layer trophoblast the bilaminar sac was and represents 25 pm. Stained with haematoxylin and eosin. yolk placenta weakly PAS-positive amylase resistant but alcian blue-negative.

an unattached sac gestation: (1) bilaminar, avascular yolk which Discussion occupied the uterine lumen enveloping the embryo and which, over much of its surface, followed the contours of the folded This study has extended previous findings on the placental endometrium; (2) a very attenuated avascular yolk sac placenta histology of the fat-tailed dunnart, Sminthopsis crassicaudata, closely apposed to the uterine luminal epithelium; and (3) a that were based on a single pregnant female (Hughes, 1974). In trilaminar, vascular, yolk sac placenta (Fig. 7b, c). Each embryo the study reported here, eight pregnant females at various lay separately in its own yolk sac some distance from the other developmental stages were investigated. By comparing pla¬ embryos. cental structure in the dunnart with that of a didelphid The unattached bilaminar, or avascular, yolk sac had oc¬ marsupial, the grey short-tailed opossum, Monodelphis casional large trophoblast cells with a squamous endoderm domestica, our investigations emphasize the variation in chorio- lining its inner surface. A more attenuated region of the vitelline placental structure between representatives of two bilaminar yolk sac was found closely apposed to the uterine primitive marsupial families, particularly with regard to the luminal epithelium (Fig. 7d). The trophoblast in this region was presence and location of trophoblast giant cells and the extent squamous, and its cytoplasm contained eosinophilic granules. of invasion of fetal tissue into the endometrium. Similar material was found in layers between the trophoblast The endometrium in pregnant dunnarts, like that in euth- and endoderm in some parts of this region. erian mammals, has a simple columnar epithelium. Padykula The trilaminar, or vascular, yolk sac placenta was composed and Taylor (1971) and Krause and Cutts (1985) reported that of an inner attenuated endodermal layer, an overlying vascular there is a pseudostratified, columnar, luminal epithelium in the mesodermal layer and an outer layer of trophoblast giant cells pregnant uterus of Didelphis virginiana. However, this con¬ that, in discontinuous areas, appeared to be attached to the clusion may have been based on observations of tangential uterine epithelium (Fig. 7b, c). The largest trophoblast giant sections of the epithelium that could give the impression that it cells were cuboidal, and their diameter was about 50 pm. Those is pseudostratified when, in fact, it is of a simple columnar type. Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access pregnancies can be obtained as ovulation follows 14—16 h after the onset of oestrous behaviour (Baggott et al, 1987). By the late presomite-early somite stage (about day 10), dunnart embryos have completely shed the shell membrane and implantation, which is centric, has begun. Hughes (1974) found that the shell membrane was present in S. crassicaudata embryos at the primitive streak stage which suggests that, in our study, the shell membrane had just broken down in the late presomite embryo. The stage of embryonic development at the time of the loss of the shell membrane has been recorded as 13—15 somite stage in Sminthopsis macroura (Selwood and Woolley, 1991) and 25 somite stage in Schoinobates volans (Bancroft, 1973) and Macropus rufogriseus (Walker and Rose, 1981). Our study shows that the shell membrane of the opossum is lost later in development, as patches of shell membrane were found between the chorionic vesicle and the maternal uterine epithelium when the embryo had paddle- shaped hind limbs. In the tammar wallaby, the action of acid and alkaline proteinases secreted by the fetal endoderm and trophoblast, as well as by endometrial gland and stromal cells, at about days 18—19 of the 27 day pregnancy, erodes the shell membrane, allowing the trophoblast to make contact with the uterine luminal epithelium (Denker and Tyndale-Biscoe, 1986). Presumably these proteinases are also present in dunnarts and opossums at implantation. Although no direct evidence of trophoblast invasion into the uterine luminal epithelium at the late presomite stage was observed in dunnarts, the cobblestone appearance (or dome shaped cell apices) of the maternal epithelium seen by light microscope studies is a characteristic feature of uterine epi¬ thelial cells at early implantation stages in several eutherian Fig. 7. Placenta of domestica, (a) The sac follows Monodelphis yolk rats (Tachi et al, 1970) and et al, highly folded contours of the simple columnar endometrial epithelium species, including pigs (King 1982). There are also a few ultrastructural features of the (E) beneath which many glands are found. Gland (G); yolk sac cavity (YSC); shell membrane fragments (arrowheads). Scale bar represents uterine luminal epithelium, common to most eutherian species, that have been seen 100 pm. (b) In the trilaminar yolk sac (TYS), trophoblast (T) is attached at the apposition—adhesion stage in to the uterine luminal epithelium (E) in discontinuous areas. Extra¬ dunnarts (C. T. Roberts and W. G. Breed, unpublished obser¬ embryonic endoderm (En); fetal erythrocytes (arrows); embryo (Em). vations), which support our conclusion that implantation has Scale bar represents 50 pm. (c) Uterine luminal epithelium (E) is begun at this time. continuous with at sites of attachment to Scale trophoblast (T) TYS. Our results suggest that implantation in dunnarts, as in bar represents 25 pm. (d) The bilaminar yolk sac placenta (BYS) is another Sminthopsis species (Selwood and Woolley, 1991) and closely to maternal epithelium (E) and is very attenuated; apposed in Didelphis virginiana (Krause and Cutts, 1985), occurs as late both trophoblast and endoderm are squamous and indistinguishable. as 3 before birth, from which time organo¬ Scale bar represents 25 pm. Stained with haematoxylin and eosin. days amniogenesis, génesis and embryonic/fetal growth must proceed at a rapid rate. In 3.5 days, dunnart embryos develop from the presomite Flynn (1930) found that early in pregnancy, in Bettongia stage to neonates which, although extremely small, can haul gaimardi, the uterine epithelial cells, although composed of a themselves up to the pouch unaided, digest milk, excrete urine single layer, appeared very 'crowded' after having undergone via mesonephric kidneys and breathe air (Tyndale-Biscoe and extensive proliferation at pro-oestrus and oestrus. As preg¬ Rentree, 1987). nancy progressed, the endometrium became very folded, with We have shown that implantation in dunnarts is not a considerably increased surface area and a simple columnar 'rudimentary', as has been suggested by Hughes (1974), epithelium. Our observations were similar to those of Flynn. despite his own description of a late pregnant female of this We could not time pregnancies accurately in the dunnarts, species. Hughes appears to have assumed that implantation because of the lack of synchrony between female receptivity, occurs only if epithelial penetration takes place and has not mating and ovulation. Females may copulate with a male for up mentioned the appositional and adhesive phases of the process to 3 days before ovulation. In addition, storage of spermatozoa (Schlafke and Enders, 1975). in the isthmus of the female can occur for up to 3 days before In dunnarts, the fetal trophoblast erodes the maternal epi¬ ovulation (Selwood, 1987; Breed et al, 1989). Consequently, thelium in the region of the avascular, bilaminar, yolk sac estimations of the stage of pregnancy based on the time placenta and comes to surround the maternal endothelium. In of mating of these animals can be inaccurate. However, this is our study, the groups of degenerating maternal uterine epi¬ not the case for the opossum, in which accurately timed thelial cells found surrounded by trophoblast in late pregnancy Downloaded from Bioscientifica.com at 10/07/2021 07:15:44PM via free access indicate that the trophoblast replaces, and presumably phago¬ Further work is required on selected marsupial species to cytoses, them. Enlarged trophoblast cells of the bilaminar yolk investigate the variations of implantation and placentation in sac placenta of the only other dasyurid species investigated, this mammalian group. As marsupial reproduction is character¬ Dasyurus viverrinus, apparently erode the maternal epithelium ized by a shorter gestation followed by a longer period of and surround 'maternal blood sinuses' (Hill, 1900), although it lactation than in eutherians, it may be that there has not been is not clear whether the maternal endothelial cells are actually strong selective pressure to develop a chorioallantoic placenta engulfed by trophoblast. in most marsupial families as the yolk sac forms the definitive In the trilaminar yolk sac placenta, the maternal epithelium placenta. Clearly, to imply that these animals are 'aplacental' in remains intact, although the nuclei of all cell layers between the contrast to the 'placental' eutherians is quite erroneous. fetal and maternal capillaries are displaced immediately above maternal capillaries such that the two bloodstreams come to lie The authors thank J. H. Bennett for supplying the dunnarts and in close proximity with only thin cytoplasmic processes of D. Golding for their maintenance. The Monodelphis material was obtained when one of the authors was on a DITAC intervening cell layers between them. This is also seen in the working overseas collaborative at the of Wisconsin, trilaminar yolk sac of the Pseudocheirus project University placenta petaurids, Madison. The authors also thank C. and G. Hermanis for (Sharman, 1961; et al, 1965), and Schoinobates Leigh peregrinus Hughes technical assistance. This work was in volans and in the supported part by grants (Bancroft, 1973) macropod, Bettongia gaimardi from the Australian Research Council and the University of 1930). In the chorioallantoic of fetal and (Flynn, placenta pigs, Adelaide. C. T. Roberts was in receipt of an Australian Postgraduate maternal capillaries indent the epithelia above them, thus Research Award. reducing the distance for diffusion of oxygen and carbon dioxide (see Enders, 1982). The same situation in the marsupial yolk sac placenta also presumably facilitates exchange. References The results of the PAS- and alcian blue-staining suggest that for the first two-thirds of pregnancy the endometrial glands are Baggott LM, Davis-Butler S and Moore HDM (1987) Characterization of oestrus actively secreting neutral glycoproteins that presumably com¬ and timed collection of oocytes in the grey short-tailed opossum, Monodel¬ prise some of the histotrophic nutrition for the early embryo. phis domestica Journal of Reproduction and Fertility 79 105—114 After the of the the cells of Bancroft BJ (1973) Embryology of Schoinobates volans (Kerr) (Marsupialia: development placenta, trophoblast Petauridae) Australian Journal of Zoology 21 33—52 all of the sac a PAS- and alcian blue- regions yolk produce Bennett JH, Breed WG, Hayman DL and Hope RM (1990) Reproductive positive (pH 2.5), amylase resistant secretion that coats them as and genetical studies in a laboratory colony of the dasyurid marsupial well as the maternal epithelium. This is presumably a carboxy- Sminthopsis crassicaudata Australian Journal of Zoology 37 207—222 lated glycoprotein possibly like the sialomucins (fibrinoid) that Bradbury S, Billington WD and Kirby DRS (1965) A histochemical and electron study of the hbrinoid of the mouse Journal of the coat the in the mouse et microscopical placenta trophoblast placenta (Bradbury al, Royal Society 84 199—211 and Harder found similar Microscopical 1965). 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