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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.