Yolk Sac Placenta Research Articles

Developmental potential and survival of glycolysis-deficient cells in fetal mouse chimeras.

Mouse embryos homozygous for a null allele of Gpi1 fail to complete gastrulation and die around E7.5. We produced E12.5 chimeric mouse conceptuses, composed of wild-type and homozygous Gpi1m/m null mutant cells to test whether the presence of wild-type cells allowed mutant cells to survive and, if so, whether they survived better in some tissue locations than others. Fourteen homozygous Gpi1m/m<-->Gpi1c/c chimeras were identified and these contained low levels of homozygous mutant cells in most tissues tested. Homozygous Gpi1m/m cells contributed better to the yolk sac endoderm and placenta than to the epiblast derivatives tested (retinal pigment epithelium, brain, tail, amnion, and yolk sac mesoderm). The depletion of mutant cells confirms that the gene acts cell autonomously, but the GPI deficiency is not always cell-lethal. When mixed with wild-type cells in chimeras, homozygous mutant cells can differentiate into many different cell types and survive until at least E12.5.

Comparative Biology of the Vertebrate Placenta—A Workshop Report

The topic of this workshop was first proposed by Professor William C. Hamlett (Notre Dame, USA) who unfortunately could not attend the meeting. We are grateful for the initial work done by him in organizing the workshop and regret that his contribution on shark placentation will be missing. The major goals of the workshop were to provide new data about the biology of vertebrate placentation over a broad range of vertebrates. What are the similarities and differences in structures as well as in regulatory factors in the organs most directly concerned with gestation? What is their possible biological significance and can this provide an understanding of how viviparity evolved? We know that different species have solved similar problems relating to gestation in different ways. However, achieving more insight might show the possible potential of using unconventional vertebrate species as new models in placental research. Carolyn Jones (Manchester, UK) showed how lectin histochemistry can be used to compare the glycosylation of the yolk sac placenta of the shark (Mustelus canis) with mammalian epitheliochorial placentae from the pig, camel, alpaca and horse, which in that order show an increasing complexity in their histological structure. All are, however, of the epitheliochorial type. It was demonstrated that each species had its own specific ‘glycotype’ with respect to glycans expressed at the feto-maternal interface, which is richly glycosylated on both the maternal epithelium and the trophoblast. The shark placenta differed from the mammalian placentae in having an egg envelope separating the fetal from the maternal tissues, and so was not exactly analogous to the mammalian placentae, but in other ways shared many similarities including the expression of all classes of glycans. Direct cell-cell contact between the shark yolk sac placenta and maternal epithelium was not possible due to the presence of the egg envelope, whereas close fetomaternal contacts in the form of microvillous interdigitations are crucial for successful implantation in mammals. It was pointed out that appropriate glycan-glycan interactions are therefore important elements in the establishment of viable pregnancies and probably also contribute to the prevention of interspecies hybridization. Luana Paulesu (Siena, Italy) explained and demonstrated that placentation in reptiles has numerous parallels with that of mammals. These may include the assembly of extra embryonic membranes as well as the amount of nutrient transfer between

Trophoblast-Specific Expression and Function of the Integrin α7 Subunit in the Peri-implantation Mouse Embryo

For implantation and placentation to occur, mouse embryo trophoblast cells must penetrate the uterine stroma to make contact with maternal blood vessels. A major component of the uterine epithelial basement membrane and underlying stromal matrix with which they interact is the extracellular matrix protein laminin. We have identified integrin α7β1 as a major receptor for trophoblast–laminin interactions during implantation and yolk sac placenta formation. It is first expressed by trophectoderm cells of the late blastocyst and by all trophectoderm descendants in the early postimplantation embryo through E8.5, then disappears except in cells at the interface between the allantois and the ectoplacental plate. Integrin α7 expression is a general characteristic of the early differentiation stages of rodent trophoblast, given that two different cultured trophoblast cell lines also express this integrin. Trophoblast cells interact with at least three different laminin isoforms (laminins 1, 2/4, and 10/11) in the blastocyst and in the uterus at the time of implantation. Outgrowth assays using function-blocking antibodies show that α7β1 is the major trophoblast receptor for laminin 1 and a functional receptor for laminins 2/4 and 10/11. When trophoblast cells are cultured on substrates of these three laminins, they attach and spread on all three, but show decreased proliferation on laminin 1. These results show that the α7β1 integrin is expressed by trophoblast cells and acts as receptor for several isoforms of laminin during implantation. These interactions are not only important for trophoblast adhesion and spreading but may also play a role in regulating trophectoderm proliferation and differentiation.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice.

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12-20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

Development of the immune system and immunological protection in marsupial pouch young

At birth the tissues of marsupial immune system are underdeveloped. The young animal is not immunocompetent. Histological and immunohistochemical studies of pouch young epithelial tissues provide a clear picture of tissue development but the timing of onset of immunocompetence awaits definition. The survival of the neonatal marsupial in a microbially rich environment is dependent on maternal strategies, including immunoglobulin transfer via milk and, in some species, prenatally via the yolk sac placenta. It is also likely that pouch secretions play a role. This review summarizes our current knowledge of the pathway of immunological development in marsupials and the protection and threats afforded by the pouch environment.

Examination of normal and abnormal placentation in the mouse.

Placental development, also known as placentation, is orchestrated by precise molecular and cellular interactions between extraembryonic cells and cells of the pregnant uterus. The major extraembryonic cell type of the placenta is the trophoblast cell, which arises from the trophectoderm cell lineage (). Trophoblast cells make the physical connection between the embryo and the maternal environment and play important roles in the implantation process and placental function (). In mice, trophoblast cells lead the embryonic invasion into the uterine stroma to achieve implantation, and contribute to the formation of a temporary yolk-sac placenta (choriovetelline placenta) during early postimplantation stages. Trophoblast cells eventually provide the major extraembryonic cell lineage that contribute to the formation of the mature chorioallantoic placenta (). In the chorioallantoic placenta, the fetal and maternal circulatory systems are brought into close apposition to allow for a range of physiological interactions that are critical for fetal growth and development. Abnormal placentation is often associated with early embryonic mortality (-) and can lead to serious pregnancy disorders such as preeclampsia ().

Ontogeny of Expression of Insulin-like Growth Factor (IGF) and IGF Binding Protein mRNAs in the Guinea-pig Placenta and Uterus

To determine the temporal and spatial distribution of insulin-like growth factor (IGF) and its family of binding proteins (IGFBPs), guinea-pig yolk sac and chorioallantoic placentae were collected at 15, 20, 25, 29, 44–45, 55 and 65–66 days of gestation. Messenger RNAs for IGF I, IGF II and IGFBP 1–6 were identified in tissue sections by in situ hybridization, using35S-cRNA probes. Epithelial and mesenchymal cell types were identified by immunohistochemistry for cytokeratin and vimentin, respectively. At 15 days of gestation, IGF-II mRNA was expressed in ectoplacental mesoderm, cytotrophoblasts and syncytiotrophoblast, and IGFBP-5 mRNA was detected in the syncytiotrophoblast. In the mid-gestation placenta, IGFBP-5 mRNA was expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth. Near term, when expansion of the labyrinth was complete, IGFBP-5 mRNA was coexpressed with IGF-II mRNA in the marginal and interlobular syncytium. These observations suggest that interaction between IGF-II and IGFBP-5 plays a role in the vascularization of the placenta by fetal vessels. IGF-II mRNA was not expressed in the maternal tissues at any gestational age. IGFBP-2, -3 and -5 mRNAs were expressed in the endometrial stroma at 7–12 days of gestation but, following establishment of the placenta, IGFBP mRNAs were more abundant in the myometrium than in the decidua. IGF-II mRNA was detected in trophoblasts invading the walls of maternal vessels, and the endothelium of the preplacental vessels expressed IGFBP-4 mRNA, while IGFBP-2 and IGFBP-5 mRNAs were present in the tunica media of mesometrial arteries that had not been invaded by trophoblast. These findings suggest that IGF-II produced by the trophoblast acts in an autocrine and/or paracrine fashion to promote trophoblast invasion and that this process is modulated by interaction with IGFBPs present in maternal tissues.

Production of prostaglandin f2alpha and its metabolite by endometrium and yolk sac placenta in late gestation in the tammar wallaby, Macropus Eugenii.

In this study, we investigated production of prostaglandin (PG) F2alpha and its metabolite, PGFM, by uterine tissues from tammar wallabies in late pregnancy. Endometrial explants were prepared from gravid and nongravid uteri of tammars between Day 18 of gestation (primitive streak) and Day 26.5 (term) and were incubated in Ham's F-10 medium supplemented with glutamine and antibiotics for 20 h. PGF2alpha and PGFM in the medium were assayed by specific, validated RIAs. Control tissues (leg muscle) did not produce detectable amounts of either PG. Both gravid and nongravid endometria secreted PGF2alpha, and production increased significantly in both gravid and nongravid uteri towards term. PGFM was produced in small amounts by both gravid and nongravid uteri, and the rate of production did not increase. Neither oxytocin nor dexamethasone stimulated PG production in vitro in any tissue at any stage. Thus, the surge in peripheral plasma PGFM levels seen at parturition may arise from increased uterine PG production, but further study is needed to define what triggers this release.

Immunohistochemical identification of epithelial and mesenchymal cell types in the chorioallantoic and yolk sac placentae of the guinea-pig

To define the epithelial and mesenchymal cell types of the guinea-pig placenta, immunostaining patterns were determined for the intermediate filament proteins cytokeratin and vimentin. Chorionic and yolk sac placentae were studied at 15, 20, 25, 29–30, 44–45, 55 and 65 days of gestation. Immunohistochemistry was performed on 5-μm thick sections of paraffin embedded tissue using specific antibodies against cytokeratin, a marker for epithelial cells, including trophoblast, and vimentin, a marker for mesenchymal cells and stromal decidua. Immunostaining was identified by the avidin-biotin-peroxidase technique with diaminobenzidine as the chromogen. Most of the surface of the placenta is covered by the columnar epithelium of the parietal yolk sac, beneath which is found a layer of chorionic giant cells. In the guinea-pig, a sheet of mesenchymal cells interposed between these cell layers immunostained for vimentin, a protein that is expressed only intracellularly, and had nuclei orientated parallel to the surface of the placenta. This cell layer is quite different from Reichert's membrane in the rat or mouse, which is acellular. Within the main placenta, cytokeratin immunostaining demonstrated that the trophoblasts lining the large maternal blood sinuses are different in character from the surrounding syncytiotrophoblast, confirming earlier ultrastructural observations. In the subplacenta, some trophoblast did not immunostain for cytokeratin and there was non-specific staining of cellular debris, so that immunostaining for vimentin provided the clearest indication of the maternal-fetal interface. In later stages of gestation (30–55 days), trophoblasts invading the walls of maternal arteries immunostained for cytokeratin and were vimentin negative. In early gestation, however, trophoblast invasion of the maternal vessels was indicated by cells that were immunoreactive for both cytokeratin and vimentin.

Mesotocin receptors during pregnancy, parturition and lactation in the tammar wallaby.

Mestocin receptor concentrations in membrane preparations from reproductive tissues of the tammar Macropus eugenii throughout gestation and lactation were assessed using [3H]-oxytocin as the ligand. There was a single binding site which bound both mesotocin and oxytocin with high and similar affinities. Mesotocin receptor concentrations in the myometrium were low (708 +/- 199 fmol mg-1 protein) in early and middle gestation but increased significantly on day 23 of pregnancy of the 26-day gestation period to 1921 +/- 552 fmol mg-1 protein. Myometrial receptors reached a peak of 2483 +/- 575 fmol mg-1 protein on days 25 and 26 of gestation, but returned to basal levels about an hour after birth. Receptor concentrations in the contralateral non-gravid uterus were much lower (605 +/- 75 fmol mg-1) and did not significantly increase throughout the period of gestation but dropped one day before birth. Mesotocin receptors were undetectable in the endometrium, the yolk sac placenta and the lateral, median and anterior vagina of all animals tested. In the lactating mammary gland after birth mesotocin receptors were initially high (588 +/- 38 fmol mg-1) but decreased after 200 days and by late lactation were 224 +/- 55 fmol mg-1 protein on day 240, close to the time of weaning. Mesotocin receptors in the ipsilateral non-lactating gland were also high in early lactation (430 +/- 153 fmol mg-1) and declined in late lactation (62 +/- 20 fmol mg-1). The changing concentrations of mesotocin receptors in pregnancy and lactation demonstrate that they are specifically regulated in tammar reproductive tissues. The increase in mesotocin receptors in gravid, but not in the non-gravid myometrium three days before birth may make the uterus responsive to the surge of mesotocin at birth. Since this rise is unilateral and only occurs in the gravid myometrium it must be due to local effects from the ipsilateral ovary or the feto-placental unit. Likewise, the down-regulation of mesotocin receptors in the contralateral, non-gravid myometrium may be due to its proximity to the developing follicle. The changing concentrations in the lactating and the adjacent, non-lactating mammary gland also reflect a differential regulation of mesotocin receptors, probably mediated via the sucking stimulus. Thus, local influences appear to be of primary importance in the regulation of mesotocin receptors during reproduction in this marsupial.

Nutritional studies of the embryo during early organogenesis with normal embryos and embryos exhibiting yolk sac dysfunction

In 1961 we reported that heterologous kidney antiserum when injected into pregnant rats resulted in wide spectrum of congenital malformations. Further studies identified that it was the IgG component of the antiserum that was teratogenic and that complement was not necessary to produce the teratogenic effect. Labeled antibody studies demonstrated that the kidney antiserum localized in the kidney and in the visceral yolk sac (VYS) and parietal yolk sac placentas. Preparation of yolk sac (YS) antiserum proved to be more potent than the kidney antiserum. Adsorption studies with VYS and parietal yolk sac antiserum revealed that the site of the teratogenic process was located in the VYS. In vitro embryo culture experiments demonstrated that direct injection of the teratogenic antibody into the amniotic or YS cavity did not injure the embryo, thus indicating that the teratogenic antibody had to come in contact with the absorptive surface of the VYS. Collaboration with Dr. John Lloyd demonstrated that teratogenic antibody interfered with the process of pinocytosis and the delivery of amino acids (AA) to the developing embryo. Our studies into the nature of the source of AA for the embryo indicated that in some instances >95% of the AA present in the developing embryo were derived from maternal proteins and the remainder from free AA in the maternal serum. We also demonstrated that embryonic methionine was derived primarily from the digestion of maternal serum proteins but that more of the methionine was diverted from the synthesis of embryonic proteins, supporting the view that it has important functions other than the synthesis of proteins. All these studies focus on the role of the YS in human development and whether human YS dysfunction may play a role in the pathogenesis of congenital malformations. Further studies on the delivery of AA to the embryo are warranted to determine whether certain AA are in short supply in maternal serum and place the embryo at risk if nutritional alterations in the maternal environment occurs. Furthermore, the YS may be an organ whose role might offer opportunities for pregnancy control. (J Pediatr 1998;132:S6-S16.)

Hydroxylated polychlorinated biphenyls : distribution in the pregnant mouse

1. In the pregnant C57BL mouse the disposition of a single, intravenous low dose of 14C-labelled 4-hydroxy-3,5,3',4'-tetrachlorobiph enyl (4-OH-TCB) or 4-hydroxy3,5,2',3',4'-pentachloro- biphenyl (4-OH-PeCB1) was monitored by liquid scintillation counting and whole-body autoradiography. The compounds were placentally transferred and accumulated in the foetal tissues (e.g. plasma and liver). Also, maternal accumulation was observed in selected tissues, including liver, adrenal gland, adipose tissue and yolk sac placenta. 2. The foetal concentration of both hydroxy- PCBs increased with time up 24 h postexposure and the foetal plasma concentration with at this time-point two-fold of that in maternal plasma. Chemicalanalysis of maternal plasma and liver showed no metabolismof the administered compounds. 3. In the pregnant C57BL mouse at late gestation, exposure to 4-OH-TCB generally resulted in a higher foetal and maternal tissue retention than did 4-OH-PeCB1. The estimated elimination half-lives (t1/2) of 4-OH-TCB in maternal liver and plasma were 69 and 13 h respectively, and for 4-OH-PeCB1 were 17 and 13 h. 4. No differences in foetal tissue concentration of 4-OH-TCB were observed between the C57BL and NMRI mouse. In contrast, earlier studies have shown that the PCB congener CB-77, the parent compound of 4-OH-TCB, resulted in a C57BL NMRI foetal ratio of 1:5.

Retinoid binding proteins in mouse yolk sac and chorio-allantoic placentas.

In the adult, as well as in the embryo, a number of specific extra- and intracellular binding proteins such as the plasma retinol binding protein (RBP), the cellular retinol binding protein type I (CRBP I), and also the cellular receptors for RBP are thought to regulate transport and metabolism of retinol (vitamin A). Since the regulation of materno-fetal transport of vitamin A is not well understood, we examined the localization of these proteins during the development of the mouse chorio-allantoic and yolk sac placentas. The labyrinthine region of the chorio-allantoic placenta, where exchange of substances can occur between the maternal and fetal circulations, did not contain RBP (mRNA or protein) or antigen(s) similar to the bovine RBP-receptor p63, whereas the visceral endoderm of the yolk sac placenta, the second site for materno-fetal transport, did. Furthermore, only the endodermal cells of the visceral yolk sac appeared to strongly accumulate radiolabelled retinoids. The cellular retinol binding protein (CRBP I) was detected both in the trophoblast layer of the placental labyrinth closest to the fetal endothelium (layer III), and in the visceral endoderm of the yolk sac. Together, these findings suggest that the yolk sac placenta mediates retinol transfer to the embryo/fetus throughout the entire gestation. The chorio-allantoic placenta, on the other hand, does not appear to have this capacity, while the presence of CRBP I does suggest a retinol-metabolizing capability.

Carbon monoxide and the embryo.

Mammals are homeotherms and expend considerable energy maintaining their body temperatures. The temperature of a mammalian embryo on the other hand is maintained by the mother and the embryo can devote its metabolic energy to growth and development. The mammalian embryo is acting as a poikilotherm and its energy needs are thus considerably less than if it were a comparably sized homeotherm. The energy requirements of the preimplantation rat embryo are generated by anaerobic metabolism. As it grows, aerobic metabolism develops. In culture, the addition of carbon monoxide to the perfusing gas for early rat embryos has a much smaller effect than decreasing the oxygen concentration. Carbon monoxide appears to be a relatively mild toxicant until the embryo is much larger, is depending much more on transport of oxygen by red blood cells, and the fraction of required metabolic energy produced by anaerobic metabolism has become quite small. The effect from smoking during gestation may be either by the concomitant reduction in food intake or a more direct toxic effect from some components in the smoke. Carbon monoxide does not seem to be the culprit. The possible mitigating effect of a compensatory increase in fetal hematocrit in response to any hypoxia must also be considered. Humans have no yolk sac placenta as rodents do, but if the switch from anaerobic to aerobic metabolism is correlated with the stage of development, then carbon monoxide exposure should not represent any significant risk to the human embryo until later in gestation.

Morphogenesis of placental membranes in the viviparous, placentotrophic lizard Chalcides chalcides (Squamata: Scincidae).

In the scincid lizard Chalcides chalcides, females ovulate small ova and supply most of the nutrients for development by placental means. The yolk is enveloped precocially by extraembryonic ectoderm and endoderm during the gastrula stage, establishing a simple bilaminar yolk sac placenta. The shell membrane begins to degenerate at this time, resulting in apposition of extraembryonic and maternal tissues. A true chorioplacenta has developed by the early pharyngula stage, as has a choriovitelline placenta and the first stages of an omphaloplacenta. Although the choriovitelline membrane disappears rapidly, the omphaloplacenta spreads to occupy the entire abembryonic pole. The yolk cleft is not confluent with the exocoelom, and no omphalallantoic placenta develops. By the limb-bud stage, an allantoplacenta has been established, with a mesometrial placentome composed of interdigitating ridges of chorioallantois and uterine mucosa. The discovery of five distinct placental arrangements in this species, three of which are transitory and two of which have not previously been recorded in reptiles, emphasizes the need for accounts that specify ontogenetic stages and the precise identity and composition of squamate placental membranes. Contrary to previous interpretations, the pattern of extraembryonic membrane development in C. chalcides is evolutionarily conservative, despite the presence of a reduced yolk mass and cytological specializations for nutrient transfer. Our observations indicate that substantial placentotrophy can evolve in squamates without major modifications of morphogenetic patterns. J Morphol 232:35-55, 1997. © 1997 Wiley-Liss, Inc.

Morphogenesis of placental membranes in the viviparous, placentotrophic lizard Chalcides chalcides (Squamata: Scincidae)

In the scincid lizard Chalcides chalcides, females ovulate small ova and supply most of the nutrients for development by placental means. The yolk is enveloped precocially by extraembryonic ectoderm and endoderm during the gastrula stage, establishing a simple bilaminar yolk sac placenta. The shell membrane begins to degenerate at this time, resulting in apposition of extraembryonic and maternal tissues. A true chorioplacenta has developed by the early pharyngula stage, as has a choriovitelline placenta and the first stages of an omphaloplacenta. Although the choriovitelline membrane disappears rapidly, the omphaloplacenta spreads to occupy the entire abembryonic pole. The yolk cleft is not confluent with the exocoelom, and no omphalallantoic placenta develops. By the limb-bud stage, an allantoplacenta has been established, with a mesometrial placentome composed of interdigitating ridges of chorioallantois and uterine mucosa. The discovery of five distinct placental arrangements in this species, three of which are transitory and two of which have not previously been recorded in reptiles, emphasizes the need for accounts that specify ontogenetic stages and the precise identity and composition of squamate placental membranes. Contrary to previous interpretations, the pattern of extraembryonic membrane development in C. chalcides is evolutionarily conservative, despite the presence of a reduced yolk mass and cytological specializations for nutrient transfer. Our observations indicate that substantial placentotrophy can evolve in squamates without major modifications of morphogenetic patterns. J Morphol 232:35–55, 1997. © 1997 Wiley-Liss, Inc.

Embryonic development and pattern formation.

During embryogenesis, information encoded in the genome is translated into cell proliferation, morphogenesis, and early stages of differentiation. Embryonic pattern arises from the spatial and temporal regulation and coordination of these events. The vitamin A (retinol) derivative retinoic acid (RA) is essential for normal development. Mammalian embryos are protected against vitamin A deficiency by maternal retinoid homeostasis until stored retinoids fall to very low levels. Retinol binding protein, which is synthesized in the yolk sac placenta of rodent embryos and in the syncytiotrophoblast of the human placenta, is essential for access of retinol to the embryo. Synthesis and metabolism of RA may involve cytoplasmic binding proteins, but the observation that mutants lacking these proteins are normal or near-normal suggests that they are not essential. Severe congenital vitamin A deficiency results in a spectrum of malformations including defects of the eye, lungs, cardiovascular system, and urogenital system. Extreme deficiency results as well in forelimb abnormalities and cleft face, but the embryos are not viable. Similar abnormalities are observed in embryos lacking two retinoid receptors, but loss of one receptor results in either normal development or mild abnormalities. Two single-receptor null mutants, RARgamma-/- and RXRalpha-/-, show regional pattern-specific resistance to teratogenic levels of RA. Mutations leading to abnormality of the structure or regulation of RA signaling pathway genes may be an important cause of human congenital abnormality.

Reproduction of a marsupial: From uterus to pouch

Parturition in marsupials is a remarkable process because the maternal endocrine system must be able to respond to signals from the tiny altricial neonate which may be as small as 5 mg (honey possum) or as ‘large’ as 800 mg (red and grey kangaroos). From the relatively few studies that have been conducted, it is clear that many features involved in the control of birth are held in common with those of eutherians. Based on current data on parturition in one species, the tammar wallaby, it is now clear that prostaglandins F 2α and PGE 2 (PGs) are the key regulators. The active oxytocic peptide in tammars is mesotocin (MT), which appears to facilitate birth. In the second half of gestation the myometrium becomes sensitive to mesotocin. As the fetus nears term, PG production in both the endometrium and yolk-sac placenta increases. A surge of PG occurs at term which stimulates uterine contractions. It induces stereotyped parturient behaviour a few minutes before emergence of the fetus, so that it can safely climb from the urogenital sinus to the pouch. Treatment with prostaglandin sythetase inhibitors through late pregnancy prevents birth. The fetus determines the timing of birth and premature birth can be induced with the glucocorticoid agonist dexamethasone. Thus, maturation of the fetal hypothalamo-pituitary-adrenal axis may lead to release of cortisol. Cortisol, in turn, may increase PG production by the uterus or placenta eventually leading to the parturient surge of this hormone in the plasma. Rising PG levels, either alone or in concert with MT, presumably lead to increased uterine contractions to ensure completion of the birth process once it is started. The small size of the term fetus (under 400 mg) and the low mass of the placenta (under 100 mg) suggests that paracrine mechanisms between the fetus, placenta and uterus may play a more important role in regulating PG production or uterine oxytocin receptor levels than occurs in most eutherians. However, the nature of the feto-placental signals and the interactions between PGs and MT remain enigmatic.

Sources of amino acids for protein synthesis during earlyorganogenesis in the rat. 4. Mechanisms before envelopment of the embryo by the yolk sac

It was previously shown that uptake and digestion of protein by the visceral yolk sac supplies almost all of the amino acid neededby the 9.5–11.5-day rat conceptus cultured in vitro. Our aim was to test the hypothesis that protein uptake and digestion may not be as important as an amino acid source in the 8.5–9.5-day period, a stage of development before the yolk sac placenta envelops the embryo and before the vitelline circulation is established. Eight and a half-day rat conceptuses were cultured in serum supplemented with trace amounts of free [ 3H]leucine, [ 3H]leucine-containing serum proteins, free [3H]methionine or [ 3H]methionine-containing serum proteins. The incorporation of radiolabelled amino acid into acid-soluble and acid-insoluble fractions of the conceptus was determined. Leucine from either source was incorporated principally into proteins of the conceptus, but a greater proportion of the methionine incorporated was found in the low molecular weight fraction. It is estimated that 88 per cent of the leucine and 96 per cent of the methionine used by the conceptus was derived from protein in the culture serum; free amino acid comprised a minor supply source. We conclude that, despite the different anatomic relationships, the majority of amino acid incorporated into newly synthesized proteins of the conceptus very early in organogenesis is supplied by the digestion of protein in extraembryonic tissue, most likely the visceral yolk sac.

Changes in structure of the trophectoderm of a marsupial in Mid-pregnancy up to the time of implantation.

Pre- and peri-implantation embryos of the dasyurid marsupial Sminthopsis crassicaudata were examined for morphological differentiation of the trophectoderm. The cells of unilaminar blastocysts were all squamous and stained intensely with toluidine blue. In bilaminar blastocysts and embryos at the early embryonic-disc stage, the trophectoderm was similar in appearance to, but stained more lightly than, the underlying endoderm. Trophoblast differentiation did not appear to occur until the mesoderm had begun to migrate between the trophoblast and endoderm beyond the embryonic disc. At this stage, trophoblasts had three distinct morphologies: (1) vacuolated, tall and columnar cells in the trilaminar region; (2) large cuboidal cells in the adjacent bilaminar region; and (3) squamous cells in the abembryonic pole of the bilaminar region. These variations in cell structure correlate with differences in subsequent functional activity in these three regions of the yolk sac placenta.