Polar Trophoblast Research Articles

Polar trophoblast (Rauber's layer) of the rabbit blastocyst

The germinal area of the rabbit blastocyst between 108 h postcoitum (pc) and 168 h pc has been examined by scanning electron and transmission electron microscopy. At 108 h and 120 h pc the polar trophoblast (Rauber's layer) is an intact epithelium overlying the epiblast of the inner cell mass. By 132 h pc the polar trophoblast cells begin to separate at multiple foci, exposing the underlying epiblast. Most of the polar trophoblast cells have become individually separated at 144 h pc. The villous, electron-dense polar trophoblast cells can be easily distinguished from the cells of the epiblast, which have smooth apical surfaces. By 162 h pc the polar trophoblast cells have disappeared from the germinal area. Before the polar trophoblast breaks up, the underlying epiblast cells are only loosely attached to one another. Concurrent with the disintegration of the trophoblast epithelium, the epiblast cells change in shape so that their lateral borders become closely apposed, and junctions develop to form a new epithelium. The epiblast becomes contiguous with the mural trophoblast, and thus the blastocyst does not lose its turgidity as the permeability seal is maintained. There are two classical theories on the fate of the polar trophoblast: the cells die, or they become incorporated into the epiblast as living cells. In newly exposed epiblast the presence of very large phagosomes, which are not found when the polar trophoblast is still intact, favors the first hypothesis and indicates that in the rabbit the epiblast is involved in the phagocytosis of the polar trophoblast.

Differentiation of trophoblast of the baboon blastocyst.

The structure of trophoblast of the baboon blastocyst undergoes a number of maturational changes from the early blastocyst to the late blastocyst stage. The striking expansion of the blastocyst that occurs during the preimplantation period is accompanied by the development of an extensive endocytic apparatus. Cationized ferritin labels coated depressions and vesicles near the apical cell surface, numerous uncoated tubules and larger apical vesicles, and multivesicular bodies within trophoblast cells. Basally and laterally the labeled components are primarily small uncoated vesicles and tubules. Small, discrete clusters of ferritin particles were seen within the basolateral compartment between trophoblast and its basal lamina and beneath trophoblast cells that do not have a basal lamina. the results indicate that ingested materials may be directed in two pathways, one involving breakdown within the lysosomal system and one involving transcytosis. The zona pellucida is a trilaminar structure consisting of a fibrillar outer layer that often contains spermatozoa, an intermediate zone, and an inner layer containing columns of dense zonal material. Loss of the zona occurs after expansion of the blastocyst and development of the endocytic organelles. During the late blastocyst stage, syncytial trophoblast differentiates at the margin of the polar trophoblast. Because blastocysts were flushed from the uterus, it could not be determined whether azonal blastocysts had been adherent to the uterine surface prior to collection.

DNA synthesis in the mouse blastocyst during the beginning of delayed implantation.

The spatiotemporal pattern of DNA synthesis in the mouse embryo at the beginning of metabolic dormancy was examined. Embryos were recovered from females at intervals following ovariectomy at 1100 hours on day 4 of pregnancy, incubated in vitro for 1 h in the presence of [3H]thymidine, and prepared for light microscopic autoradiography. The proportion of labeled cells in the embryo remained high (40-60%) for 18 h after ovariectomy and then declined gradually to 12% by 96 h. However, analysis of individual cell subpopulations showed that the decline was not uniform in all regions of the blastocyst. Labeling was high over the inner cell mass (ICM) during all time intervals in the study, while labeling over the mural trophoblast cells declined sharply by 24 h after ovariectomy. Labeling over the polar trophoblast also declined but had values that were intermediate between the ICM and mural trophoblast regions of the blastocyst. These regional differences in DNA synthesis during the arrest of development suggest that intermediate steps are involved in control of DNA synthesis in the embryo and that the ICM may play a role in the different responses of the trophoblast cell populations.

Loss of polar trophoblast during differentiation of the blastocyst of the horse.

Twelve blastocysts, collected 7-12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to greater than 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cells, forming an irregular layer of cells capping the epiblast. With continued increase in blastocyst size, polar trophoblast cells became isolated but maintained their characteristic apical endocytic structures. By Days 10-12, the scattered trophoblast cells showed evidence of deterioration, and vacuoles containing cell debris were common within the epiblast. It is suggested that polar trophoblast cells become scattered, rather than withdrawing as a unit, because they become more adherent to subjacent epiblast cells than to adjacent trophoblast cells. It is further suggested that most of the isolated cells are eventually phagocytosed by epiblast cells.

Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts.

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.

Morphology of mouse embryo cultured in a newly established culture system using collagen gel layer.

A new in vitro culture system of embryo using collagen gel as the substrate was developed, in which mouse blastocysts were cultured. Morphological analyses including electron microscopic observation disclosed that after hatchig, the mouse blastocysts were attached to the collagen gel layer and were satisfactorily developed and differentiated. In the embryo observed on the 3rd or 4th day after culture, cellular processes and villi had penetrated the collagen gel from the mural trophoblast. While there were many vacuoles in the cytoplasm and a large number of destroyed cells in the mural trophoblast mass, some steroid producing cells were also observed. The polar trophoblasts, though having a small number of vacuoles, partially exhibited characteristic activities of steroid production. Between the mural and the polar trophoblasts, a sort of desmosome or intermediate junction was observed. There were no morphological differences among the cells derived from the inner cell mass. From these results, it was concluded that this new embryo culture system using collagen gel layer as the substrate can be of value in studies on development and differentiation of the embryo; moreover, this system can be used to replicate several phenomena similar to those appearing in in utero implantation.

Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation.

Blastocysts collected from the spotted skunk during delay of implantation, early activation and late activation demonstrate three-tiered growth and developmental changes. The slow-growing blastocyst from the several months of delay is small (less than 1.1 mm) with a rounded inner cell mass consisting of clusters of rounded, lipid-filled cells. During the several days of early activation, the lipid in both inner cell mass and trophoblast diminishes, polyribosomes increase in number, and the endodermal layer differentiates as the blastocyst grows (1.2-1.6 mm). At activation the inner cell mass flattens, becomes uncovered by polar trophoblast, and forms a disc of columnar epiblast cells. The blastocyst expands rapidly during the last 24-48 h prior to implantation to 1.7-2.0 mm, and the trophoblast becomes cuboidal with a marked endocytotic apparatus. The morphological evidence, together with previous studies of protein and RNA synthesis, suggests a tooling-up period during early activation with progressive increases in rates of growth and differentiation in the last hours as implantation approaches.

Blastocyst attachment and morphogenesis of ectoplacental cone in mouse

Mouse blastocyst attaches on the antimesometrial side of the uterus through mural trophoblasts. Later the polar trophoblasts begin proliferation, and rapid multiplication towards the mesometrial side of the uterus occurs resulting in the formation of an excrescence designated as ectoplacental cone. The morphogenesis of ectoplacental cone, viewedin utero, initiates on day 6post-coitum when microvilli of the trophoblast and the uterine epithelial cells are lost and as a result of this opposing membranes appear interlocked with each other. Soon following the invasion by surrounding trophoblasts the necrosis of the epithelial cells starts. Mitochondriae of the epithelial cells, at this stage, are shrunken and lack well defined cristae. Several leucocytes are seen at the site and few electron dense structures appear wedged between the trophoblasts and epithelial cells. At places the cell membrane is studded with the basement membrane of the uterine epithelium giving an impression of a bristle coated membrane. By day 7post-coitum the basement membrane has almost disappeared leaving trophoblast cells to develop close contact with stromal cells. Collagen fibres appeared between the trophoblasts and the stromal cells, many large inclusions of high electron density representing engulfed necrotic epithelial cells are discernible. On day 8post-coitum the ectoplacental cone is fully developed. Four types of trophoblast cells can be identified in it: (i) basal cells lying on the base of the cone, are polyhedral and compactly arranged. They have a large nucleus and well developed nucleoli, (ii) central cells forming the middle area of the cone are of two types; one contained several osmiophilic granules enclosing translucent area (eccentric) and a well developed golgi complex around the nucleus, while the other has many heterophagosomes, vacuoles and residual bodies and (iii) peripheral cells contained several pleomorphic structures resembling secondary lysosomes. Minute dense granules and band of microfibrils on the apical region of these cells are seen. Dense granules probably release lytic proteins at the site and microfibrils help in forming cytoplasmic projections.

Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material

A human autoantibody from a schleroderma patient was found to immunostain interphase and mitotic centrosomes in a variety of vertebrate cells. Electron microscopic immunocytochemistry localized this antigen in dense pericentriolar material (PCM) surrounding the centrioles. The meiotic spindle of the mouse egg has no centriole but it exhibited a broad PCM band at each pole. This pattern was also found from the first through fourth mitotic divisions. During this time PCM was found assembled at a single locus in the cell and exclusively in mitotic cells; it was not observable in interphase cells. In the blastocyst, only polar trophoblast cells had characteristic centrosomes throughout the cell cycle. Results suggest PCM can exist, disperse, and reorganize during the cell cycle independently of the centriole, and its distribution in the embryo differs in cells having different fates.

Resumption of DNA synthesis in delayed implanting mouse blastocysts during activation in vitro.

The resumption of DNA synthesis in delayed implanting mouse embryos undergoing metabolic activation in vitro was examined. Blastocysts were recovered from ovariectomized mice, incubated for various intervals in basal Eagle's medium, exposed to 3H-thymidine, and prepared for light microscopic autoradiography. Following incubation the proportion of labeled cells increased from 4% at 1 hr to 30% by 24 hr. This increase in labeling was not uniform in all regions of the blastocyst, i.e., labeling was initially highest over the inner cell mass (ICM) but remained low over the polar and proximal mural trophoblast for 6 and 12 hr, respectively, and then began to increase. This pattern in the resumption of DNA synthesis during activation in vitro is similar to that reported in vivo (Given and Weitlauf, '81) and suggests that the mechanism responsible in intrinsic to the blastocyst rather than being a differential response to the intrauterine milieu. Furthermore, it appears that the ICM may play an essential role in the resumption of synthesis in the surrounding trophoblast.

Resumption of DNA synthesis during activation of delayed implanting mouse blastocysts.

Cell division ceases in mouse blastocysts during the extended dormant period associated with delayed implantation but resumes following activation of the embryos by administration of 17 beta-estradiol to the mother. To determine the temporal and spatial aspects of the resumption of DNA synthesis during activation, blastocysts were recovered from delayed implanting females at various intervals after an injection of 17 beta-estradiol, incubated with 3H-thymidine in vitro, and prepared for light microscopic autoradiography. Although less than 4% of the cells were labeled in delayed implanting embryos, the proportion of labeled cells increased soon after the administration of 17 beta-estradiol and reached a maximum of over 50% by 24 hours. This increase in labeling was not uniform in all regions of the embryo, i.e., the labeling index of the inner cell mass began a steady increase immediately after the injection of 17 beta-estradiol while labeling of the polar and proximal mural trophoblast remained depressed for 6 and 12 hours, respectively, and only then began to increase. No labeling was present over the distal mural trophoblast in delayed implanting or activated blastocysts although cytological changes characteristic of primary giant cell transformation were present in activated embryos. These results indicate that the resumption of DNA synthesis is part of the overall increase in metabolic activity associated with activation. Furthermore, the sequential pattern of resumption of synthesis suggests that the ICM may influence the initiation of DNA synthesis in the surrounding trophoblast.

Differentiation of the Blastocyst of the rhesus monkey.

A method of flushing the oviduct and/or uterus of rhesus monkeys was used to obtain a number of preimplantation stages, of which four cleavage stages and seven blastocysts that were judged to be normal were studied cytologically using transmission electron microscopy. In addition to the usual changes in mitochondria and endoplasmic reticulum that accompany differentiation of the blastomeres, the blastocysts with zonae showed sequestration of areas of cytoplasm. The first indications of junctional complexes were short stretches of parallel membrane with a slightly increased density found in the morula stage. Blastocysts developed typical apical junctional complexes, but in addition showed extensive gap junctions linking trophoblast and inner cell mass, and epiblast and differentiating endoderm. Endodermal differentiation occurred at about the same time that a basal lamina was found under mural trophoblast and epiblast (but not polar trophoblast or endoderm). Enlarged torn zonae were found in association with one blastocyst and unaccompanied by blastocysts, including a case in which the animal subsequently prove to be pregnant. This observation suggests that hatching is a normal feature of zonal escape in this species. The trophoblast of blastocysts without zonae had well-formed apical absorptive areas and, in some instances, long irregular microvilli in the area near the inner cell mass. Cell debris, vacuoles containing debris and isolated cells, although variable, were common features of the preimplantation stage in the rhesus monkey.

Electron microscopic studies of chimeric blastocysts experimentally produced by aggregating blastomeres of rat and mouse embryos

Xenogeneic chimeras between rat and mouse were produced by aggregating embryos at the 8- to 12-cell stages. Of 114 combinations we have made so far, 26 chimeric embryos developed into blastocysts. The origin of each cell in the composite embryos can be identified unambiguously by the ultrastructural appearance of the cytoplasmic inclusions. Both the rat- and the mouse-derived cells differentiated equally well into either ICM or trophoblast cells. However, the mouse-derived cells gave rise to ICM cells more frequently than the rat-derived cells. Furthermore, when the ratderived cells formed trophoblast cells, they were predominantly mural trophoblast cells, while the mouse-derived cells differentiated predominantly into the polar trophoblast cells. Cells of the same species tend to remain as a group in the chimeric blastocysts.

Monozygotic twin formation in mouse embryos in vitro.

Monozygotic twins developed from cultured murine blastocysts at the ratio of approximately 1:100. The locus at which the denuded blastocysts attached to the culture dish was usually a random section of their mural trophoblasts, in which case single egg cylinders developed unilaterally. However, in those few blastocysts attaching with their antipolar mural trophoblasts, the inner cell mass became subdivided into two parts because of restrictions imposed on its growth by the apically situated polar trophoblasts and the plastic substrate. Each subdivision apparently incorporated totipotent cells, resulting in the bilateral formation of two egg cylinders sharing the same ectoplacental cone.

Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage

Correlative scanning electron, transmission electron, and light microscopy were utilized to study the morphogenic events occurring during mouse blastocyst outgrowth and early-egg-cylinder development in vitro. After hatching and attachment of blastocysts on the plastic surface, the blastocoelic cavity collapses as the mural trophoblasts spread and migrate outward. The inner cell mass is covered with a differentiated endoderm on the blastocoelic cavity side and by the polar trophoblasts on the medium side at this stage. As the endoderm-covered inner cell mass proliferates, being physically restricted from further downward expansion by the plastic coverslip and by lack of space in the collapsed blastocoelic cavity, it migrates upward and protrudes into the culture medium in a break between the polar and mural trophoblast cells. Polar trophoblast cells apposed to the base of the egg cylinder continue to proliferate forming the ectoplacental cone. Thus, the early egg cylinder lacking a trophoblast barrier begins inverting its growth pattern from towards the culture dish surface to a more upright position. Egg-cylinder development in vitro from the inner cell mass and polar trophoblast cells closely paralleled in vivo. The functional nature of various embryonic cell types observed in these embryos was revealed by scanning electron microscopy. These studies as well as those of Wiley and Pedersen (1977) suggest that blastocysts can serve as a source of in vitro developing early mouse egg cylinders that appear to resemble their in vivo counterparts and can be used in experimental studies of mouse embryogenesis.

Maternal X chromosome expression in mouse chorionic ectoderm

AbstractAn electrophoretic variant of the X‐linked enzyme phosphoglycerate kinase (PGK‐1) has been used to study regulation of X chromosome expression in the diploid derivatives of the trophectoderm at 8–8.5 days post coitum in the mouse. These derivatives included the chorionic ectoderm and the polar trophoblast. The biochemical analysis suggests that only the maternally derived X chromosome (Xm) is expressed in the diploid trophectoderm derivatives. Cell selection and maternal tissue contamination were ruled out as possible causes of the observed Xm expression. From these and other results, we conclude that all derivatives of the trophectoderm, along with the primitive endoderm, express only Xm, whereas derivatives of the primitive ectoderm show random X chromosome expression.

Morphology of mouse egg cylinder development in vitro: A light and electron microscopic study

AbstractPhase contrast, light, and electron microscopy showed that mouse egg cylinder development from the blastocyst stage in vitro closely resembled the egg cylinder stages of the fourth to seventh days of gestation in vivo. Primary endoderm and ectoderm formed on day 2 of culture, and on day 3 visceral and parietal endoderm could be distinguinshed. Mesoderm formed on days 6.5‐7 by delaminating from a thickened region of embryonic ectoderm. Extraembryonic ectoderm developed as single layer of cuboidal epithelial cells that appeared to originate from polar trophoblast cells. By day 8 of culture egg cylinders had an ectoplacental cavity, an exocoelom, a proamnionic cavity, a chorion, and an allantois and resembled egg cylinders in vivo after seven days' gestation. However, cultured blastocysts took longer to develop to these egg cylinder stages than embryos in vivo (8 days in vitro vs. 4 days in vivo) and were shorter (600 μm in vitro vs. 700 μm in vivo). The development of mural trophoblast, parietal endoderm, Reichert's membrane, and ectoplacental cone also differed from that of their counterparts in vivo. Nevertheless, because the egg cylinder itself, which produces the embryo proper, develops similarly in vitro as in vivo, we suggest that cultured mouse blastocysts provide an excellent model for studies on early postimplantation embryogenesis.

Development of trophoblast and placenta of the mouse

At 5 days post conceptionem (p.c.) shortly after implantation, giant cell transformation starts at the abembryonic pole of the blastocyst, spreading over the mural trophoblast; 1 day later, the first ectoplacental giant cells appear at the base of the fast growing ectoplacental cone (derived from the polar trophoblast). Giant cell transformation expands over it periphery. Thus, by the 8th day p.c., the conceptus is separated from the maternal tissue by a continuous layer of giant cells, variable in thickness. Giant cells reach their greatest size by 10 days p.c. in the mural tophoblast and by 12 days p.c. in the chorioallantoic placenta. They are probably no longer formed after that stage. Around the 8th day p.c., the allantois reaches contact with the ectoplacental cone, which develops into the chorioallantoic (definitive) placenta. At 9 days p.c., its four zones can already be discriminated: chorionic plate, labyrinth, junctional zone (trophospongium), and zone of giant cells, respectively. Within the next day, the chorioallantoic placental circulation is established. The yolk sac placental circulation is established by the 9th day p.c. The villi of the proximal layer of the yolk sac increase in size and number, and their capillary network becomes more dense until the 12th to 14th day p.c. This provides evidence that the yolk sac placenta exerts its function--to a certain extent--beyond the establishment of the definitive placenta. Around the 14th day p.c., the placental labyrinth reaches its definitive features. Fetal capillaries in the labyrinth, branching from unbilical blood vessels within the septa of connective tissue are surrounded by trophoblast cells. They form a dense vascular network bathing in maternal blood. The structures of the placental zones remain almost the same during further development, the borders becoming sometimes little blurred. Adjacent to the chorionic plate, subchorionic clefts appear at the 14th day p.c. These clefts become confluent to form the intraplacental space, regularly communicating with the yolk sac cavity. At the end of gestation (19th day p.c.) there is a considerable amount of eosinophilic material ('fibrinoid') between the zone of giant cells and the decidua, probably produced by the giant cells.

Implantation and early postimplantation development of the bank vole Clethrionomys glareolus, Schreber

ABSTRACT The development of the bank vole Clethrionomys glareolus is described from implantation to the formation of the foetal membranes. The embryonic development of this species combines features of primitive rodent species, for example Geomys bursarius and highly specialized ones, for example Mus musculus. The egg-cylinder is formed by invagination into the blastocoelic cavity of the inner cell mass and polar trophoblast overlying it; this resembles in many respects the early stages of development of primitive species. The fully formed egg-cylinder, however, resembles that of the mouse and the formation of foetal membranes is also similar to that in Muridae. It is concluded that in the bank vole and also in other rodents, the extra-embryonic ectoderm of the egg-cylinder is derived from the polar trophoblast rather than from the inner cell mass.

The development of primordial and definitive amniotic cavities in early Rhesus monkey and human embryos.

Re-examination of early rhesus monkey and human embryos in the collection of the Carnegie Institution of Washington suggests that the mechanism of amniogenesis in both is basically similar to that of the hedgehog and vespertilionid bats. A primordial amniotic cavity develops by cavitation within the embryonic mass of 10-day rhesus monkey, and 7-day human, blastocysts. This primordial cavity has no relationship initially with the overlying trophoblast, contrary to earlier reports. Subsequently, there is a thinning and peripheral spreading of the epiblastic roof of the primordial cavity, resulting in partial opening of the roof and formation of a slightly cupped embryonic disc. The resulting space is not homologous with the primordial amniotic cavity; instead, it is a transitory tropho-epiblastic cavity. The definitive amniotic epithelium forms by the upfolding and mitotic proliferation of the margins of the epiblastic disc; this process is completed in 11-day rhesus, and 9-day human, blastocysts. Amniogenesis by cavitation is associated with the persistence of polar trophoblast following implantation, and it is suggested that this cavitation process may be essential for providing a free epithelial surface for the morphogenetic movement of epiblastic cells during subsequent formation of the primitive streak.