Mural Trophoblast Research Articles

Expression of the c- fms proto-oncogene and of the cytokine, CSF-1, during mouse embryogenesis

The c-fms gene encodes the cell surface receptor of the colony-stimulating factor, CSF-1. CSF-1 has recently been shown to be expressed in the maternal uterine endometrium of pregnant mice. The ontogenetic and spatial patterns of expression of the murine proto-oncogene c- fms were analyzed in the developing mouse placenta by the technique of in situ hybridization. c- fms expression was not detected in fetally derived tissues until 9.5 days postcoitum (pc) when expression first appeared in the mural trophoblast giant cells. Expression persisted at high levels in trophoblast cells throughout gestation. In the mature placenta from 13.5 days pc on, c- fms was expressed chiefly in the spongiotrophoblast layer and, to a lesser extent, in the labyrinthine trophoblast. CSF-1 expression was first detectable in the uterine epithelium at 8.5 days pc which loosely correlated with the appearance at 7.5 days of c- fms in the decidual cells around the developing egg cylinder. The time course and spatial pattern of expression of these two genes suggest a functional role for the c- fms receptor and its ligand, CSF-1, in trophoblast development and differentiation.

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.

Ultrastructure of trophoblast giant cell transformation during the invasive stage of implantation of the mouse embryo.

The fine structure of abembryonic and mural trophoblast cells of mouse embryos was analyzed during the initial stages of invasion of the endometrial stroma by the embryo (days 6-8 of pregnancy). On day 6 of pregnancy, most trophoblastic cells are flat and have spindle-shaped nuclei. A few large, round trophoblastic cells (giant cells) are present at the abembryonic pole. As pregnancy proceeds through days 7 and 8, the area occupied by the trophoblast becomes larger because of an increase in the trophoblastic cell population, growth of giant cells, and rearrangement of the latter cells into a network containing maternal blood. As flat cells transform into giant cells, their content of ribosomes, granular endoplasmic reticulum, Golgi complexes, lysosomelike bodies, and heterophagosomes increases. Reichert's membrane is always lined by cell bodies or by laminar processes of trophoblastic cells that are provided with small pores. Transformation of flat cells into giant cells is associated with an activation of the giant cells and their acquisition of invasive behavior.

The dynamic structure of rabbit blastocyst coverings. II. Ultrastructural evidence for a role of the trophoblast in neozona formation.

The neozona is the innermost layer of the complex blastocyst coverings of the rabbit and is located between the mucoprotein layer and the trophoblast. As shown previously the neozona begins to replace the zona pellucida from the 4th day post coitum (d p.c.) on (Denker and Gerdes 1979). In the present study, rabbit blastocyst coverings were checked for regional differences in their composition, comparing the embryonic and the abembryonic pole of the blastocyst, at 5 and 6 d p.c. These two stages were chosen because at 5 d p.c. a complete trophoblast layer is still present at both the embryonic disc (Rauber's layer) and the extraembryonic regions (mural and abembryonic pole trophoblast), whereas at 6 d p.c. Rauber's layer has largely degenerated. Correlation of regional differences in blastocyst coverings structure with presence or absence of an intact trophoblast is taken as suggestive evidence for a role of the trophoblast in the formation or the structural modification of blastocyst coverings components. Blastocysts of both stages were fixed in glutaraldehyde with and without ruthenium red and processed for TEM. The neozona was found to be almost equally well developed in all regions at 5 d p.c. On contrast, at 6 d p.c. (Rauber's layer defective) the neozona is consistently found to be much thinner at the embryonic disc than in the extraembryonic regions where the trophoblast is still intact. This is the first report on regional differences of the structural composition of blastocyst covering within the same blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Trophoblast giant cell release of placental lactogens: Temporal and regional characteristics

Placental lactogen (PL) production by rat trophoblast giant cells was studied using in vitro methods. The influence of trophoblast giant cell location within the conceptus and day of trophoblast giant cell isolation on the type of PL released in vitro were investigated. The effect of trophoblast giant cell location on the amount of PL, progesterone, and testosterone released in vitro was also evaluated. Trophoblast giant cells release two types of PLs in vitro; a high-molecular-weight lactogen, PL-1, and a low-molecular-weight lactogen, PL-2. The type of PL released by trophoblast giant cells was not influenced by their location within the conceptus at the time of dissection. Location did influence the amount of hormone produced by trophoblast giant cells. Mural trophoblast giant cells were more active in the production of PL, progesterone, and testosterone. The type of PL released by trophoblast giant cells is highly dependent upon the day of gestation the cells are removed for study. Trophoblast giant cells isolated on Day 10 of gestation release predominantly PL-1, while those cells isolated 24 hr later (Day 11 of gestation) release predominantly PL-2. The switch from PL-1 to PL-2 production that occurs in vivo does not occur under the in vitro conditions employed in this report.

Changes in the distribution of a spectrin-like protein during development of the preimplantation mouse embryo.

The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte alpha-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.

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.

Morphodynamics of outgrowths of mouse trophoblast in the presence and absence of a monolayer of uterine epithelium.

Mouse blastocysts were cultured in the presence and absence of a confluent uterine, luminal, epithelial monolayer in order to assess (10 comparative developmental abilities and (2) the existence and nature of embryo-uterine intercellular contacts. Embryonic development and mural trophoblast outgrowth were examined by light, transmission and scanning electron microscopy, and by time-lapse cinematography. The results demonstrate that cultured mouse blastocysts are capable of development that is essentially equivalent to the early egg cylinder stage in the presence and absence of a uterine epithelial substratum. Penetration of the endometrium and displacement of uterine luminal epithelial cell in vivo is paralleled in vitro by the displacement of uterine cells from the advancing edge of outgrowing mural trophoblast. The ability of mural trophoblast to continue to outgrow and displace uterine cells persists for a finite period of time. The findings are discussed with respect ot (1) the nature of embryo-uterine contact in vivo and (2) mechanisms proposed to explain trophoblastic displacement of uterine luminal epithelial cells during normal implantation.

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.

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.

Expression of the maternally derived X chromosome in the mural trophoblast of the mouse

ABSTRACT Only the maternally derived allelic form of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1) is observed in the mural trophoblast of heterozygous female progeny in F1 and backcross matings. We have demonstrated that this expression of the maternally derived PGK-1 is not a result of maternal tissue contamination nor of selection of cells expressing the maternal X chromosome (Xm). Our results suggest that the expression of Xm in mural trophoblast is a consequence of nonrandom X-chromosome inactivation in trophectoderm cells.

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.