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.     

Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~ E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.     

Giant cell formation in ectopic mouse trophoblast

Segmenting mouse ova, grafted beneath the kidney capsule of syngenic adult recipients, result in a growth of trophoblast, which changes from small, actively-dividing cells into giant trophoblast cells which degenerate 15 days after grafting. Similar giant cells are found in normal mouse placentas. Radioautography with ³H-thymidine, uridine, and leucine revealed cessation of DNA synthesis after day 8, with decline in RNA synthesis from day 10, and continued protein synthesis through day 15. Treatment with Colcemid reduced the graft size but failed to suppress giant cell formation. Treatment on days 4–7 of grafting with 5-fluorodeoxyuridine (FUdR), cyclohexamide, or actinomycin D resulted in giant cell suppression with the maintenance of healthy-appearing small trophoblast cells. These results confirm the early withdrawal of trophoblast grafts from the mitotic pool and the non-mitotic increase of trophoblast DNA, and demonstrate the apparent need for RNA and protein synthesis to support the development of trophoblast giant cells.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

In vitro selection and regeneration of cotton resistant to high temperature stress

Summary Cell suspension cultures of cotton (Gossypium hitirsutum L. cv. Coker 312) were exposed to various temperature:time treatments in order to select cell lines resistant to high temperature stress. Cells were exposed to 45°C for 3 h each day until the total accumulated hours of stress were: 0 h, 10 h, 75 h, 100 h, or 105 h (81 h pulsed then 24 h continuous). After the stress treatments, the cells were plated onto embryo development medium and plants were recovered. The embryogenic calli that were recovered were subcultured monthly for 6 months and tested for increased resistance to the temperature:time treatments previously determined to be lethal and to water stress as imposed by PEG. All of the selected cell lines were more resistant to both types of stress than the control cell lines. Leaf tissue of stress selected (Ro) formed and maintained callus growth when incubated at 38°C; whereas, tissue excised from nonselected controls rarely formed callus and calli which did form quickly became necrotic. These cells and plants will provide a tool for determining the mechanisms involved in resistance to high temperature stress.     

Development of Eimeria auburnensis in Cell Cultures

SYNOPSIS. Monolayer established cell line cultures of bovine kidney (Madin-Darby) and human intestine (Intestine 407), as well as embryonic bovine tracheal and embryonic spleen cell line cultures were inoculated with E. auburnensis sporozoites and observed for a maximum of 22 days. Mature 1st generation schizonts developed in the kidney, tracheal and spleen cells. In the intestine cells, trophozoites were seen in 3 of 4 experiments, but schizonts were not found. Sporozoites penetrated cells, beginning within a few minutes after inoculation. Penetration was usually accomplished within 10 seconds, and the body of the sporozoite underwent a slight constriction as it passed thru the host cell membrane. Some sporozoites left cells. Numerous intracellular sporozoites were observed in kidney, tracheal and spleen cultures. Crescent bodies were seen in the parasitophorous vacuole as early as 1 day after inoculation. At this time, the nuclei of most intracellular sporozoites had changed from vesicular to compact. Beginning 4 days after inoculation, enlarged sporozoites and parasites having a sporozoite shape, but with 2-5 nuclei, were frequently seen. These enlarged sporozoites and sporozoite-shaped schizonts evidently transformed into trophozoites and spheroidal schizonts by means of lateral outpocketings. Few trophozoites were seen. More immature schizonts developed in kidney cells than in the other cell types. The numbers of mature schizonts observed in kidney and tracheal cells were similar, but development occurred less consistently in the latter. Few immature and mature schizonts developed in spleen cells. Mature schizonts, first seen 9 days after inoculation, were considerably smaller than those reported from calves. Some motile merozoites were seen; evidently no development beyond these occurred. The nucleus and nucleolus of host cells were enlarged; this enlargement was not as pronounced as in infections in calves. Multiple host cell nuclei were frequently observed. Degenerative changes in the cultured cells and in the parasites usually occurred, beginning 9-17 days after inoculation; these were more pronounced in the spleen cells than in the others.     

Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.     

Altered downstream target gene expression of the placental Vitamin D receptor in human idiopathic fetal growth restriction

Fetal growth restriction (FGR) affects up to 5% of pregnancies and is associated with significant perinatal complications. Maternal deficiency of vitamin D, a secosteroid hormone, is common in FGR-affected pregnancies. We recently demonstrated that decreased expression of the vitamin D receptor (VDR) in idiopathic FGR placentae could impair trophoblast growth. As strict regulation of cell-cycle genes in trophoblast cells is critical for optimal feto-placental growth, we hypothesised that pathologically decreased placental VDR contributes to aberrant regulation of cell-cycle genes. The study aims were to (i) identify the downstream cell-cycle regulatory genes of VDR in trophoblast cells, and (ii) determine if expression was changed in cases of FGR. Targeted cell-cycle gene cDNA arrays were used to screen for downstream targets of VDR in VDR siRNA-transfected BeWo and HTR-8/SVneo trophoblast-derived cell lines, and in third trimester placentae from FGR and gestation-matched control pregnancies (n = 25 each). The six candidate genes identified were CDKN2A, CDKN2D, HDAC4, HDAC6, TGFB2 and TGFB3. TGFB3 was prioritised for further validation, as its expression is largely unknown in FGR. Significantly reduced mRNA and protein expression of TGFB3 was verified in FGR placentae and the BeWo and HTR-8/SVneo trophoblast cell lines, using real-time PCR and immunoblotting respectively. In summary, decreased placental VDR expression alters the expression of regulatory cell-cycle genes in FGR placentae. Aberrant regulation of cell-cycle genes in the placental trophoblast cells may constitute a mechanistic pathway by which decreased placental VDR reduces feto-placental growth.     

Quantitative investigation of reproduction of condensed chromatin of sex chromosomes during trophoblast cell polyploidization and endoreduplication in the East European field vole Microtus rossiaemeridionalis

Simultaneous measurement of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB) has been performed in two trophoblast cell populations of the East-european field vole Microtus rossiaemeridionalis, namely in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two gonosomal chromatin bodies have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female), In the proliferative trophoblast cell population, characterized by low ploidy levels (2c-16c), and in the highly polyploid population of secondary giant trophoblast cells (16c-256c), the total DNA content in GCB increased proportionally to the ploidy level. In separate bodies, the DNA content rose also in direct proportion with the ploidy level seen in the nuclei with both one and two GCBs in the two trophoblast cell populations. A certain increase in percentage of the nuclei with 2-3 GCBs was shown in the nuclei of the junctional zone of placenta; this may be accounted for by genome multiplication via uncompleted mitoses. In the secondary giant trophoblast cell nuclei (16c-256c), the number of GCBs did not exceed 2, and the share of nuclei with two GCBs did not increase, thus suggesting the polytene nature of sex chromosome in these cells. At different poloidy levels, the ratio of DNA content in the nucleus to the total DNA content in GCB did not change significantly giving evidence of a regular replication of sex chromosomes in each cycle of genome reproduction. In all classes of ploidy, the mean total DNA content in trophoblast cell nuclei with single heterochromatic body was less than in the nuclei with two and more GCBs. This may indicate that a single GCB in many cases does not derive from the fusion of two GCBs. To put it another way, in the nuclei with one GCB and in those with two or more GCBs, different chromosome regions may undergo heterochromatization. The regularities observed here are, most probably, associated with the peculiarities in the structure of X- and Y-chromosomes in a range of species of Microtus (M. agrestis, M. rossiaemeridionalis, M. transcaspicus). As a result, gonosomal chromatin bodies may include large blocks of both constitutive heterochromatin of X- and Y-chromosomes (in male and female embryos) and inactivated euchromatin of "lyonized" X-chromosome in female embryos. Therefore the presence of two or more GCBs in trophoblast cells of M. rossiaemeridionalis may be accounted for by both polyploidy and functional state of the nucleus, in which gonosomal constitutive heterochromatin and inactivated euchromatin form two large chromocenters rather than one. The differences in DNA content in GCBs in the nuclei with one and two GCBs seem to be an indirect indication that the two chromocenters may be formed by two different gonosomes, with the extent of their heterochromatization being higher than that in the nuclei with one GCB. GCBs in the trophoblast cells of M. rossiaemeridionalis are observed not only at the early developmental stages, as it was observed in rat at the first half of pregnancy (Zybina and Mosjan, 1967), but also at the later stages, up to the 17th day of gestation. At these stages, the nuclei with non-classical polytene chromosomes rearrange to those with a great number of endochromosomes, probably because of disintegration of chromosomes into oligotene fibrils. However, it does not seem unlikely that this process may involve heterochromatized gonosomal bodies, since only one or two large GCBs can be seen in the nuclei as before. The presence of prominent blocks of constitutive heterochromatin seems to favor a closer association of sister chromatids in polytene chromosomes, which prevents their dissociation into endochromosomes with the result that polyteny of sex chromosomes in the field vole trophoblast is probably retained during a longer period of embryonic development.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Genetic insights into trophoblast differentiation and placental morphogenesis

The placenta is comprised of an inner vascular network covered by an outer epithelium, called trophoblast, all designed to promote the delivery of nutrients to the fetus. Several specialized trophoblast cell subtypes arise during development to promote this function, including cells that invade the uterus to promote maternal blood flow to the implantation site, and other cells that fuse into a syncytium, expand and fold to increase the surface area for efficient transport. Mutation of many genes in mice results in embryonic mortality or fetal growth restriction due to defects in placental development. Several important principles about placental development have emerged from these studies. First, distinct molecular pathways regulate the differentiation of the various trophoblast cell subtypes. Second, trophoblast proliferation, differentiation and morphogenesis are highly regulated by interactions with adjacent cell types. Finally, the specific classes of mutant phenotypes observed in the placenta of knockout mice resemble those seen in humans that are associated with preeclampsia and intrauterine growth restriction.     

Transfection with Different Connexin Genes Alters Growth and Differentiation of Human Choriocarcinoma Cells

To examine the role of cell–cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell–cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively.In vitrogrowth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by β-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of β-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell–cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects.     

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.     

Identification and characterization of monolayer cultures of sheep trophoblast cells maintained in bicameral culture chambers

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

Endogenous formation of prostanoids and the levels of long-chain fatty acyl CoA derivatives in suspensive and adhesive cells from in vitro embryoid bodies of teratoma OTT 6050

Attachment of the cell surface to a substratum may play a critical role in initiating some cellular developmental commitments and in sustaining differentiation of cells that have already been specialized. Embryoid bodies of teratoma OTT6050 were divided, on day 10 of initial culture, into myogenic adhesive cells which were already (at day 6) characterized by endogenous prostaglandin (PG)I₂ formation and little-specialized suspensive cells which formed only thromboxane (TX)B₂ in the same culture system. Since at day 10 both cell types reached a stationary phase in which the nature of each cell was mature enough for the analyses with mass fragmentographic technique and gas chromatography- mass spectrometry (GCMS), the total levels of predominant long-chain fatty acyl CoA (acyl CoA) derivatives could be measured comparatively as methyl esters after methanolysis. It was found as a result of major differentiation that adhesive cells had a rather low ratio of arachidonyl CoA to stearyl CoA, although adhesive cells accumulated a larger total amount of acyl CoA derivatives than that accumulated in suspensive cells.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

Ontogenetic development of neural responses in the olfactory bulb of laboratory mice

Summary In laboratory mice (strain NMRI) the ontogenetic development of single unit activity in the olfactory bulb was investigated. From postnatal day 10 on, spontaneously active neurons were recorded with glass-microelectrodes, and their responses to olfactory stimuli were tested (butyric acid, geraniol, grass- and nest-odour).From day 10 to 13 only very few neurons were recordable (and most of these elements were too weak and were lost before being stimulated). At day 14 the number of recordable neurons increased rapidly, and by day 15 spontaneously active neurons reached adult level in terms of incidence and electric properties.There were 3 types of neurons: 1. respiration synchronous elements; 2. bursting neurons not correlated with respiration; 3. continuously, but randomly, firing elements (about 60% of all neurons). Reactions to odour stimuli (excitation, ca. 50%; inhibition, ca. 34%; complex reactions, ca. 12%; change in activity pattern, ca. 4%) occurred as soon as the cells were stable enough for testing. The reaction patterns showed no age specific differences; the duration of the responses varied from 100 ms to 100 s.In younger animals (P11–P14) the percentage of responses was slightly smaller (47%) than in the older ones (P30–P50; 64% response to olfactory stimulation). For some of the odours tested the proportion of responding cells differed depending on age (for instance grass odour evoked a response in 40% of the cells in young ones, but in 65% in adults).Abbreviations AP action potential - In interneuron - MTc mitral or tufted cell - P10 postnatal day 10