Electron microscopic study of the chorioallantoic placenta of the rock hyrax (Heterohyrax brucei)

The interhaemal membrane consisted of only two cellular elements: a single layer of cellular trophoblast and the fetal capillary endothelium. The hyrax is therefore one of the few mammals known to possess the cellular haemomonochorial type of placenta. The trophospongium was also cellular while the basal trophoblastic cells were strongly phagocytic. The giant multinucleate cells at the feto-maternal junction were ultrastructurally different from the trophoblast cells and showed no signs of degeneration. Their appearance suggests that they are of maternal rather than fetal origin.     

Trophoblast giant-cell differentiation involves changes in cytoskeleton and cell motility

Trophoblast giant-cell differentiation is well-characterized at the molecular level, yet very little is known about how molecular changes affect the cellular functions of trophoblast in embryo implantation. We have found, using both explanted E7.5 mouse embryo ectoplacental cone and the rat choriocarcinoma (Rcho-1) cell line, that trophoblast differentiation is distinguished by dramatic changes in cytoarchitecture and cell behavior. Undifferentiated trophoblast cells contain little organized actin and few small, peripheral focal complexes and exhibit high membrane protrusive activity, while differentiated trophoblast giant cells contain prominent stress fibers, large internal as well as peripheral focal adhesions, and become immotile. The dramatic changes in cell behavior and cytoskeletal organization of giant cells correlate with changes in the activities of the Rho family of small GTPases and a decrease in tyrosine phosphorylation of focal adhesion kinase. Together, these data provide detailed insight into the cellular properties of trophoblast giant cells and suggest that giant-cell differentiation is characterized by a transition from a motile to a specialized epithelial phenotype. Furthermore, our data support a phagocytic erosion, rather than a migratory infiltration, mechanism for trophoblast giant-cell invasion of the uterine stroma.     

Erythrophagocytosis in the caprine trophoblast

The mechanism of iron transfer by extravasation of maternal blood in the maternal-fetal interface and the subsequent phagocytosis of the erythrocytes by the trophoblast cells was described in ovine, bovine and other species. This research was performed due to the absence of studies on this process in the goat. Fragments of placentomes were obtained from 9 adult goats of an unspecified breed and were separated into Groups A, B and C, based on 90, 120 and 150 d of gestation, respectively. Fragments of 3 placentomes were obtained from each animal of all groups. The first of them was removed from the base, the second from the middle part and the third from the end of the gestational uterine horn. The fragments were fixed in Bouin solution and histologically processed according to the usual paraffin inclusion techniques. The slides were stained by hematoxilin-eosin and potassium ferrocyanide and examined under light microscopy The erythrophagocytosis (EP) process of the trophoblast was identified in all the examined histological sections, throughout the gestation and for each placentome. An accumulation of spots of blood was observed only in the maternal-fetal interface located in the arcade zone of the placentome. The main cells of the trophoblast of these areas presented an intense phagocytic activity and the observation of erythrocytes with hemossiderin pigments in their citoplasm was possible. The trophoblast epithelium of these areas was thicker and with a columnar aspect than the cubic aspect observed in other areas. The trophoblast binucleate cells do not seem to participate in EP. Our findings indicate that the phagocitosis process of the maternal erythrocites by the trophoblast also occurs in caprine species, indicating that this could be a mechanism of transplacentary transfer of iron in this species.     

Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.     

Effects of hydroxyurea on the phagocytosis of microspheres in V79 cells

Phagocytosis of fluorescent microspheres in V79 cells arrested in the S phase by hydroxyurea (HU) was investigated by flow cytometry. The phagocytic activity increased with the exposure time of HU. No dependency on the HU concentration was observed in the concentration used. Cell size (projection area) and membrane fluidity, two factors effected by HU treatment, were examined in relation to phagocytic activity, and were found to be significantly increased. The elevation in phagocytic activity could be interpreted in terms of the alteration in the two physiological factors.     

Polyploidy and polyteny in the trophoblast cells of the mink]

According to cytophotometry, trophoblast cells in the mink placenta are both diploid and polyploid, the ploidy level ranging from 2c to 64c. A great number of mink trophoblast cells were seen to divide mitotically. In addition to the ordinary mitotic figures, polyploid mitoses as well as abnormal mitotic figures were observed. Non-classic polytene chromosomes, peculiar to the mammalian trophoblast, appeared in the mink trophoblast cells to have the highest ploidy. A relatively low ploidy degree is due, probably, to a lesser invasive activity of the mink trophoblast cells as compared to the rodent giant trophoblast cells.     

Histoenzymatic and immunocytochemical characteristics of extravillous trophoblast cells of placental basal plate as parameter of their function in hypertensive pregnancy

An intense activity of enzymes which actively participate in the renin-angiotensin-aldosterone system was shown in extravillous trophoblast cells which are involved in the performing of spiral arteries into uteroplacental vessels. The hydrolase activity in villous trophoblast underwent important variations, but it was constant in cells of the extravillous trophoblast. Activity of lysosomal hydrolases, of leucine aminopeptidase and N-acetyl glucosaminidase type, was markedly positive in X-cells, while negative in the villous trophoblast. Beta glucuronidase activity has shown moderate activity in cells of extravillous trophoblast, while in villous trophoblast it was weakly emphasized or negative. Intense activity of prostaglandin E2 dehydrogenase in the way of strongly emphasized microsomal reaction was noted exclusively in extravillous cells of basal plate, especially in perivascular cell groupings. Within all examined enzymes activities, only the membranous activity of alkaline phosphatase was of the same intensity in cells of extravillous trophoblast. Lacking of penetration of these cells into the spiral arteries wall in EPH-gestosis, which also means loss of their close contact with the blood of a pregnant, implicates the practical meaning of these observations.     

Phagocytic activities of the gorgonian coral Swiftia exserta

The cellular response component of body defense in gorgonians and other cnidarians is thought to be carried out by cells with phagocytic capabilities. To test for the phagocytic character of cells, the introduction of foreign particles was employed and observed in both living cells and histological preparations of the gorgonian coral Swiftia exserta. Observations of untreated tissues revealed normal cells and tissue morphologies. A microscopic observation of living cells following the introduction of particles in a cut revealed that only a mixed population of colorless cells phagocytized the particles. Also particles or clumps of particles were seen on the surface of the colorless cells. Subsequent histological observations allowed identity of colorless cells to be inferred as granular amoebocytes, ectodermal cells, and gastrodermal cells. Cells stained for localization of peroxidase (indicative of phagocytic activity) demonstrated the presence of peroxidase-positive cells. Histological preparations revealed that major phagocytosis of particles was associated with tissue trauma. When particles were introduced by means of a cut or inserted thread, phagocytic activity was detected within 2 h. However, it was confined to the granular amoebocytes in the immediate site of trauma. After 24 h, extensive phagocytosis spread throughout a relatively large area surrounding the wound. At that later time, phagocytic cell types included granular amoebocytes, epidermal cells, sclerocytes, mesogleal cells, and gastrodermal cells of the solenia. Observations suggest that trauma induces phagocytosis in cells not normally phagocytic in S. exserta. No localization of phagocytic cells and no mitotic cells were observed at either 2 or 24 h after particle introduction.     

Murine trophoblast resists cell-mediated lysis. II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.     

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.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells.

The ability of cultured midgestation mouse trophoblast cells to synthesize progesterone from pregnenolone has been monitored by radioimmunoassay or chromatography and crystallization. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of Δ⁵,3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Since there is little or no further metabolism of the progesterone formed, the ability of trophoblast cells to convert pregnenolone to progesterone in vitro is an accurate reflection of the activity of 3β-HSD in these cells.Midgestation cultures of giant trophoblast cells have a substantially higher 3β-HSD specific activity than the smaller ectoplacental cone cells. Neither giant trophoblast nor ectoplacental cone cell cultures show an increased 3β-HSD specific activity in response to a variety of hormones, including gonadotrophins. In fact, regardless of the gestation age at which the trophoblast cultures are initiated, 3β-HSD activity inevitably follows the same temporal pattern observed in vivo. Taken together, these facts suggest that the levels of 3β-HSD in trophoblast cells are intrinsically controlled and that, unlike the ovary, progesterone production by trophoblast cells in vivo is not influenced by gonadotrophic hormone levels.     

Changes in the leukocyte phagocytic activity of donor blood after its UV irradiation. I. Its relation to the irradiation dose and initial level of phagocytic activity

Phagocytic activity of human mono- and granulocytes increased markedly after UV blood irradiation in the apparatus "Izolda" used in hospitals of the USSR for medical treatment. With the rise of irradiation dose the ratio of cells ingesting latex particles increased, although the average number of particles ingested per cell decreased. The integrative phagocytic index poorly depended on the irradiation dose. In patients with a low initial level of phagocytic index, after UV blood irradiation it became more pronounced than in those with the initial elevated level. The enhancement of phagocytic activity is the result of a direct UV-stimulation of cells. This stimulation not mediated by irradiated blood plasma is known to inhibit the phagocytic activity of leucocytes. A possible mechanism of phagocytic activity stimulation is discussed.     

Macrophage activation in the immune response

In inbred white rats, immunized with sheep erythrocytes, contents and phagocytic activity of the spleen and pulmonary macrophages have been studied on the 3d, 4th, 5th, 6th, 7th, 11th, 14th and 20th days of the experiment in the light, scanning and transmissive electron microscope, as well as the effect of the cells mentioned on proliferation of lymphoid elements. Maximal phagocytic activity of the splenic and alveolar macrophages is observed on the 7th day of the experiment. At the same time, certain drop in the lymphoid cells proliferation takes place. The change in the macrophages contents also influences proliferation of the lymphoid cells.     

Phagocytic activity of three Naegleria strains in the presence of erythrocytes of various types

The phagocytic activities of N. lovaniensis (Aq/9/1/45D) and N. gruberi (1518/1f and 1518/1e) were studied in the presence of erythrocytes of various species: chicken, rabbit, goat, and human (A+, B+, and AB+ were tested). The percentage of amoebae with ingested red cells, the phagocytic index (PhI), can be considered as an expression of phagocytic activity. Under given conditions (erythrocyte concentration, incubation time, age of amoebic cultures) each strain of Naegleria prefers one erythrocyte type. Thus, for 72-h cultures, N. lovaniensis ingested more A+ type erythrocytes than did N. gruberi strains but had very low affinity for rabbit red cells except when very high concentrations were tested. Naegleria gruberi 1f was the most active of the three strains towards rabbit and B+ and AB+ human erythrocytes, but very low PhIs were obtained with goat erythrocytes. Naegleria gruberi 1e exhibited high phagocytic activity for every erythrocyte type except for rabbit red cells.     

Distinct activities of the anaphase-promoting complex/cyclosome (APC/C) in mouse embryonic cells

The first differentiation event in mammalian development gives rise to the blastocyst, consisting of two cell lineages that have also segregated in how the cell cycle is structured. Pluripotent cells of the inner cell mass divide mitotically to retain a diploid DNA content, but the outer trophoblast cells can amplify their genomes more than 500-fold by undergoing multiple rounds of DNA replication, completely bypassing mitosis. Central to this striking divergence in cell cycle control is the E3 ubiquitin-ligase activity of the anaphase-promoting complex or cyclosome (APC/C). Extended suppression of APC/C activity during interphase of mouse pluripotent cells promotes rapid cell cycle progression by allowing stabilization of cyclins, whereas unopposed APC/C activity during S phase of mouse trophoblast cells triggers proteasomal-mediated degradation of geminin and giant cell formation. While differential APC/C activity might govern the atypical cell cycles observed in pre-implantation mouse embryos, geminin is a critical APC/C substrate that: (1) escapes degradation in pluripotent cells to maintain expression of Oct4, Sox2 and Nanog and (2) mediates specification and endoreduplication when targeted for ectopic destruction in trophoblast. Thus, in contrast to trophoblast giant cells that lack geminin, geminin is preserved in both mouse pluripotent cells and non-endoreduplicating human cytotrophoblast cells.Key words: APC/C, geminin, Emi1, cell cycle, pluripotency, trophoblast, endoreduplication, DNA damage     

Distinct activities of the anaphase-promoting complex/cyclosome (APC/C) in mouse embryonic cells

The first differentiation event in mammalian development gives rise to the blastocyst, consisting of two cell lineages that have also segregated in how the cell cycle is structured. Pluripotent cells of the inner cell mass divide mitotically to retain a diploid DNA content, but the outer trophoblast cells can amplify their genomes more than 500-fold by undergoing multiple rounds of DNA replication, completely bypassing mitosis. Central to this striking divergence in cell cycle control is the E3 ubiquitin-ligase activity of the anaphase-promoting complex or cyclosome (APC/C). Extended suppression of APC/C activity during interphase of mouse pluripotent cells promotes rapid cell cycle progression by allowing stabilization of cyclins, whereas unopposed APC/C activity during S phase of mouse trophoblast cells triggers proteasomal-mediated degradation of geminin and giant cell formation. While differential APC/C activity might govern the atypical cell cycles observed in pre-implantation mouse embryos, geminin is a critical APC/C substrate that: (1) escapes degradation in pluripotent cells to maintain expression of Oct4, Sox2 and Nanog; and (2) mediates specification and endoreduplication when targeted for ectopic destruction in trophoblast. Thus, in contrast to trophoblast giant cells that lack geminin, geminin is preserved in both mouse pluripotent cells and non-endoreduplicating human cytotrophoblast cells.     

Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.