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H J Kliman

Publications and source records attributed to H J Kliman.

34 records · Page 2Linked to original sources
PubMed1990

Aggregation of dispersed human cytotrophoblastic cells: lessons relevant to the morphogenesis of the placenta.

The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.

Cell Aggregation↗
PubMed1990

Human trophoblast-extracellular matrix (ECM) interactions in vitro: ECM thickness modulates morphology and proteolytic activity.

Trophoblast invasion of the uterine extracellular matrix, a critical process for human implantation and uteroplacental vascular development, is a striking example of controlled invasiveness. To examine cellular behavior relevant to this process, human trophoblasts were cultured on (i) Millicell filters prelayered with Matrigel and (ii) coverslips precoated with a gentle slope of Matrigel (Matribeach). Histologic sections of the Millicell system demonstrated significant invasion. However, on Matribeach the cells exhibited markedly different characteristics depending on the thickness of the Matrigel. On zone 1 (1-4 microns thick), flat aggregates and syncytia were seen. In contrast, cells on zone 2 (4-14 microns) formed rounded aggregates with intercellular processes. In this zone, prominent degradation of pericellular Matrigel proteins was assessed by both light microscopy and scanning electron microscopy. Treatment with 8-bromo-cAMP inhibited this proteolytic process. On zone 3 (14-60 microns), unicellular trophoblasts or small aggregates caused minimal matrix degradation. JEG-3 human choriocarcinoma cells exhibited similar morphologic and degradative properties on Matribeach, but zone 2 proteolysis was not affected by 8-bromo-cAMP. Our results suggest that extracellular matrix thickness has profound effects on cellular morphology and proteolytic activity. Furthermore, while both normal and malignant human trophoblasts can degrade extracellular matrix proteins, only normal trophoblast extracellular matrix degradation is inhibited by 8-bromo-cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Plasminogen activator inhibitor types 1 and 2 in human trophoblasts. PAI-1 is an immunocytochemical marker of invading trophoblasts.

Trophoblast implantation, vascular remodeling, and maintenance of intervillous blood flow may depend on the regulated production of proteolytic enzymes such as plasminogen activator (PA). Since the functional activity of plasminogen activators is determined not only by the quantity of protease but also by levels of specific plasminogen activator inhibitors (PAI), we examined trophoblasts both in vitro and in vivo for the presence of two PAIs, PAI-1 and PAI-2. Cytotrophoblasts were isolated from first trimester or term placentae, cultured, and immunocytochemically stained using specific anti-PAI antibodies. The antiserum against PAI-1 demonstrated prominent cell-surface staining and some cytoplasmic staining. The antiserum generated against PAI-2 revealed a cytoplasmic localization, with some trophoblasts staining intensely, whereas others had no apparent reactivity. We also found that cultured cytotrophoblasts contain the mRNAs for PAI-1 and PAI-2. Immunohistochemical analysis of tissue sections from 8-, 16-, and 40-week implantation sites using antisera against PAI-1 demonstrated weak staining of villous syncytiotrophoblasts but prominent cytoplasmic staining of trophoblasts invading the decidua and myometrium. Antisera against PAI-2 stained the cytoplasm of villous syncytiotrophoblasts, but no staining was evident in villous cytotrophoblasts or in invading trophoblasts. We conclude that 1) human trophoblasts can express both PAI-1 and PAI-2 in vitro and in vivo and 2) prominent PAI-1 immunostaining defines invading trophoblasts, whereas PAI-2 is the predominant PAI accumulated in villous syncytiotrophoblasts. Thus, the various trophoblast forms have distinctive patterns of PAI expression.

Cells, Cultured↗
PubMed1989

Control of the steroidogenic machinery of the human trophoblast by cyclic AMP.

Human cytotrophoblasts express adenylate cyclase activity and possess membrane-bound regulatory proteins that bind guanine nucleotides (G proteins). Stimulation of the cyclase by forskolin or addition of 8-bromo-cAMP augments progesterone secretion by cultured cytotrophoblasts at least in part, by promoting accumulation of components of the cholesterol side-chain cleavage system. This is the consequence of increased synthesis of the proteins participating in steroidogenesis as a result of the 8-bromo-cAMP-provoked increase in their respective mRNAs. We propose that progesterone synthesis by cytotrophoblasts is up-regulated by cyclic AMP, which acts to increase expression of genes encoding the steroidogenic machinery. Paracrine or autocrine factors may initiate this cascade by stimulating the cytotrophoblast adenylate cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Biosynthesis and turnover of a 34-kDa protein growth factor in human cytotrophoblasts.

Recently we isolated a new protein growth factor of 34 kDa from synctial membranes of human placenta. In its polypeptide molecular mass, antigenic structure, receptor binding specificity and partial amino acid sequence, it is unlike several known growth factors, hormones and other proteins. Here we report studies on its biosynthesis and turnover in cultured cytotrophoblasts from term human placenta. Expression of the 34-kDa protein in these cells was studied by immunoprecipitation and Western blot analyses using a highly specific antibody. The experiments have produced the following results. a) Immunostaining and Western blot analyses have demonstrated the presence of immunoreactive 34-kDa protein in isolated cytotrophoblasts. The protein is present in both freshly isolated cells and in cells that have fused in culture to form multinuclear syncytiotrophoblasts. b) Trophoblastic biosynthesis of the protein has been demonstrated by in vitro translation of cellular mRNA and by metabolic labelling experiments with intact cells. c) Pulse-chase experiments show that biosynthesis of the protein does not involve any detectable precursors of higher or lower molecular mass. d) Studies on turnover indicate that the synthesized protein is unusually stable with a half-life of 50-70 h.

Cells, Cultured↗
PubMed1988

The human villous cytotrophoblast: interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation.

Human syncytiotrophoblasts are derived from villous cytotrophoblasts by cell fusion. Coincident with this morphologic transformation, trophoblasts acquire specific endocrine functions, including elaboration of chorionic gonadotropin (hCG). We wondered if syncytia formation was a prerequisite for biochemical differentiation or simply was one part of the differentiation program. By growing purified human cytotrophoblasts under serum-free conditions and manipulating the culture surface, we were able to disassociate morphologic from biochemical differentiation. We have shown previously (Endocrinology 1986, 118:1567) that human cytotrophoblasts grown in the presence of fetal calf serum flatten out, aggregate, and form functional syncytiotrophoblasts in vitro over 24-96 hr. Here we demonstrate that when grown in the absence of serum, the cells do not undergo these morphologic changes, but remain as individual spherical cells. If the culture surface was precoated with fibronectin or a variety of collagens, but not albumin, the cells regained their ability to flatten, aggregate, and form syncytia. Attachment to and syncytia formation on fibronectin was blocked by the addition of the R-G-D-S-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro. Attachment to and syncytia formation on type I collagen was blocked by anti-human fibronectin F(ab')2 fragments, while association with type IV collagen was not affected by this antibody, suggesting that fibronectin mediates trophoblast association with type I collagen, but not type IV. Although syncytia formation did not occur when cytotrophoblasts were cultured under serum-free conditions in the absence of ECM proteins, biochemical differentiation was not affected. These cells secreted hCG at the same rate under serum-free conditions whether they were plated on plastic only--which prevented syncytia formation--or fibronectin, laminin or, type IV collagen--which allowed syncytia formation to occur. Furthermore, cytoplasmic differentiation in the absence of syncytia formation was confirmed by performing transmission electron microscopy on cytotrophoblasts grown under serum-free conditions in the presence of 8-bromo-cAMP. We conclude that syncytia formation is not a prerequisite for biochemical differentiation, but simply part of the trophoblast differentiation program.

Cell Adhesion↗
PubMed1987

HLA antigen expression and induction by gamma-interferon in cultured human trophoblasts.

The expression and regulation of HLA antigens were examined in isolated human trophoblasts. Immunocytochemical studies that used monoclonal antibodies against class I (HLA-A, B, and C) antigens revealed that both cytotrophoblasts and syncytiotrophoblasts can express HLA heavy chains and beta 2-microglobulin. Expression of these antigens increased with time in culture. The addition of recombinant gamma-interferon (1000 U/ml) to the cultures significantly increased cellular staining for these antigens over controls throughout the 72-hour time course studied. In addition, we noted that gene expression for HLA class I heavy chain was also markedly augmented by the addition of gamma-interferon. Expression of class II (HLA-DR) antigens was not detected in any of the experiments. These results suggest that during in vitro differentiation, cytotrophoblasts can express class I HLA antigens and the genes controlling their production can be regulated. Alterations in the expression or suppression of these antigens during pregnancy could be responsible for some pregnancy complications.

Cell Differentiation↗
PubMed1987

8-Bromo-adenosine 3',5'-monophosphate regulates expression of chorionic gonadotropin and fibronectin in human cytotrophoblasts.

Addition of 8-bromo-cAMP to primary cultures of human placental cytotrophoblasts results in significant alterations in the synthesis of secreted proteins, as detected by labeling with pulses of [35S]methionine. Using immunoprecipitation techniques, we demonstrated that exposure to 8-bromo-cAMP prevented the de novo synthesis and secretion of the extracellular matrix component fibronectin, but enhanced the production of hCG subunits. The effects of the cyclic nucleotide on synthesis and secretion of these proteins were evident within 24 h. 8-Bromo-cAMP increased the cellular content of mRNA encoding the hCG alpha- and beta-subunits and prevented the increase in fibronectin mRNA, as determined by blot hybridization analysis using specific cDNA probes. These findings demonstrate that cyclic nucleotides regulate the synthesis of several specific proteins in cultured human trophoblast by regulating levels of the mRNAs encoding the proteins. The actions of cyclic nucleotides in this regard may be essential for the normal expression of trophoblast endocrine function.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Gonadotropins and cyclic adenosine 3',5'-monophosphate (cAMP) alter the morphology of cultured human granulosa cells.

Morphological changes in human granulosa cells in culture were observed by phase, fluorescent, scanning electron and transmission electron microscopy following the addition of human chorionic gonadotropin (hCG), luteinizing hormone (LH), 8-bromocyclic adenosine 3',5'-monophosphate (cAMP) and cytochalasins B and D. In response to these agents, polygon-shaped granulosa cells with granular cytoplasm became rounded, leaving fingerlike processes attached to the substratum and adjacent cells. The changes in cell shape were accompanied by a centripetal movement of mitochondria and lysosomes to a perinuclear location. The morphological alterations appeared to be mediated by cyclic AMP and to be the result of a dismantling and reorganization of microfilament-containing stress fibers. Follicle-stimulating hormone (FSH), prolactin (PRL), growth hormone (GH), and human placental lactogen (hPL) did not provoke cell shape changes. We conclude that tropic hormones capable of stimulating progestin secretion by luteinized granulosa cells cause a change in cell structure in vitro which leads to a redistribution of organelles involved in steroid synthesis. The possible relationship of the cytoskeleton to steroidogenesis is considered.

Actin Cytoskeleton↗
PubMed1986

Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.

Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.

Actinin↗
PubMed1986

8-Bromo-3',5'-adenosine monophosphate stimulates the endocrine activity of human cytotrophoblasts in culture.

Cytotrophoblasts, purified from human term placentae, were cultured in the absence or presence of 8-bromo-cAMP or 8-bromo-cGMP. 8-Bromo-cAMP provoked a dose-dependent increase in the secretion of hCG and progesterone within 24 h. After 48 h, hCG secretion increased by more than 200-fold, and progesterone secretion increased nearly 5-fold. 8-Bromo-cGMP had no effect on hCG secretion. In culture in serum-supplemented medium, the mononuclear cytotrophoblasts aggregated and fused to form syncytia. This morphological transformation was not affected by 8-bromo-cAMP. Immunocytochemical studies of the alpha- and beta-subunits of hCG in control and 8-bromo-cAMP-stimulated cultures demonstrated that the cyclic nucleotide analog promoted the synthesis of both subunits in all cellular forms, including single mononuclear cells, cell aggregates, and syncytia. In serum-free medium, the cytotrophoblasts did not aggregate or form syncytia, yet they responded to 8-bromo-cAMP with an increase in hCG secretion. We conclude that the endocrine function of cytotrophoblasts can be stimulated by a cAMP-dependent mechanism which can be initiated independently of the formation of a syncytium.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component.

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.

Animals↗
PubMed1980

Association of glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane. A kinetic analysis.

A rapid filtration technique was used to analyze the kinetics of the binding reaction between glyceraldehyde-3-P dehydrogenase and the band 3 protein of the human erythrocyte membrane. Saponin was used to eliminate the membrane as a rate-limiting barrier. The re-equilibration of the enzyme following dilution of membranes in buffer took only a few seconds. Dissociation rates were greatly stimulated and association rates were reduced by increasing ionic strength. A Brönsted-Bjerrum analysis suggested that two or three charges of opposite sign on each protein were involved in binding. NADH appeared to elute the enzyme by interaction with its catalytic site, but the band 3 binding site extended beyond the NADH site, permitting the formation of a ternary NADH . enzyme . membrane complex. The release of the enzyme from fresh erythrocytes immediately following saponin lysis showed kinetics similar to the release of enzyme from ghosts. The extrapolated zero time intercepts of these reactions suggested that two-thirds of cellular glyceraldehyde-3-P dehydrogenase was membrane bound prior to hemolysis. This value is similar to that calculated for the association of the enzyme with band 3 in the intact red cell when the high ionic strength and enzyme-eluting compounds in the cytoplasm are taken into account.

Erythrocyte Membrane↗
PubMedDate not supplied

Human trophoblast-endometrial interactions in an in vitro suspension culture system.

We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium. Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator. When endometrium was cultured alone it was able to remain viable for up to 3 days. When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers. When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments. The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted. A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts. We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts. Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.

Cell Communication↗
PubMedDate not supplied

Mouse ascites golgi (MAG) mucin expression and regulation by progesterone in the rat uterus.

OBJECTIVE: To study the regulation of the blood group A-related high-molecular weight mucin glycoprotein epitope (mouse ascites golgi, MAG)-a menstrual cycle-dependent marker of endometrial receptivity-in a non-human endometrium model. METHODS: Immature Sprague-Dawley rats were injected with 1 microg of estradiol, 100 microg of testosterone, 100 microg of dexamethasone, 2.5 mg of progesterone (P), 0.325 mg of RU486, P and RU486, 100 microg of tamoxifen, or vehicle for 3 days, sacrificed, and the uteri were stained for MAG. Immunohistochemistry and blood analysis were the measurements used to compare the specimens from the exogenous hormonal and endogenous hormonal groups. Electron microscopy was used to locate the MAG epitope in one pseudopregnant adult Sprague-Dawley rat. RESULTS: The MAG epitope was present in endometrial glands of Sprague-Dawley rats, with maximal expression during proestrus and diestrus. Electron microscopy confirmed the Golgi location of this MAG epitope. In the untreated group, less than 0.5% of endometrial glands stained for MAG. The MAG was seen only in the glands of the P-treated rats and RU486 blunted this stimulatory effect by more than 95%. As little as 0.1 mg of P promoted MAG expression, with maximal response at 2.5 mg. Staining was seen 24 hours after P treatment, peaked at 72 hours, then declined. Induction of endogenous P by superovulation with pregnant mare serum gonadotropin (PMSG) and hCG (pseudopregnancy) also resulted in strong MAG glandular staining. CONCLUSION: Our results suggest that the MAG epitope is cyclically expressed and induced by P in rat endometrial glands.

ABO Blood-Group System↗