Nonenzymatic glucosylation of human placental trophoblast basement membrane collagen (relation to diabetic placenta pathology)

Nonenzymatic glucosylation is a reaction in which glucose binds nonenzymatically to hemoglobin, serum protein and glomerular basement membrane collagen etc. It has been thought that nonenzymatic glucosylation results in functional and chemical changes in those substances (hemoglobin etc) and contributes to the pathological changes in diabetes mellitus. This time we investigated whether nonenzymatic glucosylation occurred in human placental trophoblast basement (TrBM) collagen or not. The ability of glucose to interact with TrBM collagen (nonenzymatic glucosylation of TrBM collagen) was examined by incubating TrBM collagen with 3H-D-glucose in vitro. As a result it was shown that nonenzymatic glucosylation occurred in TrBM collagen and nonenzymatic glucosylation of TrBM collagen depended on the glucose concentration, reaction time and reaction temperature. These results indicate that possibly hyperglycemia, via nonenzymatic glucosylation modifies the function and chemistry of TrBM collagen and is related to the placental pathological changes in diabetic pregnancy.     

A cell membrane antigen expressed by both human breast carcinoma cells and normal human trophoblast

Immunohistological studies on frozen sections of human placentae and breast carcinoma tissue using heteroantisera raised against both trophoblast microvillous plasma membrane preparations isolated from normal placentae and irradiated MCF-7 breast carcinoma cells have demonstrated a cell membrane antigen expressed by both normal human trophoblast and human breast carcinoma cells. The heteroantisera used in this study had all been previously adsorbed with immobilized human term pregnancy serum, placental alkaline phosphatase and placental ferritin preparations, as well as with human peripheral blood leucocytes. The oncoplacental membrane antigen would appear not to be represented in other normal tissues, but is represented on Jar choriocarcinoma cells and to a lesser degree on AV3 amniotic cells. Adsorption experiments have demonstrated that this antigen may not be dominant within the range of antigenic specificities of heteroantisera raised against MCF-7 cells or isolated trophoblast microvillous plasma membrane preparations.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72?h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24?h, with a maximal effect after 72?h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Effects of diacylglycerol and gonadotropin-releasing hormone on human chorionic gonadotropin release by cultured trophoblast cells.

The effect of activation of calcium- and phospholipid-dependent protein kinase (protein kinase C) on human chorionic gonadotropin (hCG) release by cultured trophoblast cells was studied and a role of protein kinase C in the GnRH-mediated hCG release was also evaluated. Both GnRH and 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, stimulated hCG release after 3 h incubation in a dose-dependent manner with ED50 of 55 nmol/l and 4.0 nmol/l, respectively. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated hCG release while two non-tumor-promoting compounds, phorbol and 4 alpha-phorbol, failed to stimulate hCG release. hCG release by maximal effective dose of GnRH (10 mumol/l) or OAG (1 mumol/l) was further stimulated when cells were incubated with same concentrations of GnRH and OAG. OAG-stimulated hCG release was completely inhibited by a protein kinase C inhibitor, H-7, with ID50 of 23 nmol/l while H-7 did not affect GnRH-mediated hCG release. These results indicate that GnRH-stimulated hCG release is not mediated by protein kinase C pathway, however, the secretion of hCG is also regulated by the mechanism that involves protein kinase C activation.     

Collagenase IV and laminin receptors in cultured trophoblast cells]

Human trophoblast cells were obtained from a term placenta and cultured through several stages. Using specific antibodies marked using an avidin biotin system and given the characteristic "invasiveness" of placental tissue, the Authors investigated the possible presence of those markers which have been found to be correlated with invasive and metastatic tumour cells. The positivity shown by cultured trophoblast cells towards laminin receptors and collagenase IV may have important implications which might explain the strange formation and maintenance of the human uteroplacental circulation in which embryonal tissue is in direct contact with maternal blood.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Long-term culture of human trophoblast cells

A technique is presented for the preparation of cultures of replicating human trophoblast cells from term placentas. The adherent cells obtained were very slow growing (doubling time 12.5 days) as measured by the rate of increase in cell protein, [14C]-leucine uptake and cell number. Cells from individual placentas have been maintained in continuous culture for up to 1 year (10-12 passages) and have been successfully recultured after storage in liquid nitrogen. Cultured cells showed positive immunofluorescent staining for human placental lactogen, human chorionic gonadotrophin, transferrin and type IV collagen. The adenine nucleotide content indicated that energetically the cells were in balance even after prolonged culture.     

Long-term culture of human trophoblast cells

Summary. A technique is presented for the preparation of cultures of replicating human trophoblast cells from term placentas. The adherent cells obtained were very slow growing (doubling time 12.5 days) as measured b y the rate of increase in cell protein, [¹⁴C]-leucine uptake and cell number. Cells from individual placentas have been maintained in continuous culture for up t o 1 year (10–12 passages) and have been successfully recultured after storage in liquid nitrogen. Cultured cells showed positive immunofluorescent staining for human placental lactogen, human chorionic gonadotrophin, transferrin and type IV collagen. The adenine nucleotide content indicated that energetically the cells were in balance even after prolonged culture.     

Heme oxygenase expression in cultured human trophoblast cells during in vitro differentiation: effects of hypoxia

Heme oxygenases (HO-1 and HO-2) are responsible for the production of carbon monoxide, a vasodilator. HO is important in controlling placental blood flow and expression can be sensitive to oxygen. We previously reported a reduction in HO-2 expression in placentae obtained from patients with pre-eclampsia or living at high altitude, both associated with placental hypoxia. Thus we hypothesized that HO expression in cultured trophoblasts would be altered by exposure to hypoxia. HO-1 and HO-2 expression was assessed in trophoblast cell cultures following exposure to different oxygen environments. Western blot analyses showed that HO-1 expression in syncytiotrophoblast was significantly lower than in cytotrophoblasts in standard conditions (p < 0.05). There was no difference in HO-1 expression in cytotrophoblasts transferred to 2% O2 for various times. However, exposure of syncytiotrophoblast cultures to hypoxia for 12 h resulted in a significant reduction in HO-1 expression (p < 0.05). HO-2 expression was not affected by exposure to hypoxia in either cytotrophoblast or syncytiotrophoblast cultures. Possible interpretations of these findings are that chronic hypoxia alone is not responsible for reduced HO-2 expression or a much longer exposure to chronic hypoxia (perhaps months) is required. This study also reinforces the complexities of HO regulation by oxygen.     

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas.

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.     

Derivation of human triploid trophoblast stem cells

Journal of Assisted Reproduction and Genetics - Human trophoblast stem cells (hTSCs) are counterparts of the precursor cells of the placenta and are valuable cell models for the study of placental...     

Vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50muM) and E (50muM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.     

Human trophoblast cells cultured in modified medium and supported by extracellular matrix

This paper describes a culture procedure which consistently yields 80 to 90 per cent trophoblast from human first-trimester placentae. The trophoblast cells are selected and maintained but there is no increase in proliferation. The cultured cells are found to resemble extravillous rather than villous trophoblast in their immunocytochemical characteristics. This technique provides a means of obtaining human trophoblast cells with a sufficient degree of homogeneity and viability to be used for in vitro experiments.     

Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Mr of 68 000. H310 and H316 both recognise an antigen with a subunit Mr of 34 000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Mr 62 000. It is unknown whether this 62 000 dalton component is a dimer of the 34 000 dalton protein with either itself or a second protein chain of presumed Mr around 28 000. H315 recognises an antigen with subunit Mr of 36 000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.