Punicalagin promotes human villous trophoblast differentiation

Poor differentiation of trophoblasts is associated with placental dysfunction, predisposing women to multiple pregnancy disorders. Punicalagin, a prominent ellagitannin in pomegranate juice has been shown to exert anti-apoptosis and anti-oxidative effects in human trophoblasts. We hypothesized that punicalagin modulates trophoblast differentiation. We found that punicalagin-treated primary trophoblast showed reduced E-cadherin, higher Syncytin 1, more β−hCG, and increased GCM1, an upstream regulator of β−hCG. Punicalagin exposure of villous explants enhanced the number of cytotrophoblasts expressing the proliferation marker Ki67. We conclude that punicalagin enhances trophoblast differentiation and speculate that punicalagin might be used therapeutically in pregnancies at risk for placental dysfunction.     

The role of human endogenous retroviruses in trophoblast differentiation and placental development

A major portion of the human genome appears to be of retroviral origin. These endogenous retroviral elements are expressed in a variety of normal tissues and during disease states, such as autoimmune and malignant conditions. Recently, potential roles have been described for endogenous retroviral envelope proteins in normal differentiation of human villous cytotrophoblast into syncytiotrophoblast. This article provides a brief critical review of the current state of knowledge concerning the expression of the env regions of three endogenous retroviral elements: ERV-3, HERV-W, and HERV-FRD. A testable model of villous cytotrophoblast differentiation is constructed, in which a complementary expression of endogenous retroviral envelope proteins initiates hCG production, decreased cell proliferation, and intercellular fusion.     

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.     

The relationship between trophoblast differentiation and the production of bioactive hCG

We previously showed that a significant number of failing pregnancies are associated with production of human chorionic gonadotropin (hCG) having relatively low bioactivity. The present study was designed to compare the secretion of intact, immunoreactive hCG to the secretion of bioactive hCG during trophoblast differentiation, and to test the hypothesis that the lower bioactive: immunoreactive hCG ratios in failing pregnancies are related to reduced or impaired trophoblast differentiation. Cytotrophoblast cells were isolated from term placentas and cultured under conditions that induced or did not induce syncytiotrophoblast formation. Culture media were collected at regular intervals up to 72 h and levels of immunoreactive and bioactive hCG were measured. The differentiation of cytotrophoblast cells to multinucleated syncytiotrophoblast was monitored by immunocytochemistry and electron microscopy. During the 72 h culture period, concentrations of immunoreactive and bioactive hCG increased in both differentiating and non-differentiating cells. However, the concentrations of immunoreactive and bioactive hCG were higher under culture conditions that promoted trophoblast differentiation. Furthermore, the ratio of bioactive hCG to immunoreactive hCG was higher in differentiating cultures. When differentiation was inhibited by dimethyl sulfoxide, the secretion of bioactive hCG was reduced and the bioactive: immunoreactive hCG ratio did not change. These findings are consistent with the idea that production of bioactive hCG accompanies syncytiotrophoblast formation.     

Expression of DAB2IP in human trophoblast and its role in trophoblast invasion

Objective: DAB2IP is a growth inhibitor present in many types of cancer cells and is associated with epigenetic regulations controlling tumor development. The primary objective of this study is to determine whether DAB2IP participates in the invasion and migration of trophoblasts during placental development.

Methods: The expressions of DAB2IP in human placentas (10 villi, 18 term placentas and 20 pre-eclampsia placentas) were determined by immunohistochemistry, Western blotting and quantitative RT-PCR. HTR8/SVneo cells were treated with hypoxia–reoxygenation (H/R) to test how DAB2IP expression would affect the invasion and migration of trophoblasts. JEG-3 andHTR8/SVneo cells were treated with 5-aza-2-deoxycytidine (5-aza-dC) to study the role of DAB2IP promoter methylation in trophoblasts.

Results: DAB2IP was strongly expressed in human villi and extravillous trophoblasts as well as in HTR8/SVneo cells, but not in pre-eclampsia placentas. DAB2IP expression increased after H/R treatment, but the invasive and migratory abilities of trophoblasts were reduced. DAB2IP expression in JEG-3 cells also increased after treatment with 5-aza-dC.

Conclusions: These findings strongly suggest that DAB2IP is an important negative regulator at the maternal–fetal interface during early pregnancy. Excessive oxidative stress can increase DAB2IP expression in trophoblasts. The mechanism of DNA methylation may involve in its function during the development of pathologic pregnancy.     

Antiphospholipid antibodies prevent extravillous trophoblast differentiation

OBJECTIVE: We investigated the hypothesis that antiphospholipid antibodies (aPL) have a detrimental effect on human extravillous trophoblast (EVT) differentiation into giant multinucleated cells "in vitro." DESIGN: The EVT were isolated from the placental chorion using enzymatic digestion and Percoll gradient centrifugation. After 24, 36, and 48 hours in culture, giant multinuclear cells (GMC) were identified by immunohistochemistry using antibodies to cytokeratin 7 and counted. SETTING: An academic research laboratory. PATIENT(S): Placentas were donated by women having an elective cesarean section for a normal pregnancy at term. MAIN OUTCOME MEASURE(S): This model was then used to investigate the effect of two different monoclonal aPL to beta2-glycoprotein 1 (IIC5 and ID2), and control mouse IgG antibody on EVT differentiation. RESULT(S): Freshly isolated EVT were nonproliferative but moved together losing their intervening cell walls and differentiated into GMC. Maximal numbers of GMC were detected after 48 hours of culture. The aPL, IIC5, and ID2 significantly inhibited GMC formation, whereas the mouse IgG control had no effect. CONCLUSION(S): Antiphospholipid antibodies can inhibit EVT differentiation and GMC formation "in vitro" suggesting that a failure of trophoblast differentiation and subsequent uteroplacental development may be an underlying pathology in antiphospholipid syndrome-associated pregnancy loss.     

PP2 regulates human trophoblast cells differentiation by activating p38 and ERK1/2 and inhibiting FAK activation

Throughout gestation, fetal growth and development depend, in part, on placental transfer of nutrients from the maternal circulation. This latter function depends on multinucleated, terminally differentiated syncytiotrophoblasts. In vitro, freshly isolated cytotrophoblast cells differentiate spontaneously into syncytiotrophoblast in the presence of fetal bovine serum (FBS). We have previously showed that trophoblast differentiation is regulated by ERK1/2 and p38. Moreover, we showed that PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine], a Src family kinase (SFK) specific inhibitor, stimulates biochemical trophoblast cells differentiation while it inhibits cell adhesion and spreading without affecting cell fusion. Therefore, we examined the mechanisms by which PP2 modulates trophoblast cells differentiation. This study shows that PP2 stimulates ERK1/2 and p38 activation after 24h of treatments and up to 3 days while it inhibits focal adhesion kinase (FAK) phosphorylation at many sites including Tyr-397, 407, 576 and 577. Furthermore, we showed that transient activation of ERK1/2 by FBS is independent of SFK and that PP2 induces rapid activation of p38. Moreover, the kinase activity of SFK is negatively regulated by the phosphorylation of their carboxy (C)-terminal regulatory tyrosines by specific proteins called carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK). We showed the expression of Csk and CHK in human trophoblast cells. In summary, this study showed that PP2 stimulates the biochemical differentiation of trophoblast cells by stimulating p38 and ERK1/2 while it inhibits the morphological differentiation by inhibiting FAK activation.     

Expression of transferrin receptors during differentiation of human placental trophoblast cells in vitro.

In most cell types, transferrin receptor expression is correlated with the proliferation rate, being increased by growth stimulation, or decreased by induction of terminal differentiation. In the human placenta the multinucleated syncytiotrophoblast, in direct contact with maternal blood, is derived by differentiation from mononucleated cytotrophoblast. In this study we examined changes in transferrin receptor expression during in vitro differentiation of trophoblast. Cells cultured in Ham's/Waymouth's medium (HWM) remained primarily mononuclear throughout the study, whereas incubation in keratinocyte growth medium (KGM) led to formation of multinucleate masses within 2-3 days of culture. Cell surface binding of 125I-labelled transferrin increased fivefold between days 1-5 of culture in both media and surface receptors were saturated at 7-14 micrograms/ml (90-200 fM). At saturation, the amount of transferrin bound to syncytiotrophoblast was 37 per cent lower than in cytotrophoblast. Scatchard analysis revealed a reduction in the number of surface transferrin receptors in syncytiotrophoblast compared to cytotrophoblast. A corresponding 29 per cent reduction in the binding of transferrin to intracellular sites was observed in syncytiotrophoblast. Distribution of receptors between surface and intracellular sites was therefore similar in both cytotrophoblast and syncytiotrophoblast. The affinity of transferrin for transferrin receptors was 3.7-fold higher in syncytiotrophoblast when compared to cytotrophoblast. Observed differences between the two cell types were not due to the presence of growth factors or higher iron levels in KGM. Expression of a high number of surface transferrin receptors in syncytiotrophoblast (1.5 x 10(12)/mg protein), along with a high affinity of these receptors for iron-saturated transferrin, could help explain the efficient transport of large amounts of iron from mother to fetus.     

The role of galectin-1 in trophoblast differentiation and signal transduction

Galectins are proteins with the ability to bind β-galactosides through a conserved carbohydrate recognition domain. Galectin-1 exerts its biological effects by binding glycan ligands on proteins involved in cell adhesion and growth regulation. Galectin-1 inhibits trophoblast cell proliferation and induces syncytium formation. Its down-regulation in the syncytiotrophoblast has been associated with early pregnancy loss. In the choriocarcinoma-derived BeWo cells the galectin-1 induced growth inhibition is apoptosis-independent, but rather appears to be mediated by binding to cell surface receptors, such as the receptor tyrosine kinases REarranged during Transfection (RET) and Janus Kinase (JAK) 2 as well as vascular endothelial growth factor receptor 3. On the syncytiotrophoblast and extravillous trophoblast galectin-1 binds the Thomsen-Friedenreich disaccharide on mucin-1. The cell differentiation processes induced by binding to these receptors ultimately lead to the inhibition of proliferation and syncytium formation.     

Antigenicity of the human trophoblast]

In this a rabbit immunological serum against human trophoblast was obtained after triple intramuscular superficial villi extract injection from chorion of 6--7 week pregnancy with Freund's adjuvant. In immunofluorescent indirect reaction to this serum made on chorion and placenta sections some most intense specific fluorescence was obtained in cytoplasm of primordial cyto- and synytiotrophoblast. Some favourable influence of antigenic differences between ovum and mother especially implantation has been suggested.     

Biology of human trophoblast

In order to elucidate the regulation of placental growth, we have characterized the expression of proliferating cell nuclear antigen (PCNA), apoptotic DNA fragmentation and bc1-2 protein in human placenta during pregnancy. PCNA and bc1-2 protein expression were examined by immunohistochemical techniques, while the occurrence of apoptotic DNA fragmentation was assessed by in situ analysis of DNA 3′-end labeling method. Both PCNA expression and apoptotic DNA fragmentation were found in cytotrophoblasts (C-cells), being most abundant in early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, bc1-2 protein expression was found in syncytiotrophoblasts (S-cells), being least abundant in early placenta, less abundant in midterm placenta and most abundant in term placenta. These data indicate that early placenta is characterized by the highly proliferative activity of C-cells associated with the increased occurrence of apoptosis, whereas term placenta is characterized by the abundant expression of bc1-2 protein in S-cells.Furthermore, effects of EGF on the proliferative activity and differentiated function of trophoblasts were investigated using an organ culture system. Expiants of trophoblastic tissues were cultured with or without EGF, in the presence or absence of 10⁻⁸M triiodo-L-thyronine (T3) in a serum-free condition. In 4—5 week placentas, EGF and EGF receptor were almost exclusively localized in C-cells, and EGF augmented the proliferative activity of C-cells without affecting the ability to secrete hCG and hPL. By contrast, in 6—12 week placentas, EGF and EGF receptor were predominantly localized in S-cells, and EGF stimulated hCG and hPL secretion without affecting the proliferative activity of C-cess. The addition of T₃ (10⁻⁸M) resulted in an increased secretion of immunoreactive EGF by cultured placental explants. These findings suggest that EGF acts as a local factor in regulating early placental growth and function in synergy with thyroid hormone. On the other hand, progesterone selectively inhibited pleise hCG (α, β) mRNAs expression and decreased hCG secretion in normal placental tissues, whereas choriocarcinoma did not respond to progesterone. This suggests that inhibitory regulation of hCG synthesis in choriocarcinoma is different from normal placenta. It was also found that in molar trophoblasts and choriocarcinoma cells PCNA expression was high, but both bc1-2 protein and apoptotic signal expression were low. Characterization of choriocarcinoma hCG revealed that there are striking differences in carbohydrate structures between normal hCG and choriocarcinoma hCG. Sialic acid content in choriocarcinoma hCG was extremely lower compared to that in normal hCG. The detection of the alteration in hCG sugar chains is useful for biochemical diagnosis of choriocarcinoma.