D. Evain-Brion

PPARγ/RXRα Heterodimers Are Involved in Human CGβ Synthesis and Human Trophoblast Differentiation

Recent studies performed with null mice suggested a role of either RXRα or PPARγ in murine placental development. We report here that both PPARγ and RXRα are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPARγ (but not for PPARα or PPARδ) increase both human CGβ transcript levels and the secretion of human CG and its free β-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPARγ ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPARγ/RXRα heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CGβ gene (β5) were found to bind RXRα and PPARγ from human cytotrophoblast nuclear extracts, suggesting that PPARγ/RXRα heterodimers directly regulate hu...

Overexpression of copper zinc superoxide dismutase impairs human trophoblast cell fusion and differentiation.

The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast formation and a decrease in the production of pregnancy-specific hormones. Due to the role of oxygen free radicals in trophoblast cell differentiation, we investigated the role of the key antioxidant enzyme, copper/zinc superoxide dismutase, encoded by chromosome 21 in in vitro trophoblast differentiation. We first observed that overexpression of superoxide dismutase in normal cytotrophoblasts impaired syncytiotrophoblast formation. This was associated with a significant decrease in mRNA transcript levels and secretion of hCG and other hormonal markers of syncytiotrophoblast. We confirmed abnormal cell fusion by overexpression of green fluorescence protein-tagged superoxide dismutase in cytotrophoblasts. In addition, a significant decrease in syncytin transcript levels was observed in superoxide dismutase-transfected cells. We then examined superoxide dismutase expression and activity in isolated trophoblast cells from trisomy 21-affected placentas. Superoxide dismutase mRNA expression (P < 0.05), protein levels (P < 0.01), and activity (P < 0.05) were significantly higher in trophoblast cells isolated from trisomy 21-affected placentas than in those from normal placentas. These results suggest that superoxide dismutase overexpression may directly impair trophoblast cell differentiation and fusion, and superoxide dismutase overexpression in Downs syndrome may be responsible at least in part for the failure of syncytiotrophoblast formation observed in trisomy 21-affected placentas.

Review: hCGs: Different sources of production, different glycoforms and functions

Human chorionic gonadotropin (hCG) is the first hormonal message from the placenta to the mother. It is detectable in maternal blood two days after implantation and behaves like an agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two glycosylated subunits. The α-subunit is identical to the pituitary gonadotropin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). By contrast the β-subunits are distinct in each of the hormones and confer receptor and biological specificity. The hCG β-subunit contains two sites of N-glycosylation and four sites of O-glycosylation and is encoded by a cluster of genes (CGB). In this review, we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its source of production and in the pathology. It is well established that hCG is mainly secreted by the syncytiotrophoblast into maternal blood where it peaks around 8-10 weeks of gestation (WG). The invasive extravillous trophoblast also secretes hCG, and in particular like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In maternal blood hCG-H is high during early first trimester. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor. In contrast, hCG-H stimulates trophoblast invasion and angiogenesis by interacting with the TGFβ receptor 2. hCG is largely used in antenatal screening and hCG-H represents a serum marker of early trophoblast invasion. Other abnormally glycosylated hCG are described in aneuploidies. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

Effect of Matrigel on Human Extravillous Trophoblasts Differentiation: Modulation of Protease Pattern Gene Expression

Abstract The human placenta is characterized by extensive trophoblast invasion of the uterus. Indeed, extravillous cytotrophoblast cells invade the decidua and the upper third of uterine spiral arteries in the myometrium. This invasion is reflected in situ by the expression of specific markers. In order to study this invasion process, we have established an in vitro culture model of human extravillous trophoblast isolated from first trimester chorionic villi. The aim of this study was to investigate the effect of a composite matrix, the Matrigel required for the culture of this homogenous population of extravillous trophoblasts (EVCT), on their in vitro differentiation. The effect of Matrigel was studied on different markers characterized by immunocytochemistry and by real-time polymerase chain reaction assay of transcripts. In addition, the expression of 12 different matrix metalloproteases and their inhibitors were investigated. We show that human extravillous cytotrophoblasts acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest to study the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.

Review: Annexin-A5 and cell membrane repair

Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.

Expression and Regulation by PPARγ of hCG α- and β-subunits: Comparison between Villous and Invasive Extravillous Trophoblastic Cells

During human placental development trophoblast follows two differentiation pathways: the extravillous (EVCT) and the villous cytotrophoblasts (VCT) that display different phenotypes and functions. It is well established that human chorionic gonadotropin hormone (hCG) is mainly secreted by the endocrine VCT (syncytiotrophoblast) into the maternal compartment and stimulates the formation of the syncytiotrophoblast (ST) in an autocrine manner. We recently reported that the invasive EVCT also produces hCG that promotes trophoblast invasion in vitro. Herein, we compared hCG gene expression in primary culture of villous and extravillous trophoblasts obtained from the same first trimester human chorionic villi and differentiated in vitro into ST and invasive EVCT, respectively. Total hCG, free alpha and free beta subunits were quantified in cell supernatants by immunometric assays and normalized to DNA content. alpha and beta transcript levels were quantified by Q-PCR and normalized to cytokeratin 7. We show that free alpha-, free beta-subunits and total hCG are differently expressed and secreted by the two trophoblast subtypes during their differentiation in vitro. We found an alpha/beta ratio 100 times lower in invasive EVCT in comparison to the ST suggesting that beta subunit may not be step limiting for hCG production in EVCT. Finally we investigated the regulation of hCG gene expression by PPARgamma, a nuclear receptor that controls trophoblast differentiation and invasion. Interestingly, activation of PPARgamma by the agonist rosiglitazone gave opposite results in the endocrine VCT and invasive EVCT: alpha and beta subunit transcript levels and protein secretions were up regulated in VCT, whereas they were down regulated in EVCT. Our results demonstrated that hCG gene expression is differentially regulated in the two trophoblast lineages during their in vitro differentiation and modulated in an opposite way by PPARgamma.

Drug transporter expression during in vitro differentiation of first-trimester and term human villous trophoblasts

Drug transporters interfere with drug disposition during pregnancy by actively transporting drugs from mother to fetus, and vice versa. Data on their placental expression are scarce, especially during the first trimester of pregnancy. The aim of our study was to assess mRNA expression of more than 80 drug transporters by using an RT-qPCR array in primary cytotrophoblastic cells isolated from first-trimester and term human placentas and cultured for 72 h to form syncytiotrophoblasts. This original expression panel of human placental drug transporters should help to understand transplacental drug transfer and to ensure more rational drug use during pregnancy.

Hormones placentaires et croissance fœtale

Fetal growth is controlled by the placenta which mediates fetal nutritional supplies. During pregnancy, the placenta secretes a specific placental growth hormone which replaces the pituitary growth hormone in the maternal circulation. Placental growth hormone is one example of the materno-placental cooperation which is impaired in cases of intra-uterine growth retardation.