Differences in Glycolysis and Mitochondrial Respiration between Cytotrophoblast and Syncytiotrophoblast In-Vitro: Evidence for Sexual Dimorphism

In the placenta the proliferative cytotrophoblast cells fuse into the terminally differentiated syncytiotrophoblast layer which undertakes several energy-intensive functions including nutrient uptake and transfer and hormone synthesis. We used Seahorse glycolytic and mitochondrial stress tests on trophoblast cells isolated at term from women of healthy weight to evaluate if cytotrophoblast (CT) and syncytiotrophoblast (ST) have different bioenergetic strategies, given their different functions. Whereas there are no differences in basal glycolysis, CT have significantly greater glycolytic capacity and reserve than ST. In contrast, ST have significantly higher basal, ATP-coupled and maximal mitochondrial respiration and spare capacity than CT. Consequently, under stress conditions CT can increase energy generation via its higher glycolytic capacity whereas ST can use its higher and more efficient mitochondrial respiration capacity. We have previously shown that with adverse in utero conditions of diabetes and obesity trophoblast respiration is sexually dimorphic. We found no differences in glycolytic parameters between sexes and no difference in mitochondrial respiration parameters other than increases seen upon syncytialization appear to be greater in females. There were differences in metabolic flexibility, i.e., the ability to use glucose, glutamine, or fatty acids, seen upon syncytialization between the sexes with increased flexibility in female trophoblast suggesting a better ability to adapt to changes in nutrient supply.

Abstract P227: Gq Signaling In The Placental Syncytiotrophoblast Layer During Preeclampsia

Hormones implicated in preeclampsia (PE) such as angiotensin, endothelin, and vasopressin signal via receptors coupled to the Gq cascade, and Regulator of G protein Signaling-2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human PE placenta, and reducing RGS2 in placenta causes development of key features of PE in mice. New in situ hybridization data indicate that in both humans and mice, RGS2 is abundant among many cell types in the placenta, including the syncytiotrophoblast (STB) layer. In addition, RGS2 expression in the human STB layer is reduced during PE. As this layer is strongly implicated in PE, these data lead us to hypothesize a critical Gq-buffering role for RGS2 in STB cells to prevent PE. To explore the effect of excess Gq signaling within the STB layer, we utilized a Cre-Lox approach to cause expression of the Gq-coupled hM3Dq DREADD throughout the fetoplacental unit (dam: hM3Dq+, sire: Actb-Cre+) or only within the STB layer (dam: hM3Dq+, sire: Gcm1-Cre+), and then activated the hM3Dq receptor via clozapine N-oxide (CNO, 0.5 to 2 mg/kg) injection in mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gαq activation throughout the fetoplacental unit (Actb-Cre model) severely restricted fetoplacental growth compared to saline-injected controls (n=2 vs 3; placenta: 0.027±0.006 vs 0.115±0.021 g; p<0.05, and fetus: 0.048±0.007 vs 0.268±0.010 g; p<0.05). Similarly, placentas expressing hM3Dq only in STB cells (Gcm1-Cre model) had reduced placental (n=3 0.116±0.022 vs 0.201±0.036 g; p=0.05) and possibly fetal (n=3 0.1112±0.036 vs 0.247±0.028 g; p=0.06) masses after CNO. Vascularization (assessed by CD31 immunostain) was disproportionately reduced in the labyrinth layer of the Actb-Cre model after CNO (n=2 vs 3; 20.189±3.382 vs 35.762±1.976 % area; p<0.05), despite no relative change in layer (ie, decidua/junctional zone/labyrinth) thicknesses. Preliminary results indicate similar findings in the Gcm1-Cre model (n=1 17 vs 25 % area). These data highlight the pathological consequence of excess Gq signaling in the STB layer. Ongoing studies are aimed at characterizing maternal phenotypes in these models and the consequence of STB-specific deletion of RGS2 upon sensitivity to Gq stimulators.

P–376 S100P in syncytiotrophoblast regulates lipid metabolism during early pregnancy

Study question Does S100P involve in the regulation of lipid metabolism in trophoblast syncytialization during the early stage of pregnancy? Summary answer S100P suppressed lipid droplets overloading in trophoblast syncytialization during the early stage of pregnancy. What is known already S100P is a 95-amino acid protein belongs to the large family of S100 calcium-binding proteins and is exclusively expressed in syncytiotrophoblast layer of placenta, enhancing trophoblast cells proliferation, motility and invasion. Recent studies indicated S100P was aberrantly expressed in various malignant tumors, promoting their proliferation, survival, the formation and invasion of vessels, and has a close relationship with their grade malignancy, hormone dependency and the drug-resistance. Syncytialization is trophoblast cells differentiated from cytotrophoblasts fused into multinucleated cell clusters with cell boundaries deficiency. Study design, size, duration Tissues were collected from elective first trimester pregnancy terminations of healthy people and patients with recurrent spontaneous abortion. Human trophoblast stem cells (hTSCs) were isolated from Week 6 to Week 8 of fresh placental tissues (n = 8) and use forskolin to induce syncytialization. Participants/materials, setting, methods Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of S100P. The level of S100P protein and gene was measured by Real-time quantitative PCR (RT-qPCR), western blot, ELISA and immunofluorescence in hTSC induced ST(2D)-TSCT. The lipid droplets were assessed by Oil red O stain, BODIPY and electron microscope. Serum S100P concentration in normal and recurrent miscarriage patients were measured by ELISA. Main results and the role of chance S100P was exclusively localized in syncytiotrophoblast layer of the placental villous from gestational weeks 6–8 (n = 4); S100p serum concentration was increased markedly as the gestation progressed (n = 40, p &lt; 0.05); the protein abundance of S100P was increased during syncytialization of hTSCs; Lipid droplets increased during syncytialization and knockdown of S100P leads to trophoblast cell death with the overload of lipid droplet; The expression of S100P was down regulated and the amount of lipid droplets apparently accumulated in villus of recurrent miscarriage patients (n = 4). Limitations, reasons for caution The role of S100P on placenta lacks the evidence from animal models. Wider implications of the findings: S100P is an important factor facilitating in trophoblast syncytialization in the first trimester of pregnancy and could be a biomarker of the early pregnancy sustenance. Trial registration number Not applicable

Maternal Platelets of the Human Placenta: Friend or Foe?

Human pregnancy relies on hemochorial placentation, including implantation of the blastocyst and deep invasion of fetal trophoblast cells into maternal uterine blood vessels, enabling direct contact of maternal blood with placental villi. Hemochorial placentation requires fast and reliable hemostasis to guarantee survival of the mother, but also for the neonates. During human pregnancy, maternal platelet count decreases gradually from first, to second, and third trimester. In addition to hemodilution, accelerated platelet sequestration and consumption in the placental circulation may contribute to a decline of platelet count throughout gestation. Local stasis, turbulences, or damage of the syncytiotrophoblast layer can activate maternal platelets within the placental intervillous space and result in formation of fibrin-type fibrinoid. Perivillous fibrinoid is a regular constituent of the normal placenta which is considered to be an important regulator of intervillous hemodynamics, as well as having a role in shaping the developing villous trees. However, exaggerated activation of platelets at the maternal-fetal interface can provoke inflammasome activation in the placental trophoblast, and enhance formation of circulating platelet-monocyte aggregates, resulting in sterile inflammation of the placenta and a systemic inflammatory response in the mother. Hence, the degree of activation determines whether maternal platelets are a friend or foe of the human placenta. Exaggerated activation of maternal platelets can either directly cause or propagate the disease process in placenta-associated pregnancy pathologies, such as preeclampsia.

Synthesis, Assembly, and Processing of the Env ERVWE1/Syncytin Human Endogenous Retroviral Envelope

ABSTRACT Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation.

Maternal Smoking, Intrauterine Growth Restriction, and Placental Apoptosis

Pregnant women who smoke are at greater risk of delivering a growth-restricted infant than nonsmoking mothers. We wanted to see if apoptosis could be involved in the mechanisms behind smoke-induced growth restriction, and our aim was to compare apoptosis in the placenta of smoking mothers giving birth to growth-restricted infants and nonsmoking mothers with infants of appropriate weight. The project was conducted at the Magee—Womens Hospital and Magee—Womens Research Institute, University of Pittsburgh, PA. Histological sections from 20 placentas were selected from smoking mothers who had given birth to small-for-gestational-age infants (birth weight ≤ 2 SD). The controls were gestational-age matched nonsmoking mothers with infants having appropriate-for-gestational-age weight. The TUNEL method was used to demonstrate DNA fragmentation in nuclei, and a monoclonal antibody M30, specific for a neo-epitope on cytokeratin 18, was used to identify apoptotic epithelial cells. The positive nuclei (TUNEL) and positive cells (M30-positive cytoplasm) were counted blindly both in villous tissue and in decidual/basal plate tissue. M30-positive cells in villous tissues were significantly increased in placentas from smoking mothers compared to nonsmoking mothers. When evaluated by the TUNEL method, the difference between the two groups of women was not significant. Our study shows that apoptosis was increased in the placentas of smoking mothers with growth-restricted infants. The difference between the two groups was mainly in the syncytiotrophoblast layer and in connection with perivillous fibrin deposition. Cigarette smoke with reduction in blood flow has previously been shown to increase apoptosis, and it is possible that this could be one of the mechanisms playing a role in the growth restriction.

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased ( P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.

GCMa Regulates the Syncytin-mediated Trophoblastic Fusion

The human placental trophoblast cell can be classified as either a cytotrophoblast or a syncytiotrophoblast. Cytotrophoblasts can function as stem cells for the development of the syncytiotrophoblast layer via cell fusion. Anenvelopegene of the human endogenous retrovirus family W (HERV-W) calledsyncytinis specifically expressed in the syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein; therefore, it can mediate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this process by regulatingsyncytingene expression. In this report, we demonstrate that GCMa was able to regulatesyncytingene expression via two GCMa-binding sites upstream of the 5′-long terminal repeat of thesyncytin-harboring HERV-W family member in BeWo and JEG3 cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhancedsyncytingene expression and syncytin-mediated cell fusion in BeWo and JEG3 cells but not in HeLa cells. Therefore, the integration site of thesyncytin-harboring HERV-W family member in the human genome is close to the functional GCMa-binding sites by which GCMa can specifically transactivatesyncytingene expression in trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.