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Introduction Syncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. Methods We generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. Results The human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. Discussion These results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

Introduction To explore the involvement of signal transducers and activators of transcription (STATs) in trophoblast differentiation. Methods and results First, the localization of STATs in human placentas was detected via immunohistochemistry (IHC) and immunofluorescence (IF). Cytotrophoblasts (CTBs) expressed both STAT1 and 3, but syncytiotrophoblasts (STBs) did not. Staining for these two proteins showed a distinct upregulation from the proximal part to the distal end of cell columns. STAT5B was mainly expressed in the STBs, low in the CTBs, and absent in the extravillous trophoblasts (EVTs). Next, the 44 placenta samples were tested via western blot (WB) and quantitative real time polymerase chain reaction (qRT-PCR). We found a decrease in STAT1 and 3 and an increase in STAT5B as gestation increased from five to 10 weeks. Then, an in vitro co-culture model of placenta with or without decidua stromal cells (DSCs), as detected via flow cytometry, revealed an increase in the human leukocyte antigen (HLA)-G positive rate in trophoblasts from placentas co-cultured with DSCs, accompanied by an increase in p-STAT1 and 3 and a decrease in p-STAT5 and STAT5B. Finally, mRNA of matrix metalloproteinases (MMPs) and integrins after STAT silencing in HTR-8/SVneo was detected via qRT-PCR. STAT1 silencing decreased MMP9 expression, STAT3 silencing decreased MMP9, integrin α6, and β4 expression, and STAT5B silencing increased MMP2 and integrin β1 expression. Discussion Different trophoblasts showed distinct STAT expression profiles which were related to their MMP and integrin expression. DSCs promoted trophoblast differentiation into EVTs, possibly by regulating the STAT expression of the trophoblasts.

Introduction Placental transport is the first step in chemotherapeutic safety evaluations during pregnancy. However, a well-established in vitro model is not available. We previously reported that a trophoblast layer model using differentiating choriocarcinoma JEG-3 cells (DJEGs) can be used for placental drug transport studies. However, it was necessary to increase the similarities between the syncytiotrophoblast, the main layer of the placental barrier, and the in vitro evaluation model in order for the model to be useful for placental drug transport evaluations. We focused on in vivo similarities of differentiating induced pluripotent stem cells (iPSCs). iPSCs can achieve a syncytiotrophoblast-like form and secrete human chorionic gonadotropin (hCG) following bone morphogenetic protein 4 (BMP4) treatment. However, BMP4-treated iPSCs can differentiate into several cell types. In the placental transport model, a dense syncytiotrophoblast cell layer is necessary for appropriate differentiation. Methods The conditions permitting differentiation of iPSCs into syncytiotrophoblasts with retinoic acid (RA) treatment without BMP4 were investigated. The presence of syncytiotrophoblast-like cells was confirmed by measurement of mRNA expression levels of breast cancer resistance protein (BCRP) and paternally expressed 10 (PEG10) in syncytiotrophoblasts. In addition, immunofluorescence imaging of cytokeratin 7 (CK7) induced in trophoblasts was performed. Results and Discussion: RA-induced iPSCs exhibited these syncytiotrophoblast-like features and hCG secretion was maintained for at least 28 days after treatment with RA (500 nM) without BMP4. These results suggest that RA-induced iPSCs are a suitable in vitro syncytiotrophoblast model that can be used as an indicator of drug placental transport for pharmacotherapy during pregnancy.

Trophoblast fusion into syncytiotrophoblasts is a specialized yet enigmatic cellular process, which is essential for placental development and function. To facilitate mechanistic understanding of this critical process, here we re-purposed a widely used fluorescent membrane potential dye, Di-8-ANEPPS, to stably label the plasma membrane of live BeWo trophoblast cells. Compared to the methods currently available to quantify trophoblast fusion, our new fluorescent labeling method is simple, economical, robust and versatile, enabling quick and accurate quantification of fusion index in living cells.

Introduction Murine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands. Methods Notch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta. Results Notch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells. Discussion Canonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.

The placenta is an organ that secretes prostaglandin (PG) E2 into the fetal-placental circulation to regulate both vascular tone and remodeling of the fetal ductus arteriosus. Placental PGE2 synthesis might be mediated by microsomal PGE synthase-1 (mPGES-1), in addition to cyclooxygenase (COX) isoforms. Thus, the purpose of this study is to clarify the temporal and spatial expression patterns of mPGES-1, together with COX-1 and COX-2, in murine placenta. We found that mPGES-1 and COX-1 protein levels continuously increased in the placental labyrinth from gestational day (GD) 13.5 to GD19.5, becoming higher than in the decidua or the junctional zone by GD17.5. The PGE2 level at GD17.5 was also highest in the labyrinth. Immunofluorescence stainings for mPGES-1 and COX-1 in the labyrinth at GD17.5 overlapped and were located on the fetal side of the signals for connexin 26, which forms gap junctions between maternal-facing (SynT-I) and fetal-facing (SynT-II) syncytiotrophoblast layers, and on the maternal side of the signals for glucose transporter 1 on the basal plasma membrane of SynT-II. On the other hand, the signals for COX-2 did not overlap with those for mPGES-1. These results indicate that COX-1 and mPGES-1 are co-localized in murine placental SynT-II, facing the fetal-placental circulation. Therefore, SynT-II could contribute to placental synthesis of PGE2 for release into the fetal-placental circulation.

Increased production of soluble fms-like tyrosine kinase-1 (sFlt-1) from placenta is one of the major contributors to the development of preeclampsia. Our previous study has shown that hydrogen sulfide (H2S) inhibits sFlt-1 release in placenta. In the present study, we sought to investigate whether endogenous H2S affects sFlt-1 production and elucidate which H2S-producing enzyme is responsible for its effect in placenta. It was found that, besides cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), 3-mercaptopyruvatesulfurtransferase (3-MST) was identified in human placenta and mainly localized in syncytiotrophoblasts. There was no significant difference in expression level of 3-MST among preeclamptic and normal placentas. Treatment of cultured syncytiotrophoblasts with NaHS and l-cysteine suppressed sFlt-1 mRNA expression and caused a decrease in sFlt-1 protein content in culture media of the cells. Transfection of syncytiotrophoblasts with CBS siRNA and CSE siRNA reversed the above effects of l-cysteine. Furthermore, NaHS and l-cysteine treatment decreased the half-life of sFlt-1 mRNA and increased the expression of miR-133b targeting sFlt-1. MiR-133b expression was downregulated in preeclamptic placentas and correlated with the level of CBS and CSE. These results indicate that H2S is an important regulatory factor in sFlt-1 production in placenta. Reduced H2S generation in placenta contributes to development of preeclampsia by enhancing sFlt-1 production.

Introduction Forkhead box P3 (Foxp3) is necessary for induction of the immunosuppressive functions in regulatory T cells. Recent data indicate that some non-lymphoid cells, such as certain tumor cells, also express Foxp3, which participates in tumorigenesis and is related to the invasive and proliferative behavior of the tumor. However, the expression of Foxp3 in trophoblasts has not been studied previously. Methods Localization of Foxp3 in the villi and decidua in the first trimester was determined using immunohistochemistry. Foxp3 expression was detected by flow cytometry, and the expression levels in the first trimester placenta were compared with those in the term placenta. Additionally, the Foxp3 expression in trophoblasts in recurrent pregnancy loss (RPL) was analyzed by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and Western blot. Results Foxp3 is expressed by trophoblasts in early pregnancy in the villi and decidua. Foxp3 is expressed in the nuclei of both trophoblast cell columns and cytotrophoblasts. However, low Foxp3 expression was observed in syncytiotrophoblasts. Foxp3 was also expressed by the extravillous trophoblasts in early pregnancy in decidua. The Foxp3 expression in trophoblasts in the term placenta was significantly decreased compared with that in the normal early pregnancy. Moreover, decreased levels of Foxp3 mRNA and protein were observed in women with RPL. Discussion The selective expression of Foxp3 in human trophoblasts suggest that Foxp3 expression may be associated with the proliferation and invasion behavior of trophoblasts. The decreased Foxp3 expression in the trophoblasts in RPL may contribute to better understanding of the pathological characteristics of RPL.

Aims The human placental syncytiotrophoblast (STB) cells play essential roles in embryo implantation and nutrient exchange between the mother and the fetus. STBs are polyploid which are formed by fusion of diploid cytotrophoblast (CTB) cells. Abnormality in STBs formation can result in pregnancy-related disorders. While a number of genes have been associated with CTB fusion the initial events that trigger cell fusion are not well understood. Primary objective of this study was to enhance our understanding about the molecular mechanism of placental cell fusion. Methods FACS and microscopic analysis was used to optimize Forskolin-induced fusion of BeWo cells (surrogate of CTBs) and subsequently, changes in the expression of different cell cycle regulator genes were analyzed through Western blotting and qPCR. Immunohistochemistry was performed on the first trimester placental tissue sections to validate the results in the context of placental tissue. Effect of Cyclin Dependent Kinase 1 (CDK1) inhibitor, RO3306, on BeWo cell fusion was studied by microscopy and FACS, and by monitoring the expression of human Chorionic Gonadotropin (hCG) by Western blotting and qPCR. Results The data showed that the placental cell fusion was associated with down regulation of CDK1 and its associated cyclin B, and significant decrease in DNA replication. Moreover, inhibition of CDK1 by an exogenous inhibitor induced placental cell fusion and expression of hCG. Conclusion Here, we report that the placental cell fusion can be induced by inhibiting CDK1. This study has a high therapeutic significance to manage pregnancy related abnormalities.

Introduction Perfluorooctane sulfonate (PFOS) is widely used as surfactants, lubricants, adhesives, fire retardants and propellants. Animal experiments have shown that PFOS can potentially influence reproductive function. The objective of the present study was to investigate the effects of PFOS on the endocrine function of human placental syncytiotrophoblasts. Methods Primary human placental cytotrophoblasts were isolated from term placenta. After syncytialization, the levels of aromatase and apoptosis-related proteins including caspase3, Bcl-2 and Bax were examined after treatment with PFOS from 0.0001 μM to 1 μM or PFOS (0.1 μM) in the presence and absence of apoptosis inhibitor Z-VAD-FMK (30 μM) for 24 h. Results PFOS suppressed aromatase level and the secretion of estradiol, hCG and progesterone in a concentration-dependent manner from 0.0001 μM to 1 μM with a significant inhibition at 0.001 μM and above in human placental syncytiotrophoblasts. Furthermore PFOS reduced cell viability and induced apoptosis in human placental syncytiotrophoblasts as revealed by increases of pro-apoptosis proteins such as Bax and cleaved-caspase3, and decreases of pro-caspase3 and anti-apoptosis protein Bcl-2. The apoptosis induced by PFOS was further illustrated by increased DNA fragmentation and nuclear condensation. Blocking apoptosis with pan-caspase inhibitor Z-VAD-FMK, the impairment of placental endocrine function by PFOS was restored. Discussion These results indicate that PFOS may disrupt the secretion of hCG, progesterone and estradiol by human placental syncytiotrophoblasts via induction of apoptosis.

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.

The intracellular pH (pHi) of syncytiotrophoblasts is regulated, in part, by Na+/H+ exchanger (NHE)-1, NHE-2, and NHE-3. Failures in pHi homeostasis could alter critical cellular functions such as water transport and cell volume. Here, we evaluated whether alterations in syncytiotrophoblast pHi could modify water uptake mediated by aquaporins (AQPs) and the contribution of NHEs to this mechanism. We showed that changes in syncytiotrophoblast pHi did not affect water uptake in the presence of functional NHEs. However, inhibition of NHEs alters transcellular water transport mediated by AQPs in acidosis. These results suggest an interaction between placental AQPs and NHEs in the regulation of water uptake during acidotic states. Fil: Dietrich, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Damiano, Alicia Ermelinda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Objectives Well-controlled trophoblast migration and invasion at the maternal–foetal interface are crucial events for normal placentation and successful pregnancy. Growing evidence has revealed that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) participates in tumour migration and invasion as a tumour suppressor. However, the expression and function of Gadd45α in trophoblasts is unknown. This study aimed to determine the Gadd45α expression and function in the human first trimester placenta and identify the underlying mechanisms. Methods The expression of Gadd45α in human first trimester placenta was determined using immunohistochemistry. HTR8/SVneo cell line was used to investigate the effects of Gadd45α on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP)2/9 activities, and tissue inhibitor of metalloproteinase (TIMP)1/2 expression using cell proliferation assays, flow cytometric analysis, transwell migration/invasion assays, gelatin gel zymography, and western blotting, respectively. Moreover, a placental villous explant model was employed to verify its functions in placentation. Results Gadd45α was strongly expressed in syncytiotrophoblasts and trophoblast columns of human placental villi, extravillous trophoblast cells and glandular epithelium within the maternal decidua. Gadd45α knockdown significantly promoted migration and invasion of HTR8/SVneo cells, whereas it did not affect cell proliferation or apoptosis. Silencing Gadd45α also enhanced trophoblast outgrowth and migration in placental explants. These effects were related to increased activities of MMP2/9 and the decreased expression of TIMP1/2. Discussion and conclusion Gadd45α may be involved in human trophoblast migration and invasion and may function as an important negative regulator at the foetal–maternal interface during early pregnancy by directly or indirectly regulating MMP2/9 activities.

Introduction Increased estrogen production in placenta towards the end of gestation plays a pivotal role in the onset of human labor. Estrogen transforms myometrium from a quiescent to a contractile status. Glucocorticoids have been shown to induce estrogen production through the transcription factor specificity protein 1 (Sp1)-mediated induction of aromatase transcription upon elevation of cyclic adenosine mono-phosphate (cAMP) level in human placental syncytiotrophoblasts. However, it is unclear how glucocorticoids activate cAMP pathway thereby inducing aromatase expression in human placental syncytiotrophoblasts. Material and methods We investigated this issue in cultured primary human placental syncytiotrophoblasts prepared from placentas collected at term without labor. Results We demonstrated that cortisol (0.01–1 μM) dose-dependently increased corticotropin-releasing hormone (CRH) and human chorionic gonadotropin (hCG) α/β subunit expression and their production in the syncytiotrophoblasts. The induction of intracellular cAMP level, Sp1 expression, Sp1 enrichment at the aromatase promoter as well as aromatase expression by cortisol could be partially attenuated by either hCG antibody (1:100) or CRH receptor antagonist α-helical-CRH (1 μM), and further attenuated by combination of hCG antibody and α-helical-CRH. Conclusions Cortisol increases aromatase expression via induction of CRH and hCG production and subsequent elevation of cAMP level and enrichment of Sp1 at the aromatase promoter in human placental syncytiotrophoblasts. These findings may account for the parallel increases of cortisol and estrogen production prior to the onset of parturition.

Sulfate is an important nutrient for fetal growth and development. The fetus has no mechanism for producing its own sulfate and is therefore totally reliant on sulfate from the maternal circulation via placental sulfate transport. To build a model of directional sulfate transport in the placenta, we investigated the relative abundance of the 10 known sulfate transporter mRNAs in human placenta from uncomplicated term pregnancies. SLC13A4 and SLC26A2 were the most abundant sulfate transporter mRNAs, which localized to syncytiotrophoblast and cytotrophoblast cells, respectively. These findings indicate important physiological roles for SLC13A4 and SLC26A2 in human placental sulfate transport.

Human placental villi are surfaced by the syncytiotrophoblast, a multinucleated, epithelial-cell layer that functions in maternal-fetal exchange. Mononucleated cytotrophoblasts are subjacent to the syncytiotrophoblast. Using confocal fluorescence microscopy of third-trimester villi, we previously found that cytotrophoblasts are often interdigitated into the syncytiotrophoblast, that cytotrophoblasts undergo caspase-mediated apoptosis, and that apoptosis is much lower, and perhaps completely inhibited, in intact syncytiotrophoblast lacking fibrin-type fibrinoid. Previous analysis of primary cultures of human trophoblasts also indicated lower levels of apoptosis in syncytiotrophoblast compared to cytotrophoblasts. Here, using confocal microscopy we find that cultured cytotrophoblasts and syncytiotrophoblasts display complex structural relationships, as in vivo, and that apoptosis of a cytotrophoblast or syncytiotrophoblast does not induce apoptosis of adjacent trophoblasts. Using live-cell imaging of mitochondrial depolarization and nuclear condensation in cultured syncytiotrophoblasts, we show apoptosis initiates in a localized region and propagates radially at ~five μm/min with no loss of velocity until the entire syncytium has undergone apoptosis. The rate of propagation is similar in cases of spontaneous apoptosis and in apoptosis that occurs in the presence of cobalt chloride or rotenone, two inducers of apoptosis. We suggest that inhibition of syncytiotrophoblast apoptosis in vivo is important to prevent widespread syncytiotrophoblast death, which would result in placental dysfunction and contribute to poor pregnancy outcomes.

The purpose of this study is to assess the role of the protein kinase A (PKA) in regulating uptake of dehydroepiandrosterone sulfate (DHEAS), an estrogen precursor, by syncytiotrophoblasts. Forskolin, a PKA activator, significantly increased [(3)H]DHEAS uptake and the mRNA expression levels of organic anion transporter (OAT) 4 and CYP19A1 in choriocarcinoma JEG-3 cells, while other steroid sulfate transporters present in the placenta showed no change in expression level. KT5720, a PKA inhibitor, attenuated these effects of forskolin. Accordingly, the PKA pathway appears to play an important role in estrogen synthesis by cooperatively regulating OAT4 and steroidogenic enzymes in syncytiotrophoblasts.

Thyroid hormones (TH) are important for the development of the human fetus and placenta from very early gestation. The transplacental passage of TH from mother to fetus and the supply of TH into trophoblasts require the expression of placental TH plasma membrane transporters. We describe the ontogeny of the TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 in a large series (n = 110) of normal human placentae across gestation and describe their expression changes with intrauterine fetal growth restriction (IUGR n = 22). Quantitative RT-PCR revealed that all the mRNAs encoding TH transporters are expressed in human placenta from 6 weeks gestation and throughout pregnancy. MCT8, MCT10, OATP1A2 and LAT1 mRNA expression increased with gestation. OATP4A1 and CD98 (LATs obligatory associated protein) mRNA expression reached a nadir in mid-gestation before increasing towards term. LAT2 mRNA expression did not alter throughout gestation. Immunohistochemistry localised MCT10 and OATP1A2 to villous cytotrophoblasts and syncytiotrophoblasts, and extravillous trophoblasts while OATP4A1 was preferentially expressed in the villous syncytiotrophoblasts. Whilst MCT8 protein expression was increased, MCT10 mRNA expression was decreased in placentae from IUGR pregnancies delivered in the early 3rd trimester compared to age matched appropriately grown for gestational age controls. No significant change was found in the mRNA expression of the other transporters with IUGR. In conclusion, several TH transporters are present in the human placenta from early 1st trimester with varying patterns of expression throughout gestation. Their coordinated effects may regulate both transplacental TH passage and TH supply to trophoblasts, which are critical for the normal development of the fetus and placenta. Increased MCT8 and decreased MCT10 expression within placentae of pregnancies complicated by IUGR may contribute to aberrant development of the fetoplacental unit.

In non-gestational tissues, emerging data indicate that the FoxO1 family of Forkhead transcription factors play diverse roles in many cellular processes coordinating programs of gene expression that regulate apoptosis, oxidative stress resistance, and immune cell homeostasis. Successful outcome of human parturition rely on many of these processes, however there is no data available on FoxO1 proteins in human intrauterine tissues, nor their role in pregnancy complications such as pre-eclampsia. Thus the aim of this study was (i) to characterises the localisation and expression of FoxO1, acetylated (ac)-FoxO1 and phosphorylated (p)-FoxO1 in human placenta and fetal membranes obtained from term Caesarean sections (n=5); and (ii) to compare the expression of FoxO1 proteins in term placental samples from normal and pre-eclamptic pregnancies (n=5 per group). In placenta, weak FoxO1 staining was localised to the syncytiotrophoblast layer, whereas ac-FoxO1 and p-FoxO1 staining was mainly localised in the syncytiotrophoblasts and cytotrophoblasts. In fetal membranes, FoxO1, ac-FoxO1 and p-FoxO1 were localised to the trophoblast layer of the chorion, amnion epithelium and decidual cells. Quantitative RT-PCR (qRT-PCR) analysis showed a 6-fold and 12-fold higher mRNA expression in the choriodecidua compared to placenta and amnion, respectively. In both amnion and choriodecidua, FoxO1 protein expression was higher in the cytoplasmic fractions than in the nuclear fractions. On the otherhand, ac-FoxO1 and p-FoxO1 protein expression was higher in the nuclear fractions for all three tissues. There was no difference in the mRNA or protein expression of FoxO1 proteins in placental samples from normal and pre-eclamptic term pregnancies. The exact role of FoxO1 proteins in human pregnancy are unknown, however the finding that they are expressed in human gestational tissues warrants further research into their function in these tissues.

Contains fulltext : 81950.pdf (Publisher’s version ) (Closed access) Placental corticotropin-releasing factor (CRF) are thought to induce labor via activation of CRF receptor type 1 (CRF-R1) leading to several feed forward mechanisms in the placental, fetal and maternal compartments. Recently, receptor type 2 (CRF-R2) selective ligands called urocortin 2 and 3 (Ucn 2, Ucn 3) were characterized as neuropeptides in the brain. We studied the expression of Ucn 1, 2 and 3 in feto-placental tissues qualitatively (by immunohistochemistry) and quantitatively (by radioimmunoassay) and compared these with expression of placental CRF. The presented placental Ucn 2 and 3 peptide quantification, characterization and ex-vivo release results are novel. Reversed-phase HPLC fractionation of placental extracts revealed several peaks containing immune-reactive (ir)-like Ucn 2 or 3, of which the main peaks had the same retention time as the synthetic Ucn 2 and 3 peptides. Placental tissues contained between 6 and 10 times more ir-CRF than ir-Ucn 1, 2 or 3. The placental Ucn 1, 2 and 3 peptide contents correlated with each other. Our immunohistochemical results showed that all urocortins were mainly localized in the syncytiotrophoblasts of the placental villi. Placental urocortins were actively released during ex-vivo perfusion of cotyledons. From these results it can be concluded that Ucn 2 and 3 peptides are present in placental and fetal membrane tissues, and released by ex-vivo perfused cotyledons. Therefore, placental urocortins may function as paracrine or endocrine messengers during pregnancy and parturition.

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12 h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

We previously investigated tissue specificity and temporal changes in expression of five human endogenous retroviruses (HERVs), including syncytin/ERVWE1 and syncytin 2. In the current study, we examined the cellular localization and quantified the transcripts of five HERVs, syncytin, syncytin 2, HERV-H7/F(XA34), HERV-Fb1, and HERV-HML6-c14, in order to elucidate their physiological and etiological roles in the placenta and in pregnancy-induced hypertension (PIH), respectively. In situ hybridization revealed trophoblast-specific transcription of all five HERVs. Consistent with a previous immunohistochemical analysis, syncytin 2 transcripts were detected only in cytotrophoblasts. All the HERVs except HERV-HML6-c14 (HML6-c14) were predominantly localized in the cytoplasm of syncytiotrophoblasts and/or cytotrophoblasts. Quantitative analysis showed that transcriptional levels of these four HERVs were lower in placentas obtained from pregnant women with PIH (n=22) than in those from normotensive pregnant women (n=87) and that the differences were statistically significant (p=0.001, 0.01

Highly purified (>99.99%) primary villous cytotrophoblasts from uncomplicated pregnancies and pregnancies complicated with intrauterine growth restriction (IUGR) alone, IUGR with pre-eclampsia (IUGR-PE) and PE alone were cultured for 5 days and the extent of differentiation into syncytiotrophoblasts measured in terms of syncytialisation and secretion of chorionic gonadotropin (hCG) and placental lactogen (hPL). Three separate phenotypes were observed: (1) normal and IUGR-PE cells showed low syncytialisation and secretion of hCG and hPL, (2) IUGR cells showed the highest levels of syncytialisation and secretion and (3) PE cells showed high syncytialisation but low secretion. These results strongly suggest IUGR, IUGR-PE and PE to be distinct conditions in which villous cytotrophoblasts are either exposed to different environments or are genetically different.

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1 kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Recently, the gene encoding a new stress-induced protein termed reducing agent and tunicamycin-responsive protein (RTP) was identified. The function of RTP is unknown, however, the strong upregulation of RTP during cellular differentiation, and exposure to stress conditions including hypoxia suggests a specific role for RTP in these processes. In pre-eclampsia, impaired spiral artery remodelling and reduced perfusion may reduce oxygen tension in the placenta and thereby alter trophoblast differentiation and function. We therefore hypothesized that the expression of RTP mRNA is altered in the placentae of women with pre-eclampsia. The aims of this study were to determine the regional distribution and cellular localization of RTP mRNA expression and compare mRNA abundance in different regions of normotensive control and pre-eclamptic placentae. In normal and pre-eclamptic placentae, RTP mRNA was expressed in the syncytiotrophoblasts and in the intermediate trophoblasts of the basal plate. In early onset pre-eclampsia, RTP mRNA was more abundant in the chorionic villi regions. A further increase was localized to the syncytial knots and to the trophoblasts in the peri-infarct regions. The increased RTP expression may reflect lower oxygen tension and/or other stress stimuli in the placenta in pre-eclampsia.

Transport of iron from the mother to the fetus is essential for the normal development of the fetus and abnormalities in the transferrin receptor (TFR) on the placental trophoblasts might have some crucial effects on the fetal iron metabolism. The present study was undertaken to determine whether there are any changes in the expression of the transferrin receptor in the placenta from pre-eclamptic mothers. An immunohistochemical study using antibodies specific for C-terminus and N-terminus regions of the TFR revealed that TFR expression by syncytiotrophoblasts around chorionic villi is markedly reduced in placentae from pre-eclamptic pregnancies compared to those from normal pregnancies and pregnancies at early gestational age that terminated by abortion. The same result, although to a lesser extent, was obtained even in trophoblasts which were located around atrophic villi with fibrotic changes in the interstitium, or which invaded into the deciduas. The expression of human chorionic gonadotropin (HCG) on those cells was observed to the same extent in the normal and pre-eclamptic pregnancy groups. The concentration of TFR in the peripheral blood also decreased in pre-eclampsia. These results suggest that TFR synthesis in the pre-eclampsia, especially in the placental trophoblasts, is decreased.

Oncofetal fibronectin (onfFN) is an extracellular matrix (ECM) protein synthesized by tumours and fetal tissue including placenta. The appearance of onfFN and other cellular FNs in cervico-vaginal secretions and maternal blood is associated with adverse pregnancy outcomes. In the current study, we used dual (maternal and fetal) perfusion of human term placentae and primary cultures of syncytiotrophoblasts (SCTs) to determine whether the human placenta releases onfFN. ELISA and Western blotting revealed that onfFN is preferentially released to the maternal perfusate in a time-dependent manner. Immunodetection with FDC-6, extra domain A (EDA) and cell binding domain-specific antibodies revealed onfFN in maternal perfusate to be a high molecular weight (>450 kDa) protein dimer, similar to that found in amniotic fluid. This suggested that onfFN was released intact from placenta and not cleaved from an ECM. In addition, a similar high molecular weight dimeric onfFN species was noted in conditioned media from cultures of SCTs. Since SCTs directly release proteins to the intervillous space, this suggests that SCTs may be a source of onfFN detected in maternal perfusate. These results indicate that onfFN is released from human placenta and thus levels in maternal sera may provide insight into placental pathophysiology.

Placental protein 5 (PP5)/tissue factor pathway inhibitor-2 (TFPI-2), a serine proteinase inhibitor, is homologous to tissue factor pathway inhibitor (TFPI) and commonly found in peripheral blood of pregnant woman. Although TFPI is well known to be synthesized primarily in endothelium and to play an important role in regulation of the extrinsic pathway of blood coagulation, the function of PP5/TFPI-2 remains unclear. Our previous report demonstrated that PP5/TFPI-2 expression is ubiquitous and extremely high in growing placental tissue. Using newly generated polyclonal anti-PP5/TFPI-2 antibody, and by immunohistochemistry and immunoelectromicroscopy, we examined precise localization of PP5/TFPI-2 in placenta especially in syncytiotrophoblasts, which had been shown to produce PP5/TFPI-2 mRNA by our previous study using in situ hybridization. Immunoelectromicroscopy revealed PP5/TFPI-2 localizing on the surface of the microvilli and the membrane of endoplasmic reticulum of syncytiotrophoblasts. To confirm the cell surface association of PP5/TFPI-2, placental villi were incubated with heparin and resultant soluble fraction was analysed by Western blotting. Heparin liberating PP5/TFPI-2 from villi suggested that PP5/TFPI-2 might be retained on the microvilli surface through the binding to membrane-anchored proteoglycans such as glypican and/or syndecan family members. We also examined the relationship between the presence of cell layer of syncytiotrophoblasts and the coagulation using clinical specimens, and revealed that the fibrin depositions were specifically observed on the regions lacking syncytiotrophoblasts cell layer in placental villi. Therefore, it is likely that during pregnancy PP5/TFPI-2 may be retained on the surface of placental villi via proteoglycans, and may play an important role to maintain intervillous blood flow and the patency of microvasculature in feto-maternal blood system mediated by the inhibition of serine proteinases involved in the blood coagulation.

Placental trophoblasts undergo apoptosis as part of normal epithelial turnover and placental ageing. Classically, the induction of apoptosis in in vitro preparations has utilized the cytokines TNFalpha and IFNgamma and has been measured using the TUNEL technique. The aim of this study was to compare apoptotic susceptibility of mononucleated and differentiated trophoblasts using a range of cytotoxic agents. To achieve this, an in vitro model of syncytialization was used, along with isolated placental cytotrophoblasts and an extravillous cytotrophoblast derived cell line (SGHPL-4). Cytotrophoblasts from term placentae (n=12), syncytiotrophoblasts (n=12) and SGHPL-4s (n=8) were cultured under reduced oxygen or with TNFalpha/IFNgamma, dexamethasone or staurosporine. Apoptosis assessments were made using TUNEL, Annexin V binding, fluorescence microscopy and ATP/ADP measurements. Each cytotoxic agent increased apoptosis in all three cell populations. For untreated cells, cytotrophoblasts showed the greatest levels of apoptosis in culture. With stimulation, these levels were significantly elevated using dexamethasone, TNFalpha/IFNgamma and staurosporine and further raised under hypoxic conditions. SGHPL-4 cells showed similar trends to those of cytotrophoblasts, however the syncytiotrophoblasts, although responsive to dexamethasone and TNFalpha/IFNgamma, showed lower levels of apoptosis with staurosporine and hypoxia. ADP : ATP measurements gave similar results to the other techniques and ratios of less than 1.0 were correlated with Annexin V measurements on the flow cytometer (P< 0.001). The typical morphological features of apoptosis i.e. chromatin margination, membrane blebbing and apoptotic body formation were detected in cytotrophoblasts and SGHPL-4 cells. However, only chromatin condensation could be recognized in syncytiotrophoblast preparations. Necrotic cell numbers were also increased under all cytotoxic conditions. Although elevated with dexamethasone, staurosporine and hypoxia, these levels were markedly raised in cytotrophoblasts and SGHPL-4 cells following incubations with TNFalpha/IFNgamma. These observations show variations in apoptosis between mononuclear trophoblasts and differentiated multinucleated syncytiotrophoblasts. Differential effects of stimuli may suggest disparate apoptotic pathways. These variations may reflect functional differences between placental cellular and syncytial components and may highlight the importance of exogenous stimulation in various stages of placental development.

We demonstrated the subcellular localization of endothelial nitric oxide synthase (eNOS) in human placental terminal villous trophoblasts at near term period, and compared the distribution pattern with that in pre-eclamptic trophoblasts, using immunogold electron microscopy. Immunolabelling for eNOS was visible markedly in the syncytial microvilli and syncytial cytoplasm. Semiquantitative analysis showed that the concentration and the distribution pattern of gold particles for eNOS did not significantly differ between normal and pre-eclamptic placental trophoblasts. These results indicated that syncytiotrophoblastic microvilli and cytoplasm were the subcellular localization sites of syncytium-derived eNOS in terminal villi, and that there were no significant differences in this eNOS subcellular distribution pattern between normal and pre-eclamptic syncytiotrophoblasts in regard to immunohistochemically detectable eNOS.

This review summarizes the experimental evidence supporting the concept that oestrogen has a central integrative role in modulating the communication that occurs between the placenta and the fetus which results in primate fetal–placental development. Thus oestrogen, acting within placental trophoblasts, regulates the functional differentiation of syncytiotrophoblasts, manifested as an upregulation of key components of the progesterone biosynthetic pathway and the 11β-hydroxysteroid dehydrogenase (11β-HSD)-1 and -2 enzymes controlling cortisol–cortisone interconversion. The increase in 11β-HSD expression results in the switch in the qualitative and quantitative patterns of transplacental corticosteroid metabolism that induces maturation of the primate fetal hypothalamic pituitary adrenocortical axis. The studies outlined in this review, therefore, provide new insight into the role that oestrogen plays during the course of primate pregnancy and demonstrate that an oestrogen-dependent signalling system exists in utero that coordinates the placental and fetal dialogue critical to development of the placenta and endocrine systems underlying neonatal self-sufficiency.

The purpose of the present study was to localize glucose-6-phosphatase (G6Pase) activity in the human placenta at various gestational stages and to compare them to pre-eclamptic placenta activity. Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicative of G6Pase activity were observed in the endoplasmic reticulum and the nuclear envelope of the syncytiotrophoblasts in near-term placenta obtained from women with normal pregnancies. In placenta taken from women with severe pre-eclampsia, the localization pattern, enzyme activity intensity, and morphology of the endoplasmic reticulum did not differ from normal pregnancies. Stringent control experiments were performed also to ensure specific detection of G6Pase activity. The results indicate that cytochemically detectable G6Pase is present in the human placenta. This enzyme may play significant roles in carbohydrate metabolism in the human placenta.

Syndecan-1 is a cell surface heparan sulphate proteoglycan, which binds to the extracellular matrix (ECM), growth factors and antithrombin III. The early expression of syndecan-1 during mouse embryonic development suggests a potential role in the communication between the embryo and the ECM of decidua. Using immunohistochemical methods, the present study showed that the expression of syndecan-1 in the trophoblast cells changes along trophoblast differentiation. The syncytiotrophoblasts in the chorionic villi exhibited an apical expression of syndecan-1. This suggests that the expression is restricted to non-migrating, non-proliferating trophoblasts. The mode of syndecan-1 expression by human placental trophoblasts is independent of gestational age. The expression is not changed in miscarriages. In pre-eclampsia, the staining for syndecan-1 on the villous syncytiotrophoblast is weaker compared to normal pregnancy, but in placental bed the expression is similar. The unique apical localization of syndecan-1 in chorionic villi, not detected in any other tissues, suggests a potential role in fetomaternal communication probably via growth factor binding and in anticoagulation of intervillous circulation.

Human placentation is mediated by fetal trophoblastic cells which penetrate into the decidualized uterine endometrium. Trophoblast invasion requires the precisely regulated secretion of specific proteinases able to degrade the endometrial basement membranes and extracellular matrix. To document further the involvement of these proteinases during human placentation, we evaluated in vivo the expression of stromelysin-3, a member of the metalloproteinase family, during the first and third trimesters of pregnancy, by means of immunohistochemistry, in situ hybridization and Northern blot analysis. Human extravillous trophoblasts invading the maternal decidua produced stromelysin-3 during both, the first and third trimesters of pregnancy, but to a lesser extent during the latter. In floating villi, stromelysin-3 expression was restricted to the syncytiotrophoblasts that line intervillous vascular spaces. In conclusion, stromelysin-3 is expressed by differentiated, non-proliferative villous and extravillous trophoblastic cells in early and late placental beds and villi, and its pattern of expression evolves during pregnancy. Our observations suggest that stromelysin-3 could play a role in human placentation.

We have shown that oestrogen has a central integrative role in regulating key components of the progesterone biosynthetic and corticosteroid metabolic pathways within syncytiotrophoblasts that govern placental function and maturation of the fetal pituitary-adrenocortical axis. Studies utilizing classic binding procedures and RNAse protection have demonstrated that human placental villous tissue exhibits specific high affinity oestrogen binding and expresses the mRNA for the oestrogen receptor. However, it is not known whether the oestrogen receptor is expressed specifically in syncytiotrophoblasts. Therefore, the present study determined whether the oestrogen receptor protein was detectable by immunocytochemistry in cultured human syncytiotrophoblast maintained in a low oestrogen/progestin environment. Cytotrophoblasts were isolated from human term placentae by trypsin dispersion and Percoll gradient centrifugation and cultured for 5, 7 or 10 days. Incubation of syncytiotrophoblast with 5-10 micrograms/ml of the anti-oestrogen receptor rat monoclonal antibody D-75, which is specific for the primate oestrogen receptor, resulted in identification of the oestrogen receptor in the nuclei of these cells. In contrast, there was no reactivity of the trophoblasts to either rat IgG or an irrelevant rat monoclonal antibody IgG2a against mouse common leukocyte antigen T200. Collectively, these findings indicate that oestrogen receptor is expressed in the nuclei of human placental syncytiotrophoblasts and support the suggestion that the syncytiotrophoblast is an oestrogen-responsive tissue.

The invasion of extravillous trophoblasts (EVTs) to the decidua and spiral arteries in early pregnancy is a crucial step for a successful pregnancy; however, its mechanisms are not fully understood. Lipocalin2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), reportedly enhanced invasiveness via the activation of matrix metalloproteinase-9 (MMP-9) in several cancer cells. In this study, the expression and function of LCN2 in early placenta were analyzed.Early placental tissues between 7 and 10 weeks of gestation were obtained from normal pregnant women who underwent elective termination. The expression of LCN2 was examined using immunostaining and RT-PCR. EVTs isolated from these placental tissues and a choriocarcinoma cell line (JAR) were used to investigate the effects of LCN2 on proliferation, invasion potential, and MMP-9 activity under hypoxia using a WST-1 assay, Matrigel invasion assay, and gelatin gel zymography, respectively.The immunohistochemical expression of LCN2 was observed in the cytoplasm of EVTs, cytotrophoblasts and the decidua, but not in syncytiotrophoblasts. The addition of recombinant LCN2 did not affect proliferation, but enhanced the invasiveness (500 ng/mL, p0.01) and MMP-9 activity of primary cultured EVTs and JAR in a dose-dependent manner. Silencing LCN2 using shRNA reduced the invasiveness (p0.01) and MMP-9 activity of JAR. In addition, the hypoxic condition (2% O₂) increased LCN2 expression (p0.01), MMP-9 activity, and invasive ability (p0.01).LCN2 was involved in the invasiveness of EVTs, especially under hypoxia, via increased MMP-9 activity.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) act as paracrine factors at or close to the sites of biosynthesis, i.e. cellular sites of expression of specific mRNAs. To determine the developmental pattern of expression of IGF-I, IGF-II, IGF-I R and IGFBP 1–6 mRNAs in the rhesus monkey placenta, in situ hybridization histochemistry was performed in placentae and fetal membranes from 65 days until term (165 ± 5 days). IGF-I mRNA was not detectable in any of the specimens examined. IGF-11 mRNA was localized abundantly in the placenta (syncytiotrophoblasts of the chorionic villi and the anchoring villi, and the extravillous cytotrophoblasts), and in the fetal membranes (chorion and amnion). IGF-I R mRNA was expressed predominantly in the decidua. All IGFBP mRNAs (IGFBP-1 to −6) were expressed in the maternal decidua in variable abundance. Only some IGFBP mRNAs, notably IGFBP-3 mRNA, was expressed in the fetal tissues, such as the chorionic mesoderm, some extravillous cytotrophoblasts and in the amnion and chorion. Gestational age did not alter the localization or relative abundance of all of the mRNAs studied. These findings suggest a role for IGF-11 in the regulation of nutrient transport or placental hormone synthesis and/or secretion in the syncytiotrophoblasts, and a role for IGF-1I and IGFBPs in the cell to cell communication and interaction at the feto-maternal interface.

The present study determined whether the elevation in oestrogen, which occurs with advancing baboon pregnancy, is associated with a developmental increase in expression of the placental enzymes catalysing progesterone synthesis. The mRNA levels for P-450 cholesterol side-chain cleavage (P-450scc), adrenodoxin, and delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) were assessed by Northern blot analysis in placental syncytiotrophoblasts isolated from baboons in early (days 58-65), mid- (days 97-113) and late (days 161-175), gestation (term = 184 days). Placental villous tissue was dispersed and subjected to 50 per cent Percoll density gradient centrifugation to obtain primarily syncytiotrophoblasts. Mean (+/- S.E.) P-450scc mRNA level, expressed as a ratio of beta-actin in the syncytiotrophoblast-rich fraction, progressively increased with advancing pregnancy to a level in late gestation (1.81 +/- 0.28 arbitrary units) that was approximately sixfold (P0.01) greater than in early gestation (0.31 +/- 0.08) and approximately twofold greater (P0.05) than in mid-gestation (0.97 +/- 0.24). In contrast adrenodoxin mRNA expression was similar at early (0.97), mid- (1.14 +/- 0.12) and late (1.16 +/- 0.13) gestation. Syncytiotrophoblast 3 beta-HSD mRNA levels also remained constant in early (1.69), mid- (1.89 +/- 0.41) and late (1.34 +/- 0.41) gestation. On the basis of these findings, we propose that villous syncytiotrophoblasts undergo functional/biochemical differentiation, resulting in a coordinated upregulation of specific components of the steroid biosynthetic pathway required for progesterone biosynthesis during the course of primate pregnancy.

Human trophoblast differentiates in vivo and in vitro by the fusion of cytotrophoblastic cells to form syncytiotrophoblasts. A large amount of human chorionic gonadotrophin (hCG) is produced by the syncytiotrophoblasts, which express hCG luteinizing hormone (LH) receptors. Since recent investigations with electrophysiological techniques support the conclusion that hormonal effects can be mediated by modulations of the membrane ionic conductances of the cells, a perforated patch-clamp technique was used to investigate the possible presence of a chloride current evoked by hCG. The perifusion of hCG (500 mIU/ml) activated a time-independent current, which presents a linear current-voltage (I/V) relationship in symmetrical chloride concentrations. The reversal potential was − 1.8 mV with 142 mm Cl − external solution and 134 m m cl − internal solution. This reversal potential shifted with changes in the transmembrane Cl − gradient. Moreover, this hCG-induced current was sensitive to 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50 μ m ), to diphenylalamine-2-carboxylic acid (DPC) (0.5 m m ) and to 9-AC (1 m m ), three known chloride channel blockers. These results confirm the autocrine action of hCG in the physiology of the trophoblast.

Placental protein 4 (PP4) is a soluble placental tissue protein which was isolated from human placenta. The aim of the present study was to demonstrate the localization of cells containing PP4 in human placenta and in various female genital tissues under normal conditions. PP4 immunoreactive structures were demonstrated by using the peroxidase-antiperoxidase immunohistochemical technique. The samples were obtained from normal human placenta, umbilical cord, uterine cervix, endometrium, ovary and vulva. The most differentiated trophoblastic cells, the syncytiotrophoblasts, as well as the intermediate trophoblast cells contained PP4. PP4 immunoreactivity was present in umbilical cord as well. Occasionally PP4 was detected in normal ovarian, endometrial or vulvar tissue samples. Cervix and myometrium were free of PP4 immunoreactive material. PP4 staining was cytoplasmic. Our findings indicate that PP4 cannot be considered specific for the placenta since it is present in some human adult tissues as well.

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

High temperature requirement A (HtrA) family proteins are serine proteases that may serve in the quality control of misfolded or mislocalized proteins. Recently, possible involvements of HtrA1 in the normal development of the placenta and in the pathogenesis of pre-eclampsia were reported. In this study, we characterized HtrA4, a previously uncharacterized HtrA protein family member, in pre-eclampsia. Elevated expression levels of placental HtrA4 in pre-eclampsia patients were observed by qRT-PCR. Western blotting also showed an increased production of HtrA4 at the protein level in pre-eclamptic placentas. In normal chorionic villi, HtrA4 protein was more abundant in the cytoplasm of cytotrophoblasts than in syncytiotrophoblasts. In contrast, the amount of HtrA4 protein in syncytiotrophoblasts was dramatically increased in pre-eclamptic placentas. Circulating HtrA4 was detected at higher levels in sera from women with pre-eclampsia than from those with normotensive pregnancies. Serum HtrA4 levels were higher in patients with early onset and inversely correlated with the weights of the newborn and placenta. Furthermore, serum levels correlated with serum PAPP-A and PAPP-A2 levels, indicating a functional role for HtrA4 in the common pathway. These data suggest that increased HtrA4 may be involved in the onset of pre-eclampsia, and elevated levels in sera imply a potential application as a biomarker for this disorder.

We describe here for the first time the characterization of family member of netrins, netrin-4 and its receptor neogenin, during the development of the placenta. By using western blots and RT-PCR, we demonstrated the presence of netrin-4 and its receptor neogenin protein as well as their transcripts. Using immunohistochemistry, we studied the distribution of netrin-4 and neogenin in both the first trimester and term placenta. We observed staining of netrin-4 in villous and extravillous cytotrophoblasts, syncytiotrophoblast, and endothelial cells whereas staining in stromal cells was faint. In decidua, we observed netrin-4 labelling in glandular epithelial cells, perivascular decidualized cells, and endothelial cells. However, neogenin was absent in villous and extravillous cytotrophoblasts and was expressed only on syncytiotrophoblast and placental stromal cells in the first trimester and at term placenta. The pattern of distribution suggests that a functional netrin-4-neogenin pathway might be restricted to syncytiotrophoblasts, mesenchymal cells, and villous endothelial cells. This pathway function might vary with its localization in the placenta. It is possibly involved in angiogenesis, morphogenesis, and differentiation.

MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. Study design: MFG-E8 protein and its receptor integrin αvβ3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. Main outcome measures: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. Results: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. Conclusions: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals. Fil: Bocca, Silvina M.. Eastern Virginia Medical School; Estados Unidos Fil: Anderson, Sandra. Eastern Virginia Medical School; Estados Unidos Fil: Amaker, Barbara. Sentara Norfolk General Hospital; Estados Unidos Fil: Swanson, R. James. Old Dominion Univeristy; Estados Unidos Fil: Franchi, Nilda Anahi. Eastern Virginia Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Lattanzio, Frank A.. Eastern Virginia Medical School; Estados Unidos Fil: Oehninger, Sergio Carlos. Eastern Virginia Medical School; Estados Unidos

Forkhead box O (FoxO) proteins regulate inflammation, extracellular matrix (ECM) remodelling and apoptosis. We have previously identified FoxO1 proteins in human gestational tissues, and demonstrated a link between FoxO1 and rupture of fetal membranes. There is, however, no data available on the expression and localisation of FoxO3 and FoxO4 in human intrauterine tissues. Thus the aim of this study was to characterise the localisation and expression of FoxO3 and FoxO4 in (i) human placenta and fetal membranes before term spontaneous labour onset, and (ii) supracervical site (SCS) and distal site (DS) fetal membranes from non-labouring women. Immunohistochemistry, Western blotting and quantitative RT-PCR (qRT-PCR) was used to localise and quantitate FoxO3 and FoxO4 protein and mRNA expressions. Cytoplasmic and nuclear FoxO3 was localised in the syncytiotrophoblast layer, chorionic trophoblasts, amnion epithelium and decidua. Cytoplasmic FoxO4 was localised in the syncytiotrophoblasts and chorionic trophoblasts. No or very little FoxO4 protein and mRNA was present in amnion epithelium. The intensity and extent of staining of FoxO3 and FoxO4 was greater in fetal membranes obtained from the SCS compared to DS. Presence of FoxO3 and FoxO4 are expected to contribute to apoptosis and/or cell cycle regulation associated with fetal membrane rupture.

Summary The regulatory mechanism(s) for the production of human placental peptide hormones are poorly understood and two theories currently prevail: an autonomous production which varies according to the changing ratio of cyto- to syncytiotrophoblasts throughout pregnancy an autocrine-paracrine control mediated through receptor binding events. We have favored the latter hypothesis and postulated a functional analogy between the regulation of peptide hormone production by the pituitary with that by human placenta. Trophoblastic cells obtained by trypsin digestion of placentas from mid-term (13–18 weeks) and term (38–41 weeks) normal gestations were rested for 36–48 hours prior to study. A-HCG Studies . We observed the presence of specific, moderate affinity (Ka 10 8 M −1 low capacity LHRH receptor sites which could be up- and down-regulated depending on the endocrine milieu in vitro . Photoaffinity labeling studies showed that the molecular weight of this placental receptor for LHRH was comparable to that observed in rat pituitary (60 kd) and structure-activity relationships indicated that it bound both agonists and antagonists of LHRH. The concentrations of these receptor sites decreased four-fold from mid-gestation to term which was paraleled by a decreased of LHRH-induced production of bioactive hCG. Ca 2+ and cAMP, but not membrane lipid hydrolysis, mediated this LHRH-induced hCG production in human placentae. B-hPL Studies . We also found that its production by human placental cells was regulated by cellular events comparable to those responsible for prolactin production by the pituitary. Specific opioids and angiotensin II (AII) receptor sites were observed in human placentae with characteristics of high affinity (Ka 10 −10 M) and low capacity for ethylketocyclazocine (EKC) and AII. The concentrations of these receptors increased from midpregnancy to term and was manifested by an increased ability to release hPL which was stimulated 2-fold by 10 −8 M AII and 2.2 fold by 10 −7 M EKC. Dopamine (DA), naloxone, and AII antagonist (SAR-ALA II) completely inhibited this hPL production (>85%). AII stimulated inositol phosphate (IP) production whereas EKC had no effect. DA was without effect on basal IP production but inhibited the AII-induced accumulation of IP. The production of cAMP was inhibited by EKC (55%), stimulated by DA (600%), but unaffected by AII. Extracellular Ca 2+ did not influence basal or AII stimulated hPL release but was essential for DA effects. We conclude that the placental production of hCG and hPL is under the control of paracrine influences.