Early Human Trophoblast Research Articles

Marmoset and human trophoblast stem cells differ in signaling requirements and recapitulate divergent modes of trophoblast invasion

Early human trophoblast development has remained elusive due to the inaccessibility of the early conceptus. Non-human primate models recapitulate many features of human development and allow access to early postimplantation stages. Here, we tracked the pre- to postimplantation transition of the trophoblast lineage in superficially implanting marmoset embryos invivo. We differentiated marmoset naive pluripotent stem cells into trophoblast stem cells (TSCs), which exhibited trophoblast-specific transcriptome, methylome, differentiation potential, and long-term self-renewal. Notably, human TSC culture conditions failed to support marmoset TSC derivation, instead inducing an extraembryonic mesoderm-like fate in marmoset cells. We show that combined MEK, TGF-β/NODAL, and histone deacetylase inhibition stabilizes a periimplantation trophoblast-like identity in marmoset TSCs. By contrast, these conditions differentiated human TSCs toward extravillous trophoblasts. Our work presents a paradigm to harness the evolutionary divergence in implantation strategies to elucidate human trophoblast development and invasion.

Modelling early human trophoblast development in vitro

Modelling early human trophoblast development in vitro

Pigment epithelium-derived factor (PEDF): a novel trophoblast-derived factor limiting feto-placental angiogenesis in late pregnancy.

The rapidly expanding feto-placental vasculature needs tight control by paracrine and endocrine mechanisms. Here, we focused on paracrine influence by trophoblast, the placental epithelium. We aimed to identify differences in regulation of feto-placental angiogenesis in early versus late pregnancy. To this end, the effect of conditioned media (CM) from early and late pregnancy human trophoblast was tested on network formation, migration and proliferation of human feto-placental endothelial cells. Only CM of late pregnancy trophoblast reduced network formation and migration. Screening of trophoblast transcriptome for anti-angiogenic candidates identified pigment epithelium-derived factor (PEDF) with higher expression and protein secretion in late pregnancy trophoblast. Addition of a PEDF-neutralizing antibody restored the anti-angiogenic effect of CM from late pregnancy trophoblast. Notably, human recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two key players in VEGF-induced proliferation and migration, revealed that PEDF altered VEGF signaling, while PEDF alone did not affect phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and targets to modulate the intracellular effect of VEGF.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-016-9513-x) contains supplementary material, which is available to authorized users.

Wingless ligand 5a is a critical regulator of placental growth and survival.

The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival.

Urokinase-type plasminogen activator (uPA) system and invasion of early human extravillous trophoblast is regulated by hypoxia induced factor (HIF) pathway

Objective:Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human pregnancy. We have been reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Methods: Human placental villi of 5–10 weeks gestation (obtainedafter informedconsent onartificial abortion) were minced, digested and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel® for 24h under 20%, 5% and 0.1% (three times of one hour hypoxic stimulation) oxygen condition with siRNA designed for knock-down of HIF. The invasive capacity of EVT (Matrigel-coated invasion assay), localization of HIF (immunofluorescence staining), and productions of HIF, uPA and receptor uPAR protein on EVT (Western Blot and ELISA) were studied. Results: HIF protein was increased under 5% oxygen condition than 20%. Invasive capacity was significantly decreased in the presence of siRNA for HIF. Productions of uPA and uPAR were also decreased in the presence of the siRNA. Conclusions: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock-down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that uPA system in human trophoblast was induced by HIF and enhanced invasive capacity under hypoxic conditions.

Invasiveness and protease system of early human extravillous trophoblast is regulated by hypoxia induced factor (HIF) pathway

Invasiveness and protease system of early human extravillous trophoblast is regulated by hypoxia induced factor (HIF) pathway

Iodide transporters expression in early human invasive trophoblast

ContextThe placenta plays an essential role in the fetomaternal exchanges of iodine and thyroid hormones. Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism during the first trimester of pregnancy. Little is known on the expression of iodide transporters in invasive human trophoblast and the possible effect of PTU on this early phase of human placental development. ObjectiveTo analyze during early pregnancy expression of sodium/iodide symporter (NIS) and pendrin at the feto–maternal interface in situ in first trimester placentas, in vitro during human trophoblastic cell differentiation in presence or not of PTU. DesignNIS and pendrin immunodetection were performed on 8–10 WG placental tissue sections and in primary cultures of first trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (EVCT). The effect of PTU (1 mM) was tested in EVCT on iodide transporters expression, cell invasion, and hCG secretion. ResultsNIS and pendrin were present in early human trophoblast at the maternofetal interface. Their expression was modulated with in vitro trophoblast differentiation. Early invasive EVCT were characterized by higher expression of NIS than pendrin. In vitro PTU did modify significantly neither EVCT iodide transporters expression nor EVCT biological functions: i.e. invasive properties and hCG secretion. ConclusionThis study reveals that NIS is highly expressed in early human trophoblast at the feto–maternal interface. PTU has no effect on early human trophoblast invasion.

Hyperglycosylated hCG Is a Marker of Early Human Trophoblast Invasion

Human chorionic gonadotropin (hCG) is the major pregnancy glycoprotein hormone whose maternal concentration and glycan structure change all along pregnancy. hCG is mainly secreted by the syncytiotrophoblast covering the chorionic villi, but little is known about the source of hyperglycosylated hCG (hCG-H) production. The objective of the study was to analyze expression and secretion of hCG and hCG-H in vitro during human trophoblastic cell differentiation, in situ in first-trimester placentas, and in maternal sera during early pregnancy. hCG and hCG-H were measured in cell supernatants from primary cultures of first-trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (evct). hCG-H immunodetection were performed on 9 wk gestation (WG) placental tissue sections. Total hCG and hCG-H were quantified by chemiluminometric assay in 539 maternal sera collected between 9 and 19 WG during normal pregnancies. In vitro, hCG secretion reached 37 ng/ml per μg DNA during syncytiotrophoblast formation but contained few hCG-H (2-5% of total hCG). In contrast, hCG secretion (20 ng/ml per μg DNA) in evct supernatants contained 10-20% hCG-H. In situ, hCG-H immunostaining was strong in invasive and endovascular evct, weaker in mononucleated villous cytotrophoblasts, but negative in the syncytiotrophoblast. In maternal sera, hCG-H concentrations continuously decreased during pregnancy from 406 ± 222 ng/ml at 9 WG to 8 ± 6 ng/ml at 19 WG, whereas total hCG picked up at 11 WG and then decreased. This study suggests that the high levels of hCG-H observed in first-trimester maternal sera are mainly from invasive evct origin, reflecting the early trophoblast invasion process.

311. REGULATION OF THE RENIN ANGIOTENSIN SYSTEM (RAS) IN A TROPHOBLAST CELL LINE BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND 5'-AZA-2'-DEOXYCYTIDINE (AZA)

Renin and renin-like activities have been detected in early gestation placentae1,2. To explore what regulates RAS expression in trophoblasts we studied the effects of cAMP and AZA (DNA demethylating agent) on the expression of mRNAs for prorenin receptor (ATP6AP2), prorenin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE2, angiotensin II type 1 receptor (AGTR1), and a downstream target of the prorenin receptor, cyclooxygenase-2 (PTGS2) in the early human trophoblast cell line HTR-8/SVneo. Cells were cultured for 24 or 48 h with vehicle, 0.5 mM cAMP or 15 mM AZA. Messenger RNA abundances were measured relative to Alien RNA using real-time qRT-PCR. All mRNAs were detected except for ACE and ACE2. REN mRNA abundance was increased by cAMP and AZA (P < 0.0001). However, cAMP was more effective than AZA at both 24 and 48 h (P < 0.0001; P < 0.05) at the concentrations employed. AZA significantly increased AGT mRNA levels at 24 h (P = 0.001) and AGTR1 mRNA levels at 48 h (P < 0.0001), while cAMP had no effect. P TGS2 mRNA expression was increased by cAMP at 24 h (P < 0.0001) and by AZA at 48 h (P < 0.0001). cAMP and AZA had no effect on ATP6AP2 abundance. Thus, prorenin may function directly through the prorenin receptor, since cAMP upregulated both REN and its downstream target PTGS2 in HTR-8/SVneo cells, especially considering that ACE and ACE2 were not expressed. Our findings also suggest that expression of the RAS in trophoblast is regulated by DNA methylation as there was sustained over expression of REN, AGT, AGTR1 and PTGS2 with AZA. The ATP6AP2 promoter possesses CpG islands; however, its expression was not affected by AZA. This may suggest that either DNA methylation is not involved in the regulation of this gene, or that the over expressed prorenin binding to its receptor is causing suppression of ATP6AP2 abundance via a previously identified negative feedback pathway. (1) Pringle et al., Perinatal Society of Australia & New Zealand (PSANZ) 2010, Wellington, New Zealand (Abstract).(2) Itskovitz et al., Journal of Clinical Endocrinology and Metabolism, 1992, 75: 906–10.

Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for De Novo Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas

Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity in infected cells. We demonstrate that the PPARgamma antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARgamma, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARgamma in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARgamma human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.

Difference in Progesterone-Receptor Isoforms Ratio Between Early and Late First-Trimester Human Trophoblast Is Associated with Differential Cell Invasion and Matrix Metalloproteinase 2 Expression

The expression profile of the progesterone-receptor isoforms and progesterone regulation of matrix metalloproteinase 2 (MMP2) were investigated in early and late first-trimester trophoblast cells. Human trophoblast cells were obtained from legal abortions (6-12 wk of gestation). Purity of 95-98% was verified using immunohistochemistry with specific antibodies. Evaluation of cell count was performed with XTT Reagent kit, and invasion was tested using Matrigel invasion assay. Zymography was used to detect proteolytic activity, and Western blot immunoassay was used to study protein concentration. Gene expression of PGRB, PGR, and MMP2 was studied using reverse transcription-polymerase chain reaction with the housekeeping gene GAPDH used for normalization. Promoter activity was determined using luciferase reporter assay. Differential progesterone-receptor profile was documented with the dominance of PGRB in early trophoblast and the dominance of PGRA in late trophoblast. This differential profile is compatible with the inverse effect of progesterone on the two cell populations, decreasing invasion and gelatinase expression in the early first-trimester trophoblast and increasing invasion and gelatinase expression in the late first-trimester trophoblast. A decrease in MMP2 promoter activity in early trophoblast cells exposed to progesterone suggests that MMP2 expression is regulated by progesterone at the transcriptional level as well. Early trophoblast cells transfected with expressing vector for PGR encoding PGRA revealed less MMP2 activity and reversal of its response to progesterone similar to the effect observed in late trophoblast cells.

Bi-potential Behaviour of Cytotrophoblasts in First Trimester Chorionic Villi

Murine trophoblast stem (TS) cells express fibroblast growth factor receptor 2 (FGFR2) and are maintained in their proliferative state by fibroblast growth factor 4 (FGF4). We show in this report that in the first trimester human placenta FGFR2 expression is similarly found in a subset of villous cytotrophoblast and in proximal anchoring columns. Western analysis demonstrated declining FGFR2 protein expression as gestation advanced, suggesting a similar role for FGF in early human trophoblast proliferation. Mouse TS cell differentiation is known to occur along two distinct transcriptionally-regulated pathways; extravillous trophoblast (EVT) cells invade the uterine wall to promote maternal blood flow whilst syncytiotrophoblast lines chorionic villi in the labyrinth. Similar differentiation steps occur in the human placenta though the fate of human trophoblast stem cells is presently unknown. To investigate the mechanisms underlying human cytotrophoblast differentiation we have developed a novel cultured floating first trimester villous explant model in which denuded first trimester villi spontaneously regenerate syncytiotrophoblast following 48 h of culture. Addition of FGF4 and heparin inhibited syncytiotrophoblast regeneration in favor of forming clumps of cytotrophoblast. Proximal cells in these clumps were FGFR2 immuno-reactive and proliferative, intermediate parts expressed alpha5beta1-integrin, while the distal portion expressed HLA-G and the invasive integrin alpha1beta1 indicating differentiation to the EVT phenotype. In contrast, non-denuded villi exposed to FGF4 exhibited similar proliferation of the cytotrophoblast; however, these cells did not express any of the invasive EVT markers. We conclude that FGFR2-positive chorionic cytotrophoblasts exhibit bi-potential behaviour, being capable of forming either syncytiotrophoblast or EVT. We suggest bipotential trophoblast progenitor cells persist during first trimester human placental development.

Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and their inhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy

Early human trophoblast shows dramatic invasive properties during early pregnancy. A tightly-regulated activation of matrix metalloproteinases (MMPs) is considered to be of critical importance for the control of trophoblast invasion. The aim of the present study was to determine MMP-2, MMP-9, TIMP-1 and TIMP-2 protein expression in decidual endometrium during the first trimester of pregnancy (22-42 days post coitus) with special attention to their expression patterns in endometrial compartments. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. We observed that MMP-2, particularly in the fourth week, appeared to be expressed more strongly in extravillous trophoblasts (EVTs) and vascular endothelial cells in the first trimester of pregnancy. Therefore, MMP-2 is likely to be the primary mediator in invasion of the trophoblast into the decidual endometrium, as well as in vascular remodeling and angiogenesis in the first trimester of pregnancy. The high expression of TIMP-1 and TIMP-2 in EVTs and glandular epithelium suggests that a restricted and balanced expression of these molecules is important for matrix remodeling and controlled trophoblast invasion during placentation. We conclude that (1) MMP-2 and MMP-9 and their inhibitors TIMP-1, and TIMP-2 determine the invasive behavior of trophoblast into the endometrium, and in particular, (2) MMP-2 may be the key regulator of trophoblast invasion in early human pregnancy.

Alpha-fetoprotein Gene Expression in Early and Full-term Human Trophoblast

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunologial studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts.These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.

Independent regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-3 in human endometrial stromal cells by gonadotropin-releasing hormone: implications in early human implantation.

Early human trophoblast shows dramatic invasive properties during early pregnancy. The simultaneous synthesis of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) in both human trophoblast and decidual membranes suggests that their controlled and balanced expression is crucial for the rapid matrix remodeling and controlled invasion during early pregnancy. Recently, we have described the presence of an extrahypothalamic GnRH immunologically, biologically and chemically identical to the hypothalamic hormone in periimplantation human embryos. Moreover, the production of this decapeptide by the human trophoblast during the early stages of placentation is well documented. TIMP-1 and -3 messenger ribonucleic acid (mRNA) expression in cultured stromal cells and protein secretion into the medium were significantly decreased by GnRH agonist compared to that in control groups. Moreover, expression of TIMP-1 was affected to a greater extent than that of TIMP-3. GnRH antagonist ablated the down-regulation of TIMPs by the GnRH agonist. MMP-9 mRNA expression was not detected in the control groups or in the groups treated with GnRH analogs. Our results provide evidence that trophoblastic GnRH may play an important role in placental tissue organization and in the early embryo-maternal dialogue by enhancing trophoblast invasion through the specific inhibition of TIMPs.

Toxicity of Cadmium in Human Trophoblast Cells (JAr Choriocarcinoma): Role of Calmodulin and the Calmodulin Inhibitor, Zaldaride Maleate

Cadmium (Cd), the heavy metal, is toxic to the placenta. The objectives of this study were to determine if Cd toxicity is due to inhibition of placental or trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). Cd can replace calcium and thus interfere with CaM's function. Also, CaM inhibitors reverse selected toxic effects of Cd. The CaM inhibitor, zaldaride maleate, was used to determine if Cd inhibits trophoblast cell proliferation through interactions with CaM. JAr choriocarcinoma cells, a neoplastic trophoblast cell line which is similar to early human trophoblast cells, were selected to study this question. Cd (20 and 40 μm) inhibits JAr cell proliferation, as measured by cell number and BrdU incorporation. Zaldaride (10 and 20 μm) inhibits proliferation to a lesser extent; 100 μmis lethal. To determine if zaldaride alters actions of Cd, zaldaride and Cd are added simultaneously. Zaldaride (20 μm) and Cd (20 μm) together inhibit proliferation less than Cd alone, thus partially protecting cells. Metallothionein is induced in cells exposed to Cd, while zaldaride does not cause induction of this cellular defense mechanism protein. To determine if Cd inhibits proliferation through alterations of cell cycle, JAr cells enriched for G0/G1 phase were exposed to 20 μmCd, 20 μmzaldaride, or 20 μmCd plus 20 μmzaldaride for 24 hr. Cells remain in G0/G1 following Cd exposure; cells treated with 20 μmzaldaride progress through S phase and into G2. Zaldaride and Cd together allow JAr cells to leave G1 and enter S phase, partially relieving the cycle block produced by Cd. This study demonstrates a role for calmodulin in mediating the toxicity of Cd in trophoblast cell proliferation.

A study of the mechanism of hCG release by inducing IL-4 and IL-7 from early pregnancy human trophoblast cells

A study of the mechanism of hCG release by inducing IL-4 and IL-7 from early pregnancy human trophoblast cells

Differentially expressed genes in human trophoblast cells with different degrees of invasiveness

s: R.T.C. and T.G.W.M.S. Canada 1996 A.15 Differentially Expressed Genes in Human Jrophoblast Cells with Different Degrees of Invasiveness. G. Huch, H.-P. Hohn, H.-W. Denker; Institute for Anatomie, Universrty of Essen, Medical School, D-45122 Essen, Germany. The majority of molecular control mechanisms of trophoblast invasiveness are unknown so far. We have analyzed gene expression of normal trophoblast cells and of JAr malignant trophoblast cells with differing invasive potential by DDRTPCR (differential-display-RT-PCR). In JAr cells, high invasiveness was induced with phorbol-12-my ristoyl-13-acetate while for low invasiveness cells were treated with dibutyryl-CAMP (Placenta 14, 1993, A44). Normal trophoblast cells were isolated from first trimester (highly invasive) and term placentae (“non”-invasive), According to DDRT-PCR, major differences’in mRNA-patterns were seen between normal and malignant trophoblast cells, between early and term trophoblast cells, but not between JAr cells with different treatments Differentially generated PCR-products of normal trophoblast were reamplified, cloned into the vector pCRII, and sequenced. Data base searches identified only a few of the cloned fragments. One of them isolated from first trimester trophoblast showed 100% homology to j31-integrin which has been suggested on the protein level to be involved in directing trophoblast invasion (Damsky et al., 1994, Development 120, 3657). This result demonstrates that DDRT-PCR is a reliable tool to detect changes in gene expression as related to regulation of trophoblast differentiation/invasiveness. Since RNA probes for cloned PCR-fragments usually representing 3’-regions (mostly untranslated) did not give reliable results in in-situ-hybridizations we are constructing a cDNA library from first trimester human placentae. This and another one from term placenta is being screened for the respective coding regions as a basis for the construction of appropriate probes. Supported by Dr:Mildred-Scheel-Stiftung fiir Krebsforschung Maternal Growth Hormone Treatment Increases Placental Diffusion Capacity but not Fetal or Placental Growth in Sheep. J.E.Harding, P.C.Evans and P.D.Gluckman, Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Auckland, New Zealand. Growth hormone (GH) treatment increases circulating concentrations of glucose, fatty acids and IGF-1. We hypothesised that maternal GH treatment may enhance fetal and placental growth. Twelve chronically catheterised pregnant sheep were treated with GH O.lmg/Kg SC bd for 10d from 125f0.3d (meanfSE), while 11 controls received saline. GH increased placental capacity for simple diffusion, measured by steady state 14C-urea clearance. Maternal and fetal blood urea fell, but there were no other changes in fetal or placental metabolism. Fetal and placental weights were similar in GH and control groups at 134f0.3d (fetus 4.3f0.2 vs 4.3+0.3Kg, placenta 441f36 vs 411*42g). These data suggest placental diffusion capacity does not limit fetal growth under these circumstances. Human MASH2 (HASH2) Maps lo Chromosome 11~15 and is Expressed in Extravillus Trophoblast. :,Oudeians, M.Alders, J.Postmus, Lvan Wijk, H.Hodgess, A.K.Hadjantonskis, J.Bliek, M.de Meulemeester, A.Westerfeld, P.Little, and MMannens. Departments of Clinical Chemistry, Free University Hospital, Amsterdam, Human Genetics, Univ. of Amsterdam, and Biochemistry, Imperial College London. Mice deficient for the Mash-2 gene coding for the lineage-specific transcription factor of the basic helix-loop-helix (bHLH) family die at 10 days post coitum due to placental failure as a consequence of deficient spongiotrophoblast formation. By screening of chromosome 1 l-specific cosmids with a degenerate FCR probe, the human Mash2 (HASHI) was isolated. By pulsed field gel electrophoresis, HASH2 was found to map to chromosome 11~15.5 proximal to and in close vicinity of IGF.2 in the syntenic human chromosome region of mouse chromosome 7. This region harbours a cluster of imprinted genes (Mash2, Ins-2, IGF-2, and H19). In the bHLH-region, the human gene was 92.7% and 98% homologous to mouse MASH2 at the DNA and peptide levels, respectively. HASH2 expression was studied by non-radioactive RNA in situ hybridization of early human trophoblast cells of normal and androgenetic (molar) origin. in early human placentae, HASH2 expression was found in extravillus trophoblast cells only. Chorionic villas trophoblast, placental stromal cells as well as maternal placental bed giant cells were negative. In trophoblast cells of androgenetic origin, a differential distribution pattern was observed. Extravillus trophoblast cells with malignant potential (invasive moles) were positive, while extravillus trophoblast cells from complete moles without malignant potential were negative. These expression data suggest, although indirectly, that HASH2 is genetically imprinted with preferential activation of the

A study of mechanism of hCG release by inducing IL-4 and IL-7 from early pregnant human trophoblast cells

A study of mechanism of hCG release by inducing IL-4 and IL-7 from early pregnant human trophoblast cells