Fusion Of Cytotrophoblast Cells Research Articles

Involvement of the HERV-derived cell-fusion inhibitor, suppressyn, in the fusion defects characteristic of the trisomy 21 placenta

Suppressyn (SUPYN) is the first host-cell encoded mammalian protein shown to inhibit cell–cell fusion. Its expression is restricted to the placenta, where it negatively regulates syncytia formation in villi. Since its chromosomal localization overlaps with the Down syndrome critical region and the TS21 placenta is characterized by delayed maturation of cytotrophoblast cells and reduced syncytialization, we hypothesized a potential link between changes in SUPYN expression and morphologic abnormalities in the TS21 placenta. Here we demonstrate that an increase in chromosomal copy number in the TS21 placenta is associated with: (1) reduced fusion of cytotrophoblast cells into syncytiotrophoblast in vivo, (2) increased SUPYN transcription, translation and secretion in vivo, (3) increased SUPYN/syncytin-1 receptor degradation in vivo, (4) increased SUPYN transcription and secretion ex vivo, (5) decreased cytotrophoblast cell fusion ex vivo, and (6) reciprocal response of changes in SUPYN and CGB in TS21 placental cells ex vivo. These data suggest direct links between immature placentation in Down syndrome and increased SUPYN. Finally, we report a significant increase in secreted SUPYN concentration in maternal serum in women with pregnancies affected by Down syndrome, suggesting that SUPYN may be useful as an alternate or additional diagnostic marker for this disease.

THBS1 regulates trophoblast fusion through a CD36-dependent inhibition of cAMP, and its upregulation participates in preeclampsia

Preeclampsia is a pregnancy complication which threatens the survival of mothers and fetuses. It originates from abnormal placentation, especially insufficient fusion of the cytotrophoblast cells to form the syncytiotrophoblast. In this study, we found that THBS1, a matricellular protein that mediates cell-to-cell and cell-to-matrix interactions, is downregulated during the fusion of primary cytotrophoblast and BeWo cells, but upregulated in the placenta of pregnancies complicated by preeclampsia. Also, THBS1 was observed to interact with CD36, a membrane signal receptor and activator of the cAMP signaling pathway, to regulate the fusion of cytotrophoblast cells. Overexpression of THBS1 inhibited the cAMP signaling pathway and reduced the BeWo cells fusion ratio, while the effects of THBS1 were abolished by a CD36-blocking antibody. Our results suggest that THBS1 signals through a CD36-mediated cAMP pathway to regulate syncytialization of the cytotrophoblast cells, and that its upregulation impairs placental formation to cause preeclampsia. Thus, THBS1 can serve as a therapeutic target regarding the mitigation of abnormal syncytialization and preeclampsia.

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACTThe placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.

The cAMP-responsive element binding protein (CREB) transcription factor regulates furin expression during human trophoblast syncytialization

IntroductionThe multinucleated syncytiotrophoblast is formed and maintained by cytotrophoblast cell fusion and serves multiple functions to ensure a successful pregnancy. We have previously reported that the proprotein convertase furin is required for trophoblast syncytialization by processing type 1 insulin-like growth factor receptor (IGF1R). MethodsUtilizing trophoblast cell fusion models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured term cytotrophoblast cells, the expression of furin was evaluated by quantitative real-time PCR, Western blotting and immunofluorescence. The key transcription factor regulating the FUR gene promoter and critical responsive elements were identified by luciferase reporter assays, truncated mutants analysis, site-directed mutagenesis and ChIP. ResultsWe demonstrated that the levels of FUR mRNA were significantly stimulated by cAMP/PKA signaling pathway during spontaneous fusion of cytotrophoblast cells and forskolin-induced fusion of BeWo cells. cAMP-responsive element binding protein (CREB) was proven to be the key transcription factor which regulated the FUR P1 promoter during forskolin-induced BeWo cell fusion, and two critical cAMP-responsive elements (CREs) in the P1 promoter were further identified. Finally, we showed that CREB mediated endogenous furin activation and that CREB siRNA attenuated forskolin-induced furin expression and cell fusion in BeWo cells. DiscussionThis provides the first evidence of the upstream regulator of furin during trophoblast cell fusion. ConclusionsThe above results suggest that the FUR transcription is activated by CREB-dependent stimulation of the FUR P1 promoter during human trophoblast syncytialization.

Exocyst complex protein expression in the human placenta

IntroductionProtein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal–fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. ObjectiveWhile the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. MethodsA two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. ResultsThe eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Discussion/conclusionOur findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion.

The proprotein convertase furin is required for trophoblast syncytialization

The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development.

Viability of primary term cytotrophoblast cell culture in normoxia and hypoxia

Cytotrophoblast (CT) cells isolated and purified from term placenta are able to differentiate into syncytiotrophoblast cells. Previous reports suggested that hypoxia is an inhibitor of this differentiation and also increases apoptosis. As visual observations of our CT cell cultures indicated a better development in hypoxia than in normoxia (defined as 2.5 and 21% O(2), respectively), we decided to assess the effect of low oxygen tension on in vitro CT cell differentiation by measuring cell viability, apoptosis and CT cell fusion and differentiation. We observed a 45% decrease in cell viability 24 h after plating both in normoxia and in hypoxia but no difference between the two oxygen conditions. Cell viability remained stable during the 4-day culture. Apoptosis also did not increase in hypoxia. Apoptotic index and caspase activation were even lower in hypoxia than in normoxia at Day 1 and Day 4 of the culture. Finally, we observed a 5-fold and 6-fold increase in Syncytin-1 mRNA expression in normoxia and in hypoxia, respectively, indicating that hypoxia did not inhibit CT cell fusion. CT cells differentiated as well in hypoxia as an increase in inhibin α subunit mRNA was evidenced during the 4-day culture. This increase was even higher in hypoxia than in normoxia. In conclusion, hypoxia defined as 2.5% O(2) based on first trimester placental pO(2) did not decrease term primary CT cell viability and did not increase apoptosis. Moreover, it did not inhibit either CT cell fusion or differentiation.

317. DIFFERENTIAL EFFECTS OF cAMP AND CRH IN CELL VIABILITY AND CELL FUSION OF CYTOTROPHOBLASTIC CELLS

Cell fusion of cytotrophoblasts is critical for a successful pregnancy, and is marked biochemically by proteins such as human chorionic gonadotrophin (hCG) and syncytin, and morphologically as large multi-nucleated cells. Placental corticotrophin releasing hormone (CRH) plays diverse roles during pregnancy and has been linked to the length of gestation. It is well accepted that CRH expression could be upregulated by some hormones, such as hCG via cellular cAMP, and that trophoblastic cell fusion can be induced by Forskolin (increases cAMP). We have shown, by reporter gene assay in human primary placental cells, that 8-Br-cAMP increased CRH gene expression. Thus, it is of interest to determine the roles of 8-Br-cAMP and CRH on trophoblast cell viability and cell fusion. Treatment with 8-Br-cAMP for 72 h significantly inhibited BeWo cell viability (MTT assay) by 29.7% (50 μM) and 54.8% (100 μM), and increased both apoptotic and necrotic cells (FACS analysis). 8-Br-cAMP (100 μM, 48 h) resulted in the appearance of nuclear fragments (DAPI stain) and Fas ligand gene expression by 6.8-fold (real-time RT-PCR). Syncytin 1 mRNA increased by 3.2-fold (real-time RT-PCR) and hCG secretion increased by 4-fold (ELISA assay). The formation of syncytium (CellMask and Hoechst co-stain) could clearly be seen by 72 h. CRH mRNA increased with 8-Br-cAMP treatment of BeWo cells (15-fold at 48 h). Treatment with CRH (100 nM) had a mild inhibitory effect on cell growth (~18%) but no effect on cell viability, up-regulated the expression of syncytin 1 mRNA (2.3-fold at 48 h) and induced cell fusion (72 h), but had no effect on hCG secretion. In summary, we show that 8-Br-cAMP and CRH are both potential inducers of cytotrophoblast cell fusion, and there is a dissociation between morphological and biochemical differentiation. Our data also indicates that the process of cell fusion can be associated with both apoptotic and non-apoptotic events.

K+channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal–fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41 aa) and CHR (34 aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-helix protein exists as a trimer and is highly α-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis.

207. The expression of caspase-14 in the human placenta

Pro-apoptotic genes have a role in the differentiation process in the placenta leading to the fusion of cytotrophoblast cells to form the protective syncytiotrophoblast layer. The mechanisms of apoptosis in the human placenta are not clearly understood. However, a major placental apoptotic-signalling pathway is known to involve the caspases. Caspase-14 is the most recently discovered member of the caspase family members and has not previously been examined in the human placenta. The aim of the present study was to detect caspase-14 in the human placenta and study its role in apoptosis. Human placentae were collected from first trimester and term gestation. The study consisted of two parts. In the first part, first trimester and term placentae were assessed for caspase-14 by western blotting and mRNA analysis and localised with immunohistochemical studies. In the second part, apoptosis in first trimester placenta was inhibited in an in-vitro model of explant villi culture with superoxide dismutase (SOD) treatment and the genes assessed. The first study demonstrated caspase-14 to be a cytoplasmic protein localised in the cytotrophoblast cells, the mesenchyme and in the syncytiotrophoblast of the first trimester. In the term placenta, caspase-14 was expressed weakly in the syncytiotrophoblast. The immunostaining data suggest a higher expression of caspase-14 in the first trimester compared to the term placenta, and this observation was later confirmed by western blot analysis. Using the SOD in-vitro explant culture model, no significant difference in the caspase-14 protein levels were seen in either the SOD or control group. This novel study demonstrates for the first time that caspase-14 protein and mRNA are present in the human placenta. The function of caspase-14 in the human placenta is unclear.

Critical role for transcription factor AP-2alpha in human trophoblast differentiation.

To examine whether AP-2alpha is a critical component of the genetic program that directs human trophoblast differentiation, we used DNA microarray analyses to characterize the effects of a dominant-negative form of the AP-2 protein upon in vitro differentiating cytotrophoblast cells. Human cytotrophoblast cells (>95% pure) were cultured for 3 days in the presence of control medium or medium containing an adenovirus that expresses a dominant-negative mutant of AP-2 (Ad2.AP-2D/N) or an adenovirus lacking the AP-2 mutant gene (Ad.WT). DNA microarray analyses using Affymetrix human U95Av2 GeneChips were performed on RNA extracted from the three groups of cells immediately prior to and after 3 days of cell culture. Cells infected with Ad2.AP-2D/N or Ad2.WT underwent morphological differentiation similar to that of uninfected cells, with greater than 90% of the cells in each group fusing to form multinucleated syncytiotrophoblast cells. However, Ad2.AP-2D/N markedly inhibited the induction or repression of many genes that were regulated in the noninfected and Ad2.WT-infected cells during differentiation. Eighteen of the 25 most induced genes and 17 of the 20 most repressed genes during differentiation were AP-2 dependent, with the majority of these related to extracellular organization, cellular communication, and signal transduction. Taken together, these findings strongly suggest that AP-2 plays a critical role for both the induction and repression of genes that comprise postsyncytialization gene expression programs of trophoblast differentiation and maturation. AP-2, however, is not required for the fusion of cytotrophoblast cells to form a syncytium or the expression of syncytin.

Syncytial fusion of human trophoblast depends on caspase 8.

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.

Requirement of Gap Junctional Intercellular Communication for Human Villous Trophoblast Differentiation1

During pregnancy, the villous trophoblast develops from the fusion of cytotrophoblastic cells (CT) into a syncytiotrophoblast (ST), supporting the main physiological functions of the human placenta. Connexin43 (Cx43) is demonstrated in situ and in vitro in the villous trophoblast between CT and between CT and ST. Moreover, the presence of a gap junctional intercellular communication (GJIC) during in vitro trophoblast differentiation was previously demonstrated. Because the exchange of molecules through gap junctions is considered to play a major role in the control of cell and tissue differentiation, we studied the effects of a gap junctional uncoupler, heptanol, on morphological and functional trophoblast differentiation and on GJIC measured by the fluorescence recovery after photobleaching method. We found that when the GJIC was interrupted, CT still aggregated but fused poorly. This morphological effect was associated with a significant decrease of trophoblastic-specific gene expression (beta human chorionic gonadotropin and human chorionic somatomammotropin). This blocking action was reversible as demonstrated by recovery of GJIC and trophoblast differentiation process after heptanol removal. Moreover, the inhibition of the trophoblast differentiation did not affect Cx43 transcript expression and Cx43 protein expression. These data suggest that the molecular exchanges through gap junctions preceding cellular fusion are essential for trophoblast differentiation generating the multifunctional syncytiotrophoblast.

Hormones and human trophoblast differentiation: a review.

In the human, fetal cytotrophoblastic cells play a key role in the implantation process and in placental development. With the progression of placentation, two pathways of differentiation lead to the formation of two distinct phenotypes. In the villous trophoblast (fusion phenotype), the trophblast differentiates from the fusion of mononuclear cytotrophoblastic cells into a syncytium, the syncytiotrophoblast. Bathing the maternal blood, the syncytiotrophoblast is involved in maternal-fetal exchanges and in placental endocrine functions. In the extravillous trophoblast (proliferative/invasive phenotype), the cytotrophoblastic cells proliferate and migrate into the decidua, remodeling the pregnant endometrium and its vasculature. This review summarizes our current knowledge of the key step of villous differentiation-the cell-cell fusion of the cytotrophoblastic cells--and on the invasion process of extravillous trophoblast. Experimental evidence demonstrates that the genetic differentiation/invasion programs of cytotrophoblastic cells could be modulated by their environment: oxygen, extracellular matrix, and soluble factors (cytokines, growth factors, and hormones). Cytotrophoblastic cells fusion and the functional differentiation of villous trophoblast are specifically stimulated by estradiol, glucocorticoids, and human chorionic gonadotropin (hCG) whereas progesterone is ineffective. Because these hormones are temporally secreted in large amounts and present at the fetomaternal interface, they are in good position to play a physiologic role in trophoblast differentiation. hCG may be important very early in pregnancy, when production of this glycoprotein is maximal, whereas estrogen increasingly produced by the fetoplacental unit and cortisol secreted from the fetal adrenal may be implicated in the end-stage maturation and aging of the trophoblast.

Human Villous Macrophage-Conditioned Media Enhance Human Trophoblast Growth and Differentiation In Vitro

In human chorionic villi, numerous macrophages, so-called Hofbauer cells, are located adjacent to trophoblasts. To determine the role of the macrophages in the proliferation and differentiation of trophoblasts, cytotrophoblast cells were cultured in serum-free culture-conditioned media of villous macrophages (VMCM), peritoneal macrophages (PMCM), and villous fibroblasts (VFCM). In VMCM, proliferation of cytotrophoblast cells was detected at 24 h by immunocytochemistry with Ki-67-antibody. A large number (P < 0.001) of multinucleated syncytia was formed in VMCM. In VMCM, cytotrophoblast cell fusion was completed by 96 h, which coincided with the peak of hCG secretion and initiation of human placental lactogen (hPL) release. Levels of hCG (P < 0.001) and hPL (P < 0. 001) secretion from syncytial cells were significantly higher in VMCM than in PMCM or in VFCM. Concentrations of macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) analyzed by ELISA were greater in VMCM than in PMCM or in VFCM, whereas monocyte chemoattractant protein-1 (MCP-1) concentration was high in PMCM. The expression patterns of M-CSF, VEGF, and MCP-1 in villous macrophages and peritoneal macrophages by reverse transcriptase-polymerase chain reaction were similar to their secretion patterns. Thus, villous macrophages have a greater ability to stimulate hCG and hPL secretion than do peritoneal macrophages. This study suggests that macrophages within the villous stroma may stimulate the growth and differentiation of trophoblasts through their secreted substances.

Oestradiol Stimulates Morphological and Functional Differentiation of Human Villous Cytotrophoblast

Trophoblast differentiation is a complex process involving interactions of cytotrophoblastic cells with their evolutive milieu. During pregnancy, the feto-placental unit produces large amounts of steroids. Progesterone and oestradiol are increasingly produced when the syncytiotrophoblast is highly differentiated. Furthermore, receptors to these hormones are expressed by the trophoblast. This led us to test the hypothesis that steroid production could affect the morphological and functional differentiation of the trophoblast during gestation.The fusion of cytotrophoblastic cells into syncytiotrophoblast was assessed using fluorescence recovery after photobleaching for gap junctional communication analysis (gap-FRAP), desmoplakin immunostaining and connexin 43 expression. In parallel, functional differentiation was assessed by β-human chorionic gonadotrophin (βhCG) production and human chorionic somatomammotropin (hCS) expression analysis. The presence of oestradiol, 1μm, increased the percentage of coupled cells (3.8-fold), connexin 43 expression and stimulated the syncytium formation. In parallel, oestradiol (1, 3 and 5μm) induced a significant increase in the daily hCG production. The steroid action was specific, as the stimulatory effects were inhibited by tamoxifen. Oestradiol also stimulated hCS expression (51 per cent compared to control after 3 days). As trophoblastic differentiation is specifically stimulated by hCG, oestradiol could act via the stimulation of hCG production or via a direct action. In the presence of an efficient concentration of hCG antibody, oestradiol still stimulated hCS expression, suggesting a self-sufficient effect of the steroid. Physiological concentrations of progesterone were ineffective in modulating trophoblast differentiation.In conclusion, oestradiol could be implicated in the maturation and aging of the trophoblast.

Effects of endothelin on villous trophoblast differentiation and free intracellular calcium

Trophoblast differentiation is a complex process involving interactions of cytotrophoblastic cells with their evolutive milieu. The trophoblastic layer differentiates from the fusion of cytotrophoblastic cells into the syncytiotrophoblast which expresses specific hormonal productions. The intracellular and extracellular signals which trigger and mediate the fusion of cytotrophoblastic cells still need to be investigated. Regarding their endocrine and paracrine activities, endothelins (ETs) are considered as peptide hormones and growth factors. ET A and ET B endothelin receptors subtypes are expressed on the trophoblast and the biosynthesis of ET by the syncytiotrophoblast (ST) was previously demonstrated. The exact mechanisms whereby endothelins cause biological effects after binding to trophoblastic cell membrane receptors have never been investigated. Furthermore, little is known regarding Ca ++ homeostasis. Therefore, by means of microspectrofluorimetry, we have measured the effects of ETs on cytosolic [Ca ++ ]. To monitor the ET −1 action on trophoblast differentiation, βhCG secretion, hCS expression, proliferative cell nuclear antigen and desmoplakin immunostainings were used. The presence of ET −1 (10 −8 M) for 3 days partially inhibited ST formation as confirmed by desmoplakin immunostaining. Interestingly, PCNA (Proliferative Cell Nuclear Antigen) immunostaining was detected in cytotrophoblastic cell aggregates cultured in the presence of 10 −8 M Et −1 , whereas it was absent in control conditions. In parallel, βhCG release and hCS expression were significantly reduced by ET −1 . Moreover, our study demonstrated that ET −1 could induce a biphasic increase of [Ca ++ ] i : an initial transient rise sometimes followed by a sustained plateau higher than the basal level. Endothelin responses are mainly mediated by ET β receptors. The persistence of the rapid transient [Ca ++ ] i rise in Ca ++ free extracellular medium together with patch clamp studies argue for the release of intracellular Ca ++ store rather than Ca ++ entry through voltage-sensitive channels in the response to endothelin stimulation. We conclude that endothelin impairs human trophoblast differentiation and behaves as a typical calcium mobilizing peptide in trophoblast.

Regulation of gap junctional communication during human trophoblast differentiation.

During pregnancy, the trophoblast, supporting the main functions of the placenta, develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. Gap junction channels consisting of connexins link the cytosols of cells in contact. Gap junctional communication has been involved in the control of cell and tissue differentiation. Recently, a gap junctional communication was demonstrated in trophoblast cell culture by means of the fluorescence recovery after photobleaching (gap-FRAP) technique. This gap junctional communication appeared to be stimulated by human chorionic gonadotropin (hCG). Therefore, the specificity of hCG action and the signalling mechanisms implicated in gap junctional communication were investigated by means of gap-FRAP. In culture, cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 1-2 days after plating. During this in vitro differentiation, gap junctional communication was measured, and the maximum percentage of coupling between adjacent cells occurred on the fourth day. In the presence of 500 mIU/ml hCG, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days instead of 4 days in control conditions. The hCG action was specific, since the addition of heat-inactivated hCG of oFSH or of bTSH did not affect gap junctional communication in trophoblastic cells. The addition of a polyclonal hCG antibody decreased basal gap junctional communication as well as the response to exogenous hCG. Moreover, the presence of 8Br-cAMP (0.5 or 1 mM) mimicked the stimulation by hCG. Interestingly, H89 (2 microM), a specific protein kinase-A inhibitor, dramatically decreased the responses to hCG (500 mIU/ml) and the 8Br-cAMP (0.5 mM) stimulation of trophoblastic gap junctional communication. Calphostin (1 or 2 microM), a specific protein kinase-C inhibitor, strongly stimulated gap junctional communication. In conclusion, the demonstration by means of the gap-FRAP method of a gap junctional communication preceding cellular fusion could be considered as an objective and physiological criterion to mark the beginning of trophoblast differentiation. hCG, a hormone produced by the trophoblast, and two signalling mechanisms are implicated in this phenomenon.

Structural and functional changes in mitochondria associated with trophoblast differentiation: methods to isolate enriched preparations of syncytiotrophoblast mitochondria.

The syncytiotrophoblast of the human placenta is derived from the fusion of cytotrophoblast cells. The syncytiotrophoblast and cytotrophoblast cells have different functional properties. Here, we document that syncytiotrophoblast mitochondria have a distinct phenotype that differs from that of the mitochondria ofcytotrophoblast cells. Syncytiotrophoblast mitochondria are small and have a dense matrix and vesicular cristae. They contain the machinery to convert cholesterol into pregnenolone. The larger cytotrophoblast mitochondria have lamellar cristae and do not have detectable P450scc. These observations imply that trophoblast mitochondria undergo morphological and functional changes as cytotrophoblast cells differentiate into syncytiotrophoblast. Structural changes in mitochondria and accumulation of P450scc were induced in a clonal line of BeWo choriocarcinoma cells by treatment with 8-Br-cAMP, which promotes formation of syncytial structures in these cultures. We conclude that the terminal differentiation program of trophoblast cells includes major changes in the architecture and function of mitochondria. Based on the unique features of syncytiotrophoblast mitochondria, we developed a method to prepare highly enriched syncytiotrophoblast mitochondria from term placenta using differential centrifugation and density gradient centrifugation.