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Evidence for expression of both classical and non-classical major histocompatibility complex class I genes in bovine trophoblast cells.

PROBLEM: During the third trimester of pregnancy bovine trophoblast cells in the interplacentomal and arcade regions of the placenta express major histocompatibility complex class I (MHC-I) antigens. At parturition immunological recognition of MHC-I antigens appears to contribute to normal placental release. Therefore, we hypothesized that during late pregnancy bovine trophoblast cells express polymorphic, classical MHC-I antigens. METHOD OF STUDY: Cloning, microarray screening and sequencing of cDNA were used to study transcription of MHC-I genes in peripheral blood mononuclear cells (PBMC) and interplacentomal, trophoblast cells. Real-time reverse transcription, polymerase chain reaction (RT-PCR) was used to compare the abundance of MHC-I transcripts in PBMC and trophoblast cells. RESULTS: Screening of cloned MHC-I cDNA on MHC-I microarrays indicated that in PBMC 90-98% of MHC-I transcripts were encoded by classical MHC-I genes with the remainder encoded by non-classical MHC-I genes. In contrast, 21-66% of MHC-I transcripts from interplacentomal trophoblast cells were from classical genes and 34-79% were from non-classical genes. Transcripts from four non-classical MHC-I loci were identified by sequence analysis. Real-time RT-PCR indicated that the overall levels of MHC-I gene expression in PBMC and trophoblast were similar. CONCLUSION: Bovine interplacentomal trophoblast cells express both classical and non-classical MHC-I genes, but the relative level of expression varies considerably.

Animals↗

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy.

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.

Cell Communication↗

Src family kinases play multiple roles in differentiation of trophoblasts from human term placenta.

Tyrosine phosphorylation plays a major role in controlling many biological processes in different cell types. Src family kinases (SFKs) are one of the most studied groups of tyrosine kinases and can mediate a variety of signalling pathways. However, little is known about the expression of SFKs in human term placenta and their implication in trophoblast differentiation. Therefore, we examined the expression profile of SFK members over time in culture and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultured in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that resemble phenotypically mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). In this study, we showed that trophoblasts expressed all SFK members and some of them are expressed as different splice variants. Moreover, using real-time PCR, this study showed two different expression profiles of SFKs in human trophoblasts during culture. In addition, the protein level and phosphorylation status of Src were evaluated using specific antibodies. Src was rapidly phosphorylated at Tyr-416 and dephosphorylated at Tyr-527 after FBS addition. Surprisingly, inhibition of SFKs by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) or herbimycin A had different effects on trophoblast differentiation. While herbimycin A inhibited morphological and hormonal differentiation, PP2 stimulated hormonal differentiation and inhibited cell adhesion and spreading with no effect on cell fusion. In summary, this study showed that SFKs play different roles in trophoblast differentiation, probably depending on SFK members activated. Thus, this study increases our knowledge and understanding of pathology related to impaired trophoblast differentiation such as pre-eclampsia and trophoblast neoplasm.

Benzoquinones↗

Pathogenic role of anti-beta 2-glycoprotein I antibodies in antiphospholipid associated fetal loss: characterisation of beta 2-glycoprotein I binding to trophoblast cells and functional effects of anti-beta 2-glycoprotein I antibodies in vitro.

BACKGROUND: Antiphospholipid antibodies reacting with beta2-glycoprotein I (beta 2GPI) have been associated with recurrent fetal loss and pregnancy complications. OBJECTIVE: To investigate whether specific mutations in the phospholipid binding site of beta 2GPI might affect its binding to trophoblast and in turn the anti-beta 2GPI antibody induced functional effects. METHODS: beta 2GPI adhesion to trophoblast was evaluated as human monoclonal IgM or polyclonal IgG anti-beta 2GPI antibody binding to trophoblast monolayers cultured (1) in complete medium; (2) in serum-free medium; (3) after serum starvation in the presence of purified human beta 2GPI; or (4) in the presence of beta 2GPI with single or multiple mutations in the amino acid loop Cys(281)-Lys-Asn-Lys-Glu-Lys-Lys-Cys(288). The effect of anti-beta 2GPI binding to trophoblast was evaluated as chorionic gonadotropin (hCG) mRNA expression, and protein release by RT-PCR and radioimmunoassay, respectively. RESULTS: beta 2GPI adhesion to trophoblast and its consequent recognition by the specific antibodies were inversely proportional to the mutation number in the phospholipid binding site. Anti-beta 2GPI antibodies reduced gonadotropin release, hormone dependent hCG mRNA expression, and protein synthesis in the presence of beta 2GPI, while the addition of the mutants or the absence of beta 2GPI had no effect. CONCLUSIONS: beta 2GPI binds to trophoblast in vitro through its fifth domain, as reported for endothelial cells, and can be recognised by anti-beta 2GPI antibodies; the antibody binding downregulates trophoblast hCG synthesis and secretion. Such a mechanism might contribute to defective placentation in women with fetal loss associated with the antiphospholipid syndrome.

Antiphospholipid Syndrome↗

Uterine spiral artery remodeling involves endothelial apoptosis induced by extravillous trophoblasts through Fas/FasL interactions.

OBJECTIVE: Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. METHODS AND RESULTS: Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. CONCLUSIONS: Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.

Apoptosis↗

Decidua produces a protein that inhibits choriogonadotrophin release from human trophoblasts.

To test the hypothesis that uterine decidua may modulate trophoblast function, trophoblasts and decidual cells were isolated from term placentas by enzymatic digestion and Percoll gradient centrifugation. Placental trophoblasts were cocultured with decidual cells and trophoblasts or JEG-3 choriocarcinoma cells were incubated with medium conditioned by decidual cells (DCM) for 72-96 h. In cocultures decidual cells inhibited choriogonadotropin (hCG) release from trophoblasts by 75% in comparison with controls (P less than 0.001). The DCM contained a factor that markedly inhibited hCG release from trophoblasts and JEG cells in vitro compared with controls. The inhibitory effect of the factor on hCG release was dose dependent, and could be eliminated by boiling the DCM for 30 min or proteolytic enzyme treatment. Ultrafiltration and Sephadex G-50 fractionation of the DCM indicated that the apparent molecular mass was 7,000-10,000 D. DCM also inhibited the stimulatory effect of exogenous cAMP on hCG secretion by JEG-3 cells, suggesting that DCM may interfere with activation of the cAMP-dependent protein kinases or transcription of hCG genes. These results suggest that the release of trophoblast hCG is under local paracrine control, regulated in part by a protein released by decidual cells.

Cells, Cultured↗

Differentiation of human trophoblast populations involves alterations in cytokeratin patterns.

Cytokeratins (CKs) are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villous and extravillous trophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravillous trophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CK13 showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblasts were also positive for CK7 and CK13. CK17 was found related to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblasts in cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti-CK13 antibodies in the first trimester but was positive at term. CK4 and CK16 were not found in the placenta. Our study shows for the first time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specific patterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast.

Cell Differentiation↗

Two mechanisms for IgG uptake in cultured human trophoblast: evidence for a novel high affinity Fc receptor.

The mechanism of IgG transport by the placental trophoblast was examined by studying IgG uptake by purified trophoblast maintained in culture. This model retains the ability to bind and endocytose human IgG from human serum. Comparison of the relative IgG uptake by the trophoblast among the four subclasses of both human and mouse IgG indicates that the trophoblast IgG receptor has different affinities from those described for the three known human Fc gamma receptors, FcR gamma I, FcR gamma II, and FcR gamma III. These results suggest the presence of a novel trophoblast Fc gamma receptor. Although Fc gamma RIII has been reported to be present on trophoblasts, immunocytochemical studies failed to detect binding to the cell surface of antibody-specific for Fc gamma RIII, 3G8 MAb. In addition, blocking studies with MAb 3G8 did not interfere with IgG uptake. Scatchard analysis of human IgG uptake revealed a biphasic curve consistent with two distinct mechanisms for the transport of IgG by the trophoblast. The first is a higher affinity system (Ka = 1.7 x 10(7) M-1, 1.7 x 10(4) binding sites/cell) which exhibits IgG subclass and species specificity, and the second is a low affinity system (Ka = 6.9 x 10(3) M-1, 7.5 x 10(7) binding sites/cell).

Animals↗

Expression of the pregnancy-specific beta 1-glycoprotein gene in cultured human trophoblasts.

Human pregnancy-specific beta 1-glycoprotein (PS beta G), the major placental glycoprotein, shares strong sequence similarity with carcinoembryonic antigen and is a member of the immunoglobulin superfamily. To understand the role of PS beta G in placental ontogeny during pregnancy, we examined its synthesis and regulation in primary cultures of trophoblast cells. Freshly plated (12 h) cytotrophoblasts expressed little PS beta G transcripts; however, within 24 h of culture, PS beta G mRNAs became detectable. PS beta G synthesis and mRNA expression increased with time in culture, and maximal synthesis was achieved at 4 days, indicating that primary trophoblasts continue differentiating in vitro. Molecular cloning revealed that PS beta G is an extremely polymorphic protein. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity in the 5' (designated PSG-5') and coding regions, but differ in sequences at the 3' region. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three mRNAs of 2.3, 2.2, and 1.7 kilobases in primary trophoblasts and human term placental tissue. Ribonuclease protection analysis demonstrated that primary trophoblasts expressed most of the placental PS beta G transcripts. However, culturing in vitro altered PS beta G gene expression, and the level of PS beta G transcripts containing a PSG95-3' sequence was preferentially increased in primary trophoblasts. Moreover, primary trophoblasts synthesized a 64K PS beta G polypeptide in variable amounts and three PS beta Gs of 72K, 62K, and 54K in roughly equal amounts, whereas purified human term placental PS beta G consists of a major polypeptide of 72K and two minor ones of 64K and 54K. PS beta G gene expression in primary trophoblasts was slightly reduced by 8-bromo-cAMP, but was markedly inhibited by sodium butyrate.

8-Bromo Cyclic Adenosine Monophosphate↗

Macrophage-colony stimulating factor (M-CSF) regulates the expression of fibronectin and its alpha5 integrin receptor in human trophoblasts.

Macrophage-colony stimulating factor (M-CSF, CSF-1) is secreted by human trophoblasts as well as endometrial cells, while its receptor c-fms is abundantly expressed by the extravillous trophoblasts anchoring the placenta to the uterus, suggesting a role for M-CSF at the maternal-fetal interface. We investigated the effect of M-CSF on the expression of fibronectin and its receptor, the alpha5 integrin, in human trophoblasts. Exposure of trophoblasts to M-CSF produced a two to three-fold increase in fibronectin and alpha5 mRNA abundance at both 24 and 72 hours of culture. A dose-dependent increase in cellular fibronectin secretion into the culture medium was detected at both time points. Immunocytochemistry showed co-localization of cellular fibronectin and alpha5 in the cells, suggesting that attachment of trophoblasts to fibronectin is mediated in part by the alpha5beta1 integrin. We conclude that M-CSF increases fibronectin expression and secretion by the human trophoblasts, and up-regulates its specific receptor, the alpha5 integrin. We hypothesize that M-CSF may partake in the autocrine/paracrine mechanisms regulating trophoblast invasion during implantation.

Antigens, CD↗

The role of apoptosis in the regulation of trophoblast survival and differentiation during pregnancy.

Apoptosis is important for normal placental development, but it may also be involved in the pathophysiology of pregnancy-related diseases. Normal placental development is dependent upon the differentiation and invasion of the trophoblast, the main cellular component of the placenta. Trophoblast apoptosis increases in normal placentas as gestation proceeds, and a greater incidence of trophoblast apoptosis has been observed in pregnancies complicated by preeclampsia or intrauterine growth retardation (IUGR). In response to different stimuli, apoptosis may be initiated extrinsically by the death receptor pathway or intrinsically by the mitochondrial pathway. The central executioners of apoptosis are the caspases, which cleave numerous vital cellular proteins to affect the apoptotic cascade. By inhibiting caspase activation, several endogenous inhibitors, including flice-like inhibitory proteins (FLIPs), inhibitors of apoptosis (IAPs), and antiapoptotic Bcl-2 family members, can prevent further propagation of the death signal. Macrophages present at the maternal-fetal interface may also contribute to trophoblast survival by removing apoptotic cells and producing cytokines and growth factors, which influence the progression of the apoptotic cascade. This review focuses on the role of apoptosis in trophoblast development and differentiation, the molecular mechanisms by which normal trophoblast apoptosis can occur, and how it is regulated to prevent excessive trophoblast apoptosis and possible pregnancy complications.

Apoptosis↗

Characterization of morphological and cytoskeletal changes in trophoblast cells induced by insulin-like growth factor-I.

IGF-I and IGF-II were appeared to play major roles in the adhesive and migratory events that are considered to be crucial in the implantation process. The purpose of this study was to determine the effects of IGF-I on trophoblast adhesion to extracellular matrix. Trophoblast cells obtained from early gestation at artificial abortion were incubated with the indicated doses of IGF-I at the indicated times. Trophoblast cells were treated with IGF-I in the presence or absence of RGD peptide and an antibody against alpha-subunit of IGF-I receptor (alphaIR3). Morphometric and morphological changes were studied using light and electron microscopy. Furthermore, vinculin, actin stress fibers, phosphorylated focal adhesion kinase (FAK), phosphotyrosine, and paxillin were immunolocalized in trophoblast cells after IGF-I treatment in the presence or absence of alphaIR3. Immunoprecipitation and anti-phosphotyrosine immunoblotting were carried out to detect the phosphorylated FAK and phosphorylated paxillin contents of the IGF-I-treated and untreated trophoblast cells. The results showed that IGF-I promoted trophoblast adhesion to fibronectin substrate in a time- and dose-dependent manner, and addition of RGD peptide and alphaIR3 monoclonal antibody abolished the effects of IGF-I in these cells. Morphological studies exhibited an increase in the lamellipodia formation upon IGF-I treatment, and confocal images of immunofluorescent staining revealed localization of phosphorylated FAK, paxillin, and vinculin at focal adhesions as well as redistribution of actin microfilaments and formation of actin stress fibers inside the cell. Western blotting, using antiphosphotyrosine demonstrated proteins with molecular masses of 125 kDa (FAK) and 68 kDa (paxillin) present in the IGF-I-treated cells, which were lacking in the control groups. In conclusion, these findings suggest that IGF-I can stimulate lamellipodia formation and promote adhesion of trophoblast cells to extracellular matrix by activating their adhesion molecules that must be activated within the implantation window.

Cell Adhesion↗

Trophoblast and placental villous core production of lipid peroxides, thromboxane, and prostacyclin in preeclampsia.

Placentas obtained from women with preeclampsia produce more lipid peroxides and more thromboxane, but less prostacyclin, than normal. The tissue compartments within the placenta that are responsible for this are not known. The placenta is a heterogeneous tissue compartmentalized into trophoblast cells and villous core tissue that is comprised of stromal and vascular tissue. In this study we determined the placental compartments responsible for increased production of lipid peroxides and thromboxane in preeclampsia. Placentas were obtained from six normally pregnant women and seven women with preeclampsia. Trophoblast cells and villous core tissues were isolated and incubated in Dulbecco's Modified Eagle's Medium for 48 h. Samples were collected at 0, 2, 6, 16, 28, and 48 h of incubation and analyzed spectrophotometrically for lipid peroxides by a peroxide equivalent assay and for thromboxane and prostacyclin by RIA of their stable metabolites, thromboxane-B2 and 6-keto-prostaglandin-F1 alpha. Trophoblast cells isolated from preeclamptic placentas produced significantly more lipid peroxides (1972 +/- 502 vs. 1102 +/- 335 pmol/micrograms protein after 48 h of incubation), more thromboxane (328 +/- 57 vs. 153 +/- 53 pg/microgram at 48 h), and more prostacyclin (50 +/- 11 vs. 13 +/- 3 pg/microgram at 48 h, respectively) than trophoblast cells isolated from normal placentas. Villous core tissue isolated from preeclamptic placentas produced significantly more lipid peroxides (455 +/- 107 vs. 241 +/- 34 pmol/microgram) and more thromboxane (148 +/- 51 vs. 76 +/- 14 pg/microgram) than normal villous core tissue, but there was no difference in prostacyclin production (36 +/- 11 vs. 40 +/- 9 pg/microgram). Because of the increase in thromboxane production, the ratio of thromboxane to prostacyclin was higher in preeclamptic than normal villous core tissue (6.29 vs. 2.17). Comparison of production by different compartments within the placenta demonstrated that lipid peroxides and thromboxane were primarily produced by the trophoblast cells and stromal tissue, whereas prostacyclin was primarily produced by the vascular tissue. We conclude that increased placental production of lipid peroxides and thromboxane in preeclampsia originates from both the trophoblast cell and the villous core compartments. As the placenta secretes lipid peroxides, the trophoblast cells could be a source of increased lipid peroxides in the maternal circulation of women with preeclampsia. The increased ratio of thromboxane to prostacyclin in the villous core could be responsible for increased placental vasoconstriction.

6-Ketoprostaglandin F1 alpha↗

The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells.

Activation of Lyn, a Src-related nonreceptor tyrosine kinase, in trophoblast cells is associated with trophoblast giant cell differentiation. The purpose of the present work was to use Lyn as a tool to identify signaling pathways regulating the endocrine differentiation of trophoblast cells. The Src homology 3 domain of Lyn was shown to display differentiation-dependent associations with other regulatory proteins, including phosphatidylinositol 3-kinase (PI3-K). PI3-K activation was dependent upon trophoblast giant cell differentiation. The downstream mediator of PI3-K, Akt/protein kinase B, also exhibited differentiation-dependent activation. Lyn is a potential regulator of the PI3-K/Akt signaling pathway, as are receptor tyrosine kinases. Protein tyrosine kinase profiling was used to identify two candidate regulators of the PI3-K/Akt pathway, fibroblast growth factor receptor-1 and Sky. At least part of the activation of Akt in differentiating trophoblast giant cells involves an autocrine growth arrest-specific-6-Sky signaling pathway. Inhibition of PI3-K activities via treatment with LY294002 disrupted Akt activation and interfered with the endocrine differentiation of trophoblast giant cells. In summary, activation of the PI3-K/Akt signaling pathway regulates the development of the differentiated trophoblast giant cell phenotype.

Animals↗

Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.

Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells.

Amino Acid Sequence↗

Effects of epidermal growth factor and transforming growth factor alpha on the mouse trophoblast outgrowth in vitro.

In order to analyze the involvement of growth factors in the implantation mechanism, we examined the direct effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) on trophoblast outgrowth of the mouse blastocyst in vitro. ICR mouse blastocysts were cultured for 4 days on a culture plate in medium containing EGF or TGF-alpha or conditioned medium obtained from cultured endometrial epithelial cells. Blastocysts were also co-cultured with endometrial epithelial cells. The trophoblast outgrowth of these cultured blastocysts was observed daily and the percentage of outgrowing embryos was calculated and analyzed statistically by the chi-squared test. Analysis for the specific binding of 125I-EGF in outgrown trophoblasts was carried out by autoradiography. The co-culture (days 3 and 4) and the presence of EGF (10 ng/ml, day 4), TGF-alpha (1 ng/ml, day 3; 10 ng/ml, days 2 and 3; 50 ng/ml, days 2-4) or conditioned medium (days 3 and 4) significantly stimulated the rate of trophoblast outgrowth. Preincubation of the conditioned medium with monoclonal anti-EGF or anti-TGF-alpha antibody suppressed the stimulatory effect of the conditioned medium on trophoblast outgrowth. The specific 125I-EGF binding in outgrown trophoblasts was demonstrated by autoradiography. These results suggest that EGF and TGF-alpha play an important role in the implantation process by directly stimulating trophoblast development.

Animals↗

Support for the development of bovine embryos in vitro by secretions of bovine trophoblastic vesicles derived in vitro.

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.

Animals↗

Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast.

The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-beta-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated beta-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with beta-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and up-regulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating in-hibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear beta-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the path-way could contribute to trophoblastic hyperplasia and local invasion.

Cell Differentiation↗