A. Malassiné

Hypoxia impairs cell fusion and differentiation process in human cytotrophoblast, in vitro

During human pregnancy, the trophoblast develops from differentiation of cytotrophoblast cells into an endocrine active syncytiotrophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form a syncytium, reproducing the in vivo process. In this study, we examined the effect of low oxygen tension (approximately 9%, hypoxia) compared to standard conditions (approximately 19% oxygen, normoxia) on these cellular events. Under hypoxia, syncytial formation was less frequently observed, cell staining and electron microscopy revealed that cytotrophoblasts remain aggregated, with a positive proliferative cell nuclear antigen (PCNA) immunostaining. Desmoplakin and E‐cadherin, both known to disappear with cytotrophoblast fusion, showed persistent expression in hypoxic cells after 3 days of culture. In contrast, the expression of actin and ezrin, two cytoskeletal proteins, was unchanged. hCG secretion and hPL expression were both decreased in hypoxic cells, reflecting a reduced syncytial formation. Thus, on day 3, the mean values for hCG secretion were 1,100 ± 155 and 289 ± 26 mlU/mL in normoxic and hypoxic conditions, respectively. The reduced cell fusion process as well as hCG secretion and hPL expression under hypoxia were reversed by reoxygenation of the cells. We conclude that under hypoxia, the formation of functional syncytiotrophoblast is impaired due to a defect in the cytotrophoblast fusion process. This may explain the observation of a higher number of cytotrophoblast cells and a reduced syncytial layer in placentas of some pathological pregnancies.

Increase in expression and activity of thrombomodulin in term human syncytiotrophoblast microvilli.

A comparative study of thrombomodulin (TM), a potent natural anticoagulant, was performed in first trimester and term human placentae. Immunoreactive TM was observed on fetal vascular endothelium and syncytiotrophoblast at both gestational ages. Staining was stronger in term than in early placentae, particularly along the microvillous apical membrane of the syncytiotrophoblast. Similarly, a higher level of TM mRNA was detected by RT-PCR (P<0.02) and Northern blot analysis in extracts of whole term placentae. The localization of TM on syncytial microvilli was confirmed by electron microscopy after immunogold labelling. When isolated microvilli were compared at both gestational ages; a significant 2.3-fold increase in TM protein was observed in term microvilli as compared to first trimester microvilli by Western blot analysis (P<0.005) and ELISA (P<0.05). This higher level of TM in term microvilli was associated with an increase in its ability to activate protein C, from 3.7 +/- 1.2 to 8.7 +/- 4.2 mOD/min/microg protein +/- s.d. (P<0.01) in first trimester and term microvilli, respectively. The modulation of biologically active TM at the syncytial membrane exposed to maternal blood according to the length of gestation suggests that TM may be involved both in maternal haemostasis within the intervillous spaces, and also in the trophoblast differentiation process.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

Effects of endothelin on villous trophoblast differentiation and free intracellular calcium

Summary 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.

Transforming growth factor β1 (TGFβ1) inhibits gap junctional communication during human trophoblast differentiation

Summary It was recently established that, during trophoblast differentiation, gap junctional communication (GJC) precedes the formation of the syncytiotrophoblast and is required for trophoblast cell fusion. Therefore, the end of cell-to-cell communication through gap junctions appears as a judicious criterion of cell fusion. Although GJC was seen to be stimulated by human Chorionic Gonadotropin (hCG), its regulation remains poorly understood. Transforming Growth Factor-β1 (TGF-β1), a multifunctional cytokine has been shown to modulate trophoblast differentiation. Therefore, the effects of TGF-β1 on intercellular dye diffusion have been investigated in cultured trophoblast of human term placenta, by means of the Fluorescence Recovery After Photobleaching (gap FRAP) technique. In parallel, trophoblast differnetiation, hCG production and human Chorionic Somatomammotropin (hCS, a specific syncytiotrophoblast hormonal product) expression were assessed. The presence of TGF-β1 (5 or 10 ng/ml) in the culture medium for two days partially inhibited syncytium formation. The percentage of coupled cells was significantly decreased (2.8 times) after two days in presence of 10 ng/ml of TGF β1. Simultaneously, hCG release in culture medium was reduced (at this concentration, to 0.65 and 0.79 after respectively two and three days). In these conditions, Western blot analysis of trophoblast cellular proteins revealed that, after two days, hCS expression was reduced by 40% compared to control. Furthermore, the stimulation of trophoblastic GJC by exogenous hCG (500 mIU/ml) was considerably reduced by simultaneous exposure to TGF-β1 (10 mg/ml). The addition of a polyclonal hCG antibody in excess decreased basal GJC. In the presence of hCG antibody, no significant additive inhibition by TGFβ1 was observed. In conclusion, TGF-β1 was found to inhibit intercellular communication and, subsequently, differentiation and concomitant placental hormone secretions.