Françoise Ferré

Isolation and characterization of the rolipram-sensitive cyclic AMP-specific phosphodiesterase (type IV PDE) in human term myometrium

On the basis of the potencies of classical selective modulators of cyclic nucleotide phosphodiesterase (PDE) activities, five cyclic nucleotide PDE isoforms have been isolated and characterized in the cytosolic fraction of human term myometrium. By means of successive ion-exchange chromatographies, a calcium-calmodulin sensitive isoform, a cyclic GMP-stimulated isoform, a cyclic GMP-inhibited isoform, a rolipram-sensitive cyclic AMP-specific isoform and a cyclic GMP-specific isoform, corresponding to PDE I, PDE II, PDE III, PDE IV and PDE V, respectively, have been identified. We found that near term, human myometrium contains a higher proportion of the rolipram-sensitive type IV PDE isoform (about 50% of total cyclic AMP hydrolytic activity) than the type III cyclic GMP-inhibited PDE isoform (only 10%). Type IV PDE displays simple Michaelis-Menten kinetics with a high affinity for cyclic AMP (Km approximately 4.4 microM) and is selectively and competitively inhibited by rolipram (K(i) approximately 0.9 microM) and Ro 20-1724 (K(i) approximately 2.6 microM). The predominance of type IV PDE at the end of pregnancy suggests that this isoform contributes, via a modulation of the intracellular cyclic AMP level, to local control of uterine motility and thus could help the myometrium prepare for pronounced contractile activity at the time of parturition.

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR—target of the CTCF insulator—is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

Endothelin-1 binding sites and immunoreactivity in the cultured human placental trophoblast: evidence for an autocrine and paracrine role for endothelin-1.

In human placenta, endothelin (ET) could derive from maternal, fetal, and/or endogenous sources. Therefore, localization of ET-1 was investigated by immunohistochemistry in human term placenta and in cultured trophoblasts. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblasts of the villi. ET-1 IR was also detected in the decidual cells and in the extravillous cytotrophoblasts of the basal plate. The extravillous cytotrophoblasts of the chorionic plate and of the placental septa also exhibited strong ET-1 IR. For trophoblast culture, cytotrophoblastic cells were obtained from placental villi by trypsin-DNAse dispersion, further purified on a Percoll gradient, and enriched by employing a monoclonal anti-HLA class I antibody. After different periods of culture of purified cytotrophoblastic cells (1-5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblasts. The presence of ET-1,2 immunoreactivity (ET-1,2 IR) in the culture media was demonstrated by radioimmunoassay. A uniform daily production of the peptide was observed over at least 5 days (approximately 50 fmol/10(6) cells/24 h). Furthermore, trophoblastic cells that had been cultured for 5 days contained significant amounts of ET-1,2 IR (24 fmol/10(6) cells). These results suggest a trophoblastic origin for ET-1 and support the hypothesis of a paracrine and autocrine action of the peptide in the physiology of the trophoblast and placenta.

Higher endothelin concentrations in the fetoplacental unit of pregnant women of African ancestry

Immunoreactive endothelin was assayed in maternal and fetal biologic fluids of women of African and European ancestry with normal singleton pregnancies undergoing cesarean section at term for obstetric reasons. Endothelin concentration was found to be higher in the umbilical vein and artery blood of women of African origin. Higher production of endothelins in the fetoplacental unit may place these women at a greater risk of preeclampsia.

Trophoblastic localization of [125I] endothelin-1binding sites in human placenta

Summary Specific and high affinity binding sites for [125I] ET-1 have been previouslyidentified in human crude placental preparations and in membranes of human placental vessels. Nevertheless the precise localization of these receptors is unknown. In order to find an answer to this problem, placental cryostat sections (20 μm) and trophoblast in culture were processed for “in vitro” receptor autoradiography. Furthermore, a biochemical study was performed on well-defined plasma membranes isolated from fresh placenta and from trophoblast in culture. [125I] ET-1 shows specific, high affinity, and saturable binding to stem villi vessel membranes, membranes of trophoblast in culture, microvillous membrane of syncytiotrophoblast (facing maternal blood space), and basal plasma membrane of syncytiotrophoblast (facing fetal circulation). [125I] ET-1 binding affinities for microvilli (23±6 pM, n=3) and basal plasma membrane (12±2 pM, n=3) were identical; however the number of binding sites is 3.5 time higher for basal plasma membrane (147±5 fmol/mg protein, n=3) than for microvilli (40±19 fmol/mg protein, n=3). In placental sections, autoradiography showed the association of numerous grains on the smooth muscle cells of vessels and on the trophoblastic layer of the chorionic villus. In trophoblast in culture, labeling is observed mainly on cellular aggregates and on multinucleated syncytiotrophoblast. Labeling was abolished by coincubating [125I] ET-1 with an excess of unlabeled ET-1 (1 μM). This results suggest that ET-1 may be involved in the regulation of fetoplacentalcirculation and in the physiological control of trophoblast activity.