Denis Gallot

Ontogeny of Aquaporins in Human Fetal Membranes

ABSTRACT It has been proposed that four members of the aquaporin family (AQPs 1, 3, 8, and 9) are involved in the control of amniotic fluid (AF) homeostasis, as illustrated by their differential expression patterns in normal and pathological human term fetal membranes. However, there are no data available to date on their ontogeny throughout pregnancy. Our objective was to determine spatiotemporal expression profiles of the mRNA and proteins of all 13 members of this transmembrane channel family. For this purpose, we used healthy fetal membranes from the first, second, and third trimesters of pregnancy. Total mRNA and proteins were extracted from total membranes and from separated amnion and chorion. Quantitative PCR, Western blot, and immunohistochemistry experiments were carried out to determine the presence of AQPs and to quantify their spatiotemporal expression patterns throughout pregnancy. The WISH cell line was tested to propose a cellular model for the role of AQPs in the amnion compartment. AQP11 expression was established in amniotic membranes at term. Aquaporins 1, 3, 8, 9, and 11 mRNA and proteins were present in amnion and chorion throughout human gestation. Each AQP has a time-specific expression pattern, with AQP1 presenting the highest variation in terms of mRNA and protein levels. The WISH cell line also expressed the same five AQPs. Taken together, these results indicate that AQPs are expressed and potentially involved in the regulation of AF homeostasis throughout pregnancy. This also clearly supports the hypothesis that abnormal expression could occur at any time during pregnancy, ultimately leading to obstetrical pathologies such as polyhydramnios or oligohydramnios.

Retinoids regulate human amniotic tissue-type plasminogen activator gene by a two-step mechanism.

The collagenolytic effects of the tissue‐type plasminogen activator (t‐PA) leading to extracellular matrix degradation are clearly involved in the physiopathology of human foetal membranes rupture. Nevertheless, the regulation of t‐PA gene expression in extraembryonic developmental contexts remains unknown. The aim of our study is to propose the retinoic acids (RAs) as molecular regulators of t‐PA expression in foetal membranes. RA induced t‐PA mRNA and proteins in a time‐dependent manner in amniotic membrane explants and Wistar Institute Susan Hayflick (WISH) cells. Furthermore, the use of cycloheximide revealed a two‐step regulation of t‐PA gene. Gene reporter assays confirmed that the RA‐induced t‐PA gene expression occurred through interactions of retinoid receptors (RARs and RXRs) with a DR5 response element located at –7 kb from the transcription site. Site‐directed mutagenesis of this region of the t‐PA promoter showed that SP1 factor was also retinoid‐mediated induction, and immunoprecipitation assays revealed that SP1 and RAR/RXR interacted physically. Chromatin immunoprecipitation demonstrated that interactions between RARs, RXRs and t‐PA promoter were time dependent: RAR‐α/RXR‐α bound DR5 motif before and up to 12 hrs of RA exposure, and RAR‐β/RXR‐α bound DR5 response element after 12 hrs of RA treatment. Finally, experiments using shRNA and RAR‐β‐specific antagonist revealed that reducing RAR‐β induction decreased t‐PA induction. Altogether, our results established that the RA‐mediated regulation of t‐PA in human foetal membranes occurred through two steps, with a major role played by RAR‐β.

All-trans-retinoic acid regulates aquaporin-3 expression and related cellular membrane permeability in the human amniotic environment

INTRODUCTIONnThe aquaporins (AQP1, 3, 8, 9 and 11) are known to be expressed, and involved in the transport of water and small molecules through fetal membranes. To exert these crucial functions, these AQPs have to be finely regulated. All-trans-retinoic acid (atRA) was previously found to regulate some genes in this environment, raising the question of whether these AQPs were regulated by atRA.nnnMETHODSnExplants, and primary and established amniotic cells were cultured to determine which AQP were transcriptionally modified by atRA, using the qRT-PCR strategy. Immunohistochemistry and glycerol uptake tests were used to determine the impact of atRA on AQP protein expression and function. Specific agonists of retinoic acid receptors were used to identify the molecular mechanisms of AQP promoter activation. A classical gene AQP promoter study was also used to identify DR5 retinoic acid receptor elements (RAREs).nnnRESULTSnBeyond these AQPs, only one specific atRA-dependent increase in AQP3 transcripts and proteins level was established in amnion (not in chorion) and in related primary and established cells. We found three DR5-RAREs essential for inducing this transcriptional AQP3 through RARα. This transactivation of the AQP3 coding gene was functionally related to an increase of AQP3 permeability tests by a glycerol uptake assay.nnnDISCUSSIONnOur data support an atRA regulatory model of AQP3 expression leading to an increased cellular permeability in the epithelial amniotic environment. We cast new light on AF regulation in healthy pregnancy, and advance new hypotheses for obstetrical complications linked to impairment of the retinoic signaling pathway.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Retinoic acid and tracheal occlusion for diaphragmatic hernia treatment in rabbit fetuses

Lung hypoplasia and pulmonary arterial hypertension in congenital diaphragmatic hernia lead to a high perinatal mortality. Although sustained fetoscopic tracheal occlusion (TO) improves lung development, a major side effect is abnormal pneumocyte differentiation. This study evaluated the potential ability of intratracheal retinoic acid (RA) administration to reduce adverse effects of sustained TO in a rabbit model of diaphragmatic hernia.

Laryngotracheoesophageal cleft, a rare differential diagnosis of esophageal atresia

A laryngotracheoesophageal cleft, commonly called laryngeal cleft (LC), is a congenital malformation of the posterior part of the larynx creating an abnormal communication between the laryngotracheal axis and the pharyngoesophageal axis. The prenatal ultrasonographic features associating absent stomach, polyhydramnios and mediastinal pouch sign are usually considered pathognomonic for esophageal atresia. This observation demonstrates that they can also correspond to a severe form of laryngotracheoesophageal cleft extending to the carina.

Cigarette smoke condensate affects the retinoid pathway in human amnion

INTRODUCTIONnThe preterm premature rupture of membranes (PPROM) is a frequent pathology responsible of more than 30% of preterm births. Tobacco smoking is one of the most frequently described risk factors identified and contributes to the pre term weakening of fetal membranes. As previously demonstrated, all-trans retinoic acid (atRA) regulates several genes involved in the extracellular matrix dynamics, an essential actor in fetal membrane ruptures. We hypothesized that cigarette smoke may affect this pathway in human amnion.nnnMETHODSnAmnion was obtained from full-term fetal membranes collected from non-smoking women after cesarean births and used either as explants or for the isolation of derived epithelial cells. The pro-healing and transcriptomic effects of atRA were studied by a scratch assay experiment and quantitative RT-PCR, respectively, after treatment with dimethyl sulfoxyde (DMSO), atRA, DMSOxa0+xa0cigarette smoke condensate (CSC), or atRAxa0+xa0CSC.nnnRESULTSnOur results show a strong alteration of the retinoid pathway after CSC treatment on amnion-derived epithelial cells and explants. We first demonstrated that CSC inhibits the activity of the RARE reporter gene in amnion-derived epithelial cells. Then, atRAs effects on both the transcription of its target genes and wound healing were demonstrated to be inhibited or at least decreased by the CSC in human amnion epithelial cells.nnnDISCUSSIONnHere, we demonstrated that CSC altered the retinoid signal, already known to have roles in fetal membrane physiopathology. These results highlight a potential negative action of maternal smoking on the retinoid pathway in human amnion and more generally on pregnancy.