Geoffroy Marceau

Metabolism of Retinol During Mammalian Placental and Embryonic Development

Retinol (vitamin A) is a fat-soluble nutrient indispensable for a harmonious mammalian gestation. The absence or excess of retinol and its active derivatives [i.e., the retinoic acids (RAs)] can lead to abnormal development of embryonic and extraembryonic (placental) structures. The embryo is unable to synthesize the retinol and is strongly dependent on the maternal delivery of retinol itself or precursors: retinyl esters or carotenoids. Before reaching the embryonic tissue, the retinol or the precursors have to pass through the placental structures. During this placental step, a simple diffusion of retinol can occur between maternal and fetal compartments; but retinol can also be used in situ after its activation into RA(1) or stored as retinyl esters. Using retinol-binding protein knockout model, an alternative way of embryonic retinol supply was described using retinyl esters incorporated into maternal chylomicrons. In the embryo, the principal metabolic event occurring for retinol is its conversion into RAs, the active molecules implicated on the molecular control of embryonic morphogenesis and organogenesis. All these placental and embryonic events of retinol transport and metabolism are highly regulated. Nevertheless, some genetic and/or environmental abnormalities in the transport and/or metabolism of retinol can be related to developmental pathologies during mammalian development.

Retinoid Pathway and Congenital Diaphragmatic Hernia: Hypothesis from the Analysis of Chromosomal Abnormalities

Background/Objectives: Although there is strong evidence implicating genetic factors in congenital diaphragmatic hernia (CDH) pathogenesis, few causal genes have been identified. Many studies suggest that early disruption of the retinoid signaling pathway during gestation may contribute to CDH etiology. Chromosome abnormalities are detected in 10–20% of CDH cases. Chromosomal regions that are involved in balanced translocations or are recurrently deleted or duplicated in patients with CDH are of particular interest to researchers because they are more likely to harbor genes that cause or predispose one to the development of CDH. The aim of this review was to select chromosome loci which have been shown to be associated with CDH and to investigate if these loci contain candidate genes involved in the retinoic signaling pathway. Data Sources: We have re-examined the known CDH-critical chromosomal loci and searched in available databases, such as the UCSC Genome Browser and OMIM, to see whether candidate genes related to the retinoid pathway were present within these loci. Results: Twelve retinoid-related genes have been proposed as potential candidates. Among them, COUP-TFII, FOG2 and GATA4 have already been well studied, especially in animal models. We propose other candidates such as STRA6, LRAT, CRBP1, CRBP2 and CRABP1 are directly implicated in retinoic acid metabolism. Conclusion: The identification of CDH-related genes and pathways affecting a normal diaphragm will contribute to the understanding of the pathophysiology of this severe embryopathy and might help to facilitate prenatal management and devise more individual treatment strategies. Further studies are necessary to screen large cohorts of patients with CDH for microimbalances or de novo mutations in these candidate genes. Moreover, functional analyses are needed to establish their exact role in CDH etiology.

Absence of Nuclear Receptors for Oxysterols Liver X Receptor Induces Ovarian Hyperstimulation Syndrome in Mice

Ovarian hyperstimulation syndrome is a frequent complication occurring during in vitro fertilization cycles. It is characterized by a massive ovarian enlargement associated with an accumulation of extra vascular fluid. Here we show that liver X receptor (LXR)-alpha and LXR-beta deficient mice present many clinical and biological signs of ovarian hyperstimulation syndrome: ovarian enlargement, hemorrhagic corpora lutea, increased ovarian vascular permeability, and elevated estradiol. Ovulation stimulation resulted in excessive ovarian response to exogenous gonadotropins because follicle number and estradiol production were higher in transgenic mice. LXR deficiency also leads to perturbations in general inflammatory status, associated with ovarian il-6 deregulation. Upon treatment with the synthetic LXR agonist T09101317, serum estradiol and expression of star and cyp11a1 genes were markedly increased in wild-type mice, showing that LXRs are key regulators of ovarian steroidogenesis. These results suggest that LXRs control the ovulation by regulating endocrine and vascular processes.

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.

Effects of maternal retinoic acid administration in a congenital diaphragmatic hernia rabbit model

Maternal retinoid administration has beneficial effects on lung development in the nitrofen rodent toxic model of congenital diaphragmatic hernia (DH). We wanted to investigate the effects in a surgical model, where the retinoid signaling pathway is not primarily disrupted by the toxic agent. We created DH in fetal rabbits at day 23 of gestation, administrated to the does all trans‐retinoic acid (ATRA) or vehicle (VHC) intramuscularly for 8 consecutive days and harvested normal and operated (DH) fetuses at 31 d (nu2009=u20097 in each group). Normal lungs exposed to ATRA had increased surfactant protein mRNA levels without change in type II pneumocyte density. There was no measurable effect on lung‐to‐body weight ratio and airway morphometry by ATRA. In DH lungs (DH/VHC) surfactant protein mRNA levels were increased, as well as the density of type II pneumocytes. When supplemented with ATRA (DH/ATRA) these parameters returned to normal (VHC). Cell proliferation or apoptosis were not influenced by ATRA supplementation. In conclusion, maternal ATRA supplementation does not affect gross anatomic, morphologic or proliferation indices in hypoplastic lungs related to surgically induced DH in rabbit. However, ATRA lowers surfactant protein expression and normalizes type I/II pneumocyte ratio to what is observed in normal lungs. Pediatr Pulmonol. 2008; 43:594–603.

Identification of GCN2 as new redox regulator for oxidative stress prevention in vivo.

Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet. We investigated the response of GCN2+/+ and GCN2-/- mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2-/- mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage.

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.

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.