The placental sulfate transporter, Slc13a4, is critical for skeletal development in mice

s / Placenta 36 (2015) A1eA60 A5 NI2.2. THE PLACENTAL SULFATE TRANSPORTER, SLC13A4, IS CRITICAL FOR SKELETAL DEVELOPMENT IN MICE. Joanna Rakoczy , Paul Dawson , David Simmons . 1 School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia; Mater Research Institute, Brisbane, Queensland, Australia Objectives: Sulfate is an important nutrient for various cellular and metabolic processes necessary for fetal development. The fetus, having a minimal capacity to produce its own sulfate, is dependent on the mother’s circulating sulfate supply, which is transported to the fetus via placental sulfate transporters. Previously, we discovered that the sulfate transporter Slc13a4 is expressed in both the human and mouse placenta, where it is restricted to the syncytiotrophoblast layer or second layer of syncytiotrophoblast respectively. Since Slc13a4 is situated in the transporting syncytia of both mouse and human placenta, and is not widely expressed in the fetus itself, we chose to investigate its role in placental sulfate transport and fetal development. Methods: We created a mouse line containing an Slc13a4 “knockout first” allele (ES cells obtained from KOMP) to analyze the effects of Slc13a4 deletion. We then converted the knockout first allele into a conditional allele using FLPeR mice to allow for tissue-specific gene deletion. Results: Slc13a4+/crosses yielded no Slc13a4-/mice, indicating that global loss of Slc13a4 is embryonic lethal. While Slc13a4-/placentae appeared normal, by E14.5 Slc13a4-/fetuses exhibited a multitude of developmental phenotypes. One major phenotype was the complete undermineralisation of the skeleton at E16.5. Further analysis revealed that Slc13a4 fetuses have a lack of vascular invasion into the long bones and osteoblast maturation is arrested. Importantly, all these phenotypes were rescued when we used an Slc13a4 conditional knockout mouse approach (crossing with Sox2-Cre), to retain Slc13a4 expression in the placenta, but achieve deletion in the fetus, indicating that the phenotypes observed in the global knockout can be attributed to a loss of placental Slc13a4 expression. Conclusion: Our novel mouse model is providing valuable insight into the critical role of placental sulfate transporters in fetal development. NI2.3. MATERNAL CORTICOSTERONE EXPOSURE IN THE MOUSE CAUSES SEX SPECIFIC ALTERATIONS IN PLACENTAL OGT AND O-LINKED GLYCOSYLATION. James Cuffe, Sarah Steane, Kathryn McMahon, Karen Moritz, Marie Pantaleon. The University of Queensland, Brisbane, Australia Objectives: Maternal stress is known to program adult disease in a sexually dimorphic manner. It is known that placentas of male and female fetuses respond differently to the same maternal challenge rendering male offspring vulnerable to diseases in adulthood. We have previously demonstrated that short term Corticosterone (Cort) exposure (60h) in the mouse induces sex specific placental adaptations and cardiovascular outcomes in offspring. Cellular ability to respond to multiple forms of stress is associated with increased capacity to uptake and metabolise glucose for use in hexosamine signalling (HS) and O-linked glycosylation (O-GlcNAcylation). These pathways are essential for cell survival in response to stress. Interestingly, O-linked-B-acetylglucosamine transferase (OGT) is an X linked gene known to be a placental biomarker of maternal stress. Methods: Using our Cort exposure model, we examined placental levels of key enzymes involved in HS and O-GlcNAcylation. Results: Firstly, we demonstrated that placentas of female fetuses had higher mRNA expression of Ogt than males. Moreover, maternal Cort exposure increased mRNA and protein levels of the stress responsive gene HSP90 in males and females indicating a stress response in both sexes. However Ogt as well as Gftpt1 (rate-limiting regulator of HS) were upregulated to a much greater extent in placentas of female fetuses compared to males. We additionally demonstrated that Cort increased expression of proteins known to be regulated by O-GlcNAcylation. Cort increased the protein expression of the stress responsive pro-survival factor AKT2 in placentas of female but not male fetuses. Conclusion: Our data demonstrates sex-specific alterations in placental HS and OGT levels as well as downstream regulators of placental development in response to Cort. These findings may underlie the sex specific placental adaptations that act to protect the developing female fetus and in turn minimises the incidence of programmed disease outcomes in female offspring. NI2.4. EARLY ALCOHOL EXPOSURE ALTERS PLACENTAL TROPHOBLAST DIFFERENTIATION. Jacinta Kalisch-Smith, Marie Pantaleon, David Simmons, Karen Moritz. The University of Queensland, Brisbane, QLD, Australia Objectives: Maternal periconceptional alcohol (PC-EtOH) exposure causes foetal growth restriction and sex-specific changes to rat placental morphology in late gestation. This may derive from perturbations to preimplantation embryo development and its subsequent capacity to form a placenta. This study aimed to examine cell allocation in the pre-implantation embryo and trophectodermal derivatives after PC-EtOH exposure. Methods: Sprague Dawley dams were administered 12.5% v/v EtOH or control diet from 4 days prior (E-4) to 4 days after conception (E4) in liquid form. Cell number and allocation in the pre-implantation embryo was assessed at E5 and embryos were cultured in vitro for 6 days and trophoblast outgrowth area assessed. To determine whether EtOH can directly affect the differentiation of placental trophoblast stem (TS) cells, one male (RS26) and one female (EGFP) mouse line were cultured in vitro for 6 days (N1⁄43 per treatment) in 0%, 0.2% or 1% EtOH and assayed for lineage-restricted trophoblast subtype markers. RNA was extracted for qPCR and gene expression specific to the labyrinth (Ctsq, Syna) and junctional zones (Tpbpa, Prl7a2, Prl7b1, Prl2c1, Prl3d1) analysed. Results: There were no differences in pre-implantation total cell number, allocation, or outgrowth area (18-51 embryos per treatment) after in vivo PC-EtOH exposure. In vitro EtOH exposure caused dose dependent reductions in the expression of Syna (syncytiotrophoblast layer 1 [SynT-I], P<0.01), Prl7b1 (spiral-artery associated trophoblast giant cells [SpA-TGCs] and glycogen cells [GlyT], P<0.01) and Prl7a2 (spongiotrophoblast [SpT] cells, P<0.01). Conclusion: Gene expression changes suggest EtOH exposure may either delay TS cell differentiation or alter lineage allocation. In vivo this may lead to reduced surface area for nutrient transport in the labyrinth, while aberrant differentiation of junctional zone-derived subtypes may affect trophoblast invasion or endocrine activity. Changes in trophoblast differentiation can impact on placental development, causing foetal growth restriction and programming of adult disease. NI2.5. THE MISSING LINK e HOW THE PLACENTA PROGRAMS THE MATERNAL BRAIN Hugo Creeth, Simon Tunster, Jess Eddy, Anthony Isles, Rosalind John. Cardiff University, Cardiff, UK Objectives: Pregnancy is an exciting yet challenging time. In addition to the substantial physiological alterations required to support fetal growth, there are also changes in the maternal brain that induce appropriate maternal behaviour. The significance of placental endocrine signalling in this induction has not been directly assessed. The aim of this study was to provide experimental evidence that the placenta programs the maternal instinct and the generation maternal behaviour via an epigenetic process. Methods: To ask whether placental signalling induces maternal behaviour, we utilised a mouse model in which a key endocrine lineage was modulated by genetically increasing or decreasing the dosage of the imprinted gene, Phlda2. Three cohorts of wild type dams carrying fetuses that possessed either a 50% reduction or a 200% increase in the spongiotrophoblast lineage were generated using recipient embryo transfer. Half of each cohort was left to give birth and the mother’s behaviour was studied. The other half was sacrificed at E16.5, the peak of placental