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Loss of placental sulphate transporter Slc13a4 causes severe developmental defects and embryonic lethality
A96
Abstracts / Placenta 35 (2014) A1eA112
placental tissue. While this model can be highly predictive, there are several challenges associated with it. The maternal and fetal cannulations can be technically challenging to set up, and leakage from the fetal reservoir can result in early termination of the experiment. The objective of this study was to evaluate the success rate at several checkpoints for the dual perfusion of the human placenta at the Motherisk laboratory.
Figure 2. Analytical dependence of the normalized optimal uptake Fmax / F0 on the ratio of characteristic transport times g ¼ ttr / tabs. For the stream tube model, F0 is defined as F0 ¼ c0upR2, where u is the maternal blood velocity, R is the radius of the stream tube and c0 is the concentration of oxygen at the entrance to the stream tube. Shaded regions show values expected in a normal pregnancy.
P2.112-N. EFFECTS OF PARTIAL PRESSURE OF OXYGEN ON THE MODULATION OF SYNCYTIOTROPHOBLAST POTASSIUM CHANNELS BY REACTIVE OXYGEN SPECIES Paula Díaz, Colin P. Sibley, Susan L. Greenwood Maternal and Fetal Health Research Centre, University of Manchester, Manchester, UK Objectives: Altered placental partial pressure of oxygen (pO2), elevated reactive oxygen species (ROS) and dysregulated renewal of syncytiotrophoblast are characteristic features of pre-eclampsia. We showed that potassium (K+) channels regulate trophoblast differentiation and fusion to form syncytia in vitro, processes involved in syncytiotrophoblast renewal1. In many tissues K+ channel expression/activity is modulated by pO2 and ROS. Here we test the hypothesis that ROS alter syncytiotrophoblast K+ channel activity in a pO2-dependent manner. Methods: Placental villous explants from normal pregnancy were cultured at 21%pO2 (hyperoxia; n¼10) and 6%pO2 (normoxia; n¼10) for 6 days. On days 35, explants were untreated (control) or treated with 5mM 4-aminopyridine (4-AP) or 5mM tetraethylammonium (TEA), blockers of pO2-sensitive voltagegated K+ channels (KV), 100QM flupirtine, blocker of KV7 pO2-insensitive K+ channels or H2O2 (10QM-1mM) to induce oxidative stress. 86Rb efflux from explants was measured on day 6 to estimate K+ channel activity. The %86Rb efflux/min, and the rate constant of cellular 86Rb, was determined over 16min. Results: Basal %86Rb efflux was 15% higher (p<0.0001) from explants cultured in 21%pO2 as compared to 6%pO2. 4-AP (-18%, p<0.001) and TEA (-25%, p<0.001) reduced 86Rb efflux from explants cultured at 21%pO2 but had no effect at 6%pO2. Flupirtine did not affect 86Rb efflux at either pO2. Compared to the corresponding control, H2O2 (100QM/1mM) increased the 86 Rb efflux rate constant in explants cultured at 6%pO2 but significantly reduced the rate constant in explants cultured at 21%pO2 (both p<0.05). Conclusion: pO2-sensitive KV channels are functionally expressed in syncytiotrophoblast and the effect of ROS on K+ channel activity is pO2dependent. Inhibition of K+ channels could contribute to dysregulated syncytiotrophoblast renewal when ROS and pO2 are elevated, conditions that are believed to prevail in pre-eclampsia2. 1 Díaz et al. PLoS One (2014) 9(3): e90961 2 Huppertz et al. J Reprod Immunol (2013) 101-102: 74-79
P2.113-N. EVALUATING THE SUCCESS RATE OF THE DUAL PERFUSION OF THE HUMAN PLACENTA EX VIVO Janine Hutson a, b, Reuven Kedar a, Howard Priya Bapat a, b, c, b a, b a The Hospital for Sick Children, Toronto, Berger , Gideon Koren Canada; b University of Toronto, Toronto, Canada; c St. Michael's Hospital, Toronto, Canada Objectives: Dual perfusion of a single placental lobule ex vivo is a useful model to study placental transfer of substances in organized human
Methods: Placentae were collected with informed consent following cesarean delivery of uncomplicated term pregnancies for a 3-hour perfusion of various test substances over a one-year period (March 2013 to March 2014). Upon completion of each experiment, placentae were assigned one of four checkpoints to determine the success rate at various stages of the experiment: Checkpoint 0: unable to set up fetal cannulation; Checkpoint 1: successful fetal cannulation and proceeded to isolate the cotyledon; Checkpoint 2: started perfusion with closed maternal and fetal circulations; Checkpoint 3: successful perfusion with final antipyrine F:M ratio above 0.75 and fetal volume loss less than 3 ml per hour. Results: 170 placentae were collected over a one-year period. 77 placentae (45%) did not have an adequate artery-vein pair for fetal cannulation or had several tears on the maternal side. A fetal artery-vein pair was successfully cannulated in 93 placentae (55%). After isolating the single cotyledon, the perfusion was started with closed maternal and fetal circulations in only 23 placentae (12.5%), and 12 (52%) of these perfusions were successful; corresponding to 7.1% of all placentae received. Conclusions: The results of this report show an overall success rate of 7.1% for the placenta perfusion model at the Motherisk laboratory, and suggest that the fetal cannulation may be a crucial stage in ensuring a successful perfusion.
P2.114-N. LOSS OF PLACENTAL SULPHATE TRANSPORTER SLC13A4 CAUSES SEVERE DEVELOPMENTAL DEFECTS AND EMBRYONIC LETHALITY Joanna Rakoczy a, b, Paul Dawson a, b, David Simmons a a The University of Queensland, St. Lucia, Queensland, Australia; b Mater Research Institute, Woolloongabba, Queensland, Australia Objectives: Sulphate is a critical nutrient for numerous cellular and metabolic processes important for foetal development. As the foetus has a minimal capacity to produce its own sulphate, it is dependent on the mother’s circulating sulphate supply, which is transported to the foetus via placental sulphate transporters. The sulphate 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 foetus itself, we chose to investigate its role in placental sulphate transport and foetal development. Methods: We created a mouse line containing an Slc13a4 “knockout first” allele (ES cells obtained from KOMP) to analyze the affects 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-/- foetuses exhibited a multitude of developmental phenotypes including impaired development of vascular and lymphatic vessels, and a complete loss of bone ossification. 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 foetus, indicating that the phenotypes observed in the global knockout can be attributed to a loss of placental Slc13a4 expression. Interestingly, the severe phenotype of Slc13a4-/- embryos encompass the phenotypes reported for individual
Abstracts / Placenta 35 (2014) A1eA112
mouse knockout models of impaired sulfonation of structural molecules important for foetal development. Conclusion: Our novel mouse model is providing valuable insight into the critical role of placental sulphate transporters in meeting foetal nutrient demands during gestation.
P2.115-N. PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE Benjamin P. Lüscher a, b, Michael Fine c, Camilla Marini a, Benjamin Clemençon c, Christiane Albrecht c, Matthias A. Hediger c, Daniel V. Surbek a, b, Marc U. Baumann a, b a Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; b Department of Clinical Research, University of Bern, Bern, Switzerland; c Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland Objectives: Materno-fetal transplacental transport is crucial for the fetal well-being. The altered expression of placental transport proteins under specific pathophysiological conditions may affect the intrauterine environment. Pre-eclampsia is often associated with high maternal uric acid serum levels. The regulation of the placental uric transport system and its transporter glucose transporter (GLUT)-9 are not fully understood yet. The aim of this study was to investigate the placental urate transport and to characterize its transporter GLUT9. Methods: In this study we used a transepithelial transport (Transwell®) model to assess uric acid transport activity. Electrophysiological techniques and radioactive ligand up-take assays were used to measure transport activity of GLUT9 expressed in Xenopus oocytes. Results: In the Transwell/ model uric acid is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min at the linear stage. We could successfully over-express GLUT9 using the Xenopus laevis oocytes expression system. Chloride modulates the urate transport system: interestingly replacing chloride with iodine resulted in a complete loss of urate transport activity. We determined the IC50 of iodine at 30uM concentration. In radioactive up-take experiments iodine had no effect on uric acid transport. Conclusions: In vitro the “materno-fetal” transport of uric acid is slow. This indicates that in vivo the child is protected from short-term fluctuations of maternal uric acid serum concentrations. The different results regarding iodine-mediated regulation of GLUT9 transport activity between electrophysiological and radioactive ligand uptake experiments may suggest that iodine does not directly inhibit uric acid transport, but changes the mode of up-take from an electrogenic to an electroneutral transport. GLUT9 is not an uric acid uniporter, there are more ions involved in the transport. This may allow regulating uric acid transport by the change from an active to a passive transport.
P2.116. UPTAKE MECHANISM OF AN ESTRIOL PRECURSOR, 16a-HYDROXY DEHYDROEPIANDROSTERONE SULFATE, AT THE BASAL PLASMA MEMBRANE OF HUMAN TERM PLACENTA Masatoshi Tomi a, Hiromi Eguchi a, Tomohiro Nishimura a, Tetsuo Maruyama b, Emi Nakashima a a Keio University Faculty of Pharmacy, Tokyo, Japan; b Keio University School of Medicine, Tokyo, Japan Objectives: Estriol is mostly secreted from placental syncytiotrophoblasts during pregnancy. 16a-Hydroxy dehydroepiandrosterone sulfate (16a-OH DHEAS) is an estriol precursor, which is synthesized by fetal liver-specific CYP3A7 (16a-hydroxylase). To achieve placental estriol synthesis, syncytiotrophoblasts require organic anion transporter(s) for 16a-OH DHEAS uptake at the basal plasma membrane (BM) facing fetal circulation. The purpose of this study is to clarify the mechanism underlying 16a-OH DHEAS uptake at the BM of human placental syncytiotrophoblasts. Methods: The uptake of [3H]16a-OH DHEAS was characterized using basal-enriched plasma membrane vesicles (BMVs) derived from human
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term placenta. The expression of transporters in BMVs was determined by western blot analysis. Results: Protein expressions of organic anion transporter (OAT) 4 (SLC22A11) and organic anion transporting polypeptide 2B1 (SLCO2B1) were enriched in human placental BMVs compared with crude membrane fractions, which is consistent with immunohistochemical observations of the basal localization of these transporters in syncytiotrophoblasts. Uptake of [3H]16a-OH DHEAS by BMVs was sodium- and chloride-independent and was trans-stimulated by preloaded glutarate. The uptake of [3H]16a-OH DHEAS was significantly cis-inhibited by DHEAS, estrone sulfate, and bromosulfophthalein, but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, and cimetidine. These transport characteristics of [3H]16a-OH DHEAS are in good agreement with those in human OAT4-transfected COS7 cells. Conclusion: OAT4 expressed at the basal plasma membrane of the syncytiotrophoblast appears to play a predominant role in the placental uptake of 16a-OH DHEAS from the fetal circulation and contribute to estriol synthesis during pregnancy.
P2.117. NANOPARTICLE INTERACTION WITH PLACENTAL BARRIER MATTERS?
EARLY
HUMAN
PLACENTA:
Liudmila Nikitina a, Gottfried Dohr a, Herbert Juch a, b a Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria; b Institute of Human Genetic, Medical University of Graz, Graz, Austria Objectives: Reproductive nanotoxicology is still in its infancy and almost no data exists on nanoparticle (NP) mediated effects on human reproduction. Due to the size of NPs, its trafficking is expected to depend more on the specific structure of the biological barriers. Recently the ability of silica NPs to cross the placental barrier, was shown in the placental perfusion model and in trophoblast cell monolayers. Due to the generally higher vulnerability to toxic agents at the embryonic stage of prenatal development (6-10 weeks of pregnancy) and the significant structural differences between early and term placenta we investigated NP exposure in 1st trimester human placental explants. Methods: Placental explants from early pregnancy and BeWo cells were cultivated and exposed to different types of NPs: 5nm crystalline, 30nm and 100 nm amorphous silica NPs (labeled) for 6, 24, 48h. Localisation of NPs was then evaluated by light, fluorescence and electron microscopy. Results: Cultivated BeWo cells demonstrated massive uptake of silica NPs with preferable localization in the perinuclear area independent on NP type, while in placental explants no crystalline silica NPs could be detected in the tissue.NP agglomerates were localized outside the syncytium. Single amorphous silica NPs were observed in cytoplasm of syncytium layer but we did not see a transfer of NPs to the cytotrophoblasts or the mesenchymal core of the placental villi. Conclusion: The placental barrier of early placenta seems to provide better protection against NPs than single trophoblast cells and term placenta probably due to its hemo dichorial nature. Therefore cell culture experiments using standard trophoblastic cell lines like BeWo might be misleading, when used to assess developmental toxicity of NP exposures.
P2.118-N. MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Michael Lüthi a, b, Edgar Ontsouka a, b, Marc Xiao Huang a, b, Baumann b, c, Daniel Surbek b, c, Christiane Albrecht a,b a Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland; b Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland; c Department of Obstetrics and Gynecology, University Hospital Bern, Bern, Switzerland Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionally
Abstracts / Placenta 35 (2014) A1eA112
placental tissue. While this model can be highly predictive, there are several challenges associated with it. The maternal and fetal cannulations can be technically challenging to set up, and leakage from the fetal reservoir can result in early termination of the experiment. The objective of this study was to evaluate the success rate at several checkpoints for the dual perfusion of the human placenta at the Motherisk laboratory.
Figure 2. Analytical dependence of the normalized optimal uptake Fmax / F0 on the ratio of characteristic transport times g ¼ ttr / tabs. For the stream tube model, F0 is defined as F0 ¼ c0upR2, where u is the maternal blood velocity, R is the radius of the stream tube and c0 is the concentration of oxygen at the entrance to the stream tube. Shaded regions show values expected in a normal pregnancy.
P2.112-N. EFFECTS OF PARTIAL PRESSURE OF OXYGEN ON THE MODULATION OF SYNCYTIOTROPHOBLAST POTASSIUM CHANNELS BY REACTIVE OXYGEN SPECIES Paula Díaz, Colin P. Sibley, Susan L. Greenwood Maternal and Fetal Health Research Centre, University of Manchester, Manchester, UK Objectives: Altered placental partial pressure of oxygen (pO2), elevated reactive oxygen species (ROS) and dysregulated renewal of syncytiotrophoblast are characteristic features of pre-eclampsia. We showed that potassium (K+) channels regulate trophoblast differentiation and fusion to form syncytia in vitro, processes involved in syncytiotrophoblast renewal1. In many tissues K+ channel expression/activity is modulated by pO2 and ROS. Here we test the hypothesis that ROS alter syncytiotrophoblast K+ channel activity in a pO2-dependent manner. Methods: Placental villous explants from normal pregnancy were cultured at 21%pO2 (hyperoxia; n¼10) and 6%pO2 (normoxia; n¼10) for 6 days. On days 35, explants were untreated (control) or treated with 5mM 4-aminopyridine (4-AP) or 5mM tetraethylammonium (TEA), blockers of pO2-sensitive voltagegated K+ channels (KV), 100QM flupirtine, blocker of KV7 pO2-insensitive K+ channels or H2O2 (10QM-1mM) to induce oxidative stress. 86Rb efflux from explants was measured on day 6 to estimate K+ channel activity. The %86Rb efflux/min, and the rate constant of cellular 86Rb, was determined over 16min. Results: Basal %86Rb efflux was 15% higher (p<0.0001) from explants cultured in 21%pO2 as compared to 6%pO2. 4-AP (-18%, p<0.001) and TEA (-25%, p<0.001) reduced 86Rb efflux from explants cultured at 21%pO2 but had no effect at 6%pO2. Flupirtine did not affect 86Rb efflux at either pO2. Compared to the corresponding control, H2O2 (100QM/1mM) increased the 86 Rb efflux rate constant in explants cultured at 6%pO2 but significantly reduced the rate constant in explants cultured at 21%pO2 (both p<0.05). Conclusion: pO2-sensitive KV channels are functionally expressed in syncytiotrophoblast and the effect of ROS on K+ channel activity is pO2dependent. Inhibition of K+ channels could contribute to dysregulated syncytiotrophoblast renewal when ROS and pO2 are elevated, conditions that are believed to prevail in pre-eclampsia2. 1 Díaz et al. PLoS One (2014) 9(3): e90961 2 Huppertz et al. J Reprod Immunol (2013) 101-102: 74-79
P2.113-N. EVALUATING THE SUCCESS RATE OF THE DUAL PERFUSION OF THE HUMAN PLACENTA EX VIVO Janine Hutson a, b, Reuven Kedar a, Howard Priya Bapat a, b, c, b a, b a The Hospital for Sick Children, Toronto, Berger , Gideon Koren Canada; b University of Toronto, Toronto, Canada; c St. Michael's Hospital, Toronto, Canada Objectives: Dual perfusion of a single placental lobule ex vivo is a useful model to study placental transfer of substances in organized human
Methods: Placentae were collected with informed consent following cesarean delivery of uncomplicated term pregnancies for a 3-hour perfusion of various test substances over a one-year period (March 2013 to March 2014). Upon completion of each experiment, placentae were assigned one of four checkpoints to determine the success rate at various stages of the experiment: Checkpoint 0: unable to set up fetal cannulation; Checkpoint 1: successful fetal cannulation and proceeded to isolate the cotyledon; Checkpoint 2: started perfusion with closed maternal and fetal circulations; Checkpoint 3: successful perfusion with final antipyrine F:M ratio above 0.75 and fetal volume loss less than 3 ml per hour. Results: 170 placentae were collected over a one-year period. 77 placentae (45%) did not have an adequate artery-vein pair for fetal cannulation or had several tears on the maternal side. A fetal artery-vein pair was successfully cannulated in 93 placentae (55%). After isolating the single cotyledon, the perfusion was started with closed maternal and fetal circulations in only 23 placentae (12.5%), and 12 (52%) of these perfusions were successful; corresponding to 7.1% of all placentae received. Conclusions: The results of this report show an overall success rate of 7.1% for the placenta perfusion model at the Motherisk laboratory, and suggest that the fetal cannulation may be a crucial stage in ensuring a successful perfusion.
P2.114-N. LOSS OF PLACENTAL SULPHATE TRANSPORTER SLC13A4 CAUSES SEVERE DEVELOPMENTAL DEFECTS AND EMBRYONIC LETHALITY Joanna Rakoczy a, b, Paul Dawson a, b, David Simmons a a The University of Queensland, St. Lucia, Queensland, Australia; b Mater Research Institute, Woolloongabba, Queensland, Australia Objectives: Sulphate is a critical nutrient for numerous cellular and metabolic processes important for foetal development. As the foetus has a minimal capacity to produce its own sulphate, it is dependent on the mother’s circulating sulphate supply, which is transported to the foetus via placental sulphate transporters. The sulphate 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 foetus itself, we chose to investigate its role in placental sulphate transport and foetal development. Methods: We created a mouse line containing an Slc13a4 “knockout first” allele (ES cells obtained from KOMP) to analyze the affects 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-/- foetuses exhibited a multitude of developmental phenotypes including impaired development of vascular and lymphatic vessels, and a complete loss of bone ossification. 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 foetus, indicating that the phenotypes observed in the global knockout can be attributed to a loss of placental Slc13a4 expression. Interestingly, the severe phenotype of Slc13a4-/- embryos encompass the phenotypes reported for individual
Abstracts / Placenta 35 (2014) A1eA112
mouse knockout models of impaired sulfonation of structural molecules important for foetal development. Conclusion: Our novel mouse model is providing valuable insight into the critical role of placental sulphate transporters in meeting foetal nutrient demands during gestation.
P2.115-N. PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE Benjamin P. Lüscher a, b, Michael Fine c, Camilla Marini a, Benjamin Clemençon c, Christiane Albrecht c, Matthias A. Hediger c, Daniel V. Surbek a, b, Marc U. Baumann a, b a Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; b Department of Clinical Research, University of Bern, Bern, Switzerland; c Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland Objectives: Materno-fetal transplacental transport is crucial for the fetal well-being. The altered expression of placental transport proteins under specific pathophysiological conditions may affect the intrauterine environment. Pre-eclampsia is often associated with high maternal uric acid serum levels. The regulation of the placental uric transport system and its transporter glucose transporter (GLUT)-9 are not fully understood yet. The aim of this study was to investigate the placental urate transport and to characterize its transporter GLUT9. Methods: In this study we used a transepithelial transport (Transwell®) model to assess uric acid transport activity. Electrophysiological techniques and radioactive ligand up-take assays were used to measure transport activity of GLUT9 expressed in Xenopus oocytes. Results: In the Transwell/ model uric acid is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min at the linear stage. We could successfully over-express GLUT9 using the Xenopus laevis oocytes expression system. Chloride modulates the urate transport system: interestingly replacing chloride with iodine resulted in a complete loss of urate transport activity. We determined the IC50 of iodine at 30uM concentration. In radioactive up-take experiments iodine had no effect on uric acid transport. Conclusions: In vitro the “materno-fetal” transport of uric acid is slow. This indicates that in vivo the child is protected from short-term fluctuations of maternal uric acid serum concentrations. The different results regarding iodine-mediated regulation of GLUT9 transport activity between electrophysiological and radioactive ligand uptake experiments may suggest that iodine does not directly inhibit uric acid transport, but changes the mode of up-take from an electrogenic to an electroneutral transport. GLUT9 is not an uric acid uniporter, there are more ions involved in the transport. This may allow regulating uric acid transport by the change from an active to a passive transport.
P2.116. UPTAKE MECHANISM OF AN ESTRIOL PRECURSOR, 16a-HYDROXY DEHYDROEPIANDROSTERONE SULFATE, AT THE BASAL PLASMA MEMBRANE OF HUMAN TERM PLACENTA Masatoshi Tomi a, Hiromi Eguchi a, Tomohiro Nishimura a, Tetsuo Maruyama b, Emi Nakashima a a Keio University Faculty of Pharmacy, Tokyo, Japan; b Keio University School of Medicine, Tokyo, Japan Objectives: Estriol is mostly secreted from placental syncytiotrophoblasts during pregnancy. 16a-Hydroxy dehydroepiandrosterone sulfate (16a-OH DHEAS) is an estriol precursor, which is synthesized by fetal liver-specific CYP3A7 (16a-hydroxylase). To achieve placental estriol synthesis, syncytiotrophoblasts require organic anion transporter(s) for 16a-OH DHEAS uptake at the basal plasma membrane (BM) facing fetal circulation. The purpose of this study is to clarify the mechanism underlying 16a-OH DHEAS uptake at the BM of human placental syncytiotrophoblasts. Methods: The uptake of [3H]16a-OH DHEAS was characterized using basal-enriched plasma membrane vesicles (BMVs) derived from human
A97
term placenta. The expression of transporters in BMVs was determined by western blot analysis. Results: Protein expressions of organic anion transporter (OAT) 4 (SLC22A11) and organic anion transporting polypeptide 2B1 (SLCO2B1) were enriched in human placental BMVs compared with crude membrane fractions, which is consistent with immunohistochemical observations of the basal localization of these transporters in syncytiotrophoblasts. Uptake of [3H]16a-OH DHEAS by BMVs was sodium- and chloride-independent and was trans-stimulated by preloaded glutarate. The uptake of [3H]16a-OH DHEAS was significantly cis-inhibited by DHEAS, estrone sulfate, and bromosulfophthalein, but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, and cimetidine. These transport characteristics of [3H]16a-OH DHEAS are in good agreement with those in human OAT4-transfected COS7 cells. Conclusion: OAT4 expressed at the basal plasma membrane of the syncytiotrophoblast appears to play a predominant role in the placental uptake of 16a-OH DHEAS from the fetal circulation and contribute to estriol synthesis during pregnancy.
P2.117. NANOPARTICLE INTERACTION WITH PLACENTAL BARRIER MATTERS?
EARLY
HUMAN
PLACENTA:
Liudmila Nikitina a, Gottfried Dohr a, Herbert Juch a, b a Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria; b Institute of Human Genetic, Medical University of Graz, Graz, Austria Objectives: Reproductive nanotoxicology is still in its infancy and almost no data exists on nanoparticle (NP) mediated effects on human reproduction. Due to the size of NPs, its trafficking is expected to depend more on the specific structure of the biological barriers. Recently the ability of silica NPs to cross the placental barrier, was shown in the placental perfusion model and in trophoblast cell monolayers. Due to the generally higher vulnerability to toxic agents at the embryonic stage of prenatal development (6-10 weeks of pregnancy) and the significant structural differences between early and term placenta we investigated NP exposure in 1st trimester human placental explants. Methods: Placental explants from early pregnancy and BeWo cells were cultivated and exposed to different types of NPs: 5nm crystalline, 30nm and 100 nm amorphous silica NPs (labeled) for 6, 24, 48h. Localisation of NPs was then evaluated by light, fluorescence and electron microscopy. Results: Cultivated BeWo cells demonstrated massive uptake of silica NPs with preferable localization in the perinuclear area independent on NP type, while in placental explants no crystalline silica NPs could be detected in the tissue.NP agglomerates were localized outside the syncytium. Single amorphous silica NPs were observed in cytoplasm of syncytium layer but we did not see a transfer of NPs to the cytotrophoblasts or the mesenchymal core of the placental villi. Conclusion: The placental barrier of early placenta seems to provide better protection against NPs than single trophoblast cells and term placenta probably due to its hemo dichorial nature. Therefore cell culture experiments using standard trophoblastic cell lines like BeWo might be misleading, when used to assess developmental toxicity of NP exposures.
P2.118-N. MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Michael Lüthi a, b, Edgar Ontsouka a, b, Marc Xiao Huang a, b, Baumann b, c, Daniel Surbek b, c, Christiane Albrecht a,b a Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland; b Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland; c Department of Obstetrics and Gynecology, University Hospital Bern, Bern, Switzerland Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionally