Expression of aquaporin 9 in human chorioamniotic membranes and placenta

OBJECTIVE: Aquaporin 9 (AQP9) is one of the recently identified water channels that is also permeable to neutral solutes including urea. To investigate the molecular mechanism of intramembranous pathway of amniotic fluid regulation, we sought to determine whether AQP9 is expressed, and the cellular localization of AQP9 expression in human fetal membranes. STUDY DESIGN: Fetal membranes from 5 normal term human pregnancies were studied. Northern analysis was used to determine the tissue AQP9 messenger RNA (mRNA) expression. In situ hybridization and immunohistochemical staining with specific anti-AQP9 antibody was used for cellular AQP9 localization in the human fetal membranes. RESULTS: Northern analysis detected AQP9 mRNA expression in human amnion, chorion, and placenta. In situ hybridization revealed AQP9 mRNA expression in epithelial cells of the amnion, chorion cytotrophoblasts, and syncytiotrophoblasts and cytotrophoblasts of placenta. Further immunohistochemical study confirmed the AQP9 protein expression in these cell types of fetal membranes. CONCLUSION: This study demonstrated the expression of AQP9 mRNA and protein in human chorioamniotic membranes and placenta. The AQP9 expression in fetal membranes suggests that AQP9 may be an important water channel in intramembranous amniotic fluid water regulation.     

水通道蛋白9在人胎盘和胎膜中的表达

目的 检测正常人胎盘与胎膜中水通道蛋白9（aquaporin 9，AOP9）的表达。方法 收集5例足月剖宫分娩的胎盘和胎膜样本，运用RT—PCR方法从mRNA水平检测AQP9在胎盘与胎膜的表达；运用免疫组织化学和Western印迹方法检测AQP9蛋白在胎盘与胎膜中的表达。结果 RT—PCR结果显示AQP9mRNA在胎盘和胎膜均有表达；Western印迹显示两条带在相对分子量为30kD及45kD左右；免疫组织化学显示AQP9表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜滋养细胞。结论 AQP9在母胎液体交换、胎儿代谢物的排出及羊水平衡等的分子机制中可能发挥重要作用。     

Quantitative comparison of placental expression of three aquaporin genes

Three water channel proteins, aquaporins, have been shown to be expressed in the placentae of humans and sheep-AQP1, 3, 8; AQP1 is in the vasculature, whereas AQP3, 8 are in the trophoblast cells. In this study we used the sensitive and reproducible technique of real-time PCR to compare the level of expression of the mRNAs for AQP1, 3 and 8 in the ovine placenta at five stages of gestation (27, 45, 66, 100 and 140 days-where term approximately 150 days). AQP3 was quantitatively the most highly expressed AQP at 66, 100, and 140 days). At 27 days before significant trophoblast development had occurred, the only AQP present was AQP1, in the vasculature. The expression of these aquaporins underlie the high water and urea permeability of the ovine placenta in the last half of pregnancy.     

Expression of Erythropoietin mRNA, Protein and Receptor in Ovine Fetal Membranes

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid.     

Role of aquaporin 1 in fetal fluid homeostasis

Abstract

Introduction: The homeostasis of maternal-fetal fluid exchange is critically important in pregnancy. We sought to investigate the function of aquaporin 1 (AQP1) during pregnancy by examining fluid compartments of AQP1 knockout (AQP1-KO) mice.

Methods: Homozygous pregnant AQP1 knockout (KO) mice and control pregnant wild type CD1 mice were sacrificed. Placenta and fetal membranes were examined by immunohistochemical staining for expression of AQP1. The total number of embryos, atrophic embryos, and fetal and placental weight was recorded in each subgroup. Amniotic fluid amount in each sac was measured and amniotic fluid composition was determined. Analysis of variance of factorial design and one-factor analysis of variance were used for statistics.

Results: Immunohistochemistry performed on CD1, though not AQP1-KO, placenta revealed that AQP1 was expressed at the vascular endothelial cell, trophocyte, and amnion epithelial cell. In AQP1-KOs, the number of embryos decreased with advancing gestational age. Although the fetal weight of AQP1-KO mice was significantly lower than wild type, amniotic fluid amount was increased in AQP1-KO mice. The AQP1-KO placenta demonstrated increased degeneration with evidence of altered blood vessel structure and increased syncytiotrophoblast nodules.

Conclusions: These findings demonstrate a critical role of AQP1 in placental and fetal growth and maternal-fetal fluid homeostasis.     

Inducing proliferation of human amnion epithelial and mesenchymal cells for prospective engineering of membrane repair

OBJECTIVE: To prepare a tissue engineering approach to fetal membrane repair after premature rupture of the membranes (PROM) by characterizing the proliferation potential of human amnion epithelial and mesenchymal cells from preterm and term placenta in primary culture. METHODS: Amnion epithelial and mesenchymal cells from 15 preterm (23-36 week) and 27 term placentas collected at cesarean section were separated enzymatically, characterized immunohistochemically (anti-cytokeratin 18 and anti-E-cadherin, and anti-vimentin, respectively), and their ratio determined. Proliferation on tissue culture polystyrene (TCPS) or collagen in one medium and on TCPS in four different media after 14 days was measured photometrically and compared in preterm vs. term placenta. For statistical analysis the Mann-Whitney test was used. RESULTS: Preterm and term epithelial:mesenchymal cell ratios were 4.3:1 and 7.8:1. Term epithelial cells proliferated similarly on TCPS or collagen. Mesenchymal cells proliferated only with fetal bovine serum (FBS). Proliferation of term amnion cells in medium containing FBS, epithelial growth factor (EGF), insulin, transferrin and triidothyronine(T3) was significantly increased (p < 0.001) compared with the other three media, and percentage proliferation was slightly higher in preterm cells. CONCLUSION: Characterization of human amnion epithelial and mesenchymal cells identified the most potent proliferation-inducing medium yet. Studies of the wound-healing potential of these cells are needed, examining their behavior and proliferation on fibrin microbeads and other extracellular matrixes as the next step towards engineering membrane repair in PROM.     

Developmental expression of vascular endothelial growth factor (VEGF) receptors and VEGF binding in ovine placenta and fetal membranes

The receptor tyrosine kinases, kinase-insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), and their ligand vascular endothelial growth factor (VEGF) are essential for the development and maintenance of placental vascular function during pregnancy. To further understand the role of VEGF in mediating angiogenesis and vascular permeability during development, the cellular localization of KDR and Flt-1 mRNA and protein, and the distribution of(125)I-VEGF binding sites in placenta, chorion and amnion of ovine fetuses were examined at three different gestational ages. In placentae at 62, 103 and 142 days, the predominant site of KDR mRNA and protein, and VEGF binding was the maternal vascular endothelium. In addition, a specific, although weak, signal for KDR mRNA was found in the maternal epithelium. At 103 and 142 days but not 62 days gestation, KDR mRNA and protein as well as VEGF binding sites were abundantly present in the endothelium of villous blood vessels. In the fetal membranes at 62, 103 and 142 days gestation, KDR mRNA and protein were expressed in the amniotic epithelium and intramembranous blood vessel endothelium, where binding of(125)I-VEGF was strong. There was no KDR mRNA or VEGF binding in the chorionic cytotrophoblast. Flt-1 expression was not detectable in placentae or fetal membranes at the three ages studied.In summary, the results demonstrated that VEGF receptors are present in the maternal and fetal vasculatures of the ovine placenta. This expression is consistent with a capillary growth-promoting function of KDR and its ligand VEGF. Further, the presence of KDR and VEGF binding sites in ovine fetal membranes suggests a role for VEGF in promoting intramembranous vascularity and permeability throughout gestation.     

水通道蛋白1和水通道蛋白3在羊水过少孕妇胎盘和胎膜的表达及其意义

目的 研究水通道蛋白1(AQP1)和AQP3在羊水过少孕妇胎盘、胎膜中的表达及分布,探讨其在羊水平衡途径中的作用. 方法 选取30例孤立性羊水过少的足月孕妇为研究组,同期分娩的30例正常羊水量的足月孕妇为对照组.分别采用实时荧光定量PCR和免疫组织化学SP法,检测两组胎盘、胎膜组织中AQP1和AQP3 mRNA和蛋白的表达及分布. 结果 AQP1mRNA在研究组羊膜组织中的表达为对照组的0.31,AQP1蛋白在研究组羊膜组织中的表达为0.14±0.02,较对照组的0.25±0.03明显下调(P<0.05).而在胎盘、绒毛膜中的分布及表达强度两组间差异无统计学意义(P>0.05).AQP3 mRNA在研究组羊膜和绒毛膜中的表达分别为对照组的0.31和0.37,而胎盘中的表达为对照组的7.36.AQP3蛋白在研究组羊膜和绒毛膜中的表达分别为0.18±0.05和0.18±0.04,均较对照组的0.26±0.03和0.29±0.06明显下调(P<0.05),而在胎盘组织中的表达研究组为0.47±0.09,较对照组0.28±0.01明显上调(P<0.05). 结论 AQP1和AQP3在羊水过少孕妇胎盘、胎膜组织中表达的改变为羊水减少后的代偿性反应.     

Effect of labor on glucocorticoid receptor (GR(Total), GRalpha,and GRbeta) proteins in ovine intrauterine tissues

OBJECTIVE: Activation of the fetal hypothalamic-pituitary-adrenal axis and a concurrent increase in plasma cortisol concentration plays a crucial role in parturition in sheep. The effects of cortisol on intrauterine tissues are mediated, in part, by glucocorticoid receptors (GR). In the present study, we determined the temporal and tissue-specific expression of GR(Total), GRalpha, and GRbeta isoforms in ovine placenta, fetal membranes, and uterus during the onset and progression of labor. METHODS: Intrauterine tissues were collected from following three groups of pregnant ewes at term (147-149 days of gestation): animals not in labor (NIL; 140-145 days; n = 5), early labor (EL; 143-149 days; n = 6), and active labor (L; 145-149 days; n = 6). They were analyzed for GR(Total), GRalpha, and GRbeta by immunohistochemical and Western blot analyses. RESULTS: GR(Total), GRalpha, and GRbeta proteins were present in trophoblast cells but not maternal tissue of the placentome. GRalpha and beta were distinctly expressed in trophoblast nuclei and cytosol, respectively. GR(Total), GRalpha, and GRbeta were also detected in amnion epithelial cells and chorionic epithelium of the fetal membranes, and the maternal endometrial epithelium. GR(Total) and GRalpha were also present in amnion mesenchymal and glandular epithelium. GR(Total) and GRalpha (95 kDa) protein levels were significantly (P < .05) higher during labor; GRbeta (90 kDa) levels remained unchanged. CONCLUSION: The temporal and tissue-specific pattern of GR expression within ovine intrauterine tissues during labor implicates GR in regulating cortisol action to induce enzymatic changes controlling labor in sheep.     

Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta

OBJECTIVE: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. STUDY DESIGN: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. RESULTS: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor(164) being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. CONCLUSION: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes.     

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.     

Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.     

Placental and membrane aquaporin water channels: correlation with amniotic fluid volume and composition

OBJECTIVES: To assess the role of aquaporins (AQPs) in the regulation of amniotic fluid (AF) volume, we determined AF volume and composition and placental and fetal membrane AQP expression throughout the second half of murine gestation. METHODS: Pregnant CD1 mice were sacrificed at e10-19 and AF volume and composition determined. Placenta and fetal membranes were screened for AQP gene expression. AQP gene expression was quantified by real-time RT PCR and protein location determined by immunohistochemistry. Changes in AF volume were correlated with AQP expression. RESULTS: Both membranes and placenta demonstrated expression of AQP1, -3, -8 and -9. Advancing gestation was associated with increased AF volume from e10 to e16, with a marked decrease in AF volume from e16 to e19. By immunohistochemistry, AQP1 was localized to placental vessels and AQP3 to trophoblast. AF volume was negatively correlated with fetal membrane AQP1 and placental AQP1 and AQP9 expression, and positively correlated with placental AQP3 expression. CONCLUSION: Changes in AQPs with advancing gestation, and their correlation with AF volume, suggest a role in mediating placental and membrane water flow and ultimately AF volume. AQP1 appears to regulate fetal membrane water flow, and AQP3 is a likely candidate for the regulation of placental water flow.     

水通道蛋白-1和血管内皮生长因子的表达与羊水量的关系

目的:研究水通道蛋白-1(AQP1)、血管内皮生长因子(VEGF)与羊水量的关系。方法:应用免疫组织化学SP法分别检测20例羊水过少、22例羊水正常、17例羊水过多的胎盘、胎膜组织中AQP1、VEGF的表达,并对其表达强度行半定量分析。同时每组随机抽取10例用Western blot分别检测胎盘、胎膜组织中AQP1、VEGF蛋白表达量。结果:免疫组化显示AQP1、VEGF在3组胎盘、胎膜中均有阳性表达,胎膜中随着羊水量增加,其表达量相应上调,且具有显著性差异(P<0.05),胎盘中AQP1、VEGF表达量均无显著差异。Western blot检测蛋白的表达也有相同结果。且胎膜上AQP1和VEGF表达强度呈正相关。结论:AQP1和VEGF均与羊水量异常有关,AQP1和VEGF上调可能促进了羊水的跨膜转运。     

Review: Water channel proteins in the human placenta and fetal membranes

It has been established that the permeability of the human placenta increases with advancing gestation. Indirect evidence has also proposed that aquaporins (AQPs) may be involved in the regulation of placental water flow but the mechanisms are poorly understood. Five AQPs have been found in the human placenta and fetal membranes [AQP1, 3, 4, 8 and 9]. However, the physiological function(s) and the regulation of these proteins remain unknown. Emerging evidence has shown that human fetal membrane AQPs may have a role in intramembranous amniotic fluid water regulation and that alterations in their expression are related to polyhydramnios and oligohydramnios. In addition, we have observed a high expression of AQP3 and AQP9 in the apical membrane of the syncytiotrophoblast. Moreover, AQP9 was found to be increased in preeclamptic placentas, but it could not be related to its functionality for the transport of water and mannitol. However, a significant urea flux was seen. Since preeclampsia is not known to be associated with an altered water flux to the fetus we propose that AQP9 might not have a key role in water transport in human placenta, but a function in the energy metabolism or the urea uptake and elimination across the placenta. However, the role of AQP9 in human placenta is still speculative and needs further studies. Insulin, hCG, cAMP and CFTR have been found to be involved in the regulation of the molecular and functional expression of AQPs. Further insights into these mechanisms may clarify how water moves between the mother and the fetus.     

Hypoxia modulation of caveolin-1 and vascular endothelial growth factor in ovine fetal membranes

During normal pregnancy, amniotic fluid is absorbed from the amniotic compartment into fetal blood through the intramembranous blood vessels in the fetal membranes. It has been hypothesized that this transport process is mediated by transcytosis of caveolae-like vesicles. Because fetal hypoxia increases intramembranous absorption, the authors explore the effects of hypoxia on the gene expression of caveolin-1, a structural protein of caveolae, in ovine fetal membranes and cultured amnion cells. Near-term ovine fetuses were rendered hypoxic for 4 days. Caveolin-1 mRNA and protein levels were significantly reduced in the amnion and chorion but not in the placenta. In cultured ovine amnion cells incubated in 2% oxygen for 24 hours, hypoxia did not significantly alter caveolin-1 mRNA or protein expression. Vascular endothelial growth factor mRNA levels were increased in response to hypoxia in the fetal membranes as well as in cultured amnion cells. The results indicate that hypoxia does not augment but instead down-regulates or has no effect on caveolin-1 gene expression in the amnion and chorion, suggesting that caveolin-1 may play a role as a negative regulator of amnion transport function under hypoxic conditions.     

Expression of aquaporin water channels in canine fetal adnexa in respect to the regulation of amniotic fluid production and absorption

Amniotic fluid (AF) is created by the flow of fluid from the fetal lung and bladder and reabsorbed in part by fetal swallowing and partly by the transfer across the amnion to the fetal circulation. Placental water flux is an important factor in determining AF volume and fetal hydration. In addition the fetal membranes might be involved in the regulation of fluid composition. To understand the mechanisms responsible for maintaining a correct balance of AF volume we evaluated the expression of aquaporins (AQPs) in canine fetal adnexa. AQPs are a family of integral membrane proteins permitting passive but physiologically rapid transcellular water movement. The presence of AQP1, 3, 5, 8 and -9 was immunohistochemically assessed in canine fetal adnexa, collected in early, middle and late-gestation during ovario-hysterectomies performed with fully informed owners' consent. Changes in AF volume and biochemical composition were also evaluated throughout pregnancy. Our results show distinct aquaporin expression patterns in maternal and extraembryonic tissues in relation to pregnancy period. AQP1 was localized in placental endothelia, allantochorion, amnion, allantois and yolk sac. AQP3 was present in the placental labyrinth, amnion, allantois and yolk sac. AQP8 was especially evident on the epithelia lining the glandular chambers, the amniotic and allantois sacs. AQP9, a channel highly permeable to water and urea, was observed in epithelia of amnion, allantois and yolk sac. In summary, AQP1, 3, 5, 8 and -9 have distinct expression patterns in canine fetal membranes and placenta in relation to pregnancy period, suggesting an involvement in mediating the AF changes during gestation.