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.     

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。     

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.     

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.     

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.     

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 of Aquaporin 1 and Aquaporin 3 in Fetal Membranes and Placenta in Human Term Pregnancies with Oligohydramnios

ObjectiveTo explore the pathophysiology of oligohydramnios, the association between the expression of aquaporin 1 and aquaporin 3 in fetal membranes and placenta and oligohydramnios was investigated.MethodsSixty patients underwent elective cesarean sections at term were studied, 30 patients with isolated oligohydramnios and the other 30 with normal amniotic fluid volume (AFV). Real-time polymerase chain reaction and immunohistochemistry were employed to determine expression and localization of aquaporin 1 and aquaporin 3 in amnion, chorion and placenta, respectively.ResultsThe expression of aquaporin 1 and aquaporin 3 was detected in amnion, chorion and placenta using real-time RT-PCR. By immunohistochemistry, aquaporin 1 and aquaporin 3 protein expressions in amnion epithelia and chorion cytotrophoblasts were identified. In placenta, aquaporin 1 was detected in placental vessels, while aquaporin 3 was found in trophoblast cells. In comparison to normal AFV group, there was a significant decrease of aquaporin 1 expression in amnion in oligohydramnios group, but no significant difference in chorion and placenta between the two groups. The expression of the aquaporin 3 in amnion and chorion in oligohydramnios group was significantly decreased, while expression in placenta was significantly increased compared with that in normal AFV group.ConclusionsAlteration of aquaporin 1 and aquaporin 3 expression in fetal membranes and placenta may be important in the pathophysiology of isolated oligohydramnios.     

Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.     

Reciprocal expression of peroxisome proliferator-activated receptor-gamma and cyclooxygenase-2 in human term parturition

OBJECTIVE: The objective of this study was to test the hypothesis that peroxisome proliferator activated receptor-gamma (PPAR-gamma) is expressed in intrauterine tissues before active term human parturition, and that its repression is associated with up-regulation of cyclooxygenase-2 (COX-2). STUDY DESIGN: Specimens were collected from women with term singleton pregnancies after spontaneous labor or cesarean section before labor, prepared for immunoblot and immunohistochemical analysis, and probed for PPAR-gamma or COX-2. RESULTS: PPAR-gamma expression was prominent in fetal membranes and placenta before active labor. After labor, PPAR-gamma expression was significantly reduced in fetal membranes, but not in placenta. The ratio of COX-2:PPAR-gamma was significantly elevated in fetal membranes with labor. PPAR-gamma immunostaining was prominent in syncytiotrophoblast, extravillous cytotrophoblasts, and cells of the amnion and chorion. COX-2 immunostaining was abundant in the amnion and rare in the placenta. CONCLUSION: PPAR-gamma is highly expressed in term intrauterine tissues. In fetal membranes, PPAR-gamma levels are reduced once active labor commences, coincidental with a relative increase in COX-2 expression.     

Vascular endothelial growth factor activation of intramembranous absorption: a critical pathway for amniotic fluid volume regulation

OBJECTIVE: The purpose of this review is to propose a critical role for vascular endothelial growth factor (VEGF) in mediating the transfer of amniotic fluid from the amniotic compartment through the fetal membranes and fetal surface of the placenta into fetal blood. METHODS: Experimental findings in humans and animal models on the action of VEGF in mediating fluid transfer are reviewed and interpreted in order to postulate a proposed mechanism for VEGF regulation of amniotic fluid absorption through the fetal membranes and placenta. RESULTS: Recent scientific advances suggest that up-regulation of VEGF gene expression in the amnion and chorion is associated with increased transfer of amniotic fluid into fetal blood. The possible mechanisms of action for VEGF appear to involve regulation of intramembranous blood vessel proliferation and membrane transport via passive permeation as well as nonpassive transcytotic vesicular movement of fluid. CONCLUSION: Currently evolving concepts suggest that amniotic fluid volume is regulated through modulation of the rate of intramembranous absorption of amniotic fluid by both passive and nonpassive mechanisms. The permeability factor VEGF appears to be a critical regulator of amniotic fluid transport in the fetal membranes.     

原发性羊水过多产妇胎膜和胎盘组织中水通道蛋白8的表达

目的 探讨水通道蛋白8(AQP8)在原发性羊水过多产妇胎膜和胎盘组织中的表达.方法 收集2005年10月-2007年5月重庆医科大学附属第一医院和重庆市妇幼保健院行足月剖宫产分娩(孕37～40周)的原发性羊水过多产妇12例为羊水过多组,同期因社会因素行剖宫产分娩的正常产妇12例为对照组.采用RT-PCR技术检测两组产妇胎膜和胎盘组织中的AQP8 mRNA表达水平;采用免疫组化技术检测两组产妇胎膜和胎盘组织中的AQP8蛋白表达水平.结果 (1)羊水过多组羊膜、绒毛膜和胎盘组织中的AQP8 mRNA表达水平分别为0.78±0.13、0.58±0.10、0.86±0.15,对照组分别为0.39±0.07、0.45±0.09、0.34±0.09,羊水过多组各组织中AQPS mRNA表达水平均高于对照组,两组分别比较,差异均有统计学意义(P<0.05).(2)羊水过多组羊膜、绒毛膜和胎盘组织中的AQP8蛋白表达水平分别为0.195±0.024、0.170±0.028、0.193±0.024,对照组分别为0.151±0.018、0.156±0.024、0.152±0.023,其中羊水过多组羊膜、胎盘组织中AQP8蛋白表达水平均高于对照组,差异有统计学意义(P<0.05),而羊水过多组绒毛膜组织中AQP8蛋白表达水平虽高于对照组,但两组比较,差异无统计学意义(P>0.05).结论 原发性羊水过多产妇羊膜和胎盘组织中AQP8mRNA及蛋白的表达水平均显著高于正常产妇,提示AQP8在产妇羊水量的调节中发挥重要作用.     

Regulation of amniotic fluid volume by intramembranous absorption in sheep: role of passive permeability and vascular endothelial growth factor

OBJECTIVE: During long-term intravascular fluid infusion in the ovine fetus, a large increase in fetal urinary flow rate occurs while amniotic fluid volume increases only slightly because of increased intramembranous absorption. The current study tested the hypotheses that passive intramembranous permeability increases in response to fetal intravascular saline solution infusion and that the increased intramembranous absorption occurs in parallel with an increase in vascular endothelial growth factor gene expression in the amnion, chorion, and placenta. STUDY DESIGN: Chronically catheterized fetal sheep that average 126 +/- 1 (SE) days of gestation either were infused intravascularly with 7 L of normal saline solution over 3 days (n = 8 sheep) or served as time controls (n = 6 sheep). Amniotic fluid volume and fetal urinary flow rate were measured daily. Intramembranous diffusional permeability was estimated daily as being equal to the clearance of intra-amniotically injected technetium 99m. Vascular endothelial growth factor messenger RNA abundance in the amnion, chorion, and placenta was determined by Northern blot analysis. Statistical analyses included analysis of variance. RESULTS: In the infused fetuses, amniotic fluid volume and urinary flow increased (P <.01) by 891 +/- 144 mL and 3488 +/- 487 mL per day, respectively, on infusion day 3 compared with no changes over time in the control fetuses. In the infused fetuses, estimated intramembranous absorption increased by 4276 +/- 499 mL during the 3-day infusion. Intramembranous technetium 99m permeability was similar over time in the two groups. In the infused group, vascular endothelial growth factor messenger RNA levels in the amnion, chorion, and placenta increased 2- to 4-fold compared with the control group (P <.001). CONCLUSION: The up-regulation of vascular endothelial growth gene expression may mediate the increase in the intramembranous absorption that is induced by volume-loading diuresis; however, this does not occur by passive mechanisms. We speculate that vascular endothelial growth mediates the increased intramembranous absorption by increasing vesicular transport.     

水通道蛋白3、9在特发性羊水过多产妇胎盘和胎膜中的表达变化及意义

目的 探讨水通道蛋白(AQP)3、9在特发性羊水过多产妇胎盘和胎膜中的表达、分布及其在特发性羊水过多发病巾的作用.方法 2006年6月至2008年3月,选择在温州医学院附属第二医院产科住院并分娩的21例特发性羊水过多的足月产妇为研究组,同期分娩的30例羊水量正常的足月产妇为对照组.采用实时荧光定量PCR技术,检测两组产妇胎盘、胎膜中AQP3、9 mRNA的表达及分布,采用免疫组化链霉菌抗生物素蛋白.过氧化物酶(SP)连接法枪测两组产妇胎盘、胎膜中AQP3、9蛋白的表达及分布.结果 (1)两组产妇羊膜、绒毛膜、胎盘中均可检测到AQP3、9 mRNA的表达.AQP3、9主要表达于羊膜上皮细胞、绒毛膜滋养细胞、胎盘滋养细胞.(2)研究组羊膜上皮细胞AQP3、9 mRNA的表达分别为对照组的5.00倍和3.25倍,而研究组绒毛膜滋养细胞AQP3、9 mRNA的表达分别为对照组的2.03倍和2.08倍,分别比较,差异均有统计学意义(P<0.01);但是研究组胎盘滋养细胞AQP3、9 mRNA的表达均明显低于对照组,差异也有统计学意义(P<0.01).(3)AQP3、9蛋白在研究组羊膜上皮细胞的表达强度为7.5±2.0、11.1±1.8,对照组为5.3 ±1.6、5.6±2.3,两组比较,差异有统计学意义(P<0.05);AQP3、9蛋白在研究组绒毛膜滋养细胞的表达强度为7.5±2.0、10.0±1.6,对照组为5.4±2.2、5.6±2.1,分别比较,差异也有统计学意义(P<0.05),但是AQP3、9蛋白在胎盘滋养细胞的表达强度(3.5±1.4、4.0 ±2.5)较对照组(5.6±1.3、7.1±2.9)明显下调(P<0.05).结论 AQP3、9在特发性羊水过多产妇胎盘和胎膜中的表达变化可能为羊水增加后的代偿性反应,其调节机制尚待进一步研究.     

Changes in expression of the cyclooxygenase gene in human fetal membranes and placenta with labor.

OBJECTIVE: Our objective was to examine the expression of the gene coding for cyclooxygenase, the central enzyme in prostaglandin synthesis, in human placenta and fetal membranes during pregnancy and before and after labor at term. STUDY DESIGN: Expression of the gene for cyclooxygenase was examined with Northern hybridization to ribonucleic acid from human placenta throughout pregnancy and human amnion and chorion decidua in the late third trimester. RESULTS: Expression was undetectable in trophoblast during the first and second trimesters. Expression in amnion and trophoblast increased 3.5- and 2.5-fold, respectively, in association with labor. CONCLUSIONS: Our results suggest that the increase in prostaglandin synthesis within the uterus that is seen with the onset of labor is associated with an increase in the expression of the gene cyclooxygenase.     

Localization of the Fas-Fas ligand system in human fetal membranes

OBJECTIVE: To determine if fetal membranes might be one of the sources of Fas and Fas ligand in amniotic fluid. STUDY DESIGN: Human fetal membranes from elective cesarean section (n = 6) were fixed in paraformaldehyde. Rolls of paraffinembedded fetal membranes were cut into 5-micron sections. After blocking with horse and goat sera, sections were incubated overnight with primary antibodies followed by the appropriate secondary antibodies. Avidin-biotin complex and diaminobenzidine were used for immunoperoxidase localization. Expression of Fas and Fas ligand was read by light microscopy. RESULTS: Both Fas and Fas ligand were localized in amnion, chorion and decidual layers. In amnion, Fas and Fas ligand were expressed predominantly in epithelial cells and fibroblasts, while there was no immunostaining in the subepithelial compact connective tissue. In the chorion, the expression was mainly in the chorionic trophoblast, with inconsistent expression in the reticular layer. In the decidua, the expression of Fas and Fas ligand was less prominent than in amnion and chorion. CONCLUSION: Localization of Fas and Fas ligand in human fetal membranes suggests that fetal membranes could be one of the sources of soluble Fas and Fas ligand in amniotic fluid.