Ovine Fetal Membranes Research Articles

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion.

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARβ and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARβ messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARβ mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARβ during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

Transport-associated pathway responses in ovine fetal membranes to changes in amniotic fluid dynamics.

Current evidence suggests that amniotic fluid volume (AFV) is actively regulated by vesicular transport of amniotic fluid outward across the amnion and into the underlying fetal vasculature in the placenta. Our objective was to determine whether gene expression profiles of potential stimulators, inhibitors, and mediators of vesicular transport are altered in response to changes in intramembranous absorption (IMA) rate. Samples of ovine amnion and chorion were obtained from fetal sheep with normal, experimentally reduced or increased AFVs and IMA rates. Amnion and chorion levels of target mRNAs were determined by RT‐qPCR. In the amnion, caveolin‐1 and flotillin‐1 mRNA levels were unchanged during alterations in IMA rate. However, levels of both were significantly higher in amnion than in chorion. Tubulin‐α mRNA levels in the amnion but not in chorion were reduced when IMA rate decreased, and amnion levels correlated positively with IMA rate (P < 0.05). Dynamin‐2 mRNA levels were not altered by experimental conditions. Vascular endothelial growth factor (VEGF 164 and VEGF 164b) mRNA levels increased during both increases and decreases in IMA rate, whereas soluble Flt‐1 levels did not change. Neither HIF‐1α nor PBEF mRNA levels in the amnion were correlated with VEGF 164 expression levels and were not related to IMA rate. Collectively, our findings suggest that changes in amnion microtubule expression may be important in the regulation of transcellular vesicular transport of amniotic fluid and thus modulate IMA rate. Further, our results are consistent with the concept that the amnion is the rate‐limiting layer for amniotic fluid transport.

Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes

Intrauterine infection and inflammation are responsible for the majority of early (<32 weeks) spontaneous preterm births (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (n=5). Using a Transwell model, they were stimulated ex vivo with γ-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE2 and cytokines (human interleukin 6 (IL6), IL10 and TNFα; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20 h). In human membranes, the IKKβ inhibitor TPCA-1 (7 μM) and p38 MAPK inhibitor SB239063 (20 μM) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE2 in the fetal compartment. NAC (10 mM) inhibited accumulation of PGE2 and IL10 only; NBDI (10 μM) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB.

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.

Cloning and cellular expression of aquaporin 9 in ovine fetal membranes

Amniotic fluid (AF) absorption across fetal membranes is essential for AF volume homeostasis, balancing fetal swallowing, urine flow, and lung liquid production. In sheep, AF is absorbed primarily across the amniotic membrane into fetal vasculature situated between the amnion and chorion. Aquaporins (AQPs) are cell membrane proteins that serve as water channels. Recent studies have demonstrated the expression of AQP 1, 3, 8, and 9 in human chorioamniotic membranes and placenta. As AF dynamics continued to be explored primarily in the ovine model, we sought to clone and characterize the expression of ovine AQP9 in fetal membranes. Ovine AQP9 gene was cloned with the use of homology reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR and Northern analysis were used to determine AQP9 gene expression, and immunohistochemistry (IHC) used to localize AQP9 protein expression in ovine fetal membranes. A 2085-base pair (bp) full-length complementary DNA (cDNA) sequence of ovine AQP9 was cloned. The ovine AQP9 cDNA is 86%, 82%, and 82%, and the predicted amino acid sequence (295 amino acids) is 77%, 71%, and 69% identical to human, rat, and mouse AQP9, respectively. RT-PCR and Northern analysis detected AQP9 messenger RNA expression in ovine amnion and allantois, but not in placenta, chorion, or umbilical cord. Immunohistochemistry localized AQP9 protein in epithelia of amnion and allantois. The presence of significant AQP9 messenger RNA and protein expression in ovine fetal membranes suggests that AQP9 may be a major water channel for intramembranous AF resorption in sheep. The cloning of ovine AQP9 and the demonstration of AQP9 expression in amnion and allantois significantly enhances our understanding of ovine AF regulation and offers the potential for therapeutic approaches for the treatment of oligohydramnios and polyhydramnios.

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 factor164 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. (Am J Obstet Gynecol 2002;186:303-10.)

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.

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 of125I-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 of125I-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.

The Transfer of Prostaglandin E2 Across Ovine Fetal Membranes In Vivo

This study investigated whether prostaglandin E2 (PGE2) can cross intact fetal membranes in vivo. We have developed a novel ovine model using in vivo dialysis systems, positioned between the fetal membranes and decidua and in the amniotic cavity. Sheep were given 25 microCi, 50 ng [3H]PGE2 or 1 mg PGE2 to the amniotic cavity, and radioactivity or PGE2/PGEM concentrations in dialysates were determined. Endogenous basal PGE2 concentrations were significantly higher in extra-amniotic than in intra-amniotic dialysates (715 +/- 436 versus 80 +/- 31 pg/mL; mean +/- standard error of the mean; P < .05). After injection of [3H]PGE2 into the amniotic cavity, no radioactivity was detected in the extraamniotic dialysate, and this was not influenced by labor induced by ACTH administration to the fetus. In contrast, intra-amniotic injection of 1 mg PGE2 resulted in marked transfer of PGE2 across the fetal membranes to the extra-amniotic dialysis system. These results suggest that PGE2 can cross the ovine fetal membranes in vivo, escaping metabolism in the chorion.