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.     

Leptin Alters Adrenal Responsiveness by Decreasing Expression of ACTH-R, StAR, and P450c21 in Hypoxemic Fetal Sheep

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO(2) ～15?mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96?hours. An ACTH challenge was performed at 72?hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96?hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH(1-24) challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.     

Ontogenesis of beta-adrenergic receptors in the ovine placenta

Radioligands with high specific activity and high affinity have recently become available to study the beta-adrenergic receptor (BAR). BAR may be important in the placenta for mediating a variety of metabolic and hemodynamic effects of catecholamines including placental hormone synthesis and secretion, placental glycogenolysis, and placental blood flow. Little is known regarding the development of the BAR. We have used the tritiated radioligand dihydroalprenolol to study the ontogenesis of BAR in the fetal and maternal portions of the ovine placenta. The receptor number decreased from 415 fmoles . mg-1 of protein at 120 days' gestation to 226 fmoles . mg-1 at 145 days; this trend was highly significant (r = -0.822, p less than 0.01). Similar changes were noted in the dissociation constant. Downward regulation as a result of fetal neurosympathetic maturation is proposed as an explanation for this observation.     

Ontogeny of imidazoline binding sites in the human placenta

INTRODUCTION: Non-adrenergic imidazoline binding sites (IBS) were described as pharmacologically distinct from alpha2-adrenergic receptors. Recently it was shown that the human placenta is the richest source of IBS, however, no function has been assigned to this new putative receptor. As concerns the presence of alpha2-adrenoceptors in the human placenta, it was reported that no alpha2-receptors were detected in human placental membranes with the radiolabelled alpha2-adrenoceptor antagonist [3H]rauwolscine or the alpha2-adrenoceptor agonist [3H]clonidine. This scientific contradiction has been solved when the authors have recently demonstrated that IBS and alpha2-adrenoceptors coexist in human term placental membranes. STUDY OBJECTIVE: Scientific literature does not provide any information regarding the ontogeny of IBS. The present study intended to determine the density of IBS and alpha2-adrenoceptors in correlation with gestational age. MATERIALS AND METHODS: Human first and second trimester placentas (6-10 and 14-18 weeks of gestation, respectively) were obtained immediately following the interruption of gestation, third trimester placentas (38-40 weeks) were obtained after normal vaginal delivery. Human placental membrane fractions were prepared and radioligand binding assays were performed in duplicate, using [3H]RX 821002 and [3H]RX 781094 (idazoxan) as radioligands. RESULTS: According to the results of the binding assays, the concentration of alpha2-adrenoceptors decreased with the advancing gestational age. In contrast with this pattern, the density of IBS significantly increased. CONCLUSION: Our present results demonstrated that the density of IBS shows a significantly increasing tendency throughout gestation. The unique position of the placenta between maternal and fetal circulations determines its function as a mediator in transport mechanisms. The increasing expression of IBS in the growing placenta might suggest a role for these sites in the mediation of the transport of nutrients, ions, etc.     

Ontogeny of amino acid transport system A in rat placenta

Amino acid transport System A has previously been demonstrated in apical membranes derived from rat placenta, as well as in apical and basal membranes derived from human placenta. We have studied Na⁺-dependent α-(methylamino)isobutyric acid (MeAIB) transport in apical and basal predominant membrane fractions prepared from 14 and 20 day gestation rat placenta. Marker enzyme recoveries did not differ significantly between age groups. Markers for intracellular organelles were also found to be comparable. Na⁺-dependent MeAIB transport was not sensitive to freezing and could be found in all membrane components tested. Kinetic parameters were studied-K_m=852 ± 215 μm, V_max=718 ± 126 pmol/5 sec/mg protein-20 day apical; K_m=748 ± 269 μm, V_max=610 ± 176 pmol/5 sec/mg protein—20 day basal-predominant; K_m614 ± 261 μm, V_max=123 ± 45 pmol/5 sec/mg protein—14 day apical. Kinetic parameters could not be determined in the 14 day gestation basal-predominant fraction because of the small amount of uptake present. We conclude that System A like activity is found in both apical and basal predominant membrane fractions derived from rat placenta, and that this activity increases over the last one third of gestation.     

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.     

Ontogeny and distribution of cells expressing HLA-B locus-specific determinants in the placenta and extraplacental membranes

Both the trophectoderm and the inner cell mass of the blastocyst contribute to the cell populations found in the placenta and extraplacental membranes. Previous studies have shown differences between those two embryologically distinct populations of cells in their expression of class I HLA, and further differences among trophectoderm-derived trophoblast cell subpopulations. Binding patterns for antibodies to both monomorphic and allotypic determinants on class I heavy chains have been reported. In the present study, extraembryonic cells were evaluated by immunohistology for binding of the monoclonal antibody 4E, which identifies locus-specific determinants on HLA-B. Some inner cell mass-derived cells (mesenchymal cells) acquired high levels of HLA-B as gestation progressed and other continued to express low levels at late stages of gestation (amnion cells). In contrast, throughout gestation both villous and extravillous trophoblast cells failed to express detectable HLA-B. The binding patterns for 4E followed the patterns established by a monoclonal antibody to class I HLA heavy chains (61D2), and those reported for antisera to allotypic determinants. The findings support the suggestion that trophoblast cells forming the fetal component of the maternal-fetal interface exert highly effective regulation over the expression of class I HLA.     

Morphologic alterations in ovine placenta and fetal liver following induced severe placental insufficiency

OBJECTIVES: Umbilical-placental embolization with microspheres has been used as a model of placental insufficiency and intrauterine growth restriction (IUGR). However, the effects of embolization on placental structure and organ morphology of the resulting IUGR fetus are relatively unexplored. In this study using ovine fetuses, we determined the location and distribution of microspheres within the placenta and explored the extent of placental and fetal organ morphologic changes induced by placental embolization. We hypothesized that microspheres administered into the umbilical circulation over 4 days would cause placental damage without significant morphologic alterations in fetal kidney or liver. METHODS: Eleven pregnant sheep at 118 +/- 1 (SE) days' gestation were studied. In six fetuses, embolization was induced by injections of 15-microm diameter microspheres on 4 successive days into the fetal descending aorta proximal to the umbilical arteries. Five fetuses served as time controls. RESULTS: In embolized fetuses, microspheres were detected in the placenta embedded in the fetal cytotrophoblastic layer or maternal parenchyma adjacent to villous cytotrophoblasts. Fetal cytotrophoblasts appeared normal except for loss of distinct separation between fetal and maternal cell layers. Microspheres were also detected in the fetal membranes within capillaries. The body weights of embolized fetuses were lower than controls, as were the body weight-normalized liver but not kidney weights. In the liver of the embolized fetuses, the number of hematopoietic cell clusters was markedly reduced, whereas the fetal kidneys appeared normal. CONCLUSIONS: We conclude that after 4 days of umbilical-placental embolization, microspheres were concentrated at the fetal villi proximal to the apical maternal-fetal interface and in the fetal membranes. There were noticeable morphologic changes in the embolized placentas, with no apparent gross damage to the placenta. The reduction in fetal liver weight and liver extramedullary hematopoietic cell abundance associated with embolization may predispose the fetus to alterations in liver function that could persist after birth.     

Comparison of leucine, serine and glycine transport across the ovine placenta

To estimate the transport rate of maternal glycine across the placenta [1-¹³C]glycine and L-[1-¹³]serine were infused intravenously in pregnant sheep using both continuous and bolus infusions. Each tracer was infused together with L-[1-¹³C]leucine, to enable a comparison with the placental transport of an essential amino acid. At steady state, fetal plasma leucine enrichment was 40 per cent of maternal enrichment, indicating that approximately 60 per cent of the entry rate of leucine into fetal plasma is derived from protein breakdown in the placenta and fetus. Fetal plasma glycine enrichment was 11 per cent of maternal and there was no detectable fetal serine enrichment. The direct flux of maternal leucine into the fetal circulation was approximately 3.0 (bolus experiments) to 3.6 (continuous infusion experiments) pmol/min (kg fetus) and greater than the estimated 1.4 μmol/min (kg fetus) direct flux of maternal glycine, despite the fact that the net umbilical uptake of glycine exceeds that of leucine. This supports the conclusion that placental glycine production is a quantitatively important contribution to fetal glycine uptake via the umbilical circulation. The fetal glycine supply from the placenta is provided by a relatively small direct maternal glycine transplacental flux and a larger contribution derived from serine utilization within the placenta for glycine production.     

Technetium Tc 99m rapidly crosses the ovine placenta and intramembranous pathway

OBJECTIVE: This study examined the movement of the soluble ion technetium Tc 99m across the ovine placenta and intramembranous pathway. STUDY DESIGN: Nineteen fetal sheep at 131 ± 1 (SE) days' gestation were studied. After a 1-hour control period technetium Tc 99m was injected into either a fetal vein (n = 7), the amniotic cavity (n = 5), or a maternal vein (n = 5). Maternal and fetal blood, fetal urine, and amniotic and allantoic fluid were sampled during the control period and for 8 hours after the injection. Fetal urine was drained externally throughout the experiment. In five animals technetium Tc 99m was injected intraamniotically after the fetus was killed with air emboli and sampled as described. RESULTS: Intrafetally injected technetium Tc 99m rapidly crossed the placenta; then it entered and was concentrated in the amniotic cavity. Intraamniotically injected technetium Tc 99m rapidly entered into the fetal circulation. The maternally injected technetium Tc 99m rapidly crossed the placenta into the fetus, suggesting a half-time for placental exchange of <50 minutes. The technetium Tc 99m injected into the dead fetus group demonstrated significantly less maternal absorption than in the live fetus group. CONCLUSIONS: The soluble ion technetium Tc 99m demonstrated a much more rapid movement in both directions across the ovine placenta then previously demonstrated for the smaller ion sodium. Technetium Tc 99m rapidly crossed the intramembranous pathway bidirectionally, suggesting a high permeability of the intramembranous pathway. Minimal maternal absorption of technetium Tc 99m in the dead fetus group suggests little transmembranous absorption by the mother. (Am J Obstet Gynecol 1996;175:1557-62.)     

Characterization of the down-regulated genes identified in preeclampsia placenta

Objectives: We focus our attention on placental genes that are repressed in preeclampsia. Methods: A search was conducted through the online PubMed database. Results: A majority of the down-regulated genes appear to be evolved as the defense and protective mechanism for the mother, namely maternal fitness genes. A half of the down-regulated genes are also located within and in close proximity to known imprinted genes. Conclusion: Preeclampsia may be associated with either: (1) down-regulated expression of genes located in close proximity to known imprinted genes, (2) an imbalance in the maternal-fetal genetic response, or (3) an interaction of the two.     

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.