Multiomics analyses of vesicular transport pathway-specific transcripts and proteins in ovine amnion: responses to altered intramembranous transport.

Amniotic fluid volume (AFV) is determined by the rate of intramembranous (IM) transport of amniotic fluid (AF) across the amnion. This transport is regulated by fetal urine-derived stimulators and AF inhibitors. Our objective was to utilize a multiomics approach to determine the IM transport pathways and identify the regulators. Four groups of fetal sheep with experimentally induced alterations in IM transport rate were studied: control, urine drainage (UD), urine drainage with fluid replacement (UDR), and intra-amniotic fluid infusion (IA). Amnion, AF, and fetal urine were subjected to transcriptomics (RNA-Seq) and proteomics studies followed by Ingenuity Pathway Analysis. The analysis uncovered nine transport-associated pathways and four groups of differentially expressed transcripts and proteins. These can be categorized into mediators of vesicular uptake and endocytosis, intracellular trafficking, pathway activation and signaling, and energy metabolism. UD decreased IM transport rate and AFV in conjunction with enhanced expression of vesicular endocytosis regulators but reduced expression of intracellular trafficking mediators. With UDR, IM transport rate decreased and AFV increased. Energy metabolism activators increased while trafficking mediators decreased in expression. IA increased IM transport rate and AFV together with enhanced expressions of vesicular endocytosis and trafficking mediators. We conclude that IM transport across the amnion is regulated by multiple vesicular transcytotic and signaling pathways and that the mediators of intracellular trafficking most likely play an important role in determining the rate of IM transport. Furthermore, the motor protein cytoplasmic dynein light chain-1, which coexpressed in AF and fetal urine, may function as a urine-derived IM transport stimulator.

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 RARß and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARß 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, RARß 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 RARß during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

Regulation of amniotic fluid volume: insights derived from amniotic fluid volume function curves.

Recent advances in understanding the regulation of amniotic fluid volume (AFV) include that AFV is determined primarily by the rate of intramembranous absorption (IMA) of amniotic fluid across the amnion and into fetal blood. In turn, IMA rate is dependent on the concentrations of yet-to-be identified stimulator(s) and inhibitor(s) that are present in amniotic fluid. To put these concepts in perspective, this review 1) discusses the evolution of discoveries that form the current basis for understanding the regulation of AFV, 2) reviews the contribution of IMA to this regulation, and 3) interprets experimentally induced shifts in AFV function curves and amnioinfusion function curves in terms of the activity of the amniotic fluid stimulator and inhibitor of IMA. In the early 1980s, it was not known whether AFV was regulated. However, by the late 1980s, IMA was discovered to be a "missing link" in understanding the regulation of AFV. Over the next 25 years the concept of IMA evolved from being a passive process to being an active, unidirectional transport of amniotic fluid water and solutes by vesicles within the amnion. In the 2010s, it was demonstrated that a renally derived stimulator and a fetal membrane-derived inhibitor are present in amniotic fluid that regulate IMA rate and hence are the primary determinants of AFV. Furthermore, AFV function curves and amnioinfusion function curves provide new insights into the relative efficacy of the stimulator and inhibitor of IMA.

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 (VEGF164 and VEGF164b) 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 VEGF164 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.

Aquaporins in ovine amnion: responses to altered amniotic fluid volumes and intramembranous absorption rates.

Aquaporins (AQPs) are transmembrane channel proteins that facilitate rapid water movement across cell membranes. In amniotic membrane, the AQP-facilitated transfer of water across amnion cells has been proposed as a mechanism for amniotic fluid volume (AFV) regulation. To investigate whether AQPs modulate AFV by altering intramembranous absorption (IMA) rate, we tested the hypothesis that AQP gene expression in the amnion is positively correlated with IMA rate during experimental conditions when IMA rate and AFV are modified over a wide range. The relative abundances of AQP1, AQP3, AQP8, AQP9, and AQP11 mRNA and protein were determined in the amnion of 16 late-gestation ovine fetuses subjected to 2 days of control conditions, urine drainage, urine replacement, or intraamniotic fluid infusion. AQP mRNA levels were determined by RT-qPCR and proteins by western immunoblot. Under control conditions, mRNA levels among the five AQPs differed more than 20-fold. During experimental treatments, mean IMA rate in the experimental groups ranged from 100 ± 120 mL/day to 1370 ± 270 mL/day. The mRNA levels of the five AQPs did not change from control and were not correlated with IMA rates. The protein levels of AQP1 were positively correlated with IMA rates (r(2) = 38%, P = 0.01) while the remaining four AQPs were not. These findings demonstrate that five AQPs are differentially expressed in ovine amnion. Our study supports the hypothesis that AQP1 may play a positive role in regulating the rate of fluid transfer across the amnion, thereby participating in the dynamic regulation of AFV.

Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms.

Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport.

Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep.

We hypothesized that prostaglandin E2 (PGE2) stimulates amniotic fluid transport across the amnion by upregulating vascular endothelial growth factor (VEGF) expression in amnion cells and that amniotic PGE2 concentration correlates positively with intramembranous (IM) absorption rate in fetal sheep. The effects of PGE2 at a range of concentrations on VEGF 164 and caveolin-1 gene expressions were analyzed in cultured ovine amnion cells. IM absorption rate, amniotic fluid (AF) volume, and PGE2 concentration in AF were determined in late-gestation fetal sheep during control conditions, isovolumic fetal urine replacement (low IM absorption rate), or intra-amniotic fluid infusion (high IM absorption rate). In ovine amnion cells, PGE2 induced dose- and time-dependent increases in VEGF 164 mRNA levels and reduced caveolin-1 mRNA and protein levels. VEGF receptor blockade abolished the caveolin-1 response, while minimally affecting the VEGF response to PGE2. In sheep fetuses, urine replacement reduced amniotic PGE2 concentration by 58%, decreased IM absorption rate by half, and doubled AF volume (P < 0.01). Intra-amniotic fluid infusion increased IM absorption rate and AF volume (P < 0.01), while amniotic PGE2 concentration was unchanged. Neither IM absorption rate nor AF volume correlated with amniotic PGE2 concentration under each experimental condition. Although PGE2 at micromolar concentrations induced dose-dependent responses in VEGF and caveolin-1 gene expression in cultured amnion cells consistent with a role of PGE2 in activating VEGF to mediate AF transport across the amnion, amniotic PGE2 at physiological nanomolar concentrations does not appear to regulate IM absorption rate or AF volume.

Ovine fetal swallowing responses to polyhydramnios.

Abstract Swallowing of amniotic fluid by late gestation fetuses increases when amniotic fluid volume (AFV) is elevated. Our objectives were to quantitatively characterize fetal swallowing when AFV is elevated above normal to polyhydramniotic levels and to explore the mechanisms that mediate these changes. Late gestation fetal sheep were studied under basal conditions and during intra-amniotic infusion of lactated Ringer's solution. Control AFV averaged 631 ± 214 mL (SE, n = 6), swallowed volume was 299 ± 94 mL/day, and there were 5.7 ± 1.8 bouts/day of rapid swallowing. During intra-amniotic infusion, AFV (3065 ± 894 mL) and daily swallowed volume (699 ± 148 mL/day) increased (P < 0.05) and the number of bouts reached a maximum of 13.7 ± 2.0 bouts/day when AFV exceeded 1500 mL. Unexpectedly, the volume swallowed per bout (57.3 ± 5.8 mL, n = 102) did not vary with AFV (r = 0.023, P = 0.81). Neither the number of swallows/day nor the volume/swallow changed consistently with elevated AFV. Daily swallowed volume increases and reaches a maximum of twice normal as AFV approaches polyhydramniotic levels. Mechanistically, the increase in swallowing was achieved primarily by an increase in the number of bouts of swallowing per day rather than the expected passive increase in volume per bout. This implies changes in fetal behavior as AFV was elevated. Furthermore, swallowed volume was four times more sensitive to increases in AFV than reported previously.

Inhibitor of intramembranous absorption in ovine amniotic fluid.

Intramembranous absorption increases during intra-amniotic infusion of physiological saline solutions. The increase may be due partly to the concomitant elevation in fetal urine production as fetal urine contains a stimulator of intramembranous absorption. In this study, we hypothesized that the increase in intramembranous absorption during intra-amniotic infusion is due, in part, to dilution of a nonrenal inhibitor of intramembranous absorption that is present in amniotic fluid. In late-gestation fetal sheep, amniotic fluid volume and the four primary amniotic inflows and outflows were determined over 2-day intervals under three conditions: 1) control conditions when fetal urine entered the amniotic sac, 2) during intra-amniotic infusion of 2 l/day of lactated Ringer solution when urine entered the amniotic sac, and 3) during the same intra-amniotic infusion when fetal urine was continuously replaced with lactated Ringer solution. Amniotic fluid volume, fetal urine production, swallowed volume, and intramembranous absorption rate increased during the infusions independent of fetal urine entry into the amniotic sac or its replacement. Lung liquid secretion rate was unchanged during infusion. Because fetal membrane stretch has been shown not to be involved and because urine replacement did not alter the response, we conclude that the increase in intramembranous absorption that occurs during intra-amniotic infusions is due primarily to dilution of a nonrenal inhibitor of intramembranous absorption that is normally present in amniotic fluid. This result combined with our previous study suggests that a nonrenal inhibitor(s) together with a renal stimulator(s) interact to regulate intramembranous absorption rate and, hence, amniotic fluid volume.

Regulation of intramembranous absorption and amniotic fluid volume by constituents in fetal sheep urine.

Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine, and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2-day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer solution or lactated Ringer solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement, whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume.

Fetal swallowing as a protective mechanism against oligohydramnios and polyhydramnios in late gestation sheep.

UNLABELLED: Our objectives were to (1) quantify the relationship between daily swallowed volume and amniotic fluid volume (AF volume) in late gestation ovine fetuses and (2) use the resulting regression equation to explore the role of swallowing in regulating AF volume. Daily swallowed volume ranged from 36 to 1963 mL/d while experimental AF volume ranged from 160 to 6150 mL (n = 115). Swallowed volume was near zero when AF volume was far below normal, a maximum of 635 ± 41 (standard error) mL/d when AF volume was 1682 ± 31 mL and did not increase further with higher AF volumes. Computer simulations predicted that fetal swallowing would (1) return AF volume to normal in 5 to 6 days following an acute volume change in the absence of changes in other amniotic inflows or outflows and (2) stabilize AF volume in 4 to 8 days following sustained alterations in amniotic inflows or outflows other than swallowing. CONCLUSIONS: The volume of AF swallowed each day by the fetus is a strong function of AF volume and reaches a maximum when mild polyhydramnios develops. With deviations in AF volume from normal, changes in fetal swallowing protect against oligohydramnios and polyhydramnios because the changes in swallowing over time reduce the extent of the AF volume change. However, with experimental changes in AF volume stabilizing in 1 to 2 days, it appears that swallowing is not the major regulator of AF volume.

The placenta in the integrated physiology of fetal volume control.

Almost all water that enters the conceptus of the sheep enters via the placenta. The forces that drive water are hydrostatic and osmotic. The placental channels that allow water to cross into the fetus have not been identified by microanatomic means. Although an "equivalent pore" system can account for the diffusional entry of small hydrophilic solutes, it can be calculated that the filtration coefficient of this system is too small to account for the demonstrated trans-placental water flows. It is possible that a second much less numerous system of large pores permits the flow of water, but that is by no means certain. The placenta does not control the amount of water that enters the conceptus; nor does any other single fetal structure. And water entry is not dependent on the volume of water already present. However, the combined physiological properties of the fetal heart, kidneys, somatic tissues and placenta constitute a consistent explanation of fetal water volume control.

Intramembranous solute and water fluxes during high intramembranous absorption rates in fetal sheep with and without lung liquid diversion.

OBJECTIVE: To examine mechanisms that mediate increased intramembranous solute and water absorption. STUDY DESIGN: Intramembranous solute and water fluxes were measured in fetal sheep under basal conditions and after intraamniotic infusion of lactated Ringer's solution of 4 L/d for 3 days with and without lung liquid diversion. RESULTS: Intramembranous sodium, potassium, chloride, calcium, glucose, and lactate fluxes increased 2.5- to 7.9-fold, were linearly related to volume fluxes (r = 0.83-0.99), and were unaffected by lung liquid. All clearance rates, except that of lactate, increased to equal the intramembranous volume absorption rate during infusion. CONCLUSION: Under basal conditions, passive diffusion makes a minor and bulk flow a major contribution to intramembranous solute absorption. During high absorption rates, the increase in solute absorption above basal levels appears to be due entirely to bulk flow and is unaffected by lung liquid. The increased bulk flow is consistent with vesicular transcytosis.

Responses of amniotic fluid volume and its four major flows to lung liquid diversion and amniotic infusion in the ovine fetus.

We designed experiments to allow direct measurement of amniotic fluid volume and continuous measurement of lung liquid production, swallowing, and urine production in fetal sheep. From these values, the rate of intramembranous absorption was calculated. Using this experimental design, the contribution of lung liquid to the control of amniotic fluid volume was examined. Fetuses were assigned to 1 of 4 protocols, each protocol lasting 3 days: control, isovolemic replacement of lung liquid, supplementation of amniotic fluid inflow by 4 L/day, and supplementation of amniotic inflow during isovolemic replacement of lung liquid. We found no effect of lung liquid replacement on any of the known flows into and out of the amniotic fluid. Although intramembranous absorption increased greatly during supplementation, the amniochorionic function curves were not altered by isovolemic lung liquid replacement. We conclude that lung liquid does not appear to contain a significant regulatory substance for amniotic fluid volume control.

Sequential growth of fetal sheep cardiac myocytes in response to simultaneous arterial and venous hypertension.

While the fetal heart grows by myocyte enlargement and proliferation, myocytes lose their capacity for proliferation in the perinatal period after terminal differentiation. The relationship between myocyte enlargement, proliferation, and terminal differentiation has not been studied under conditions of combined arterial and venous hypertension, as occurs in some clinical conditions. We hypothesize that fetal arterial and venous hypertension initially leads to cardiomyocyte proliferation, followed by myocyte enlargement. Two groups of fetal sheep received intravascular plasma infusions for 4 or 8 days (from 130 days gestation) to increase vascular pressures. Fetal hearts were arrested in diastole and dissociated. Myocyte size, terminal differentiation (%binucleation), and cell cycle activity (Ki-67[+] cells as a % of mononucleated myocytes) were measured. We found that chronic plasma infusion greatly increased venous and arterial pressures. Heart (but not body) weights were approximately 30% greater in hypertensive fetuses than controls. The incidence of cell cycle activity doubled in hypertensive fetuses compared with controls. After 4 days of hypertension, myocytes were (approximately 11%) longer, but only after 8 days were they wider (approximately 12%). After 8 days, %binucleation was approximately 50% greater in hypertensive fetuses. We observed two phases of cardiomyocyte growth and maturation in response to fetal arterial and venous hypertension. In the early phase, the incidence of cell cycle activity increased and myocytes elongated. In the later phase, the incidence of cell cycle activity remained elevated, %binucleation increased, and cross sections were greater. This study highlights unique fetal adaptations of the myocardium and the importance of experimental duration when interpreting fetal cardiac growth data.

Effects of intravascular infusions of plasma on placental and systemic blood flow in fetal sheep.

Six singleton fetal sheep of 118-122 days gestational age were instrumented with flow sensors on the brachiocephalic artery, the postductal aorta, and the common umbilical artery and with arterial and venous intravascular catheters. At 125-131 days of gestation, we started week-long continuous recordings of flows and pressures. After control measures had been obtained, the fetuses were given continuous intravenous infusions of adult sheep plasma at an initial rate of 229 ml/day. After 1 wk of infusion, fetal plasma protein concentrations had increased from 34 to 78 g/l, arterial and venous pressures had increased from 42 to 64 and from 2.7 to 3.7 mmHg, and systemic resistance (exclusive of the coronary bed) had increased from 0.047 to 0.075 mmHg.min(-1).ml(-1), whereas placental resistance had increased from 0.065 to 0.111 mmHg.min(-1).ml(-1). Fetal plasma renin activities fell as early as 1 day after the start of infusion and remained below control (all changes P < 0.05). All flows decreased slightly although these decreases were not statistically significant. Thus the increase in arterial pressure was entirely due to an increase in systemic and placental resistances.

Intravascular infusions of plasma into fetal sheep cause arterial and venous hypertension.

Fetal volume control is driven by an equilibrium between fetal and maternal hydrostatic and oncotic pressures in the placenta. Renal contributions to blood volume regulation are minor because the fetal kidneys cannot excrete fluid from the fetal compartment. We hypothesized that an increase in fetal plasma protein would lead to an increase in plasma oncotic pressure, resulting in an increase in fetal arterial and venous pressures and decreased angiotensin levels. Plasma or lactated Ringer solution was infused into each of five twin fetuses. After 7 days, fetal protein concentration was 71.2 +/- 4.2 g/l in the plasma-infused fetuses compared with 35.7 +/- 6.3 g/l in the lactated Ringer-solution-infused fetuses. Arterial pressure was 68.0 +/- 3.6 compared with 43.4 +/- 1.9 mmHg in the lactated Ringer solution-infused fetuses (P < 0.0003), whereas venous pressure was 4.8 +/- 0.3 mmHg in the plasma-infused fetuses compared with 3.3 +/- 0.4 mmHg in the lactated Ringer solution-infused fetuses (P < 0.036). Six fetuses were studied on days 0, 7, and 14 of plasma protein infusion. Fetal protein concentration increased from 31.1 +/- 1.5 to 84.8 +/- 3.8 g/l after 14 days (P < 0.01), and arterial pressure increased from 43.1 +/- 1.8 to 69.1 +/- 4.1 mmHg (P < 0.01). Venous pressure increased from 3.0 +/- 0.4 to 6.2 +/- 1.3 mmHg (P < 0.05). Fetal heart rate did not change. Angiotensin II concentration decreased, from 24.6 +/- 5.6 to 2.9 +/- 1.3 pg/l, after 14 days (P < 0.01). Fetal plasma infusions resulted in fetal arterial and venous hypertensions that could not be corrected by reductions in angiotensin II levels.

Absorption of amniotic fluid by amniochorion in sheep.

Swallowing of amniotic fluid and lung fluid inflow were eliminated in 10 chronically instrumented fetuses. The urachus was ligated, and fetal was urine drained to the outside. At the beginning and the end of 21 experiments of 66 +/- 5 (SE) h duration, all amniotic fluid was temporarily drained to the outside for volume measurement and sampling. Amniotic fluid osmolalities and oncotic pressures were experimentally controlled. Amniochorionic absorption of amniotic fluid depended strongly on the osmolality difference between amniotic fluid and fetal plasma (P < 0.001), but at zero osmolality difference there still was a mean absorption rate of 23.8 +/- 4.7 (SE) ml/h (P < 0.001). Absorption was unaffected by the protein concentration difference between amniotic fluid and fetal plasma, but infused bovine albumin in the amniotic fluid was absorbed at a rate of 1.8 8 +/- 0.4 g/h (P < 0.001), corresponding to a volume flow of fluid of 33.8 8 +/- 6.1 ml/h (P < 0.001). Fluid absorption in the amniochorion is driven in part by crystalloid osmotic pressure, but about 25 ml/h is absorbed by a path that is permeable to protein. That path has the physiological characteristics of lymphatic drainage, although no anatomic basis is known to exist for a lymphatic system in the amniochorion.