Prostaglandin biosynthesis stimulatory and inhibitory substances in human amniotic fluid during pregnancy and labor

Human amniotic fluid has been separated into two fractions; one fraction inhibits prostaglandin biosynthesis and the other fraction is stimulatory. The activity of the stimulatory fraction increased with increasing gestational age and was greater still during labor. The activity of the inhibitory fraction decreased with increasing gestational age and was smaller still during labor. We speculate that these changes may play a significant role in parturition.     

Changes of Enzymes Involved in Prostaglandin Metabolism and Prostaglandin Binding Proteins in Rat Brain During Development and Aging

In the developing rat brain, the enzymatic formation of prostaglandin D2 from prostaglandin H2 increased 60-fold from day 12 of gestation to birth. The activity still rose gradually to the highest level (90 nmol/min/g wet tissue) at day 7 after birth. The activities of prostaglandin E2 and F2 alpha synthetases in rat brain were highest at gestational age 19 days (30 nmol/min/g wet tissue), respectively. The specific activity of NADP-dependent 15-hydroxy-prostaglandin D2 dehydrogenase in rat brain was highest at the earliest gestational age we examined (day 12 of gestation). The specific bindings of prostaglandin D2 and E2 to the crude mitochondrial fraction of rat brain were observed from day 16 of gestation and increased to day 7 after birth. Although the activities of the enzymes responsible for prostaglandin metabolism were unchanged postmaturationally, the maximal concentrations of the binding sites on the synaptic membrane for both prostaglandins D2 and E2 decreased with constant affinity to less than one-sixth with age from 1 week to 24 months after birth. These results indicate that prostaglandins may play important roles during maturation and aging in rat brain.     

Microsomal 20 alpha-hydroxysteroid dehydrogenase activity for progesterone in human placenta

Placental 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity was studied in order to evaluate the mechanism of continuation of pregnancy and initiation of labor. The placentas obtained at various gestational weeks were homogenized and fractionated into "nuclear", "mitochondrial", "microsomal" and "supernatant" fractions. Each fraction was incubated with 14C-progesterone and a hydrogen donor. Enzymatic activity was measured by the conversion of progesterone to 20 alpha-dihydroprogesterone. The highest activity of 20 alpha-HSD for progesterone was found to be localized in "microsomal" fraction. The Km constant of 20 alpha-HSD was 4.5 X 10(-6)M for progesterone in "microsomal" fraction. It was found that placental microsomal 20 alpha-HSD required NADPH as well as NADH. 20 alpha-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20 alpha-HSD activity for progesterone significantly, suggesting that the steroid produced by the feto-placental unit may be involved in the metabolism of progesterone in human placenta.     

Changes in Interleukin 1 Levels in Human Amniotic Fluid with Gestational Ages and Delivery

In order to understand the role of interleukin 1 (IL-1) in pregnancy, the amount of IL-1 in normal human amniotic fluid (AF) from various gestational ages and delivery was measured using an ELISA. AF samples were divided into three groups of varying gestational ages. Group 1 of AF was collected by amniocentesis from gestational ages <24 weeks (n = 13). Group 2 was collected transvaginally during delivery following labor ≥36 weeks (n = 36). Group 3 was transabdominally collected from elective cesarean section without labor ≥36 weeks (n ?8). IL-1α was present in AF of early gestational age, 19.2±21.7 pg/ml, in group 1, and appeared to increase with gestational age, 63.4±50.1 pg/ml, in group 3. In contrast, IL-1β was not detectable in either group 1 or 3. However, the concentration of IL-1 in group 2 was extremely high (IL-lα, 233.1±351.9 pg/ml; IL-1β, 1,093.5±1,369.7 pg/ml) compared to the other groups. Moreover, these concentrations tended to increase with the duration of labor. IL-1α and IL-1β concentrations in AF were intimately related. These findings suggest that IL-1 has some roles during pregnancy and especially during labor.     

Expression and localization of the contractile prostaglandin F receptor in pregnant rat myometrium in late gestation, labor, and postpartum

A polyclonal antibody was raised against amino acids 7-18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to alpha-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.     

Premature labor and indomethacin

Administration of indomethacin to 29 women in the 26th–37th week of gestation with premature labor contractions was followed in 26 by a significant decrease of uterine activity. The effect of therapy was monitored by serial external tocometry recordings and by plasma concentrations of estriol, h.P.L., alpha-fetoprotein, and estriol/creatinine ratio in urine. The labor was monitored by cardiotocography; the newborn infants were examined by the pediatrician. Following oral indomethacin treatment (25 mg every 6 Hours for 5 days) no untoward effects was observed on maternal and fetal wellbeing during pregnancy and labor. Four out 29 newborn infants, all appropriate for gestational age of 36–40 weeks, had one-minute Apgar scores less than 7, cyanosis, tachypnea, hypoxemia and serious oxygen dependency for several days. All the infant survived. These abnormalities may be due to short-term exposure to indomethacin and consequent inhibition of cyclo-oxygenase, impairement of prostaglandin-dependent physiological regulation of vessel tone during fetal life and circulatory disorders at birth.     

Premature labor and indomethacin.

Administration of indomethacin to 29 women in the 26th--37th week of gestation with premature labor contractions was followed in 26 by a significant decrease of uterine activity. The effect of therapy was monitored by serial external tocometry recordings and by plasma concentrations of estriol, h.P.L., alpha-fetoprotein, and estriol/creatinine ratio in urine. The labor was monitored by cardiotocography; the new born infants were examined by the pediatrician. Following oral indomethacin treatment (25 mg every 6 Hours for 5 days) no untoward effects was observed on maternal and fetal wellbeing during pregnancy and labor. Four out 29 newborn infants, all appropriate for gestational age of 36--40 weeks, had one-minute Apgar scores less than 7, cyanosis, tachypnea, hypoxemia and serious oxygen dependency for several days. All the infant survived. These abnormalities may be due to short-term exposure to indomethacin and consequent inhibition of cyclo-oxygenase, impairement of prostaglandin-dependent physiological regulation of vessel tone during fetal life and circulatory disorders at birth.     

Effects of gestational age and labor on the expression of prostanoid receptor genes in pregnant baboon cervix

We sought to determine whether expression of genes encoding prostaglandin receptors varied with advancing gestational age and in association with the onset of spontaneous labor in the cervix of pregnant baboons. We performed cesarean hysterectomy on 14 pregnant baboons, five during spontaneous labor. Expression of genes was quantified by Northern analysis. Clear signals which were similar in estimated size to the human genes were detected by Northern analysis for the genes encoding the EP1, EP2, EP3, EP4, FP, IP and TP receptors. Expression of the gene encoding the prostanoid EP1 receptor increased with advancing gestational age prior to labor (r2 = 0.8, P = 0.007). There was a 4 fold lower level of expression of the EP2 receptor gene among animals in labor compared with animals not in labor (P = 0.006) and approximately 2-fold lower levels of expression of the FP and TP receptor genes (P < 0.0001 and P = 0.0002, respectively). We conclude that variation in the relative expression of prostanoid receptor types and sub-types may have a role in cervical dilatation in primate parturition.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human fetal membranes and decidua vera

We have previously reported that platelet-activating factor (PAF) is present in human amniotic fluid obtained from women in labor. We have also demonstrated that PAF, lyso-PAF, and alkyl acyl-sn-glycero-3-phosphocholine (AA-GPC) are present in human amnion tissue. In the reported study, we have investigated the enzymes involved in PAF metabolism in amnion tissue and their regulation. A phospholipase A2 activity has been demonstrated in amnion tissue which cleaves alkyl acyl (long-chain) sn-glycero-3-phosphocholine. The enzyme activity is not altered by Ca2+ and is distinctly different from the phospholipase A2 that we have previously characterized in this tissue. Amnion tissue contains acetyltransferase activity which requires Ca2+ and is associated with the microsomal fraction. Acetylhydrolase is also present in the cytosolic fraction of amnion tissue. Acetylhydrolase activity has also been demonstrated in amniotic fluid. The affinities of acetyltransferase (for lyso-PAF) and acetylhydrolase (for PAF) were unaffected by Ca2+. In the presence of Ca2+, however, the specific activity of acetyltransferase was increased four- to fivefold while that of acetylhydrolase was unaffected. Acetyltransferase and acetylhydrolase activities in fetal membranes and decidua were similar and were unchanged with gestational age. The possible role of PAF in the initiation of human parturition is discussed.     

Proteolytic enzymes in human fetal membranes and amniotic fluid. A comparison of normal and premature ruptured membranes

The amniotic and chorionic membranes obtained at term and term amniotic fluid contain a soluble protease activity which cleaves [14C]-labeled globin at acid pH. In contrast, a salt extract of the pellet fraction obtained from the fetal membranes displays only negligible protease activities at the pH range of 4-8. Specific activities of the proteases in the soluble and salt-extractable fractions of fetal membranes which were intact before onset of labor were not significantly different from the respective activities in cases of premature rupture of fetal membranes (PROM). However, the protease activity of the amniotic fluid was found to increase with advancing gestational age and to reach maximal activity at term. A heat-sensitive and nondializable protease inhibitory activity was found in term amniotic fluid. This inhibitory activity acted on the cytosolic protease of amniotic membranes from control and PROM cases, but not on the soluble protease of chorionic membranes, and had a similar potency in fluids from PROM cases or fluids collected at term. These results do not support a role for fetal membrane proteases, amniotic fluid proteases, or amniotic fluid protease inhibitory activities in the etiology of PROM. However, the observed changes in amniotic fluid protease activity with fetal age suggest a physiological role for the enzyme in normal fetal development.     

Plasma and erythrocyte selenium and glutathione peroxidase activity in preterm infants at risk for bronchopulmonary dysplasia

The purpose of this study was to examine the relationships between selenium status, as measured by plasma and erythrocyte selenium and glutathione peroxidase (GPx) activity, and other postnatal factors, including selenium intake, gestational age, and oxygen dependence in preterm infants at risk for bronchopulmonary dysplsia. Eighteen preterm infants of 30 wk gestational age or less were included. At postnatal wk 1 and 4, selenium concentrations and GPx activity were measured and oxygen dependence and daily selenium intakes were determined from the medical chart. Plasma and erythrocyte selenium concentrations decreased from wk 1 to wk 4, whereas erythrocyte GPx activity increased. Increased selenium intakes during wk 1 were associated with increased erythrocyte GPx activity at both time-points, as well as a decreased need for supplemental oxygen on d 28. Preterm infants display increasing erythrocyte GPx activity despite declines in plasma and erythrocyte selenium. GPx activity might be enhanced by very early selenium supplementation.     

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.     

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.     

DNA and DNA-polymerase activity in chicken brain regions during ontogeny

Abstract—

• 1 The DNA content of cerebral hemispheres, optic lobes, cerebellum and remainder was determined in chicken brains from the 11th day of embryonic life to 6 weeks after hatch. Each region showed a characteristic pattern of variation during development. The cerebellum showed the most rapid and the optic lobes the least rapid rate of DNA increase during the period studied. The concentration of DNA within these regions decreased continuously with age except for that of the cerebellum which passed through a maximum just before hatching.
• 2 The nature of the DNA-polymerase activity in soluble extracts from these brain regions seemed to be similar to the properties reported for this enzyme activity in other vertebrate tissues. Glycerol was stimulatory and denatured DNA was preferred to native DNA as primer. The requirements for magnesium ions and DNA were absolute. The requirement for deoxynucleoside triphosphates indicated this to be a replicative rather than a terminal addition enzyme. At nearly every age the level of enzyme activity was highest in extracts from the embryonic cerebellum.
• 3 The particulate fraction from brain homogenates decreased the DNA-polymerase activity observed in soluble brain extracts. Data are presented which indicate that this inhibition was the result of dephosphorylation of the deoxynucleoside triphosphate substrates by an ATPase in the brain particulate fraction whose activity increases during ontogeny.

     

Amniotic fluid levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 before and after the onset of labor in normal pregnancies.

Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells and promotes the regulation of trophoblast growth and invasion. Tumor necrosis factor receptor 1 (TNF-R1) is a receptor for TNF-alpha, and soluble TNF-R1 (sTNF-R1) is present in amniotic fluid after receptor shedding. We evaluated whether amniotic fluid TNF-alpha and sTNF-R1 levels during labor differed from those before the onset of labor in normal pregnancies. This study enrolled 34 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these subjects, 17 went into labor and had subsequent term deliveries (the labor group), and the other 17 underwent cesarean section without labor (the nonlabor group). The average gestational age at entry was 38-39 weeks of gestation. Maternal ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the TNF-alpha and sTNF-R1 levels were determined by the ELISA method. Each of these levels was compared between the two groups. There was a significant increase in amniotic fluid TNF-alpha levels in the labor group. However, amniotic fluid sTNF-R1 levels did not differ significantly between the two groups. Amniotic fluid TNF-alpha may promote the onset of labor at term and/or term labor contributing to subsequent delivery may induce the production and secretion of TNF-alpha into the amniotic cavity. There was no pregnancy-associated increase in receptor shedding or cell apoptosis at the onset of labor.     

Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification.

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.     

Identification of Staphylococcus aureus Colony-Spreading Stimulatory Factors from Mammalian Serum

Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.     

The nature of the cytosolic activators of the adrenal steroid 21-hydroxylation system

The steroid 21-hydroxylase activity present in the microsomes of bovine adrenals is stimulated by components of the cytosol. The nature of these activators has been examined by two procedures. The first consisted of treating cytosol with increasing amounts of acetone. When the concentration of the organic solvent reached 50%, a precipitate, presumably proteinaceous, formed. The portion of the precipitate that was redissolvable in 0.05 m potassium phosphate buffer, pH 7.2, contained 7–15% of the stimulatory activity originally present in the cytosol. When the acetone concentration was raised to 90%, another active material precipitated. It was identified as oxidized glutathione (GSSG) and it accounted for about 5% of the activity in the cytosol. In an attempt to avoid the harmful effects of acetone, the second procedure employed only gel filtration and ion exchange resin chromatography. By these means the cytosol was separated into 11 protein fractions and a small molecular weight material. Forty six percent of the proteins and the same fraction of the stimulatory activity present in the original cytosol were recovered. Because all 11 protein fractions contained some stimulatory activity, the results suggested that the protein constituents of these fractions were relatively nonspecific. Yet, of the several known proteins which were tested for activity (bovine serum albumin, ovalbumin, human γ-globulin, bovine pancreatic ribonuclease, and pig insulin) only bovine serum albumin proved to be active. An additional 8% of the stimulatory activity of the cytosol was present in the fraction containing the low molecular weight components and this was all attributable to its GSSG content.