Wen Xuan Wu

Differential Expression of Myometrial Oxytocin Receptor and Prostaglandin H Synthase 2, But Not Estrogen Receptor α and Heat Shock Protein 90 Messenger Ribonucleic Acid in the Gravid Horn and Nongravid Horn in Sheep during Betamethasone-Induced Labor1

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor α (ERα), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130–140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101–110 dGA (n = 3), 111–120 dGA (n = 3)...

Changes in estrogen receptor messenger ribonucleic acid in sheep fetal and maternal tissues during late gestation and labor

OBJECTIVEnOur purpose was to investigate whether there is an increase in messenger ribonucleic acid for estrogen receptor in critical maternal or fetal tissues in the last third of pregnancy and during labor in sheep.nnnSTUDY DESIGNnEstrogen receptor messenger ribonucleic acid was measured by Northern hybridization analysis in fetal-placental and maternal uterine tissues during the last third of pregnancy and during spontaneous or cortisol-induced labor in sheep. Statistical differences were assessed with two-way analysis of variance.nnnRESULTSnNo estrogen receptor messenger ribonucleic acid was observed in amnion or chorion in any animal studied. There were no gestational age related changes in estrogen receptor messenger ribonucleic acid in any tissues between 100 and 145 days gestation. During spontaneous and cortisol-induced labor estrogen receptor messenger ribonucleic acid increased significantly (p < 0.05) in myometrium, endometrium, and cervix. No increase was observed in the fetal placental cotyledon and mesometrium. Estrogen receptor messenger ribonucleic acid was markedly decreased (p < 0.05) in myometrium and endometrium after fetal adrenalectomy.nnnCONCLUSIONnAn increase in estrogen receptor messenger ribonucleic acid in association with labor may contribute part of the mechanism by which estrogens exert their influence on the process of parturition.

Tissue-specific ontogenic expression of prostaglandin H synthase 2 in the ovine myometrium, endometrium, and placenta during late gestation and at spontaneous term labor.

OBJECTIVEnThe purposes of this study were to determine (1) whether uterine tissues as well as the fetal placenta are involved in the development of prostaglandin-synthesizing capacity associated with impending labor in pregnant sheep and (2) whether the key enzyme of prostaglandin synthesis, prostaglandin H synthase 2, is differentially expressed in the different intrauterine tissues during late gestation and in association with labor.nnnSTUDY DESIGNnMyometrium, endometrium, and fetal placenta were removed from ewes at 95 days gestation, (n = 3), 101 to 110 days gestation (n = 3), 111 to 120 days gestation (n = 3), 121 to 130 days gestation (n = 3), 131 to 140 days gestation (n = 3), and 141 to 145 days gestation (n = 4) and from ewes in spontaneous term labor at 143 to 147 days gestation (n = 4). Expressions of prostaglandin H synthase 2 messenger ribonucleic acid and protein were determined by Northern blot and Western blot analyses. Prostaglandin H synthase 2 was localized in the fetal placenta by immunohistochemical means.nnnRESULTSnLevels of both prostaglandin H synthase 2 messenger ribonucleic acid and protein increased gradually from 115 days gestation in the fetal placenta and from 131 days gestation in the endometrium. A further and more significant increase in prostaglandin H synthase 2 concentration occurred in the placenta and endometrium during spontaneous term labor. In contrast, myometrial concentrations of prostaglandin H synthase 2 messenger ribonucleic acid and protein remained at steady basal levels during the course of pregnancy and increased only during labor. Prostaglandin H synthase 2 was localized in the trophoblast cells of the fetal placenta.nnnCONCLUSIONSnTissue-specific ontogenic expression of prostaglandin H synthase 2 was observed in myometrium, endometrium, and placenta during late ovine gestation and spontaneous term labor. Fetal placenta and endometrium showed increased expression of prostaglandin H synthase 2 messenger ribonucleic acid and protein during late ovine gestation, whereas myometrial prostaglandin H synthase 2 concentration remained low throughout late gestation. Prostaglandin H synthase 2 concentrations in the myometrium, endometrium, and placenta are all upwardly regulated during labor.

Changes in prostacyclin synthase in pregnant sheep myometrium, endometrium, and placenta at spontaneous term labor and regulation by estradiol and progesterone.

OBJECTIVEnOur purpose was to investigate, first, whether there were changes in the abundance of prostacyclin synthase protein in intrauterine tissues of pregnant ewes in association with spontaneous term labor. Second, we examined the effect of either estradiol or progesterone, or both, on regulation of prostacyclin synthase protein abundance in uterine tissues using an ovariectomized nonpregnant sheep model.nnnSTUDY DESIGNnThe abundance of prostacyclin synthase protein was quantified by Western blot analysis in the myometrium, endometrium, and placenta of pregnant ewes in spontaneous term labor (n = 6) and term control ewes not in labor (n = 6). The changes of prostacyclin synthase in the myometrium and endometrium of 20 ovariectomized nonpregnant sheep (n = 5 for each group) were evaluated after treatment with estradiol, progesterone, or both.nnnRESULTSnProstacyclin synthase protein was present in pregnant and nonpregnant sheep myometrium, endometrium, and placenta at a molecular weight of about 55 kd. At spontaneous term labor the level of prostacyclin synthase decreased in endometrium (P <.05), increased in myometrium (P <.05), and remained unchanged in placenta. Estradiol and progesterone had no effect on prostacyclin synthase protein abundance in nonpregnant ovine endometrium and myometrium.nnnCONCLUSIONSnThe decrease in prostacyclin synthase in pregnant sheep endometrium during labor may indicate paracrine interactions between the endometrium, the myometrium, fetal membranes, or a combination of these. The significant increase of prostacyclin synthase in pregnant sheep myometrium at spontaneous term labor may contribute to the increased uterine sensitivity to oxytocin or stimulate vasodilatation during labor to increase myometrial blood flow. Neither estradiol nor progesterone at the dosages studied changed prostacyclin synthase expression in the nonpregnant myometrium and endometrium. The molecular mechanism or mechanisms that differentially regulate prostacyclin synthase expression in pregnant uterine tissues merit further study.

Differential effects of natural and synthetic glucocorticoids on cytochrome 17α-hydroxylase (P-45017α) and cytochrome P-450 side-chain cleavage (P-450scc) messenger ribonucleic acid in the sheep placenta

OBJECTIVEnOur purpose was to determine differential effects of natural and synthetic glucocorticoids on cytochrome 17alpha-hydroxylase and cytochrome P-450 side-chain cleavage messenger ribonucleic acid in the sheep placenta and to determine whether feed-forward effects during labor are involved in further inducing placental cytochrome 17alpha-hydroxylase.nnnSTUDY DESIGNnSheep underwent placement of myometrial electromyogram electrodes while they were under general anesthesia at 117 days gestation. At 125 days gestation either saline solution (early control animals not in labor, n = 5), 0.48 mg betamethasone during 48 hours (n = 7), 0.48 mg dexamethasone during 48 hours (n = 7), or 55 mg cortisol during a maximum of 96 hours (n = 4) was directly administered intravenously to the fetus. Necropsies were performed at 127 to 129 days gestation. We also studied 6 ewes in spontaneous term labor at 143-147 days gestation, 6 term control animals not in labor at 140 to 147 days gestation, and 6 sheep in which myometrial activity was inhibited by intravenous infusion to the ewe of the selective cyclooxygenase 2 inhibitor nimesulide 9 hours after the onset of labor beginning at 147 to 148 days gestation. Total fetal placental ribonucleic acid was analyzed by Northern blot with complementary deoxyribonucleic acid probes for cytochrome 17alpha-hydroxylase, cytochrome P-450 side-chain cleavage, and 18S ribosomal ribonucleic acid to correct for loading.nnnRESULTSnPlacental cytochrome 17alpha-hydroxylase messenger ribonucleic acid was detectable neither in term control animals not in labor nor in early control animals not in labor. Placental cytochrome 17alpha-hydroxylase messenger ribonucleic acid was induced in spontaneous term labor and all cortisol-infused sheep in labor with respect to term control animals not in labor and early control animals not in labor (P <.01). All betamethasone-infused sheep had myometrial contraction activity; however, only 4 of 7 had detectable placental cytochrome 17alpha-hydroxylase messenger ribonucleic acid. Placental cytochrome 17alpha-hydroxylase messenger ribonucleic acid was not detected in dexamethasone-infused sheep, even the 2 that had myometrial contractions. After reversal of the progression of spontaneous labor with nimesulide placental cytochrome 17alpha-hydroxylase messenger ribonucleic acid was significantly lower than the spontaneous term labor group (without nimesulide treatment). The placentas from all animals expressed cytochrome P-450 side-chain cleavage messenger ribonucleic acid, but no changes were associated with either gestational age studied (130 versus > 140 days gestation) or glucocorticoid-induced premature labor and spontaneous term labor.nnnCONCLUSIONSn(1) In sheep the expression of placental cytochrome 17alpha-hydroxylase is tightly associated with spontaneous term labor, and active synthesis of placental cytochrome 17alpha-hydroxylase is required during the progression of labor. (2) Cortisol is a more potent stimulator of placental cytochrome 17alpha-hydroxylase messenger ribonucleic acid in sheep than are synthetic glucocorticoids. (3) Betamethasone has a greater effect in inducing labor in sheep than does dexamethasone, possibly mediated through placental cytochrome 17alpha-hydroxylase.

The effects of fetal adrenalectomy at 110 days gestational age on AVP and CRH mRNA expression in the hypothalamic paraventricular nucleus of the ovine fetus

AVP and CRH produced in the parvocellular neurons of the paraventricular nucleus (PVN) have both been implicated in the regulation of anterior pituitary ACTH synthesis and secretion. In sheep, fetal ACTH secretion increases around 120 days gestational age (dGA). Little is known about adrenal regulation of AVP and CRH immediately prior to this critical period. We investigated the effects of adrenalectomy and subsequent cortisol (F) administration on PVN AVP and CRH mRNA in the fetal sheep PVN at 109-125 dGA. At 109-113 dGA, fetal sheep adrenals were removed (ADX)(n = 8); or sham surgery performed (CONT)(n = 4). From day 6 post ADX, maternal plasma cortisol and fetal plasma ACTH and cortisol levels were determined daily by radioimmunoassays. From day 7 post ADX, cortisol (4 micrograms/min) was continuously infused intravenously to four ADX fetuses (ADX + F). Fetal hypothalami were collected at 123-125 dGA, and studied by in-situ hybridization and quantitative autoradiography for AVP and CRH mRNA. Plasma cortisol levels remained low in CONT and ADX fetuses (< 4.9 ng/ml), while during cortisol infusion to ADX + F fetuses, plasma F increased to 16.4 +/- 2.2 and 22.3 +/- 3.2 ng/ml (mean +/- S.E.M.) on day 10 and 13, respectively. Plasma ACTH levels increased significantly in ADX compared with CONT fetuses. This ACTH increase was completely suppressed in ADX + F fetuses. AVP mRNA abundance in the whole PVN was the same in all three groups, however, a separate analysis of AVP mRNA abundance in parvocellular and magnocellular regions of the PVN revealed that AVP mRNA in the parvocellular PVN showed a significant increase in ADX and suppression in ADX + F fetuses when compared to CONT. AVP mRNA in the magnocellular PVN remained unchanged. PVN CRH mRNA expression was augmented in ADX and suppressed in ADX + F when compared to CONT fetuses. We conclude that in fetal sheep at 109-125 dGA: AVP and CRH mRNA abundance in the parvocellular region of the PVN are increased by adrenalectomy and that cortisol inhibits this increase.

Increase in Prostaglandin H synthase 2, but not Prostaglandin F2α synthase mRNA in intrauterine tissues during betamethasone-induced premature labor and spontaneous term labor in sheep

OBJECTIVE: Prostaglandin F synthase (PGFS) catalyzes reduction of prostaglandin H2 to PGF2α,. No information exists on PGFS expression and regulation during pregnancy, either mRNA or protein, in relation to labor in uterine tissues in any species. We characterized PGFS mRNA expression in ovine myometrium, endometrium, maternal and fetal placenta in betamethasone-induced premature labor and spontaneous term labor using our cloned ovine PGFS riboprobe. Prostaglandin H synthase (PGHS) 2 mRNA was evaluated simultaneously as a control whose pregnancy related changes are well known. METHODS: Poly-A or total RNA from fetal placenta, myometrium, and endometrium in control ewes at 143-147 days of gestational age (dGA, TCNL, n = 6), and ewes in spontaneous term labor at 145-147 dGA (STL, n = 6) and endometrium and maternal and fetal placenta in early control ewes not in labor (ECNL, n = 6) and betamethasone induced labor at 128-135 dGA (BL, n = 6) were analyzed for PGHS2 and PGFS mRNA. RESULTS: PGFS mRNA did not change at spontaneous term labor in myometrium, endometrium, and fetal placenta. PGFS mRNA decreased during betamethasone-induced premature labor in endometrium and maternal placenta (P < .05), but remained unchanged infetal placenta and myometrium. PGHS2 mRNA increased in endometrium, placenta, and myometrium during betamethasone-induced premature labor and spontaneous term labor. CONCLUSION: Increased PGHS2, but not PGFS mRNA was tightly associated with the onset of betamethasone-induced premature labor as well as spontaneous term labor in the endometrium, placenta, and myometrium. Transcription of PGFS mRNA may not be the rate-limiting step in the pathway contributing to increased PGF2α, at labor.

Maternal intravenous administration of long chain n-3 polyunsaturates to the pregnant ewe in late gestation results in specific inhibition of prostaglandin h synthase (PGHS) 2, but not PGHS1 and oxytocin receptor mRNA in myometrium during betamethasone-induced labor.

Objective: Both the onset of labor and time to delivery during betamethasone-induced delivery are delayed by omega-3 polyunsturated fatty acid (PUFA) administration to pregnant sheep. That fatty acid also inhibits the labor-related increase in maternal plasma estradiol and maternal and fetal prostaglandin E2. To evaluate the mechanism of inhibition of prostaglandin production and delay of onset of labor and time of delivery in PUFA-treated sheep, we determined the effect of PUFA on myometrial prostaglandin H synthase (PGHS) 1 and 2 and oxytocin receptor mRNA levels in betamethasone-induced labor. Methods: At 124 days gestation, a 20% emulsion of either intralipid (IL, n = 6) or PUFA (n = 6) was infused continuously (3 mL/kg per day) intravenously (IV) to the ewe. At 125 days gestation, betamethasone was administered IV (10 μg/h over 48 hours) to fetuses of both intralipid- and PUFA-treated ewes. Myometrium was collected at necropsy either during betamethasone-induced labor as evaluated by myometrial electromyography or within 5 days of the termination of betamethasone infusion, if delivery did not occur after fetal betamethasone infusion. Total myometrial RNA was analyzed by Northem blot for oxytocin receptor and PGHS1 and 2 mRNA normalized for 18s. Results: Treatment with PUFA decreased myometrial PGHS2 mRNA but did not alter myometrial PGHS1 and oxytocin receptor mRNA after betamethasone administration. Conclusion: This finding provides a mechanism whereby PUFA delays betamethasone-induced delivery in sheep and suggests a potential role of PUFA as an effective tocolytic agent in human pregnancy.

Distribution of arachidonic, eicosapentaenoic, docosahexaenoic and related fatty acids in ovine endometrial phospholipids in late gestation and labor

The quantitative distribution of phospholipid (PL) fatty acids from ovine endometrial tissues taken at 105 (n = 3) and 131 and 147 (n = 5) days of gestation age (dGA) and in spontaneous labor (SL, n = 3) is reported. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC), were separated by thin layer chromatography (TLC) and analyzed for fatty acid composition by quantitative gas chromatography (GC). Saturates are found mainly in PS and PI and unsaturates predominantly in PC and PE. The major long-chain polyunsaturated fatty acids (LC-PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are found primarily in PC, PE, and PI. AA accumulates in PC, PI and PS (p < 0.05) from late gestation to term and significantly declines in PC and PS (p < 0.02) during labor, suggesting that ovine endometrium is a possible source of prostaglandin (PG) precursors. EPA decreases significantly from around 105 dGA to term and at labor in PC (p < 0.02) and in PI (p < 0.01), which may indicate the involvement of 3-series PGs in the regulation of uterine contraction. Unsaturation index (UI) and total PUFA increase from late gestation to term in PE (p < 0.05) and decrease during labor (p < 0.05). The ratios of n-6/n-3 PUFA increase in PI (p < 0.05) and in PC (p < 0.01) during labor mainly due to the decline of EPA in these PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary phospholipid saturation increases with glucocorticoid receptor mRNAs in late gestation but not due to labor in the fetal rhesus monkey

We report here changes in abundance of pulmonary cytosolic phospholipase A2 (cPLA2) and glucocorticoid receptor (GR) mRNAs and phospholipid (PL) fatty acids (FA) with gestational age, in either spontaneous or androstenedione-induced premature labor for 15 fetal rhesus monkeys and 1 neonate. Pulmonary RNA and lipids were extracted from the lungs of the rhesus monkeys, gestational ages ranging from 140 days to term. Northern hybridization analysis was performed for both cPLA2 and GR mRNAs. The results demonstrated the transcript of the cPLA2 constantly expressed and that of GR increased gradually in the lungs during the gestational stages studied. Phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) were separated by thin layer chromatography and the fatty acid composition in these PL was quantified by gas chromatography (GC). Regression analysis demonstrated that the expression of GR, mRNAs increased significantly with gestational age (P < 0.01). Furthermore, the level of GR mRNA is significantly correlated with that of cPLA2, with Pearson correlation coefficient of 0.83 (P < 0.01). The PC palmitate percentage increased significantly with gestational age (P < 0.01), while the PC unsaturation index and total polyunsaturated FA decreased during late gestation (P < 0.01). Changes in PG FA with gestational age were similar to those in PC, while PI FA did not change with gestational age. There was no effect of either spontaneous or induced labor on mRNA levels of cPLA2 and GR or the FA composition of pulmonary PL studied. We conclude that the pulmonary PL FA compositions are under developmental control.