PGHS-2 Expression Research Articles

The Long-Term Effects of Prenatal Hypoxia on Coronary Artery Function of the Male and Female Offspring.

Prenatal hypoxia predisposes the offspring to the development of cardiovascular (CV) dysfunction in adult life. Using a rat model, we assessed the effect of prenatal hypoxia on vasoconstrictive and vasodilative mechanisms in left anterior descending coronary arteries of 4- and 9.5-month-old offspring. Endothelium-dependent relaxation to methylcholine and vasoconstriction responses to endothelin-1 (ET-1) were assessed by wire myography. Prenatal hypoxia impaired endothelium-dependent vasodilation in 4- and 9.5-month-old offspring. Inhibition of nitric oxide (NO) synthase prevented coronary artery relaxation in all groups. Inhibition of prostaglandin H synthase (PGHS) improved relaxation in prenatally hypoxic males and tended to improve vasorelaxation in females, suggesting that impaired vasodilation was mediated via increased PGHS-dependent vasoconstriction. An enhanced contribution of endothelium-dependent hyperpolarization to coronary artery vasodilation was observed in prenatally hypoxic males and females. No changes in endothelial NO synthase (eNOS) and PGHS-1 expressions were observed, while PGHS-2 expression was decreased in only prenatally hypoxic males. At 4 months, ET-1 responses were similar between groups, while ETB inhibition (with BQ788) tended to decrease ET-1-mediated responses in only prenatally hypoxic females. At 9.5 months, ET-1-mediated responses were decreased in only prenatally hypoxic females. Our data suggest that prenatal hypoxia has long-term similar effects on the mechanisms of impaired endothelium-dependent vasodilation in coronary arteries from adult male and female offspring; however, coronary artery contractile capacity is impaired only in prenatally hypoxic females. Understanding the mechanistic pathways involved in the programming of CV disease may allow for the development of therapeutic interventions.

Antenatal Suppression of IL-1 Protects against Inflammation-Induced Fetal Injury and Improves Neonatal and Developmental Outcomes in Mice.

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1β induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1β, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1β, IL-6, and IL-8, increased IL-1β, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.

Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A

Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

6.3 The Commonalities and Differences in the Molecular Expression of Different Phenotype-specific Causes of Preterm Labour in Comparison to Term Labour

Preterm birth comprises of several distinct clinical phenotypes. The commonalities and differences of the genes altered in the different phenotypes of preterm labour (PTL) have yet to be elucidated.Myometrial biopsies...

Corticotropin-releasing hormone interacts with interleukin-1β to regulate prostaglandin H synthase-2 expression in human myometrium during pregnancy and labor.

Context:The onset of labor appears to involve the activation of myometrial inflammatory pathways, and transcription factors such as nuclear factor-κB (NF-κB) control expression of the contraction-associated proteins required to induce a procontractile phenotype. These responses might involve CRH, which integrates immune and neuroendocrine systems.Objectives:In human myometrium we investigated cyclooxygenase 2 (PGHS2) expression and regulation by CRH and the proinflammatory cytokine IL-1β before and after labor.Design:Myometrial tissues obtained from pregnant women at term before (n = 12) or during labor (n = 10) and pathological cases of choriamnionitis-associated term labor (n = 5) were used to isolate primary myocytes and investigate in vitro, CRH effects on basal and IL-1β regulated p65 activation and PGHS2 expression.Results:In nonlaboring myometrial cells, CRH was unable to induce NF-κB nuclear translocation; however, it altered the temporal dynamics of IL-1β-driven NF-κB nuclear entry by initially delaying entry and subsequently prolonging retention. These CRH-R1-driven effects were associated with a modest inhibitory action in the early phase (within 2 hours) of IL-1β stimulated PGHS2 mRNA expression, whereas prolonged stimulation for 6–18 hours augmented the IL-1β effects. The early-phase effect required intact protein kinase A activity and was diminished after the onset of labor. The presence of chorioamnionitis led to exaggerated PGHS2 mRNA responses to IL-1β but diminished effects of CRH.Conclusions:CRH is involved in the inflammatory regulation of PGHS2 expression before and during labor; these actions might be important in priming and preparing the myometrium for labor and cellular adaptive responses to inflammatory mediators.

PL.01 Molecular Markers of Early and Established Labour in Human Myometrium

The normal physiological end point of pregnancy is signalled by the onset of myometrial contractions. However, the biochemical processes may have already occurred at or before term via a series...

Abstract 1635: Effect of dietary fish oil on rat colonic prostaglandin (PG) E2 metabolism

Abstract PGE2, an eicosanoid generated from arachidonic acid (AA) by the cyclooxygenase function of prostaglandin H synthases (PGHS)-1 and 2, has been linked to the risk of many cancers including colorectal carcinogenesis. Targeting colonic PGE2 homeostasis by utilizing nutritional products such as fish oil may reduce endogenous PGE2 that, in turn, may decrease subsequent inflammatory events that lead to carcinogenesis. Rats were fed high fat fish oil diets contained different ratios of eicosapentaenoic acid (EPA) and n-6 fatty acid. The colon was horizontally divided into three equal length transections, first (proximal end), second (transverse) and third (distal end). Enzymes involved in PGE2 metabolism (15-hydroxyprostaglandin dehydrogenase (15-PGDH) and PGE synthase (mPGES)) were measured using quantitative PCR and semi-quantitative Western Blotting. GC-MS analysis was performed to detect fatty acid levels in serum and colon. LC-MS/MS analysis was used to examine eicosanoid levels. Our results showed that the colonic epithelial mucosa contained less PGE2 per mg of protein than did the whole colon. Colonic PGE2 and several eicosanoids including 13-Hydroxyoctadecadienoic acid(13HODE), 5-Hydroxyeico-satetraenoic acid (5 HETE), 15-Hydroxyeicosatetraenoic acid (15 HETE) and 12-Hydroxyeicosatetraenoic acid (12 HETE) were distributed differentially in different part of the colon. Dietary fish oil intake increased tissue levels of EPA in the colon. PGHS-2 gene was affected the most by dietary fish oil among all the genes tested in normal colon. Fish oil dose-dependently decreased PGHS-2 mRNA expression in all three colon sections. mRNA expression of 15-PGDH was higher in the third colonic section than the first two sections; however it was not altered by dietary fish oil. In conclusion, different parts of the colon have different eicosanoid profiles. The decreases in PGE2 in normal colon observed with fish oil feeding do not appear to be related to expression of PGHS-1, mPGES and 15-PGDH but are most closely correlated with PGHS-2 despite that PGHS-2 levels are already low in the normal colon. These data support the concept of anatomic variability in colonic carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1635. doi:1538-7445.AM2012-1635

Chemoreflex Activity Increases Prostaglandin Endoperoxide Synthase mRNA Expression in the Late-Gestation Fetal Sheep Brain

Fetal sheep defend blood pressure, blood volume, and blood gases using baro- and chemoreflexes that influence autonomic and neuroendocrine responses. The local generation of prostanoids within the fetal brain is also an important component in activating hormone responses to these stimuli, but the relationship between the reflexes and prostanoid biosynthesis is unclear. The present study was performed to test the hypothesis that the abundances of prostaglandin biosynthetic enzymes in the fetal brain are dependent upon the activity of the baro- and chemoreflex pathways. We subjected chronically catheterized fetal sheep in late gestation to a 10-minute period of brachiocephalic occlusion (BCO), a stimulus that provokes brisk cardiovascular and neuroendocrine responses. We compared the central nervous system abundance of prostaglandin endoperoxide synthases 1 and 2 (PGHS-1 and PGHS-2) after BCO to (1) fetal sheep that had been subjected to BCO after chronic sinoaortic denervation plus bilateral vagotomy and (2) fetal sheep in which the N-methyl d-aspartate (NMDA) receptor antagonist, ketamine, had been administered prior to BCO. Abundances of messenger RNA (mRNA) for PGHS-1 and of mRNA and protein for PGHS-2 in fetal hippocampus were reduced significantly by either prior denervation or ketamine administration. Prostaglandin endoperoxide synthases 1 and 2 mRNA in pituitary were decreased and increased, respectively, by ketamine pretreatment. The results of this study are consistent with the conclusion that the expression of PGHS-1 and -2 in fetal hippocampus and pituitary are influenced by the baro- and/or chemoreflex pathways within the fetal brain in late gestation.

Evaluation of the uterine environment early in pregnancy establishment to characterise cows with a potentially superior ability to support conceptus survival

During previous investigations, the capacity of the cow to secrete prostaglandin in response to oxytocin has been linked with pregnancy outcome. The objective of the present study was to evaluate the predictive value of prostaglandin release to identify groups of cows as potentially superior (SR, low prostaglandin release) or inferior (IR, high prostaglandin release) for pregnancy outcome and to utilise these cows to investigate factors that contribute to optimum uterine conditions for early pregnancy. Animals were synchronised and received an in vitro-derived blastocyst on Day 7 post-oestrus. Tissues (trophoblast and endometrial) and uterine luminal fluid (ULF) were recovered 10 days later. Pregnancy rates were 94 and 78% for SR and IR cows, respectively. Of the pregnant SR cows, 69% had larged conceptuses (>24 cm) in contrast to 43% IR of cows. IR cows with small conceptuses (<12 cm) had significantly lower mean Day 3 and 5 post-oestrous progesterone concentrations than cows with large conceptuses. The expression of factors involved in the prostaglandin pathway, pregnancy and conceptus development were analysed via quantitative RT-PCR and Western blot analysis. Investigation of 16 endometrial gene transcripts indicated no differences between IR and SR cows except for osteopontin expression which, in uteri with large conceptuses, was 2-fold greater in SR than IR cows (P=0.02). There was greater expression of CTGF, OXTR, PGES, PGHS2 and UTMP mRNA in uteri of SR and IR cows that had large compared with small conceptuses (P<0.05). More IFNT protein was recovered in SR compared with IR ULF (P<0.03). SR cows with large conceptuses had less TIMP2 and legumain protein in their gravid, compared with their non-gravid horns (P≤0.02) whereas IR cows did not. The predictive value of prostaglandin release in response to oxytocin challenge does not appear to be an effective indicator of subsequent pregnancy rates in cows. Differences between the two groups appear to be associated with subtle differences in progesterone and uterine protein concentrations that may be related to differences in conceptus size.

A study of the product profile and substrate specificity of prostaglandin endoperoxide H synthases

Prostaglandin endoperxide H synthase (PGHS), which catalyzes the first and committed step in prostaglandin synthesis, exhibits both a cyclooxygenase and a peroxidase activity. Initially two molecules of O2 are incorporated into arachidonic acid to form the hydroperoxy endoperoxide PGG2, which is reduced to form the hydroxyl endoperoxide PGH2. There are two isozymes (PGHS‐1 and ‐2), encoded by different genes. Both are structurally and catalytically similar, but show different expression pattern. PGHS‐1 is constitutively expressed, but PGHS‐2 expression is transient and inducible in response to several mitogenic and pro‐inflammatory stimuli. Besides their different expression mode, coupling of each isozyme with the downstream prostanoid synthases is distinctive. For instance, thromboxane A2 is synthesized by functional coupling between PGHS‐1 and thromboxane synthase in platelet cells, which do not express PGHS‐2. Depending on the efficiency of their coupling, not only the designated prostaglandin is formed, but a combination of other prostaglandins can be produced. The product profile was shown to be even more complicated in the presence of reduced glutathione at physiological concentrations, which altered drastically arachidonic acid metabolism. Secondly, the peroxidase active site of PGHS, which is located across from the cyclooxygenase site and hydrophobic dome‐shaped, was investigated to dissect the essential residues for PGG2 specificity.

Purinergic agonists flex vas deferens muscle

The vas deferens is described typically as a muscular tube that functions as a conduit to move sperm from the epididymis to the urethra. A growing body of evidence, however, shows that the system is far more complex. Smooth muscle tone and contractions are affected by a number of neuocrine, endocrine and perhaps lumicrine agents. Likewise, epithelial activity is modified by a similar host of agents. Stimulation of the hypogastric nerve, which innervates the male reproductive ducts, results in the co-release of noradrenaline, ATP, and other neurotransmitters that induce or modulate smooth muscle contraction and can induce epithelial ion transport. In fact, it has been suggested that purinergic receptors in the reproductive duct might be targeted for the development of non-steroidal male contraceptives (Gur et al. 2007). Thus, there is great interest in determining regulatory cascades that link purinergic stimulation to smooth muscle contraction or relaxation. One caveat in exploiting purinergic pathways to manage male fertility is the apparently conflicting observations that ATP exposure can, depending upon conditions, lead to either smooth muscle contraction or relaxation. Thus, multiple receptors and signalling cascades are probably involved in these responses. Both ionotropic P2X and metabotropic P2Y receptors are present on vas deferens smooth muscle cells with rapid muscle contraction typically associated with P2X-mediated Ca2+ entry. The cascade accounting for relaxation appears to be more complex. In a recent issue of The Journal of Physiology, Ruan et al. (2008) provide keen insight on a mechanism that probably contributes to muscle relaxation, in vitro. Their work builds on the observation that vas deferens epithelium contributes to the response of smooth muscle to adrenergic stimuli (Okpalaugo et al. 2002). This observation suggests that vas deferens smooth muscle activity can be modified by paracrine agents released from the epithelium. It is becoming more widely recognized that neurotransmitters and hormones can modify the activity of epithelial cells lining the epididymes and vas deferens. Indeed, neurotransmitters, steroid hormones, peptide hormones and autocoids have been shown to modify epithelial ion transport. HCO3− secretion is particularly important because it initiates and supports increased sperm motility. Importantly, noradrenaline, ATP and adenosine have been shown to induce anion secretion. The Ruan paper adds purinergic modulation of smooth muscle tone to the activities probably mediated by the epithelium lining the reproductive duct. Prostaglandin E2 is a key element in the epithelium-dependent response to ATP. This observation has far-reaching impact because prostaglandin H synthase 2 (PGHS2; formerly known as COX2) expression in vas deferens epithelial cells is dependent upon testosterone. In both rats (McKanna et al. 1998) and humans (Kirschenbaum et al. 2000), vas deferens epithelial PGHS2 expression correlates with serum testosterone. PGHS2 immunoreactivity is present during gestation and following puberty, but absent pre-pubertally or following castration. Recently, Pierucci-Alves & Schultz (2007) demonstrated that testosterone exposure enhances bradykinin-induced vas deferens epithelial anion secretion in vitro, a response mediated by prostaglandins. Thus, testosterone-induced epithelial PGHS2 expression provides the basis for androgen-dependent sperm development and delivery. The results provided by Ruan and colleagues are the foundation of a model that brings many seemingly disparate elements together with the culmination of viable sperm delivery. ATP released from the hypogastric nerve can rapidly promote smooth muscle contraction by the activation of P2X receptors while co-released noradrenaline can promote a sustained response by activating α1 receptors on smooth muscles. These same neurotransmitters can affect epithelial HCO3− secretion by interacting with epithelial P2Y and β2 receptors, respectively. In epithelial cells exposed to testosterone, PGHS2 can be stimulated downstream from the P2Y receptor resulting in PGE2 production. PGE2 then interacts with sperm to promote motility, with prostanoid receptors on epithelial cells to further enhance anion secretion, and with prostanoid receptors on adjacent smooth muscle cells to promote hyperpolarization and smooth muscle relaxation. Thus, the work presented in this issue provides compelling evidence that the vas deferens is much more that just a muscular tube. The vas deferens is a complex tissue in which many component parts contribute to the timely delivery of mature active sperm. Dr Schultz receives research support from the National Institutes of Health (R01HD058398).

Blockade of Estrogen Receptors Decreases CNS and Pituitary Prostaglandin Synthase Expression in Fetal Sheep

Background/Aims: Both prostaglandin E₂ (PGE₂) and estradiol stimulate fetal ACTH secretion and augment fetal ACTH responses to stress. We have reported that estradiol increases prostaglandin endoperoxide synthase-2 (PGHS-2), and we have proposed that there is a positive feedback relationship between estrogen and fetal hypothalamus-pituitary-adrenal (HPA) axis activity that is dependent upon PGHS activity in the fetal brain. The present study was designed to test the hypothesis that blockade of estrogen receptors in the fetal brain decrease PGHS-2 expression and reduces fetal HPA axis activity. Methods: In study 1, six time-dated pregnant ewes with chronically-catheterized twin fetuses were used. In each pregnancy, one twin was treated intracerebroventricularly (icv) with the estrogen receptor antagonist ICI 182,780 (25 µg/day; n = 6) while the other twin served as an age-matched control. In study 2, plasma samples were drawn from 10 singleton chronically-catheterized fetuses on alternating days until the time of spontaneous parturition. Results: ICI infusion caused significantly decreased PGHS-2 mRNA abundance in fetal central nervous system and pituitary, with the greatest decreases occurring in hippocampus and pituitary. There were no statistically significant changes in PGHS-1 mRNA. ICI infusion did not significantly change fetal plasma concentrations of pro-opiomelanocortin (POMC), ACTH, or cortisol in fetuses 130–134 days ges- tation (study 1) but did decrease the preparturient rise in plasma pro-opiomelanocortin concentrations in study 2. Conclusion: We conclude that PGHS-2 expression in the late-gestation fetal brain is in part stimulated by circulating estrogens in fetal plasma. Blockade of CNS estrogen receptors reduces preparturient plasma concentrations of POMC, but does not reduce fetal HPA axis activity in 130–134 day fetal sheep.

Grape polyphenols affect mRNA expression of PGHS-2, TIS11b and FOXO3 in endometrium of heifers under ACTH-induced stress

Stress activates the hypothalamo–pituitary–adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol ( P < 0.001) and progesterone concentrations ( P < 0.01) compared to CTR group. PGHS-2 was significantly ( P < 0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 ( P < 0.05), FOXO3 and TIS11b ( P < 0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was suggested.

Differential expression of prostaglandin (PG) synthesis enzymes in conceptus during peri-implantation period and endometrial expression of carbonyl reductase/PG 9-ketoreductase in the pig

Prostaglandins (PGs) play a pivotal role in luteolysis, maternal recognition of pregnancy, and implantation. In many species, including pigs, both conceptus (embryo and associated membranes) and endometrium synthesize PGE(2), which may antagonize PGF(2alpha) by playing a luteotropic/antiluteolytic role. Previously, we have reported expression profiles of PG G/H synthases (PGHS-1 and PGHS-2), PGE synthase (mPGES-1), and PGF synthase (PGFS) in the endometrium of cyclic and pregnant pigs. In the present study, expression of above-mentioned PG synthesis enzymes and PG 9-ketoreductase (CBR1), which converts PGE(2) into PGF(2alpha), and the PGE(2)/PGF(2alpha) ratios were investigated in porcine peri- and post-implantation conceptuses. Furthermore, expression of CBR1 was examined in the endometrium. PGHS-2 and mPGES-1 were upregulated, and PGHS-1, PGFS, and CBR1 were downregulated in conceptuses during trophoblastic elongation. A second increase of mPGES-1 mRNA occurred after days 20-21 of pregnancy. After initiation of implantation, expression of PGHS-1, PGFS, and CBR1 in conceptuses increased and remained higher until days 24-25 of pregnancy. Comparison of the endometrial CBR1 protein expression in cyclic and pregnant gilts revealed upregulation on days 16-17 of the cycle and downregulation on days 10-11 of pregnancy. In conclusion, reciprocal expression of PGHS-2, mPGES-1, PGFS, and CBR1 in day 10-13 conceptuses and decrease of endometrial CBR1 may be important in increasing the PGE(2)/PGF(2alpha) ratio during maternal recognition of pregnancy. This study indicates that PGE(2) produced via PGHS-2 and mPGES-1 in conceptus may be involved in corpus luteum control. Moreover, high expression of conceptus PGHS-1, mPGES-1, PGFS, and CBR1 after initiation of implantation suggests their significant role in placentation.

Labor-Associated Regulation of Prostaglandin E and F Synthesis and Action in the Ovine Amnion and Cervix

Prostaglandins (PGs) are key regulators of cervical dilatation and membrane breakdown at the onset of labor. PG synthase and receptor expression has been previously documented in uterine tissues; however, mechanisms governing the changes occurring in the cervix and amnion are less well established. The aim of the current study was to determine the level of expression of PG synthetic enzymes and receptors in these tissues in association with induced labor in sheep. Labor was induced in sheep at 135 days of gestation by continuous fetal dexamethasone infusion. Amnion and cervical tissue was obtained before and after labor for measurement of mRNA encoding enzymes (cytosolic phospholipase A2 [cPLA2], PGH synthase-2 [PGHS-2], PGF synthase [PGFS], and PGE synthase [PGES]) and receptors (FP and EP1-4) by real-time polymerase chain reaction (PCR). cPLA2 expression increased significantly in cervical tissue at labor onset, whereas expression of the other enzymes measured did not change. There was a marked rise in EP3 expression in the cervix, but abundance of this receptor was lower than EP2 and FP expression, which did not change. The amnion exhibited a labor-associated decrease in PGHS-2, PGFS, and FP mRNA expression. The regulation of PG synthesis and action occurring in the amnion and cervix in association with labor appear to differ markedly between the two tissues, indicating tissue-specific roles for PGs. The data support a role for increased PG synthesis and action in the cervix and suggest a decrease in PG production and action in the amnion, in sharp contrast to the pattern reported in human amnion.

Differential impact of prostaglandin H synthase 1 knockdown on platelets and parturition

Platelet activation is a hallmark of severe preeclampsia, and platelet PGH synthase 1-derived (PGHS1-derived) thromboxane A(2) (TxA(2)) has been implicated in its pathogenesis. However, genetic disruption of PGHS1 delays parturition. We created hypomorphic PGHS1 (PGHS1(Neo/Neo)) mice, in which the substantial but tissue-dependent variability in the inhibition of PGHS1-derived eicosanoids achieved by low-dose aspirin treatment is mimicked, to assess the relative impact of this strategy on hemostatic and reproductive function. Depression of platelet TxA(2) by 98% in PGHS1(Neo/Neo) mice decreased platelet aggregation and prevented thrombosis. Similarly, depression of macrophage PGE(2) by 75% was associated with selectively impaired inflammatory responses. PGF(2alpha) at 8% WT levels was sufficient to induce coordinated temporal oxytocin receptor (OTR) expression in uterus and normal ovarian luteolysis in PGHS1(Neo/Neo) mice at late gestation, while absence of PGHS1 expression in null mice delayed OTR induction and the programmed decrease of serum progesterone during parturition. Thus, extensive but tissue-dependent variability in PG suppression, as occurs with low-dose aspirin treatment, prevents thrombosis and impairs the inflammatory response but sustains parturition. PGHS1(Neo/Neo) mice provide a model of low-dose aspirin therapy that elucidates how prevention or delay of preeclampsia might be achieved without compromising reproductive function.

Induction of Uterine Prostaglandin H Synthase 2 by Estradiol Following Fetal Adrenalectomy

In sheep, fetal cortisol stimulates the conversion of progesterone to estradiol in late gestation initiating labor. It is unclear whether an intact fetal hypothalamic-pituitary-adrenal (HPA) axis is required to induce the estradiol-triggered subsequent endocrine changes including enhanced intrauterine prostaglandin (PG) synthesis associated with the onset of labor. We have shown that maternal estradiol administration stimulates PG H synthase (PGHS)-2 expressions in pregnant ovine intra-uterine tissues. The current study was undertaken to determine whether the fetal adrenal mediates estradiol's stimulation of the intrauterine PGHS-2 in pregnant sheep. Placenta, myometrium, and endometrium were collected from two groups of ewes at 123-127 d of gestational age (dGA) after fetal adrenalectomy and vehicle treatment (ADX; n = 5); or fetal ADX and maternal estradiol administration (5 mg twice a day for 2 d, ADX+E2, n = 5). PGHS-2 mRNA and protein were analyzed by Northern and Western Blot analyses in both groups and presented as the ratios to beta actin mRNA for Northern and G protein beta subunit for Western blot analysis. Fetal plasma cortisol was measured by radioimmunoassay. Data were analyzed by Student's t test. Fetal plasma cortisol levels were low in ADX and ADX+ E2 groups (<6 ng/mL). The cervix of all ADX+E2 treated ewes was dilated at necropsy. Three out of five ADX+ E2-treated ewes delivered within 48 h. The cervix was closed in all fetal ADX ewes at necropsy. PGHS-2 mRNA and protein increased (p < 0.05) in myometrium and endometrium, but not placenta in ADX+E2-treated ewes compared with ADX group. These data provide the first in vivo evidence for estradiol upregulation of intrauterine PGHS-2 in late gestation in the absence of an intact fetal HPA axis. Thus, the fetal adrenal is not required to mediate estradiol's stimulation of uterine PGHS-2 expression associated with the onset of labor.

Changes in the expression of prostaglandin E and F synthases at induced and spontaneous labour onset in the sheep.

The differential production of prostaglandin (PG) F(2 alpha) and PGE(2) within the uterine compartment may play a role in controlling myometrial contraction. We hypothesized that the enzymes downstream of PG endoperoxide synthase-2 (PGHS-2) determine the ratio of PGF(2 alpha) and PGE(2) in the utero-ovarian vein plasma and the time of normal and preterm labour onset. The aim of this study was to simultaneously determine the expression of PGF and PGE synthases (PGFS and PGES) in gestational tissues at spontaneous and induced-preterm labour in sheep. Myometrial, endometrial and placental tissue were obtained from ewes in dexamethasone-induced preterm labour, age-matched control ewes, and ewes in spontaneous term labour for analysis of mRNA expression by real-time PCR. PGFS mRNA expression was significantly increased following dexamethasone-induced and spontaneous labour onset in placentome (P<0.01) but was unchanged in the myometrium and endometrium. In contrast, PGES mRNA expression remained unchanged or decreased. PGHS-2 mRNA expression was increased in all tissues examined in both dexamethasone-induced and spontaneous labour (P<0.001). Plasma PGE(2) and PGF(2 alpha) concentrations rose in both dexamethasone-induced and spontaneous labour with the ratio of PGF(2 alpha):PGE(2) increased with labour onset (P<0.05). These results are consistent with the hypothesis that the increased expression, of PGFS is responsible for the increased PGF(2 alpha):PGE(2) ratio and this, together with increased PGHS-2 expression, accounts for myometrial activity at labour onset. The findings point to PGFS expression as a key factor in regulating the uterotonic process in the sheep.

Effects of chronic PGHS-2 inhibition on PGHS-dependent vasoconstriction in the aged female rat.

In menopause, aging and decreased estrogen levels are risk factors contributing to impaired vascular function. Previously, in a young ovariectomized rat model, we demonstrated an increase in prostaglandin H synthase (PGHS)-dependent vasoconstriction that could be prevented by estrogen replacement. Subsequently, we found that, with aging, estrogen acts through suppression of the PGHS-2 isoform. Chronic PGHS-2 inhibition reduces PGHS-dependent vasoconstriction in aging. Ovariectomized, aged (12 months) Sprague-Dawley rats were treated with a placebo (n=7), or the PGHS-2 inhibitor (NS-398, s.c. 3 mg/kg) for 1 week (n=6) or 4 weeks (n=6). Methacholine (endothelium-dependent dilator) and phenylephrine (adrenergic constrictor) were used to assess vascular function in the absence or presence of the nonselective PGHS inhibitor, meclofenamate (1 micromol/l) or the specific PGHS-2 inhibitor NS-398 (10 micromol/l). One week of chronic PGHS-2 inhibition abolished a PGHS-dependent shift in methacholine-induced relaxation, while modulation was still observed in phenylephrine constriction. Surprisingly, 4 weeks of PGHS-2 inhibition enhanced PGHS-dependent modulation of vasoconstriction (P<0.05). PGH2/thromboxane inhibition (U-51605, 50 micromol/l) mimicked the results observed with PGHS inhibition among the groups. PGHS-2 expression increased with chronic PGHS-2 inhibition compared to control (P<0.05). Our data indicate a paradoxical increase in PGHS-dependent vasoconstriction and PGHS-2 expression with prolonged inhibition of PGHS-2 activity. Hence, inhibitors of PGHS-2 activity may not be beneficial in counteracting the vascular dysfunction seen with aging and menopause.

Cortisol Infusion Decreases Renin, But Not PGHS-2, EP2, or EP4 mRNA Expression in the Kidney of the Fetal Sheep at Days 109–116

Renal prostaglandins (PG), renin, and cortisol are necessary for normal kidney development and function during fetal life. We examined the effects of cortisol infusion before completion of nephrogenesis (d 109-116 gestation; 2.0-3.0 mg hydrocortisone succinate/24 h) on the renal mRNA expression of PGHS-2, the PGE(2) receptors, EP(2) and EP(4), and renin in fetal sheep. Cortisol infusion raised plasma cortisol levels to 42.8 +/- 6.0 nmol/L compared with saline infusion levels of 1.5 +/- 0.5 nmol/L (p < 0.001), but had no effect on fetal body weight, proportional kidney mass, or blood gases. Cortisol decreased significantly the relative expression of renin mRNA (saline: 0.93 +/- 0.06 units; cortisol: 0.32 +/- 0.03 units, p < 0.05), however it had no effect upon the expression of PGHS-2, EP(2), or EP(4) mRNA in fetal sheep kidney. Although there is substantial evidence that PGE(2) acting through either the EP(2) or EP(4) receptor stimulates renin synthesis in the adult kidney, our results have demonstrated that before the completion of nephrogenesis, cortisol down-regulation of renin mRNA expression is independent of any change in the expression of PGHS-2, EP(2), or EP(4) mRNA expression. During nephrogenesis, the insensitivity of PGHS-2, EP(2), and EP(4) expression to down-regulation by cortisol may permit continued PG regulation of renal development and urine formation.