Sexual dimorphism in miR-210 expression and mitochondrial dysfunction in the placenta with maternal obesity.

BACKGROUND: Maternal obesity is a major problem in obstetrics, and the placenta is involved in obesity-related complications via its roles at the maternal-fetal interface. We have recently shown a causative role for micro(mi)RNA-210, a so called 'hypoxamir' regulated by HIF-1α, in mitochondrial dysfunction in placentas from women with preeclampsia. We also reported mitochondrial dysfunction in placentas with maternal obesity. Here we hypothesized that expression of miR-210 is dysregulated in the placentas with obesity. METHODS: Placentas from uncomplicated pregnancies were collected at term from healthy weight or control (CTRL, pre-pregnancy body mass index (BMI)<25), overweight (OW, BMI=25-24.9) and obese (OB, BMI>30) women following C-section with no labor. Expression of miRNA-210 and its target genes was measured by reverse transcription-PCR and Western Blot, respectively. Mitochondrial respiration was assessed by Seahorse Analyzer in syncytiotrophoblast (ST) 72 h after cytotrophoblast isolation. RESULTS: Expression of miR-210 was significantly increased in placentas of OB and OW women with female but not male fetuses compared with CTRL placentas of females. However, expression of HIF-1α in these placentas remained unchanged. Levels of tumor-necrosis factor-alpha (TNFα) were increased in OW and OB placentas of females but not males, and in silico analysis suggested that activation of miR-210 expression in these placentas might be activated by NFκB1 (p50) signaling. Indeed, chromatin Immunoprecipitation assay showed that NFkB1 binds to placental miR-210 promoter in a fetal sex-dependent manner. Female but not male STs treated with TNFα showed overexpression of miR-210, reduction of mitochondrial target genes and decreased mitochondrial respiration. Pre-treatment of these STs with small interfering RNA to NFkB1 or antagomiR-210 prevented the TNFα-mediated inhibition of mitochondrial respiration. CONCLUSIONS: Our data suggest that the inflammatory intrauterine environment associated with maternal obesity induces an NFκB1-mediated increase in miR-210 in a fetal sex-dependent manner, leading to inhibition of mitochondrial respiration and placental dysfunction in the placentas of female fetuses.

Prenatal vitamin C and E supplementation in smokers is associated with reduced placental abruption and preterm birth: a secondary analysis.

OBJECTIVE: Smoking and pre-eclampsia (PE) are associated with increases in preterm birth, placental abruption and low birthweight. We evaluated the relationship between prenatal vitamin C and E (C/E) supplementation and perinatal outcomes by maternal self-reported smoking status focusing on outcomes known to be impacted by maternal smoking. DESIGN/SETTING/POPULATION: A secondary analysis of a multi-centre trial of vitamin C/E supplementation starting at 9-16 weeks in low-risk nulliparous women with singleton gestations. METHODS: We examined the effect of vitamin C/E by smoking status at randomisation using the Breslow-Day test for interaction. MAIN OUTCOME MEASURES: The trial's primary outcomes were PE and a composite outcome of pregnancy-associated hypertension (PAH) with serious adverse outcomes. Perinatal outcomes included preterm birth and abruption. RESULTS: There were no differences in baseline characteristics within subgroups (smokers versus nonsmokers) by vitamin supplementation status. The effect of prenatal vitamin C/E on the risk of PE (P = 0.66) or PAH composite outcome (P = 0.86) did not differ by smoking status. Vitamin C/E was protective for placental abruption in smokers (relative risk [RR] 0.09; 95% CI 0.00-0.87], but not in nonsmokers (RR 0.92; 95% CI 0.52-1.62) (P = 0.01), and for preterm birth in smokers (RR 0.76; 95% CI 0.58-0.99) but not in nonsmokers (RR 1.03; 95% CI 0.90-1.17) (P = 0.046). CONCLUSION: In this cohort of women, smoking was not associated with a reduction in PE or the composite outcome of PAH. Vitamin C/E supplementation appears to be associated with a reduction in placental abruption and preterm birth among smokers.

Can changes in angiogenic biomarkers between the first and second trimesters of pregnancy predict development of pre-eclampsia in a low-risk nulliparous patient population?

OBJECTIVE: To determine if change in maternal angiogenic biomarkers between the first and second trimesters predicts pre-eclampsia in low-risk nulliparous women. DESIGN: A nested case-control study of change in maternal plasma soluble Flt-1 (sFlt-1), soluble endoglin (sEng) and placenta growth factor (PlGF). We studied 158 pregnancies complicated by pre-eclampsia and 468 normotensive nonproteinuric controls. SETTING: A multicentre study in 16 academic medical centres in the USA. POPULATION: Low-risk nulliparous women. METHODS: Luminex assays for PlGF, sFlt-1 and sEng performed on maternal EDTA plasma collected at 9-12, 15-18 and 23-26 weeks of gestation. Rate of change of analyte between first and either early or late second trimester was calculated with and without adjustment for baseline clinical characteristics. MAIN OUTCOME MEASURES: Change in PlGF, sFlt-1 and sEng. RESULTS: Rates of change of PlGF, sEng and sFlt-1 between first and either early or late second trimesters were significantly different in women who developed pre-eclampsia, severe pre-eclampsia or early-onset pre-eclampsia compared with women who remained normotensive. Inclusion of clinical characteristics (race, body mass index and blood pressure at entry) increased sensitivity for detecting severe and particularly early-onset pre-eclampsia but not pre-eclampsia overall. Receiver operating characteristics curves for change from first to early second trimester in sEng, PlGF and sFlt-1 with clinical characteristics had areas under the curve of 0.88, 0.84 and 0.86, respectively, and for early-onset pre-eclampsia with sensitivities of 88% (95% CI 64-99), 77% (95% CI 50-93) and 77% (95% CI 50-93) for 80% specificity, respectively. Similar results were seen in the change from first to late second trimester. CONCLUSION: Change in angiogenic biomarkers between first and early second trimester combined with clinical characteristics has strong utility for predicting early-onset pre-eclampsia.

Effect of increasing maternal body mass index on oxidative and nitrative stress in the human placenta.

Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta.

Prostaglandin F2-alpha receptor regulation in human uterine myocytes.

Investigations of the modulation of prostaglandin F(2alpha) receptor (FP) expression in primary cultures of human uterine myocytes showed that FP mRNA expression was reduced by progesterone, unaltered by cAMP (8-bromo cAMP or forskolin), but increased by the PKA antagonist H89. Interleukin (IL)-1beta, tumour necrosis factor-alpha and oxytocin increased FP mRNA expression and IL-6 and prostaglandin E(2) reduced FP mRNA expression. The changes in FP protein levels were similar to the mRNA responses. We found that the IL-1beta-induced increase in FP expression was mediated at least in part via protein kinase C (PKC), but was independent of mitogen-activated protein kinase, phospholipase C and PI3 kinase. Since IL-1beta activates NFkappaB, AP-1 and C/EBP, we over-expressed these transcription factors alone and in combination and found that only NFkappaB alone increased FP mRNA expression. Finally, we found that the IL-1beta-induced increase in FP expression was unaffected by progesterone and/or cAMP, but was accentuated by H89. These data suggest that the pregnancy-induced down-regulation in myometrial FP expression is mediated by progesterone and cAMP and that the increase with labour is induced by inflammatory cytokine activation of PKC and NFkappaB.

Protein nitration in placenta - functional significance.

Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a "molecular fingerprint" of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings.

Perfusion with lipopolysaccharide differently affects the secretion of interleukin-1 beta and interleukin-1 receptor antagonist by term and preterm human placentae.

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1beta into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p<0.001). However, there were no significant differences between IL-1beta levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1beta into the maternal compartment (p<0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p<0.001 and p=0.017, respectively). However, the capacity of preterm placentae to release IL-1beta and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1beta was expressed by both term and preterm placentae before and after perfusion (+/- LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/- LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/- LPS) did not affect IL-1Ra expression. The localization of IL-1beta and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1beta and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS.

Post-Translational Modifications of the P2X(4) purinergic receptor subtype in the human placenta are altered in preeclampsia.

P2X(4) receptors are activated by extracellular ATP to raise intracellular calcium, thus altering cell signalling. ATP release occurs under pathophysiological, stress and adverse cell conditions; these are all increased in preeclampsia. Although P2X(4) is abundantly expressed in normal placenta neither the differences in the amount of protein nor its post-translational modifications have been studied in placentae from pregnancies complicated by preeclampsia. Thus we examined P2X(4) protein expression, localization and post-translational modifications in normotensive controls, term and preterm preeclamptic placentae. Densitometric analysis of Western blots showed a significant increase in P2X(4) protein expression in both term (p=0.002) and preterm preeclamptic (p=0.0008) placental samples compared to normotensive controls however the tissue localization of this receptor subtype was unaltered across the groups. Our data showed that P2X(4) is a nitrated protein in the placenta and this nitration is upregulated in preterm preeclamptic placenta compared to normotensive controls (p=0.03). We also demonstrated that P2X(4) is heavily glycosylated in the placenta by deglycosylation with PNGase F which reduced the protein product size by 23 kDa. We propose that P2X(4) acts within the syncytiotrophoblast to alter intracellular calcium and subsequent signalling pathways thereby restoring placental cell homeostasis following ATP-induced changes during pathophysiological conditions such as preeclampsia. We also propose that the post-translational modifications of nitration and glycosylation are required for the normal functioning of P2X(4).

Expression of NADPH oxidase isoform 1 (Nox1) in human placenta: involvement in preeclampsia.

Increased oxidative stress in the placenta has been associated with preeclampsia (PE), a clinical syndrome involving placental pathology. The enzymatic sources of reactive oxygen species in the human placenta are as yet unidentified. We hypothesized that NADPH oxidase is a main source of reactive oxygen species in the placenta and its expression may change in PE. Employing RT-PCR, we have amplified a novel NADPH oxidase isoform Nox1 from human choriocarcinoma BeWo cells. Using polyclonal anti-peptide antiserum recognizing unique Nox1 peptide sequences, we identified by immunohistochemistry and cell fractionation that Nox1 protein localizes in the BeWo cell membrane structures. Immunohistochemistry of normal placental tissues showed that Nox1 was localized in syncytiotrophoblasts, in villous vascular endothelium, and in some stromal cells. At the immunohistochemical level Nox1 expression was significantly increased in syncytiotrophoblast and endothelial cells in placentas from patients with preeclampsia as compared to gestational age-matched controls. Western blot analysis of whole placental homogenate confirmed this increase. Our data suggests that increased Nox1 expression is associated with the increased oxidative stress found in these placentas.

Dexamethasone fails to inhibit the induction of cytosolic phospholipase A(2) expression by interleukin-1beta in cultured primary human amnion fibroblasts.

OBJECTIVE: To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts. DESIGN AND METHODS: Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity. RESULTS: Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts. CONCLUSION: The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.

Determination of non-bilayer phospholipid arrangements and their antibodies in placentae and sera of patients with hypertensive disorders of pregnancy.

Studies suggest that preeclampsia (PE) originates in the placenta and is associated with deficient trophoblast invasion of spiral arteries. The direct cause remains unknown, but preeclampsia is often associated with circulating factors that can induce generalized endothelial dysfunction. Antiphospholipid antibodies (APA) in circulation are also associated with vascular diseases. Although the quantification of APA is not currently used as a prognostic of the risk of PE, studies suggest that thrombophilias play a role in PE pathogenesis. In fact, the pathology of placentae from PE and Antiphospholipid syndrome patients is similar; atherosis, thrombosis and infarction, and endothelium activation represent the pathological mechanisms. We identified a new antibody which recognizes non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, and which triggered an autoimmune-like disease in mice. We evaluated the presence of NPA in the placentae and in sera, and whether NPA induced NPA antibodies in patients with hypertensive disorders of pregnancy (HDP). Results showed increased levels of NPA in the syncytiotrophoblast, extravillous cytotrophoblast, syncytial knots and the amnion epithelial cell membranes of the placenta, as well as increases in NPA and NPA antibodies in sera from HDP patients, when compared with controls. This suggests that NPA derived from placenta could be one of multiple factors associated with pregnancy pathologies.

IFPA meeting 2015 workshop report I: placental mitochondrial function, transport systems and epigenetics.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.

Activation of oestrogen receptor complexes: evidence for the distinct regulation of ligand and oligonucleotide binding sites.

The activation of the rat uterine oestrogen receptor has been measured in vitro by its binding to oligodeoxythymidylate cellulose (oligo(dT] and was found to be sensitive to the time and temperature of prior incubation of cytosol with oestradiol. The presence of 20 mM dithiothreitol promoted receptor activation and was partially inhibited by 10 mM molybdate; molybdate also inhibited the time- and temperature-dependent activation of receptor. The nucleotides GTP, ATP, ADP, CTP and UTP all promoted receptor activation; the effect of GTP was significantly greater than that of ATP. It is unlikely that phosphate donation is involved in receptor activation as the effects of GTP could be reproduced by p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate), while PPi was also effective in activating receptor. The results provide evidence for the distinct regulation of the oligonucleotide- and ligand-binding domains, since manipulations which promoted binding to oligo(dT) did not affect either ligand binding capacity or the rate constant and composition the biphasic dissociation of the ligand receptor complex.

Heme oxygenase expression in cultured human trophoblast cells during in vitro differentiation: effects of hypoxia.

Heme oxygenases (HO-1 and HO-2) are responsible for the production of carbon monoxide, a vasodilator. HO is important in controlling placental blood flow and expression can be sensitive to oxygen. We previously reported a reduction in HO-2 expression in placentae obtained from patients with pre-eclampsia or living at high altitude, both associated with placental hypoxia. Thus we hypothesized that HO expression in cultured trophoblasts would be altered by exposure to hypoxia. HO-1 and HO-2 expression was assessed in trophoblast cell cultures following exposure to different oxygen environments. Western blot analyses showed that HO-1 expression in syncytiotrophoblast was significantly lower than in cytotrophoblasts in standard conditions (p < 0.05). There was no difference in HO-1 expression in cytotrophoblasts transferred to 2% O2 for various times. However, exposure of syncytiotrophoblast cultures to hypoxia for 12 h resulted in a significant reduction in HO-1 expression (p < 0.05). HO-2 expression was not affected by exposure to hypoxia in either cytotrophoblast or syncytiotrophoblast cultures. Possible interpretations of these findings are that chronic hypoxia alone is not responsible for reduced HO-2 expression or a much longer exposure to chronic hypoxia (perhaps months) is required. This study also reinforces the complexities of HO regulation by oxygen.

Increased nitrotyrosine in the diabetic placenta: evidence for oxidative stress.

OBJECTIVE: To evaluate the presence of nitrotyrosine (NT) residues in placental villous tissue of diabetic pregnancies as an index of vascular damage linked to oxidative stress. RESEARCH DESIGN AND METHODS: Villous tissue was collected and flash frozen after delivery from 10 class C and D IDDM patients (37.9+/-3.2 weeks) and 10 normotensive pregnant individuals (37.5+/-3.8 weeks). Serial sections of tissue were immunostained with specific antibodies to NT, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and manganese superoxide dismutase (MnSOD). Sections were scored for intensity of immunostaining (0-3) by three observers blinded to the identity of tissue. RESULTS: All tissues demonstrated immunostaining for eNOS in both syncytiotrophoblast and stem villous vascular endothelium with no apparent differences between groups. Immunostaining for iNOS was seen in the villous stroma, but again was not different between the two groups. Significantly more intense NT staining was apparent in vascular endothelium and villous stroma (both P < 0.02) of diabetic placentas. The endothelium of large villous vessels of diabetic tissues also showed more intense immunostaining for MnSOD (P < 0.01). CONCLUSIONS: In these diabetic pregnancies, we were unable to show increased eNOS, unlike previous findings in preeclamptic pregnancies. The presence of NT may indicate vascular damage in the diabetic placenta due to peroxynitrite action formed from increased synthesis/interaction of nitric oxide and superoxide. The apparently paradoxical increase in MnSOD expression may be an adaptive response to increased superoxide generation.

Nitrotyrosine residues in placenta. Evidence of peroxynitrite formation and action.

The interaction of nitric oxide and superoxide produces peroxynitrite anion, a strong, long-lived oxidant with pronounced deleterious effects that may cause vascular damage. The formation and action of peroxynitrite can be detected by immunohistochemical localization of nitrotyrosine residues. We compared the presence and localization of nitrotyrosine and of the endothelial isoform of nitric oxide synthase in placental villous tissue from normotensive pregnancies (n = 5) with pregnancies complicated by preeclampsia (n = 5), intrauterine growth restriction (n = 5), and preeclampsia plus intrauterine growth restriction (n = 4), conditions characterized by increases in fetoplacental vascular resistance, fetal platelet consumption, and fetal morbidity and mortality. In all tissues, absent or faint nitrotyrosine immunostaining but prominent nitric oxide synthase immunostaining were found in syncytiotrophoblast. In tissues from normotensive pregnancies, faint nitrotyrosine immunostaining was found in vascular endothelium, and nitric oxide synthase was present in stem villous endothelium but not in the terminal villous capillary endothelium. In contrast, in preeclampsia and/or intrauterine growth restriction, moderate to intense nitrotyrosine immunostaining was seen in villous vascular endothelium, and immunostaining was also seen in surrounding vascular smooth muscle and villous stroma. The intensity of nitrotyrosine immunostaining in preeclampsia (with or without intrauterine growth restriction) was significantly greater than that of controls. Intense nitric oxide synthase staining was seen in endothelium of stem villous vessels and the small muscular arteries of the terminal villous region in these tissues and may be an adaptive response to the increased resistance. The presence of nitrotyrosine residues, particularly in the endothelium, may indicate the formation and action of peroxynitrite, resulting in vascular damage that contributes to the increased placental vascular resistance.

Prostaglandin F2alpha potentiates cortisol production by stimulating 11beta-hydroxysteroid dehydrogenase 1: a novel feedback loop that may contribute to human labor.

In human pregnancy, cortisol and PGs are involved in the onset of labor and play an important role in the mechanisms leading to parturition. Recent studies have shown that at term, cortisol increases PG synthesis and decreases PG metabolism in chorion trophoblast (CT) cells. In CT, 11 beta-hydroxysteroid oxidase type 1 (11 beta-HSD1) converts biologically inactive cortisone to cortisol to regulate cortisol availability. In the present study, we have investigated whether 11 beta-HSD1 activity could be influenced by PGs. We have shown that in CT, PGF2alpha rapidly increased 11 beta-HSD1 reductase activity in a dose-dependent manner via the PGF2alpha receptor, localized in the fetal membranes. PGF2alpha stimulated 11 beta-HSD1 activity through increased intracellular calcium mobilization, activation of PKC, and the phosphorylation of the 11 beta-HSD enzyme. We propose that within CT there is a novel feed forward loop by which PGF2alpha acts to promote cortisol production from cortisone through increases in 11beta-HSD1, and this in turn leads to further net PG output for the onset of labor and birth.

Differential localization of superoxide dismutase isoforms in placental villous tissue of normotensive, pre-eclamptic, and intrauterine growth-restricted pregnancies.

Several isoforms of superoxide dismutase (SOD), including copper/zinc (cytosolic) and manganese (mitochondrial), exist. In the human placenta, SOD may prevent excessive superoxide accumulation and any potential deleterious oxidative effects. In pre-eclampsia, increased levels of lipid peroxide and decreased SOD activity have been described in the placenta. Oxidative stress such as occurs in pre-eclampsia can alter expression of SOD isoforms. The objective of this study was to localize the copper/zinc and manganese SOD isoforms in the placenta using immunohistochemistry and to compare localization and intensity of immunostaining in tissues from normotensive pregnancies with those from pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR). Western blotting with specific antibodies recognized a 17-kD copper/zinc and a 23-kD manganese SOD subunit in placental homogenates. Intense immunostaining for the manganese SOD isoform was seen in villous vascular endothelium, but only faint staining was found in the syncytiotrophoblast or villous stroma. In serial sections, intense immunostaining for copper/zinc SOD was seen in certain cells of the villous stroma but only faint immunostaining in syncytiotrophoblast and vascular endothelium. No apparent differences in localization or intensity of immunostaining for either isoform were seen between tissues of normotensive or pre-eclamptic pregnancies, with or without IUGR. The different cellular localizations of the SOD isoforms suggest that they fulfill different functional roles within the placenta.

Alterations in progesterone receptors in the rat uterus bearing an intra-uterine device during the oestrous cycle and early pregnancy.

The binding characteristics, content and intracellular distribution of cytosolic and nuclear progesterone receptors have been investigated, using [3H]progesterone as ligand, in the rat uterus bearing a unilateral intra-uterine device (IUD) during the oestrous cycle and from days 3 to 6 of pregnancy. The dissociation constants of nuclear and cytosolic progesterone-receptor complexes for IUD-containing and control uterine horns were similar. Cytosolic receptor concentrations in the IUD-containing uterus were always lower but changed in a manner similar to the control during the periods studied. Nuclear receptor concentrations in the control horn reflected changes in hormone levels during the oestrous cycle although concentrations measured were greater than previously reported. However, in IUD-containing uteri the pattern was completely reversed with minimal levels at pro-oestrus. Nuclear receptor concentrations were little different in both horns during early pregnancy. Total progesterone receptor synthesis determined between metoestrus and pro-oestrus in IUD-containing horns was significantly less than that of control horns. This correlated with the attenuated rise of nuclear oestrogen receptor levels previously observed between these times in IUD-containing uterine horns.