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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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one-third of the preterm births ( Slattery & Morrison 2002 ). Therefore, delineating the mechanism underlying membrane rupture may provide novel targets for the prevention of preterm birth. Human fetal membranes are composed of amnion and chorion. The
Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
Mothers and Babies Research Centre, Department of Obstetrics and Gynaecology, University of Newcastle, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
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. 2006 , Li et al . 2011 ). We have assessed the methylation status of these genes in amnion collected from normal pregnancies at 11–17 weeks of gestation, at term (37–41 weeks) in the absence of labour and following term labour. Decidua was also
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, People’s Republic of China
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developed. The human fetal membranes, composed of the amnion and chorion, possess the largest capacity of cortisol regeneration through the reductase action of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) among fetal tissues ( Murphy 1977 , 1981
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We have examined the expression of the intercellular adhesion molecule-1 (ICAM-1) mRNA in primary and established amnion-derived cell cultures and regulation of this expression by tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta. TNF-alpha (50 ng/ml) and IL-1beta (1.0 ng/ml) induced 18- and 11-fold increases respectively in expression of the ICAM-1 mRNA in WISH cells (an amnion epithelium-derived cell line). The increase was detectable within one hour of treatment and peaked by two hours. The protein synthesis inhibitor, cycloheximide (10 microg/ml) did not inhibit this induction. Increased levels of ICAM-1 protein were detected in the cells within 4 h after initiation of treatment with either cytokine. By 16 h of treatment with IL-1beta or TNF-alpha ICAM-1 reached 40 and 73 pg/microg cellular protein, representing 6- and 11-fold stimulations respectively. In primary amnion cells, basal expression of ICAM-1 mRNA was undetectable. However, TNF-alpha (50 ng/ml) induced ICAM-1 mRNA within two hours, peak expression being reached between four and eight hours after initiation of treatment. The present report demonstrates for the first time that amnion derived cells can express ICAM-1 and, further, that this expression is regulated by pro-inflammatory cytokines. This has implications for the amnion as a possible source for soluble ICAM-1, for this gene product as a marker for preterm labour, and for participation of the amnion, additional to its reported secretory role, in inflammatory processes of the fetal membranes.
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We have evaluated the mechanism by which tumour necrosis factor-alpha (TNF-alpha) induces increased prostaglandin (PG) biosynthesis in amnion-derived WISH cells. WISH cells were treated with 50 ng/ml TNF-alpha or vehicle for 0-24 h. PGE2 production was stimulated by TNF-alpha within 2 h and continued to accumulate for at least 24 h. Increased prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression was evident within 30 min and was highest by 1 h, returning to unstimulated levels by 2 h. The PGHS-2 mRNA was re-induced at 8 h and was also elevated at 16 h. Immunoreactive PGHS-2 protein was nearly undetectable in control cells. However, within 30 min of TNF-alpha treatment, PGHS-2 protein was elevated and was induced for at least 16 h suggesting rapid production of both the PGHS-2 mRNA and protein. Transcription run-on assays indicated that the initial increase in the PGHS-2 mRNA was due to a 20-fold increase in the rate of transcription. The PGHS-2 mRNA decayed with an apparent half-life of 1 h in TNF-alpha-stimulated WISH cells. Induction of PGHS-2 expression proceeded in the presence of 10 microg/ml cycloheximide which agrees with the classification of PGHS-2 as an immediate early gene. These results indicate that a bi-phasic induction of the PGHS-2 mRNA is due, in part, to an initial transcriptional activation which results in rapid and continued synthesis of the PGHS-2 protein. This may be a unique characteristic of amnion cells which may be partially responsible for increased PG concentrations in the amniotic fluid during infection-associated preterm labour.
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Increased prostaglandin biosynthesis during intrauterine infection may be a possible mechanism by which preterm labour is initiated. Inflammatory cytokines and growth factors are known to stimulate prostaglandin production through an increase in prostaglandin endoperoxide H synthase (PGHS)-2 synthesis and activity. Interleukin-4 (IL-4), an anti-inflammatory cytokine, can downregulate PGHS-2 expression and inhibit prostaglandin production. Therefore, the aims of the current study were to determine the effects of IL-4 on PGHS-1 and PGHS-2 expression in amion-derived WISH cells treated with inflammatory cytokines and growth factors. In WISH cells, near-maximal production of the PGHS-2 mRNA occurred using 5 ng/ml EGF, 1 ng/ml IL-1beta or 50 ng/ml TNF-alpha. Time-course experiments determined that the PGHS-2 mRNA was induced maximally by these stimuli by 1 h. Pretreatment of WISH cells with IL-4 reduced PGHS-2 mRNA levels at 1 h by 67% in cells treated with EGF, 62% in cells treated with IL-1beta and 54% in cells treated with TNF-alpha. Pretreatment with IL-4 more effectively inhibited PGHS-2 expression than simultaneous addition with EGF or IL-1beta but not TNF-alpha. Immunoblot analysis showed a correlation between inhibition of mRNA levels and levels of PGHS-2 protein, although stimulation of PGHS-2 protein production by EGF was undetectable. Levels of PGHS-1 protein and mRNA remained unchanged in all experiments. Increased production of prostaglandin E2 (PGE2) in response to TNF-alpha and IL-1beta treatment was attenuated by IL-4 pretreatment, by 52% and 72%, respectively. No attenuation of EGF-stimulated PGE2 levels was seen. We conclude that IL-4 inhibits PGHS-2 mRNA and protein production in cytokine-stimulated WISH cells, but does not affect EGF-stimulated PGE2 production, suggesting that EGF can induce prostaglandin biosynthesis by a mechanism other than through increased PGHS-2 expression.
Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
National Research Centre for Growth and Development, University of Auckland, New Zealand
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Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
National Research Centre for Growth and Development, University of Auckland, New Zealand
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Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
National Research Centre for Growth and Development, University of Auckland, New Zealand
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Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
National Research Centre for Growth and Development, University of Auckland, New Zealand
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Tissues Total RNA preparations and total cellular protein lysates were derived from a previously prepared tissue bank of gestational tissues ( Keelan et al. 1999 , Blumenstein et al. 2002 ) stored at −75 °C. RNA and protein samples of amnion
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During human pregnancy, parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor are produced by the uterus, placenta, fetal membranes (amnion and chorion) and developing fetus. PTHrP alternative 3' mRNA splicing results in transcripts which encode three PTHrP isoforms and have been identified in amnion. Uteroplacental PTHrP expression is greatest in amnion and increases dramatically during late pregnancy. The aims of this study were to determine PTH/PTHrP receptor mRNA expression at preterm and term gestations and to determine 3' alternative splicing patterns in placenta, amnion and choriodecidua at preterm and term gestations. Using semiquantitative reverse transcription-polymerase chain reaction, PTHrP and PTH/PTHrP receptor transcripts were identified in preterm (n=5) and term (n=7) gestational tissues. PTH/PTHrP receptor mRNA expression did not differ between tissue types or change with advancing gestation. In contrast, PTHrP expression in the same tissues increased with advancing gestation and was significantly greater in amnion than in placenta and choriodecidua. Thus PTHrP, although produced predominantly in amnion, may act in amnion and other tissues including placenta, choriodecidua and myometrium. In amnion over placenta, transcripts encoding PTHrP 1-139 and 1-173 were detected in some preterm and all term samples and those encoding PTHrP 1-141 were detected in all samples. Similar results were obtained for reflected amnion. In placenta and choriodecidua, PTHrP 1-139 and 1-173 transcripts were undetectable or of low abundance. PTHrP 1-141 transcripts were detected in some placenta and choriodecidua samples. In summary, transcripts encoding PTHrP 1-141 appeared to be more abundantly expressed than those encoding PTHrP 1-139 or 1-173. However, the up-regulation of PTHrP expression in amnion at term may involve each of the alternative 3' mRNA splicing pathways since transcripts for each isoform appeared to be more consistently expressed at term.
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ABSTRACT
While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.
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Human labour is associated with increased prostaglandin synthesis within the fetal membranes. We have studied the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2), in human fetal membranes throughout pregnancy, at mRNA, protein and activity levels. COX-1 mRNA expression was low in human amnion and chorion-decidua and did not change with gestational age. COX-2 mRNA expression in fetal membranes increased with gestational age, with significant up-regulation prior to the onset of labour and in association with labour. Protein concentrations of COX-1 did not change, whilst concentrations of COX-2 increased from the first to the third trimester. COX activity increased with gestational age and in association with labour, although prostaglandin production in fetal membranes collected after labour was reduced, suggesting reduced substrate supply. These data suggest that it is up-regulation of COX-2, rather than of COX-1, which mediates increased prostaglandin synthesis within the fetal membranes at term. Much of the increase in COX-2 expression precedes the onset of labour, suggesting that it is a cause, rather than a consequence, of labour.
