scholarly journals Localisation and temporal changes in prostaglandin G/H synthase-1 and -2 content in ovine intrauterine tissues in relation to glucocorticoid-induced and spontaneous labour

2000 ◽  
Vol 165 (2) ◽  
pp. 399-410 ◽  
Author(s):  
WJ McLaren ◽  
IR Young ◽  
GE Rice
Keyword(s):  
Time Course ◽  
Polyclonal Antibodies ◽  
Placental Tissue ◽  
Fold Increase ◽  
Fetal Membranes ◽  
Western Blots ◽  
Protein Levels ◽  
Tissue Extracts ◽  
Term Labour ◽  
Labour Onset

Parturition in the ewe is preceded by an increase in the synthesis of prostaglandins (PGs) by gestational tissues. To establish the uterine source of these PGs, placental cotyledons, fetal membranes and maternal uterine tissues were collected from ewes (n=6) at spontaneous parturition. Solubilised tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PG G/H synthase-1 and -2 (PGHS-1 and PGHS-2). PGHS-1 was expressed by all intrauterine tissues at term labour. Densitometric analysis of Western blot autoradiographs showed that the fetal membranes and maternal cervix contained the largest amounts of PGHS-1. PGHS-1 enzyme content of ovine amnion was significantly greater than that of either chorion or allantois (P<0.05). PGHS-1 protein content of myometrial, endometrial and cotyledonary tissue extracts was minimal. Formation of the PGHS-2 isozyme was confined to placental tissue at term labour. PGHS-2 protein levels in sheep placenta were significantly higher than those of PGHS-1 in all intrauterine tissues examined. This result supports the hypothesis that PGHS-2 is a major contributor to PG formation at term labour. To elucidate the developmental changes in PGHS-1 and PGHS-2 relative to labour onset, an experimental paradigm of glucocorticoid-induced delivery was used. Previous characterisation and validation of this labour model demonstrated that direct, transabdominal, intrafetal injection of the synthetic glucocorticoid betamethasone (5.7 mg in 1 ml aqueous vehicle) on day 131 of gestation induced labour onset in 56.6+/-0.8 h (mean+/-s.e.m.). As the latent period to induced-labour was known, the time course of enzyme formation could be ascertained. Sheep (n=20) were killed by barbiturate injection at various time intervals post-injection (0, 14, 28, 42 and 56 h). Tissue extracts collected at post-mortem examination were prepared and analysed by Western blots. PGHS-2 was induced in ovine cotyledon in a time-dependent fashion following glucocorticoid injection (P<0.05). There was a 12-fold increase in abundance between the time of betamethasone administration (0 h) and established labour (56 h). The PGHS-2 isozyme was not detected in any of the other tissues examined. In contrast, formation of the PGHS-1 isozyme did not change in relation to induced-labour in any of the intrauterine tissues. This finding is consistent with constitutive formation of PGHS-1. Previous studies have demonstrated a rise in PG production in association with glucocorticoid-induced labour and spontaneous delivery. The results of the present study indicate that this rise in PG production is due to increased formation of the PGHS-2 isozyme in ovine cotyledon. PGHS-2 appears to be induced by exogenous glucocorticoid administration and/or the mechanisms controlling ovine parturition. The role of PG formation by the fetal membranes is yet to be elucidated.

Expression of prostaglandin G/H synthase-1 and -2 in ovine amnion and placenta following glucocorticoid-induced labour onset

1996 ◽  
Vol 151 (1) ◽  
pp. 125-135 ◽  
Author(s):  
W J McLaren ◽  
I R Young ◽  
M H Wong ◽  
G E Rice
Keyword(s):  
Polyclonal Antibodies ◽  
Statistical Significance ◽  
Late Gestation ◽  
Saline Control ◽  
Electromyographic Activity ◽  
Western Blots ◽  
Fetal Sheep ◽  
Enhanced Expression ◽  
Term Labour ◽  
Labour Onset

Abstract Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized. This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection. Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N2 and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset. In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour. These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues. Journal of Endocrinology (1996) 151, 125–135

Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium

1997 ◽  
Vol 273 (6) ◽  
pp. C1937-C1946 ◽  
Author(s):  
James F. Collins ◽  
Hua Xu ◽  
Pawel R. Kiela ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Polyclonal Antibodies ◽  
Membrane Vesicle ◽  
Age Groups ◽  
Fold Increase ◽  
Jejunal Mucosa ◽  
Mrna Levels ◽  
Adult Rats ◽  
Intestinal Brush Border Membrane ◽  
Protein Levels

Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 μM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant ( K i) = 5 μM in PS120 fibroblasts] and NHE3 ( K i = 650 μM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 μM HOE-694 was 38–45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.

Labour-associated changes in adrenomedullin content in human placenta and fetal membranes

2003 ◽  
Vol 105 (4) ◽  
pp. 419-423 ◽  
Author(s):  
A. AL-GHAFRA ◽  
N. M. GUDE ◽  
S. P. BRENNECKE ◽  
R. G. KING
Keyword(s):  
Human Placenta ◽  
Mode Of Delivery ◽  
Placental Tissue ◽  
Fetal Membrane ◽  
Fetal Membranes ◽  
Tissue Extracts ◽  
Human Labour ◽  
Labour Group

The aim of the present study was to determine the effects of labour and mode of delivery on human placental and fetal membrane content of adrenomedullin (AdM). Placentas and fetal membranes were collected either at term or pre-term gestation from women either in labour or not in labour, and AdM was measured in tissue extracts by specific RIA. There were significant increases in AdM concentrations in amnion and choriodecidua for the in-labour group compared with the not-in-labour group for both pre-term and term gestations. There was no difference in AdM concentration in placental tissue between labour groups. This study provides evidence that fetal membrane AdM is increased in amniotic and choriodecidual tissues in response to labour, and suggests that it may play a role during human labour.

Measurement of interleukin-1 alpha and interleukin-6 in pregnancy-associated tissues

1996 ◽  
Vol 8 (7) ◽  
pp. 1069 ◽  
Author(s):  
L Gunn ◽  
P Hardiman ◽  
S Tharmaratnam ◽  
D Lowe ◽  
T Chard
Keyword(s):  
Amniotic Fluid ◽  
Interleukin 1 ◽  
Elective Caesarean Section ◽  
Maternal Blood ◽  
Fetal Membranes ◽  
Fetal Blood ◽  
Tissue Extracts ◽  
Arterial Blood ◽  
Term Labour ◽  
In Pregnancy

The concentrations of interleukin-1 alpha (IL-1 alpha) and IL-6 in pregnancy-associated tissues were investigated in term labour and delivery in the absence of labour (elective Caesarean section). Samples of amniotic fluid, placenta, fetal membranes, umbilical venous and, where possible, umbilical arterial blood were collected at delivery (37-41 weeks of gestation). Maternal blood was sampled during labour. Fluid and tissue extracts were assayed for IL-1 alpha and IL-6 by radioimmunoassay. Placenta and membranes were examined histologically for evidence of infection. Concentrations of IL-1 alpha and IL-6 in amniotic fluid and membrane extract, and IL-1 alpha in maternal and fetal blood, were raised after the onset of labour. Concentrations of both cytokines in the placenta remained unchanged. There was a good correlation between concentrations of both cytokines in amniotic fluid and membranes. There was also a significant correlation between concentrations of IL-1 alpha and IL-6 in amniotic fluid, placenta and membranes. It is suggested that the fetal membranes or maternal decidua, but not the placenta, internal fetal or maternal tissues, are the main sources of IL-1 alpha and IL-6 during labour.

Labour-associated increase in interleukin-1 α release in vitro by human gestational tissues

1996 ◽  
Vol 150 (3) ◽  
pp. 515-522 ◽  
Author(s):  
N Laham ◽  
S P Brennecke ◽  
K Bendtzen ◽  
G E Rice
Keyword(s):  
Amniotic Fluid ◽  
Time Course ◽  
Interleukin 1 ◽  
Maternal Plasma ◽  
Bacterial Endotoxin ◽  
Tissue Explants ◽  
Gestational Tissues ◽  
Term Labour ◽  
Labour Onset

Abstract The aims of this study were to investigate the concentration and release of interleukin-1α (IL-1α) at the time of human term labour, and to study the regulation of IL-1α release from human gestational tissue explants by bacterial endotoxin. Immunoreactive IL-1α concentrations in maternal plasma, amniotic fluid and conditioned media from human amniotic, choriodecidual and placental explants were quantified before and after spontaneous term labour-onset and delivery. Furthermore, the effects of a bacterial endotoxin, lipopolysaccharide (LPS), on the release of IL-1α from human gestational tissue explants over a time course of 24 h (n=3) and LPS concentrations ranging from 10–107 pg/ml (n=3) were investigated. IL-1α concentrations in maternal plasma and amniotic fluid did not change significantly with spontaneous term labour-onset. In contrast, IL-1α was released in detectable amounts from human amniotic and choriodecidual explants only in association with term labour-onset and delivery. Similarly, placental release of IL-1α was increased significantly in explant cultures in association with term labour-onset and delivery. LPS increased IL-1α release significantly only from human placental explants from both term not-in-labour and term after-labour tissues. The data demonstrate differential regulation of IL-1α release from human gestational tissues in association with labour and LPS treatment and the observations support the hypothesis that the labour-associated increase in IL-1α release from the fetal membranes is independent of exposure to bacterial endotoxin. Journal of Endocrinology (1996) 150, 515–522

scholarly journals PGAM-M expression is regulated pretranslationally in hindlimb muscles and under altered loading conditions

1999 ◽  
Vol 86 (1) ◽  
pp. 236-242 ◽  
Author(s):  
Rachel Kell ◽  
Heather Pierce ◽  
Steven J. Swoap
Keyword(s):  
Enzymatic Activity ◽  
Soleus Muscle ◽  
Fold Increase ◽  
Firefly Luciferase ◽  
Phosphoglycerate Mutase ◽  
M Gene ◽  
Specific Expression ◽  
Western Blots ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Enzymatic activity from the muscle-specific isoform of phosphoglycerate mutase (PGAM-M) is higher within glycolytic skeletal muscles than in oxidative muscles. The hypothesis that PGAM-M is regulated pretranslationally among muscles of the hindlimb was tested using enzymatic assays, Western blots, and Northern blots. We further investigated the regulatory level(s) at which PGAM-M gene expression is controlled during hindlimb unweighting. PGAM-M mRNA and immunoreactive protein levels were fourfold lower in the rat soleus muscle than in the tibialis anterior (TA), plantaris, and extensor digitorum longus muscles. Four weeks of unweighting induced a 2.5-fold increase in PGAM enzymatic activity within the soleus muscle, a 1.8-fold increase in PGAM-M immunoreactivity, and a 3.5-fold increase in PGAM-M mRNA. To examine potential transcriptional regulatory mechanisms, the proximal 400 bp of the rat PGAM-M promoter were linked to a firefly luciferase and injected into normal and unweighted TA and soleus muscles. Firefly luciferase activity was elevated two- to threefold in the TA and the unweighted soleus over the normal soleus muscle. These data suggest that PGAM-M expression is pretranslationally regulated among muscle types and within unweighted slow-twitch muscle. Furthermore, the proximal 400 bp of the PGAM-M promoter contains cis-acting sequences to allow muscle-type-specific expression of a reporter gene and responsiveness to soleus muscle unweighting.

scholarly journals Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF.

1993 ◽  
Vol 123 (3) ◽  
pp. 741-747 ◽  
Author(s):  
E Wilson ◽  
Q Mai ◽  
K Sudhir ◽  
R H Weiss ◽  
H E Ives
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Thymidine Incorporation ◽  
Polyclonal Antibodies ◽  
Mechanical Strain ◽  
Fold Increase ◽  
Cell Number ◽  
Neonatal Rat ◽  
Western Blots ◽  
Fourfold Increase

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF-A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.

Differential release of interleukin-6 from human gestational tissues in association with labour and in vitro endotoxin treatment

1996 ◽  
Vol 149 (3) ◽  
pp. 431-439 ◽  
Author(s):  
N Laham ◽  
S P Brennecke ◽  
K Bendtzen ◽  
G E Rice
Keyword(s):  
Amniotic Fluid ◽  
Interleukin 6 ◽  
Elective Caesarean Section ◽  
Fold Increase ◽  
Dependent Manner ◽  
Explant Cultures ◽  
Gestational Tissues ◽  
Term Labour ◽  
Labour Onset

Abstract In this study, we quantified interleukin-6 (IL-6) concentrations in amniotic fluid at term and preterm labour, and determined the gestational tissue source of IL-6. In addition, aspects of the regulatory mechanisms involved in IL-6 release at the time of term labour and in response to bacterial endotoxin, lipopolysaccharide (LPS), have been established. IL-6 concentrations were 2-fold higher in amniotic fluid collected at term compared with preterm gestation, with an additional 2-fold increase in association with term labour. IL-6 was released from all choriodecidual and placental explants but was detected in only 33% of amniotic explant cultures of tissues obtained before labour onset. In contrast, IL-6 was detected in all amniotic, choriodecidual and placental cultures of tissues obtained after term labour onset and delivery, and the mean IL-6 release was significantly higher than that measured in explant cultures of both amniotic (80-fold increase, P<0·0001) and choriodecidual (3-fold increase, P<0·02) but not placental explants taken at the time of elective Caesarean section at term before labour onset. LPS significantly (P<0·05) increased the release of IL-6 from human choriodecidual and placental explants but not amniotic explants, in a time- and dose-dependent manner. IL-6 is a physiological constituent of amniotic fluid and its production by gestational tissues is differentially regulated by LPS and spontaneous labour onset and delivery. Journal of Endocrinology (1996) 149, 431–439

Synthesis of gill Na+-K+-ATPase in Atlantic salmon smolts: differences in α-mRNA and α-protein levels

2000 ◽  
Vol 278 (1) ◽  
pp. R101-R110 ◽  
Author(s):  
Helena D'Cotta ◽  
Claudiane Valotaire ◽  
Florence le Gac ◽  
Patrick Prunet
Keyword(s):  
Atlantic Salmon ◽  
Atpase Activity ◽  
Early Stage ◽  
Polyclonal Antibodies ◽  
Mrna Abundance ◽  
Activity Measurement ◽  
Mrna Levels ◽  
Western Blots ◽  
Α Subunit ◽  
Protein Levels

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na+-K+-ATPase activity of Atlantic salmon smolts. A major α-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The α-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in α-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na+-K+-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in α-mRNA abundance and had basal levels of α-protein and enzyme activity. Measurement of the binding of [3H]ouabain to Na+-K+-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20–23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in α-mRNA levels after 24 h with a rise in Na+-K+-ATPase activity occurring only after 11 days. No qualitative change in α-expression was revealed at either the mRNA or protein level. Immunological identification of the α-protein was performed with polyclonal antibodies directed against the rat α-specific isoforms, revealing that parr, freshwater, and seawater smolts have an α3-like isoform. This study shows that the increase in Na+-K+-ATPase activity in smolt gills depends first on an increase in the α-mRNA expression and is followed by a slower rise in α-protein abundance that eventually leads to a higher synthesis of Na+-K+pumps.

scholarly journals Activin A and activin receptors in gestational tissue from preeclamptic pregnancies

2001 ◽  
Vol 171 (1) ◽  
pp. 57-64 ◽  
Author(s):  
U Manuelpillai ◽  
M Schneider-Kolsky ◽  
A Dole ◽  
EM Wallace
Keyword(s):  
Activin A ◽  
Maternal Serum ◽  
Receptor Protein ◽  
Fetal Membranes ◽  
Maternal Circulation ◽  
Western Blots ◽  
Activin Receptor ◽  
Protein Levels ◽  
A Subunit ◽  
Gestational Tissues

Maternal serum activin A levels are elevated in women with preeclampsia. To explore whether this could be due, at least in part, to increased production by the gestational tissues, we have measured activin A in the serum of women with (n=23) or without preeclampsia (n=62) at 29-40 weeks of gestation and in placenta and fetal membranes from preterm preeclamptic (PT-PE, n=8), term preeclamptic (T-PE, n=10) and healthy term controls (T-C, n=10). We have also explored if there are associated changes in activin receptor Alk2, ActRII and ActRIIB in these tissues. The relative amounts of receptor proteins were measured by densitometry on Western blots and receptors and activin beta(A) subunit localised by immunohistochemistry in PT-PE, T-PE and T-C gestational tissues (n=8-10/group). Maternal serum activin A levels were significantly elevated in women with preeclampsia (multiples of the normal median (MoM)=3.5, P<0.0001, Mann-Whitney U test) compared with healthy women (median MoM=1.0). Compared with control tissues, the activin A content was significantly higher in preeclamptic placentae (P=0.001 and P=0.0005 for PT-PE and T-PE respectively, Mann-Whitney U test), but significantly lower in the amnion (P=0.005 and P=0.014 for PT-PE and T-PE respectively) and choriodecidua (P=0.009 for T-PE). The maternal serum activin A level in women with preeclampsia was significantly correlated with elevated placental production (P=0.01, Pearson's correlation). Receptor Alk2 protein levels were significantly elevated in T-PE placentae (P=0.0006, Mann-Whitney U test), ActRIIB levels were significantly lower in PT-PE placentae (P=0.01) and ActRII levels were significantly lower in PT-PE choriodecidua (P=0.0002) compared with controls. There were no apparent differences in the distribution of the beta(A) subunit and receptors Alk2, ActRII and ActRIIB between control and preeclamptic tissues. These findings suggest that elevated levels of activin A in the maternal circulation in association with preeclampsia are due, at least in part, to increased placental production, and that the regulation of activin synthesis in placenta and fetal membranes is differentially regulated. Further, the differences in activin receptor protein levels between preeclamptic and control placenta and choriodecidua suggest that activin A-induced regulation may be altered in preeclampsia.

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