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

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.

Glucose transporter protein responses to selective hyperglycemia or hyperinsulinemia in fetal sheep

2001 ◽  
Vol 281 (5) ◽  
pp. R1545-R1552 ◽  
Author(s):  
Marianne S. Anderson ◽  
Judy Flowers-Ziegler ◽  
Utpala G. Das ◽  
William W. Hay ◽  
Sherin U. Devaskar
Keyword(s):  
Glucose Transporter ◽  
Statistical Significance ◽  
Late Gestation ◽  
The Other ◽  
Utilization Rate ◽  
Fetal Sheep ◽  
Glut 1 ◽  
Transporter Protein ◽  
Glut 4 ◽  
Sustained Increase

The acute effect of selective hyperglycemia or hyperinsulinemia on late gestation fetal ovine glucose transporter protein (GLUT-1, GLUT-3, and GLUT-4) concentrations was examined in insulin-insensitive (brain and liver) and insulin-sensitive (myocardium and fat) tissues at 1, 2.5, and 24 h. Hyperglycemia with euinsulinemia caused a two- to threefold increase in brain GLUT-3, liver GLUT-1, and myocardial GLUT-1 concentrations only at 1 h. There was no change in GLUT-4 protein amounts at any time during the selective hyperglycemia. In contrast, selective hyperinsulinemia with euglycemia led to an immediate and persistent twofold increase in liver GLUT-1, which lasted from 1 until 24 h with a concomitant decline in myocardial tissue GLUT-4 amounts, reaching statistical significance at 24 h. No other significant change in response to hyperinsulinemia was noted in any of the other isoforms in any of the other tissues. Simultaneous assessment of total fetal glucose utilization rate (GURf) during selective hyperglycemia demonstrated a transient 40% increase at 1 and 2.5 h, corresponding temporally with a transient increase in brain GLUT-3 and liver and myocardial GLUT-1 protein amounts. In contrast, selective hyperinsulinemia led to a sustained increase in GURf, corresponding temporally with the persistent increase in hepatic GLUT-1 concentrations. We conclude that excess substrate acutely increases GURf associated with an increase in various tissues of the transporter isoforms GLUT-1 and GLUT-3 that mediate fetal basal glucose transport without an effect on the GLUT-4 isoform that mediates insulin action. This contrasts with the tissue-specific effects of selective hyperinsulinemia with a sustained increase in GURfassociated with a sustained increase in hepatic basal glucose transporter (GLUT-1) amounts and a myocardial-specific emergence of mild insulin resistance associated with a downregulation of GLUT-4.

scholarly journals Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

2012 ◽  
Vol 302 (12) ◽  
pp. E1483-E1492 ◽  
Author(s):  
Anne M. Maliszewski ◽  
Monika M. Gadhia ◽  
Meghan C. O'Meara ◽  
Stephanie R. Thorn ◽  
Paul J. Rozance ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Uptake ◽  
Late Gestation ◽  
Saline Control ◽  
Acid Mixture ◽  
Prolonged Infusion ◽  
Fetal Sheep ◽  
Leucine Oxidation ◽  
Pregnant Sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min−1·kg−1, P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min−1·kg−1 in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min−1·kg−1 in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min−1·kg−1 in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg−1·min−1, P < 0.05). The glucose-O2 quotient also decreased over time in the AA group ( P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon ( r2 = 0.38, P < 0.05), cortisol ( r2 = 0.31, P < 0.05), and NE ( r2 = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.

Decreases in ovine fetal cartilage sulfate uptake and serum sulfate during gestation

1985 ◽  
Vol 249 (1) ◽  
pp. E115-E120
Author(s):  
F. H. Morriss ◽  
R. N. Marshall ◽  
S. S. Crandell ◽  
B. J. Fitzgerald ◽  
L. Riddle
Keyword(s):  
Human Serum ◽  
Costal Cartilage ◽  
Full Term ◽  
Late Gestation ◽  
In Vitro Assays ◽  
Sulfate Uptake ◽  
Fetal Sheep ◽  
Ovine Fetal ◽  
Serum Sulfate

In vitro assays for [35S]sulfate uptake by ovine fetal costal cartilage were used to assess gestational changes in cartilage metabolism. Addition of 20% normal human serum to the incubation medium increased fetal cartilage [35S]sulfate incorporation into glycosaminoglycans. Both basal and human serum-stimulated uptakes of [35S]sulfate by fetal sheep cartilage decreased from midgestation to full term. The incremental response in [35S]sulfate uptake that was stimulated by human serum decreased as gestation proceeded to full-term. Fetal serum sulfate concentration decreased logarithmically during gestation, raising the possibility that cartilage sulfate uptake might become substrate limited as full term is approached. Perfusion of seven late gestation sheep fetuses for 7 days with Na2SO4 to achieve serum sulfate concentrations similar to those observed earlier in gestation resulted in a 33% increase in mean cartilage [35S]sulfate uptake compared with that of control twin fetuses, but uptake was not increased to values that occurred spontaneously earlier in gestation. These results suggest that the decreasing rate of [35S]sulfate uptake by fetal cartilage during the last half of gestation is associated only minimally with decreasing serum sulfate levels and is most consistent with intrinsic change in resting chondrocyte metabolism during gestation.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

Effects of Maternal Nutrient Restriction During Gestation on LH Production in the Female Bovine Fetus

2021 ◽  
Vol 99 (Supplement_2) ◽  
pp. 25-26
Author(s):  
Sterling H Fahey ◽  
Sarah West ◽  
John M Long ◽  
Carey Satterfield ◽  
Rodolfo C Cardoso
Keyword(s):  
Protein Content ◽  
Fetal Development ◽  
Monozygotic Twins ◽  
Late Gestation ◽  
Nutrient Restriction ◽  
Western Blots ◽  
Physiological Processes ◽  
Individual Potential ◽  
The Individual

Abstract Gestational nutrient restriction causes epigenetic and phenotypic changes that affect multiple physiological processes in the offspring. Gonadotropes, the cells in the anterior pituitary that secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are particularly sensitive to nutritional changes during fetal development. Our objective herein was to investigate the effects of gestational nutrient restriction on LH protein content and number of gonadotropes in the fetal bovine pituitary. We hypothesized that moderate nutrient restriction during mid to late gestation decreases pituitary LH production, which is associated with a reduced number of gonadotropes. Embryos were produced in vitro with X-bearing semen from a single sire then split to generate monozygotic twins. Each identical twin was transferred to a virgin dam yielding four sets of female twins. At gestational d 158, the dams were randomly assigned into two groups, one fed 100% NRC requirements (control) and the other fed 70% of NRC requirements (restricted) during the last trimester of gestation, ensuring each pair of twins had one twin in each group. At gestational d 265, the fetuses (n = 4/group) were euthanized by barbiturate overdose, and the pituitaries were collected. Western blots were performed using an ovine LH-specific antibody (Dr. A.F. Parlow, NIDDK). The total LH protein content in the pituitary tended to be decreased in the restricted fetuses compared to controls (P &lt; 0.10). However, immunohistochemistry analysis of the pituitary did not reveal any significant changes in the total number of LH-positive cells (control = 460±23 cells/0.5 mm2; restricted = 496±45 cells/0.5 mm2, P = 0.58). In conclusion, while maternal nutrient restriction during gestation resulted in a trend of reduced LH content in the fetal pituitary, immunohistological findings suggest that these changes are likely related to the individual potential of each gonadotrope to produce LH, rather than alterations in cell differentiation during fetal development.

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