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Medical Research Council Group in Fetal and Neonatal Health and Development; Departments of Physiology and Obstetrics and Gynecology, University of Toronto (W.L.W., A.C.H., S.J.L., J.R.G.C.), Toronto, Ontario, Canada M5A 1A8; and Department of Obstetrics and Gynecology, University of Ottawa W.G.), Ottawa, Canada
Address all correspondence and requests for reprints to: Dr. W. L. Whittle, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5A 1A8. E-mail: wendy.whittle{at}utoronto.ca
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were quantified by RIA.
PGHS-II messenger RNA (mRNA) and protein expression were determined by
in situ hybridization and Western blot analysis,
respectively. Data were analyzed by ANOVA (P 
0.05). Labor-type uterine contractions were present after 68 h of
cortisol infusion and had increased significantly by 80 h.
Labor-type uterine contractions were induced after 68 h of
cortisol plus 4-OHA infusion, but the contraction frequency remained
less than that in the cortisol-treated animals. Fetal cortisol infusion
increased fetal and maternal plasma E2 concentrations and
decreased the maternal plasma P4 concentration
significantly; concurrent 4-OHA infusion attenuated the increase in
fetal and maternal plasma E2, but not the decrease in
maternal plasma P4. The fetal plasma PGE2
concentration increased after both cortisol and cortisol plus 4-OHA
infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2
concentration rose after fetal cortisol infusion, but not after
cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and
protein expression were increased significantly after both cortisol and
cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein
expression increased after cortisol infusion, but not after cortisol
plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine
activity pattern, and intrauterine PGHS-II expression were not altered
in either control group. We conclude that these data suggest distinct
pathways of intrauterine PG synthesis: a
cortisol-dependent/E2-independent mechanism within
trophoblast tissue leading to elevations in fetal plasma
PGE2, and an E2-dependent mechanism within
maternal endometrium that leads to increased maternal plasma
PGF2 and appears necessary for uterine activity and
parturition. |
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, as well as triggers the expression of a
specific cassette of contraction-associated proteins (CAPs) within the
myometrium (2). Consequently, myometrial contractility is
stimulated, and labor and delivery of the fetus ensue. However, recent
evidence has led us to question this concept. Studies examining the natural ontogeny of intrauterine PG production during late gestation and the onset of labor have found that the rise in fetal plasma PGE2 concentration occurred with a time course similar to that of the rise in fetal plasma cortisol and preceded the rise in both fetal and maternal plasma E2 concentrations (3). The expression of P450C17hydroxylase, the rate-limiting enzyme of E2 synthesis from C21 precursors in the ovine placenta, did not increase until the onset of early labor, well after the rise in fetal plasma PGE2 concentration and the increase in placental PGH synthase II (PGHS-II) expression. In addition, intrafetal E2 infusion failed to increase the expression of PGHS-II messenger RNA (mRNA) in the sheep placenta, although intrafetal administration of cortisol did increase placental PGHS-II expression and plasma PG levels (4, 5). These observations suggested that cortisol, but not E2, might increase placental trophoblast PGHS-II expression and activity to produce PGE2.
In related studies we also found that changes in maternal plasma
concentrations of 13,14-dihydro-15-keto-PGF2
(PGFM) were correlated with increased endometrial PGHS-II expression,
maternal plasma E2 levels, and uterine activity
(3, 6). Recently, E2 has been shown
to increase PGHS-II expression significantly in nonpregnant ovine
myometrium and in nonpregnant ovine endometrium after progesterone
priming (7). These observations suggested that placental
E2 may stimulate nontrophoblast intrauterine
tissue PGHS-II expression/activity to produce
PGF2, which, in turn, may contribute to
uterine activity.
Therefore, we hypothesized that there might be two separate pathways of intrauterine PG production: a cortisol-dependent/ E2-independent pathway within fetal placental trophoblast tissue, and an E2-dependent pathway within maternal intrauterine tissues. To test this hypothesis, we infused late gestation sheep fetuses with cortisol in the presence and absence of the aromatase inhibitor 4-hydroxyandrostendione (4-OHA) and determined changes in placental and uterine PGHS-II expression and PG output.
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Materials and Methods |
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Experimental protocol
Beginning on days 125–128 of gestation, fetuses received a
continuous infusion of saline (3 ml/h; n = 10) or cortisol (1.35
mg/h, in the same volume of infusate; n = 10; Steraloids, Wilton,
NH). After 24 h of infusion, five animals in each group received
an additional intrafetal infusion of Lentaron (1.44 mg/h 4-OHA, a
competitive, suicide inhibitor of the
P450aromatase enzyme; Ciba-Geigy;
Basel, Switzerland) (9). Fetal and maternal arterial blood
samples were collected at 12-h intervals beginning 24 h before the
start of the infusion protocol and continued through the infusion
period. Blood to be used for the determination of
E2 and progesterone (P4)
was collected into syringes rinsed previously with heparinized saline;
blood to be used for the determination of PGE2
and 13,14-dihydro15-keto-PGF2 (PGFM) was
collected in heparinized syringes and then transferred to vials
containing indomethacin (200 µl 1 mg/ml INDOCID; Merck Sharp and
Dohme, Kirkland, Canada). Plasma was separated from blood cells by
centrifugation at 1500 x g for 10 min at 4 C. Plasma
samples were frozen at -20 C for subsequent assay.
Uterine muscle electromyogram activity was processed by a Grass
wide-band AC preamplifier (model 7P511J) and was recorded using a Grass
78D EEG and polygraph data recording system (Grass Instruments, Quincy,
MA). Uterine activity monitoring began 24 h before the start of
the infusion protocol and continued for the infusion period. Uterine
contractures were defined as a low amplitude uterine activity pattern
(duration, 5–8 min; frequency, 
2–3/h); uterine contractions were
defined as a high amplitude activity pattern (duration, <1 min;
frequency, 
30/h) (3).
From preliminary studies (data not shown) it was determined that an intrafetal cortisol infusion (1.35 mg/h) for a period of about 80 h was sufficient to induce a uterine contraction pattern consistent with labor. After completion of an 80-h infusion period, a terminal plasma sample was taken, then each animal was killed with an overdose of Euthanyl (sodium pentobarbital, MTC Pharmaceuticals, Cambridge, Canada), and intrauterine tissues were collected. Placental cotyledons were dissected from the uterine muscle and fetal membranes; a single piece of intercotyledonary endometrium was bluntly peeled from the myometrium at the level of the miduterine horn. An adjacent cross-sectional piece of uterine tissue, including myometrium, endometrium, chorion, and amnion, was cut, rolled, and collected. Tissues were snap-frozen in liquid nitrogen for subsequent Western blot analysis and slow frozen over dry ice for in situ hybridization studies.
RIA
The extraction of plasma samples and the RIA for fetal plasma
PGE2 and maternal plasma PGFM and
P4 were conducted as previously described and
validated (10, 11, 12). The intraassay coefficients of
variation were 8%, 4%, and 10%, respectively. The RIA for fetal and
maternal plasma E2 was performed using a
commercially available125I RIA kit (ImmuChem
Double Antibody 17ßEstradiol 125I RIA Kit,
ICN Biomedicals, Inc., Costa Mesa, CA) validated for use
with ovine plasma. The intraassay coefficient of variation was 3%.
Fetal plasma androstenedione concentrations were measured using a
commercially available 125I RIA kit (ImmuChem
Double Coated Tube Androstenedione 125I RIA Kit,
ICN Biomedicals, Inc.). The sensitivity of the assay was
0.12 pg/ml. The specificity was provided by the manufacturer; the main
cross-reacting steroids were dehydroepiandrosterone, androsterone,
testosterone, and estrone, with 2.08%, 1.96%, 0.83%, and 0.2%
cross-reactivities, respectively. The intraassay coefficient of
variation was 12%.
In situ hybridization
Sense and antisense probes based on the structure of the human
PGHS-II gene were synthesized by the University of Ottawa Molecular
Biology Department (Ottawa, Canada) using an Oligo1000 DNA synthesizer
(Beckman Coulter, Inc., Mississauga, Canada). The
oligonucleotide sequence for the PGHS-II probe was GGG ACA GCC CTT CAC
GTT ATT GCA GAT GAG AGA CTG AAT TGA GGC AGT GT, corresponding to
nucleotides 1734–1783 of the human PGHS-II. Northern analysis was used
to confirm that the probe recognized the 4.5-kb transcript of the
PGHS-II. The probe was labeled with terminal deoxynucleotidyl
transferase (Life Technologies, Inc., Burlington, Canada)
and 33P-labeled deoxy--thio-ATP (1300 Ci/mmol;
NEN Life Science Products, DuPont Canada, Inc.,
Mississauga, Canada). The probe was used at a concentration of
approximately 600 cpm/µl.
The method used for in situ hybridization has been described previously (13). Briefly, tissue sections (10 µm) were mounted on Fisher SuperFrost glass slides (Fisher Scientific, Nepean, Canada), fixed with 4% paraformaldehyde, dehydrated through graded ethanol, and stored in 95% ethanol at 4 C. Slides were removed from ethanol and allowed to air-dry at room temperature. Tissues were incubated overnight in a moist incubation chamber at 42 C with the radiolabeled oligonucleotide PGHS-II probe diluted in hybridization buffer. Hybridization buffer was composed of 4 x SSC (single strength 1 x SSC: 150 mM sodium chloride, 15 mM sodium citrate; Sigma; St. Louis, MO), 50% deionized formamide (Life Technologies, Inc., Burlington, Canada), 0.02% BSA (Roche Molecular Biochemicals, Dorval, Canada), 10% dextran sulfate (Pharmacia Biotech, Baie d’Urfe, Canada), 200 µg hydrolyzed salmon sperm DNA/ml, 0.02% polyvinylpyrrolidone, 40 mM dithiothreitol, and 50 mM sodium phosphate (pH 7.0; Sigma). After incubation, slides were washed with 1 x SSC at room temperature for 30 min and then with 1 x SSC at 45 C for 45 min. Slides were washed with decreasing strength SSC, dehydrated in ethanol, air-dried, and exposed to x-ray film ( Eastman Kodak Co., Rochester, NY). Placental tissue was exposed for 38 h, and myometrium/fetal membranes were exposed for 270 h. The autoradiographic films were developed using standard procedure. The linearity of the mRNA signals was established by simultaneous exposure of the samples with 14C-labeled standards in the appropriate range (RPA504, Amersham Pharmacia Biotech, Aylesbury, UK), and the OD of PGHS-II mRNA expression was determined relative to a curve established by these 14C-labeled standards. Nonspecific binding was established using a 45-mer nonsensical sequence oligonucleotide probe, and the signal was subtracted from the antisense PGHS-II mRNA signal; the specificity of the antisense PGHS-II probe was established by incubation with positive tissue controls (term ovine placentome and cultured human amnion cells, both previously shown to express PGHS-II mRNA) (3, 4). The autoradiograms were then analyzed using computerized image analysis software (Image Research, Inc., St. Catherines, Canada; Laser Scanner, Molecular Dynamics, Inc., Sunnyvale, CA; ImageQuant software, Becton Dickinson and Co., Mountain View, CA). The relative OD of placental and endometrial PGHS-II expression was assessed using nine tissue sections per animal.
Western blot analysis
Frozen tissue samples were homogenized on ice for 1 min in RIPA
lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1% (wt/vol) sodium deoxycholate, 0.1% sodium lauryl sulfate
(SDS), 100 µM sodium orthovanadate (Sigma),
1% (vol/vol) Triton X-100 (Fisher Chemicals, Fairlawn, NJ), and
Complete MiniEDTA-free protease inhibitors (Roche Molecular Biochemicals; Dorval, Canada)]. Homogenates were centrifuged at
4 C at 15,000 x g for 15 min, and supernatants were
collected. Protein concentrations were determined by the Bradford assay
(14) using BSA (Bio-Rad Laboratories, Inc.,
Richmond, CA) as the standard and protein absorbance at 595 nm.
Protein samples (25–100 µg) were separated by polyacrylamide gel (4–10% gradient) electrophoresis as described by Laemmli (15). Proteins were electrophoretically transferred to a 0.45-µm pure nitrocellulose membrane (Bio-Rad Laboratories, Inc.); transfer was confirmed by protein visualization with Ponceau S solution (Sigma). Blots were washed with PBS-T (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM NaH2PO4, and 0.1% Tween-20, pH 7.5; Sigma) and incubated overnight with blocking solution (5% skim milk powder in PBS-T). Blots were incubated with primary antibody for PGHS-II (1:500 dilution in blocking solution; PG27, Oxford Biomedical Research, Inc., Oxford, MA) or PGHS-I (1:4000 dilution in blocking solution; PG19, Oxford Biomedical Research Instruments, Inc.). Blots were then rinsed five times for 5 min each time with PBS-T and incubated with secondary antiserum conjugated with horseradish peroxidase for 1 h (1:1000 dilution in blocking serum; Amersham Pharmacia Biotech). Blots were washed six times for 5 min each time, and the antibody-antigen complex was detected using the Amersham Pharmacia Biotech ECL detection system (Amersham Pharmacia Biotech). Blots were exposed to x-ray film (Eastman Kodak Co.) for serial exposure times to determine the appropriate concentration of protein loading and exposure time to ensure that the protein signal was within the linear response range. The intensity of the protein signal was quantified using computerized image analysis software (Image Research, Inc.; laser scanner from Molecular Dynamics, Inc.; ImageQuant software).
Statistical analysis
Uterine contractility data were analyzed by two-way ANOVA
(repeated measures) followed by post-hoc Tukey’s test and
Student’s t test. Plasma data were analyzed by two-way
ANOVA (repeated measures), followed by post-hoc Tukey’s
test. PGHS-II mRNA and protein data were analyzed by one-way ANOVA
followed by post-hoc Tukey’s test. Significance was set at
P 0.05. Data are presented as the mean ±
SEM for n = 4 or 5/group.
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Results |
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). Labor-type uterine
contractions were present after 68 h of cortisol plus 4OHA
infusion and had increased significantly by the time of death (80 h;
Fig. 1). However, during the last 2 h before death, the
cortisol-treated animals had a significantly greater contraction
frequency (56.6 ± 6.7 contractions/2 h) compared with the animals
treated with cortisol and 4-OHA (26.1 ± 9.0 contractions/2 h).
The uterine contracture pattern was not altered at any time during the
infusion in either control group (Fig. 1).
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); the maternal
plasma E2 concentration was increased from
15 ± 2 to 217 ± 59 pg/ml (Fig. 3). The increases in both fetal and
maternal plasma E2 concentrations were attenuated
significantly by coinfusion with 4-OHA (Figs. 2 and 3). At the time of
death, the fetal plasma E2 concentration was 64%
less, and the maternal plasma E2 concentration
was 54% less than those in the cortisol infusion group. Fetal plasma
E2 (Fig. 3) and maternal plasma
E2 were not altered at any time during the
infusion period in either control group. The androstenedione
concentration in fetal plasma was 342 ± 41 pg/ml before the start
of the infusion. The concentration of androstenedione rose to 5340
± 1427 pg/ml in the animals treated with cortisol and 4-OHA, but did
not change significantly in any of the other animal groups (data not
shown). The mean maternal P4 concentration was
decreased significantly from 33 ± 3 to 9 ± 3 ng/ml at the
time of death in the cortisol-treated animals. Maternal plasma
P4 also decreased significantly in the cortisol-
plus 4-OHA-treated animals (Fig. 3). The ratio of
E2/P4 in maternal plasma
was increased from 0.46 to 23.4 by cortisol infusion. The
E2/P4 ratio change in the
animals coinfused with 4-OHA was attenuated and rose from 0.32 to only
2.3. There was no significant change in maternal plasma
P4 before and at the end of the infusion period
in either control group of animals (data not shown).
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) to 1618 ±
471 and 1241 ± 91 pg/ml, respectively; these values were not
statistically different from each other. The mean maternal plasma PGFM
concentration increased significantly from 83 ± 40 to 423 ±
85 pg/ml at the time of death in the cortisol-infused animals. However,
in the cortisol plus 4-OHA-treated animals, the basal PGFM
concentration was not increased significantly at the end of the
infusion period (basal PGFM concentration, 149 ± 22 pg/ml;
terminal PGFM concentration, 157 ± 49 pg/ml; Fig. 5). The plasma PGE2
and PGFM concentrations in the control animals did not change
throughout the infusion period (Figs. 4 and 5).
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); there was a corresponding increase in
placental PGHS-II immunoreactive protein expression in both of these
groups of animals (Fig. 7). PGHS-II mRNA
expression in the intercotyledonary endometrium was increased
significantly after cortisol infusion, but not after cortsiol plus 4OHA
infusion. Similarly, endometrial immunoreactive PGHS-II protein
expression was increased after cortisol, but not after cortisol plus
4-OHA, infusion (Fig. 7). Placental and endometrial immunoreactive
PGHS-II protein migrated an equal distance under electrophoresis (data
not shown). Levels of immunoreactive PGHS-I protein expression in
placenta and endometrium were not significantly different among the
four groups of animals (Fig. 8).
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Discussion |
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production are dependent upon
increased placental E2 synthesis. Estrogen
synthesis is also required for the development of a full labor-like
pattern of uterine contractility.
We used Lentaron (4-OHA) to inhibit placental aromatase activity. 4-OHA
is a competitive inhibitor that irreversibly binds to the active site
of the aromatase enzyme (9). Nathanielsz et al.
(16) showed that maternal iv 4-OHA infusion inhibited
androgen-induced E2 synthesis in pregnant rhesus
monkeys, suggesting that 4-OHA could inhibit placental aromatase
activity. France et al. (17) showed that 4-OHA
could inhibit ovine placental aromatase activity in vitro
with a Ki of 0.05 µM; we
presume that this is the mechanism of the 4-OHA effect in the present
study. There was no effect on basal steroid or PG concentrations in the
animals receiving 4-OHA but not cortisol. The androstenedione
concentration was increased in the plasma of fetuses treated with
cortisol and 4-OHA. This increase was not observed in the other
treatment groups, consistent with precursor build-up after cortisol
infusion in the presence of 4-OHA and effective inhibition of placental
aromatase activity. Using this protocol we were able to block the
cortisol-induced increase in placental E2
production, and thereby we were able to determine the effects of
intrafetal cortisol on intrauterine PGHS-II expression and PG output in
the presence and absence of increased placental
E2 production. However, we cannot exclude the
effects of basal placental E2 production, nor can
we exclude the possibility that the increase in androstenedione
production and/or 4-OHA may have influenced endometrial PGHS-II
expression and PGF2 output in the animals
treated with cortisol plus 4-OHA.
Recently, glucocorticoids (GC) have been found to up-regulate PGHS-II
expression and activity in some trophoblast-derived cells
(18, 19, 20, 21), although inhibition of PGHS-II in amnion WISH
cells treated with GC has also been reported (22). The
effects of cortisol on ovine placental PGHS-II expression and
PGE2 output were independent of an increase in
placental E2 production. Ovine placental PGHS-II
mRNA and protein expression have been localized previously to the
mononuclear trophoblast cells (23), which also express the
glucocorticoid receptor (24). The postreceptor mechanism
by which GCs could regulate PGHS-II is not well understood; a specific
glucocorticoid response element within the 5'-promoter region of the
PGHS-II gene has been reported by two groups of investigators
(25, 26). The PGHS-II promoter also contains other
transcription factor-binding sites including two nuclear factor-
B
(NFB) sites (25, 26, 27). Although GCs have been shown to
have a suppressor effect at the NFB site, recent evidence has
suggested that GCs may interact with the NFB site to induce
expression of acute phase hepatic reactant genes (28). A
similar interaction could occur with PGHS-II. Alternatively, GCs could
increase PGHS-II expression through interference with repressor
transcription factors, by stimulation of promotional transcription
factors, or by affecting an increase in the stability of PGHS-II mRNA
(20, 21, 28, 29). Our present findings mimic the effects
of GCs observed in human amnion epithelial cells (18) and
amnion fibroblast cells (19). Mixed cultures of human
amnion cells increased PGE2 output in response to
cortisol and dexamethasone stimulation (20, 21, 29). This
stimulatory effect was receptor dependent and involved an increase in
PGHS-II expression. Thus, we suggest that the prepartum increase in
fetal adrenal cortisol output increases the expression of PGHS-II
within fetal ovine placental trophoblast cells, leading to
PGE2 production; this PG synthesis occurs
independently of an increase in placental E2
output.
In addition, we found that endometrial PGHS-II expression and
intrauterine PGF2 output, reflected in
maternal PGFM concentrations, were dependent upon placental
E2 synthesis. Previously, we were unable to
demonstrate an increase in sheep placental PGHS-II expression with
intrafetal E2 infusion (4). In
addition, PGHS-II expression and activity could not be stimulated by
E2 in cultured human trophoblast-derived cells
(30). However, PGHS-II expression can be stimulated by
E2 in other tissues, including human monocytes,
bovine oviduct, nonpregnant ovine endometrium, and myometrium (7, 31, 32). These data support a role for E2
in the regulation of PG synthesis by intrauterine, nontrophoblast
tissue, while arguing against a role for E2 in
the regulation of trophoblast PGHS-II. Recent studies have reported the
presence of the estrogen receptor (ER) within nontrophoblast
intrauterine tissues, including maternal placental villi, endometrium,
and myometrium of the sheep in late gestation. The ER was absent from
placental trophoblast cells (33). These data further
preclude an E2 effect on PGHS-II within the fetal
trophoblast tissue and are consistent with ER-mediated
E2 regulation of PGHS-II within the endometrium
of pregnant sheep. The PGHS-II promoter does not contain an estrogen
response element, but does contain the transcription factor-binding
site AP-1 (25, 26, 27). E2 has been
shown to interact with the AP-1 site to induce gene expression
(28). Thus, we suggest that placental
E2 up-regulates maternal endometrial PGHS-II
expression and PGF2 output at the onset of
ovine parturition; this effect may be direct, mediated by the ER. In
addition, early studies using nonpregnant sheep showed that
P4 treatment increased intrauterine PG synthetic
activity and was a prerequisite for the additional effects of
E2 (7, 34). Therefore, we must not
exclude the role P4 plays in the regulation of PG
production.
In addition to the observed changes in PGHS-II expression within the
fetal trophoblast and maternal endometrial tissue, we cannot exclude
possible changes in the expression and activity of other key enzymes in
the PG biosynthetic pathway, including PGE isomerase, PGF synthase, and
PG dehydrogenase (PGDH). To date, little information is available
regarding the expression and activity of these enzymes within ovine
intrauterine tissues. PGF synthase mRNA has recently been identified in
the ovine maternal placenta, endometrium, and myometrium
(35). The expression of this enzyme decreased only within
the endometrium during betamethasone-induced preterm labor and remained
unchanged in all three tissues with spontaneous term labor
(35). These data suggest that the increase in PGHS-II
expression may be more important than PGF synthase in the regulation of
PGF2 production at the onset of labor. PGDH
has been identified within the ovine fetal trophoblast and
maternal endometrium during pregnancy, and its activity increases
within the placenta at the time of active labor (36, 37).
In contrast, human chorionic and placental PGDH expression and activity
have been found to decrease with the onset of labor (38).
Using cultured chorionic and placental trophoblast cells,
P4 has been found to maintain PGDH
expression/activity, and cortisol has been shown to decrease PGDH
expression/activity (38). Evidence has suggested that at
term increasing cortisol concentrations compete with
P4 in the regulation of PGDH; the resultant
effect is a net decrease in PGDH expression, leading to an overall
increase in intrauterine PG production (39). In our
animals plasma fetal cortisol increased, and maternal plasma
progesterone decreased, suggesting that PGDH expression and activity
within the intrauterine tissues may decrease and contribute to the rise
of PGs. Given that maternal plasma PGFM levels did not increase in the
cortisol- plus 4-OHA-treated animals, increased cortisol and decreased
P4 may not be sufficient to suppress endometrial
PGDH expression/activity and lead to increased
PGF2 output. In addition,
E2 may play a role in the regulation of ovine
intrauterine PGDH expression/activity. The regulation of these key
enzymes requires further investigation.
Placental PGE2 production might play an important role in mediating fetal hypothalamic-pituitary-adrenal (HPA) axis activation and placental steroidogenesis at the onset of labor. Fetal plasma cortisol and PGE2 concentrations increase with a similar time course over the last 20 days of gestation and are associated in a positive feedback manner (40). Intrafetal PGE2 infusion increased fetal plasma cortisol and ACTH hormone concentrations in late gestation (41, 42). Recently, we have shown that specific inhibition of PGHS-II blocked the increase in fetal plasma cortisol and ACTH concentrations induced by RU486 administration in late gestation sheep (43). These data suggest that placental PGE2 may be important for sustaining activation of the HPA axis at the end of gestation and the onset of labor. In sheep, placental E2 production is mediated by the rate-limiting action of P450C17 hydroxylase, which catalyzes the conversion of C21 steroids to the C19 steroid precursors that will be aromatized to form estrogen (44, 45). Expression of placental P450C17 hydroxylase at the onset of labor occurs well after the increases in placental PGHS-II expression and PGE2 production (3, 6, 46). We propose that toward the end of gestation, fetal adrenal cortisol induces placental PGHS-II expression and PGE2 production; in turn, PGE2 may direct placental estrogen synthesis and act in a positive feedback loop to maintain fetal HPA activation through the onset of labor.
The frequency of uterine contractions was attenuated in the absence of
placental E2 production, although we did not find
a delay in the activation of uterine contractility from contractures to
contractions. Myometrial contractility is associated with induction of
a specific cassette of CAPs, including connexin 43, oxytocin receptor,
ion channels, and PG receptors (2); these proteins are
responsible for the evolution of uterine activity from quiescence to
contractility. We suggest that endometrial PGHS-II may also be
considered a CAP, contributing to this evolution of uterine activity.
Once CAP expression has been initiated, the uterus can be stimulated to
contract by a variety of uterotonins, in particular oxytocin and PGs
(1, 2, 4). CAP expression appears to be regulated by a
ratio of E2/P4 in late
gestation (2). In the present study the
E2/P4 ratio in the
cortisol-treated animals increased 24-fold at the end of the infusion
period, whereas the E2/P4
ratio of the cortisol- plus 4OHA-treated animals increased only 2-fold.
Thus, the lack of change in the
E2/P4 ratio and/or the lack
of increase in intrauterine PGF2 production
(7) observed in the cortisol- plus 4-OHA-treated animals
may have failed to induce CAP expression, thereby attenuating uterine
activity. Alternatively, CAPs may have been induced but uterine
contractility not initiated because the production of
PGF2 did not increase. These possibilities
remain to be evaluated.
Based on the observations of the present study we propose a new model
for the onset of parturition in sheep (Fig. 9). We suggest that toward the end of
gestation there is a gradual and sustained increase in the placental
trophoblast expression of PGHS-II expression and
PGE2 production under the regulation of fetal
cortisol produced from the maturation of the fetal HPA axis. Placental
PGE2, in turn, mediates an autocrine/paracrine
increase in placental P450C17hydroxylase
expression/activity to promote placental estrogen production and also
acts to sustain fetal HPA axis activation. Estrogen up-regulates the
expression of maternal endometrial PGHS-II and
PGF2 output as well as induces the expression
of CAPs. Consequently, myometrial contractility is stimulated, and
labor ensues. This hypothesis follows a tissue-specific progression of
parturition events from a fetal signal to a maternal labor
response.
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Acknowledgments |
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Footnotes |
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Received February 14, 2000.
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References |
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B
and steroid receptor-signaling pathways. Endocr Rev 20:435–459 and endothelin-1 from cow
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in betamethasone (bM) induced
premature labor (BL) and spontaneous term labor (STL) in pregnant
sheep. J Soc Gynecol Invest 7:267A (Abstract 801)
-hydroxylase/C-17,20 lyase,
aromatase and sulfatase in dexamethasone-induced and natural
parturition. J Endocrinol 122:351–359[Abstract]
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 Q. Zhang, V. Collins, K. Chakrabarty, R. F. Wolf, N. Unno, D. Howe, J. C. Rose, and W. X. Wu Regulation of Membrane-Associated Prostaglandin E2 Synthase 1 in Pregnant Sheep Intrauterine Tissues by Glucocorticoid and Estradiol Endocrinology, August 1, 2006; 147(8): 3719 - 3726. [Abstract] [Full Text] [PDF]
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G Schuler, G R Ozalp, B Hoffmann, N Harada, P Browne, and A J Conley Reciprocal expression of 17{alpha}-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis. Reproduction, April 1, 2006; 131(4): 669 - 679. [Abstract] [Full Text] [PDF]
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 H. K. Palliser, J. J. Hirst, G. T. Ooi, G. E. Rice, N. L. Dellios, R. M. Escalona, H. C. Parkington, and I. R. Young Prostaglandin E and F Receptor Expression and Myometrial Sensitivity at Labor Onset in the Sheep Biol Reprod, April 1, 2005; 72(4): 937 - 943. [Abstract] [Full Text] [PDF]
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 C. E. Wood Estrogen/Hypothalamus-Pituitary-Adrenal Axis Interactions in the Fetus: the Interplay Between Placenta and Fetal Brain Reproductive Sciences, February 1, 2005; 12(2): 67 - 76. [Abstract] [PDF]
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 Z Cheng, M Elmes, S E Kirkup, E C Chin, D R E Abayasekara, and D C Wathes The effect of a diet supplemented with the n-6 polyunsaturated fatty acid linoleic acid on prostaglandin production in early- and late-pregnant ewes J. Endocrinol., January 1, 2005; 184(1): 165 - 178. [Abstract] [Full Text] [PDF]
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 V. Kumarasamy, M. D. Mitchell, F. H. Bloomfield, M. H. Oliver, M. E Campbell, J. R. G. Challis, and J. E. Harding Effects of periconceptional undernutrition on the initiation of parturition in sheep Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R67 - R72. [Abstract] [Full Text] [PDF]
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W. X. Wu, X. H. Ma, T. Coksaygan, K. Chakrabarty, V. Collins, J. Rose, and P. W. Nathanielsz Prostaglandin Mediates Premature Delivery in Pregnant Sheep Induced by Estradiol at 121 Days of Gestational Age Endocrinology, March 1, 2004; 145(3): 1444 - 1452. [Abstract] [Full Text] [PDF]
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S. Gupta, S. Gyomorey, S. J. Lye, W. Gibb, and J. R. G. Challis Effect of Labor on Glucocorticoid Receptor (GRTotal, GR{alpha}, and GR{beta}) Proteins in Ovine Intrauterine Tissues Reproductive Sciences, April 1, 2003; 10(3): 136 - 144. [Abstract] [PDF]
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G. Ptak, M. Clinton, M. Tischner, B. Barboni, M. Mattioli, and P. Loi Improving Delivery and Offspring Viability of In Vitro-Produced and Cloned Sheep Embryos Biol Reprod, December 1, 2002; 67(6): 1719 - 1725. [Abstract] [Full Text] [PDF]
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R.L. Martin, W.L. Whittle, A.C. Holloway, S. Gyomorey, W. Gibb, S. Lye, and J.R.G. Challis Ontogeny and Regulation of Ovine Placental Prostaglandin E2 Synthase Biol Reprod, September 1, 2002; 67(3): 868 - 873. [Abstract] [Full Text] [PDF]
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