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Laboratory of Pregnancy and Newborn Research (W.X.W., X.H.M., P.W.N.), Physiology Department, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; Department of Obstetrics and Gynecology (N.U.), Faculty of Medicine, University of Tokyo, Tokyo, Japan
Address all correspondence and requests for reprints to: Dr. Peter W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401. E-mail: pwn1{at}cornell.edu
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Introduction |
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Several investigators have demonstrated that activation of gene expression associated with onset of labor is not restricted to the uterine myometrium. Our own data from several studies in pregnant sheep consistently demonstrate increased abundance of messenger RNA (mRNA) for ER (4, 5), Hsp 90 and 70 (6), OTR (9), cytosolic phospholipase A2 (cPLA2), and prostaglandin H synthase-2 PGHS-2 (21), occurring coincidentally in labor in the endometrium simultaneously with critical biochemical changes in the myometrium. Similar intrauterine changes in labor-related genes are precipitated by glucocorticoid induced labor (4, 5, 6, 21). Increased PGHS-2 was also found in the placenta in later gestation and during labor (22, 23). In addition to endometrial and myometrial changes associated with labor, several reports indicate that the fetal membranes are involved in PG production during human parturition (24, 25, 26, 27, 28).
Based on those observations, we and others have proposed that the onset of labor is associated with preparation of the various uterine tissues with resultant changes in a cassette of genes (up- or down-regulation). Each member of the cassette of genes contributes to some extent in the switch in myometrial contractility patterns from contractures and contractions as well as the progression of established labor. Alteration of this cassette of genes in uterine tissues is a prerequisite for labor and delivery. Several uterine tissues, including myometrium, endometrium, placenta, amnion, and chorion, appear to play critical roles in connecting fetal signals that indicate fetal readiness for birth to maternal factors that carry labor forward to a successful conclusion.
The current study was designed to examine the connection of PG production with the expression of uterine labor-related genes. PGs are a major uteronic stimulator during labor and probably act in an autocrine and paracrine as well as an endocrine manner. We hypothesize that PG acts as a critical and indispensable link in the positive feed-forward regulation of myometrial contraction during labor and leads to recruitment of many other positive feed-forward systems (including up-regulation of PG synthesis) that are indispensable to the promotion of the delivery.
To test our hypothesis, we characterized the effects of inhibiting PG production by nimesulide during spontaneous term labor on ER, OTR, Hsp 70 and 90, cPLA2, and PGHS-2 mRNA expression in pregnant ovine myometrium, endometrium and placenta. Nimesulide (CAS 51803–78-2, N-(4-nitro-2-phenoxyphenyl)-methanesulfonilamide), a nonsteroidal antiinflammatory drug, has been shown not to affect PG synthesis in the bronchial tree (29), where constitutive PGHS-1 exerts a bronchoprotective role (30) and preserves integrity of the gastric mucosa (31, 32, 33). By contrast, nimesulide markedly affects PGHS-2 mediated PG production. Recently nimesulide has been shown to inhibit PGHS-2 with selectivity over PGHS-1 activity of the order of 70 (34, 35). We infused nimesulide into pregnant ewes that were already in established labor as shown by myometrial contractility pattern. We monitored PG production during nimesulide infusion period by measuring fetal PG production. Results were compared with data obtained from appropriate age matched controls not in labor.
Premature labor is not predictable in most clinical circumstances and appropriate treatments to arrest the progress of labor that has already commenced is a very important clinical goal. In addition, a majority of the previous studies did not dissect mechanisms that occur before labor and after establishment of labor. Thus, our current study focused on the regulation of the expression of uterine labor-related genes during the progression of labor.
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Materials and Methods |
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Care of animals
At 120 days of gestational age (dGA), ewes from which tissues
were obtained were instrumented with electromyogram (EMG) leads sewn
into the myometrium to enable monitoring of myometrial contractility
patterns (1). Fetal and maternal carotid arterial and jugular venous
catheters were placed to permit infusion into and/or sampling from both
ewe and fetus. Frequent fetal arterial blood was taken to ensure that
fetuses were in good condition throughout the study (1). From 140 dGA,
myometrial EMG was recorded continuously. Labor was defined as having
occurred when the myometrial EMG record showed a clear switch from
contractures (long duration, low amplitude epochs of myometrial
activity lasting longer than 3 min) to short labor contractions
followed by well established contraction activity for at least 5 h
(36).
Nimesulide infusion
Nimesulide was kindly provided by Dr. Bennett (London, UK) and
prepared in DMEM (D-5648, Sigma Chemical Co., St. Louis, MO) at a
concentration of 1.25 mg/ml. Nimesulide infusion to the ewe iv (30 mg
bolus, followed by 6 h infusion, 30 mg/h) commenced 9 h after
onset of labor at 147–148 dGA. Myometrium, endometrium, and fetal
cotyledon were collected at necropsy under halothane general anesthesia
at the end of infusion. Tissues were also collected from six
chronically instrumented vehicle infused pregnant ewes in spontaneous
term labor (STL) and six term control ewes not in labor (TCNL) at
143–147 dGA. Myometrium, endometrium, and fetal cotyledon were
dissected rapidly from the similar positions in the middle of the
uterine body, submerged immediately in liquid-N2, and
stored at -80 C for later RNA analysis.
RIA of PGE2
Fetal plasma PGE2 concentration were determined by
RIA using an antibody, kindly provided by Dr. Leslie Myatt (University
of Cincinatti, Cincinatti, OH), generated in rabbits to a
PGE2-BSA conjugate. The cross-reactivity of this antibody
with PGE2, PGD2, PGF2
, 6-keto-PGE, and TXB2
was 100%, 1.8%, 1.5%, <2.2% and <1%, respectively (37). One
hundred microliters of plasma were equilibrated with acid for 2–3 h,
extracted twice with diethyl ether, and reconstituted with 500 µl of
0.1% gelatin in 0.1 M PBS. Aliquots of 100 µl
reconstituted samples were assayed in duplicate. Tritiated
PGE2 served as the labeled ligand (DuPont NEN, Boston, MA)
and dextran-charcoal PBS solution was used to separate bound from free
ligand. Prostaglandin E2 standard (Cayman Chemical Co. Ann
Arbor, MI) used at 3.13–400 pg/tube were diluted in the assay buffer
(0.1% gelatin in 0.1 M PBS). Tritiated PGE2 at
5000 cpm and PGE2 antiserum diluted 1:15,000 were added in
100-µl aliquots each to standards and extracted samples and incubated
at 4 C overnight. A 15-min incubation with 1 ml dextran-charcoal
solution at 4 C was followed with centrifugation at 2,400 x
g for 15 min at 4 C. The supernatant containing the bound
ligand was decanted into scintillation vials, and scintillation fluid
was added and counted. Extraction recovery of tritiated
pGE2 added to plasma was 79.5 ± 2.1% (mean ±
SEM) (n = 6), and the solvent blank was negligible.
Assay values were not corrected for recovery. Plasma pools were made by
adding 0.5, 1, 2, and 8 ng/ml exogenous PGE2 to
charcoal-extracted pooled fetal sheep plasma. When aliquots of 100 µl
of these levels were extracted and assayed, 0.58 ± 0.11,
0.74 ± 0.09, 1.61 ± 0.13 and 5.69 ± 0.41 ng/ml were
measured, respectively (n = 10). The assay sensitivity was 3.13 pg
per tube, which is equivalent to 0.157 ng/ml when 20 µl of plasma was
extracted. The intraassay CV and the interassay CV were 8.8% and
8.1%, respectively.
Northern blot analysis
Total RNA was prepared from individual tissues as described
previously (5). Briefly, total RNA was extracted from frozen
myometrium, endometrium, and fetal cotyledon and separated on
1.0–1.4% agarose/0.66 M formaldehyde gel. After
electrophoresis, RNA was transferred to a nylon membrane (Gene Screen
Plus, DuPont NEN) and hybridized to ER, OTR, Hsp 90 and 70, PGHS-2
complementary DNA (cDNA) probe, and cPLA2 oligo probe under the
hybridization conditions described previously in detail (4, 5, 6, 9, 21).
cDNA probes
Human ER cDNA containing the entire coding region was kindly
made available by Dr. Pierre Chambon (University of Strasbourg,
Strasbourg, France). A 131-bp cDNA probe encoding part of the sheep
endometrial OTR, generated by PCR, was kindly made available to us by
Dr. Flint (University of Nottingham, Leicestershire, UK) (38). Human
Hsp 90- (catalog no. 78313) and 70–1 (catalog no. 78318) cDNA were
purchased from American Type Culture Collection (Rockville, MD). Human
PGHS-2 cDNA was purchased from Oxford Biomedical Research, Inc. (Oxford
MI) (catalog no. R91-PGHS-2, R74-PGHS-1). Each of the cDNA probes was
labeled with -32P dCTP using the random priming method
(DuPont NEN) to specific activity of approximately 1 x
109 cpm/µg and used at a final concentration of 1 x
106 cpm specific probe/ml of hybridization solution.
Hybridization was carried out at 42 C for 20 h in hybridization
solution containing each of the specific probes. Membranes were washed
sequentially in 2 x SSC (1 x SSC is 0.15 M NaCl
and 0.015 M Na Citrate, pH 7.0) at room temperature for 10
min, and 0.5 x SSC with 0.1% SDS at 65 C for 30 min.
Oligonucleotide probes
The oligo nucleotide probes were synthesized by the Cornell
University Biotechnology Program, Oligonucleotide Synthesis Facility
(Ithaca, NY) according to the reported human cPLA2 cDNA by Clark
et al. (39) and Sharp et al. (40). The sequence
of the oligonucleotide probe-1 for cPLA2 is:
5'-CCCACCTGAACCCAATATGGCTACCACAGGCACATCACGTGC-3', and that of probe-2
for cPLA2: 5'-TGCCTTCATCACACCAGAGAATCCCACCATGGCTCGGAAACC-3',
without interruption of nucleotide sequences, which corresponds to
nucleotides 550–633 of cDNA encoding cPLA2 (32). Two oligonucleotide
probes were simultaneously used to increase the sensitivity of the
Northern blot analysis. Specificity of Northern blotting for the cPLA2
was validated as described previously (21). Membrane was prehybridized
and hybridized at similar conditions to those described above with the
exception that 40% formamide was used and the temperature of the
prehybridization (5 h) and hybridization (24 h) was 40 C. The
concentration of the cPLA2 oligo probe used was 1–2 x
106 cpm/ml. Membranes were initially washed at room
temperature in 2 x SSC; 0.1% SDS for 10 min, followed by
stringent wash at 55 C in 0.5 x SSC; 0.1% SDS for 15 min and
then briefly at 23 C in 0.2 x SSC; 0.1% SDS.
Membranes were stripped of cDNA or oligo probes by boiling in
0.1 x SSC with 0.1% (wt/vol) SDS for 30 min and rehybridized
with -32P-labeled 18S cDNA probe (kindly provided by Dr.
A. Berndtson, Cornell University) to normalize each mRNA level. Kodak
X-OMAT AR film was exposed to the membrane with intensifying screens at
-80 C. Exposure durations were varied to achieve hybridization signals
within the limited linear range for densitometry (2–7 days).
Autoradiograph signals were analyzed and quantified with a scanner
(ScanMaker II, Microtek), and data were analyzed in a power Macintosh
computer (8100/80) with a densitometry program-Scan Analysis (Biosoft,
Cambridge, UK).
Statistical analysis
One-way ANOVA was used to test for significant differences among
the three groups (i.e. term control not in labor,
spontaneous term labor and nimesulide treated) followed by multiple
comparison using a Tukey-Kramer procedure. Data are presented as
mean ± SEM.
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Results |
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). This
decrease was sustained during the nimesulide infusion period.
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) and myometrium (Fig. 3). OTR and ER mRNA in endometrium
decreased significantly after nimesulide infusion (P <
0.05), but the fall in Hsp 90 and 70 mRNA was not significant (Fig. 2).
In the myometrium, Hsp 70 and OTR mRNA decreased significantly
following nimesulide (P < 0.05) and the abundance of
Hsp 90 and ER mRNA did not change significantly (Fig. 3).
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) by Northern blot
analysis. PGHS-2 mRNA abundance increased in all three tissues
(P < 0.001) during spontaneous term labor (Fig. 4).
cPLA2 mRNA abundance was significantly increased (P <
0.001) in ovine endometrial tissue in spontaneous term labor (Fig. 5). Abundance of cPLA2 mRNA was also
detected in the myometrium, but no difference was found according to
the status of myometrial activity or treatment in those tissues (data
not shown). Following nimesulide infusion, PGHS2 mRNA abundance in the
myometrium and placenta (Fig. 4) was significantly decreased below the
level seen in spontaneous term labor (P < 0.05) and
was suppressed to levels similar to term control ewes not in labor
(P > 0.05). Following nimesulide infusion, PGHS-2 was
significantly decreased below levels seen in spontaneous term labor in
endometrium (Fig. 4) (P < 0.05) but remained
significantly higher than term control ewes not in labor
(P < 0.01). Compared with values seen in spontaneous
term labor, cPLA2 did not change (P > 0.05) in
endometrium after nimesulide infusion (Fig. 5).
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Discussion |
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Our observations strongly suggest that PG synthesis is an essential component of the critical positive feedforward system in the parturition process, at the level of the control of the two key enzymes: cPLA2 (to provide arachidonic acid substrates for PG synthesis) and PGHS-2 (to convert arachidonic acid into PGH2, the intermediate precursor for all PGs). Although there is controversy whether cPLA2 activity is one of the limiting factors in PG synthesis, any stimulus to PG production must also increase the activity of PGHS because this enzyme has a short half-life and is destroyed after a limited number of reactions (42). PGHS-2 expression markedly increased in uterine tissues of pregnant women (23) and sheep (21) in association with labor. However, the precise timing of the increase in different uterine tissues is poorly characterized in vivo in any species.
We recently demonstrated that both estradiol and progesterone are involved in the regulation of PGHS in vivo in ovariectomized nonpregnant sheep. Estradiol up-regulated PGHS-2 abundance in the myometrium, whereas progesterone was without effect. In contrast, progesterone was a more potent stimulator of endometrial PGHS-2 abundance than estradiol. Estradiol and progesterone did not alter PGHS-1 abundance in either endometrium or myometrium (43). These data suggest that the rise in progesterone between 0.6–0.7 of ovine pregnancy, which is thought to play a critical role in ovine parturition by several investigators (44, 45, 46), may be associated with increased PGHS-2. In addition, elevated fetal and maternal plasma estrogen concentrations at the end of pregnancy (47, 48) and immediately before the onset of labor may be responsible for the induction of increased PGHS-2 abundance associated with the onset of labor.
The current study is an extension of our previous study and
provides the first evidence that PGs act as positive signals for
uterine PGHS-2 mRNA expression in vivo during the
progression of labor. This conclusion is supported by the recent
demonstration that PGF2 induces expression of PGHS-2 in the ovine
corpus luteum (49). Inhibition of PGHS-2 with resultant decreases in
myometrial contraction power (41) and decreased PG production resulted
in the decreased expression of PGHS-2 mRNA in all three uterine tissues
examined. Our current data provide further firm evidence that PGs are
major driving factors for the progression of labor. They also suggest
that PG may stimulate further PG production by either autocrine and/or
paracrine mechanisms.
Oxytocin and PGs are two major uterotonic systems that regulate myometrial contraction during parturition. Those two systems are intertwined in a positive feed forward loop that may undergo rapid and strong sustained self-activation (10). In the present study, we only inhibited the PG system. As we have shown, the effect of nimesulide was to decrease OTR mRNA abundance in both endometrium and myometrium.
It is unlikely that nimesulide had a direct effect to interfere with the transcription of uterine labor-related genes at the mRNA level. The mechanism of action of nonsteroid antiinflammatory drugs (NSAIDs) studied so far indicates that they bind at the critical functional site of PGHS, close to the putative catalytic residue Tyr385 (50). The binding of the inhibitor results in irreversible inactivation by acetylation of the cyclooxygenase component. A second mechanism of the inhibitor action is the reversible binding that competes with substrate (51). Therefore, the inhibition of PGHS-2 by nimesulide with subsequent suppression of uterine labor-related gene expression is most likely due to the decreased production of PGs. PGs may act as a critical and indispensable link, possibly even as transcription factors, in the positive feed forward loop for myometrial contraction during labor. This positive feed forward loop has an explosive "runaway" effect on the progression of labor eventually leading to delivery by the recruitment of other positive feed-forward systems (such as ER, OTR, Hsp90 and 70, PGHS, or estrogen and progesterone production and secretion).
Recently, Sugimoto and colleagues demonstrated that absence of the FP PG receptor did not abolish induction of the uterine OTR following lutectomy in FP knock-out mice (52). However, it is still possible that other PG systems, such as PGE, is involved in the regulation of uterine OTR induction during normal parturition. Our observations do indicate a role for PG in production of the OTR. Further investigations are necessary to evaluate the various regulatory roles and mechanisms that the different eicosanoids play via their several receptors.
Conclusions
PGHS-2 inhibition resulted in sustained myometrial
contraction and decreased PGE2 level in fetal plasma. The
decreased expression of uterine labor-related cassette gene expression
in the utero-placental unit following nimesulide inhibition may result
from the altered production of PGs.
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Acknowledgments |
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Footnotes |
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Received November 12, 1997.
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induces expression of prostaglandin G/H synthase-2 in the ovine corpus
luteum: a potential positive loop during luteolysis. Biol Reprod 57:1016–1022[Abstract]
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) or vehicle infusion (
). Values are % baseline
± SEM; n = 4 in each group. *, P
< 0.05 compared with baseline, and corresponding vehicle infusion.
