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The Perinatal Research Centre, Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Canada T5H 3V9
Address all correspondence and requests for reprints to: Dr. B. F. Mitchell, Department of Obstetrics and Gynecology, No. 205 CSC, 10240 Kingsway Avenue, Edmonton, Alberta, Canada T5H 3V9. E-mail: brymitch{at}gpu.srv.ualberta.ca
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Abstract |
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Pregnant rats were given a single injection of RU486 (2.5 mg/rat in
oil) on day 15 of pregnancy (normal delivery occurs on day 22). Control
animals received injections of oil only. Groups of animals (n = 5
in each group) were euthanized at 0, 6, 12, 24, and 48 h after
injection and during labor (immediately after delivery of the first
pup). Maternal serum estradiol (E2), P4 and
uterine OT, and PGE2 concentrations were measured by RIA.
Prostaglandin F2
and estrogen receptor levels were
measured by enzyme immunoassay (EIA). OTR and P4 receptor
(PR) were measured using radioligand-binding assays. OT, OTR, and
estrogen receptor mRNAs were measured with ribonuclease protection
assays.
The average time to delivery, after RU486 injection, was 27.0 ±
1.2 h. Serum E2 and P4 levels were
increased slightly, but significantly, at 24 h after RU486. In
controls, OT mRNA increased significantly, and this increase was
blocked in the RU486 treatment group. OTR mRNA levels increased within
6 h of RU486 and remained elevated until delivery. OTR peptide was
increased by 12 h. PGE2 and PGF2 were
increased 3-fold and 16-fold, respectively, but not until after the
increase in OTR had occurred.
We conclude that the mechanism of action of RU486 is to inhibit the P4 suppression of OTR synthesis, allowing increased expression of OTR, which may directly stimulate myometrial contractions or act indirectly through increased synthesis of PGs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the rat, synthesis of OT and OTR is primarily regulated by estrogen and progesterone (P4) (3, 4, 5). In most animal species, including the rat, parturition is preceded by a significant increase in estrogen and decrease in P4 concentrations, resulting in a marked increase in the estrogen/P4 ratio (5, 6, 7). This change favors synthesis of OT and OTR within intrauterine tissues. Evidence also suggests that PGs play a role in the regulation of the uterine activity, and it has been proposed that part of the role of OT is mediated by stimulation of decidual PG synthesis (8).
We recently demonstrated that administration of an antiestrogen will significantly delay the increase in uterine OT mRNA and peptide and OTR and, consequently, delay parturition (7). The present studies were undertaken to determine the effects of an antiprogestin compound (RU486) on intrauterine levels of OT, OTR, and PGs and to observe the effects on the process of parturition in the rat. A clearer understanding of the intrauterine paracrine network involving sex steroids, OT, OTR, and PGs may yield new strategies for controlling the time of birth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment groups
The rats were divided into two groups. The experimental group
was treated once with RU486 (2.5 mg/rat in 0.4 ml oil) at day 15 of
pregnancy. sc injections were performed into the back of the rat, using
a 26.5-gauge needle. Animals (n = 5) were euthanized at 0, 6, 12,
24, or 48 h after treatment and during labor (after delivery of
the first pup). The control group received only injections with vehicle
(0.4 ml oil). The rats were injected between 0900 and 1000 h.
Samples were taken for measurement of serum concentrations of estradiol
(E2) and P4, uterine OT mRNA and peptide
levels, uterine cytosol concentrations of estrogen receptor (ER), ER
mRNA and PR, uterine OTR mRNA and protein levels, and uterine
PGE2 and prostaglandin F2
(PGF2) levels. For the final results, there were five
animals in each group, except the 6-h controls (n = 3) and the
48-h RU486 group (n = 4) and the delivery group (n = 6).
RIAs
The RIA procedures and antisera for the E2 and
P4 have been characterized previously (7). The samples
were extracted from sera and incubated with radiolabeled E2
or P4 and antibody overnight at 4 C. Free and bound
steroids were separated with dextran-coated charcoal. After
centrifugation, the supernatant was counted by scintillation
spectrometry.
OT peptide was determined by RIA according to the procedure described previously (7). The antibody for OT was generously provided by Dr. A. P. F. Flint. It does not cross-react significantly with vasopressin or other known peptides. Briefly, the uterine tissues were homogenized in acid buffer (5% formic acid, 10% trifluoroacetic acid, and 1% NaCl in 1.0 N HCl) and centrifuged at 1000 x g for 30 min. The supernatant treated with acid buffer was passed through C-18 Sep-pak cartridges (Waters, Milford, MA). OT was eluted from the column with 75% acetonitrile in 0.01 M trifluoroacetic buffer. The extracts were dried and then dissolved in RIA buffer containing 50 mM sodium phosphate and 10 mM EDTA. A 25- to 50-µl aliquot was incubated with 5000 cpm [125I]OT (New England Nuclear-DuPont, Boston, MA) and OT antibody overnight at 4 C. After incubation with second antibody (rabbit antisheep serum) and normal sheep serum, the samples were centrifuged at 1000 x g for 15 min. The supernatant was aspirated, and the pellet was counted in a Geiger spectrometer (model 1275, Minigamma, Pharmacia, Wallac OY, Turku, Finland).
Uterine concentrations of PGE2 were measured in the cytosolic fraction. Uterine tissues were homogenized in Tris-EDTA buffer containing monothioglycerol and centrifuged at 1,000 x g for 15 min. The supernatant was centrifuged at 105,000 x g for 1 h, and the supernatant after this centrifugation was termed cytosol extract. Samples of 0.5 ml of this supernatant, with 3 ml citrate buffer containing 15% ethanol, were passed through C-18 Sep-pak cartridges preequilibrated with 4 ml 95% ethanol and 4 ml double distilled H2O. The column was washed with 2 ml H2O and 3 ml petroleum ether. PGs were eluted from the column with 3 ml ethylacetate. The eluted PGs were dried and reconstituted with sodium phosphate buffer containing gelatin. The PGE2 assay has been described previously (7). The antiserum specific for PGE2 was provided by Dr. Leslie Myatt from the University of Cincinnati. A 50- to 100-µl aliquot was incubated with [3H]PGE2 (New England Nuclear-DuPont) and antibody overnight at 4 C. Free and bound PGE2 were separated with dextran-coated charcoal. After centrifugation, the supernatant was counted by scintillation spectrometry.
EIA
PGF2 concentrations in uterine tissue cytosols
were measured by EIA. The assay used an ACE EIA kit (Cayman Chemical
Co., Ann Arbor, MI) and was performed according to the manufacturer’s
instruction. The ER assay was performed using a monoclonal antibody kit
(Abbott Laboratories, Mississauga, ON) and following the
manufacturer’s instructions. This antibody measures both occupied and
unoccupied cytosolic receptors, and the ER measurements are not
affected by the presence of physiological concentrations of other
steroids.
Receptor-binding assays
OTR in uterine tissues was measured using a modification of a
published OT-binding assay, as we have previously described (7).
Briefly, the pellet obtained after the 105,000 x g
centrifugation step was washed, resuspended in Tris buffer, and
incubated for 1 h with 0.6 nM [3H]OT
(New England Nuclear-DuPont) and increasing concentrations of
nonradioactive OT (0.0–9.4 nM). Nonspecific binding was
measured after the addition of 100 nM nonradioactive OT.
Incubation was terminated by filtering the suspension through a glass
microfibre filter (GF/C, Whatman, Springfield Mill, Maidstone, England)
and rinsing with cold Tris buffer. The filters (carrying receptor-bound
[3H]OT) were counted by scintillation spectrometry. The
total number of OTR and dissociation constant in each sample were
determined by Scatchard analysis using six concentrations of OT.
P4 receptors were measured in the cytosolic fraction, as previously described (7). Samples were incubated with a mixture containing increasing concentrations of [3H]promegestone ([3H]R-5020, 0.2 nM to 5 nM; New England Nuclear-DuPont), in the presence of 10-fold concentrations of dexamethasone and dihydrotestosterone, to prevent binding to glucocorticoid or androgen receptors. Nonspecific binding was measured after the addition of 50 nM nonradioactive R-5020. After incubation overnight, free and bound ligand were separated with dextran-coated charcoal. After centrifugation, the supernatants were counted in a scintillation spectrometer.
OT, OTR, and ER mRNA assay
Uterine tissues were homogenized in 4 M guanidinium
thiocyanate, and total RNA was purified by phenol-chloroform at acidic
conditions (pH 4.0). The concentration of RNA was determined by
spectrophotometry at A260. OT, OTR, and ER mRNA levels in the uterine
tissues were measured by ribonuclease protection assay. A specific
probe for rat OT mRNA was prepared from a rat genomic clone kindly
provided by Dr. Hartwig Schmale from the University of Hamburg (9). The
antisense complementary RNA (cRNA) probe corresponded to exon C and
part of the second intron of the rat OT gene. The cRNA probe for OTR
was prepared from a rat genomic clone (kindly provided by Dr. Stephen
J. Lye from the University of Toronto) and corresponded to exon 2 and 3
of OTR gene, which encodes transmembrane domains 3–7. The rat ER cRNA
probe was prepared using PCR with primers to portions of exons 4 and 6
(complementary DNA Cycle Kit, Invitrogen Corp., San Diego, CA). The
resultant 439 nucleotide probe was subcloned using pCR-Script AmpSK(+)
cloning kit (Stratagene, La Jolla, CA).
The ribonuclease protection assays were performed as previously described (7). Briefly, 10 µg (OT), 20 µg (ER), or 40 µg (OTR) samples of total RNA were hybridized to gel-purified antisense 32P-labeled OT, OTR, or ER RNA probes in 80% formamide and 5-fold concentrated salts containing 200 mM 1,4-piperazinediethanesulfonic acid, 2 M NaCl, and 5 mM EDTA for 18 h at 55 C. After incubation with 0.75 µg ribonuclease A and 300 units ribonuclease T1 (both from Boehringer Mannheim Canada, Laval, QB) for 30 min at 30 C, protected fragments were analyzed on 6% or 8% denaturing polyacrylamide gels. An RNA sample from one animal at each time point from each group, chosen at random, was run on each gel. The gel for OT, OTR, or ER was exposed to XAR x-ray film (Eastman Kodak, Rochester, NY) for 1 h or 16 h. A rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) probe (Ambion Inc. Austin, TX) was used in all samples as an internal control (10). Autoradiograms were quantitated using laser densitometry, and data were presented as the ratio of arbitrary densitometric units of OT, OTR, or ER mRNA to GAPDH mRNA.
Statistical analysis
Data are presented in text and graphs as the mean ±
SEM. The results were first analyzed by one-way ANOVA
(InStat, GraphPad Software, San Diego, CA) to detect changes with
advancing gestational age. Post hoc comparisons of the means
were performed using the Fisher’s Protected LSD test (SuperANOVA,
Abacus Concepts Inc., Berkeley, CA). Differences between the
experimental and control groups were sought using the two-tailed
unpaired Student’s t test. Differences were considered to
be significant when a P value less than 0.05 was obtained.
If Bartlett’s test revealed nonhomogeneity of variance, the
corresponding nonparametric test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum concentrations of E2 and P4 did not
change significantly in the control group throughout the 48 h of
observation (Fig. 1
). However, in the RU486-treated
animals, there was a significant increase in serum E2 and
P4 by the time of delivery of the first fetus, compared
with time zero. In the treated group that had not delivered by 48
h, the serum E2 increase was similar to that of the group
that had delivered. However, serum P4 concentrations in the
treated, but undelivered animals, did not increase as did the group
that delivered prematurely.
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) and did not change significantly after RU486
treatment. The levels of PR were low, but detectable, and did not
change in the control group, remaining in the range of 200 fmol/mg
tissue protein. Levels of PR were undetectable in the RU486-treated
group.
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), but concentrations of OT peptide remained unchanged
(Fig. 3b). RU486 abolished this increase in OT mRNA, and levels
remained low after treatment, even during premature labor and delivery.
There was no effect of RU486 treatment on uterine OT peptide
concentrations. Expression of GAPDH did not change with gestational age
or RU486 treatment.
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). By 6 h after RU486 treatment,
OTR mRNA concentrations increased significantly and remained high until
delivery. The measurements of OTR binding increased in parallel to the
levels of OTR mRNA (Fig. 4b). In the group that did not deliver by
48 h, OTR mRNA and peptide levels were increased to a level
similar to those of animals that delivered.
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remained at constant
low levels in the control group (Fig. 5). In contrast,
the concentrations of PGE2 in the RU-treated group
increased gradually, achieving maximal levels after 24 h of
treatment and remaining high until delivery. This increased
concentration after RU486 was even more pronounced for
PGF2, where a 16-fold increase was observed between
12 h and 24 h after treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Parturition in most animal models proceeds normally only when P4 concentrations decrease. However, in our experiments, both maternal serum E2 and P4 concentrations were increased at the time of premature parturition in the RU486-treated animals. This is in keeping with the data of Garfield et al., though the changes in that study did not achieve statistical significance (11). We interpret the increase in sex steroids to indicate that the antiprogestin has interfered with a negative feedback loop and upregulated ovarian production of E2 and P4. This would be compatible with previous studies where treatment with an anti-P4 monoclonal antibody increased serum E2 levels in early pregnancy in the hamster (15). RU486 also may have a direct effect on ovarian P4 synthesis, at this stage of pregnancy, by stimulating activity of 3ß-hydroxysteroid dehydrogenase activity (16).
On day 15 of pregnancy, uterine cytosol concentrations of PR were low but detectable. After RU486, PR was undetectable. We are uncertain whether this effect is an artifact caused by the ability of RU486 to compete for [3H]P binding to rat uterine cytosolic PR (17) or whether the decrease is real. However, in T47D human breast cancer cells, RU486 caused a transient reduction in basal levels of PR mRNA and protein (18). Additionally, our assay measured only cytosolic PR, and the apparent decrease could have been caused by an RU486-induced shift of PR into the nuclear subcellular fraction.
We detected no change in cytosolic ER or ER mRNA after RU486. Indeed, there was a consistent trend to decreased ER mRNA with RU486, though this did not reach statistical significance in any group. This was a surprising result, because it is known that P4 interferes with replenishment of cytosolic ER (19). P4 also increases the turnover of nuclear ER in hamster decidual cells (20). When rhesus macaques were treated with RU486 in late gestation, cytosolic and nuclear ER levels increased significantly in both decidua and myometrium, changes that are not usually seen in normal spontaneous term labor (21). Our failure to demonstrate an RU486-induced increase in ER in rats during late pregnancy may be caused by limiting our measurements to the cytosolic compartment. However, the lack of change in ER mRNA suggests that this is not the case. It also is possible that our relatively short observation period did not permit evaluation of the full affects of RU486, though it is noteworthy that the biological affect of parturition did occur. Finally, it is possible that there are interspecies differences in the influence of RU486 on ER during late pregnancy.
Concentrations of OT mRNA within the uterus of the control group increased significantly between day 15 and day 17 of pregnancy without any change in uterine OT peptide concentrations. This confirms our previous findings (7) of increased concentrations of OT mRNA between day 14 and day 18 of pregnancy without changes in peptide levels. RU486 blocks this normal increase in OT mRNA, suggesting that P4 may have a positive influence on uterine OT synthesis. This would support previous findings in the nonpregnant rat, where E2 and P4 had a synergistic effect on uterine OT gene expression (3). Our previous studies (22), using term human chorio-decidua, demonstrated that E2 stimulates synthesis of OT mRNA, but P4 alone was without effect. In concert, these data support the contention that uterine OT gene expression depends on a synergistic action between E2 and P4.
Tissue concentrations of OT remained constant in the control rats despite an increase in OT mRNA on day 16. We have noted this disparity between OT mRNA and OT peptide concentrations previously (7). The initial translation product from OT mRNA requires further modification before synthesis of mature amidated OT recognized by our RIA (23). Thus, the discrepancy between OT mRNA and peptide levels may be secondary to delays in translation or posttranslational processing of OT mRNA.
There is a marked increase in OTR approximately at the time of parturition in the rat, and this parallels an increase in the sensitivity of the myometrium to OT (24). In the human, OTR also increases markedly as parturition approaches (25), and a similar increase occurs in uterine OTR mRNA concentrations (26). In the present studies, a significant increase in OTR occurred within 6 h of RU486 treatment, and this was immediately followed by an increase in OTR peptide. This strongly suggests that P4 plays an important role in inhibition of OTR synthesis and confirms earlier findings (4, 5). Recently, Larcher et al. (27) have demonstrated that P4 treatment results in a much greater suppression of OTR binding than of OTR mRNA. Conversely, our results with RU486 reveal a greater relative effect on OTR mRNA (approximately 10-fold increase), compared with OTR binding (approximately 5-fold increase). The reason for the differences is not clear, but their studies were performed in ovariectomized, nonpregnant rats using a synthetic OT analogue as ligand, whereas our studies used radiolabeled OT with tissues from intact pregnant animals. In any case, these data suggest that control of OTR translation may be an important regulatory step and that the mechanisms may be different in the pregnant and nonpregnant states. It is noteworthy that in both of our experimental models [prolonged gestation by tamoxifen (7) and preterm labor by RU486], parturition occurred when OTR peptide reached a threshold value without the changes in uterine OT gene expression.
The mechanisms mediating the effects of sex steroids on OTR gene remain unclear. Recently, the promoter region of the OTR gene in several species has been sequenced, and several half-palindromes of estrogen response elements were found (28). It is possible that these half-EREs confer estrogen sensitivity. However, despite strong evidence that P4 down-regulates OTR gene expression, the OTR promoter region is devoid of response elements that are known to interact with PR. This suggests that the antagonism of P4 on uterine OTR gene expression is mediated by an indirect genomic or a nongenomic mechanism. As noted by Larcher et al. (27), the disparate effects of P4 on OTR mRNA and binding measurements suggests that at least part of the effect of P4 may be at the translational or posttranslational level.
Basal uterine tissue levels of PGF2 and PGE2
were similar. However, after RU486, PGE2 increased
approximately 4-fold and PGF2 increased more than
10-fold. The time course of the increases seemed to follow those for
OTR. This is in agreement with previous studies showing that PGs
increased in rats after RU486 only in those that were aborting and only
after labor had started (12). Rat endometrium in late gestation
produces PGE2 and PGF2 in response to OT
stimulation (8) and thus, in our experiments, may be a result of the
increased OTR. These PGs may play a role in parturition by being
directly utertonic or by stimulating further synthesis of OTR (29).
Earlier studies by Chan et al. (8, 29) have demonstrated a
potential positive feedback loop between PGs and OTR. Our results do
not allow conclusions as to whether the rise in PGs is a cause or
consequence of labor. Finally, our data are compatible with previous
findings that RU486 stimulates production of PGE2 and
PGF2 by human endometrial stromal cells (30).
All four RU486-treated animals that remained undelivered at 48 h were bleeding vaginally at the time they were killed. Their uterine concentrations of OT, OTR, and PGs were indistinguishable from those who delivered. Indeed, the only way in which they differed from the delivered group was that their serum P4 levels were significantly reduced, compared with the treated animals at the time of delivery or with the controls at 48 h. We speculate that these animals had completed the biochemical changes that initiated labor, were in active labor, and would have delivered shortly after the end of the experiment at 48 h. This distribution of delivery times would be similar to that observed previously in rats administered the same dose of RU486 at a similar time in gestation (11).
In summary, these data have demonstrated that the antiprogestin RU486 stimulates premature delivery in the rat by evoking changes in OTR and PGs similar to those seen in normal term parturition. Our findings strongly support a major role for P4 in suppressing OTR gene expression in late pregnancy, before myometrial activation, around the time of labor onset. The data also suggest that the increase in PGs occurs secondarily to the increase in OTR. The process of RU486-induced premature delivery occurred without an increase in uterine OT gene expression, suggesting that regulation of the receptor is a more critical step than regulation of the ligand. Further investigation into regulation of uterine OTR may provide information on which to develop improved methods for controlling the timing of parturition.
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Footnotes |
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Received October 8, 1996.
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