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00/0 Endocrinology Copyright 2000 by The Endocrine Society

Vol. 141, No. 1 Printed in U.S.A.

Uterine Expression of Prostaglandin H2 Synthase in Late Pregnancy and during Parturition in Prostaglandin F Receptor-Deficient Mice*
KAZUHITO TSUBOI, YUKIHIKO SUGIMOTO, AYA IWANE, KEI YAMAMOTO, SHOZO YAMAMOTO, AND ATSUSHI ICHIKAWA
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University (K.T., Y.S., A.Iw., A.Ic.), Kyoto 606-8501; and the Department of Biochemistry, University of Tokushima School of Medicine (K.Y., S.Y.), Tokushima 770-8503, Japan
ABSTRACT PG production in uterine tissues is important for many physiological processes in late pregnancy, including parturition. We examined the expression of the PGH2 synthases, cyclooxygenase-1 (COX-1) and COX-2, in uterine tissues during late pregnancy, using PGF receptordeficient (FP / ) mice. Female FP / mice are unable to deliver normal fetuses at term, as they do not undergo luteolysis necessary for parturition. In wild-type mice, COX-1 messenger RNA (mRNA) was expressed in the endometrial epithelium, myometrium, and decidua throughout late pregnancy. The expression of COX-1 mRNA in the endometrial epithelium and myometrium decreased both in wild-type mice undergoing natural parturition and in FP / mice undergoing ovariectomy-induced parturition, but expression of COX-1 mRNA was enhanced in FP / mice at the expected term. In wild-type mice, COX-2 mRNA was not expressed in the myometrium before parturition, but was markedly induced during parturition. This induction of COX-2 was absent in FP / mice at the expected term, but was found during ovariectomy-induced parturition in these mice. Expression of COX-2 proteins was confirmed by immunohistochemical analysis. Thus, in uterine tissues, myometrial expression of COX-2 is closely associated with the occurrence of parturition, but uterine expression of COX-1 is induced much earlier and kept at a high level until parturition occurs. These results suggest that COX-1-derived PGs are responsible for the induction of luteolysis, and that COX-2derived PGs play a role in the final pathway of parturition. (Endocrinology 141: 315324, 2000

Gs ARE involved in various mammalian reproductive processes, including the induction of parturition (1 4). PGH2 synthase, generally referred to as cyclooxygenase (COX), is the rate-limiting enzyme in the biosynthetic pathway of various PGs from arachidonic acid (5). Of the two isozymes, COX-1 is generally considered to be the constitutive enzyme, playing housekeeping roles in many animal tissues. In contrast, COX-2 is thought to be more tightly regulated than COX-1, playing various physiological and pathological roles (6). Aspirin-like drugs, which inhibit enzymatic activities of the COX isozymes, are known to cause delayed parturition in many species (7). Indeed, a large amount of PGs, especially PGE2 and PGF2 , are produced and released in uterine tissues during parturition (8). As both PGE2 and PGF2 have potent uterotonic activity (9), these PGs have been thought to play roles in the physiological parturition process. However, the specific roles these PGs play in parturition remain unclear. The actions of PGs are mediated by specific receptors on the surface of cells (10). To examine the physiological roles of the PGF receptor (FP) in parturition, we generated FPReceived May 17, 1999. Address all correspondence and requests for reprints to: Atsushi Ichikawa, Ph.D., Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: aichikaw@pharm.kyoto-u.ac.jp. * This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists (to K.T.).

deficient (FP / ) mice (11). As reported previously, FP / mice are unable to deliver normal fetuses at term, although they are normal in other aspects of reproduction physiology. These mice did not show the normal decline of serum progesterone levels that precedes parturition. Ovariectomy on day 19 of pregnancy permitted successful delivery in these FP / mice, indicating that parturition is initiated when PGF2 interacts with FP in the ovary of pregnant mice, inducing luteolysis. It has just recently been reported that COX1-deficient mice also show phenotypes of delayed parturition (12). This report stated that the significant increase in uterine PGF2 production on day 19 was lost in COX-1-deficient mice, and that the administration of PGF2 restored successful parturition. Hence, uterine induction of COX-1 before parturition appears important for inducing luteolysis. On the other hand, a number of reports have shown that a large amount of PG production and induction of COX-2 are detected in uterine tissues during parturition (8, 13). In humans, these events that occur in uterine tissues were found only during natural parturition and not upon cesarean section (14). Thus, it is likely that PGs produced by COX-2 take part in parturition through their uterotonic activity. From these results we speculated that uterine expression of the COX isozymes is regulated by different mechanisms, and that COX-1 and COX-2 preferentially contribute to the induction of luteolysis and myometrial contractility, respectively. To examine this possibility, we used FP / mice, because luteolysis is impaired in late pregnancy, and parturition can be artificially initiated by ovariectomy treatment. Here we show the uterine expression of COX-1 and COX-2 in FP / mice
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during late pregnancy and during ovariectomy-induced parturition. The results observed suggest that these two isozymes are regulated by parturition signals in very different ways.
Materials and Methods Animal and tissue preparations
Female wild-type and FP / mice, with the chimeric background (129/Ola C57BL/6), were maintained at 23 C under a 12-h light, 12-h dark cycle (11). To obtain timed pregnant mice, wild-type or FP / virgin female mice (9 14 weeks of age) were housed overnight with males and checked the following morning for vaginal plugs. The day a vaginal plug was observed was counted as day 1 of pregnancy or day 1 after conception. In wild-type females, natural parturition was observed on day 20. Uterine horns were isolated on day 17 or 20 for Northern analysis; on day 15, 17, or 20 for in situ hybridization analysis; and on day 20 for immunohistochemical analysis. For Northern analysis of COX messenger RNAs (mRNAs) throughout late pregnancy in wildtype mice (Fig. 1), C57BL/6 mice were used (Japan SLC, Inc., Hamamatsu, Japan). For the series of ovariectomy experiments, FP / mice were anesthetized by ether and bilaterally ovariectomized on day 19 of pregnancy. Ovariectomy-induced parturition occurred about 20 h after the operation. Uterine horns were isolated 12, 16, 20 (during parturition), or 36 h after ovariectomy for Northern analysis and 20 h after ovariectomy for in situ hybridization analysis. FP / mice that were sham-operated or left untreated served as controls.

abdominal aorta on the indicated day after conception. Cells were removed by centrifugation, and plasma was stored at 20 C until use. Plasma concentrations of estradiol and progesterone were measured by RIA (Diagnostic Products, Los Angeles, CA). Samples were analyzed in two separate experiments. Data were expressed as the mean sem (n 4 6).

Northern blot analysis

Uterine horns were dissected, freed from the conceptuses and placentas, immediately frozen in liquid N2, and stored at 80 C until use. Total RNA was extracted from both uterine horns derived from one animal by the acid guanidinium thiocyanate-phenol-chloroform method (15). Total RNA (15 g) was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane (Biodyne-A, Pall, Port Washington, NY). Hybridization was performed with 32P-labeled complementary DNA (cDNA) fragments specific for COX-1 (16), COX-2 (16) (corresponding to the first 409 amino acids of the coding region), and the oxytocin receptor (OTR) (11) at 65 C in 6 SSC (1 SSC is composed of 0.15 m NaCl and 0.015 m sodium citrate), 0.5% SDS, and 5 Denhardts solution. After hybridization, filters were washed at 65 C in 2 SSC-1% SDS, and the hybrids were detected by autoradiography. The filters were then rehybridized with a 32P-labeled cDNA fragment specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; CLONTECH Laboratories, Inc., Palo Alto, CA). Autoradiograms were subjected to densitometric analyses for quantification of COX-1, COX-2, or OTR mRNA levels relative to GAPDH mRNA levels using NIH Image software. For each group of tissues, three animals were analyzed, and data were expressed as the mean sem.

Measurement of plasma steroid concentrations

Measurement of plasma concentrations of progesterone and estradiol was performed as reported previously (11). Blood was drawn from the

FIG. 1. Uterine expression of COX-2 (a) and COX-1 (b) mRNA in wild-type mice during late pregnancy. Uterine horns were collected on the indicated days of pregnancy (days 12, 15, 17, and 20) or postpartum (days 2 and 4) and subjected to Northern blot analysis. The positions of the major bands are indicated by arrowheads. The same blots were rehybridized with a 32P-labeled cDNA probe for GAPDH. On day 20 of pregnancy, mice undergoing parturition were analyzed (part.). The lower panels show quantified and normalized COX-2 and COX-1 mRNA levels (mean SEM; n 3). The mRNA levels were expressed as the fold of the level on day 12 (COX-2) or 15 (COX-1) of pregnancy (for COX-2; **, P 0.01 vs. day 17; for COX-1, and *, P 0.05 vs. days 15 and 17, respectively). Northern blot experiments were independently repeated three times with different animals, and similar results were obtained. Representative results are shown in the upper panels.

UTERINE COX EXPRESSION IN PGF RECEPTOR-DEFICIENT MICE In situ hybridization

In situ hybridization was carried out as described previously (17). Uterine horns, dissected and freed from the fetuses and placentas (for Fig. 5) or containing the fetuses and placentas (for Fig. 6) were immediately frozen. Sections 10 m in thickness were cut on a Jung Frigocut 3000E cryostat (Leica Instruments, Nussloch, Germany) and thawmounted onto poly-l-lysine-coated glass slides. Antisense riboprobes were synthesized by transcription with T3 RNA polymerase (Stratagene, La Jolla, CA) in the presence of [ -35S]CTP for in situ hybridization. The sections were fixed with 4% formalin and acetylated with 0.25% acetic anhydride. Hybridization was carried out in a buffer containing 50% formamide, 2 SSC, 10 mm Tris(hydroxymethyl)aminomethane (Tris)Cl (pH 7.5), 1 Denhardts solution, 10% dextran sulfate, 0.2% SDS, 100 mm dithiothreitol, 500 g/ml sheared single stranded salmon sperm DNA, and 250 g/ml yeast transfer RNA. The riboprobes were added to the hybridization buffer at 1.5 105 cpm/ l. After incubation at 60 C for 5 h, the slides were washed for 1 h in 2 SSC. The sections were treated with 20 g/ml ribonuclease A, followed by an additional wash in 0.1 SSC at 60 C for 1 h. The slides were then dipped in nuclear track emulsion (NTB3, Eastman Kodak Co., Rochester, NY). After exposure for 5 weeks at 4 C, the dipped slides were developed, fixed, and counterstained with hematoxylin and eosin. The specificity of the signals for each probe was verified by their disappearance when the sense probe was hybridized (see examples in Fig. 5j for COX-2 and Fig. 5k for COX-1, not shown for others) or an excess amount of unlabeled probe was added (data not shown). These experiments were repeated two or three times with different animals, and similar results were obtained.

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creased by 29.5-fold from that on day 17 (P 0.01). Thereafter, COX-2 mRNA levels gradually decreased between days 2 and 4 postpartum. Hence, expression of COX-2 mRNA was highly induced during parturition. In contrast, COX-1 mRNA was expressed during an earlier period of pregnancy. COX-1 mRNA (the major transcript was 2.8 kb in size) was not detectable on day 12, but the expression of COX-1 mRNA progressively increased between days 15 and 17, reaching maximal levels on day 17 (3.7-fold of that on day 15; P 0.05). By day 20, during parturition, expression of COX-1 mRNA had decreased greatly (P 0.05), resulting in low levels being detected on days 2 and 4 postpartum. Hence, the uterine expression of COX-2 mRNA appeared to be closely associated with parturition, whereas the expression of COX-1 mRNA occurred much earlier than this process.
Expression of COX-1, COX-2, and OTR mRNA in late pregnancy and parturition in FP / mice

Immunohistochemistry
Immunohistochemical analyses were performed essentially using the method described previously (18). Uterine horns were freed from embryos and placentas and treated with Bouins fixative (Muto Pure Chemicals, Tokyo, Japan) for 48 h at 4 C. Sections 20 m in thickness were cut on a Jung Frigocut 3000E cryostat. The free floating sections were incubated with goat polyclonal primary antibody raised against COX-2 (1:8000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 days at 4 C. After incubation with biotinylated secondary antibody against goat IgG (1:2000; Vector Laboratories, Inc., Burlingame, CA) for 2 h, the antibodies were detected using avidin-biotin-peroxidase complexes (diluted 1:4000; Vector Laboratories, Inc.). Labeling was revealed by incubation with 50 mm Tris-Cl (pH 7.6) containing 0.02% 3,3 -diaminobenzidine, 0.0045% H2O2, and 0.6% nickel ammonium sulfate for 3 min to obtain brown-stained products. The sections were counterstained with kernechtrot to give violet staining of the nuclei (19). The specificity of the signals in the immunohistochemical analyses was confirmed by their disappearance when 1) the primary antibody was preabsorbed by antigen (Santa Cruz Biotechnology, Inc.; Fig. 7d), or 2) the sections were incubated with normal goat serum instead of the primary antibody (data not shown). These experiments were repeated two or three times with different animals, and similar results were obtained.

As reported previously (11), whereas the plasma concentration of progesterone of wild-type mice decreased rapidly in late pregnancy, FP / mice did not show such a decrease (Fig. 2). Plasma estradiol levels rose on day 19, compared with those on day 17, in both wild-type and FP / mice (P 0.05) and were not significantly different between the two types of mice during this period (for days 17, 19, 21, and 22, P 0.65, 0.91, 0.16, and 0.60, respectively). Therefore, in wild-type mice, the calculated estradiol/progesterone ratio

Statistical analysis
For the statistical analyses of plasma steroid concentrations and quantified data of Northern blot analyses, one-way ANOVA followed by Students t test were used to evaluate differences between individual groups. Values were considered statistically significant at P 0.05.

Results Expression of COX-1 and COX-2 mRNA in late pregnancy and parturition in wild-type mice

Uterine expression of mRNAs encoding COX-1 and COX-2 was examined during late pregnancy and parturition in wild-type mice (Fig. 1). The major COX-2 transcript was detected as a 4.1-kb band. COX-2 mRNA was hardly detected on days 12, 15, and 17 of pregnancy, but was markedly induced during parturition on day 20; the mRNA level in-

FIG. 2. Plasma concentrations of progesterone, estradiol, and the estradiol/progesterone ratio (E/P) in late pregnancy and postpartum in wild-type mice ( / , closed circles) and during late pregnancy in FP / mice ( / , open circles). Plasma concentrations of progesterone and estradiol were measured by RIA, and E/P was calculated. Values are expressed as the mean SEM (n 4 6). **, P 0.01 for FP / mice vs. wild-type mice.

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markedly increased on days 19 and 21, whereas it remained constant up to day 22 in FP / mice. We then investigated the uterine expression of COX-2 and COX-1 mRNAs on days 17 and 20 of pregnancy in FP / mice (Fig. 3). COX-2 mRNA was highly induced during parturition on day 20 in wild-type mice (to levels 20-fold of the level on day 17). In FP / mice, however, no such induction of COX-2 mRNA was found on day 20. In wildtype mice, COX-1 mRNA was seen to decrease during parturition on day 20. In contrast, no such decrease in COX-1 mRNA was found on day 20 in FP / mice. As ovariectomy of FP / mice on day 19 of pregnancy has been shown to result in the successful delivery of fetuses 20 h after treatment (11), we examined the mRNA expression of COX-1 and COX-2 compared with that of the OTR, a key factor in parturition, during ovariectomy-induced parturition in FP / mice (Fig. 4). OTR mRNA was markedly induced by 12 h after ovariectomy, began to decrease by 16 h (P 0.05), and became minimal at 36 h after treatment (Fig. 4c). In contrast, expression of COX-2 mRNA progressively increased, reached maximal expression at 20 h after ovariectomy (during parturition), and decreased again by 36 h (P 0.05; Fig. 4a). Sham-operated or untreated mice did not show such an induction of either OTR or COX-2 mRNA. Expression of COX-1 mRNA decreased significantly by 16 h and was minimal by 36 h after ovariectomy (Fig. 4b). Sham-operated or untreated mice did not show such a decrease. Thus, ovariectomy resulted in the mRNA expression of COX-1 very different from that of COX-2 and OTR in these FP / mice. In the Northern analysis of COX-2 mRNA, an additional transcript with a higher mol wt (6.5 kb) was detected at 20 h after ovariectomy in FP / mice (Fig. 4a). As this band was

also detected upon Northern analysis with another fragment of the coding region as well as the 3 -untranslated region of COX-2 cDNA, but not with an intron sequence as a probe (data not shown), the band is probably one of the mature COX-2 gene products. In addition, amplification of the fulllength coding sequence of COX-2 mRNA by RT-PCR in this sample resulted in a single band with the predicted size (data not shown), suggesting that the higher band is presumably one of the different COX-2 mRNA variants in the region other than the coding sequence.
Cellular localization of COX-1 and COX-2 in uterine tissues of FP / mice

Distribution of COX-1 and COX-2 mRNA was examined in uterine tissues isolated from wild-type and FP / mice throughout late pregnancy and parturition. The distribution of COX-1 and COX-2 in uterine tissues isolated from FP / mice on days 15 and 17 of pregnancy was identical to that in wild-type mice. The results obtained by in situ hybridization analysis of COX-1 and COX-2 mRNA are summarized in Table 1. We compared the distribution of COX-1 and COX-2 in uterine tissues on day 20 of pregnancy among the three mice groups: 1) wild-type mice undergoing natural parturition, 2) nontreated FP / mice without parturition, and 3) ovariectomized FP / mice undergoing parturition (Figs. 5 and 6). Strong hybridization signals for COX-2 mRNA were observed in the circular layer of the myometrium in wild-type mice (Fig. 5b). Hybridization was not observed in nontreated FP / mice (Fig. 5e), but was again observed in ovariectomized FP / mice (Fig. 5h). In contrast, hybridization signals

FIG. 3. Uterine expression of COX-2 (a) and COX-1 (b) mRNA in late pregnancy in wild-type and FP / mice. Uterine horns were collected from wild-type ( / ) and FP / ( / ) mice on day 17 (17), from wild-type mice undergoing parturition (part.), or from FP / mice on day 20 of pregnancy (20) and were subjected to Northern blot analysis. The same blots were rehybridized with a GAPDH probe. In the lower panels, COX-2 and COX-1 mRNA levels were quantified and normalized to GAPDH mRNA levels (mean SEM; n 3). The mRNA levels were expressed as the fold of the level on day 17 in wild-type mice (**, P 0.01 for part. vs. day 17; , P 0.01 for FP / mice vs. wild-type mice). Experiments were independently repeated three times with different animals, and similar results were obtained. Representative results are shown in the upper panels.

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FIG. 4. Effects of ovariectomy on uterine expression of COX-2 (a), COX-1 (b), and OTR (c) mRNA in FP / mice. FP / mice were ovariectomized bilaterally (ovx), sham-operated (sham), or left untreated ( ) on day 19 of pregnancy; their uterine horns were collected at the indicated hours after treatment; and their RNA was subjected to Northern blot analysis. Quantification of COX-2, COX-1, and OTR mRNA levels normalized to GAPDH mRNA levels is shown in the lower panels (mean SEM; n 3; * and **, P 0.05 and P 0.01 vs. untreated mice, respectively). Levels were expressed as the fold of the mRNA level in untreated mice. Experiments were independently repeated three times with different animals, and similar results were obtained. Representative results are shown in the upper panels.

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TABLE 1. Uterine distribution of COX-1 and COX-2 mRNA in late pregnancy of wild-type ( / ) and FP / ( / ) mice
COX-1 COX-2

( / ), ( / ) 15d, 17d Circular myometrium Decidua Endometrial epithelium ( / ) 20d (undergoing natural parturition) Circular myometrium Decidua Endometrial epithelium ( / ) 20d (without parturition) Circular myometrium Decidua Endometrial epithelium ( / ) 20d (undergoing ovariectomy-induced parturition) Circular myometrium Decidua Endometrial epithelium Relative signal intensities: , very high; , high; , moderate; , background level. Genotype, day of pregnancy (d), and occurrence or absence of parturition are indicated at the top of each set of uterine tissues.

could not be detected for COX-1 mRNA in the circular myometrium in wild-type or ovariectomized FP / mice (Fig. 5, c and i), but COX-1 signals were observed in nontreated FP / mice (Fig. 5f). COX-1 signals were detected in the endometrial epithelium in every group, but extremely high endometrial epithelial expression of COX-1 was detected in FP / mice (Fig. 5, c, f, and i). No hybridization signals were observed when sense probes of COX-2 and COX-1 were used as controls (Fig. 5, j and k, for control of b and c, respectively; not shown for others). In all three groups of mice, signals for both COX-1 and COX-2 mRNA were detected in the decidua and the placental labyrinth (Fig. 6). COX-1 and COX-2 distribution in sham-operated FP / mice was identical with that in nontreated FP / mice (data not shown). Thus, on day 20 of pregnancy, the uterine localization of COX-1 and COX-2 mRNA in FP / mice was quite different from that in wild-type mice. However, the uterine expression of COX-1 and COX-2 in ovariectomized FP / mice was identical to that in wild-type mice. Northern blot and in situ hybridization analyses demonstrated that COX-2 expression in the myometrium is closely associated with the occurrence of parturition. To confirm this expression at the protein level, COX-2 expression was determined by immunohistochemistry (Fig. 7). Intense positive signals for the anti-COX-2 antibody were detected in the circular myometrium in wild-type and ovariectomized FP / mice, both of which underwent parturition. In contrast, no signals were found in nontreated FP / mice. When primary antibodies preabsorbed by antigens were used, all signals were abolished.
Discussion COX-1 expression in late pregnancy

PGF2 plays a crucial role in the induction of parturition. Based on endocrinological analyses, luteolytic PGF2 is thought to be derived from intrauterine tissues. It was recently reported that COX-1-deficient female mice have a defect similar to that of FP / mice, which can be restored by the administration of PGF2 (12). This study also showed a lack of PGF2 synthesis from intrauterine tissues during late pregnancy in COX-1-deficient mice. It is therefore likely that PGF2 synthesized by COX-1 in intrauterine tissues plays a crucial role in the induction of luteolysis. In wild-type female mice, COX-1 mRNA was induced on day 15, reached peak levels on day 17, and decreased on day 20 when parturition occurred. On day 17, COX-1 mRNA was highly expressed in the epithelial layer of the endometrium and the circular layer of the myometrium, but COX-1 expression in endometrial epithelial cells decreased and that in myometrial cells disappeared on day 20, during parturition. These results agreed with previous reports using COX-1specific antibodies (20). In FP / mice, high levels of COX-1 mRNA expression were also observed on day 17, but unlike in wild-type mice, this expression was still high on day 20. In these mice, COX-1 mRNA was persistently expressed in both the endometrial epithelium and the myometrium on day 20, as shown by in situ hybridization. In ovariectomized FP / mice, COX-1 mRNA expression began to decrease by 16 h after ovariectomy and reached minimal levels by 36 h, as observed on day 20 in wild-type mice. As the failure of parturition in FP / mice is due to incomplete luteolysis, the persistent expression of COX-1 mRNA in endometrial epithelial and myometrial cells may reflect the result of a persistent production of progesterone in FP / mice (11). Hence, progesterone can be considered to be one of the possible positive regulators of COX-1 expression in these cells. Indeed, administration of progesterone was sufficient to elicit persistent expression of COX-1 mRNA in ovariectomized FP / mice (our unpublished data). In this case, if this COX-1 expression is responsible for luteolytic PGF2 synthesis, progesterone withdrawal could serve as a negative feedback system of uterine PG synthesis. On the other hand, COX-1 induction was observed on day 15 in intrauterine tissues, which is later than the increase in plasma progesterone. Some other additional regulators are probably necessary for the induction of COX-1 gene expression.
COX-2 expression during parturition

In a previous report we showed that FP / mice demonstrate a failure in parturition, and that this is due to a defect in luteolysis during late pregnancy, resulting in high levels of plasma progesterone (11). These results indicated that

In contrast to the abundant expression of COX-1 mRNA, expression of COX-2 mRNA in intrauterine tissues was not as high throughout late pregnancy. However, on day 20, COX-2 expression was highly induced in the myometrium of wild-type females. One of the most remarkable findings in this study is the absence of COX-2 expression in the myometrium of FP / mice. As plasma estradiol levels in FP / mice are comparable to those in wild-type mice throughout pregnancy (Fig. 2), the absence of COX-2 expression on day 20 in FP / mice is the result of a persistent production of progesterone. When these mice were subjected to ovariectomy, COX-2 as well as OTR expressions were induced in the myometrium (Fig. 5 and data not shown). Both COX-2 and OTR expressions are known to be positively regulated by

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FIG. 5. Myometrial and endometrial epithelial expression of mRNAs for COX-2 (b, e, h, and j) or COX-1 (c, f, i, and k) in wild-type mice undergoing natural parturition (upper panels), in FP / mice with (bottom panels) or without (middle panels) ovariectomy-induced parturition. Uterine sections were subjected to in situ hybridization analyses. ai, Hybridization signals by antisense probes. j and k, Sense probe controls for b and c, respectively. Brightfield (a, d, and g) and darkfield (b, c, e, f, h, i, j, and k) photomicrographs are shown. LM, Longitudinal smooth muscle layer; CM, circular smooth muscle layer; E, endometrial epithelium. Scale bars, 150 m.

estradiol in nonpregnant animals, but progesterone did not have an inhibitory effect on the estradiol-induced expression of either gene (21, 22), suggesting that induction of COX-2 and OTR is mediated by some factors downstream of ovariectomy-induced progesterone withdrawal. However, OTR

expression reached maximal levels by 12 h after ovariectomy, whereas COX-2 expression was initiated by 16 h after treatment and was greatly enhanced during parturition. Such a different profile of COX-2 induction indicates that the mechanisms underlying COX-2 expression are different from

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FIG. 6. Decidual expression of mRNAs for COX-2 (b, e, and h) or COX-1 (c, f, and i) in wild-type mice undergoing natural parturition (upper panels), in FP / mice with (bottom panels) or without (middle panels) ovariectomyinduced parturition. Uterine sections were subjected to in situ hybridization analyses. Brightfield (a, d, and g) and darkfield (b, c, e, f, h, and i) photomicrographs are shown. D, Decidua; P, placental labyrinth. Scale bar, 150 m.

those for OTR gene induction, and that the expression of COX-2 in the myometrium is closely associated with the occurrence of parturition. It has been shown that the in vivo and in vitro production of PGs from uterine tissues is markedly elevated by the stimulation of oxytocin or interleukin-1 (IL-1 ) (13, 20, 23). The induction of OTR preceding COX-2 expression in the myometrium suggested that COX-2 expression may be initiated by oxytocin signaling. On the other hand, IL-1 is also produced locally from immune cells within uterine tissues; this is inhibited by progesterone (24 26). Hence some cytokines are postulated to be potential signaling molecules working during the onset of labor (27). Indeed, IL-1 was shown to induce prominent COX-2 ex-

pression at the transcriptional level in rat myometrial cells (20). It is therefore likely that locally synthesized cytokines such as IL-1 initiate or enhance myometrial COX-2 expression during parturition. Alternatively, it is still possible that the delivery of fetuses itself enhances myometrial COX-2 expression. What is the role of uterine COX-2-derived PGs in parturition? As the expression of COX-2 in the myometrium is closely associated with the occurrence of parturition, COX2-derived PGs seem to play a role in the final pathway of parturition. The facts that PGE2 and PGF2 are the major products of uterine COX and that both are known to have potent uterotonic actions (9) suggest that COX-2-derived PGs

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FIG. 7. Immunohistochemical analysis of the COX-2 protein in the circular myometrium. a, Wild-type mice undergoing natural parturition. b and c, FP / mice without or with ovariectomy-induced parturition, respectively. d, Wild-type mice undergoing natural parturition, stained with the primary antibody preabsorbed by antigen. Signals for the anti-COX-2 antibody (brown) were evident in a and c and were absent in b and d. Sections were counterstained with kernechtrot to stain the nuclei (violet). Scale bar, 25 m.

may be responsible for myometrial contraction. However, in FP / mice, ovariectomy can restore parturition; hence, the uterotonic action of PGF2 is dispensable for parturition (11). Uterine PGE2 has also been thought to be involved in cervical ripening (28). The roles of the uterotonic and nonuterotonic actions of PGE2 should be addressed using mice deficient in each of the subtypes of PGE receptors. The present study indicates that uterine expression of the COX enzymes is biphasic during the periparturient period and is under the control of progesterone. Interestingly, down-regulation of COX-1 expression and induction of COX-2 were also found in human intrauterine tissues during labor (14, 29, 30), although the human circumstance is more complicated; ovarian progesterone production does not continue, luteolysis does not occur, and the systemic progesterone concentration does not decrease prepartum. A decrease in the local concentration of progesterone by alteration of its metabolism has been proposed as a possible mechanism underlying the onset of human labor (31), but it remains to be tested whether expression of the COX enzymes is regulated under the influence of progesterone or by different mechanisms. However, the association of COX-2 induction in intrauterine tissues with the occurrence of labor may re-

flect the importance of COX-2-derived PGs in the natural parturition process in humans.
Acknowledgments
We thank Dr. M. Katsuyama, Ms. H. A. Popiel, and Mr. S. Takami for their helpful discussions and generous support.

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