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BOR - Papers in Press, published online ahead of print March 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013870
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BIOLOGY OF REPRODUCTION 69, 195–201 (2003)
DOI: 10.1095/biolreprod.102.013870
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Role of Prostaglandin H2 Synthase 2 in Murine Parturition: Study on Ovariectomy-Induced Parturition in Prostaglandin F Receptor-Deficient Mice1

Kazuhito Tsuboi, Aya Iwane, Sayako Nakazawa, Yukihiko Sugimoto, and Atsushi Ichikawa2

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606–8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine the prostaglandin (PG) H2 synthase (generally referred to as cyclooxygenase [COX]) isozyme responsible for producing uterotonic PGs during parturition, we used PGF2{alpha} receptor-deficient mice, which exhibit parturition failure due to impaired withdrawal of serum progesterone at term. On ovariectomy-induced parturition in these mice, uterine COX-2 mRNA expression was drastically induced in the myometrium, whereas COX-1 mRNA expression in the endometrial epithelium decreased. The concomitant administration of progesterone with ovariectomy resulted in a delay in parturition and the disappearance of both the increase in COX-2 mRNA and the decrease in COX-1 mRNA. Thus, the expression of myometrial COX-2 and the occurrence of parturition are closely associated in this model. Furthermore, administration of the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522, effectively delayed ovariectomy-induced parturition in these mice. These findings suggest that COX-2-derived PGs contribute to the onset of parturition after the decrease in serum progesterone level.

female reproductive tract, parturition, pregnancy, progesterone, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanism that determines the timing of parturition remains elusive despite intensive studies. Preterm parturition, resulting from the disruption of normal parturition signaling, is considered to be the major cause of neonatal mortality. Therefore, coming to an understanding of the molecular events that trigger parturition is essential. Prostaglandins (PGs) 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]. Aspirin-like drugs, which inhibit the enzymatic activities of the COX isozymes, have been known to cause a delay in parturition in many species, suggesting that PGs are important mediators of the onset of parturition [6]. Of the two isozymes of COX, COX-1 is mostly expressed constitutively, playing housekeeping roles in many tissues, whereas COX-2 is induced on various pathophysiological stimuli such as by cytokines [7].

In the induction of parturition of rodents, a decrease in serum progesterone levels precedes uterine contraction. We previously reported that PGF2{alpha} receptor-deficient (FP-/-) mice exhibit a phenotype of loss of parturition due to impaired luteolysis and persistently high serum progesterone levels at term [8]. Gross et al. [9] reported that COX-1-deficient mice exhibit similar parturition defects with impaired luteolysis. These studies together indicate that COX-1-derived PGF2{alpha} is essential for luteolysis, which is a trigger for the induction of murine parturition. On the other hand, along with luteolytic action of PGF2{alpha}, PGE2 and PGF2{alpha} have been reported to have potent uterotonic activities in the periparturient uterus [10], suggesting that these PGs may contribute to the parturition process by directly contracting myometrial smooth muscle after the decrease in serum progesterone levels. However, it remains unclear as to which COX isozyme is responsible for producing these uterotonic PGs. We have previously reported that the uterine expression of COX-2 mRNA is induced and the expression of COX-1 mRNA is decreased during parturition in wild-type mice, suggesting the involvement of COX-2 in producing uterotonic PGs during parturition [11]. A significant amount of COX-1 is expressed in intrauterine tissues at term, and the role of COX-2 has been obscured by the observation that a COX-2-selective inhibitor delayed murine parturition only at high doses [12]. COX-2-deficient mice have not been used for the analysis of parturition due to their infertility [13].

To determine the isozyme responsible for producing uterotonic PGs during parturition, we used an ovariectomy-induced parturition model in FP-/- mice. As stated herein, FP-/- mice do not undergo parturition due to persistently high serum progesterone levels. Ovariectomy on the day before the expected term date is able to induce both successful parturition and uterine COX-2 mRNA expression at 20 h after the treatment [11]. In the present study, we show that the expression of COX-2 mRNA in the myometrium is closely correlated with the occurrence of parturition, since myometrial COX-2 mRNA expression at 20 h after ovariectomy greatly decreased when parturition was delayed by treatment with progesterone. Furthermore, both COX-nonselective and COX-2-selective inhibitors effectively delayed ovariectomy-induced parturition in FP-/- mice. Taken together, we propose that COX-2 is the major isozyme responsible for the production of PGs, contributing to the onset of parturition downstream of the decrease in serum progesterone level, presumably via their uterotonic activities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal and Tissue Preparations

Female FP-/- mice, with the chimeric background (129/Ola x C57BL/6), were maintained at 23°C under a 12L:12D cycle [8]. To obtain timed-pregnant mice, FP-/- virgin female mice (8 to 12 wk of age) were housed overnight with C57BL/6 males and checked the following morning for vaginal plugs. The day when a vaginal plug was observed was counted as Day 1 of pregnancy. FP-/- mice were anesthetized by ether and bilaterally ovariectomized at 2130–2230 h on Day 19 of pregnancy as previously described [8, 11]. Because not only progesterone withdrawal but also an increase in estradiol level appear necessary for induction of parturition, we investigated the effect of progesterone or estradiol on the ovariectomy-induced parturition and COX gene expression, and we also added both steroid-administrated groups to examine possible interactions of each effect. FP-/- mice were treated subcutaneously with vehicle (0.1 ml of sesame oil), progesterone (1 mg; Research Biochemicals International, Natick, MA), estradiol (17ß-estradiol, 250 ng; Research Biochemicals International), or both estradiol and progesterone immediately after the ovariectomy. First, the time of onset of parturition after ovariectomy was examined by infrared videorecording. The onset of parturition was defined as the time of complete delivery of the first pup in each mother. Second, uterine horns were isolated 20 h after ovariectomy and subjected to Northern blot analysis or in situ hybridization analysis. Control animals received a sham operation followed by vehicle treatment. For the groups administered with COX inhibitors, animals were treated subcutaneously with vehicle (0.1 ml of 3.5% dimethyl sulfoxide in sesame oil), the COX-nonselective inhibitor, indomethacin (5 mg/kg; Sigma, St. Louis, MO), or COX-2-specific inhibitors, Dup-697 (5 mg/kg; Cayman Chemical, Ann Arbor, MI) [14] or JTE-522 (15 mg/kg; 4-[4-cyclohexyl-2-methyloxazol-5-yl]2-fluorobenzenesulphonamide; a gift from Japan Tobacco Inc., Osaka, Japan) [15] at 12 h after ovariectomy. Thereafter, the onset of parturition was monitored by infrared videorecording. All mouse breeding and experiments were performed according to the guideline for animal experiments of Kyoto University.

Northern Blot Analysis

Uterine horns were dissected, freed from the conspectuses 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 [16]. Total RNA (15 µg) was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane (Biodyne-A, Pall, Port Washington, NY). Hybridizations were performed with 32P-labeled cDNA fragments specific for COX-1 [17], COX-2 [17], and connexin-43 (Cx-43) (1.2-kilobase fragment corresponding to the entire coding region) [18] at 65°C in 6 x SSC (1 x SSC is composed of 0.15 M NaCl and 0.015 M sodium citrate), 0.5% SDS, and 5 x Denhardt solution. After hybridization, filters were washed at 65°C in 0.5 x SSC and 0.25% 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, Palo Alto, CA). Autoradiograms were subjected to densitometric analyses for quantification of COX-1, COX-2, or Cx-43 mRNA levels relative to GAPDH mRNA levels using the National Institutes of Health Image software. For each group of tissues, three animals were analyzed and data were expressed as mean ± SEM.

In Situ Hybridization

In situ hybridization was performed as described previously [11, 19]. Uterine horns were dissected, freed from the fetuses and placentas, and immediately frozen. Sections 10 µm in thickness were cut on a Jung Frigocut 3000E cryostat (Leica Instruments, Nussloch, Germany) and thaw mounted onto poly-L-lysine-coated glass slides. Mouse cDNAs for COX-1 and COX-2 [17] were prepared in the pBluescript II vector (Stratagene, La Jolla, CA), and antisense or sense riboprobes specific for COX-1 and COX-2 were synthesized by transcription with T3 or T7 RNA polymerase (Stratagene) in the presence of [{alpha}-35S]CTP. The sections were fixed with 4% formalin and acetylated with 0.25% acetic anhydride. Hybridization was performed in a buffer containing 50% formamide, 2 x SSC, 10 mM tris(hydroxymethyl)aminomethane-Cl (pH 7.5), 1 x Denhardt solution, 10% dextran sulfate, 0.2% SDS, 100 mM dithiothreitol, 500 µg/ml of sheared single-stranded salmon sperm DNA, and 250 µg/ml of yeast tRNA. The antisense riboprobes were added to the hybridization buffer at 1.5 x 105 cpm/µl. After incubation at 60°C for 5 h, the slides were washed for 1 h in 2 x SSC. The sections were treated with 20 µg/ml of ribonuclease A, followed by an additional wash in 0.1 x SSC at 60°C for 1 h. The slides were then dipped in nuclear track emulsion (NTB3, Eastman Kodak, Rochester, NY). After exposure for 1.5 (for COX-1) or 4 (for COX-2) wk at 4°C, the dipped slides were developed, fixed, and counterstained with hematoxylin-eosin. These experiments were repeated two or three times with different animals and similar results were obtained.

Statistical Analysis

For the statistical analyses of the time of onset of parturition after ovariectomy and the quantified data of Northern blot analyses, one-way ANOVA followed by the Student or Welch t-test were used to evaluate differences among individual groups. Some statistical analyses were performed after logarithmic transformations of the data when they were appropriate. Values were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Steroid Treatment on the Onset of Parturition after Ovariectomy at Term in FP-/- Mice

We examined the effects of progesterone and/or estradiol treatments of FP-/- mice on the timing of ovariectomy-induced parturition by an infrared videorecording system. FP-/- mice treated with vehicle exhibited parturition at 19.9 ± 2.3 h (n = 4) after ovariectomy on the evening of Day 20 (1800 h on the average). On the other hand, the onset of parturition was delayed to 43.6 ± 4.2 h (n = 4) after ovariectomy in the progesterone-treated mice on the evening of Day 21 (1800 h on the average) (Fig. 1). However, treatment of FP-/- mice with estradiol had no effect on the onset of parturition either in the presence or absence of progesterone treatment. These results demonstrated that progesterone but not estradiol has a retardant effect on the onset timing of parturition in this model.



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  		FIG. 1. Effects of the treatment with ovarian steroids on the onset of parturition after ovariectomy in FP-/- mice. FP-/- mice on Day 19 of pregnancy were ovariectomized and immediately treated with vehicle (–), progesterone (P), estradiol (E), or both estradiol and progesterone (EP). The time of onset of parturition after ovariectomy was examined. Data are expressed as mean ± SEM. The number of mice used in each group is indicated in parentheses. **P < 0.01 vs. vehicle

Expression of COX-1 and COX-2 mRNAs after Ovariectomy at Term in FP-/- Mice

To assess possible contribution of COX isozymes to the onset of ovariectomy-induced parturition, we examined uterine expression levels of mRNAs for COX-1, COX-2, and Cx-43, a myometrial major gap junction protein, at 20 h after each treatment by Northern blot analyses (Fig. 2). At this point, vehicle- or estradiol-treated mice underwent parturition but other groups did not (Fig. 1). In FP-/- mice, the levels of uterine COX-1 mRNA expression were persistent during late pregnancy, as reported previously [11]. Ovariectomy on Day 19 resulted in a significant decrease in COX-1 mRNA levels compared with the levels on sham treatment and on Day 17. Estradiol treatment enhanced the decrease in COX-1 mRNA expression. However, the inhibition of ovariectomy-elicited parturition by progesterone or both steroid treatments reversed the COX-1 gene expression to the levels on sham treatment. In contrast to COX-1, ovariectomy led to highly induced uterine COX-2 mRNA levels (on Day 20, 23.8- and 43.3-fold of the levels on sham treatment, and on Day 17, respectively; P < 0.01 for both), accompanied with the induction of parturition. The expression of uterine COX-2 mRNA was down-regulated to faint levels, when the parturition was inhibited by progesterone treatment. Estradiol treatment had no effect on uterine COX-2 mRNA levels in either the presence or absence of progesterone treatment. The change in expression levels for Cx-43 mRNA was similar to but showed more modest changes than that for COX-2 mRNA on treatment with ovarian steroids. Thus, the expression of uterine COX-2 mRNA and the occurrence of parturition were closely associated.



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  		FIG. 2. Effect of ovariectomy and ovarian steroid treatment on uterine expression of COX-1 (a), COX-2 (b), and Cx-43 (c) mRNAs in late pregnant FP-/- mice. FP-/- mice were ovariectomized bilaterally (ovx) or sham operated (sham) on Day 19 of pregnancy and were treated with vehicle (–), progesterone (P), estradiol (E), or both estradiol and progesterone (EP). Uterine horns were collected 20 h after the treatment and subjected to Northern blot analysis (20). Uterine horns of FP-/- mice on Day 17 of pregnancy without treatment were also subjected to Northern blot analysis (d 17). The positions of the major bands are indicated by arrowheads. The same blots were rehybridized with a 32P-labeled cDNA probe for GAPDH. The lower panels show quantified and normalized COX-1, COX-2, and Cx-43 mRNA levels relative to GAPDH mRNA levels (mean ± SEM; n = 3). Values were expressed as the fold of the level of sham-operated mice treated with vehicle. *P < 0.05 and **P < 0.01 vs. sham-operated mice treated with vehicle. {dagger}P < 0.05 and {dagger}{dagger}P < 0.01 vs. ovariectomized mice treated with vehicle

Cellular Localization of COX-1 and COX-2 mRNAs after Ovariectomy at Term in FP-/- Mice

We next examined the distribution of COX-1 and COX-2 mRNAs in uterine tissues at 20 h after ovariectomy and administration of ovarian steroids in FP-/- mice (Figs. 3 and 4). In situ hybridization analyses revealed a marked spatial separation between COX-1 and COX-2 mRNAs in these tissues. Signals for COX-1 mRNA were found in the endometrial epithelium in vehicle-, progesterone-, and both estradiol- and progesterone-treated mice (Fig. 3b, c, and e). Consistent with the results of the Northern blot analysis, COX-1 signals were weak in estradiol-treated mice (Fig. 3d). In contrast to COX-1 signals, hybridization signals for COX-2 mRNA were observed in circular myometrium in vehicle-treated mice (Fig. 4b). However, consistent with the results of the Northern blot analysis, these signals disappeared when parturition was inhibited by progesterone treatment (Fig. 4c). Estradiol treatment had no effect on the distribution and signal intensity of COX-2 mRNA in the absence of progesterone treatment (Fig. 4d) and did not induce COX-2 mRNA signals in the presence of progesterone treatment (Fig. 4e). Hybridization signals were abolished when an excess amount of unlabeled probe was added (Figs. 3f and 4f, for controls of Figs. 3b and 4b, respectively; not shown for others) or the sense probe was hybridized (data not shown).



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  		FIG. 3. Bright-field (a) and dark-field (b–f) photomicrographs showing hybridization signals for COX-1 mRNA in uterine tissues after ovariectomy and administration of ovarian steroids in late pregnant FP-/- mice. FP-/- mice on Day 19 of pregnancy were ovariectomized and treated with vehicle (a, b, f), progesterone (c), estradiol (d), or both estradiol and progesterone (e). Uterine horns were collected at 20 h after the treatment, and sections were subjected to in situ hybridization analyses. The specificity of hybridization signals was verified by disappearance of the signals in (b) in the presence of an excess amount of unlabeled probe (f) or by absence of the signals with the sense probe (data not shown). LM, Longitudinal smooth muscle layer; CM, circular smooth muscle layer; E, endometrial epithelium. Bar = 150 µm



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  		FIG. 4. Bright-field (a) and dark-field (b–f) photomicrographs showing hybridization signals for COX-2 mRNA in uterine tissues after ovariectomy and administration of ovarian steroids in late pregnant FP-/- mice. FP-/- mice on Day 19 of pregnancy were ovariectomized and treated with vehicle (a, b, f), progesterone (c), estradiol (d), or both estradiol and progesterone (e). Uterine horns were collected at 20 h after the treatment, and sections were subjected to in situ hybridization analyses. The specificity of hybridization signals was verified by disappearance of the signals in (b) in the presence of an excess amount of unlabeled probe (f) or by absence of the signals with the sense probe (data not shown). LM, Longitudinal smooth muscle layer; CM, circular smooth muscle layer; E, endometrial epithelium. Bar = 150 µm

Delaying Effects of COX-Nonselective or COX-2-Selective Inhibitors on the Onset of Parturition after Ovariectomy in FP-/- Mice

To determine whether COX-2-derived PGs have a role in the onset of parturition after ovariectomy in FP-/- mice, animals were treated with COX-nonselective or COX-2-selective inhibitors at term after ovariectomy, and the onset of parturition was monitored. The time to delivery after ovariectomy was significantly prolonged by treatment with the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522 (Fig. 5), suggesting a role of COX-2-derived PGs in the onset of parturition in this system.



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  		FIG. 5. Delaying effects of COX-nonselective and COX-2-selective inhibitors on the onset of parturition after ovariectomy in FP-/- mice. FP-/- mice were ovariectomized on Day 19 of pregnancy. At 12 h after ovariectomy, animals were treated with vehicle (–), the COX-nonselective inhibitor, indomethacin (Indo), or the COX-2-selective inhibitors, Dup-697 (Dup) or JTE-522 (JTE). The time of onset of parturition after ovariectomy was examined. Data are expressed as mean ± SEM. The number of mice used in each group is indicated in the parentheses. *P < 0.05 and **P < 0.01 vs. vehicle, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The principal finding in the present study is that induction of the COX-2 gene in the myometrium is closely associated with the onset of ovariectomy-induced parturition in FP-/- mice, whereas expression of the COX-1 gene in the uterus is down-regulated at that time. This observation supports the important role of COX-2, which is induced after the decline of serum progesterone levels in the signaling pathway of murine parturition. Indeed, COX-2-selective inhibitors were able to delay ovariectomy-induced parturition in FP-/- mice. Reese et al. [12] recently proposed that COX-2 may not be involved in mouse parturition because its mRNA is undetectable in the myometrium on the morning of Day 19 of pregnancy. Although we have a similar result that shows the absence of the COX-2 mRNA in the myometrium of wild-type mice on the morning of Day 19, strong signals for COX-2 mRNA were detected in the myometrium during both natural parturition on Day 20 in wild-type mice and ovariectomy-induced parturition in FP-/- mice, occurring an average of 20 h after operation. Indeed, the expression of COX-2 mRNA could be induced 16 h but not 12 h after ovariectomy in FP-/- mice [11]. Thus, induction of COX-2 gene in the myometrium presumably starts only a few hours before the onset of parturition. Based on these results, we concluded that COX-2-derived PGs other than PGF2{alpha} contribute to the onset of parturition after the decrease in circulating progesterone levels.

The role of COX-2 in preterm parturition has been proposed by earlier studies. Specifically, COX-2 mRNA has been induced in the uterus on lipopolysaccharide- or ethanol-induced preterm parturition [20, 21]. Furthermore, the pharmacological inhibition of COX-2 but not the genetic ablation of COX-1 delayed lipopolysaccharide-induced preterm parturition [20, 22]. Thus, COX-2 may have an important role in preterm parturition and in our ovariectomy-induced term parturition in FP-/- mice. On the other hand, Reese et al. [12] reported that a COX-2-selective inhibitor did not significantly delay murine spontaneous term parturition, except at high doses that may also inhibit COX-1. They thus suggested that COX-2 does not play a primary role in the signaling pathway of parturition, which is inconsistent with our results. This discrepancy may have arisen from the higher sensitivity of our system for detecting the tocolytic effects of the COX-2-selective inhibitor than by the analysis of spontaneous parturition. The lower variability in the time of ovariectomy-induced parturition on vehicle treatment in our system than that seen with spontaneous parturition potentiates the sensitivity for detecting the tocolytic effects and thus should be useful for studies such as this that investigate potential tocolytic treatments.

Although COX inhibitors are an effective treatment for preterm parturition in humans, their use is limited by the adverse effects of inducing fetal ductus arteriosus (DA) closure in utero [23]. It has been reported that 100% of mice lacking both COX-1 and COX-2 died within 12 h of birth due to an abnormality in the DA, whereas the mortality rate was only 35% in the COX-2-deficient mice [24]. This result, together with our studies, suggests that the use of COX-2-selective inhibitors should provide a therapeutic advance toward the treatment of preterm parturition with fewer adverse effects than COX-nonselective inhibitors if the extent of the roles of each COX isozyme on DA are conserved across animal species. Indeed, the COX-2 inhibitor has been reported to possibly prevent human preterm delivery [25]. However, caution is needed on using COX-2 inhibitors for the treatment of preterm labor due to the remaining risk on the DA and the reported adverse effects on the fetal kidney [26].

One of the intriguing findings in this study is that administration of progesterone was sufficient to completely suppress uterine COX-2 mRNA induced by ovariectomy in FP-/- mice. Therefore, periparturient withdrawal of circulatory progesterone may be crucial for the induction of uterine COX-2 mRNA in wild-type and ovariectomized FP-/- mice. In contrast, administration of estradiol failed to show any effects on ovariectomy-induced and progesterone-suppressed COX-2 expression, as observed in the timing of parturition. These results may reflect that the controls of gene expression by progesterone predominate those by estradiol during late pregnancy. Indeed, in the present study, COX-2 showed a similar expression pattern to that of Cx-43, a proposed important gap junctional protein during parturition, which was demonstrated to be induced by progesterone withdrawal in the periparturient uterus [27]. The negative regulation of COX-2 mRNA by progesterone in vivo has also been suggested by Critchley et al. [28], who reported that the cessation of progesterone administration elevates COX-2 mRNA expression in the nonpregnant human uterus. However, progesterone did not decrease COX-2 mRNA levels in bovine myometrial or epithelial cells in vitro [29, 30]. These differences suggest that the negative regulation of COX-2 mRNA by progesterone is by an indirect fashion and may require some paracrine factors.

The result that COX-2 localizes in the myometrium during parturition, which is mostly consistent with previous findings [31], may be of help in considering the possible contribution of immune cells and paracrine factors in the negative regulation of COX-2 gene expression by progesterone. Mackler et al. [32, 33] reported that macrophages are accumulated in murine myometrium, especially stroma surrounding muscle bundles in late pregnancy. Intrauterine macrophages are known to produce interleukin-1ß (IL-1ß), which is locally acting on neighboring cells, but this cytokine production is inhibited by progesterone [34–36]. Furthermore, like IL-1ß, it was suggested that macrophage-derived chemokines, such as monocyte chemotactic protein 1 and RANTES (regulated on activation, normal T-cell expressed and secreted), are present within uterine tissues, and their expression was also suppressed by progesterone [37, 38]. Such suppression in paracrine factor production in macrophages may mediate the negative regulation of COX-2 gene expression by progesterone. Indeed, IL-1ß has been shown to induce COX-2 expression at the transcriptional level in rat myometrial cells [39].

In our study, ovariectomy treatment decreased uterine COX-1 mRNA expression, which was restored by the administration of progesterone, suggesting that progesterone is necessary for elevated COX-1 mRNA expression during late pregnancy. This stimulatory effect of progesterone is consistent with other reports using nonpregnant animals. Specifically, antiprogestin treatment was found to abolish COX-1 mRNA expression in the baboon endometrial epithelium during the luteal phase of the menstrual cycle [40], and the administration of progesterone elicited a modest induction of uterine COX-1 mRNA expression in ovariectomized nonpregnant mice [41]. However, this stimulatory action of progesterone may have species-specific differences because the treatment of progesterone had no effect on uterine COX-1 in ovariectomized nonpregnant sheep [42]. In contrast to progesterone, the administration of estradiol greatly decreased uterine COX-1 mRNA in ovariectomized FP-/- mice. We previously reported that uterine COX-1 mRNA decreases sharply during spontaneous parturition in wild-type mice, in which serum estradiol levels rise at term [11]. Thus, a rise in serum estradiol concentrations, in addition to the withdrawal of progesterone, is thought to be required for the rapid down-regulation of uterine COX-1 mRNA during spontaneous parturition. The inhibitory effect of estradiol on COX-1 mRNA is surprising because estradiol has been reported to increase COX-1 mRNA in the uterus of ovariectomized nonpregnant mice [41, 43]. It is unknown why this difference arises, but it may suggest that the regulation of COX-1 mRNA by estradiol is indirect and is mediated by other factors specific for each condition.

In summary, our results suggest that COX-2 is induced in the myometrium and COX-2-derived PGs other than PGF2{alpha} play a pivotal role in the onset of parturition under the control of progesterone withdrawal. However, the exact mechanism by which COX-2-derived PGs contribute to the parturition process requires further investigation.


   ACKNOWLEDGMENTS
 
We thank Drs. N. Eguchi and Y. Urade for their generous assistance with the infrared videorecording technique. We also thank Ms. H. A. Popiel for her careful reading of the manuscript and Ms. S. Terai for secretarial assistance.


   FOOTNOTES
 
1 This work was supported in part by the grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research; the Sankyo Foundation of Life Science, the Takeda Science Foundation, and Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology of Japan. Back

2 Correspondence: Atsushi Ichikawa, Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606–8501, Japan. FAX: 81 75 753 4557; aichikaw{at}pharm.kyoto-u.ac.jp Back

Received: 27 November 2002.

First decision: 28 December 2002.

Accepted: 26 February 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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