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Endocrinology Vol. 143, No. 7 2593-2598
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Coordinate Regulation of Prostaglandin Metabolism for Induction of Parturition in Mice

Sandra K. Winchester, Takuji Imamura, Gil A. Gross, Lisa M. Muglia, Sherri K. Vogt, Jason Wright, Kikuko Watanabe, Hsin-Hsiung Tai and Louis J. Muglia

Departments of Pediatrics, Molecular Biology and Pharmacology, and Obstetrics and Gynecology (S.K.W., T.I., G.A.G., L.M.M., S.K.V., J.W., L.J.M.), Washington University School of Medicine, St. Louis, Missouri 63110; Division of Applied Life Sciences (K.W.), University of East Asia, Shimonoseki, Yamaguchi, Japan 751-8503; and Division of Pharmaceutical Sciences (H.-H.T.), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Louis J. Muglia, M.D., Ph.D., Washington University School of Medicine, 660 Euclid Avenue, Department of Pediatrics, Box 8208, St. Louis, Missouri 63010. E-mail: . muglia_l{at}kids.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins are essential for the initiation of parturition in mice. The peak in uterine prostaglandin F2{alpha} levels occurs at d 19.0 of gestation, just before the onset of labor. Our studies set out to determine the important regulatory step(s) involved in this increase of prostaglandin F2{alpha}. We show that cytosolic phospholipase A2 mRNA, protein, and activity do not significantly vary during mouse gestation. Rather, our studies demonstrate that cyclooxygenase-1 mRNA is abruptly induced at d 15.5 of gestation, but cyclooxygenase-1 protein levels only gradually increase throughout gestation. In contrast, cyclooxygenase-2 protein remains constant during gestation. We find that prostaglandin F synthase protein increases significantly during gestation reaching peak levels between d 15.5 and d 17.5 of gestation. We also find that the level of prostaglandin dehydrogenase, responsible for degradation of prostaglandins, decreases during late gestation. Taken together these results suggest that the regulation of prostaglandin F2{alpha} is a complex process involving the coordinate induction of synthetic enzymes along with a decrease in degradative enzymes involved in prostaglandin metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRETERM LABOR AND delivery of infants is a frequent, and often devastating, complication of pregnancy that has remained refractory to medical intervention. The limited ability to treat preterm labor chiefly stems from inadequate understanding of the mechanisms controlling the normal onset of labor and the potential ways in which these mechanisms can malfunction. Although many families of molecules have the capacity to promote uterine contractions (1), including prostaglandins, neuropeptides such as oxytocin, and inflammatory cytokines, how these factors are induced at the appropriate point during gestation and result in appropriately timed labor is unknown.

Genetic studies in mice have proven extremely valuable for unequivocal identification of essential components of the pathway necessary for labor in this species (2). The action of prostaglandin F2{alpha} (PGF2{alpha}) on the corpus luteum of the ovary, to cause luteolysis and a fall in plasma progesterone, is integral to the process of labor initiation (3). As would be expected from this finding, mutations in enzymes altering PGF2{alpha} production have dramatic effects on the timing of parturition. Parturition studies in mice deficient in cytosolic phospholipase A2 (cPLA2) (4, 5) and cyclooxygenase-1 (COX-1) (6, 7) have demonstrated these to be the specific prostaglandin (PG) synthetic isoforms required for release of arachidonic acid from plasma membranes and conversion to prostaglandin H2 (PGH2), the requisite intermediate for PGF2{alpha} synthase (Fig. 1Go). Despite knowing that these isoforms are essential for parturition, how their activity is altered to result in the precisely timed increase in PGF2{alpha} for luteolysis has not been determined. Indeed, previous studies have revealed that COX-1 mRNA is induced at least 2 d before the onset of PGF2{alpha} elevation that is required for parturition (7). The dissociation of COX-1 mRNA and PGF2{alpha} induction suggests other mechanisms must be brought into play for establishing the timing for parturition. These mechanisms could include increasing substrate for COX-1 to act upon, dissociation of COX-1 mRNA and protein production, specific regulation of PG F synthase (PGFS) expression, or alteration in PGF2{alpha} degradation. To determine the important regulatory step(s) in the induction of uterine PGF2{alpha} in the mouse, we analyzed expression of each component of the prostaglandin synthetic and degradative pathway.



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  		Figure 1. Schematic diagram of the PG synthesis pathway. Overview of the PG synthesis pathway for the formation of PGF2{alpha}.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal husbandry
Wild-type and COX-1 knockout (6) mice were maintained on a 12-h light/12-h dark cycle and provided with ad libitum access to food and water. All protocols used were in accordance with the NIH guidelines and approved by the Animal Care and Use Committee at Washington University School of Medicine. Matings of estrous females with stud males were determined by the presence of a copulation plug on the morning following introduction into the male cage. After detection of a copulation plug, females were removed from the male cage, with the morning of plug detection designated as 0.5 d gestation to obtain accurate gestational timing.

Plasma progesterone measurements
Plasma progesterone levels were measured by RIA according to the kit instructions (Diagnostics Products, Los Angeles, CA). Blood samples were obtained by retroorbital phlebotomy and measurements averaged from two to four wild-type mice on specific days of gestation.

RNA hybridization analysis
Uterine tissue, separated from cervical tissue, was harvested from wild-type or COX-1 knockout mice and immediately frozen in liquid nitrogen. RNA was prepared by the guanidinium thiocyanate-cesium chloride method (8). Briefly, 10 µg of uterine RNA was subjected to electrophoresis on 1.2% agarose-formaldehyde gels and transferred overnight to nitrocellulose membranes. The membrane was hybridized with [32P]dUTP-labeled RNA probes specific to either murine cPLA2 (407 bp EcoRI fragment in pBluescript SKII+) or murine COX-1 mRNA (1298-bp ScaI fragment in pBluescript SKII+). The blots were hybridized overnight at 60 C in 50% formamide-containing buffer. The filters were then washed and the hybridized probes were quantitated on a phosphor imager. Each mRNA hybridization signal was corrected for loading by normalization to a cyclophilin A probe.

Total membrane protein preparation
Uterine tissue was harvested from gravid wild-type mice and frozen in liquid nitrogen. Total membrane extracts were prepared by homogenization in 20 mM Tris (pH 7.6) 2 mM MgCl2. The resulting homogenate was centrifuged at 600 x g for 2 min. The supernatant was then spun at 30,000 x g for 20 min at 4 C. The supernatant was aliquoted and the remaining membrane pellet was resuspended in homogenization buffer. Protein concentrations were determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL).

Microsomal protein extraction
Microsomal protein extracts were prepared according to the method of Johnson et al. (9). Uterine tissue was homogenized in 0.1 M phosphate buffer, 10 mM EDTA, 250 mM mannitol, 300 µM diethyl dithiocarbamate. The tissue homogenates were then spun at 10,000 x g for 20 min. The resulting supernatant was centrifuged at 100,000 x g for 1 h at 4 C. The microsomal pellet was resuspended in 1% Tween, 80 mM Tris (pH 8.0), 300 µM diethyl dithiocarbamate, 500 µM EDTA. Protein concentrations were determined by a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Cytosolic protein preparation
Cytosolic protein extracts were prepared by homogenization of uterine tissue in 0.25 M sucrose, 50 mM HEPES buffer (pH 7.4). The protein homogenates were spun at 133,000 x g for 1 h at 4 C. The resulting supernatant was collected and protein concentrations were determined by the BCA protein assay (Pierce Chemical Co.).

Protein immunoblot analysis
Cytosolic phospholipase A2.
Ten micrograms total membrane protein extracts were subjected to electrophoresis on an 8% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked in Tween-Tris-buffered saline (TTBS; 0.1% Tween 20, 25 mM Tris, 137 mM NaCl, 3 mM KCL) containing 5% dry milk and then incubated with cPLA2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:1000 overnight at 4 C.

Cyclooxygenase-1 and -2.
Five micrograms microsomal protein extracts were subjected to electrophoresis on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked in TTBS containing 5% dry milk and incubated with COX-1 (1:200) or COX-2 (1:1000) antibody (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature.

PGFS and PG dehydrogenase.
Ten micrograms cytosolic uterine protein extracts were subjected to electrophoresis on a 15% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked in TTBS containing 5% dry milk and incubated with antisera to either liver or lung PGFS (10, 11), 1:3000 or with antisera to prostaglandin dehydrogenase (12), 1:1000 overnight at 4 C.

After incubation with primary antibody, the membranes were washed in TTBS three times for 15 min each and then exposed to horseradish peroxidase-conjugated antimouse or antirabbit IgG secondary antibody (1:1000) at room temperature for 1 h. Proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Equivalent loading and transfer of protein was determined by incubation of membranes with antisera to ß-actin (Sigma, St. Louis, MO), 1:1000 at room temperature for 30 min or with antisera to binding protein (BiP) (BD Transduction Sciences, San Diego, CA), 1:1000 at room temperature for 1 h.

Cytosolic phospholipase A2 protein activity assay
The cPLA2 activity assays were performed according to the manufacturer’s instructions (Cayman Chemical Co., Ann Arbor, MI). Briefly, 50 µg lyophilized total membrane uterine extracts were resuspended in cPLA2 assay buffer (80 mM HEPES, pH 7.4; 150 mM NaCl; 10 mM CaCl2; 4 mM Triton X-100; 30% glycerol; 1 mg/ml BSA). The reaction was initiated with the addition of arachidonyl thiophosphatidylcholine substrate and incubated at room temperature for 1 h. Following the incubation, 5,5'-dithio(2-nitrobenzoic acid)/EGTA was added to each well to terminate the reaction and develop color. The plate was incubated for 5 min at room temperature and then read at 405 nm.

Statistical analysis
Statistical analyses of protein immunoblots and the cPLA2 activity assays were determined using one-way ANOVA. Values were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of plasma progesterone concentration during murine gestation
Our previous studies have shown that uterine PGF2{alpha} levels remain low through early and midgestation and then increase sharply at approximately d 18.5 of gestation to exert their effects on the corpus luteum (7). To determine the temporal relationship between this rise in uterine PGF2{alpha} and the fall in circulating progesterone, we measured plasma progesterone concentration at daily intervals during pregnancy. As shown in Fig. 2Go, the plasma progesterone concentration rises in the mouse during early and midgestation. Plasma progesterone is maintained at a high concentration until a sharp decline in concentration occurs at d 18.5. The temporal association of the increase in uterine PGF2{alpha} and the decline in serum progesterone, together with previous gene ablation and pharmacological studies (2, 3, 7, 13), define the regulated induction of uterine PGF2{alpha} production as a key event for the timing of labor in the mouse.



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  		Figure 2. Plasma progesterone levels in the gestational mouse. Blood was collected from gravid mice at specific gestational days during murine pregnancy (n = 2–4 mice per time point). The solid line represents the mean plasma progesterone concentration, with individual points represented as crosses. Duplicate points at d 10.5 and d 15.5 overlap.

 
Analysis of cPLA2 in the uterus during murine gestation
The first step in the prostaglandin synthesis pathway is the liberation of arachidonic acid from membrane phospholipids through the action of cPLA2. To determine whether increased PGF2{alpha} production occurs by induction of cPLA2, thereby increasing substrate availability for later steps, we examined the expression of cPLA2 during pregnancy in the uterus. As shown in Fig. 3AGo, there is minimal variation of cPLA2 mRNA expression during gestation. In addition, the mRNA levels of cPLA2 during gestation remain similar to the levels expressed in both the nongravid and the postpartum uterus. Although these studies used whole uterus rather than isolated endometrial RNA, further in situ hybridization analyses failed to detect more subtle changes restricted to the endometrium (data not shown). Analysis of cPLA2 protein during pregnancy demonstrated that the magnitude of expression remains similar in the nongravid uterus through d 17.5 (Fig. 3BGo). The expression of cPLA2 slightly decreases starting at d 18.5 and remains at this level through the end of gestation and postpartum at d 19.5 (Fig. 3BGo).



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  		Figure 3. No increase in cPLA2 abundance and activity in the uterus during gestation. A, Northern blot analysis of cPLA2 in the uterus during pregnancy. Total RNA was collected from nongravid (NG) or gravid female mice at the gestational day indicated. RNA was hybridized to a radiolabeled cPLA2 cDNA probe with cyclophilin hybridization serving as a loading control. B (top panel), Western blot analysis was performed on total membrane protein fractions. Samples were collected from nongravid or gravid female mice at the gestational age in days indicated. Samples were run on an SDS-PAGE gel and immunoblotted with antisera to cPLA2. Equivalent loading and transfer of protein was determined by immunoblotting with antisera to BiP. The blot is representative of results from three separate experiments. Bottom panel, Densitometric analysis of uterine cPLA2 protein during mouse gestation. Data are from n = 3 mice per time point and are presented as the mean ± SEM. Differences among groups were not statistically significant. C, Total membrane protein samples were isolated from nongravid and gravid females at the gestational age in days indicated and cPLA2 activity was measured. The data from n = 3 mice per time point are presented as the mean ± SEM. Differences among groups were not statistically significant.

 
Even though the amount of cPLA2 does not increase during gestation, there remains the possibility that the activity of the cPLA2 enzyme changes. Because we were unable to detect a shift in cPLA2 that might represent a change in the phosphorylation by Western blot analysis, we chose instead to directly analyze cPLA2 activity. The results of cPLA2 activity assays also indicated that the enzyme activity is not significantly altered during murine pregnancy. Figure 3CGo demonstrates that cPLA2 activity in gravid mice is relatively constant and similar to the activity found in nongravid mice. Furthermore, at d 18.5, cPLA2 activity slightly decreases, consistent with our Western blot analysis (Fig. 3BGo).

Expression of COX-1 and -2 during murine gestation
COX-1 and COX-2 are the enzymes that convert arachidonic acid to PGH2. COX-1 mRNA is low early in gestation followed by prominent induction at d 15.5 and remains elevated through d 19.0 of gestation (Fig. 4AGo). These results are consistent with previous studies that have indicated that the regulation of COX-1 is important for the initiation of parturition in the mouse (7, 14). Surprisingly, a different pattern of expression is demonstrated by uterine COX-1 protein. The amount of COX-1 protein slowly increases throughout gestation reaching maximal levels just before the initiation of labor (Fig. 4BGo). In contrast to COX-1, the expression of COX-2 protein in the uterus does not significantly change during murine gestation (Fig. 4BGo).



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  		Figure 4. Expression of COX-1 and -2 in the uterus during gestation. A, Northern blot analysis of COX-1 mRNA in the uterus during pregnancy. Total RNA was collected from nongravid or gravid female mice at the gestational days indicated. RNA was hybridized to a radiolabeled probe for COX-1. Equivalent loading and transfer was confirmed by hybridization of the same membrane to a cyclophilin probe. B, Western blot analysis was performed on microsomal protein samples obtained from nongravid or gravid female mice at the indicated gestational day. Samples were run on an SDS-PAGE gel and immunoblotted with antisera to either COX-1 or COX-2. Equivalent loading and transfer of protein was determined by immunoblotting with antisera to BiP.

 
Expression of PGFS during murine gestation
In the final steps of the pathway, PGH2 is converted into the different PGs by their specific synthases. Because PGF2{alpha} is the primary PG involved in the induction of luteolysis, we examined PGFS protein expression in the uterus during pregnancy. Initial examination of PGFS expression revealed that the two different PGFS isoforms (designated liver and lung type) were both present in the mouse uterus and each of these isoforms exhibited different patterns of expression. Western blot analysis using liver-type PGFS antisera indicated that the abundance of liver-type PGFS in the nongravid uterus is initially very low and remains low early in gestation (Fig. 5AGo). Levels of liver-type PGFS begin to increase at d 14.5 of gestation and remain elevated until d 19.0 when they begin to slightly decline. We also noted a shift in the protein size between the nongravid and gravid uterus. Immunoreactive liver-type PGFS from nongravid mice has a molecular weight of 40 kDa, but in midpregnancy, beginning at d 12.5, there is a shift in the protein size to 41 kDa (Fig. 5AGo). Protein analysis of PGFS with the lung-type-specific antisera exhibited a slightly different pattern of expression in the gravid uterus (Fig. 5BGo). Lung-type PGFS levels were low in nongravid and d 12.5 mice. The amount of lung-type PGFS protein begins to increase at d 14.5 of pregnancy and reaches peak abundance at d 17.5. Following d 17.5, levels of PGFS remain elevated relative to nongravid mice through the end of pregnancy.



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  		Figure 5. Prostaglandin F synthase expression in the uterus during gestation. Uterine tissue was collected from nongravid or gravid female mice at the gestational day indicated and Western blot analysis was performed on cytosolic protein fractions. Samples were run on a SDS-PAGE gel and immunoblotted with antisera to either lung or liver PGFS. Equivalent loading and transfer of protein was determined by immunoblotting with antisera to ß- actin. A (top panel), Western blot analysis of uterine protein immunoblotted with antisera to liver PGFS. The blot is representative of results from three separate experiments. Bottom panel, Densitometric analysis of uterine protein immunoblotted with antisera to liver PGFS. Data are from n = 3 mice per time point and are presented as the mean ± SEM. P <= 0.05 for d 15.5–19.5 vs. nongravid control. B, (top panel), Western blot analysis of uterine protein immunoblotted with antisera to lung PGFS. The blot is representative of results from three separate experiments. Bottom panel, Densitometric analysis of uterine protein immunoblotted with antisera to lung PGFS. Data are representative of n = 3 mice per time point and are presented as the mean ± SEM. P <= 0.03 for d 14.5–19.5 vs. nongravid control.

 
Decline in PG dehydrogenase expression during murine gestation
In addition to enzymes that regulate the production of PGs, certain enzymes modulate PG levels by catalyzing the degradation of PGs into inactive metabolites. We evaluated alterations in the efficiency of PG degradation by examining the expression of 15-hydroxyprostaglandin dehydrogenase (PGDH). Examination of results by one-way ANOVA indicated that levels of PGDH differed significantly from nongravid mice (P < 0.00001). PGDH protein is high in nongravid uterus, but begins to decrease early in gestation. The levels of PGDH reach their lowest levels at d 15.5 and continue to stay low for the remainder of pregnancy. Following labor at d 19.5, the levels of PGDH again begin to increase (Fig. 6Go).



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  		Figure 6. Decrease in PGDH expression in the uterus during gestation. Top panel, Western blot analysis was performed on cytosolic protein fractions. Samples were collected from nongravid or gravid female mice at the gestational day indicated. Samples were run on an SDS-PAGE gel and immunoblotted with antisera to PGDH. Equivalent loading and transfer of protein was determined by immunoblotting with antisera to ß-actin. The blot is representative of results from three separate experiments. Bottom panel, Densitometric analysis of PGDH uterus protein during mouse gestation. Data are from n = 3 mice per time point and are presented as the mean ± SEM. P <= 0.01 for d 15.5–19.5 vs. nongravid control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we define the regulation of key PG synthetic enzymes demonstrated to be essential for normal parturition in the mouse to better understand the mechanism involved in the regulation of PGF2{alpha} induction and the onset of labor. Interestingly, no one component of the PG synthetic or degradative pathway recapitulates the pattern of uterine PGF2{alpha} induction. Although essential for normal parturition, cPLA2 does not appear to vary in abundance or activity through gestation in mice. This finding is in accord with previous studies evaluating human cPLA2 activity in the myometrium (15) but differs from studies in ovine pregnancy (16). Consistent with previous studies demonstrating that COX-1 is essential for labor in mice (6, 7, 14) and induced to high levels in the gravid uterine epithelium, we find substantial up-regulation of COX-1 mRNA. This up-regulation occurs at d 15.5 of gestation, which is 4 d before the onset of labor and 2–3 d before the induction of PGF2{alpha} expression required for luteolysis. Interestingly, the patterns of induction for COX-1 mRNA and protein differ substantially, with COX-1 protein gradually, rather than abruptly, increasing in abundance. This dissociation could reflect translational control of COX-1 mRNA or different half-lives of mRNA and protein conferring differences on when new steady-state levels of each are reached. Consistent with one previous study (17), we find no change in uterine COX-2 expression before the onset of labor. Although COX-2 activity is required for the successful establishment of pregnancy (18), COX-2 appears dispensable for the initiation of labor.

To our knowledge, this report is the first characterization of PGFS expression in the uterus during murine pregnancy. The PG synthases are members of a large family of proteins collectively identified as aldo-keto reductases (19). The mouse PGFS genes have not yet been cloned, in part because of the difficulty discerning among various members of the aldo-keto reductase family by cross-species homologies because of the very high degree of homology between proteins with different enzymatic activities within a given species. Both the liver-type (11) and lung-type (10) antisera recognize proteins with distinct patterns of expression and substantial induction during gestation. Again, this induction occurs long before an increase in uterine PGF2{alpha} concentration is measured. Intriguingly, PGDH expression decreases in a pattern reciprocal to the pattern of induction of COX-1 and PGFS. Reduction in PGDH expression late in gestation also occurs in human pregnancy (20, 21) and may prove a fundamental component of labor initiation by allowing greater access of labor-promoting PGs to relatively distant sites, such as the corpus luteum in mice.

One plausible model for the mechanism by which PGF2{alpha} increases abruptly during late gestation, given our findings that no one molecule in the PG biosynthetic pathway recapitulates this pattern of expression, is first that cPLA2 activity is present in excess throughout gestation. Then as COX-1 protein gradually increases during late gestation, it provides increased PGH2 to PGFS, which has already been induced. Just before term, COX-1 activity increases to an extent that the threshold for efficient degradation of PGF2{alpha} by a diminished amount PGDH is exceeded. The limited PGDH activity would allow greater steady-state levels of PGF2{alpha} to transit from the uterus to the corpus luteum and precipitate labor. Identifying the regulatory factors conferring this coordinate pattern of gene induction and repression should provide critical insight into the clock timing the onset of parturition.


   Acknowledgments
 
We thank Drs. Kathleen Bethin and David Rudnick for manuscript review and members of the Muglia laboratory for helpful discussions.


   Footnotes
 
This work was supported by grants from the March of Dimes and the Rockefeller Brothers Fund (to L.J.M.) and the Lalor Foundation and NIH Grant 1-F32-HD-41292-01 (to S.K.W.).

Abbreviations: BiP, Binding protein; COX, cyclooxygenase; PG, prostaglandin; PGDH, 15-hydroxyprostaglandin dehydrogenase; PGF2{alpha}, prostaglandin F2{alpha}; PGFS, prostaglandin F synthase; PGH2, prostaglandin H2; TTBS, Tween-Tris-buffered saline.

Received February 11, 2002.

Accepted for publication April 2, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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