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BOR - Papers in Press, published online ahead of print April 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037820
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BIOLOGY OF REPRODUCTION 73, 343–350 (2005)
DOI: 10.1095/biolreprod.104.037820
© 2005 by the Society for the Study of Reproduction, Inc.

Sufficient Progesterone-Priming Prior to Estradiol Stimulation Is Required for Optimal Induction of the Cervical Prostaglandin System in Pregnant Sheep at 0.7 Gestations1

Wen Xuan Wu 2 3, Turhan Coksaygan 3, Kaushik Chakrabarty 4, Valta Collins 4, James C. Rose 3, and Peter W. Nathanielsz 5

Department of Obstetrics and Gynecology,3 Wake Forest University SOM, Winston-Salem, North Carolina 27157 Department of Obstetrics and Gynecology,4 University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 Department of Obstetrics and Gynecology,5 University of Texas Health Sciences Center, San Antonio, Texas 78229


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106–108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121–123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE2 receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

cervix, estradiol, parturition, PG, progesterone, sheep


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parturition is a highly organized and regulated process. Although myometrial contraction is the most dramatic sign of labor, cervical ripening and dilation before and during labor are essential processes in preparation for and completion of parturition. However, our understanding of biomedical mechanisms of cervical ripening and dilation is limited. Prostaglandins (PGs) are known to have a key role in the control of cervical compliance in human pregnancy [1] and in other species [2–4]. A large number of randomized controlled trials have demonstrated that PGE2 is an effective agent in priming the cervix and that its use before the induction of labor reduces the risk of failed induction and operative delivery [5]. The anatomical and biochemical changes of cervical ripening, such as shortening of the cervix, degradation of collagen, and alterations in proteoglycan composition [6], can be reproduced by the direct application of PGE2 to the cervix [6–8]. However, the control of cervical PG production and function throughout gestation and during labor is still unclear.

The production of PGs is regulated by prostaglandin-endoperoxide synthase, and two types of the enzyme, COX1 and COX2, have been characterized in mammalian cells [9–11]. COX1 is constitutively expressed in most tissues, and COX2 is an inducible enzyme that responds to inflammatory cytokines and growth factors and is increased in intrauterine tissues during premature and spontaneous term labor [12, 13]. We have recently demonstrated that cervical glandular epithelial cells are the major cell type containing COX2 mRNA and protein in pregnant baboon, which peaks near term [14]. Our data suggest that the cervix is not only the target of PGs but also a site for producing PGs. However, the exact hormonal signals that regulate production and function of cervical PGs are not understood.

The effects of PGs are mediated through a family of G-protein-coupled receptors. At least four receptors (PTGER 1–4) encoded by separate genes exist for PGE2 [15–18]. These four receptors work through multiple second messenger systems to produce different downstream events and determine the tissue-specific response to PGE2. PTGER1 and PTGER3 receptors are coupled to calcium mobilization and inhibition of adenylate cyclase, respectively, both of which enhance smooth muscle contraction [15–18]. In contrast, PTGER2 (also known as EP2) and PTGER4 (also known as EP4) receptors both stimulate adenylate cyclase [15–18] and tend to decrease smooth muscle contractility. Of the four subtype receptors, PTGER2 and PTGER4 are the most likely receptors responsible for PGE2-induced cervical ripening and dilation because a softened, relaxed, and dilated cervix is required for fetal delivery.

Cervical ripening occurs in two steps, a slow stage extending over the major part of pregnancy and a final rapid process just preceding labor. Progesterone is the major circulating hormone in maternal plasma throughout gestation, and estradiol is not elevated until labor in many experimental animal models. Also estradiol-induced premature labor before 0.9 gestations is often associated with cervical dystocia [3, 19, 20]. Based on these facts, we hypothesized that sufficient progesterone priming was required before estradiol induction of the cervical PG system. To test our hypothesis, we analyzed the separate and interactive functions of progesterone and estrogen in regulating the cervical PG system in pregnant sheep at 0.6–0.8 gestations before the progesterone concentration in maternal plasma reached its term level.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Pregnant Rambouillet-Dorset ewes bred on a single occasion and carrying fetuses of known gestational age were studied. Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee and conducted in facilities approved by the American Association for the Accreditation of Laboratory Animal Care.

At 106–108 dGA, ewes from which tissues were obtained were instrumented with fetal and maternal carotid arterial and jugular venous catheters. The fetal and maternal carotid arterial and jugular venous catheters were implanted as we described previously [21]. Maternal and fetal arterial blood samples were taken daily for the determination of pH and blood gases to evaluate maternal and fetal well-being. One day after surgery, ewes were treated with sesame oil or progesterone 100 mg twice a day, administered intramuscularly for 14 days to produce term levels of progesterone in maternal plasma (n = 5 for each group) or sesame oil for 12 days followed by estradiol 5 mg twice a day, administered intramuscularly for 2 days to produce labor levels of estradiol in maternal plasma (n = 5) [20], or estradiol plus progesterone with progesterone 100 mg administered intramuscularly twice a day for 10 days and then 2 days of sesame oil followed by 2 days of estradiol (5 mg intramuscularly, twice a day). At 121–123 dGA, necropsies were performed under halothane anesthesia after which the animals were killed. The cervix was collected for later RNA and protein analysis. Maternal arterial blood samples were collected on the day before vehicle or steroid treatment, the 10th day of progesterone treatment, and the day at necropsy. Plasma was separated by centrifugation at 1500 x g for 10 min at 4°C. Plasma samples were frozen at –20°C for subsequent assays.

RIA

The RIAs for maternal plasma estradiol and progesterone were performed using commercially available 125I RIA kits (Diagnostic Products Co., Los Angeles, CA) as we described previously [20]. The sensitivity of the assays was 8 pg/ml for estradiol and 20 pg/ml for progesterone. The specificity was provided by the manufacturer. The intra-assay coefficients of variation of estradiol and progesterone were 8% and 4%.

Northern Blot Analysis

Polyadenylated RNA was extracted from cervix by oligo dT cellulose affinity chromatography using a commercial kit (Invitrogen, San Diego, CA). Samples of polyadenylated RNA (2 µg) were separated by electrophoresis on a 1.4% (wt/vol) agarose-0.66 M formaldehyde gel and transferred onto a nylon membrane (NEN Life Science) and then subjected to Northern blot analysis for COX2, PTGER2, PTGER4, and ESR1 mRNAs as described previously [13, 14, 20, 22]. Cyclophilin mRNA was used to control RNA loading.

In Situ Hybridization

Frozen sections (4 µm thick) cut onto commercially prepared poly-L-lysine-coated slides (Sigma Chemical Company, St. Louis, MO) were fixed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (20 min), washed twice in 0.1 M phosphate buffer, immersed in triethanolamine-HCl (3.71 g TEA, 2 ml 6M NaOH, and 198 ml of water), pH 8.0, and then TEA and acetic anhydride (0.25%) for 10 min. They were then washed in 2x SSC for 5 min and briefly in 70% ethanol and allowed to air dry. The specimens were incubated for 2 h in a humidified container (55°C) with 70 µl of prehybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl [pH 8.0], 5 mM EDTA, 10 mM sodium phosphate buffer [pH 8.0], 1x Denhardt solution) and for at least 16 h in hybridization buffer (i.e., prehybridization buffer plus probe = 1 x 106 cpm per specimen in 70 µl). Control slides were hybridized in the presence of an excess of unlabelled antisense RNA or labeled sense RNA. Slides were then washed three times in 4x SSC and 4 mM dithiothreitol (DTT, sigma), three times in NTE buffer (0.5 M NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH 8.0) at 37°C (second NTE wash was for 30 min with 30 µg/ml Ribonuclease A), 2x SSC and 1 mM DTT for 45 min, 0.1x SSC and 1 mM DTT at 60°C for 30 min, and finally 0.1x SSC at 37°C for 30 min. The slides were then dehydrated in a graded series of ethanol plus 0.3 M ammonium acetate. They were air dried and exposed to autographic films for 2–4 days and then dipped in emulsion (Kodak NTB2; VWR, South Plainfield, NJ) and exposed for 1–4 wk at 4°C. Following developing and fixing, they were counterstained with hematolxylin and eosin, mounted, and covered with a glass coverslip.

Synthesis of Probes

Our cloned ovine COX2 cDNA (20) in pCR II vector (Invitrogen), which include promoters for phage polymerases SP-6 to produce antisense probe and T-7 to produce sense probe, was linearized by an appropriate restriction enzyme. PTGER2 and PTGER4 probes were made from full length of human cDNAs obtained from Dr. M. Abramovitz of Merck Frosst (Quebec, Canada). The PTGER2 and PTGER4 cDNAs had been cloned into pcDNA3 vector (Invitrogen), which included promoters for phage polymerases SP-6 to produce antisense probe and T-7 to produce sense probe. The application of human prostanoid receptors in sheep tissues was characterized by our previous study [22]. The plasmid (TRIscript) containing cyclophilin cDNA with RNA polymerase promoters was purchased from Ambion (Ambion, Inc., Austin, TX). The antisense and sense riboprobes were synthesized using a commercial kit (MAXIscript; Ambion) and labeled with [{alpha}-32P] UTP for Northern blot analysis or [{alpha}-35S] UTP for in situ analysis (NEN Life Science). Recombinant human ESR1 cDNA containing the entire coding region was kindly made available by Dr. Pierre Chambon (University of Strasbourg, France). ESR1 cDNA probes were labeled with {alpha}-32P dCTP (NEN Life Science) for Northern blot analysis.

Solubilized Cell Membrane Extraction and Western Blot Analysis

To prepare solubilized cell extracts, approximately 1 g cervical tissue was homogenized for 1 min (Polytorn, Brinkman Instruments, Inc., Westbury, NY) on ice in TE buffer (50 mM Tris and 10 mM EDTA) containing 2 mM octyl glucoside and 0.2 mM phenylmethylsulfony fluoride and centrifuged at 30 000 x g for 1 h at 4°C. The crude pellets (membrane, nuclei, and mitochondria) were sonicated in 1 ml TE sonication buffer (20 mM Tris and 50 mM EDTA) containing 45 mM octyl glucoside and 0.2 mM phenylmethylsulfony fluoride. The supernatants were centrifuged at 13 000 x g for 25 min at 4°C. The recovered supernatants (solubilized cell extract) were stored at –70°C until electrophoretic analysis. The protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Hercules, CA). The solubilized proteins (50 µg/lane) separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were electrophoretically transferred to a nylon membrane (Imobilon; Millipore Corp., Bedford, MA), using a Bio-Rad transfer blot cell. The protein bands were visualized using an enhanced chemiluminescence Western blotting detection kit (ECL; Amersham Life Sciences, Arlington Heights, IL). The molecular sizes of the proteins were determined by running standard molecular weight marker proteins (Bio-Rad) in an adjacent lane. G protein ß subunit (Gß) was detected on each blot to control protein loading. Chemiluminescence signals were analyzed and quantified with the scanner, and data were analyzed with a densitometry program (Scan Analysis) and quantified against an arbitrary scale in the plot.

Immunocytochemistry

Frozen sections (4 µm) of the pregnant sheep cervix were immunostained for COX2 using the avidin-biotin immunoperoxidase method as described previously [20] to localize the cellular distribution of COX2 protein in intrauterine tissues. Specificity of immunostaining for the COX2 was confirmed by two approaches: 1) omission of the primary antibody and 2) incubation of the slides with the normal rabbit serum instead of the primary antibody [13, 14, 20, 23].

Antibodies for Western Blot Analysis and Immunocytochemistry

A rabbit polyclonal antibody for human COX2 (Oxford Biomedical Research, Inc., Oxford, MI) was used at 1:1000 dilutions and incubated at 4°C for 20 h. This COX2 antibody has been characterized in our previous studies in application of sheep tissues [13, 14, 20, 23]. Rabbit polyclonal antibodies for human PTGER2 and PTGER4 receptors (Cayman Chemical Co.) were used at 1:500 and 1:1000 dilutions and incubated at 4°C for 20 h. Specificity of Western blot analysis for PTGER2 and PTGER4 antibodies was confirmed by preabsorpting PTGER2 and PTGER4 antibodies with synthetic polypeptides that were used for generating the antibodies (Cayman Chemical). Rabbit polyclonal antibody for bovine Gß was purchased (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A second antibody, horseradish peroxidase conjugated donkey anti-rabbit IgG, was used for Western (Amersham), and a biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) was used for immunocytochemistry.

Statistical Analysis

Comparison of two means was made with the Student t-test. Comparison of three or more means was made by ANOVA and multiple post hoc comparisons with the Tukey method for 95% confidence interval of pairwise differences. Statistical significance was assumed at the 5% level. Data were presented throughout as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estradiol and Progesterone Concentrations in Maternal Plasma

Maternal plasma estradiol concentration measured in conscious animals at the end of the study before necropsy was significantly higher in the estradiol (340.02 ± 64.65 pg/ml) and estradiol-plus-progesterone-treated groups (405 ± 103.86 pg/ml) than the control group (42.86 ± 21.85 pg/ ml, P < 0.05). Muscular injection of 100 mg progesterone twice a day increased progesterone concentrations in maternal plasma to the high range of levels found at term in progesterone treated ewes (97.62 ± 19.04 ng/ml) compared with controls (20.56.4 ± 2.20 ng/ml).

Effects of Estradiol or/and Progesterone on COX2, PTGER2, PTGER4, and ESR1 mRNA Expression in the Pregnant Sheep Cervix

In the pregnant sheep cervix, the ratio of COX2 or PTGER4 over cyclophilin mRNA, analyzed by Northern blot analysis, increased significantly after combined estradiol and progesterone treatment (Figs. 1 and 2; P < 0.05). Progesterone or estradiol alone had no significant effect on COX2 and PTGER4 mRNA levels in the cervix. In contrast, progesterone either alone or combined with estradiol significantly stimulated ESR1 mRNA expression in the pregnant sheep cervix (Fig. 2; P < 0.05). Estradiol alone had no significant effect on cervical ESR1 mRNA expression (Fig. 2; P > 0.05). There was no change in PTGER2 mRNA abundance after any steroid treatment, either alone or in combination (Fig. 3; P > 0.05).



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  		FIG. 1. Northern blot analysis of COX2 (A) and PTGER4 (B) mRNA in the pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1–5), estradiol (E; lanes 6–10), progesterone (P; lanes 11– 15), and progesterone and estradiol (EP; lanes 16–20) at 121–123 dGA. C) Cyclophilin mRNA in each corresponding lane. D) Densitometric analysis of COX2 and PTGER4 to cyclophilin mRNA ratio in Cont and E-, P-, and EP-treated groups (n = 5 in each group). COX2 and PTGER4 mRNA increased only in the cervix in EP group (*P < 0.05 compared with Cont, E, or P). Values are presented as mean ± SEM



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  		FIG. 2. Northern blot analysis of ESR1 mRNA (A) in the pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1–5), estradiol (E; lanes 6–10), progesterone (P; lanes 10–15), and progesterone and estradiol (EP; lanes 16–20) at 121–123 dGA. B) Cyclophilin mRNA in each corresponding lane. C) Densitometric analysis of ESR1 to cyclophilin mRNA ratio in Cont and E-, P-, and EP-treated groups (n = 5 in each group). ESR1 mRNA significantly increased in the cervix of P and EP groups (*P < 0.05 compared with C). Values are presented as mean ± SEM



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  		FIG. 3. Northern blot analysis of PTGER2 mRNA (A) in the pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1– 5), estradiol (E; lanes 6–10), progesterone (P; lanes 10–15), and progesterone and estradiol (EP; lanes 16–20) at 121–123 dGA. B) Cyclophilin mRNA in each corresponding lane. C) Densitometric analysis of PTGER2 to cyclophilin mRNA ratio in Cont and E-, P-, and EP-treated groups (n = 5 in each group). PTGER2 mRNA remained the same after E or P or EP treatment compared with controls. Values are presented as mean ± SEM

Effects of Estradiol or/and Progesterone on COX2, PTGER2, and PTGER4 Protein Expression in the Pregnant Sheep Cervix

Compared with the control group, there was a concurrent rise of cervical COX2 and PTGER4 proteins observed by Western blot analysis associated with elevated COX2 and PTGER4 mRNA levels in animals that received the combined estradiol and progesterone treatment (Fig. 4; P < 0.05), whereas progesterone or estradiol alone did not produce any significant effect on cervical COX2 and PTGER4 protein concentrations (Fig. 4; P > 0.05). Consistent with the mRNA levels, cervical PTGER2 protein levels unchanged after any combination of the steroid treatment (Fig. 5; P > 0.05), COX2 was expressed as two bands about 70 and 72 kDa (Fig. 4). Both immunoreactive bands of COX2 were induced in the EP group. This observation is consistent with the report that COX2 is partially N-glycosylated, and all known COX2 enzymes have consensus N-glycosylation sites at an analogous position [11, 12]. PTGER4 and PTGER2 proteins were both expressed as a single band about 52 kDa (Figs. 4 and 5). The stainings for PTGER2 and PTGER4 proteins on Western blot analysis were abolished by incubation with the preabsorbed PTGER2 and PTGER4 antibodies (data not shown).



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  		FIG. 4. Western blot analysis of COX2 (A) and PTGER4 (B) protein in the pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1–5), estradiol (E; lanes 6–9), progesterone (P; lanes 10–13), and progesterone and estradiol (EP; lanes 14–18) at 121–123 dGA. C) Gß protein in each corresponding lane. D) Densitometric analysis of COX2 and PTGER4 to Gß ratio in Cont and E-, P-, and EP-treated groups (n = 4–5 in each group). COX2 and PTGER4 proteins only increased in the cervix in EP treated group (*P < 0.05 compared with Cont, E, or P). Values are presented as mean ± SEM



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  		FIG. 5. Western blot analysis of PTGER2 (A) protein in the pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1– 5), estradiol (E; lanes 6–9), progesterone (P; lanes 10–13), and progesterone and estradiol (EP; lanes 14–18) at 121–123 dGA. B) Gß in each corresponding lane. C) Densitometric analysis of PTGER2 to Gß ratio in Cont and E-, P-, and EP-treated groups (n = 4–5 in each group). PTGER2 protein remained the same after E or P or EP treatment. Values are presented as mean ± SEM

Cellular Localization of COX2, PTGER2, and PTGER4 mRNA and Protein in the Pregnant Sheep Cervix

COX2 mRNA and protein analyzed by in situ hybridization (Fig. 6) and immunocytochemistry (Fig. 6) were localized only in cervical glandular epithelial cells. No positive detection of COX2 mRNA or protein was found in cervical smooth muscle cells or fibroblast cells. In contrast, PTGER2 and PTGER4 proteins were localized in smooth muscle cells (Fig. 7). PTGER4 protein was also localized in the smooth muscle cells of blood vessels and cervical glandular epithelial cells (Fig. 7). No hybridization signal or staining was observed when the cervical sections were reacted with COX2 sense probe (Fig. 6) or stained in absence of primary COX2, PTGER2, or PTGER4 antibodies, which were replaced by normal rabbit serum (Figs. 6 and 7). We attempted to identify the cell types expressing PTGER2 and PTGER4 receptor genes in the pregnant sheep cervix using in situ hybridization but could not reliably detect signals above background because of diffuse low levels of expression.



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  		FIG 6. In situ (A–C) and immunocytochemical (D–F) analysis of cellular distribution of COX2 mRNA (A, bright field, and B, dark field) and protein (D, x400 magnification, and E, x200 magnification) in pregnant sheep cervix. COX2 mRNA and protein were localized in epithelial cells of the cervical glands in pregnant sheep. The specific in situ hybridization and immunostaining signals for COX2 mRNA and protein were abolished when COX2 antisense riboprobe was replaced by sense riboprobe (C) or in the absence of the primary COX2 antibody (F) in the cervix. Original magnification: A–D and F; x400; E x200



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  		FIG. 7. Immunocytochemical analysis of cellular distribution of PTGER4 (A–C) and PTGER2 (D) protein in the pregnant sheep cervix. Both PTGER2 (B) and PTGER4 (D) were localized in the cervical smooth muscle cells. PTGER4 was also localized in the blood vessels (C) and epithelial cells of cervical glands (A). The specific stainings for PTGER4 and PTGER2 were abolished in the absence of the primary PTGER4 (E) and PTGER2 (F) antibodies. Original magnification: x200


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Intrauterine tissues from pregnant sheep [24, 25], baboons [26], and women [27] have increased PG production in late gestation, a time when progesterone concentration is rising in maternal plasma. In many experimental animal models studied, including pregnant sheep, the cervix undergoes considerable ripening throughout gestation, when progesterone is the major circulating hormone and estradiol is low in maternal plasma [28]. Estrogen-induced premature labor in pregnant sheep is often associated with cervical dystotia [3, 19, 20]. Based on these observations, we propose that progesterone exerts a facilitatory role on cervical PG synthesis and that the priming effect of progesterone optimizes the stimulatory action of estrogen on the cervical PG system associated with onset of labor.

Our study provides firm evidence for the first time in support of our hypothesis that optimal induction of the cervical PG system can be achieved only in animals that received sufficient progesterone priming before estradiol stimulation at 0.7 dGA. Progesterone is a potent stimulator of nonpregnant sheep endometrial COX2 [23, 29, 30]. To our knowledge, the priming effect of progesterone on the uterine or cervical PG system in pregnant animals has not been determined in any species. Furthermore, the interactive function of estrogen and progesterone on regulating the cervical PG system is even less defined. The pregnant sheep is an excellent animal model to dissect out the separate and interactive functions of estradiol (the major estrogen in pregnant sheep) and progesterone because progesterone increases in maternal plasma throughout pregnancy, while estradiol remains low. During the surge of estradiol at labor, progesterone falls. This switch in levels of the two hormones provides a unique opportunity to use an in vivo approach to determine the individual and interactive functions of each steroid in preparation for and completion of parturition. Our results indicate that an increase in estradiol alone cannot account for all the cervical changes that occur in labor.

In addition to inducing the key enzyme responsible for the production of cervical PG, additional priming with progesterone at 0.7 gestations before stimulation with estradiol also enhanced PTGER4 expression, a relaxing subtype of PGE2 receptor [15–18]. Parallel evaluation of PTGER2 receptor, the other relaxant receptor of PGE2, was also performed. Estradiol and progesterone, either alone or in combination, did not alter cervical PTGER2 receptor mRNA and protein abundance, suggesting a differential regulation of relaxant PGE2 receptor subtypes by progesterone and estrogen in the pregnant cervix. In the current study, we focused on the two cyclic adenosine monophosphate (cAMP)-coupled relaxing PGE2 receptors based on the assumption that an effaced and dilated rather than contracted cervix is essential for fetal delivery during labor. In addition, cervical ripening involves the remodeling of the extracellular matrix [31]. Matrix metalloproteinases (MMPs) are the primary enzymes responsible for collagen and extracellular matrix remodeling. PGE2 has been shown to mediate MMP secretion through a cAMP second messenger system [32]. Of the four PGE2 receptors, both PTGER2 and PTGER4 signal through cAMP system [15–18, 32]. Evidence suggests that PTGER4 activation is most likely responsible for regulating tissue remodeling [33–35]. PTGER4 knockout animals were shown to produce lower levels of MMP2 and MMP13, both known to degrade collagen types I and III, which are the two dominant collagens found within the cervix [6, 7]. Furthermore, PTGER4 mRNA and protein expression peak on the day of parturition in the pregnant rat cervix [36]. However, the regulation of PTGER2 and PTGER4 expression by estrogen and progesterone in the cervix in pregnancy has not been evaluated in any species. This is the first study demonstrating the stimulation of PTGER4 expression by estradiol in the sufficient progesterone primed cervix at 0.7 gestations, suggesting an interactive role of progesterone and estrogen in regulating PTGER4 receptor expression in the pregnant sheep cervix. The increase in PTGER4 coincident with the increase in cervical COX2 level in the current study suggests that sufficient progesterone priming not only promotes PG synthesis but also enhances PGE2's function in the pregnant cervix.

The effect of estradiol alone on the cervical PG system was not significant, which may represent one of the biochemical mechanisms that lead to the cervical dystocia often associated with estradiol-induced premature labor [3, 19, 20]. Relating the current observation to our previous studies, there are interesting differences. Estradiol alone was able to induce COX2 expression in pregnant sheep myometrium [20] and endometrium [20], indicating that estrogen regulation of the PG system is tissue specific. Similar tissue specificity has been observed for glucocorticoids and gonadotropin. Glucocorticoids stimulate COX2 expression in cultured human amnion cells [37] but inhibit COX2 expression in numerous other cell types [38]. In addition, gonadotropin stimulates COX2 expression in rat ovary [39] but not in other tissues studied. These results indicate that cell type-specific subcellular signals are important to direct responses to steroid hormone regulation in some cell types while leaving the PG synthetic activity of other cells unaltered. This form of differential regulation is of fundamental importance for PG synthesis, which appears ubiquitous if not universal in cell function.

In the uterus, progesterone is known to negatively regulate the expression of ESR1 [18, 40], and progesterone withdrawal results in an induction of ESR1 expression. Therefore, one mechanism by which progesterone priming before estradiol stimulation facilitates induction of the cervical PG system is through an increase in expression of ESR1. To test this hypothesis, we measured cervical ESR1 mRNA levels in all four groups. Surprisingly, progesterone alone up-regulated cervical ESR1 mRNA in pregnant sheep. Estradiol could stimulate ESR1 mRNA expression only after sufficient progesterone priming. Our data once again suggest that tissue- and cell type-specific regulations are important mechanisms in directing responses of different tissues to steroid hormones.

Progesterone is acknowledged to play a central role in controlling uterine function during pregnancy. On the one hand, progesterone, acting as a gene suppressor, down-regulates a number of genes that are essential for myometrial contraction, including the gap junction protein [41, 42], calcium channels, steroid receptor and associated proteins [40, 43], and oxytocin receptor [41, 44]. On the other hand, progesterone can also promote intrauterine PG production [23, 29, 30, 45]. High progesterone concentrations are temporally associated with the development of intrauterine PG production in pregnant sheep and women [24, 27]. Over many years this finding has led to the speculation that progesterone regulates intrauterine PG production throughout pregnancy. Progesterone is a more potent stimulator than estradiol on endometrial COX2 expression in nonpregnant sheep [23, 30]. Therefore, we have proposed dual functions for progesterone in the control of parturition, inhibition of myometrial contractility, and facilitation of intrauterine PG production. Although progesterone alone did not alter the cervical COX2 and PTGER4 expression, our data clearly indicate that sufficient progesterone treatment to produce levels seen at term is essential for optimized estradiol stimulation of the cervical PG system.

Steroid-receptor complexes induce or repress the expression of genes by interacting with regulatory DNA sequences and transcription factors. Studies with the genomic organization of the rat PTGER4 gene [36] demonstrated that a total of three Sp1 sites lie upstream of the PTGER4 transcriptional start site. The estrogen receptor has been shown to interact with Sp1 to regulate gene transcription through GC-rich cis-acting elements [46]. Progesterone is also clearly involved in the cervical ripening process [41, 44]. These Sp1 sites may also be a mechanism by which progesterone may influence cervical PTGER4 receptor expression. The potential molecular mechanisms of the interactions of estrogen and progesterone in COX2 gene regulation are unclear. Although steroid response elements have not been identified in COX2 gene, it is possible that other sequence motifs in COX2 gene may be associated with estrogen or progesterone action.

Consistent with our findings in the pregnant baboon cervix [14], COX2 mRNA and protein were localized only in the cervical glandular epithelial cells. These glandular structures are in fact deep slitlike invaginations of the surface epithelium, with blind-ended tubules [47]. Thus, there is a large surface area for the production of cervical mucus, which fills the endocervical canal. By secreting mucus, cervical glandular cells can provide a high PG environment within the entire lower birth canal. Indeed, the amount of PGE2 recovered from vaginal fluid (by lavage) is higher during labor than before labor [48]. This localized high PG environment will be able to induce all the necessary changes that must occur in the lower birth canal for the proper completion of labor.

In contrast, PTGER4 and PTGER2 proteins are localized in almost every type of cellular component in the cervix including blood vessels. This suggests that PGE2 may influence a variety of cellular functions in the cervix during pregnancy, although the distinct roles of each cell type in control of cervical ripening during pregnancy are still not clear.

In conclusion, these data suggest that additional progesterone priming at 0.7 dGA in pregnant sheep synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 expression and support our hypothesis that estradiol's stimulation is optimized by sufficient progesterone's priming in the pregnant sheep cervix.


   FOOTNOTES
 
1 Supported by NIH HD 39247. Back

2 Correspondence: Wen Xuan Wu, Department of Obstetrics and Gynecology, Wake Forest University, Baptist Medical Center, Winston-Salem, NC 27157. FAX: 336 716 6937; wenwu{at}wfubmc.edu Back

Received: 8 November 2004.

First decision: 15 December 2004.

Accepted: 12 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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