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Reproduction (2007) 133 1027-1034
DOI: 10.1530/REP-06-0328
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Regulation of the prostaglandin enzymatic system by estradiol and progesterone in nonpregnant sheep cervix

Qi Zhang, Valta Collins1, Kaushik Chakrabarty1, James C Rose and Wen Xuan Wu

Department of Obstetrics and Gynecology and the Center of Research for Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA and 1 The University of Oklahoma Health Sciences Center, RP1, Suite 470, 800 N. Research park, Oklahoma City, Oklahoma 73104 USA

Correspondence should be addressed to W X Wu at Department of Obstetrics and Gynecology, Wake Forest University Baptist Medical Center, Winston Salem, North Carolina 27157, USA; Email: wenwu{at}wfubmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
In the present study, we examined the in vivo effects of estradiol (E2) and progesterone on cyclooxygenase (COX) 2, prostaglandin F synthase (PTGFS, also known as PGFS), and membrane-associated prostaglandin E synthase 1 (mPTGES1) expression at both mRNA and protein levels using a nonpregnant ovariectomized (OVX) sheep model. Sixteen ewes were OVX shortly after ovulation. After 40 days, ewes were treated with saline (Cont, n=5), or E2 infused intravenously for 2 days (50 µg/day, n=5) or intravaginal progesterone (P) sponges for 10 days (0.3 g P, n=6). Cervical COX2, PTGFS, and mPTGES1 mRNA and protein were quantified by northern and western blot analyses respectively. In situ hybridization and/or immunocytochemistry were used to localize the cellular distribution of COX2, PTGFS, and mPTGES1 mRNAs and proteins. COX2 mRNA abundance increased significantly in the cervix after E2 treatment (P<0.05). However, progesterone was a more potent stimulator than E2 of COX2 mRNA and protein abundance in the cervix (P<0.01). In contrast, PTGFS and mPTGES1 mRNA and protein concentrations did not change after E2 or progesterone treatment (P>0.05). COX2, PTGFS, and mPTGES1 mRNA and protein were only localized in cervical glandular epithelial cells. This study shows that increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E2 and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E2. The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was confined in the cervical glandular epithelial cells of nonpregnant sheep.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
The production of prostaglandins is largely regulated by prostaglandin cyclooxygenase (COX), and two types of the enzyme, COX1 and COX2, have been characterized in mammalian cells (Dewitt & Smith 1988, Hla & Neilson 1992, Otto & Smith 1994). We have repeatedly shown that COX1 is constitutively expressed in pregnant and nonpregnant uterine tissues (Zhang et al. 1996, Wu et al. 1999). COX2 is an inducible enzyme that responds to inflammatory cytokines, growth factors (Diaz et al. 1992, Hempel et al. 1994), and steroid hormones (Wu et al. 1997, 2004a, 2005b) and is increased in intrauterine tissues, including the cervix during premature and spontaneous term labor (Wu et al. 1999, 2004a, 2005a). Therefore, COX2 is the focal point of our present study. We have recently demonstrated that cervical glandular epithelial cells are the major cell type containing COX2 mRNA and protein in pregnant baboon and sheep (Wu et al. 2004b, 2005a). Our data suggest that the cervix is not only the target of prostaglandins but also a site for producing prostaglandins. However, the exact hormonal signals that regulate production and function of cervical prostaglandins are not understood.

In addition to COX, post-COX enzymes, such as prostaglandin E2 synthases (PTGES, also known as PGES) and prostaglandin F2{alpha} synthase (PTGFS, also known as PGFS), determine the exact mix of two major prostaglandins involved in reproduction and parturition. PTGES acts downstream of COX to catalyze the conversion of PGH2 into PGE2 (Jakobsson et al. 1999, Watanabe et al. 1999). At least three forms of human PTGES have been cloned and characterized, including membrane-associated PTGES1 (mPTGES1) (Jakobsson et al. 1999), mPTGES2 (Tanikawa et al. 2002), and cytosolic PTGES (cPTGES; Tanioka et al. 2000). Among these, mPTGES1 shows coordinated induction with COX2 by inflammatory stimuli in various cells and tissues (Jakobsson et al. 1999, Murakami et al. 2002), including pregnant intrauterine tissues (Zhang et al. 2006), which, therefore, is the focus of the present study. PTGFS is responsible for converting PGH2 into PGF2{alpha}. Hence, information regarding the regulation of post-COX enzymes is also required for a better understanding of the control of the cervical prostaglandin production.

During pregnancy, the cervix grows ten times in length and ripens through the major part of gestation (Stevens & Lowe 1992, Poma 1999). However, the biochemical mechanisms underlying the anatomic changes of the pregnant cervix are far from clear. In pregnant sheep and many other experimental animal models, progesterone is the major circulating hormone in maternal plasma throughout gestation and estradiol (E2) is not elevated until labor (Challis 1971, Liggins et al. 1973). In addition, E2-induced premature labor prior to 0.9 gestation is often associated with cervical dystocia (Hindson et al. 1967, Liggins et al. 1973, Wu et al. 2004a). Based on these facts, we hypothesized that progesterone may be an important regulator of the cervical prostaglandin system. Indeed, additional progesterone priming prior to E2 stimulation at 0.7 gestation optimized the expression of the cervical prostaglandin system induced by E2 in pregnant sheep (Wu et al. 2005a). However, it is impossible to determine the individual progesterone or E2 impact at the physiological level on regulation of the cervical prostaglandin enzymatic system in pregnant sheep because of the indispensable relationship between placental progesterone production and pregnancy maintenance. Therefore, in the present study, we used ovariectomized (OVX) nonpregnant sheep as a model to analyze the separate functions of progesterone and estrogen in regulating the cervical prostaglandin enzymatic system. In situ hybridization and immunocytochemical staining of COX, mPTGES1, and PTGFS were applied to determine the cell types which contained COX2, PTGFS, and mPTGES1 in the cervix.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Animals and tissue collection
Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee. All facilities were approved by the American Association for the Accreditation of Laboratory Animal Care. In order to reduce endogenous ovarian steroid levels to a minimum, 16 nonpregnant ewes were OVX on the day of ovulation following the removal of progesterone sponges used to synchronize estrus. After 40 days, ewes were treated with one of the following regimens: saline controls (Cont, n=5), E2 infused intravenously for 2 days (50 µg/day, n=5), or intravaginal progesterone (P) sponge for 10 days (containing 0.3 g P, n=6). Progesterone sponges were purchased from Carter Holt Harvey Plastic Products (Hamilton, New Zealand) to produce plasma concentrations ranging from 1.5 to 2 ng/ml (data provided by the manufacturer). The dose of E2 used produces physiological increments in plasma E2 concentrations, which stimulated uterine COX2 and estrogen receptor mRNA and protein expression (our pilot study, Wu et al. 1996, 1997). Two days of estrogen and ten days of progesterone treatment were chosen to simulate the physiological estrous cycle in vivo. All animals in the control group were infused with the same rate of physiological saline.

Tissues were removed while the ewes were under halothane general anesthesia. Cervix was rapidly dissected and divided into three portions (up, middle, and lower portions) and frozen in liquid nitrogen for later RNA and protein extractions. Middle portion of the cervix was used in the present study. Frozen tissues were stored at –80 °C until extracted for RNA and proteins. Another similar portion of the cervix was slowly frozen in liquid nitrogen-cooled isopentane for later in situ hybridization and immunocytochemistry analysis.

Polyadenylated RNA preparation and northern blot analysis
Polyadenylated RNA was extracted from frozen cervix by oligo dT cellulose affinity chromatography using a commercial kit (Fast Track 2.0, Invitrogen). 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, Wilmington, DE, USA) and then subjected to northern blot analysis for COX2, mPTGES1, and PTGFS. Cyclophilin 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) were fixed in freshly prepared 4% para-formaldehyde in 0.1 M phosphate buffer (20 min), washed twice in 0.1 M phosphate buffer, immersed in triethanolamine–HCl (3.71 g triethylamide (TEA), 2 ml of 6 M NaOH, and 198 ml 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 prehybridization buffer (50% freshly deionized formamide, 0.3 M NaCl, 20 mM Tris–HCl (pH 8.0), 5 mM EDTA, 10 mM sodium phosphate buffer (pH 8.0), and 1x Denhardt’s solution), and for at least 16 h in hybridization buffer (i.e. prehybridization buffer plus probe=1x106 c.p.m. per specimen in 70 µl). Control slides were hybridized with the labeled sense riboprobe. Slides were then washed thrice in 4x SSC and 4 mM dithiothreitol (DTT, Sigma); thrice 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 RNase A (Sigma); 2x SSC and 1 mM DTT; 0.1x SSC and 1 mM DTT at 60 °C; and finally 0.1x SSC at room temperature. The slides were then dehydrated in a graded series of ethanol plus 0.3 M ammonium acetate. They were then dipped in emulsion (Kodak NTB2) and exposed for 1–4 weeks at 4 °C. Following developing and fixing, they were counterstained with hematoxylin and eosin, mounted, and covered with a glass cover slip.

Synthesis of probes
Our cloned ovine COX2, mPTGES1, and PTGFS cDNAs in pCR II vector (Invitrogen), which include promoters for phage polymerases SP-6 to produce antisense probe and T-7 to produce sense probe, were linearized by appropriate restriction enzymes and antisense and sense riboprobes were synthesized using a commercial kit (MAXIscript, Ambion, Austin, TX, USA) labeled with [{alpha}-32P]UTP for northern blot analysis or [{alpha}-35S]UTP for in situ hybridization (NEN Life Science). The plasmid containing cyclophilin cDNAs with RNA polymerase promoters was purchased from Ambion.

Solubilized cell membrane extraction and western blot analysis
Solubilized cell membrane extracts from cervix were prepared as described previously (Wu et al. 1999, 2004a). The protein concentration was determined by the method of Bradford (Bio-Rad Laboratories). The solubilized proteins (100 µg/lane) were then separated on 10% SDS-PAGE and electrophoretically transferred to the nylon membrane (Immobilon, Millipore Corp., Bedford, MA, USA), using a Bio-Rad transfer blot cell. Western blot analysis was performed as before (Wu et al. 1999, 2004a). The protein bands were visualized using an ECL Western Blotting Detection Kit (ECL, Amersham Life Sciences). Different lengths of X-ray exposure time were conducted to determine the linear range with the amount of mRNA and protein of cervical COX2, PTGFS, and mPTGES1. The molecular sizes of the proteins were determined by running standard molecular weight marker proteins (Bio-Rad Laboratories) in an adjacent lane. 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.

Immunocytolocalization for COX2, PTGFS, and mPTGES1
Frozen sections (4 µm) of ovine cervical tissues were immunostained for COX2, mPTGES1, and PTGFS using the avidin–biotin immunoperoxidase method. Unless otherwise specified, all slides were sequentially incubated for various times at room temperature with each of the following reagents: (1) 3% (v/v) H2O2 in methanol for 30 min to block endogenous peroxidase activity; (2) 25% (v/v) normal goat serum and 5% (w/v) BSA in 0.05 M Tris–Cl with 0.15 M NaCl (pH 7.6) for 1 h; (3) rabbit anti-human COX2 polyclonal serum or rabbit anti-bovine PTGFS, or rabbit anti-human mPTGES1 for 20 h at 4 °C; (4) biotinylated goat anti-rabbit immunoglobulin G (IgG; Vector, Burlingame, CA, USA) for 0.5 h; (5) avidin–biotin complex (Vector): 40 µl each in 5 ml of 0.05 M Tris–Cl (pH 7.6) for 0. 5 h; and 6) 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) for 10 min: 4 µg/10 ml of 0.05 M Tris buffer (pH 7.6) to which was added 0.2 ml of 3% H2O2. After each incubation, the slides were washed with TBS for 15 min except for step (2). The slides were then counterstained with hematoxylin and mounted in Permount. Specificity of immunostaining for the enzymes was confirmed by three approaches: (1) omission of the primary antibody, (2) incubation of the slides with the normal rabbit serum instead of the primary antibodies, and (3) western blot analysis.

Antibodies for COX2 and mPTGES1 and PTGFS used for western blot analysis
A rabbit polyclonal antibody for COX2 raised against a synthetic peptide corresponding to a distinct C-terminal region of human COX2 (Oxford Biomedical Research Inc., Oxford, MI, USA) was used at 1:1000 dilution and incubated at 4 °C for 20 h. The COX2 antibody used in the present study was able to bind a highly purified ovine COX2 (Oxford Biomedical Research; Cat. number: NP04) demonstrated in our previous study (Wu et al. 1997). A rabbit polyclonal antibody for human mPTGES1 (Cayman Chemical Com, Ann Arbor, MI, USA) was used at 1:500 dilution and incubated at 4 °C for 20 h. A rabbit polyclonal antibody for bovine PTGFS kindly provided by Dr Watanabe (University of East Asia, Yamaguchi, Japan) was used at 1:500 dilution at 4 °C for 20 h. The primary antibodies used for immunocytochemistry were the same antibodies used for western analysis. A rabbit polyclonal antibody for bovine Gß was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A second antibody, horseradish peroxidase-conjugated donkey anti-rabbit IgG, was used for western blot analysis (Amersham Life Sciences) and a biotinylated goat anti-rabbit IgG (Vector) was used for immunocytochemistry. The specificity of each antibody was determined by: (1) parallel analysis of the positive control protein; (2) preabsorbing antibody with synthetic polypeptides which were used for generating the antibody; and (3) incubating the blots with the normal rabbit serum instead of the antibody.

Statistical analysis
Following normalization of the abundance of COX2, PTGFS, and mPTGES1 to cyclophilin (for northern) or Gß (for western) in individual samples, COX2, PTGFS, and mPTGES1 mRNA/protein concentrations in each western blot were expressed as a ratio of COX2, PTGFS, and mPTGES1 to cyclophilin or Gß respectively. One-way ANOVA was used to test for significant differences among the three groups (Cont, E2, and P) followed by multiple comparisons using the Tukey–Kramer procedure. Data were presented as means ± S.E.M.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Acknowledgements
 References
 
Regulation of COX2 mRNA and protein by E2 and progesterone
COX2 mRNA (Fig. 1Go) abundance increased significantly in the cervix after E2 treatment (P<0.05). In contrast, cervical COX2 protein level was not induced in the present experimental system (Fig. 2Go). There was a significant increase in both COX2 mRNA and protein levels in the cervix after progesterone treatment. Progesterone was a more potent stimulator than E2 of COX2 mRNA and protein abundance in the cervix (P<0.01, Figs 1Go and 2Go).


Figure 1
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  		Figure 1 (A) COX2 mRNA in the cervix of OVX nonpregnant ewes: saline-treated controls (Cont, lanes 1–5), progesterone treated (P, lanes 6–11), and estradiol treated (E2, lanes 12–16). Each lane represents one animal. (B) The same blot was hybridized with a cyclophilin (CP) cRNA probe to demonstrate relative amounts of RNA in each lane. (C) Northern blot signals for COX2 mRNA in the cervix of OVX nonpregnant ewes were quantified by densitometry and expressed as a ratio of COX2 to CP mRNA (mean ± S.E.M., n=5 for C and E2 groups, n=6 for P group). COX2 mRNA was significantly increased (*P<0.05) in the cervix following E2 treatment compared with Cont group. P was a more potent stimulator of cervical COX2 mRNA expression than E2 (+P<0.01 compared with Cont and E2).

 

Figure 2
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  		Figure 2 (A) Western blot analysis of COX2 protein in the cervix of OVX nonpregnant ewes: control saline-treated ewes (Cont, lanes 1–5), estradiol-treated ewes (E2, lanes 6–10), and progesterone-treated ewes (P, lanes 11–15). Each lane represents one animal. (B) Western blot signals for COX2 protein in the cervix of OVX nonpregnant ewes were quantified by densitometry (mean ± S.E.M., n=5 for each group). COX2 protein was increased in the cervix following P treatment compared with Cont and E2 (*P<0.01).

 
Regulation of PTGFS mRNA and protein by E2 and progesterone
In contrast, PTGFS mRNA (Fig. 3Go) and protein (Fig. 4Go) concentrations did not change after E2 or progesterone treatment (P>0.05).


Figure 3
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  		Figure 3 (A) PTGFS and (B) mPTGES1 mRNA in the cervix of OVX nonpregnant ewes: saline-treated controls (Cont, lanes 1–5), progesterone treated (P, lanes 6–11), and estradiol treated (E2, lanes 12–16). Each lane represents one animal. (C) The same blot was hybridized with a cyclophilin (CP) cRNA probe to demonstrate relative amounts of RNA in each lane. (D) Northern blot signals for PTGFS and mPTGES1 mRNA in the cervix of OVX nonpregnant ewes were quantified by densitometry and expressed as a ratio of PTGFS (solid bar) and mPTGES1 (open bar) mRNA to CP (mean ± S.E.M., n=5 for C and E2 groups, n=6 for P group). PTGFS and mPTGES1 mRNA remained unchanged after E2 or P treatment in the OVX nonpregnant sheep cervix (P>0.05, mean ± S.E.M.).

 

Figure 4
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  		Figure 4 Western blot analysis of (A) PTGFS and (B) mPTGES1 protein in the cervix of OVX nonpregnant ewes: control saline-treated ewes (Cont, lanes 1–5), estradiol-treated ewes (E2, lanes 6–10), and progesterone-treated ewes (P, lanes 11–15). Each lane represents one animal. (C) Gß protein in each corresponding lane. (D) Western blot signals for the ratio of PTGFS (solid bar) and mPTGES1 (open bar) over Gß proteins in the cervix of OVX nonpregnant ewes were quantified by densitometry (mean ± S.E.M., n=5 for each group). PTGFS and mPTGES1 proteins were unchanged in the cervix following P and E2 treatment.

 
Regulation of mPTGES1 mRNA and protein by E2 and progesterone
Additionally, mPTGES1 mRNA (Fig. 3Go) and protein (Fig. 4Go) concentrations remained unchanged after E2 or progesterone treatment (P>0.05).

In situ hybridization and immunohistochemistry analysis of COX2, PTGFS, and mPTGES1 mRNA and protein in the cervix
Both COX2 mRNA (Fig. 5AGo) and protein (Fig. 5CGo) were only localized in cervical glandular epithelial cells. In addition, PTGFS mRNA (Fig. 5EGo) and protein (Fig. 5GGo) were also confined to the cervical glandular epithelial cells. mPTGES1 mRNA level was below the sensitivity of our in situ hybridization system; however, the protein of mPTGES1 was clearly present in the epithelial cells of the cervix (Fig. 5IGo). When the sense probes in the replacement of antisense probes, the hybridization signals for COX2 (Fig. 5BGo) and PTGFS (Fig. 5FGo) mRNA were abolished. Similarly, the brown immunostaining, produced by enzymatic conversion of diaminobenzadine substrate into brown pigments, was absent when the primary antibodies for COX2, PTGFS, and mPTGES1 were replaced by normal rabbit serum (Fig. 5D, H and JGo).


Figure 5
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  		Figure 5 In situ and immunocytochemical analysis of cellular distribution of COX2, PTGFS, and mPTGES1 mRNA and protein in OVX nonpregnant sheep cervix. COX2 mRNA (A) and protein (C) were localized in epithelial cells of the cervical glands of the OVX nonpregnant sheep. Similarly, PTGFS mRNA (E) and protein (G) and mPTGES1 protein (I) were also confined to the cervical glandular epithelial cells. The specific in situ hybridization and immunostaining signals for COX2 (B), PTGFS (F) mRNA and COX2 (D), PTGFS (H) and mPTGES1(J) proteins were abolished when COX2 and PTGFS antisense riboprobes were replaced by sense riboprobes (B and F) or in the absence of the primary PTGFS antibody (D, H, and J) in the cervix. The black scale bar is 20 µm except in C, E, G, H, and I in which it represents 40 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Acknowledgements
 References
 
The present study is the first to simultaneously examine the regulatory effect of E2 and progesterone on COX2, PTGFS, and mPTGES1 expression at both mRNA and protein levels in the cervix to provide a more complete picture on the control of the prostaglandin enzymatic system in the cervix by E2 and progesterone. E2 and progesterone are two major steroids during estrous cycle and throughout pregnancy which regulate the uterine growth and remodeling partly by controlling prostaglandins production and function. This is the first study to show that both E2 and progesterone significantly elevated the abundance of cervical COX2, but not PTGFS and mPTGES1, mRNA. Progesterone, but not E2, also stimulated COX2 expression at protein level. More strikingly, progesterone is a more powerful stimulator on cervical COX2 expression than E2 at both mRNA and protein levels. Since prostaglandins are compounds with a very short half life, it is important to characterize local prostaglandin production capability. It has been very well documented, clinically and experimentally, that prostaglandins are effective agents inpriming the cervix in several species including sheep (Aiken 1972, Novy et al. 1974, Owiny & Fitzpatrick 1990, Sharma et al. 2005). Therefore, characterization of regulation of the key prostaglandin synthetic enzymes is required to provide a better understanding of the control of prelabor cervical ripening.

It is impossible to characterize the separate function of E2 and progesterone on control of the cervical prostaglandin system during pregnancy due to the indispensable relationship between placental steroid production and pregnancy maintenance; thus, we conducted the present study using the nonpregnant sheep model. The control of the cervical prostaglandin system in nonpregnant sheep model may not replicate the exact mechanisms which occur in pregnant sheep; however, it will certainly assist our understanding of the regulation of the pregnant cervical prostaglandin system. We have previously demonstrated that additional progesterone priming in pregnant sheep facilitated the cervical COX2 expression at both mRNA and protein levels (Wu et al. 2005a). Due to the presence of endogenously placental-produced E2 and progesterone, we were not able to define the separate function of E2 or progesterone on the control of the cervical prostaglandin enzymatic system in a hormone-free system, which is the goal of the present study and is an extension of our previous publications.

Our present experiment provided direct evidence for progesterone’s stimulation of the cervical COX2 expression at both mRNA and protein levels. Our observations strongly suggested that both E2 and progesterone were the positive stimulators for cervical COX2 expression. The stimulating effect of progesterone on cervical COX2 was much more powerful than E2 at both mRNA and protein levels. Interestingly, COX2 enzyme mRNA level was significantly elevated in the ovine cervix after E2 treatment, although the level of COX2 protein was hardly detectable in the same tissue by western blot analysis. A potential explanation for the low COX2 enzyme protein expression after E2 treatment is that cervical COX2 may be controlled more strictly at the transcriptional level than at the translational level. Or the change of cervical COX2 protein was under the detection sensitivity of western analysis we used.

Regulation of COX2 has been extensively studied. A series of COX2 inducers have been identified, e.g. cAMP (Wong et al. 1989), interleukin-1 (Kawaguchi et al. 1994), tumor necrosis factor (Diaz et al. 1992), transforming growth factor ß (Kester et al. 1994), phorbol ester (Habib et al. 1993), endothelin (Kester et al. 1994), and glucocorticoids (Whittle et al. 2000, Wu et al. 2001). Unfortunately, most studies have been conducted in in vitro cell culture systems. Such paradigms do not reflect the totality of events present in the intact structure of the tissue and in particular in vitro studies do not reflect local or blood-borne in vivo cell-to-cell communication. Therefore, they cannot completely reproduce the physiological response of the tissues as in vivo. A few studies have characterized in vivo regulation of COX2 in rat ovary (Sirois et al. 1992) by human chorionic gonadotropin and in nonpregnant and pregnant sheep uterus by E2 and progesterone (Charpigny et al. 1997, Wu et al. 2004a, 2005a, 2005b). Results from our and others’ previous studies demonstrated that progesterone could induce COX2 expression (Charpigny et al. 1997, Wu et al. 1997) and activated prostaglandin production (Louis et al. 1976) in nonpregnant and pregnant sheep uterus (Wu et al. 2005a). However, the regulation of cervical COX2, PTGFS, and mPTGES in vivo by progesterone and E2 has not been determined in any species.

It is very well documented that prostaglandin production is mainly controlled at COX2 level (Dewitt & Smith 1988, Diaz et al. 1992, Hla & Neilson 1992, Hempel et al. 1994); however, recent studies have suggested that post-COX enzymes may also determine the exact mix of the final prostaglandin product (Jakobsson et al. 1999, Murakami et al. 2002, Palliser et al. 2006). Therefore, parallel studies were performed to compare and contrast the changes of cervical COX2, mPTGES1, and PTGFS mRNA and protein in the same animals. In contrast to increased cervical COX2 levels after E2 and progesterone treatment, the expression of PTGFS and mPTGES1 mRNA and protein levels were not significantly different in ewes that received E2 or progesterone treatment compared with those of the control group, suggesting distinct regulatory roles of E2 and progesterone on different prostaglandin enzymes. Cervical prostaglandin production in sheep is mainly controlled at COX level. Because of the central location of COX in the biosynthetic pathway of prostaglandins and the rapid catalytic inactivation of COX, this enzyme is a logical control point and a rate-limiting step in the formation of prostaglandins. In contrast to the stimulation of endometrial mPTGES1 by E2 in pregnant sheep at 0.7 gestation (Zhang et al. 2006), there was no response of cervical mPTGES1 to E2 in the present study, which provided another example of tissue-specific regulation of the prostaglandin enzymatic system. Palliser et al.(2006) have recently reported that there was no labor-associated induction of PTGFS in pregnant sheep cervix which also suggested that the labor associated rise of E2 may not be able to stimulate cervical PTGFS expression. Consistent with Palliser’s finding, we failed to induce the cervical PTGFS expression by E2 in nonpregnant sheep.

Similar to the finding in pregnant sheep and baboon, COX2 mRNA and protein were both confined to the epithelial cells in nonpregnant sheep cervix. In addition, cervical PTGFS and mPTGES1 had a similar cellular distribution highlighting the importance of cervical epithelial cells in prostaglandin production. The junction between the cervix and the uterine body is the internal orifice of the cervix uteri, and at this point the nature of the epithelium and uterine wall changes. The endocervical canal runs between the uterine and vaginal cavities and is lined by a single layer of tall columnar mucus-secreting epithelium that extends into the underlying stroma (endocervical mucus glands). These glandular structures are in fact deep slit-like invaginations of the surface epithelium, with blind-ended tubules (Stevens & Lowe 1992). Thus, there is a large surface area for the production of cervical mucus, which fills the endocervical canal. To our surprise, it was this glandular structure that stained intensively for COX2, PTGFS, and mPTGES1 mRNA and/or protein. In contrast, cervical smooth muscle cells had almost negligible staining for all the three prostaglandin synthetic enzymes studied. By secreting mucus, cervical glandular cells can spread prostaglandins within the entire lower birth canal.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Acknowledgements
 References
 
Increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E2 and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E2. The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was all confined in the cervical glandular epithelial cells of nonpregnant sheep.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
References
 
Supported by NIH HD 39247 and 21350. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 15 November 2006
First decision 4 January 2007
Accepted 23 January 2007


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 

Aiken JW 1972 Aspirin and indomethacin prolong parturition in rats: evidence that prostaglandins contribute to expulsion of foetus. Nature 240 21–25.[CrossRef][Medline]

Challis JRG 1971 Sharp increase in free circulating oestrogen in immediately before parturition in sheep. Nature 229 208.[CrossRef][Medline]

Charpigny G, Reinaud P, Tamby JP, Creminon C, Martal J, Maclouf J & Gullomot M 1997 Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy. Endocrinology 138 2163–2171.[Abstract/Free Full Text]

Dewitt DL & Smith WL 1988 Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. PNAS 85 1412–1416.[Abstract/Free Full Text]

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