Separate and synergistic effect of progesterone and estradiol on induction of annexin 2 and its interaction protein p11 in pregnant sheep myometrium

in Journal of Molecular Endocrinology

To search for myometrial candidate genes regulated by progesterone, we isolated annexin 2 cDNA by subtractive hybridization and cloning. We also examined the effect of estradiol and/or progesterone, individually or combined, on expressions of annexin 2 and its ligand protein, p11 in pregnant sheep intrauterine tissues. Annexin 2 is a Ca2+-dependent phospholipid-binding protein which interacts with p11 to form a bivalent heterotetramer. The heterotetramer was indicated in the production of prostaglandins through the regulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Thus, annexin 2 and p11 could be the important players in Ca2+ signaling and prostaglandin production in uterine smooth muscles. Twenty-two ewes were treated with vehicle (n = 6), or 5 mg estradiol administered intramuscularly twice a day for 2 days (n = 6), or 100 mg progesterone administered intramuscularly twice a day for 14 days (n = 5), or estradiol plus progesterone with 100 mg progesterone administered intramuscularly twice a day for 10 days (n = 5) and then with vehicle for 2 days followed by estradiol for 2 days (5 mg administered intramuscularly twice a day). At 121 days of gestation age (dGA), endometrium, myometrium, placenta, and cervix were obtained under halothane anesthesia. Subtractive hybridization was used to isolate differentially expressed mRNAs from myometrial tissues treated with progesterone, which was confirmed by western blot at protein level. Annexin 2 and p11 interaction was validated by immunoprecipitation and their expressions in intrauterine tissues were determined by western blot analysis. Data were analyzed by ANOVA. A cDNA clone from myometrial-subtracted library representing differentially expressed mRNA in the pregnant sheep myometrium with progesterone treatment was identified as annexin 2 by sequence analysis and BLASTN search. Annexin 2 was present in both cytosolic and membrane-associated fractions of intrauterine tissues. In contrast, p11 was detectable only in myometrial cytosolic fraction. Both annexin 2 and p11 significantly increased in myometrial cytosolic fraction after progesterone or progesterone plus estradiol treatment. Furthermore, estradiol and progesterone combined had a more profound effect on induction of annexin 2 than progesterone alone. Annexin 2 was immunoprecipitated specifically by p11 antibody from the myometrium. This study indicates that progesterone may play an important role in the control of myometrial contractility by modifying protein expression associated with Ca2+ and prostaglandin signaling systems during pregnancy. Enhanced expression of myometrial annexin 2 in progesterone plus estradiol group supports our hypothesis that estrogen’s stimulation is optimized by progesterone’s priming in the pregnant sheep uterus.

Abstract

To search for myometrial candidate genes regulated by progesterone, we isolated annexin 2 cDNA by subtractive hybridization and cloning. We also examined the effect of estradiol and/or progesterone, individually or combined, on expressions of annexin 2 and its ligand protein, p11 in pregnant sheep intrauterine tissues. Annexin 2 is a Ca2+-dependent phospholipid-binding protein which interacts with p11 to form a bivalent heterotetramer. The heterotetramer was indicated in the production of prostaglandins through the regulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Thus, annexin 2 and p11 could be the important players in Ca2+ signaling and prostaglandin production in uterine smooth muscles. Twenty-two ewes were treated with vehicle (n = 6), or 5 mg estradiol administered intramuscularly twice a day for 2 days (n = 6), or 100 mg progesterone administered intramuscularly twice a day for 14 days (n = 5), or estradiol plus progesterone with 100 mg progesterone administered intramuscularly twice a day for 10 days (n = 5) and then with vehicle for 2 days followed by estradiol for 2 days (5 mg administered intramuscularly twice a day). At 121 days of gestation age (dGA), endometrium, myometrium, placenta, and cervix were obtained under halothane anesthesia. Subtractive hybridization was used to isolate differentially expressed mRNAs from myometrial tissues treated with progesterone, which was confirmed by western blot at protein level. Annexin 2 and p11 interaction was validated by immunoprecipitation and their expressions in intrauterine tissues were determined by western blot analysis. Data were analyzed by ANOVA. A cDNA clone from myometrial-subtracted library representing differentially expressed mRNA in the pregnant sheep myometrium with progesterone treatment was identified as annexin 2 by sequence analysis and BLASTN search. Annexin 2 was present in both cytosolic and membrane-associated fractions of intrauterine tissues. In contrast, p11 was detectable only in myometrial cytosolic fraction. Both annexin 2 and p11 significantly increased in myometrial cytosolic fraction after progesterone or progesterone plus estradiol treatment. Furthermore, estradiol and progesterone combined had a more profound effect on induction of annexin 2 than progesterone alone. Annexin 2 was immunoprecipitated specifically by p11 antibody from the myometrium. This study indicates that progesterone may play an important role in the control of myometrial contractility by modifying protein expression associated with Ca2+ and prostaglandin signaling systems during pregnancy. Enhanced expression of myometrial annexin 2 in progesterone plus estradiol group supports our hypothesis that estrogen’s stimulation is optimized by progesterone’s priming in the pregnant sheep uterus.

Keywords:

Introduction

There is abundant in vivo and in vitro evidence that progesterone and estrogen are key factors in regulating myometrial contractility associated with the maintenance of pregnancy and the onset of labor across species. The generally accepted view is that progesterone maintains uterine quiescence (Csapo 1977), whereas estrogen promotes uterine contractility (Mecenas et al. 1996). According to this view, the course of pregnancy depends on the balance of these two factors and the onset of labor is associated with the removal of the progesterone block and the replacement of block by estrogen promotion. However, this view is likely to be too simplistic for a complex process such as pregnancy maintenance and termination. In many experimental animal models, including pregnant sheep, the uterus and the cervix undergo considerable growth and ripening throughout gestation, and the intrauterine prostaglandin system develops throughout the course of pregnancy when progesterone is the major circulating hormone and estradiol is constantly low in maternal plasma (Liggins et al. 1972, 1973).

Based on the above observation, we have proposed that progesterone exerts a facilitatory role on uterine growth and remodeling by altering a cassette of uterine genes (up- or down-regulation). Each member of the cassette of genes contributes to some extent in the preparation and maturation of the uterus. Altered abundance of members of this cassette of genes in the uterus is a prerequisite for labor and delivery. To test our hypothesis, we employed suppression subtractive hybridization (SSH), a sensitive and powerful technique to identify unknown and differentially expressed genes, to characterize myometrial genes regulated by progesterone. Improved understanding of the previously undescribed genes or known genes with unknown functions will advance our knowledge of the key regulatory mechanisms involved in the preparation of the uterus and maintenance of pregnancy by progesterone.

Using SSH, we identified a marked increase in annexin 2 mRNA associated with progesterone treatment. Annexins are a family of cytosolic Ca2+-dependent phospholipid-binding proteins that form an evolutionarily conserved multigene family being expressed throughout plant and animal species (Gerke & Moss 2002). The family of proteins are characterized structurally by a highly α-helical and tightly packed protein core domain responsible for Ca2+ binding, which enable them to peripherally dock onto negatively charged cytosolic membrane phospholipids in their Ca2+-bound conformation, and a variable N-terminal hydrophilic interaction domain for binding protein ligands, such as p11 (Gerke et al. 2005). Annexin 2 exists both as a monomer and a bivalent heterotetramer, in which two annexin 2 molecules bind to a dimer of p11 (also known as S100A10), the natural protein ligand of annexin 2. p11 belongs to S100 proteins, which bind Ca2+ at two binding sites of the EF-hand type per molecule, and is involved in the regulation of different Ca2+-dependent intracellular signaling events (Gerke & Weber 1985). The heterotetramer is anchored to the cytosolic plasma membrane as a result of the interaction of the annexin 2 with the membrane phospholipids, while the p11 subunit protrudes away from the membrane into the cytosol (Gerke et al. 2005).

It is the property of peripheral docking onto the membrane that links the annexin 2 and p11 heterotetramer complex to many membrane-related intra-cellular events. The target membrane of annexin 2 includes plasma membrane and membranes of endosomes. Thus, annexin 2 has been suggested to function in membrane organization and traffic (Babiychuk & Draeger 2000, Draeger et al. 2005), endocytosis and exocytosis (Zobiack et al. 2003), tight junction assembly (Lee et al. 2004), regulation of plasma membrane ion channels (Girard et al. 2002, Okuse et al. 2002, Poon et al. 2004), and in lipid-related cellular signaling (Wu et al. 1997a, Huang et al. 2002). p11 has been shown to regulate prostaglandin production by altering cPLA2 activity and release of arachidonic acid in rat gastric epithelial cells (Akiba et al. 2000). Therefore, the heterotetramer of annexin 2 and p11 could be an important mediator in Ca2+ signaling and prostaglandin synthesis in uterine smooth muscles. Understanding the link between the hormonal and the cellular signaling in myometrium is critical for understanding the mechanisms which control myometrial contraction. In the present study, the specific interaction of annexin 2 and p11 in the myometrium was determined by immunoprecipitation (IP). Furthermore, we examined intrauterine annexin 2 and p11 in response to the individual and/or combined estradiol and/or progesterone treatment. In addition, the tissue-specific expression and the subcellular distribution of annexin 2 and p11 were also determined in the endometrium, myometrium, placenta, and cervix.

Materials and methods

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 day of gestation (dGA), ewes from which tissues were obtained were catheterised with fetal and maternal carotid arterial catheters. We intentionally set the animal experiment at a time point in gestation before the increase in fetal cortisol takes place. The fetal and maternal carotid arterial catheters were implanted as we described previously (Wu et al. 2004a). 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 (C; n = 6) or 100 mg progesterone administered intramuscularly twice a day (P; n = 5) for 14 days to produce term levels of progesterone in maternal plasma, or sesame oil for 10–12 days followed by 5 mg estradiol administered intramuscularly twice a day (E; n = 6) for 2 days to produce labor levels of estradiol in maternal plasma, (EP; n = 5) or with 100 mg progesterone administered intramuscularly twice a day for 10 days, and then 2 days with sesame oil followed by 2 days with estradiol (5 mg administered intramuscularly twice a day). At 121 dGA, necropsies were performed under halothane anesthesia after which the animals were killed. The tissues of myometrium, intercaruncular endometrium, maternal placenta (MP; caruncular endometrium), fetal placenta (FP; cotyledons), and cervix were collected for RNA and protein analysis.

mRNA extraction, RT-PCR cloning, and rapid amplification of cDNA ends (RACE)

mRNA was extracted from ovine uterine myometrium using the Micro-FastTrack kit (Invitrogen). cDNAs were synthesized from the mRNA using a kit of SuperScript III First-Strand Synthesis System (Invitrogen). SMART RACE cDNA Amplification Kit (Clontech) was used to obtain the 5′ and 3′ ends of the cDNA sequences. The primers for 3′-RACE were designed from the original cDNA sequences of subtraction clones and the primers of 5′-RACE from long-range PCR products. Touchdown PCR for RACE was done under the following conditions: 94 ° C for 30 s for initial denaturing, 94 ° C for 5 s, 72 ° C for 3 min for 5 cycles; 94 ° C for 5 s, 70 ° C for 3 min for 5 cycles; and 94 ° C for 5 s and 68 ° C for 3 min for 25 cycles. The PCR products were analyzed on 1.2% agarose gels and cloned into pCR2.1-TOPO cloning vector (Invitrogen). For RT-PCR, cDNA fragments were amplified from the reverse transcription by PCR kit with high proofreading DNA polymerases (Invitrogen). Positive clones were identified by hybridization using cDNA probes or by PCR screening. The DNA sequencing was done at the core facility of Wake Forest University Baptist Medical Center (Winston-Salem, NC, USA).

Construction of subtracted cDNA library

The subtracted cDNA library was constructed using a commercial kit (Clontech Laboratories, Inc.). Briefly, the mRNAs were extracted from the myometrial tissues of the ewes treated with progesterone (n = 5) and ewes of the age-matched animals treated with vehicle (n = 5). The extraction of the two mRNA population samples was done simultaneously using the same reagents and protocol. The cDNAs were synthesized from 2 μ g mRNA from the myometrial samples, followed by second-strand synthesis and adaptor ligation. For construction of a library enriched for transcripts with elevated expression level in the progesterone-treated myometrium relative to the control, myometrium from control was designated as ‘driver’ and from progesterone treated as ‘tester’. The cDNA clones were subject to differential screening using a kit (Clontech Laboratories, Inc.) to select positive ones for further analysis (see below).

Protein extraction and western blot analysis

Approximately, 1 g of tissue was homogenized for 1 min (Polytron, Kinematica AG, Switzerland) on ice in TE buffer (50 mM Tris and 10 mM EDTA) containing 2 mM octyl glucoside and 0.2 mM phenyl-methylsulfonyl fluoride and centrifuged at 30 000 g for 1 h at 4 ° C to collect the soluble cytosolic fraction. 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 phenylmethylsulfonyl fluoride. The supernatants were centrifuged at 13 000 g for 25 min at 4 ° C. The recovered supernatants (solubilized membrane fraction) were stored at − 70 ° C until electrophoresis analysis. The protein concentration was determined by Bradford’s method (Bio-Rad Laboratories).

The proteins (25 μ g/lane) separated on 10% Tris–glycine SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Imobilon, Millipore Corp., Bedford, MA, USA), and blocked with 6% nonfat milk for 1 h at room temperature. A rabbit polyclonal antibody against amino acids 1–50 of human annexin 2, a shared polypeptide sequence between human and sheep, was purchased from Santa Cruz Biotechnology (Cat. no. sc-9061; Santa Cruz, CA, USA). A mouse monoclonal antibody produced against bovine p11 was obtained from BD Transduction Laboratories (Cat. no. A14120-050). The primary antibodies were used at 1:1000 dilution for overnight incubation at 4 ° C. Nonimmune normal rabbit (for annexin 2) or nonimmune normal mouse (p11) IgG was used to control the specificity of respective antibodies. The secondary antibodies, anti-rabbit or mouse IgG linked with horseradish peroxidase, were purchased from Amersham. The membrane was washed three to four times (1 h) with TTBS buffer (150 mM NaCl, 0.1% Tween 20, and 10 mM Tris–HCl (pH 7.5)). The secondary antibody (1:5000 dilution) was incubated with the membrane for 1 h at room temperature and then washed again with TTBS buffer for 1 h.

The protein bands were visualized using a Super-Signal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). The molecular sizes of the proteins were determined by running a pre-stained molecular marker (See Blue plus, Invitrogen) in an adjacent lane. β-Actin (Santa Cruz Biotechnology, Cat. no. sc-47778) and Gβ subunit (Santa Cruz Biotechnology, Cat. no. sc-261) were detected on each blot to control cytosolic and membrane-associated protein loading respectively. Chemiluminescence signals from X-ray films were analyzed and quantified with the scanner and the data were analyzed with a densitometry program – Scan Analysis and quantified against an arbitrary scale in the plot.

Immunoprecipitation

Myometrial cytosolic proteins (1 mg) from progesterone- and estradiol-treated and control animals were centrifuged at 16 000 g for 30 min at 4 ° C to pellet tissue debris and insoluble materials. The cleared supernatants were transferred to fresh microcentrifuge tubes, diluted to 1 μ g/μ l proteins with IP buffer (1% Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 1 mM EDTA), and incubated with 50 μ l protein A/G plus agarose (Santa Cruz Biotechnology) for 3 h at 4 ° C with gentle rotation. The supernatants were centrifuged at 1000 g for 5 min at 4 ° C to remove the protein A/G agarose beads. The pre-cleared supernatant was divided equally into two microcentrifuge tubes for each sample. Two micrograms of monoclonal p11 antibody were added to one tube and 2 μ g nonimmune mouse IgG to another tube. The tubes were mixed with gentle rotation at 4 ° C overnight. Then, 25 μ l protein A/G were added to each tube and the tubes were mixed for 1 h at 4 ° C. The immunoprecipitates were collected by centrifugation at 1000 g for 5 min and washed five times, each with 0.5 ml IP buffer. The immunoprecipitates were solubilized in sample buffer for SDS-PAGE and western blot with antibodies against proteins of annexin 2 and p11.

Bioinformatics

The acquired nucleotide and deduced amino acid sequences were aligned against the GenBank databases (nt and protein) at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) using BLAST to search for sequence matches. The alignments were done using the Clustal W program (http://www2.ebi.ac.uk/ClustalW/).

Statistical analysis

Following normalization of the abundance of annexin 2 or p11 to β-actin (cytosolic protein) or Gβ (membrane protein) in individual samples, annexin 2 or p11 protein concentration in each western blot was expressed as a ratio of annexin 2 or p11 to β-actin or Gβ respectively. Differences between different groups for western blot analysis were analyzed by ANOVA followed by multiple comparison using a Tukey–Kramer procedure. Data are presented as the mean ± s.e.m.

Results

Analysis of progesterone up-regulated myometrial genes by SSH

Following subtractive hybridization, differentiating screening and BLAST search after DNA sequencing, annexin 2 was identified as a positive gene up-regulated at mRNA level in the uterine myometrial tissues treated with progesterone (Fig. 1). We then cloned the ovine annexin 2 cDNA and compared the evolutionary conservation of annexin 2 with other species (Fig. 2).

Cloning and characterization of ovine annexin 2 cDNA

The ovine annexin 2 cDNA sequence obtained by RT-PCR and RACE was 1079 nucleotides (GenBank accession no. DQ358998) with an open reading frame of 1020 nucleotide, which was translated to a protein of 339 residues. Examination across species reveals that annexin 2 was highly conserved between the species, which has 100, 99, 97, 96, and 97% identity to annexin 2 proteins from cow, dog, mouse, human, and rat respectively. Most of the discrepancy residues were conserved substitutions (Fig. 2), reflecting a functional constraint during evolution.

Progesterone inducing cytosolic annexin 2 and p11 in uterine myometrial tissues

Since SSH revealed induced annexin 2 at mRNA level, we then used western blot analysis to determine whether the mRNA change of myometrial annexin 2 induced by progesterone was carried to the protein level. In order to differentiate the individual and combined effect of estradiol and progesterone on the expression of annexin 2 in pregnant sheep myometrium, we examined annexin 2 and p11 protein level in four groups of animals: treated with estradiol alone, progesterone alone, EP, and the control treated with vehicle. We analyzed annexin 2 expression in both cytosolic and membrane fractions. Immunoreactive annexin 2 appeared as a single band with an approximate molecular weight of 36 kDa which was absent when the same myometrial samples were subject to the nonimmune normal rabbit IgG (Fig. 3). Annexin 2 was greatly increased in myometrial cytosolic fractions from sheep treated with progesterone, and further elevated in the myometrium from animals treated with both estradiol and progesterone (Fig. 4). On the other hand, annexin 2 could not be detected in the cytosolic fractions of myometrium from animals treated with estradiol alone or vehicle (Fig. 4). Annexin 2 was also detected in the cervical cytosolic fractions with no difference between the different treatment groups (Fig. 5). In contrast, annexin 2 was not found in the cytosolic fraction of the endometrium, FP and MP (data not shown). Annexin 2 could be detected in the membrane fractions in all intrauterine tissues, including myometrium, cervix, endometrium, and FP and MP. However, there were no differences found between the control and the treatment groups in membrane fractions (Figs 6 and 7).

Since p11 was reported as a natural protein ligand of annexin 2, parallel studies were performed on the same animals to analyze p11 expression. Immunoreactive p11 appeared as a single band with an approximate molecular weight of 11 kDa (Fig. 8). p11 expression displayed a similar pattern to annexin 2 in cytosolic fraction of myometrial protein (Fig. 8). There was no detectable expression of myometrial p11 from control and estradiol-treated animals. On the contrary, progesterone alone or progesterone plus estradiol significantly induced p11 expression in the myometrium (Fig. 8). The cytosolic p11 protein was below the detection level in the cervix, endometrium, FP, and FP (data not shown). In addition, membrane-associated p11 was not found in any of the intrauterine tissues studied.

The binding of annexin 2 and p11 was verified by IP

We performed three independent experiments of IP using myometrial cytosolic protein samples from five animals (treated with progesterone and estradiol or control). The IP using p11 antibody is shown in Fig. 9. As expected, p11 protein was not detected from the supernatant of the myometrium obtained from controls (both IgG and p11 groups). In contrast, p11 was identified from animals treated with estradiol and progesterone (IgG and p11). The p11 protein was precipitated by its antibody from the cytosolic fraction treated with progesterone plus estradiol, but not with nonimmune mouse IgG. Using the same blot, we detected annexin 2 from p11 immunoprecipitates of the animals treated with estradiol and progesterone, but not from the IgG (Fig. 9).

Discussion

Annexin family proteins have been studied in different cell types, such as epithelial (Yao et al. 1999, Akiba et al. 2000), endothelial (Hajjar & Acharya 2000, Deora et al. 2004), and skeletal and smooth muscle cells (Babiychuk et al. 1999, Draeger et al. 2005). Even though the cell biology of annexin 2 and p11 has been extensively studied at the molecular and cellular levels in various cells and tissues, changes in annexin 2 and p11 in intrauterine tissues have not been studied in relation to pregnancy and parturition in any species.

Throughout pregnancy, the uterus undergoes extensive biochemical and physiological changes. Although myometrial activity initially remains relatively quiescent throughout pregnancy, the sensitivity of the uterus to various uterotonins, such as oxytocin and prostaglandins, is increased through the course of pregnancy due to increased respective receptors (Fuchs et al. 1983, 1984, Baguma-Nibasheka et al. 1998, Gimpl & Fahrenholz 2001, Wu et al. 2001) with no further augmentation at labor (Palliser et al. 2005). In addition, the uterus increases ten times in weight and the cervix extends ten times in length during pregnancy (Cumningham et al. 2001). Along with uterine anatomic changes, there is a dramatic increase in the activity of the intrauterine prostaglandin system. For example, intrauterine prostaglandin cyclo-oxygenase 2 (COX2) mRNA and fetal plasma prostaglandin E2(PGE2) increase throughout the second half of pregnancy in pregnant sheep (Rice et al. 1995, Wu et al. 1999) and pregnant women (MacDonald & Casey 1993). The increased prostaglandin production reflected by well-described higher forebag (the small amniotic sac below the fetal presenting part) prostaglandins in primate (MacDonald & Casey 1993) is associated with increased COX2 mRNA abundance in the surrounding lower uterine segment and cervical tissues (MacDonald & Casey 1993, Wu et al. 2004b). More interestingly, all of these anatomical, physiological, and biochemical changes in pregnant uterus develop when maternal plasma progesterone concentration is constantly high. In some animal models, such as in sheep, pregnant uterus matures and cervix ripens when progesterone is the major circulating hormone and estradiol is constantly low in maternal plasma (Liggins et al. 1973, Challis 1971).

In nonpregnant sheep, we have demonstrated that progesterone is a more potent stimulator than estradiol in stimulating endometrial and cervical COX2 mRNA and protein expression (Wu et al. 1997b). Based on the above observation obtained from pregnant and non-pregnant uterus, we hypothesize that progesterone can facilitate the expression of many intrauterine gene expression throughout pregnancy, which is a prerequisite for the maximal stimulation of the uterus by estrogen at the end of pregnancy in order to induce onset of labor (Wu et al. 2005). However, the genes facilitated by progesterone are poorly characterized. Many of these progesterone-induced genes alter gradually through the course of pregnancy; therefore, it is difficult to analyze them with routine technology. SSH is a sensitive and effective technique for rapidly identifying the changes in the abundance of mRNAs for individual genes during physiological events.

In this study, we identified annexin 2 and p11 as the up-regulated genes in pregnant sheep myometrium treated with progesterone alone or progesterone plus estradiol and confirmed their specific interaction in pregnant myometrium by IP. We then used western blot analysis to determine the individual and interactive functions of estradiol and progesterone on the control of myometrial annexin 2 and p11 genes. Our study provides firm evidence in support of our hypothesis (Wu et al. 2005) that progesterone can prepare the uterus by facilitating the array of gene expression, such as annexin 2 and p11. However, optimal induction of annexin 2 can only be achieved in animals that received combined progesterone and estradiol treatment, indicating that the presence of estradiol influences progesterone function which may be achieved through induced estrogen (Wu et al. 1996, 2005) and progesterone receptors (Spencer et al. 2004). Our results also suggested that an increase in estradiol alone could not account for all the uterine changes that occurred in labor if the uterus was not fully prepared by progesterone. Without prior progesterone priming, estradiol alone was not able to stimulate myometrial annexin 2.

Steroid receptor complexes induce or repress the expression of genes by interacting with regulatory DNA sequences and transcription factor(s). The upstream promoter region of human p11 gene has been analyzed (Huang et al. 2003). Although p11 promoter sequence lacks estrogen and progesterone response elements, two AP-1 sites, which may serve as the indirect transcription regulatory elements by estrogen and progesterone (Huang et al. 2003), have been located on p11 promoter region. Relatively few vertebrate annexins have been subjected to detailed promoter analysis and none of the annexin 2 promoter sequence has been cloned in any species. Based on the promoter sequence analysis of other members of annexins’ family, the presence of SP1-binding sites in most annexin promoters so far examined, is consistent with their broad patterns of expression, but the expression of other regulatory elements, such as T cell-specific silencer in annexin 6 (Donnelly & Moss 1998), suggests that under certain circumstances tight transcriptional control may be exerted. Our study provides solid data for tissue-specific induction of annexin 2 and p11 by progesterone in pregnant sheep myometrium, while leaving annexin 2 and p11 in all other intrauterine tissues unchanged. Further study on the ovine annexin 2 and p11 genes are necessary to prove or disprove the existence of estrogen and progesterone response elements in the corresponding promoters or other sequence motifs associated with estrogen and progesterone action which will enhance our understanding of the regulatory mechanisms of myometrial annexin 2 and p11 expression throughout pregnancy.

Annexin 2 was present in both cytosolic and membrane fractions in all intrauterine tissues examined. In contrast, p11 was detectable only in the myometrial cytosolic fraction by western analysis. However, it was only the cytosolic annexin 2 and p11 that were significantly induced by progesterone or progesterone plus estradiol. Progesterone exerted no effect on membrane-associated annexin 2 in any of the intrauterine tissues studied. The mechanism and significance of such a tight control of annexin 2 and p11 located in different subcellular compartments are not clear. Membrane-associated annexin 2 and p11 complex might function either as ion channels and/or ion channel regulators (Gerke & Moss 2001, Girard et al. 2002, Okuse et al. 2002, Poon et al. 2004), whereas cytosolic annexin 2 and p11 complex interact with cPLA2 and alters its activity, thereby changing the arachidonic acid release and prostaglandin synthesis (Wu et al. 1997a, Huang et al. 2002, Hayes et al. 2004). Both the proteins have been suggested to be involved in the intracellular Ca2+ signaling and their function is tightly correlated with the intracellular calcium concentration. The physiological significance of induced myometrial annexin 2 and p11 in the cytosolic fraction by progesterone is intriguing. Further studies are needed to determine the functions of annexin 2 and p11 in both cytosolic and membrane-associated fractions of uterine myometrium during pregnancy and labor, especially in the processes associated with the intracellular prostaglandin production and Ca2+ signaling in the smooth muscle cells.

Conclusion

This study demonstrates that progesterone facilitated the expression of myometrial cytosolic annexin 2 and p11 and priming with sufficient progesterone prior to estradiol’s stimulation further enhanced annexin 2 expression in myometrial tissue when compared with progesterone or estradiol alone. Our data indicates that progesterone may play an important role in myometrial Ca2+ and prostaglandin signaling systems by altering relevant gene expression during pregnancy.

Figure 1
Figure 1

Dot blot screen analysis of up-regulated gene clones after progesterone-treated myometrial SSH. Replicate dot blots were hybridized with both (A) forward-subtracted and (B) reverse-subtracted probes. Dots hybridized with reverse-subtracted probes corresponded to background (B). Clones hybridized only with forward-subtracted probes represent mRNAs that were up-regulated in the tester (progesterone treated) population of myometrium (A). Two of these clones shown by the red arrows were identified as annexin 2 by sequence analysis and BLASTN search. Note: B5 and B10 in (A) were identical samples to B5 and B10 in (B).

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 2
Figure 2

Alignment of annexin 2 protein sequences across several species. The deduced ovine annexin 2 amino acid sequence (GenBank accession no. DQ358998) is aligned with its orthologs from cow (NP_777141), dog (NP_001002619), human (AAH23990), mouse (AAH03327), and rat (NP_063970). Four annexins’ repeated domain signatures are highlighted in the aligned sequences. Symbols: identical residues (*); conserved substitution (:); and semi-conserved substitution (.).

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 3
Figure 3

Western analysis of two EP-treated myometrial cytosolic samples with annexin 2 (A2) antibody (A) or nonimmune normal rabbit IgG (B). (A) A single band with an approximate molecular weight of 36 kDa was detected when the rabbit polyclonal A2 antibody was applied on the blot (lanes 1 and 2). (B) There were no detectable bands on the blot when the identical myometrial samples were subjected to the nonimmune normal rabbit IgG in the replacement of A2 antibody (lanes 3 and 4). (C) and (D) β-Actin used as the quality control of the loaded proteins was detectable on both blots.

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 4
Figure 4

Western blot analysis of cytosolic annexin 2 (A2) protein (A) in pregnant sheep myometrium collected from controls treated with vehicle (Cont, lanes 1–6), progesterone (P, lanes 7–11), estradiol and progesterone (EP, lanes 12–16), and estradiol (E, lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of A2 to actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). A2 protein increased in P-treated myometrium. Sufficient P priming prior to E stimulation further enhanced myometrial A2 expression (*P < 0.05 when compared with Cont, E, or EP; P < 0.05 when compared with P).

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 5
Figure 5

Western blot analysis of cytosolic A2 protein (A) in pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of A2 to actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Cervical cytosolic A2 protein remained unchanged after E, P, or EP treatment.

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 6
Figure 6

Western blot analysis of membrane-associated A2 protein in pregnant sheep fetal placenta (FP; A) and maternal placenta (MP; C) collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) and (D) Gβ protein in each corresponding lane for fetal placenta (B) and maternal placenta (D). Densitometric analysis of fetal placenta (E) and maternal placenta (F) A2 to Gβ ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Membrane-associated A2 protein remained unchanged after E, P, or EP treatment in fetal and maternal placenta when compared with Cont.

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 7
Figure 7

Western blot analysis of membrane-associated A2 protein (A) in pregnant sheep myometrium (A), endometrium (C), and cervix (E) collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. Gβ protein in each corresponding lane for myometrium (B), endometrium (D), and cervix (F). Densitometric analysis of myometrial (G), endometrial (H), and cervical (I) A2 to Gβ ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Membrane-associated A2 protein remained unchanged after E, P, or EP treatment in myometrium, endometrium, and cervix when compared with Cont.

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 8
Figure 8

Western blot analysis of cytosolic p11 protein (A) in pregnant sheep myometrium collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of p11 to β-actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). p11 protein increased in P- and EP-treated myometrium (*P < 0.05 when compared with Cont and E).

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

Figure 9
Figure 9

Immunoprecipitation (IP) of the annexin 2–p11 complex from myometrium treated with progesterone and estradiol (EP) or vehicle (Cont). Supernatants (lanes 1–4) and the protein A/G agarose beads representing immunoprecipitated proteins (lanes 5–8) were loaded at 20 μ g proteins/lane. The precipitated proteins of annexin 2 (A) or p11 (B) were detected by western blot analysis. Positive myometrial control (+ve) was loaded in lane 9. Lanes 1 and 8: Cont IgG (vehicle-treated myometrial samples reacted with IgG only); lanes 2 and 7: Cont p11 (vehicle-treated myometrial samples reacted with p11 antibody); lanes 3 and 6: EP-treated IgG (EP-treated myometrial samples reacted with IgG only); note: both annexin 2 and p11 were detected only in the supernatants, but not in protein A/G agarose beads; lanes 4 and 5: EP-treated p11 (EP-treated myometrial samples reacted with p11 antibody); note: both annexin 2 and p11 were detected in protein A/G agarose beads suggesting that annexin 2 and p11 complex was precipitated by p11 antibody; please note that unprecipitated annexin 2 was also detectable in the supernatant of EP-treated myometrium (A, lane 4), indicating that some unbound or free annexin 2 still existed in myometrial cytosol after immunoprecipitation.

Citation: Journal of Molecular Endocrinology 38, 4; 10.1677/jme.1.02143

This work was supported by NIH 39247. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • AkibaS Hatazawa R Ono K Hayama M Matsui H & Sato T 2000 Transforming growth factor-alpha stimulates prostaglandin generation through cytosolic phospholipase A(2) under the control of p11 in rat gastric epithelial cells. British Journal of Pharmacology1311004–1010.

    • Search Google Scholar
    • Export Citation
  • BabiychukEB & Draeger A 2000 Annexins in cell membrane dynamics. Ca (2+)-regulated association of lipid microdomains. Journal of Cell Biology1501113–1124.

    • Search Google Scholar
    • Export Citation
  • BabiychukEB Palstra RJ Schaller J Kampfer U & Draeger A 1999 Annexin VI participates in the formation of a reversible membrane-cytoskeleton complex in smooth muscle cells. Journal of Biological Chemistry27435191–35195.

    • Search Google Scholar
    • Export Citation
  • Baguma-NibashekaM Wentworth RA Green LR Jenkins SL & Nathanielsz PW 1998 Differences in the in vitro sensitivity of ovine myometrium and mesometrium to oxytocin and prostaglandin E2 and F2α . Biology of Reproduction5873–78.

    • Search Google Scholar
    • Export Citation
  • ChallisJR1971 Sharp increase in free circulating oestrogens immediately before parturition in sheep. Nature229208.

  • CsapoAI1977 The ‘see–saw’ theory of parturition. Ciba Foundation Symposium159–210.

  • CumninghamFG Gant NF Leveno KJ Gilstrap LC Hauth JC Wenstrom KD 2001 Anatomy of the reproductive tract 21. In: Williams Obstetrics. edn 21 chap. 3 pp 31–61 New York Chicago: McGraw-Hill.

  • DeoraAB Kreitzer G Jacovina AT & Hajjar KA 2004 An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface. Journal of Biological Chemistry27943411–43418.

    • Search Google Scholar
    • Export Citation
  • DonnellySR & Moss SE 1998 Functional analysis of the human annexin I and VI gene promoters. Biochemical Journal15681–687.

  • DraegerA Wray S & Babiychuk EB 2005 Domain architecture of the smooth-muscle plasma membrane: regulation by annexins. Biochemical Journal387309–314.

    • Search Google Scholar
    • Export Citation
  • FuchsAR Periyasamy S & Soloff MS 1983 Systemic and local regulation of oxytocin receptors in the rat uterus and their functional significance. Canadian Journal of Biochemistry and Cell Biology61615–624.

    • Search Google Scholar
    • Export Citation
  • FuchsAR Fuchs F Husslein P & Soloff MS 1984 Oxytocin receptors in the human uterus during pregnancy and parturition. American Journal of Obstetrics and Gynecology150734–741.

    • Search Google Scholar
    • Export Citation
  • GerkeV & Moss SE 2001 Annexins: from structure to function. Physiological Reviews82331–371.

  • GerkeV & Moss SE 2002 Annexins: from structure to function. Physiological Reviews82331–371.

  • GerkeV & Weber K 1985 The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells. Embo Journal42917–2920.

    • Search Google Scholar
    • Export Citation
  • GerkeV Creutz CE & Moss SE 2005 Annexins: linking Ca2+ signaling to membrane dynamics. Nature Reviews. Molecular Cell Biology6449–461.

    • Search Google Scholar
    • Export Citation
  • GimplG & Fahrenholz F 2001 The oxytocin receptor system: structure function and regulation. Physiological Reviews630–683.

  • GirardC Tinel N Terrenoire C Romey G Lazdunski M & Borsotto M 2002 p11 an annexin II subunit an auxiliary protein associated with the background K+ channel TASK-1. Embo Journal214439–4448.

    • Search Google Scholar
    • Export Citation
  • HajjarKA & Acharya SS 2000 Annexin II and regulation of cell surface fibrinolysis. Annals of the New York Academy of Sciences902265–271.

    • Search Google Scholar
    • Export Citation
  • HayesMJ Merrifield CJ Shao D Ayala-Sanmartin J Schorey CD Levine TP Proust J Curran J Bailly M & Moss SE 2004 Annexin 2 binding to phosphatidylinositol 45-bisphosphate on endocytic vesicles is regulated by the stress response pathway. Journal of Biological Chemistry27914157–14164.

    • Search Google Scholar
    • Export Citation
  • HuangXL Pawliczak R Cowan MJ Gladwin MT Madara P Logun C & Shelhamer JH 2002 Epidermal growth factor induces p11 gene and protein expression and down-regulates calcium ionophore-induced arachidonic acid release in human epithelial cells. Journal of Biological Chemistry27738431–38440.

    • Search Google Scholar
    • Export Citation
  • HuangX Pawliczak R Yao XL Madara P Alsaaty S Shelhamer JH & Cowan MI 2003 Characterization of the human p11 promoter sequence. Gene310133–142.

    • Search Google Scholar
    • Export Citation
  • LeeDB Jamgotchian N Allen SG Kan FW & Hale IL 2004 Annexin A2 heterotetramer: role in tight junction assembly. American Journal of Physiology. Renal Physiology287F481–F491.

    • Search Google Scholar
    • Export Citation
  • LigginsGC Grieves SA Kendall JZ & Knox BS 1972 The physiological roles of progesterone oestradiol-17 and prostaglandin F 2 in the control of ovine parturition. Journal of Reproduction and Fertility. Supplement1685–100.

    • Search Google Scholar
    • Export Citation
  • LigginsGC Fairclough RJ Grieves SA Kendall JZ & Knox BS 1973 The mechanism of initiation of parturition in the ewe. Recent Progress in Hormone Research29111–159.

    • Search Google Scholar
    • Export Citation
  • MacDonaldPC & Casey ML 1993 The accumulation of prostaglandins (PG) in amniotic fluid is an after effect of labor and not indicative of a role for PGE2 or PGF2 alpha in the initiation of human parturition. Journal of Clinical Endocrinology and Metabolism761332–1339.

    • Search Google Scholar
    • Export Citation
  • MecenasCA Giussani DA Owiny JR Jenkins SL Wu WX Honnebier BO Lockwood CJ Kong L Guller S & Nathanielsz PW 1996 Production of premature delivery in pregnant rhesus monkeys by androstenedione infusion. Nature Medicine2443–448.

    • Search Google Scholar
    • Export Citation
  • OkuseK Malik-Hall M Baker MD Poon WY Kong H Chao MV & Wood JN 2002 Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature417653–656.

    • Search Google Scholar
    • Export Citation
  • PalliserHK Hirst JJ Ooi GT Rice GE Dellios NL Escalona RM Parkington HC & Young IR 2005 Prostaglandin E and F receptor expression and myometrial sensitivity at labor onset in the sheep. Biology of Reproduction72937–943.

    • Search Google Scholar
    • Export Citation
  • PoonWY Malik-Hall M Wood JN & Okuse K 2004 Identification of binding domains in the sodium channel Na(V)1.8 intracellular N-terminal region and annexin II light chain p11. FEBS Letters558114–118.

    • Search Google Scholar
    • Export Citation
  • RiceGE Freed KA Aitken MA & Jacobs RA 1995 Gestational- and labour-associated changes in the relative abundance of prostaglandin G/H synthase-1 and -2 mRNA in ovine placenta. Journal of Molecular Endocrinology14237–245.

    • Search Google Scholar
    • Export Citation
  • SpencerTE Johnson GA Burghardt RC & Bazer FW 2004 Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

    • Search Google Scholar
    • Export Citation
  • WuWX Owiny JR & Nathanielsz PW 1996 Regulation of estrogen receptor mRNA and its peptide in the non-pregnant sheep uterus. Biology of Reproduction55762–768.

    • Search Google Scholar
    • Export Citation
  • WuT Angus CW Yao XL Logun C & Shelhamer JH 1997a P11 a unique member of the S100 family of calcium-binding proteins interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2. Journal of Biological Chemistry27217145–17153.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Zhang Q Buchwalder L & Nathanielsz PW 1997b Regulation of prostaglandin endoperoxide H synthase 1 and 2 by estradiol and progesterone in nonpregnant ovine myometrium and endometrium in vivo. Endocrinology1384005–4012.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH & Nathanielsz PW 1999 Tissue-specific ontogenic expression of prostaglandin H synthase 2 in the ovine myometrium endometrium and placenta during late gestation and at spontaneous term labor. American Journal of Obstetrics and Gynecology1811512–1519.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Zhang Q & Nathanielsz PW 2001 Characterization of topology- gestation- and labor-related changes of a cassette of myometrial contraction-associated protein mRNA in the pregnant baboon myometrium. Journal of Endocrinology171445–453.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Coksaygan T Chakrabarty K Collins V Rose JR & Nathanielsz PW 2004a Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age. Endocrinology1451444–1452.

    • Search Google Scholar
    • Export Citation
  • WuWX Smith GC Rose J & Nathanielsz PW 2004b Characterization of the concentration gradient of prostaglandin H synthase 2 mRNA throughout the pregnant baboon uterus. Journal of Endocrinology182241–248.

    • Search Google Scholar
    • Export Citation
  • WuWX Coksaygan T Chakrabarty K Collins V Rose JC & Nathanielsz PW 2005 Sufficient progesterone–priming prior to estradiol-stimulation is required for optimal induction of the cervical prostaglandin system. Biology of Reproduction73343–350.

    • Search Google Scholar
    • Export Citation
  • YaoXL Cowan MJ Gladwin MT Lawrence MM Angus CW & Shelhamer JH 1999 Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity. Journal of Biological Chemistry27417202–17208.

    • Search Google Scholar
    • Export Citation
  • ZobiackN Rescher U Ludwig C Zeuschner D & Gerke V 2003 The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes. Molecular Biology of the Cell144896–4908.

    • Search Google Scholar
    • Export Citation

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  • View in gallery

    Dot blot screen analysis of up-regulated gene clones after progesterone-treated myometrial SSH. Replicate dot blots were hybridized with both (A) forward-subtracted and (B) reverse-subtracted probes. Dots hybridized with reverse-subtracted probes corresponded to background (B). Clones hybridized only with forward-subtracted probes represent mRNAs that were up-regulated in the tester (progesterone treated) population of myometrium (A). Two of these clones shown by the red arrows were identified as annexin 2 by sequence analysis and BLASTN search. Note: B5 and B10 in (A) were identical samples to B5 and B10 in (B).

  • View in gallery

    Alignment of annexin 2 protein sequences across several species. The deduced ovine annexin 2 amino acid sequence (GenBank accession no. DQ358998) is aligned with its orthologs from cow (NP_777141), dog (NP_001002619), human (AAH23990), mouse (AAH03327), and rat (NP_063970). Four annexins’ repeated domain signatures are highlighted in the aligned sequences. Symbols: identical residues (*); conserved substitution (:); and semi-conserved substitution (.).

  • View in gallery

    Western analysis of two EP-treated myometrial cytosolic samples with annexin 2 (A2) antibody (A) or nonimmune normal rabbit IgG (B). (A) A single band with an approximate molecular weight of 36 kDa was detected when the rabbit polyclonal A2 antibody was applied on the blot (lanes 1 and 2). (B) There were no detectable bands on the blot when the identical myometrial samples were subjected to the nonimmune normal rabbit IgG in the replacement of A2 antibody (lanes 3 and 4). (C) and (D) β-Actin used as the quality control of the loaded proteins was detectable on both blots.

  • View in gallery

    Western blot analysis of cytosolic annexin 2 (A2) protein (A) in pregnant sheep myometrium collected from controls treated with vehicle (Cont, lanes 1–6), progesterone (P, lanes 7–11), estradiol and progesterone (EP, lanes 12–16), and estradiol (E, lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of A2 to actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). A2 protein increased in P-treated myometrium. Sufficient P priming prior to E stimulation further enhanced myometrial A2 expression (*P < 0.05 when compared with Cont, E, or EP; P < 0.05 when compared with P).

  • View in gallery

    Western blot analysis of cytosolic A2 protein (A) in pregnant sheep cervix collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of A2 to actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Cervical cytosolic A2 protein remained unchanged after E, P, or EP treatment.

  • View in gallery

    Western blot analysis of membrane-associated A2 protein in pregnant sheep fetal placenta (FP; A) and maternal placenta (MP; C) collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) and (D) Gβ protein in each corresponding lane for fetal placenta (B) and maternal placenta (D). Densitometric analysis of fetal placenta (E) and maternal placenta (F) A2 to Gβ ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Membrane-associated A2 protein remained unchanged after E, P, or EP treatment in fetal and maternal placenta when compared with Cont.

  • View in gallery

    Western blot analysis of membrane-associated A2 protein (A) in pregnant sheep myometrium (A), endometrium (C), and cervix (E) collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. Gβ protein in each corresponding lane for myometrium (B), endometrium (D), and cervix (F). Densitometric analysis of myometrial (G), endometrial (H), and cervical (I) A2 to Gβ ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). Membrane-associated A2 protein remained unchanged after E, P, or EP treatment in myometrium, endometrium, and cervix when compared with Cont.

  • View in gallery

    Western blot analysis of cytosolic p11 protein (A) in pregnant sheep myometrium collected from controls treated with vehicle (Cont; lanes 1–6), progesterone (P; lanes 7–11), estradiol and progesterone (EP; lanes 12–16), and estradiol (E; lanes 17–22) at 121 dGA. (B) β-Actin protein in each corresponding lane. (C) Densitometric analysis of p11 to β-actin ratio in Cont, P-, EP-, and E-treated groups (n = 5–6 in each group). p11 protein increased in P- and EP-treated myometrium (*P < 0.05 when compared with Cont and E).

  • View in gallery

    Immunoprecipitation (IP) of the annexin 2–p11 complex from myometrium treated with progesterone and estradiol (EP) or vehicle (Cont). Supernatants (lanes 1–4) and the protein A/G agarose beads representing immunoprecipitated proteins (lanes 5–8) were loaded at 20 μ g proteins/lane. The precipitated proteins of annexin 2 (A) or p11 (B) were detected by western blot analysis. Positive myometrial control (+ve) was loaded in lane 9. Lanes 1 and 8: Cont IgG (vehicle-treated myometrial samples reacted with IgG only); lanes 2 and 7: Cont p11 (vehicle-treated myometrial samples reacted with p11 antibody); lanes 3 and 6: EP-treated IgG (EP-treated myometrial samples reacted with IgG only); note: both annexin 2 and p11 were detected only in the supernatants, but not in protein A/G agarose beads; lanes 4 and 5: EP-treated p11 (EP-treated myometrial samples reacted with p11 antibody); note: both annexin 2 and p11 were detected in protein A/G agarose beads suggesting that annexin 2 and p11 complex was precipitated by p11 antibody; please note that unprecipitated annexin 2 was also detectable in the supernatant of EP-treated myometrium (A, lane 4), indicating that some unbound or free annexin 2 still existed in myometrial cytosol after immunoprecipitation.

  • AkibaS Hatazawa R Ono K Hayama M Matsui H & Sato T 2000 Transforming growth factor-alpha stimulates prostaglandin generation through cytosolic phospholipase A(2) under the control of p11 in rat gastric epithelial cells. British Journal of Pharmacology1311004–1010.

    • Search Google Scholar
    • Export Citation
  • BabiychukEB & Draeger A 2000 Annexins in cell membrane dynamics. Ca (2+)-regulated association of lipid microdomains. Journal of Cell Biology1501113–1124.

    • Search Google Scholar
    • Export Citation
  • BabiychukEB Palstra RJ Schaller J Kampfer U & Draeger A 1999 Annexin VI participates in the formation of a reversible membrane-cytoskeleton complex in smooth muscle cells. Journal of Biological Chemistry27435191–35195.

    • Search Google Scholar
    • Export Citation
  • Baguma-NibashekaM Wentworth RA Green LR Jenkins SL & Nathanielsz PW 1998 Differences in the in vitro sensitivity of ovine myometrium and mesometrium to oxytocin and prostaglandin E2 and F2α . Biology of Reproduction5873–78.

    • Search Google Scholar
    • Export Citation
  • ChallisJR1971 Sharp increase in free circulating oestrogens immediately before parturition in sheep. Nature229208.

  • CsapoAI1977 The ‘see–saw’ theory of parturition. Ciba Foundation Symposium159–210.

  • CumninghamFG Gant NF Leveno KJ Gilstrap LC Hauth JC Wenstrom KD 2001 Anatomy of the reproductive tract 21. In: Williams Obstetrics. edn 21 chap. 3 pp 31–61 New York Chicago: McGraw-Hill.

  • DeoraAB Kreitzer G Jacovina AT & Hajjar KA 2004 An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface. Journal of Biological Chemistry27943411–43418.

    • Search Google Scholar
    • Export Citation
  • DonnellySR & Moss SE 1998 Functional analysis of the human annexin I and VI gene promoters. Biochemical Journal15681–687.

  • DraegerA Wray S & Babiychuk EB 2005 Domain architecture of the smooth-muscle plasma membrane: regulation by annexins. Biochemical Journal387309–314.

    • Search Google Scholar
    • Export Citation
  • FuchsAR Periyasamy S & Soloff MS 1983 Systemic and local regulation of oxytocin receptors in the rat uterus and their functional significance. Canadian Journal of Biochemistry and Cell Biology61615–624.

    • Search Google Scholar
    • Export Citation
  • FuchsAR Fuchs F Husslein P & Soloff MS 1984 Oxytocin receptors in the human uterus during pregnancy and parturition. American Journal of Obstetrics and Gynecology150734–741.

    • Search Google Scholar
    • Export Citation
  • GerkeV & Moss SE 2001 Annexins: from structure to function. Physiological Reviews82331–371.

  • GerkeV & Moss SE 2002 Annexins: from structure to function. Physiological Reviews82331–371.

  • GerkeV & Weber K 1985 The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells. Embo Journal42917–2920.

    • Search Google Scholar
    • Export Citation
  • GerkeV Creutz CE & Moss SE 2005 Annexins: linking Ca2+ signaling to membrane dynamics. Nature Reviews. Molecular Cell Biology6449–461.

    • Search Google Scholar
    • Export Citation
  • GimplG & Fahrenholz F 2001 The oxytocin receptor system: structure function and regulation. Physiological Reviews630–683.

  • GirardC Tinel N Terrenoire C Romey G Lazdunski M & Borsotto M 2002 p11 an annexin II subunit an auxiliary protein associated with the background K+ channel TASK-1. Embo Journal214439–4448.

    • Search Google Scholar
    • Export Citation
  • HajjarKA & Acharya SS 2000 Annexin II and regulation of cell surface fibrinolysis. Annals of the New York Academy of Sciences902265–271.

    • Search Google Scholar
    • Export Citation
  • HayesMJ Merrifield CJ Shao D Ayala-Sanmartin J Schorey CD Levine TP Proust J Curran J Bailly M & Moss SE 2004 Annexin 2 binding to phosphatidylinositol 45-bisphosphate on endocytic vesicles is regulated by the stress response pathway. Journal of Biological Chemistry27914157–14164.

    • Search Google Scholar
    • Export Citation
  • HuangXL Pawliczak R Cowan MJ Gladwin MT Madara P Logun C & Shelhamer JH 2002 Epidermal growth factor induces p11 gene and protein expression and down-regulates calcium ionophore-induced arachidonic acid release in human epithelial cells. Journal of Biological Chemistry27738431–38440.

    • Search Google Scholar
    • Export Citation
  • HuangX Pawliczak R Yao XL Madara P Alsaaty S Shelhamer JH & Cowan MI 2003 Characterization of the human p11 promoter sequence. Gene310133–142.

    • Search Google Scholar
    • Export Citation
  • LeeDB Jamgotchian N Allen SG Kan FW & Hale IL 2004 Annexin A2 heterotetramer: role in tight junction assembly. American Journal of Physiology. Renal Physiology287F481–F491.

    • Search Google Scholar
    • Export Citation
  • LigginsGC Grieves SA Kendall JZ & Knox BS 1972 The physiological roles of progesterone oestradiol-17 and prostaglandin F 2 in the control of ovine parturition. Journal of Reproduction and Fertility. Supplement1685–100.

    • Search Google Scholar
    • Export Citation
  • LigginsGC Fairclough RJ Grieves SA Kendall JZ & Knox BS 1973 The mechanism of initiation of parturition in the ewe. Recent Progress in Hormone Research29111–159.

    • Search Google Scholar
    • Export Citation
  • MacDonaldPC & Casey ML 1993 The accumulation of prostaglandins (PG) in amniotic fluid is an after effect of labor and not indicative of a role for PGE2 or PGF2 alpha in the initiation of human parturition. Journal of Clinical Endocrinology and Metabolism761332–1339.

    • Search Google Scholar
    • Export Citation
  • MecenasCA Giussani DA Owiny JR Jenkins SL Wu WX Honnebier BO Lockwood CJ Kong L Guller S & Nathanielsz PW 1996 Production of premature delivery in pregnant rhesus monkeys by androstenedione infusion. Nature Medicine2443–448.

    • Search Google Scholar
    • Export Citation
  • OkuseK Malik-Hall M Baker MD Poon WY Kong H Chao MV & Wood JN 2002 Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature417653–656.

    • Search Google Scholar
    • Export Citation
  • PalliserHK Hirst JJ Ooi GT Rice GE Dellios NL Escalona RM Parkington HC & Young IR 2005 Prostaglandin E and F receptor expression and myometrial sensitivity at labor onset in the sheep. Biology of Reproduction72937–943.

    • Search Google Scholar
    • Export Citation
  • PoonWY Malik-Hall M Wood JN & Okuse K 2004 Identification of binding domains in the sodium channel Na(V)1.8 intracellular N-terminal region and annexin II light chain p11. FEBS Letters558114–118.

    • Search Google Scholar
    • Export Citation
  • RiceGE Freed KA Aitken MA & Jacobs RA 1995 Gestational- and labour-associated changes in the relative abundance of prostaglandin G/H synthase-1 and -2 mRNA in ovine placenta. Journal of Molecular Endocrinology14237–245.

    • Search Google Scholar
    • Export Citation
  • SpencerTE Johnson GA Burghardt RC & Bazer FW 2004 Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

    • Search Google Scholar
    • Export Citation
  • WuWX Owiny JR & Nathanielsz PW 1996 Regulation of estrogen receptor mRNA and its peptide in the non-pregnant sheep uterus. Biology of Reproduction55762–768.

    • Search Google Scholar
    • Export Citation
  • WuT Angus CW Yao XL Logun C & Shelhamer JH 1997a P11 a unique member of the S100 family of calcium-binding proteins interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2. Journal of Biological Chemistry27217145–17153.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Zhang Q Buchwalder L & Nathanielsz PW 1997b Regulation of prostaglandin endoperoxide H synthase 1 and 2 by estradiol and progesterone in nonpregnant ovine myometrium and endometrium in vivo. Endocrinology1384005–4012.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH & Nathanielsz PW 1999 Tissue-specific ontogenic expression of prostaglandin H synthase 2 in the ovine myometrium endometrium and placenta during late gestation and at spontaneous term labor. American Journal of Obstetrics and Gynecology1811512–1519.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Zhang Q & Nathanielsz PW 2001 Characterization of topology- gestation- and labor-related changes of a cassette of myometrial contraction-associated protein mRNA in the pregnant baboon myometrium. Journal of Endocrinology171445–453.

    • Search Google Scholar
    • Export Citation
  • WuWX Ma XH Coksaygan T Chakrabarty K Collins V Rose JR & Nathanielsz PW 2004a Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age. Endocrinology1451444–1452.

    • Search Google Scholar
    • Export Citation
  • WuWX Smith GC Rose J & Nathanielsz PW 2004b Characterization of the concentration gradient of prostaglandin H synthase 2 mRNA throughout the pregnant baboon uterus. Journal of Endocrinology182241–248.

    • Search Google Scholar
    • Export Citation
  • WuWX Coksaygan T Chakrabarty K Collins V Rose JC & Nathanielsz PW 2005 Sufficient progesterone–priming prior to estradiol-stimulation is required for optimal induction of the cervical prostaglandin system. Biology of Reproduction73343–350.

    • Search Google Scholar
    • Export Citation
  • YaoXL Cowan MJ Gladwin MT Lawrence MM Angus CW & Shelhamer JH 1999 Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity. Journal of Biological Chemistry27417202–17208.

    • Search Google Scholar
    • Export Citation
  • ZobiackN Rescher U Ludwig C Zeuschner D & Gerke V 2003 The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes. Molecular Biology of the Cell144896–4908.

    • Search Google Scholar
    • Export Citation