Differential Expression of Inositol 1,4,5-Trisphosphate Receptor Types ...

BIOLOGY OF REPRODUCTION 63, 532–537 (2000)

Differential Expression of Inositol 1,4,5-Trisphosphate Receptor Types 1, 2, and 3 in Rat Myometrium and Endometrium During Gestation1 Jose´ E. Mesonero,3 Zahra Tanfin,4 Mauricette Hilly,3 Pascal Colosetti,3 Jean-Pierre Mauger,2,3 and Simone Harbon4 INSERM U442,3 Signalisation Cellulaire et Calcium, Signalisation et Re´gulations Cellulaires,4 CNRS, IFR-FR 46, Universite´ Paris-Sud, F-91405 Orsay Cedex, France

Ca21 concentration is an important determinant of smooth muscle contractility [4]. Stimulation of the phosphoinositide–PLC pathway by contractile agonists that activate Gprotein-coupled receptors has been demonstrated in various myometrial preparations [5–9]. IP3 binds to an intracellular IP3 receptor (IP3R) located on the surface of intracellular Ca21 stores, e.g., endoplasmic reticulum, and then releases Ca21 into the cytoplasm [1, 10]. Thus, the IP3R plays a central role in the conversion of IP3 signals produced by external stimuli into intracellular Ca21 signals. Molecular cloning studies have demonstrated the existence of at least three types of IP3Rs, encoded by genes designated type 1 [11, 12], type 2 [13], and type 3 [14]. These three IP3R types have different primary structures and tissue distributions, and their amino acid sequences are approximately 60–70% identical. IP3Rs also differ in their affinity for IP3, in the order IP3R-2 . IP3R-1 . IP3R-3 [15]. We have recently shown that, in rat myometrium, the responsiveness of the PLCb3 pathway associated with receptor and/or direct G-protein activation increased during gestation and coincided with the increase in the amount of Gq/G11 [8]. Little is known about the IP3 receptor isoforms present in the rat uterus and their possible regulation in relation to pregnancy and increases in the production of inositol phosphates. We addressed this question by assessing IP3 binding capacity and the expression of IP3R isoform genes in terms of mRNA and corresponding protein levels in the myometrium and endometrium at the middle (Day 12) and end (Day 21) of gestation compared to estrogenprimed rats (Day 0). We found that 1) the number of IP3 binding sites increased in both tissues with advancing gestation; 2) IP3R-1, -2, and -3 genes were coexpressed, albeit to different extents, in the nonpregnant myometrium and endometrium; and 3) the expression of IP3R-1 and IP3R-3 increased over the course of gestation, whereas the level of IP3R-2 increased only in the endometrium at term.

ABSTRACT The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP3Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF42 was enhanced with advancing gestation. 3[H]IP3 binding sites in membranes also increased during pregnancy (Day 21 . Day 12 . Day 0). The mRNAs encoding for three isoforms of IP3R as well as their corresponding proteins, IP3R-1, IP3R-2, and IP3R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP3Rs increased with advancing gestation, except for IP3R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP3Rs. The difference noted between the two uterine tissues suggests that IP3Rs may have cell-specific functions.

calcium, parturition, pregnancy, signal tranducers, signal transduction, uterus

INTRODUCTION

The uterus undergoes remarkable changes in growth and function during pregnancy. The biochemical and morphological changes that take place in the endometrium are thought to be a prerequisite for implantation and adequate development of the fetus. The function of the myometrium gradually changes during gestation. It is initially quiescent so as to maintain the growing fetus and then becomes active at term to expel the mature fetus. Cellular processes as diverse as cell motility, contraction, secretion, and cell proliferation are regulated by intracellular Ca21 [1, 2], and this may be of crucial importance for uterine cells during gestation. The initial response of many cells to Ca21-mobilizing receptor agonists is the activation of phospholipase C (PLC) catalyzing the hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2) leading to an increase in inositol 1,4,5trisphosphate (IP3) production [3]. It is known that the generation of IP3 and the associated increase in intracellular

MATERIALS AND METHODS

Materials

[3H]IP3 (17–21 Ci/mmol) was obtained from New England Nuclear Product division (Dupont de Nemours, Les Ulis, France) and IP3 from Calbiochem (La Jolla, CA). Myo-[2-3H]inositol (10–20 Ci/mmol), Hybond membranes, enhanced chemiluminescence (ECL) detection system, and the Megaprime DNA labeling kit were obtained from Amersham International (Les Ulis, France). AG1-X8 and Bradford reagent were from Bio-Rad (Ivry, France). Rabbit polyclonal antibody against a synthetic peptide corresponding to the 16 C-terminal residues of IP3R-2 was prepared by Covalab (Oullins, France) [16]. Monoclonal antibody against IP3R-3 was obtained from Transduction Laboratories (San Diego, CA) and rabbit polyclonal antibody against

J.E.M. is the recipient of ‘‘Poste Vert from INSERM’’ and ‘‘Conseil Re´gional d’Ile-de-France.’’ The first two authors contributed equally to the work presented here. 2 Correspondence: Jean Pierre Mauger, INSERM U442, Signalisation Cellulaire et Calcium, Universite´ Paris-Sud, Baˆtiment 443, F-91405 Orsay Cedex, France. FAX: 33 1 69 15 58 93; e-mail: [email protected] 1

Received: 31 December 1999. First decision: 11 February 2000. Accepted: 28 March 2000. Q 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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PREGNANCY-RELATED CHANGES IN IP3 RECEPTORS

IP3R-1 was obtained from Affinity Bioreagents (Goldon, CO). Antirabbit IgG and antimouse IgG antibodies were from Sanofi Diagnostics Pasteur (Marnes-La-Coquelte, France). A full-length cDNA encoding type 1 IP3R (pCMVI-9) [12] and a rat type 2 partial cDNA (pCMVI-102) [13], were kindly provided by Dr. T.C. Su¨dhof (Howard Hughes Medical Institute, Dallas, TX). cDNA (pCBG1IP3R3) [14] was obtained from Prof. G.I. Bell (Howard Hughes Medical Institute, University of Chicago). Lithium chloride, carbamoylcholine chloride (carbachol), b-estradiol 3-benzoate, leupeptin, aprotinin, and PMSF were from Sigma Chemical Co. (St. Louis, MO). RNeasy Mini Kit and QIAshredder columns were from QIAGEN (Les Ulis, France). Animals and Tissue Processing

Pregnant rats (Wistar) were used at Days 12 and 21 of gestation. Immature female rats (Wistar 5 wk old) were treated with 30 mg estradiol for 2 days and were used the following day. These estradiol-primed animals were considered as Day 0. Rats were killed by decapitation, and the uterine horns were quickly cleared of adhering fat and excised out longitudinally. After removal of fetuses, their sites of attachment, and placental tissues, myometrium was separated by stripping away the endometrium as previously described [5, 8]. Histological sections of myometrial preparations showed the samples to consist almost exclusively of longitudinal muscle. For each specific experiment we minimized variation by using tissues pooled from uteri from at least 12 (Day 0), 6 (Day 12), and 3 (Day 21) rats. Determination of [3H]Inositol Phosphates

Myometrial and endometrial strips (;30 mg) were labeled with 5 mCi of myo-[3H]inositol (0.4 mM) [5, 8]. Tissues were washed three times with nonradioactive Krebs buffer and were incubated with 1 ml of fresh buffer, containing 10 mM LiCl before exposure to the agents indicated. Reactions were stopped by immersing the strips in 1.5 ml of cold 7% (w:v) trichloroacetic acid (TCA), followed by homogenization and centrifugation at 3000 3 g for 20 min at 48C. The TCA-soluble supernatants were extracted with diethylether, neutralized with Tris base, and applied to a column (0.7 3 2 cm) of anion-exchange AG1-X8. Total inositol phosphates (IP3 1 IP2 1 IP1) were eluted together in a single step with 12 ml of 1 M ammonium formate/0.1 M formic acid. The [3H]phosphoinositides present in the TCA pellets were extracted by the method of Bligh and Dyer [17], essentially as previously described [6]. The 3H content of the various samples was determined by scintillation spectrometry. The production of [3H]inositol phosphates was expressed as a percentage of radioactivity incorporated into phosphoinositides obtained from the corresponding sample. Western Blot Analysis

Endometrial and myometrial membranes were prepared as previously described [8] and stored at 2808C. Protein concentration was determined by the method of Bradford [18]. Protein samples were analyzed by SDS-PAGE in 5% acrylamide gels as described by Laemmli [19], and the proteins were then transferred to Hybond-ECL membranes that were then blocked 60 min in 5% nonfat dried milk in Trisbuffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 500 mM NaCl). The blots were probed with rabbit polyclonal anti-

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IP3R-1 antibody or rabbit polyclonal antibody against a synthetic peptide corresponding to the 16 C-terminal residues of IP3R-2 [16], both used at 1:1000 dilution. Monoclonal antibody against IP3R-3 was used at 1:2000 dilution. The primary antibodies against IP3R-1 and IP3R-2 were detected with an antirabbit IgG antibody coupled to horseradish peroxidase, and the primary antibody against IP3R-3 was detected with an antimouse IgG antibody (dilution 1: 5000), using the ECL detection system. In certain experiments, the blots were reprobed after stripping in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM b-mercaptoethanol for 60 min at 608C. Nitrocellulose sheets were rinsed in TBS and then reblotted for 60 min in 5% nonfat dried milk in TBS before being sequentially probed with other antibodies. The developed blots were quantified by densitometry with a Molecular Dynamics Densitometer. We verified that the amplitude of the signal was proportional to the amount of proteins loaded on the gels, at least within the investigated range (10–50 mg). Thus, the binding of primary and secondary antibodies as well as the ECL reaction were not limiting. Equilibrium [3H]IP3 Binding Studies

For IP3 binding studies, the myometrial and endometrial membrane fractions (300 mg) were incubated for 6 min on ice in 500 ml of ice-cold cytosol-like medium consisting of 110 mM KCl, 20 mM NaCl, 1 mM Na2HPO4, 1 mM EDTA, 25 mM HEPES/KOH, pH 7.5, supplemented with 1 mg/ml BSA and 1 nM [3H]IP3 (20 Ci/mmol), with or without unlabeled IP3, to give a final concentration of 1, 5, and 50 nM, as previously described [16, 20]. Nonspecific binding was determined in the presence of 5 mM IP3. At the end of incubation, 400 ml of the sample was layered onto a Whatman GF/C glass-fiber filter and washed with 1 ml of ice-cold medium containing 250 mM sucrose, 10 mM Na2HPO4, at pH 8. Radioactivity retained on the filter was counted by scintillation spectrometry. IP3 binding was expressed in each sample as femtomoles per milligram of protein. cDNA Probes and Northern Blot Analysis

IP3R-1 was detected with a 2.5-kilobase (kb) probe [12], obtained by digestion with BamHI (4126–6658) of the fulllength rat cDNA encoding type 1 IP3R (pCMVI-9). IP3R2 was detected with a 1.7-kb probe [13] obtained by digestion with BamHI and PstI (1804–3475) of the rat type 2 partial cDNA (pCMVI-102). IP3R-3 was detected with a 1.9-kb probe [14] obtained by digestion with SacI (5247– 7143) of the corresponding rat cDNA (pCBG1IP3R-3). Probes were 32P-labeled with the Megaprime DNA labeling kit. Total RNA was extracted from isolated uterine tissues using the RNeasy Mini Kit and QIAshredder columns, according to the manufacturer’s protocols. Samples of total RNA were denatured in 1 mM glyoxal [21] and fractionated by electrophoresis in 1% agarose gels. The RNA was then transferred to Hybond-N membranes and probed with the 32P-labeled cDNA fragments as described elsewhere [21]. The membranes were washed in high-stringency conditions with a final 15-min wash in 0.13 standard saline citrate/ 0.1% SDS at 608C. Radioactivity in the membrane was quantified with a PhosphoImager scanner using Imagequant 3.2 software (both from Molecular Dynamics) before the membrane was placed against x-ray film. RNA levels were standardized by reprobing the blots with 18S rRNA probes.

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TABLE 1. Pregnancy-related changes in basal, carbachol- and AlF42-induced generation of [3H]inositol phosphates.* [3H]inositol phosphates 3 100 [3H]inositol phospholipids Myometrium Addition Control (basal) Carbachol AlF42

Day 0 4.5 6 0.3 47 6 3.6 101 6 9.3

Endometrium

Day 12 8.5 6 1.0† 79 6 8.1† 154 6 12†

Day 21

Day 0

12 6 1.3† 123 6 11† 245 6 22†

3.1 6 0.3 32 6 2.4 73 6 6.8

Day 12 7.8 6 0.8† 81 6 7.2† 157 6 14†

Day 21 9.7 6 0.8† 128 6 11† 244 6 23†

* [3H]Inositol-labeled myometrial and endometrial strips from Days 0, 12, and 21 of gestation were incubated as described in Materials and Methods without agonist (basal) or stimulated with 100 mM carbachol for 15 min or with 20 mM NaF 1 20 mM AlCl3 (AlF42) for 20 min. Values are means 6 SEM from three experiments, each performed in triplicate. † P , 0.05 significantly different from the corresponding Day 0 values.

Data Analysis

The results are expressed as means 6 SEM. Means were compared using Student’s t-test. P , 0.05 was considered to be significant. RESULTS

Increase in the Generation of [3H]Inositol Phosphates in Rat Myometrium and Endometrium During Gestation

We have previously demonstrated that pregnancy leads to a marked enhancement in phosphoinositide breakdown with an increased production of IP3 in rat myometrium [8]. We found in this study that this is also the case for the endometrium (Table 1). In both myometrial and endometrial preparations, there was a basal level of inositol phosphates generation. Inositol phosphate levels were two and three times higher at Days 12 and 21, respectively, than at Day 0 for both tissues. Similarly, the induction by carbachol (100 mM) of inositol phosphate production increased during gestation (Day 21 . Day 12 . Day 0). At this concentration (100 mM), the muscarinic agonist caused a maximal inositol phosphate response in both tissues (data not shown). The pattern of the increase in inositol phosphate generation (Day 21 . Day 12 . Day 0) was similar if the stimulation was triggered by AlF42, a direct activator of G-proteins. IP3 Binding Sites at Days 0, 12, and 21 in the Myometrium and Endometrium

Specific IP3 binding sites were detected in membranes derived from Day 0, Day 12, and Day 21 myometrium and endometrium (Table 2). During pregnancy, the amount of IP3 bound in both myometrial and endometrial membranes, as determinated in the presence of 1, 5, and 50 nM [3H]IP3, increased significantly (Day 0 , Day 12 , Day 21). There was 50% more IP3 binding at Day 21 than at Day 0 in both

myometrium and endometrium. Homologous competition experiments (n 5 4) performed in myometrial membranes revealed that IC50 values (8.8 6 3.5, 7.9 6 1.2, and 9.9 6 4.6 nM for Day 0, Day 12, and Day 21, respectively) were not significantly different. The resulting slope factors (0.70 6 0.10, 0.69 6 0.1, and 0.68 6 0.06 for Day 0, Day 12, and Day 21, respectively) suggested the presence of a heterogeneous population of IP3Rs in these preparations. Pregnancy-Related Expression of IP3R Proteins in Rat Myometrium and Endometrium We used specific IP3R-1, IP3R-2, or IP3R-3 antibodies to investigate the expression and possible quantitative changes in these receptors in rat myometrium and endometrium during gestation. We performed immunoblots with myometrial and endometrial membranes derived from various stages of gestation (0, 12, and 21 days), resolved by SDS-PAGE, and stained with specific antibodies (Fig. 1). The three types of IP3R, all about 260 kDa in size, were present at day 0 and throughout gestation in both uterine tissues. Quantitative analysis of various blots indicated that IP3R-1 was more strongly expressed (3.8 times higher levels, n 5 3, P , 0.01) in myometrial than in endometrial membranes for Day 0 rats. IP3R-1 levels were higher in both tissues during pregnancy than at Day 0. In the myometrium, IP3R-1 protein levels were 1.5 and 3 times higher at Days 12 and 21, respectively, than at Day 0 (n 5 3, P , 0.01). In the endometrium IP3R-1 levels were 3 and 7 times higher at Days 12 and 21, respectively than at Day 0 (n 5 3, P , 0.01). IP3R-2 was detected in both myometrium and endometrium. The level of expression of this isoform did not change during pregnancy in the myometrium but was upregulated (2 times higher, n 5 3, P , 0.01) at term in the endometrium. IP3R-3 levels were upregulated during pregnancy in both endometrium and myometrium (2 and 4 times higher at Days 12 and 21, respectively, than at Day 0, n 5 3, P , 0.01). The IP3R-3 isoform was preferentially expressed

TABLE 2. Pregnancy-related changes in IP3 binding to myometrial and endometrial membranes.* Specific [3H]IP3 binding (femtomoles/mg protein) Myometrium [IP3] 1 nM 5 nM 50 nM

Endometrium

Day 0

Day 12

Day 21

Day 0

Day 12

Day 21

62 6 4 208 6 14 626 6 47

86 6 5† 263 6 16† 760 6 44†

100 6 6† 319 6 20† 860 6 64†

33 6 3 185 6 10 747 6 40

49 6 6† 216 6 12† 923 6 103†

73 6 9† 249 6 19† 1182 6 125†

* Myometrial and endometrial strips from Days 0, 12, and 21 were incubated in the presence of 1 nM, 5 nM, or 50 nM [3H]IP3 as described in Materials and Methods. The data were obtained from three independent experiments involving different membrane preparations, each performed in triplicate. Results are expressed as means 6 SEM. † P , 0.05 significantly different from the corresponding Day 0 values.


FIG. 1. Immunodetection of IP3Rs in myometrium and endometrium during pregnancy. Membranes (20 mg) from isolated myometrium and endometrium at Days 0, 12, and 21 of gestation were separated by 5% SDS-PAGE. IP3R isoforms were detected with anti-IP3R-1, -IP3R-2, and -IP3R-3 antibodies as indicated. A typical Western blot for each IP3R is shown. Histograms (myometrium, open bars; endometrium, hatched bars) represent the densitometric quantification of each IP3R type from three blots derived from different membrane preparations (arbitrary units 6 SEM relative to Day 0 myometrium 5 100). *P , 0.01 significantly different from Day 0 myometrium values. #P , 0.01 significantly different from Day 0 endometrium values.

in the endometrium. The above quantitative variations could be observed for different amounts of proteins up to 50 mg. Additionally, when the blots (Fig. 1) were reprobed with antibodies specific to calreticulin, a calcium-related protein, the immunologic signal remained identical for all myometrial and endometrial preparations, reflecting that the changes observed were specific for IP3Rs (data not shown). Thus, in both the myometrium and the endometrium, the three types of IP3R are expressed but are differentially regulated during the course of gestation. Pregnancy-Related Expression of IP3R mRNA in Rat Myometrium and Endometrium

We assessed the amount of mRNA present for each type of IP3R by Northern blotting using specific probes obtained from the respective cDNAs cloned from rats (Fig. 2). Northern blots were carried out with 25 mg of total RNA from myometrium or endometrium obtained from Day 0 rats or from pregnant rats at Days 12 and 21 of gestation. We detected a single ;10-kb transcript for each type of

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FIG. 2. Differential expression of IP3R-1, IP3R-2, and IP3R-3 mRNAs in myometrium and endometrium during pregnancy. Northern blot analysis of 25 mg of total RNA extracted from rat myometrium and endometrium at Days 0, 12, and 21 of gestation probed with IP3R-1, IP3R-2, and IP3R-3 cDNA fragments as indicated. The blots were reprobed with 18S rRNA for quantification. A typical Northern blot of the IP3R mRNA types is shown. Histograms (myometrium, open bars; endometrium, hatched bars) represent the radioactive signal quantified from three individual blots derived from different RNA preparations (arbitrary units 6 SEM relative to Day 0 myometrium 5 100). *P , 0.01 significantly different from Day 0 myometrium values. #P , 0.01 significantly different from Day 0 endometrium values.

IP3R. IP3R-1 mRNA levels at Day 0 were 4 times lower in the endometrium than in the myometrium (n 5 3, P , 0.01). The pattern of IP3R-1 mRNA expression during gestation differed in myometrium and endometrium. In the myometrium, IP3R-1 mRNA levels were 3 times higher (n 5 3, P , 0.01) at midgestation than Day 0 levels, and decreased to Day 0 levels at Day 21. However, in the endometrium, IP3R-1 mRNA expression increased progressively throughout gestation, with levels 4 times higher at Day 21 than at Day 0 (n 5 3, P , 0.01). IP3R-2 mRNA expression was barely detectable in Day 0 myometrium,

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with no apparent change at Days 12 and 21. In the endometrium, IP3R-2 mRNA levels were very low at Day 0 but consistently increased and were 2.5 times higher at the end of gestation (n 5 3, P , 0.01). The IP3R-3 isoform was detected in both myometrium and endometrium. At Day 0, the level of mRNA for this isoform in the endometrium was twice that in the myometrium, and the level of this mRNA progressively increased in both tissues during gestation, being 3 times higher at Day 21 (n 5 3, P , 0.01). DISCUSSION We have previously reported that there is a progressive increase during gestation in the generation of inositol phosphates triggered by receptor and G-protein activation in the rat myometrium [8]. We demonstrate herein that the PIP2/ PLC signaling pathway is similarly upregulated in the endometrium throughout pregnancy (Day 21 . Day 12 . Day 0). In both uterine tissues, the higher levels of inositol phosphates at Days 12 and 21 of gestation were associated with an increase in the capacity for binding IP3 to its receptors on the corresponding membrane preparations. Northern blot hybridization experiments demonstrated the presence in the myometrium and endometrium of three different mRNAs encoding for the three IP3R (IP3R-1, -2, and -3) isoforms. Immunoblot experiments with isoform-specific antibodies confirmed the presence of IP3R-1, -2, and -3 proteins in both the myometrium and endometrium. The expression IP3R-1 and IP3R-3 increased during the course of gestation in both tissues, whereas the expression of IP3R-2 increased only in the endometrium at term. There was a strong correlation between the levels of mRNA and protein except for IP3R-1 in the myometrium. IP3R-1 mRNA levels peaked at Day 12 and then declined at Day 21, whereas the corresponding protein level increased progressively (Day 21 . Day 12 . Day 0). Such a lack of correlation between mRNA and protein levels may reflect differences in half-time degradation for mRNA and proteins. IP3R-1 appeared to be expressed preferentially in the myometrium, whereas IP3R-3 levels were higher in the endometrium, and IP3R-2 was poorly expressed in the myometrium as compared to the endometrium. These differences between the two uterine tissues suggest that IP3 receptor isoforms have different cell-specific functions. Several uterine functions are regulated during gestation. These include the cell proliferation that is associated with the hyperplasia of both uterine tissues, active cell death that increases as pregnancy progresses, the secretory function of the epithelial cells of the endometrium that contribute to the output of key regulatory factors necessary for the initiation and maintenace of pregnancy, and finally the coordinated muscle contraction needed to expel the fetus. All these functions are controlled by an increase in the cytosolic Ca21 concentration in the target cells. The variety of the spatiotemporal pattern of the Ca21 signaling has been implicated in this versatility of Ca21-regulated functions. The frequency and the amplitude of the Ca21 oscillations can be decoded to allow specific gene expression [22]. Using cell-permeant caged IP3 ester, it was shown that Ca21 spike frequency can optimize gene expression [23] or efficient activation of calmodulin-dependent protein kinase II [24]. Recent work demonstrated that specific Ca21 signaling patterns can be encoded by differential expression of IP3R subtypes [25]. Smooth muscle contraction is generally thought to be activated by IP3-induced Ca21 release from sarcoplasmic reticulum [1, 4]. Because IP3R-1 levels are highest in the myometrium, it is likely that the type 1 IP3R

may be one of the suppliers of the Ca21 required for activation of uterine contractions. This interpretation is supported by the upregulation of IP3R-1 levels at midpregnancy, culminating near term. The simultaneous increase in IP3 generation and IP3R-1 expression near term may be of physiological relevance because this would make it possible for the myometrial cells to produce the appropriate coordinated contractions needed to expel the fetus. Worth considering are the reports demonstrating high levels of IP3R-1 in human [26] and mouse [27] myometrium. IP3R-3 is expressed and upregulated (Day 21 . Day 12 . Day 0) in both endometrial and myometrial cells. A potential role for IP3R-3 in the Ca21 regulation of myometrial contractility can similarly be retained. Ca21 is also known to play a key role in smooth muscle cell proliferation, so it was not surprising that the amounts of uterine IP3 receptors, mainly types 1 and 3, increased during gestation, which is associated with the proliferation and hyperplasia of both uterine tissues. The IP3R-2 receptor was hardly detectable in the myometrium at Day 0 and throughout gestation; it was expressed in the endometrium where it was up-regulated at term, suggesting a functional role for this type of IP3 receptor in the endometrium but its minimal role in controlling myometrial activites. Pregnancy-related changes in the PLC signaling cascade in rat myometrium, operating at the level of Gq/G11 have been reported [8]. This study demonstrates that the IP3 receptor system is a further example of a key signaling component of the PLC pathway whose expression and binding capacities are affected by the hormonal status of the uterus. The increased amounts of IP3 receptors and their mRNA levels in uterine tissues are probably due to changes in the estrogen/progesterone balance occurring during gestation. A potential regulation of the expression of IP3 receptors by sex steroid hormones can thus be postulated. In line with this proposal is the recent report demonstrating the presence of a steroid element on the promotor of the gene for IP3R-1 [28]. It has also recently been reported that in addition to sex steroids, the mechanical stretch imparted by the growing uterine content may contribute to differential control of gene expression in the pregnant uterus [29]. Our data concerning the differential expression of IP3R in the endometrium and myometrium may allow the encoding of a different spatiotemporal pattern of Ca21 signaling in the uterine cell types, resulting in the ultimate control of specific processes that occur in the uterus during gestation. ACKNOWLEDGMENTS We thank Dr. T.C. Su¨dhof (Howard Hughes Medical Institute, Dallas), who kindly provided us with the rat IP3R-1 and IP3R-2 cDNA and Prof. G.I. Bell (Howard Hughes Medical Institute, University of Chicago) for providing the rat IP3R-3 cDNA. We also thank G. Thomas for excellent technical assistance.

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