| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pregnancy |
Physiologie Animale, INRA,3 Centre de Recherches de Jouy, 78352 Jouy en Josas cedex, France
INSERM U 361,4 75014 Paris, France
CNRS, UMR 8619,5 Université Paris Sud, 91405, Orsay cedex, France
CNRS, UMR 7079,6 Université Pierre et Marie Curie 4, 75252 Paris cedex 05, France
Maternité Port-Royal, Hopital-Cochin,7 Université René Descartes, 75014 Paris, France
INSERM U469,8 34094 Montpellier Cedex 5, France
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
female reproductive tract, gene regulation, parturition, pregnancy, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-IL-1ß and premature human childbirth was proposed [2]. During pregnancy, the uterus is also the site of intense cell proliferation and growth controlled by many growth factors, G protein-coupled receptor agonists, and steroid hormones. The use of myometrial cells in primary culture has demonstrated that the signal transduction pathways controlling proliferation involve many of the molecular partners required in the regulation of contraction during parturition [3–6]. This suggests that subtle tuning of the expression of these partners throughout pregnancy may steer the signaling pathways toward the appropriate physiological response.
However, change in gene expression has been assessed for a limited number of genes during pregnancy. The development of gene array technology offers the opportunity to identify and to monitor simultaneous variations in the gene expression of multiple transcripts. This strategy generates a global view of molecular mechanisms underlying the regulation of physiological or pathological events. In the context of pregnancy, such an approach has been used recently to identify transcripts specifically related to bacterially induced preterm parturition in mice [7], to generate differential expression profiles for inflammatory genes in human gestational membranes [8], and to identify a novel panel of genes potentially involved in human uterine contraction during pregnancy and labor [9].
In the present study, we extended the analysis to a more important number of the genes involved in different myometrial functions that may orchestrate pregnancy and parturition. We used two DNA macroarrays, each containing 1176 different human complementary DNAs (cDNA), to investigate myometrial modifications in gene expression in tissues sampled at three late stages of human pregnancy: preterm, term not in labor, and term in labor.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Myometrial tissue (
1 g) was taken from the upper edge of the hysterotomy during Caesarean section, in the transverse lower uterine segment. The Hospital Ethics Committee approved the study. All patients gave their informed consent in writing. Patients ranged from 28 and 32 (median = 30) yr. The data were analyzed for three groups of four women: at term (range 38.5–40 wk gestation) in women who were in labor and those who were not and preterm (28–31.5 wk gestation) (Table 1). The indication for Caesarean section in the non-labor group was a previous Caesarean delivery. No patients were on medication. The groups not in labor showed no sign of uterine contractions or cervical changes, and the fetal heart rate anomalies observed in two patients were unrelated to uterine activity. Myometrial tissue from the women in labor was collected during emergency Caesarean section. All the patients in this group had spontaneous regular uterine contractions (five times/10 min) with associated cervical dilatation (>4 cm). None of the women received any therapeutic intervention for their labor. Myometrial tissue from the outer uterine wall at an extraplacental site was immediately removed by sharp dissection, leaving behind the decidua, and then carefully minced with fine scissors and rapidly snap frozen in liquid nitrogen. Tissues were stored at -80°C until used.
|
RNA Isolation, Probe Labeling, and Hybridization
Total RNA was extracted by the phenol/chloroform method [10], and subsequent steps leading to hybridization were according to Clontech guidelines. Briefly, RNA were precipitated with isopropanol and purified by RNase-free DNAse I (Clontech Laboratories Inc., Palo Alto, CA). The RNA quantity was determined by absorbance at 260 nm. Each RNA extract was also analyzed by 0.8% agarose gel electrophoresis and ethidium bromide staining to estimate its integrity.
The mRNA of each sample was converted to cDNA using Atlas cDNA synthesis protocol (Atlas Pure Total RNA Labeling System kit, Clontech), in the presence of dNTPs and 50 µCi alpha 33P-labeled dATP (Amersham, Les Ulis, France) (specific activity 3000 Ci/mmol). The radioactive cDNA probes were purified using NucleoSpin Extraction Spin column (Clontech) to remove unincorporated nucleotides and reagents. Probes synthesized using this procedure were 3.8 x 106 (±1.5 x 106) cpm/20 µg retrotranscribed RNA and 5.9 x 106 (±1.7 x 106) cpm/20 µg retrotranscribed RNA for Atlas 1.2 and Atlas 1.2II array, respectively.
Complementary DNA probes were hybridized to Atlas Human 1.2 (#7850-1) and Atlas Human 1.2II (#7852-1) array nylon membranes (Clontech). These membrane arrays contained two sets of known genes (1176 in each), with almost no overlap in their content (list of genes available at http://atlasinfo.clontech.com/genelists/hu1.2.xls and http://atlasinfo.clontech.com/genelists/hu1.2II.xls). Prehybridization and hybridization were performed according to the protocol described by the manufacturer. The hybridization solutions were discarded, and the membranes were then washed four times for 30 min at 68°C with 2x saline-sodium citrate (SSC) containing 1% SDS and then once with 0.1x SSC containing 0.5% SDS. The membranes were finally washed in 2x SSC at room temperature and wrapped in a Saran film. They were exposed to phosphor imaging screens for 7 days, and then the signals were scanned with a phosphor imager (Fuji-film FLA 3000, Paris, France). The raw data obtained after phosphor imaging are accessible on-line at http://data.jouy.inra.fr/unites/bdr/charpigny-germain.
Analysis of Array Data
The hybridization images of each array were imported into Advanced Image Data Analyzer software (Raytest, Strasbourg, France) for densitometry measurement of spot intensity, global, and local background levels. Local background was subtracted from the raw data for all genes. To minimize experimental variations and make it possible to compare the experiments to each other, we normalized our data. The raw value of a specific gene was divided by the sum of spot intensities for the membrane. This was calculated for all replicates to generate what is referred to as the relative intensity of a specific gene.
Data were filtered by one-way analysis of variance (ANOVA) applied to the replicates in the three groups for each gene on the array membrane. Only the genes found to be significant by the ANOVA analysis (P < 0.10 considered to be significant) were kept for the clustering analysis. Before clustering, the normalized medians of values of the genes were log 2 transformed. Average-linkage hierarchical clustering of an uncentered Pearson correlation similarity matrix was applied, using the CLUSTER program designed by Eisen et al. [11], and the results were displayed by using TREEVIEW (software available at http://genome-www4.stanford.edu/MicroArray/SMD/restech.html).
In the final second step of analysis, the data points from relative intensity values were averaged for quadruplicates in each group, and any nonreproducible data (based on ANOVA and ad hoc post Student t-test, P < 0.05 considered to be significant) were discarded. In all the data shown in Tables 2–8, only changes in RNA levels by more than twice the value for the preterm group were observed.
|
Identity of genes plotted on each array membrane was verified by using an independent RT-PCR protocol [12]. Eight genes were selected at random among the very weakly, moderately, and highly expressed transcripts. In all cases, sequencing of PCR cDNA products revealed a sequence and size corresponding to that of the expected gene (data not shown).
Genes are referred to throughout the text according to both their GenBank accession number and their HUGO gene nomenclature (The Human Genome Organization, http://www.gene.ucl.ac.uk/hugo).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We established the expression profile of 2 x 1176 genes in human myometrial tissues at three stages of gestation: preterm, full term not in labor, and full term in labor. Figure 1 describes, according to their level of expression, the distribution of the set of genes revealed on the type 1.2II macroarray membranes. For most of the genes, the level of relative intensity fell in the 0.1–10/10 000 range of the total intensity of the hybridization signal computed for each membrane. The distribution of the expression levels in the preterm group was significantly different from that in the other two groups. In contrast, the distribution profile was almost the same in the two term groups (in labor and not in labor).
|
Hierarchical clustering analysis was applied to the populations of genes that exhibited significant differential expression in the three groups of patients (ANOVA, P < 0.10). The results illustrated in Figure 2 showed that the expression of genes was different and was either up-regulated or down-regulated, depending on the clinical status of the patients. Data obtained with type 1.2 membranes displayed two clusters of genes, which were almost equivalent in size, and showed opposite patterns of change from preterm to term (Fig. 2A). The difference between term in labor and term not in labor was less important. Interestingly, double hierarchical cluster analysis based on both the gene axis and the patient axis allowed the CLUSTER software to reconstruct the experimental groups of preterm patients and term-in-labor patients according to their exact clinical status (Fig. 2B). Similar results were obtained for the hybridizations on the type 1.2II membranes (data not shown).
|
The second step of the analysis consisted in examining the differential expression of every gene on the basis of a classical ANOVA analysis, followed by a post Student t-test at risk levels of P < 0.05. It is worth noticing that only 4% of the genes investigated displayed significantly different expression in the preterm and term groups (with or without labor). The populations of genes statistically selected from 1.2 and 1.2II membranes are shown in Tables 2–8.
Genes Down-Regulated in Patients at Term Not in Labor
A first set of 50 genes, of which the expression was significantly lower in patients at term not in labor than in preterm patients, was selected (Tables 2 and 3). These genes were assigned to four groups according to their physiological function and their known target cells, determined by consulting the OMIM database (Online Mendelian Inheritance in MAN, OMIM [13]). The main group contains 52% of genes, involved in the control of cell proliferation and differentiation in a variety of tissues (Table 2). This group also includes six genes related to cell populations of the immune system. Most of the latter group of genes displayed a decrease by more than 10-fold.
|
Marked down-regulation was observed in a second group of 10 genes that are potentially involved in the contractile function of the smooth muscle and the uterine autonomic nervous system (Table 3). In this group, it should be pointed out that G protein-coupled receptors (GPCRs) and their associated kinases predominate.
We combined the remaining 13 down-regulated transcripts in a group named "miscellaneous" (Table 3), containing genes to which it was very difficult to assign any putative function in the myometrium or because their expression was totally unexpected in this tissue.
Genes Down-Regulated at Term in Labor
Tables 4 and 5 list the 27 genes that were significantly lower in subjects at full term in labor than preterm. The same groups of genes specified previously were down-regulated, with a predominance of those mediating cell proliferation, oncogenesis, and immunomodulation. It is interesting to note that the data in Tables 4 and 5 show a concomitant down-regulation of the integrins, different PLC isoforms, transcriptional factors, and oncogenes, which are required for harmonious smooth muscle cell growth. We also observed a marked decrease of the GABA A receptor, which is involved in uterine relaxation.
|
|
Genes Up-Regulated at Term, Without and With Labor
Tables 6 and 7 show sets of 18 and 10 genes that were significantly up-regulated in the myometrium of the groups at term not in labor and at term in labor, as compared to the preterm group. Our data revealed the presence of an additional family of genes (Tables 6 and 7) associated with inflammatory processes. It is worth noting that the total number of up-regulated genes was smaller than their down-regulated counterparts. Moreover, the magnitude of the differential expression of these up-regulated genes was globally less pronounced than that of their down-regulated homologues.
|
|
Some genes that had not previously been reported in the pregnant human myometrium are listed as associated to the control of the smooth muscle cells and/or their innervations (e.g., GLRB and DLG2, Table 6). The global picture emerging from Tables 6 and 7 illustrates the importance of the up-regulation of genes that contribute to immunoinflammatory processes at the end of gestation.
Table 8 lists the few genes that are differentially expressed in the term-not-in-labor and term-in-labor groups. Only two of them in the list were already detected in the comparison of preterm and term-in-labor groups. The MEL oncogene is down-regulated and the tumor protein TP53 is up-regulated with the advancing process of parturition. For these two genes, the magnitude of differential expression was similar to that obtained between preterm and term in labor (Tables 4 and 7, respectively).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Contraction-Associated Genes
Pharmacological and biochemical studies have demonstrated that the expression of some GPCRs in association with many partners of signal pathways controlling relaxation was down-regulated in the myometrium at term, when muscle hyperactivity is required for parturition [19]. In this study we confirmed some of these results at the level of gene expression. We observed a noticeable reduction in the expression of the CGRP (calcitonin gene-related peptide)-receptor transcript at term. According to Anouar et al. [20] and to Dong et al. [21], the loss of CGRP response at term could contribute to initiation of rat and human labor. An up-regulation of a calmodulin-dependent phosphodiesterase (PDE-1) was observed at term as it has been clearly established in human term myometrium [22]. These data indicate that hydrolysis of cAMP/cGMP via PDE families may damper the actions of uterorelaxant agents at the end of pregnancy. Our data demonstrated also the down-regulation of some GPCR genes that have not yet been linked to human parturition. We reported a marked decrease in the expression of the GABA A receptor at term. GABA A receptors mediate myometrial relaxation and are expressed in the nonpregnant human myometrium [23]. A decrease of GABA A receptors has been reported at term in the rat myometrium, where they seem to play an important role in maintaining a quiescent state during pregnancy [24]. Thyrotropin-releasing hormone (TRH) receptors have been detected in the uterus [25], but their function in this organ remains unknown so far. The dramatic 40-fold decrease observed at term for TRH receptors suggests that they have a physiological function during pregnancy.
Our analysis also revealed that down-regulation of GPCR was accompanied by a significant decrease of GRK1 and 6 expression (GPCR kinase). Gestational-related changes operating at the level of GRK have been reported in the rat [26] and human myometrium [27]. Such a decrease could help to prevent desensitization of GPCR leading to uterine contraction.
Inflammation-Associated Genes
The role of inflammatory-immune mediators in parturition has been proposed. Although IL1 production has been reported to be associated with premature parturition [28], in the present study, we did not detect any significant up-regulation of the IL1 transcript. Moreover, the decrease of IRAK1 (an IL1 receptor-associated kinase [29]) transcripts we observed at term casts doubt on any important role for IL1 during normal parturition. However, in our data one third of genes up-regulated at term are linked to inflammatory-immune pathways. Our findings are convergent with the recent data obtained in human myometrium in labor using suppression subtractive hybridization approach [15]. All together, these results strongly suggest a role of inflammatory processes in parturition. Recent evidence also suggests that cell adhesion molecules have diverse biochemical and physiologic functions, including the regulation of inflammatory processes, cell differentiation, gene transcription, angiogenesis, apoptosis, and cell signaling [30–32]. Cell adhesion molecule expression (ICAM1) increases in the human myometrium during late pregnancy and parturition. These changes can be attributed to leukocytes infiltrating these tissues [33]. We noted that the increased expression of ICAM at term was not restricted to ICAM1 but also involved ICAM2.
Proliferation-Associated Genes
Most of the genes that displayed a marked down-regulation in the term groups were known, among other possible functions, to drive cell proliferation and differentiation. Some of the proliferation-associated genes that were down-regulated in our experiments had already been reported to be related to uterine cell tumor development. This is true for the high-mobility group protein HMGIC, which is involved in the development of uterine leiomyomas [34], and osteocalcin, which has been found in osteosarcomas developed in the uterine corpus [35]. The list of down-regulated genes also includes the LIFR (leukemia inhibitory factor receptor) and gp130, a component of the LIF receptor (also known as interleukin-6 signal transducer, IL6ST). LIF acts via a receptor complex, in which signaling is triggered by heterodimerization with the LIF-Rb/gp190 protein (IL11, LIF) of gp130 [36]. Thus, the decrease in LIFR seen in the patients at term not in labor and that of the IL6ST in the patients at term in labor might contribute to the disappearance of the effects of the LIF in the myometrium at the end of human gestation.
In the human myometrium, late pregnancy is a time where substantial remodeling of the extracellular matrix occurs, including increased collagenolysis, the formation of elastic fibers within the circular muscle of the myometrium, as well as marked increases in the expression of fibronectin and in the thickness of the continuous basement membranes [37]. So far, no information is available concerning the differential expression of integrins in the human uterus at term. However, the present study demonstrates that the expression of integrin alpha 5 and integrin beta 3 was markedly reduced at term during labor and that of integrin beta 6 was reduced at term. Integrin alpha 5 and beta 3 can form a complex that interacts with platelet-derived growth factor receptor, leading to potentiation of its proliferative effect [38]. We also demonstrated the concomitant down-regulation of the integrin receptor tyrosine kinase, growth factor receptor (epidermal growth factor), Ras family member, PKC zeta, phosphoinositide 3-kinase, phospholipases (gamma 1, gamma 2, and epsilon), and several transcription factors. Some of the genes associated with the inhibition of cell proliferation and differentiation were increased at the end of gestation (e.g., tumor protein p53, maspin). All these signaling molecules are able to form a regulating network that may be involved in controlling the exact time course of uterine growth and cell survival.
In conclusion, the idea that emerges from our functional genomics approach is that the mechanism of parturition is preceded by a massive down-regulation of a large panel of developmental, cell adhesion molecule, and proliferation-related genes. This process may be necessary to allow the organ to develop its full contractile capacity. We suggest that labor can best be regarded as a coordinated regulation of uterine proliferation and contraction, involving a complex network of genes. The hypothesis of local actions of signaling factors originating from intrauterine and/or adjacent tissues in the control of uterine functions now needs to be explored. Global changes in gene expression in term and pregnant women including studies on decidual-myometrial crosstalk should be investigated. Such a wealth of data may make it possible to construct comprehensive pathways of the molecular and biochemical changes that lead to preterm labor and would allow the development of novel strategies to prevent premature delivery.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 33 1 34 65 23 64; germain{at}jouy.inra.fr
Received: 21 November 2002.
First decision: 16 December 2002.
Accepted: 24 January 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
vß3 integrins. EMBO J 2002 21:1957-1966.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  M. Serrano-Sanchez, Z. Tanfin, and D. Leiber Signaling Pathways Involved in Sphingosine Kinase Activation and Sphingosine-1-Phosphate Release in Rat Myometrium in Late Pregnancy: Role in the Induction of Cyclooxygenase 2 Endocrinology, September 1, 2008; 149(9): 4669 - 4679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhong, D. A. Murtazina, J. Phillips, C.-Y. Ku, and B. M. Sanborn Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells Biol Reprod, June 1, 2008; 78(6): 1007 - 1017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O'Brien, J. J. Morrison, and T. J. Smith Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 Gene Expression in Human Myometrium at Labor Reproductive Sciences, April 1, 2008; 15(4): 382 - 393. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Tattersall, N. Engineer, S. Khanjani, S. R Sooranna, V. H Roberts, P. L Grigsby, Z. Liang, L. Myatt, and M. R Johnson Pro-labour myometrial gene expression: are preterm labour and term labour the same? Reproduction, April 1, 2008; 135(4): 569 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Lirussi, Z. Rakotoniaina, S. Madani, F. Goirand, M. Breuiller-Fouche, M.-J. Leroy, P. Sagot, J. J. Morrison, M. Dumas, and M. Bardou ADRB3 Adrenergic Receptor Is a Key Regulator of Human Myometrial Apoptosis and Inflammation During Chorioamnionitis Biol Reprod, March 1, 2008; 78(3): 497 - 505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Chevillard, A. Derjuga, D. Devost, H. H Zingg, and V. Blank Identification of interleukin-1{beta} regulated genes in uterine smooth muscle cells Reproduction, December 1, 2007; 134(6): 811 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-J. Leroy, E. Dallot, I. Czerkiewicz, T. Schmitz, and M. Breuiller-Fouche Inflammation of Choriodecidua Induces Tumor Necrosis Factor Alpha-Mediated Apoptosis of Human Myometrial Cells Biol Reprod, May 1, 2007; 76(5): 769 - 776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Jeng, V. R. Suarez, M. G. Izban, H.-Q. Wang, and M. S. Soloff Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1110 - E1121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Garcia-Verdugo, D. Leiber, P. Robin, E. Billon-Denis, R. Chaby, and Z. Tanfin Direct Interaction of Surfactant Protein A with Myometrial Binding Sites: Signaling and Modulation by Bacterial Lipopolysaccharide Biol Reprod, April 1, 2007; 76(4): 681 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Buhimschi, I. A. Buhimschi, G. Zhao, E. Funai, G. Peltecu, G. R. Saade, and C. P. Weiner Biomechanical Properties of the Lower Uterine Segment Above and Below the Reflection of the Urinary Bladder Flap Obstet. Gynecol., March 1, 2007; 109(3): 691 - 700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Soloff, M. G. Izban, D. L. Cook Jr, Y.-J. Jeng, and R. C. Mifflin Interleukin-1-induced NF-{kappa}B recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells Mol. Hum. Reprod., October 1, 2006; 12(10): 619 - 624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Breuiller-Fouche and G. Germain Gene and protein expression in the myometrium in pregnancy and labor. Reproduction, May 1, 2006; 131(5): 837 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M Lindstrom and P. R Bennett The role of nuclear factor kappa B in human labour Reproduction, November 1, 2005; 130(5): 569 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cammas, P. Reinaud, O. Dubois, N. Bordas, G. Germain, and G. Charpigny Identification of Differentially Regulated Genes During Elongation and Early Implantation in the Ovine Trophoblast Using Complementary DNA Array Screening Biol Reprod, April 1, 2005; 72(4): 960 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Havelock, P. Keller, N. Muleba, B. A. Mayhew, B. M. Casey, W. E. Rainey, and R. A. Word Human Myometrial Gene Expression Before and During Parturition Biol Reprod, March 1, 2005; 72(3): 707 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bailey and G N. Europe-Finner Identification of human myometrial target genes of the c-Jun NH2-terminal kinase (JNK) pathway: the role of activating transcription factor 2 (ATF2) and a novel spliced isoform ATF2-small J. Mol. Endocrinol., February 1, 2005; 34(1): 19 - 35. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
