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research-article |
University Health Network,7 Toronto General Research Institute, Toronto, Ontario, Canada M5G 2C4
Samuel Lunenfeld Research Institute,4 Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
Departments of Physiology5 and Obstetrics and Gynecology,6 University of Toronto, Toronto, Ontario, Canada M5S 1A1
ABSTRACT
While the insulin-like growth factor (IGF) system is known to regulate uterine function during the estrous cycle, there are limited data on its role in myometrial growth and development during pregnancy. To address this issue, we defined the expression of the Igf hormones (1 and 2), their binding proteins (Igfbp 1–6), and Igf1r receptor genes in pregnant, laboring, and postpartum rat myometrium by real-time PCR. IGF family genes were differentially expressed throughout gestation. Igf1 and Igfbp1 mRNA levels were upregulated during proliferative phase (Days 6–12) of rat gestation. Igfbp3 gene expression also was elevated in proliferating smooth muscle cells (SMCs) and was highest at the time of transition between proliferative and synthetic phases (Days 12–15). Igfbp6 gene expression profile paralleled plasma progesterone (P4) concentrations, peaking during the synthetic phase (Days 17–19) and decreasing thereafter. Administration of P4 at late pregnancy (starting from Day 20) to maintain elevated plasma P4 concentrations blocked the onset of labor and prevented the fall in Igfbp6 mRNA levels. In contrast, the treatment of pregnant rats with the P4 receptor antagonist RU486 on Day 19 induced preterm labor and the premature decrease of Igfbp6 gene expression. Igfbp2 gene expression was transiently upregulated during the contractile phase of gestation (Days 21–23) solely in the gravid horn of unilaterally pregnant rats, but it was not affected in P4- or RU486-treated animals, supporting a role for mechanical stretch imposed by the growing fetuses. Igfbp5 gene was induced during postpartum involution. Our results suggest the importance of the IGF system in phenotypic and functional changes of myometrial SMCs throughout gestation in preparation for labor.
insulin-like growth factor receptor, parturition, pregnancy, progesterone, uterus
Insulin-like growth factor (IGF) ligands (IGF1 and IGF2) play a pivotal role in tissue homeostasis, regulating cell proliferation, differentiation, and survival during embryonic development and in normal adult physiology [1, 2]. IGFs signal through two types of cell membrane receptors. The actions and bioavailability of the IGFs are modulated extracellularly by a family of six secreted high-affinity binding proteins (IGFBPs) [3]. IGFBPs are found both in the systemic circulation and in the local tissue environment (reviewed in Le Roith [2]). In the circulation, IGFBPs act as carrier proteins, providing a large, readily available reserve of IGFs, whereas at the local level they modulate IGF activity. Approximately 80% of IGFs in the plasma are bound to a 150-kDa complex that includes IGFBP3 and an acid labile subunit, which protects the IGFs from protease degradation and prolongs their circulating half-life [4]. The remainder is largely bound to other binding proteins, and about 1% of IGFs are circulating freely. IGF1 and IGF2 have an exquisitely high affinity for IGFBPs that exceeds their affinity for IGF receptors. Consequently, IGFBPs are viewed as proteins involved in the sequestration of ligand from the IGFIR, thereby inhibiting mitogenesis, differentiation, cell survival, and other IGF-stimulated events [5]. IGFBP proteolysis can reverse this inhibition or generate IGFBP fragments with novel bioactivity. Alternatively, IGFBP interaction with cell or matrix components may concentrate IGFs near their receptor, thus enhancing IGF activity. Recent studies (reviewed in Firth and Baxter [3] and Rajaram et al. [4]) have shown that in addition to IGF-dependent functions, IGFBPs also may act in an IGF-independent manner.
A growing body of evidence suggests that the IGF system plays an essential role during the reproductive cycle. IGFs and IGFBPs demonstrate specific expression profiles in the myometrium during the menstrual cycle of humans [6] and in the estrous cycle of animal species such as rat [7], pig [8], cow [9], and sheep [10]. IGF1 and IGF2 contribute to the development of placenta [11], preimplantation blastocysts [12], trophoblast invasion [13], and decidualization [14]. Numerous studies also have implicated IGF1 as a mediator of uterine growth; for example, mice carrying a null mutation in the Igf gene have very small uteri and are infertile [15]. Overexpression of IGFBP1 in transgenic mice reduced IGF1 to basal levels, caused general growth fetal retardation, and specifically blocked estrogen-induced uterine growth [16, 17], whereas smooth muscle-targeted overexpression of IGF1 caused hyperplasia of the myometrium and increased uterine horn length in nonpregnant (NP) mice [18].
The pregnant rodent uterus is a remarkably plastic system that undergoes a program of rapid growth and differentiation in order to support the growth and development of embryos and then rapidly resets itself after parturition [19]. We have reported previously that during pregnancy the myometrium undergoes gradual changes in phenotype characterized by an early proliferative phase, an intermediate phase of cellular hypertrophy and matrix elaboration, and the final contractile/labor phase [20]. We have proposed that phenotypic modulation of uterine myocytes is the result of integrated endocrine signals and mechanical stimulation on the uterus by the growing fetus. We have demonstrated already that both of these signals are important in regulating the onset of labor [21]; however, their role in myometrial smooth muscle differentiation is unknown.
Based on the diverse roles of the IGF signaling pathway in reproduction, we hypothesize that the IGF system is involved directly in the phenotypic modulation of pregnant myometrium during the different phases of differentiation from early to late pregnancy and to the onset of labor. Furthermore, we suggest that the mechanical and hormonal stimuli may play a role in regulating IGF system gene expression in uterine tissue during pregnancy. In the present study, we defined the mRNA expression profile of the various components of the IGF axis in the rat myometrium during normal pregnancy, spontaneous term labor, progesterone (P4)-delayed labor, and RU486-induced preterm labor using real-time PCR. We also investigated the effect of mechanical stretch on the expression of some IGF family constituents using empty and gravid horns of unilaterally pregnant rats. IGFBP protein levels were detected using an immunoblotting technique.
Wistar rats (Charles River Co., St. Constance, QC, Canada) were housed individually under standard environmental conditions (12L:12D cycle) and fed Purina Rat Chow (Ralston Purina, St. Louis, MO) and water ad libitum. Female virgin rats were mated with male rats. Day 1 of gestation was designated as the day a vaginal plug was observed. The average time of delivery under these conditions was during the morning of Day 23. Our criteria for labor were based on delivery of at least one pup. The Samuel Lunenfeld Research Institute Animal Care Committee approved all animal experiments.
Normal pregnancy and term labor. Animals were killed by carbon dioxide inhalation, and myometrial samples were collected on Gestational Days 0 (NP), 6, 8, 10, 12, 14, 15, 17, 19, 21, 22, and 23 (labor), or 1 and 4 days postpartum. Tissue was collected at noon on all days, with the exception of the labor sample (d23L), which was collected once the animals had delivered at least one pup (n = 4).
Progesterone-delayed labor. To determine whether high plasma levels of P4 might modulate the expression of IGF family genes, pregnant rats were randomized to receive daily subcutaneous injections of either P4 (medroxyprogesterone acetate, 16 mg/kg in 0.4 ml sterile saline; Pharmacia Canada Inc., Mississauga, ON, Canada) or vehicle starting on Day 20 of gestation. Animals (n = 4 at each time point for each treatment) were killed on Days 21, 22, or 23 during labor in the vehicle-treated group or on Days 21, 22, 23, or 24 in the P4-treated group.
RU486-induced preterm labor. On Day 19 of gestation, two groups of rats were treated with either RU486 (10 mg/kg, s.c., at 1000 h, in 0.5 ml corn oil containing 10% EtOH, mifepristone; 17ß-hydroxy-11ß-[4-dimethylaminophenyl]-17-[1-propynyl]-estra-4,10-dien-3-one; Biomol International, Plymouth Meeting, PA) or vehicle. Myometrial samples were collected from both groups of animals on Day 20, when the RU486-treated animals had delivered at least one pup (n = 4).
Unilaterally pregnant rats. Under general anesthesia virgin female rats underwent tubal ligation through a flank incision to ensure that they subsequently became pregnant in only one horn [21]. Animals were allowed to recover from surgery for at least 7 days before mating. Pregnant myometrial samples from empty and gravid horns were collected on Days 6, 12, 14, 15, 17, 19, 21, 22, and 23, or Postpartum Day 1 (n = 4).
Animals were killed by carbon dioxide inhalation. For RNA and protein extraction the uterine horns were placed into ice-cold PBS, bisected longitudinally, and dissected away from both pups and placentas. The endometrium was carefully removed from the myometrial tissue by mechanical scraping on ice which, as we have previously shown, removes the entire luminal epithelium and the majority of the uterine stroma [22]. The myometrial tissue was flash frozen in liquid nitrogen and stored at –70°C. For each day of gestation, tissue was collected from four different animals.
Total RNA was extracted from the frozen tissues using TRIZOL (Gibco BRL, Burlington, ON, Canada) according to manufacturer's instructions. RNA samples were column purified using RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada), and treated with 2.5 µl DNase I (2.73 Kunitz units/µl; Qiagen) to remove genomic DNA contamination. Reverse transcription (RT) and real-time PCR were performed to detect the mRNA expression of IGFs and IGF-related genes in rat myometrium (Table 1). Total RNA (2 µg) was primed with random hexamers to synthesize single-strand cDNAs in a total reaction volume of 100 µl using the TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA) as described previously [23]. The thermal cycling parameters of RT were modified according to the Applied Biosystems manual. Hexamer incubation at 25°C for 10 min and RT at 42°C for 30 min were followed by reverse transcriptase inactivation at 95°C for 5 min. Complementary DNA (20 µg) from the previous step was subjected to real-time PCR using specific sets of primers (Table 1) in a total reaction volume of 25 µl (Applied Biosystems). RT-PCR was performed in an optical 96-well plate with an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) using SYBR Green detection chemistry. The run protocol was as follows: initial denaturation stage at 95°C for 10 min, and 40 cycles of amplification at 95°C for 15 sec and 60°C for 1 min. After PCR, a dissociation curve was constructed by increasing temperature from 65°C to 95°C for detection of PCR product specificity. In addition, a no-template control (H2O control) was analyzed for possible contamination in the master mix. A cycle threshold (Ct) value was recorded for each sample. PCR reactions were set up in triplicates, and the mean of the three Cts was calculated. A comparative Ct method (
Ct method) was applied to the raw Ct values to find a relative gene expression across normal gestation. To obtain experimental results, the expression of individual genes at every gestational day (1) was normalized to ribosomal 18S mRNA, and (2) had a fold change calculated relative to the expression of the same gene in a corresponding NP sample using an arithmetic formula (see ABI User Bulletin no. 2). For unilaterally pregnant animals, gene expression was shown as fold change relative to Day 6 gravid horn mRNA level, and for P4- and RU486-treated animals, gene expression was shown as fold change relative to the vehicle sample. Validation experiments were performed to ensure the PCR efficiencies between the target genes and 18S were approximately equal.
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Total protein was extracted from the frozen tissues using radioimmunoprecipitation assay lysis buffer as described previously [23]. Protein samples (40–50 µg) were resolved by electrophoresis on a 12%–15% SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) in 25 mM Tris-HCl, 250 mM glycine, and 0.1 % (wt/vol) SDS, pH 8.3, for 18 h at 30 V at 4°C. Protein expression levels of IGFBPs 1, 2, 3, and 6 were measured by Western blot analysis using anti-IGFBP1 (1:300; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-IGFBP2, anti-IGFBP3, and anti-IGFBP6 (1:3000; Gropep, Adelaide, Australia) rabbit primary antibody. To avoid cross-reactivity between antibodies, detection for different IGFBPs was performed at all times using different PVDF membranes. PVDF membranes were stripped and reprobed with anti-smooth muscle calponin (also known as CNN1; 1:3000; clone hCP; Sigma-Aldrich, Oakville, ON, Canada) mouse primary antibodies to control the loading variations. Probed membranes were exposed to x-ray film (Kodak XAR; Eastman Kodak, Rochester, NY) and analyzed by densitometry. Calponin is constitutively expressed in NP and pregnant rat tissue under the same protein extraction conditions [24].
Gestational profiles were subjected to a one-way ANOVA followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine differences between groups. P4 (Days 21, 22, and 23) and tubal ligation data were analyzed by two-way ANOVA followed by pairwise multiple comparison procedures as described above. The Day 24 P4-treated group was compared to the Day 23 vehicle group using a t-test. RU486 results were compared to vehicle using a one-way ANOVA. Where required, the data were transformed by the appropriate method to obtain a normal distribution. Statistical analysis was carried out using SigmaStat version 2.01 (Jandel Corp., San Rafael, CA), with the level of significance for comparison set at P < 0.05.
Relative abundance of the Igf1 and Igf2 gene transcripts in the myometrium is shown in Figure 1. Igf1 mRNA was detected in NP myometrial samples. Igf1 gene expression was induced predominantly during early pregnancy, the proliferative phase of myometrial growth. The transcript levels of Igf1 increased starting on Day 6 (2.43 ± 0.22-fold increase vs. NP, P < 0.05), peaked at Day 12 (6.37 ± 0.66-fold increase vs. NP, P < 0.05), and returned to the NP level during late gestation and labor (Fig. 1A). Igf1 transcript levels were induced again in postpartum myometria (4.35 ± 0.38-fold increase at Postpartum Day 4 vs. NP, P < 0.05). The Igf2 gene expression was relatively unchanged throughout pregnancy (P > 0.05; Fig. 1B). IGF1 exerts its actions through the cell membrane receptor IGF1R. Igf1r mRNA was detected in pregnant rat myometrium and did not show a significant change across gestation, but it increased at Postpartum Day 4 (2.86 ± 0.30-fold increase vs. NP, P < 0.05; Fig. 1C).
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The mRNA profile of Igfbps in the pregnant rat myometrium exhibited dramatic changes associated with the phase of myometrial differentiation (Fig. 2). Igfbp1 mRNA abundance increased remarkably after implantation (366.2 ± 17.56-fold increase on Day 6 vs. NP, P < 0.05), and this high level of gene expression was maintained throughout the whole proliferative phase of gestation (174.2 ± 10.42-fold increase on Day 14 vs. NP, P < 0.05). Subsequently, Igfbp1 transcript levels gradually decreased and were almost undetectable during late gestation, through labor and the postpartum period. Expression of the major IGF carrier protein, IGFBP3, followed a profile similar to that of the Igf1 gene. Igfbp3 mRNA levels were upregulated on Day 6 (7.85 ± 1.05-fold increase vs. NP, P < 0.05) and were maintained at high levels through Day 12 (13.07 ± 1.78-fold increase vs. NP, P < 0.05), after which there was a gradual decrease, reaching NP levels on Day 19 and the remainder of pregnancy. In contrast, Igfbp6 mRNA levels increased mainly during the second half of gestation (8.03 ± 1.06-fold increase on Day 12 vs. NP, P < 0.05), reaching a peak at Day 19 (26.13 ± 3.45-fold increase vs. NP, P < 0.05), and then gradually decreasing to NP levels on Postpartum Day 1 (Fig. 2). Igfbp2 mRNA levels also remained low throughout early gestation, increased gradually after midgestation (10.45 ± 2.27-fold increase on Day 14 vs. NP, P < 0.05), and peaked on Day 22 (99.91 ± 19.41-fold increase vs. NP, P < 0.05) before exhibiting a precipitous decrease during postpartum period (0.91 ± 0.11-fold increase at Postpartum Day 4 vs. NP, P < 0.05; Fig. 2). Igfbp4 transcript levels remained statistically unchanged throughout the gestational and postpartum periods. Igfbp5 exhibited a distinct profile in that expression was relatively low and unchanged throughout pregnancy but was dramatically increased during the postpartum period (7.62 ± 1.14-fold increase at Postpartum Day 1 vs. NP, P < 0.05). Thus, four of the six Igfbps studied showed significant changes in expression profile associated with distinct phases of myometrial differentiation.Western immunoblot analysis was performed using specific anti-IGFBP1, anti-IGFBP2, anti-IGFBP3, and anti-IGFBP6 antibodies to determine whether protein expression of these IGFBPs paralleled the changes in mRNA. All proteins were detected as single bands of a similar size (Fig. 3); therefore, analysis of IGFBPs was performed using different PVDF membranes. With the exception of IGFBP3, the pattern of IGFBP protein expression (normalized to that of CNN1) followed that of the mRNA profiles (Figs. 2 and 3). Thus, IGFBP1 protein levels were significantly upregulated at early gestation (4.2-fold increase on Days 6–8 relative to NP, P < 0.05), whereas IGFBP6 and IFGBP2 were elevated during the second part of gestation (Fig. 3, A, B, and D). IGFBP6 protein expression was elevated from Day 17 of pregnancy (5.2-fold increase relative to NP, P < 0.05) and maintained a high expression level throughout late gestation (Fig. 3D), whereas IGFBP2 was upregulated specifically at late gestation and during labor (Days 21–23), followed by a sharp decline during postpartum period (Fig. 3B).
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Modulation of Igfbp Gene Expression by Progesterone
Only two Igfbps (Igfbp2 and Igfbp6) exhibited an expression profile that might suggest modulation by progesterone. To investigate this possibility we experimentally modulated progesterone exposure either by administration of exogenous P4 to prevent the normal fall in this hormone at term or by administration of RU486 to induce preterm withdrawal on Day 19. Administration of exogenous P4 delayed the onset of labor by more than 24 h and prevented the fall of Igfbp6 mRNA levels on Day 23 compared with vehicle-treated animals delivering at term (P < 0.05; Fig. 4). Moreover, the mRNA levels of Igfbp6 in P4-treated rat myometrium remained high on Day 24 compared with vehicle-treated animals on Day 23 (laboring sample; P < 0.05). Treatment of pregnant rats with the P4 receptor antagonist, RU486, on Day 19 caused the onset of preterm labor within 24 h and a significant 3.6-fold decrease in expression of the Igfbp6 gene (P < 0.05; Fig. 5). The levels of Igfbp2 gene were unaffected by modulation of P4 exposure.
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Gravidity Modulates the Expression of Igfbps in Unilaterally Pregnant Rat Myometrium
We compared Igfbp2 and Igfbp6 gene expression in myometrial tissue from empty and gravid horns of unilaterally pregnant rats to assess the influence of mechanical stretch on these genes. The mRNA profiles for both genes in the gravid horn were similar to those of normal pregnant animals (Figs. 2 and 6). Although transcript levels of Igfbp6 were lower in empty than in gravid horns, its expression increased progressively in both horns, reaching a peak on Days 17–19 of pregnancy (P < 0.05) and then decreasing gradually to low levels by Postpartum Day 1 (Fig. 6). In contrast, while expression of the Igfbp2 gene remained at very low levels in the empty horn throughout gestation, expression in the gravid horn was increased dramatically during late pregnancy and at term (Day 22, 95-fold increase in gravid vs. empty horn, P < 0.05). Interestingly, on Postpartum Day 1 there was an instantaneous drop in gravid horn Igfbp2 mRNA to a level comparable to that detected in the empty horn (Fig. 6).
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Comparative Characteristics of Igfbp Gene Expression in NP, Pregnant, and Postpartum Rat Myometrium.
A comparative Ct method (Ct method) was used in our study to find the relative gene expression for each Igfbp across gestation (Fig. 2); this approach allowed us to compare relative gestational changes of all six IGFBP genes expressed in rat myometrium. Igfbp1 gene in NP rat myometrium was found to be the lowest among all IGFBPs; therefore, it has been chosen as a calibrator for all Igfbp relative gene expression study. Analysis of these data revealed two groups of Igfbps characterized by very different mRNA abundances (Fig. 7). The expressions of Igfbp2, Igfbp5, and Igfbp6 genes (Fig. 7A) were considerably higher than Igfbp1, Igfbp3, and Igfbp4 (Fig. 7B) throughout gestation. Igfbps expressed at low levels were upregulated at early gestation (except Igfbp4), whereas highly expressed genes were elevated at late gestation. Of all the Igfbps we studied, the Igfbp6 gene demonstrated the most dramatic gestational changes and the highest expression levels in pregnant rat myometrium. Likewise, the Igfbp2 gene changed significantly during the second half of gestation, switching from levels typical of lowly expressed to those of highly expressed genes (such as Igfbp6 and Igfbp5). Igfbp5 mRNA also was highly abundant throughout gestation and during postpartum involution compared with other Igfbp genes. This might suggest that during early gestation, small absolute changes in gene expression could trigger substantial changes in uterine growth and function, whereas at late gestation greater changes are necessary to achieve significant effects.
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The IGF system is involved in many important biological processes, including muscle growth and differentiation, both in vitro [25] and in vivo [26, 27]. Our data demonstrate that expression of the IGF system in the pregnant myometrium is finely controlled in a manner that suggests it may modulate the pattern of differentiation of this tissue. In the uterus, IGFs are essential for normal uterine development and play a central role during implantation and the establishment of pregnancy in all species studied [6–12]. We have noticed that IGF2 was expressed evenly in the myometrium throughout pregnancy. During human pregnancy, there was little change in IGF2 concentration in maternal serum compared with preconception values, whereas IGF1 concentration increased from 30 to 50 nmol/l [28]. Uterine Igf1 gene expression is regulated by the steroid hormones estrogen and progesterone (P4), and IGFs are thought to mediate many of the actions of these steroid hormones on uterine growth and development [16]. The level of uterine Igf1 mRNA was significantly reduced following ovariectomy, but it increased to levels higher than those in intact animals following administration of estrogen alone [29] or estrogen and progesterone [30]. IGF1 has been shown to stimulate DNA synthesis in primary myometrial SMCs of both human and rat in vitro [31, 32] and may be responsible for pathological myometrial growth in vivo [33]. Our finding that Igf1 gene expression is upregulated in the rat myometrium during the first half of gestation, when myocyte proliferation is high, is consistent with these observations (Figs. 1 and 8). A similar upregulation of Igf1 gene in pregnant gilt myometrium in early gestation suggests that this relationship may be common across species [8]. Others have shown that paracrine overproduction of IGF1 in NP transgenic mice resulted in hyperplasia of the muscular layers of the uterus, leading to longitudinal growth [18]. Therefore, we suggest that elevated IGF1 expression, triggered by pregnancy-related sex steroid hormones, is responsible for the early proliferative phase of myometrial differentiation.
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Current knowledge of the gestational changes in the expression of Igfbp genes in the myometrium is limited [19, 34–36]. These genes have been localized in uterine tissues by in situ hybridization during the estrous cycle and early pregnancy in rodents [7, 37]. Our study is the first comprehensive examination of the myometrial gene expression pattern of six Igfbps throughout pregnancy, labor, and postpartum involution, and it demonstrates their temporal association with the phenotypic modulation of the pregnant myometrium. Moreover, we provide evidence that Igfbp gene expression is regulated by the steroid hormone P4 and by the mechanical stretch of the uterus imposed by the developing fetus(es).
IGFBP1 is a major secretory product of the human and primate decidualized endometrium [14, 36], whereas in rodents it is associated with predecidual transformation of the uterus and is therefore regarded as a more generalized pregnancy-associated peptide than as a decidual factor [19]. While we found elevated levels of Igfbp1 in myometrium in early pregnancy, the precise role that it plays is unclear. Studies also have shown that IGFBP1 is capable of modulating the action of IGF1 in both a positive and negative manner (reviewed in Lee et al. [38, 39]), which may be dependent upon the phosphorylation status of this binding protein. IGF1 has been shown to regulate IGFBP1 production by first-trimester decidua in a biphasic manner; low concentrations stimulate IGFBP1 secretion, whereas high concentrations are inhibitory [40]. It is possible that the increased levels of IGFBP1 in early gestation stimulate IGF action (providing a biochemical basis for myometrial proliferation), whereas the increase in IGF1 expression by midgestation inhibit IGFBP1 expression (thus preventing excessive hyperplasia). IGFBP3 was another binding protein with a gene expression that was induced during the proliferative phase of myometrial differentiation (Fig. 2). IGFBP3 is the major carrier protein of IGFs and can significantly extend the half-life of hormones in serum [19]. The elevated expression of Igfbp3 during early pregnancy suggests that it may contribute to the myocyte proliferation induced by IGF1. A genome-wide microarray study in pregnant mouse myometrium found that Igfbp6 and Igfbp2 mRNA were upregulated from midgestation, and both were functionally categorized as regulators of cell growth [41]. We have previously documented that myometrial hypertrophic growth during the synthetic phase of myocyte differentiation in midpregnancy is dependent upon P4 and mechanical stretch. Our finding that Igfbp6 mRNA expression parallels the profile of plasma P4, together with our demonstration that P4 modulates Igfbp6 (but not Igfbp2) expression raise the possibility that IGFBP6 mediates the actions of P4 on myometrial growth. The elevated level of Igfbp2 mRNA during late pregnancy and labor was restricted to the gravid horn (in unilaterally pregnant rats), suggesting that, in contrast to Igfbp6, mechanical stretch may contribute to its expression. Cerro and Pintar [19] demonstrated by in situ hybridization that Igfbp2 gene expression increased significantly at late gestation but was restricted to the circular layer of myometrium. Our previous studies have shown that the circular layer of the myometrium is more responsive to mechanical stretch than the longitudinal [23]. The circular muscle would provide the primary contractile response during labor, whereas the role of the longitudinal layer is to shorten the uterus upon expulsion of each fetus. IGFBP2 was associated with smooth muscle hypertrophy in vascular [42] and visceral (urinary bladder) rat SMCs [43]. Based on these observations, we speculate that IGFBP2 mediates stretch-induced myometrial hypertrophy, whereas IGFBP6 mediates progesterone-induced hypertrophy. In contrast to other IGFBPs, Igfbp5 gene expression was specifically increased postpartum. Postpartum uterine involution is a critical event because it completes the reproductive cycle following pregnancy and labor by returning the uterus to its NP state so that the females can remain fertile (Fig. 8). Uterine involution involves several processes similar to those of development and wound healing, specifically substantial tissue reorganization, matrix metalloproteinase induction, extracellular matrix degradation, and apoptosis processes in which IGFs and IGFBP5 also have been implicated [44–46]. IGFBP5 has been implicated in mammary gland involution, in which it interacts with components of the ECM involved in tissue remodeling and apoptosis. In the present study we found an increase in myometrial Igf1, Igfbp5, and Igf1r gene expression after parturition, suggesting a role for these proteins in the process of postpartum involution. It is possible that IGFBP5 acts to accumulate large quantities of IGF1 on the cell surface, eventually releasing it site specifically during postpartum remodeling; in this way, IGFBP5 may promote SMC mitogenesis and preserve the integrity of the myometrial tissue architecture.
In summary, we suggest that members of the IGF system are key participants in tissue growth and remodeling during pregnancy, as they have demonstrated a distinct expression pattern correlating to each phase of myometrial differentiation (Fig. 8). The temporal association between the expression of specific members of this family further suggests that they may mediate the program of myometrial phenotypes ("myometrial programming" [20]), that supports the growth of the myometrium throughout pregnancy, the initiation and process of labor, and the critical remodeling of this tissue postpartum.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by a grant from the Canadian Institutes of Health Research (CIHR).
Correspondence: 2Stephen J. Lye, Samuel Lunenfeld Research Institute at Mount Sinai, 600 University Ave., Suite 870, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8740; e-mail: lye{at}mshri.on.ca
Received: 6 September 2006.
First decision: 3 October 2006.
Accepted: 7 November 2006.
REFERENCES
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