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Reproduction (2007) 134 503-511
DOI: 10.1530/REP-07-0004
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

The expression of transforming growth factor ß in pregnant rat myometrium is hormone and stretch dependent

Oksana Shynlova1, Prudence Tsui1,2, Anna Dorogin1, B Lowell Langille4 and Stephen J Lye1,2,3

1 Samuel Lunenfeld Research Institute at Mount Sinai, Mount Sinai Hospital, 600 University Avenue, Suite 870, Toronto, Ontario, Canada M5G 1X5, 2 Departments of Physiology and 3 Obstetrics and Gynecology, University of Toronto, Toronto, Canada M5S 1A1 and 4 Toronto General Research Institute, University Health Network, Toronto, Canada M5G 2C4

Correspondence should be addressed to S J Lye; Email: lye{at}mshri.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor ß (TGFß) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFßs. The expression of TGFß1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfß1-3 genes were expressed differentially in pregnant myometrium. Tgfß2 gene was not affected by pregnancy, whereas the Tgfß1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfß3 gene expression throughout pregnancy. Tgfß3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfß3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20–23) caused a significant decrease in the expression of Tgfß3 gene. In addition, Tgfß3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFß family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The transforming growth factor ß (TGFß) superfamily is composed of five related peptides, three of which are found in mammalian tissues, namely TGFß1, TGFß2, and TGFß3 (Massague et al. 1994). They exert their actions via three receptors: type I, type II, and type III (Massague et al. 1994). The intracellular signaling cascade linking TGFß family ligands and TGFß receptors to a cellular response is extremely complex and includes a large group of SMAD proteins carrying signals from the cell surface directly to the nucleus (Attisano & Wrana 2002). TGFß isoforms have been studied extensively in the reproductive system. They are multifunctional growth factors expressed by fetoplacental, cervical, and uterine tissues, where they regulate (1) cellular proliferation, (2) tissue remodeling, and (3) inflammatory response (Barnard et al. 1990, Lawrence 1996). The mRNA of all three TGFß isoforms is present in human term placenta in the syncytiotrophoblastic layer, chorionic plate, and in cells of the extravillous trophoblast (Schilling & Yeh 2000). TGFßs were amongst the first identified regulators of invasive trophoblast differentiation since they inhibited the proliferation of first trimester cytotrophoblasts (Graham et al. 1992). Furthermore, TGFßs exert anti-invasive effects on trophoblasts by increasing tissue inhibitor of matrix metalloproteinase (TIMP) expression and blocking matrix metalloproteinase (MMP) activity (Graham & Lala 1991, Ma & Chegini 1999). In placental trophoblasts and myometrial smooth muscle cells (SMCs), TGFß has been shown to up-regulate the expression of fibronectin, a marker of preterm labor (Goldenberg et al. 1997, Chegini et al. 1999). TGFßs mRNA and proteins are expressed in the human myometrium during menstrual cycle (Chegini et al. 1994) and pregnancy (Chegini et al. 1999, Kuscu et al. 2001), and have been suggested to play a central role regulating excitability and contractility in the human myometrium at term (Hatthachote & Gillespie 1999).

We proposed earlier that the ability of the myometrium to contract at term can be defined biochemically as an increase in expression of a cassette of genes encoding ‘contraction-associated proteins’ (CAPs), which control the contractile activity and responsiveness of the myometrium during labor (Challis 1994). We have shown that the timely expression of putative CAPs (Cx43, oxytocin receptor, prostaglandin receptor) is regulated by the integration of fetal endocrine and growth signals underlying myometrial activation (Ou et al. 1997, 1998). In addition, we have reported that the activation of CAP genes at term correlated with the increase in extracellular matrix (ECM) protein expression (Shynlova et al. 2004). It has been reported by others that the CAP and ECM expression could be regulated by cytokines, such as TGFß1, which may play a role in preparing the myometrium for parturition (Hatthachote et al. 1998). There is an evidence for the involvement of other cytokines in the modulation of myometrial function. Specifically, pro-inflammatory cytokines increase in human myometrium at term, suggesting that a cascade of cytokine interactions might prepare the myometrium for spontaneous preterm or term labor (Romero et al. 1991, 2006).

While several studies have reported the immunolocalization of TGFßs and their receptors in pregnant term and preterm human myometrium (Hatthachote et al. 1998, Chegini et al. 1999, Kuscu et al. 2001), very limited information is available on the expression of TGFß ligands and their function throughout pregnancy. We hypothesized that cytokines, specifically TGFßs, may assist in the myometrial activation at labor by mediating changes in pregnant uterine smooth muscle during consecutive phases of myometrial differentiation (Shynlova et al. 2006). We further proposed that mechanical and hormonal stimuli might regulate the expression of TGFßs in pregnant myometrium. In this study, we defined the expression profile of TGFß1-3 in the rat myometrium during normal pregnancy, spontaneous term labor, and post partum using real-time PCR, immunoblotting, and immunohistology techniques. We also investigated the effect of progesterone (P4) on the expression of Tgfß3 gene using a P4-delayed labor and RU486-induced preterm labor models. In addition, the effect of gravidity on the expression and localization of TGFß3 was investigated using a unilateral tuballigation rat model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Wistar rats (Charles River Co., St Constance, Canada) were housed individually under standard environmental conditions (12 h light:12 h darkness cycle) and fed Purina Rat Chow (Ralston Purina, St Louis, MO, USA) 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.

Experimental design
Normal pregnancy and term labor
Animals were killed by carbon dioxide inhalation and myometrial samples were collected on gestational days 0 (non-pregnant, NP), 6, 8, 10, 12, 14, 15, 17, 19, 21, 22, 23 (labor), or 1 and 4 day post partum (PP). Tissue was collected at 1200 h on all days with the exceptions of the labor sample (day 23L) that was collected once the animals had delivered at least one pup (n = 4).

P4-delayed labor
To determine whether high plasma levels of P4 might modulate the expression of TGFß family genes, pregnant rats were randomized to receive daily s.c. injections of either P4 (medroxyprogesterone acetate, 16 mg/kg in 0.4 ml sterile saline, Pharmacia Canada Inc.) 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 23L in the vehicle-treated group or 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, Ply-mouth Meeting, PA, USA) 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 (Ou et al. 1998). 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, 23, or 1PP (n = 4).

Tissue collection
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 we have previously shown removes the entire luminal epithelium and the majority of the uterine stroma (Piersanti & Lye 1995). The myometrial tissue and decidua were flash-frozen in liquid nitrogen and stored at –70 °C. For immunohistochemical studies the intact uterine horns were placed in ice-cold PBS and fixed immediately in 4% paraformaldehyde solution at 4 °C for 48 hours. For each day of gestation, tissue was collected from four different animals.

Real-time-PCR analysis
Total RNA was extracted from the frozen tissues using TRIZOL (Gibco BRL) according to manufacturer’s instructions. RNA samples were column purified using RNeasy Mini Kit (Qiagen), and treated with 2.5 µl DNase I (2.73Kunitz unit/µl, Qiagen) to remove genomic DNA contamination. RT and real-time PCR (RT-PCR) was performed to detect the mRNA expression of TGFßs and TGFß-related genes in rat myometrium. 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 RT Kit (Applied Biosystems, Foster City, CA, USA) as described earlier (Shynlova et al. 2005). cDNA (20 ng) from the previous step was subjected to real-time PCR using specific sets of primers (see legend to Fig. 1Go) 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 the SYBR Green detection chemistry. The run protocol was as follows: initial denaturation stage at 95 °C for 10 min, 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min. After PCR, a dissociation curve was constructed by increasing temperature from 65 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. PCRs were set up in triplicates and the mean of the three Cts was calculated. A comparative Ct method ({Delta}{Delta}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 gene at every gestational day (1) was normalized to ribosomal 18S mRNA and (2) a fold change was calculated relative to the expression of the same gene in corresponding NP sample using an arithmetic formula (see ABI User Bulletin #2). For unilaterally pregnant animals, the gene expression was shown as fold change relative to day 6 gravid horn mRNA level, whereas that of P4- and RU486-treated animals was shown as a fold change relative to the vehicle sample. Validation experiments were performed to ensure that the PCR efficiencies between the target genes and 18S were approximately equal.


Figure 1
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  		Figure 1 The effects of gestational age on the expression of TGFb1-3 genes in the pregnant rat myometrium. Total RNAs were extracted from frozen myometrial tissues, single-strand cDNAs were synthesized as described under ‘Materials and Methods’ and mRNA levels were analyzed on the indicated days of gestation by real-time PCR. Specific forward and reverse primers were designed using Primer Express software, version 2.0.0 (Applied Biosystems) as follows: TGFb1 mRNA, 5'-AGAAGTCACCCGCGTGCTAAT-3' (sense primer) and 5'-CACTGCTTCCCGAATGTCTGA-3' (antisense primer; GenBank accession #: NM_021578); TGFb2 mRNA, 5'-TTCA-GAATCGTCCGCTTCGAT-3' (sense primer) and 5'-TTGTTCAGC-CACTCTGGCCTT-3' (antisense primer; GenBank accession #: NM_031131); TGFß3 mRNA, 5'-ATACAACACCCTGAACCCGGA-3' (sense primer) and 5'-CGACTTCACCACCATGTTGGA-3' (antisense primer; GenBank accession #: NM_013174); 18S, 5'-GCGAAAG-CATTTGCCAAGAA-3' (sense primer) and 5-‘GGCATCGTT-TATGGTCGGAAC-3' (antisense primer; GenBank accession #: X01117). TGFb1-3 mRNA levels were normalized to 18S mRNAs and expressed in fold changes relative to a corresponding non-pregnant sample. The bars represent mean ± S.D. (n = 4 at each time point). Data labeled with different letters are significantly different from each other (P < 0.05).

 
Western immunoblot analysis
Total protein was extracted from the frozen tissues using RIPA lysis buffer as described earlier (Shynlova et al. 2005). 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, USA) in 25 mM Tris–HCl (pH 8.3), 250 mM glycine, 0.1% (wt/vol) SDS, for 18 h at 30 V and 4 °C. The protein expression levels of TGFß1 and TGFß3 were measured by western analysis using primary antibody (TGFß1: mouse monoclonal, 1:2000; TGFß3: rabbit polyclonal, 1:2000; Abcam International, Cambridge, CA, USA). PVDF membranes were stripped and reprobed with anti-tubulin (1:3000, clone DM 1A; Sigma–Aldrich) and anti-calponin (1:3000, clone hCP; Sigma) mouse primary antibodies to control the loading variations. Probed membranes were exposed to X-ray film (Kodak XAR, Eastman Kodak) and analyzed by densitometry (ImageJ software program; National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/). Calponin is constitutively expressed in non-pregnant and pregnant rat tissue under the same protein extraction conditions (Williams et al. 2005). Tubulin is present in almost all eukaryotic cells and is commonly used as a housekeeping protein (Vemuganti et al. 2004).

Immunohistochemistry
The fixed myometrial tissues were sectioned into 10 µm thickness and collected on Superfrost Plus slides (Fisher Scientific Ltd., Nepean, ON, Canada). The frozen sections were immersed in 0.3% hydrogen peroxide (Fisher Scientific, Fair Lawn, NJ, USA). Antigen retrieval was performed by cooking the tissues at 90 °C after 5 min, followed by blocking with 5% normal goat serum and incubation with primary antibodies overnight at 4 °C. Primary antibody was rabbit anti-TGFß3 (1:100, Abcam International). For the negative controls, ChromPure non-specific rabbit IgGs (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) were used at the same concentration and sections were also incubated with secondary antibodies in the absence of primary antibodies. Secondary antibodies used for detection were biotinylated goat anti-rabbit (1:200; Vector, Burlingame, CA, USA). Final visualization was achieved using Vectastain Elite ABC Kit (Vector). Counterstaining with Harris Hematoxylin (Sigma diagnostics) was carried out before slides were mounted with Cytoseal XYL (Ricard-Allan Scientific, Kalamazoo, MI, USA). Myometrial cells from each of the three tissue sets were observed on a Leica DMRXE microscope (Leica Microsystems, Richmond Hill, ON, Canada). A minimum of five fields were examined for each gestational day and uterine horn for each set of tissue, and representative tissue sections were photographed with Sony DXC-970 MD (Sony Ltd., Toronto, ON, Canada) 3CCD color video camera.

Statistical analysis
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 tuballigation data were analyzed by two-way ANOVA followed by pairwise multiple comparison procedures as described above. The day 24 P4 treated group was compared with the day 23 vehicle group using a t-test. RU486 results were compared with 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, USA) with the level of significance for comparison set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Figure 1Go illustrates the expression of Tgfß1-3 gene throughout pregnancy and post partum. Relative abundance of the Tgfß1 mRNA was significantly increased starting at mid-gestation (3.01 ± 0.21-fold increase on day 12 vs NP, P < 0.05) and was maintained elevated until labor and post partum (5.22 ± 0.80-fold increase on day 1PP versus NP, P < 0.05). No significant changes were found for Tgfß2 myometrial transcript levels across gestation (P > 0.05). In contrast, a dramatic bimodal change in Tgfß3 mRNA abundance was observed throughout pregnancy. We detected a transient activation of myometrial Tgfß3 gene expression on gestational days 14–15 (11.30 ± 1.30- and 12.36 ± 1.91-fold increase versus NP, P < 0.05), a subsequent decrease at late gestation (6.92 ± 0.96-fold increase on day 19 versus NP, P < 0.05), and a second dramatic increase at labor (45.31 ± 8.73-fold increase on day 23 versus NP, P < 0.05). During post partum involution Tgfß3 transcript levels quickly decreased (8.37 ± 1.84-fold increase on 1PP versus NP, P < 0.05). We applied immunoblotting technique to analyze if protein expression of TGFß1 and TGFß3 reflects their gene expression. As expected from the mRNA data, the expression of TGFß1 and TGFß3 proteins started increasing from mid-gestation (P < 0.05 for day 17 versus NP), was further up-regulated at late gestation and during labor (three- to fourfold increase on days 21–23 versus NP for TGFß1 and TGFß3 proteins, P < 0.05; Fig. 2Go) and decreased thereafter. From these observations, it appears that the expression of TGFß1 and TGFß3 in rat myometrium was clearly dependent on gestational age.


Figure 2
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  		Figure 2 The effect of gestational age on the expression of TGFß1 (A) and TGFß3 (B) proteins in the pregnant rat myometrium. Representative western blots and densitometric analysis of TGFß1 and TGFß3 protein levels throughout normal gestation and post partum. TGFß1 and TGFß3 protein expression levels were normalized to {alpha}-tubulin (A) or calponin (B). Bar graphs showing the mean ± S.E.M. ROD (n = 3–4 at each time point). Data labeled with different letters are significantly different from each other (P < 0.05).

 
We also studied the temporal and spatial distribution of TGFß3 protein in the myometrium across gestation (Fig. 3Go). According to our observation, the TGFß3 staining in the rat myometrium was significantly altered during pregnancy. Immunostaining of TGFß3 in uterine smooth muscle of non-pregnant and early pregnant (day 6) animals was extremely weak. However, starting from day 15 the immunoreactivity of the rat myometrium increased dramatically (Fig. 3Go). Consistent with our gene and protein expression results the most intense staining was found in laboring samples. In addition, we detected more intense TGFß3 immunostaining at late gestation in the circular myometrial layer when compared with the longitudinal myometrial layer (Fig. 3Go). TGFß3 protein was always detected in the cytoplasm of myometrial SMCs and this spatial distribution was similar in both uterine muscle layers.


Figure 3
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  		Figure 3 TGFß3 immunolocalization in pregnant rat myometria during gestation. Immunohistochemical examination was performed on sections of uterus from non-pregnant (NP) (A), 6 days (B), 15 days (C), 17 days pregnant (D), laboring (day 23) (E), and 1 day post partum (F) animals. Expression of TGFß3 in circular myometrial layers increased significantly during late gestation (C–E, arrows). Relatively weak immunostaining was present in longitudinal myometrial layers (C–E). The lack of immunostaining after incubation of myometrial tissue with non-specific rabbit IgGs on gestational day 14 (G) or with anti-rabbit secondary antibodies in the absence of primary antibody on labor (day 23) is shown in H. Magnification, 200x; scale bar A–E = 50 µm.

 
P4 is the major hormone of pregnancy in the rat. In our study, artificial maintenance of elevated P4 levels at late gestation (from day 20 to day 23) by daily injections of hormone caused a failure to initiate labor. We showed before that this treatment prevented the increase in the expression of CAPs, AP-1, and ECM genes in the rat myometrium (Piersanti & Lye 1995, Shynlova et al. 2004). We have now shown that the administration of P4 also prevented the increase of Tgfß3 mRNA levels at term when compared with vehicle-treated animals (Fig. 4AGo). Tgfß3 gene remained low in myometrium of P4-treated rats on gestational day 22, day 23 (labor), and day 24 (1 day after normal delivery) when compared with controls (P < 0.05). On the contrary, treatment of pregnant rats with the P4 receptor antagonist RU486 on day 19 caused the onset of preterm labor within 24 h and a 3.6-fold increase in Tgfß3 gene expression (P < 0.05, Fig. 4BGo). These results demonstrated that decreased P4 signaling during late pregnancy caused by RU486 led to Tgfß3 gene induction, whereas maintenance of high plasma P4 levels prevented this increase.


Figure 4
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  		Figure 4 The effects of progesterone (A) and RU486 (B) on myometrial TGFß3 expression. Real-time PCR analysis of TGFß3 mRNA levels in pregnant rat myometrium during progesterone-delayed (A) and RU486-induced preterm (B) labor normalized versus 18S mRNA and expressed in fold change relative to a vehicle day 21 (A) or day 20 (B) samples. Shown are vehicle (black bars) and P4- or RU486-treated (white bars) samples. Values represent mean ± S.D. (n = 4 at each time point). A significant difference is indicated by *P < 0.05 or ***P < 0.001.

 
Since TGFß1 and TGFß3 protein expression increased specifically at the second half of gestation when mechanical stretch of uterine walls imposed by growing fetuses was apparent, we decided to study Tgfß3 gene and protein expression using the unilaterally pregnant rat model (Fig. 5Go). This model enables us to distinguish the effects of endocrine and mechanical stimuli, given that both gravid and empty horns were subjected to the same hormonal environment. Tgfß1 mRNA levels were not statistically different between empty and gravid horns (data not shown). We found that Tgfß3 gene expression in the empty horn was very low throughout gestation. In contrast, Tgfß3 transcript levels were dramatically increased in the gravid uterine horns, showing a profile similar to that of normal pregnant animals. Relative quantification indicated a transient induction of Tgfß3 gene in the gravid horn at mid-pregnancy (12.4-fold increase on day 14 when compared with day 6) and at term (22.4-fold increase on day 23L when compared with day 6; Fig. 5Go). This increase in Tgfß3 mRNA of the gravid horn was statistically different from the corresponding empty horn (two-way ANOVA, P < 0.05). We also found a dramatic increase in TGFß3 protein immunoreactivity in the gravid horns of unilaterally pregnant rats on late gestation (Fig. 6A, C and EGo) when compared with their pairing empty horns (Fig. 6B, D and FGo).


Figure 5
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  		Figure 5 Expression of TGFß3 gene in the myometrium of unilaterally pregnant rats during gestation. mRNA levels were analyzed on the indicated days of gestation by real-time PCR using specific primers (see Fig. 1Go). TGFß3 gene expression levels in empty (white bars) and gravid (black bars) were normalized to 18S mRNAs and expressed in fold changes relative to a day 6 gravid sample. The bars represent mean ± S.D. (n = 4 at each time point). A significant difference between gravid and empty horn of the same gestational day is indicated by *P < 0.05 or ***P < 0.001.

 

Figure 6
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  		Figure 6 TGFß3 localizes within the circular layer SMCs of the gravid myometrium from unilaterally pregnant rats. Uterine tissue was collected from empty and gravid horns of unilaterally pregnant rats on different gestational days, including day 17 (A and B), day 19 (C and D), and day 23 (E and F). Tissues were labeled with anti-TGFß3 antibody and light microscopy images of cross-sections were collected. TGFß3 localized mainly within the cytoplasm of circular muscle SMCs of the gravid uterine horn (arrows) and was much less abundant in the empty horn. No gestational changes in localization were observed. Magnification, 200x; scale bar A–H = 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
We have previously reported that the myometrium undergoes gradual changes in phenotype throughout gestation. These stages are characterized by an early proliferative phase, an intermediate phase of cellular hypertrophy and matrix elaboration, and the final contractile phase (Shynlova et al. 2006). We proposed that phenotypic modulation of uterine myocytes is the result of integration of endocrine signals and mechanical stimulation of the uterus by the growing fetus. We have also demonstrated that these signals are important in regulating the onset of labor (Lye et al. 2001). In this report we showed that cytokines, specifically the TGFßs, were (1) differentially expressed in the pregnant rat myometrium during specific phases of gestation and labor and (2) regulated by gravidity and ovarian hormones.

Numerous studies have shown that TGFßs control a remarkable diversity of cellular functions, many of which are directly related to cell growth. We found that TGFß mRNA and protein levels were low during the early phase of gestation when rat myometrial SMCs undergo hyperplasia, but increased during the synthetic phase when the proliferative activity of myometrial SMCs was substantially reduced. Consistent with our results, total TGFß1 levels (Hatthachote et al. 1998) and TGFß3 immunoreactive proteins (Kuscu et al. 2001) have been reported to be elevated in the pregnant human myometrium when compared with non-pregnant tissues. TGFß1 is well-known for its bimodal and dose-dependent effects on the growth of SMCs. Arici and colleagues demonstrated that low concentrations of TGFß1 stimulate cell proliferation in leiomyoma (Arici & Sozen 2003) and vascular cells (Battegay et al. 1990), while these stimulatory effects disappear at high TGFß1 concentrations. It has been also shown that increased TGFß1 gene expression induced by angiotensin II led to de novo protein synthesis in cultured vascular SMC (Koibuchi et al. 1993). New protein synthesis is a property of hypertrophic cells. We have previously documented myometrial hypertrophic growth during the second part of gestation (Shynlova et al. 2006). Our finding that TGFß1 and TGFß3 expression was induced during that specific time period raises the possibility that TGFßs may support cellular hypertrophy in late pregnant uterus.

Interestingly, we observed two periods of transient myometrial induction of TGFß3 gene during rat gestation. The first increase in TGFß3 gene expression occurred at mid-gestation (around day 14). As was shown before (Reynolds 1949) at that time fetal growth mediates an acute stretch of the uterine walls creating a transient hypoxemia of myometrial SMCs. We have previously shown that at this time there is a transient activation of the stress-induced (intrinsic) apoptotic pathway in the myometrium, a characteristic signal that we believe stops myometrial proliferative activity and promotes smooth muscle differentiation to a synthetic and later to a contractile phenotype (Shynlova et al. 2006). Interestingly, we found that the expression of hypoxia-induced transcription factor (HiF-1{alpha}) gene was up-regulated in the rat myometrium around day 14 of gestation and later at term, supporting the occurrence of two periods of hypoxia during gestation (Shynlova, Lye, unpublished). It has been shown in human placental explants that TGFß3 expression correlates closely with the expression of HiF-1{alpha} (Caniggia et al. 1999, 2000). In addition, HiF-1{alpha} has been shown to directly regulate TGFß3 gene expression in mouse trophoblast cells in vitro (Schaffer et al. 2003). Taken together, we suggest that the activation of TGFß3 gene expression at mid- and late gestation is likely mediated by HiF-1{alpha}. It is also plausible that a similar molecular mechanism (mechanical stretch imposed by growing fetuses on myometrial SMCs) is responsible for the second period of TGFß3 gene induction in the rat myometrium before and during parturition. A number of in vitro studies have shown up-regulation of TGFß by mechanical stretch in a variety of cell types such as vascular SMCs (Li et al. 1998), intestinal SMCs (Gutierrez & Perr 1999), pulmonary arterial SMCs (Mata-Greenwood et al. 2005), and cardiomyoctyes (van Wamel et al. 2001). These data correspond well with our in vivo studies using the unilaterally pregnant rats where we demonstrated that TGFß3 gene and protein expression was induced specifically in the gravid horns but not in the empty horns of late pregnant and laboring animals. Our results are further supported by a study using a similar experimental approach in which mRNA and protein expression of components of the TGFß-signaling axis are up-regulated by a stretch in the unilateral ureteric obstruction model in fetal sheep (Yang et al. 2001).

Furthermore, we found a difference in spatial distribution of TGFß3 protein in a gravid horn; the circular myometrial layer showed more intense immunoreactivity than the longitudinal. Our previous studies have reported that the circular layer of the myometrium is more responsive to mechanical stretch than longitudinal based on the fact that the expression of the connexin43 (Doualla-Bell et al. 1995; a putative CAP gene) and {gamma}-actin (Shynlova et al. 2005; a component of SM contractile apparatus) were increased in uterine circular muscle at late gestation. Others have also reported different responses to stretch, nor-adrenaline, and estrogen stimulation in circular versus longitudinal muscle (Matsumoto 1980, Doualla-Bell et al. 1995). This suggests that the two myometrial layers play different roles in labor contractions. The circular muscle primarily contracts rhythmically, while the longitudinal layer shortens the uterus upon expulsion of each fetus. We believe that stretch-induced activation of TGFß signaling in the circular myometrial layer is one of the factors preparing the myometrium for labor. In other independent studies using cultured vascular SMCs, mechanical stretch not only stimulated TGFß mRNA expression in a time- and elongation-dependent manner, but also up-regulated expression of type I and type IV collagen, and fibronectin genes, which was largely inhibited by addition of neutralizing antibody against TGFß (Li et al. 1998, Joki et al. 2000). TGFß3 treatment was also found to stimulate fibronectin expression in human cultured leiomyoma cells (Arici & Sozen 2000). We have reported earlier that expression of fibronectin, as well as major components of basement membrane, namely type IV collagen and laminin, are dramatically up-regulated prior to and during labor in rats (Shynlova et al. 2004). Thus, the parallel increase of TGFß3 and ECM components at late gestation suggests that TGFß3 may mediate ECM induction, providing the mechanism to anchor hypertrophied uterine myocytes in order to produce coordinated, forceful labor contractions.

It has been shown in vitro that the TGFß-signaling system can be regulated by ovarian hormones in human myometrial cells (Chegini et al. 1996, Awad et al. 1997). In the non-pregnant ovariectomized mouse uterus, the expression of TGFß can be transiently increased by the in vivo injection of estrogen (Das et al. 1992). In the present study we found a negative correlation between plasma P4 levels and the expression of TGFß3 at late pregnancy, suggesting additional hormonal regulation of this gene. We speculate that the decrease in P4 levels, followed by an increase in estrogen plasma levels and mechanical stimulation of myometrium are all responsible for the activation of TGFß3 at term.

To date, the physiological role of TGFßs in preparing the myometrium for labor is not fully understood. Among the three TGFß isoforms we studied, TGFß1 and TGFß3 genes show significant changes across gestation. We suggest that at mid-gestation TGFß3 may influence the transition from proliferative and synthetic myometrial phenotypes. We also suggest that at late gestation both TGFß1 and TGFß3 proteins (1) support myometrial cellular hypertrophy and (2) play a role in the preparation of myometrium for labor contractions. Our results support and expand the understanding of myometrial phenotypic modulation during pregnancy and demonstrate a significant role for members of the TGFß family in this process.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by a grant from the CIHR. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 3 January 2007
First decision 26 January 2007
Revised manuscript received 22 May 2007
Accepted 1 June 2007

O Shynlova and P Tsui contributed equally to this work


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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