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Research Article |
Samuel Lunenfeld Research Institute,3 Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
Departments of Physiology4
and Obstetrics & Gynecology,5 University of Toronto, Ontario M5S 1A1, Canada
ABSTRACT
In the present study, we determined the contribution of myometrial hyperplasia, hypertrophy, and apoptosis to uterine growth during pregnancy. The changes in two endogenous markers of cell replication, proliferating cell nuclear antigen (PCNA) protein expression and bromodeoxyuridine (BrdU) incorporation, were studied. Myocyte hypertrophy was assessed by measuring the protein:DNA ratio. The expression levels of antiapoptotic regulatory proteins (BCL2 and BCL2L1) and enzymes involved in apoptosis (caspases 3, 6, 7, 9, and 10) were assessed by immunoblotting throughout gestation and postpartum. Myometrial cell apoptosis was determined by TUNEL staining and DNA fragmentation assays. Both BrdU incorporation and PCNA labeling were elevated in early pregnant myometrium and decreased dramatically after midgestation, with a simultaneous increase in cellular hypertrophy. Levels of BCL2 were high during early gestation, followed by significantly elevated levels of BCL2L1 at midgestation. The expression of caspase 10 in myometrial samples declined from a high nonpregnant level to a complete loss at early gestation. The cleaved forms of caspases (CC) 3, 6, 7, and 9, as well as poly(ADP-ribose)polymerase-1, were undetectable in the myometrial samples at early or late gestation but were transiently elevated at midgestation. Immunohistochemical staining of CC3 confirmed the activation of the caspase cascade, but TUNEL-positive staining or the increase in DNA fragmentation was not detected. Collectively, two distinct phases of myometrial growth were observed: myocyte hyperplasia associated with an increase in antiapoptotic proteins during the first half of gestation, and cellular hypertrophy during the second part of gestation. The transition between these phases was associated with transient activation of the caspase cascade that triggered the differentiation of uterine smooth muscle.
apoptosis, pregnancy, uterus
INTRODUCTION
Uterine growth during pregnancy represents one of the most remarkable events in reproduction. Depending on the species, the uterus can exhibit a 500- to 1000-fold increase in volume and a 15-fold or greater increase in weight. The human uterus increases from a weight of approximately 50 g in the nonpregnant state to approximately 1200 g at term, representing a 24-fold increase in mass [1]. Physiological growth of the pregnant uterus occurs by two mechanisms: an increase in cell number (hyperplasia), and an increase in cell size (hypertrophy). Hypertrophy is one of the primary cellular adaptations that uterine smooth muscle undergoes during pregnancy and usually is characterized by 1) increase in cellular protein synthesis, including thin, thick, and intermediate filaments; 2) extracellular matrix protein synthesis; 3) increase in cell organelles, including mitochondria as well as smooth and rough endoplasmic reticulum; and 4) transitions in contractile protein content and organization (for review, see [2]). Our previous data have suggested potential hypertrophic changes in late-pregnant myometrium, with significantly increased expression of extracellular matrix proteins during the second part of gestation [3]. Additionally, we have found that ACTG2 (also known as 
-actin), a marker of hypertrophied urinary bladder smooth muscle [4], was elevated in late-pregnant rat myometrium [5].
Factors known to influence uterine growth include steroid hormones and growth factors. It has been shown that in myometrial leiomyoma cells, both estrogen and progesterone upregulate cell proliferating activity, whereas only estrogen induced proliferation of normal myometrial smooth muscle cells (SMCs) [6]. Moreover, Maruo et al. [7] have found that epidermal growth factor and insulin-like growth factor (IGF)-I play a crucial role in prompting uterine leiomyoma growth by stimulating the proliferative potential and inhibiting apoptosis of cultured human leiomyoma cells [7]. The Igf1 receptor mRNA is coexpressed with Igf1 in primate uterine smooth muscle, setting the stage for paracrine/autocrine IGF regulation of myometrial growth.
The increase in total cell mass generally depends on the net result of cell production through cell division and cell loss through programmed cell death (apoptosis). The contributory role and regulation of cell proliferation versus prolonged cell survival during uterine growth is extremely important, because growth of uterine leiomyoma is associated with increased mitotic activity and decreased apoptosis in humans and rats [8, 9]. Apoptosis is the physiological process by which excess or dysfunctional cells are eliminated during development or normal tissue homeostasis, and nowhere is it more dramatic than in the reproductive system. For example, apoptosis occurs cyclically in human nonpregnant endometrium, throughout pregnancy in mouse [10] and human decidua [11], human placenta [12], amnion epithelial cells [13, 14], rat cervical SMCs during later pregnancy [15], and mammary glands during weaning [16, 17]. At least two pathways activate apoptosis (for review, see [18]). The first is a mechanism that involves activation of a group of tumor necrosis factor receptors, such as Fas (ligand-receptor pathway). The second (exogenous stimulus pathway) is a parallel, mitochondria-dependent route activated by physiological stimuli (lack of growth factors, changes in hormonal environment, hypoxia, and hypoglycemia) and/or environmental stimuli (exposure to cytotoxic compounds, radiation, and viral infection) that is transmitted independently of surface membrane receptors and is governed by BCL2 family proteins. The execution phase of apoptosis uses a common pathway of cytosolic cystein proteases (caspases), which are present in most cells in inactive proenzyme form that must be cleaved autocatalytically or by other caspases [19]. Triggering of apoptosis results in the activation of a caspase cascade in which the last caspases to be activated are downstream effector caspases, such as caspase (CASP) 3 (also called Yama and apopain) that digest cellular substrates, resulting in morphological changes and/or cell death.
Despite the remarkable nature of uterine growth during pregnancy, little information is available regarding the mechanisms that initiate and regulate this growth. Furthermore, no data precisely maps the contribution of hyperplasia, hypertrophy, and apoptosis to uterine growth from early to late pregnancy. The role of cell apoptosis in myometrial growth is largely unknown. Therefore, the goal of the present study was to gain insight regarding the pattern of myometrial growth across pregnancy and to determine whether apoptosis contributes to specific elements of this growth. In the present study, we used a rat model to investigate the proliferative activity of the pregnant myometrium by examining protein expression levels of proliferating cell nuclear antigen (PCNA) and its immunohistochemical staining that appears exclusively in dividing cells as well as incorporation of bromodeoxyuridine (BrdU), a nonradioactive analog of thymidine and marker of individual cell proliferation. Myocyte hypertrophy was assessed by measuring the protein:DNA ratio. We have examined the protein expression levels of major enzymes involved in apoptosis; initiator and effector CASP3, CASP6, CASP7, CASP9, and CASP10; as well as major apoptosis-regulating proteins BCL2 and BCL2L1 (also known as Bcl-xL) in pregnant and postpartum rat myometrium. Apoptotic activity of pregnant myometrium also was visualized using TUNEL and DNA fragmentation assays, which detect the characteristic internucleosomal DNA strand breaks.
MATERIAL AND METHODS
Animals
Wistar rats (Charles River Co.) were housed individually under standard environmental conditions (12L:12D) and fed Purina Rat Chow (Ralston Purina) and water ad libitum. Female virgin rats were mated with male Wistar rats. Gestational Day 1 was designated as the day a vaginal plug was observed. The average time of delivery under these conditions was during the morning of Gestational Day 23. Animals were killed by carbon dioxide inhalation, and myometrial samples were collected on Gestational Days 0 (nonpregnant), 6, 8, 10, 12, 14, 15, 17, 19, 21, 22, and 23 (labor) as well as on Postpartum Days 1 and 4. The Samuel Lunenfeld Research Institute Animal Care Committee approved all animal experiments.
Tissue Collection
Tissue was collected at 1000 h on all days with the exception of the labor sample (Gestational Day 23), which was collected once the animals had delivered at least one pup. For protein extraction, the uterine horns were placed into ice-cold PBS, bisected longitudinally, and dissected away from pups, placentas, and mesometrial triangles. The endometrium was carefully removed from the myometrial tissue by mechanical scraping on ice, which we have shown previously removes the entire luminal epithelium and the majority of the uterine stroma [20]. The whole myometrial tissue from each animal was flash-frozen in liquid nitrogen and stored at –70°C. To minimize intra-animal variation, each whole-frozen myometrial tissue sample was pulverized individually under liquid nitrogen with a mortar and pestle for protein extraction. For immunohistochemical studies, the intact uterine horns were placed in ice-cold PBS buffer, cut into segments (thickness, 3–10 mm) using a scalpel blade and fixed immediately in 4% paraformaldehyde solution at 4°C for 48 h. These segments were further cross-sectioned or sectioned longitudinally. For each day of gestation, tissue was collected from three or four different animals and processed for different experimental purposes.
BrdU Staining
Cellular proliferation was calculated by measuring the rate of incorporation of BrdU (Sigma), a nonradioactive thymidine analog, into the replicating DNA during the S phase of the cell cycle. The BrdU was injected into the peritoneum (30 mg/kg in 0.9% saline) at 17, 9, and 1 h before death on Gestational Days 6, 12, 18, and 23 (labor). The animals were anesthetized and perfusion-fixed with 10% buffered formalin pumped to the heart via an intravenous butterfly tube. Uterine horns were excised, washed in saline, and fixed with 10% buffered formalin overnight. In each horn, a 1-cm segment containing a fetal sac was dehydrated and embedded in paraffin. Sections were cut and processed according to a previously suggested protocol [21]. Briefly, tissue sections were cut on a microtome and mounted on microscope slides (Fisher Scientific Ltd.). Sections were then permeabilized using 2.5% trypsin in PBS buffer (pH 7.4) for 30 min followed by DNA denaturing by immersion in 2 M HCl for 1 h at 37°C. Sections were incubated with primary anti-BrdU monoclonal antibody (1:15; BD Biosciences) overnight at 4°C followed by a wash in PBS and incubation with secondary biotinylated anti-mouse immunoglobulin (1:200; DAKO) for 1 h. The sections were then washed and incubated with ABC reagent (Vector Laboratories) for 80 min. The peroxidase reaction was developed by adding 0.02% 3,3'-diaminobenzinidine and 0.005% H2O2 in 0.05 M Tris buffer (pH 7.6). Cells were counterstained with Harris Hematoxylin (Sigma) to allow differentiation between positive and negative nuclei. In negative-control experiments, tissue from rats not exposed to BrdU was processed and stained as described above. Sections were then placed under Leica DMRXE microscope (Leica Microsystems) and counted according to a suggested protocol: using x200 magnification, stained and total consecutive nuclei were counted from five noncontiguous, randomly selected fields of all layers of myometrium. Percentages of BrdU-positive nuclei were determined by the number of cells having positively stained nuclei divided by total number of cells in the field (labeled and unlabeled) and multiplied by 100.
Protein:DNA Ratio for Cellular Hypertrophy
For protein extraction, the frozen myometrial tissue sample weight was recorded, and the tissue was pulverized under liquid nitrogen with a mortar and pestle. The pulverized tissue was homogenized for 1 min in RIPA lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% [v/v] Triton X-100, 1% [v/v] sodium deoxycholate, and 0.1% [w/v] SDS) supplemented with 100 µM sodium orthovanadate and protease-inhibitor cocktail tablets (Complete Mini; Roche). Samples were centrifuged at 12000 x g for 15 min at 4°C, and the supernatant was collected and stored at –80°C. Protein concentrations were determined using a protein assay (Bio-Rad). Total protein per horn values were obtained according to the following formula: total protein per horn = [(protein concentration [mg/ml] x total RIPA buffer used)/sample weight] x total myometrial wet weight. The DNA was extracted according to a previously suggested protocol for phenol extraction of nucleic acids [22]. Myometrial samples were left to rotate at 55°C overnight in DNA lysis buffer (0.02 M Tris-HCl, 1% [w/v] SDS, and 5 mM EDTA [pH 4]) with 0.2 mg/ml of proteinase K (Roche) followed by centrifugation at 15000 x g for 5 min at 4°C. The supernatant was transferred to a new tube and incubated with 0.04 mg/ml of RNase A (Sigma) for 1 h at 37°C. Phenol:chloroform (1:1, v:v) was added, and samples were centrifuged at 15000 x g for 5 min at 4°C. The upper phase was transferred to a new tube, and DNA was precipitated with 100% ethanol. Pellets were washed with 70% ethanol, followed by resuspension in Tris-EDTA buffer (10 mM Tris-HCl [pH 7.5] and 1 mM EDTA). The samples were assayed spectrophotometrically using a wavelength of 260 nm for the peak absorption of DNA. A solution containing 50 µg/ml of double-stranded DNA has an absorbance of 1 at 260 nm. Total DNA per horn was calculated according to the following formula: DNA (mg/horn) = [(DNA concentration [mg/ml] x total lysis buffer used)/sample weight] x total weight of myometrium.
Western Immunoblot Analysis
Four sets of proteins were extracted from a frozen myometrial tissues as described above (see Protein:DNA Ratio for Cellular Hypertrophy). Protein samples (40–60 µg) were resolved by electrophoresis on a 12%–15% SDS-PAGE. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore) in 25 mM Tris-HCl, 250 mM glycine, and 0.1% (w/v) SDS, overall pH 8.3, for 18 h at 30 mV at 4°C. Membranes were blocked in Tris-buffered saline (50 mM Tris and 150 mM NaCl, overall pH 7.4) with 0.1% (v/v) Tween-20 (TBST) supplemented with 5% nonfat dry milk and then incubated with PCNA mouse primary antisera (1:1000; Oncogene); Bcl-2 (1:1000; Santa Cruz Biotechnology, Inc.); Bcl-xL (1:1000; Bcl-2 Family Antibody Sampler Kit; Cell Signaling, Inc.); cleaved CASP3, CASP6, CASP7, CASP9, and CASP10; and cleaved PARP-1 (1:1000; Cleaved Caspase Antibody Sampler Kit; Cell Signaling, Inc.) rabbit primary antisera. After primary antibody incubation, membranes were washed with TBST buffer, followed by incubation with a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:1000; Amersham), and exposed to enhanced chemiluminescence reagent (Amersham). To confirm that equal amounts of PCNA; BCL2; BCL2L1; cleaved CASP3, CASP6, CASP7, CASP9, and CASP10; and poly(ADP-ribose)polymerase-1 (ADPRT) proteins were electrophoresed in each reaction, the blot was stripped and reprobed with anti-calponin antibody at a 1:3000 dilution (Sigma-Aldrich Chemie Gmbh) using the probing conditions described above. Probed membranes were exposed to radiographic film (Kodak XAR; Eastman Kodak). The intensity of the 36-kDa band of PCNA, 26-kDa band of BCL2, 30-kDa band of BCL2L1, 17- and 19-kDa bands of cleaved CASP3, 18-kDa band of cleaved CASP6, 20-kDa band of cleaved CASP7, 17- and 38-kDa bands of cleaved CASP9, 65-kDa of CASP10, and 89-kDa bands of cleaved ADPRT were quantified by densitometry, and their protein levels were normalized to the calponin (also known as CNN1) protein levels (34 kDa) and expressed as relative optical density (ROD) units. To avoid cross-reactivity between antibodies, detection for different caspases was performed at all times using different polyvinylidene difluoride membranes.
Immunohistochemistry
The fixed myometrial tissues were gradually dehydrated in ethanol and embedded in paraffin. Sections (thickness, 5 µm) were collected on Superfrost Plus slides (Fisher Scientific Ltd.). Paraffin sections were deparaffinized and rehydrated. After quenching in 0.3% hydrogen peroxide (Fisher Scientific), the sections to be stained for anti-Bcl-2 rabbit (1:100; Santa Cruz Biotechnology, Inc.), and anti-cleaved CASP3 rabbit primary antibodies (1:200; Cell Signaling, Inc.) were heated in a microwave oven for 10 min in 10 mM sodium citrate and blocked with 5% normal goat serum (ABC rabbit kit; Vector Laboratories). Sections to be stained with anti-PCNA mouse primary antibodies (1:1800; Ab-1; Oncogene) were pretreated in 4 N HCl solution at room temperature for 10 min and blocked with 5% normal horse serum (ABC mouse kit; Vector Laboratories). All sections were incubated with primary antibodies overnight. For the negative controls, normal rabbit or normal mouse serum (ABC mouse kit) were used at the same concentration as primary antibodies, and sections also were incubated with secondary antibodies in the absence of primary antibodies. Secondary antibodies used for detection were biotinylated anti-mouse or biotinylated anti-rabbit (1:200; ABC mouse kit). Final visualization was achieved using Vectastain Elite Kit (Vector Laboratories). Counterstaining with Harris Hematoxylin (Sigma) was carried out before slides were mounted with Cytoseal XYL (Richard-Allan Scientific). For the assessment of staining intensity, tissue sections from each of the three sets were observed on a Leica DMRXE microscope (Leica Microsystems). At minimum, five fields were examined for each gestational day and uterine horn for each set of tissue, and representative tissue sections were photographed with a Sony DXC-970 MD 3CCD color video camera (Sony Ltd.).
TUNEL Staining
Paraffin sections were processed the same way as described for immunohistochemistry. After permeabilization with proteinase K (10 µg/ml for 10 min at room temperature; Roche), sections were blocked with 2% BSA/20% fetal bovine serum in PBS for 15 min and preincubated for 10 min with One-Phor-All buffer (Amersham). Biotinylated nucleotides were incorporated into 3-OH DNA fragments by incubating with FLPCpure Terminal deoxynucleotidyl Transferase enzyme (TdT; Amersham) combined with Biotin-16-dUTP (Roche) for 2 h in a moist chamber at 37°C. Biotinylated nucleotides were then detected using Streptavidine Horseradish VectaStain ABC mouse kit (Vector Laboratories) followed by diaminobenzidine color-developing for 15 min and Hematoxylin (Sigma) counterstaining for 2 min.
DNA Laddering Assay
Twenty-five milligrams of frozen tissue were ground to a fine powder and homogenized using a minipestle and a microtube (Kontes) on ice in 500 µl of homogenizing buffer (0.1 M NaCl, 0.01 M EDTA [pH 8], 0.3 M Tris-HCl [pH 8], and 0.2 M sucrose). After adding 10 µl of 10% SDS and 10 µl of a 20 mg/ml solution of proteinase K (Roche), homogenates were vigorously shaken and incubated for 40 min at 55°C. Next, 22 µl of 10% SDS were added to the tubes, incubated for 30 min at 65°C, precipitated with 88 µl of 8% potassium acetate, and centrifuged at 4000 x g for 10 min at 4°C. Then, 600 µl of phenol:chloroform:isoamyl alcohol (25:24:1; v:v:v; Invitrogen) were added to the supernatant and centrifuged at 4000 x g for 10 min at 4°C. The aqueous phase was transferred to new tubes, and DNA was precipitated with absolute alcohol at –70°C overnight and then pelleted at 20000 x g for 30 min, air-dried, dissolved in Tris-EDTA buffer (10 mM Tris-HCl [pH 8] and 1 mM EDTA) containing DNase-free RNase (Amersham) and incubated at 37°C for 1 h. Next, 60 µl of phenol:chloroform:isoamyl alcohol (Invitrogen) were added and centrifuged at 4000 x g for 10 min at 4°C. The DNA from the aqueous phase was precipitated overnight at –70°C by adding 3 M sodium acetate and cold absolute ethanol. Samples were pelleted for 30 min at 20000 x g, washed in ice-cold 80% ethanol, and resuspended in Tris-EDTA buffer (pH 8). The DNA concentration was determined spectrophotometrically, and 20 µg of DNA samples were loaded in 0.75% agarose gel and run at 70 V for 3 h. Pictures were taken under ultraviolet light.
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. When 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.), with the level of significance for comparison set at P < 0.05.
RESULTS
Cellular Hyperplasia
The proliferative rates of myometrial tissue throughout gestation and labor were determined by BrdU incorporation followed by immunostaining of early pregnant (Gestational Day 6), late-pregnant (Gestational Day 18), and laboring (Gestational Day 23) rat myometrium (Fig. 1A). Smooth muscle 
-actin (also known as ACTA2) immunostaining was used to distinguish between smooth muscle and connective tissue proliferation (data not shown). A peak of DNA synthesis occurred during early gestation. The percentage of stained nuclei (stained/total x 100) in the gravid myometria was high at the beginning of pregnancy (26.5% ± 6.3% [values are mean ± SEM throughout] at Gestational Day 6, P < 0.003 vs. Gestational Day 23), decreased slightly by Gestational Day 12 (22.0% ± 8.0%), decreased significantly by Gestational Day 18 (11.8% ± 4.7%), and remained low at Gestational Day 23 (3.7% ± 1.4% at labor) (Fig. 1B). Interestingly, in early pregnant myometrium, a significantly (P < 0.001) increased number of positive nuclei were observed in the longitudinal muscle layer (33.3% ± 5.9%) compared with the circular muscle layer (3.9% ± 2.8%), suggesting a differential regulation of cellular proliferation between the two layers of uterine muscle (Fig. 1C). To further confirm the proliferative status of pregnant rat myometrium, we used PCNA, an endogenous marker of cell replication. The PCNA protein expression assessed by immunoblotting of myometrium from pregnant rats at selected time points throughout gestation and postpartum indicated a high level of proliferative activity during early pregnancy (4.6-fold increase at Gestational Day 6 vs. nonpregnant, P = 0.002, n = 4) that was maintained until midgestation but decreased to low levels at late gestation and postpartum (Fig. 2). Moreover, immunohistochemical data establish a similar pattern of PCNA staining throughout gestation, with strong immunoreactivity at early gestation and very weak staining in nonpregnant and late-pregnant myometrial samples (data not shown). This strong immunostaining of PCNA in SMCs was only detected in the longitudinal uterine muscle layer, compared with lower staining in circular uterine muscle. Both Western blot and immunostaining data were consistent with our BrdU method of assessing myometrial cellular proliferation (Fig. 1).
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Cellular Hypertrophy
Hypertrophic changes in the pregnant rat myometrium were determined biochemically by measuring the protein:DNA ratio at different gestational time points (Fig. 3). Total myometrial DNA content per horn increased slightly with gestation, whereas total myometrial protein contents per horn increased dramatically. Collectively, these data indicate a marked increase in myometrial cell size in the second half of pregnancy (starting after Gestational Day 15). Histologically, myometrial SMCs also showed hypertrophic changes throughout pregnancy. According to our observation, the sizes of individual myocytes and their nuclei clearly increased after Gestational Day 15 compared to those of early pregnancy (Figs. 1, 5, and 6).
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Apoptosis-Regulating Proteins
Interestingly, the apoptosis-regulating proteins BCL2 and Bcl-xL (also known as BCL2L1) were expressed differently in pregnant rat myometrium. Immunoblotting shows that BCL2 protein expression was abundant in nonpregnant and early pregnant myometrium (up to Gestational Day 12), then decreased dramatically during late gestation (17-fold decrease at Gestational Days 21 and 22 vs. nonpregnant, P < 0.001, n = 4) and returned to a high nonpregnant level by Postpartum Day 4 (Fig. 2). This expression pattern was very similar to that of the marker of cell replication PCNA (Fig. 2). Immunohistochemical staining demonstrated that BCL2 protein localized in the cytoplasm of myometrial cells (data not shown). A noticeable increase was found in the number of BCL2-positive myocytes in the longitudinal muscle layer compared with the circular muscle layer; this coincided with higher PCNA labeling and BrdU incorporation in the longitudinal muscle layer of the myometrium. Collectively, these data suggest differential regulation of myometrial proliferative activity between the two muscle layers and a determinant role of the antiapoptotic protein BCL2 during the proliferative phase of myometrial growth. In contrast, the antiapoptotic protein BCL2L1 showed a different protein expression profile, demonstrating a significant increase after midgestation on Gestational Days 14–17 (4- to 5.7-fold increase vs. nonpregnant, P < 0.001, n = 4) and a decrease at late pregnancy (Fig. 2).
Caspase Cascade Activation
We have examined the potential involvement of two different apoptotic pathways in myometrial growth. We have assessed the protein expression levels of initiator CASP10 (extrinsic pathway) and CASP9 (intrinsic pathway) throughout gestation. Our findings demonstrate that the expression of CASP10 in myometrial samples gradually declined from a high level in nonpregnant rats to a complete disappearance by Gestational Day 10 (Fig. 4). Conversely, cleaved CASP9 immunosignals were transiently elevated from Gestational Day 12 to Gestational Day 15, with the most intense signals being detected on Gestational Day 14 (28-fold increase vs. nonpregnant, P < 0.001, n = 4), followed by a sharp decline to almost undetectable levels by late gestation and postpartum (Fig. 4). The downregulation of CASP10 followed by a rapid elevation of cleaved CASP9 protein levels led us to conclude that the mitochondrial (intrinsic, stress-induced) pathway of apoptosis was preferentially activated in pregnant rat myometrium at midgestation. Once the initiator caspases are activated, they process and cleave downstream effector caspases, such as CASP3, CASP6, and CASP7. Western immunoblot analysis demonstrated a transient induction of active CASP3 proteins between Gestational Day 12 and Gestational Day 15, with a reduction to an undetectable level at late gestation and before labor (Fig. 4). Two downstream effectors, CASP6 and CASP7, showed the same expression profile (Fig. 4). In apoptosis, CASP3 is the main executioner caspase. We studied the immunohistochemical staining of active CASP3 in myometrial tissue sections. Cleaved CASP3 immunoreactivity was selectively found in myometrial SMCs at Gestational Days 12–14, confirming our protein expression data (Fig. 5C). To investigate further caspase involvement in downstream physiological changes occurring throughout gestation, we analyzed ADPRT, known previously as PARP-1, a caspase substrate frequently used as an early marker for cells undergoing apoptosis. We found that protein samples extracted from rat myometrial tissues collected on Gestational Days 12–15 contained a faint, 89-kDa protein band recognized by antibodies directed toward cleaved ADPRT catalytic fragment (Fig. 4).
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Apoptosis
The cleavage of genomic DNA into discrete fragments before membrane disintegration is a major hallmark of apoptosis. To characterize further the event of myometrial apoptosis, we used DNA ladder analysis to measure potential nonrandom DNA fragmentation. Our data showed a small amount of purified DNA fragments with the distinctive ladder pattern at midgestation, indicating a very mild increase in apoptosis at the time of the transient activation of the caspase cascade (data not shown). This result emphasizes the need to study the apoptosis in situ (i.e., in tissue sections), because apoptotic cells in vital organ tissue are rapidly and efficiently removed. We found a good correlation with the low levels of DNA laddering using in situ TUNEL labeling, which determines the cleavage of the genomic DNA into discrete fragments before membrane disintegration. Pregnant myometrium samples collected on different gestational days were negative for TUNEL staining. Figure 6 demonstrates TUNEL incorporation in pregnant rat uterus and shows that only one to three TUNEL-labeled nuclei were identified among the myometrial cells in each field of all layers of pregnant myometrium throughout gestation, mostly in areas close to the perimetrium. An untreated control coverslip and liver tissue samples produced no TUNEL-positive nuclei, whereas pregnant endometrium showed a massive apoptosis indicated by TUNEL-positive nuclei (Fig. 6, G and H).
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DISCUSSION
The normal turnover of uterine tissue represents a complex process involving cell proliferation, differentiation, and apoptosis, which together maintain tissue homeostasis. We have shown that myometrial hyperplasia is high during early gestation, likely reflecting endocrine-induced processes, and decreases dramatically later. Myometrial hypertrophy exhibits the opposite pattern, being low at the beginning of pregnancy and increasing considerably with gestational age. The transition from myometrial hyperplasia to hypertrophy is associated with transient activation of caspase cascade but not with biochemical or morphological features of apoptosis.
During the last 15 years, immunohistochemistry for the detection of BrdU incorporated into DNA has been widely used to identify proliferating cells in the S phase of the cell cycle [21]. The alternative method for the detection of cellular proliferation is PCNA cyclin expression and immunolocalization; PCNA is a nonnuclear auxiliary protein of DNA polymerase-delta and is fundamental in the initiation of cell proliferation, found predominantly in dividing cells, and relatively undetectable in resting cells (for review, see [15]). In the present study, PCNA protein expression and BrdU incorporation identified a distinct proliferative phase of uterine growth that endured for the entire first half of gestation. Using these methods, we also found that myometrial hyperplasia was most profound in the longitudinal muscle layer. Studies of uterine growth in pregnant rats have shown that initial growth involves lengthening of the uterine tube; this is consistent with the higher proliferative rate in the longitudinal muscle layer observed in the present study. Although other investigators have shown that an increase in cell division occurs during pregnancy, the present study is, to our knowledge, the first to characterize directly the pattern and extent of smooth muscle cell proliferation in pregnant myometrium. Whereas the mechanisms underlying the proliferative phase of myometrial growth remain to be determined, steroid hormones and/or growth factors likely mediate this response. The uterus undergoes sex steroid-regulated growth during each menstrual/estrous cycle. The maximal rate of proliferation in the myometrium of peripubertal rats occurs on the day of proestrus, the estrogen-dominant phase of the cycle, thereby suggesting that cell division in this tissue is driven by estrogen [9]. Moreover, myometrial cell proliferation is significantly increased in estrogen-treated primates, and it is increased even further in those treated with estrogen plus progesterone [23].
A growing body of evidence suggests that the mitogenic action of steroids (especially estrogen) in their target tissue is mediated, in part, through local production of growth factors, such as epidermal growth factor, IGF-I, and transforming growth factor, acting through paracrine and/or autocrine mechanisms [24]. Several in vivo and in vitro studies in different systems, including the uterus, have advocated this hypothesis [25–27]. Therefore, we speculate that the well-defined proliferative phase of myometrial growth at early gestation is sex steroid/growth factor-dependent and contributes to uterine growth by creating a pool of cells sufficient in number to undergo hypertrophy later, under the proper conditions.
Using protein:DNA ratios as a biochemical marker of hypertrophy, we have shown a marked increase in cell size during the second half of gestation. Moreover, we visually observed an increase in the size of myocytes after Gestational Day 15 in the course of our immunohistological studies. We estimated the high rate of myometrial cellular hypertrophy based on the fact that cell diameters were doubled and cell density was markedly decreased in late-pregnant myometrium. It is worth noting that myometrial hypertrophy occurs specifically during the second half of gestation, when fetal growth is maximal. Several groups in the past have suggested that in addition to endocrine factors, the enlarging fetus might induce uterine growth by stretching the uterine wall. For example, inflating the uterine horn of a nonpregnant rat with a balloon resulted in myometrial growth by a combination of both hypertrophy and hyperplasia [28]. This study reported an approximate doubling of cell size (protein:DNA ratio) and a 50% increase in cell number [28]. In unilaterally pregnant rats, three parameters of uterine growth (protein, RNA content, and DNA content) were dramatically increased in the gravid horn (subjected to stretch by growing fetus) on Gestational Days 14 and 16, while remaining unchanged in the nongravid horn [29]. Interestingly, the region of the uterus immediately surrounding the enlarging fetus and, consequently, experiencing the most significant degree of stretch demonstrated greater cellular hypertrophy (protein:DNA ratio) compared to that found in nonfetal sites [29]. These studies confirm that stretch plays an important and, perhaps, dominant role in promoting the growth of the uterus during pregnancy. The protein:DNA ratio is a widely used marker for hypertrophy. In the context of the uterus, however, this measurement may underestimate the true degree of myometrial hypertrophy, because polyploidal nuclei appear in the myometrium during pregnancy [30]. Consistent with this new finding, our previous studies have established that ACTG2 (also known as -actin) expression levels are consistently elevated from mid to late pregnancy [5], a time that we now reveal corresponds with a hypertrophic phenotype of the myometrial cells. A similar positive relationship between hypertrophy and increased expression of -actin has been reported in bladder (visceral) [4] and in hepatic portal vein (vascular) muscle [31].
In the present study, we show that around Gestational Days 12–14 in the rat, a sequential activation of the stress-induced apoptotic pathway in myometrium occurs, with a transient, overlapping induction of CASP3, CASP6, CASP7, and CASP9. The activation of caspase cascade was of particular interest to us, because the timing of the activation coincided with the major phenotypic modulation of the myometrium, the transition between hyperplasia and hypertrophy. We also found that the caspase substrate, the cleaved (inactive) form of ADPRT, was present in pregnant myometrial samples from Gestational Days 12–14. The ADPRT is involved in DNA repair predominantly in response to environmental stress and is essential for the maintenance of genomic integrity and survival. Intriguingly, we found no evidence of wide-scale apoptosis and no significant increase in synchronized nonrandom fragmentation of DNA using both in situ (TUNEL) and biochemical (DNA laddering) methods. This led us to hypothesize that transiently elevated caspase cleavage at midgestation may not stimulate a programmed cell death but, additionally, may be a characteristic signal to stop myometrial proliferative activity and to promote smooth muscle differentiation.
We suppose that transient activation of exogenous stimulus (intrinsic) apoptotic pathway in the myometrium at midgestation caused a cleavage of a vast variety of their substrate proteins. Cleavage of these substrates can have different consequences, such as cell-cycle arrest or cellular senescence. Generally, caspases in higher organisms have acquired functions in cellular processes other than apoptosis, namely cell survival and cell cycling [32]. Recent reports have revealed that at least part of the enzymatic machinery of the apoptosis cascade is engaged in normal differentiation: CASP3 has been shown to be essential in lens fiber differentiation [33] and in terminal differentiation of erythroid cells [34], and CASP3 and CASP14 have been described as being crucial for normal epidermal differentiation of keratinocytes [35]. Peptide inhibition of CASP3 activity or homologous deletion of Casp3 (Casp3-null mice) leads to a dramatic reduction of both myofibril formation and expression of muscle-specific proteins in skeletal myoblasts [32]. Further evidence to support a role for apoptotic signaling in skeletal muscle differentiation came from the results of Fernando et al. [36], who showed that in striated muscle, the cellular alterations associated with skeletal muscle differentiation share a high degree of similarity with key phenotypic changes usually ascribed to apoptosis. For example, actin fiber disassembly/reorganization is a conserved feature of both apoptosis and differentiating myoblasts, and a muscle-contractile protein, myosin light-chain kinase, is required for the apoptotic feature of membrane blebbing. Those authors also reported that CASP3 is required for skeletal muscle differentiation and acts through the myocyte regulatory factors, MYOD1 and MYOG (also known as myogenin) [36]. Collectively, these data, in combination with our own results, led us to hypothesize that apoptotic signaling in the pregnant uterine muscle might play a role similar to that described in skeletal muscle differentiation by mediating the end of the proliferative phase and the transition to the hypertrophic phase of uterine growth.
Interestingly, the major antiapoptotic protein BCL2 has been shown to prevent an activation of the caspase cascade [37]. It is an integral mitochondrial membrane protein that blocks apoptosis by preventing loss of mitochondrial membrane potential and release of cytochrome c [38]. In the present study, we found a significant decline in the expression of BCL2 protein at midgestation, the time of transition from proliferative to synthetic myometrial phenotype and transient activation of the caspase cascade. Interestingly, the expression profile of BCL2 protein was very similar to that of PCNA (Fig. 2). It is reasonable to speculate that BCL2 proteins could be protecting myometrial SMCs from premature termination of the proliferative phase of uterine growth. Another antiapoptotic protein, BCL2L1, showed a completely different expression profile, being upregulated in the period following activation of caspases. Known as one of the most important members of the antiapoptotic group of proteins, BCL2L1 may play a role similar to that of BCL2 by preventing the development of true apoptosis in myometrial tissue, which is incompatible with the function of this organ.
Despite the clear transformation that takes place in the myometrium and the possible importance of the different phases of uterine growth throughout pregnancy and labor, little is known about the causes and mechanisms of this transition from myometrial hyperplasia to hypertrophy. We believe that such transformation is triggered by the process called uterine conversion, an adaptive mechanism to accommodate the growing fetus [39]. During early pregnancy, the shape of the fetus is spherical, and maternal blood supply is abundant. The process of fetal growth continues until a critical time, specific to each species, when the conceptus reaches a maximum spherical radius and the uterine tissue is stretched. Tension is so great that it creates ischemia, resulting in circulatory stasis, which is detrimental to maternal blood flow (Fig. 7). The conversion of embryo shape from a sphere to a cylinder, which requires only a few hours, causes a release of uterine tissue tension and reestablishment of the maternal blood supply throughout the uterus [40]. Notably, late gestation is accompanied by rapid growth of the fetus, and it also is marked by a second period of mechanical stretch, which ends at parturition. It is a reasonable hypothesis that uterine conversion can cause transient ischemia in a stretched myometrium that can lead to a hypoxic response in this tissue and activation of the intrinsic apoptotic pathway. However, we showed that under normal circumstances, caspase cascade activation does not occur at term, when the uterine stretch is maximal. This leads us to conclude that the specific hormonal environment at late gestation blocks or attenuates the activating effects of mechanical stretch on the caspase cascade. Additionally, we cannot rule out the possibility that the transition from hyperplastic to hypertrophic growth at midgestation and, specifically, the activation of caspase cascade could be triggered by other factors derived from maternal decidua or the fetus itself.
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In summary, our data demonstrate that the myometrium undergoes gradual changes in phenotype from early to late pregnancy. These stages may be characterized by 1) an early proliferative phase, 2) the transition period characterized by caspase cascade activation, 3) an intermediate phase of cellular hypertrophy and matrix elaboration, and 4) the final phase in which the cells assume a contractile phenotype. We hypothesize 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 showed previously that these signals are important in regulating the onset of labor; however, their role in regulating earlier myometrial smooth muscle differentiation was ill-defined. We speculate that uterine conversion leads to ischemia, which in turn could activate the apoptotic cascade, and that might be the trigger for the switch from hyperplastic to hypertrophic growth. A detailed analysis of the mechanisms behind the transition of the myometrium between phenotypes and, specifically, the exact causes of caspase cascade activation might lead us to new ways to treat disorders of pregnancy associated with inadequate or premature myometrial transformation, thus preventing premature birth.
FOOTNOTES
1 Supported by a grant from the Canadian Institute of Health Research. 
2 Correspondence: Stephen J. Lye, Samuel Lunenfeld Research Institute at Mount Sinai, 600 University Avenue, Suite 870, Toronto, Ontario M5G 1X5, Canada. FAX: 416 586 8740; lye{at}mshri.on.ca
Received: 3 October 2005.
First decision: 1 November 2005.
Accepted: 11 January 2006.
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