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Department of Molecular and Integrative Physiology3
College of Medicine,4 University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, cervix, estradiol, pregnancy, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Information regarding the hormonal regulation of cervical growth during rat pregnancy is more limited. Previously, we demonstrated that relaxin promotes marked cervical growth [3, 6] and that it does so in part by increasing the rates of proliferation and decreasing the rates of apoptosis of both epithelial and stromal cells [17, 18]. Moreover, we recently demonstrated that the most dramatic effects of relaxin on these processes occur during late pregnancy [17].
Estrogen also likely contributes to growth of the cervix during pregnancy. Studies conducted with ovariectomized nonpregnant rats demonstrated that estrogen alone promotes marked cervical growth [19, 20]. Estrogen, like relaxin, may contribute to the accumulation of cervical cells during pregnancy by increasing the rate of cell proliferation and by decreasing the rate of apoptosis. Mitogenic actions of estrogen on the cervix and uterus in nonpregnant mice have been reported [21–24]. A study by Berman et al. [25] demonstrated an inverse correlation between serum estrogen levels and the percentage of cells undergoing apoptosis in the vagina of cycling rats. Moreover, bilateral ovariectomy resulted in an increase in the rate of apoptosis of vaginal cells, which was reversed following estrogen-replacement treatment [25]. To date, no study has examined the influence of endogenous estrogen on either cervical cell proliferation or apoptosis during pregnancy in any species. In rats, serum concentrations of estrogen steadily increase during the second half of pregnancy (Fig. 1A) [8], and estrogen likely plays an important role in regulating the rates of proliferation and apoptosis of cervical cells as pregnancy progresses.
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Limited evidence indicates that the elevated serum levels of progesterone during the second half of pregnancy (Fig. 1A) [9] may also regulate cervical growth. A recent study from our laboratory demonstrated that the administration of the progesterone antagonist RU486 into ovariectomized pregnant rats on Day 22 resulted in a tendency for increased cervical wet weight [15]. This is consistent with an earlier report that the administration of RU486 to rats on Day 18 of pregnancy caused a statistically significant increase in the cervical wet weight [26]. These limited findings in pregnant rats indicate that progesterone may restrain cervical growth, perhaps by inhibiting the accumulation of cervical cells. However, as is the case with estrogen, endogenous progesterone has not been reported to influence either cell proliferation or apoptosis of cervical cells during pregnancy in any species.
Based on the studies described above, we hypothesized that, like relaxin, estrogen increases the rate of proliferation and decreases the rate of apoptosis of cervical cells during the second half of rat pregnancy and that progesterone has the opposite actions during the same period. These hypotheses were tested in cervical tissues obtained from pregnant rats in which the actions of endogenous estrogen were blocked with the estrogen antagonist ICI 182,780 (ICI) and the actions of progesterone were blocked with the progesterone antagonist RU486. Serum concentrations of estrogen and progesterone as well as ratios of the concentrations of the hormones change as the second half of pregnancy progresses. Accordingly, the effects of blocking the actions of estrogen and progesterone on cervical cell proliferation and apoptosis were examined at 3-day intervals throughout the second half of rat pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal Treatment 1: Blocking the Actions of Endogenous Relaxin and Estrogen
Figure 1B shows the experimental design for examining estrogen's effects on the rates of cell proliferation and apoptosis of cervical cells during pregnancy. There were three treatments: PBS control, monoclonal antibody against rat relaxin (MCA1) and MCA1 + ICI. The PBS vehicle control was included to obtain baseline rates of proliferation and apoptosis. These rats were injected via tail vein with monoclonal antibody vehicle 0.5 ml PBS at 0900 h daily from Day 8 until cervices were collected at 0900 h on Days 10, 13, 16, 19, and 22 of pregnancy and Day 3 postpartum. At 0900 h on the 2 days before tissue collection, rats also received s.c. injections of 0.2 ml ICI vehicle sesame oil.
It is known that relaxin's effects on cervical growth in rodents are dependent upon estrogen priming [19, 20]. Antiestrogen treatment alone would be expected to block not only estrogen's actions but also relaxin's actions on cervical growth, and this treatment would not enable determination of the effects of estrogen alone. Accordingly, rats treated with the monoclonal antibody MCA1 against rat relaxin [29] were included to determine the extent to which relaxin alone contributes to the rates of cervical cell proliferation and apoptosis on the selected days. Treatment of the MCA1 group was the same as that of the control group except that rats were injected with 5 mg of MCA1 dissolved in 0.5 ml of PBS.
Finally, to determine estrogen's effects independent of those mediated in combination with relaxin, rats were treated with both MCA1 and ICI. At 0900 h on the 2 days before tissue collection, estrogen action was blocked with s.c. injections of ICI (Tocris, www.tocris.com) at a dose of 2 mg/kg. The 2-day ICI treatment duration was based on a preliminary study in which ICI treatment of Day 16 pregnant rats for 2 consecutive days effectively reduced the rates of proliferation of cervical stromal cells and maximally increased the rates of apoptosis of both epithelial and stromal cells (data not shown).
To evaluate cell proliferation, 800 µg of 5'-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich Corp., www.sigmaaldrich.com) was injected s.c. 8 h before the cervices were collected as previously described [17]. There were a total of six groups for each of the three treatments and six rats were used per group.
Animal Treatment 2: Blocking the Actions of Endogenous Progesterone
Figure 1C shows the experimental design for examining progesterone's effects on the rates of cell proliferation and apoptosis of cervical cells during pregnancy. There were two treatments: vehicle and RU486. The vehicle control provided baseline rates of proliferation and apoptosis of cervical cells during pregnancy. These rats were injected s.c. with 0.4 ml ethanol:sesame oil (1:7) 20 h before cervical tissues were collected at 0900 h on Days 7, 10, 13, 16, 19, and 22 of pregnancy and Day 3 postpartum. RU486 treatment was the same as the control treatment except that the rats received RU486 (Sigma-Aldrich Corp.) at a dose of 15 mg/kg. The dose and duration of RU486 treatment were based on a preliminary study in which cervices from Day 18 pregnant rats treated with RU486 at a dose of 15 mg/kg showed significant effects on the rates of proliferation of both epithelial and stromal cells (data not shown). We chose to treat the animals with RU486 for 20 h because data obtained by Ikuta et al. [26] and our laboratory [15] indicate that a majority of rats start to abort shortly after 20 h of RU486 treatment.
The rats were injected with BrdU 8 h before the cervices were collected as described in the estrogen study. There were a total of seven groups for each treatment, and six rats were used per group.
Tissue Collection and Processing
Animals were killed and cervical tissues were collected at 0900 h on the days described above. After trimming extraneous tissues, cervices were weighed and prefixed in 10% neutral buffered formalin for 2 h. Each cervix was then bisected in cross section to obtain cephalic and caudal halves. Fixation was continued in fresh neutral buffered formalin for a total of 24 h. After fixation, cervices were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin [30]. The cervical tissue blocks were sectioned at 5 µm thickness, mounted on positively charged slides (Superfrost Plus, Fisher Scientific, www.fishersci.com) and air-dried overnight. Tissue sections from each tissue block were at least 50 µm apart to ensure that different cervical cells were analyzed. Two sections from each block were analyzed.
BrdU Immunohistochemistry for Detection of Cell Proliferation
BrdU immunostaining was done as previously described [17]. Briefly, BrdU incorporation into proliferating cells was determined immunohistochemically using a mouse monoclonal antibody (clone NCL-BrdU; Vector Laboratories, Inc., www.vectorlabs.com) and a Vectastain Elite ABC kit (Vector Laboratories, Inc.). All incubations, unless otherwise noted, were performed at 25°C. Slides were cleared in xylene and rehydrated in a descending series of ethanol. DNA was denatured by incubation in aqueous 2 N HCl for 30 min at 37°C. After this and all other incubations, slides were rinsed in three changes of PBS (pH 7.5; 5 min/rinse). Antibody penetration was improved by incubation in 0.01% trypsin (Sigma-Aldrich Corp.) in PBS for 15 min at 37°C. Tissue sections were immersed in 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity. After incubation with blocking buffer (3% normal horse serum in PBS; Vector Laboratories, Inc.) for 30 min, anti-BrdU antibody (1:200 dilution in blocking buffer) was applied, and slides were incubated overnight (
16 h) in humidified chambers at 4°C. Biotinylated horse antimouse IgG and avidin-biotin-peroxidase complex were prepared as directed (Vectastain Elite ABC kit; Vector Laboratories, Inc.). Biotinylated horse antimouse IgG (1:65 dilution in blocking buffer) was applied for 30 min. The avidin-biotin-peroxidase complex was then applied for 30 min. Antibody binding sites were visualized using a 3,3'-diaminobenzidine peroxidase substrate (Peroxidase Substrate Kit; Vector Laboratories, Inc.) that was prepared as directed and applied to sections for 2–3 min. Slides were rinsed with distilled water for 10 min and counterstained with Ehrlich's hematoxylin for 10 min. Slides were washed with tap water for 10 min, then dehydrated in ethanol, cleared, and coverslipped with Permount (Fisher Scientific). For negative control slides, either anti-BrdU antibody was omitted (blocking buffer only) or nonspecific mouse IgGs were substituted (1:300 in blocking buffer; Sigma-Aldrich Corp.).
TUNEL Immunohistochemistry for Detection of Apoptosis
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling in conjunction with morphometric analysis was employed as previously described [18] to detect and quantify cells undergoing apoptosis. In brief, sections were stained immunocytochemically by TUNEL using the method described by Gavrieli et al. [31]. A commercial kit (ApopTag in situ Apoptosis Detection; Serologicals Corporation, www.serologicals.com) that links digoxigenin-nucleotide to DNA by terminal deoxynucleotidyl transferase (TdT) was used. Sections were deparaffined with xylene, rehydrated with a descending series of ethanol, incubated with proteinase K, immersed in 3% aqueous hydrogen peroxide, and then pretreated with equilibration buffer. DNA was labeled at the 3' end by incubating sections with a mixture of digoxigenin deoxynucleotide triphosphate, unlabeled deoxynucleotide triphosphate and TdT enzyme at 37°C for 1 h. Slides were washed with PBS, and then incubated with antidigoxigenin antibody conjugated to peroxidase at room temperature for 30 min. Slides were washed again in PBS, incubated with 3,3'-diaminobenzidine peroxidase substrate, counterstained with methyl green, mounted, and sealed. Positive control slides were treated with deoxyribonuclease I before the labeling reaction. Negative control slides were incubated with labeling reaction solution devoid of TdT enzyme.
Morphometric Analysis
To determine the rates of proliferation and apoptosis of cervical cells, the labeling index (LI) was determined from BrdU- and TUNEL-labeled sections, respectively. The LI was obtained by dividing the number of labeled cells by the total number of cells analyzed per section and multiplying by 100. Sections were examined morphometrically at 400x magnification with a BH-2 light microscope (Olympus Corp., www.olympus.com) equipped with a video camera and connected to a personal computer running a Stereo Investigator program (MicroBrightField, Inc., www.microbrightfield.com). The Stereo Investigator program automatically controls the movement of the microscope stage to permit unbiased selection of fields of analysis. Epithelial cells and subepithelial stromal cells were analyzed independently. The subepithelial stromal cells, those that are found between the epithelial cell layer and the circular smooth muscle cell layer, consist primarily of fibroblasts and cells associated with blood vessels. The circular and longitudinal smooth muscle cells found in the outer perimeter of the cervix were excluded from morphometric analysis because these cells showed little, if any, evidence of treatment effects. Data were obtained from four sections (two sections from each cervical half) and at least 300 epithelial cells and 500 stromal cells were analyzed per section. Thus, at least 1200 epithelial cells and 2000 stromal cells were analyzed for each of the six rats per group.
Statistics
Data were expressed as the mean ± SEM. To determine if there were significant differences in the BrdU LI and the TUNEL LI of vehicle-treated controls on different days they were tested using one-way ANOVA followed by a Tukey test. Statistical analysis of treatment effects was assessed using two-factor ANOVA, followed by preplanned least squares means comparison. The level of significance was set at P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The baseline rates of proliferation (see Figs. 2 and 4) and apoptosis (see Figs. 3 and 5) of cervical epithelial and stromal cells obtained with vehicle controls in the two experiments were similar and consistent with those we and others previously described [17, 18, 32]. Accordingly, subsequent description of results focuses on the treatment effects, that is, changes in the rates of proliferation and apoptosis of cervical cells that occur following treatments that block the actions of relaxin, estrogen, and progesterone.
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Effects of Blocking the Actions of Relaxin and Estrogen
Cervical wet weights following the blocking the actions of relaxin and relaxin plus estrogen are shown in Table 1. There was a progressive increase in the cervical weight during the second half of pregnancy in the PBS vehicle controls. This finding is consistent with several earlier reports [5, 18, 33]. When relaxin was immunoneutralized with MCA1, cervical wet weights were lower than in PBS controls on Days 16, 19, and 22 of pregnancy (P 0.01). Following combined blocking of the actions of relaxin and estrogen with MCA1 + ICI, cervical weight was lower than in group MCA1 on Days 13, 19, and 22 and there was a tendency (P = 0.08) for it to be lower on Day 16.
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The results of morphometric analysis of blocking the actions of relaxin and estrogen on cervical epithelial and stromal cell proliferation rates during pregnancy are shown in Fig. 2, A and B, respectively. Neutralization of endogenous relaxin with MCA1 significantly decreased the proliferation rate in both cellular compartments throughout the second half of pregnancy. Consistent with our previous study, its effects were increasingly pronounced as pregnancy approached term [17]. There was no effect of treatment either on Day 10 or on Day 3 postpartum; serum levels of relaxin are low on both days. When estrogen's actions were also blocked with ICI in MCA1-treated animals the proliferation rate in both cellular compartments was dramatically lower than in rats in which only the actions of relaxin were blocked. Unlike the neutralization of relaxin's actions, however, the effects of estrogen on cervical cell proliferation do not become increasingly pronounced as pregnancy approaches term. On Day 3 postpartum, when serum estrogen levels are low [8], the administration of ICI did not influence the rate of cervical cell proliferation.
The results of morphometric analysis of blocking the actions of relaxin and estrogen on cervical epithelial and stromal cell apoptosis rates during pregnancy are shown in Fig. 3, A and B, respectively. Consistent with our previous report [17], neutralization of relaxin with MCA1 increased the rate of apoptosis in both cellular compartments during the second half of pregnancy, with the most profound effects occurring during late pregnancy. There were no effects of treatment on Day 10 of pregnancy or on Day 3 postpartum. When estrogen's actions were also blocked, the apoptosis rate in both cellular compartments was far greater than in rats in which only the actions of relaxin were blocked. Blocking estrogen's effects on Day 10 and on Day 3 postpartum was without effect on the rates of apoptosis in both cellular compartments.
Effects of Blocking the Actions of Progesterone
Cervical wet weights following blocking of the actions of progesterone are shown in Table 2. Unlike the blocking of the actions of relaxin and estrogen, RU486 treatment increased the cervical weight on days 16, 19, and 22 of pregnancy.
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The results of morphometric analysis of RU486 treatment on cervical epithelial and stromal cell proliferation rates during pregnancy are shown in Fig. 4, A and B, respectively. The rate of cell proliferation in both cellular compartments was markedly lower than vehicle controls on Days 16, 19, and 22 of pregnancy but did not differ from controls on other days examined.
The effects of blocking the actions of progesterone on apoptosis in cervical cells were more complex (Fig. 5, A and B). As was the case after blocking the actions of relaxin and estrogen, blocking the actions of progesterone markedly increased the apoptosis rate in the stroma during late pregnancy. However, treatment with RU486 was without effect on the rate of apoptosis of epithelial cells during late pregnancy and actually decreased the rate of apoptosis in both cellular compartments on Day 7 and in the epithelium during the early stages (Days 10 and 13) of the second half of pregnancy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because estrogen promotes proliferation of cervical epithelial cells on Day 10 of pregnancy, it is reasonable to think that the circumference of the cervical lumen increases at this stage of pregnancy. This does not happen. Harkness and Harkness reported that the circumference of the rat cervix remains unchanged until Day 12 of pregnancy and then steadily increases during the rest of pregnancy to result in the 3-fold enlargement by term [5]. It may be that the estrogen-induced proliferation at midpregnancy is counterbalanced by progesterone-mediated apoptosis of cervical epithelial cells on the same day, with an outcome of increased cervical cell turnover and little or no change in the total cervical cell content. In support of this view, our data demonstrate that blocking the actions of progesterone inhibits apoptosis of cervical cells during midpregnancy (Days 7–10). By Day 13, our results indicate that both estrogen and relaxin promote cervical epithelial cell proliferation, whereas progesterone exerts little or no influence on the rates of proliferation and apoptosis on the same day. The combined effects of these three hormones likely bring about the beginning of the enlargement of cervical luminal circumference that occurs at this stage of rat pregnancy [5].
Caution must be taken when postulating hormonal responses based on the serum concentration of a hormone or the expression levels of its cognate receptor in target tissues. Whereas serum levels of estrogen rise as the second half of pregnancy progresses [8] and there is a tendency for increasing estrogen receptor 
(ESR1) expression in cervical subepithelial stromal cells during the second half of rat pregnancy [32], estrogen's effects on proliferation and apoptosis do not increase as pregnancy approaches term. Similar discrepancies are observed with progesterone. Despite the fact that the serum levels of progesterone decline rapidly at functional luteolysis on Days 21–22 of pregnancy (Fig. 1A), the hormone continues to promote cervical cell proliferation during this period. In contrast, during the middle of pregnancy, when serum progesterone levels are far higher than those found during Days 21–22, the hormone does not influence the cervical cell proliferation rates. Also, there seems to be no correlation between the expression levels of cervical progesterone receptors [32] and the magnitude of progesterone receptor-induced proliferation during rat pregnancy observed in the present study. The mechanism(s) by which cervical responsiveness to hormone is modulated is not known. It may be attributable to changes in the availability of coregulatory proteins at different stages of pregnancy [35].
An interesting finding with respect to progesterone's actions is that the hormone exerts inconsistent actions on apoptosis of cervical cells during pregnancy. Progesterone promotes apoptosis of cervical cells during midpregnancy (Days 7–10) but inhibits apoptosis of cervical stromal cells during late pregnancy. Whereas we cannot provide a mechanism that explains progesterone's opposite roles, this finding underscores the importance of examining the actions of hormones on a target tissue of interest throughout pregnancy rather than selecting a particular stage of pregnancy. Had this study focused on examining the effects of progesterone during late rat pregnancy, it would have failed to observe the opposing actions of progesterone on apoptosis of cervical cells.
The observation that RU486 treatment increased cervical wet weight on Days 16, 19, and 22 of pregnancy may seem inconsistent with the findings that the same treatment reduced the rates of cell proliferation and increased the rates of apoptosis of cervical cells on Days 19 and 22 of pregnancy. We postulate that the increase in cervical wet weight that occurs following RU486 administration [26] is attributable to an increase in the water content that more than compensates for loss of weight attributable to the reduction in the cervical cell content.
The present study lays important groundwork for subsequent studies aimed at gaining a better understanding of the hormonal regulation of cervical growth during late pregnancy. It does so by demonstrating that a comprehensive understanding of this phenomenon requires analysis of the roles of relaxin, estrogen, and progesterone. Additionally, by demonstrating that all three hormones have dramatic effects on cell proliferation and apoptosis, our study affords physiologically meaningful endpoints to be utilized for subsequent studies. For example, these endpoints might be used to gain a better understanding of the roles receptors for each of the three hormones play in regulating proliferation and apoptosis in the cervix. Receptors for relaxin [1, 36, 37], estrogen [32, 38, 39], and progesterone [32, 40, 41] have been reported to be present in epithelial cells and subepithelial stromal cells in rodent reproductive tract. One important objective that can likely be attained is the identification of the cellular components within the cervix that contain the receptors that mediate the effects of relaxin, estrogen, and progesterone on cell proliferation and apoptosis. Tissue reconstitution experiments using reproductive tissues (uterus and vagina) from wild-type and Esr1 knockout mice have demonstrated the roles of stromal and epithelial ESR1 in estrogen-induced responses [42–44].
In conclusion, this study provides evidence that estrogen and progesterone as well as relaxin contribute to the increase in the cervical cell content during late pregnancy by both promoting proliferation and inhibiting apoptosis of cervical cells. Their coordinated effects that lead to an increase in cervical cell numbers likely contribute to a rapid and safe delivery of the fetuses at term.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O. David Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801. FAX: 217 333 1133; od-sherw{at}uiuc.edu
Received: 20 May 2005.
First decision: 9 June 2005.
Accepted: 20 June 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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