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Regular Article |
a Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ER and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ER and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ER were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ER was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ER and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.
apoptosis, cervix, parturition, pregnancy, steroid hormone receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß and progesterone receptor (PR) expression, depending on the uterine cell type examined [2–7]. Comparatively little attention has been paid to the steroid receptor dynamics in the epithelial and stromal compartments of the uterine cervix. While some of the studies mentioned above considered the uterine cervix only as an extension of the uterus, it has been shown that the cervix presents different histologic and functional properties [8–11]. The uterine cervix is a dynamic structure with a high capacity to adapt to different, even opposing, roles during the sequence of physiological events of gestation (acting as a barrier to retain the fetus during pregnancy and afterwards dilating to allow a normal delivery) and has differential biological responses to modifications to the hormonal milieu [12–15]. These different responses imply many cellular and extracellular events, e.g., E2-mediated eosinophil infiltration [16, 17], steroid-hormone receptor expression [18], collagen metabolism [12, 19, 20], and fibroblastic cell plasticity [21]. The influence of ovarian steroid hormones on these events is still not fully understood.
In rodents, many studies have addressed cell proliferation and regression of the decidualized endometrium and its hormonal regulation throughout gestation [22–25]. It has also been demonstrated that, in the rat uterine cervix, both the accumulation and death of epithelial and stromal cells during the second half of pregnancy and at term are under hormonal influence [26–29].
There is a need to establish the normal range of physiological changes in the uterine cervix to facilitate the accurate interpretation of hormone replacement models and to better understand the biology of this organ. In the present study, the rate of proliferation and apoptosis in association with the dynamic expression of sex steroid hormone receptors in the rat uterine cervix during the estrous cycle, pregnancy, and postpartum were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female adult rats (over 200 g body weight) of a Wistar-derived strain bred at the Department of Human Physiology (Santa Fe, Argentina) were used. Animals were maintained under a controlled environment (22 ± 2°C; lights-on from 0600 to 2000 h) and had free access to pellet laboratory chow (Constantino, Córdoba, Argentina) and tap water. Vaginal smears were obtained daily, and only those animals showing three consecutive 4-day cycles were used [30]. To obtain pregnant specimens, females in proestrous were caged overnight with males of proven fertility. The day that sperm were found in the vagina was designated Day 1 (D1) of pregnancy. In our colony, delivery occured on D23 between 1230 and 1400 h. All rats were handled in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences.
Tissue Samples
Rats were randomly assigned to each of the different experimental groups (n = 3 or 4 animals per group). Two hours before being killed, rats were injected i.p. with 6 mg/100 g body weight bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO), a thymidine analogue. Animals were killed by decapitation. Uterine cervical tissue was obtained a) at noon of estrus and diestrus II, b) during pregnancy (D5, D9, and D12–D23), or c) in early postpartum (on the first day [PPD1] after parturition). Tissue samples were fixed by immersion in 10% buffered formalin for 6 h at room temperature (RT) and embedded with Paraplast paraffin (Sigma). Serial sections (4 µm in thickness) of whole cervices were taken along the cervical canal and mounted on 3-aminopropyl triethoxysilane-coated slides (Sigma) and dried for 24 h at 37°C. The evaluation of immunostaining in the uterine cervix was performed by dividing each section of tissue into three zones: epithelium, subepithelial stroma (an area adjacent to epithelium, 110 µm wide from basement membrane toward the outer layers), and muscular stroma. These three zones were chosen based on different cellular phenotypes and subepithelial fibroblastic cell plasticity [21]. Cellular turnover in blood vessels and its modulating mechanisms were excluded in this study, though ongoing experiments address this issue.
Immunohistochemistry
For immunohistochemistry (IHC), sections were deparaffinized and dehydrated in graded ethanols. BrdU incorporation to detect cells in the S phase of the cell cycle was evaluated as previously described [31]. In brief, an acid hydrolysis for DNA denaturation and microwave (MW) pretreatment for antigen retrieval were performed previous to routine immunohistochemical procedures. Sections used for immunostaining of ER and PR were also subjected to a MW pretreatment following the same previously described protocol [32]. Endogenous peroxidase activity and nonspecific binding sites were blocked. Primary antibodies were incubated overnight at 4°C. For BrdU labeling, a mouse monoclonal antibody against BrdU (clone 85-2C8, Novocastra, Newcastle upon Tyne, U.K.) was used at a dilution of 1:400. Immunostaining of ER was performed using a mouse monoclonal antibody raised to full-length recombinant human ER (clone 6F11, Novocastra) at a dilution of 1:60. PR was immunolocalized by applying a monoclonal antibody diluted 1:100 (clone AT, Affinity Bioreagents, Golden, CO). Reactions were developed using a streptavidin-biotin peroxidase method and diaminobenzidine (DAB) (Sigma) as a cromogen substrate. Samples were counterstained with Harris hematoxylin (Biopur, Rosario, Argentina) and mounted with permanent mounting medium (PMyR, Buenos Aires, Argentina).
Each immunohistochemical run included positive and negative controls. For negative controls, the primary antibody was replaced with nonimmune mouse serum (Sigma) or the IHC was performed in samples from animals that did not receive BrdU. Suppliers have tested specificity of the primary antibodies used.
Double IHC
To identify the immunophenotype of the proliferating stromal cell subtypes, double immunohistochemistry was performed after BrdU using anti-vimentin or anti--smooth muscle actin (-SMA) as a second primary antiserum. After BrdU immunostaining as described above, sections were MW heated (1 min at maximal power setting, followed by 4 min at 40% of full power and 10 additional min after power was turned off to wash out any remaining antibody from the former assay) [33]. Sections were rinsed with phosphate buffered saline (PBS) and preincubated again with normal goat antiserum for 30 min. Samples were then incubated overnight at 4°C with the second primary antibody, either anti-vimentin (clone V9, Novocastra) 1:100 or anti--SMA (clone sm-1, Novocastra) 1:50. Reactions were developed using a streptavidin-biotin-peroxidase method. Visualization of these antigens was achieved by the nickel-intensified DAB technique [34]. The DAB solution (2.3 mg DAB, 4 ml 0.05 M Tris-HCl buffer [pH 7.5], 15 µl 30% H2O2, and 460 µl 1% nickel chloride) was added to the samples. After 10 min at RT, the samples were rinsed in running water. Slides were counterstained with Nuclear Fast Red and mounted with permanent mounting medium. Proliferating stromal cells exhibited brown-stained nuclei (BrdU positive) and black-stained cytoplasm (either vimentin or -SMA positive).
In Situ Detection of Apoptosis
Sections were analyzed for in situ detection of cells with DNA strand breaks (apoptotic cells) using the TUNEL technique (ApopTag, Intergen Co., Purchase, NY). Manufacturer's instructions were followed. In brief, after deparaffinization and dehydration, sections were incubated with proteinase K (20 µg/ml) (Intergen) for 15 min at 37°C and then treated with hydrogen peroxide in PBS for 10 min at RT to quench endogenous peroxidase activity. Sections were then incubated with a mixture containing digoxigenin-labeled deoxynucleotide triphosphate (DIG-dNTP), unlabeled dNTP, and TdT enzymes in a humidified chamber at 37°C for 1 h. Slides were subsequently rinsed with PBS and incubated with anti-digoxigenin-peroxidase for 30 min at RT and substrate-chromogen mixture (DAB, Sigma) for 6 min. Samples were counterstained with Mayer hematoxylin, dehydrated, and mounted with permanent mounting medium. Negative control slides were run using the same procedures except that distilled water was added instead of TdT enzyme. As a positive control, involuting rat prostate after the second day of castration was processed in an identical manner to the experimental samples.
Evaluation of Cell Proliferation and Apoptosis
Tissue sections were evaluated using an Olympus BH2 microscope (illumination: 12-V tungsten-halogen lamp, 100 W, equipped with a stabilized light source; Olympus Optical Co., Ltd., Tokyo, Japan), with a Dplan 100x objective (numerical aperture = 1.25) (Olympus). Proliferative and apoptotic indices were obtained considering either the percentage of positive cells (for epithelial cells) or the volume fraction of positive cells (for stromal cells).
Percentage of proliferating epithelial cells was examined after BrdU incorporation (2000 cells were counted/tissue section). Proliferating subepithelial fibroblastic-like cells or smooth muscle cells were identified using double IHC for BrdU and vimentin or BrdU and -SMA, respectively. Volume fractions were calculated by applying the formula given by Weibel [35]: Vv = Pi/P, where Vv is the estimated volume fraction of the object, Pi is the number of incident points over positive cells, and P is the number of incident points over all cells in the studied population. To obtain the data for the point-counting procedure, a glass disk with a squared grid was inserted into a focusing eyepiece [36]. Vessel cells and infiltrating inflammatory cells, e.g., neutrophils, macrophages, and eosinophils, were excluded in all analyses.
Apoptotic index was expressed as a percentage of epithelial cells counted in randomly selected microscopic fields on a total of 2000 epithelial cells. Apoptosis in the stroma was evaluated in each defined region (subepithelium and muscular region) by applying the volume fraction formula as previously described.
Quantification of ER and PR Expression
In order to estimate the expression levels of ER and PR in each particular cell compartment of the cervix (epithelial, subepithelial, and muscular stroma), three quantitative measures were obtained: a) the percentages of stromal and epithelial cells expressing PR and ER, regardless of intensity; b) the intensity of the positive staining for both ER and PR (these numbers represent the relative amount of receptor protein per positive nucleus) [37]; and c) a score that represents the relative amount of receptor protein in each cellular compartment (this score was obtained by multiplying the average staining-intensity grade of each specimen by the percentage of positively stained cells).
Evaluation of the epithelium was done in 2000 cells per section. The percentage of stromal cells that expressed steroid receptors in the subepithelial and muscular stroma (a total of 1000 cells in each area) was analyzed. To evaluate staining intensity (optical density, OD) of both types of hormone receptors, an image analysis was performed following a previously described protocol [21, 38]. Briefly, images were recorded using a Dplan 100x objective (numeral aperture = 1.25) of systematic randomly selected fields of stromal and epithelial cells (n > 40 fields) with an Olympus BH2 microscope and a Sony ExwaveHAM color video camera (Sony Electronics, Inc., Park Ridge, NJ). Image analysis was performed using the Image Pro-Plus 4.1.0.1 system (Media Cybernetics, Silver Spring, MD). Several internal controls were established to maximize the level of accuracy and robustness of the method [37]. The microscope was set up properly for Koehler illumination, a reference image of an empty field was recorded for the correction of unequal illumination (shading correction), and the measurement system was calibrated with a reference slide before any measurements were taken.
Using Auto-Pro macro language, an automated standard sequence operation was created to measure OD. In this automated analysis, images of immunostained slides were converted into an eight-bit gray scale and calibrated so that the background staining of the histological slide was regarded as zero. The OD was measured as an average gray, being equal to the sum of the gray intensity of each pixel divided by the number of pixels measured. The resolution of the images was set to 640 x 480 pixels and the final screen resolution was 0.103 µm/pixel. Means for each rat were calculated and used for statistical analysis.
Statistics
Statistical analyses were performed using the Kruskal-Wallis one-way analysis of variance. Significance between groups was determined by Dunn posttest, and correlations were performed using Pearson analysis [39].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BrdU incorporation in the epithelium exhibited significant differences between diestrus II and estrus (Fig. 1A); increased cell proliferation was apparent in diestrus. Apoptotic indices showed a reciprocal pattern to proliferative indices (Fig. 2A; r = -0.85, P < 0.05); more epithelial cells were in apoptosis during estrus than during diestrus II (Fig. 1D). The subepithelial region is mainly composed of two groups of cells: fibroblast-like cells and blood vessels cells. As was mentioned in Materials and Methods, vascular cells were not included in the stromal quantitation. BrdU incorporation in the stroma was restricted to the subepithelial region (Fig. 1B); there was no evidence of proliferation in the muscular region (Fig. 1C). As in the epithelium, fibroblastic cell proliferation was higher in diestrus II than during estrus (Fig. 1B). Apoptotic indices in the subepithelial region showed no differences between diestrus II and estrus (Fig. 1E). Proliferative and apoptotic indices in the muscular region were the lowest compared with other histological areas studied, and no differences were found between estrus and diestrus II (Fig. 1, C and F).
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During the Estrous Cycle, ER and PR Expression Exhibit Specific Variations According to Cell Subtype Studied
Steroid receptor scores in the studied compartments of uterine cervix are shown in Figure 1, G–L. In the epithelium, higher values of ER expression were observed in diestrus II versus estrus (Fig. 1G). Staining intensity patterns were significantly different, while the percentage of positive cells showed no variation between the stages (Table 1). We found that ER expression correlated negatively and significantly with programmed cell death in epithelial cells of the uterine cervix during the estrous cycle (Fig. 2B, r = -0.89, P < 0.01). The ER score in the subepithelial stromal compartment showed no statistical differences between the stages studied (Fig. 1H). As in the epithelium, the ER score in the muscular region was significantly higher during diestrus II (Fig. 1I). The PR score in epithelial cells was higher in estrus compared with diestrus II (Fig. 1J); different scores were due to changes in the percentages of positive cells (Table 1). Whereas more than 90% of cell nuclei in the epithelium expressed PR during estrous, a low percentage of positive cells were found in diestrus II (Table 1). A positive correlation was observed between PR expression and apoptosis in epithelial cells (Fig. 2C, r = 0.91, P < 0.001). No differences were observed in PR expression in either stromal compartment (Fig. 1, K and L). Even though no differences in PR scores were found in the stroma, a significantly higher percentage of positive cells (fibroblastic and muscular) were observed in diestrus II versus estrus (see Table 1).
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While High Proliferative Activity Is Observed Throughout Pregnancy, High Apoptotic Indices Are Present Exclusively at Postpartum
As is shown in Figure 3A, epithelial BrdU incorporation showed remarkable differences throughout pregnancy with two significant peaks, one in the middle of gestation (D12–D14) and one close to the end of gestation (D20 and D21). At term (D23), proliferating epithelial cells showed the lowest value found during pregnancy; however, 24 h after delivery (PPD1), proliferation was even lower. Representative photomicrographs are shown in Figure 4, A–C.
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In the subepithelial layer, the proliferative index exhibited a significant increase in the second third of pregnancy, while very low proliferative activity was observed at the end of pregnancy and PPD1 (Fig. 3B). Representative photomicrographs are shown in Figure 4, A–C. Besides fibroblast-like cells, a significant number of endothelial cells incorporated BrdU throughout pregnancy, but as was mentioned previously, they were not quantified in this study. In both the circular and longitudinal layers of smooth muscle cells, very low proliferative activity was observed during pregnancy and postpartum (Fig. 3C).
Throughout pregnancy, apoptotic activity in the epithelium never exceeded 1.8%, with the highest scores for programmed cell death on D5 and the lowest indices between D13 and D23 (day of parturition) (Fig. 4, D–F). A dramatic increase in epithelial apoptosis was observed on the day after parturition (PPD1), reaching values of 9% (Figs. 3D and 4F). In the subepithelial layer as well as in the muscular compartment, low apoptotic scores were observed throughout pregnancy (Fig. 3, E and F). On PPD1, both stromal compartments showed increased apoptotic indices, as was already described for the epithelium; however, the apoptotic rate in the stroma was always lower than in the epithelium (Fig. 3, D–F; Fig. 4, D–F). Apoptosis was not observed in endothelial cells.
During Pregnancy and Postpartum, ER and PR Expression Differ in the Regions Studied
In the epithelium, ER expression changed throughout pregnancy (Fig. 3G). All score variations were due to fluctuations in ER staining intensity, while no changes were observed in the percentage of positive cells (Table 1). A significant increase in ER expression was found at the middle of gestation, while 24 h after parturition, expression was very low. Epithelial ER scores were correlated in a negative manner with the apoptotic index (Fig. 5A, r = -0.77; P < 0.001).
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In the subepithelial layer, ER scores showed no statistical differences throughout pregnancy or postpartum (Fig. 3H). In contrast, the muscular region showed a significant decrease in ER expression during PPD1 (Fig. 3I). Differential expression of ER is illustrated in Fig. 4, G–L.
PR scores in epithelial cells throughout pregnancy were remarkably low, with a significant increase at term (D23) (Fig. 3J; Fig. 4, M and O). This high PR expression in epithelial cells at term was maintained through PPD1 (Figs. 3J and 4Q). Changes in the percentage of PR-positive cells accounted for all variations on PR scores (Table 1). Image analysis showed that the intensity of immunostaining, evaluated as OD, showed no change during the studied periods. No differences were observed in PR expression in subepithelial cells during pregnancy or postpartum (Fig. 3K; Fig. 4, M, O, and Q). In contrast, muscular cells exhibited an increase in receptor expression on D13 that was maintained until D23 and decreased significantly at PPD1 (Fig. 3L; Fig. 4, N, P, and R). When muscular PR expression and apoptotic index were compared (Fig. 5B), a strong negative correlation arose between both variables (r = -0.90, P < 0.0001).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During pregnancy, the basal proliferative activity of the epithelium is modified, exhibiting two well-differentiated peaks. The first is observed at the middle of gestation (D12–D14) and the second close to delivery (D20 and D21). It has recently been reported that relaxin (Rlx) promotes a dramatic increase in the accumulation of new epithelial cells in the rat cervix [26]. In agreement with those results, we observed an epithelial proliferation pattern temporally associated with that exhibited by Rlx levels during pregnancy [42]. Moreover, immediately after delivery when serum Rlx drops, epithelial proliferation decreased in parallel. However, results obtained during the estrous cycle (diestrus II and estrus) suggested that, besides Rlx, other hormones might also be involved in modulating the proliferative activity of the cervical epithelium.
BrdU incorporation in cervical stroma in all studied physiological stages was always greater in the subepithelial layer than in deeper layers (muscular layers). It seems likely that these results are attributable to differences in the phenotype of cells that populate both regions [21]. P4 appears to be an essential mitogen for stromal cell in the decidua basalis during early pregnancy (D8–D10), but this action is lost by D14 [24]. In the present study, we found that, during pregnancy, fibroblastic cells were the predominant BrdU-incorporating cellular subtype in the stroma. Evidence of an association between high P4 levels and fibroblastic proliferation was observed. This suggests that the mitogenic effect of P4 could be mediated through its own receptor since there is a trend of increased expression of PR following the described pattern of P4 serum levels [43]. Similar evidence for the proliferative effects of P4 in stromal cells has been reported for the mouse uterus [44]. The pattern of stromal cell proliferation during pregnancy differed from that of epithelial cells.
Different results concerning changes in the rate of rat cervical cell apoptosis during pregnancy have been reported [27, 29, 45]. While Leppert's group [27, 45] described apoptosis as increasing progressively in cervical smooth muscle cells and fibroblasts during the second half of pregnancy, a recent report [29] and the present study revealed that the apoptotic index declines during the second half of pregnancy in both cervical epithelium and stroma. Moreover, apoptosis is the predominant event during postpartum involution and could contribute to the recovery of the uterine cervical structure after delivery.
In several adult endocrine-dependent organs, apoptosis is reported to take place after hormone withdrawal [46, 47]. Cell death occurs in all cellular compartments during the estrous cycle, indicating that apoptosis is a physiological phenomenon in rat reproductive tracts [48, 49]. Our results in the luminal epithelium of the uterine cervix at estrus are coincident with previous reports of the rat uterus, where apoptotic cells correlate with the reduction of E2 plasma levels [49, 50]. Moreover, we have found a significant inverse correlation between cell death and cell proliferation in rat cervical epithelial cells during the estrous cycle.
PR was highly expressed in fibroblastic and smooth muscle components of the cervical stroma during pregnancy, whereas low levels of PR immunoreactivity were observed in cervical epithelium. At parturition (D23), a dramatic increase in epithelial PR-positive cells occurred and was maintained during postpartum. In contrast, in smooth muscle cells, a significant decrease in PR immunoreactivity was found after delivery. Taken together, these results suggest that, during pregnancy and postpartum, there was a differential regulation of PR expression in the different compartments studied. High levels of P4 during pregnancy could be responsible for a downregulation of its own receptor in the epithelium of the uterine cervix. On the other hand, at the end of pregnancy, when very low serum levels of P4 are coincident with elevated levels of E2, PR is highly expressed in the epithelium. These results are in agreement with those reported by Tibbetts et al. [3] for the mouse uterine glandular epithelium. In the smooth muscle compartment, PR expression could not have been downregulated by P4 during pregnancy; however, the decrease in PR immunoreactivity at PPD1 could be explained by the rise of E2 and P4 withdrawal. In this sense, E2 and P4 could exert both positive and negative effects on PR expression in the uterine cervix depending on the cell types examined.
Furthermore, no difference in ER immunoreactivity was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease was observed in the epithelial compartment after delivery. These results are coincident with a recent study in human cervix where it was shown that ER mRNA and protein expression in the epithelium were significantly decreased after parturition when compared with term or nonpregnant women [51].
In the cervical epithelium, high expression of PR and a decreased immunoreactivity of ER form the steroid receptor profile that enclose very low levels of proliferation and widespread apoptosis during the postpartum period. This steroid receptor profile is extended to the estrous cycle since, during estrus, higher apoptotic indices are correlated with low levels of ER immunoreactivity and elevated expression of PR. The significance of and mechanisms underlying these observations still need to be clarified. Because ER and PR protein may have a long half-life, time-dependent correlations between hormone receptor status versus proliferative/apoptotic indices could be better understood by studying ER and PR mRNA expression using RT-PCR and in situ hybridization. These methods might prove more suitable for studying short-term changes in gene expression during hypervariable endocrine periods, like the rat estrous cycle and peripartum period.
It is obvious that the rat estrous cycle, pregnancy, and postpartum are physiologically distinct. We have additionally shown that there were differential responses to signals that modulate proliferation and/or apoptosis in the same physiological stage according to the cellular compartment evaluated. We believe that these types of studies will develop the background knowledge necessary to elucidate the biological meaning of cell turnover and steroid receptor dynamics observed in hormone replacement experiments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants from the Argentine National Council for Science and Technology (CONICET) (PIP 528/98) and the Argentine National Agency for the Promotion of Science and Technology (ANPCyT) (PICT-99 No. 5-7001). J.V. and J.G.R. are Fellows and E.H.L. is Career Investigator of the CONICET. 
2 Correspondence: Mónica Muñoz-de-Toro, Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Casilla de Correo 242, 3000 Santa Fe, Argentina. FAX: 54 342 4575207; monicamt{at}fbcb.unl.edu.ar
Accepted: April 3, 2002.
Received: December 22, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß in the rat uterus. Biol Reprod 1999 61::955-964 and ß in the female reproductive tract of the rat during the estrous cycle. Biol Reprod 2000 63:1331-1340 and ß in the human cervix at term pregnancy. Mol Hum Reprod 2001 7:293-300This article has been cited by other articles:
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C. P Read, R A. Word, M. A Ruscheinsky, B. C Timmons, and M. S Mahendroo Cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in mice Reproduction, August 1, 2007; 134(2): 327 - 340. [Abstract] [Full Text] [PDF]
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