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Regular Article |
a Department of Human Physiology, Faculty of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina
b Department of Histology and Embryology, Institute of Biology, UNICAMP, Campinas, 13083-970 São Paulo, Brazil
c Laboratory for Cell Biology, The University of São Paulo School of Medicine, 01246-903 São Paulo, Brazil
ABSTRACT
Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and
-smooth muscle actin (-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never -SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.
cervix, female reproductive tract, myofibroblast, parturition, pregnancy, uterus
INTRODUCTION
During pregnancy the uterine cervix acts as a barrier, retaining the fetus and preventing the entry of physical and microbiological agents that perturb the normal development of the conceptuses [1]. Compared to other parts of the reproductive tract, the uterine cervix of the rat is a rather compact structure that can be easily distinguished, although its transition to the uterine horns is somewhat gradual. Histologically, connective tissue represents 50–65% of all the tissue in the rat [2]. Smooth muscle cells are sparsely represented in the exocervical portion but increase cranially [3]. At the time of parturition, dramatic changes, such as a widespread collagen remodeling and changes in proteoglycan metabolism, polymorphonuclear infiltration, and water imbibition, have been observed [4–8]. These histological modifications are associated with the cervical ripening that permits the dilation and effacement of the organ to allow a normal delivery [9, 10]. These characteristics show that the cervix is a dynamic structure with a high capacity to adapt to different and conflicting physiological situations.
Most of the above-mentioned studies have focused on changes at the extracellular matrix (ECM) of the cervical stroma. Comparatively little attention has been paid to the cellular compartment, composed of fibroblasts, smooth muscle cells, and capillary buds. The fibroblastic cells in the rat cervical stroma are distributed in the fibrous ring that surrounds the mucous layer and are also scattered between the longitudinal and transversal layers of smooth muscle cells [2, 11].
In many physiological or pathological situations, marked alterations in the structural and biochemical architecture of fibroblast and fibroblast-like stromal cells were observed, suggesting an important phenotypic plasticity that permits adaptation [12, 13]. As part of this cellular transformation, one might also expect changes in cytoskeletal elements. Desmin, vimentin, and -smooth muscle actin (-SMA) display cell-specific localization and are frequently expressed in a specific pathway of differentiation [14, 15].
It has been suggested that different organs contain fibroblasts with specific features [16, 17]. Komuro [18] has proposed a classification of fibroblasts into subtypes depending on their main functions: 1) fibrogenesis, 2) tissue skeleton or barrier, 3) intercellular communication system, 4) gentle contractile machinery, 5) endocrine activity, and 6) vitamin A storage. Other specific functions or features could be added to this list [19], indicating the complexity of fibroblast biology. Immunohistochemical studies disclosed heterogenous cytoskeletal phenotypes among all these fibroblast-like stromal cells (myofibroblasts). In normal human and animal tissues the most frequent phenotypes found are represented by cells positive for vimentin and desmin (VD phenotype) and by cells expressing vimentin and -SMA (VA phenotype) [13, 19, 20]. Whereas cells expressing vimentin, -SMA, and desmin (VAD phenotype) and cells positive for vimentin, -SMA, desmin, and smooth muscle-myosin heavy chains (VADM phenotype) are observed only in pathological situations [13, 19, 20].
The aim of this study was to investigate the morphological and immunohistochemical characteristics of fibroblastic cells of the fibrous ring that surrounds the mucous layer of uterine cervix in cycling, pregnant, and postpartum rats. For the first time, we report here differential expression of the intermediate filament desmin in the fibroblastic cells of the rat uterine cervix under different physiological conditions. In addition, we observed some ultrastructural features characteristic of myofibroblasts in the lamina propria of the uterine cervix in late pregnancy. Considering the functions listed above, fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.
MATERIALS AND METHODS
Animals
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 controlled environment (22 ± 2°C; lights on from 0600 to 2000 h). Animals had free access to pellet laboratory chow (Nutric, Córdoba, Argentina) and tap water. Vaginal smears were used to confirm normal cycling and for the identification of specific stages of the estrous cycle [21]. To obtain pregnant specimens, proestrous females 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 occurs on D23 between 1230 and 1400 h. Postpartum samples were collected from lactating mothers on the first (D24) and second days (D25) after parturition.
All animal work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Academy of Sciences (USA).
Tissue Sample Collection and Processing for Light and Electron Microscopy
Rats were randomly assigned to each of the different experimental groups (three or four animals per group). Uterine cervical tissue was taken on the morning of two different stages of the estrous cycle (estrous and diestrus), during pregnancy (D5, D9, and D12 through D23), and during postpartum involution (D24 and D25). Tissue samples were fixed for light microscopy by immersion in 10% buffered formalin for 6 h at room temperature (RT). For electron microscopy the samples were obtained under a stereoscopic microscope and fixed in 2% glutaraldehyde (Sigma Chemical Co., St. Louis, MO) and 0.1% tannic acid (Sigma) in a 0.1 M cacodylate buffer (pH 7.4) for 2 h at RT.
For light microscopy, fixed tissues were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Serial sections (5 µm in thickness) of whole cervices were taken along the cervical canal, mounted on 3-aminopropyl triethoxysilane (Sigma)-coated slides, and dried for 24 h at 37°C.
For electron microscopy, fixed cervices were rinsed in saline solution, postfixed with 1% OsO4 (Sigma), dehydrated with acetone, and embedded in Epon 812 resin (Polyscience, Warrington, PA). Semithin sections were cut and stained with methylene blue. Representative areas of the lamina propria (using the epithelium and the basement membrane as a guide) were selected. Ultrathin sections were studied under an LEO 906 (LEO Electron Microscopy Ltd., Oberkochen, Germany) transmission electron microscope.
Immunohistochemical Detection of Vimentin, Desmin, and -SMA
Immunostaining was done as previously described [22]. In brief, after deparaffination, microwave pretreatment (antigen retrieval) was performed. The endogen peroxidase activity was inhibited, and nonspecific binding was blocked. Sections were incubated with monoclonal antibodies for 3 h at RT. The following mouse monoclonal antibodies were used: anti-desmin (clon DER-11; Novocastra, Newcastle upon Tyne, UK) 1:50; anti-vimentin (clon V9; Novocastra) 1:100 and anti--SMA (clon sm-1; Novocastra) 1:100. Visualization of antigens was achieved by the nickel-intensified diaminobenzidine (DAB) technique [23]. The DAB solution was composed of 2.3 mg DAB (Sigma), 4 ml 0.05 M Tris-HCl buffer (pH: 7.5), 15 µl 30% H2O2, and 460 µl 1% nickel chloride. Chromogen substrate was used for 10 min at RT, then rinsed in running water. Samples were mounted with permanent mounting medium (P.M.Y.R., Buenos Aires, Argentina).
Measurement of Vimentin, Desmin, and -SMA Expression by Image Analysis
Image analysis was performed using the Image Pro-Plus 4.1.0.1 system (Media Cybernetics, Silver Spring, MD). The images were recorded with a Sony ExwaveHAM color video camera (Sony Electronics Inc., ParkRidge, NJ) attached to an Olympus BH2 microscope (illumination: 12-V halogen lamp, 100 W, equipped with a stabilized light source) (Olympus Optical Co., Ltd, Tokyo, Japan), using a Dplan 100x objective (NA = 1.25). 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 measurement started.
Using Auto-Pro macro language, an automated standard sequence operation was created to measure the optical density (OD). In this automated analysis process, images of immunostained slides were converted to an eight-bit gray scale, and the operator calibrated the gray level so that the background staining of the histological slide was regarded as zero. The optical density 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.
Expression of the cytoskeletal proteins was quantified in the lamina propria of the uterine cervix, in a 110-µm wide area adjacent to the epithelium (from the basement membrane toward the outer layers). Microscopic fields covering the whole subepithelial cervical stroma (more than 40 fields) from each section were recorded. Three different sections were evaluated for each cervical specimen. Means for each rat were calculated and used for statistical analysis.
Statistics
Differences between groups were evaluated by the Kruskal-Wallis one-way analysis of variance. Probabilities were assigned using the Mann-Whitney U-test [24].
RESULTS
Expression of Vimentin, Desmin, and -SMA in Cervical Fibroblastic Cells
In all physiological stages studied (i.e., cycling, pregnant, and postpartum) fibroblastic cells of the rat cervical tissue were always positively stained with anti-vimentin and anti-desmin but never reacted with anti--SMA antiserum. Immunohistochemical expression for vimentin was fairly constant throughout pregnancy and showed no differences with the fibroblastic cells from cycling and postpartum uterine cervices (Table 1)().
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Whereas vimentin expression did not change in intensity in any stage studied, desmin intensity varied dramatically during pregnancy (Fig. 1). The staining pattern for desmin corresponded to a halo surrounding the nucleus at low expression, and radiating through the cytoplasm up to the cell membrane when maximal expression was achieved (Fig. 1).
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Very low expression of desmin was observed in fibroblastic cells of cycling rats (estrus and diestrus) and during postpartum (D24 and D25) (Fig. 2). As shown in Figure 2, during the first half of pregnancy (D5, and from D9 through D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found in uterine cervical fibroblasts from D15 to D22 of pregnancy. Maximal expression of desmin occurred at the day of parturition (D23 of pregnancy) with an acute decline after delivery. Due to this transient expression of desmin, fibroblastic cells achieved a significant coexpression of vimentin and desmin during the second half of pregnancy.
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Ultrastructural Features of Cervical Fibroblastic Cells
As well as light microscopic studies, electron microscopic observations were perfomed on the fibrous layer adjacent to the epithelium (lamina propria) of cervices from pregnant, postpartum, and cycling rats. Coincident with the transient desmin expression described above, ultrastructural features showed typical characteristics of myofibroblasts in late pregnancy (Fig. 3). These cells displayed remarkable alterations in their size, shape, and distribution of their organelles. An increase in total cell volume was observed, as well as a change in shape (from fusiform to rounded-ovoid) with large euchromatic nuclei. Fibroblastic cells of cervices from pregnant animals presented bundles of intermediate filaments, and in some cases, the cells had long cytoplasmic extensions (Fig. 3). The abundant cytoplasm is well endowed with rough endoplasmic reticulum and Golgi complex (Fig. 3). The presence of junctional complexes between the interior of these fibroblastic cells and the adjacent stroma is evident (Fig. 4); these structures have the characteristics of the so-called fibronexus, which is a transmembrane complex involving intracellular microfilaments in apparent continuity with ECM fibrillary structures. These junctions are believed to be concerned with the transmission of contractile force within the tissue and are one of the distinctive characteristics of myofibroblastic cells. Other ultrastructural features found in cervical myofibroblastic cells are a few pinocytosis vesicles alongside the linear densities and discontinuous thin strips of basal lamina-like material adjacent to the cell membrane. These ultrastructural features, taken together, resemble the myofibroblastic phenotype defined in previous morphological descriptions that suggested both secretory and contractile roles [13, 25–27].
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Fibroblastic cells from cycling rats displayed features of the different stages of fibroblastic function. Fibroblasts from early stages of pregnancy shared these ultrastructural characteristics, displaying a full range spectrum from relatively undifferentiated (inactive) fusiform fibroblasts (Fig. 5), through fibroblasts actively synthesizing ECM, to myofibroblastic-like cells. While pregnancy progresses, many fibroblasts are activated, showing increased secretory characteristics (Fig. 6) and reaching the features of typical myofibroblasts at the end of pregnancy (Fig. 4).
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DISCUSSION
Understanding the complex molecular biology underlying the dynamic function of the uterine cervix is a basic problem in the physiology of mammalian gestation and parturition. During pregnancy, extensive tissue remodeling involves both the ECM and cells of the cervical tissue [7, 28]. The cellular and extracellular compartments must adapt themselves, in a very short period of time, to new functional demands imposed by gestation and parturition and subsequently restore themselves during the following involution period [3]. The present study gives a detailed description of the morphological and immunohistochemical features observed in the fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats.
To our knowledge, this is the first report that addresses the presence of cells with structural and biological properties between those of resident fibroblasts and those of smooth muscle cells in the lamina propria of the rat uterine cervix. These cells were defined as myofibroblasts, because the ultrastructural features and the immunohistochemical expression of both vimentin and desmin agree with the descriptions made by other authors [19, 25]. Myofibroblasts have been found in a variety of organs such as rat intestinal villi [29], periodontal ligament [30], rat and mouse adrenal gland capsule [31], rat testicular capsule [32], etc. In all these organs, myofibroblasts showed smooth-muscle differentiation features, suggesting that some normal stromal cells are equipped with smooth-muscle structures.
In the uterus, regionally specific changes might be expected, because at the end of pregnancy, the different parts of the uterus should serve the dual but conflicting functions of acting as a barrier to retain the conceptuses and opening the cervix at the time of delivery. This dynamic role of the uterine cervix is achieved by the interplay between both the ECM and the cellular compartment. The ECM is susceptible to undergoing profound changes such as a widespread collagenolysis, changes in proteoglycan metabolism, and water imbibition [5, 33, 34]. However, in this changing environment, the cellular compartment (composed mainly of fibroblastic and smooth muscle cells) has to maintain the basic structure of the organ, including epithelial continuity and blood supply.
The variable expression of desmin found during different days of pregnancy might contribute to the cellular compartment adaptation to the changes of the regional forces produced by alterations in the ECM composition. Therefore, given the role that microtubules, microfilaments, and intermediate filaments play in determining cellular morphology and function, it is possible that the temporal increase of desmin expression in myofibroblasts could account for the rapid transformation of cell shape and organelle distribution as a consequence of increased synthetic activity of collagen and elastin fibers during pregnancy [15]. Whereas the persistence of vimentin, in either nonpregnant or pregnant rats, may reflect nonspecific cell integrity roles, such as centralization of nuclei and abutment of nuclear membrane, rather than playing a specific role during pregnancy adaptation [35].
An important aspect of the differential expression of desmin is its mechanism of control. It is interesting to mention that desmin expression increased in parallel with the plasma levels of relaxin described for pregnant rats [36]. Relaxin reaches significant plasma levels from D15 of pregnancy, showing a significant elevation 25–30 h before parturition. Desmin increased its expression from D15, and the highest levels of expression are reached just before parturition. Moreover, immediately after delivery, both relaxin and desmin reach basal levels. On the other hand, it was also demonstrated that progesterone promotes differentiation and growth of the stromal cells in uterine decidua [37]. The possible role of progesterone and/or relaxin on the regulation of fibroblastic plasticity observed in the uterine cervix needs to be investigated.
Based on the differential expression of desmin and the ultrastructural features, the foregoing results demonstrate that myofibroblasts are present in the fibrous ring of the rat uterine cervix, illustrating fibroblastic cell plasticity. Ongoing studies are addressing hormonal regulation and the functional significance of the above-mentioned event. Studies that investigate the correlations between the morphological changes of the cervical fibroblasts and the dynamic changes of the mechanical environment are required to understand the role of these cells in cervical dilation during parturition.
ACKNOWLEDGMENTS
We are very grateful to Dr. Julio Piva (Santa Fe, Argentina) for helpful discussion during research protocol design and to Janine Calabro (Tufts University School of Medicine, Boston, MA) for grammatical revision of the manuscript.
FOOTNOTES
First decision: 11 January 2001.
1 This study was supported by grants from the Argentine National Council for Science and Technology (CONICET) (PIP 528/98), the Argentine National Agency for the Promotion of Science and Technology (PICT-99 5-7001), and the Agreements: CONICET/Brazilian National Council for Scientific and Technological Development (CNPq) (1786) and SECYT-CAPES (BR04/99/OG). J.V. is a Fellow of the CONICET; J.G.R. is a recipient of an R. Carrillo-A. Oñativia fellowship from the Argentine Ministry of Health; G.S.M. is a Career Investigator of the CNPq; and E.H.L. is a Career Investigator of the CONICET. 
2 Correspondence: Enrique H. Luque, Department of Human Physiology,
Faculty of Biochemistry and Biological Sciences, Casilla de Correo 242,
3000 Santa Fe, Argentina. FAX: 54 342 4550944; eluque{at}fbcb.unl.edu.ar
Accepted: March 13, 2001.
Received: December 1, 2000.
REFERENCES
-Smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990; 63:21-29[Medline]This article has been cited by other articles:
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  S. M. Yellon, C. A. Ebner, and Y. Sugimoto Parturition and Recruitment of Macrophages in Cervix of Mice Lacking the Prostaglandin F Receptor Biol Reprod, March 1, 2008; 78(3): 438 - 444. [Abstract] [Full Text] [PDF]
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J. G. Ramos, J. Varayoud, V. L. Bosquiazzo, E. H. Luque, and M. Munoz-de-Toro Cellular Turnover in the Rat Uterine Cervix and Its Relationship to Estrogen and Progesterone Receptor Dynamics Biol Reprod, September 1, 2002; 67(3): 735 - 742. [Abstract] [Full Text] [PDF]
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