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a Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33101
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies of the female reproductive tract have demonstrated the production of multiple mucins with different patterns of expression and possible multiple functions in different tissues [7–9]. In the distal reproductive tract, cervical mucins not only create a barrier to sperm and pathogen entrance to the endometrium but also provide a protective covering for the vaginal epithelium. In humans, the rheologic and viscoelastic properties of human cervical mucus have been shown to vary during the menstrual cycle in a hormone-dependent manner [10]. MUC4 and MUC5B are reported to be the major mucin genes expressed by the endocervix [11]. Mucins also cover the surface of the uterine epithelium [12, 13]. In the rodent uterus, the expression of the membrane mucins Muc1 and Muc4 (sialomucin complex) is regulated by ovarian hormones during the estrous cycle. In the normal cycling rodent, membrane mucin expression in the uterus changes during the estrous cycle, increasing with high levels of estrogen in the plasma and decreasing at times that correspond with high plasma progesterone levels [9, 12].
Sialomucin complex (SMC/Muc4) is one of two membrane mucins widely expressed in various epithelia [14]. It is a heterodimeric glycoprotein that was originally isolated and characterized from highly metastatic 13762 rat mammary adenocarcinoma ascites tumor cells [15]. SMC/Muc4 is composed of a highly O-glycosylated mucin subunit ASGP-1 [16] noncovalently attached to the membrane via a transmembrane subunit ASGP-2 [17], which contains two epidermal growth factor-like sequences [18]. Since SMC is a highly O-glycosylated protein with rigid, elongated structures projecting above the luminal cell surface, it has been hypothesized that it acts as a protective anti-adhesive and anti-recognition agent at the cell surface [6]. The expression of SMC has been shown to prevent cell-cell adhesion and cell-matrix interaction when transfected into melanoma cells [6]. Recent studies from our laboratory have shown that SMC/Muc4 is expressed in many epithelia, including the trachea [19, 20], colon [19], lactating mammary gland [19, 21], and cornea and conjunctiva [22].
The potential protective function of apical cell surface mucins presents a dilemma in the uterus, since the presence of mucins inhibits cell-cell interactions necessary for blastocyst implantation [13]. Mucins must be lost from the surface to achieve the required adhesive interactions between the apical plasma membrane of trophoblast cells of the blastocyst and the apical plasma membrane of the uterine epithelium that are necessary for implantation [23–28]. SMC is expressed in the pregnant rat uterus between Days 1 and 4 postcoitus, but it disappears abruptly during Day 5, at the time of onset of the receptive period [12]. These results indicate that SMC may act as a barrier to prevent implantation in the prereceptive period, similar to the mechanism proposed for Muc1 in the rat [29]. SMC must be lost from the apical surface of the rat uterine lining in order to generate the receptive state for uterine implantation.
These observations on the expression of SMC in the rat uterus and its changes during pregnancy raise the question of what happens in other regions of the female reproductive tract, such as the cervix and vagina, that are more accessible and vulnerable to infection. SMC might be regulated differently in these tissues, since there is no requirement for down-regulation for blastocyst implantation. However, these tissues do undergo physiological and structural changes that are sensitive to the estrous cycle. The present study demonstrates the expression of SMC/Muc4 in the oviduct, cervix, and vagina at similar levels throughout the estrous cycle. Moreover, SMC expression can also be observed in the cervix and vagina in ovariectomized rats, in which uterine expression is lost. These results indicate that regulation of SMC expression in these tissues is different from that in the uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rabbit polyclonal antisera directed against intact rat tumor ASGP-2 [30] and the COOH-terminal cytoplasmic domain of ASGP-2 (anti-C-pep) [19] were used for immunoprecipitations. Monoclonal antibody (mAb) 4F12 directed against the N-terminus of ASGP-2 [19] was used for immunoblotting and immunohistochemical staining. Goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate was purchased from Promega (Madison, WI). Biotinylated goat anti-rabbit and anti-mouse antibodies and avidin-HRP conjugate were purchased from Dako (Carpinteria, CA). Protein A agarose, estradiol-17ß, progesterone, diaminobenzidine (DAB), and general laboratory chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Tissue Collection and Preparation
Experiments were conducted in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals. Adult Fischer 344 rats (6–8 wk old; Charles River Laboratories, Wilmington, MA) were housed 2 per cage in a controlled environment. Before animals were killed, the estrous cycle was staged by performing vaginal smears. The determination of the stage of the cycle was later confirmed by histological changes in the uterine and vaginal epithelia that occur normally in cycling rats. Oviduct, uterus, cervix, and vagina were dissected from adult Fischer 344 rats and used for either immunoblotting, immunoprecipitations, or immunostaining. For immunoblotting and immunoprecipitations, the tissues were pulverized with a mortar and pestle in liquid N2 and stored as a powder at -80°C. For immunoblotting, tissue powders were solubilized in a 1% SDS buffer. For quantitation of total protein, solubilized protein was boiled and clarified by centrifugation at 12 000 x g. Protein concentrations were determined using the Modified Lowry Assay kit (Pierce, Rockford, IL). For immunoprecipitations, tissue powders were solubilized in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) and homogenized with a probe sonicator. The lysates were centrifuged at 2000 x g for 10 min, and the supernate (2S) was added to protein A-agarose-bound antiserum. For immunostaining, tissues were fixed in methacarn (60% methanol, 30% inhibsol, 10% acetic acid) overnight and embedded in paraffin [31].
Ovariectomized rats (3 per group) were used for hormone studies. The rats were injected s.c. with 2.5 µg of estradiol-17ß and 4 mg of progesterone in 0.2 ml sesame oil for 3 consecutive days. Control animals received only sesame oil. On the fourth day, the uterus, cervix, and vagina were harvested from each animal and used for immunoblotting and immunostaining.
Immunoblotting
SDS-PAGE was performed under reducing conditions using 6% polyacrylamide gels and the mini-Protean II system (Bio-Rad, Hercules, CA). Proteins (10 µg) were resolved on SDS-PAGE and transferred to nitrocellulose filters, which were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline with 0.5% Tween 20. After a 1-h incubation in primary antibody diluted 1:10 000 in 1% BSA/Tris-buffered saline with 0.5% Tween 20, the filters were incubated with HRP-conjugated goat anti-mouse IgG diluted 1:14 000. The signal was detected with the Renaissance Enhanced Chemiluminescence Kit (DuPont NEN, Boston, MA).
Immunoprecipitations
Anti-ASGP-2 and anti-C-pep immunoprecipitations were performed with 20 µl of protein A-agarose and 20 µl of antiserum. All immunoprecipitations were rotated overnight at 4°C and washed six times for 10 min each in RIPA. Bound proteins were released by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with 4F12 mAb.
Uterine Rinse SMC
Uterine horns, cervix, and vagina were dissected from rats that were in the estrous or diestrous stage (3 each) and rinsed briefly in PBS. The tissues were cut longitudinally and opened to expose the luminal epithelium. Sequential rinses with single-strength PBS (3 times) and double-strength PBS, 1.5 M urea, and 0.05% Triton X-100 in single-strength PBS (2 times each) were performed by adding 2 ml of the solution to a 15-ml conical tube containing the slit tissues. After gentle rocking for 1 min, the solution was collected. All rinses were reduced in volume to 0.1 ml by centrifugation in Centricon filters (Amicon, Beverly, MA) with a 500-kDa cut-off. The volumes were then brought to 2 ml with 0.05% Triton X-100 in single-strength PBS, and the cycle was repeated 3 times. The rinsed tissues were processed to give a 2S fraction. All samples were then immunoprecipitated with anti-ASGP-2, and the precipitations were analyzed by SDS-PAGE and immunoblotted with 4F12 mAb.
Immunostaining
Paraffin blocks containing the various regions of the female reproductive tract were sectioned (5 µm thick) and routinely stained with hematoxylin and eosin. Immunostaining was performed by first dewaxing the sections in xylene and dehydrating in absolute ethanol. Endogenous peroxidase was inactivated by incubating in methanol-H2O2 for 20 min. The sections were washed in tap water for 5 min and then incubated for 1 h in primary antibody (anti-ASGP-2 4F12 mAb) diluted 1:600 in 0.5% BSA in PBS. The sections were washed in PBS for 5 min and incubated for 45 min in biotinylated anti-mouse secondary antibody diluted 1:400 in 0.5% BSA in PBS. After a 5-min wash in PBS, the sections were incubated for 45 min in avidin-HRP diluted 1:400 in 0.05 Tris-HCl, pH 7.4. The sections were washed in PBS, in 0.05 Tris-HCl, pH 7.4 (5 min each), then incubated in DAB substrate for 7 min, and subsequently washed in water and counterstained in hematoxylin for 15 sec. The sections were washed in water, dehydrated with ethanol, cleared in xylene, and mounted with Permount (Fisher Chemicals, Pittsburgh, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous work in our laboratory demonstrated that SMC/Muc4 is expressed in the rat uterus in a hormonally regulated manner [12]. The presence of SMC in the uterus raises the possibility that SMC/Muc4 provides a protective mechanism in other regions of the female reproductive tract that are accessible and vulnerable to infection. In an initial effort to determine which other tissues of the female reproductive tract express SMC/Muc4, lysates were prepared from the ovaries, oviduct, uterus, cervix, and vagina of rats that were in the estrous stage, which had been previously shown to have the highest level of expression during the estrous cycle [12]. The lysates were analyzed by Western blotting using the mAb 4F12, which recognizes rat ASGP-2 (Fig. 1). The results indicated that a considerably higher level of SMC/Muc4 was present in the cervix and vagina than in the uterus (Fig. 1). A much lower level of SMC/Muc4 was present in the oviduct, and SMC/Muc4 was not observed in the ovaries (Fig. 1).
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Lack of Variation of Cervical and Vaginal SMC/Muc4 During the Estrous Cycle
In the uterus, the level of SMC in the rat varies significantly during the estrous cycle, with the highest level during the estrous stage [12]. SMC is also lost immediately prior to implantation of the blastocyst from the uterine surface. Thus, it was of interest to know whether there are also variations in expression of SMC in the more accessible lower regions of the female reproductive tract. As shown in Figure 1B, no substantial differences were observed in the expression levels of SMC/Muc4 in the cervix and vagina of rats between the estrous and diestrous stages.
Soluble Form of SMC/Muc4
In most normal tissues that express SMC/Muc4, including the uterus [12], the glycoprotein complex is found in both membrane and soluble forms [14]. To determine which form(s) of SMC/Muc4 is present in the cervix and vagina, a sequential immunoprecipitation analysis was performed on tissue homogenates of rats that were in either the estrous or the diestrous stage. Tissue homogenates were immunoprecipitated with anti-C-pep and with anti-ASGP-2 antisera, the former recognizing the membrane form of SMC via the COOH-terminus of ASGP-2. Between 40% and 60% of SMC/Muc4 protein was immunoprecipitated by the anti-C-pep antisera in the vagina (Fig. 2) and cervix (data not shown) of rats in the estrous stage, calculated as described previously [12, 20]. Consistent with this estimate, approximately 40% of SMC was removed from the vaginal lumen by sequentially rinsing slit vaginal canals with various solutions for 1 min each (Fig. 3). The mild conditions and short rinse times were used to distinguish loosely bound SMC from the membrane-bound SMC, which cannot be easily washed from the luminal surface. Most of the SMC that could be rinsed from the vagina (Fig. 3) and cervix (data not shown) without disrupting cell or membrane structures was removed during the two rinses with isotonic saline, indicating that it was loosely bound, as in the uterus [12] and trachea [20]. These results indicated that SMC was present in both nonmembrane and transmembrane forms on the apical surface of cervical and vaginal epithelia, and that the soluble form was loosely adsorbed to the luminal surface—similar to results reported previously for the uterus [12], trachea [20], and cornea [21].
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Immunohistochemical Localization of SMC/Muc4
Immunohistochemical techniques were used to determine the localization of SMC/Muc4 in the various tissues of the female reproductive tract. In order to have a protective function, SMC/Muc4 must be present at the luminal surface of epithelia. The pattern of SMC/Muc4 expression in the oviduct, uterus, cervix, and vagina was examined from rats that were at the diestrous, proestrous, estrous, and metestrous stages using 4F12 antibody (Fig. 4). Three rats were examined to determine the immunohistochemical localization of SMC in different tissues for each stage of the estrous cycle. The expression of SMC was uniform in all the rats examined, with minimal variation between animals. In the uterus, the staining was localized at the apical aspects of the luminal (Fig. 4, a–d) and glandular epithelial cells (not shown). Staining of the uterine luminal epithelium was the most intense during the estrous (Fig. 4c) and metestrous (Fig. 4d) stages; staining decreased during the diestrous (Fig. 4a) and proestrous (Fig. 4b) stages. During the estrous and metestrous stages, SMC appeared to be localized not only at the apical surface of uterine epithelia but also intracellularly (Fig. 4). No significant differences were observed in the staining patterns of SMC/Muc4 in glandular epithelia during the estrous cycle (data not shown), indicating that SMC is differentially regulated in the luminal, as compared to the glandular, epithelia.
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In contrast to observations in the uterus, vaginal expression of SMC/Muc4 was not substantially quantitatively altered during the estrous cycle (Fig. 1). However, the localization pattern of vaginal staining with 4F12 varied substantially during the cycle (Fig. 4, k–o). At the diestrous stage, there was strong staining at the apical surface of the columnar stratified layer (Fig. 4k). At the proestrous stage, the luminal epithelium comprises two layers of cuboidal cells, both of which stained with 4F12. The underlying squamous epithelium, which is separated from the columnar epithelial cells by cornified cell layers, showed intense staining with 4F12. The underlying columnar cells did not express SMC/Muc4 (Fig. 4l). During the estrous stage, the superficial columnar cells from the proestrous stage are sloughed off, exposing the stratified squamous epithelium, which is covered by a thick cornified layer. During this stage, the stratified squamous epithelium, composed of 3–4 cell layers, showed intense staining (Fig. 4m). Toward the end of the estrous stage, the cornified layers begin sloughing off. The cornified layer is completely detached from the underlying viable epithelium during the metestrous stage, and the epithelium is infiltrated by leukocytes. During this stage, distinct superficial layers of the stratified squamous epithelium stained intensely for SMC/Muc4 (Fig. 4n).
SMC expression in the endocervix and ectocervix was quantitatively similar to that in the vagina during the estrous cycle, and the localization patterns also changed substantially. During the diestrous stage of the endocervix, 2–3 layers of the superficial stratified columnar epithelium stained intensely with 4F12 (Fig. 4f). Likewise, in the ectocervix, a superficial layer of stratified cells stained positive. In the proestrous stage, 1–2 layers of the stratified columnar epithelial cells of the endocervix stained intensely (Fig. 4g). The number of cell layers in the ectocervix staining with 4F12 increased to about 4–5 stratified columnar cells, though the staining appeared somewhat discontinuous. During the estrous stage, 5–6 layers of the stratified squamous epithelium in the endocervix stained intensely with 4F12 (Fig. 4m). During the metestrous stage, about 4–5 layers of the stratified columnar cells stained strongly. The number of layers staining with the 4F12 antibody increased to the full thickness of the epithelium in the metestrous endocervix. The most superficial layer of the luminal epithelium showed weak staining, and the underlying layers of about 5–6 cells stained intensely with 4F12. This superficial layer is infiltrated with leukocytes. In the ectocervix, there is a transition to about 3–4 stratified squamous cells that stained with 4F12 (Fig. 4i).
Although there were differences in the staining patterns among different regions of the oviduct, the expression level did not appear to change during the estrous cycle. The isthmus of the oviduct, which is composed of highly branched folds whose epithelium consists of a single layer of ciliated and secretory columnar cells, stained intensely at the apical surface. In addition, weak staining was observed intracellularly (Fig. 5a). The infundibulum and ampulla of the oviduct are also composed of branched folds, whose epithelium is lined with a single layer of ciliated and secretory columnar cells. SMC expression in cells of the infundibulum (data not shown) and ampulla (Fig. 5b) appeared weak and localized at the apical surface of these cells.
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SMC/Muc4 Expression in Ovariectomized Rats
Blastocyst implantation involves direct interaction between the apical surfaces of the endometrial epithelium and the trophoblast, and thus requires that the uterus be in a receptive state [24]. Receptivity is regulated by the ovarian hormones estrogen and progesterone. Previous work in our laboratory has shown that SMC/Muc4 expression is lost in the uteri of ovariectomized rats and regained upon estrogen treatment. Estrogen-induced expression of SMC/Muc4 in the uterus is blocked in the presence of progesterone [12]. Existing data on the role of estrogen and progesterone in uterine SMC/Muc4 expression raise the question of whether the effect of these hormones is specific to the uterine epithelium or whether it also applies to other regions of the female reproductive tract, such as the cervix and vagina. In this study, ovariectomized rats were treated with either sesame oil (as a control), estrogen, or progesterone for 3 consecutive days. Lysates of the uteri, cervix, and vagina were prepared and analyzed by Western blotting using 4F12 antibody (Fig. 6). Consistent with the previously reported data, SMC/Muc4 expression was lost in the uterus of ovariectomized rats and regained upon treatment with estrogen, while progesterone failed to induce SMC expression in the uterus. In contrast, SMC/Muc4 was expressed in the cervix and vagina of ovariectomized rats at levels similar to those found in untreated animals (Fig. 6). Moreover, the levels of expression of the glycoprotein complex did not appear to change upon estrogen or progesterone treatment. These results suggest that, in contrast to the uterus, the cervix and vagina constitutively express SMC/Muc4 and do not require the presence of estrogen for expression.
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Although no significant differences were observed in the expression level of SMC/Muc4 in the lower female reproductive tract of ovariectomized rats under different hormonal treatments, it was important to analyze possible differences in the localization patterns of SMC/Muc4 with the different hormonal treatments because of the substantial effects of the hormones on tissue histology. SMC/Muc4 expression pattern was analyzed in the uterus, cervix, and vagina immunohistochemically using the monoclonal 4F12 antibody. In the uterus of estrogen-treated, ovariectomized animals, SMC/Muc4 expression was intense in the luminal epithelial cells (Fig. 7a), resembling that of the early estrous stage (see Fig 4c). However, SMC/Muc4 expression was abolished at the luminal epithelium of progesterone-treated and control animals (Fig. 7, b and c). SMC/Muc4 expression was present in the uterine glandular epithelium of estrogen-treated (data not shown), progesterone-treated (Fig. 7b), and control rats (data not shown), suggesting that SMC/Muc4 may be regulated by different mechanisms in uterine luminal and glandular epithelia.
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The pattern of SMC/Muc4 localization in the cervix appears to be influenced by estrogen treatment. In ovariectomized rats, the cervical lumen is lined by a single layer of simple columnar cells that stained strongly at the apical surface (Fig. 7f). Upon estrogen treatment, the cervical lumen is lined by a stratified squamous epithelium composed of about 3–4 cells (Fig. 7d), which stained intensely. Progesterone treatment did not appear to significantly change the SMC/Muc4 expression, which was similar to that of control animals. These results indicate that SMC/Muc4 is constitutively expressed in the cervix of ovariectomized rats; however, the localization of expression changed in response to estrogen. Similar to the situation in the cervix, the pattern of SMC/Muc4 vaginal localization changed in response to estrogen. In the ovariectomized rats, there was strong staining at the apical surface of the columnar layer lining the vaginal epithelium (data not shown). Upon estrogen treatment, strong staining was evident in the vaginal luminal surface comprising two layers of columnar cells. In addition, weak staining was observed in the stratified squamous epithelium underlying the superficial columnar epithelial cells. The process of cornification was evident below the squamous epithelial layer. This pattern of staining in the vagina of the ovariectomized rat closely resembled that of the proestrous stage in the estrous cycle. Progesterone treatment did not appear to affect SMC/Muc4 expression in the vagina.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the estrous cycle, the uterus undergoes multiple changes that are coordinated with the levels of the ovarian hormones estrogen and progesterone in the plasma. Work on Muc1 in the mouse uterus showed the highest expression level of this glycoprotein in the proestrous and estrous stages, which correspond with high estrogen levels, while the lowest levels of Muc-1 were observed in the metestrous and diestrous stages, which correspond with high progesterone levels [9]. Previous work by McNeer et al. [12] indicated the modulation of SMC/Muc4 expression by estrogen and progesterone in hormone replacement experiments such that SMC/Muc4 was up-regulated in ovariectomized rats treated with estrogen and down-regulated by progesterone. In contrast, other regions of the female reproductive tract constitutively express SMC/Muc4 and are unresponsive to ovarian hormones (summarized in Table 2). Even the uterine glandular epithelium appears unresponsive. These studies indicate that the hormonal regulation of SMC/Muc4 applies only to the uterine luminal epithelium in the female reproductive tract and suggest that the uterus has evolved a special regulatory mechanism for the luminal cell mucins, presumably to facilitate the down-regulation of the anti-adhesive effects of the mucins at the time of implantation.
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The novel finding of SMC/Muc4 in the oviduct raises questions about its function in a tissue where protection would appear to be less important. Several studies have focused on the expression of glycoproteins in the oviduct. The oviduct functions as a tube for the transport of gametes and also is an active secretory organ, whose secretions maintain a suitable environment for continued maturation of male gametes, interaction between gametes, and early embryonic development [36]. Previous studies showed the presence of a high molecular weight glycoprotein that is secreted by epithelial cells of the oviduct in the goat [37], baboon [38], and sheep [36]. These oviductal glycoproteins appear to be modulated by ovarian steroid hormones, and may be estrogen-dependent [38]. In addition, localization of these glycoproteins was the highest in the oviductal epithelium at the follicular phase when levels of plasma estrogen were high [37, 38]. In our study, oviductal SMC/Muc4 expression did not appear to be modulated during the estrous cycle. However, its expression appears to have regional specificity. SMC/Muc4 is expressed at a higher level in the epithelia of the isthmus as compared to the ampulla or infundibulum. Perhaps the expression pattern is related to the variety of reproductive and developmental events that occur along the length of the oviduct.
We also report the novel finding of SMC/Muc4 protein in the rat cervix and vagina in a constitutive manner during the estrous cycle. Previous studies have shown rheological changes in the human reproductive tract mucus in response to menstrual cycle phase. At ovulation, the mucus is watery, but it becomes viscous after ovulation [10]. Many groups have attempted to determine changes in glycoproteins that may contribute to the alterations in the mucus character. Work by Vrcic et al. [39] showed that distinct lectin reactivity patterns accompany the cyclic changes in the morphology of the vaginal epithelium. Studies by Gipson et al. [7] indicated the presence of MUC4 and MUC1 message in the ectocervical and vaginal epithelium, but they did not show any variations during different phases of the menstrual cycle. These findings suggest that viscosity changes in human cervical mucus are likely due to the presence of other mucins, probably of the gel-forming class.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kermit L. Carraway, Department of Cell Biology and Anatomy (R-124), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101. FAX: 305 243 4431; kcarrawa{at}mednet.med.miami.edu
Accepted: July 12, 1999.
Received: May 3, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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