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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 883-891
Copyright © 2004 by The Endocrine Society

Estrogen Enhances Cystatin C Expression in the Macaque Vagina

Ov D. Slayden, Kevin Hettrich, Rebecca S. Carroll, Lesley N. Otto, Amanda L. Clark and Robert M. Brenner

Oregon National Primate Research Center (O.D.S., K.H., R.S.C., R.M.B.), Beaverton, Oregon 97006; and Division of Urogynecology and Reconstructive Pelvic Surgery (L.N.O., A.L.C.), Oregon Health and Science University, Portland, Oregon 97239

Address all correspondence and requests for reprints to: Ov D. Slayden, Ph.D., Division of Reproductive Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: slaydeno{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cystatin C is a secreted inhibitor of cysteine proteinases that participates in extracellular matrix remodeling. Whether hormones affect its expression in the vagina was unknown. Consequently, we examined the effects of estradiol (E2), progesterone (P), and raloxifene on vaginal cystatin C in rhesus macaques. In experiment 1, ovariectomized animals were treated sequentially with E2 (14 d) and E2 + P (14 d) to induce 28-d menstrual cycles. Vaginal samples were collected on d 6, 8, 14, and 28 of the induced cycle. Some cycled animals were deprived of both E2 + P for 28 d. In experiment 2, ovariectomized animals were treated for 5 months with E2 alone, E2 + P, raloxifene, or left untreated. Total RNA from the vaginal wall was analyzed for the cystatin C transcript with a commercially prepared cDNA array and semiquantitative RT-PCR. Vaginal cryosections were analyzed by in situ hybridization for cystatin C transcript and by immunocytochemistry for the protein. E2 treatment significantly (5-fold; P < 0.05) increased expression of cystatin C transcript over the levels in the hormone-deprived controls, and cotreatment with P (E2 + P) blocked this effect. Raloxifene treatment did not affect cystatin C expression. In situ hybridization and immunocytochemistry revealed that cystatin C was localized in fibroblasts and smooth muscle cells throughout the vaginal wall but not in smooth muscle cells of arteries or levator ani myocytes. In summary, E2 increased vaginal cystatin C expression in the fibroblasts and smooth muscle bundles, P suppressed this effect, and raloxifene had no effects on cystatin C. Elevated cystatin C, by suppressing cysteine proteinase activity, may strengthen the vaginal wall and mitigate the potential for pelvic floor prolapse.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PELVIC ORGAN PROLAPSE is a prevalent disorder in women that involves structural failure of the connective tissues of the pelvic floor and vaginal wall (1, 2). Risk factors associated with pelvic organ prolapse include age (1, 3), parity (1, 3), and instrumental delivery, and a prevalent theory is that injury as a result of childbirth is a major factor contributing to the disease (4, 5, 6, 7). However, the majority of cases of prolapse occur years after childbirth, suggesting that factors other than injury contribute to the disease (2). Because menopause leads to a surge in the incidence of prolapse (2), many believe that ovarian steroid hormones, especially estrogens, influence the strength of pelvic floor connective tissues (8). However, the mechanism through which estradiol (E2) may protect the pelvic floor is still poorly understood. In women, it is difficult to separate the effects of aging and menopause because they occur simultaneously. In an animal model, the variables of age and parity can be controlled to isolate the effects of hormonal treatment.

Nonhuman primates, including rhesus macaques (Macaca mulatta), share many features with women that make them excellent experimental models for study of hormone action on the pelvic floor and vagina (9). For instance, the effects of E2 and progesterone (P) on the reproductive tract have been well documented in macaques (10) and are nearly identical in women and monkeys. Other similarities include a semiupright posture, similar pelvic floor anatomy, similar parturition (11), and occurrence of vaginal prolapse in multiparous animals (9). In rhesus macaques, E2 induces epithelial cell proliferation that leads to a thickened, cornified vaginal epithelium. At the same time, E2 treatment stimulates hypertrophy of the vaginal smooth muscle and an increase in the extracellular matrix (ECM), resulting in a greatly thickened vaginal tube. Together, these effects of E2 may result in a vaginal tube that is more resistant to breakage than the vaginal tube of ovariectomized, hormone-deprived macaques (9).

Selective estrogen receptor modulators (SERMs) have recently been developed that have differential estrogenic effects in various organ systems (12). Typically, SERMs fail to induce classic estrogen-stimulated cell proliferation in the uterus but have significant estrogen-like effects on structural tissues including bone (13). Recent studies report conflicting results on the effects of SERMs on pelvic floor connective tissues (14, 15). Development was discontinued for two SERMs under investigation, levormeloxifene and idoxifene, after pelvic organ prolapse was noted as an adverse affect associated with therapy (15). Raloxifene, however, has not been associated with a greater risk of surgery for prolapse (14).

In a preliminary study, we screened samples of macaque vaginal RNA with commercially prepared pathway-targeted macro cDNA arrays (macroarray) for regulators of ECM molecules (16). Compared with other regions of the reproductive tract, expression of cystatin C was markedly elevated in the vagina of rhesus monkeys after 6 d of E2 treatment. Cystatin C is a secreted inhibitor of lysosomal cysteine proteinases including cathepsin B, L, and H and is reported to suppress the action of these enzymes on proteolysis during normal tissue processes (17). The acid cathepsins contribute to collagen degradation as they depolymerize collagen fibers by cleaving near cross-link sites (18). Because proteolysis of collagen and other proteins in the vaginal wall would weaken vaginal integrity, estrogen induction of a proteolytic inhibitor could favor vaginal strength and mitigate the tendency for prolapse. We, therefore, set a goal of fully characterizing the effects of E2 and P on the expression and localization of cystatin C in the rhesus vagina. In addition, we determined whether or not raloxifene had any estrogen-like effects on vaginal cystatin C expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Animal care and husbandry was provided by the veterinary staff of the Division of Animal Resources of the Oregon National Primate Research Center (ONPRC) at the Oregon Health and Science University (OHSU). All studies and procedures were reviewed and approved by the ONPRC/OHSU Institutional Animal Care and Use Committee. The entire vagina was collected at necropsy from nulliparous, young adult rhesus macaques. In experiment 1 (short term), 12 ovariectomized macaques were treated sequentially with Silastic capsules containing E2 for 14 d, and then E2 + P for 14 d to induce artificial menstrual cycles as described previously (19). In this model, removal of the P implant at the end of the artificial cycle induces menstruation and begins the next cycle. The implants used in these experiments produced serum levels of 80.7 ± 43 pg E2 and 4.9 ± 1.1 ng P per milliliter, which are within the normal range for cycling rhesus macaques. Tissues were collected during the proliferative phase (d 6, 8, and 14 of E2 alone; n = 6), and the late secretory phase (d 28; E2 + P; n = 4) of the induced cycle. Tissues were also collected from three animals that had both E2 and P implants removed (hormone deprived) for 28 d. Experiment 2 (long term) was a 5-month study in which 15 ovariectomized rhesus macaques were treated either with Silastic capsules releasing E2 (n = 4), Silastic capsules releasing E2 + P (n = 3), raloxifene (5 mg/kg, by oral administration; n = 4), or no hormones (n = 4; hormone-deprived). Samples were collected at the end of the 5-month treatment. Samples of macaque endometrium and rectus abdominis muscle were also collected from some of the animals listed above for comparison to the vagina.

Immediately after necropsy, the vagina and pelvic floor ligaments were dissected grossly as described by Otto et al. (9). The urinary bladder and urethra were removed from the anterior vaginal tube, and the colon was separated from the posterior wall by sharp dissection. Full-thickness samples of the anterior and posterior wall from the upper third of the vagina were weighed and frozen in liquid nitrogen for isolation of total RNA. Slices transecting the lateral wall from the vaginal lumen to the levator ani muscle were frozen in Tissue Tek OCT (Miles Inc., Elkhart, IN) and cryosectioned for in situ hybridization (ISH) or fixed in 4% paraformaldehyde and embedded in paraffin for histological analysis and immunocytochemistry (ICC).

Isolation of total RNA

Samples were thawed in 10 volumes of TRIzol reagent (Invitrogen, Carlsbad, CA), immediately homogenized with a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY), and total RNA isolated following the standard TRIzol protocol. The TRIzol-extracted sample was precipitated with ethanol and then combined with RNeasy lysis buffer (Qiagen, Valencia, CA) for additional purification with the RNeasy midi kit. RNA bound to the RNeasy filters was treated with Rnase-free Dnase (catalog no. 79254; Qiagen) on the filter following the manufacturer’s instructions. Concentrations of the total RNA in the final extract were quantified by UV absorbance on a 640B spectrophotometer (Beckman Instruments Inc., Fullerton CA), and RNA integrity was determined by fractionation on denaturing agarose gels stained with ethidium bromide.

Macro gene array

Total RNA was analyzed on low-density (macro) arrays (GEArray Q Series human ECM and adhesion molecule kit; SuperArray, Bethesda, MD) following the manufacturer’s instructions. These arrays contained 96 target cDNAs associated with cell adhesion and regulation of the extracellular matrix, plus three sets of housekeeping genes (cyclophilin, RPL-13a, and ß-actin) and control DNA (PUC 18) to detect nonspecific hybridization. The array membranes were prehybridized in 2 ml hybridization solution for 2 h at 60 C. Total RNA (5 µg) was reverse transcribed with MMLV reverse transcriptase (200 U/ml; Promega, Madison WI), the GEA primer mix (SuperArray) and [32P] dCTP (NEN Life Science Products, Boston, MA). One microliter of the resulting [32P]-labeled cDNAs was electrophoresed on a 1% agarose gel, the gel was exposed to x-ray film, and lanes were compared to confirm similar labeling of the samples. The remaining cDNA probe was denatured at 68 C and hybridized to the arrays in a roller hybridization incubator (model 1000; Robbins Scientific, Sunnyvale CA) overnight in 0.8 ml of hybridization solution at 60 C. After high-stringency washing [0.1x saline sodium citrate (SSC) at 60 C], the arrays were visualized by phosphor imaging with a BioRAD Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA) and quantified with ScanAlyze (Michael Eisen, Stanford University, Stanford, CA). For each array, the mean intensity for the negative controls (PUC-18 and no cDNA) was calculated, and this signal level plus 2 SD of the mean intensity was considered the background level on the blot. The background was subtracted from signal for each target cDNA, and the relative signal for each gene on the membrane was calculated as ratio of intensity of target gene to the intensity of RPL-13a.

RT-PCR

Two micrograms of total RNA were reverse transcribed with the Omniscript reverse transcription kit (Qiagen), oligo-dT primer (Invitrogen), and RNAsin (Promega). The resulting cDNAs were PCR amplified using HotStarTaq master kit (Qiagen) and human cystatin C-specific (20) or human RPL-13a primers (21) (Table 1Go). PCR amplification was performed with 1.75 mM MgCl2 and 0.25 µM primers and optimized for maximum yield for both products individually (33 cycles at 94 C for 45 sec, 54 C for 45 sec, and 72 C for 1 min, followed by a final extension at 72 C for 10 min). The amplified PCR products were electrophoresed on 1.5% agarose-ethidium bromide gels, isolated, and sequenced by the ONPRC Molecular Biology Core to verify the identity of PCR products. A semiquantitative RT-PCR assay for cystatin C transcript was validated by conducting PCR amplifications with both sets of primers in each reaction, and the resulting products were separated on 1.5% agarose gels stained with ethidium bromide. Both cystatin C and RPL-13a were found to amplify in a linear relationship for up to a maximum of 30 cycles. Gels were photographed, and the intensity of each band was quantified with ScanAlyze. Each tissue sample was assayed in duplicate. Relative cystatin C expression was determined as the ratio of cystatin C to RPL-13a.


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  		TABLE 1. PCR primers

 
ISH

ISH of frozen sections of macaque vaginal wall was conducted with macaque-specific riboprobes labeled with [35S]UTP (DuPont NEN Life Science Products, Boston, MA) prepared from sequenced PCR products described above that were ligated to T7 RNA polymerase promoters. Probes were prepared from PCR-amplified monkey-specific cDNA in two steps. First, a T7 RNA polymerase promoter was ligated to sense and antisense strands of PCR product using the Lig-N-Scribe no-cloning promoter addition kit (Ambion, Austin, TX). Second, the riboprobes were synthesized with the MAXIscript In vitro transcription kit (Ambion). Probes for cystatin C were initially tested by Northern blot hybridization. For Northern blotting, total RNA from macaque vaginal tissue was fractionated on 1% denaturing formaldehyde agarose gels and transferred to supported nitrocellulose membranes (Life Technologies, Inc.). The membranes were hybridized with 32P-labeled riboprobes (SA, 106 cpm/ml). Hybridization was performed according to the manufacturer’s instructions with ExpressHyb hybridization solution (CLONTECH Laboratories, Inc., Palo Alto, CA). The blots were exposed to Kodak BioMax MS film with TranScreen-LE intensifying screen at -70 C (Eastman Kodak Co., Rochester, NY). This test revealed probe hybridization to a single, specific 700-bp band consistent with the expected size of cystatin C transcript.

Techniques for ISH with 35S-labeled probes were published previously (22). Briefly, frozen sections, 10 µm thick, were mounted on Super Frost Plus slides (Fisher Scientific, Pittsburgh, PA) and fixed in 4% paraformaldehyde in PBS for 10 min at 4 C. The tissue sections were rinsed in 2x SSC, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min and then air dried. One slide per tissue group was treated with RNase A [20 mg/ml, 0.5 M NaCl, 0.01 M Tris, 1 mM EDTA (pH 8.0)] as a negative control. All the slides were prehybridized for 1 h at 42 C in 10 mM dithiothreitol, 0.3 M NaCl, 20 mM Tris (pH 8.0), 5 mM EDTA, 1x Denhardt solution, 10% dextran sulfate, and 50% formamide. Then sections were incubated at 55 C overnight in the same solution containing the appropriate concentration of the antisense probe (5 x 106 cpm/ml). Representative slides were also incubated with sense probe as another negative control. After hybridization, all the slides were treated with RNase A at 37 C for 30 min to inactivate nonhybridized probe, rinsed in a descending series of SSC (2x SSC, 1x SSC, 0.5x SSC), and then washed in 0.1x SSC at 65 C (high stringency) for 30 min. Sections were dehydrated in an ascending series of alcohol dilutions, vacuum dried, coated with NTB2 autoradiographic emulsion (Eastman Kodak), stored at 4 C for 2 wk, developed in aqueous D-19 (Eastman Kodak), lightly counterstained with hematoxylin, dehydrated in an ascending series of alcohol dilutions, cleared with xylene, and coverslipped with Permount (Fisher Scientific).

Silver grains in macaque ISH preparations were counted over epithelium, lamina propria mucosa, smooth muscle, arteries, connective tissue fibroblasts, and the myocytes of the levator ani muscle in sections hybridized with radiolabeled probe. The counts were made with Image Pro-Plus (Media Cybernetics Inc., Silver Springs, MD) on images captured at x250 original magnification. A region of the slide away from the section was similarly counted as background. Background counts were subtracted from each tissue-specific counts and the abundance of silver grains was expressed as the number of silver grains per square micrometer.

ICC

Consecutive 5-µm sections were cut and mounted on Superfrost Plus (Fisher Scientific) or poly-L-lysine-coated slides. The slides were deparaffinized in xylene and then rehydrated stepwise in ethanol and rinsed briefly in deionized H2O. Antigen retrieval was performed by heating sections in citrate buffer (BioGenex, San Ramon, CA) for 10 min in a household-type pressure cooker. The antigen-retrieved slides were allowed to cool to room temperature, rinsed once in PBS, and then immediately used for ICC. The slides bearing antigen-retrieved sections were incubated in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidases and rinsed once in deionized H2O and then three times in PBS. The slides were treated for 20 min at 4 C with horse serum and then incubated overnight at room temperature with polyclonal (goat) anti-cystatin C antibody (Strategic Biosolutions, Newark, NJ). The standard working concentration of cystatin C antibody (6 µg/ml) was determined by serial dilution. As a control for nonspecific staining, some sections were incubated with normal goat serum. After incubation with primary antibody, the slides were rinsed with 0.1% gelatin, 0.075% polyoxyethylene 23 lavrylether (BRIJ) in PBS (4 C), and reincubated with horse serum and then with a biotinylated second antibody (horse antigoat) for 30 min (25 C). The slides were rinsed with 0.1% gelatin, 0.075% BRIJ in PBS and the biotinylated antibody complexes were reacted with an avidin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA). The slides were then treated with 0.05% osmium tetroxide for 1 min, lightly counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific).

Histology and photomicroscopy

Paraffin sections (5 µm) were placed on SuperFrost Plus slides and deparaffinized by standard methods. For general histology and staining of connective tissue elements, a standard Masson-Trichrome method was used followed by dehydration and mounting in Permount.

Low-power micrographs were photographed with an Olympus OM-system 38 mm macro lens (Olympus Optical, Tokyo, Japan) on Ektachrome 64-T film (Eastman Kodak) and digitized with a Polaroid SprintScan 35 scanner. High-power micrographs were captured through Zeiss planapochromatic lenses with the Optronics DEI-750TD CCD camera (Optronics Engineering, Goleta, CA). Digital images were adjusted for sharpness and contrast with Adobe Photoshop (Adobe Systems, Seattle, WA), and photomicrographs were printed with a Stylus Photo 1200 printer (Epson, Tokyo, Japan).

Statistical analysis

Numerical data from ISH grain counts and the expression of cystatin-C transcript detected by macroarray and RT-PCR were analyzed by one-way ANOVA with post hoc analysis by Fisher’s least significant differences test (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Vaginal histology

Figure 1Go shows Masson-Trichrome-stained sections of the lateral vaginal wall from rhesus macaques after being either hormone deprived or treated with E2, E2 + P, or raloxifene for 5 months. The vaginal epithelium, lamina propria, muscularis, and paravaginal attachment, which inserts into the levator ani muscle (9), were clearly evident as distinct zones under all conditions. Treatment with E2 for 5 months clearly resulted in a thickened vaginal tube compared with animals deprived of hormones. This increase included a thickening of the epithelium with prominent spines and increased thickness of the lamina propria and muscularis. Cotreatment with P (E2 + P) suppressed the effect of E2 on vaginal thickness. The vagina of animals treated with raloxifene was atrophied and did not differ from that of the hormone-deprived animals.



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  		FIG. 1. Photomicrographs of Masson-Trichrome-stained paraffin sections of lateral vaginal wall from macaques that were hormone deprived (A) or treated with E2 alone (B) or E2 + P (C) or raloxifene alone (D), all for 5 months. EP, Epithelium; LP, lamina propria; SM, muscularis; PVA, paravaginal attachment; LA, levator ani muscle. Bar, 3 mm.

 
Hormonal regulation of cystatin C transcript

Macroarrays. Figure 2Go compares the effects of hormones on cystatin C mRNA as detected by macroarray in the vagina, endometrium, and rectus abdominis after both short- and long-term treatment. Quantitative analysis of the ECM and cell adhesion cDNA macroarray data showed a strikingly strong signal for cystatin C only in vaginal arrays from animals treated with E2 alone. Compared with ovariectomized control animals receiving no hormone, short-term treatment of macaques with E2 resulted in a significant 5-fold increase in cystatin C signal (P < 0.05). Cystatin C mRNA abundance was similar on d 6, 8, and 14, so results from these days were combined. Cystatin C expression was also maximal after long-term (5 months) treatment with E2, and did not significantly differ from the effect seen with short-term E2 treatment. Both short- and long-term treatment with E2 + P blocked most of the effects of E2, so that the cystatin C signal was reduced to that observed in the hormone-deprived control animals. Vaginal cystatin C expression under raloxifene treatment did not differ from the hormone-deprived controls, an indication that this SERM did not act as an estrogen on cystatin C in the primate vagina.



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  		FIG. 2. Mean (±SE) expression of cystatin C transcript (ratio of cystatin C/RPL-13a) detected in the macaque vagina by macro cDNA array. Means with different superscripts are statistically different (P < 0.05; n = 4/group).

 
Cystatin C mRNA was only minimally detectable in hormone-deprived endometrium and rectus muscle, and treatment with E2 or E2 + P had no effect on cystatin C mRNA expression in these tissues.

RT-PCR analysis. Analysis of vaginal RNA by RT-PCR for cystatin C and RPL-13a is shown in Fig. 3Go. The RT-PCR technique clearly detected a single 330-bp cystatin C and a single 378-bp RPL-13a band on ethidium bromide stained agarose gels (Fig. 3AGo). The PCR products were isolated from the gels and sequenced. The macaque cystatin C PCR product (GenBank accession no. AY288103) was 93%, identical to bp 4958–5291 of the human cystatin C (GenBank accession no. X52255). The RPL13A sequence was 98% identical to bp 278–667 of the human sequence (GenBank accession no. NM012423). Quantitation of ethidium bromide-stained gels revealed that the linear range for PCR amplification with both primers was 26–30 cycles, and the range of 28 cycles was selected as optimal. Analysis of increasing amounts of vaginal total RNA (Fig. 3BGo) revealed a low limit of detection when 100 ng total RNA were assayed. Figure 3CGo shows an ethidium bromide-stained gel bearing amplified PCR products from the short-term study of hormone-deprived, E2- and E2 + P-treated animals, and Fig. 3DGo represents the ratios of cystatin C/RPL 13A in quantitative scans of gels from the long-term treatment group. These observations confirm and extend the macroarray results and indicate that the vaginal cystatin C transcript was low in the hormone deprived, significantly elevated by E2 treatment (P < 0.05), suppressed by treatment with E2 + P, and unaffected by raloxifene.



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  		FIG. 3. Semiquantitative RT-PCR analysis of cystatin C expression in total RNA from rhesus macaque vagina. PCR products amplified with cystatin C, or RPL-13a-specific primers for 33 cycles revealed single distinct bands (A). PCRs containing both cystatin C and RPL 13a primers revealed detection at 100 ng total RNA (B) and clearly show the effect of hormone treatment on cystatin C expression (C). Quantitation of the bands on gels from experiment 2 (D; P < 0.05 n = 3).

 
Localization of cystatin C

ISH. Figure 4Go, A–G, shows photomicrographs of ISH for cystatin C mRNA with 35S-labeled riboprobes in the macaque vagina from an animal treated with E2 for 14 d during the artificial proliferative phase. No specific signal was detected in similar sections after RNase treatment (Fig. 4HGo) or after hybridization with sense probe (data not shown). A strong hybridization signal was clearly seen in all layers of the vagina (Fig. 4AGo) with particularly intense hybridization detected in the lamina propria and muscularis (Fig. 4Go, B and C) and the fibroblasts of the levator ani muscle (Fig. 4Go, F and G). No specific signal was detected in vascular smooth muscle (Fig. 4Go, D and E) or the skeletal muscle myocytes (Fig. 4Go, F and G). In contrast, hybridization for cystatin C in animals treated with E2 + P during the artificial secretory phase was minimal in the fibroblasts and smooth muscle cells (data not shown). A moderate hybridization signal was detected in the stratified squamous epithelium of the vagina, but this signal did not vary with hormonal treatment.



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  		FIG. 4. ISH for cystatin C in the macaque vagina after experiment 1. A bar representing 3 mm (A) shows the magnification for the low-power photographs (A and H). All other micrographs were made at x200 original magnification, and a 20-µm bar is shown (F). The signal is clearly evident in fibroblasts (B and G) and smooth muscle cells (SM) (C). A white line has been drawn (B) to separate the squamous epithelial layer (Ep) from the lamina propria (LP). No specific cystatin C signal was detected in vascular smooth muscle cells (D and E; ART, artery). Phase-contrast (F) and dark-field (G) photographs of the levator ani muscle (LA) show that the cystatin C signal was localized to the fibroblasts (arrows) between the skeletal muscle fibers, not the myocytes. RNase treatment is shown as a negative control (H).

 
Figure 5Go, A–D, shows a comparison of ISH from ovariectomized animals that were either hormone deprived or treated long term (5 months) with E2 alone, E2 + P, or raloxifene. Analysis of silver grain counts from these animals is shown in Fig. 6Go. In the hormone-deprived control, there was a basal level of cystatin C mRNA detected in the vaginal epithelium, lamina propria, muscularis, and connective tissues of the paravaginal attachment. Treatment with E2 for 5 months resulted in a maximal increase in cystatin C expression in the smooth muscle myocytes and connective tissue fibroblasts. No significant increase in cystatin C expression was detected in arterial smooth muscle or in the myocytes of the levator ani skeletal muscle, but the fibroblasts scattered throughout the levator ani muscle clearly showed an estrogen-dependent increase in cystatin C expression. Treatment with E2 + P significantly suppressed this effect of E2 on cystatin C expression in these tissues (Fig. 6Go; P < 0.05; also compare Fig. 4Go, B and C). Vaginal cystatin C transcript levels in raloxifene-treated animals did not differ from the hormone-deprived controls.



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  		FIG. 5. Cystatin C ISH in macaque vagina from experiment 2. Ep, Epithelium; LP, lamina propria; SM, smooth muscle; LA, levator ani. Bar, 3 mm.

 


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  		FIG. 6. Mean (±SE) silver grain counts of ISH from experiment 2. a, Means that are significantly higher than all others (P < 0.05).

 
ICC. Figure 7Go presents photomicrographs of cystatin C immunostaining in the macaque vagina from ovariectomized animals that were either hormone deprived or treated with either E2 alone or E2 + P for 5 months. In the hormone-deprived animals, specific cystatin C staining was barely detectable in epithelium, lamina propria, muscularis, and fibroblasts of the levator ani muscle (Fig. 7Go, A, E, I, and M). Treatment with E2 increased the intensity of cystatin C immunostaining in the smooth muscle of the muscularis and fibroblasts but not in the epithelium (Fig. 7Go, B and F) or the vascular smooth muscle (Fig. 7IGo). Treatment with E2 + P blocked the ability of E2 to increase expression of cystatin C protein (Fig. 7Go, C, G, and O). In the estrogen-treated levator ani, only the fibroblasts, not the skeletal myocytes, expressed cystatin C protein (Fig. 7NGo).



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  		FIG. 7. Photomicrographs showing immunostaining for cystatin C in the macaque vagina after experiment 2. The plate is arranged in four columns from left to right: no hormone, E2 alone, E2 + P, and control (normal goal serum; E2-treated animal). The rows show epithelium (Ep) and lamina propria (LP), muscularis, an artery (Art), and the levator ani (LA). All sections were counterstained lightly with hematoxylin. Bar (in P), 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cystatin C is a secreted inhibitor of cysteine proteinases including cathepsins B, H, L, and C (24, 25). Like other members of the family 2 cystatins (e.g. cystatin D, S, SA, and SN), cystatin C is reported to regulate cysteine proteinase action during normal body processes including bone resorption (26) and tissue remodeling. Cystatin C deficiency and increased cysteine proteinase activity is associated with numerous pathological conditions associated with ECM degradation including cancer progression (20, 27) and atherosclerosis (28). Expression of cystatin C is reported for all human organs, and cystatin C is present in all body fluids (25). However, mRNA levels vary greatly between different tissue types (29). High levels of cystatin C transcript have been reported for prostate, salivary gland, and colorectal cancer.

In this study, we demonstrated that E2 acts to increase cystatin C expression in the macaque vagina and that cotreatment with P (E2 + P) inhibited E2-stimulated cystatin C mRNA and protein expression. Studies by our laboratory and others have demonstrated that nuclear estrogen receptors (ERs) and progesterone receptors are present in basal epithelial cells, fibroblasts, and smooth muscle cells of the macaque vagina. Therefore, cystatin C expression follows a pattern similar to many other E2-stimulated factors in the reproductive tract (10). However, E2 treatment failed to increase cystatin C expression in the endometrium, a well-characterized target for estrogen action. Therefore, there may be cell-specific factors that either interact with the ER or are indirectly influenced by E2 action through ERs to induce cystatin C expression in the vagina. Structural analysis of the human cystatin C gene (29) revealed features common with those of housekeeping genes and that the 5'-flanking sequences lacked putative hormone response elements. However, the mouse cystatin C has two adjacent activator protein-1 binding sites in the promoter region (30), and in mouse decidua, cystatin C transcription is induced by TGFß and epithelial growth factor. The macaque cystatin C gene promoter has not yet been analyzed.

Treatment with estrogen results in a thickening of all components of the vaginal wall (31). This thickening is associated with both cell hypertrophy and increases in ECM. The major fibrous proteins of the ECM, collagen and elastin, provide structural integrity and mechanical strength to the vagina (18). Extracellular proteases including matrix metalloproteinases (MMPs) (32) and cathepsins (18) that degrade these structural components are likely to play significant roles in the turnover of ECM. Expression of MMP-2 and MMP-9 is decreased by estrogen treatment of fibroblasts isolated from arcus tendineous from women (32). In preliminary studies our macro gene array analysis (data not shown) revealed evidence for gene expression of structural genes (e.g. collagen1A and 4A, fibronectin-1) and matrix-modifying enzymes including gelatinase A, MMP-26, and cathepsin B and D in vaginal tissues; expression of these genes may affect overall balance of synthesis vs. degradation of ECM proteins. Activated cathepsin B but not procathepsin B is increased in the vagina of women with pelvic floor prolapse (18). In the macaque vagina, E2-induced cystatin C could act to suppress the actions of activated cathepsin B or other cysteine proteinases, effectively shifting the balance in favor of ECM maintenance. Blockade of cystatin C expression, either through hormone deprivation (as associated with menopause) or suppression of E2 action by P, may lead to reduced levels of fibrous protein in the ECM accompanied by thinning and, perhaps, weakening of the vaginal wall. In this study, we showed that raloxifene treatment failed to elevate vaginal cystatin C expression; elsewhere we reported that raloxifene failed to maintain vaginal thickness (31); clearly, this SERM is not estrogenic in the macaque vagina. Other SERMs (e.g. levormeloxifene and idoxifene) may have unwanted effects on the pelvic floor (14, 15). Therefore, further study of specific SERM compounds is needed to characterize the action of these novel compounds in women.

In summary, the cysteine protease inhibitor, cystatin C, is increased by E2 and suppressed by P in the fibroblasts and smooth muscle cells of the rhesus macaque vagina. Because of the need to improve our understanding of pelvic floor disorders, the physiological role of vaginal cathepsins and cystatin C, their effects on collagen and elastin, and their impact on the strength and integrity of the pelvic floor are high-priority issues for additional research.


   Footnotes
 
This work was supported by National Institutes of Health Grants RR00163 (to O.D.S. and R.M.B.), HD38673 (to A.L.C.), HD01243 (to L.N.O.), and U54 HD 18185 as part of the Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: E2, Estradiol; ECM, extracellular matrix; ER, estrogen receptor; ICC, immunocytochemistry; ISH, in situ hybridization; MMP, matrix metalloproteinase; P, progesterone; SERM, selective estrogen receptor modulator; SSC, saline sodium citrate.

Received July 3, 2003.

Accepted November 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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