2 Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390; 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; 4 Department of Pathology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; 5 Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195
Received on June 4, 2004; revised on August 9, 2004; accepted on August 9, 2004
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Abstract |
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Key words:
5-reductase type 1
/
cervical ripening
/
hyaluronan
/
hyaluronan synthase 2
/
parturition
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Introduction |
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The cervix is composed of an extracellular matrix consisting predominantly of collagen, elastin, and proteoglycans, and a cellular portion consisting of epithelium, smooth muscle, stromal cells, and blood vessels (Leppert, 1995). The cervical softening and remodeling process is complex and involves properly timed biochemical cascades that result in tissue growth, increased cervical secretions, changes in the composition and structure of the extracellular matrix, and infiltration of the cervical stroma matrix by inflammatory cells. Regulation of these processes and the interactions between the cellular component, inflammatory cells, and extracellular matrix remain unclear.
Associated with the changes in tensile properties of the cervix during ripening are changes in the collagen and glycosaminoglycan components of the cervical connective tissue. Collagen, because of its cross-linked, three-dimensional fibrillar structure, contributes greatly to the stiffness of the cervix. The organization, distribution, and structure of the collagen fibrils in the cervix are altered during pregnancy. Microscopic examinations of cervical tissue from numerous species demonstrate that in the nonpregnant animal, collagen is present in densely packed, large bundles of fibrils with little intervening extracellular matrix material (Bryant et al., 1968; Buckingham et al., 1962
; Csoka et al., 1999
; Leppert, 1995
; Rimmer, 1973
; Theobald et al., 1982
; Winkler and Rath, 1999
). Near term, and associated with increased extensibility of the cervix, the collagen bundles become smaller, more dispersed, and randomly oriented.
Concomitant with the changes in collagen structure is a marked increase in the glycosaminoglycan (GAG) content of the cervix in human and rat (Downing and Sherwood 1986; Osmers et al., 1993
). In particular, the GAG hyaluronan (HA) increases markedly in the cervix during late pregnancy in human, sheep, guinea pig, rabbit, and rat (Anderson et al., 1991
; Downing and Sherwood, 1986
; El Maradny et al., 1997
; Rajabi et al., 1992
). At the onset of labor, HA is the predominant GAG in the cervix. Studies in humans suggest increased serum concentrations of HA in women in labor as compared to pregnant women who are not in labor (Kobayashi et al., 1999
). Immediately after delivery, the concentration of cervical HA decreases to that of the nonpregnant state. HA also increases in cervices primed with prostaglandin E2 (Rath et al., 1993
), antiprogesterone (Cabrol et al., 1991
), and relaxin (Downing and Sherwood, 1986
), suggesting regulation by steroid and peptide hormones as well as prostaglandins. A higher concentration of HA has been reported in the cervical mucus of women with clinical indication of threatened preterm labor compared to normal pregnant women, implicating a role for HA in parturition and as a potential marker for prediction of impending preterm labor (Ogawa et al., 1998
). Additionally, increased HA of low molecular weight is reported in cervical mucus of women with normal pregnancies in the first stages of labor (Obara et al., 2001
).
The biological roles of HA in cervical remodeling are hypothesized but not well defined. HA is localized to the stromal extracellular matrix in the rabbit cervix (Maradny et al., 1997). Because cervical maturation is accompanied by an increase in water content (El Maradny et al., 1997
; Laurent and Fraser, 1992
) and HA has a high affinity for water molecules, a proposed role for HA in cervical remodeling is thought to be in promotion of tissue hydration. The accumulation of HA and water molecules in the interstices between the collagen fibrils may promote dispersion or prevent aggregation of the collagen fibrils, thus weakening the tensile strength of the matrix. Although numerous studies document regulated expression of HA in the ripening cervix, the enzymes controlling synthesis and degradation of HA and their regulation during cervical ripening at parturition have not been described.
HA is a polymer of repeating disaccharides of D-glucuronic acid ß-1, 3-N-acetylglucosamine-ß1, 4 (Lee and Spicer, 2000; Tammi et al., 2002
). Molecules of HA have a high molecular mass, ranging from 105 to 107 Da but can also exist as smaller fragments and oligosaccharides under certain physiological and pathophysiological conditions. HA is synthesized by one or more of three related isoenzymes in mammalian species, named hyaluronan synthase 1, 2, and 3 (HAS1, HAS2, HAS3) (Itano and Kimata, 1996
; Spicer et al., 1996
, 1997
). These enzymes are integral plasma membrane proteins that coordinately polymerize and translocate HA out of the cell. This is in contrast to other GAGs that are synthesized by resident Golgi enzymes and covalently attached to protein cores. The three HAS isoenzymes have distinct tissue and temporal expression patterns that dictate their varied functions. Genetic deletions of the three HAS genes indicate that only HAS2 is vital for development, resulting in death at embryonic day 10 due to failure of normal heart development (Camenisch et al., 2000
; Spicer et al., 2002
). The specific roles of HAS1 and HAS3 have not yet been documented.
The uptake and catabolism of HA appears to be as important as its synthesis in tissue morphogenesis and tissue homeostasis. HA is normally degraded by hyaluronidase enzymes. Five hyaluronidase genes and one pseudogene have been identified that vary in their tissue-specific expression (Csoka et al., 1999; Stern, 2003
). Hyaluronidase 1 (Hyal1) and hyaluronidase 2 (Hyal2) are proposed to be the major hyaluronidases of somatic tissues. Hyal1 appears to be a lysosomal enzyme, whereas Hyal2 is anchored to membranes, including the plasma membrane. Cellular uptake of HA is in part mediated by the cell surface receptors CD44 and RHAMM (Turley et al., 2002
). A balance in the regulation of HA synthesis and catabolism is crucial to normal tissue function.
In the current study we identify the transcription profiles of enzymes responsible for HA synthesis and degradation in the mouse and human cervix and define their temporal mRNA expressions in pregnancy. Additionally, we report aberrant expression of HAS2 in a mouse model with defects in cervical ripening: the 5-reductase type 1 deficient mouse (5
R1KO) (Mahendroo et al., 1996
, 1997
, 1999
). Based on the results of this study, we propose increased transcription of HAS2 to be the key regulator of increased HA content in cervical tissue during ripening in the mouse. Additionally, we show in the human an increase in HAS2 transcripts in cervical tissue obtained from women in labor as compared to pregnant women that are not in labor, suggesting that regulation of HA synthesis is a conserved process in both mouse and human cervical remodeling.
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Results |
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As compared to wild-type cervix at gestation day 18, HAS2 was identified as a gene with decreased expression in the cervix of the 5R1KO mouse model in a screen using the Affymetrix mouse oligonucleotide microarray chip A (data not shown). Verification of the chip data was done using quantitative real-time PCR methods (Figure 3A). The mRNA expression of HAS2 at gestation day 18, 1 day prior to parturition, was reduced by 70% in the 5
R1KO cervix as compared to wild type. In contrast to HAS2 transcripts, the other two synthases, HAS1 and HAS3, and the HA catabolic enzymes Hyal1 and Hyal2 were expressed in gestation day 18 cervix of the mutant at a level similar to those in wild-type controls (Figure 3A).
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A balance in the regulation of HA synthesis and catabolism is critical to normal tissue homeostasis of HA. HA is degraded by a family of enzymes known as hyaluronidases. To evaluate HA catabolism in the cervix, the expression of Hyal1 and Hyal2 was determined in the mouse cervix from gestation day 11 to 19 by quantitative real-time PCR. Transcripts for both enzymes are expressed in the pregnant cervix with Hyal1 transcript abundance greater than Hyal2 on gestation day 15 and 18 (Figure 4A). Hyal1 and Hyal2 mRNA expression remain fairly constant until day 19, at which time both mRNAs increase in amount (Figure 4B, 4C). This temporal pattern of expression suggests the increased HA content prior to parturition is due primarily to increased synthesis. The elevated hyaluronidase expression after birth may facilitate the removal of HA as the cervix is remodeled to the nonpregnant state. Hyal1 and Hyal2 expression are similar in the cervix of 5R1KO at gestation day 18 as compared to wild type, again supporting HA synthesis rather than catabolism as the key regulatory step in elevation of tissue HA content during cervical ripening (Figure 3A).
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Discussion |
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Cervical remodeling can be divided into two distinct but overlapping phases: (1) softening, and (2) ripening (Liggins, 1978). Cervical softening is a gradual process that occurs several days or weeks prior to parturition (gestation day 12 in the rat [Harkness and Harkness, 1959
] and during the second trimester of pregnancy in the human [Leppert, 1995
]) and is characterized by cervical growth, changes in the tensile properties of the cervix, and changes in the composition of the extracellular matrix. During softening, the cervix must initiate changes required for parturition yet maintain the structure in a tightly closed state that is resistant to mechanical forces. Cervical ripening, which occurs in the hours (rodents) and days (women) preceding parturition, is characterized by hydration and further growth, decreased tensile strength, increased cervical secretions and lubrication, disorganization of collagen fibrils, further changes in the composition of GAGs, and infiltration of inflammatory cells. During this phase the cervix must completely efface and dilate to allow passage of the fetus.
Cervical softening and ripening are influenced by the local endocrine milieu, as well as interactions and cross-talk between the cellular components (stroma and epithelium), inflammatory cells, and extracellular matrix. The suppressive effect of progesterone on HAS2 gene expression exemplifies the influence of the local endocrine milieu. Direct regulation of HAS2 by progesterone would be supported by rescue of HAS2 expression in the 5R1KO by administration of the progesterone receptor antagonist onnapristone (ZK98299) because inactivation of progesterone receptor function by agonists accelerates cervical ripening (Chwalisz, 1994
). However, onnapristone did not overcome the inhibition of HAS2 expression in the 5
R1KO mouse nor accelerate cervical ripening (unpublished data). This result suggests that the suppression of HAS2 mRNA synthesis in the mouse was not due to direct interaction of the progesterone receptor with regulatory elements of the HAS2 promoter.
Transcription of HAS2 mRNA during pregnancy is detected from the earliest time point measured, day 11. Thus basal transcription of HAS2 occurs in a progesterone-rich environment, but an enhancement of transcription occurs on progesterone withdrawal on day 18. Previous studies report increased cervical HA content in animals treated with prostaglandin E2 or the peptide hormone relaxin and a decrease with treatment by progesterone receptor antagonists (Cabrol et al., 1991; Downing and Sherwood, 1986
; Rath et al., 1993
). These observations suggest that the temporal increase in HAS2 mRNA expression on gestation day 18 may be regulated in a tissue-specific expression by prostaglandin E2, relaxin, or other molecules expressed in pregnancy. This hypothesis is supported by the observation that the expression of HAS2 in the 5
R1KO mice is normal in other tissues that express HAS2, such as skin, lung, heart, and spleen (unpublished data). Additionally, the 5
R1KO mice have normal fertility rates (Mahendroo et al., 1997
), implying that HAS2 expression within the cumulus cells during cumulus oocyte expansion at ovulation are normal.
The observation that HAS2 mRNA is expressed in the mouse cervical epithelium yet HA is localized to both the matrix surrounding the stroma and epithelial cells shows the role the epithelium plays in influencing the composition and structure of the stromal extracellular matrix and suggests that HA may have additional functions during cervical remodeling in addition to a structural role in the stromal matrix.
The diverse physiological functions of HA in cervical softening and ripening remain to be elucidated. In other cell systems, HA plays a structural role and mediates signaling events via interactions with cell surface receptors, such as RHAMM and CD44 (Turley et al., 2002). Within the cervix, HA is postulated to carry out a structural role by promoting tissue hydration and collagen disorganization of the matrix. We propose that HA must have multiple functions in the cervical ripening process. This hypothesis is supported by the observation that the 5
R1KO mice have a normal increase in tissue hydration (determined by measurement of cervical wet and dry weight) on gestation day 18 despite the 67% decrease in tissue HA content (Table I). We predict that during cervical softening, HA's role may be to modify the tissue architecture to allow for an increase in tissue volume, creation of cell-free spaces, and modification of the stiff collagen matrix. Similar to HA's function in cartilage, HA would facilitate a softened (elastic) structure but provide strength and load-bearing capabilities required of the cervix during this period (Watanabe et al., 1997
). During cervical ripening the accelerated increase in HA may promote tissue hydration, leading to disorganization of collagen fibrils as well as influence the recruitment or activation of inflammatory cells in the cervix.
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Recent studies have shown a role for extracellular matrix components in activation of inflammatory cells in models of chronic tissue inflammation and wound healing (Horton et al., 1998; McKee et al., 1996
, 1997
; Wang et al., 2002
). Low-molecular-weight HA (<2 x 105 Da) binds to the cell surface receptor, CD44, and stimulates macrophages that are recruited to sites of inflammation to produce chemokines that in turn facilitate maintenance of an inflammatory response through attraction of other inflammatory cells. Low-molecular-weight HA is generated either through synthesis or through catabolism of high-molecular-weight HA to lower-molecular-mass products by hyaluronidase enzymes. We hypothesize that HA of low molecular weight may also influence macrophage activation and neutrophil migration during cervical ripening as it does during tissue inflammation and wound healing (Savani et al., 2000
). On activation the leukocytes and macrophages would then release molecules that in turn could facilitate further reorganization of the cervical extracellular matrix or perhaps function to serve as a cleanup crew in preparation for remodeling of the cervix back to the nonpregnant state immediately after birth.
Finally, the observation that HAS2 transcripts are elevated in IL women relative to pregnant NIL women suggests that HA function and homeostasis during cervical ripening is conserved between mouse and human. No HAS2 gene expression was detected in a small group of cervices from nonpregnant women (unpublished data). Previous reports describe an increase in HA in maternal serum with advancing gestation (Kobayashi et al., 1999) and increased HA in cervical mucus of women in the first stage of labor (Obara et al., 2001
). In addition to increased HA in mucus from women in early stages of labor, this study reports an increase in lower-molecular-weight HA and increased hyaluronidase activity in this group, suggesting that HA size may be important functionally during cervical ripening (Obara et al., 2001
). Another report suggests increased HA in cervical mucus of women with threatened preterm labor as compared to women with uncomplicated pregnancies (Ogawa et al., 1998
). However, cervical mucus HA was measured at a single time point in gestation that varied significantly between the two groups. These observations lead us to suggest that HA not only plays a role in cervical ripening but also that a premature elevation in HA during pregnancy may be a factor in initiation of premature labor. Our future studies will address the regulation of the HAS2 gene during cervical ripening as well as the physiological roles HA plays in the remarkable process of cervical remodeling during parturition.
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Materials and methods |
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Human tissues
Cervical tissues in this study were obtained from women undergoing cesarean hysterectomies due to complications such as placenta previa/acceta, uterine rupture, and leiomyomas. Tissues were obtained from these surgical specimens with appropriate documentation of the state of uterine contractions, presence or absence of clinical infection, stages of labor, use of oxytocin and prostaglandins, and cervical dilation and effacement. These criteria allow the accurate assessment of the sample as coming from a woman who is NIL and one who is IL. Cervical tissue was dissected into stromal-enriched and epithelium-enriched fractions and frozen at 80°C until used for RNA extraction. These tissues are collected by the Obstetrics and Gynecology Tissue and Biological Procurement Facility at University of Texas Southwestern headed by Dr. Ann Word. All protocols for obtaining tissue have been approved by the institutional review committee. Tissues are obtained following completion of informed consent forms from women prior to surgery.
RNA measurements and quantitative real-time PCR
Total RNA was extracted from frozen mouse or human tissue using RNA Stat 60 (Tel-Test B, Friendswood, TX). Subsequently, total RNA was treated with DNase I to remove any genomic DNA using DNA-free (Ambion, Austin, TX). CDNA synthesis was performed using 2 µg total RNA in a 100 µl volume (TaqMan cDNA synthesis kit, Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using SYBR Green and a PRISM7900HT Sequence Detection System (Applied Biosystems). Aliquots (20 ng) of cDNA were used for each quantitative PCR reaction, and each reaction was run in triplicate. Relative mRNA abundance was estimated based on comparisons of the cycle threshold (CT) value using the formula (2CT). Relative gene expression between experimental groups was determined using the delta delta CT method as described in User Bulletin #2 (Applied Biosystems). In the mouse, cyclophillin was used as the normalizer housekeeping gene, whereas in the human study h36B4 was used as the normalizer gene.
In the mouse studies, 2 to 3 cervix/genotype/timepoint were analyzed individually and used to determine an average ± SE. In human tissues, each sample was analyzed individually. Data are presented as the average relative gene expression ± SE.
Tissue harvest and preparation
Cervices for in situ hybridization and immunohistochemistry were harvested from anesthetized mice and fixed via transcardial perfusion with 4% paraformaldehyde. Subsequent paraffin processing, embedding, and sectioning were done by standard procedures (Sheehan and Hrapchak, 1980; Woods and Ellis, 1996
).
RNA in situ hybridization
Hybridizations were done by the Molecular Pathology Core Laboratory. 35S-labeled sense and antisense probes were generated by SP6 and T7 RNA polymerases, respectively, from linearized cDNA templates by in vitro transcription using the Maxiscript kit (Ambion). The plasmids contain a complementary DNA fragment encoding 500 nucleotides of the mouse HAS2 gene (Spicer et al., 1996).
Radioisotopic in situ hybridization was done as previously described (Shelton et al., 2000). Briefly, sections of cervix were deparaffinized, permeabilized, and acetylated prior to hybridization at 55°C with riboprobes diluted in a mixture containing 50% formamide, 0.3 M NaCl, 20 mM TrisHCl, pH 8.0, 5 mM ethylenediamine tetra-acetic acid, pH 8.0, 10 mM NaPO4, pH 8.0, 10% dextran sulfate, 1x Denhardt's, and 0.5 mg/ml tRNA. Following hybridization, the sections were rinsed with increasing stringency washes, subjected to RNase A (2 µg/ml, 30 min at 37°C) and dehydrated prior to dipping in K.5 nuclear emulsion gel (Ilford, UK). Autoradiographic exposure was for 28 days.
Review and photography of all histologic preparations was done on a Leica Laborlux-S photomicroscope equipped with brightfield and incident angle darkfield illumination. Photomicrography was achieved using this microscope and an Optronics VI-470 CCD camera interfaced with a Scion CG-7 framegrabber. Images were captured using Scion Image 1.62c acquisition and analysis software and processed with Adobe Photoshop 5.5.
Immunohistochemistry
HA was detected by a specific binding probe, hyaluronan-binding protein (HABP-b) that is biotinylated (Seikagaku 400763, Falmouth, MA). Five-micrometer paraffin sections were dewaxed and rehydrated in a graded series of alcohol solutions. The sections were treated with 0.6% hydrogen peroxide in phosphate buffered saline (PBS) for 20 min to eliminate endogenous peroxidase activity. Blocking was done for 20 min with 2% bovine serum albumin (BSA) in PBS. Serial sections were subjected to either HABP-b (3.3 µg/ml in 2% BSA/PBS) or PBS for 16 h at 4°C. Sections were subsequently incubated with peroxidase-conjugated streptavidin (1:500) (Vector Labs SA5704, Burlingame, CA) for 30 min at room temperature. The slides were incubated for 5 min twice with diaminobenzidine (Vector Labs), which produces a brown precipitate. Sections were counterstained in a solution of hematoxylin. The coverslips were attached with Permount.
Specificity of staining was verified by pretreatment with Streptomyces hyaluronidase (200 TRU/ml PBS) (Seikagaku 100740) for 2 h at 60°C prior to application of HABP-b.
FACE
FACE methodology as described previously was adapted for GAG analysis in the cervix (Calabro et al., 2000, 2001
). Cervices were excised and wet weight determined. Cervices were frozen in liquid nitrogen and subsequently lyophilized in a Speed-Vac for a minimum of 6 h. The freeze-dried cervix was weighed to determine dry weight. The water content was derived from the weight of the samples before and after lyophilization. To isolate GAGs, each dried tissue sample was put in 900 µl 100 mM ammonium acetate, 0.0005% phenol red, pH 7.0, and digested with proteinase K for 4 h at 37°C. The sample was boiled to inactivate the proteinase K and pelleted by centrifugation to remove any undigested material. The sample was divided into aliquots and lyophilized. An enzyme/buffer sham sample containing only the buffer was also digested with proteinase K as a negative control.
One aliquot was redissolved in 210 µl 100 mM ammonium acetate, 0.0005% phenol red, pH 7.0, and then split into 2 100-µl aliquots. One 100-µl aliquot was left untreated, and the other was sequentially digested with the following enzymes: 10 mU hyaluronidase SD for 1 h at 37°C (Seikagaku 1007411A), 10 mU chondroitinase ABC (Seikagaku 1003301A) for 1 h at 37°C, and 1 U of alkaline phosphatase (Sigma P6772, St. Louis, MO) together with 0.5 U glucoamylase (Sigma A7420) for 2 h at 37°C.
After enzyme digestion, the samples were fluorescently derivatized by addition of 40 µl of 12.5 mM 2-aminoacridone hydrochloride (500 nmol) in 85% dimethyl sulfoxide/15% acetic acid followed by incubation for 15 min at room temperature (Molecular Probes A-6289, Eugene, OR). Then 40 µl of 1.25 M sodium cyanoborohydride (Aldrich 156159, Milwaukee, WI) (50,000 nmol) in ultrapure water was added followed by incubation for 16 h at 37°C. After derivitization, 20 µl of glycerol (20% final concentration) was added to each sample prior to electrophoresis. All derivatized samples were stored at 70°C in the dark.
Five microliters of each sample and a standard were run on a monosaccharide gel in 1x Tris borate ethylenediamine tetra-acetic acid buffer. The gels were illuminated with UV light (365 nm) from an Ultra Lum Transilluminator, imaged with a Quantix cooled CCD camera from Roper Scientific/Photometrics, and analyzed using Gel-Pro (Media Cybernetics, Silver Spring, MD) as described (Calabro et al., 2001). Values were quantified as micrograms of HA and normalized to the dry weight of cervix.
Statistical analysis
The Student t-test was used to determine significant differences between groups. For all statistical analyses employed, p < 0.05 indicates significance.
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Acknowledgements |
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Abbreviations |
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References |
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