Regulation of hyaluronan expression during cervical ripening

Kelly J. Straach2, John M. Shelton3, James A. Richardson4, Vincent C. Hascall5 and Mala S. Mahendroo1,2

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


1 To whom correspondence should be addressed; e-mail: mala.mahendroo{at}utsouthwestern.edu.

Received on June 4, 2004; revised on August 9, 2004; accepted on August 9, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In preparation for birth, the uterine cervix undergoes a remarkable transformation from a closed, rigid structure to a distensible, remodeled configuration that stretches to allow passage of a fetus. Cervical ripening requires changes in the composition and structure of the extracellular matrix. These include an increase in the glycosaminoglycan hyaluronan (HA) prior to parturition. We show that the increase in cervical HA with advancing gestation correlates with the temporal increase in transcription of hyaluronan synthase 2 (HAS2) in the mouse. On gestation day 18, 1 day prior to birth, HAS2 transcripts are most abundant and begin to decline after birth. The steroid 5{alpha}-reductase type 1 deficient mouse, which fails to undergo cervical remodeling, has decreased expression of HAS2 mRNA and decreased tissue HA. HAS2 transcripts are expressed by cervical epithelium, and HA is localized to the matrix surrounding the stroma and to a lesser extent around the epithelium. HAS2 expression is suppressed in mice treated with progesterone. The mRNA expression levels of HA metabolizing enzymes hyaluronidase 1 and 2 were unchanged during pregnancy but increased after birth. Thus the net increase in HA content at term correlates with increased transcription of HAS2. Regulation of HA content is conserved in women because HAS2 transcripts are up-regulated in cervices of women in labor as compared to pregnant women not in labor. These results provide insights into the regulation of HA biosynthesis during cervical ripening and underscore the physiological role of HA in this essential process.

Key words: 5{alpha}-reductase type 1 / cervical ripening / hyaluronan / hyaluronan synthase 2 / parturition


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The molecular mechanism controlling the initiation of parturition remains one of the fundamental unanswered questions in reproductive biology. Successful parturition in all mammalian species is characterized by two essential processes. First, the uterus must be converted from a quiescent structure with dis-synchronous contractions to an active, coordinately contracting organ. Second, the cervix must undergo a transformation from a closed, rigid, nondistensible structure to a soft, distensible ring permitting passage of a term fetus. Successful delivery requires that these independent events be coordinated and timely.

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, 1995Go). 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., 1968Go; Buckingham et al., 1962Go; Csoka et al., 1999Go; Leppert, 1995Go; Rimmer, 1973Go; Theobald et al., 1982Go; Winkler and Rath, 1999Go). 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 1986Go; Osmers et al., 1993Go). 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., 1991Go; Downing and Sherwood, 1986Go; El Maradny et al., 1997Go; Rajabi et al., 1992Go). 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., 1999Go). 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., 1993Go), antiprogesterone (Cabrol et al., 1991Go), and relaxin (Downing and Sherwood, 1986Go), 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., 1998Go). 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., 2001Go).

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., 1997Go). Because cervical maturation is accompanied by an increase in water content (El Maradny et al., 1997Go; Laurent and Fraser, 1992Go) 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, 2000Go; Tammi et al., 2002Go). 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, 1996Go; Spicer et al., 1996Go, 1997Go). 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., 2000Go; Spicer et al., 2002Go). 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., 1999Go; Stern, 2003Go). 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., 2002Go). 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{alpha}-reductase type 1 deficient mouse (5{alpha}R1KO) (Mahendroo et al., 1996Go, 1997Go, 1999Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Quantitative real-time polymerase chain reaction (PCR) was used to determine the relative abundance and temporal expression of HAS1, HAS2, and HAS3 in the mouse cervix from gestation day 11 to day 19, the day of birth. All three HASs are expressed in the mouse cervix, but with varying degrees of abundance. In Figure 1A an estimate of the relative mRNA abundance of the three isozymes at gestation day 15 and 18 is indicated. The abundance is estimated from the log of the average CT (cycle threshold) for each time point and is expressed relative to HAS2 expression on day 18. HAS2 has the greatest mRNA expression on day 18. Relative to HAS2 abundance at this time point, HAS1 mRNA on days 15 and 18 is only 1%, HAS2 on day 15 is 30%, and HAS3 mRNA expression on days 15 and 18 is 10%. HAS1 expression is low (CT range: 26.8–27.9), followed by HAS3 with intermediate expression (CT range: 23.0–25.5), and finally HAS2 with the greatest expression (CT range: 20.0–22.5). The expression of HAS1 increases on gestation day 18 compared with earlier time points. However, the overall transcription is low compared with the other HAS enzymes, suggesting that this is not the key enzyme responsible for HA synthesis in the cervix (Figure 1A, 1B). In contrast, HAS2 is expressed throughout this period with peak expression on gestation day 18 followed by a decline in expression on day 19 (Figure 1C). HAS3 is expressed in the cervix from day 11 to 19. However, the temporal pattern differs from HAS2 with little change in expression until birth, at which time expression declines (Figure 1D). Together these data indicate that HAS2 mRNA levels increase the day prior to parturition and that HAS2 may be the most abundant HAS in the mouse cervix.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Relative abundance and temporal expression of HAS1, HAS2, and HAS3 transcripts in the pregnant mouse cervix. Quantitative real-time PCR was done using cervical tissue from gestation day 11 to 19. Two to three cervices per time point were used. (A) Relative abundance of transcripts for HAS1, HAS2, and HAS3 on day 15 and 18 of gestation ± SE. Temporal expressions of the three HASs through the second half of gestation are indicated: HAS1 (B), HAS2 (C), and HAS3 (D).

 
To determine if the increase in HAS2 mRNA correlates with an increase in HA content in the cervix, tissue HA levels were measured using fluorophore-assisted carbohydrate electrophoresis (FACE). The tissue content of HA was measured in wild-type mice on gestation days 15, 16, 17, 18, 19, and 1 day postpartum (d1pp). For each time point, micrograms of HA was normalized to the dry tissue weight. HA is present at all time points; however, as indicated in Figure 2, the increases in cervical HA on gestation days 18 and 19 correlate well with the increases in HAS2 expression (Figure 1C).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 2. HA content in cervix. HA synthesis occurs throughout the latter days of gestation with the greatest content on gestation days 18 and 19. HA content was measured by FACE and values normalized to cervical dry weight. Data represent an average and SD for 3 cervices per time point.

 
The importance of HAS2 in cervical HA synthesis was further emphasized through investigation of mice with defective cervical ripening. Mouse models with defects in cervical ripening that fail to initiate parturition are studied in our laboratory. One model has a targeted mutation in the steroid 5{alpha}-reductase type 1 gene (5{alpha}R1KO mouse) (Mahendroo et al., 1996Go). The steroid 5{alpha}-reductase enzyme catalyzes the reduction of a double bond at the 4, 5 position of the A ring of steroids such as progesterone, testosterone, and androstenedione (Russell and Wilson, 1994Go). Inadequate metabolism of progesterone to less active steroids by 5{alpha}-reductase type 1 leads to elevated progesterone concentrations within the cervical tissue. This results in a compromise in cervical ripening at term despite normal uterine contractility in the 5{alpha}R1KO mice. In this mouse model, pregnancy progresses normally, but the animals fail to deliver young. The pups die in utero and eventually are delivered or resorbed 2 to 3 days after the expected day of delivery. In an attempt to identify genes that may play a role in the cervical ripening process, the expression profile of genes in wild-type cervix was compared to that of the cervix of 5{alpha}R1KO mice in microarray screens.

As compared to wild-type cervix at gestation day 18, HAS2 was identified as a gene with decreased expression in the cervix of the 5{alpha}R1KO 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{alpha}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).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. Decreased HAS2 mRNA expression and HA content on gestation day 18 in cervical ripening defective 5{alpha}R1KO mouse. (A) HAS2, HAS1, HAS3, Hyal1, and Hyal2 expressions were determined by real-time PCR in wild type (WT), and 5{alpha}R1KO mice. Four to five cervices were measured per genotype and an average ± SE is indicated by the error bars. (B) FACE analysis of HA content on gestation day 18 in WT and 5{alpha}R1KO mice. Each sample is treated with (+) or without (–) hyaluronidase SD and chondroitinase ABC to cleave the GAGs into disaccharides specific for HA as well as other GAGs. Migration of the HA-specific product is indicated by an arrow. The micrograms of HA are normalized to cervical dry weight. The values indicated below the gel are average values from 3 to 4 animals per group ± SE.

 
To determine if the decrease in HAS2 mRNA expression observed in the cervical ripening defective mice correlated with a decrease in tissue HA, FACE analysis was done to quantitate HA levels (Figure 3B). HA content was measured in gestation day 18 cervix from wild-type and 5{alpha}R1KO mice. The total micrograms of HA is normalized to the cervical dry weight and expressed as an average of 3 to 4 cervices per group. The cervical content of HA was reduced by 68% in the 5{alpha}R1KO as compared with wild type. These data are consistent with the reduced expression of HAS2 mRNA in the mutant mouse.

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 5{alpha}R1KO 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).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Relative abundance and temporal expression of Hyal1 and Hyal2 transcripts in the pregnant mouse cervix. Quantitative real-time PCR was performed using cervical tissue from gestation day 11 to 19. Two to three cervices were measured per time point. (A) Relative mRNA abundance of Hyal1 and Hyal2 on days 15 and 18 of pregnancy. (B and C) Temporal expression of Hyal1 and Hyal2 through the second half of gestation. SE is represented by the error bars.

 
To begin to address the in vivo regulation of HAS2 in the cervix, we initiated studies in wild-type mice in which progesterone was administered from day 17 of pregnancy (Figure 5). Cervices were collected from these mice late on gestation day 18, and expression of HAS2 was measured by PCR. Progesterone administration to wild-type mice inhibited the temporal rise in HAS2 expression on day 18 suggesting that this steroid suppressed HAS2 expression. HAS1 expression declined further in the presence of progesterone. In contrast to HAS2 expression, HAS3, Hyal1, and Hyal2 remained unchanged.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Progesterone suppresses the temporal increase in cervical HAS2 mRNA expression on gestation day 18. Real-time PCR was performed using gestation day 18 cervices from untreated animals and animals administered a time-released progesterone pellet on day 17 to give a circulating dose of 110 ng/ml. In each group, 3 cervices were used and data represent an average ± SE.

 
To determine the cell-specific expression of HAS2 mRNA in the pregnant cervix, an antisense probe specific to the mouse HAS2 mRNA was generated and hybridized to cervical sections from a wild-type mouse at gestation day 18 (Figure 6A). Expression was detected primarily in the epithelium of cervix and vagina. The striking pattern of expression of HAS2 within the epithelium indicates mRNA for HAS2 within the superficial terminally differentiated epithelium but not within the less differentiated basal epithelium layer (Figure 6B and 6C). In contrast, HA is localized primarily to the matrix surrounding the stroma as determined by staining of HA using a biotinylated-HA-binding protein probe (HABP-b) on wild-type cervix at gestation day 18 (Figure 7A and 7B). Additionally, some staining is evident in the pericellular matrix surrounding the cervical epithelium (data not shown).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 6. HAS2 transcripts expressed in terminally differentiated, mucus-secreting cervical epithelium. In situ hybridization using an antisense probe specific for mouse HAS2 hybridized to day 18 wild-type cervix. The o indicates the cervical os and the f, fornix, indicates the cervical–vaginal junction. (A) Low magnification of darkfield image. Bar of measure indicates 200 microns. (B) High magnification of darkfield image. Region shown in B is indicated by a box in A. Delineation of the basal epithelium from the superficial epithelium is indicated by a short and long bracket, respectively. (C) Corresponding brightfield image at high magnification.

 


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 7. Distribution of HA in gestation day 18 mouse cervix. Sections were incubated with a biotinylated HA-binding protein in the absence (A) and presence (B) of pretreatment with Streptomyces hyaluronidase. Large bracket indicates stroma and small bracket indicates the epithelium in A while S, E, O indicate stroma, epithelia, and cervical os, respectively, in B. Bar of measure indicates 200 microns.

 
The data reveal increased HA at the end of pregnancy in the mouse cervix and suggest that the increase is due to transcriptional up-regulation of HAS2 in the epithelium as parturition approaches. We next sought to determine if the increase in HA in pregnant women in labor as compared to pregnant women not in labor correlated with alterations in expression of either HAS1, HAS2, HAS3, or Hyal1. Cervical tissues obtained from cesarean hysterectomies from women in labor (IL) or women not in labor (NIL), were dissected into stroma-enriched and epithelium-enriched fractions, and total RNA was isolated and used to measure expression of HAS1, HAS2, HAS3, and Hyal1 by PCR. To compare relative mRNA abundance of the three synthases, expression in each group is relative to in labor epithelial HAS2 expression. The three HAS synthases were expressed in the pregnant cervix (Figure 8A). Transcript expression was greatest for HAS2 IL in both the stroma and epithelium as compared to the NIL group. HAS1 mRNA expression in the stroma showed a similar pattern of expression, though reduced abundance as compared to HAS2 expression. In the human cervix, mRNA transcripts for HAS2 are in greater abundance in both epithelium and stroma. No significant difference in Hyal1 expression was observed in the stroma or epithelium when comparing expression of the IL group versus the NIL group (Figure 8B). The results of these experiments indicate a role of HAS2, and perhaps to a lesser extent HAS1, in regulation of HA content in the human cervix during remodeling.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8. Transcription of HAS2 is the most abundant of the three synthases and is induced in labor in humans. (A) HAS1, HAS2, HAS3 expression in epithelium enriched tissue (NIL, n = 11, IL, n = 8) and stromal enriched tissue (NIL, n = 9, IL, n = 10). (B) Hyal1 expression in stroma (NIL, n = 7, IL, n = 8) and epithelium (NIL, n = 11, IL, n = 8). Asterisks (*) indicate significance between the NIL and IL group (p < 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The observation that HA increases in the cervix at the end of gestation has been known for over 30 years, yet the regulation of its biosynthesis and physiological function have not been investigated (Danforth et al., 1974Go). Our studies suggest tissue-specific and temporal regulation of HA biosynthesis by HAS2 that correlates with cervical remodeling during parturition. Although we cannot rule out the possibility that modifications of synthase or hyaluronidase enzyme activity are regulatory, several lines of evidence suggest that HAS2 transcription is the key regulator of the increase in cervical HA accumulation at the end of pregnancy. First, in the mouse, HAS2 transcription is temporally upregulated in the hours preceding the onset of labor. Second, the mRNA expression of HAS2 during pregnancy is greater than that of the other hyaluronan synthases, HAS1 and HAS3. Third, the elevation in cervical tissue HA content correlates with the temporal expression of HAS2 mRNA. Fourth, the 5{alpha}R1KO mouse, which fails to initiate parturition due to a defect in cervical ripening, has decreased mRNA expression of HAS2 and decreased HA content in the cervix at gestation day 18. Fifth, the mRNA expression of hyaluronidases that breakdown HA (Hyal1 and Hyal2) is not changed during pregnancy. Sixth, in humans, HAS2 was the most abundant HA synthase transcribed in the pregnant cervix, and its mRNA expression was increased in IL women.

Cervical remodeling can be divided into two distinct but overlapping phases: (1) softening, and (2) ripening (Liggins, 1978Go). Cervical softening is a gradual process that occurs several days or weeks prior to parturition (gestation day 12 in the rat [Harkness and Harkness, 1959Go] and during the second trimester of pregnancy in the human [Leppert, 1995Go]) 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 5{alpha}R1KO by administration of the progesterone receptor antagonist onnapristone (ZK98299) because inactivation of progesterone receptor function by agonists accelerates cervical ripening (Chwalisz, 1994Go). However, onnapristone did not overcome the inhibition of HAS2 expression in the 5{alpha}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., 1991Go; Downing and Sherwood, 1986Go; Rath et al., 1993Go). 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{alpha}R1KO mice is normal in other tissues that express HAS2, such as skin, lung, heart, and spleen (unpublished data). Additionally, the 5{alpha}R1KO mice have normal fertility rates (Mahendroo et al., 1997Go), 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., 2002Go). 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{alpha}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., 1997Go). 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.


View this table:
[in this window]
[in a new window]
 
Table I. Summary of average cervix weight and water content

 
In other cell systems where HA plays a crucial role, the formation and stability of the HA rich extracellular matrix requires interaction of HA with proteins and other GAGs (Tammi et al., 2002Go; Turley et al., 2002Go). In the developing cardiovascular system, for example, a composite matrix of HA and a chondroitin sulfate proteoglycan, versican, are essential for provision of a hydrated matrix for endothelial cell migration and transformation during cardiac embryogenesis as shown in mice deficient in synthesis of HAS2 or versican (Camenisch et al., 2000Go; Mjaatvedt et al., 1998Go). Similarly, stabilization of the HA matrix requires interaction with aggrecan and cartilage link protein in cartilage and with the heavy chain subunits of the inter alpha trypsin inhibitor during expansion of the cumulus oocyte matrix during ovulation (Day, 1999Go; Zhuo et al., 2001Go). Interactions of HA with its cell surface receptor, CD44, are reported to mediate cell signaling events, activate immune cell function, and control uptake and intracellular degradation of HA (Turley et al., 2002Go). Identification of HA-associated molecules in the cervix will be important for assessing the biological function of HA in this tissue. The HA binding molecules CD44, versican, and tumor necrosis factor–stimulated gene-6 are reported to be expressed in the cervix of some species (Fujimoto et al., 2002Go; Maradny et al., 1997Go; Osmers et al., 1993Go).

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., 1998Go; McKee et al., 1996Go, 1997Go; Wang et al., 2002Go). 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., 2000Go). 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., 1999Go) and increased HA in cervical mucus of women in the first stage of labor (Obara et al., 2001Go). 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., 2001Go). Another report suggests increased HA in cervical mucus of women with threatened preterm labor as compared to women with uncomplicated pregnancies (Ogawa et al., 1998Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Mice
Animals were housed at 22°C under a 12-h light cycle (lights on, 0600–1800 h). All mice used in these studies were of mixed strain (C57BL6/129SvEv). 5{alpha}R1KO mice were generated and genotyped as described previously (Mahendroo et al., 1996Go). Females housed overnight with males were checked at midday for vaginal plugs. Plug day was counted as day 0, and birth occurred on day 19. All studies were conducted in accordance with the standards of humane animal care described in the NIH Guide for the Care and Use of Laboratory Animals using protocols approved by an institutional animal care and research advisory committee.

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 (2–CT). 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, 1980Go; Woods and Ellis, 1996Go).

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., 1996Go).

Radioisotopic in situ hybridization was done as previously described (Shelton et al., 2000Go). 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 Tris–HCl, 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., 2000Go, 2001Go). 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 100741–1A), 10 mU chondroitinase ABC (Seikagaku 100330–1A) 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., 2001Go). 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.


    Acknowledgements
 
We thank R. Ann Word for helpful discussions, Sivling Lopez for technical assistance, Aniq Darr for teaching us FACE methodology, and David W. Russell for critical reading of the manuscript. We thank Dr. R. Ann Word and the Human Tissue Procurement Facility in the Department of Obstetrics and Gynecology for human cervical tissues. This work was aided by Grant 0203 from the Burroughs Wellcome Fund to M.S.M.


    Abbreviations
 
BSA, bovine serum albumin; FACE, fluorophore-assisted carbohydrate electrophoresis; GAG, glycosaminoglycan; HA, hyaluronan; HAS, hyaluronan synthase; IL, in labor; NIL, not in labor; PBS, phosphate buffered saline; PCR, polymerase chain reaction


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Anderson, J., Raynes, J., Fitzpatrick, R., and Dobson, H. (1991) Increased hyaluronate synthesis and changes in glycosaminoglycan ratios and molecular weight of proteoglycan: synthesised by cultured cervical tissue from ewes at various stages of pregnancy. Biochim. Biophys. Acta, 1075, 187–190.[CrossRef][ISI][Medline]

Bryant, M.W., Greenwell, A., and Weeks, P. (1968) Alterations in collages organization during dilation of the cervix uteri. Surg. Gynecol. Obstet., 126, 27.[ISI][Medline]

Buckingham, J., Selden, R., and Danforth, D. (1962) Connective tissue changes in the cervix during pregnancy and labor. Ann. NY Acad. Sci., 97, 733–742.[ISI][Medline]

Cabrol, D., Carbonne, B., Bienkiewicz, A., Dallot, E., Alj, A.E., and Cedard, L. (1991) Induction of labor and cervical maturation using mifepristone (RU 486) in teh late pregnant rat. Influence of a cyclooxygenase inhibitor (Diclofenac). Prostaglandins, 42, 71–79.[CrossRef][Medline]

Calabro, A., Hascall, V.C., and Midura, R.J. (2000) Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage. Glycobiology, 10, 283–293.[Abstract/Free Full Text]

Calabro, A., Midura, R., Wang, A., West, L., Plaas, A., and Hascall, V.C. (2001) Fluorophore-assisted carbohydrate electrophoresis (FACE) of glycosaminoglycans. Osteoarth. Cartil., 9, S16–S22.[CrossRef][ISI]

Camenisch, T.D., Spicer, A.P., Brehm-Gibson, T., Biesterfeldt, J., Augustine, M.L., Calabro, A. Jr., Kubalak, S., Klewer, S.E., and McDonald, J.A. (2000) Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J. Clin. Invest., 106, 349–360.[Abstract/Free Full Text]

Chwalisz, K. (1994) The use of progesterone antagonists for cervical ripening and as an adjunct to labour and delivery. Hum. Reprod., 9, 131–161.[ISI][Medline]

Csoka, A., Scherer, S., and Stern, R. (1999) Expression analysis of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31. Genomics, 60, 356–361.[CrossRef][ISI][Medline]

Danforth, D., Veis, A., Breen, M., Weinstein, H., Buckingham, J., and Manalo, P. (1974) The effect of pregnancy and labor on the human cervix: changes in collagen, glycoproteins, and glycosaminoglycans. Am. J. Obstet. Gynecol., 120, 641.[ISI][Medline]

Day, A.J. (1999) The structure and regulation of hyaluronan-binding proteins. Biochem. Soc. Trans., 27, 115–121.[ISI][Medline]

Downing, S.J. and Sherwood, O.D. (1986) The physiological role of relaxin in the pregnant rat. IV. The influence of relaxin on cervical collagen and glycosaminoglycans. Endocrinology, 118, 471–479.[Abstract]

El Maradny, E., Kanayama, N., Kobayashi, H., Hossain, B., Khatun, S., Liping, S., Kobayashi, T., and Terao, T. (1997) The role of hyaluronic acid as a mediator and regulator of cervical ripening. Hum. Reprod., 12, 1080–1088.[CrossRef][ISI][Medline]

Fujimoto, T., Savani, R.C., Watari, M., Day, A.J., and Strauss, J.F. III (2002) Induction of the hyaluronic acid–binding protein, tumor necrosis factor-stimulated gene-6, in cervical smooth muscle cells by tumor necrosis factor-{alpha} and prostaglandin E2. Am. J. Pathol., 160, 1495–1502.[Abstract/Free Full Text]

Harkness, M.L.R. and Harkness, R.D. (1959) Changes in the physical properties of the uterine cervix of the rat during pregnancy. J. Physiol., 148, 524–547.[ISI][Medline]

Horton, M.R., McKee, C., Bao, C., Liao, F., Farber, J.M., Hodge-DuFour, J., Puré, E., Oliver, B.L., Wright, T.M., and Nobel, P.W. (1998) Hyaluronan fragments synergize with interferon-{gamma} to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages. J. Biol. Chem., 273, 35088–35094.[Abstract/Free Full Text]

Itano, N. and Kimata, K. (1996) Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase. J. Biol. Chem., 271, 9875–9878.[Abstract/Free Full Text]

Kobayashi, H., Sun, G.W., Tanaka, Y., Kondo, T., and Terao, T. (1999) Serum hyaluronic acid levels during pregnancy and labor. Obstet. Gynecol., 93, 480–484.[Abstract/Free Full Text]

Laurent, T. and Fraser, J. (1992) Hayaluronan. FASEB J., 6, 2397–2404.[Abstract/Free Full Text]

Lee, J.Y. and Spicer, A.P. (2000) Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curr. Opin. Cell Biol., 12, 581–586.[CrossRef][ISI][Medline]

Leppert, P.C. (1995) Anatomy and physiology of cervical ripening. Clin. Obstet. Gynecol., 38, 267–279.[ISI][Medline]

Liggins, G. (1978) Ripening of the cervix. Semin. Perinatol., 2, 261–271.[ISI][Medline]

Mahendroo, M., Cala, K., and Russell, D. (1996) 5{alpha}-Reduced androgens play a key role in murine parturition. Mol. Endocrinol., 10, 380–392.[Abstract]

Mahendroo, M.S., Cala, K.M., Landrum, C.P., and Russell, D.W. (1997) Fetal death in mice lacking 5{alpha}-reductase type 1 caused by estrogen excess. Mol. Endocrinol., 11, 917–927.[Abstract/Free Full Text]

Mahendroo, M., Porter, A., Russell, D., and Word, R.A. (1999) The parturition defect in steroid 5{alpha}-reducase type 1 knockout mice is due to impaired cervical ripening. Mol. Endocrinol., 13, 981–992.[Abstract/Free Full Text]

Maradny, E., Kanayama, N., Kobayashi, H., Hossain, B., Khatun, S., Liping, S., Kobayshi, T., and Terao, T. (1997) The role of hyaluronic acid as a mediator and regulator of cervical ripening. Hum. Reprod., 12, 1080–1088.[CrossRef][ISI][Medline]

McKee, C., Penno, M., Cowman, M., Burdick, M., Strieter, R., Bao, C., and Nobel, P.W. (1996) Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. J. Clin. Invest., 98, 2403–2413.[Abstract/Free Full Text]

McKee, C., Lowenstein, C., Horton, M.R., Wu, J., Bao, C., Chin, B.Y., Choi, A.M.K., and Nobel, P.W. (1997) Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor {kappa}B-dependent mechanism. J. Biol. Chem., 272, 8013–8018.[Abstract/Free Full Text]

Mjaatvedt, C.H., Yamamura, H., Capehart, A.A., Turner, D., and Markwald, R.R. (1998) The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev. Biol., 202, 56–66.[CrossRef][ISI][Medline]

Obara, M., Hirano, H., Ogawa, M., Hiromitsu, T., Hosoya, N., Yoshida, Y., Miyauchi, S., and Tanaka, T. (2001) Changes in molecular weight of hyaluronan and hyaluronidase activity in uterine cervical mucus in cervical ripening. Acta Obstet. Gynecol. Scand., 80, 492–196.[CrossRef][ISI][Medline]

Ogawa, M., Hirano, H., Tsubaki, H., Kodama, H., and Tanaka, T. (1998) The role of cytokines in cervical ripening: correlations between the concentrations of cytokines and hyaluronic acid in cervical mucus and the induction of hyaluronic acid production by inflammatory cytokines by human cervical fibroblasts. Am. J. Obstet. Gynecol., 179, 105–110.[ISI][Medline]

Osmers, R., Rath, W., Pflanz, M.A., Kuhn, W., Stuhlsatz, H.W., and Szeverényi, M. (1993) Glycosaminoglycans in cervical connective tissue during pregnancy and parturition. Obstet. Gynecol., 81, 88–92.[Abstract]

Rajabi, M., Quillen, E.W., Nuwayhid, B.S., Brandt, R., and Poole, A.R. (1992) Circulating hyaluronic acid in nonpregnant, pregnant, and postpartum guinea pigs: elevated levels observed in parturition. Am. J. Obstet. Gynecol., 166, 242–246.[ISI][Medline]

Rath, W., Osmers, R., Adelmann-Grill, B.C., Stuhlsatz, H.W., Szerveny, M., and Kuhn, W. (1993) Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E2. Prostaglandins, 45, 375–384.[CrossRef][Medline]

Rimmer, D. (1973) The effect of pregnancy on the collagen of the uterine cervix of the mouse. J. Endocrinol., 57, 413–418.[ISI][Medline]

Russell, D. and Wilson, J. (1994) Steroid 5{alpha}-reductase: two genes/two enzymes. Ann. Rev. Biochem., 63, 25–61.[CrossRef][ISI][Medline]

Savani, R.C., Bagli, D.J., Harrison, R.E., and Turley, E.A. (2000) The role of hyaluronan-receptor interactions in wound repair. In H. Garg and M. Longaker (Eds.), Scarless wound healing. Marcel Dekker, New York, pp. 115–142.

Sheehan, D.C. and Hrapchak, B.B. (1980) Theory and practice of histotechnology. Battelle Press.

Shelton, J.M., Lee, M., Richardson, J., and Patel, S. (2000) Microsomal triglyceride transfer protein expression during mouse development. J. Lipid Res., 41, 532–537.[Abstract/Free Full Text]

Spicer, A.P., Augustine, M.L., and McDonald, J.A. (1996) Molecular coning and characterization of a putative mouse hyaluronan synthase. J. Biol. Chem., 271, 23400–23406.[Abstract/Free Full Text]

Spicer, A.P., Olson, J.S., and McDonald, J.A. (1997) Molecular cloning and characterization of a cDNA encoding the third putative mammalian hyaluronan synthase. J. Biol. Chem., 272, 8957–8961.[Abstract/Free Full Text]

Spicer, A.P., Tien, J.L., Joo, A., Joo, A., Spicer, A.P., Tien, J.L., and Bowling, J. (2002) Investigation of hyaluronan function in the mouse through targeted mutagenesis. Glycoconj. J., 19, 341–345.[CrossRef][ISI][Medline]

Stern, R. (2003) Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology, 13, 105R–1115.[Abstract/Free Full Text]

Tammi, M.I., Day, A.J., and Turley, E.A. (2002) Hyaluronan and homeostasis: a blancing act. J. Biol. Chem. 277, 4581–4588.[Free Full Text]

Theobald, P., Rath, W., Kuhnle, H., and Kuhn, W. (1982) Histological and electronmicroscopic examinations of collagenous connective tissue of teh non-pregnant cervix, pregnant cervix, and the pregnant prostaglandin-treated cervix. Arch. Gynecol., 231, 241–245.[ISI][Medline]

Turley, E.A., Noble, P.W., and Bourguignon, L.Y.W. (2002) Signaling properties of hyaluronan receptors. J. Biol. Chem., 277, 4589–4592.[Free Full Text]

Wang, Q., Teder, P., Judd, N.P., Nobel, P.W., and Doerschuk, C.M. (2002) CD44 deficiency leads to enhanced neutrophil migration and lung injury in Escherichia coli pneumonia in mice. Am. J. Pathol., 161, 2219–2228.[Abstract/Free Full Text]

Watanabe, H., Nakata, K., Kimata, K., Nakanishi, I., and Yamada, Y. (1997) Dwarfism and age-associated spinal degeneration of heterozygote cmd mice defective in aggrecan. Proc. Natl Acad. Sci. USA, 94, 6943–6947.[Abstract/Free Full Text]

Winkler, M. and Rath, W. (1999) Changes in the cervical extracellular matrix during pregnancy and parturition. J. Perinat. Med., 27, 45–61.[CrossRef][ISI][Medline]

Woods, A.E. and Ellis, R.C. (1996) Laboratory hystopathology, a complete reference. Churchill-Livingston Press.

Zhuo, L., Yoneda, M., Zhao, M., Yingsung, W., Yoshida, N., Kitagawa, Y., Kawamura, K., Suzuki, T., and Kimata, K. (2001) Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice. J. Biol. Chem., 276, 7693–7696.[Abstract/Free Full Text]