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Biol Reprod 2003, 10.1095/biolreprod.102.014704
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BIOLOGY OF REPRODUCTION 70, 385–390 (2004)
DOI: 10.1095/biolreprod.102.014704
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Thrombospondin 2 Deficiency in Pregnant Mice Results in Premature Softening of the Uterine Cervix1

Robert Kokenyesi2,3, Lucas C. Armstrong4, Azin Agah4, Raul Artal3, and Paul Bornstein4

Department of Obstetrics, Gynecology, and Women's Health,3 Saint Louis University, St. Louis, Missouri 63117 Department of Biochemistry,4 University of Washington, Seattle, Washington 98195


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gradual disorganization of collagen fibers in the stromal connective tissue of the uterine cervix is characteristic of progressive cervical softening during pregnancy. A lack of thrombospondin (TSP) 2 has been shown to be associated with altered collagen fibril morphology of connective-tissue-rich organs such as skin and tendon. The goal of this study was to determine the role of TSP2 in cervical softening by studying a TSP2-null mouse line. Creep testing showed that, in the nonpregnant animal and on Day 10 of pregnancy, there was no difference between the cervical extensibility of the wild-type and the TSP2-deficient mice. However, by Day 14 of pregnancy, the TSP2-null mice showed 4.5-fold increase in cervical extensibility, and by Day 18, a 6.1-fold increase, when compared with wild-type mice. A further indicator of compromised cervical integrity was that, on Days 14 and 18 of pregnancy, the cervix of TSP2-null mice broke rapidly under standard loading conditions that did not break the cervix of wild-type mice. Western blotting showed that TSP2 was expressed in the cervix of mice on Days 14 and 18 of pregnancy but not on Day 10 or in the nonpregnant animal. As determined by immunohistochemistry, the amount of matrix metalloproteinase 2 (MMP2) in the cervix of TSP2-null mice increased 11-fold on Day 14 of pregnancy and 19-fold on Day 18. Thus, TSP2-null mice provide an animal model to assist in the understanding of the molecular basis of spontaneous, premature softening of the uterine cervix.

cervix, female reproductive tract, parturition, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The uterine cervix functions as a connective-tissue-rich sphincter to ensure the retention of the fetus in the uterus during pregnancy [1, 2]. In the nonpregnant female or very early in pregnancy, the cervical canal is very narrow, and the cervix resists mechanical force to open it. By the end of the pregnancy, however, the cervix softens and becomes highly extensible to permit dilatation and effacement that allows the passage of the newborn through the birth canal in human [3], rat [4], and mouse [5]. Premature cervical softening during the first and early second trimesters may lead to a condition known as incompetent cervix, resulting in recurrent pregnancy loss [6, 7]. Most studies have focused on the late stage of cervical softening, the dilatation and effacement during and around labor; several regulatory factors have been identified, including steroid hormones [8], prostaglandins [9], cytokines [10], and nitric oxide [11]. However, the molecular basis for the structural changes during physiological and premature softening has remained poorly understood.

The universal feature of physiological cervical softening is the gradual remodeling of the stromal extracellular matrix. Characteristically, the cervical collagen fibers and bundles are progressively dissociated and disorganized during pregnancy in several species, including human [12–15] and mouse [16, 17]. The time course of such reorganization in mice correlates with altered mechanical properties of the tissue, such as decreased stiffness [5]. The stromal matrix has been shown to be composed of structural proteins, such as types I and III collagen and elastin, as well as proteoglycans and hyaluronan [12, 18]. Increased amounts of collagen-binding proteins, such as the proteoglycan decorin, have been found to correlate with increased cervical extensibility [4, 19, 20]. However, it is unclear whether altered expression of any extracellular matrix protein can be the cause of cervical softening. Thrombospondin (TSP) 2 is a multimeric extracellular matrix glycoprotein that is capable of a wide variety of molecular interactions [21, 22] and is functionally distinct from TSP1 [23]. Studies of TSP2-null mice have shown that a lack of TSP2 is associated with an altered morphology of fibrillar collagen fibers in skin and tendon [24, 25]. In addition, an increased amount and activity of matrix metalloproteinase 2 (MMP2) were found in granuloma tissue formed in the TSP2-null mice [26]. The expression pattern and physiological role of TSP2 have not been studied in uterine cervix.

We therefore studied the cervix of the TSP2-null mice because we wanted to determine if the altered state of collagen in these mice affected cervical softening.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The TSP2-null mice have previously been described [24]. In our studies, we initially used mice on both a pure 129/SvJ and on a mixed 129/SvJ/C57BL/6 background. Further experimentation was done on 129/SvJ/C57BL/6 mice. Timed pregnant females were used in which the presence of a cervical plug was considered to be Day 0 of pregnancy. All protocols involving experimental animals were reviewed and approved by the Saint Louis University Animal Care Committee.

Biomechanical Testing

The creep testing apparatus was built based on the design of Hollingsworth and Isherwood [27], and measurements were carried out according to a previously described procedure [4, 19]. The freshly excised cervix was mounted on the creep testing apparatus as previously described, except that both steel pins were inserted through the same cervical canal. Such placement of the pins provides a loading similar to that of a pup passing through the cervix during delivery. The cervix was immersed in Ham F12 medium during the test. A 50-g weight was used to provide constant loading during the 40-min test. Creep tests were carried out on at least 6 cervices at each gestational age with similar results. Statistical analysis of the extensibility values were done by Student t-test.

Extraction Protocol

Cervices were excised, frozen in liquid nitrogen, ground to a powder, and preextracted with TBS (20 mM Tris/HCl buffer, pH 7.4 containing 0.15 M NaCl) and protease inhibitors (10 mM EDTA, 1 mM PMSF, 10 mM N-ethylmaleimide, 5 µg/ml pepstatin) for 30 min in a cold room. The mixture was centrifuged at 15 000 x g for 30 min, the supernatant was removed for storage, and the residue was used for collagen extractions. The extraction method of sequential solubilization was used [28] because this approach results in the extraction of noncrosslinked collagen with 1 M NaCl followed by the extraction of acid-labile, aldimine-crosslinked collagen with 0.5 M acetic acid. After preextraction, the residue was extracted with 1 M NaCl (at 250 µl/10 mg wet weight) with the same protease inhibitors (stirring for 24 h at 4°C), and the residue was extracted with 0.5 M acetic acid (250 µl/10 mg wet weight). The residue from the acetic acid extraction was then boiled for 15 min with 1% SDS and 5% ß-mercaptoethanol to solubilize the heat-labile keto-amine-crosslinked collagen and the collagen-associated proteins.

Western Blotting

Extracts representing 3% of the wet weight of the cervix were run on a 6% polyacrylamide gel. The proteins were then electro-transferred onto an Immobilon P membrane (Millipore, Billerica, MA) for 3 h at 300 mA constant current. The membrane was blocked with 5% dry milk in TBS for 1 h at room temperature and was then incubated with a 1:500 dilution of anti-mouse TSP2 monoclonal antibody [29] for 12 h at 4°C. The membrane was then washed three times with TBST (TBS containing 0.3% Tween-20) and incubated with a 1:1000 dilution of an alkaline phosphatase-conjugated goat anti-mouse IgG for 1 h at room temperature. Following a series of washes with TBST, the bound antibody was visualized by the BCIP/NBT color development system (BioRad, Hercules, CA).

Immunohistochemistry

Paraffin-embedded sections were stained with antibodies against the N-terminal fragment of TSP2 (rabbit anti-mouse) and MMP2 (rabbit anti-human; Chemicon, Temecula, CA) as previously described [26]. Briefly, the sections were dewaxed with xylene and rehydrated through graded concentrations of ethanol. Sections were then treated with 0.1% H2O2 in methanol for 30 min to block endogenous peroxidase. The slides were then washed in PBS and blocked with 1% BSA in PBS for 30 min. To detect MMP2 levels, sections were incubated with antibodies to MMP2 (1:200 dilution) for 1 h. After an incubation with the primary antibody, the sections were washed in PBS and incubated with biotinylated secondary antibody. Color development was achieved by incubating the slides with avidin-biotin-horseradish peroxidase complex (ABC-HRP, Vector Laboratories, Burlingame, CA) followed by treatment with 3-3'-diaminobenzidine (Vector Laboratories). Sections were counterstained with methylene green, dehydrated, and mounted. All steps were carried out at room temperature. Sections were examined with the aid of an Eclipse 800 light microscope (Nikon, Tokyo, Japan).

Staining intensity of MMP2 was assessed by histomorphometry using MetaMorph software [26]. Quantitation was performed on two cervices for each genotype on Day 14 of pregnancy and three cervices for each genotype on Day 18. For each cervix, four sections and 10 fields per section were analyzed. Statistical significance was evaluated by Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biomechanical Properties of the Cervix in TSP2-Null Mice

Cervices from wild-type mice generated a creep curve similar to that obtained from a rat cervix [4]. There was an initial, rapid extension of the cervix during the first 5–10 min after loading, followed by a slower, linear extension until the end of the test (Fig. 1). The extensibility and the inner circumference values of the cervices from wild-type mice showed a gradual increase from the nonpregnant state to Day 18 of pregnancy, indicating that a progressive cervical softening is readily detectable by creep testing. The two phases of the creep curve were present when the wild-type cervix was tested during all stages in pregnancy and in the nonpregnant state.



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  		FIG. 1. Measurements of creep properties. Representative creep curves are shown for each stage of pregnancy (wild-type: black circles; TSP2-null: white triangles)

The extensibility values of the cervices of nonpregnant TSP2-null mice and at Day 10 of pregnancy were similar to that of the wild-type counterparts. However, beginning on Day 14 of pregnancy, the cervices of the TSP2-null mice showed significant deviation from the cervices of wild-type mice at the same gestational stage (Fig. 1). Most noticeably, the cervices of TSP2-null mice broke during creep testing, a phenomenon not observed with wild-type cervices at any stage of pregnancy. The mechanical failure of the TSP2-null cervices occurred relatively early during creep testing (Fig. 1), with the average time to failure of 9.6 min ± 1.9 min (SEM) on Day 14 of pregnancy and 2.2 min ± 0.4 min (SEM) on Day 18. The creep curves from Day 14 of pregnancy exhibited the initial, rapid extension phase, but these cervices broke before the transition to the linear portion of the curve. The creep curves from Day 18 of pregnancy showed that these cervices broke even before completing the initial, rapid extension phase. The mean extensibility values of the TSP2-null cervices were calculated using the available extension before failure, and these values were compared with the values of wild-type cervices from the same time frame. The data showed the expected gradual increase in extensibility in the wild-type animals during pregnancy (Fig. 2; black bars). The extensibility values for wild-type cervices increased significantly during pregnancy (P < 0.05 for nonpregnant to Day 10; P < 0.001 from Day 10 to Day 14 and from Day 14 to Day 18). However, cervices from TSP2-null animals showed a 4.5-fold increase by Day 14 and a 6.1-fold increase by Day 18 of pregnancy over the extensibility values of the corresponding wild-type cervices (Fig. 2; gray bars). Cervical wet weight was not significantly different in nonpregnant (19 ± 2 mg vs. 21 ± 1 mg) mice, and at Day 10 (20 ± 1 mg vs. 18 ± 2 mg), Day 14 (32 ± 3 mg vs. 34 ± 3 mg), and Day 18 (45 ± 3 mg vs. 50 ± 3 mg) of pregnancy in wild-type and TSP2-null animals, respectively.



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  		FIG. 2. Cervical extensibilities. Extensibilities were calculated from creep curves. n = 6 for each bar; SEM is indicated by error bars. Significant differences between wild-type and TSP2-null as indicated: *;thP < 0.01

TSP2 Expression in the Cervix during Pregnancy

We used uterine cervices of wild-type mice to determine if TSP2 is expressed in this portion of the reproductive tract. TSP2 was detected in cervices from Day 14 and Day 18 pregnant mice, but not from cervices of nonpregnant or Day 10 pregnant mice (Fig. 3). On Day 14 of pregnancy, TSP2 was coextracted only with noncrosslinked collagen. However, on Day 18 of pregnancy, TSP2 was coextracted with both the noncrosslinked and the irreversibly crosslinked collagen fractions. These results were reproducible in three independent tissue extractions and several Western blots. Immunohistochemical staining of cervices confirmed that TSP2 was detectable by Day 14 of pregnancy.



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  		FIG. 3. Western analyses of TSP2 expression in the wild-type cervix during pregnancy. NaCl extracts (N), acetic acid extracts (Ac), and SDS extracts (S) were run on a 6% polyacrylamide gel. TSP2 was detected using a monoclonal antibody

MMP2 in the TSP2-Deficient Cervix

Because increased levels of MMP2 have been implicated as a possible cause of the TSP2-null phenotype in reaction to injury [26], we wished to determine whether increased levels of MMP2 are also detectable in the prematurely softened cervix on Days 14 and 18 of pregnancy. Immunohistochemical analysis showed that MMP2 staining intensity was indistinguishable between wild-type and TSP2-null mice on Day 10 of pregnancy (Fig. 4, A and D). However, the staining appeared more intense and more widespread in the cervix of TSP2-null mice on Days 14 and 18 of pregnancy compared with those of wild-type mice (Fig. 4, B, C, E, and F). We performed histomorphometric analysis of the stained sections (Fig. 5) and found that the TSP2-null cervix had an 11-fold increase (P < 0.001) in MMP2 staining on Day 14 of pregnancy and a 19-fold increase (P < 0.001) on Day 18 compared with the wild-type cervix.



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  		FIG. 4. MMP2 expression in the cervix. Paraffin-embedded sections were stained with antibodies to MMP2 and visualized by the peroxidase reaction. Representative sections of the external os area are depicted. At Day 10 of pregnancy, MMP2 staining intensity was indistinguishable between the wild-type (A) and TSP2-null (D) mice. At Days 14 and 18 of pregnancy, MMP2 levels were elevated in TSP2-null cervix (E and F) compared with the wild-type cervix (B and C), respectively. Bar = 50 µm



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  		FIG. 5. Histomorphometric analysis of MMP2 expression. Sections of wild-type and TSP2-null cervices at Days 14 and 18 of pregnancy were stained with antibodies against MMP2. MMP2 levels were measured by histomorphometric analyses of immunohistochemical sections. Wild-type, light gray bars; TSP2-null, dark gray bars. Values are arbitrary units. Bars indicate the mean ± SD


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report represents the first attempt to use knockout mice to evaluate the role of a specific extracellular matrix component in cervical softening. We focused on the matricellular glycoprotein, TSP2, that has been demonstrated to affect the morphology of type I collagen fibrils, and, concomitantly, cause skin and tendon laxity [24, 25]. Our aim was to determine whether TSP2 was expressed in connective-tissue-rich stroma of the uterine cervix and whether TSP2 deficiency would affect the mechanical properties of the cervix during pregnancy.

Our data demonstrate that TSP2 expression in the uterine cervix starts between Day 10 and Day 14 of pregnancy. Thus, TSP2 is the first extracellular matrix molecule whose expression has been detected in the cervix only during pregnancy. This finding suggests that a specific role of cervical TSP2 is to maintain the integrity of the uterine cervix during the second half of pregnancy, and raises the possibility that other extracellular matrix molecules may be induced in a manner similar to TSP2 during pregnancy. Therefore, the remodeling of uterine cervix may not only be characterized by an altered balance of biosynthesis and degradation of existing matrix components but also by the appearance of newly induced matrix proteins. The absence of TSP2 in the nonpregnant animal and on Day 10 of pregnancy was not due to resistance of the tissue to the extraction because decorin, a known collagen fibril-associated proteoglycan, was detected in all extracts by Western blotting (data not shown). Induction of TSP2 has been observed during wound healing [30], where granulation tissue is under the influence of growth factors and cytokines. The uterine cervix at term has been described as a site of heightened immune reactivity [31] because of the presence of cytokines [10, 13] and the invasion by eosinophils and neutrophils and macrophages [31–33]. The increased expression of TSP2 on Day 18 of pregnancy coincides with the timing of increased invasion of the cervix by inflammatory cells, and this raises the possibility that TSP2 expression may, at least partially, be regulated by factors secreted from those cells.

Our findings demonstrate that cervices of TSP2-null mice have significantly increased extensibility by Day 14 of pregnancy, and this extensibility is further increased by Day 18 of pregnancy. These observations provide evidence for a significant, premature cervical softening by early third trimester and for a more severe premature softening by late third trimester. The cervices that showed increased extensibility also broke during mechanical testing.

The cervical extracellular matrix can be considered a fiber-reinforced composite material with load-bearing types I and III collagen fibers embedded in a viscous matrix of proteoglycans and hyaluronan [34]. The resistance to mechanical stress in a creep test is generated by the friction between the collagen fibers and the surrounding matrix during the slow movement of the fibers through the matrix [35]. In the case of the skin, a tissue with creep properties similar to that of the cervix [36], it has been shown that, after application of a load, the collagen fibers first align in the direction of the load [37], and this period corresponds with the rapid, initial extension phase. The linear phase of the creep curve is thought to reflect the slippage of the collagen fibrils as they pass each other. With the advance of pregnancy, the collagen fibers become more randomly oriented in the cervix [38, 39]. By analogy, the rapid, initial extension phase and the linear phase of the cervical creep curve are likely to be generated during the alignment of randomly oriented collagen fibers followed by the slippage of the fibers. The lack of the linear phase of the creep curve in the cervix of TSP2-null mice on Day 14 and the lack of the linear phase and most of the initial extension phase on Day 18 of pregnancy suggest that the cervical stroma is restructured in a way that permits the very rapid slippage of the collagen fibrils, leading to the failure of the tissue. Thus, our data from creep testing suggest that the lack of TSP2 results in decreased friction between collagen fibrils and the noncollagenous matrix. Our preliminary study of the cervical ultrastructure did not reveal striking differences between wild-type and TSP2-null mice; a more sophisticated, quantitative morphometric evaluation of the electron micrographs is necessary to determine what ultrastructural feature(s) might correlate with the altered mechanical properties in the TSP2-null mice.

TSP2 has been implicated in reducing the levels of active MMP2 in fibroblast cultures [40] and in sponge granulomas [41]; however, extracts of adult skin have not shown elevated MMP2 activity in TSP2-null mice [40]. Our data demonstrate that elevated levels of MMP2 are present in the TSP2-null cervix on Day 14 and on Day 18 of pregnancy. This finding suggests that the increase in cervical MMP2 is likely to contribute to the increased extensibility and to the tendency of the cervix to break under loading. These observations are important because they indicate a similarity between the mechanism of cervical softening in the mouse and in the human. Investigations of MMPs in human lower uterine segment, a transitional tissue between the uterus and the cervix, have shown that increased proteolytic activity by MMP8 and MMP9 is characteristic of the last phase of the cervical softening, the cervical dilatation and effacement during labor [42, 43].

Pregnant TSP2-null females do not experience spontaneous preterm deliveries. This finding is likely because other crucial factors, such as strong uterine contractions, need to coincide with premature cervical softening to start preterm delivery. Our observations are consistent with previous findings in the rat showing that the onset of uterine contractions is independent of cervical softening [44, 45]. Thus, TSP2-null mice represent a useful animal model for a milder form of spontaneous, premature cervical softening, as determined by the onset at late second trimester. Because this kind of cervical softening is thought to be the cause of decreased cervical length in women with elevated risk of preterm delivery [46, 47], further studies of the TSP2-null mice are likely to lead to a better understanding of the detailed molecular mechanism of premature cervical softening.

In summary, we have described the expression pattern of TSP2 in the mouse uterine cervix during pregnancy and have evaluated the role of TSP2 in cervical softening by studying a TSP2-deficient mouse line. Our studies demonstrate that TSP2 is the first reported example of an extracellular matrix protein whose expression is induced de novo in the cervix during pregnancy. We also show that an absence of TSP2 causes premature cervical softening. Thus, TSP2-null mice represent a useful animal model for the study of molecular mechanisms involved in the spontaneous, premature softening of the uterine cervix.


   ACKNOWLEDGMENTS
 
We thank Stan Davis, Meribeth Broadway, and Christina Zander for assistance with animal husbandry at Saint Louis University Department of Comparative Medicine.


   FOOTNOTES
 
1 This work was supported by NIH grants HD41036 to R.K. and AR45418 to P.B. Back

2 Correspondence: Robert Kokenyesi, Department of Obstetrics, Gynecology, and Women's Health, Saint Louis University, 6420 Clayton Rd., Suite 290, St. Louis, MO 63117. FAX: 314 645 6173; kokenyr{at}slucare1.sluh.edu Back

Received: 16 December 2002.

First decision: 29 December 2002.

Accepted: 12 September 2003.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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