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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 5 2231-2235
Copyright © 2003 by The Endocrine Society

Decreased Type I Collagen Expression in Human Uterine Cervix during Pregnancy

Masaaki Iwahashi, Yasuteru Muragaki, Akira Ooshima and Naohiko Umesaki

Departments of Obstetrics and Gynecology (M.I., N.U.) and Pathology (Y.M., A.O.), Wakayama Medical University, Wakayama 641-0012, Japan

Address all correspondence and requests for reprints to: Masaaki Iwahashi, M.D., Department of Obstetrics and Gynecology, Wakayama Medical University, Kimiidera 811-1, Wakayama 641-0012, Japan. E-mail: masaaki{at}wakayama-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To provide some insight into the mechanism of cervical ripening, the expression of type I collagen was investigated in human uterine cervical tissues obtained from the first (n = 4) and third (n = 3) trimesters of normal pregnancy. Indirect immunofluorescent staining was performed for type I collagen, and Northern blot analysis was done to assess expression of mRNA for the {alpha}1(I) chain. Collagens were also extracted from the human cervical tissues in the first and third trimesters of pregnancy. Immunohistochemical analysis revealed loose distribution of type I collagen in the uterine cervix of the first trimester compared with the third trimester of pregnancy. The relative levels of various collagens were evaluated by SDS-PAGE. The ratios of the intensity of the band of {alpha}1(I) to that of total collagen {alpha}1 chain in cervical tissues of the third trimester were significantly lower than those in cervical tissues of the first trimester of pregnancy (P < 0.05). In contrast, the ratios of the intensity of the band of {alpha}1(III) to that of total collagen {alpha}1 chain in cervical tissues of the third trimester were significantly higher than those in cervical tissues of the first trimester of pregnancy (P < 0.05). Northern blot analysis revealed that the cervical expression of mRNA for the {alpha}1(I) chain was significantly reduced in the third trimester compared with the first trimester of pregnancy (P < 0.01). These results suggest that type I collagen might play an important role in the maintenance of pregnancy and that decreased expression of this collagen could be associated with the process of uterine cervical ripening.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXTRACELLULAR MATRIX (ECM) is considered to play an important role in the stability of tissues and in regulating the growth and differentiation of cells (1, 2). Synthesis, accumulation, and catabolism of the ECM are involved in wound healing and in the initiation and progression of numerous diseases (3).

Human cervix is made up mainly of fibrous connective tissues in which collagen (4) and glycosaminoglycans (5) predominate. The physiological properties of the cervix depend on interplay between collagen and glycosaminoglycan molecules (6). During ripening, marked biochemical changes take place in the cervix, causing it to become soft and dilatable at the time of parturition (7, 8). The most striking changes are the decreases in concentration of collagen and glycosaminoglycans (9, 10), together with a marked increase in hyaluronic acid (11, 12, 13).

Collagen is the main component of cervical connective tissue, giving it rigidity (4). Therefore, collagen might play a pivotal role in the structure and function of human uterine cervix. In the present study, we investigated the uterine cervical expression of type I collagen, a major component of the ECM, in the first and third trimesters of pregnancy by immunofluorescence staining, SDS-PAGE, and Northern blot analysis.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This project was approved by the Committee on Investigations Involving Human Subjects of Wakayama Medical University. Informed consent was obtained from each subject after the purpose and nature of the study had been fully explained.

Tissues

Pregnant cervical tissues (ectocervix, not lower uterine segment) were obtained from seven women aged 33–41 yr, four cases in the first trimester (9–14 wk) and three cases in the third trimester (36–38 wk), without labor by abdominal hysterectomy for gynecological application and were immediately frozen in liquid nitrogen. The details of the subjects studied in this investigation are given in Table 1Go. We excluded necrotic tissue from analysis by histologic examination. Gestational age was determined by the date of the last menstrual period and by ultrasonographic measurements performed in early pregnancy.


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  		Table 1. Clinical details of the subjects

 
Primary antibody

Monoclonal antibody (mAb) against the {alpha}1(I) chain of human type I collagen was used. Preparation of the antibodies has been described previously (14). In brief, BALB/C mice were immunized with human type I collagen, after it had been extracted from human placentas. The spleen cells of these mice were then hybridized with myeloma cells. After HAT (hypoxanthine-aminopterine-thymidine) selection, positive hybrids were identified by enzyme-linked immunosorbent assay. The specificity of the antibody was determined by immunoblotting or by inhibition in an enzyme-linked immunosorbent assay. This antibody showed no cross-reaction with type III, IV, and VI collagen or fibronectin.

Immunohistochemistry

Immunohistochemical analysis was performed by the standard indirect immunofluorescence method. In brief, 3-µm frozen sections were rehydrated in PBS at room temperature and then incubated with the primary antibody (diluted 1:100 in PBS) for 12 h at 4 C in a humidified chamber. After incubation, the sections were washed twice in PBS for 3 min. Each section was then incubated for 1 h at room temperature with human plasma-preabsorbed, fluorescein isothiocyanate-conjugated goat antibodies against mouse Ig diluted 1:100 in PBS (Organon Teknik Co., West Chester, PA). Subsequently, the sections were washed again in PBS, mounted in buffered glycerol, and examined under a fluorescence microscope (Olympus Corp., Tokyo, Japan).

SDS-PAGE of pepsin-solubilized collagens from the human cervical tissues

Minced samples of human cervical tissues were washed overnight in cold distilled water and freed of blood. Tissues were homogenized with a Polytron homogenizer in 50 volumes of 0.5 M acetic acid that contained 1 mg/ml pepsin (Sigma Chemical Co., St. Louis, MO). Collagens were extracted by previously described methods (15).

The solubility of the tissue collagen from each cervical sample was estimated by comparing the hydroxyproline content of the initial homogenate with that of the final solution of collagen (16). Type V collagen was isolated by salt precipitation from pepsin digests of human uterine cervical tissues by the methods described elsewhere (17, 18). The extracted type V collagen was also lyophilized. Estimation of the relative abundance of the {alpha}1(III) chain and {alpha}1(V) chain was performed by interrupted gel electrophoresis (15). Electrophoresis was performed in an 8% polyacrylamide slab gel (Sigma Chemical Co.) as previously described (19).

Lyophilized samples of collagens and type V collagen were dissolved at a concentration of 0.2 mg/ml and denatured by heating in the gel buffer that contained 1% SDS at 60 C for 30 min. Aliquots of 25 ml of each solution of denatured collagens and 5 ml of denatured type V collagen were applied to the gel and subjected to electrophoresis at 80 mA. After 1.5 h, the current was switched off, and sample wells were filled with a solution of 20% ß-mercaptoethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan) to cleave the intramolecular disulfide bonds of type III collagen, [{alpha}1(III)]3. Then electrophoresis was resumed and allowed to continue for another 1 h. Each collagen {alpha} chain was stained with Coomassie brilliant blue (Sigma Chemical Co.) and quantitated by densitometry. The relative amounts of {alpha}1(I), {alpha}1(III), and {alpha}1(V) chains were calculated by dividing the intensities of band areas under densitometric peaks of {alpha}1(I), {alpha}1(III), and {alpha}1(V) by total intensities of bands of {alpha}1 chains.

Northern blot analysis

The {alpha}1(I) collagen probe was a 1.1-kb EcoR1-EcoR1 fragment from human cDNA (20). DNA inserts were isolated as previously described (21). A riboprobe transcription kit (Promega Corp., Madison, WI) was used for transcription, and the transcripts were labeled with [32P]CTP for Northern blot analysis.

Total RNA was isolated from the cervical tissues, followed by size-fractionation on 1% denaturing agarose-formaldehyde gels and transfer to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by overnight capillary blotting in 20x sodium chloride-sodium citrate (SSC) solution. For normalization of {alpha}1(I) mRNA levels, duplicate membranes were prepared from the same RNA samples for separate hybridization to the {alpha}1(I) probe and a probe for ß-actin mRNA. Before Northern blot analysis, the transferred RNA was covalently cross-linked to the nitrocellulose membranes with a UV cross-linker (Stratagene, La Jolla, CA). Northern blots were prehybridized for 3 h at 65 C [{alpha}1(I)] or 42 C (ß-actin) in the presence of 50% (vol/vol) formamide under standard conditions, followed by hybridization with the appropriate radiolabeled probe at the same temperature for 16 h. Membranes were washed in 2x SSC/0.1% sodium dodecyl sulfate for 10 min at room temperature and then washed twice in 0.1x SSC/0.1% sodium dodecyl sulfate at 65 C [{alpha}1(I)] or at 55 C (ß-actin). Then, the membranes were exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) for 18 h at -70 C. Autoradiographs were analyzed by densitometry to quantitate differences in transcript levels between the first and third trimesters of pregnancy.

Statistical analysis

The ratios of {alpha}1(I) chains and {alpha}1(V) chains to total {alpha}1 chains, as estimated by densitometry, were represented as the means ± SEM. Densitometric analysis of the expression of type I collagen mRNA was conducted after normalization for the level of ß-actin mRNA in each sample. When two transcripts were detected, both were analyzed densitometrically.

Results were expressed as the mean ± SEM. Mean values were compared by Student’s t test or ANOVA, using a StatView software program (SAS Institute, Inc., Cary, NC) on a Macintosh computer. Two-tailed P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical analysis of the cervix

Control sections were stained with goat antibodies against mouse IgG without prior application of the primary antibody (Fig. 1AGo). When the mAb was first allowed to react with an excess of the {alpha}1(I) chain, no immunostaining was observed.



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  		Figure 1. Immunofluorescence micrographs of human cervical tissues obtained from the first and third trimesters of pregnancy with immunostaining by a mAb specific for the {alpha}1(I) chain. No immunofluorescence was recognized in the control section (A). Strong immunofluorescence specific for type I collagen was diffusely distributed in the stroma of the cervical tissues in the first trimester of pregnancy (B); however, in the third trimester, this collagen was distributed with dissociated pattern in the edematous stroma of cervix. (C). Original magnification, x250.

 
Immunostaining with the mAb against type I collagen showed a fibrillar pattern in the dense fibrous connective tissues of cervical stroma in the first trimester of pregnancy (Fig. 1BGo). In contrast, there was diffuse and dissociated immunostaining for type I collagen in the edematous cervical stroma in the third trimester (Fig. 1CGo).

Interrupted SDS-PAGE of pepsin-solubilized collagens from the cervix

The interrupted SDS-PAGE revealed that the relative level of {alpha}1(I) decreased in the cervical tissues of the third trimester of pregnancy, as compared with the first trimester (Fig. 2Go). The ratio of intensities of bands of each {alpha}1 chain to total {alpha} chains in the first and third trimesters of pregnancy is shown in Table 2Go. The ratio of intensities of bands of {alpha}1(I) to total {alpha} chains in the third trimester was significantly lower than that in the first trimester of pregnancy (P < 0.05). In contrast, the ratio of intensities of bands of {alpha}1(III) to total {alpha} chains in the third trimester was significantly higher than that in the first trimester of pregnancy (P < 0.01).



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  		Figure 2. SDS-PAGE of pepsin-solubilized collagens of the cervical tissues from the first trimester (lanes 1–4), the third trimester of pregnancy (lanes 5–7), and type V collagen extracted from the uterine cervical tissues (lane 8). The lane number represents the patient number. Samples of heat-denatured collagen (5 mg, lanes 1–7; 1.0 mg, lane 8) were subjected to electrophoresis on a slab gel for 1 h. After reduction in situ with ß-mercaptoethanol for 30 min, electrophoresis was resumed for 1 h. In this study, the {alpha} chains derived from type IV collagen could not be detected because of their low level in the sample. Note that the type V collagen obtained from the uterine cervical tissues by differential salt precipitation was composed predominantly of the {alpha}1(V) chain.

 

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  		Table 2. Relative abundance of the each {alpha}1 chain of collagen as compared with the total {alpha}1 chain in the cervical tissues obtained from the first trimester and the third trimester of pregnancy

 
No significant difference in the ratio of intensities of bands of {alpha}1(V) to total {alpha} chains was detected between the two groups.

Northern blot analysis of {alpha}1(I) mRNA

Northern blot analysis was done to determine the expression of the {alpha}1(I) gene in cervical tissues from the first and third trimesters of pregnancy (Fig. 3Go). The results of densitometric analysis of {alpha}1(I) mRNA expression are shown in Fig. 4Go. In cervical tissues from the third trimester, {alpha}1(I) mRNA expression was significantly lower than in tissues from the first trimester (0.43 ± 0.18 vs. 5.37 ± 2.11 densitometry units; P < 0.01). In contrast, the ß-actin mRNA level was similar in cervical tissues from both the first and third trimesters of pregnancy.



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  		Figure 3. Northern blot analysis of expression of the {alpha}1(I) chain gene in cervical tissues obtained from the first trimester (lanes 1–4) and the third trimester of pregnancy (lanes 5–7). The lane number represents the patient number. Twenty micrograms of total RNA from each source was analyzed. The positions of size markers (28S and 18S rRNA) are indicated. Cervical expression of the {alpha}1(I) chain mRNA was markedly reduced in the third trimester compared with the first trimester of pregnancy. In contrast, the level of ß-actin mRNA was identical.

 


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  		Figure 4. Densitometric analysis of the {alpha}1(I) chain mRNA in cervical tissues obtained from the first trimester and the third trimester of pregnancy. Results are represented as the mean ± SEM. **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated expression of type I collagen in the human cervical tissues obtained from the first and third trimesters of pregnancy. We were able to solubilize 75–85% of collagen in the human cervical tissues, as measured by reference to levels of hydroxyproline (data not shown). Therefore, it was postulated that the extracted collagen might accurately reflect the entire complement of collagen in the tissues.

The reduced cervical levels of type I collagen in the process of cervical softening suggest at least two possible mechanisms for regulation of the turnover of this collagen. First, the synthesis of type I collagen by cervical stromal cells might be reduced at the gene level in the process of cervical softening, as demonstrated in the present study by the decrease of {alpha}1(I) mRNA. Second, the degradation of cervical type I collagen might be intensified in the process of cervical softening. This finding suggests that type I collagenolytic enzyme activity might be increased in the human cervical tissues of pregnancy in the third trimester. Type I collagen-degrading enzyme is thought to be produced by the cervical stromal cells themselves (22, 23) or by neutrophil (24) accumulated in the cervical tissues during ripening. It is suggested that during pregnancy, collagenase in the cervical connective tissues might be controlled by hormones such as estrogen (25, 26, 27), dehydroepiandrosterone (28), and prostaglandins (29, 30), and/or cytokines such as IL-I (31) and IL-8 (13, 32, 33, 34, 35). It is also possible that decreased cervical synthesis and increased degradation of type I collagen occur simultaneously in the process of cervical ripening. Recent studies have shown that nitric oxide (NO) might participate in cervical ripening during pregnancy (36, 37) and that pharmacological manipulation of the NO bioavailability may have considerable clinical application (38, 39, 40, 41). Chwalisz et al. (39) reported that direct application of an NO donor (sodium nitroprusside) to the cervix can induce the biomechanical and anatomical changed characteristic of cervical ripening in guinea pigs. These observations are consistent with those recently reported by Thomson et al. (41, 42) in humans. In addition, it is suggested that NO production is increased in the cervix of rats in term and preterm labor (36) and that NO might directly control various metalloproteinases (43).

The ECM of the cervix is a fiber-reinforced compositive viscoelastic material made up of fibrillar collagen (two thirds type I, one third type III) and proteoglycans (hyaluronic acid, chondroitin sulfate, keratan sulfate, and dermatan sulfate; Refs. 44 and 45). Thus, our study suggested that a decreased ratio of type I collagen and increased ratio of type III collagen in the uterine cervix at term might induce cervical softening before labor and that decreased expression of type I collagen, the main macromolecular component of the ECM of the cervix, might be a key event in cervical dilatation at parturition.

The number of subjects in our study was very low. It was ethically difficult to obtain sufficient specimens from human cervical tissue of appropriate size during pregnancy and before labor. However, this study provides some clues to understanding the physiology of cervical ripening in terms of the ECM metabolism. Further work is needed to elucidate the mechanisms regulating the cervical expression of genes for other types of collagen in normal pregnancy, preterm cervical dilatation, and premature delivery.


   Footnotes
 
This work was partly supported by a research grant from the 1998 Wakayama Medical Award for Young Researchers.

Abbreviations: ECM, Extracellular matrix; mAb, monoclonal antibody; NO, nitric oxide; SSC, sodium chloride-sodium citrate.

Received August 1, 2002.

Accepted February 3, 2003.


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 Introduction
 Materials and Methods
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 Discussion
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