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Endocrinology Vol. 142, No. 6 2221-2229
Copyright © 2001 by The Endocrine Society

ARTICLES

Evidence That Relaxin Inhibits Apoptosis in the Cervix and the Vagina during the Second Half of Pregnancy in the Rat1

Shuangping Zhao, P. A. Fields and O. D. Sherwood

Department of Molecular and Integrative Physiology (S.Z., O.D.S.) and College of Medicine (O.D.S.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Structural/Cellular Biology (P.A.F.), University of South Alabama College of Medicine, Mobile, Alabama 36688

Address all correspondence and requests for reprints to: Dr. O. D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: od-sherw{at}uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19–21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE SECOND half of 23-day rat pregnancy, the cervix and vagina grow and become more extensible (1, 2, 3, 4, 5, 6, 7). Cervical wet weight increases about 3-fold (1, 3, 5), and vaginal wet weight increases about 2-fold (5). It is known that the protein hormone relaxin plays a major role in promoting growth of the lower reproductive tract. Relaxin, which is secreted by the corpora lutea throughout the second half of pregnancy (8), induces more than a 40% increase in the wet weight of the cervix and the vagina by term (3, 5, 6, 7, 9). Our laboratory previously demonstrated that relaxin-dependent growth of the cervix and vagina is accompanied by an increase in their cell content (5, 6, 7, 9). Relaxin increases the number of epithelial cells, and this contributes to the increase in the circumference of the cervical and vaginal lumina (7, 9, 10). Relaxin also increases the number of stromal cells in the cervix and the vagina (7, 9). This increased cellular content of the lower reproductive tract likely contributes to relaxin’s vital role in facilitating rapid and safe delivery of the pups (11).

The cellular mechanism(s) that regulates the accumulation of cervical and vaginal cells during the second half of pregnancy is not known. Homeostatic control of cell number is thought to result from the dynamic balance between programmed cell death (apoptosis) and cell proliferation (12). Apoptosis affects scattered single cells and involves cell shrinkage, chromatin condensation, and the formation of apoptotic bodies that contain nuclear fragments (13, 14). A striking feature that occurs in nearly all cases of apoptosis is the activation of calcium/magnesium-dependent endonuclease activity, which specifically cleaves cellular DNA between regularly spaced nucleosomal units. The end result is the generation of DNA fragments that can be stained immunohistochemically and localized in situ (15). There are reports that apoptosis occurs in the female reproductive tract in both normal cycling (16, 17) and pregnant rats (18, 19).

The first hypothesis tested in this study is that the rate of apoptosis in cervical and vaginal cells declines during the second half of rat pregnancy. Finding that it does, we tested the hypothesis that relaxin inhibits apoptosis by determining whether the rate of apoptosis in the cervix and the vagina increases after the passive neutralization of circulating endogenous relaxin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Primiparous Sprague Dawley-derived rats, which were bred at about 75 days of age (Exp 1) or 90 days of age (Ex. 2), were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The day that sperm were found in the vagina was designated day 1 of pregnancy. The animals, which arrived on day 3, were housed individually and maintained in a light-controlled room with alternating 14 h of light (0700–2100 h) and 10 h of darkness, at a temperature of 23-25 C. From day 8, the light was shifted to 2100–1100 h to synchronize more precisely the stage of gestation among rats (20). With this shift in the light schedule, rats deliver in the early morning hours on day 23 of pregnancy, which is also designated as day 1 postpartum. Teklad 6% mouse/rat diet 8664 (Harlan/Teklad, Madison, WI) and water were available ad libitum. The animal experimentation described in this study was approved by the University of Illinois at Urbana-Champaign Laboratory Animal Care Advisory Committee.

Exp 1: Rate of apoptosis during pregnancy
To test the hypothesis that the rate of apoptosis in the lower reproductive tract declines during the second half of pregnancy, cervices and vaginas were collected throughout most of gestation and early postpartum. Four rats were anesthetized with ether and killed by cervical dislocation between 0900–1100 h on each of days 5, 10, 15, 18, and 21 of pregnancy and days 1 and 2 postpartum. The cervices and vaginas were quickly removed, cleaned, and weighed. Tissues were fixed for 24 h in 10% neutral buffered formalin. After fixation, tissues were dehydrated in an ascending series of ethanol, cleared with xylene, embedded in paraffin, and sectioned to obtain 5-µm sections.

Exp 2: Influence of endogenous relaxin on apoptosis during late pregnancy
To test the hypothesis that relaxin inhibits the rate of apoptosis in cervical and vaginal cells, endogenous relaxin was passively neutralized. A monoclonal antibody for rat relaxin designated MCA1 (21) was administered on days 19–21 of pregnancy, when relaxin levels are maximal in the peripheral circulation (8). The rationale for this experiment is that immunoneutralization of the putative survival factor relaxin, during the period when its effects are likely maximal, will cause pronounced cell death in the cervix and the vagina. Additionally, the neutralization of relaxin, at least 3 days before delivery, enabled analysis of the effect of treatment over a 3-day period.

On day 9 of pregnancy, rats were laparotomized under ether anesthesia, and the number of implantation sites was determined. Only rats with eight or more implantation sites were used because serum relaxin levels are directly related to the number of conceptuses in rats with small litters (22). Animals were randomly divided into three treatments, with 20 rats per treatment. Unanesthetized rats were placed in a restraining device and injected via tail vein. One group received 10 mg MCA1 at 0900 h daily from day 19 to day 21 of pregnancy. The control groups received either 10 mg monoclonal antibody for fluorescein (MCAF, monoclonal antibody control; 11), or 1 ml PBS (vehicle control).

To determine the time course over which apoptosis occurs after the withdrawal of relaxin, cervices and vaginas from four rats per treatment were collected 6, 12, 24, 48, and 72 h after the initial injection of MCA1, MCAF, and PBS. Skeletal muscle, which is not a target for relaxin, was also collected from two rats per group, 24 h after treatment, as a negative tissue control. Tissues were fixed, embedded, and sectioned as described for Exp 1.

In situ localization of apoptosis in cervical and vaginal cells
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling (TUNEL), in conjunction with morphometric analysis, was employed to detect and quantify cells undergoing apoptosis. Sections were stained immunocytochemically by TUNEL using the method described by Gavrieli et al. (15). A commercial kit (ApopTag in situ Apoptosis Detection; Intergen, Purchase, NY), which links digoxigenin-nucleotide to DNA by terminal deoxynucleotidyl transferase (TdT), was used. Sections were deparaffined with xylene, rehydrated with a descending series of ethanol, incubated with proteinase K, immersed in 3% aqueous hydrogen peroxide, and then pretreated with equilibration buffer. DNA was labeled at the 3'-end by incubating sections with a mixture of digoxigenin deoxynucleotide triphosphate, unlabeled deoxynucleotide triphosphate, and TdT enzyme at 37 C for 1 h. Slides were washed with PBS and incubated with antidigoxigenin antibody conjugated to peroxidase at room temperature for 30 min. Slides were washed again in PBS, incubated with 3,3' diaminobenzidine, counterstained with methyl green, mounted, and sealed. Positive control slides were treated with deoxyribonuclease I before the labeling reaction. Negative control slides were incubated with labeling reaction solution devoid of TdT enzyme.

Sections were examined morphometrically with a BH-2 light microscope (Olympus Corp., Mellville, NY) equipped with a video camera and connected to a personal computer running a Stereo Investigator program (MicroBrightField, Inc., Colchester, VT). The Stereo Investigator program automatically controls the movement of the microscope stage to provide unbiased determination of the fields of analysis. For both the cervix and the vagina, epithelial cells and stromal cells were analyzed independently. The percentage of TUNEL-labeled cells (labeling index, LI) was determined by counting TUNEL-labeled cells divided by total cells in the same field and then multiplying by 100. Data were obtained from 4 rats/group, 3 sections (approximately 20µm apart)/rat, and at least 500 cells/section. Thus, at least 6000 cells were analyzed per group for the epithelium, and 6,000 cells were analyzed per group for the stroma.

Ultrastructural analysis of apoptosis in cervical and vaginal cells
To confirm that cervices and vaginas demonstrating elevated TUNEL-labeling contain apoptotic cells, transmission electron microscopy was used to examine the morphological characteristics of cells. Cervices and vaginas were collected 24 h after the initial injection of MCA1 or MCAF, on day 19 of pregnancy, as described for Exp 2. Evaluation of cervical tissue was conducted by collecting 3 areas of tissue from 2 MCA1-treated and 2 MCAF-treated pregnant rats. The small pieces of tissue were placed in Karnovsky’s fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS) for 24 h, washed 3 times in 0.1 M cacodylate buffer, and then shipped to the University of South Alabama College of Medicine. There, the pieces of tissue were cut in half and processed for electron microscopy evaluation as previously described for the rat uterus (23). Thin sections were cut from 3 depths of each tissue half, and 50 epithelial cells and 50 stromal cells were evaluated from each section, for a total of 900 epithelial cells and 900 stromal cells per animal. Two areas of vaginal tissue were collected from one MCA1-treated and one MCAF-treated pregnant rat and handled as described above. Therefore, 600 epithelial cells and 600 stromal cells were evaluated per animal. A nucleus had to be observed in the cell in order for the cell to be counted. The percent of apoptotic cells was calculated.

Statistical analysis
All data were analyzed by one-way ANOVA and Tukey’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: Rate of apoptosis during pregnancy
Mean cervical and vaginal wet weights are shown in Fig. 1Go. The cervical and vaginal wet weights did not differ on days 5 and 10. However, the tissue weights increased progressively between days 10 and 23 of pregnancy (P < 0.05). On the day of delivery (day 23 of pregnancy or day 1 postpartum), the cervical and vaginal wet weights were maximal, and they decreased dramatically by the following day (day 2 postpartum).



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  		Figure 1. Mean (+ SE) cervical (A) and vaginal (B) wet weights during rat pregnancy and early postpartum (n = 4). Different superscript letters indicate a significant difference (P < 0.05).

 
The rate of apoptosis in both cervical compartments varied among days of pregnancy (P < 0.05). The LI of TUNEL-labeled cells in the cervical epithelium (Fig. 2AGo) was approximately 2% on days 5 and 10, declined to approximately 0.5% on days 15–21, and then rose dramatically to 18% by the second day after delivery. In the cervical stroma, the LI was significantly lower on day 21 than earlier in pregnancy or days 1 and 2 postpartum. As with the epithelium, the LI increased dramatically during the first 2 days postpartum (Fig. 2BGo).



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  		Figure 2. Mean (+ SE) LI of TUNEL-labeled cells in the cervical epithelium (A) and stroma (B) during pregnancy and early postpartum (n = 4). Different superscript letters indicate a significant difference (P < 0.05).

 
Similar results were observed in the vagina (Fig. 3Go). The LI in the vaginal epithelium and stroma were lower on days 18 and 21 than either days 5 and 10 of pregnancy or days 1 and 2 postpartum.



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  		Figure 3. Mean (+ SE) LI of TUNEL-labeled cells in the vaginal epithelium (A) and stroma (B) during pregnancy and early postpartum (n = 4). Different superscript letters indicate a significant difference (P < 0.05).

 
Exp 2: Influence of endogenous relaxin on apoptosis during late pregnancy
Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). When endogenous relaxin was neutralized with MCA1 on days 19–21 of pregnancy, the LI of TUNEL-labeled cells in the epithelium of the cervix was greater than in the epithelium of controls 6, 12, 24, and 48 h after the initiation of treatment (Fig. 4AGo). The maximal LI occurred 12 and 24 h after treatment. Whereas the LI in the cervical stroma did not increase as markedly as in the epithelium, it was greater than in controls 6 and 12 h after the neutralization of endogenous relaxin (Fig. 4BGo). The LI in vaginal epithelial cells was greater 6, 12, and 24 h after initiation of neutralization of endogenous relaxin than it was in controls. The maximal LI occurred 24 h after treatment (Fig. 5AGo). Treatment did not influence the LI in the vaginal stroma (Fig. 5BGo). The LI in skeletal muscle, 24 h after treatment, was only about 0.05%, and there was no difference among treatment groups (data not shown).



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  		Figure 4. Mean (+ SE) LI of TUNEL-labeled cells in the cervical epithelium (A) and stroma (B) after PBS, MCAF, and MCA1 treatment (n = 4). An asterisk indicates a significant difference from controls (P < 0.05). The superscript letter indicates a significant difference (P < 0.01) from other MCA1-treated groups that have no superscript letter.

 


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  		Figure 5. Mean (+ SE) LI of TUNEL-labeled cells in the vaginal epithelium (A) and stroma (B) after PBS, MCAF, and MCA1 treatment (n = 4). An asterisk indicates a significant difference from controls (P < 0.05). The superscript letter indicates a significant difference (P < 0.01) from other MCA1-treated groups that have no superscript letter.

 
Cervical and vaginal wet weights were significantly lower than those in PBS and MCAF controls 48 h and 72 h, and 72 h, respectively, after initiation of MCA1 treatment (Fig. 6Go).



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  		Figure 6. Mean (+SE) cervical (A) and vaginal (B) wet weights after PBS, MCAF, or MCA1 treatment (n = 4). Two asterisks indicate a significant difference from controls (P < 0.01).

 
Ultrastructural analysis of apoptosis in cervical cells
A cell was classified as apoptotic if it contained a nucleus with condensed chromatin and/or a shrunken nucleus as indicated by separation from the nuclear envelope. Results obtained with electron microscopy were consistent with those obtained with the TUNEL method. In cervical tissue from MCAF-treated rats, apoptotic cells were not evident. In contrast, in cervical tissue from MCA1-treated rats, apoptotic cells were observed in 6.5% (117 of 1800) of the epithelial cells and 2.5% (45 of 1800) of the stromal cells evaluated (Fig. 7Go). Cytoplasmic organelles, such as mitochondria, were intact. The apoptotic stromal cells seemed to be fibroblasts, as indicated by the large amount of rough endoplasmic reticulum and by a lack of dense filaments found in smooth muscle cells. The cervical epithelium of all samples from MCA1-treated animals seemed disrupted, as indicated by spacing of adjacent epithelial cells (Fig. 8AGo). This was not observed in the tissues from MCAF-treated animals (Fig. 8BGo). In the epithelium, an occasional apoptotic cell seemed to have been phagocytized by an adjacent cell (Fig. 8AGo).



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  		Figure 7. Cervical tissue from a MCA1-treated rat. A, An apoptotic fibroblast cell (ap). Note the nucleus (N) with condensed chromatin, separated nuclear envelope (single arrows), rough endoplasmic reticulum (double arrows), mitochondria (M), and collagen (C). B, An apoptotic epithelial cell (ap). Note the nucleus (N) with condensed chromatin, rough endoplasmic reticulum (small arrows), and mitochondria (M). A = 18,200x; B = 9,000x.

 


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  		Figure 8. Cervical tissue from a MCA1-treated rat. A, An apoptotic cervical epithelial cell (ap) with fragmented chromatin material (short arrows) that appears to have been phagocytized by an adjacent epithelial cell. Note the increased space (long arrows) between cervical epithelial cells of the MCA1-treated rat, compared with the epithelium in an MCAF-treated rat (Fig. 8BGo). L, Cervical lumen. A = 5,000x; B = 5,000x.

 
Similar results were obtained with the vagina. In vaginal tissue from MCAF-treated rats, apoptotic cells were not evident. In contrast, in vaginal tissue from MCA1-treated rats, apoptotic cells were observed in 3% (18 of 600) of the epithelial cells and 2% (12 of 600) of the stromal cells evaluated (Fig. 9Go).



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  		Figure 9. Vaginal tissue from a MCA1-treated rat. A, Apoptotic fibroblast cells (ap), nucleus (N), and collagen (C). B. An apoptotic epithelial cell (ap), nucleus (N). A = 3,800x; B = 5,000x.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides evidence that there is a decline in the rate of apoptosis in epithelial and stromal cells in both the cervix and vagina during the second half of rat pregnancy. Moreover, this study provides evidence that this reduction in the rate of apoptosis is attributable, at least in part, to the hormone relaxin.

One function of apoptosis is to maintain steady cell numbers in tissues (12). However, when tissues must grow, as is the case during pregnancy, a mechanism that can contribute to increased cell numbers is the reduction in the rate at which cells undergo apoptosis. This report indicates that during the first half of rat pregnancy, when the lower reproductive tract is not growing extensively, the rate of apoptosis is moderate and steady. However, during the second half of pregnancy, when cervical and vaginal growth increase progressively, the rate of apoptosis is reduced severalfold. Consistent with the view that apoptosis plays a role in regulating cell numbers is the observation that the rate of apoptosis is extremely high after parturition, when the size of the cervix and vagina decrease markedly.

Whereas the roles that hormones play to reduce the rate of apoptosis in the cervix and vagina during the second half of rat pregnancy are poorly understood, this study provides evidence that relaxin may act as a cell survival factor during this period. The observation that the rates of apoptosis are relatively low during the second half of pregnancy, when relaxin is secreted into the blood, provides indirect evidence that relaxin may inhibit the rate of apoptosis. The strongest evidence that relaxin is a cell survival factor is the finding that the rate of apoptosis increases markedly after the neutralization of endogenous relaxin during late pregnancy. Additionally, the finding that both cervical and vaginal wet weights were lower than controls after neutralization of relaxin is consistent with the view that relaxin promotes growth of the lower reproductive tract, at least in part, by inhibiting apoptosis.

Findings from the analysis of the effects of relaxin on apoptosis in cervical and vaginal tissue by electron microscopy were in general agreement with those obtained by the TUNEL method. There was an increase in the rate of apoptosis in epithelial cells and stromal cells in both the cervix and the vagina after immunoneutralization of relaxin. The rates of apoptosis after immunoneutralization of relaxin tended to be somewhat higher when determined by electron microscopy than when determined by the TUNEL method. A likely explanation for this is that fragmentation of the chromosomes has not occurred in nuclei with condensed chromatin. An increase in the rate of apoptosis after neutralization of relaxin was found in vaginal stromal cells by means of electron microscopy but not by the TUNEL method.

Relaxin is not the only hormone that regulates growth of the cervix and vagina in rats. Studies with ovariectomized nonpregnant rats demonstrated that relaxin’s growth-promoting effects on the cervix are estrogen-dependent (24, 25). Moreover, estrogen alone promotes growth of the cervix (24, 25) and vagina (26) in nonpregnant rats. Estrogen has also been demonstrated to prevent apoptosis in the rat vagina (16, 17, 27, 28). During the second half of rat pregnancy, developing ovarian follicles secrete increasing quantities of estrogen (29). Therefore, it seems likely that estrogen not only contributes to relaxin’s role as a cell survival factor but also, in and of itself, is a cell survival factor in the lower reproductive tract during rat pregnancy.

This report may provide insight concerning a mechanism associated with relaxin’s effects on other rat tissues and/or target tissues in other species during pregnancy. Specifically, relaxin-induced growth of the rat nipple (30, 31) and pig vagina, cervix, uterus, and mammary glands (32, 33, 34) during pregnancy may be attributable, at least in part, to a reduction in the rate of apoptosis.

Presently, the possibility that relaxin also increases the rate of cell proliferation in the cervix and vagina during rat pregnancy cannot be ruled out. Indeed, studies of porcine ovarian cells by Bagnell and co-workers (35, 36) provide evidence that relaxin promotes cell proliferation in an in vitro biological system. These workers reported that relaxin increased the rate of [3H]thymidine incorporation into cultures of porcine follicular granulosa cells and thecal cells. The hypothesis that relaxin both inhibits the rate of apoptosis and increases the rate of cell proliferation in the rat cervix and vagina is attractive because it provides complimentary mechanisms to account for the dramatic growth of the lower reproductive tract that occurs during the second half of pregnancy.

Studies concerning the changes that occur in the rate of cervical cell apoptosis during rat pregnancy are not in agreement. Previously, another laboratory reported that cervical DNA content declines steadily and by about 80% between day 12 and term (18). This laboratory reported that apoptosis increases progressively in cervical smooth muscle and fibroblast cells during the second half of rat pregnancy (18, 19). About 5% of both smooth muscle and fibroblast cells were reported to be apoptotic on day 5 of pregnancy, and the rate of apoptosis in both cell types was in the range of 30–50% by days 18 and 21 of pregnancy. In contrast, we previously reported that the cervical DNA content increases by more than 50% during the second half of pregnancy (5). Additionally, in the present study, we report that the rate of apoptosis declines during the second half of rat pregnancy in both the cervical epithelium and the cervical stroma. We report that the LI is not greater that 4% during pregnancy, and we found no evidence that smooth muscle cells undergo notable apoptosis. We cannot presently account for the differences in the findings between the two laboratories.

In summary, this study provides evidence that the rate of apoptosis in the cervix and vagina declines during the second half of rat pregnancy. Moreover, this study provides evidence that the reduction in the rate of apoptosis is attributable, at least in part, to the influence of relaxin. Therefore, it is concluded that relaxin-induced growth of the cervix and the vagina is likely attributable, at least in part, to inhibition of apoptosis.


   Acknowledgments
 
The authors thank Ms. L. Hung for assistance with tissue sectioning and cell counting; B. Sylavong, in the School of Life Sciences Animal Care Facility, for supervision of animal care; the School of Life Science Artist Services for preparing figures; and the College of Medicine Document Management Center for assistance with the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grant USPHS HD-08700 (to O.D.S.) and a predoctoral fellowship from Reproductive Biology Training Grant PHS 5T32-HD-07028 (to S.Z.) Back

Received June 19, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harkness MLR, Harkness RD 1959 Changes in the physical properties of the uterine cervix of the rat during pregnancy. J Physiol 148:524–547
  2. Hwang J-J, Sherwood OD 1988 Monoclonal antibodies specific for rat relaxin. III. Passive immunization with monoclonal antibodies throughout the second half of pregnancy reduces cervical growth and extensibility in intact rats. Endocrinology 123:2486–2490[Abstract]
  3. Hwang J-J, Shanks RD, Sherwood OD 1989 Monoclonal antibodies specific for rat relaxin. IV. Passive immunization with monoclonal antibodies during the antepartum period reduces cervical growth and extensibility, disrupts birth, and reduces pup survival in intact rats. Endocrinology 125:260–266[Abstract]
  4. Downing SJ, Sherwood OD 1985 The physiological role of relaxin in the pregnant rat. III. The influence of relaxin on cervical extensibility. Endocrinology 116:1215–1220[Abstract]
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