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Endocrinology, doi:10.1210/en.2007-1176
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Endocrinology Vol. 149, No. 5 2072-2079
Copyright © 2008 by The Endocrine Society

Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and Inhibit Apoptosis in the Mouse Cervix and Vagina

LiJuan Yao, Alexander I. Agoulnik, Paul S. Cooke, Daryl D. Meling and O. David Sherwood

Department of Molecular and Integrative Physiology (L.Y., O.D.S.), College of Medicine (O.D.S.), and Department of Veterinary Biosciences (P.S.C., D.D.M.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Obstetrics and Gynecology (A.I.A.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. O. David 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 objective of this study was to determine whether stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild-type (wt) and LGR7 knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep, and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17β (designated d 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10 of treatment. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before cervices and vaginas were collected at 1000 h on d 10. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling was used to quantify apoptosis. Relaxin markedly increased proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P < 0.01) but had no effect on tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of cervix and vagina, whereas epithelial LGR7 does not affect these processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RELAXIN IS SECRETED by the corpora lutea during the second half of pregnancy in rats, mice, and pigs (1). Relaxin stimulates marked growth and extensibility of the lower reproductive tract and thereby facilitates parturition in these species (1, 2, 3, 4, 5, 6). Relaxin-dependent growth of the cervix and vagina in rats is accompanied by increases in both stromal and epithelial cells (4, 5, 7, 8). Moreover, we found that relaxin causes cell accumulation by promoting cell proliferation and inhibiting apoptosis (9, 10, 11).

Cellular and molecular mechanisms of relaxin actions on cervix and vagina during pregnancy are not well understood. A fundamental step toward further understanding of these mechanisms is to identify specific cells that contain relaxin receptors, i.e. to identify the cells that initiate relaxin’s effects. Early efforts to do so in the 1990s used immunohistochemical localization of biotinylated relaxin at the light microscope level (4, 12, 13). Whereas relaxin binding was observed in epithelial cells and smooth muscle cells deep within the stromal compartment, relatively little, if any, apparent binding was observed in subepithelial stroma cells. In 2002 the orphan receptor relaxin receptor (LGR7) (14) was identified as the relaxin receptor. The discovery of LGR7, which is now also designated RXFP1 (15), made possible additional approaches toward the identification of cells that contain relaxin receptors. Limited studies that used antibodies to LGR7 (14), in situ hybridization of LGR7 transcripts (16), and staining for β-galactosidase activity driven by an IRES-LacZ reporter cassette in the LGR7 gene (17) reported relaxin receptors were located in subepithelial stroma cells and smooth muscle layers in rodent vaginal and/or cervical tissues. Relaxin receptors were not observed in cervical and/or vaginal epithelial cells (14, 16, 17).

The objective of this study was to use relaxin bioactivity as a means of identifying the tissue compartment(s) that initiates relaxin’s actions on the cervix and vagina. Cooke et al. (18) and Buchanan et al. (19) previously used an estrogen receptor (ER)-{alpha} knockout mouse (20) in combination with a tissue separation/recombination methodology to determine the respective roles of stromal and epithelial cell ER{alpha} in estrogen-induced epithelial cell proliferation in mouse uterine and vaginal tissues. The present report describes the use of LGR7-knockout mice (21) with a similar tissue separation/recombination method to obtain functional evidence that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in mouse cervix and vagina.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All animal experiments described here were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Illinois Laboratory Animal Care Advisory Committee.

Animal treatment experiment 1a: relaxin’s effects on mouse cervix and vagina
Because mouse cervix and vagina were used in the tissue separation/recombination studies to identify cellular compartments that are responsive to relaxin, a preliminary study was conducted to determine whether relaxin promotes cell proliferation and inhibits apoptosis in the mouse lower reproductive tract, as it does in rats.

Virgin female ICR mice weighing 21–24 g (4–5 wk of age) were obtained from Harlan Sprague Dawley (Indianapolis, IN). Animals were housed in 27- x 41- x 15-cm polycarbonate cages in groups of six to eight mice per cage. Mice were maintained under constant conditions of lighting (12 h light, 12 h dark) and temperature (23 C) and were provided with Teklad 6% mouse/rat diet 7002 (Harlan/Teklad, Madison, WI) and water ad libitum. Figure 1Go is a diagram of experiment 1. On d 1 of treatment, the mice were anesthetized ip with xylazine and ketamine and bilaterally ovariectomized. All animals received progesterone (P) and estradiol-17β (E) continuously from d 1 through d 10 of treatment in doses previously demonstrated to provide physiological levels in mice (22, 23, 24). The P and E were placed in silicone rubber tubing implants (SF Medical, Hudson, MA). Sixteen-millimeter lengths of the SILASTIC brand silicon tubing (inner diameter 1.57 mm, outer diameter 3.18 mm) were first sealed with a 3-mm SILASTIC brand medical adhesive plug (Dow Corning, Midland, MI) at one end. Implants were then filled by injection to an effective length of 10 mm with either a P suspension (1 g/ml; Sigma Chemical Co., St. Louis, MO) or an E solution (60 µg/ml; Sigma) in sesame oil (Sigma). The open end of each implant was then closed with an additional 3-mm SILASTIC brand adhesive plug. The P implants were rinsed two times in 100% ethanol followed by distilled water. Both P and E implants were incubated in PBS [0.14 M NaCl, 0.01 M PO4 (pH 7.0)] at 37 C for 24 h before surgical insertion. The ethanol rinse and PBS incubation removes residual steroid from the outside of the implant, thereby reducing the transitory surge of hormone after implant insertion (25). Immediately after ovariectomy, a P implant was placed sc over one flank and an E implant was placed sc over the other.


Figure 1
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  		FIG. 1. Protocol for the animal surgery and hormonal treatments in experiments 1a, 1b, and 2. See Materials and Methods for details.

 
Porcine relaxin purified as previously described (26) was used. Beginning at 0600 h on d 8 and terminating at 0600 h on d 10 of treatment, animals were injected sc at 6-h intervals with either relaxin at a dose of 4 µg in 0.2 ml PBS (group OPER; n = 6) or relaxin vehicle (group OPE; n = 8). Ten hours before tissue collection, i.e. at 2400 h on d 9, all mice were injected sc with 5-bromo-2'-deoxyuridine (BrdU) at a dose of 100 µg per 0.2 ml PBS.

Animal treatment experiment 1b: relaxin’s effects on mouse cervix require LGR7
Because the planned tissue separation/recombination experiments were dependent on relaxin acting through LGR7, a preliminary study was conducted to determine whether relaxin promotes cell proliferation and inhibits apoptosis in the cervix of LGR7-knockout mice.

LGR7-deficient mice (Lgr7–/–) on a C57BL/6J background developed as described previously (21) were used. Heterozygous Lgr7+/– females were bred to either heterozygous Lgr7+/– or homozygous Lgr7–/– males to generate Lgr7+/+ [wild-type (wt)], Lgr7+/– heterozygote, and Lgr7–/– homozygote knockout (ko) offspring; only wt and Lgr7–/– mice were used in experiment 1b. Animals were genotyped neonatally using DNA from ear punches as previously described (21). There were four treatment groups (Fig. 1Go): OPEwt, n = 7; OPERwt, n = 7; OPEko, n = 5; and OPERko, n = 6.

Animal treatment experiment 2: relaxin’s effects on cervical and vaginal tissue recombinants
The procedure used for separation and recombination of epithelium and stroma from neonatal mouse cervix and vagina has been described previously (18, 19, 27). Briefly, cervices and vaginas from neonatal wt and Lgr7–/– littermate mice (3–5 d old) were separated with a fine scalpel, dissected free of adherent connective tissue and fat, placed in Hanks’ balanced salt solution. Tissues were enzymatically dissociated by placing them in a solution of 1% Trypsin 250 (Difco Laboratories, Detroit, MI) in calcium- and magnesium-free Hanks’ balanced salt solution for 90 min at 4 C. After trypsin digestion, cervices and vaginas were cut open, and then stroma (St) and epithelium (Ep) were physically separated using fine surgical instruments. Four types of both cervical and vaginal tissue recombinants were prepared: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep, and ko-St+ko-Ep. The stroma and epithelium were recombined on nutrient agar plates and allowed to adhere during overnight culture at 37 C. After overnight culture, the tissue recombinants were grafted under the renal capsules of syngeneic wt female C57BL/6J mice. Approximately 3 wk after grafting, all hosts were ovariectomized and received hormone treatments as described for experiment 1a and 1b (Fig. 1Go). Six replicates of each of the four types of cervical and vaginal tissue recombinants were obtained from group OPE as well as from group OPER.

Tissue collection and processing
At 1000 h (±30 min) on d 10 of treatment, mice were killed with CO2. For experiments 1a and 1b, reproductive tracts were removed and trimmed of fat and connective tissue. Cervices and vaginas were separated with a scalpel and weighed on a Sartorius balance. For experiment 2, tissue recombinant grafts were collected after killing host animals. Cervices, vaginas, and tissue grafts were processed as previously described (8, 28). Grafts were prefixed in neutral buffered formalin for 2 h. Fixation was continued in fresh neutral buffered formalin for a total of 24 h. After fixation, tissues were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. All samples were serially sectioned (5 µm), mounted on positively charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), and air dried overnight. To avoid the possibility of evaluating the same cells on consecutive slides, there was a distance of at least 20 µm between sections on adjacent slides.

Tracking of cell lineages using transgenic mice expressing green fluorescent protein (GFP)
Although either the stromal or epithelial tissue fraction obtained by the tissue separation and recombination method used in this study has been shown to be reliably devoid of contamination with the other (29), it is always critical to demonstrate that the epithelium present in a particular tissue recombination was derived from the recombined epithelium, rather than contaminating epithelium that was not completely removed from the stroma during tissue separation. To address this, stroma from wt mice was used in tissue recombination with epithelium derived from transgenic C57BL/6F1 mice expressing an enhanced GFP under the control of a chicken β-actin promoter and cytomegalovirus enhancer (Jackson Laboratories, Bar Harbor, ME). With these GFP mice, all tissues, with the exception of erythrocytes and hair, appear green under excitation light or when stained with an antibody for GFP and thus can be used to unequivocally trace cell lineages in the tissue recombinants (30). The wt-St+GFP-Ep tissue recombinants were then grafted under the renal capsule of a syngeneic wt female mouse. Approximately 3 wk after grafting, the host was ovariectomized and received sc implants containing P and E as described in Fig. 1Go. At 1000 h (±30 min) on d 8 of treatment, the host was killed and tissue recombinants were collected. Tissue recombinants were frozen onto a sectioning stub using Tissue-Tek O.C.T. compound (Sigma), and 15-µm sections were cut using a Reichert-Jung Cryostat 1800 cryomicrotome (Leica, Deerfield, IL). Direct fluorescence was then visualized using a BX51 fluorescent microscope (Olympus Corp., Melville, NY) with a filter having an emission wavelength of 488 nm.

Relaxin effects on stromal and epithelial cell proliferation
BrdU incorporation into proliferating cells was determined immunohistochemically using a mouse monoclonal antibody (clone NCL-BrdU; Vector Laboratories, Inc., Burlingame, CA) and a Vectastain Elite ABC kit (Vector Laboratories) according to procedures previously described (8, 31).

Morphometric analysis was conducted in the blind to determine the cellular proliferation rate of the cells. The labeling index of BrdU-positive cells was determined by dividing the number of BrdU-positive cells by the total number of cells analyzed per section and multiplying by 100. Sections were examined morphometrically at x400 magnification with a BH-2 light microscope (Olympus) 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 permit unbiased selection of fields of analysis. Epithelial cells and stromal cells were analyzed independently. Because previous studies demonstrated that relaxin does not influence rates of either proliferation or apoptosis of smooth muscle cells (8, 9, 10), only nonsmooth muscle cells were analyzed within the stroma. Data were obtained from four sections per tissue (cervix, vagina, or tissue recombinant), and at least 500 epithelial cells and 500 stromal cells were analyzed per section. Thus, at least 2000 epithelial cells and 2000 stromal cells were analyzed for each tissue sample.

Analysis of relaxin’s effects on stromal and epithelial cell apoptosis
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling immunostaining was used with a commercial kit (ApopTag in situ apoptosis detection; Serologicals Corp., Norcross, GA), which links digoxigenin-nucleotide to DNA by terminal deoxynucleotidyl transferase, according to procedures previously described (9, 32). Morphometric analysis of relaxin’s effects on epithelial and stromal cell apoptosis was done blindly as described above for morphometric analysis of cell proliferation.

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

Staining for β-galactosidase activity
Because of the surgical and hormonal treatments associated with experiment 2, the physiological circumstances under which the cervical and vaginal tissues were analyzed differ from those when relaxin normally acts on reproductive tissues. Accordingly, tissue location of LGR7-bearing cells in the cervix and vagina was determined during late pregnancy in mice by a different experimental approach. In the creation of the Lgr7–/– mice used for this study (21), an IRES-LacZ reporter cassette was inserted into the mutant allele in the correct orientation, which allows the expression of the reporter gene. For histological analysis of LacZ expression, cervical and vaginal tissues from d 17.5 pregnant wt and Lgr7+/– mice were processed for β-galactosidase activity as described previously (21, 33). In brief, after fixation for 2 h [PBS containing 0.2% glutaraldehyde, 2% formaldehyde], samples were rinsed for 90 min in PBS containing 0.02% Nonidet P-40, 2 mM MgCl2, and 0.01% sodium deoxycholate and stained with X-gal (1 mg/ml) staining solution (PBS containing 0.02% NP-40, 2 mM MgCl2, 0.01% sodium deoxycholate, 5 mM potassium ferricyamide, 5 mM potassium ferrocyamide) at 37 C in a humidified chamber overnight. Tissues were processed through increasing concentrations of ethanol, dehydrated, and paraffin wax embedded. Seven-micrometer sections were cut and counterstained with eosin. Photomicrographs were taken at x400 magnification with a Nikon-TMS inverted microscope equipped with an Olympus DP70 digital imaging system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: relaxin’s effects on mouse cervix and vagina
Relaxin not only increased wet weights (Fig. 2AGo) but also markedly increased cell proliferation (Fig. 2BGo) and decreased apoptosis (Fig. 2CGo) in both epithelium and stroma (P < 0.01) of cervix and vagina of wt mice. In contrast, relaxin did not influence these processes in Lgr7–/– cervices (Fig. 3Go). Although the difference in the wet weight of the tissues was only slightly higher in wt sibling control females in this mouse strain after relaxin treatment, there was a significant increase in cell proliferation and decrease in apoptosis as in ICR mice. No relaxin-induced changes in these parameters were observed in Lgr7–/– mice.


Figure 2
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  		FIG. 2. Influence of relaxin on cervical and vaginal wet weight (A), proliferation (B), and apoptosis (C) of epithelial and stromal cells in cervix and vagina. Four sections were analyzed per cervix and per vagina, and at least 500 of both epithelial and stromal cells were analyzed per section. Each column represents the mean (+ SEM). **, Significant difference (P < 0.01) between relaxin-treated mice (OPER, n = 6) and their PBS controls (OPE, n = 8).

 

Figure 3
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  		FIG. 3. Effect of relaxin on cervical wet weight (A), cell proliferation (B), and apoptosis (C) in cervices of wt and Lgr7–/– (ko) mice. Four sections were analyzed per cervix, and at least 500 of both epithelial and stromal cells were analyzed per section. Each column represents the mean (+SEM). **, Significant difference (P < 0.01) between relaxin-treated mice (OPERwt, n = 7) and their PBS controls (OPEwt, n = 7). Groups OPERko (n = 6) and OPEko (n = 5) did not differ.

 
Tracking of cell lineages using transgenic mice expressing GFP
Tissue recombinants composed of wt-St+GFP-Ep had epithelium, but not stroma, that fluoresced brightly, indicating GFP expression only in the epithelial compartment (Fig. 4Go, A and B). These results indicate the completeness and accuracy of the tissue separation procedure and show that epithelium in the grafts was derived from that added at the time of tissue recombination, rather than contaminating epithelium not completely separated from the underlying stroma after initial trypsinization.


Figure 4
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  		FIG. 4. Fluorescence (A) and light microscopy (B) of a 15-µm section of a cervical tissue recombination wt-St+GFP-Ep. Intermittent white lines indicate the stromal border. Bar (A), 200 µm. Both micrographs are the same magnification. Ep, Epithelium; St, stroma.

 
Experiment 2: relaxin’s effects on cervical and vaginal tissue recombinants
Relaxin dramatically increased epithelial and stromal cell proliferation in wt-St+wt-Ep and wt-St+ko-Ep cervical tissue recombinants (Fig. 5Go, A and B; P = 0.01). In contrast, relaxin had no effect on either epithelium or stroma of tissue recombinants lacking wt stroma (ko-St+wt-Ep and ko-St+ko-Ep). The epithelial origin (either wt or ko) had no effect on the relaxin response of either stromal or epithelial cells. Figure 6Go contains representative micrographs of two of the four cervical tissue recombinants from treatment group OPER stained to detect cell proliferation. In tissue recombinants that contained wt-St, relaxin induced substantial proliferation of basal epithelial cells as well as proliferation of cells throughout stromal tissue.


Figure 5
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  		FIG. 5. Influence of relaxin on labeling indices for proliferation (A and B) and apoptosis (C and D) in the cervical tissue recombinants. Four sections were analyzed per recombinant, and at least 500 of both epithelial and stromal cells were analyzed per section. Each column represents mean (+SEM) of six recombinants. The OPER recombinants wt-St+wt-Ep and wt-St+ko-Ep did not differ from each other but differed from all other groups (**, P < 0.01).

 

Figure 6
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  		FIG. 6. Histology of wt-St+ko-Ep (A) and ko-St+wt-Ep (B) tissue recombinants from group OPER mice. Sections were stained for BrdU (brown nuclei) to identify proliferating cells and with hematoxylin (blue nuclei) to determine cells that were not proliferating after BrdU administration. Arrows indicate BrdU-positive cells. Bar (in panel A), 200 µm. Both micrographs are the same magnification.

 
Consistent with cell proliferation findings, relaxin decreased epithelial and stromal cell apoptosis equally in tissue recombinants wt-St+wt-Ep and wt-St+ko-Ep (Fig. 5Go, C and D; P < 0.01) but had no effect on tissue recombinants lacking wt stroma. Similar to the proliferation results, epithelial origin (either wt or ko) had no effect on the antiapoptotic relaxin response in either stroma or the epithelium.

Results from morphometric analysis of vaginal tissue recombinants (Fig. 7Go) were highly consistent with those obtained with cervical tissue recombinants. Relaxin markedly and equally increased proliferation and decreased apoptosis of epithelial and stromal cells in those recombinants that contained wt stroma (P = 0.01). As in cervical tissue recombinants, epithelial origin (either wt or ko) had no effect on proliferative or antiapoptotic relaxin responses.


Figure 7
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  		FIG. 7. Influence of relaxin on labeling indices for proliferation (A and B) and apoptosis (C and D) in the vaginal tissue recombinants. Four sections were analyzed per recombinant, and at least 500 of both epithelial cells and stromal cells were analyzed per section. Each column represents the mean (+SEM) of six recombinants. The OPER recombinants wt-St+wt-Ep and wt-St+ko-Ep did not differ from each other, but they differed from all other groups (**, P < 0.01).

 
LGR7 expression in stromal cells of cervix and vagina
β-Galactosidase activity in cervical and vaginal tissue of Lgr7–/–mice on d 17.5 of pregnancy are shown in Fig. 8Go, A and C, respectively. In both organs strong staining was observed in stroma just beneath the basal layer of the epithelium. Moderate staining was associated with deeper smooth muscle bundles. Staining was not observed in the epithelium.


Figure 8
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  		FIG. 8. LGR7 expression in cervical and vaginal stromal cells. β-Galactosidase activity in cervix (A and B) and vagina (C and D) from mice on d 17.5 of pregnancy. In Lgr7+/– mice (A and C), strong staining was observed in subepithelial stroma (large arrows). Modest staining was seen in smooth muscle bundles (small arrows). Staining was not observed in either cervix or vagina of wt mice (B and D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are several novel findings in the present study. The first is that relaxin promotes proliferation and inhibits apoptosis of both epithelial and stromal cells in mouse cervix and vagina. These cellular actions of relaxin on the mouse lower reproductive tract are dramatic and comparable with those in the rat lower reproductive tract (7, 8, 9, 10, 11). Both increased cell proliferation and decreased apoptosis likely contribute to the relaxin-dependent cervical and vaginal growth during the second half of pregnancy in both species (1, 2, 3, 4, 5, 6). A second finding is that relaxin’s actions on cell proliferation and apoptosis are totally dependent on LGR7. The finding that LGR7 is the only receptor that mediates these actions is consistent with earlier studies that demonstrated that nipple development is severely impaired in females with deletion of LGR7 (17, 21, 34). The findings that relaxin acting through LGR7 has marked effects on the rates of cell proliferation and apoptosis in the mouse lower reproductive tract form the biological foundation for this study. Determination of rates of cell proliferation and apoptosis are both highly suitable means of investigating relaxin’s actions in grafted tissue recombinants.

The present study, which is the first to investigate which tissue compartment(s) relaxin acts through to produce its critical effects on epithelial and stromal proliferation and apoptosis, demonstrates that relaxin receptor-expressing cells within the stroma are necessary and sufficient to mediate relaxin-induced proliferation of stromal and epithelial cells in mouse cervix and vagina. The tissue recombination data provide no evidence that epithelial LGR7 contributes to these biological actions. These findings are in agreement with the limited studies that used antibodies to LGR7 (14), in situ hybridization of LGR7 transcripts (16), and staining for LGR7-specific β-galactosidase activity (17) to report relaxin receptors in stromal but not epithelial cells in rodent vaginal and/or cervical tissues. The expression of relaxin receptor in lower reproductive tract of pregnant mice reported here also provides evidence that LGR7 is located in stroma but not epithelium. Within the stromal compartment, prominent β-galactosidase staining was confined to cells located just below the epithelium in both the cervix and vagina. These findings in the cervix are novel, and those in the vagina are consistent with an earlier report that used this methodology to locate LGR7 expression in the vagina of a nonpregnant LGR7 knockout mouse (17).

The tissue recombinations in combination with the histological findings provide strong evidence that relaxin’s marked effects on epithelial proliferation and apoptosis of the mouse lower reproductive tract are initiated through actions on subepithelial stromal cells. It is well established that stroma, or its embryonic precursor, mesenchyme, interacts with epithelia to promote hormonal effects on differentiation, growth, and morphogenesis of many reproductive organs (35). In some cases, full hormonal response requires hormone receptors in both stromal and epithelial compartments. For example, induction of mouse uterine epithelial secretory proteins lactoferrin and complement component C3 require both stromal and epithelial ER{alpha} (36). In other cases, and consistent with the present study, actions of hormones on mouse stromal cell receptors are necessary and sufficient to bring about full hormonal responses in epithelia via paracrine action(s). For example, it has been shown that estrogen promotes uterine and vaginal epithelial proliferation (18, 19) and uterine P receptor expression (37) through its effects on stromal ER{alpha}. Similarly, stromal P receptors mediate the inhibitory effects of P on estrogen-induced uterine epithelial proliferation (38), and mesenchymal androgen receptors trigger androgen-induced regression of mammary epithelium in male mouse fetuses (39). This is the first report of protein hormone acting through its stromal receptor to elicit epithelial responses in adult developed reproductive tract via a paracrine mechanism. An earlier report provided evidence that Müllerian-inhibiting substance, from fetal Sertoli cells, causes Müllerian duct regression by a similar paracrine mechanism in male rat fetus (40). Neither the number nor nature of paracrine factors produced by cervical and vaginal stromal cells in response to relaxin is known. Moreover, it is not known whether the same paracrine factor(s) act on the epithelium to concomitantly promote cell proliferation and inhibit apoptosis.

Our data indicate that relaxin’s actions on stromal cells are attributable, at least in part, to paracrine actions. Whereas the great majority of the cells containing LGR7 lie just beneath the epithelium, relaxin promotes proliferation and inhibits apoptosis of stromal cells spread throughout the stroma.

Results of this study raise fundamental questions. Do the same paracrine factors secreted by the subepithelial LGR7-bearing cells promote proliferation and inhibit apoptosis in both epithelial and stromal compartments? Is there a single paracrine factor that not only promotes proliferation but also inhibits apoptosis? Finally, the close proximity of the prominent stromal LGR7-bearing cells to the epithelium tempts the speculation that the epithelium plays a role in the development of these relaxin-responsive cells. There is precedent for such a phenomenon. Dürnbereger and Kratochwil (39) demonstrated that androgen responsiveness of male mouse mammary mesenchyme could be initiated only at the epithelial surface and only when mammary epithelium was present. Thus, it was concluded that mammary epithelium induced androgen receptor expression in adjacent mesenchymal cells (35, 39).

The influence of relaxin on cell proliferation and apoptosis was determined in both the cervix and vagina for two reasons. First, it is well established that cervical and vaginal growth in the mouse and rat are markedly stimulated by relaxin (1, 2, 3, 4, 5, 6). Accordingly, it was of interest to determine whether relaxin-induced growth of both organs is regulated by similar cellular mechanisms. Additionally, whereas tissue recombination technique had been used previously with the vaginal tissue, its applicability to the smaller cervix had not been reported. As the findings indicate, the technique is highly applicable to both cervical and vaginal tissue. The similar findings in both cervical and vaginal recombinants support the view that relaxin promotes cell proliferation and inhibits apoptosis via similar mechanisms in both portions of the lower reproductive tract.

The use of ovariectomized mice treated with E plus P was based on the following rationale. Relaxin’s actions on the female reproductive tract are estrogen dependent. Relaxin has little, if any, effect on reproductive tract growth in ovariectomized rodents in the absence of estrogen treatment (41, 42), and the reproductive tract fails to respond to relaxin in ER{alpha} knockout female mice (Cooke, P. S., D. LoTurco, and O. D. Sherwood, unpublished observation). Thus, treatment with estrogen is required. Moreover, treatment with estrogen is sufficient. The actions of relaxin on the female rodent reproductive tract have been demonstrated in ovariectomized nonpregnant rodents treated with estrogen only (43). Nevertheless, it seemed prudent to treat the animals with E and P because mice are exposed to both steroids during the second half of pregnancy when relaxin brings about its effects on the female reproductive tract. P does influence the response to relaxin in the model used. In preliminary experiment 1, which used ICR mice, groups primed with only E were also used (groups OE and OER). The use of P in combination with E attenuated the magnitude of responses. However, the ratios of the magnitude of the effects of relaxin on cell proliferation and apoptosis in mice given E plus P were similar to those obtained in mice given only E. Based on this, both steroids were used. Simply stated, the animal model was chosen because it provided physiological amounts of E and P that maintain pregnancy (22, 23, 24), and it worked effectively.

Finally, the tissue separation/recombination technique in conjunction with the subsequent hormonal treatments and cellular activity indicators offers a promising experimental method for additional efforts to determine cellular and molecular mechanisms associated with relaxin’s actions in these sites. This is the case because relaxin’s effects on cell proliferation and apoptosis in stroma and epithelium are both dramatic and precise. This experimental method is being used for the following ongoing study. Relaxin’s growth-promoting effects on the lower reproductive tract in rodents are dependent on E acting through ER{alpha} (1). ER{alpha} is located in both epithelium and stroma in the rodent lower reproductive tract (44, 45, 46). Tissue recombinants obtained from wt and ER{alpha} ko mice are being used to identify the cellular compartment(s) that mediates E effects on relaxin’s actions.

In conclusion, the present report, which uses Lgr7–/– and wt mice in conjunction with a tissue separation/recombination techniques, provides conclusive evidence that LGR7 in the stroma is both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in cervix and vagina. The method described here is a promising tool for future efforts to determine the cellular and molecular mechanisms of relaxin’s actions.


   Acknowledgments
 
We thank Anne Truong for technical support and Edwin Hadley for assistance with graphics.


   Footnotes
 
This work was supported by National Institutes of Health Grant RO1 HD40448 (to O.D.S.) and the Billie A. Field Endowment (to P.S.C.). The work in the Department of Veterinary Biosciences at the University of Illinois was conducted in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR16515 from the National Center for Research Resources, National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 24, 2008

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E, estradiol-17β; Ep, epithelium; ER, estrogen receptor; GFP, green fluorescent protein; ko, knockout; LGR7, relaxin receptor; P, progesterone; St, stroma; wt, wild type.

Received August 24, 2007.

Accepted for publication January 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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