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Biology of Reproduction 61, 1452-1459 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Expression of Connexin-26, -32, and -43 Gap Junction Proteins in the Porcine Cervix and Uterus During Pregnancy and Relaxin-Induced Growth1

Judy A. Lenharta, Peter L. Ryana, Kathleen M. Ohletha, and Carol A. Bagnell2,a

a Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connexin (CX) proteins participate in growth, differentiation, and tissue remodeling. Relaxin-stimulated reproductive tissue growth and remodeling may be facilitated by enhanced intracellular communication. This study was an examination of the effects of relaxin in vivo on expression of CX-26, CX-32, and CX-43 in the cervix and uterus of prepubertal pigs. In addition, expression of these proteins was monitored in the sow uterus during pregnancy. Relaxin was administered to prepubertal gilts every 6 h for 54 h. CX expression was characterized by immunoblotting and localized by immunofluorescence. Significant increases in all three CXs were observed in the cervix following relaxin treatment (P < 0.05). Uterine CX proteins were also significantly higher (P < 0.05) in relaxin-treated animals compared to controls. The CX protein level in relaxin-treated animals was similar to that observed during the second half of pregnancy, but below levels found in mature, nonpregnant sows. This is the first evidence for specific CX expression in the porcine cervix, and the first study to show that relaxin increases the expression of CX proteins in the porcine uterus and cervix. The data show that CX proteins are differentially regulated in the uterus of the pig during pregnancy. These data support a role for CX-mediated communication during relaxin-induced reproductive tissue growth and remodeling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Relaxin changes the mechanical properties of reproductive tissue, stimulating the growth and remodeling of the porcine uterus and cervix [1, 2]. However, the means by which relaxin promotes reproductive tissue growth and remodeling are not fully understood. Changes in uterine and cervical connective tissue matrix composition [3] and proteolytic enzyme profile [4, 5] are associated with relaxin-induced growth. In addition, relaxin's uterotropic effects in vivo involve changes in the uterine insulin-like growth factor system [6]. It has also been reported that relaxin modulates cell-cell communication in uterine myometrial [7] and endometrial stromal cells [8] in vitro. Given the importance of cell-cell communication in regulating tissue growth [9, 10], the possibility that relaxin facilitates growth by enhancing intracellular communication should be considered.

Gap junctions are involved in the regulation of cellular proliferation and tissue homeostasis. As mediators of intercellular communication, they permit the exchange of nutrients, ions, and small regulatory proteins between adjoining cells [5, 10]. Gap junction proteins belong to an evolutionarily conserved, multigene family of channel-forming proteins called connexins (CXs), which assemble as hexameric hemichannels (connexons) between the membranes of adjacent cells [9, 11]. These CXs form a family of at least 13 proteins that are named on the basis of their molecular size [12]. In the uterus, connexin-26 (CX-26) and connexin-32 (CX-32) are the major gap junction proteins in the endometrium [13, 14], while CX-43 is the predominant CX in myometrial smooth muscle cells [15, 16].

Tissue-specific regulation of CX expression by steroid hormones has been demonstrated in the uterus on the basis of studies of pregnant animals [16–19]. Progesterone suppresses myometrial/endometrial CX-26 and CX-43 during pregnancy, while estrogen induces CX-43 expression in the myometrium. In addition to steroid hormones, growth factors that activate myometrial protein kinase pathways have been implicated in the regulation of myometrial CX-43 gene expression [20]. Relaxin activates uterine protein kinases [21, 22] and thus may contribute to changes in CX protein expression and/or activity. In contrast, factors regulating cervical gap junction-mediated, cell-cell coupling have yet to be reported. While gap junctions between muscle cells of the human uterine cervix have been reported [23], expression of specific CX proteins in cervical tissues has not been characterized. Additionally, whether gap junction expression is altered during the extensive, hormone-driven remodeling of the cervix during pregnancy has not been investigated.

The objective of these studies was to characterize CX protein expression during uterine and cervical growth and remodeling in the pig. Changes in CX expression were studied in prepubertal gilts treated with relaxin to induce uterine and cervical growth in the absence of steroids. In addition, pregnant sows were utilized to characterize CX expression during physiological uterine growth and remodeling.


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

Purified porcine relaxin (CM-A fraction; 3000 U/mg) was prepared at the Department of Biomedical Sciences (University of Guelph, ON, Canada) by extraction and purification from ovaries of pregnant sows [24]. Purity was confirmed by SDS-PAGE, which revealed a single band at approximately 6.2 kDa. The biological activity of the relaxin preparation was ascertained by inhibition of spontaneous uterine motility in vitro [25], and immunoreactivity was verified by RIA [26]. Zymed Laboratories (South San Francisco, CA) was the supplier of monoclonal anti-rat CX-26, CX-32, and CX-43 antibodies; CX-26, CX-32, and CX-43 peptides; purified normal mouse immunoglobulins (IgG); and fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse IgG. Goat anti-mouse IgG horseradish peroxidase-conjugated antibody was purchased from Transduction Laboratories (Lexington, KY). Normal goat serum (NGS) and normal swine serum (NSS) were acquired from Vector Labs. (Burlingame, CA). Renaissance Western Blot Chemiluminescence Reagent Plus was obtained from NEN Life Science (Wilmington, DE). Autoradiographic film (Hyperfilm-ECL) was purchased from Amersham (Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and Gibco-BRL Life Technologies (Gaithersburg, MD), unless otherwise specified.

Animals

Prepubertal (~115 days old) Yorkshire-Landrace gilts (Swine Unit of the New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ) were injected i.m. with porcine relaxin (0.5 mg/0.5 ml saline; n = 4) or saline (0.5 ml; n = 5) every 6 h for 54 h [2]. Uterine and cervical tissues were collected and processed as described by Wang-Lee et al. [4]. Briefly, this involved cutting 1-cm segments from the uterus and the uterine, center, and vaginal portions of the cervix. Additionally, skeletal muscle, which is fused to form a functional syncytium and does not contain gap junctions [12], was collected and frozen in liquid nitrogen for use as a negative control in immunoblot analysis. The marked trophic effects of relaxin on the uterus and cervix, and the systemic and local concentrations of relaxin achieved after in vivo relaxin administration in this animal model, have been reported [2, 4, 6]. The prepubertal status of the gilts was confirmed by the absence of estradiol-17ß in the plasma and uterine flushes of all animals before and after the treatment regimen [6]. In addition, follicular estradiol levels from control and relaxin-treated gilts (1.18 ± 0.42 and 0.51 ± 0.10 ng/ml, respectively) [6] were an order of magnitude lower than in cyclic animals (10–500 ng/ml) [27].

In other experiments, multiparous Duroc sows that exhibited at least two estrous cycles averaging 21 days were checked for estrus in the presence of a boar, and the first day of estrus was designated Day 0 of the cycle. Pregnant sows were obtained by mating animals on the first day of estrus, which was then designated Day 0 of pregnancy. Uteri were collected from pregnant sows on Days 40, 90, and 110 of pregnancy [28]. At the time of tissue collection, sows were stunned and exsanguinated. In addition, uteri from nonpregnant mature pigs were obtained from a local abattoir. All tissues were immediately frozen in liquid nitrogen and stored at -80°C until use. The animal experimentation procedures described here were reviewed and approved by the Rutgers University Animal Care Advisory Committee.

Progesterone RIA

Progesterone was measured in plasma and uterine flushes after extraction with petroleum ether [29]. 4-Pregnen-3,20-dione antibody and 1,2,6,7-3H progesterone were obtained from Steraloids (Wilton, NH) and Amersham, respectively. Intra- and interassay coefficients of variation for the assay were 3.4 ± 1.4% and 11.1 ± 3.8%, respectively, and assay sensitivity was 0.4 ng/ml.

Immunoblotting

Protein was extracted from tissue samples as described previously [30]. Briefly, tissues were homogenized in boiling lysis buffer, sonicated to reduce viscosity, and centrifuged to remove insoluble material. Protein samples (15 µg) were resolved on 10% Bis-Tris-HCl-buffered polyacrylamide electrophoresis gels (Novex, San Diego, CA) under reducing conditions and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked in 5% BSA in Tris-buffered saline (TBST); they were then incubated with anti-CX-26, CX-32, or CX-43 antibody (1 µg/ml) in TBST/1% BSA overnight at 4°C. After primary antibody incubation, membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000 in TBST/5% nonfat dry milk) for 1 h at room temperature (RT). After washing in TBST, membrane-bound antibodies were detected by chemiluminescence. Antiserum specificity was confirmed in separate immunoblot experiments by absorption of each CX antibody (1 µg/ml) with excess CX-specific peptide (10 µg/ml) for 10 min at RT prior to use. In these experiments, membranes were blocked in TBST/5% nonfat dry milk (2 h at RT) and then incubated with the antibody/peptide solutions (1 h at RT).

Immunohistochemistry

Frozen cervical tissue sections were hydrated in PBS (0.015 M) for 5 min at RT prior to immunostaining. Paraffin-embedded uteri were processed for immunohistochemistry as described in Lenhart et al. [30], with modifications. Tissue sections were incubated in 0.1% buffered trypsin (porcine pancreas type II) for 10 min at RT to unmask antigens. Cervical and uterine tissues were blocked in 3% BSA, 3% NGS, and 3% NSS in PBS (BGS-PBS) for 1 h at RT; they were then incubated overnight at 4°C with either CX-26, CX-32, or CX-43 antibodies (4 µg/ml in PBS). Normal mouse IgG served as the negative control for all antibodies. Sections were rinsed in PBS (5 times, 3 min each) and then incubated with FITC-conjugated goat anti-mouse IgG (1:25 in BGS-PBS) for 1 h at RT in a light-tight, humidified box. Tissues were washed in PBS (5 times, 3 min each) and then mounted in an antifade reagent (Slowfade; Molecular Probes, Eugene, OR). Fluorescent staining was viewed using a Zeiss Axioskop (Carl Zeiss, Thornwood, NY) microscope equipped with filters for fluorescence microscopy and photographed using T-MAX 400 film. In addition, immunolabeled sections were examined by confocal laser scanning microscopy using a Zeiss Laser Scanning Microscope 410 Invert, equipped with an argon/krypton laser and fitted with the appropriate filter blocks for the detection of fluorescein. Images were taken using consistent brightness and contrast settings and a line (8) averaging function. Nearby sections were stained with hematoxylin and eosin to confirm uterine and cervical histology.

Densitometry and Statistical Analysis

CX proteins were quantified in the immunoblots by scanning densitometry (Sigma Gel; SPSS, Chicago, IL). Data are expressed as the mean ± SEM of samples from pregnant and nonpregnant sows and control and relaxin-treated gilts using at least 3 animals per group. Data were examined by ANOVA and tested for differences by Fisher's least significant difference. A value of P < 0.05 was accepted as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Progesterone RIA

Progesterone was monitored to determine whether systemic or local progesterone was contributing to the effects observed. Prepubertal animals were used so that the effect of relaxin on CX expression could be analyzed independently of the effects of systemic and/or local steroids. Progesterone was undetectable in the plasma and uterine flushes of all animals before and after the treatment regimen.

Effect of Relaxin on CX-26, CX-32, and CX-43 Protein Expression in the Cervix

Maximal growth and remodeling activity in the uterine and vaginal portions of the cervix takes place at different times during pregnancy [31]. When tissue homogenates from uterine (UC), center (CC), and vaginal (VC) portions were analyzed, immunoreactive bands corresponding to CX-26 (~28 kDa), CX-32 (~31 kDa), and CX-43 (~43 kDa) were observed throughout the cervix of control and relaxin-treated prepubertal gilts. Relaxin administration significantly (P < 0.05) increased cervical expression of all three CX proteins examined in comparison to the control values (Fig. 1, A–C). While no difference in CX-26 or CX-32 protein was observed between the UC, CC, and VC, CX-43 protein expression was greater in the VC of relaxin-treated animals when compared to the UC (P < 0.05). No immunoreactivity was detected in skeletal muscle samples, even with longer exposure (data not shown).



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  		FIG. 1. Effect of relaxin on CX expression in the porcine cervix. Protein samples from the uterine and vaginal portions of the cervix of control and relaxin-treated prepubertal pigs were resolved by SDS-PAGE, and CX proteins were detected using monoclonal CX-specific antibodies. Values with different letters were significantly different (P < 0.05)

Localization of CX-26, CX-32, and CX-43 in the Cervix of Control and Relaxin-Treated Prepubertal Gilts

Figure 2, A and B, shows representative brightfield micrographs of the center cervix from control (Fig. 2A) and relaxin-treated (Fig. 2B) prepubertal gilts. The fluorescence micrographs of the cervix (Fig. 2, C–H) illustrate areas similar to those shown in brightfield. In cervices from prepubertal, relaxin-treated animals, CX-26 (Fig. 2D) as well as CX-32 (Fig. 2F) immunofluorescence was associated with the luminal epithelial cell layer. Specifically, CX-26 immunofluorescence was more prominent in epithelial cells (E) bordering the lumen (L), while immunoreactive CX-32 was more concentrated in epithelial cells adjacent to the stroma. In prepubertal controls, a similar pattern of CX-26 and CX-32 staining was observed (Fig. 2, C and E); however, expression was more intense in the cervices of relaxin-treated animals. On the other hand, CX-43 immunoreactivity was not evident in the luminal epithelium of either control or relaxin-treated prepubertal gilts. Instead, intense, punctate CX-43 immunofluorescence was observed in the cervical stroma of both groups (Fig. 2, G and H).



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  		FIG. 2. Localization of CX-26, -32, and -43 proteins in the cervix of control and relaxin-treated prepubertal gilts. A, B) Representative micrographs of cervices from control (A) and relaxin-treated (B) gilts, stained with hematoxylin and eosin to illustrate tissue composition. C–H) Cervical CX protein in tissue sections from control and relaxin-treated animals, incubated with monoclonal CX-specific antibodies (4 µg/ml) and visualized using an FITC detection system. Cervical epithelium (E) with borders indicated by white line (i———i), lumen (L), stroma (S). C, D) CX-26 protein was detected in the epithelium bordering the lumen of control (C) and relaxin-treated animals (D), while CX-32 protein was more evident in the epithelium adjacent to the stroma (E and F). Immunoreactive CX-43 was absent in epithelial cells of the cervix; however, intense CX-43 labeling was observed in the cervical stroma of control (G) and relaxin-treated (H) gilts. Bar = 100 µm

Effect of Relaxin and Pregnancy on CX-26, CX-32,and CX-43 Protein Expression in the Porcine Uterus

Uterine CX-26 expression throughout pregnancy (Days 40, 90, and 110) was similar to expression in prepubertal animals (Fig. 3A). Uterine CX-26 immunoreactivity in prepubertal pigs was significantly higher in relaxin-treated animals than in prepubertal controls and pregnant pigs at Day 40 and Day 90 (P < 0.05). In contrast, uterine CX-26 expression in Day 110 pregnant pigs and relaxin-treated prepubertal animals was similar. CX-26 protein in the uteri of mature, nonpregnant sows was significantly greater (P < 0.01) than in pregnant, prepubertal, or relaxin-treated prepubertal gilts. Uteri from pregnant animals at Day 40 and Day 90 had significantly higher (P < 0.05) amounts of CX-32 than those of animals at Day 110 (Fig. 3B). By Day 110 of pregnancy, expression of CX-32 protein was attenuated to levels similar to those in control prepubertal animals. Relaxin administration enhanced uterine CX-32 protein expression in prepubertal pigs when compared to controls (P < 0.05) and pregnant pigs at all three time points analyzed (P < 0.01). As observed for CX-26, immunoreactive CX-32 protein was significantly higher (P < 0.05) in the uteri of nonpregnant mature sows than in pregnant, prepubertal, or relaxin-treated prepubertal animals. While uterine CX-26 and CX-32 proteins appeared as broad, immunoreactive bands at 26 and 32 kDa, respectively (Fig. 3, A and B), uterine CX-43 expression was characterized by multiple immunoreactive proteins, ranging in size from 43 to 47 kDa (Fig. 3C). The 43-kDa-immunoreactive band was observed in uterine tissue from all animals, regardless of treatment. In contrast, the higher molecular size bands (see * in Fig. 3C), which corresponded to the phosphorylation pattern of CX-43 observed in other tissues [32], were detected in all but the immature control animals. Total CX-43 protein expression was quantified by adding densitometric values for all bands in a given lane. The pattern of CX-43 immunoreactivity in porcine uteri (Fig. 3C) was comparable to that observed for CX-26 (Fig. 3A), in that uterine CX-43 expression in pregnant sows and immature gilts was similar. Likewise, treatment of prepubertal gilts with relaxin resulted in a significant increase (P < 0.05) in immunoreactive CX-43 when compared with that in controls. In addition, CX-43 expression in uteri from nonpregnant sows was significantly higher (P < 0.05) than in any other experimental group.



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  		FIG. 3. Immunoblot analysis of CX expression in the porcine uterus. Uterine proteins from pregnant (Day 40, Day 90, Day 110), mature nonpregnant, prepubertal, and relaxin-treated prepubertal pigs were resolved by SDS-PAGE, and CX proteins were detected using monoclonal CX-specific antibodies. Representative blots are shown; multiple bands of higher molecular weight that cross-reacted with the CX-43 antibody are indicated with an asterisk. Values with different letters were significantly different (P < 0.05)

Localization of CX-26, CX-32, and CX-43 in the Uterus of Control and Relaxin-Treated Prepubertal Gilts

The brightfield micrographs shown in Figure 4, A and B, are representative of the uterine tissues in which CX proteins were localized after relaxin treatment of prepubertal pigs. The confocal fluorescence micrographs of the endometrium (Fig. 4, C–F) illustrate areas similar to that shown in the brightfield view (Fig. 4A). Immunoreactive CX-32 was scarce in the endometrium of control animals (Fig. 4C), while punctate endometrial CX-32 was associated with the epithelial lining of the endometrial glands and surrounding stromal tissue of relaxin-treated gilts (Fig. 4D). Scattered CX-43-immunopositive plaques were identified in the endometrial stroma of control and relaxin-treated animals. In addition, intense CX-43 immunoreactivity was observed in association with the endothelial layer of the blood vessels in the endometrial stroma (Fig. 4, E and F). There was little or no evidence for specific CX-26 immunolabeling in the endometrium of control or relaxin-treated prepubertal gilts (data not shown). Punctate myometrial CX-26, CX-32, and CX-43 immunofluorescence was observed in both control and relaxin-treated prepubertal gilts. While the images in Figure 4, G and H, illustrate myometrial CX-26 immunostaining, they are representative of the low-level staining observed for all three CXs in control and relaxin-treated pigs. The punctate areas of CX-26 immunoreactivity in the myometrial micrographs (Fig. 4, G–H) were chosen at a point where the endometrial stroma (S) meets the myometrium (M) in order to demonstrate positive myometrial CX immunofluorescence alongside areas of stromal tissue that did not cross-react with the antibodies. Endometrial and myometrial CX protein expression was low, even after relaxin administration, and when compared to CX immunofluorescence in the cervix (Fig. 2, C–H). No specific immunolabeling was detected when tissues were incubated with control mouse immunoglobulin (data not shown).



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  		FIG. 4. Localization of CX-26, -32, and -43 proteins in the uterus of relaxin-treated prepubertal gilts. A, B) Representative micrographs of endometrium (A) and myometrium (B), stained with hematoxylin and eosin. C–H) Uterine CX protein in tissue sections from control and relaxin-treated prepubertal gilts, incubated with monoclonal CX-specific antibodies (4 µg/ml) and visualized using an FITC detection system. Endometrial gland (EG), blood vessel (BV), stroma (S), myometrium (M). C, D) CX-32 protein was lightly scattered in the endometrium of control gilts (C) and evident in the epithelium of the endometrial glands of relaxin-treated animals (D). E, F) CX-43 labeling in the endometrium was found predominantly in stromal fibroblasts of control animals (E) and more intensely associated with the stromal blood vessels of gilts after relaxin administration (F). G, H) Myometrial CX-26 immunostaining is representative of CX protein abundance in uterine myocytes before (G) and after (H) exposure to relaxin. Bar = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hall and colleagues [2, 33] first reported relaxin-mediated growth and remodeling of the prepubertal pig uterus and cervix. In the present study, relaxin's growth-promoting effect, confirmed by studies in our laboratory [4, 6], was associated with significant changes in the expression of gap junction proteins CX-26, CX-32, and CX-43 in both the uterus and cervix of the prepubertal gilt. Spatial and temporal regulation of CX protein expression during pregnancy in the mammalian uterus has been documented [14, 16, 34] and has been correlated with changes in estrogen and progesterone levels [19, 35, 36]. The data presented here support and extend those observations by demonstrating that relaxin enhances expression of these proteins in the absence of endogenous steroids. In addition, we report here for the first time that CX-26, -32, and -43 gap junction proteins are expressed in the porcine cervix and that relaxin increases the expression of these CXs in the cervix of the prepubertal pig. Moreover, we demonstrate that cervical CX-26, -32, and -43 proteins in the relaxin-treated prepubertal gilt are restricted to specific tissue compartments.

Relaxin plays a major role in promoting growth and softening of the cervix during late pregnancy in many mammalian species [37]. While high-affinity relaxin-binding sites have been identified in cervical tissues of the pig [38] and rat [39], the mechanism by which relaxin binding is translated into cervical remodeling is not fully elucidated. Our data establish that porcine cervical gap junctions are composed of at least three different CX proteins—CX-26, -32, and -43—and that one of the actions of relaxin on the porcine cervix is to increase the expression of these three proteins. The tissue-specific expression of CX isotypes we observed in the cervix, with CX-26 and CX-32 localized in luminal epithelial cells and CX-43 in stromal and muscle tissue, is comparable to their distribution in the uterus under the influence of estrogen [16, 23]. However, the physiological significance of an increase in these cervical CX proteins in response to relaxin is unclear. In other tissues, alterations in the temporal and spatial patterns of CX expression occur during development and adaptive physiological processes [40, 41]. The relaxin-mediated transformation of the cervix involves extensive remodeling of the cervical connective tissue matrix [33, 42, 43] and an increase in connective tissue enzyme activity [4, 44]. In addition, relaxin stimulates cervical secretory activity [44, 45] and enhances luminal epithelial cell proliferation [46]. Thus, relaxin may enhance CX protein expression in order to coordinate cervical connective tissue remodeling. Our data also suggest that, by increasing cervical CX protein expression, relaxin may mediate cell-cell communication between endothelial cells and the surrounding stroma and smooth muscle, thereby inducing proliferation of quiescent endothelial cells in the cervix. A potential role for relaxin in the proliferation of cervical endothelial cells is supported by several lines of evidence. First of all, endothelial cells of the cervix contain relaxin-binding sites in both the rat and the pig [38, 39]. Secondly, relaxin has been reported to increase arterial cross-sectional area of the porcine cervix [3]. Finally, cell proliferation in the cervical endothelium of the rat was enhanced following relaxin treatment [46]. Likewise in the uterus, relaxin may facilitate growth of endothelial cells through gap junction communication, as CX-43 immunostaining is associated with blood vessels and relaxin significantly increases uterine CX-43 protein expression (Fig. 3C). This is supported by histological studies showing that relaxin significantly enhances uterine vascularization [47], and endothelial cell proliferation [48] in a manner similar to that reported for the cervix.

While we found a relaxin-induced increase in cervical gap junction proteins, relatively little information is available concerning steroidal control of cervical gap junction expression. In contrast to the situation for gap junction expression in the myometrium, there is no correlation between cervical gap junction density and the estrogen/progesterone ratio in women [23]. In addition, no increase in the number of cervical gap junctions was observed immediately before or during labor, as is seen in the myometrium [23], suggesting that formation of myometrial and cervical gap junctions does not occur simultaneously during pregnancy. The absence of a relationship between changing steroid concentrations and cervical gap junction formation implies that other factors, such as relaxin, may be involved in the regulation of cervical gap junction communication. In this study, we present evidence to support the hypothesis that relaxin may coordinate cervical remodeling via enhanced CX protein expression and that this effect is independent of ovarian steroids.

The importance of gap junctional communication in facilitating implantation [13, 14, 34] and controlling myometrial contraction at the time of parturition is well documented [19, 35, 49]. However, the significance of cell-cell communication during uterine growth, which occurs throughout the course of pregnancy, is less clear. Channels composed of CX-43 are important for electrical coupling between muscle fibers, such as in the myometrium [50]; CX-26 channels are found in nonexcitable cell types, such as uterine epithelial cells [14, 35], where they are thought to be important contributors to metabolic coupling. Growth factors such as relaxin, which raise cAMP levels [51] and activate protein kinase pathways [21, 22], can affect gap junction communication [7] and have been implicated in the regulation of CX expression in uterine myocytes [20]. Our immunoblotting data demonstrate an increase in uterine CX protein expression in response to relaxin. While the functional significance of this increase in CX protein is unknown, given relaxin's established uterotropic effects [1,2], these findings suggest that relaxin may enhance metabolic coupling between cells in the porcine uterus and contribute to the exchange of growth-promoting regulatory molecules. Furthermore, the immunolocalization data presented here are consistent with the observation that the growth-promoting effects of relaxin in the pig are not dependent on prior exposure to estrogen [2]. These localization studies allowed us to determine the cell types in the porcine uterus that expressed CX-26, -32, and -43. However, the immunostaining was not sensitive enough to unequivocally demonstrate differences in uterine CX expression due to relaxin treatment in specific cell types, as was the case in porcine cervical tissues. The intensity of uterine CX immunostaining in response to relaxin (Fig. 4) could best be described as intermediate between the high level of CX expression observed after exposure to estrogen and the significant attenuation of CX expression seen in response to progesterone [16, 17, 23, 52]. Thus, we propose that relaxin may act in concert with estrogen and progesterone to regulate CX-mediated communication in the uterus.

In the pig, circulating progesterone is elevated throughout gestation; it then declines precipitously at the time of parturition [53]. In contrast, plasma estrogens are undetectable until Day 80, then rise steadily in later gestation, with peak levels achieved just before parturition [53]. This results in an increase in the estrogen/progesterone ratio in the sow as pregnancy progresses. On the other hand, relaxin is secreted throughout the 114-day gestation period in the pig and is detectable within 1 wk of conception. Systemic relaxin increases to about 10 ng/ml by Day 110 of pregnancy, followed by a surge 2 days prior to parturition [54]. Thus, the observation that uterine CX expression was not significantly different in sows between Day 40 and Day 110 of pregnancy was unexpected, given the impact of estrogen treatment on CX expression in other species [16, 19, 35] and the effects of relaxin reported here. These data suggest that in pregnant sows, progesterone domination of the uterus through Day 110 of gestation is sufficient to inhibit the positive impact of circulating estrogens and relaxin on CX expression.

In conclusion, these studies demonstrate that CX-26, CX-32, and CX-43 are expressed in the cervix of the prepubertal pig in a spatially distinct fashion and that relaxin enhances the expression of cervical gap junction proteins. While we show that relaxin also enhances uterine expression of CX-26, CX-32, and CX-43 gap junction proteins, we found that cervical CX protein expression in response to relaxin was greater than that of the uterus. Our data revealed that immunoreactive CX protein expression during relaxin-mediated uterine growth approached levels observed toward the end of pregnancy, even in the absence of circulating steroids. Given relaxin's ability to promote growth and remodeling in the reproductive tract, we propose that relaxin increases cell-cell communication between the diverse cellular components of the porcine cervix and uterus, thus coordinating cervical growth and uterine accommodation in the pig.


   ACKNOWLEDGMENTS
 
The authors would like to thank Drs. Edward Zambraski, Patricia Schoknecht, and Sylvie Ebner for their assistance with animal surgery; the employees at the Swine Facility of the New Jersey Agricultural Experiment Station; Dr. John P. McMurtry, USDA Agricultural Research Service, Beltsville, MD, for providing uterine tissue from pregnant sows; Ms. Susan Becker and Dr. Heather Billings for assistance with the progesterone RIA; and Ms. Rebecca McConochie and Ms. Naomi Nomura for technical assistance.


   FOOTNOTES
 
1 This research was supported by USDA Grant #93-37203-8979 and New Jersey Agricultural Experiment Station Grant #D-06136-1. Back

2 Correspondence: Carol A. Bagnell, Dept. of Animal Sciences, Rutgers University, 84 Lipman Drive, New Brunswick, NJ 08901. FAX: 732 932 6996: bagnell{at}aesop.rutgers.edu Back

Accepted: July 14, 1999.

Received: April 26, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Min G, Hartzog MG, Jennings RL, Winn SR, Sherwood OD. Evidence that endogenous relaxin promotes growth of the vagina and uterus during pregnancy in gilts. Endocrinology 1997; 138:560–565.[Abstract/Free Full Text]
  2. Hall JA, Cantley TC, Day BN, Anthony RV. Uterotropic actions of relaxin in prepubertal gilts. Biol Reprod 1990; 42:769–774.[Abstract]
  3. Winn RJ, O'Day-Bowman MB, Sherwood OD. Hormonal control of the cervix in pregnant gilts. IV. Relaxin promotes changes in the histological characteristics of the cervix that are associated with cervical softening during late pregnancy in gilts. Endocrinology 1993; 133:121–128.[Abstract]
  4. Wang-Lee JL, Lenhart JA, Ohleth KM, Ryan PL, Bagnell CA. Regulation of urokinase- and tissue-type plasminogen activator by relaxin in the uterus and cervix of the prepubertal gilt. J Reprod Fertil 1998; 114:119–125.[Abstract]
  5. Too CKL, Bryant-Greenwood GD, Greenwood FC. The effect of oestrogen and relaxin on uterine and cervical enzymes: collagenase, proteoglycanase and B-glycuronidase. Acta Endocrinol 1986; 111:394–403.
  6. Ohleth KM, Lenhart JA, Ryan PL, Radecki SV, Bagnell CA. Relaxin increases insulin-like growth factors (IGFs) and IGF-binding proteins of the pig uterus in vivo. Endocrinology 1997; 138:3652–3658.[Abstract/Free Full Text]
  7. Cole WC, Garfield RE. Evidence for physiological regulation of myometrial gap junction permeability. Am J Physiol 1986; 251:C411-C420.
  8. Lane B, Oxberry W, Mazella J, Tseng L. Decidualization of human endometrial stromal cell in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins. Hum Reprod 1994; 9:259–266.[Abstract/Free Full Text]
  9. Bruzzone R, White TW, Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 1996; 238:1–27.[Medline]
  10. Yamasaki H, Naus CC. Role of connexin genes in growth control. Carcinogenesis 1996; 17:1199–1213.[Free Full Text]
  11. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 1996; 65:475–502.[CrossRef][Medline]
  12. Dermietzel R, Hwang TK, Spray DS. The gap junction family: structure, function and chemistry. Anat Embryol 1990; 182:517–528.[Medline]
  13. Day WE, Bowen JA, Barhoumi R, Bazer FW, Burghardt RC. Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium. Anat Rec 1998; 251:277–285.[CrossRef][Medline]
  14. Winterhager E, Grummer R, Jahn E, Willecke K, Traub O. Spatial and temporal expression of connexin26 and connexin43 in rat endometrium during trophoblast invasion. Dev Biol 1993; 157:399–409.[CrossRef][Medline]
  15. Rezapour M, Kilarski WM, Severs NJ, Gourdie RG, Rothery S, Backstrom T, Roomans GM, Ulmsten U. Quantitative immunoconfocal analysis of human myometrial gap junction connexin43 in relation to steroid hormone concentrations at term labour. Hum Reprod 1997; 12:159–166.
  16. Risek B, Clier FG, Phillips A, Hahn DW, Gilula NB. Gap junction regulation in the uterus and ovaries of immature rats by estrogen and progesterone. J Cell Sci 1995; 108:1017–1032.[Abstract]
  17. Jahn E, Classen-Linke I, Kusche M, Beier HM, Traub O, Grummer R, Winterhager E. Expression of gap junction connexins in the human endometrium throughout the menstrual cycle. Hum Reprod 1995; 10:2666–2670.[Abstract/Free Full Text]
  18. Granot I, Dekel N. Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem 1994; 269:30502–30509.[Abstract/Free Full Text]
  19. Lye SJ, Nicholson BJ, Mascarenhas M, MacKenzie L, Petrocelli T. Increased expression of connexin-43 in the rat myometrium during labor is associated with an increase in the plasma estrogen:progesterone ratio. Endocrinology 1993; 132:2380–2386.[Abstract]
  20. Geimonen E, Boylston E, Royek A, Anderson J. Elevated connexin-43 expression in term human myometrium correlates with elevated c-jun expression and is independent of myometrial estrogen receptors. J Clin Endocrinol Metab 1998; 83:1177–1185.[Abstract/Free Full Text]
  21. Sanborn BM, Sherwood OD. Effect of relaxin on bound cAMP in rat uterus. Endocr Res 1981; 8:179–192.
  22. Kalbag SS, Roginsky MS, Jelveh Z, Sulimovici S. Phorbol ester, prolactin and relaxin cause translocation of protein kinase C from cytosol to membranes in human endometrial cells. Biochim Biophys Acta 1991; 1094:85–91.[Medline]
  23. Saito T, Oyamada M, Yamasaki H, Mori M, Kudo R. Coordinated expression of connexins 26 and 32 in human endometrial glandular epithelium during the reproductive cycle and the influence of hormone replacement therapy. Int J Cancer 1997; 73:479–485.[CrossRef][Medline]
  24. Sherwood OD, O'Byrne EM. Purification and characterization of porcine relaxin. Arch Biochem Biophys 1974; 160:185–196.[CrossRef][Medline]
  25. Wiqvist N, Paul KG. Inhibition of spontaneous uterine motility in vitro as a biological assay of relaxin. Acta Endocrinol (Copenh) 1958; 29:135–146.[Medline]
  26. Porter DG, Ryan PL, Norman L. Lack of effect of oxytocin output from the porcine neural lobe in vitro or in lactating sows in vivo. J Reprod Fertil 1992; 96:251–260.[Abstract]
  27. Howard HJ, Ford JJ. Relationships among concentrations of steroids, inhibin, insulin-like growth factor-1 (IGF-1), and IGF-binding proteins during follicular development in weaned sows. Biol Reprod 1992; 47:193–201.[Abstract]
  28. Bagnell CA, Tashima L, Tsark W, Ali SM, McMurtry JP. Relaxin gene expression in the sow corpus luteum during the cycle, pregnancy and lactation. Endocrinology 1990; 126:2514–2520.[Abstract]
  29. Billings HJ, Katz LK. Progesterone facilitation and inhibition of estradiol-induced sexual behavior in the female goat. Horm Behav 1997; 31:47–53.[CrossRef][Medline]
  30. Lenhart JA, Downey BR, Bagnell CA. Connexin 43 gap junction protein expression during follicular development in the porcine ovary. Biol Reprod 1998; 58:583–590.[Abstract/Free Full Text]
  31. Eldridge-White R, Easter RA, Heaton DM, O'Day MB, Petersen GC, Shanks RD, Tarbell MK, Sherwood OD. Hormonal control of the cervix in pregnant gilts. I. Changes in the physical properties of the cervix correlate temporally with elevated serum levels of estrogen and relaxin. Endocrinology 1989; 125:2996–3002.[Abstract]
  32. Warn-Cramer BJ, Cottrell GT, Burt JM, Lau AF. Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase. J Biol Chem 1998; 273:9188–9196.[Abstract/Free Full Text]
  33. Hall JA, Anthony RV. Influence of ovarian steroids on relaxin-induced distensibility and compositional changes in the porcine cervix. Biol Reprod 1993; 48:1348–1353.[Abstract]
  34. Risek B, Gilula NB. Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during pregnancy. Development 1991; 113:165–182.[Abstract]
  35. Risek B, Guthrie S, Kumar N, Gilula NB. Modulation of gap junction transcript and protein expression during pregnancy in the rat. J Cell Biol 1990; 110:269–282.[Abstract/Free Full Text]
  36. Grummer R, Chwalisz K, Mullholland J, Traub O, Winterhager E. Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biol Reprod 1994; 51:1109–1116.[Abstract]
  37. Sherwood OD. Relaxin. In: Knobil E, N JD (eds.), The Physiology of Reproduction. Second Edition. New York: Raven Press; 1994: 926–940.
  38. Min G, Sherwood OD. Identification of specific relaxin-binding cells in the cervix, mammary glands, nipples, small intestine and skin of pregnant pigs. Biol Reprod 1996; 55:1243–1252.[Abstract]
  39. Kuenzi MJ, Sherwood OD. Immunohistochemical localization of specific relaxin-binding cells in the cervix, mammary glands and nipples of pregnant rats. Endocrinology 1995; 136:1367–1373.[Abstract]
  40. Kilarski WM, Fu X, Backstrom T, Roomans GM. Progesterone, estradiol and oxytocin and their in vitro effect on maintaining the number of gap junction plaques in human myometrium at term. Acta Physiol Scand 1996; 157:461–469.[CrossRef][Medline]
  41. Grummer R, Winterhager E. Regulation of gap junction connexins in the endometrium during early pregnancy. Cell Tissue Res 1998; 293:189–194.[CrossRef][Medline]
  42. Luque EH, Munoz de Toro MM, Ramos JG, Rodriguez HA, Sherwood OD. Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissues at term. Biol Reprod 1998; 59:795–800.[Abstract/Free Full Text]
  43. Winn RJ, Baker MD, Sherwood OD. Individual and combined effects of relaxin, estrogen and progesterone in ovariectomized gilts. I. Effects on the growth, softening and histological properties of the cervix. Endocrinology 1994; 135:1241–1249.[Abstract]
  44. Hwang JJ, Macinga D, Rorke EA. Relaxin modulates human cervical stromal cell activity. J Clin Endocrinol Metab 1996; 81:3379–3384.[Abstract]
  45. Huang C, Li Y, Anderson LL. Stimulation of collagen secretion by relaxin and effect of oestrogen on relaxin binding in uterine cervical cells of pigs. J Reprod Fertil 1993; 98:153–158.[Abstract]
  46. Burger LL, Sherwood OD. Relaxin increases the accumulation of new epithelial and stromal cells in the rat cervix during the second half of pregnancy. Endocrinology 1998; 139:3984–3995.[Abstract/Free Full Text]
  47. Vasilenko P, Mead JP, Weidmann JE. Uterine growth-promoting effects of relaxin: a morphometric and histological analysis. Biol Reprod 1986; 35:987–995.[Abstract]
  48. Dallenbach-Hellweg G, Dawson AB, Hisaw FL. The effect of relaxin on the endometrium of monkeys: histological and histochemical studies. Am J Anat 1966; 119:61–78.[CrossRef]
  49. Orsino A, Taylor CV, Lye SJ. Connexin-26 and connexin-43 are differentially expressed and regulated in the rat myometrium throughout late pregnancy and with the onset of labor. Endocrinology 1996; 137:1545–1553.[Abstract]
  50. Miyoshi H, Boyle MB, MacKay LB, Garfield RE. Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor. Biophys J 1996; 71:1324–1334.[Abstract/Free Full Text]
  51. Sanborn BM, Kuo HS, Weisbrodt NW, Sherwood OD. The interaction of relaxin with the rat uterus. I. Effects on cyclic nucleotide levels and spontaneous contractile activity. Endocrinology 1980; 106:1210–1215.[Abstract]
  52. Ou C, Orsino A, Lye S. Expression of connexin-43 and connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals. Endocrinology 1997; 138:5398–5407.[Abstract/Free Full Text]
  53. Robertson HA, King GJ. Plasma concentrations of progesterone, oestrone, oestradiol-17ß and of oestrone sulphate in the pig at implantation, during pregnancy and at parturition. J Reprod Fertil 1974; 40:133–141.[Medline]
  54. Sherwood OD, Chang CC, BeVier GW, Dzuik PJ. Radioimmunoassay of plasma relaxin levels throughout pregnancy and parturition in the pig. Endocrinology 1975; 97:834–837.[Abstract]



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