| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Obstetrics, Gynecology & Women's Health, New Jersey Medical School, Newark, New Jersey 07103
b Department of Obstetrics, Gynecology and Reproductive Medicine, State University of New York Health Sciences Center at Stony Brook, Stony Brook, New York 11794
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
decidua, implantation, pregnancy, progesterone, relaxin
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Enhanced chemiluminescence reagents were purchased from Amersham (Arlington Heights, IL). Mouse monoclonal antibody to human proMMP-1 and peroxidase-conjugated goat anti-mouse immunoglobulin (Ig)G were from Calbiochem, San Diego, CA (i.e., Oncogene Research Products). Sheep anti-human proMMP-3 antibodies and pure proMMP-1 and proMMP-3 proteins were kindly provided by Dr. Hideaki Nagase, University of Kansas Medical Center, Kansas City, KS. Peroxidase-conjugated donkey anti-sheep IgG, polyvinylidine difluoride (PVDF) membranes, and electrophoresis reagents were from Sigma-Aldrich (St. Louis, MO). Pure porcine relaxin was a gift from Dr. David Sherwood, University of Illinois. Medroxyprogesterone acetate (MPA) was from Steraloids Inc. (Wilton, NH). The VEGF enzyme-linked immunosorbent assay (ELISA) was from R&D Systems (Minneapolis, MN). The GeneAmp RNA-PCR kit was from PE Applied Biosystems (Foster City, CA). PCR primers were synthesized by Sigma-Genosys (The Woodlands, TX). The QuantamRNA 18S internal standard kit was from Ambion, Inc. (Austin, TX). GelStar nucleic acid stain was from FMC Bioproducts (Rockland, ME).
Endometrial Cell Culture and Treatment
Human endometrium was obtained from premenopausal women at various phases of the menstrual cycle who had undergone hysterectomy for medical reasons such as uterine prolapse, uterine fibroids, or adenomyosis, but not endometrial hyperplasia or endometrial cancer. Endometrial samples were diagnosed histologically and classified as either proliferative or secretory endometrium. Protocols were approved by the Human Subjects Committee of the State University of New York Health Sciences Center at Stony Brook in accordance with U.S. Department of Health and Human Services regulations. All samples were obtained after informed consent was provided by the subjects. Primary cultures of stromal and glandular epithelial cells were prepared from the samples using well-established methods previously described in detail [1–7, 19–21]. Each experiment used tissue from a single individual. Replicate wells of cells were incubated without or with pure porcine relaxin (3000 GPU/mg) at 50 or 100 ng/ml or MPA (0.1 µM) for 1 or 2 days. Conditioned medium samples were collected and maintained frozen at -80°C.
Assessment of Relaxin mRNA by Reverse Transcriptase-Polymerase Chain Reaction
Determination of human relaxin specific mRNA in primary cultures of endometrial stromal and glandular epithelial cells was performed using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) method. Cultured cells of endometrial tissue from 6 women taken during the secretory (n = 3) or proliferative phase (n = 3) were studied.
Total RNA was isolated from the endometrial cells using an acid guanidinium thiocyanate extraction procedure as described previously [7]. Total RNA from each sample was reverse transcribed into first-strand cDNA using cloned MuLV reverse transcriptase and random hexamers. The resulting cDNA was used as the template to amplify (by PCR) a 306-base pair (bp) DNA segment corresponding to amino acids 31–132 in the C peptide of the prorelaxin protein.
The primer pair we used, which spanned the exon 1-intron-exon 2 boundary such that products of the correct size could only have arisen from mRNA templates, was a 19-nucleotide forward primer with the sequence 5'-TCTCTGAGCCAGGAAGATG-3', corresponding to amino acids 31–36, and a 22-nucleotide reverse primer with the sequence 5'-CTTAGGCTTGGATACTCATTCT-3', which was complementary to the 3' end of the relaxin mRNA amino acids 126–132.
RNA from human corpus luteum taken at term pregnancy was used as a positive control. RNA from human lung (Clontech Inc., Palo Alto, CA) was used as a negative control. Two percent of the resulting DNA product was further amplified using the following pair of nested exon-intron boundary spanning primers: an 18-nucleotide forward primer with the sequence 5'-CCAGTGGCAGAAATTGTG-3', corresponding to amino acids 43–48; and a 22-nucleotide reverse primer with the sequence 5'-GATTCCAGTCTTCTCTTTGAAG-3', corresponding to amino acids 96–102. This nested PCR reaction yielded a 181-bp DNA product (corresponding to amino acids 43–102). The location of the primers used is shown in Figure 1.
|
Primer pairs were selected using the Wisconsin Package version 9.1 (Genetics Computer Group, Madison, WI) software using stringent specifications for the intron/exon junction spanning region of human prorelaxin cDNA. The primers were selected based on their guanine cytosine content and melting and annealing temperatures. In addition, because the primer pair was not intended to distinguish between the H1 and H2 transcripts, the pair used for the first PCR reaction was one that would amplify both H1 and H2 relaxin cDNA, and the sequence of the primer pair used for the nested reaction corresponded to H2 cDNA.
To normalize for RNA variability, an internal standard PCR reaction for 18S RNA was performed. Relative quantitative PCR was performed by amplifying 18S RNA using QuantamRNA 18S internal standards in a reaction setup parallel to the second (nested) relaxin reaction. The products of both the relaxin amplification (181 bp) and the 18S amplification (488 bp) were resolved on 2% agarose gels containing GelStar nucleic acid stain and photographed under UV light. Image analysis of the gels was performed using an AlphaImager 2000 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). Nucleotide sequences of the purified PCR products from endometrial and corpus luteum samples were authenticated by determining the precise nucleotide sequence using an ABI Prism automated DNA Sequencer. Sequences were compared with the reported sequences of human H2 relaxin listed in GenBank.
Immunoassays
Relaxin concentrations in conditioned medium samples were determined using a human relaxin-specific radioimmunoassay previously described [22], which uses recombinant human H2 relaxin protein (kindly provided by Genentech Inc., South San Francisco, CA), 125I-labeled H2 human relaxin as radioligand, and a rabbit polyclonal anti-human H2 relaxin antibody. All samples were assessed in two assays. The sensitivity of the assay is 10–25 pg/tube. The intraassay variation was 8.7% (n = 11 observations).
VEGF content in conditioned medium samples was assessed using a human VEGF-specific ELISA (R&D Systems). Interassay and intraassay variations were 5.0% (n = 5 assays) and 4.1% (n = 10 observations), respectively, and the precision of the assay was 94%–98%.
Western Blot Analysis
Expression of procollagenase (proMMP-1) and prostromelysin (proMMP-3) in conditioned medium were determined by Western blot analysis using specific, well-characterized, primary antibodies and methods described previously [23]. Briefly, conditioned medium samples were treated with SDS-PAGE buffer and electrophoresed on 10% SDS-PAGE gels. Broad-range kaleidoscope molecular weight markers were used to estimate molecular weights. Pure proMMP-1 and proMMP-3 proteins were used as positive controls. Proteins were electroblotted onto PVDF membranes, which were then blocked with 3% BSA-Tris-buffered saline, Tween 20 (TBST) for 30 min at room temperature. After washing twice with TBST, the blots were incubated overnight at 4°C with primary antibody diluted in 2% BSA-TBST. Membranes were then washed three times with TBST during a 30-min period and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. Subsequently, the blots were washed three times with TBST over a 30-min period and developed by the enhanced chemiluminescence method. Intensities of the signals obtained on developed films were determined using a computing densitometer (Molecular Dynamics 300B, Sunnyvale, CA) using the volume integration method with appropriate corrections for background absorption, as we described previously [23, 24].
Statistical Analyses
Densitometric values for control untreated conditioned cell media were set at 100, and values for relaxin-treated conditioned cell media are expressed as a percentage of control. Data determined to be normally distributed after assessment using the Shapiro-Wilk test were then analyzed using two-tailed t-tests. All data were normally distributed, except for the proMMP-1 levels from glandular epithelial cells isolated from tissue taken during the secretory phase. These data were therefore assessed using the Wilcoxon signed-rank test. All comparisons were performed using JMP statistical software (SAS Institute, Inc., Cary, NC) written for the Macintosh Computer (Apple Computers, Cupertino, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Total RNAs from cultured human endometrial stromal and glandular epithelial cells from tissue taken at both proliferative and secretory phases were subjected to RT-PCR, and the 182-bp relaxin-specific product was seen following reactions programmed by RNA from all 6 endometrial samples and corpus luteum as shown in Figure 2. This product was not detectable when RNA from human lung was used, although the 488-bp 18S RNA-specific product was clearly detected. Nucleotide sequences of the PCR products from endometrium and corpus luteum RNAs were determined to be sequences of the H2 human relaxin gene. Because the PCR primers spanned the intron in the relaxin gene, products of the correct size could have arisen only from mRNA templates, not from DNA contamination.
|
Detection of Relaxin Protein
Immunoreactive relaxin was undetectable (<20 pg/ml) in culture medium that had not been exposed to cells. In distinct contrast, immunoreactive relaxin was detected in conditioned medium from cultures of both endometrial stromal and glandular epithelial cells as shown in Table 1. Conditioned medium from cultures of endometrial stromal cells from tissue taken during the secretory phase contained mean levels of 63 ± 20 pg/ml (± SEM, n = 4 experiments, 6 replicate wells). Levels detected in medium from glandular epithelial cells of tissue taken during the proliferative phase were 174 ± 53 pg/ml (n = 4 experiments, each using a specimen from 1 individual, 7 replicate wells). Relaxin was detected in medium from glandular epithelial cells of tissue taken during the secretory phase in 3 (n = 6 replicate wells) of 4 experiments, each using a specimen from 1 individual. Although conditioned medium from proliferative phase glandular epithelial cells appears to have higher relaxin levels, no significant differences between these values and those of the other categories were seen.
|
Relaxin Regulation of VEGF Expression
Both stromal and glandular epithelial cells expressed ample quantities of VEGF and both cell types responded to relaxin as shown in Figure 3. In cultures of glandular epithelial cells from tissue taken during the proliferative phase, relaxin caused a significant inhibition of VEGF expression to 66.3% ± 6% of control (mean ± SEM, P = 0.03, n = 4 experiments; Fig. 3A). In distinct contrast, in cultures of glandular epithelial cells from tissue taken during the secretory phase, relaxin caused a significant increase of VEGF expression to 169.5% ± 11% of control (P = 0.003, n = 5 experiments). Similarly, in stromal cell cultures, relaxin significantly increased VEGF expression to 229% ± 28% of control (P = 0.04, n = 3 experiments; Fig. 3B).
|
Effect of Progestin on VEGF Expression
In contrast to the effect of relaxin, progestin inhibited VEGF expression from stromal cells taken during the secretory phase to 65.1% ± 11 % of control (P = 0.03, n = 6 experiments) as shown in Figure 3C.
Relaxin Regulation of MMP Expression
Relaxin significantly inhibited expression of endometrial proMMP-1, as shown in Figure 4. Relaxin significantly inhibited expression by glandular epithelial cells isolated from endometrial tissue taken at both phases of the menstrual cycle (Fig. 4A); a more marked inhibitory effect on cells isolated from tissue taken during the secretory phase (16.4% ± 5% of control (mean ± SEM), P < 0.0001, n = 5 experiments) than on cells from tissue taken during the proliferative phase (50.2% ± 6% of control, P = 0.004, n = 4 experiments) was seen. In addition, expression by stromal cells from tissue taken during the secretory phase was inhibited by relaxin to 67.2% ± 8% of control (P = 0.05, n = 3 experiments; Fig. 4B).
|
In contrast, relaxin had no effect on proMMP-3 expression from either glandular epithelial or stromal endometrial cells (Fig. 5). Expression of proMMP-3 by relaxin-treated glandular epithelial cells from tissue taken during the proliferative phase was 105% ± 12% of control (n = 4 experiments) and by cells from tissue taken during the secretory phase was 82% ± 11% of control (n = 5 experiments). Expression of proMMP-3 by relaxin treated stromal cells from tissue taken during the secretory phase was 91% ± 5% of control (n = 3 experiments).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The data presented here suggest that this function is to support endometrial conditions needed for implantation and to maintain pregnancy. The data demonstrate that relaxin stimulates VEGF expression from both stromal and glandular epithelial cells isolated from tissue taken during the secretory phase, and inhibits expression of procollagenase, with its most marked inhibitory effect on glandular epithelial cells isolated from tissue taken during the secretory phase. Our data are in agreement with those of Unemori et al. [12] who demonstrated that relaxin stimulates VEGF expression in a cell line of human endometrial cells. However, the present studies are the first to examine the effects of relaxin on VEGF expression in well-characterized, specific human endometrial cell types. Also, no previous studies have assessed the effects of relaxin on any endometrial matrix metalloproteinase. The present studies demonstrate that relaxin inhibits expression of proMMP-1, with its most marked inhibitory effect on glandular epithelial cells isolated from tissue taken during the secretory phase. Relaxin regulation of VEGF expression suggests a novel mechanism by which relaxin can regulate endometrial vascularization, and in concert with the dramatic inhibitory effects of relaxin on MMP-1 during the secretory phase, suggests a role for relaxin in endometrial support of implantation.
Results of studies by Hisaw and colleagues [13, 14] were the first to suggest a role for relaxin in endometrial vascularization. The results of these studies in the rhesus monkey suggest that relaxin induces marked proliferation of endothelial cells in endometrial blood vessels, and dilatation of the superficial endometrial blood vessels [14]. Results from these in vivo studies suggest that relaxin stimulates an intensified differentiation of the endometrial stromal cells into predecidual cells [13]. Relaxin appears to be necessary for maintaining endothelial proliferation and vascular dilatation in the endometrium of the rhesus monkey. Animals in which administration of relaxin-containing extracts was discontinued, but steroids were continued, showed reduced endothelial proliferation and dilatation [14]. Regulation of VEGF expression in endometrium provides an explanation for these findings.
Extensive and detailed studies of the effects of relaxin in human endometrial differentiation have found that relaxin appears to be a significant modulator that regulates differentiation of the human endometrium [1–12]. Relaxin stimulates the production of several secretory products, including prolactin, insulin-like growth factor, and insulin-like growth factor binding protein-1 (IGFBP-1) in progestin-primed endometrial stromal cells [2–4]. Prolactin and IGFBP-1 are considered to be the major secretory proteins of decidual cells, and the induction of expression of these secretory proteins has been widely used as a biochemical marker of decidualization of endometrial stromal cells in vitro [25]. IGFBP-1 is the major protein secreted from endometrial stromal cells during hormone stimulation. IGFBP-1 plays an essential role in regulating the mitogenic activity during growth and differentiation phases of endometrial stromal cell decidualization [21]. Detailed studies of regulation of IGFBP-1 gene promoter activity in endometrial stromal cells demonstrate that relaxin, not progestin, is the major inducer of IGFBP-1 gene transcription [5]. Thus, relaxin appears to be a more powerful regulator of human endometrial decidualization than progesterone. Studies have also shown that relaxin significantly increases total cellular protein and inhibits progestin-induced DNA synthesis, and it dramatically alters the ultrastructure of progestin-treated endometrial stromal cells [6]. No evidence of secretory activity was shown by cells treated with progesterone and estradiol, although secretory activity appears to be a prominent feature of decidualized endometrial stromal cells in vivo. Cells in the stromal cultures that were treated with relaxin in addition to progestin exhibited ultrastructural features that were characteristic of secretory cells. Thus in stromal cell cultures, progesterone alone is inadequate to induce full cellular function; relaxin is necessary as well.
Relaxin is an important agent in the remodeling of connective tissue in several reproductive tract tissues [26]. Relaxin markedly modulates the connective tissue phenotype of human fibroblasts of several target organs. Relaxin is a positive regulator of matrix metalloproteinase (MMP) expression in human cervical fibroblast cell models, increasing procollagenase and prostromelysin expression, and decreasing the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) [23]. Relaxin also decreases the synthesis and secretion of interstitial collagens in normal human dermal fibroblasts in a dose-dependent manner [27]. No previous studies have been done to determine the effects of relaxin in the modulation of endometrial connective tissue despite considerable evidence that endometrial maturation involves remodeling of the interstitial extracellular matrix [28]. The effect of relaxin on MMP expression varies with cell type; inhibition of procollagenase by relaxin in endometrial cells is in distinct contrast to the stimulatory effect of relaxin on procollagenase expression in cervical fibroblasts [23].
In summary, the findings presented here provide definitive evidence for endometrial synthesis of relaxin and support the hypothesis that relaxin, in conjunction with other regulatory agents, is involved in the secretory phase remodeling of the endometrium, which supports implantation. Relaxin appears to be an important regulator of endometrial maturation via mechanisms (in addition to the previously documented regulation of stromal cell differentiation) that involve regulation of angiogenesis and endometrial connective tissue composition.
|
FOOTNOTES |
|---|
1 This work was supported by National Institutes of Health grants HD-22338 and HD-19247 to L.T. 
2 Correspondence: Laura T. Goldsmith, Department of Obstetrics, Gynecology & Women's Health, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ, 07103. FAX: 973 972 4574; goldsmit{at}umdnj.edu
Accepted: January 8, 2002.
Received: September 17, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Heng, R. Ivell, P. Wagaarachchi, and R. Anand-Ivell Relaxin signalling in primary cultures of human myometrial cells Mol. Hum. Reprod., October 1, 2008; 14(10): 603 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mazella, S. Liang, and L. Tseng Expression of Delta-Like Protein 4 in the Human Endometrium Endocrinology, January 1, 2008; 149(1): 15 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Figueiredo, A. L. Mui, C. C. Nelson, and M. E. Cox Relaxin Stimulates Leukocyte Adhesion and Migration through a Relaxin Receptor LGR7-dependent Mechanism J. Biol. Chem., February 10, 2006; 281(6): 3030 - 3039. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Negishi, Y. Li, A. Usas, F. H. Fu, and J. Huard The Effect of Relaxin Treatment on Skeletal Muscle Injuries Am. J. Sports Med., December 1, 2005; 33(12): 1816 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Dimitriadis, C. Stoikos, M. Baca, W. D. Fairlie, J. E. McCoubrie, and L. A. Salamonsen Relaxin and Prostaglandin E2 Regulate Interleukin 11 during Human Endometrial Stromal Cell Decidualization J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3458 - 3465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. T. Nguyen and C. W. Dessauer Relaxin Stimulates Protein Kinase C {zeta} Translocation: Requirement for Cyclic Adenosine 3',5'-Monophosphate Production Mol. Endocrinol., April 1, 2005; 19(4): 1012 - 1023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tang, J. Mazella, H. Hui Zhu, and L. Tseng Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells Mol. Hum. Reprod., April 1, 2005; 11(4): 237 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.S. Hayes, E.C. Curnow, A.O. Trounson, L.A. Danielson, and E.N. Unemori Implantation and Pregnancy Following In Vitro Fertilization and the Effect of Recombinant Human Relaxin Administration in Macaca fascicularis Biol Reprod, November 1, 2004; 71(5): 1591 - 1597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Bond, L. J. Parry, C. S. Samuel, H. M. Gehring, F. L. Lederman, P. A. W. Rogers, and R. J. Summers Increased Expression of the Relaxin Receptor (LGR7) in Human Endometrium during the Secretory Phase of the Menstrual Cycle J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3477 - 3485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mazella, M. Tang, and L. Tseng Disparate effects of relaxin and TGF{beta}1: relaxin increases, but TGF{beta}1 inhibits, the relaxin receptor and the production of IGFBP-1 in human endometrial stromal/decidual cells Hum. Reprod., July 1, 2004; 19(7): 1513 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. D. Sherwood Relaxin's Physiological Roles and Other Diverse Actions Endocr. Rev., April 1, 2004; 25(2): 205 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. T. Goldsmith, G. Weiss, S. Palejwala, T. M. Plant, A. Wojtczuk, W. C. Lambert, N. Ammur, D. Heller, J. H. Skurnick, D. Edwards, et al. Relaxin regulation of endometrial structure and function in the rhesus monkey PNAS, March 30, 2004; 101(13): 4685 - 4689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Luna, A. Riesewijk, J. A. Horcajadas, R. d. van Os, F. Dominguez, S. Mosselman, A. Pellicer, and C. Simon Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle Mol. Hum. Reprod., February 1, 2004; 10(2): 85 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Bartsch, B. Bartlick, and R. Ivell Phosphodiesterase 4 Inhibition Synergizes with Relaxin Signaling to Promote Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 324 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. T. Nguyen, L. Yang, B. M. Sanborn, and C. W. Dessauer Phosphoinositide 3-Kinase Activity Is Required for Biphasic Stimulation of Cyclic Adenosine 3',5'-Monophosphate by Relaxin Mol. Endocrinol., June 1, 2003; 17(6): 1075 - 1084. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
