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Original Studies |
Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University School of Medicine (F.R., E.M.C., Y.W., H.-Y.H., M.L.P.), Stanford, California 94305; the Department of Gynecology and Obstetrics, Valencia University School of Medicine and Center for Gynecology and Obstetrics (F.R., E.M.C., F.B.-M.), Valencia, Spain; and the Department of Gynecology and Obstetrics, Lin-Kou Medical Center, Chang Gung Memorial Hospital (H.-Y.H.), Taipei, Taiwan
Address all correspondence and requests for reprints to: Francisco Raga, M.D., Center for Gynecology and Obstetrics. Pedro Aleixandre 57–7, 46006 Valencia, Spain. E-mail: cegiob{at}interbook.net
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Abstract |
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Recently, we have described the presence of an extrahypothalamic GnRH immunologically, biologically and chemically identical to the hypothalamic hormone in periimplantation human embryos. Moreover, the production of this decapeptide by the human trophoblast during the early stages of placentation is well documented.
TIMP-1 and -3 messenger ribonucleic acid (mRNA) expression in cultured stromal cells and protein secretion into the medium were significantly decreased by GnRH agonist compared to that in control groups. Moreover, expression of TIMP-1 was affected to a greater extent than that of TIMP-3. GnRH antagonist ablated the down-regulation of TIMPs by the GnRH agonist. MMP-9 mRNA expression was not detected in the control groups or in the groups treated with GnRH analogs.
Our results provide evidence that trophoblastic GnRH may play an important role in placental tissue organization and in the early embryo-maternal dialogue by enhancing trophoblast invasion through the specific inhibition of TIMPs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Once the embryo has adhered to the maternal uterine surface, it must be able to rapidly break through the endometrial basement membranes (BMs) and gain access to the maternal circulation to ensure successful implantation and placentation (2). This process of trophoblast invasion is associated with tissue remodeling of BMs, a specialized form of extracellular matrix (ECM) that separate cells from the underlying or surrounding connective tissue stroma. The major components of BMs are type IV collagen, laminin, entactin/nidogen, several proteoglycans, and SPARC (BM-40, osteonectin) (3).
The trophoblastic invasion is regulated in part by matrix metalloproteinases (MMPs), a group of enzymes involved in matrix degradation (4, 5). These enzymes share some important characteristics, such as common mode of activation, a conserved acid sequence in the putative metal-binding, active site region, and inhibition by specific proteinase inhibitors known as tissue inhibitor of metalloproteinase (TIMPs) (6).
Among MMPs, the 92-kDa type IV collagenase (MMP-9, gelatinase B) is a very important enzyme for BM degradation, capable of degrading virtually all components of the ECM, such as collagens, proteoglycans, and glycoproteins (4). MMP-9 degrades a variety of substrates, but particularly collagen type IV and V (7). It is commonly expressed by macrophages, certain transformed and tumor cells, and human cytotrophoblast from the first trimester human placenta (8, 9, 10).
Regulation of MMPs is controlled not only at the level of gene transcription and activation of the latent enzyme, but also locally within any tissue by members of a family of specific inhibitors, the TIMPs, of which four members have been cloned and sequenced (11). These naturally occurring specific inhibitors, of decidual or trophoblast cell origin, have an important physiological role in regulating trophoblast invasion (9). The best known TIMP is TIMP-1, a ubiquitous 28.5-kDa secreted glycoprotein that forms tight stoichiometric noncovalent complexes with the active forms of all known MMPs and in addition binds pro-MMP-2 (12, 13). TIMP-2 is a 21.5-kDa nonglycosylated protein that has 40% amino acid sequence identity with TIMP-1 and similar inhibitory activity against MMPs but preferentially binds to pro-MMP-2 (14, 15). TIMP-3, originally described in chicken but now also characterized in mouse and human, is a 24-kDa glycoprotein that differs from the other family members in having an affinity for the ECM (16, 17, 18). The protein encoded by the recently described gene for TIMP-4 has not yet been characterized; however, gene expression is confined primarily to the heart (19).
Human trophoblast shows dramatic invasive properties in early pregnancy, when the placenta is anchored to the uterine wall. During this process, the trophoblast invades the maternal endometrium, the spiral arteries, and even the smooth muscle layer of the uterus. This process provides a striking example of controlled invasion. Unlike tumor invasion, trophoblast invasion is precisely regulated; it is confined spatially to the uterus and temporally to early pregnancy (20).
Previous studies have demonstrated the presence of an extrahypothalamic GnRH immunologically, biologically, and chemically identical to the hypothalamic hormone in a variety of tissues (21, 22, 23). It is also a well established fact that the human placenta produces and secretes GnRH, and this immunoreactive GnRH is present in both cytotrophoblast and syncytiotrophoblast (24, 25). The placenta produces the highest concentrations of GnRH in early gestation, so it has been implicated as one of the primary regulators of the synthesis and secretion of hCG (26). It has also been demonstrated that GnRH receptor mRNA is expressed in both cytotrophoblast and syncytiotrophoblast (27).
Recently, it has been shown that immunoreactive GnRH was produced and secreted in vitro by cultured rhesus monkey embryo (28), mouse embryo (29), and human embryo (30) during the entire periimplantation period. In addition to this, there are indications that the presence of low affinity/high capacity binding sites for GnRH are present in both human endometrium and decidua (31, 32, 33, 34, 35).
We hypothesized that trophoblastic GnRH may play an important role in early trophoblast invasion at the embryo-maternal interface by regulating decidualized endometrial cell expression of MMP-9 and TIMPs. Consequently, the present study was undertaken to examine the mRNA expression pattern and protein secretion into the medium of 92-kDa type IV collagenase, TIMP-1, and TIMP-3 in cultured human stromal cells in the presence of GnRH agonist and antagonist.
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Materials and Methods |
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Endometrial samples were obtained from fertile premenopausal women, aged 23–31 yr, undergoing laparoscopic surgery in the midluteal phase of the menstrual cycle for various benign gynecological reasons. Endometriosis and pelvic inflammatory disease were excluded at the time of surgery.
Written consent from the patients and approval by the institutional committee on the use of human subjects in research at Stanford University were obtained before collection of tissue samples for this study.
Endometrial samples were taken using a Novak curette in the operating room immediately before surgical procedures. One part of the tissue was fixed with 4% paraformaldehyde and processed for routine hematoxylin-eosin staining; the rest was collected in medium (DMEM; Life Technologies, Grand Island, NY) containing antibiotics [0.5 µg/mL fungizone (Life Technologies and 50 µg/mL gentamicin (Sigma Chemical Co., St. Louis, MO)].
Isolation of human endometrial stromal and epithelial cells
Endometrial tissue samples were washed with DMEM (Life Technologies) to remove excess blood. Then the tissue samples were minced into approximately 1-mm3 pieces and digested overnight at room temperature with DMEM containing 0.1% collagenase type IA, 0.02% deoxyribonuclease type I (Worthington Biochemical Corp., Freehold NJ), and antibiotics as previously described (35, 36).
After tissue digestion, the stromal and epithelial cells were isolated as follows. The cell solution was allowed to settle for 5–10 min, then the supernatant (stroma-rich fraction) was filtered to a new tube with cell strainer (70-µm pore size nylon; Falcon, Becton Dickinson and Co., Lincoln Park, NJ).
To confirm the identity of the isolated cells, immunohistochemical localization of cytokeratin and vimentin as markers for epithelial and stromal cells, respectively, was carried out. Cell viability was established using trypan blue. The isolated stromal cells prepared by this method contained less than 0.1% endometrial epithelial or vascular cell (36, 37).
Culture and hormonal treatment
The isolated stromal cells (one to three passages) were plated at 2 x 105/well in six-well culture plates (Falcon) and cultured in standard medium [75% DMEM (Life Technologies) and 25% MCDB-105 (Sigma Chemical Co.)], containing antibiotics, 5 µg/mL insulin (Sigma Chemical Co.), and 10% charcoal-stripped FBS (Life Technologies). After confluence, cell cultures were treated with serum-free standard medium supplemented with 10 µg/mL human apo-transferrin (Sigma Chemical Co.), 50 µg/mL ascorbic acid (Sigma Chemical Co.), progesterone (1 µmol/L; Sigma Chemical Co.), estradiol (1 µmol/L; Sigma Chemical Co.), epidermal growth factor (20 ng/mL; Sigma Chemical Co.), and 1 mg/mL BSA (Irvine, Inc., CA). Control confluent cells were cultured in the same medium without steroids. Standard medium and serum-free medium were renewed every 2–3 days throughout the culture period (36).
As a marker of decidualization, PRL in conditioned medium derived from cultured cells was measured by enzyme-linked immunosorbent assay (Diagnostic System Laboratories, Webster, TX) with a detection limit of 0.14 ng/mL and intra- and interassay coefficients of variation of 5.5–9.0% and 6.6–10.4%, respectively. All samples were assayed in triplicate.
Confluent decidualized stromal cells were stimulated with either GnRH agonist (Histrelin, Sigma Chemical Co.) or GnRH antagonist (Detirelix, Sigma Chemical Co.) in a dose-dependent study (0–5 µmol/L) for 96 h. As a control for GnRH specificity, confluent decidualized cells were also treated with GnRH agonist (2 µmol/L) neutralized with GnRH antagonist at increasing doses (0–5 µmol/L) for 96 h. All the experiments were performed a minimum of four times with similar results.
Preparation of oligonucleotide primers for RT-PCR
Sequences of complementary DNA (cDNA) clones for mRNAs that should be detected in stromal cells [MMP-9 (38), TIMP-1 (39), and TIMP-3 (40)] were obtained from the GenBank database of the National Center for Biotechnology Information of the NIH. The corresponding primer sequences were constructed with the help of the program OLIGO 4.1 Primer Analysis Software (National Bioscience, Plymouth, MN) and were synthesized by the Protein, Amino Acid and Nucleic Acid Facility, Stanford University Medical Center (Stanford, CA). Amplification of human ß-actin primers (Clontech, Palo Alto, CA) served as an internal standard (41). The oligonucleotide primer sequences used in the present study are the same as those previously described by our group (36).
RNA preparation and RT
Total mRNA was extracted from cultured cells using the guanidinium isothiocyanate method (RNAzol method, Tel-Test, Inc., Friendswood, TX). The amount and purity of extracted RNA were quantitated by spectrophotometry in a GenQuant RNA/DNA calculator (Pharmacia Biotech Ltd., Cambridge, UK). The concentrations of mRNA were diluted to 1 µg/µL for RT-PCR.
For each mRNA to be detected, 19 µL RT-Mastermix were prepared [5 mmol/L MgCl2 solution, 1 x PCR buffer, 1 mmol/L deoxy (d)-ATP, 1 mmol/L dCTP, 1 mmol/L dGTP, 1 mmol/L dTTP, 2.5 µmol/L oligo(deoxythymidine)16, and 20 IU ribonuclease inhibitor (all from Perkin Elmer, Foster City, CA), 100 IU Moloney leukemia virus reverse transcriptase (Life Technologies), and 1 µg total RNA diluted in 1 µL diethylpyrocarbonate-treated H2O] and filled into a 0.5-mL thin wall PCR tube (Applied Scientific, South San Francisco, CA). RT-Mastermix in PCR tubes was covered with 50 µL light white mineral oil (Sigma Chemical Co.) and kept on ice until RT (36).
RT was carried out in a DNA Thermal Cycler 480 using a program with the
following parameters: 42 C for 15 min, 99 C for 5 min, and 4 C for
. After the reaction was completed, samples were stored at
-20 C until PCR. As negative controls, a defined volume of cultured
medium was extracted and subjected to the same RT-PCR reaction for the
different primers (36).
Quantitative competitive-PCR (QC-PCR)
Using an internal standard for QC-PCR as previously described (36, 42), quantitative mRNA expression of 92-kDa type IV, TIMP-1, and TIMP-3 in cultured stromal cells was determined. A competitive cDNA fragment was constructed by a deletion of a fragment from the target cDNA to be detected (36). The deleted cDNA fragments were obtained by PCR amplification of reverse transcribed total RNA from a luteal phase endometrial biopsy with the 5'-end original primer and 3'-end competitive primer. The PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide, and the cDNA was extracted from the gel, purified with an agarose gel extraction kit (Boehringer Mannheim, Mannheim, Germany), and quantitated by spectrophotometry (Pharmacia Biotech Ltd., Cambridge, UK). Independent sequence analysis was performed to confirm the identity of the expected sequence and the amplified cDNA.
To establish the equivalence of target cDNA to internal standard cDNA, serial dilutions of competitive cDNA were added to each PCR sample and coamplified with target cDNA (42). PCR products were stored at -20 C until 2% agarose gel electrophoresis was carried out in the presence of ethidium bromide. After completion of electrophoresis, the agarose gel was analyzed on the GelDoc 1000 system (Bio-Rad Laboratories, Inc., Hercules, CA). DNA size calculation and UV densitometry were carried out using the Molecular Analyst Software (Bio-Rad Laboratories, Inc.).
Western blot
Expression of TIMP-1 and -3 was determined at the protein level by Western blot analysis on cultured medium after 20-fold concentration with Cetricon 10 concentrators (Amicon, Inc., Beverly, MA), using 10% SDS-PAGE gels. Aliquots of each sample were applied to the gels and size-fractionated by electrophoresis according to a modified method previously described (43). Proteins were electroblotted on Hybond-ECL nitrocellulose membranes for 90 min at 1 mA/cm2. Membranes were blocked in 5% skim milk in Tris-buffered saline-Tween (10 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween-20, pH 7.8) overnight at 4 C. After blocking, membranes were incubated at room temperature for 1 h with the specific antibodies to TIMP-1 and TIMP-3 (both from Oncogen Research Products, Cambridge, MA). After three washes, membranes were incubated with the secondary peroxidase-coupled antibody at a dilution of 1:10,000 for 1 h at room temperature. An excess of the second antibody was eliminated by extensive washes in Tris-buffered saline-Tween buffer. Chemiluminescent substrate was used according to the manufacturer’s instructions (ECL Western blotting, Amersham, Les Ulis, France), and immunoreactive proteins were visualized after 0.5- to 10-min exposure to Hyperfilm-ECL (Amersham). TIMP-1 and -3 activities were determined on the films by scanning densitometry using the GelDoc 1000 system (Bio-Rad Laboratories, Inc.).
Data analysis
Statistical analysis was performed by ANOVA and independent sample t test, using the Statistical Package for Social Science (SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.
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Results |
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The effect of ovarian steroids on PRL production by human endometrial stromal cells grown to confluence in standard medium as a marker of decidualization was determined. There was no detectable level of PRL (<2 ng/106 cells) in conditioned medium from cells grown in the absence of steroid hormones (negative control). The PRL level in conditioned medium from cells grown in the presence of steroid hormones was 4.58 ± 0.3 ng/106 cells (mean ± SD of PRL levels obtained from 16 representatives experiments).
mRNA expression of TIMP-1 and TIMP-3, but not MMP-9, is modulated by GnRH in cultured stromal cells
To assess the ability of GnRH to mediate TIMP-1, TIMP-3, and 92-kDa type IV collagenase mRNA expression in stromal cells, confluent decidualized cultures were treated in the absence or presence of increasing concentrations of GnRH agonist.
QC-PCR was used to accurately determine the levels of MMP-9 and TIMP-1
and the TMP-3 mRNA expression. Figure 1
shows a ratio of target to internal standard cDNA documenting a
dose-dependent down-regulation of both TIMP-1 (Fig. 1A) and TIMP-3
(Fig. 1B) mRNA expression by increasing concentrations of GnRH agonist
(P < 0.05). Moreover, TIMP-1 and TIMP-3 mRNA
expression was enhanced in the control decidualized stromal cells,
treated with steroid hormones compared to that in the control group
treated without steroids in serum-free medium (Fig. 1).
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Modulation of TIMP-1 and TIMP-3 protein secretion into the medium by GnRH agonist: Western blot analyses
The expression of TIMP-1 and -3 was determined at the protein
level by Western blot analysis on cultured medium of the different
groups exposed to GnRH agonist as well as the negative control groups.
TIMP-1 and -3 activities determined on the films by scanning
densitometry revealed a dose-dependent down-regulation of both TIMP-1
(Fig. 2A) and TIMP-3 (Fig. 2B) protein
levels in the cultured medium of decidualized stromal cells treated
with increasing concentrations of GnRH agonist (P <
0.05).
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GnRH antagonist ablates the effect of GnRH agonist on TIMP-1 and TIMP-3 mRNA expression and protein secretion
To illustrate that the effect of the GnRH agonist was a specific receptor-mediated effect rather than a nonspecific or toxic effect, confluent decidualized stromal cells were treated with a constant dose of GnRH agonist and increasing concentrations of GnRH antagonist.
The effect of GnRH agonist on TIMP-1 (Fig. 3a) and TIMP-3 (Fig. 3b) mRNA expression
by QC-PCR in stromal cells was attenuated by increasing concentrations
of GnRH antagonist in a dose-dependent manner. Furthermore, Western
blot analyses demonstrated results at the protein level in the cultured
medium similar to those of mRNA for both TIMP-1 (Fig. 4a) and TIMP-3 (Fig. 4b). Therefore, the
reverse of the inhibitory effect of the GnRH agonist on TIMP-1 and
TIMP-3 mRNA and protein levels by increasing concentrations of GnRH
antagonist in a dose-dependent manner illustrates a specific
receptor-mediated effect of GnRH on TIMPs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is a well established fact that the human trophoblast produces and secretes GnRH, which is immunologically, biologically, and chemically identical to the hypothalamic hormone (23, 25, 45, 46). The presence of this decapeptide in both cytotrophoblast and syncytiotrophoblast has been extensively demonstrated (23, 25, 47).
The placenta produces the highest concentrations of GnRH in early gestation, before the 15th week, which correlates temporally with hCG production and maximal trophoblastic invasiveness. For this reason, placental GnRH has been implicated as one of the primary regulators of the synthesis and secretion of hCG (48). It has also been demonstrated that GnRH receptor mRNA is expressed in both cytotrophoblast and syncytiotrophoblast and that the mRNA levels exhibit changes paralleling the time course of hCG secretion during pregnancy, providing a paracrine/autocrine regulatory mechanism of hCG secretion by human placental GnRH during the first trimester of pregnancy (49).
Recently, we have demonstrated that periimplantation human embryos (morula to blastocyst stage) produce this hormone (30, 50) as well as that the human endometrium expresses the GnRH receptor in both endometrial compartments (epithelium and stroma) (35). Therefore, we hypothesize that the local trophoblastic secretion of GnRH as well as the presence of its receptor in the human decidualized endometrium may play an autocrine/paracrine role in the blastocyst-maternal dialogue during early embryonic implantation by modulating the balance between the MMPs and their natural inhibitors, the TIMPs.
In the present study we have demonstrated that treatment of decidualized stromal cells with increasing amounts of GnRH agonist resulted in a significant decrease in TIMP-1 and TIMP-3 mRNA expression and protein secretion into the medium. Moreover, this effect is a specific receptor-mediated effect, as it can be reversed in a dose-dependent manner by increasing concentrations of GnRH antagonist. This is in accord with the fact that exogenous administration of GnRH can displace the antagonist from the pituitary receptors and reestablish gonadotropin secretion, as both GnRH (native or agonist) and GnRH antagonist compete for the same receptor (51).
On the other hand, it is very interesting that GnRH agonist do not exert any effect on MMP-9 mRNA expression in decidualized endometrial stromal cells. Moreover, we were unable to detect MMP-9 mRNA expression in the control decidualized stromal cells. This is consequent with the lack of expression of all of the MMPs except the 72-kDa gelatinase in the secretory endometrium, suggesting that they may be negatively regulated by progesterone (52).
Our results in vitro are consequent with a recent report in vivo, in which GnRH agonist induced TIMP-1 mRNA inhibition in myometrium and leiomyomas, suggesting a direct effect of GnRH agonist on ECM degradation throughout a mechanism involving the balance MMPs/TIMPs (53).
These results emphasize the reciprocal nature of the decidual-placental interactions during implantation and reinforce the idea that the inhibitory function of TIMPs is crucial in keeping this invasion controlled (20). Moreover, the molecules and signals operating in the interaction between decidual membrane and invasive trophoblast remain unknown to date. However, there is evidence that hCG may play an important role in this invasive process (20), and that GnRH is the primary regulator of trophoblastic hCG (26). This strengthens the possible role of GnRH in this invasive process not only by a direct effect (54) but also by modulating other factors.
Basic research on early human implantation is of great interest to clinicians, as embryonic implantation is the major factor limiting or allowing fertility in humans (44). Recently, we have reported that the administration of GnRH agonist during the early stages of embryonic implantation in patients undergoing in vitro fertilization and embryo transfer is associated with increased pregnancy and implantation rates (30). Hence, the results of the present study may explain at least in part the beneficial effects of GnRH agonist on human implantation by the selective inhibition of TIMPs in the maternal decidua (30, 54).
We have investigated the effect of GnRH analogs (agonist and antagonist) on MMP-9, TIMP-1, and TIMP-3 mRNA expression in cultured decidualized stromal cells using QC-PCR; a well established technique whose sensitivity provide a major advantage to analyze low abundance mRNAs derived from cells (42, 55, 56, 57). We have also correlated the mRNA data with protein secretion into the medium (Western blot), reinforcing the results of the study. Thus, in the present study we have learned that GnRH appears to play a crucial role in the regulation of tissue degradation during trophoblastic invasion in implantation and early placentation by changing the balance between MMPs and TIMPs that is required for active tissue degradation.
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Acknowledgments |
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Footnotes |
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Received August 3, 1998.
Revised October 7, 1998.
Accepted October 19, 1998.
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