help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chou, C.-S.
Right arrow Articles by Leung, P. C. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chou, C.-S.
Right arrow Articles by Leung, P. C. K.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 10 4781-4790
Copyright © 2003 by The Endocrine Society

Regulatory Effects of Gonadotropin-Releasing Hormone (GnRH) I and GnRH II on the Levels of Matrix Metalloproteinase (MMP)-2, MMP-9, and Tissue Inhibitor of Metalloproteinases-1 in Primary Cultures of Human Extravillous Cytotrophoblasts

Chun-Shan Chou, Hua Zhu, Colin D. MacCalman and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An intricate balance between the production of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), modulates the overall proteolytic activity of trophoblasts during human implantation. In these studies we have examined the ability of classical GnRH I and the second form of this hormone (GnRH II) to regulate MMP-2, MMP-9, and TIMP-1 mRNA and protein levels in extravillous cytotrophoblasts propagated from explants of first trimester chorionic villi. GnRH I and GnRH II were found to increase MMP-2 and MMP-9 mRNA and protein levels in these primary cell cultures in a dose- and time-dependent manner using quantitative competitive-PCR and ELISA. In contrast, these two hormones decreased trophoblastic TIMP-1 mRNA and protein levels. Cetrorelix, a GnRH receptor antagonist, inhibited the regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 expression in these cells. Collectively, these observations suggest that GnRH I and GnRH II differentially regulate MMP-2, MMP-9, and TIMP-1 expression in human trophoblasts, possibly via distinct receptor-mediated intracellular signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INVASION OF embryonic trophoblasts into the maternal decidua and uterine vasculature, which increases blood flow to the placenta and ensures an adequate supply of nutrients and oxygen to the growing fetus, is a critical step in human pregnancy (1, 2, 3). This highly regulated developmental process is mediated, at least in part, by the spatiotemporal expression of matrix metalloproteinases (MMPs) in both decidual cells and the distinct subpopulations of trophoblasts present at the maternal-fetal interface (4, 5).

Of the multiple MMPs produced by the human placenta (6, 7, 8), MMP-2 and MMP-9 have been assigned key roles in promoting the invasive capacity of cytotrophoblasts. In particular, MMP-2 and MMP-9 are expressed primarily by extravillous cytotrophoblasts (EVTs) in vivo and in vitro. The production of these two MMPs by the placenta is down-regulated during the third trimester of pregnancy, paralleling the decline in trophoblast invasiveness associated with gestational age (9, 10, 11, 12). In addition, function-perturbing antibodies specific for MMP-9 or MMP-2 have been shown to be capable of reducing the invasive capacity of EVTs in vitro (11, 13). Reduced MMP-9 activity has also been reported in primary cultures of cytotrophoblasts isolated from placentas obtained from women diagnosed with preeclampsia (14), a disease in which trophoblast invasion into the maternal decidua and vasculature is believed to be compromised (15, 16).

MMPs are synthesized as latent precursors that must be cleaved after secretion to become activated (17). The activity of MMPs is further regulated by the secretion of specific tissue inhibitors of metalloproteinases (TIMPs) (18). TIMP-1, -2, and -3 are produced by the human placenta and decidua, suggesting an autocrine and paracrine regulation of the MMP-mediated invasion of trophoblasts (19, 20, 21). In addition, TIMP-1 has been shown to inhibit the invasive capacity of primary culture EVTs, indicating that an intricate balance between the production of proteases and their inhibitors modulates the overall proteolytic activity of trophoblasts in vivo and in vitro.

We have recently determined that GnRH is capable of increasing MMP-2 and MMP-9 mRNA levels in stromal cells isolated from first trimester decidual tissues (22). This hormone has also been shown to decrease the levels of TIMP-1 and TIMP-3 in human endometrial stromal cells allowed to undergo steroid-mediated decidualization in vitro (23). Taken together, these observations suggest that GnRH plays a key role in human implantation and placentation by regulating MMP activity at the maternal-fetal interface. In these studies we have examined the ability of GnRH (GnRH I) and the second form of mammalian GnRH (GnRH II), both of which are expressed by the human placenta and endometrium (24, 25, 26, 27, 28), to regulate MMP-2, MMP-9, and TIMP-1 mRNA and protein levels in primary cultures of EVTs propagated from first trimester placental tissues in a dose- and time-dependent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

Tissue samples of first trimester placenta were obtained from women undergoing elective termination of pregnancy. The use of these tissues was approved by the committee for ethical review of research involving human subjects, University of British Columbia. All patients provided informed written consent.

Cell isolation and culture

EVTs were propagated from first trimester placental tissue explants as described by Graham et al. (29). Briefly, chorionic villi were washed thoroughly in DMEM (Life Technologies, Inc., Burlington, Canada) containing penicillin and streptomycin (100 IU/ml and 100 µg/ml, respectively). The villi were minced finely and plated in 25-cm2 tissue culture flasks (BD Biosciences, Franklin, NJ) containing DMEM supplemented with antibiotics and 10% heated-inactivated fetal bovine serum (FBS; Life Technologies, Inc.). The fragments of chorionic villi were allowed to adhere for 2–3 d, after which any nonadherent material was removed. These tissue explants were cultured for a further 10–14 d, with the culture medium being replaced every 2 d. EVTs were separated from the villous explants by a brief (2- to 3-min) trypsin digestion [0.125% (vol/vol) trypsin, EDTA, and Ca2+- and Mg2+-free PBS] at 37 C and plated in 60-mm2 culture dishes (BD Biosciences) containing DMEM supplemented with antibiotics and 10% FBS.

The purity of the EVT cultures was determined by immunostaining with a monoclonal antibody directed against cytokeratin 8 and 18 (BD Biosciences) according to the methods described by MacCalman et al. (30). Only cell cultures that exhibited 100% immunostaining for cytokeratin were included in these studies.

All studies were performed using EVTs (passage 2) plated in 60-mm2 culture dishes at a density of 1 x 106 cells (BD Biosciences) and grown to 80% confluence. Twenty-four hours before each hormone treatment, FBS was removed from the culture medium.

Hormone treatments

EVTs were cultured in the presence of increasing concentrations of GnRH I or GnRH II (0, 0.1, 1, 10, or 100 nM) for 24 h or a fixed concentration of GnRH I or GnRH II (100 nM) for 0, 3, 6, 12, 24, or 48 h. In addition, cultures of EVTs were treated with GnRH I or GnRH II (100 nM) alone or in combination with Cetrorelix (100 nM), a GnRH I antagonist, for 24 h. Cells treated with vehicle (0.1% ethanol) served as a control for all of these experiments.

Primer design

Nucleotide sequences specific for MMP-2, MMP-9, or TIMP-1 and that also spanned different exons were identified in the human mRNA sequences deposited in GenBank (National Center for Biotechnology Information, Bethesda, MD). Forward and reverse primers corresponding to these DNA sequences and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were used to quantify and assess the integrity of the total RNA samples, were synthesized at the Nucleic Acid and Protein Synthesis Unit, University of British Columbia. To construct internal standard cDNA fragments, floating primers with a sequence complementary to a short nucleotide sequence present in the expected PCR products were generated by attaching the complementary sequences of these binding sites to the 3' end of the original reverse primers specific for MMP-2, MMP-9, or TIMP-1. We recently used this approach to examine the effects of GnRH I on MMP-2, MMP-9, and TIMP-1 mRNA levels in primary cultures of human endometrial stromal cells (22). The specific sequences of these primer sets and the expected sizes of the resultant PCR products are listed in Table 1Go.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Oligonucleotide primer sequences and optimized PCR conditions for the analysis of MMP-2, MMP-9, and TIMP-1 mRNA levels in trophoblasts

 
RNA preparation and RT-PCR

Total RNA was prepared from the EVT cultures using a RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA) using a protocol recommended by the manufacturer. The concentration of total RNA present in each of these extracts was quantified by optical densitometry (260/280 nm) using a Du-64 UV-spectrophotometer. An aliquot (1 µg) of the total RNA extracts prepared from the EVTs was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Oakville, Canada).

PCR was performed using template cDNA generated from total RNA extracts prepared from EVT cultures and the primer sets specific for MMP-2, MMP-9, or TIMP-1. The PCR conditions were as follows: 1 min at 94 C, 1 min at 56, 59, or 55 C for MMP-2, MMP-9, or TIMP-1, respectively, and 1.5 min at 72 C, followed by a final extension at 72 C for 15 min. The cycles were repeated 20–35 times. A linear relationship between the yield of the PCR products and the number of cycles performed was observed after 25 cycles for MMP-2, 30 cycles for MMP-9, and 27 cycles for TIMP-1 (data not shown).

The resultant PCR products for MMP-2, MMP-9, and TIMP-1 were separated using gel electrophoresis and visualized by ethidium bromide staining (data not shown). Aliquots of these MMP-2, MMP-9, and TIMP-1 PCR products were subcloned into the PCR II vector (Invitrogen, Carlsbad, CA) and selected clones subjected to DNA sequence to confirm the specificity of the primers. These clones were also used to generate target or internal standard MMP-2, MMP-9, or TIMP-1 cDNA fragments by standard molecular biology techniques.

Quantitative competitive-PCR (QC-PCR)

The QC-PCR strategy employed in these studies is based upon the competitive coamplification of a known amount of an internal standard specific for MMP-2, MMP-9, or TIMP-1 added to aliquots of the first strand cDNA prepared from our primary cultures of EVTs (22, 31, 32, 33).

To determine the optimal amounts of the internal standards to be used in the QC-PCR analysis, PCR mixtures containing a fixed amount of the target MMP-2, MMP-9, or TIMP-1 cDNAs (1 µl) and increasing concentrations of the corresponding internal standard cDNAs (0.3125–40 pg/µl for MMP-2, 0.075–10 pg/µl for MMP-9, or 3.125–400 pg/µl for TIMP-1, respectively) were prepared. PCR was then performed using these cDNA mixtures and the distinct MMP-2, MMP-9, or TIMP-1 primer sets under the optimized conditions described above.

An aliquot (10 µl) of the resultant MMP-2, MMP-9, or TIMP-1 PCR products was separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining (Fig. 1Go). The intensity of the ethidium bromide staining of the PCR products was analyzed using UV densitometry (Biometra, Whiteman Co., Gottingen, Germany). Volume counts (square millimeters) of the scanned PCR products were then determined using Scion Image computer software (Scion Image Co., Frederick, MD). The absorbance values obtained for each of the target and corresponding internal standard cDNAs generated by PCR were plotted against the amount of internal standard initially added to the reaction mixtures. The point of interception on these line graphs was taken as the optimal amount of internal standard to be used in the QC-PCR analysis (Fig. 1Go). Based upon these observations, MMP-2, MMP-9, or TIMP-1 internal standard cDNAs were added to aliquots of the first strand cDNA to be used in the QC-PCR analysis at concentrations of 5, 1.25, or 50 pg/µl, respectively.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. Determination of the optimal amounts of internal standard MMP-2 (A), MMP-9 (B), or TIMP-1 (C) cDNAs to be added to the QC-PCR reaction mixtures. Photomicrographs of ethidium bromide-stained gels containing PCR products generated using a fixed amount of target cDNA and increasing amounts of the corresponding internal standard (upper panels). The sizes of the resultant PCR products relative to a 100-bp ladder (MW) are indicated. The intensity of the ethidium bromide staining of these PCR products was determined by UV densitometry, and the resultant absorbance values plotted against the amount of internal standard added to each PCR in the line graphs (lower panels).

 
QC-PCR was performed using an aliquot (1 µl) of the first strand cDNA synthesized from the EVTs containing the optimized amount of the corresponding internal standard cDNA and the MMP-2, MMP-9, or TIMP-1 primer sets under the PCR conditions described above. The ratios of the intensity of ethidium bromide staining of the resultant target/internal standard PCR products were then determined as described above.

ELISA

The levels of active MMP-2, MMP-9, and TIMP-1 present in the conditioned medium of the EVTs were measured by ELISA using commercially available kits (Amersham Pharmacia Biotech, Piscataway, NY). MMP-2, MMP-9, and TIMP-1 levels were detected in the total protein extracts prepared from the conditioned culture medium with mean intra- and interassay coefficients of variation of 7 and 16.9% for MMP-2, 4.3 and 20.2% for MMP-9, and 8.9 and 13.1% for TIMP-1, respectively. All samples were assayed in duplicate.

Statistical analysis

The absorbance values obtained from the ethidium bromide-stained gels were subjected to statistical analysis using PRISM 2 computer software (GraphPad, San Diego, CA). Statistical differences between the absorbance values were assessed by ANOVA. Differences were considered significant for P <= 0.05. Significant differences between the means were determined using Dunnett’s test. The results are presented as the mean relative absorbance ± SEM obtained using five or more different cell preparations.

Statistical differences between the dose- or time-dependent effects of GnRH I or GnRH II on MMP-2, MMP-9, or TIMP-1 expression levels present in the total protein extracts prepared from the conditioned media of the EVT cultures were assessed by ANOVA, followed by Dunnett’s test. The results are presented as the mean protein levels ± SEM obtained using cultures propagated from five or more different placental tissues.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH I and GnRH II increase MMP-2 and MMP-9 mRNA and protein levels in EVTs in a dose-dependent manner

MMP-2 and MMP-9 mRNA and protein were detected in all of the EVT cultures examined in these studies (Figs. 2Go and 3Go). GnRH I increased MMP-2 and MMP-9 mRNA and protein levels in primary cultures of EVTs in a dose-dependent manner (Fig. 2Go). A significant increase (P <= 0.001) in the levels of the mRNA transcripts encoding these two MMP subtypes was only detected in EVTs cultured in the presence of the highest concentration of GnRH I (100 nM) examined in these studies. However, a significant increase (P <= 0.001–0.05) in the protein expression levels of these two MMP subtypes was first detected in EVTs cultured in the presence of lower concentrations of this hormone (1 nM).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2. MMP-2 and MMP-9 mRNA and protein levels in EVTs cultured in the presence of increasing concentrations of GnRH I. A and B, Representative photomicrographs of ethidium bromide-stained gels containing QC-PCR products generated using template cDNA synthesized from EVTs cultured in the presence of 0, 0.01, 1, or 100 nM (lanes 1–4, respectively). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked on the right of the photomicrograph. The intensity of the ethidium bromide staining of the PCR products was determined by UV densitometry, and the resultant absorbance values were used to calculate the ratio of target to the internal standard cDNA for each QC-PCR reaction. The results derived from this analysis as well as those from four other independent studies (data not shown) are represented (mean ± SEM; n = 5) in the bar graphs below (a, P < 0.001 vs. untreated control). C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. One milligram of total protein from conditioned medium was used in each reaction. Data are shown as the means of five independent assays ± SEM [a, P < 0.001; b, P < 0.05 (vs. untreated control)] in the bar graphs.

 


View larger version (27K):
[in this window]
[in a new window]
  		FIG. 3. MMP-2 and MMP-9 mRNA and protein levels in EVTs cultured in the presence of increasing concentrations of GnRH II. A and B, Representative photomicrographs of ethidium bromide-stained gels containing QC-PCR products generated using template cDNA synthesized from EVTs cultured in the presence of 0, 0.01, 1, or 100 nM (lanes 1–4, respectively). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. untreated control)]. C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. One milligram of total protein from conditioned medium was used in each reaction. Data are shown as the means of five independent assays ± SEM (a, P < 0.001 vs. untreated control) in the bar graphs.

 
GnRH II significantly increased (P <= 0.001–0.05) MMP-2 mRNA and protein levels in these primary cell cultures at all of the hormone concentrations examined in these studies (Fig. 3Go). In contrast, MMP-9 mRNA and protein levels were only significantly greater in EVTs cultured in the presence of 100 nM of this hormone (P <= 0.001).

GnRH I and GnRH II increase MMP-2 and MMP-9 mRNA and protein levels in EVTs in a time-dependent manner

A significant increase (P <= 0.001–0.05) in MMP-2 mRNA levels was detected in EVTs cultured in the presence of GnRH I for 6 h, with maximum levels of this mRNA transcript being detected after 24 h of treatment (Fig. 4Go). However, a significant increase (P <= 0.001) in the levels of active MMP-2 present in the conditioned medium of these cells was only detected after 12 h of culture under these experimental conditions. A significant increase (P <= 0.001) in MMP-9 mRNA and protein levels was also observed in EVTs cultured in the presence of GnRH I for 12 and 24 h, respectively. There was a significant decline (P <= 0.001) in the mRNA and protein levels of both of these MMP subtypes after 48 h of culture in the presence of GnRH I.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 4. Time-dependent effects of GnRH I on MMP-2 or MMP-9 mRNA and protein levels in EVTs. A and B, QC-PCR analysis of MMP-2 or MMP-9 mRNA levels in EVTs cultured in the presence of GnRH I (100 nM) for 0, 3, 6, 12, 24, or 48 h (lanes 1–6, respectively). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. untreated control)]. C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM (a, P < 0.001 vs. untreated control) in the bar graphs.

 
GnRH II caused a significant increase (P <= 0.001–0.05) in MMP-2 mRNA levels in EVTs after 12 h of culture, with maximum levels of this mRNA transcript being detected in cells cultured in the presence of this hormone after 24 h (Fig. 5Go). MMP-2 protein levels were also significantly increased (P <= 0.001) after 12 h of treatment, with maximum expression being detected in the conditioned medium of EVTs cultured in the presence of GnRH II for 24 h. Similarly, MMP-9 mRNA and protein levels were significantly higher (P <= 0.001–0.05) in EVTs cultured in the presence of GnRH II for 12 h and continued to increase until 24 h. A subsequent decline in the mRNA and protein levels of both of these MMP subtypes was observed in EVTs cultured in the presence of this hormone for 48 h.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. Time-dependent effects of GnRH II on MMP-2 or MMP-9 mRNA and protein levels in EVTs. A and B, QC-PCR analysis of MMP-2 or MMP-9 mRNA levels in EVTs cultured in the presence of GnRH II (100 nM) for 0, 3, 6, 12, 24, or 48 h (lanes 1–6, respectively). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. untreated control)]. C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM [a, P < 0.001; b, P < 0.05 (vs. untreated control)] in the bar graphs.

 
The addition of vehicle to the culture medium had no significant effect on MMP-2 and MMP-9 mRNA or protein levels in these primary cell cultures at any of the time points examined in these studies (data not shown).

GnRH-I and GnRH-II decrease TIMP-1 mRNA and protein levels in EVTs in a dose- and time-dependent manner

TIMP-1 mRNA and protein were detected in all of the EVT cultures examined in these studies. GnRH-I and GnRH-II decreased TIMP-1 mRNA and protein levels in a dose-dependent manner. A significant decrease (P <= 0.001–0.05) in TIMP-1 mRNA and protein levels was only observed in EVTs cultured in the presence of the higher concentrations of GnRH I (1 and 100 nM) used in these studies (Fig. 6Go). Similarly, only the higher concentrations of GnRH II (100 nM) were capable of significantly decreasing (P <= 0.001) TIMP-1 mRNA levels in these primary cell cultures. In contrast, GnRH II was capable of significantly decreasing (P <= 0.001–0.05) TIMP-1 protein levels at all of the hormone concentrations examined in these studies.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 6. TIMP-1 mRNA and protein levels in EVTs cultured in the presence of increasing concentrations of GnRH I or GnRH II. A and B, QC-PCR analysis of TIMP-1 mRNA levels in EVTs cultured in the presence of 0, 0.01, 1, or 100 nM (lanes 1–4, respectively). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. untreated control)]. C and D, Analysis of TIMP-1 expression in conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM [a, P < 0.001; b, P < 0.05 (vs. untreated control)] in the bar graphs.

 
A significant decrease in TIMP-1 mRNA (P <= 0.001–0.05) and protein levels (P <= 0.001) was observed in EVTs cultured in the presence of either GnRH I or GnRH II after 12 h, which was followed by a progressive increase in TIMP-1 expression in these cells until the termination of these studies at 48 h (Fig. 7Go).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 7. Time-dependent effects of GnRH I or GnRH II on TIMP-1 mRNA and protein levels in EVTs. A and B, QC-PCR analysis of TIMP-1 mRNA levels in EVTs cultured in the presence of GnRH I or GnRH II (100 nM) for 0, 3, 6, 12, 24, or 48 h (lanes 1–6, respectively). The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. untreated control)]. C and D, Analysis of TIMP-1 levels in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM (a, P < 0.001 vs. untreated control) in the bar graphs.

 
The addition of vehicle to the culture medium had no significant effect on TIMP-I mRNA or protein levels in these primary cell cultures at any of the time points examined in these studies (data not shown).

Cetrorelix inhibits the regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 mRNA and protein levels in EVTs

Cetrorelix, a GnRH receptor (GnRHR) antagonist, was capable of inhibiting the stimulatory effects of GnRH I, but not GnRH II, on MMP-2 and MMP-9 mRNA and protein levels in EVTs (Figs. 8Go and 9Go, respectively). Similarly, Cetrorelix inhibited the decrease in TIMP-1 mRNA and protein levels in EVTs cultured in the presence of GnRH I, but had no significant effect (P <= 0.05) on the GnRH II-mediated down-regulation of TIMP-1 expression levels in these cells (Fig. 10Go).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 8. Effects of Cetrorelix on MMP-2 or MMP-9 mRNA and protein levels in EVTs cultured in the presence of GnRH I. A and B, QC-PCR analysis of MMP-2 or MMP-9 mRNA levels in untreated EVTs (lane 1) or in cells cultured in the presence of a fixed amount of GnRH I (100 nM) alone (lane 2) or in combination with Cetrorelix (100 nM; lane 3). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below [a, P < 0.001; b, P < 0.05 (vs. treatment with GnRH I alone)]. C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM [a, P < 0.001; b, P < 0.05 (vs. treatment with GnRH I alone)] in the bar graphs.

 


View larger version (28K):
[in this window]
[in a new window]
  		FIG. 9. Effects of Cetrorelix on MMP-2 or MMP-9 mRNA and protein levels in EVTs cultured in the presence of GnRH II. A and B, QC-PCR analysis of MMP-2 or MMP-9 mRNA levels in untreated EVTs (lane 1) or in cells cultured in the presence of a fixed amount of GnRH II (100 nM) alone (lane 2) or in combination with Cetrorelix (100 nM; lane 3). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below. C and D, Analysis of the levels of active MMP-2 or MMP-9 present in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM in the bar graphs.

 


View larger version (24K):
[in this window]
[in a new window]
  		FIG. 10. Effects of Cetrorelix on TIMP-1 mRNA and protein levels in EVTs cultured in the presence of GnRH I or GnRH II. A and B, QC-PCR analysis of TIMP-1 mRNA levels in untreated EVTs (lane 1) or in cells cultured in the presence of a fixed amount of GnRH I or GnRH II (100 nM) alone (lane 2) or in combination with Cetrorelix (lane 3). The sizes of the resultant target and internal standard PCR products relative to a 100-bp ladder (lane M) are marked to the right of the photomicrograph. The absorbance values obtained from five independent studies are represented (mean ± SEM) in the bar graphs below (a, P < 0.00 vs. treatment with GnRH I or GnRH II alone). C and D, Analysis of TIMP-1 levels in the conditioned medium of these EVT cultures by ELISA. Data are shown as the means of five independent assays ± SEM (b, P < 0.05 vs. treatment with GnRH I or GnRH II alone) in the bar graphs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we report that GnRH I and GnRH II are capable of increasing MMP-2 and MMP-9 and concomitantly decreasing TIMP-1 mRNA and protein levels in EVTs propagated from explants of first trimester chorionic villi. The regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 expression in these cells was inhibited by Cetrorelix, a GnRHR-specific antagonist (33), suggesting that the biological actions of these two hormones on EVTs may be mediated by different receptor-mediated intracellular signaling pathways.

GnRH I expression in the human placenta is highest during early gestation (24, 25), which coincides with maximum trophoblast invasion into the decidua (1, 2, 3). Significant levels of GnRH II have also been detected in human placental tissues at all stages of gestation (27). In addition to regulating MMP-2 and MMP-9 expression in EVTs, we have recently determined that both GnRH I and GnRH II increase the expression of urokinase plasminogen activator (uPA), another protease active at the maternal fetal interface (33). GnRH I and GnRH II may also promote the proteolytic activity of EVTs in vitro by virtue of their ability to coordinately decrease the expression levels of the endogenous inhibitors of these two families of proteases, TIMP-1 and plasminogen activator inhibitor-1, respectively, in these cell cultures (33). Collectively, these observations suggest that GnRH I and GnRH II play key roles in promoting the invasive capacity of human trophoblasts.

GnRH I and GnRH II were both capable of increasing the mRNA and protein levels of MMP-2 and MMP-9 in EVTs. To date, the mechanisms underlying these two distinct regulatory effects have not been determined. However, the increase in MMP-2 and MMP-9 mRNA suggests that one level of regulation may be elicited by an increase in gene transcription and/or increased mRNA stability (34). Similarly, the increased MMP-2 and MMP-9 activity in these cell cultures may be due to an increase in the production of the latent enzyme and/or decreased degradation of these enzymes (34). Our previous observations, which indicate that uPA is coordinately regulated with MMP-2 and MMP-9 in EVTs in response to GnRH I or GnRH II, suggest that the functions of these enzymes may be linked, possibly by participation as components of a proteolytic cascade. In particular, increased uPA expression in these cell cultures may lead to the generation of plasmin from plasminogen, which, in turn, activates the latent form of MMP-9 (35). In addition, MMP-2 has been shown to activate MMP-9 activity in human cancer cells (36).

GnRH II has been shown to mimic the biological actions of GnRH I in extrapituitary tissues, including the placenta, ovary, and endometrium. In the placenta, GnRH I and GnRH II stimulate the secretion of the ß-subunit of human chorionic gonadotropin (27). However, the regulatory effects of GnRH II on the ß-subunit of human chorionic gonadotropin secretion from placental tissue explants were greater than those observed with equal concentrations of native GnRH I or its synthetic analogs (27). In addition to regulating the hormonal capacity of the human placenta, our studies suggest that both GnRH I and GnRH II may play key roles in modulating the invasive phenotype of human trophoblasts by virtue of their ability to regulate the balance between MMP and TIMP expression in these cells. Similarly, GnRH II is capable of eliciting these regulatory effects at lower hormone concentrations than GnRH I. GnRH II has also been shown to be a more potent regulator of the steroidogenic capacity of granulosa-lutein cells (37) and the proliferation of human endometrial and ovarian cancer cells in vitro (38). These observations have led to the proposal that GnRH II may be the biologically active form of this hormone in extrapituitary tissues (27).

Prolonged exposure to GnRH I resulted in a decrease in MMP-2 and MMP-9 mRNA and protein levels in the primary cultures of EVTs. Similarly, the inhibitory effects of GnRH I and GnRH II on TIMP-1 mRNA and protein levels in these primary cell cultures were reduced with time in culture. This biphasic effect may be attributed to a decrease in GnRHR-I expression levels, a biological phenomenon previously observed in GnRH I-stimulated pituitary cells and villous cytotrophoblasts isolated from first trimester placental tissues (39, 40). In addition, GnRH I and GnRH II may be subject to degradation by the high levels of C-ase-1 postproline peptidase present in human placental tissues and primary cultures of human trophoblasts (41, 42).

GnRH II has been shown to bind the GnRHR present in the human placenta with higher affinity than GnRH I, suggesting that a receptor specific for GnRH II is present in this dynamic tissue (27). The ability of Cetrorelix, to inhibit the regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 mRNA and protein levels, provides further evidence that the biological actions of these two hormones are elicited by distinct receptors. Recently, a gene encoding a second receptor for GnRH (GnRHR II) has been identified in the human genome (33, 43, 44). Although a full-length mRNA transcript encoding this second form of human GnRHR has not been isolated, GnRHR II mRNA transcripts have been detected in total RNA extracts prepared from human term placenta (33).

In summary, we have determined that GnRH I and GnRH II increase the mRNA and activity levels of MMP-2 and MMP-9 mRNA in primary cultures of EVTs propagated from first trimester placental tissues in a dose- and time-dependent manner. In contrast, these two hormones decreased the expression levels of the endogenous inhibitor, TIMP-1 in these cell cultures. These findings strengthen our hypothesis that GnRH I and GnRH II play key regulatory roles in the proteolytic degradation of the extracellular matrix that occurs at the maternal-fetal interface during early pregnancy in the human.


   Footnotes
 
This work was supported by an operating grant from the Canadian Institutes of Health Research (to P.C.K.L. and C.D.M.).

C.D.M. and P.C.K.L. contributed equally to these studies.

C.D.M. is a career investigator with the British Columbia Research Institute for Children’s and Women’s Health.

P.C.K.L. is the recipient of a distinguished senior investigatorship scholar award from the Michael Smith Foundation for Health Research.

Abbreviations: EVT, Extravillous cytotrophoblast; FBS, fetal bovine serum; GnRHR, GnRH receptor; MMP, matrix metalloproteinase; QC-PCR, quantitative competitive-PCR; TIMP, tissue inhibitor of metalloproteinases; uPA, urokinase plasminogen activator.

Received April 16, 2003.

Accepted July 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pijnenborg R, Dixon G, Robertson WB, Brosens I 1980 Trophoblastic invasion of the human decidua from 8 to 18 weeks of pregnancy. Placenta 1:3–19[Medline]
  2. Pijnenborg R, Bland JM, Robertson WB, Brosens I 1983 Uteroplacental arteriole changes related to interstitial trophoblast migration in early human pregnancy. Placenta 4:387–414
  3. Aplin JD 1991 Implantation, trophoblast differentiation and haemochorial placentation: mechanistic evidence in vivo and in vitro. J Cell Sci 99:681–692[Medline]
  4. Fata JE, Ho ATV, Leco KJ, Moorehead RA, Khoka R 2000 Cellular turnover and extracellular matrix remodelling in female reproductive tissues: functions of metalloproteinases and their inhibitors. Cell Mol Life Sci 57:77–95[CrossRef][Medline]
  5. Vu TH, Werb Z 2000 Matrix metalloproteinases:effectors of development and normal physiology. Gene Dev 14:2123–2133[Free Full Text]
  6. Graham CH, McCrae KR, Lala PK 1993 Molecular mechanisms controlling trophoblast invasion of the uterus. Trophoblast Res 7:237–250
  7. Tarrade A, Goffin F, Munuat C, Lai-Kuen R, Tricottet V, Foidart JM, Vidaud M, Frankenne F, Evain-Brion D 2002 Effect of matrigel on human extravillous cytotrophoblasts differentiation: modulation of protease pattern gene expression. Biol Reprod 67:1628–1637[Abstract/Free Full Text]
  8. Fisher S, Cui T, Zhang L, Hartman L, Grahl K, Zhang G, Tarpey J, Damsky C 1989 Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J Cell Biol 109:891–902[Abstract/Free Full Text]
  9. Polette M, Nawrocki B, Pintiaux A, Massenat C, Maquoi E, Volders L, Schnapps JP, Birembault P, Foidart JM 1994 Expression of gelatinase A and B and their tissue inhibitors by cells of early and term human placenta and gestational endometrium. Lab Invest 71:838–846[Medline]
  10. Xu P, Wang YL, Zhu SJ, Luo SY, Piao YS, Zhuang LZ 2000 Expression of matrix metalloproteinase-2, -9, and -14, tissue inhibitors of metalloproteinase-1, and matrix proteins in human placenta during the first trimester. Biol Reprod 62:988–994[Abstract/Free Full Text]
  11. Isaka K, Usada S, Ito H, Sagawa Y, Nakamura H, Nishi H, Suzuki Y, Li YF, Takayama M 2003 Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta 24:53–64[CrossRef][Medline]
  12. Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S 1994 Developmental regulation of 72 and 94 kd type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 171:832–838[Medline]
  13. Librach CL, Werb Z, Fitzgerald ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH, Fisher SJ 1991 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol 113:437–449[Abstract/Free Full Text]
  14. Graham CH, McCrae KR 1996 Altered expression of gelatinase and surface-associated plasminogen activator activity by trophoblast cells isolated from placentas of preeclamptic patients. Am J Obstet Gynecol 175:555–562[CrossRef][Medline]
  15. Robertson WB, Kong TY, Brosens I 1986 The placental bed biopsy: a review from three European centres. Am J Obstet Gynecol 155:401–412[Medline]
  16. Redline RW, Patterson P 1995 Pre-eclampsia is associated with an excess of proliferative immature intermediate trophoblast. Hum Pathol 26:594–600[CrossRef][Medline]
  17. Nagase H, Woessner Jr JF 1999 Matrix metalloproteinases. J Biol Chem 274:21491–21494[Free Full Text]
  18. Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP 1997 Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 74:111–122[Medline]
  19. Graham CH, Lala PK 1991 Mechanism of control of trophoblast invasion in situ. J Cell Physiol 148:228–234[CrossRef][Medline]
  20. Hurskainen T, Hoyhtya M, Tuuttila A, Oikarinen A, Auto-Harmainen H 1996 mRNA expressions of TIMP-1, -2, and -3 and 92-kDa type IV collagenase in early human placenta and decidual membrane as studied by in situ hybridization. J Histochem Cytochem 44:1379–1388[Abstract]
  21. Ruck P, Marzusch K, Horny HP, Diet J, Kaiserling E 1996 The distribution of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the human placenta. Placenta 17:263–266[CrossRef][Medline]
  22. Chou CS, Tai CJ, MacCalman CD, Leung PCK 2003 Dose-dependent effects of gonadotropin-releasing hormone on matrix metalloproteinase (MMP)-2 and MMP-9 and tissue specific inhibitor of metalloproteinases-1 messenger ribonucleic acid levels in human extravillous cytotrophoblasts in vitro. J Clin Endocrinol Metab 88:680–688[Abstract/Free Full Text]
  23. Raga F, Casan EM, Wen Y, Huang HY, Bonilla-Musoles F, Polan ML 1999 Independent regulation of matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and TIMP-3 in human endometrial stromal cells by gonadotropin releasing hormone:implications in early human implantation. J Clin Endocrinol Metab 84:636–642[Abstract/Free Full Text]
  24. Siler-Khodr TM, Kodhr GS 1978 Content of leutinizing hormone-releasing factor in the human placenta. Am J Obstet Gynecol 130:216–219[Medline]
  25. Pragar D, Weber MM, Herman-Bonert V 1992 Placental growth factors and releasing/inhibiting peptides. Semin Reprod Endocrinol 10:83–91
  26. Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL 1998 Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. Mol Hum Reprod 1998 4:893–898[Abstract/Free Full Text]
  27. Siler-Khodr TM, Grayson M 2001 Action of chicken II GnRH on the human placenta. J Clin Endocrinol Metab 86:804–810[Abstract/Free Full Text]
  28. Cheon KW, Lee HS, Parhar IS, Kang IS 2001 Expression of the second form of gonadotropin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod 7:447–452[Abstract/Free Full Text]
  29. Graham CH, Lysiak JJ, McCrae KR, Lala PK 1992 Localization of transforming growth factor-ß at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol Reprod 46:561–572[Abstract]
  30. MacCalman CD, Furth EE, Omigbodun A, Bronner M, Coutifaris C, Strauss III JF 1996 Regulated expression of cadherin-11 human epithelial cells: a role for cad-11 in trophoblast-endometrium interactions? Dev Dynam 206:201–211[CrossRef][Medline]
  31. Jin CF, Mata M, Fink DJ 1994 Rapid construction of deleted DNA fragments for use as internal standards in competitive PCR. PCR Methods Appl 3:252–255[Medline]
  32. Chou CS, Zhu H, Shalev E, MacCalman CD, Leung PCK 2002 The effects of gonadotropin-releasing hormone (GnRH)-1 and GnRH II on the urokinase type plasminogen activator/plasminogen activator inhibitor system in human extravillous cytotrophoblasts in vitro. J Clin Endocrinol Metab 87:5594–5603[Abstract/Free Full Text]
  33. Neill JD 2002 GnRH and GnRH receptor genes in the human genome. Endocrinology 143:737–743[Abstract/Free Full Text]
  34. Nuttall RK, Kennedy TG 2000 Epidermal growth factor and basic fibroblast growth factor increase the production of matrix metalloproteinases during in vitro decidualization of rat endometrial stromal cells. Endocrinology 141:629–636[Abstract/Free Full Text]
  35. Kleiner DE, Stetler-Stevenson WG 1993 Structural biochemistry and activation of matrix metalloproteases. Curr Opin Cell Biol 5:891–897[CrossRef][Medline]
  36. Fridman R, Toth M, Pena D, Mobashery S 1995 Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Res 55:2548–2555[Abstract/Free Full Text]
  37. Kang SK, Tai CJ, Nathwani PS, Leung PCK 2001 Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells. Endocrinology 142:182–192[Abstract/Free Full Text]
  38. Grundker C, Gunthert AR, Millar RP, Emons G 2002 Expression of gonadotropin releasing hormone II receptor in human endometrial and ovarian cancer cells and effects of GnRH II on tumor cell proliferation. J Clin Endocrinol Metab 87:1427–1430[Abstract/Free Full Text]
  39. Cheng KW, Leung PCK 2000 The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor. Can J Physiol Pharmacol 78:1029–1052[CrossRef][Medline]
  40. Currie WD, Setoyama T, Lee PS, Baimbridge KG, Church J, Ho Yuen B, Leung PCK 1993 Cytosolic free Ca2+ in human syncytiotrophoblast cells increased by gonadotropin-releasing hormone. Endocrinology 133:2220–222[Abstract]
  41. Siler-Khodr TM, Kang IS, Jones MA, Harper MJK, Khodr GS, Rhode J 1989 Characterization and purification of a placental protein that inactivates GnRH, TRH and angiotensin II. Placenta 10:283–296[Medline]
  42. Kang IS, Siler-Kodhr TM 1992 Chorionic peptidase inactivates GnRH as a post-proline peptidase. Placenta 13:81–87[Medline]
  43. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641[Abstract/Free Full Text]
  44. Morgan K, Conklin D, Pawson AJ, Sellar, Ott TR, Millar RP 2003 A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation of chromosome 1q.12. Endocrinology 144:423–436[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. L. Poon, B.-S. An, W.-K. So, G. L. Hammond, and P. C. K. Leung
Temporal Recruitment of Transcription Factors at the 3',5'-Cyclic Adenosine 5'-Monophosphate-Response Element of the Human GnRH-II Promoter
Endocrinology, October 1, 2008; 149(10): 5162 - 5171.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
C. Morimoto, Y. Osuga, T. Yano, Y. Takemura, M. Harada, T. Hirata, Y. Hirota, O. Yoshino, K. Koga, K. Kugu, et al.
GnRH II as a possible cytostatic regulator in the development of endometriosis
Hum. Reprod., November 1, 2005; 20(11): 3212 - 3218.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
G. Rajashekhar, A. Loganath, A. C. Roy, S. S. Chong, and Y. C. Wong
Hypoxia Up-regulated Angiogenin and Down-regulated Vascular Cell Adhesion Molecule-i Expression and Secretion in Human Placental Trophoblasts
Reproductive Sciences, July 1, 2005; 12(5): 310 - 319.
[Abstract] [PDF]

Home page
 Endocr. Rev.Home page
C. K. Cheng and P. C. K. Leung
Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans
Endocr. Rev., April 1, 2005; 26(2): 283 - 306.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
G S Harrison, M E Wierman, T M Nett, and L M Glode
Gonadotropin-releasing hormone and its receptor in normal and malignant cells
Endocr. Relat. Cancer, December 1, 2004; 11(4): 725 - 748.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. Ding, J. Xu, X. Luo, and N. Chegini
Gonadotropin Releasing Hormone and Transforming Growth Factor {beta} Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells
J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5549 - 5557.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
X. Luo, L. Ding, and N. Chegini
Gonadotropin-releasing hormone and TGF-{beta} activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells
Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E991 - E1001.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. S. Kauffman and E. F. Rissman
A Critical Role for the Evolutionarily Conserved Gonadotropin-Releasing Hormone II: Mediation of Energy Status and Female Sexual Behavior
Endocrinology, August 1, 2004; 145(8): 3639 - 3646.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C.-S. Chou, A. G. Beristain, C. D. MacCalman, and P. C. K. Leung
Cellular Localization of Gonadotropin-Releasing Hormone (GnRH) I and GnRH II in First-Trimester Human Placenta and Decidua
J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1459 - 1466.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chou, C.-S.
Right arrow Articles by Leung, P. C. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chou, C.-S.
Right arrow