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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 5 2335-2340
Copyright © 2003 by The Endocrine Society

Ghrelin Is Involved in the Decidualization of Human Endometrial Stromal Cells

Keisuke Tanaka, Hiroyuki Minoura, Tetsuya Isobe, Hitoshi Yonaha, Hiroaki Kawato, Dong Fang Wang, Toshimichi Yoshida, Masayasu Kojima, Kenji Kangawa and Nagayasu Toyoda

Departments of Obstetrics and Gynecology and Pathology (T.Y.), Mie University School of Medicine, Mie 514-8507, Japan; Department of Molecular Genetics, Institute of Life Science, Kurume University (M.K.), Kurume, Fukuoka 839-0861, Japan; and Department of Biochemistry, National Cardiovascular Center Research Institute (K.K.), Suita, Osaka 565-8565, Japan

Address all correspondence and requests for reprints to: Hiroyuki Minoura, M.D., Ph.D., Department of Obstetrics and Gynecology, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan. E-mail: hminoura{at}clin.medic.mie-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Successful implantation involves a complex interaction between the endometrium and the embryo. It is well known that several neuropeptides are expressed in the endometrium and placenta during embryonal implantation, suggesting an important role as chemical mediators of the feto-maternal relationship. Ghrelin has recently been identified as the endogenous ligand for the GH secretagogue receptor. Ghrelin is a peptide hormone with many physiological functions, and its expression in the human placenta has been reported. To investigate the involvement of ghrelin in embryonal implantation, we assessed the spatio-temporal expression pattern of ghrelin and its receptor in the human endometrium and placenta through the normal menstrual cycle and in early pregnancy. We also examined the effect of ghrelin on the decidualization of endometrial stromal cells (ESC). Weak expression of ghrelin mRNA was detected in the nonpregnant endometrium, and it was dramatically increased in the decidualized endometrium. A GH secretagogue receptor mRNA was detected in the endometrium throughout the normal menstrual cycle and in early pregnancy, but not in the first trimester placenta. Immunohistochemical analysis using an antighrelin antibody revealed strong signals in decidual cells and extravillous trophoblast cells. Coculture with first trimester placenta up-regulated ghrelin mRNA expression by primary cultured ESC, although sex steroids and 8-bromo-cAMP had no effect. In addition, ghrelin enhanced the decidualization of ESC induced by 8-bromo-cAMP (8-Br-cAMP) in vitro. Thus, ghrelin is a novel paracrine/autocrine factor that is involved in cross-talk between the endometrium and embryo during embryonal implantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENT DEVELOPMENTS IN assisted reproductive technology have allowed almost all patients to achieve the fertilization step of reproduction. The implantation rate (IR) of transferred embryos obtained by in vitro fertilization has improved dramatically to 15% due to the development of better embryo culture systems, such as blastocyst embryo transfer and coculture with somatic cells (1, 2). However, the IR has reached a plateau that is still far below the estimated IR (20–30%) of embryos fertilized in vivo, and the high miscarriage rate still remains a major problem. One of the keys to solving these problems would be the establishment of a method for the evaluation of endometrial embryonal receptivity. Greatly increased knowledge about preimplantation embryos and the molecular biology of the uterus have led to the development of new concepts with regard to the implantation process (2, 3, 4). However, the exact molecular events in the endometrium that are induced by embryonal implantation are still far from clear, because the factors participating in the feto-maternal relationship around the time of implantation have not been identified. Numerous molecular factors that may be involved in embryonal implantation have been described, including ovarian steroid hormones, cytokines, adhesion molecules, and neuropeptides (5, 6, 7, 8). Several neuropeptides, such as corticotropin-releasing factor and vasoactive intestinal peptide, are expressed in the endometrium and embryo during this period (9). It is becoming clear that such neuropeptides may have an important role as chemical mediators of cell to cell interactions during embryonal implantation (3, 9).

Ghrelin, a 28-amino acid peptide found in the hypothalamus and stomach, was recently identified as the endogenous ligand for the GH secretagogue receptor (GHS-R) (10). It has been shown to stimulate the release of GH from somatotrophs of the anterior pituitary through the GHS-R (10, 11, 12, 13). Recent studies have revealed that ghrelin has many physiological functions, including the regulation of gastric function, an antiproliferative effect on thyroid and breast tumors (14, 15, 16, 17), cardiovascular actions, and stimulation of insulin secretion (18, 19, 20, 21). It has also been reported that ghrelin is expressed in the human placenta (22, 23). However, there are few reports about the relationship between embryonal implantation and ghrelin.

The aim of this study was to investigate the involvement of ghrelin in embryonal implantation. First, we investigated the presence of ghrelin mRNA, its receptor mRNA, and ghrelin peptide in first trimester decidual and placental tissues as well as the spatio-temporal pattern of ghrelin expression in the endometrium throughout the normal menstrual cycle and during early pregnancy. Second, we investigated the regulation of ghrelin gene expression, especially the effect of coculture with chorionic tissue on ghrelin gene expression by endometrial stromal cells (ESC). Finally, to demonstrate that ghrelin may participate in embryonal implantation, we investigated its effect on the differentiation of ESC into decidual cells using primary cell culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

The endometrial tissues used in this study were obtained from 30 female patients with regular menstrual cycles who were taking no medication such as estradiol, progesterone, or GnRH analog and were undergoing total hysterectomy for leiomyoma or carcinoma in situ of the uterine cervix. The patients were 28–44 yr old. The menstrual stage of endometrial samples was defined by serum estradiol and progesterone levels and histological examination. The endometrial dating of the proliferative endometrial samples was d 7–10, and that of the secretory endometrial samples was from d 21–26. First trimester (from 6–8 wk gestation) decidua and placenta were obtained from 30 women undergoing therapeutic abortion by dilatation and curettage. The patients were 21–34 yr old. Written informed consent was obtained in each instance. The current study was approved by institutional review board in our center.

Extraction of total RNA

Total RNA was extracted from tissues and cultured cells by the acid guanidinium thiocyanate-phenol chloroform method (24). Total RNA concentrations were determined by spectrophotometry at 260 nm and were adjusted to 0.5 µg/µl.

RT-PCR and real-time PCR for ghrelin and PRL, and nested RT-PCR for GHS-R

First strand cDNA was synthesized from 5.0 µg total RNA. The resulting cDNA was subjected to PCR amplification with 20 µM each of the sense and antisense primers and 2.5 U AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Norwalk, NJ). The PCR primer pairs specific for ghrelin were 5'-AACACCAGAGAGTCCAGCA-3' (sense) and 5'-CAACATCAAAGGGGGCGTT-3' (antisense), and the specific primers for PRL were 5'-ACCCTTCGAGACCTGTTTGA-3' (sense) and 5'-GTGACCAGATGATACAGAGG-3' (antisense). RT-PCR signals were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin. The PCR primers specific for GAPDH were 5'-ACCCTTCGAGACCTGTTTGA-3' (sense) and 5'-GTGACCAGATGATACAGAGG-3' (antisense). The reaction volume was 40 µl, and the PCR involved 35 cycles of denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 1 min. The nested PCR was performed for detection of GHS-R. The primary PCR primer pair specific for GHS-R was 5'-TGGTCATCCTTGTCATCTGGG-3' (sense) and 5'-CGGTACTTC TTGGACATGAT-3 (antisense); the secondary PCR primer pair was 5'-CGGTGCTC TACAGTCTCATCG-3' (sense) and 5'-GGTAGAAGAGGACAAAGGA-3' (antisense). PCR involved 30 cycles of denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 1 min. Then the PCR products were electrophoresed on 2% agarose gel.

A sequencing system (ABI PRISM 7700, PE Applied Biosystems, Chiba, Japan) was used for quantitative real-time RT-PCR, and data were analyzed using the program Sequence Detector (version 1.6.3, PE Applied Biosystems). The primer pair and dye probes for human GAPDH were purchased from PE Applied Biosystems. The original sequences of the ghrelin mRNA (accession no. 9966512) and PRL (accession no. 4506104) are in GenBank. We designed the sequences of primer pairs and dye probes using by Primer Express program (version 1.0, PE Applied Biosystems) using these sequences. The sequences of the primer pairs and dye probes were as follows. The ghrelin sense primer was 5'-AGCTCTAGCAGGCTGGC TCC-3', its antisense primer was 5'-CCAGTTCATCCTCTGCCCC-3', and the dye probe was 5'-CCCGGAAGATGGAGGTCA AGCAGA-3'. The primer pair for ghrelin spans introns 1 and 2. For PRL, the sense primer was 5'-TGCCAGGTGACCCTTCGA-3', the antisense primer was 5'-GGTTATGGATGTAGTGGGACAGG-3', and the dye probe was 5'-CCTGTTTGAACCGCGCCGTCG-3'. The primer pair for PRL spans intron 1 and 2. The dye probes were labeled by 6-carboxyfluorescein at the 5' end and by 6-carboxy-tetramethyl-Rhodamin at 3' end. The internal control was GAPDH (PE Applied Biosystems). The reaction volume was 50 µl, and the PCR involved 40 cycles.

Immunohistochemical staining

Tissues were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections 3–4 µm thick were deparaffinized and processed by standard histological techniques on glass slides coated with Silan (DAKO Corp., Kyoto, Japan). Sections were incubated in 0.6% H2O2 in methanol for 30 min to block endogenous peroxidase activity, followed by equilibration in 10 mM PBS (pH 7.4) and incubation for 30 min in 10% normal goat serum (Life Technologies, Inc., Gaithersburg, MD). Antighrelin rabbit antiserum (10) was used as the primary antibody (at a dilution of 1:500 in PBS) for overnight incubation at 4 C. We used 1:500 diluted normal rabbit serum as a negative control. After washing the slides in PBS three times for 5 min each time, incubation was performed with goat antirabbit peroxidase-labeled antibody (MBL, Nagoya, Japan) for 30 min at room temperature. Color development was achieved with diaminobenzidine 4HCl/H2O2 solution, after which the sections were counterstained with hematoxylin. Negative control sections were processed with omission of the primary antibody.

Primary culture of ESC

Primary culture of ESC was performed to study the regulatory mechanism of decidualization in vitro (25, 26, 27). Luteal phase endometrial tissues obtained from patients undergoing total hysterectomy for leiomyoma or cervical carcinoma in situ were minced and digested with 2.5 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) and 20 µg/ml deoxyribonuclease I (Sigma-Aldrich) in culture medium for 60 min at 37 C. The culture medium was DMEM/Ham’s F-12 (1:1) medium (Sigma-Aldrich) containing 1% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich), and 2.5 µg/ml amphotericin B (Life Technologies, Inc.). The digested tissues were filtered through a 75-µm pore size nylon mesh and treated with erythrocyte-lysing buffer for 5 min at 37 C. After washing with culture medium, 1 x 106 cells were seeded in a 6-well cell culture plate (3 cm in diameter; Falcon, BD Biosciences, Franklin Lakes, NJ). After allowing stromal cells to attach to the plate for 30 min at 37 C, the medium was changed. The purities of the cells were checked by immunofluorescent staining of vimentin (DAKO Corp., Tokyo, Japan), desmine (DAKO Corp.), and cytokeratin (DAKO Corp.). ESCs prepared by this method were verified to contain less than 0.1% of nonstromal cells. After reaching confluence, the attached ESC were cultured in the presence or absence of various agents, with a medium change every 48 h. All experiments were performed using at least five different preparations of cultured cells.

Coculture of ESC with first trimester placenta

First trimester placental tissues (6 wk gestation) obtained from 20 patients undergoing artificial abortion were minced. Then 20 mg (wet weight) placental tissue were added on cell culture inserts (0.4 µm pore size; Falcon) for coculture with ESC for 7 d (28, 29, 30, 31). The villi were separated by culture insert from ESC.

In vitro decidualization

Sex steroids (Sigma-Aldrich; 200 pg/ml 17ß-estradiol and 100 ng/ml progesterone) and 0.5 mM 8-Br-cAMP (Sigma-Aldrich) were used to induce in vitro decidualization of ESC (31, 32). The medium was changed every 2 d during culture for 10 d. Decidualization was assessed using the expression of PRL mRNA as a differentiation marker. PRL mRNA was quantified by the real-time RT-PCR method described above. To examine the effect of ghrelin on the decidualization of ESC, we added 10-9, 10-8, 10-7, and 10-6 M human ghrelin peptide (10) to cultures. All experiments were performed at least five times.

Statistical analysis

Fisher’s protected least significant difference test was used to compare mean values, and differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ghrelin mRNA and GHS-R mRNA in early pregnancy decidual and placental tissue

First we examined the presence of ghrelin mRNA and GHS-R mRNA in early pregnancy decidual and placental tissues using RT-PCR. A ghrelin transcript corresponding to the predicted 141-bp size was present in the decidual and placental tissues (Fig. 1AGo). A GHS-R transcript corresponding to the predicted 271-bp size was present in decidual tissue, but not in placental tissue (Fig. 1BGo). Each amplification product was subcloned into the pGEM-Te vector, and the nucleotide sequences were determined by the dideoxy method (data not shown). These sequences showed 100% homology to the corresponding regions of ghrelin and GHS-R.



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  		Figure 1. A, Expression of ghrelin mRNA and GHS-R mRNA in decidua and placenta in the first trimester. The expected sizes of the amplification products were 141 and 250 bp for ghrelin and GHS-R, respectively. cDNAs from five patients were pooled. B, Expression of GHS-R mRNA in the endometrium during the normal menstrual cycle and early pregnancy. cDNA obtained from five patients were pooled. The menstrual cycle was confirmed by histological endometrial dating.

 
Changes in ghrelin mRNA expression in the endometrium through the normal menstrual cycle and early pregnancy

Ghrelin mRNA levels in the normal cycling endometrium and early pregnancy decidua were determined using quantitative real-time RT-PCR. As shown in Fig. 2Go, ghrelin expression increased from the proliferative phase (d 7–10 of menstrual cycle) to the secretory phase (d 21–26 of menstrual cycle, 5.6-fold compared with proliferative endometrium) and increased dramatically in decidualized endometrium (from 6–8 wk gestation, 58.1-fold compared with proliferative endometrium). On the other hand, in endometrium obtained from patients with ectopic pregnancy, ghrelin mRNA did not increase compared with secretory endometrium (5.8-fold compared with proliferative endometrium; Fig. 2Go).



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  		Figure 2. Quantification of ghrelin mRNA in human proliferative endometrium, secretory endometrium, first trimester deciduas, and ectopic pregnancy endometrium. The ghrelin mRNA level was quantified using real-time RT-PCR. Columns indicate the mean and SD of five samples obtained from individual patients. The average levels of ghrelin expression in proliferative endometrium were defined as 1. *, P < 0.0001.

 
Immunohistochemical localization of ghrelin in human endometrium, first trimester decidua, and placenta

On immunohistochemical analysis, ghrelin was not detected in proliferative endometrium (Fig. 3AGo). Its expression was seen in luminal and glandular epithelial cells from secretory endometrium, but not in stromal cells (Fig. 3BGo). In early pregnancy decidua, strong signals were detected in decidual cells, and weak signals were detected in luminal and glandular epithelium (Fig. 3CGo). In first trimester placenta, strong signals were detected in the extravillous trophoblast (EVT) at the tips of the chorionic villi (Fig. 3EGo). Weak signals were detected in the cytotrophoblast (Fig. 3EGo). By contrast, no signals were detected in the negative controls for the endometrium, decidua, and EVT (data not shown). In ectopic pregnancy endometrium, immunostaining was similar to that in secretory endometrium. Interestingly, no signals were detected in decidualized stromal cells from the patient with ectopic pregnancy (Fig. 3FGo). Also, no signals were determined in the negative control for decidualized stromal cells in the ectopic pregnancy (data not shown).



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  		Figure 3. Immunohistochemical localization of ghrelin in human endometrium, first trimester placenta, and decidua. A, Proliferative endometrium; B, secretory endometrium; C, first trimester decidua; D, first trimester decidua; E, first trimester placenta; F, ectopic pregnancy endometrium. Original magnitude: A–C and F, x100; D, x200; and E, x40.

 
Regulation of ghrelin mRNA expression in primary cultures of ESC

The in vitro differentiation of ESC into decidual cells, confirmed by morphological changes and PRL expression, was induced by sex steroids, 8-Br-cAMP, and coculture with first trimester placental tissue (Fig. 4Go). As shown in Fig. 4Go, the ghrelin mRNA level increased slightly after the addition of sex steroids and 8-Br-cAMP (1.9- and 1.7-fold compared with the control, respectively). Dramatic induction of ghrelin mRNA expression was seen after coculture with first trimester placental tissue (27.6-fold compared with the control).



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  		Figure 4. Induction of ghrelin mRNA expression in ESC by coculture with first trimester placenta. ESCs were cultured without any substances (control) or with sex steroids (200 pg/ml 17ß-estradiol and 100 ng/ml progesterone; EP) or 0.5 mM 8-Br-cAMP (cAMP). ESCs were cocultured with 20 mg (wet weight) first trimester placenta (co-cul). The ghrelin mRNA levels were quantified using real-time RT-PCR. Columns indicate the mean and SD of five RNA samples obtained from an individual well. These data show a typical pattern of five experiments using ESCs obtained from five individual patients. The ghrelin mRNA levels of each control sample were defined as 1. *, P < 0.0001.

 
Effect of ghrelin on in vitro decidualization of ESC

We examined the effect of ghrelin on the decidualization of ESC using PRL expression as a differentiation marker. GHS-R mRNA was detected in the cells by nested RT-PCR (data not shown). As shown in Fig. 5Go, ghrelin itself did not induce PRL mRNA expression (1.5-fold compared with the control). Ghrelin significantly enhanced the induction of PRL expression by 8-Br-cAMP in a dose-dependent manner (Fig. 5Go). However, this synergistic effect on the induction of PRL expression by sex steroids was not observed (data not shown).



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  		Figure 5. Effect of ghrelin on the in vitro decidualization of ESC. ESCs were cultured without any substances (control) or with 10-9, 10-8, 10-7, and 10-6 M ghrelin and/or 0.5 mM 8-Br-cAMP (cAMP). PRL mRNAs were quantified using real-time RT-PCR. Columns indicate the average and SD of five samples obtained from individual patients. The PRL mRNA levels of each control sample were defined as 1. *, P < 0.0001; **, P < 0.0005; ***, P < 0.01; ****, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated that ghrelin is expressed in first trimester decidual and placental tissues, whereas GHS-R mRNA is expressed in decidua, but not in placenta. Moreover, our data suggested that ghrelin enhanced the decidualization of ESC induced by 8-Br-cAMP in vitro.

Weak expression of ghrelin mRNA was detected in proliferative endometrium. Its expression was slightly increased in luteal endometrium, and a dramatic increase was observed in first trimester decidua (Fig. 2Go). On immunohistochemical analysis, ghrelin peptide was detected in the luminal and glandular epithelial cells of secretory endometrium, and strong positive signals were detected in decidual cells during early pregnancy. The human endometrium acquires receptivity for embryonal implantation under regulation of the hypothalamus-pituitary-ovarian axis. It is well known that specific products secreted by glandular and luminal epithelial cells of the endometrium at the time of embryonal attachment play a critical role in implantation (1, 2, 3). Recently, ghrelin has been reported to regulate many physiological functions in addition to GH secretion (14, 15, 16, 17, 18, 19, 20, 21). At this stage, the role of ghrelin secreted by glandular epithelial cells of the endometrium is far from clear. We detected GHS-R mRNA expression in the endometrium throughout the normal menstrual cycle and during early pregnancy (Fig. 1BGo). Taken together, these data suggest that ghrelin acts as a paracrine/autocrine factor in the secretory endometrium. It is possible that ghrelin is involved in the acquisition of endometrial embryo receptivity and that it influences the development of periimplantation embryos.

On immunohistochemical analysis, the strongest signals were seen in EVT cells from first trimester placenta. It is well known that EVT cells form the frontier of invasion into the maternal endometrium (33, 34). During placental development, human trophoblasts differentiate into two main lineages: villous and extravillous cells. EVT cells invade the maternal uterus and also migrate up the spiral arteries, transforming them into large, low resistance vessels. Gualillo et al. (23) reported that ghrelin is expressed in first trimester placenta, being found mainly in cytotrophoblasts, whereas it is negative in third trimester placenta. They suggested that ghrelin acts during embryonal implantation and placental development, but they did not mention the expression of ghrelin in EVT cells. Invading chorionic tissues send several messengers to the maternal endometrium, such as cytokines and prostaglandins. Our data suggested that ghrelin is another chemical mediator involved in cross-talk between the invading placenta and the endometrium.

The ghrelin mRNA level in early pregnancy deciduas was dramatically increased compared with that in normal cycling endometrium. Immunohistochemical analysis revealed strong signals in decidualized ESC. Interestingly, in the endometrium from patients with ectopic pregnancy, the ghrelin mRNA level did not increase, and immunoreactive ghrelin was not detected in decidual cells. These data suggested that ghrelin may be an embryo-dependent gene in ESC, so we investigated the embryo dependency of ghrelin gene expression in ESC using coculture with first trimester placenta. Decidualization can be induced by both sex steroids and 8-Br-cAMP in vitro. As shown in Fig. 4Go, ghrelin gene expression was slightly induced by sex steroids and 8-Br-cAMP, but was dramatically induced by coculture with first trimester placenta. To rule out contamination of ESC by trophoblasts, we checked for the presence of human chorionic gonadotropin mRNA; it was not detected in total RNA samples from ESC (data not shown). Identification of genes that are induced in ESC by stimulation with chorionic tissue, such as ghrelin, may open the door to the discovery of a novel cross-talk mechanism between the endometrium and the embryo.

In the present study GHS-R mRNA was detected in normal cycling endometrium and in decidual tissue by nested RT-PCR, but not in first trimester placenta. Thus, the target cells for ghrelin produced by first trimester placenta and decidua are thought to be ESC or endometrial epithelial cells. To clarify the role of ghrelin in embryonal implantation, we investigated its effect on the decidualization of ESC. It is known that ESC differentiate into active endocrine cells, decidual cells, during embryonal implantation. Decidual cells are regulated by many factors, such as sex steroids, cytokines, ILs, and prostaglandins (31, 32, 35). Interestingly, we found that ghrelin enhanced the decidualization of ESC induced by 8-Br-cAMP in vitro, so ghrelin may be important in the regulation of ESC differentiation, and it may be involved in the dramatic alterations of the endometrium induced by embryonal implantation. Future studies may help to demonstrate the intracellular molecular mechanisms for the regulation of ESC differentiation by ghrelin.

In conclusion, ghrelin is a novel paracrine/autocrine factor that is involved in cross-talk between endometrium and embryo during implantation. We believe that a study addressing the role of ghrelin during the periimplantation period may help to clarify the molecular mechanism of embryonal implantation.


   Acknowledgments
 


   Footnotes
 
Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; ESC, endometrial stromal cells; EVT, extravillous trophoblast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHS-R, GH secretagogue receptor; IR, implantation rate.

Received July 11, 2002.

Accepted February 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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