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Biology of Reproduction 60, 1468-1474 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Functional Role of Arg-Gly-Asp (RGD)-Binding Sites on ß1 Integrin in Embryo Implantation Using Mouse Blastocysts and Human Decidua1

Shigetatsu Shiokawa2,a, Yasunori Yoshimurad, Hiroki Sawab, Shinya Nagamatsuc, Hideki Hanashia, Ken Sakaia, Motomu Andoa, and Yukio Nakamuraa

a Department of Obstetrics and Gynecology, b Department of Neurosurgery, and c Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, 181-8611, Japan d Department of Obstetrics and Gynecology, Keio University School of Medicine, Shinjukuku, Tokyo, 160-8582, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amino acid residues 140–164 of integrin ß1 comprise an Arg-Gly-Asp (RGD) cross-linking region. The present study was undertaken to study the role of the RGD cross-linking region of integrin ß1 subunit in embryo implantation. Decidual cells attached to fibronectin (FN)-coated dishes. A peptide corresponding to integrin ß1[140–164] (DDL; DYPIDLYYLMDLSYSMKDDLENVKS) inhibited decidual cell attachment to FN-coated dishes in a dose-dependent manner. A variant integrin peptide in which Asp 157 and Asp 158 were replaced by Ala (AAL; DYPIDLYYLMDLSYSMKAALENVKS) did not affect decidual cell attachment to FN. Inhibition by DDL peptide was reversed by prior treatment with an RGD-containing peptide but not by prior treatment with an RGE-containing peptide. Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h of culture. Blastocysts that attached to decidual cells exhibited extensive outgrowth after 48 h. Treatment of decidual cells with synthetic peptides did not affect the rates of hatching and attachment of blastocysts. The outgrowth of embryos on decidual cells was inhibited by DDL peptide in a dose-dependent manner, but not by AAL peptide. These findings suggest that integrin ß1[140–164] on decidual cells may be important in embryonic development and differentiation following attachment.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß1 integrins are cell adhesion receptors belonging to the integrin supergene family that recognize multiple ligands, including fibronectin (FN), laminin, collagen, epiligrin, invasin, and vascular cell adhesion molecule-1 [1–6]. ß1 integrins mediate cell-cell and cell-extracellular matrix interactions. The ß subunits of integrins contribute to both ligand binding [7] and transduction of intracellular signals [8–11]. The major integrin binding site is an Arg-Gly-Asp (RGD) tripeptide, present in a variety of integrin ligands. In FN it is located in the tenth type III repeat (III10) [12, 13], the structure of which has been elucidated by nuclear magnetic resonance [14] and crystallography [15]. Studies with FN fragments and phage display libraries have suggested that other sites in this region of FN also are needed for full integrin binding activity [16–18]. Contact regions for the RGD sequence have been identified in the integrin subunits [19]. Affinity cross-linking of RGD peptides and site-directed mutagenesis demonstrate that a ligand-binding site in the {alpha}IIbß3 integrin is located between amino acid residues 109 and 172 of the ß subunit [20–24]. Studies with the {alpha}vß3 integrin, an RGD-binding site at amino acids 61–203 [25], and a similar cross-linking region involving amino acids 120–140 in the ß1 subunit has also been found to be involved in ligand binding [26]. In each case, the RGD-binding site is near or at a site that binds divalent cations. The {alpha} subunit also contains one or more ligand binding sites; as with the ß subunits, these sites localize to the divalent cation binding sequences [27–31].

Recent studies have demonstrated that the expression of ß1 integrins in human endometrium increases at the time of implantation [32–35]. Certain ß1 integrin moieties appear to be regulated throughout the endometrial cycle. We have reported that outgrowth of embryos on decidual cells, but not their attachment, is inhibited by antibodies recognizing the components of the ß1 integrin family, suggesting that ß1 integrins on decidual cells may be important in development and differentiation after attachment [36, 37]. However, the location of the epitopes of monoclonal anti-human ß1 integrin antibodies used in these studies remains unclear. The integrin ß1 subunit plays a central role in regulating integrin binding. The primary sequence of amino acids 140–202 of ß1 is highly conserved in all ß subunits of integrins. Of this segment, the amino-terminal domain is particularly conserved. The sequence ß1[140–164] is referred to as the RGD cross-linking region [38]. The identification of the ligand binding domain of ß1 integrins is important for understanding their function. In the present study, we used synthetic peptides to study the role of the RGD cross-linking region on integrins in embryo implantation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic Peptides

The soluble peptides used in this study were synthesized chemically and HPLC-purified by Sawaday Technology (Tokyo, Japan). A synthetic peptide used in this study was derived from the long arm of the ß1 chain, residue numbers 140–164 (DDL, DYPIDLYYLMDLSYSMKDDLENVKS). A variant peptide (AAL, DYPIDLYYLMDLSYSMKAALENVKS), in which both Asp 157 and Asp 158 were replaced by Ala, was also used. GRGDSP (RGD peptide) and GRGESP (RGE peptide) also were purchased from Sawaday Technology.

Tissue Preparation

Specimens of decidua were obtained from 27 women undergoing therapeutic abortion between 7 and 11 wk of gestation. The median age of these women was 29 yr (range: 22–35 yr). Gestational ages were estimated from information on the date of the last menstruation, uterine size, and measurement of crown-rump length. All women gave informed consent for collection of the tissues, and the study was approved by the Ethics Committee of Kyorin University School of Medicine, Tokyo, Japan. The specimens were placed immediately in ice-cold medium 199 (Gibco, Grand Island, NY) containing 25 mM HEPES (Sigma Chemical Co., St. Louis, MO) and 1% antibiotic-antimycotic mixture (Gibco) and were transported to the laboratory within 1 h of the procedure. After blood clots had been removed, the decidual tissue was rinsed thoroughly in ice-cold medium 199. The tissue was trimmed and cut into approximately 1-mm3 pieces using a small pair of scissors. A portion of the tissue was stained with hematoxylin-eosin for histologic examination. The remaining tissue was treated enzymatically to disperse the cells.

Decidual Cell Culture

Isolation of decidual cells was performed by the methods described by Satyaswaroop et al. [39] and Braverman et al. [40], with minor modifications. The tissue was treated with 0.1% collagenase (type IA; Sigma) and 0.1% hyaluronidase (type IS; Sigma) in Ca2+-free PBS while being stirred at 37°C for 1 h. At the end of this period, the cell suspension was filtered through nylon mesh (pore size, 105 µm) to remove undigested tissue debris. The cells were collected from the filtrate by centrifugation at 800 x g for 10 min, and the pellet was resuspended in medium 199 containing 10% fetal calf serum (FCS; Gibco). The cell suspension was filtered through a 38-µm stainless steel sieve (Spectrum, Los Angeles, CA) to retain the glandular elements as previously described [39, 41]. The stromal cells then were collected by centrifugation, washed, and resuspended in 20% isotonic Percoll solution and layered on top of 20–60% and 40–55% Percoll gradients [40]. The tubes were centrifuged for 15 min at 30 000 x g in a Beckman L3–50 ultracentrifuge set at 4°C, using a type 65 fixed-angle rotor (Beckman, Palo Alto, CA). An enriched fraction of prolactin-producing decidual cells layered as a single band with a cell density of 1.033–1.048 g/ml. The band contained a near-homogeneous population of large round mononucleated cells (> 25 µm diameter) [40]. The decidual cells were washed and suspended three times in medium 199 supplemented with 10% FCS and 1% antibiotic-antimycotic mixture. Aliquots of decidual cell suspensions were counted by the dye exclusion test using 0.4% (v:v) trypan blue dye in PBS. The stromal cells were plated at 5 x 105 cells/ml in a 35 x 10-mm plastic Petri dish (Falcon #3001; Beckton-Dickinson, Lincoln Park, NJ). The culture medium was changed every 48 h, and the cultures were maintained in humidified 95% air:5% CO2 at 37°C for 10 days.

Decidual Cell Attachment Assays

Chamber dishes (16-well) (Nalge Nunc International, Naperville, IL) were incubated with 25 µg/ml of FN (Iwaki Glass, Chiba, Japan) for 1 h at 37°C. Dishes were washed three times with PBS, and nonspecific adhesion to the culture dish was blocked with 0.2% BSA in PBS for 1 h. The dishes were washed three times with PBS before decidual cells were plated. Synthetic peptides also were co-coated with FN at concentrations of 0.1–10 µM. Decidual cells were plated on substrate-coated dishes at a density of 1 x 106/ml in medium 199 containing 10% FCS. In competitive inhibition assays, RGD or RGE peptide at 10 µM also was co-coated onto the FN-DDL coated dishes. After a 1-h incubation at 37°C, the chambers were washed to remove unattached cells. The attached cells were trypsinized and counted. All of the experiments presented here were run in quadruplicate and repeated at least four times.

Assays for Embryo Attachment and Spreading onHuman Decidua

Embryo attachment and spreading assays were performed using cultured human decidua. Female ICR mice (8 wk old; Clea Japan, Tokyo, Japan) were superovulated by an injection of 5 IU of eCG (Teikoku-zoki, Tokyo, Japan) followed after 48 h by an injection of 2.5 IU of hCG (Teikoku-zoki Co.) and then were caged with ICR males. Embryos were flushed from the uterine horns at the late morula stage or early blastocyst stage (96 h after hCG), collected, and rinsed in medium 199 supplemented with 0.4% BSA.

Decidual cells had been cultured in medium 199 supplemented with 10% FCS for 10 days. In experimental cultures, subconfluent decidual cell monolayers were incubated with either AAL or DDL peptide at a concentration of 0.1–10 µM for 12 h before addition of the embryos. As a control study, subconfluent decidual cell monolayers were used that had not been treated with either synthetic peptide. Five to eight embryos were then placed in prepared dishes with a subconfluent monolayer culture of decidual cells and cocultured for 96 h. Embryo attachment was identified by gently flushing a small amount of medium on each embryo using a glass pipette pulled to a very fine bore. Embryos that showed no movement while being observed under an Olympus inverted phase-contrast microscope (Olympus Optical Co. Ltd., Tokyo, Japan) were considered to be attached. Embryos were classified as spreading if migration of individual cells or monolayers of trophoblasts from the ectoplacental cone rudiment were observed. The extent of spreading was determined by photographing the embryos at a magnification of x200 and printing each negative at the same size. The area of outgrowth was measured using a color image analyzing system (SP500; Olympus Optical) as described by Imamura et al. [42]. The same observer (S.S.) produced each tracing. The final value for each embryo was calculated from the average of three tracings. The result of each treatment represents the average of the measurements of at least 45 embryos. Measurements of embryo attachment and spreading were made at 24 and 48 h of incubation, respectively. The area of embryo outgrowth was determined between 48 and 96 h of incubation.

Statistical Analysis

The percentages of embryo hatching, attachment, and spreading, and the area of embryo spreading are expressed as the mean ± SEM. To obtain a normal distribution, the percentages of embryo attachment and spreading were transformed using an arcsine transformation. Statistical analysis was performed by ANOVA with Scheffé's test. Differences were considered statistically significant if p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human decidual cells immediately attached to FN-coated dishes but did not attach to FN-uncoated dishes within 1 h (Fig. 1). When decidual cells were plated on a substrate consisting of a mixture of FN and DDL peptide, the percentage of attached cells was significantly inhibited compared with that in FN-coated dishes (Figs. 1 and 2). To establish whether the DDL sequence in the ß1[140–164] peptide is important for RGD binding, we synthesized a variant peptide (AAL) in which both ASP 157 and ASP 158 were replaced by Ala [DYPIDLYYLMDLSYSMKAALENVKS]. In dishes coated with a mixed substrate of FN and AAL peptide, AAL peptide did not affect decidual cell attachment to FN-coated dishes (Fig. 2). These data indicate that DDL peptide is a potent inhibitor of integrin ß1-mediated decidual attachment to FN. To determine whether the FN binding activity of decidual cells was mediated by the RGD recognition site, competitive inhibition assays were performed using the synthetic peptides, GRGDSP and GRGESP. When decidual cells were plated on a mixed substrate of FN, 10 µM DDL peptide, and 10 µM RGD peptide, the decrease in number of attached cells was significantly reversed (Figs. 3 and 4). However, RGE peptide did not affect the decrease in number of attached cells in FN and DDL-coated dishes (Figs. 3 and 4).



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  		FIG. 1. Effect of synthetic peptides on decidual cell attachment to FN-coated dishes. Culture dishes were coated with 25 µg/ml of FN alone (A) or with the various peptides—FN with 0.1 µM of DDL peptide (B), FN with 1 µM of DDL peptide (C), FN with 10 µM of DDL peptide (D), FN with 0.1 µM of AAL peptide (F), FN with 1 µM of AAL peptide (G), and FN with 10 µM of AAL peptide (H)—or with no FN coating (E). Bar = 100 µm.



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  		FIG. 2. Percentage of attached decidual cells on FN-coated dishes in the presence or absence of synthetic peptides. Bar graph) FN-uncoated dishes (control), FN-coated dishes without synthetic peptides. Line graph) DDL+FN-coated dishes (open circles); AAL+FN-coated dishes (solid circles). *, p < 0.001 vs. FN (+).



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  		FIG. 3. Effect of synthetic peptides on decidual cell attachment on FN-coated dishes in the presence of DDL. Culture dishes were coated with 25 µg/ml FN either alone (A) or with 10 µM DDL peptide (B), with 10 µM of DDL peptide and 10 µM of RGD peptide (C), or with 10 µM of DDL peptide and 10 µM of RGE peptide (D). Bar = 100 µm.



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  		FIG. 4. Percentage of attached decidual cells on FN-coated dishes in the presence or absence of synthetic peptides; the letter a above pairs of bars indicates a significant difference (p < 0.001).

Hatched mouse blastocysts cultured in vitro attach and form trophoblast outgrowth on human decidual cells, providing a model for implantation [36, 37]. The effects of DDL peptide on embryo attachment and the subsequent spreading of trophoblasts were investigated in vitro on decidual cells incubated with synthetic peptides. When decidual cell monolayers were treated with synthetic peptides in concentrations above 1 mM, minute flaws were observed in the continuity of the cellular pattern, resulting in a "stocking run" appearance. These tiny discontinuities were apparently the result of dissociation of adjacent cells and not detachment. Increasing concentrations of synthetic peptides induced progressively larger discontinuities in the decidual cell monolayers, ending with substantial holes and resulting in monolayer detachment from the culture dish by 72 h. However, synthetic peptides at concentrations less than 100 µM had no effect on monolayer integrity.

The addition of DDL or AAL peptide to the cultured decidual cells did not affect the hatching ratio. Attachment of hatched blastocysts was slightly, but not significantly, reduced in cultures treated with synthetic peptides when compared with control cultures. The spreading of trophoblasts from attached blastocysts was observed 48–96 h after coculture with decidual cells. The area of trophoblast outgrowth increased with the duration of coculture with decidual cells. Both the incidence and area of trophoblastic outgrowth from attached blastocysts were inhibited significantly by the addition of DDL peptide at concentrations of 0.1–10 µM (Figs. 5 and 6). Exposure of the decidual cells to DDL peptide inhibited the area of outgrowth of trophoblasts in a dose-dependent manner 96 h after coculture (Figs. 5 and 6). However, the addition of AAL peptide did not affect the area of trophoblastic outgrowth at concentrations of 0.1–10 µM (Figs. 5 and 6).



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  		FIG. 5. Spreading of mouse blastocysts on human decidual cells in the presence or absence of synthetic peptides. Decidual cells were cultured with synthetic peptides at a concentration of 0.1 to 10 µM. Embryos were cocultured with decidual cells treated with DDL peptide at a concentration of 0.1 (A), 1 (B), or 10 (C) µM, or with AAL peptide at a concentration of 0.1 (D), 1 (E), or 10 (F) µM, or without synthetic peptides (G) for 96 h. Bar = 100 µm.



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  		FIG. 6. Quantitative evaluation of the area of outgrowth (µm2) of embryos cultured without (open circles) or with synthetic peptide at a concentration of 0.1 µM (solid circles), 1 µM (solid triangles), or 10 µM (solid squares). Decidual cells were treated with AAL peptide (A) or DDL peptide (B). The area of outgrowth was determined 48–96 h after coculture with decidual cells. *p < 0.05, **p < 0.01, ***p < 0.001.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many integrins recognize short RGD-containing amino acid sequences. The DDL sequence is by far the most frequently isolated sequence on RGD-containing FN fragments. The DDL sequence is located in the same region of the ß subunit that was identified in earlier studies as being involved in RGD binding [20–23]. A synthetic peptide containing the DDL sequence of the ß3 subunit (amino acids 109–133) binds to an RGD peptide [43]. We hypothesized that the corresponding region in the ß1 subunit (amino acids 140–164) also is involved in ligand-integrin interactions, and we used DDL peptides to identify the function of integrin in decidual cells. A previous study demonstrated that a mutation of Asp 119 to tyrosine, while rendering the whole integrin inactive, failed to affect RGD binding by the binding region peptide [43]. Therefore, we used a variant peptide in which both Asp 157 and Asp 158 were replaced by Ala as a control peptide. Decidual cells did not attach to the FN-uncoated dishes but did attach to FN-coated dishes. This phenomenon was inhibited by DDL peptide in a dose-dependent manner but not by AAL peptide. This suggests that decidual cells attach to FN via the DDL motif in surface integrins. The inhibition by DDL peptide was blocked by RGD peptide but not by RGE peptide, implying that DDL peptide binds to RGD peptide. Thus, the DDL sequence in the integrin ß1 subunit on decidual cells is critical for decidual cell recognition of the RGD sequence in FN.

Although DDL peptide interfered with decidual cell attachment to FN, it did not affect embryo attachment to decidual cells. We previously have found that decidual cells express ß1 integrin on their surface and that attachment of embryos to the decidual cells is not inhibited by the addition of a monoclonal antibody against the ß1 integrin subunit [36, 37]. These findings suggest that decidual cells may interact with FN through DDL-RGD linkage but that blastocyst attachment to decidual cells is regulated by another mechanism. The {alpha}4ß1 integrin is a very unusual integrin found on epithelial cells. The ß4 integrin is absent from proliferative endometrium but is conspicuous in early and mid-luteal epithelium [44]. The {alpha}4ß1 integrin is detected in the endometrial epithelial cells of fertile women but is absent from the endometrium of women with unexplained infertility [45]. The {alpha}4ß1 integrin recognizes and binds to the III CS region found in alternatively spliced isoforms of FN [46]. These forms are present in the FN made by trophoblasts, with oncofetal FN being implicated as a "molecular glue" used for embryo attachment [47]. In the present study, we used the DDL peptide, which interacts with the RGD sequence. Since {alpha}4ß1 integrin interacts with non-RGD cell recognition sites, DDL peptide did not affect blastocyst attachment to decidual cells.

It has been reported recently that mouse embryos produce a broad repertoire of extracellular matrix (ECM) constituents and ECM receptors from the onset of development [33]. Primary mouse trophoblastic cells appear to interact with FN exclusively through the RGD recognition site, indicating that the FN receptor is expressed on the surface of trophoblastic cells. Mouse blastocyst outgrowth on ECM proteins is mediated by trophoblast expression of several integrin receptors, possibly in concert with one another [48]. The activity of RGD-binding integrins is critical in mediating the adhesion and migratory function of primary mouse trophoblastic cells. Normally, the first maternal cell to which the embryo attaches is an endometrial epithelial cell surface and the decidual cells come into play during trophoblast invasion. Our previous studies have demonstrated that decidual cells express ß1 integrin on the cell surface [35, 36]. Outgrowth, but not attachment, of embryos on decidual cells is inhibited in a dose-dependent manner by adding an antibody that recognizes the ß1 chain, implying that ß1 integrins are important in blastocyst development and differentiation following attachment [36, 37]. In this study, therefore, we use the decidual cells as a model for studying embryo attachment and implantation. Mouse trophoblast invasion is different from human trophoblast invasion. Since human embryos necessary to these experiments are not available ethically, we were obliged to use mouse embryos in the present study. In the present study the outgrowth of embryos on decidual cells was inhibited by addition of DDL peptide in a dose-dependent manner. These findings suggest that trophoblastic and decidual cells may interact with each other via ß1 integrin on their surfaces by DDL-RGD linkage. This linkage is involved in trophoblastic spreading on decidual cells. Takada et al. [26] have reported that CHO cells expressing integrin ß1 spread well on FN, but CHO cells expressing mutated integrin ß1 (Asp130 to Ala substitution) do not. These data in conjunction with ours indicate that highly homologous regions within ß1 integrins affect cell spreading.

An adhesive interaction of the cell with its substratum is an integral component of cell migration [49]. Contacts typically are mediated by transmembrane adhesion receptors that link the internal cytoskeleton and its associated motors to extracellular surfaces [50]. As a result of integrin-ECM binding, adhesion receptor clustering or cytoskeletal-dependent processes such as cell spreading occur. Recently, it has become clear that cell contact with even one specific ECM component, such as FN, initiates multiple signals affecting cell behavior [51]. There is abundant evidence that adhesion molecules participate in a large variety of signal transduction events important for regulating cell migration [52]. These signals are dependent on integrin-mediated adhesion. In the present study, DDL peptide blocked the RGD-DDL-mediated interaction between trophoblasts and decidual cells. DDL peptide blocked integrin-mediated adhesion, resulting in blockade of the signaling process. These findings suggest that RGD-DDL linkage between trophoblasts and decidual cells may play an important role in the outgrowth of embryos on decidual cells. It is therefore possible that trophoblastic cells utilize the DDL sequence predominantly for anchorage to ECM proteins during cell migration.

A point mutation in a highly conserved region of the ß1 subunit, resulting in an Asp130 to Ala substitution, abrogates the RGD-dependent binding of {alpha}5ß1 to FN without disrupting gross structure or heterodimer assembly [26]. The ß3 subunit in Glantzmann's thrombasthenia patients has a point mutation at position 119 (Asp to Tyr) that inactivates {alpha}IIbß3 binding to fibrinogen, underscoring the importance of the region in the vicinity of D119 in ligand-ß3 integrin interactions [50]. These data suggest that point mutations involving the integrin binding site are the cause of many diseases. There is ample evidence that analysis of integrins may provide a novel approach to the assessment of uterine receptivity in a variety of infertility states. ß1 integrins supply most of the mechanical attachment to ECM. This study suggests that a ternary peptide in a highly conserved region of the ß1 subunit plays an important role in the process of implantation. Point mutations at the ß1 integrin RGD-binding site may be a cause of unreceptive endometrium. A better understanding of binding sites on integrins in the endometrium may lead to a more directed therapeutic approach to patients with implantation failure and remove some of the uncertainty that surrounds this diagnosis.

In summary, the present study demonstrates that the DDL sequence is critical in FN-ß1 integrin interactions on decidual cells. Integrin ß1[140–164] on decidual cells may be important in development and differentiation following attachment. Finally, RGD-DDL linkage between trophoblastic and decidual cells plays an important role in embryo implantation.


   FOOTNOTES
 
1 This work was supported in part by Grants-in-Aid (C) 08671925 (to S.S.), and (B)(2) 09470365 (to Y.Y.) from the Ministry of Education, Science and Culture (Tokyo, Japan). Back

2 Correspondence: Shigetatsu Shiokawa, Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan. FAX: 422 47 3177; kyoringy{at}tk.xaxon.ne.jp Back

Accepted: January 21, 1999.

Received: October 5, 1998.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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