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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022764
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BIOLOGY OF REPRODUCTION 70, 729–736 (2004)
DOI: 10.1095/biolreprod.103.022764
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Differential Expression of Ezrin/Radixin/Moesin (ERM) and ERM-Associated Adhesion Molecules in the Blastocyst and Uterus Suggests Their Functions During Implantation1

Hiromichi Matsumoto3, Takiko Daikoku4, Haibin Wang4, Eimei Sato3, and S.K. Dey2,4,5,6

Laboratory of Animal Reproduction,3 Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan Departments of Pediatrics,4 Cell and Developmental Biology,5 Pharmacology,6 Vanderbilt University Medical Center, Nashville, Tennessee 37232


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.

early development, embryo, implantation, trophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The process of implantation involves a cascade of cell-cell communications between the two epithelial tissues: the trophectoderm and luminal epithelium. Prior to implantation, these tissue layers are two separate entities and are polarized with a continuous seal of junctional complexes and cell adhesion molecules. With the onset of the attachment reaction, the trophectoderm and the luminal epithelium first make contact at their apical borders followed by epithelial cell apoptosis and trophoblast cell invasion into the endometrium at the site of the attachment [1, 2]. For this interaction, both the receptive luminal epithelium and the trophectoderm of the implantation-competent blastocyst must alter their functional programming via changes in cell surface molecules.

CD44, CD43, ICAM-1, and ICAM-2 are all integral membrane proteins that cross-link with actin filaments by ezrin/radixin/moesin (ERM) proteins in the organization of cortical actin-based cytoskeletons including microvilli formation. Although overexpression of these ERM-binding membrane proteins (ERMBMPs) in cultured epithelial cells does not elongate microvilli, the addition of epidermal growth factor (EGF) induces a remarkable microvilli elongation in these cells [3]. These results suggest that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with ERM proteins.

The EGF family of growth factors is comprised of EGF itself, transforming growth factor-{alpha}, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, epiregulin, and neuregulins [4–7]. We previously demonstrated that HB-EGF is the earliest known molecular marker of implantation in mice because it is expressed exclusively in the uterine luminal epithelium surrounding the blastocyst several hours before the attachment reaction [8]. Evidence suggests that an interaction between uterine HB-EGF and blastocyst ErbBs is important for the attachment reaction [9–11]. Activation of EGF-related molecules requires proteolytic cleavage of the propeptide and release of the corresponding mature protein from the cell surface for binding to ErbB receptors [12]. Recent evidence suggests that matrix metalloproteinases (MMPs) are involved in proteolytic processing of pro-HB-EGF to mature HB-EGF [13–15]. CD44 is a cell surface proteoglycan that is implicated in cell adhesion and trafficking. It is also known to be involved in cell migration and invasion contributing to tumor survival and progression. CD44 binds to hyaluronan, and specific CD44 isoforms display characteristics of heparan sulfate proteoglycan (CD44HSPG). CD44HSPG can recruit proteolytically active MMP-7/matrilysin and pro-HB-EGF to form a complex on the surface of tumor cells, postpartum uterine and lactating mammary gland epithelia, and uterine smooth muscle cells [16]. The pro-HB-EGF within this complex is processed by MMP-7, and the resulting mature HB-EGF activates ErbB4 leading to cell survival. In the present study, we examined the distribution patterns of ERM-associated adhesive molecules (CD43, CD44, ICAM-1, and ICAM-2) and the ERM family members of cross-linker proteins (ezrin, radixin, and moesin) in dormant and implantation-competent blastocysts as well as in the uterus during the peri-implantation period in order to better understand the cell-cell communication between the blastocyst and uterus at the time of implantation. As observed in the present investigation, differential distribution of ERM proteins and adhesion molecules in dormant and implantation-competent blastocysts as well as in the receptive uterus prior to and during implantation suggests that selective interactions of ERM proteins with ERM-binding membrane proteins are involved in rendering dormant blastocysts competent for implantation and making the uterus ready for the attachment reaction and regulated invasion by the trophectoderm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult CD-1 mice were purchased from the Charles Rivers Laboratory (Raleigh, NC). All mice were housed in the Institutional Animal Care Facilities according to NIH and institutional guidelines for laboratory animals. Females were mated with fertile males of the same strain to induce pregnancy (Day 1 = vaginal plug). Normal blastocysts were collected on the morning (1000 h) of Day 4 of pregnancy. To induce conditions of delayed implantation, mice were ovariectomized on the morning (0800–0900 h) of Day 4 of pregnancy that was maintained with daily injections of progesterone (P4, 2 mg/mouse) from Days 5–7. To terminate delayed implantation and to initiate implantation, P4-primed delayed-implanting pregnant mice were injected with estradiol-17ß (E2, 25 ng/mouse) [17]. Dormant blastocysts were collected 12 h after the last P4 injection, while activated blastocysts were collected 12 h after the last P4 and E2 injections. All steroids were dissolved in sesame oil and injected subcutaneously (0.1 ml/mouse).

Immunodetection of Ezrin, Radixin, Moesin, CD44, CD43, ICAM-1, and ICAM-2 in Blastocysts

Immunolocalization in blastocysts was performed as previously described [18]. In brief, blastocysts were fixed in 3.7% formaldehyde in PBS at room temperature for 30 min, permeabilized in 2.5% Tween 20 in PBS for 5 min, and then incubated overnight at 4°C with goat polyclonal antibodies to radixin and CD43 at a dilution of 1:100 in PBS; rabbit polyclonal antibodies to ezrin, moesin, and ICAM-2 at a dilution of 1:200; or rat monoclonal antibodies to CD44 and ICAM-1 at a dilution of 1:50. Antibodies to radixin, CD43, and ICAM-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), ezrin and moesin from Upstate (Waltham, NY), and CD44 and ICAM from R & D Systems (Minneapolis, MN). After several washes with PBS containing 0.5% Triton X-100 and 0.5% bovine serum albumin (BSA), blastocysts were incubated with TRITC-labeled rabbit anti-goat antibody, TRITC-labeled goat anti-rabbit antibody, or TRITC-labeled donkey anti-rat antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 1 h at room temperature. Nuclei were labeled with Hoechst 33342 (1 µg/ml; Molecular Probes, Eugene, OR) for 30 min at room temperature. After several washes with PBS containing 0.1% BSA, blastocysts were mounted. Antigens labeled with TRITC were depicted in red, and nuclei stained with Hoechst showed blue staining. Incubation of blastocysts in nonimmune sera instead of primary antibodies served as negative controls (data not shown). They were then viewed in a Zeiss LSM 510 confocal scanning laser microscope (Axioplan 2 Imaging; Carl Zeiss Inc., Oberkochen, Germany) using excitation wavelengths of 543 and 364 nm for detecting red and blue fluorescences, respectively. Images shown in the Results section are representative of at least 15 blastocysts from three to four different animals.

Immunohistochemical Localization of Ezrin, Radixin, Moesin, CD44, CD43, ICAM-1, and ICAM-2 in the Uterus

Frozen sections (10 µm thick) were mounted onto poly-L-lysine-coated slides and stored at -80°C until used. Sections were fixed in cold acetone on ice for 10 min followed by washing in PBS (pH 7.4) for 10 min twice. For blocking nonspecific fluorescence, sections were incubated with nonimmune sera derived from rabbits, goats, or donkeys for the detection of radixin or CD43; ezrin, moesin, or ICAM-2; and CD44 or ICAM-1, respectively. Immunolocalization of ezrin, radixin, moesin, CD44, CD43, ICAM-1, and ICAM-2 in the uterine sections followed similar procedures as described previously for blastocysts.

In Situ Hybridization

Sense or antisense 35S-labeled cRNA probes were generated using appropriate polymerase with mouse-specific cDNAs to ezrin, radixin, and moesin. These probes were generated by PCR cloning using specific primers as follows. radixin: 5'-GCA GCT AGA AAG GGC ACA AT-3' (sense) and 5'-GCT TTT CTC TGG TGG TGG TT-3' (antisense); ezrin: 5'-CTC CTC CTT GGT TCC TTC TA-3' (sense) and 5'-CTC GGT TAC ATG GTT CTT GG-3' (antisense); and moesin: 5'-AGC GTG CTC TCC TGG AAA AT-3' (sense) and 5'-TGT GGG GAA CAA GGA AGA AG-3' (antisense).

Probes had specific activities of about 2 x 109 dpm/µg. The protocol for in situ hybridization was as previously described [8]. In brief, frozen uterine sections were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 15 min. After prehybridization, sections were hybridized with 35S-labeled antisense cRNA probes at 45°C for 4 h in 50% formamide hybridization buffer. After hybridization and washing, sections were treated with RNase A (20 mg/ml) at 37°C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Company, Rochester, NY). The slides were poststained with hematoxylin and eosin. Sections hybridized with the sense probes served as negative controls (data not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ERM Proteins and ERM-Associated Adhesion Molecules Are Differentially Expressed in Dormant and Implantation-Competent (Activated) Blastocysts

In mice, the first attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium occurs around midnight on Day 4 (Day 1 = vaginal plug) of pregnancy. Ovariectomy in the morning of Day 4 prior to preimplantation estrogen secretion results in blastocyst dormancy and delayed implantation. The conditions of delayed implantation can be maintained by continued P4 treatment but are terminated with an injection of estrogen leading to implantation-competent (activated) blastocysts and subsequent implantation. We examined the distributions of ERM proteins in normal Day 4 blastocyst as well as in P4-treated dormant and P4 plus estrogen-treated activated blastocysts. In dormant blastocysts, radixin was localized on the cell surface and to some extent in the cytoplasm of both polar and mural trophectoderm cells. In contrast, radixin was targeted primarily to the cell surface of the mural trophectoderm at a much higher level in implantation-competent (activated) blastocysts and in normal Day 4 blastocysts (Fig. 1A). The localization of ezrin was different from that of radixin. Ezrin was distinctly present in the primitive endoderm and also at a reduced level on the trophectoderm cell surface of dormant blastocysts (Fig. 1B). In normal Day 4 and activated blastocysts, ezrin was present on the cell surface of the entire trophectoderm without much region-specific localization, and the localization was not apparent in the primitive endoderm. The localization of moesin was limited primarily to the cell surface of the mural trophectoderm; the level of signal was higher in activated blastocysts than that in dormant blastocysts. In normal Day 4 blastocysts, moesin was apparently present on the polar trophectoderm cell surface (Fig. 1C). These results suggest that radixin and ezrin are involved in cellular organization of the trophectoderm during blastocyst activation prior to implantation and that radixin is particularly involved in making the mural trophectoderm, the presumptive site of attachment with the luminal epithelium, ready for implantation.



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  		PLATE 1. Expression of ERM proteins (Fig. 1) and ERM-associated adhesion molecules (Fig. 2).  FIG. 1. Expression of ERM proteins in normal Day 4 blastocysts and in implantation-competent (activated) and dormant blastocysts. Normal blastocysts were collected on Day 4 of pregnancy. Activated blastocysts were collected 12 h after an injection of E2 in P4-primed delayed implanting mice, while dormant blastocysts were retrieved 12 h after the last P4 injection. They were fixed, permeabilized, and incubated with specific primary antibodies. Localization of antigens (red) and nuclei (blue) were visualized using TRITC-conjugated secondary antibody and Hoechst 33342, respectively. Indirect immunofluorescence staining was visualized by scanning laser confocal microscopy. A) Immunolocalization of radixin.  Note that in Day 4 and implantation-competent blastocysts (activated), the outer surface of the mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass, and primitive endoderm. B) Immunolocalization of ezrin. Note that the expression of ezrin on the trophectoderm surface was higher in Day 4 and activated blastocysts, while its localization in the primitive endoderm of dormant blastocysts was distinct. C) Immunolocalization of moesin. Note that the expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts. Composite confocal images are comprised of 2.5–3.0 µm scanning of an entire blastocyst. Bar = 50 µm.  FIG. 2. Expression of ERM-associated adhesion molecules CD44, ICAM-1, CD43, and ICAM-2 in implantation-competent (activated) and dormant blastocysts. Activated or dormant blastocysts were fixed, permeabilized, and incubated with antibodies. Localization of antigens (red) and nuclei (blue) were visualized using TRITC-conjugated secondary antibody and Hoechst 33342, respectively. Indirect immunofluorescence was visualized by scanning laser confocal microscopy. Immunolocalization of (A) CD44, (B) ICAM-1, (C) CD43, and (D) ICAM-2. Note that CD44 was present both in the trophectoderm and inner cell mass, while CD43 and ICAM-2 was localized primarily to the trophectoderm. The localization of ICAM-1 was very intense in the ICM, with low levels in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. Composite confocal images are comprised of 2.5–3.0 µm scanning of an entire blastocyst. Bar = 50 µm

There were little differences in the distribution patterns of the ERM-associated adhesive molecules CD44, CD43, ICAM-1, and ICAM-2 between dormant and activated blastocysts, although their cell-specific localizations were different (Fig. 2). For example, while CD44 was present in both the trophectoderm and ICM, CD43 was displayed primarily on the trophectoderm (Fig. 2, A and B). The localization of ICAM-1 was interesting; while it was present in both the trophectoderm and ICM, it is the only adhesion molecule that showed intense signals in the ICM, suggesting that ICAM-1 may be involved in maintaining the ICM integrity both during dormancy and activation (Fig. 2C). On the other hand, ICAM-2 was present primarily on the trophectoderm cell surface (Fig. 2D). These results suggest that, in dormant blastocysts before activation, adhesive molecules associated with ERM proteins are already positioned in a cell-specific manner for interacting with radixin and/or ezrin expressed in activated blastocysts. Thus, ERM proteins expressed on trophectoderm cell surfaces of implantation-competent blastocysts should act as a cross-linker between actin and adhesive molecules to change the cell polarization and/or differentiation for adhesion and attachment with the luminal epithelium.

ERM Proteins and ERM-Associated Adhesion Molecules Are Expressed in a Cell-Specific Manner in the Receptive Uterus Prior to and During Implantation

During normal pregnancy, the luminal epithelial cells are differentiated and polarized on Day 4 prior to the attachment reaction. We observed that all three ERM proteins are present primarily in uterine luminal and glandular epithelial cells on this day of pregnancy (Fig. 3). On Day 5 following implantation, ERM proteins, particularly radixin and moesin, were also expressed in stromal cells in addition to the luminal epithelium surrounding the implanting blastocyst. However, ERM proteins in the stroma as well as in the luminal and glandular epithelium at the interimplantation sites were down-regulated as compared to implantation sites on Day 5 or to Day 4 uteri (Fig. 3). In situ hybridization results show that mRNA localization for ezrin, radixin, and moesin more or less followed the similar pattern as their protein localization at the implantation site (Fig. 4). These results of ERM expression in the luminal epithelium and stromal cells during implantation suggest that these proteins are involved in the dynamic changes in the cellular architecture that occur during this time.



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  		FIG. 3. Expression of ezrin, radixin, and moesin proteins in the receptive uterus on Day 4 or following implantation on Day 5. Localization of antigens (red) and nuclei (blue) were visualized using TRITC-conjugated secondary antibody and Hoechst 33342, respectively. Indirect immunofluorescence staining was visualized by scanning laser confocal microscopy. Note that ERM proteins were localized to the uterine epithelium on Day 4, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. IS, implantation sites; Inter-IS, interimplantation site. Bar = 100 µm



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  		FIG. 4. In situ hybridization of ezrin, radixin, and moesin mRNAs in the receptive uterus on Days 4 and 5 prior to and during implantation, respectively. Note that localization of ezrin, radixin, and moesin mRNAs more or less followed the similar pattern as their protein localization (Fig. 3). Bar = 500 µm

To investigate the possibility of potential interactions between the ERM proteins and ERM-associated adhesion molecules, cell-specific localization of CD43, CD44, ICAM-1, and ICAM-2 was examined by immunofluorescence analysis (Fig. 5). While CD44 and ICAM-1 were distributed in stromal cells, CD43 and ICAM-2 were localized in luminal and glandular epithelial cells on Day 4. On Day 5 following implantation, the accumulation of CD44, CD43, and ICAM-1 was distinctly visible in stromal cells, whereas the localization of ICAM-2 was similar to what was observed on Day 4. Of particular interest was heightened accumulation of CD44 and ICAM-1 in a few layers of stromal cells underneath the luminal epithelium surrounding the implanting blastocyst. In contrast, while stromal localization of CD44 and CD43 was absent, localization of ICAM-1 was low at the interimplantation site (Fig. 5). These results suggest that adhesive molecules CD43 and ICAM-2 are involved in regulating blastocyst attachment with the luminal epithelium and CD44 and ICAM-2 for regulating restricted trophoblast invasion into the stroma.



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  		FIG. 5. Expression of ERM-associated adhesion molecules CD44, CD43, ICAM-1, and ICAM-2 in the receptive uterus prior to and during implantation on Days 4 and 5, respectively. Localization of antigens (red) and nuclei (blue) were visualized using TRITC-conjugated secondary antibody and Hoechst 33342, respectively. Indirect immunofluorescence staining was visualized by scanning laser confocal microscopy. Note that the localization of CD44 and ICAM-1 was exclusively localized in the stroma on Day 4, while the localization of CD43 and ICAM-2 was limited to the epithelium. On Day 5, the localization of CD44 became highly concentrated in the antimesometrial stroma of the implantation chamber, and to some extent ICAM-1 showed similar localization. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. IS, implantation sites; Inter-IS, interimplantation site. Bar = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The initiation of implantation involves a series of cell-cell communications between two epithelial tissues: the trophectoderm and luminal epithelial cells. Our observation of differential expression of ERM proteins in dormant and activated blastocysts with no apparent changes in the adhesion molecules suggests that ERM proteins could be the limiting factors for interactions with the adhesion molecules in regulating blastocyst activation for implantation. The ERM proteins function as cross-linkers between plasma membranes and actin filaments [19]. These proteins are present in eukaryotic cells in organisms ranging from Caenorhabditis elegans to humans. ERM proteins normally function as cross-linkers between cortical actin filament and plasma membranes and are involved in the formation of microvilli, cell adhesion sites, ruffling membranes, and cleavage furrows. Indeed, several studies have shown localization of ERM proteins on the cell surface structures such as microvilli, ruffling membranes, and cell adhesion sites, where actin filaments associate with plasma membranes [20–23]. Furthermore, ezrin is expressed in mouse oocytes and throughout preimplantation embryo development, and it is always associated with the apical domains of outer cells during blastocyst formation [24]. Our results of increased expression of radixin on the outer surface of mural trophectoderm cells suggest that radixin acts as a main cross-linker protein between plasma membranes and actin filaments to make blastocysts competent for implantation.

Upregulation of ErbB1 and ErbB4 in implantation-competent but not dormant blastocysts with parallel expression of HB-EGF in the receptive, not the delayed, uterine luminal epithelium exclusively surrounding the blastocyst prior to the attachment reaction suggests that paracrine/juxtacrine signaling by HB-EGF is important for the attachment reaction [10, 11]. In this respect, upregulated expression of ERM proteins in activated blastocysts perhaps helps luminal epithelial HB-EGF to interact with ErbBs expressed in trophectoderm cells.

Pinopodes on the endometrial surface are suggested as ultrastructural markers for uterine receptivity for implantation [25]. Since adhesion molecules present on the luminal epithelial surface are considered to be important for the initial steps in the implantation process, it was suggested that HB-EGF and integrins present on the surface of the pinopodes are, in fact, important for implantation [26, 27]. Recently, it has been observed that HB-EGF is present both inside the luminal epithelial cells and on the surface of pinopodes of human endometrium [28]. Thus, it could be envisioned that ERM proteins and ERM-associated adhesion molecules in conjunction with HB-EGF generate pinopodes in the receptive uterus to initiate the process of implantation. We have previously shown that COX-2 is expressed in the luminal epithelial and stromal cells surrounding the implanting blastocyst [29], and COX-2-derived prostaglandins (PGs) are crucial for implantation and decidualization in mice [29]. The heightened expression of CD44 in the stroma surrounding the implanting blastocyst is coincident with similar expression of COX-2 on Day 5 [29]. Elevated COX-2 expression is associated with increased tumor angiogenesis and invasion and suppression of host immunity [30]. The stable overexpression of COX-2 in non-small cell lung cancer results in the upregulation of CD44, and CD44-dependent invasion is increased. In contrast, abrogation of tumor COX-2 expression results in decreased PGE2 production with decreased CD44 expression and tumor invasion. These results would suggest that stromal cells surrounding implanting blastocyst help in regulated invasion by trophoblast cells that behave like invading tumor cells, and PGE2-induced CD44 could be involved in this event. CD44 also coclusters with MMP-9 and promotes MMP-9 activity, tumor invasion, and angiogenesis [31, 32]. We have previously shown that MMP-9 is expressed in stromal cells exclusively at the site of implantation on Day 5 at the antimesometrial pole followed by high expression in trophoblast giant cells on Day 8 [33].

In conclusion, we suggest that upregulated expression of radixin in the mural trophectoderm of activated blastocysts contributes to cell surface changes allowing trophectoderm ErbBs to interact with luminal epithelial HB-EGF for initiating the process of implantation. At the onset of implantation on Day 5, uterine ZO-1 and E-cadherin [34] in collaboration with ERM and ERM-associated proteins help in the remodeling of cellular architecture of the endometrium permitting restricted invasion of the trophoblast into the antimesometrial stroma of the implantation chamber.


   FOOTNOTES
 
1 Supported in part by NIH grants (HD12304 and HD33994), a grant from the Kuribayashi Foundation (H.M.), and Japan Society for the Promotion of Science JSPS-13460129 and JSPS-13876059 (E.S.). S.K.D. is a recipient of an NICHD/NIH Merit Award. H.W. is a Lalor Foundation postdoctoral fellow. Back

2 Correspondence: S.K. Dey, Department of Pediatrics, Division of Reproductive and Developmental Biology, Vanderbilt University Medical Center, MCN-D4100, Nashville, TN 37232-2678. FAX: 615 322 4704; sk.dey{at}vanderbilt.edu Back

Received: 3 September 2003.

First decision: 17 September 2003.

Accepted: 5 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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