The cytoplasmic expression of E-cadherin and β-catenin in bovine trophoblasts during binucleate cell differentiation

Placenta (2005), 26, 393e401 doi:10.1016/j.placenta.2004.08.002

The Cytoplasmic Expression of E-Cadherin and b-catenin in bovine trophoblasts during binucleate cell differentiation H. Nakano*, A. Shimada, K. Imai, T. Takahashi and K. Hashizume1 Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki 305-8602, Japan Paper accepted 3 August 2004

Binucleate cells are endocrine cells generated by the acytokinesis and endoreduplication of the trophectoderm in the ruminant placenta. These cells are migratory and secrete hormones into the maternal circulation after fusing with uterine epithelial cells. In this study, we performed immunohistochemistry for E-cadherin and b-catenin in bovine placenta and a bovine trophoblast cell line (BT-1). We found that E-cadherin and b-catenin were distributed not only at the cell to cell boundary but throughout the cytoplasm in binucleate cells, although they were concentrated at the cell to cell boundary in epithelial cells in bovine placenta. Moreover, b-catenin was detected in the nuclei of binucleate cells. Binucleate cells after fusion with uterine epithelial cells (fetomaternal hybrid cells) in the maternal side showed no intracellular expression of E-cadherin and b-catenin. The transformation into binucleate cells in the BT-1 cell line was also accompanied by the cytoplasmic accumulation of E-cadherin and b-catenin. We further demonstrated that levels of cytoplasmic b-catenin were well correlated with the DNA content of binucleate cells in BT-1. The dynamic changes in the distribution of E-cadherin and b-catenin suggest an important role in binucleate cells, including the rearrangement of cadherin-mediated cell adhesions during cell migration and the onset of endoreduplication probably via the nuclear transfer of b-catenin. Placenta (2005), 26, 393e401 Ó 2004 Elsevier Ltd. All rights reserved.

INTRODUCTION Mammalian trophoblasts differentiate into phenotypes distinct in morphology and function during the formation of the placenta [1,2]. Bovine trophoblast binucleate cells are generated from the trophectoderm with its acytokinesis and constitute about 20% of the trophectoderm throughout gestation [1,3,4]. They have two large polyploid nuclei formed through endoreduplication [5], and produce hormones including the placental lactogen/prolactin-related protein family and pregnancy-associated glycoprotein family [1]. Binucleate cells migrate and fuse with uterine epithelial cells in placentomes where the interdigitation between chorionic villi and uterine caruncular crypts occurs [3,4,6]. These fetomaternal hybrid cells (mostly trinucleate cells) discharge fetal hormones into the maternal circulation and then die [3,4,6]. Binucleate cells are considered analogous to extravillous * Corresponding author address: Department of Biochemistry and Molecular Biology Health Sciences Centre 2258, 3330 Hospital Drive NW Calgary, Alberta, Canada T2N 4N1. Tel.: D403 220 7243; fax: D403 270 0737. E-mail address: [email protected] (H. Nakano). 1 Present address: Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. 0143e4004/$esee front matter

cytotrophoblasts in humans and trophoblast giant cells in rodents with respect to polyploidy and migratory/invasive properties [2,7]. The mechanism by which binucleate cells differentiate is poorly understood, however, the differentiation accompanies a loss of cytokeratin filaments and associated desmosomes [8e11] and the altered expression of integrin subtypes [12e14]. These results suggest a role for cell adhesion in the genesis and function of binucleate cells [10,14]. We have established a bovine trophoblast cell line (BT-1 [15]), and found that the use of a collagen gel as a culture substratum was effective in transforming mononucleate BT-1 cells into a binucleate cell phenotype [16]. These binucleate cells assumed a round shape and often formed multilayered clusters in monolayers of polygonal BT-1 cells [16]. The flexibility of the collagen gel may allow BT-1 cells to change their shape and affect cell adhesion, resulting in binucleate cells. E-cadherin is a transmembrane cell surface molecule that is involved in Ca2C-dependent cell to cell adhesion of epithelial cells [17]. The cytoplasmic tail of E-cadherin interacts with the actin cytoskeleton via proteins including a-, b- and g-catenins, vinculin and a-actinin [18], and this linkage strengthens cell adhesion. Beta-catenin, in addition to having a role in cell to cell adhesion, functions as an intracellular signaling molecule in the canonical Wnt pathway through its translocation from Ó 2004 Elsevier Ltd. All rights reserved.

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the cytoplasm to the nucleus [19]. E-cadherin-mediated cell adhesion is involved in the genesis of the trophectoderm in blastocysts by establishing the cell polarity [20e22], however, E-cadherin expression is not static but is dynamically regulated in parallel with further differentiation into invasive and syncytial trophoblasts [23e27]. In this study, we investigated E-cadherin and b-catenin immunofluorescence in bovine placenta and the BT-1 cell line. We found that E-cadherin and b-catenin were accumulated in the cytoplasm of binucleate cells both in vivo and in vitro, suggesting a role for intracellular E-cadherin and b-catenin in the functions of binucleate cells.

washes with PBS, the tissue sections were mounted using Perma Fluor (Shandon, Pittsburgh, PA, USA) and viewed under an epifluorescence microscope (IX70, Olympus, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera (Orca C4742-95, Hamamatsu Photonics, Hamamatsu, Japan). Fluorescent images were taken with the CCD camera under the control of image analysis software, AQUACOSMOS (Hamamatsu Photonics). For cell counts, over 150 binucleate cells from the images of 20 different fields were analyzed with each sample for E-cadherin or b-catenin immunoreactivity. Data were expressed as percentage G SD (n Z 4).

MATERIALS AND METHODS

Culture of bovine trophoblast cells (BT-1)

Tissue immunohistochemistry

The BT-1 cell line [15] was cultured in Dulbecco’s modified Eagle’s/F-12 medium (DME/F-12, GIBCO Invitrogen, Carlsbad, CA, USA) containing 100 IU/ml of penicillin and 100 mg/ml of streptomycin (Sigma), and 10% heat-inactivated fetal bovine serum (Sigma). Binucleate cell differentiation was induced by growing BT-1 cells on a type I collagen gel (Nitta Gelatin, Osaka, Japan) substratum in the culture medium as previously described [16].

Bovine placental materials were collected at the abattoir of the National Institute of Livestock and Grassland Science (Tsukuba, Japan) with the approval of the ethical committee. Placentomes from four Japanese Black cows at mid to late pregnancy (103, 202, 202 and 234 days of gestation) were cut into small pieces (5 ! 5 ! 2 mm), and fixed in 2% or 4% paraformaldehyde (PA) in 0.1 M phosphate buffer (pH 7.4) for 6 h or overnight. After several washes with phosphate-buffered saline (PBS), tissues were immersed in 10%, 20% and then 30% sucrose in PBS, and then embedded in the Tissue-Tek O.C.T. compound (Sakura Finetechnical Co., Tokyo, Japan) and frozen with liquid N2-cooled isopentane. Cryosections 6 mm thick were cut with a cryostat (HM500, MICROM, Randburg, Germany) and mounted on 3-aminopropyltriethoxysilane (APS)-coated glass slides (Matsunami, Osaka, Japan). After three washes with PBS, the tissue sections were blocked with PBS containing 10% normal goat serum and 0.3% Triton X-100 (Sigma, St Louis, MO, USA) for 30 min at room temperature. Incubation with primary antibody was carried out overnight at 4 (C. Monoclonal mouse anti-human E-cadherin (clone 36, IgG: 250 mg/ml, BD Biosciences, San Jose, CA, USA), monoclonal mouse anti-chicken b-catenin (clone 15B8, IgG: 2 mg/ml, Sigma) and polyclonal rabbit antibovine placental lactogen (PL [10]) were diluted 1:100, 1:1000 and 1:2000, respectively, in PBS containing 1% BSA and 0.05% NaN3 (1% BSA/PBS). As controls, normal rabbit serum at the corresponding dilution in 1% BSA/PBS for the polyclonal antibody, and the vehicle solution (1% BSA/PBS) for monoclonal antibodies, were used. For double labeling, either anti-E-cadherin and anti-PL, or anti-b-catenin and antiPL were incubated together. After three washes with PBS, the secondary antibody in 1% BSA/PBS was applied for 2 h at room temperature. For the secondary antibody, Alexa 488 (or Alexa 546)-conjugated goat anti-rabbit or anti-mouse IgG (Molecular Probes, Eugene, OR, USA) was used at a dilution of 1:200e400. For the staining of actin filaments (F-actin), Alexa 488-conjugated phalloidin (final concentration: 0.165 mM, Molecular Probes) was added to the secondary antibody solution. Nuclei were stained with Hoechst 33342 (final concentration: 5 mg/ml, Molecular Probes). After three

BT-1 immunocytochemistry BT-1 cells cultured on the collagen gel substratum for 12e16 days were stained as described above with some modifications. The cells were fixed with 4% PA for 15 min at 4 (C. After washes with PBS, the cells were blocked and permeabilized with PBS containing 10% normal goat serum and 0.3% Triton X-100 for 30e60 min at room temperature. Incubation with primary antibody was carried out in 1% BSA/PBS supplemented with 0.1% Triton X-100 for 2 h at room temperature. The dilution of anti-E-cadherin, anti-b-catenin and anti-PL was 1:2000, 1:2000 and 1:8000, respectively. After washes with PBS, the secondary antibody in 1% BSA/PBS supplemented with 0.1% Triton X-100 was applied for 1 h at room temperature. Nuclei were stained with Hoechst 33342. After washes with PBS, collagen gels with the cells were stripped from culture dishes, mounted on slide glasses with Perma Fluor, and viewed under the IX70 epifluorescence microscope equipped with the CCD camera. Image analysis BT-1 cells cultured on gels for 12e13 days were fixed and double-stained with anti-b-catenin and anti-PL as described above. Hoechst 33342 (DNA), Alexa 546 fluorescence (b-catenin) and Alexa 488 fluorescence (PL) images in the same field were taken with the CCD camera, and analyzed using AQUACOSMOS software. Over 100 binucleate cells from the images of 20 fields after background subtraction were analyzed for the quantification of b-catenin and DNA fluorescence intensity. A linear regression analysis was performed to examine the relationship between b-catenin and DNA levels. Three independent experiments were performed.

Nakano et al.: E-Cadherin and b-Catenin in Binucleate Trophoblasts

Western blotting 2

Confluent BT-1 cells in collagen-coated 25 cm culture flask were lysed in 2 ml of SDS sample buffer, and then boiled at 100 (C for 5 min. The cell lysate was centrifuged at 15,000 rpm for 7 min to remove an insoluble material. The BT-1 cell lysate (5 ml) or the A-431 cell lysate (a positive control for the anti-Ecadherin antibody provided by the manufacturer) was separated by SDS polyacrylamide gel electrophoresis in 7.5% polyacrylamide gels as described previously [10]. Molecular weight markers (pre-stained Precision Protein StandardsÔ) were obtained from Bio-Rad, Hercules, CA, USA. Proteins were then electrophoretically transferred to the polyvinylidene difluoride (PVDF) membrane (Hybond-P, Amersham Biosciences, Piacataway, NJ, USA) as described previously [10]. The membrane was blocked with 5% skimmed milk in Trisbuffered saline (TBS) for 1 h at room temperature, and then incubated with either the anti-E-cadherin or anti-b-catenin antibody at the dilution of 1:2500 in TBS containing 1% skimmed milk for overnight at 4 (C. After three washes with TBS containing 0.1% Tween-20 (Sigma) (TBST), the membrane was incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad) at the dilution of 1:2000 in TBS containing 1% skimmed milk for 1 h at room temperature. After three washes with TBST, the immunoreaction was visualized with alkaline phosphatase conjugate substrate kit (Bio-Rad). RESULTS Immunohistochemistry for E-cadherin and b-catenin in bovine placenta Double-labeling studies were performed with the monoclonal anti-E-cadherin and the polyclonal anti-placental lactogen (PL), or the monoclonal anti-b-catenin and anti-PL on the same sections since anti-PL specifically labeled binucleate cells in previous studies [10,11]. We found that E-cadherin was expressed not only at the cell to cell boundary but also in the cytoplasm of binucleate cells (Figure 1A, D, arrowheads), although it was concentrated at the intercellular boundary of the trophectoderm and uterine epithelium in the placentome. The cytoplasmic staining of E-cadherin was confined to PL-positive binucleate cells (Figure 1B, E, arrowheads). We obtained similar results from an examination of four different samples, and confirmed that about 60% of the PL-positive binucleate cells (63.3 G 3.8%, n Z 4, total 753 cells counted) were E-cadherin-positive both in the intercellular boundary and in the cytoplasm. Staining of b-catenin was also found at the intercellular boundary and in the cytoplasm of binucleate cells (Figure 2A, D, arrowheads), although it was concentrated at the intercellular boundary in the trophectoderm, the uterine epithelium and the endothelium in blood vessels. The cells with cytoplasmic staining of b-catenin largely corresponded to PL-positive binucleate cells (Figure 2B, E, arrowheads). However, more binucleate cells were positive for b-catenin

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in the cytoplasm than for E-cadherin; over 90% of the PLpositive binucleate cells (99.1 G 0.6%, n Z 4, total 782 cells counted) were simultaneously positive for b-catenin in the cytoplasm. b-Catenin immunoreactivity was also detected in the nuclei of binucleate cells in addition to the cytoplasm (Figure 2A, D), although the staining was more intense in the cytoplasm. Small and immature binucleate cells with little or no staining of PL had a weak but uniform staining of b-catenin in the cytoplasm and nuclei (Figure 2DeF, arrows). Binucleate cells after fusion with uterine epithelial cells (feto-maternal hybrid cells) in the maternal side appeared to shrink and were PL-negative due to the degranulation. These cells consistently lost the intracellular staining of E-cadherin and b-catenin (Figures 1GeI and 2GeI, double arrowheads). Distribution of F-actin in the bovine placenta F-actin stained with Alexa 488-phalloidin was distributed around the periphery of binucleate cells in the placentome (Figure 3A, arrows). F-actin was also distributed around the periphery of the uterine epithelium and the trophectoderm concentrating in apical microvilli (Figure 3A, arrowheads), and the cytoplasm in stromal cells. Immunocytochemistry for E-cadherin and b-catenin in BT-1 E-cadherin and b-catenin in mononucleate BT-1 cells were expressed at the cell to cell boundary, typical of polarized epithelial cells ([16] and in this study). Western blot analysis using either the anti-E-cadherin or anti-b-catenin antibody revealed a single E-cadherin (120 kDa, Figure 4A, arrowhead) and b-catenin (94 kDa, Figure 4B, arrowhead) protein species in BT-1 cells. Upon the differentiation into binucleate cells with collagen gel cultures, E-cadherin (Figure 5A, arrowheads) and b-catenin (Figure 5D, arrowheads) were accumulated in the cytoplasm of binucleate cells, and the latter was also detected in the nuclei (e.g. Figure 5G, I, arrows). E-cadherin and b-catenin in binucleate cells truly resided in the cytoplasm and not the cell surface since omitting Triton X-100 in the staining procedure caused the disappearance of both E-cadherin and b-catenin staining (data not shown). The binucleate cells with cytoplasmic E-cadherin were largely PL-positive (Figure 5AeC, arrowheads), while the binucleate cells with cytoplasmic b-catenin corresponded to both PL-positive (Figure 5GeI, asterisks) and PL-negative cells (Figure 5GeI, arrows). We noticed that the binucleate cells with strong b-catenin signals often had PL immunoreactivity and intense Hoechst 33342 fluorescence showing increased DNA content (Figure 5GeI, asterisks). We therefore performed an image analysis to quantify DNA and b-catenin levels in binucleate cells. As shown in Figure 6, regression analysis revealed a positive linear relationship between DNA and b-catenin levels in binucleate cells. PL-positive binucleate cells were distributed in the high DNA and high b-catenin region in the scatter plot (Figure 6).

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Figure 1. Double-label immunofluorescence staining for E-cadherin and placental lactogen (PL) in bovine placenta. Tissue sections of a placentome at 103 days of gestation were double-stained with a monoclonal anti-E-cadherin (A, D, G) and polyclonal anti-PL (B, E, H) antibody. Nuclei were stained with Hoechst 33342 (C, F, I). Higher power views of the area indicated by an arrow in A, B and C are shown in D, E and F, respectively. Note that binucleate cells (arrowheads) are both E-cadherin-positive and PL-positive in the cytoplasm. A feto-maternal hybrid cell in the maternal side is indicated by double arrowheads in GeI. CV: chorionic villi, UC: uterine caruncular crypts. Scale bars 100 mm (AeC), 50 mm (DeI).

DISCUSSION In this study, we demonstrated that E-cadherin and b-catenin were accumulated in the cytoplasm of binucleate cells in the bovine placenta and BT-1 cell line. E-cadherin is a transmembrane glycoprotein and is normally expressed at the intercellular boundary in epithelial cells including the bovine trophectoderm ([21,28] and in this study). E-cadherin participates in Ca2C-dependent cell to cell adhesion via its

extracellular domains, while its cytoplasmic domains interact with F-actin via the anchor proteins a-, b- and g-catenins, vinculin and a-actinin [18]. The cytoplasmic E-cadherin in binucleate cells would not interact with F-actin since F-actin was only distributed along the plasma membrane of binucleate cells in bovine placenta. Lang et al. [29] recently demonstrated that F-actin and its binding partners (vinculin and a-actinin) were co-distributed along the plasma membrane of binucleate cells in the bovine placenta, consistent with our results.

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Figure 2. Double-label immunofluorescence staining for b-catenin and PL in bovine placenta. Tissue sections of a placentome at 202 days of gestation were double-stained with a monoclonal anti-b-catenin (A, D, G) and polyclonal anti-PL (B, E, H) antibody. Nuclei were stained with Hoechst 33342 (C, F, I). Some binucleate cells are indicated by arrowheads in AeF. Note that these binucleate cells are b-cadherin-positive in the cytoplasm and nuclei. Immature binucleate cells are indicated by arrows in DeF. Feto-maternal hybrid cells in the maternal side are indicated by double arrowheads in GeI. CV: chorionic villi, UC: uterine caruncular crypts. Scale bars 100 mm (AeC), 50 mm (DeI).

The cytoplasmic staining of E-cadherin was mainly detected in mature binucleate cells containing PL granules in the placenta, but it was lost in the degranulated cells in the maternal side. Mature binucleate cells are committed to migrate and fuse with uterine epithelial cells to deliver fetal hormones into the maternal circulation [4,6]. Our results suggest that the accumulation of E-cadherin in the cytoplasm is relevant to functions of mature binucleate cells, including cell migration. The migration of binucleate cells is not typical.

Binucleate cells do cross the trophoblast cell layer but retain tight junctions with adjacent cells so that the plasma membrane of binucleate cells cannot flow through the tight junctions [6,8,30]. Electron microscopy has revealed that many small clear vesicles are present in the cytoplasm of migrating binucleate cells, and then appear to be inserted at the top of the tight junctions [30]. This insertion of new membrane forms a continuously expanding pseudopodium termed ‘‘migration front’’, and allows a close apposition to cells in the maternal

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Figure 3. Actin cytoskeleton (F-actin) distribution in bovine placenta. Tissue sections of a placentome at 202 days of gestation were double-stained with Alexa 488-conjugated phalloidin (A) and the polyclonal anti-PL antibody (B). Nuclei were stained with Hoechst 33342 (C). Binucleate cells are indicated by arrows in AeC. Arrowheads indicate apical microvilli of the trophectoderm. CV: chorionic villi, UC: uterine caruncular crypts. Scale bars 50 mm (AeC).

side [30]. E-cadherin is a transmembrane protein, and therefore the cytoplasmic E-cadherin in binucleate cells is considered to be localized in intracellular vesicles presumably by endocytosis. The endocytosis of E-cadherin is generally observed in cultured epithelial cells and is stimulated by hepatocyte growth factor, phorbol ester or Ca2C-free medium [31e34]. The endocytosed E-cadherin is then recycled to sites of new cell to cell contacts or targeted for degradation [33,35]. It is likely that vesicles containing E-cadherin may be delivered to the migrating front of binucleate cells to make contact with

the apex of uterine epithelial cells, although it should be investigated whether small clear vesicles in migrating binucleate cells would contain E-cadherin in future study. Interestingly, binucleate cells that differentiated from the BT-1 cell line also accumulated E-cadherin in the cytoplasm. The result indicated that the E-cadherin response was inherent in bovine trophoblasts during binucleate cell differentiation. The mechanism of the cytoplasmic accumulation of E-cadherin is not elucidated. However, binucleate cells are often found in multilayered clusters among the monolayer of

Figure 4. Immunoblotting analysis of E-cadherin and b-catenin in BT-1 cells. Western blot analysis was performed on the BT-1 cell lysate using the antiE-cadherin (A) or anti-b-catenin antibody (B). The A-431 cell lysate is a positive control for the anti-E-cadherin antibody. Immunoreactive signals are indicated by arrowheads.

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Figure 5. E-cadherin and b-catenin immunofluorescence in binucleate cells differentiated from BT-1 cells. BT-1 cells cultured on collagen gels were double-stained with either anti-E-cadherin (A) and anti-PL (B), or anti-b-catenin (D, G) and anti-PL (E, H). Nuclei were stained with Hoechst 33342 (C, F, I). Some binucleate cells differentiated from BT-1 cells are indicated by arrowheads in AeF. Note that these binucleate cells are both PL-positive and E-cadherin/b-catenin-positive in the cytoplasm. In GeI, PL-positive and PL-negative binucleate cells are indicated by asterisks and arrows, respectively. Scale bars 100 mm (AeF), 50 mm (GeI).

BT-1 cells [16], and therefore the formation of a multilayer might be involved in the E-cadherin response by affecting the cell polarity. Our culture system would provide a tool for elucidating the mechanism of E-cadherin internalization and its role in the migration of binucleate cells. Extravillous cytotrophoblasts in humans and trophoblast giant cells in rodents, counterparts of the bovine trophoblast binucleate cells, are terminally differentiated and invasive trophoblast phenotypes [7]. These cells lose E-cadherin during the differentiation [23,25,26]. The invasiveness of extravillous cytotrophoblasts or trophoblast giant cells extends to the decidua, while that of bovine trophoblast binucleate cells is limited to the epithelial layer of uterine caruncles [7]. Thus,

the difference in E-cadherin responses reflects the degree of migratory activity in invasive trophoblasts between species. The results of immunofluorescence microscopy indicated that in addition to E-cadherin, b-catenin was accumulated in the cytoplasm of binucleate cells both in vivo and in vitro. However, the staining pattern of b-catenin was somewhat different from that of E-cadherin. The cytoplasmic staining of b-catenin was found in both mature (PL-positive) and immature (PL-negative) binucleate cells, and moreover b-catenin was expressed in the nuclei of binucleate cells. This cytoplasmic and nuclear accumulation of b-catenin suggests that the protein, in addition to its role in cell to cell adhesion, functions as an intracellular signaling molecule best characterized in the Wnt

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Figure 6. The relationship between DNA and b-catenin levels in binucleate cells differentiated from BT-1 cells. A representative scatter plot of Hoechst 33342 (DNA) and Alexa 546 (b-catenin) fluorescent staining in 141 binucleate cells from one experiment is shown. Data on fluorescent intensity are expressed in arbitrary units (a.u.). Closed and open circles indicate PL-positive and PL-negative cells, respectively. The linear regression equation is Y Z 0.05 C 2.0X, and the correlation coefficient (R) is 0.72 (p ! 0.0001). Similar results were obtained from three independent experiments.

pathway [19]. Wnt proteins are secreted signal molecules that act as local mediators to control many aspects of cellular and developmental processes [36]. Activation of the Wnt pathway stabilizes b-catenin in the cytoplasm through the inhibition of the proteolytic degradation of b-catenin in the ubiquitineproteosome system. Stabilized b-catenin shuttles from the cytoplasm to the nucleus, where it binds to the lymphoid enhancer binding factor (LEF)-1/T cell factor (TCF) family of transcription factors and stimulates Wnt-responsive genes [19]. The cytoplasmic and nuclear accumulation of b-catenin started in immature binucleate cells, and persisted, implying a

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role for b-catenin in the maturation of binucleate cells. In this study, we demonstrated that intracellular levels of b-catenin were positively correlated with the DNA content of binucleate cells. Binucleate cells in the bovine placenta and BT-1 are polyploidy due to endoreduplication [5,16]. Since Wntresponsive genes include genes whose products promote entry into S phase such as cyclin D1 and c-myc [37e39], the intracellular b-catenin may be involved in the onset of endoreduplication in binucleate cells via the activation of such genes. In rodent trophoblasts, cyclin D1 is induced and levels of cyclin B1 are reduced with giant cell differentiation [40,41]. S-cyclins (cyclins E and A) and a cyclin-dependent kinase inhibitor, p57Kip2 appear to involve in the initiation and termination of S phase during endoreduplication cell cycles in the giant cells [41,42]. Trophoblasts derived from mutant mice deficient in the activating enzyme of NEDD8/Rub 1, (an ubiquitin-like molecule), accumulate b-catenin in the cytoplasm and nucleus due to the failure of b-catenin degradation [43]. These trophoblasts are unable to enter S phase of the endoreduplication cell cycles, and accompany with aberrant expression of cyclin E and p57Kip2 [43]. Thus, the intracellular b-catenin signaling can affect the endoreduplication cell cycles in the giant cells. The signaling may be operated to achieve polyploidization in both rodent and bovine trophoblasts. In summary, we demonstrated that E-cadherin and b-catenin were specifically accumulated in the cytoplasm of bovine trophoblasts that differentiate into binucleate cells. The latter was also detected in the nuclei of binucleate cells, and was associated with polyploidization. Our results suggest E-cadherin and b-catenin may have key roles in cell migration and endoreduplication of binucleate cells through a rearrangement of cell adhesions and an activation of the Wnt signaling.

ACKNOWLEDGEMENTS We are grateful to Mrs. M. Akiyama for technical assistance. H. Nakano is a domestic research fellow supported by a grant from the Japan Society for the Promotion of Science.

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