Epithelial cadherin distribution in abnormal human pre-implantation embryos

Mina Alikani1,2,3

1 Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia and 2 Tyho-Galileo Research Laboratories, West Orange, NJ 07052, USA

3 To whom correspondence should be addressed. E-mail: mina.alikani{at}embryos.net


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: E(epithelial)-cadherin is a vital cell adhesion protein that plays a critical role in morphogenesis. Previous studies of E-cadherin distribution in human embryos have produced equivocal results. METHODS: Immunocytochemistry in conjunction with laser scanning confocal microscopy was used to detect E-cadherin in 97 human cleavage stage embryos and 35 blastocysts from normal and abnormal fertilization. An antibody against human placental E-cadherin was used to locate the protein. RESULTS: In blastomeres of cleaving embryos on the second and third days following insemination, E-cadherin was located in the cytoplasm—mostly concentrated in the cell margins. On the fourth day of development, the protein was relocated in compacting embryos to membranes in areas of cell-cell contact. In other abnormally compacted or non-compacted embryos with extensive cytoplasmic fragmentation, cell arrest or blastomere multi-nucleation, E-cadherin relocalization was either absent or erratic. In apparently normal blastocysts, E-cadherin in the inner cells was diffuse and cytoplasmic while properly organized trophectoderm cells were surrounded by a band of membrane E-cadherin. Disorganization of trophectoderm was associated with disruption of the regular E-cadherin banding pattern. CONCLUSION: As in other mammalian species examined, E-cadherin distribution in human embryos is stage-dependent. Disturbances in the distribution of E-cadherin occur in embryos with cleavage abnormalities and suggest one path to abortive or abnormal blastulation and loss of embryonic viability. The implications of similar changes in the blastocyst are well worth investigating since they could threaten blastocyst integrity.

Key words: blastocyst/compaction/E-cadherin/fragmentation/trophectoderm


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cadherins are calcium-dependent transmembrane glycoproteins that play a critical role in regulation of morphogenesis through their involvement in junctional and non-junctional cell adhesion, cell polarity, and cell signalling (for review, see Fleming et al., 2001Go). The intracellular domain of each cadherin dimer is linked to the actin cytoskeleton by anchoring proteins (catenins), while the extracellular domain—which contains calcium binding sites—extends from the surface and can bind to another cadherin dimer on a neighbouring cell.

E(epithelial)-cadherin (also known as uvomorulin) is the first cadherin to be expressed during mammalian development. In the mouse, detergent-resistant surface expression of uvomorulin has been detected 6–11 h post-activation (Clayton et al., 1993Go), but its de-novo synthesis apparently occurs at the late two-cell stage (Vestweber et al., 1987Go), i.e. after the embryonic genome is normally activated (Flach et al., 1982Go). During subsequent stages and until compaction, uvomorulin remains uniformaly distributed on free surfaces of blastomeres but it is more concentrated at regions of cell-cell contact. During compaction, the flattening of the outer embryonic cells coincides with an increase in intercellular adhesion and uvomorulin redistribution to areas of apposing cell membranes (Johnson et al., 1979Go; 1986Go; Vestweber et al., 1987Go and Becker et al., 1992Go). At the same time, free surface protein is reduced and becomes relatively more susceptible to detergent extraction (Clayton et al., 1993Go). This distribution pattern is maintained in outer blastomeres of the mouse morula and in the blastocyst, while the inner cells continue to show diffuse cytoplasmic distribution of the protein (Vestweber et al., 1987Go).

The function of E-cadherin is thought to be regulated through protein phosphorylation, a post-translational modification (Sefton et al., 1996Go) and its ‘trafficking’ to and from the cell surface (for review, see Bryant and Stow, 2004Go).

In the human, the E-cadherin gene (CDH1) is ~100 kb and is located on chromosome 16q22 (Berx et al., 1995Go). E-cadherin has been discovered on plasma membranes of human spermatozoa and (inseminated but undivided) oocytes (Rufas et al., 2000Go).

In human embryos presumed to be normal, E-cadherin mRNA has been found throughout the pre-implantation stages including in pronuclear eggs, cleavage stages and blastocysts (Bloor et al., 2002Go). In apparent contradiction to observations in the mouse (described above) as well as in the pig (Reima et al., 1993Go) and the cow (Barcroft et al., 1998Go), however, E-cadherin expression in the limited number of human embryos examined so far—using antibodies primarily against E-cadherin from mouse carcinoma cells—has been defined as ‘cytoplasmic, punctate, extremely weak’ (Bloor et al., 2002Go) and ‘evident in isolated regions of cell contact in trophectoderm or [inner cell mass]’ (Ghassemifar et al., 2003Go).

In this study, immunocytochemistry was used in conjunction with laser scanning confocal microscopy (LSM) to examine the distribution of E-cadherin in some detail and in a large number of human embryos produced by different techniques during assisted reproduction. These embryos were of different genomic constitutions and showed a wide variety of morphologies. The reactivity of early embryonic E-cadherin with an antibody raised against human E-cadherin (of placental origin) was used.

All the embryos examined in the study had been judged to be unsuitable for clinical use based on several criteria as described below. However, at the time of fixation, some of the cleavage stage embryos did contain the number of cells appropriate for the day of development as well as cells that were mitotically active; some of these and some of the blastocysts appeared morphologically normal.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source and description of human embryos
The abnormal embryos used in these experiments were obtained from consenting patients, undergoing infertility treatment by IVF and embryo transfer (IVF/ET), and under a protocol approved by the Internal Review Board of Saint Barnabas Medical Center (Livingston, New Jersey, USA) in 1995 and 1999, revisions of which were re-approved in 2000, 2002 and 2004. The protocol concerns in-depth study of abnormal gametes and embryos generated during clinical IVF procedures and judged to be unsuitable for transfer or cryopreservation.

Fresh or frozen/thawed embryos with one or more of the following characteristics were considered to be non-viable and clinically unusable: (i) fewer than two cells on day 2 of development; (ii) fewer than five cells on day 3 of development; (iii) no division in 24 h of culture; (iv) one or more highly uneven cleavage divisions; (v) loss of >35% of the total cytoplasmic volume to fragmentation or degeneration; (vi) large fragments associated with few remaining blastomeres; (vii) one or more multi-nucleated blastomeres appearing either on day 2 or day 3 of development; and (viii) <50% of the cells remaining intact after thawing of cryopreserved embryos. Ovarian stimulation protocols and embryo culture methods are described in detail elsewhere (Alikani et al., 2003Go).

In total, 92 embryos from normally fertilized eggs (ICSI and standard insemination) were examined. A total of 38 embryos were fixed on day 3 of development, while the remaining 54 were fixed on or after day 4 (up to day 7) of development. The latter group included 29 blastocysts. In addition, 40 abnormally fertilized embryos were examined on days 2–6 of development; these included tri-pronucleate IVF and ICSI embryos, single pronucleate ICSI embryos and embryos that developed from inseminated eggs in which pronuclei were never seen. Six blastocysts were among the 40 abnormally fertilized embryos examined.

Fixation and confocal microscopy
Embryos were fixed and permeabilized for 30–60 min at room temperature in 2% formaldehyde and 0.5% Triton X100 in PIPES buffer. Following fixation, they were washed and kept overnight (or until processed) at 4°C in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) in order to minimize non-specific binding.

The primary antibody was a monoclonal mouse antibody immonoglobulin G (IgG) (IgG2a isotype; Zymed Laboratories, Inc., San Francisco, CA, USA) against E-cadherin from human placenta (diluted 1:500). Embryos were incubated with primary antibody for 60 min at 37°C; this was followed by two 10-minute washes in 0.2% Tween 20 (Sigma-Aldrich, Inc., Saint Louis, MO, USA) and a minimum of seven washes in PBS supplemented with 3% BSA (PBS/BSA).

The secondary antibody was a fluorescein isothiocyanate (FITC) conjugated goat anti- mouse IgG, whole molecule (Sigma-Aldrich, Inc., Saint Louis, MO, USA), diluted 1:200. Following 60 min incubation at 37°C with secondary antibody, the specimen was washed through a minimum of seven drops of PBS/BSA, with 0.2% sodium azide (PBS/BSA/Az). To counterstain the nuclei/DNA, 20 min of incubation in 0.06 mg/ml propidium iodide followed. Embryos incubated with secondary antibody only and stained with propidium iodide served as negative controls.

For examination, eggs and embryos were placed in 2 µl drops of PBS/BSA/Az covered with mineral oil on a glass cover–slip set in a steel chamber (Attofluor cell chamber, Molecular Probes, Eugene, OR, USA).

Laser scanning confocal microscopy was carried out either with an Olympus FluoView laser scanning confocal microscope (Olympus America, Inc., Melville, NY, USA), consisting of an IX70 fluorescence-Nomarski DIC microscope equipped with an argon laser (emitting at wavelength 488 nm), a krypton laser (emitting at wavelengths of 568 nm and 647 nm) and a transmitted light detector, or with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc, Thornwood, NY, USA) equipped with an argon laser and an HeNe laser (emitting at wavelength 543 nm). The images are presented either as single 3–5 µm thick optical sections or projections reconstructed from a series of sections.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Embryos treated with secondary antibody only (negative controls) did not show any fluorescence (not shown).

Days 2 and 3 of development
On days 2 and 3 of development, stage appropriate as well as slow growing embryos—whether resulting from normal or abnormal fertilization—showed diffuse cytoplasmic staining that was most pronounced in the cell margins, as was indicated visually and by the fluorescence profile of single optical sections of embryos (Fig. 1). In fragmented embryos, blastomeres, anucleate and some small nucleated fragments associated with them showed diffuse cytoplasmic staining (Fig. 2).



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Figure 1. Representative day-3 embryo stained for E-cadherin (green) and nuclear DNA (red). The image is a 4 µm thick single optical slice. Fluorescence intensity is graphed for the distance represented by the red arrow drawn across the embryo. E-cadherin fluorescence (green line in the graph) is concentrated in the cell margins, as shown by the graph peaks (marked by arrows). The intensity of DNA fluorescence (red line in the graph) is negligible in the same distance (i.e. no nuclei present). Scale bar = 50 µm.

 


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Figure 2. Representative day 3 embryo with extensive cytoplasmic fragmentation stained for E-cadherin (green) and nuclear DNA (red). The image is a projection of multiple 3–4 µm thick optical slices. E-cadherin localization is shown in panel (A), the nuclei in panel (B), and both in panel (C). E-cadherin appears diffuse in the three mono-nucleated cells (arrowheads in C) and in fragments (arrows in C). The differential interference contrast (DIC) image in panel (D) is a single mid-section optical slice. Scale bar = 50 µm.

 

Day 4 of development
Representative day 4 embryos are shown in Fig. 3. Changes in fluorescence pattern were first seen on this day, albeit neither uniformly nor universally. Some embryos with multiple compacted cells showed staining in areas of cell–cell contact along with cytoplasmic staining (Fig. 3A–D). Other embryos appeared compacted, but their component cells either had very limited membrane staining or lacked it altogether (Fig. 3E). Embryos with extensive cytoplasmic fragmentation mostly exhibited diffuse cytoplasmic staining, as they did during day 3 of development; however, erratic membrane staining was also present in a small proportion of the remaining nucleated cells (Fig. 3F).



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Figure 3. Representative day-4 embryos stained for E-cadherin (green) and nuclear DNA (red). (A) Compacting eight-cell embryo showing relocation of E-cadherin to areas of cell–cell contact, although fluorescence is weak (arrowheads); this embryo had only four cells on day 3 of development. (B) Compacting five-cell embryo, showing membrane localization of E-cadherin, again weakly (arrowheads); compaction appears atypical; this embryo had five cells, 10% fragmentation and one binucleate cell on day 3 of development. (C) Compacting seven-cell embryo, showing erratic membrane localization of E-cadherin (arrowheads). This embryo had five cells on day 3 of development. (D) Compacted embryo, beginning to cavitate late on day 4 of development; it shows extensive membrane-localized E-cadherin. At least two cells and one large fragment are excluded from the embryo proper (asterisks); staining is primarily cytoplasmic in these excluded cells. This embryo was an uneven and disorganized 11-cell with one or two large fragments on day 3 of development. (E) A partially compacted embryo with several multi-nucleated blastomeres and erratic membrane-located E-cadherin (arrowheads). (F) An extensively fragmented embryo, partly reconstructed from multiple optical sections, showing minimal membrane-localized E-cadherin (arrowhead). Insets show embryos just before fixation. Scale bar = 50 µm.

 

Days 5–7 of development
The morphology of embryos on days 5–7 of development varied greatly, ranging from few vacuolized cells to fully differentiated and expanded blastocysts. The pattern of staining also varied with different morphologies. Representative embryos from this group are shown in Figs 4–6.



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Figure 4. Apparently normal day 5–7 blastocysts stained for E-cadherin (green) and DNA (red). Images in A, B and C are projections of multiple 5 µm optical sections. Images in panels (ad) are single optical sections and show (a) E-cadherin, (b) differential interference contrast (DIC), (c) DNA and (d) the combination. (A) Day 7 expanded blastocyst that developed from an egg in which fertilization was not confirmed. The cells of the trophectoderm (both polar and mural) show an intense ‘belt’ of fluorescence indicating localization of E-cadherin in the membranes. Cells within the inner cell mass (ICM) (arrows in a and b) show diffuse cytoplasmic E-cadherin, but no membrane localization of the protein. Arrowhead points to a multi-nucleate cell within the polar trophectoderm (TE) overlying the ICM, presumably formed by fusion of multiple cells. (B) Hatched day 7 blastocyst from a normally fertilized egg showing the same pattern as seen in A. The area of the ICM is indicated by arrows in panel a and b. (C) A day 5 expanded blastocyst that developed from an egg in which three pronuclei were seen 19 h following ICSI. The pattern of staining is similar to that seen in A and B. The ICM is marked by arrows in a and b. Scale bar = 50 µm.

 

The overall staining pattern of normal-appearing blastocysts was consistent: inner cell mass (ICM) cells displayed cytoplasmic but not membrane staining. Properly organized and uniformly sized trophectoderm (TE) cells in expanded blastocysts with normal or near normal morphology were surrounded by a strong band of fluorescence (Fig. 4A–C). Differences in staining were not seen between mural (cells surrounding the blastocoel) and polar (cells overlaying the ICM) TE. Several hatching blastocysts were examined in which details of the embryo’s escape from the zona pellucida could be visualized. Hatching was observed to have started either in the area of the ICM or away from it (Fig. 5).



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Figure 5. Apparently normal day 5 hatching blastocyst stained for E-cadherin (green) and DNA (red). This blastocyst developed from a normally fertilized egg that was biopsied on day 3 of development and was diagnosed as a trisomy 13 aneuploid embryo. (A) Although membrane localization of E-cadherin is visible in some trophectoderm (TE) cell junctions (arrowheads), in other areas the protein appears to be mostly cytoplasmic. The inner cell mass (ICM) (arrow) shows typical cytoplasmic staining. (B) The embryo is hatching through an artificial biopsy hole in the zona pellucida (arrow). (C) Partial reconstruction of the blastocyst shows its overall morphology. Scale bar = 50 µm.

 

Representative embryos with abnormal blastulation are shown in Fig. 6. The abnormalities included an absent ICM, a disorganized TE or one composed of abnormally large cells, and a peri-vitelline space (PVS) filled with fragments and excluded cells. Atypically large cells within disorganized TE often did not show any membrane staining (Fig. 6A,C). Excluded arrested cells and fragments in the PVS did not show any cytoplasmic or other staining (Fig. 6B). Morulae that formed on day 5 rather than day 4 of development (no cavity present) still showed cell–cell contact area staining, albeit not extensively.



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Figure 6. Abnormal blastulation on day 5 of development; E-cadherin (green) and DNA (red) staining are shown. Images in AC are projections of multiple 4–5 µm optical sections. Images in (ad) are single optical sections and show (a) E-cadherin, (b) DIC, (c) DNA, and (d) the combination. (A) Day 5 embryo with very few trophectoderm (TE) cells, some showing the typical belt pattern of E-cadherin localization (arrowheads). The TE is generally disorganized (arrows in b) and there is no visible inner cell mass (ICM). (B) An embryo showing abnormal blastulation and ‘belt’ staining of TE cell membranes (arrowheads); many excluded cells and fragments which do not show any staining are visible (arrows in a and b). (C) A blastocyst with many large TE cells and limited localization of E-cadherin in TE cell junctions (arrowheads). Note the excluded fragments (arrows). Scale bar = 50 µm.

 

Embryos that developed from presumed digynic (ICSI 3PN) fertilization were often found to be compacted on day 4 of development. They showed various degrees of cell–cell contact area staining on (or after) that day; some digynic embryos blastulated apparently normally by day 5 of development (Fig. 4C). Presumably, polyspermic embryos (IVF 3PN) also showed occasional compaction and erratic membrane staining on day 4, but fewer were found to blastulate on (or after) day 5 of development.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, the distribution of the vital cell adhesion protein E-cadherin was assessed in human embryos by immunocytochemistry and confocal microscopy. The images obtained suggest that E-cadherin distribution in human embryos is stage-dependent. During the first three days in culture, in embryos with apparently normal morphology, the protein appears to be cytoplasmic and mainly concentrated in the non-contact regions of cells. On day 4 of development, it is relocated to areas of cell-cell contact. Following differentiation, E-cadherin appears to be cytoplasmic in the ICM cells while it appears as a band delineating the borders between individual TE cells, as is characteristic for epithelial cells in general.

In a study by Bloor et al. (2002)Go, only two out of eight ‘normal’ blastocysts showed ‘localization of E-cadherin to TE cell junctions’, while the remaining blastocysts showed ‘weak and punctate’ staining. Many factors may have contributed to this and other differences between our two studies. The most conspicuous is the diversity of the embryos examined (and of IVF human embryos in general). Weak staining and complete absence of membrane staining was also observed in the present study in some compacted day 4 embryos and in some blastocysts that were apparently normal. The choice of primary antibody may be another contributing factor. In the present study, a monoclonal antibody, produced in the mouse, against E-cadherin from the human placenta was used. Bloor et al. (2002)Go utilized an antibody against mouse E-cadherin. The homology between mouse and human E-cadherin molecules is between 69 and 75%, as suggested by basic local alignment search tool (BLAST 2) sequences (Tatusova and Madden, 1999Go; http://www.ncbi.nlm.nih.gov/blast/b12seq/wblast2.cgi). However, monoclonal antibodies are generally specific for a 4–5 amino acid sequence and are usually directed against the amino or carboxy terminal of a given protein (Pieczenik, 2003Go); mouse and human E-cadherin differ at their amino end. It is therefore possible that the antibodies in the study by Bloor et al. (2002)Go did not fully recognize E-cadherin in human embryos, thus producing inconsistent results.

The timely expression and distribution of E-cadherin is essential for normal compaction and blastulation. In all species of mammals examined, compaction begins after a certain number of cleavage divisions. The actual number depends on the species; in the human, compaction takes place between the eight and 16 cell stages, i.e. between the third and the fourth cleavage divisions (Nikas et al., 1996Go). The images of cleavage stage embryos with divisional asynchronies and related abnormalities as well as those of abnormal blastocysts collected for this study suggest that E-cadherin distribution is perturbed in these embryos. Specifically, redistribution of the protein to cell-cell contact areas was found to be absent or erratic. This apparent failure of abnormal embryos to properly relocate E-cadherin may be the immediate cause of their frequent failure to compact (and subsequently blastulate) normally.

The failure to relocate E-cadherin may be a consequence of developmental asynchrony or loss of physical contact between blastomeres (for instance through fragmentation or arrest of one or more cells). In the mouse, reverse translocation of E-cadherin from cell–cell contact areas to the cytoplasm has been shown to occur in decompacted embryos (Clayton et al., 1993Go; Pey et al., 1998Go). Conversely, in the human, disaggregation of fragmented embryos into their component cells and fragments and reaggregation of the cells in a ‘chimaeric’ form can, in some cases, overcome the barriers to normal compaction and lead to blastulation (Alikani and Willadsen, 2002Go).

At a more basic level, a possible reason for failure of embryos to fully engage E-cadherin in compaction is failure of genomic activation. Experimental evidence indicates that activation of the embryonic genome is a prerequisite for proper compaction. E-cadherin-null homozygous mutant mouse embryos that use residual maternal E-cadherin rather than zygotic E-cadherin to initiate compaction are unable to maintain the compacted state and fail to form blastocysts (Larue et al., 1994Go; Riethmacher et al., 1995Go).

Maintenance of compaction is dependent also on development of junctional complexes between apposing cells (for review, see Fleming et al., 2001Go). These complexes include gap junctions, tight junctions (zonula occludens), adherens junctions and desmosomes; E-cadherin plays an integral part in the latter two.

In the human, Dale et al. (1991)Go and Gualtieri et al. (1992)Go showed that TE and ICM cells are in communication through gap and tight junctions as well as desmosome-like structures. However, other evidence suggests that in some embryos, particularly those with morphological abnormalities, both the expression and the localization of junctional proteins are altered (Hardy et al., 1996Go; Ghassemifar et al., 2003Go).

The pattern of staining in abnormal blastocysts examined here was of particular interest. Some blastocysts did not show the expected localization of E-cadherin. Moreover, TE cells in some abnormal blastocysts did not exhibit the typical epithelial cell type of E-cadherin banding; in the same blastocysts, the cells of the ICM (if present) showed the diffuse cytoplasmic staining just as was seen in blastocysts with normal morphology.

The analysis of the images alone cannot provide an explanation for these observations. Nonetheless, the absence of E-cadherin banding in some TE cells is reminiscent of epithelial-to-mesenchymal cell transformation during which E-cadherin is internalized, facilitating the dissolution of adherens junctions between epithelial cells, thereby giving them mobility (Palacios et al., 2005Go). Notwithstanding a role for epithelial-to-mesenchymal transformation in interactions between uterine cells and the trophoblast during implantation (for example, Thie et al., 1996Go), under the circumstances of in vitro culture, a similar mechanism could lead to separation of TE cells from their neighbouring cells and localized or general disruption of the trophectoderm. The accidental formation of trophoblastic vesicles during hatching of human blastocysts through narrow slits left after partial zona dissection lends support to this suggestion (Cohen et al., 1990Go). The implications of abnormal regulation and distribution of E-cadherin in human blastocysts are therefore well worth investigating, especially in view of the current trend toward universal application of blastocyst culture and transfer for treatment of infertility, and the twinning complications associated with this technology (Milki et al., 2003Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The author would like to thank Dr Henry H. Malter for his assistance with confocal microscopy during the initial stages of this study. Ms Leona Cohen-Gould, Director of Electron Microscopy Core Facility of Joan and Sanford I. Weill Medical College of Cornell University is also thanked for assistance with confocal microscopy. Dr Steen Willadsen, Dr Jacques Cohen and Professor Alan Trounson are thanked for critical reading of the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Alikani M and Willadsen SM (2002) Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos. Reprod Biomed Online 5,56–58.[Medline]

Alikani M, Cekleniak NA, Walters E and Cohen J (2003) Monozygotic twinning following assisted conception: an analysis of 81 consecutive cases. Hum Reprod 18,1937–1943.[Abstract/Free Full Text]

Barcroft LC, Hay-Schmidt A, Caveney A, Gilfoyle E, Overstrom EW, Hyttel P and Watson AJ (1998) Trophectoderm differentiation in the bovine embryo: characterization of a polarized epithelium. J Reprod Fertil 114,327–339.[ISI][Medline]

Becker DL, Leclerc-David C, Warner A (1992) The relationship of gap junctions and compaction in the preimplantation mouse embryo. Dev Suppl., 113–118.

Berx G, Staes K, van Hengel J, Molemans F, Bussemakers MJ, van Bokhoven A and van Roy F (1995) Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1). Genomics 26,281–289.[CrossRef][ISI][Medline]

Bloor DJ, Metcalfe AD, Rutherford A, Brison DR and Kimber SJ (2002) Expression of cell adhesion molecules during human pre-implantation embryo development. Mol Hum Reprod 8,237–245.[Abstract/Free Full Text]

Bryant DM and Stow JL (2004) The ins and outs of E-cadherin trafficking. Trends Cell Biol 14,427–434.[CrossRef][ISI][Medline]

Clayton L, Stinchcombe SV and Johnson MH (1993) Cell surface localisation and stability of uvomorulin during early mouse development. Zygote 1,333–344.[Medline]

Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP and Wiemer K (1990) Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 5,7–13.[ISI][Medline]

Dale B, Gualtieri R, Talevi R, Tosti E, Santella L and Elder K (1991) Intercellular communication in the early human embryo. Mol Reprod Dev 29,22–28.[CrossRef][ISI][Medline]

Flach G, Johnson MH, Braude PR, Taylor RA and Bolton VN (1982) The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J 1,681–686.[ISI][Medline]

Fleming TP, Sheth B and Fesenko I (2001) Cell adhesion in the pre-implantation mammalian embryo and its role in trophectoderm differentiation and blastocyst morphogenesis. Front Biosci 6,D1000–1007.[ISI][Medline]

Ghassemifar MR, Eckert JJ, Houghton FD, Picton HM, Leese HJ and Fleming TP (2003) Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol Hum Reprod 9,245–252.[Abstract/Free Full Text]

Gualtieri R, Santella L and Dale B (1992) Tight junctions and cavitation in the human pre-embryo. Mol Reprod Dev 32,81–87.[CrossRef][ISI][Medline]

Hardy K, Warner A, Winston RM and Becker DL. (1996) Expression of intercellular junctions during pre-implantation development of the human embryo. Mol Hum Reprod 2,621–632.[Abstract]

Johnson MH, Chakraborty J, Handyside AH, Willison K, Stern P (1979) The effect of prolonged decompaction on the development of the preimplantation mouse embryo. J Embryol Exp Morphol 54, 241–261.[ISI][Medline]

Johnson MH, Maro B and Takeichi M (1986) The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres. J Embryol Exp Morphol 93,239–255.[ISI][Medline]

Larue L, Ohsugi M, Hirchenhain J and Kemler R (1994) E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci USA 91,8263–8267.[Abstract/Free Full Text]

Milki AA, Jun SH, Hinckley MD, Behr B, Giudice LC and Westphal LM (2003) Incidence of monozygotic twinning with blastocyst transfer compared with cleavage-stage transfer. Fertil Steril 79,503–506.[CrossRef][ISI][Medline]

Nikas G, Ao A, Winston R, Handyside AH (1996) Compaction and surface polarity in the human embryo in-vitro. Biol Reprod 55, 32–37.[Abstract]

Palacios F, Tushir JS, Fujita Y and D’Souza-Schorey C (2005) Lysosomal targeting of E-cadherin: a unique mechanism for the down-regulation of cell–cell adhesion during epithelial to mesenchymal transitions. Mol Cell Biol 25,389–402.[Abstract/Free Full Text]

Pey R, Vial C, Schatten G and Hafner M (1998) Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos. Proc Natl Acad Sci USA 95,12977–12982.[Abstract/Free Full Text]

Pieczenik G (2003) Are the universes of antibodies and antigens symmetrical? Reprod Biomed Online 6,154–156.[Medline]

Reima I, Lehtonen E, Virtanen I and Flechon JE (1993) The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig. Differentiation 54,35–45.[ISI][Medline]

Riethmacher D, Brinkmann V and Birchmeier C (1995) A targeted mutation in the mouse E-cadherin gene results in defective pre-implantation development. Proc Natl Acad Sci USA 92,855–859.[Abstract/Free Full Text]

Rufas O, Fisch B, Ziv S and Shalgi R (2000) Expression of cadherin adhesion molecules on human gametes. Mol Hum Reprod 6,163–169.[Abstract/Free Full Text]

Sefton M, Johnson MH, Clayton L and McConnell JM (1996) Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin. Mol Reprod Dev 44,77–87.[CrossRef][ISI][Medline]

Tatusova TA and Madden TL (1999) BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174,247–250.[CrossRef][ISI][Medline]

Thie M, Fuchs P and Denker HW (1996) Epithelial cell polarity and embryo implantation in mammals. Int J Dev 40,389–393.[ISI]

Vestweber D, Gossler A, Boller K and Kemler R (1987) Expression and distribution of cell adhesion molecule uvomorulin in mouse pre-implantation embryos. Dev Biol 124,451–456.[CrossRef][ISI][Medline]

Submitted on June 6, 2005; resubmitted on July 3, 2005; accepted on July 6, 2005.





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