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
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Abstract |
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Key words: blastocyst/compaction/E-cadherin/fragmentation/trophectoderm
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Introduction |
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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 611 h post-activation (Clayton et al., 1993), but its de-novo synthesis apparently occurs at the late two-cell stage (Vestweber et al., 1987
), i.e. after the embryonic genome is normally activated (Flach et al., 1982
). 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., 1979
; 1986
; Vestweber et al., 1987
and Becker et al., 1992
). At the same time, free surface protein is reduced and becomes relatively more susceptible to detergent extraction (Clayton et al., 1993
). 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., 1987
).
The function of E-cadherin is thought to be regulated through protein phosphorylation, a post-translational modification (Sefton et al., 1996) and its trafficking to and from the cell surface (for review, see Bryant and Stow, 2004
).
In the human, the E-cadherin gene (CDH1) is 100 kb and is located on chromosome 16q22 (Berx et al., 1995
). E-cadherin has been discovered on plasma membranes of human spermatozoa and (inseminated but undivided) oocytes (Rufas et al., 2000
).
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., 2002). In apparent contradiction to observations in the mouse (described above) as well as in the pig (Reima et al., 1993
) and the cow (Barcroft et al., 1998
), however, E-cadherin expression in the limited number of human embryos examined so farusing antibodies primarily against E-cadherin from mouse carcinoma cellshas been defined as cytoplasmic, punctate, extremely weak (Bloor et al., 2002
) and evident in isolated regions of cell contact in trophectoderm or [inner cell mass] (Ghassemifar et al., 2003
).
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.
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Materials and methods |
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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., 2003).
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 26 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 3060 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 coverslip 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 35 µm thick optical sections or projections reconstructed from a series of sections.
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Results |
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Days 2 and 3 of development
On days 2 and 3 of development, stage appropriate as well as slow growing embryoswhether resulting from normal or abnormal fertilizationshowed 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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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 cellcell contact along with cytoplasmic staining (Fig. 3AD). 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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Days 57 of development
The morphology of embryos on days 57 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 46.
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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. 4AC). 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 embryos 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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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 cellcell contact area staining, albeit not extensively.
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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 cellcell 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.
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Discussion |
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In a study by Bloor et al. (2002), 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)
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, 1999
; http://www.ncbi.nlm.nih.gov/blast/b12seq/wblast2.cgi). However, monoclonal antibodies are generally specific for a 45 amino acid sequence and are usually directed against the amino or carboxy terminal of a given protein (Pieczenik, 2003
); 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)
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., 1996). 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 cellcell contact areas to the cytoplasm has been shown to occur in decompacted embryos (Clayton et al., 1993; Pey et al., 1998
). 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, 2002
).
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., 1994; Riethmacher et al., 1995
).
Maintenance of compaction is dependent also on development of junctional complexes between apposing cells (for review, see Fleming et al., 2001). 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) and Gualtieri et al. (1992)
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., 1996
; Ghassemifar et al., 2003
).
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., 2005). Notwithstanding a role for epithelial-to-mesenchymal transformation in interactions between uterine cells and the trophoblast during implantation (for example, Thie et al., 1996
), 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., 1990
). 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., 2003
).
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Acknowledgements |
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Submitted on June 6, 2005; resubmitted on July 3, 2005; accepted on July 6, 2005.
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