1 Institute of Reproductive and Developmental Biology, Wolfson & Weston Research Centre for Family Health, Hammersmith Hospital, Du Cane Road, London W12 0NN, 2 Electron Microscopy Unit, Department of Histopathology, 6th Floor, Laboratory Block, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
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Abstract |
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Key words: co-culture in vitro/EVT/feto-maternal interface/fusion of plasma membranes
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Introduction |
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The invasive cytotrophoblast cells, commonly known as extravillous trophoblast (EVT) cells, follow two distinct pathways of differentiation (Pijnenborg et al., 1980). In the first pathway, EVT cells invade the decidualized endometrium, as well as the inner third of the myometrium. Some of these EVT cells further differentiate by fusing with one another to form the giant cells of the placental bed. It is this spatial confinement of trophoblast cells to the decidualized endometrium and the inner third of the myometrium that distinguish them from invasive cancer cells (Fisher and Damsky, 1993
). In the second pathway, EVT cells invade the uterine spiral arteries and adopt a vascular phenotype (Zhou et al., 1997
). This involves replacement of the vascular smooth muscle cells and endothelium by trophoblast, and the formation of distensible vessels for the provision of large volumes of blood to the feto-maternal interface. The initial remodelling of the uterine spiral arteries seems to involve cells migrating through the decidua to the target vessels (Pijnenborg et al., 1980
), and occurs within the first trimester of pregnancy. This is limited to the decidual portion of these vessels. Remodelling of the inner myometrial portion occurs at 1418 weeks gestation and is mediated by intravascular migration of EVT cells (Pijnenborg et al., 1980
). In some abnormalities of pregnancy such as pre-eclampsia, trophoblast invasion of the uterine spiral arteries does not proceed beyond the decidual portions of the spiral arteries (Brosens et al., 1972
; Moodley and Ramsaroop, 1989
). Conversely, in other pathological conditions of pregnancy, trophoblast invasion of the decidua and the myometrium becomes excessive, resulting in placenta accreta (Hustin et al., 1990
).
The fate of cytotrophoblast cells in the first trimester `floating villi' differs from those of the anchoring villi. In the floating villi, cytotrophoblast cells exist as polarized epithelial monolayers anchored to a basement membrane surrounding a mesenchymal stromal core containing fetal blood vessels (Boyd and Hamilton, 1970; Benirschke and Kaufmann, 1990; Aplin, 1991
). These cytotrophoblast cells are highly proliferative and differentiate to form the outer syncytial covering of the villous tree (Boyd and Hamilton, 1966
; Muhlhauser et al., 1993
). Unlike the anchoring villi, the floating villi do not come in contact with the uterine wall but are bathed in maternal blood, thus forming the placental barrier where the exchange of oxygen, nutrients and waste products occurs between the mother and the fetus.
Models of the feto-maternal interface (Vicovac et al., 1995; Babawale et al., 1996
) have shown that floating villi can readily form structures resembling anchoring villi when placed in contact with appropriate substrates, and these villi retain the potential to differentiate. This study has assessed the ultrastructural features of the interface formed in vitro, and compared these findings with previous studies on the feto-maternal interface of human pregnancy.
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Materials and methods |
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Incubation method
All incubations were carried out in 24-well culture plates (Corning Ltd, High Wycombe, UK) using RPMI 1640 culture medium supplemented with 10% fetal calf serum, 2 mmol/l L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin (Sigma-Adrich). From each of six patients, four co-cultures were set up, corresponding to 24, 48, 72 and 96 h of culture. Cultures were incubated in an atmosphere of 5% CO2:95% air at 37°C and set up as follows: a piece of villous tissue (0.5 mm3) was placed on decidual explant (2 mm3) already plated on a few drops of culture medium left undisturbed for ~30 min. Both tissue samples were from the same patient. Thereafter, 2 ml culture medium was added carefully to each culture chamber. Tissue samples were removed for processing at 24 h intervals and the culture medium in the remaining culture chambers was changed daily. Four control cultures of decidual explants alone (without the placental villi) were set up from each of the six patients, corresponding to 24, 48, 72 and 96 h of culture as per the experiment.
Tissue processing for transmission electron microscopy (TEM)
Co-cultured samples (24 samples) and control samples (24 samples) were fixed in 3% glutaraldehyde in cacodylate buffer (pH 7.2) for 30 min, post-fixed in 1% osmium tetroxide in cacodylate buffer (pH 7.2), dehydrated through ascending grades of alcohol and embedded in Araldite. Semi-thin sections were cut at 1 µm and stained in 1% Toluidine Blue in order to identify areas of interest for viewing on the electron microscope. Ultra-thin sections (6080 nm) were taken from the selected areas, stained with uranyl acetate and lead citrate. The stained sections were then examined and areas of interest photographed using a Hitachi electron microscope, model HU12A, at an accelerating voltage of 75 kV.
Immunocytochemistry/cytokeratin immunodetection
Immunolabelling of trophoblast cells was carried out with the anti-cytokeratin antibody CAM 5.2 (International Cancer Research Fund, London, UK) using the mouse avidinbiotin indirect immunoperoxidase detection method on Araldite-embedded co-cultured placental tissue blocks sectioned at 1 µm thickness. Slides were placed in sodium ethoxide for 30 min to remove the Araldite (etching procedure) after which endogenous peroxidase activity was blocked using 3% hydrogen peroxide in tap water for 30 min at room temperature. The slides were then placed in 0.1% trypsin (Sigma-Adrich) in 0.1% CaCl2, pH 7.8 at 37°C for 10 min and then rinsed in PBS.
Serum blocking was carried out with 5% normal goat serum for 15 min to eliminate non-specific binding. The tissue sections were then incubated in CAM 5.2 at 1/10 dilution overnight at 4°C. Non-immune mouse serum was substituted for CAM 5.2 in tissue sections that served as the negative control. The sections were thereafter rinsed in 3x5 min changes of PBS and incubated with biotinylated goat anti-mouse secondary antibody (Dako, High Wycombe, UK) at 1/500 dilution for 1 h. After 3x5 min further changes of PBS, the sections were incubated with peroxidase-labelled streptavidin 1/500 (Boehringer, Lewes, UK) for 1 h. A final rinse of 3x5 min changes of PBS was carried out. Peroxidase activity was demonstrated by developing the sections in 0.05% 3,3'diaminobenzidine solution (DAB; Dako), with 0.03% hydrogen peroxide as substrate.
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Results |
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Discussion |
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The tissues comprising the feto-maternal interface showed the anticipated structures in this study. The decidua contained no low-molecular weight cytokeratin-positive cells in the stromal compartment (Figure 4), and the placental villi showed a multi-layered structure of mesenchymal core, basal lamina, cytotrophoblast and syncytium (Figures 5 and 9
). Differentiation of cytotrophoblast into syncytium appears to involve fusion, dissolution, or both of the intervening plasma membranes between the cells (Figures 7 and 8
). These findings are consistent with earlier work, and indicate that they occur in vitro as a dynamic process to maintain the syncytial structural integrity and function (Enders, 1965
; Boyd and Hamilton, 1966
; Morrish et al., 1997
; Mayhew et al., 1999
).
Previous studies have shown that desmosomal proteins are expressed in human placenta and trophoblast, and these may be involved in the functional differentiation of human and guinea pig trophoblast (Firth et al., 1980; Douglas and King, 1990
; Winterhager et al., 1999
, 2000
; Cronier et al., 2001). The presence of desmosomes within the cell column (Figure 11
) suggests that cell adhesion was well maintained between the cells in culture. There was extensive cellcell contact between the trophoblast cell column and decidual cells (Figures 13, 11,13
). This was particularly apparent in Figure 13
, and seems to show desmosomes and the associated intermediate filaments connecting trophoblast and decidual cells. Further studies are needed to confirm this unexpected finding but, if correct, this is a clear indication of the lack of reaction between these genetically distinct cells. The expression of integrins changes within trophoblastic cell columns as they differentiate and invade either the decidua or the blood vessels (Damsky et al., 1994
; Vicovac et al., 1995
; Vicovac and Aplin, 1996
) and it is possible that the desmosomes, in their role in maintaining adequate cellcell adhesion, may be involved in this process.
Previous work has shown that all cytotrophoblast cells express low-molecular weight cytokeratins (Khong et al., 1986; Aplin et al., 1999
). Not all cytotrophoblast expressed CAM 5.2 in the semi-thin sections used for immunohistochemistry (Figure 3
), which was unexpected, as our earlier work on this model showed general positive staining (Babawale et al., 1996
). We attribute the apparent incompatibilities to the more rigorous procedures needed for semi-thin methodology, which affects the detection limit so that only cells with high levels of cytokeratin can be visualized.
Differentiation of cytotrophoblast to form cell columns and invasive EVT was clearly documented (Figures 13 and 1011). The process of trophoblastic cell invasion does not seem to damage the decidual structures (Figures 1013
), but rather there is continued healthy contact between invading trophoblast, decidual cells and decidual matrix. Some of the factors implicated in regulation have already been identified (matrix metalloproteinase-2 and -9, tissue inhibitors of metalloprotineases-1 and -2) (Librach et al., 1991
; Pollete et al., 1994
; Babawale et al., 1995
; Bischof et al., 1998
) and the co-culture model may be a useful technique to explore the roles of these factors further. Invasion is obviously an active process, and is consistent with the depletion of glycogen storage as well as the presence of an active nucleoli and lamellipodic processes in some of the trophoblast cells observed (Figures 11 and 14
). The nature of the lamellae observed (Figure 12
) requires further investigation, as such structures are not common in connective tissue.
In summary, our findings indicate that this model has features at the cellular and sub-cellular levels that resemble those observed in vivo in human pregnancy, and support our observations that this model is useful for the study of human placentation.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed. E-mail: mark.sullivan{at}ic.ac.uk
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References |
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Submitted on July 12, 2001; resubmitted on November 19, 2001; accepted on January 7, 2002.