University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cell aggregate/human/immunocytochemistry/placental bed giant cell/ultrastructure
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The origin of placental bed giant cells has been much disputed (Boyd and Hamilton, 1970), and has still to be established conclusively. Various investigators have described them as multilobular and multinuclear decidual cells rather than trophoblast cells (Wynn, 1967
; Boyd and Hamilton, 1970
). Robertson and Warner (1974) suggested that they are derived from the syncytiotrophoblast, while other investigators are of the opinion that they are formed by mitosis without cytokinesis of the interstitial trophoblast (Avery and Hunt, 1969
; Pijnenborg et al., 1981
; Graham et al., 1992
), and others believe that they may be formed as a result of homograft reaction at the placental bed (Park, 1965
). It has also been suggested that such cells are a result of endoreduplication, that is, replication of the nuclear material without subsequent mitosis and cell division (Zybina, 1961
). However, most embryologists agree that the mutinucleated condition is established as a result of cell fusion (Boyd and Hamilton, 1970
; Hunt and Avery, 1971
; Kliman et al., 1986
), although direct evidence to support this theory is lacking.
The most obvious evidence of cell fusion during trophoblast formation in other primates is the presence within the cytoplasm of segments of parallel cell membranes, the standard intercellular distance apart but fused at their margins, thus isolating within the cytoplasm a small portion of former intercellular `space' (Schlafke and Enders, 1975). Other features of cell fusion include gap junctions and remnants of desmosomes (Cavicchia, 1971). Gap junctions are specialized cell membrane domains consisting of clusters of channels called connexons (CX). Ultrastructural studies have shown gap junctions to be pentalaminar structures with a 2 nm gap, found between cells of the trophoblastic layers of the haemochorial placentae of rabbits, rats and mice (Metz et al., 1976
). They have been implicated in the process of giant cell formation by cell fusion in the guinea pig (Firth et al., 1980
), and have been observed between syncytiotrophoblast and cytotrophoblast cells in the human villous placenta (De Virgiliis et al., 1982
). Gap junctions link the cytoplasmic compartments of neighbouring cells, forming a pathway for direct exchange of ions and small molecules (Yeh and Kurman, 1989
). At least 12 different mammalian CX have been identified, of which two, CX32 and CX43, have been shown to be expressed in the rat endometrium (Yeh and Kurman, 1989
), and CX26 and CX43 in the rat decidua during trophoblast invasion (Winterhager et al., 1993
). In-vitro studies have demonstrated that the permeability, conductance and other properties of gap junctional channels depend on the make-up of their component connexins (Veenstra et al., 1995
). The possibility therefore exists that the expression of CX32 and CX43 on trophoblast cells may play an important role in their functional differentiation in different zones of the human endometrium, and in the formation of giant cells.
Desmosomes have also been linked to the formation of placental bed giant cells (Cronier et al., 1994). Staining for desmosomes has been reported at intercellular boundaries in aggregates of cytotrophoblastic cells isolated from normal-term placentas, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous areas of the syncytiotrophoblast (Cronier et al., 1994
).
Ultrastructural studies on human placental bed giant cells in early pregnancy are still lacking due to the difficulties in isolating the implantation site and in identifying the giant cells. In the present study, we have attempted to: (i) describe the fine structure of placental bed giant cells in the early human placental bed using transmission electron microscopy of resin-embedded sections; and (ii) examine the mode of formation and function of giant cells using confocal microscopy of formaldehyde-fixed, paraffin-embedded sections immunostained with antibodies to cytokeratin, CX32 or CX43 and placental proteins.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cryostat sections (5 µm thickness) from each batch of tissue were stained with a monoclonal antibody against cytokeratin to confirm the presence of trophoblast cells. On this basis, the decidua was classified as decidua basalis if staining for cytokeratin was present, or decidua parietalis if it was absent.
Light microscopy
Single immunostaining for cytokeratin
All incubations were carried out in a humid atmosphere, at room temperature. Paraffin sections of placental bed were de-waxed in xylene, rehydrated in an ascending series of ethanol solutions and washed thoroughly in running tap water, then in deionized water. Sections were then incubated with 50 µg/ml Proteinase-K (Boehringer Mannheim, Lewes, East Sussex, UK) for 10 min and washed thoroughly with Tris buffer pH 7.5. They were then incubated with 0.5% bovine serum albumin (BSA; Sigma Chemicals Ltd, Poole, Dorset, UK) for 10 min. Excess fluid was removed and sections were incubated for 30 min with rabbit polyclonal anti-cytokeratin antibody diluted 1:100 in 0.5% BSA in Tris buffered saline (TBS). After thoroughly rinsing in Tris buffer, sections were incubated with anti-rabbit fluoro-isothiocyanate (FITC; Dakopatts Ltd, High Wycombe, Bucks, UK) diluted 1:150 in 0.5% BSA for 30 min, or with a biotinylated anti-rabbit antibody diluted 1:100 in 0.5% BSA for 30 min, and then with a solution of Vectastain Elite ABC avidinbiotinperoxidase (Vector Laboratories Ltd, Bretton, Peterborough, UK). After thoroughly rinsing in Tris buffer and double-distilled water, the sections for fluorescent microscopy were mounted in Citifluor mounting medium and examined immediately. The sections labelled with peroxidase were incubated with 1 mg/ml 3',3-diaminobenzidine (DAB; Dako Limited, Ely, Cambs, UK) plus 0.03% (v/v) H2O2, dehydrated in ethanol, cleared in xylene and mounted in DPX.
Combined labelling for gap junction proteins (CX32 and CX43) or human placental lactogen (HPL) or human chorionic gonadotrophin (-HCG)
Following immunostaining for cytokeratin, some sections were incubated with 0.5% BSA in Tris buffer for 10 min at room temperature. Excess fluid was removed and the sections were incubated with mouse monoclonal anti-CX32 (Chemicon International Ltd, Harrow, UK) or CX43 (Chemicon International Ltd) diluted 1:1000 in 0.5% BSA overnight at 4°C or mouse monoclonal HPL (Sigma) or mouse monoclonal -HCG (Sigma) diluted 1:100 in 0.5% BSA for 30 min at room temperature. After thoroughly rinsing in Tris buffer, the sections were incubated with anti-mouse Texas red (Sigma) diluted 1:150 in 0.5% BSA for 30 min at room temperature. They were then rinsed in Tris buffer, thoroughly washed in double-distilled water, and mounted in Citifluor (Agar Scientific Ltd, Stansted, Essex, UK) before viewing in a Leica TCS-NT confocal microscope (Leica Microsystems Ltd, Knowlhill, Milton Keynes, UK). Negative controls included omission of primary antibody and positive controls of human myocardium were stained for CX43 and rat liver for CX32. Adjacent sections stained with haematoxylin and eosin were examined for comparative histology.
Ultrastructural study
Tissues were fixed in 2% glutaraldehyde in 0.1 M PIPES buffer pH 7.4 for 1 h at 4°C. They were washed in three changes of 0.1 M PIPES buffer pH 7.3, post-fixed in 1% osmium tetroxide in 0.1 M PIPES at pH 7.4 containing 1.5% potassium ferricyanide and 2 mmol/l calcium chloride for 1 h at room temperature. Tissues were then washed in 0.1 M PIPES buffer pH 7.4 for 2 min followed by 0.05 M sodium maleate buffer, pH 5.2 for 10 min at 20°C, and then immersed in 2% uranyl acetate in 0.05 M sodium hydrogen maleate buffer for 1 h. After rinsing in deionized water, tissues were dehydrated in an ascending series of ethanol solutions, treated with propylene oxide (two changes of 15 min each), and transferred to a 50:50 mixture of propylene oxide and Spurr's resin (TAAB Laboratories Ltd, Aldermaston, Berks, UK). After an overnight agitation, tissues were treated for 8 h in 25:75 propylene oxide and Spurr's resin, and four changes of 100% Spurr's resin over 36 h. They were next transferred to latex moulds filled with resin and thermally cured at 60°C for 48 h. Sections (0.51 µm thickness) were cut using a Reichart Ultracut S and stained with methylene blue for light microscopy. Areas of interest were identified and areas of necrosis selectively avoided. Ultra-thin sections of about 4060 nm (showing silver to silver-grey interference colours) were cut and mounted on copper grids. These were double stained with uranyl acetate and lead citrate for 30 s each and examined in a Philips CM100 electron microscope at an accelerating voltage of 80 kV.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental evidence of a role for cell-to-cell communication via gap junctions in the control of cell proliferation and differentiation is well established (Lo and Gilula, 1979; Loewenstein and Rose, 1992
). It has been suggested by Firth et al. (1980) that gap junctions connecting different layers of the trophoblast (cytotrophoblast and syncytiotrophoblast) could play a role in trophoblast development as a starting point for cellular fusion. One of the most important morphological features in this present study is the presence of gap junctions and junctional complexes between the mononuclear cells within cell aggregates, suggesting the potential of cell-to-cell communication. CX43 was also observed in abundance in the decidual stroma. This finding is in keeping with that of Winterhager et al. (1993), who demonstrated expression of CX43 in the rat endometrium during early pregnancy. The gap junctions may modulate cytoplasmic exchange between cells within an aggregate, or mediate the formation of morphogenetic gradients (Lo and Gilula, 1979
). The presence of gap junctions on the trophoblast cells within aggregates and their absence on the single large trophoblast cells implies a process of internalization following coalescence of individual cells. Previous studies have reported gap junctions to be internalized by endocytosis for lysosomal degradation (Severs et al., 1996
) and ubiquitin-mediated proteosomal proteolysis (Laing and Beyer, 1995
). In addition, in-vitro studies have reported gap junctions to precede fusion (Cronier et al., 1994
), and hence they may play an important role in giant cell formation.
Intercellular fusion is a rapid process, and thus is difficult to visualize on static images. However, one may infer the occurrence of such an event by the presence of intracytoplasmic cell structures such as the persistence of residual cell partitions and remnants of intracytoplasmic desmosomes. The electron microscopy results presented here show no evidence of intercellular fusion equivalent to that which occurs during formation of the syncytium (Boyd and Hamilton, 1960). There was no evidence of remnants of intracytoplasmic desmosomes or mitotic figures in the multinucleated giant cells to suggest either cell fusion or division. Thus, although strongly supportive, our results cannot provide conclusive evidence of cell fusion as the origin of giant cells.
The fine structure of placental bed giant cells suggests that they are not totally effete or passive cells (Robertson, 1987), but are involved in at least some aspects of synthetic activity. The numerous polyribosomes and mitochondria, abundant rough endoplasmic reticulum, extensive Golgi complex and cytoplasmic vesicles are indicative of protein synthesis. Giant cells have long been linked with the production of enzymes and elaboration of hormones (Boyd and Hamilton, 1960
), and our findings indicate they may elaborate protein hormones such
-HCG and HPL. This is in good agreement with Kurman et al. (1984), but contradicts the findings of Gosseye and Fox (1984) and Loke and King (1995). This discrepancy may be due to the different fixation methods used, and to the sensitivities of the immunolabelling techniques employed. Immunocytochemical studies cannot demonstrate that the products synthesized are secreted, but it is interesting that Pedersen et al. (1998) have recently reported the presence of HPL in maternal serum during normal pregnancy, and its significant decrease in diabetic mothers.
In-vitro studies of human trophoblast cells have shown them to aggregate and subsequently fuse to form a syncytium in which placenta-specific hormones such as human somatotrophin, HCG and HPL have been detected (Cronier et al., 1994; Richards et al., 1994
). HCG has also been reported to promote the expression of cadherin, a cell adhesion molecule that facilitates cellular aggregation (Shi et al., 1993
) and gap junctional communication (Cronier et al., 1994
). Thus, from our observations here and the descriptions in the literature, we suggest that cell fusion may be stimulated and reinforced by autocrine and paracrine pathways.
Unlike the villous syncytiotrophoblast, the placental bed giant cells express HLA-G class I major histocompatibility complex antigens (Loke et al., 1997). The HLA-G on giant cells may facilitate interaction with the maternal cells in the placental bed. This interaction may lead to some cells undergoing programmed cell death (apoptosis) (Al-Lamki et al., 1998
), while others may undergo terminal differentiation to giant cells. Giant cells have been shown to produce protease and protease inhibitors (Sasagawa et al., 1987
), leucine amino-peptidase (Loke and Butterworth, 1987
) and have lectin-binding affinity (Earl et al., 1990
).
It therefore seems likely that in early placental development, there are at least two possible fates for the interstitial extravillous trophoblast cells migrating into the endometrium; apoptosis and giant cell transformation. While both processes may serve to limit trophoblast invasion, the transformation of trophoblast cells into isolated masses of syncytium may also ensure adequate local production of hormones and other products that are critical to maintaining a normal pregnancy.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avery, G.B. and Hunt, C.V. (1969) The differentiation of trophoblast giant cells in the mouse studied in kidney capsule grafts. Transplant. Proc., 1, 6166.[ISI][Medline]
Boyd, J.D. and Hamilton, W.J. (1960) The giant cells of the human pregnant uterus. J. Obstet. Gynaecol. Brit. Emp., 67, 208218.[Medline]
Boyd, J.D. and Hamilton, W.J. (1970) The Human Placenta. W. Heffer, Cambridge.
Cavicchia, J.C. (1971) Junctional complexes in the trophoblast of the human full term placenta. J. Anat., 108, 339346.[ISI][Medline]
Cronier, L., Bastide, B., Hervé, J.C. et al. (1994) Gap junctional communication during human trophoblast differentiation: influence of human chorionic gonadotrophin. Endocrinology, 135, 402408.[Abstract]
De Virgiliis, G., Sideri, M., Fumagalli, G. et al. (1982) The junctional pattern of the human villous trophoblast: a freeze fracture study. Gynecol. Obstet. Invest., 14, 263272.[ISI][Medline]
Earl, U., Estlin, C. and Bulmer, J.N. (1990) Fibronectin and laminin in early human placenta. Placenta, 11, 223231.[ISI][Medline]
Firth, J.A., Farr, A. and Bauman, K. (1980) The role of gap junctions in trophoblastic cell fusion in the guinea-pig placenta. Cell Tissue Res., 205, 311318.[ISI][Medline]
Gosseye, S. and Fox, H. (1984) An immunohistological comparison of secretory capacity of villous and extravillous trophoblast in the human placenta. Placenta, 5, 329348.[ISI][Medline]
Graham, C.H., Lysiak, J.J., McCrae, K.R. et al. (1992) Immunolocalisation of transforming growth factor-ß at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol. Reprod., 46, 561572.[Abstract]
Hunt, C.V. and Avery, G.B. (1971) Increased level of deoxyribonucleic acid during trophoblast giant-cell formation in mice. J. Reprod. Fertil., 25, 8591.[Medline]
Kliman, H.J., Nestler, J.E., Sermasi, E. et al. (1986) Purification, characterisation, and in vitro differentiation of cytotrophoblast cells from human term placentae. Endocrinology, 118, 15671582.[Abstract]
Kurman, R.J., Young, H.J.N., Colleen, S. et al. (1984) Immunocytochemical localisation of placental lactogen and chorionic gonadotrophin in the normal placenta and trophoblastic tumors, and the placental site trophoblast tumor. Int. J. Gynecol. Pathol., 3, 101121.[ISI][Medline]
Laing, J.G. and Beyer, E.C. (1995) The gap junction protein connexin 43 is degraded via ubiquitin proteasome pathway. J. Biol. Chem., 270, 2639926403.
Lo, C. and Gilula, N.B. (1979) Gap junctional communication in the preimplantation mouse embryo. Cell, 18, 399409.[ISI][Medline]
Loewenstein, W.R. and Rose, B. (1992) The cellcell channel in the control of growth. Semin. Cell Biol., 3, 5979.[Medline]
Loke, Y.W. and Butterworth, B.H. (1987) Heterogeneity of human trophoblast population. In Gill, T.J. and Wegmann, J.G. (eds), Immunoregulation and Fetal Survival. Oxford University Press, New York, pp. 197209.
Loke, Y.W. and King, A. (1995) Human Implantation. Cell Biology and Immunology. Cambridge University Press, Cambridge, UK.
Loke, Y.W., King, A., Burrows, T. et al. (1997) Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antibodies, 50, 135146.
Metz, J., Heinrich, D. and Forrssman, W.G. (1976) Gap junctions in hemodichorial and hemotrichorial placenta. Cell Tissue Res., 171, 305315.[ISI][Medline]
Park, W.W. (1965) Cellular events at the feto-maternal junction. In Park, W.W. (ed) The Early Conceptus Normal and Abnormal. Livingstone, Edinburgh, pp. 7477.
Pedersen, J.F., Sorensen, S. and Molsted-Pedersen, L. (1998) Serum levels of human placental lactogen, pregnancy-associated plasma protein A and endometrial secretory protein PP 14 in first trimester of diabetic pregnancy. Acta Obstet. Gynaecol. Scand., 77, 155158.[ISI][Medline]
Pijnenborg, R., Dixon, G., Robertson, W.B. et al. (1981) The pattern of interstitial trophoblast invasion of the myometrium in early human pregnancy. Placenta, 2, 303316.[ISI][Medline]
Richards, R.G., Hartmena, S.M. and Handwerger, S. (1994) Human cytotrophoblast cells cultured in maternal serum progress to a differentiated syncytial phenotype expressing both human chorionic gonadotrophin and human placental lactogen. Endocrinology, 135, 321329.[Abstract]
Robertson, W.B. (1987) Pathology of the pregnant uterus. In Fox, H. (ed) Haines and Taylor Obstetrical and Gynaecological Pathology. Churchill Livingstone, Edinburgh, pp. 11491176.
Robertson, W.B. and Warner, B. (1974) The ultrastructure of the human placental bed. J. Pathol., 112, 203211.[ISI][Medline]
Sasagawa, M., Yamazaki, T., Endo, M. et al. (1987) Immunohistochemical localisation of HLA antigens and placental proteins (hCG, ßhCG, CTP, hPL and SP1) in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast. Placenta, 8, 515528.[ISI][Medline]
Schlafke, S. and Ender, A. (1975) Cellular basis of interaction between trophoblast and uterus at implantation. Biol. Reprod., 12, 4165.[ISI][Medline]
Severs, N.J., Dupont, E., Kaprielian, R.R. et al. (1996) Gap junctions and connexins in the cardiovascular system. In Yacoub, M.H., Carpentier, A., Pepper, J. and Fabiani, J.-N. (eds), Annual of Cardiac Surgery. 9th edn. Current Science, London, pp. 3144.
Shi, Q.J., Lei, Z.M., Rao, C.V. et al. (1993) Novel role of human chorionic gonadotrophin in differentiation of human cytotrophoblasts. Endocrinology, 132, 13871395.[Abstract]
Veenstra, R.D., Wang, H.K., Beblo, D.A. et al. (1995) Selectivity of connexin-specific gap junctions does not correlate with channel conductance. Circ. Res., 77, 11561165.
Wells, M. and Bulmer, J.W. (1988) The human placental bed. Histology, immunocytochemistry and pathology. Histopathology, 13, 483498.[ISI][Medline]
Winterhager, E., Grummer, R., Willecke, K. et al. (1993) Temporal expression of connexin-26 and connexin-43 in rat endometrium during trophoblast invasion. Dev. Biol., 157, 399409.[ISI][Medline]
Wynn, R.M. (1967) Cellular Biology of the Uterus. Appleton-Century-Crofts, New York.
Yeh, I.T. and Kurman, R.J. (1989) Functional and morphological expression of trophoblast. Lab. Invest., 61, 13.[ISI][Medline]
Zybina, E.V. (1961) Endomitosis and polyteny of the giant cells of trophoblast. Dokl. Acad. Nauk SSR, 140, 11771180.
Submitted on April 15, 1998; accepted on October 22, 1998.