1 Academic Unit of Obstetrics and Gynaecology, University of Manchester, St Mary's Hospital, Whitworth Park, Manchester M13 0JH, UK; and 2 Department of Obstetrics & Gynecology, University of Illinois at Chicago, 840 South Wood Street, Chicago IL 60612, USA3
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Abstract |
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Key words: baboon/decidua/endometrium/human chorionic gonadotrophin/pregnancy
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Introduction |
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Materials and methods |
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After excision, specimens of endometrium were carefully dissected and fixed in 3% (w/v) paraformaldehyde/1% (v/v) glutaraldehyde for 6 h at room temperature prior to postfixation in 1% osmium tetroxide and embedding in Araldite resin (Ladd Research Industries, Burlington, VT, USA). Some material was also embedded without post-fixation for immunocytochemical studies. Semi-thin sections 0.5 µm thick were cut on a Reichert OMUIII ultramicrotome and stained with toluidine blue and only those specimens with appropriate areas containing luminal epithelium were selected for ultra-thin sectioning. Three of the nine specimens were selected from group 1, five from group 2 and the specimens from groups 3, 4 and 5 for examination at the ultrastructural level. Pale gold sections were cut and mounted on copper grids, contrasted with uranyl acetate and lead citrate and examined in a Philips 301 electron microscope at an accelerating voltage of 60 kV. Some sections of selected non-osmicated specimens were cut at 0.75 µm, mounted on 3-aminopropyltriethoxy-silane (APES)-coated slides (Maddox and Jenkins, 1987) for immunocytochemistry. They were stained with monoclonal antibodies to cytokeratin (CAM 5.2; Becton Dickinson, San Jose, CA, USA) and
-smooth muscle actin (
-SMA; Dako, Carpinteria, CA, USA) as previously described (Murray and Verhage, 1985
), for examination at the light microscope level.
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Results |
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Cycling females treated with bioactive HCG (day 10 post-ovulation).
Light microscopy
Plaque formation (Figure 1b) was evident in all the specimens that contained luminal epithelium. The plaque reaction often affected the neck region of the uterine glands, also. Cells were greatly enlarged, forming nests, and often nuclei were pale and round, with mitoses evident in most of the specimens. The stromal cells were packed closely together, with little, if any, extracellular matrix visible between them.
Electron microscopy
The cells forming the plaque were greatly enlarged (Figure 3a) with oval, pale nuclei that contained dispersed chromatin and occasional mitotic figures. Prominent nucleoli, some with evidence of an internal channel-like substructure, were evident. Microvilli were generally sparse and rather short. Very few desmosomes were seen, and lateral cell membranes interdigitated with each other. Mitochondria were generally small and somewhat electron dense. Granules of glycogen were dispersed throughout the cytoplasm with only occasional masses seen basally. Round, dilated cisternae of rough endoplasmic reticulum and many Golgi bodies were evident, and smooth walled cisternae were also present in some areas; in some cells, secretory droplets were present, generally near the basal lamina but sometimes dispersed through the cytoplasm. In some sections, cells appeared to enfold each other, and some vacuoles containing cell debris were seen (Figure 3b
). The basal lamina was delicate and thin, and often difficult to detect due to the close apposition of the stromal cells which penetrated between nests of plaque cells with narrow processes (Figure 3c
).
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Electron microscopy
The apices of the columnar cells (Figure 5a) could be seen to bear microvilli of various sizes, sometimes with a feathery glycocalyx. Nuclei were oval and mainly euchromatic with a prominent nucleolus and heterochromatin dispersed peripherally. Mitochondria were rod-shaped and of moderate electron density, generally clustered at the basal regions of the cell, while the cisternae of rough endoplasmic reticulum showed some dilation. Several Golgi bodies were evident (Figure 5b
) and a few secretory droplets could be seen. The basal lamina was thin and the underlying extracellular matrix composed mainly of a flocculent, amorphous substance, with few collagen fibrils.
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Cycling female treated with recombinant FSH (day 10 post-ovulation).
Light microscopy
The luminal epithelium was composed of regular columnar cells (Figure 1d) under which was a somewhat oedematous stroma, very sparsely populated with cells in some areas but with a slightly greater density in others. The cells were generally spindle-shaped with a high nucleo:cytoplasmic ratio.
Electron microscopy
The luminal epithelium was composed of columnar cells with pale, euchromatic nuclei (Figure 6a), sometimes glycogen was seen basally. The mitochondria were rather electron lucent and sometimes branched, and there were small Golgi stacks (Figure 6b
). Cisternae of rough endoplasmic reticulum were present. Junctional complexes were seen uniting the apices of cells, and desmosomes were also evident between the lateral cell membranes, as well as interdigitations. The basal surface was smooth and even, and lay on a thin basement membrane.
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Female at day 15 of pregnancy
Light microscopy
This specimen exhibited a striking plaque response with nests of large, pale cells containing euchromatic nuclei interspersed between darker cells (Figure 1e). Mitotic figures were often seen. Dense stromal cells insinuated their processes between the nests of cells, sometimes almost reaching the luminal surface. There was an underlying stroma composed of densely packed cells and blood vessels in this area appeared to be somewhat dilated. No implantation site was evident in the area studied.
Electron microscopy
Cells of the plaque were greatly hypertrophied, forming nests basally and an irregular profile on the epithelial cell surface (Figure 7a). The plaque cells contained large, oval or slightly irregular, mainly euchromatic nuclei; occasional bi- or trinucleate cells were seen (Figure 7b
) and one example of a cell with four nuclei was present. Nucleoli were prominent and often showed signs of an internal channel-like substructure. Occasional very large nuclei were observed, suggestive of polyploidy (Figure 7c
). Mitochondria were generally small and stacks of Golgi cisternae not frequently observed (Figure 7a
), and secretory droplets were also present. Large vacuoles with contents of variable electron density were occasionally seen (Figure 7a,b
). Glycogen was seen dispersed through the cytoplasm (Figure 7a
) and also formed dense masses, especially near the basal surface (Figure 7d
); some fat droplets were present. Between the cells, junctional complexes were found apically with well-developed tonofilaments (Figure 7a
), and occasional desmosomes were also seen, but lateral membranes generally formed interdigitations with each other. The basement membrane was often difficult to detect due to the density of the underlying extracellular matrix and proximity of stromal cells. There was no evidence of any cyto- or syncytio-trophoblast in this area of the epithelium.
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Discussion |
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Early descriptions of the natural occurrence of plaque formation in pregnancy have been reviewed by Rossman who cites Selenka as first describing the phenomenon in 1900 and 1903 in several species of Old World (catarrhine) monkeys (Rossman, 1940). More recently, the plaque response has been reported in a variety of primates including the Dusky Leaf Monkey (Presbytis obscura) (Burton, 1980
), African Green monkey (Cercopithecus aethiops) (Owiti et al., 1986
) and the Rhesus monkey (Enders et al., 1985
; Enders and Schlafke, 1986
) as well as the New World monkeys (Moore et al., 1985
; Enders and Lopata, 1999
). Reports relating to the baboon have, however, been mixed (Luckett, 1974
): the plaque response was not seen in a study of the villous period in baboon pregnancy (1340 days) (Houston, 1969
) but was reported in a 20-day chacma baboon, Papio ursinus, lateral to the placental margin (Gilbert and Heuser, 1954
). More recently, remnants of plaque have been described in the baboon at days 14 and 17 of gestation (Jones et al., 2001
). It has also been pointed out (Enders et al., 1997
) that, in the baboon, plaque formation is never as extensive as that found in the Macaque and New World monkeys. The plaque response has been described at stage 5 of development (day 12 of pregnancy) in the baboon (Tarara et al., 1987
), but only at the light microscope level. In the baboon, the plaque is restricted to the epithelium immediately adjacent to the implantation site (Enders and Schlafke, 1986
; Tarara et al., 1987
; Jones et al., 2001
) whereas in the macaque it is much more extensive (Enders et al., 1983
, 1985
; Enders, 1991
). The large nuclei found in the plaque have been described by Enders and co-workers, who suggested that they were probably polyploid (Enders et al., 1985
), and in the present study very large nuclei as well as examples of bi-, tri- and multi-nucleate cells were observed. The nucleolar channels have also been described by Enders, who compared them to those seen in human uterine glandular cells on day 18 of the menstrual cycle (Enders, 1991
). Rossman, in his detailed histological study of deciduomata, illustrated examples of multinucleate giant cells as well as cells with extremely large nuclei, and discussed the possibility of cell fusion as well as cell degeneration (Rossman, 1940
). Evidence of phagocytosis, in the form of large vacuoles and the engulfment of whole cells, was also observed in the present study, suggesting that cell fusion may, on occasions, become lethal to one participant in the process.
The function of plaque formation is not clear; it is not involved in the actual process of implantation, that is, the penetration of the luminal epithelium by the blastocyst, but may provide nutrition by means of the intracellular glycogen (Rossman, 1940; Enders et al., 1985
). In this context, it is interesting to note that the Tarsier (Tarsius spectrum), descended from the epitheliochorial lemurs but too specialized in its placentation to be considered a forerunner of the Old and New World monkeys, has a type of plaque reaction involving the uterine glands which is thought to provide histiotrophic nutrition for the developing embryo (Hill, 1932
). The importance of histiotrophic nutrition with reference to the human embryo has recently been highlighted (Burton et al., 2001
) and it was suggested that glandular secretions may be taken up by the synctiotrophoblast and yolk sac epithelia prior to the establishment of haemiotrophic nutrition. It has also been suggested (Enders et al., 1985
) that the plaque response might stimulate vascular enlargement over a broad area extending beyond the developing placenta. This would bring about a precocious development of the maternal vasculature and so accelerate the development of the placenta as the lacunae communicate with the enlarged vascular bed.
In the baboon, the expression of -smooth muscle actin appears to be hormonally regulated; previous studies have shown it to be absent in the smooth muscle cells of the myometrium and blood vessels in ovariectomized animals, but to appear following oestrogen treatment (Christensen et al., 1995
). During the menstrual cycle, it is not found in the stromal fibroblasts but by day 14 of pregnancy it is apparent in stromal cells beneath the luminal epithelium, and can be demonstrated by immunocytochemistry as shown here. There is, coincident with this, a change in the morphology of the cells which develop the features of decidualisation, an attribute that is apparent within the first week after implantation (Enders, 1991
). They become larger in size with a more rounded profile and at the ultrastructural level many extensions and processes, which contain actin filaments, can be seen. There is also evidence of increased biosynthetic activity, with many strands of endoplasmic reticulum and Golgi saccules. These morphological observations confirm our previous biochemical evidence of cell specific gene expression in these stromal cells at the maternal-fetal interface (Tarantino et al., 1992
; Kim et al., 1999a
).
The decidual cells, i.e. those enlarged stromal cells showing changes associated with pregnancy, also produced increased amounts of extracellular matrix as seen by the dense material surrounding the cells under the luminal epithelium. This may coincide with an increase in collagen/laminin receptors together with their specific extracellular matrix molecules as has been observed in early pregnancy in the baboon (Fazleabas et al., 1997), with an increase in the
1,
3,
6, ß1 and
vß3 integrins as in the human. At this stage in pregnancy, the decidualized stromal cells are packed closely together, later however they become more spaced out and rounder, and a distinct pericellular basement membrane can be seen around each one from about day 40 of pregnancy ( Jones et al., 2001
). Thus, the increased expression of actin occurs in concert with changes in integrin expression and extracellular matrix secretion suggesting alterations in signal transduction pathways (Fazleabas et al., 1997
, 1999
; Kim et al., 1999b
). Similar changes in the actin cytoskeleton have previously been described in the differentiation of granulosa cells (Kranen et al., 1993
) and has been associated with cellular remodelling by actin-rich myofibroblasts in breast tumours (Brouty-Boye et al., 1991
). Such myofibroblasts have been shown to synthesise extracellular matrix components including collagen types I, III, V, fibronectin, vimentin and oncofetal fibronectin (Brouty-Boye et al., 1991
).
These studies show that the infusion of chorionic gonadotrophin can produce morphological changes indistinguishable from those observed in pregnancy, thus suggesting that the chorionic gonadotrophin (CG) produced by the blastocyst plays an important role in the response of the maternal tissues to pregnancy. In contrast, the substitution of FSH for CG produces no comparable biological response in the endometrium. This confirms in-vitro experiments with human endometrial gland cells which show them to be insensitive to FSH with respect to triggering the same downstream cascade (Zhou et al., 1999). Receptors for HCG have been identified in both glandular and luminal epithelium and in stromal cells in the human (Reshef et al., 1990
; Ziecik et al., 1992
) and in our model both glandular and stromal compartments respond in a dramatic way. Likewise, in-vitro studies have also shown stromal cells to be susceptible to the effects of CG (Moy et al., 1996
; Nemansky et al., 1998
; Han et al., 1999
). In our model, the plaque reaction produced by infusion of HCG affected the whole of the luminal epithelium in contrast to the in-vivo situation where epithelial plaque forms only adjacent to the implantation site. This suggests that there is a diffusion gradient of CG produced by the blastocyst, restricting its effect to a local response, compared with a more general effect induced by infusion of the hormone. Further studies are required to elucidate the mechanism whereby these responses occur.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 29, 2001; accepted on August 21, 2001.