Ultrastructure of epithelial plaque formation and stromal cell ...

Human Reproduction Vol.16, No.12 pp. 2680–2690, 2001

Ultrastructure of epithelial plaque formation and stromal cell transformation by post-ovulatory chorionic gonadotrophin treatment in the baboon (Papio anubis) C.J.P.Jones1,3 and A.T.Fazleabas2 1Academic

Unit of Obstetrics and Gynaecology, University of Manchester, St Mary’s Hospital, Whitworth Park, Manchester M13 0JH, UK; and 2Department of Obstetrics & Gynecology, University of Illinois at Chicago, 840 South Wood Street, Chicago IL 60612, USA

3To

whom correspondence should be addressed.

BACKGROUND: To understand factors controlling endometrial responses to pregnancy, we have established a model using the baboon and examined the effects of infused human chorionic gonadotrophin (HCG) on the preparation of the luminal epithelium and stromal cell differentiation for the establishment of pregnancy. METHODS: The ultrastructure of endometrium from normal day 10 post-ovulation animals, cycling females treated with either HCG or FSH (control), and a day 15 pregnant animal has been compared. RESULTS: In the control endometrium, the luminal epithelium was smooth and regular, with underlying spindle shaped stromal cells. In pregnancy, the luminal epithelium underwent a plaque reaction, while stromal cells enlarged and developed filamentrich cell processes. Infusion of HCG produced changes similar to those seen in pregnancy, with generalized plaque formation and stromal decidualization, while in the animal treated with FSH there was no response. CONCLUSIONS: This study indicates that infusion of HCG into the uterus can duplicate many of the responses of the endometrium to pregnancy, although in this case the plaque reaction involved the whole of the luminal epithelium, rather than only the implantation site as in pregnancy. Key words: baboon/decidua/endometrium/human chorionic gonadotrophin/pregnancy

Introduction The factors controlling endometrial responses to implantation and pregnancy in primates are not well understood. In the human, manipulation of hormonal signals, to separate those from the implanting embryo from those produced endogenously in the uterus, is not possible. We have therefore established the baboon as a primate model instead, for these types of studies. By infusing recombinant human chorionic gonadotrophin (HCG) we have been able to examine the effects of this hormone, normally produced by the blastocyst, on the luminal epithelium and the process of decidualization in preparation for pregnancy (Fazleabas et al., 1999). In this study we compare specimens of endometrium from normally cycling day 10 post-ovulation animals with tissue from animals treated with chorionic gonadotrophin, with FSH as a control, and from a day 15 pregnant animal. The aim of this investigation is to examine the ultrastructural effects of exogenous chorionic gonadotrophin and to compare it with the endometrium of the normal cycling baboon and that from a 15 days pregnant animal. In this way, it is hoped to elucidate the role that embryonic signals, in the form of HCG, play in modulating a receptive endometrium. The uterus is receptive to implantation between days 8 and 10 in the baboon, while the blastocyst 2680

arrives in the uterus at day 6 and begins to secrete HCG shortly afterwards. The corpus luteum also becomes responsive at day 6. Because it was necessary to separate out the effects of paracrine embryonic factors from those of an attachment reaction, day 15 of pregnancy was selected for our studies as this was the earliest time point that an implantation site can be obtained with confidence.

Materials and methods Uterine tissue was obtained from the following adult female baboons (Papio anubis) either at hysterectomy or endometriectomy: (i) Normally cycling females on day 10 post-ovulation (n ⫽ 9); (ii) cycling females treated with bioactive recombinant human chorionic gonadotrophin (HCG; Serono Laboratories, Norvell, MA, USA) (n ⫽ 9) or (iii) with heat-inactivated (30 min boiling) HCG (n ⫽ 1); (iv) one normally cycling female treated with Alzet-delivered FSH from day 6 post-ovulation till tissue collection on day 10; (v) a female at day 15 of pregnancy. The HCG treatment protocol has previously been described (Christensen et al., 1995; Fazleabas et al., 1999) and involves infusion of HCG between days 6 and 10 post-ovulation via a cannula attached to an Alzet minipump. All experimental procedures were approved by the Animal Care Committee of the University of Illinois, Chicago. © European Society of Human Reproduction and Embryology

EM of baboon endometrium after HCG infusion

Figure 1. Semi-thin 0.5 µm sections stained with toluidine blue showing the luminal epithelium in (a) a normal cycling baboon at day 10 post-ovulation with a regular epithelium and spindle-shaped stromal cells; (b) after infusion of HCG there is a pronounced plaque response with hypertrophic epithelial cells and closely packed stroma; (c) Infusion of heat-inactivated HCG shows no response; (d) FSH is not biologically active and the epithelium and stromal cells resemble those of the control; (e) In a 15 day pregnant animal there is a large plaque response and decidualisation of the stromal cells. Note: all the figures are printed at the same magnification. Scale bar: 50 µm.

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-aminopropyltriethoxysilane (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 α-smooth muscle actin (α-SMA; Dako, Carpinteria, CA, USA) as previously described (Murray and Verhage, 1985), for examination at the light microscope level.

Results Normally cycling female at day 10 post-ovulation. Light microscopy The luminal epithelium was regular, composed of columnar epithelial cells (Figure 1a), some of which showed apical protrusions. In some specimens, deposits of glycogen, distinguished by their deep blue coloration with toluidine blue, could be seen in the cells. The underlying stromal cells were elongated or spindle shaped, dispersed through the stroma. Occasional granular cells could be seen near the epithelial layer. In one case, the stromal cells were densely packed together but this was focal and in other areas the cells were more diffusely distributed.

Electron microscopy The luminal epithelium was composed of regularly aligned cells with domed apices adorned with short microvilli (Figure 2a). Junctional complexes were present at the apices of the cell borders and occasional desmosomes along the lateral cell borders. In some cases, interdigitations helped to bind cells together. Nuclear morphology differed between specimens, being oval and euchromatic in one case and more heterochromatic in others. Deposits of glycogen were present in the cell: In one specimen these were quite pronounced basally whereas elsewhere they were finely dispersed. Several small stacks of Golgi cisternae were present (Figure 2b). The basal surface of the epithelium was smooth and rested on a thin basal lamina, beneath which was an extracellular matrix that was mainly fluffy and amorphous with some bundles of collagen fibres (Figure 2c). Stromal cells were generally spindle-shaped and well spaced out, orientated parallel to the luminal surface (Figure 2c). Intercellular contacts were maintained via fine cell processes. Nuclei were generally oval or with a slightly irregular profile, with peripheral and dispersed clumps of heterochromatin. Narrow, and occasionally somewhat distended cisternae of rough endoplasmic reticulum and small Golgi stacks were evident, and fat droplets were occasionally seen. Mitochondria were generally small and narrow. Cycling females treated with bioactive HCG (day 10 postovulation). 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. 2681

C.J.P.Jones and A.T.Fazleabas

Figure 2. Normal cycling animals at day 10 post-ovulation. (a) Low power view of columnar epithelial cells with domed apices bearing microvilli. Masses of glycogen can be seen in some cells above the nuclei (g). Junctional complexes are present at the apices of the cells (arrowheads); (b) Stacks of Golgi cisternae and vesicles can be seen (G) together with various organelles (m ⫽ mitochondria). Occasional desmosomes (arrowheads) link adjoining cells; (c) Stromal cells lying beneath and parallel to the epithelial basement membrane (arrowheads) contain narrow cisternae of rough endoplasmic reticulum and sparse organelles in their scanty cytoplasm. The extracellular matrix is flocculent. Scale bars: 2 µm.

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 2682

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). The stromal cells were markedly altered from the control state, exhibiting a secretory phenotype similar to that


Figure 3. Cycling females treated with bioactive HCG. (a) Apical part of the plaque showing large, euchromatic nuclei and pale cytoplasm and small, electron-dense mitochondria; (b) Vacuoles containing cell debris (*) are present; (c) Stromal cell (S) processes (arrows) can be seen between nests of plaque cells—the difference in the nuclear size between plaque and stromal cells is very apparent here; (d) Stromal cell showing signs of decidualisation, with many cisternae of rough endoplasmic reticulum, large pale mitochondria (m) and masses of cytoplasmic filaments in the cell blebs and cytoplasm (arrows). Note the close contact between the cells and their irrregular profiles. Scale bars: (a), (b) and (c) 5 µm; (d) 2 µm.

associated with decidualization, being large and plump with large round nuclei, little heterochromatin and large nucleoli. Their profile was irregular with many blunt processes and blebs (Figure 3d). Mitochondria were small and dark and the cisternae of rough endoplasmic reticulum were often dilated with flocculent contents. Masses of fine cytoplasmic filaments could be seen within the cells’ processes (Figure 3d) and also running under the plasma membrane; sometimes these showed periodicity. Glycogen was dispersed throughout the cells and occasionally formed clumps, and some fat droplets were present. Cells made contact with each other via fine processes and very little extracellular matrix could be seen due to the close packing of the cellular components. Occasionally, granular cells were present (Figure 4a); these

were possibly mast cells as some of the granules had electron-lucent areas within, as described previously (Jones et al., 1988). In the deeper stroma, the cells were more widely spaced with more heterochromatic nuclei and a smaller volume of cytoplasm, resembling the stromal cells of the control animals (Figure 4b). Cycling female treated with heat-inactivated HCG (day 10 post-ovulation). Light microscopy There was no plaque formation in this specimen (Figure 1c), which had an even, regular luminal epithelium composed of cells with domed apices. The stroma was oedematous in parts, with widely spaced spindle-shaped cells. 2683


Figure 4. Cycling females treated with bioactive HCG. (a) A mast cell with characteristic granules containing electron lucent areas lies adjacent to stromal cells with well-developed cytoplasmic filaments (arrows); (b) Deeper into the stroma the widely-spaced cells assume a more normal phenotype. Scale bars: (a) 2 µm; (b) 5 µm.

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. The stromal cells were widely spaced (Figure 5c), though in contact via slender cell processes. Little cytoplasm was evident around the nuclei, and it contained mitochondria, narrow cisternae of rough endoplasmic reticulum and the occasional fat or secretory droplet. Cytoplasmic filaments were not prominent in these cells. Cycling female treated with recombinant FSH (day 10 postovulation). 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 2684

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. The extracellular matrix was fine and amorphous with occasional collagen fibrils, usually well dispersed. Cells were spaced well apart, with large, pale nuclei and little cytoplasm (Figure 6c). They were generally orientated parallel to the epithelial layer, and made contact with each other via their narrow cell processes. Electron-lucent mitochondria and short strands of rough endoplasmic reticulum were present, with occasional Golgi bodies. Some fat droplets were seen. 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


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 syncytiotrophoblast in this area of the epithelium. In the stroma, large, electron-lucent decidualized cells with many blunt processes were packed closely together under the plaque, separated by a dense extracellular matrix, and also extending up between the nests of plaque cells. Nuclei were generally large and euchromatic with prominent nucleoli (Figure 7d). Numerous parallel cisternae of rough endoplasmic reticulum were present suggesting that these cells were exhibiting a secretory phenotype, and clumps of fine intracytoplasmic filaments were seen along the cell periphery (Figure 8a) and in cell pseudopodia. Fat droplets were frequently seen in these cells, together with secretory droplets. Adjacent to the plaque, cells tended to be very closely in apposition, with adjacent plasma membranes interdigitating in places. Deeper into the stroma, cells adopted a more spindle-shaped appearance and were more dispersed (Figure 8b), and occasional large granular lymphocytes were also present. Here, the extracellular matrix was flocculent with occasional bundles of collagen. Figure 5. Cycling female treated with heat-inactivated HCG. (a) Low-power view showing columnar cells with a microvillous surface and pale nuclei. A small part of the stroma can be seen bottom left; (b) Higher power to show Golgi bodies (G), and other organelles; (c) Cells in the stroma are spindle-shaped and widelyspaced, as in the normal control. Scale bars: (a) and (c) 5 µm; (b) 1 µm.

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

Immunocytochemistry Cytokeratin (CAM 5.2) Epithelial components were stained by this antibody, and the change in the histology of the luminal epithelium from being a regular, smooth layer of columnar cells, as seen in the cycling animal treated with heat-inactivated HCG and the FSH control, to the round nests of plaque cells in the HCG treated and pregnant animals, was very striking. The epithelia of the uterine glands were also evident with this antibody (Figure 9a-d).

α-smooth muscle actin There was no significant binding of this antibody to stromal components in the cycling animal, nor in the ones treated with heat-inactivated HCG or FSH. However, in the 15-day pregnant baboon and that treated with HCG there was strong cytoplasmic binding to decidualized stromal cells under the luminal epithelium (Figure 9e-h). Discussion One of the earliest reports of plaque formation in primates as a consequence of hormonal manipulation (Hisaw et al., 1937) described the treatment of castrated macaque monkeys with oestrogen and progestin. This produced a significant increase in the size of the uterus, with marked mitotic activity in the endometrium. In some cases the endometrium was traumatised during the course of treatment, either by biopsy or by inserting threads into the body of the uterus. This resulted in a typical plaque response involving the entire luminal epithelium, with clumps of epithelial cells in which mitotic figures were common, with many cells reaching the proportions of giant cells and fusing with their neighbours. Stromal changes were also described, though these were initially mainly oedematous 2685


Figure 6. Cycling female treated with Alzet-delivered FSH. (a) Low power of columnar luminal epithelium with apical microvilli and a smooth basal surface. Apical junctional complexes can be seen (arrowheads) and the nuclei are pale and euchromatic; (b) Detail of the cytoplasmic organization showing Golgi bodies (G), mitochondria (m) and numerous desmosomes (arrowheads); (c) Stromal cells have scanty cytoplasm containing rough endoplasmic reticulum and mitochondria. The extracellular matrix is amorphous in character with few fibrillar elements. Scale bars: (a) 5 µm; (b) 1 µm; (c) 2 µm.

in nature; after 15 days, however, the endometrium became rich in closely packed, round stromal cells, suggestive of decidualization. Similar responses were also noted in the rhesus monkey after theelin (Rossman, 1940) or oestrogen and/or progesterone treatment followed by an artificial deciduogenic stimulus (Marston et al., 1971; Ghosh and Sengupta, 1989; Sengupta et al., 1990). The primate therefore differs from the rodent in that the induction of decidualization by means of artificial stimuli to produce a deciduoma, involves both the epithelial and stromal components of the uterus, whereas in the rodent the response is limited to the stromal cells (Krehbiel, 1937; O’Shea et al., 1983). These studies also show that such a response can be initiated without a contribution from the embryo, although Ghosh and Sengupta admit that while the 2686

use of such a model contributes to our understanding of the role of steroids in the initiation of pregnancy, the role of conceptus-secreted compounds in the onset of the decidual cell reaction in the primate is not elucidated by these means (Ghosh and Sengupta, 1989). 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,


Figure 7. Female at day 15 of pregnancy. (a) Plaque surface, showing a cell containing a vacuole containing cell debris (*) and many organelles, with some dispersed glycogen (g) near a junctional complex (arrowhead); (b) A binucleate plaque cell can be seen (arrows), together with cells containing secretory droplets and vacuoles containing cell debris; (c) A cell with a very large nucleus (N) within which are invaginations of cytoplasm containing glycogen masses; (d) Stromal cells (S) with masses of endoplasmic reticulum lie closely against each other and the base of the plaque (P) where three dark masses of glycogen (arrowheads) can be seen. Scale bars: (a) 5 µm; (b) and (c) 10 µm; (d) 3 µm.

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 (13–40 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 2687


Figure 8. Female at day 15 of pregnancy. (a) Intracytoplasmic filaments are present in these stromal cells (arrowheads) which have many surface protrusions and are filled with strands of rough endoplasmic reticulum; (b) Deeper parts of the stroma contain cells of a more normal phenotype, with wide spaces between them. Scale bars: (a) 1 µm; (b) 10 µm.

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. 2688

In the baboon, the expression of α-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.,


Figure 9. Immunocytochemistry: (a)–(d) Cytokeratin staining (CAM 5.2); (e)–(h) α-smooth muscle actin. (a) A normal cycling baboon at day 10 post-ovulation; (b) Infusion of HCG; (c) Treatment with FSH; (d) 15 day pregnant animal. The cytokeratin is present in the luminal epithelium which is regular in (a) and (c) but shows a marked plaque response in (b) and (d). (e) A normal cycling baboon at day 10 postovulation; (f) Infusion of HCG; (g) Treatment with FSH; (h) 15 day pregnant animal. There is little staining with the antibody in (a) and (c) but infusion of HCG (f) and pregnancy (h) induces a marked staining reaction in the stromal cells. Note: All figures are printed at the same magnification. Scale bar: 50 µm.

1997), with an increase in the α1, α3, α6, β1 and αvβ3 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.

Acknowledgements We thank Ms. Patricia Mavrogianis and Ms. Allison Brudney for their expert technical assistance and are grateful to The Wellcome Trust for a Travel Grant to CJPJ. These studies were conducted as part of the National Co-operative Program on Markers for Uterine Receptivity and funded by NIH grant HD 29964 (to ATF).

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